Marcin Renke

Transcription

Marcin Renke

GDAŃSKI UNIWERSYTET MEDYCZNY
Marcin Renke
AKTUALNE MOŻLIWOŚCI LECZENIA
NEFROPROTEKCYJNEGO – BLOKADA UKŁADU
RENINA-ANGIOTENSYNA-ALDOSTERON
I CO DALEJ?
Rozprawa habilitacyjna
Katedra i Klinika Nefrologii, Transplantologii
i Chorób Wewnętrznych Gdańskiego Uniwersytetu Medycznego
Kierownik: Prof. dr hab. med. Bolesław Rutkowski
Gdańsk 2010
Wydano za zgodą
Senackiej Komisji Wydawnictw
Gdańskiego Uniwersytetu Medycznego
Wydawca: Gdański Uniwersytet Medyczny
Druk: Dział Wydawnictw GUMed
Gdańsk, ul. Marii Skłodowskiej-Curie 3a
Zlecenie KW/10/11
SPIS TREŚCI
WYKAZ PUBLIKACJI BĘDĄCYCH PRZEDMIOTEM ROZPRAWY
HABILITACYJNEJ ................................................................................................................... 5
SPIS UŻYWANYCH SKRÓTÓW ............................................................................................ 7
1. WSTĘP .................................................................................................................................. 9
1.1. Przewlekła Choroba Nerek, epidemiologia i patogeneza ........................................... 9
1.2. Nefroprotekcja i aktualne możliwości jej optymalizacji........................................... 10
2. CEL BADAŃ ...................................................................................................................... 12
3. MATERIAŁ I METODY .................................................................................................... 13
4. OMÓWIENIE WYNIKÓW ................................................................................................ 14
4.1. Dawkowanie Inhibitorów Konwertazy Angiotensyny w nefroprotekcji ................. 14
4.2. Terapia potrójna hamująca układ Renina-Angiotensyna-Aldosteron ....................... 15
4.3. Zastosowanie N-acetylocysteiny w nefroprotekcji ................................................... 16
4.4. Zastosowanie atorwastatyny u chorych z PChN....................................................... 17
4.5. Zastosowanie pentoksyfiliny w nefroprotekcji ......................................................... 18
4.6. Bezpieczeństwo stosowania badanych schematów podawania leków potencjalnie
nefroprotekcyjnych ................................................................................................... 19
4.7. Perspektywy i dalsze badania.................................................................................... 19
4.8. Krytyczna ocena materiału i metod........................................................................... 20
5. PODSUMOWANIE ............................................................................................................ 21
6. WNIOSKI............................................................................................................................ 23
7. PIŚMIENNICTWO ............................................................................................................. 24
8. PRACE BĘDĄCE PRZEDMIOTEM ROZPRAWY .......................................................... 31
WYKAZ PUBLIKACJI BĘDĄCYCH PRZEDMIOTEM
ROZPRAWY HABILITACYJNEJ
Praca A: Tylicki L., Renke M., Rutkowski P., Larczyński W., Aleksandrowicz E., ŁysiakSzydłowska W., Rutkowski B. Dual blockade of the renin-angiotensin-aldosterone system
with high-dose angiotensin-converting enzyme inhibitor for nephroprotection: an open, controlled, randomized study. Scand. J. Urol. Nephrol.2008; vol. 42, nr 4, s. 381-388.
(IF 0,909; KBN/MNiSW 10)
Praca B: Renke M., Tylicki L., Knap N., Rutkowski P. Neuwelt A., Petranyuk A. Larczyński
W., Woźniak M., Rutkowski B.: High-dose angiotensin-converting enzyme inhibitor attenuates oxidative stress in patients with chronic kidney disease. Nephrol. Dial. Transplant. 2009;
vol. 24, nr 2, s. 689-690.
Praca C: Tylicki L., Rutkowski P., Renke M., Larczyński W., Aleksandrowicz E., ŁysiakSzydłowska W., Rutkowski B. Triple pharmacological blockade of the renin-angiotensinaldosterone system in nondiabetic CKD : an open-label crossover randomized controlled trial.
Am. J. Kidney Dis. 2008; vol. 52, nr 3, s. 486-493.
Praca D: Renke M., Tylicki L., Knap N., Rutkowski P., Neuwelt A., Larczyński W.,
Woźniak M., Rutkowski B.: Spironolactone attenuates oxidative stress in patients with chronic kidney disease. Hypertension 2008; vol. 52, s. e132-e133.
Praca E: Renke M., Tylicki L., Rutkowski P., Larczyński W., Aleksandrowicz E., ŁysiakSzydłowska W., Rutkowski B. The effect of N-acetylcysteine on proteinuria and markers of
tubular injury in non-diabetic patients with chronic kidney disease : a placebo-controlled, randomized, open, cross-over study. Kidney Blood Press. Res. 2008; vol. 31, nr 6, s. 404-410.
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Praca F: Renke M., Tylicki L., Rutkowski P., Larczyński W., Neuwelt A., Aleksandrowicz
E., Łysiak-Szydłowska W., Rutkowski B. The effect of N-acetylcysteine on blood pressure
and markers of cardiovascular risk in non-diabetic patients with chronic kidney disease: a
placebo-cotrolled, randomized, cross-over study. Med. Sci. Monit. 2010; 16, 7, s 13-18.
Praca G: Tylicki L., Renke M., Rutkowski P., Larczyński W., Aleksandrowicz E., ŁysiakSzydłowska W., Rutkowski B. Effects of N-acetylcysteine on angiotensin converting enzyme
plasma activity in patients with chronic kidney diseases. Blood Purif. 2008; 26, 4, s. 354.
Praca H: Renke M., Tylicki L., Rutkowski P., Neuwelt A., Larczyński W., Ziętkiewicz M.,
Aleksandrowicz E., Łysiak-Szydłowska W., Rutkowski B. Atorvastatin improves tubular status in non-diabetic patients witch chronic kidney disease – placebo controlled, randomized,
cross-over study. Acta Biochim. Pol. 2010; vol. 57, nr 4, s 547-552.
Praca I: Renke M., Knap N., Tylicki L., Rutkowski P. Neuwelt A., Larczyński W., Woźniak
M., Rutkowski B.: Atorvastatin attenuates oxidative stress in patients witch chronic kidney
disease. Med. Sci. Monit. 2010; vol. 16, nr 3, s. LE3.
Praca J: Renke M., Rutkowski P., Tylicki L., Ziętkiewicz M., Larczyński W., Rutkowski B.
Pentoksyfilina stary lek czy nowa nadzieja nefrologii? Przegl. Lek. 2008; 65, 7/8, s. 358-361.
(KBN/MNiSW 4)
Praca K: Renke M., Tylicki L., Rutkowski P., Knap N., Ziętkiewicz M., Neuwelt A., Aleksandrowicz E., Łysiak-Szydłowska W., Woźniak M., Rutkowski B. Effect of pentoxifylline
on proteinuria, markers of tubular injury and oxidative stress in non-diabetic patients with
chronic kidney disease : placebo controlled, randomized, cross-over study. Acta Biochim. Pol.
2010; vol. 57, nr 1, s. 119-123.
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SPIS UŻYWANYCH SKRÓTÓW
α1m
- α1-mikroglobulina
AlAT
- aminotransferaza alaninowa
AspAT
- aminotransferaza asparaginianowa
ARA
- antagonista receptora AT-1 dla Angiotensyny II
ATO
- atorwastatyna
CK
- kinaza kreatynowa
CRP
- białko C-reaktywne
GFR
- wskaźnik filtracji kłębuszkowej
eGFR
- wyliczony wskaźnik filtracji kłębuszkowej
GSH
- glutation
IKA
- Inhibitor Konwertazy Angiotensyny
KDIGO
- The Kidney Disease: Improving Global Outcomes
K/DOQI
- Kidney Disease Outcomes Quality Initiative
NAC
- N-acetylocysteina
NAG
- N-Acetylo-β-D-Glukozamina
NHANES III - Third National Health and Nutrition Examination Survey
NKF
- National Kidney Foundation
PIIINP
- aminokońcowy propeptyd prokolagenu typu III
PChN
- Przewlekła Choroba Nerek
PNN
- przewlekła niewydolność nerek
PTF
- pentoksyfilina
RAA
- układ renina-angiotensyna-aldosteron
UCK GUMed - Uniwersyteckie Centrum Kliniczne Gdańskiego Uniwersytetu Medycznego
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1. WSTĘP
1.1. Przewlekła Choroba Nerek, epidemiologia i patogeneza
Przewlekłą Chorobę Nerek (PChN) rozpoznajemy, zgodnie z zaleceniami amerykańskiej
organizacji National Kidney Foundation (NKF), gdy spełniony jest jeden z poniższych warunków: co najmniej przez 3 miesiące obserwuje się uszkodzenie nerek czynnościowe lub
strukturalne z prawidłowym lub zmniejszonym wskaźnikiem filtracji kłębuszkowej (GFR) lub
GFR wynosi w tym okresie stale poniżej 60 ml/min/1,73 m2. Klasyfikacja PChN wg NKF
została zawarta w tabeli 1.
Wyniki badań epidemiologicznych przeprowadzonych w wielu krajach na różnych kontynentach wskazują, że PChN może występować u 6 a nawet 15% badanej populacji, co stanowi 380, a nawet 870 milionów (średnio około 600 mln) ludzi na świecie. Dane na ten temat
uzyskano między innymi z badań: NHANES III, przeprowadzonego w Stanach Zjednoczonych Ameryki Północnej, AusDiab w Australii, OGHMA w Japonii i PREVEND oraz HUNT
zakończonych w Holandii i Norwegii. W Polsce wyniki badania PolNef wskazują, że problem
ten może dotyczyć nawet 4 milionów osób [14]. Nawet te szacunkowe dane wskazują, że
PChN jest istotnym problemem epidemiologicznym, który stanowi również poważny problem
ekonomiczny dla większości krajów świata. Skłania to nefrologów na całym świecie do poszukiwania skutecznych metod nefroprotekcji, które byłyby w stanie zmniejszyć liczbę chorych u których dochodzi do progresji choroby w kierunku schyłkowej niewydolności nerek.
W efekcie można by doprowadzić do zwolnienia narastania zapotrzebowania na kosztowne
leczenie nerkozastępcze. W 2006 roku w Polsce liczba osób dializowanych wzrosła o 5,24%,
a w 2007 o 6,27% (865 osób), łącznie dializowano w 2007 roku 18 214 chorych. Oznacza to,
że liczba leczonych dializami stale wzrasta i roczny przyrost jest podobny jak w większości
krajów europejskich (4-6%). Wszystko wskazuje na to, że zgodnie z przewidywaniami zawartymi w „Raporcie o Stanie Leczenia Nerkozastępczego – 2002” liczba chorych leczonych
nerkozastępczo w naszym kraju w 2010 roku wyniesie 27 000 pacjentów [38].
Warto dodać, że we wszystkich badaniach epidemiologicznych znajduje potwierdzenie to, że
czynnikami mającymi wpływ na częstsze występowanie PChN są nadciśnienie tętnicze, cukrzyca,
płeć męska, otyłość, wiek i palenie tytoniu. Skuteczne działania nefroprotekcyjne, a więc postępowanie mające na celu ochronę funkcji nerek u chorych ze stadiami PChN od I do IV, są nie–9–
zmiernie ważne i powinny być wspierane nie tylko przez nefrologów, ale również organizatorów ochrony zdrowia w naszym kraju. Poszukiwanie skutecznych metod nefroprotekcji, również farmakologicznych, jest tylko częścią trudnego zadania zahamowania epidemii chorób
cywilizacyjnych, które niewątpliwie mają wpływ na jakość i długość życia współczesnego
człowieka.
Tabela 1. Klasyfikacja przewlekłej choroby nerek według NKF K/DOQI w modyfikacji
KDIGO
Stadium Opis
1
2
3
GFR (ml/min/1,73 m2)
Uszkodzenie
nerek z prawidłowym GFR
Uszkodzenie
nerek z niewielkim ↓ GFR
Umiarkowane ↓
GFR
> 90
Inne określenia
Albuminuria, białkomocz, hematuria
T, jeżeli po
przeszczepie
nerki
Utajona PNN
60-89
30-59
4
Znaczne ↓ GFR
15-29
5
Schyłkowa niewydolność nerek
< 15 (lub dializa)
Wyrównana PNN
Zaawansowana
PNN
Niewyrównana
PNN, mocznica
D, jeżeli
dializowany
NKF K/DOQI – The National Kidney Foundation Kidney Disease Outcomes Quality Initiative, KDIGO – The Kidney Disease: Improving Global Outcomes, PNN – przewlekła niewydolność nerek. Według: Levey A.S. i wsp.: Kidney Int. 2005, 67,2089-2100. [15]
1.2. Nefroprotekcja i aktualne możliwości jej optymalizacji
Ochrona funkcji nerek powinna być brana pod uwagę, u każdego człowieka, także
zdrowego. Wielu chorobom nerek można zapobiegać, a u pacjenta z rozpoznaną PChN spowolnić jej postęp. Należy dodać, że spowalniając postęp PChN opóźnia się nie tylko moment
rozpoczęcia leczenia nerkozastępczego, ale również zmniejsza się ryzyko śmierci wskutek
powikłań sercowo-naczyniowych.
Wprowadzenie do praktyki klinicznej postępowania nefroprotekcyjnego stało się możliwe po poznaniu i analizie mechanizmów leżących u podstaw postępującego uszkodzenia ne-
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rek. Ważna okazała się hipoteza postawiona przez Brennera, który wskazał na istotną rolę
zmian hemodynamicznych wewnątrz kłębuszków nerkowych w odpowiedzi na uszkodzenie
nefronów [7]. Stało się to podstawą do badań, które potwierdziły rolę nadmiernej aktywacji
układu renina-angiotensyna-aldosteron (RAA) w postępie niewydolności nerek. Przeprowadzono również szereg badań, które udowodniły nefroprotekcyjny potencjał leków hamujących
układ RAA. Były to między innymi duże badania kliniczne potwierdzające właściwości inhibitorów konwertazy angiotensyny I (IKA) wśród chorych z nefropatią cukrzycową (Collaborative Study Group, BENEDICT) i niecukrzycową (REIN, AASK) oraz antagonistów receptora AT-1 dla angiotensyny II (ARA): IDNT, RENAAL, DETAIL i wiele innych [45]. Również w Klinice Nefrologii, Transplantologii i Chorób Wewnętrznych od końca lat 90. poprzedniego stulecia przeprowadzono szereg programów poświęconych optymalizacji leczenia
nefroprotekcyjnego przy pomocy farmakologicznej blokady układu RAA [33,34,39,49], które
były podstawą rozprawy habilitacyjnej Kolegi Leszka Tylickiego. Niewątpliwie farmakologiczna blokada układu RAA jest podstawową strategią nefroprotekcyjną stosowaną w leczeniu pacjentów z PChN. Jednak w ten sposób nie udaje się całkowicie zahamować postępu
choroby. Prowadzi to do poszukiwania uzupełniających strategii terapeutycznych i/lub modyfikacji dotychczas stosowanych. Cykl badań przeprowadzonych w ostatnich latach pod kierownictwem Pana Profesora Bolesława Rutkowskiego, który miał na celu poprawę istniejących standardów postępowania nefroprotekcyjnego u chorych z niecukrzycową przyczyną
PChN stał się podstawą niniejszej rozprawy habilitacyjnej.
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2. CEL BADAŃ
• Ocena wpływu terapii skojarzonej ARA i IKA w dawkach ponad maksymalnych na
białkomocz, biomarkery uszkodzenia cewek nerkowych, włóknienia i stresu oksydacyjnego u chorych z PChN nie będącą następstwem cukrzycy.
• Ocena wpływu terapii skojarzonej potrójnej blokującej układ RAA (IKA, ARA i antagonista aldosteronu) na białkomocz i wydalanie z moczem biomarkerów uszkodzenia
cewek nerkowych, stresu oksydacyjnego oraz włóknienia u chorych z PChN nie będącą następstwem cukrzycy.
• Ocena wpływu dodania N-acetylocysteiny (NAC) do terapii blokującej układ RAA na
wartości ciśnienia tętniczego, aktywność osoczową enzymu konwertującego angiotensynę, białkomocz, homocysteinę i biomarkery uszkodzenia cewek nerkowych u chorych z PChN nie będącą następstwem cukrzycy.
• Ocena wpływu dołączenia atorwastatyny (ATO) do terapii blokującej układ RAA na
białkomocz i wydalanie z moczem biomarkerów uszkodzenia cewek nerkowych oraz
stresu oksydacyjnego u chorych z PChN nie będącą następstwem cukrzycy.
• Ocena wpływu dodania pentoksyfiliny (PTF) do terapii blokującej układ RAA na
białkomocz i wydalanie z moczem biomarkerów uszkodzenia cewek nerkowych oraz
stresu oksydacyjnego u chorych z PChN nie będącą następstwem cukrzycy.
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3. MATERIAŁ I METODY
Przeprowadzono badania, wśród chorych w wieku od 18 do 65 lat z białkomoczem
pochodzenia niecukrzycowego, z prawidłową lub nieznacznie upośledzoną funkcją nerek będących pod stałą opieką Poradni Chorób Nerek przy UCK GUMed w latach 2004 – 2008.
Badania rozpoczynano od okresu wstępnego w którym chorzy otrzymywali leczenie nefroprotekcyjne z użyciem leków blokujących układ RAA (IKA i/lub ARB). Pacjenci byli kwalifikowani do dalszej części projektu jeżeli ich wartości ciśnienia tętniczego były niższe od
130/80 mm Hg. Następnie chorzy byli randomizowani do badania i w zależności od schematu
badawczego, szczegółowo opisanego w poszczególnych artykułach, otrzymywali dalsze leczenie. Oznaczenia wykonywano w trakcie randomizacji i po każdym okresie badania. Podczas prowadzonych badań oznaczono: ciśnienie tętnicze krwi, kreatyninę, poziom potasu w
surowicy krwi, białkomocz dobowy, albuminurię, biomarkery uszkodzenia cewek nerkowych
oznaczane w moczu: α1-mikroglobulinę (α1m) [12] i N-Acetylo-β-D-Glukozaminę (NAG)
[3] i pośredni marker włóknienia – aminokońcowy propeptyd prokolagenu typu III (PIIINP)
[41] oraz wydalanie z moczem 15-F2α-izoprostanów (biomarker stresu oksydacyjnego). Ponadto w wybranych badaniach oznaczano aktywność reninową osocza, poziom homocysteiny,
wysoko czułe białko C-reaktywne (hsCRP), aminotransferazy alaninowej (ALAT), aminotransferazy asparaginianowej (AspAT), kinazy kreatynowej (CK) oraz poziomy cholesterolu i
triglicerydów. Łącznie przeprowadzono badania u 92 chorych. Szczegółowy opis grup badanych pacjentów i stosowanych metod badawczych został zawarty w poszczególnych artykułach będących przedmiotem rozprawy habilitacyjnej, zamieszczonych w rozdziale 8.
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4. OMÓWIENIE WYNIKÓW
4.1. Dawkowanie Inhibitorów Konwertazy Angiotensyny
w nefroprotekcji
Jak do tej pory nie ustalono optymalnego nefroprotekcyjnego dawkowania IKA oraz
ARA. Udowodniono, że zarówno małe, jak i standardowo stosowane dawki IKA i ARA w
leczeniu nadciśnienia tętniczego zmniejszają białkomocz oraz mają korzystny wpływ na biomarkery uszkodzenia cewek nerkowych. Jednocześnie efekt ten jest zależny od dawki. Małe
dawki IKA, ramiprilu w badaniu DIABHYCAR, nie wpływały na zwolnienie progresji
uszkodzenia nerek, pomimo zmniejszenia albuminurii [18]. Wydawać się więc by mogło, że
w celu zapewnienia skutecznej nefroprotekcji stosować powinno się wysokie dawki leków
hamujących układ RAA, oczywiście o ile nie występują działania uboczne stosowanych preparatów. Biorąc pod uwagę badania doświadczalne, które wskazywały na potencjalnie korzystne stosowanie dawek supramaksymalnych leków blokujących układ RAA przeprowadzono badanie, które miało na celu odpowiedź na pytanie czy stosowanie ponad maksymalnych dawek IKA ma sens z punktu widzenia dalszej redukcji białkomoczu i ograniczenia
uszkodzenia cewek nerkowych. W pracy A [46] wykazano, że podwojenie dawki cilazaprilu
pomimo zwiększonej blokady układu RAA, którą określono badając aktywność reninową
osocza, nie miało wpływu na białkomocz, biomarkery uszkodzenia cewek nerkowych oznaczane w moczu: α1m i NAG i pośredni marker włóknienia - PIIINP. Wnioski wypływające z
tej pracy były zbieżne z doniesieniem Hasa i wsp., którzy stwierdzili, że stosowanie spiramilu
w dawce dwukrotnie większa niż maksymalnie stosowana do leczenia nadciśnienia tętniczego
nie miało wpływu na redukcję białkomoczu [11]. Warto dodać, że stosowanie ponad maksymalnych dawek ARA (telmisartan, losartan, irbesartan) miało korzystny wpływ na białkomocz i spowolnienie postępu przewlekłych nefropatii przebiegających z białkomoczem
[1,36,50]. Przyczyny różnego wpływu ponad maksymalnych dawek IKA i ARA na progresję
PChN są nie do końca jasne. Warto dodać, że podwojona dawka cilazaprilu miała korzystny
wpływ na redukcję parametrów stresu oksydacyjnego (praca B) [28]. Oznaczano wydalanie z
moczem 15-F2α-izoprostanów, które pod wpływem stosowanego leku zmniejszyło się istotnie
statystycznie. Być może w ten sposób stosowane dawki ponad maksymalne IKA mogą korzystnie wpływać na hamowanie progresji PChN, ponieważ uważa się, że izoprostany mają
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również pewną aktywność biologiczną, jako substancje o właściwościach obkurczających
naczynia nerkowe [42].
4.2. Terapia potrójna hamująca układ Renina-AngiotensynaAldosteron
Terapia skojarzona dwulekowa blokująca układ RAA, polegająca na jednoczesnym stosowaniu leków z grupy ARA i IKA prowadzi do lepszej ochrony nerek niż monoterapia ARA
lub IKA wśród chorych z PChN i współistniejącym białkomoczem. Na zasadność takiego
rozumowania wskazują wyniki badań eksperymentalnych, jak również analiza mechanizmów
działania obu grup leków. Udało się nam potwierdzić te przypuszczenia wykazując, że leczenie skojarzone zmniejsza białkomocz oraz ogranicza uszkodzenie cewek nerkowych w stopniu większym, niż monoterapia lekami obu grup wśród chorych z PChN i białkomoczem
[33,49]. Wnioski wypływające z naszych badań zostały potwierdzone przez innych badaczy
[35,37]. Entuzjazm stosowania terapii skojarzonej został zmącony przez wyniki badania
ONTARGET [16], które nie wykazało korzyści terapii skojarzonej ARA i IKA nad monoterapią. Obserwowano również w tym badaniu większą ilość działań niepożądanych wśród
chorych leczonych terapią skojarzoną. Należy jednak dodać, że populację badaną stanowili w
większości pacjenci bez cech PChN, co nie pozwala na proste przeniesienie wniosków na
interesującą nas grupę pacjentów. Obecnie wielu badaczy uważa, że leczenie skojarzone ARA
i IKA może być stosowane w profilaktyce rozwoju istniejącej PChN [4,8,17,25].
Ocena terapii skojarzonej potrójnej, czyli korzyści wynikających z łącznego stosowania
IKA, ARA oraz antagonistów receptora dla aldosteronu, stanowiła kontynuację dotychczas
zakończonych prac. Wydaje się, że tego typu terapia mogłaby być skuteczniejsza od terapii
podwójnej z powodu dodatkowego ograniczenia efektów działania aldosteronu, który może
być syntetyzowany drogą niezależną od osi RAA i w ten sposób nie podlegać w pełni hamującemu wpływowi IKA oraz ARA. W naszym badaniu ocenialiśmy wpływ stosowanej terapii
na białkomocz, biomarkery uszkodzenia cewek nerkowych, włóknienia i nasilenie stresu
oksydacyjnego u pacjentów z PChN w stadium od I do III. Wyniki badania przedstawiono w
pracach C [48] i D [27]. Stwierdziliśmy, że terapia potrójna w porównaniu do podwójnej blokującej układ RAA w większym stopniu zmniejsza białkomocz, wydalanie NAG i PIIINP. Do
podobnych wniosków doszli również inni badacze [6]. Warto dodać, że terapia potrójna miała
– 15 –
również korzystny wpływ na redukcję parametrów stresu oksydacyjnego (praca D). Oznaczano wydalanie z moczem 15-F2α-izoprostanów, które pod wpływem stosowanego leczenia
zmniejszyło się istotnie statystycznie. Prowadzona terapia była bezpieczna, nikt z badanych
chorych nie przerwał programu z powodu działań niepożądanych stosowanych leków.
4.3. Zastosowanie N-acetylocysteiny w nefroprotekcji
Nie ulega wątpliwości, że farmakologiczna blokada układu RAA stanowi obecnie podstawową strategię leczenia przewlekłych nefropatii. Wprowadzenie leków hamujących układ
RAA do terapii pacjentów z uszkodzeniem nerek doprowadziło do zwolnienia tempa progresji
PChN. Do tej pory nie udało się jednak całkowicie zahamować jej postępu. Skłania to do poszukiwania uzupełniających strategii terapeutycznych. Podczas prowadzonych badań naszą
uwagę zwróciło kilka preparatów o potencjalnych możliwościach nefroprotekcyjnych. Jednym z nich była N-acetylocysteina (NAC), syntetyczny prekursor zredukowanego glutationu
(GSH), który stymuluje wewnątrzkomórkową syntezę GSH m.in. w ten sposób wpływając na
ograniczenie stresu oksydacyjnego [2]. NAC w szeregu badań doświadczalnych wykazywała
m.in. właściwości hamowania aktywności tkankowej i osoczowej enzymu konwertującego
angiotensynę. Ponadto stwierdzano obniżenie poziomu aldosteronu w surowicy krwi, homocysteiny, poprawę GFR, czy systemowego ciśnienia krwi u badanych zwierząt [23,51]. W
celu zweryfikowania hipotezy o potencjalnych właściwościach nefroprotekcyjnych NAC u
chorych z PChN przeprowadzono podwójnie ślepe, krzyżowe, randomizowane badanie kliniczne, kontrolowane placebo w Klinice Nefrologii AM w Gdańsku. Oceniano wpływ 1200
mg NAC stosowanego przez okres 8 tygodni u 20 chorych z PChN i stabilnym białkomoczem. Wykazano, że NAC nie miało wpływu na badane parametry: białkomocz dobowy, albuminurię, biomarkery uszkodzenia cewek nerkowych (NAG i α1m) i włóknienia (PIIINP),
poziom homocysteiny oraz ciśnienie tętnicze krwi. Wyniki przedstawiono w pracach E [30] i
F [31]. Potwierdzono natomiast wpływ NAC na aktywność enzymu konwertującego w badanej populacji (praca G) [47]. Podsumowując należy stwierdzić, że w przeprowadzonych krótkoterminowych badaniach nie udało się jednoznacznie potwierdzić korzystnego wpływu NAC
na ochronę funkcji nerek u chorych z PChN. Być może jednym z powodów braku korzystnych efektów NAC było stosowanie leku u chorych z stabilnym, znikomym lub miernym
białkomoczem oraz prawidłowymi poziomami homocysteiny i wartościami ciśnienia tętni– 16 –
czego krwi. Mogło to mieć wpływ na negatywne wyniki przeprowadzonych badań. Wydaje
się, że dla poznania odpowiedzi na pytanie, czy potencjalne właściwości kardio- i nefroprotekcyjne NAC mają znaczenie kliniczne, konieczne jest przeprowadzenie długoterminowych,
randomizowanych badań na znacznie większej populacji chorych z PChN.
4.4. Zastosowanie atorwastatyny u chorych z PChN
Kolejnym ocenianym preparatem była atorwastatyna (ATO). Przedstawiciel grupy leków zwanej statynami, którego niewątpliwą zaletą jest siła działania, dobra tolerancja leku i
brak konieczności modyfikacji dawki w zależności od stopnia niewydolności nerek [13].
ATO należy do inhibitorów reduktazy HMGCoA, których znaczenie w leczeniu hiperlipidemii jest obecnie powszechnie znane i akceptowane. Wiadomo również, że PChN towarzyszą
zaburzenia lipidowe, które mają niekorzystny wpływ na rokowanie w tej grupie chorych
[10,40]. Ponadto, od chwili odkrycia pierwszych statyn, trwają także badania nad innymi mechanizmami działania tych leków. W badaniach eksperymentalnych zwracano m.in. uwagę na
korzystny wpływ statyn na białkomocz i hamowanie progresji niewydolności nerek. Wyniki
badań klinicznych są niejednoznaczne, część badaczy opisywała zmniejszanie się białkomoczu pod wpływem stosowanych statyn [5,43], inni stosując wysokie dawki leków opisywali
odwrotne zjawisko bez wpływu na oceniany GFR [9,19]. W celu wyjaśnienia potencjalnych
właściwości nefroprotekcyjnych ATO i rozszerzenia wskazań do stosowania tej grupy leków
u pacjentów z PChN bez hipercholesterolemii przeprowadzono randomizowane, podwójnie
ślepe, krzyżowe, kontrolowane placebo badanie kliniczne wśród 14 chorych z PChN i białkomoczem znikomym lub miernym. Po 8 tygodniowym okresie wstępnym, kiedy optymalizowano terapię lekami blokującymi układ RAA, dodawano przez okres 12 tygodni 40 mg
ATO lub placebo, a następnie po 12 tygodniowej przerwie ponownie stosowano lek badany i
placebo przez kolejne 12 tygodni. Wyniki przedstawiono w pracy H [32]. Stwierdzono korzystny, istotny statystycznie, wpływ stosowanego ATO na biomarkery uszkodzenia cewek
nerkowych (NAG i α1m) i brak takiego działania na oznaczany białkomocz dobowy i eGFR.
Przedstawione wyniki są zbieżne z badaniami doświadczalnymi przedstawionymi przez Tsujihata i współpracowników [44]. Warto dodać, że ATO miała korzystny wpływ na redukcję
parametrów stresu oksydacyjnego. Oznaczano wydalanie z moczem 15-F2α-izoprostanów,
które pod wpływem stosowanego leku zmniejszyło się istotnie statystycznie (praca I) [24].
– 17 –
Zarówno ograniczenie stresu oksydacyjnego, jak i zmniejszenie uszkodzenia śródmiąższu
nerki mogą mieć korzystny wpływ na rokowanie wśród chorych z PChN. Dowody na kardioi nefroprotekcyjne działanie statyn wśród chorych z PChN najprawdopodobniej dostarczy
duże badanie kliniczne SHARP (ponad 9000 chorych), którego wyniki mają być przedstawione pod koniec 2010 roku.
4.5. Zastosowanie pentoksyfiliny w nefroprotekcji
Istnieje szereg teoretycznych przesłanek wskazujących na słuszność hipotezy, że terapia
nefroprotekcyjna powinna być uzupełniona przez zastosowanie pentoksyfiliny (PTF). Omówiono to szczegółowo w jednej z poglądowych publikacji autora (praca J) [26]. Poza znanym
od wielu lat działaniem na układ naczyniowy PTF ma również mieć właściwości antycytokinowe, zmniejszać nasilenie stanu zapalnego, hamować syntezę kolagenu, prowadzić do ograniczenia produkcji reaktywnych form tlenu i w efekcie zmniejszenia nasilenia stresu oksydacyjnego. Pierwsze badania kliniczne przeprowadzone wśród chorych na cukrzycę i PChN
[20,21,22] wskazują na szereg korzyści płynących z tego typu leczenia. W celu weryfikacji
hipotezy czy uzupełnienie optymalnej terapii lekami blokującymi układ RAA u chorych z
PChN bez cukrzycy o PTF może przynieść dodatkowe korzyści przeprowadzono w Klinice
Nefrologii AM w Gdańsku następujące badanie. 22 chorych z PChN i białkomoczem znikomym lub miernym po 8 tygodniach terapii optymalnej blokującej układ RAA otrzymało dodatkowo zgodnie z randomizacją 1200 mg PTF lub placebo. Następnie preparaty badane odstawiono na 8 tygodni i ponownie włączono na kolejne 8 tygodni. Wyniki tego krzyżowego,
podwójnie ślepego badania kontrolowanego placebo przedstawiono w pracy K [29]. PTF dodana do terapii blokującej układ RAA zmniejszała białkomocz (o 26%) , ale wynik ten nie
osiągnął znamienności statystycznej prawdopodobnie z powodu zbyt małej liczebności grupy
badanej. Było to spowodowane stosunkowo złą tolerancją stosowanej dawki leku. Działania
niepożądane, głównie pod postacią zaburzeń żołądkowo-jelitowych wystąpiły u blisko 23%
badanych pacjentów. PTF nie miała wpływu na oceniane wskaźniki stresu oksydacyjnego
(wydalanie z moczem 15-F2α-izoprostanów) i uszkodzenia cewek nerkowych (NAG i α1m).
Pomimo częściowo negatywnych wyników tego badania wydaje się, że dopiero duże wieloośrodkowe badanie kliniczne da nam odpowiedź na pytanie czy PTF znajdzie swoje trwałe
miejsce we współczesnej nefrologii. Dotychczas przeprowadzone badania budzą nadzieję, ale
nie dają ostatecznej odpowiedzi czy warto stosować ten lek wśród chorych z PChN.
– 18 –
4.6. Bezpieczeństwo stosowania badanych schematów podawania
leków potencjalnie nefroprotekcyjnych
Podczas prowadzonych badań nie stwierdzono niekorzystnego wpływu stosowanych leków blokujących układ RAA, NAC, ATO i PTF na poziom filtracji kłębuszkowej wyrażonej
jako eGFR. Nie wystąpiło też w żadnym badaniu istotne klinicznie podwyższenie poziomu
potasu wśród leczonych pacjentów. Podczas stosowania terapii potrójnej blokującej układ
RAA notowano podwyższenie poziomu potasu u 10 spośród 18 chorych, wartości te wynosiły
maksymalnie u 2 chorych 5,7 i 5,9 mmol/L. Natomiast przy stosowaniu ponadmaksymalnych
dawek IKA u jednego z chorych stwierdzono poziom potasu 6,2 mmol/L bez objawów klinicznych, nie znaleziono też różnic istotnych statystycznie pomiędzy poziomami potasu w
badanych grupach.
Działania niepożądane, głównie pod postacią zaburzeń żołądkowo-jelitowych wystąpiły
podczas stosowania PTF. Uniemożliwiło to ukończenie badania przez 5 pacjentów, co mogło
mieć istotny wpływ na uzyskane wyniki. Dolegliwości ustąpiły u wszystkich chorych po przerwaniu terapii z wykorzystaniem PTF. Stosowanie ATO w dawce dobowej 40 mg i NAC
1200 mg nie wiązało się z wystąpieniem istotnych działań niepożądanych w badanych grupach pacjentów. Leczenie tego typu można uznać za bezpieczne przy uwzględnieniu przeciwwskazań do stosowania poszczególnych preparatów.
4.7. Perspektywy i dalsze badania
Wydaje się konieczne kontynuowanie prac nad optymalnym blokowaniem układu RAA,
który pełni kluczową rolę w utrzymaniu homeostazy ustroju. W badaniach, które są w pewien
sposób kontynuacją podjętych tematów będziemy oceniać wpływ aliskirenu (inhibitora reniny) na funkcję i strukturę nerek, oraz wykładniki stresu oksydacyjnego. Planujemy również
ocenę terapii łączonej zawierającej aliskiren i ARA oraz porównanie z innymi rodzajami terapii hamującymi układ RAA.
Niewątpliwie uzyskane wstępne wyniki stosowania PTF u chorych z PChN są zachęcające, ale wymagają przeprowadzenia dużego wieloośrodkowego badania klinicznego, które
pozwoliłoby na odpowiedź na pytanie o faktyczne miejsce tego leku we współczesnej nefro-
– 19 –
logii. Wydaje się konieczne porównanie efektów działania niskich i maksymalnych dawek
PTF, które jak wykazują doświadczenia własne są gorzej tolerowane przez część chorych, co
znacznie ogranicza stosowanie PTF w tej grupie pacjentów.
4.8. Krytyczna ocena materiału i metod
Badane grupy chorych były niejednorodne, obejmowały chorych z niecukrzycową przewlekłą chorobą nerek przebiegającą z białkomoczem, z prawidłową lub miernie upośledzoną
funkcją nerek (PChN od I do III). Liczebność badanych grup była ograniczona z powodu
szczupłości środków finansowych przeznaczonych na prowadzone badania, ale najczęściej
wystarczająca dla potwierdzenia lub zaprzeczenia stawianych hipotez badawczych. Ponadto
wykorzystywane metody badawcze mogły być obarczone błędami, m.in. zbierana przez pacjentów dobowa zbiórka moczu może być niedokładna, co może mieć wpływ na uzyskiwane
wyniki. Ponadto korzystne działanie badanych leków na biomarkery uszkodzenia cewek nerkowych, czy też włóknienia powinno być zweryfikowane przez badania histopatologiczne,
których nie wykonywano rutynowo podczas prowadzonych badań.
– 20 –
5. PODSUMOWANIE
Tabela 2. Zestawienie badań będących podstawą rozprawy habilitacyjnej z wyszczególnieniem najważniejszych wniosków z nich wypływających.
Praca
Publikacja
Główne wnioski
Praca A
[46]
Scand J Urol Nephrol Terapia skojarzona IKA i ARA, z wykorzystaniem po2008
nad maksymalnych dawek IKA nie ma wpływu na białkomocz, badane biomarkery uszkodzenia cewek nerkowych i włóknienia u chorych z PChN i białkomoczem.
Praca B
[28]
Nephrol Dial Transplant.
2009
Terapia skojarzona IKA i ARA, z wykorzystaniem ponad maksymalnych dawek IKA ma wpływ na zmniejszenie wydalania z moczem 15-F2α-izoprostanów
(wskaźnika stresu oksydacyjnego) u chorych z PChN i
białkomoczem.
Praca C
[48]
Am J Kidney Dis.
2008
Terapia potrójna blokującej układ RAA (IKA, ARA i
antagonista aldosteronu) zmniejsza białkomocz i wydalanie badanych biomarkerów uszkodzenia cewek nerkowych i włóknienia u chorych z PChN i białkomoczem.
Praca D
[27]
Hypertension
2008
Terapia potrójna blokująca układ RAA (IKA, ARA i
antagonista aldosteronu) ma wpływ na zmniejszenie
wydalania z moczem 15-F2α-izoprostanów u chorych z
PChN i białkomoczem.
Praca E
[30]
Kidney Blood Press
Res. 2008
NAC dodane do terapii blokującej układ RAA nie ma
wpływu na białkomocz i badane biomarkery uszkodzenia cewek nerkowych u chorych z PChN i białkomoczem.
Praca F
[31]
Med. Sci. Monit.
2010
Dodanie NAC do terapii blokującej układ RAA nie ma
wpływu ciśnienie tętnicze i badane markery zagrożenia
sercowo-naczyniowego u chorych z PChN i białkomoczem.
Praca G
[47]
Blood Purif.
2008
NAC dodane do terapii blokującej układ RAA zmniejsza aktywność osoczową enzymu konwertującego angiotensynę u chorych z PChN i białkomoczem.
Praca H
[32]
Acta Biochim. Pol.
2010
ATO dodane do terapii blokującej układ RAA nie ma
wpływu na białkomocz, zmniejsza natomiast wydalanie
z moczem biomarkerów uszkodzenia cewek nerkowych
u chorych z PChN i białkomoczem.
– 21 –
Praca
Publikacja
Główne wnioski
Praca I
[24]
Med. Sci. Monit.
2010
Dodanie ATO do terapii blokującej układ RAA zmniejsza wydalania z moczem 15-F2α-izoprostanów (wskaźnika stresu oksydacyjnego) u chorych z PChN i białkomoczem.
Praca J
[26]
Przegl. Lek.
2008
Praca poglądowa omawiająca rolę pentoksyfiliny w
nefrologii
Praca K
[29]
Acta Biochim Pol.
2010
PTF dodane do terapii blokującej układ RAA zmniejsza
białkomocz o 26% (wynik nie znamienny statystycznie), nie ma natomiast wpływu na wydalanie biomarkerów uszkodzenia cewek nerkowych i stresu oksydacyjnego u chorych z PChN i białkomoczem.
W przebiegu przeprowadzonej serii badań przedstawionych w powyższych rozważaniach udowodniono, że zasady optymalnego leczenia nefroprotekcyjnego podlegają ciągłym
modyfikacjom. Wynika to z poszerzającej się wiedzy dotyczącej patogenezy PChN oraz
wprowadzania nowych leków lub poszukiwania nowych zastosowań dla preparatów znanych
już od wielu lat, o których mechanizmach działania wiemy obecnie więcej niż przed laty. Pozwala to na poszukiwanie nowych form terapii łączonej, która mogłaby pełniej chronić upośledzoną funkcję nerek i skuteczniej hamować procesy prowadzące do rozwoju ich schyłkowej niewydolności. W tabeli 2 przedstawiono wnioski płynące z przeprowadzonych badań
klinicznych. Na ich podstawie można sformułować pewne zalecenia, które mogłyby wpłynąć
na modyfikacje istniejących standardów dotyczących postępowania nefroprotekcyjnego. Wydaje się, że w określonych grupach chorych z PChN powinna znaleźć zastosowanie terapia
potrójna (IKA, ARA i antagonista aldosteronu), która stosowana świadomie może przynieść
wymierne korzyści pacjentom i nie narażać ich na działania niepożądane stosowanych leków.
Naszym zdaniem również ATO lub inna statyna powinny znaleźć stałe miejsce w postępowaniu nefroprotekcyjnym.
– 22 –
6. WNIOSKI
• Terapia skojarzona ARA i IKA w dawkach ponad maksymalnych nie ma wpływu na
białkomocz, badane biomarkery uszkodzenia cewek nerkowych i włóknienia u chorych z PChN nie będącą następstwem cukrzycy. Leczenie to zmniejsza wydalanie z
moczem izoprostanów. Świadczy to o ograniczeniu stresu oksydacyjnego, ale również
może mieć bezpośredni korzystny efekt na naczynia nerkowe.
• Terapia potrójna blokująca układ RAA (IKA, ARA i antagonista aldosteronu) zmniejsza białkomocz i wydalanie z moczem badanych biomarkerów uszkodzenia cewek
nerkowych, stresu oksydacyjnego oraz włóknienia u chorych z PChN nie będącą następstwem cukrzycy.
• NAC dodane do terapii blokującej układ RAA nie ma wpływu na białkomocz, wartości ciśnienia tętniczego, homocysteinę i biomarkery uszkodzenia cewek nerkowych u
chorych z PChN nie będącą następstwem cukrzycy. Dołączenie NAC do terapii nefroprotekcyjnej zmniejsza aktywność osoczową enzymu konwertującego angiotensynę w
w./w. grupie chorych.
• Dodanie ATO do terapii blokującej układ RAA zmniejsza wydalanie z moczem badanych biomarkerów uszkodzenia cewek nerkowych i izoprostanów u chorych z PChN
nie będącą następstwem cukrzycy. Tego typu leczenie nie ma dodatkowego wpływu
na białkomocz w w./w. grupie chorych.
• PTF dodana do terapii blokującej układ RAA zmniejsza białkomocz o 26% (wynik
nieznamienny statystycznie), nie ma natomiast wpływu na wydalanie z moczem izoprostanów oraz biomarkerów uszkodzenia cewek nerkowych u chorych z PChN nie
będącą następstwem cukrzycy.
• Leczenie nefroprotekcyjne podlega i nadal będzie podlegać indywidualizacji i optymalizacji w celu pełniejszej ochrony funkcji nerek.
– 23 –
7. PIŚMIENNICTWO
1. Aranda P., Segura J., Ruilope L.M., Aranda F.J., Frutos M.A. Lopez V.: Long-term
renoprotective effects of standard versus high doses of telmisartan in hypertensive
nondiabetic nephropathies. Am. J. Kidney Dis. 2005, 46, 1074-1079.
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8. PRACE BĘDĄCE PRZEDMIOTEM ROZPRAWY
Scandinavian Journal of Urology and Nephrology, 2008; 42: 381388
ORIGINAL ARTICLE
Scand J Urol Nephrol Downloaded from informahealthcare.com by (ACTIVE) Karolinska Institutet University Library on 11/30/10
For personal use only.
Dual blockade of the renin angiotensin aldosterone system with
high-dose angiotensin-converting enzyme inhibitor for
nephroprotection: An open, controlled, randomized study
LESZEK TYLICKI1, MARCIN RENKE1, PRZEMYSLAW RUTKOWSKI1,
WOJCIECH LARCZYŃSKI1, EWA ALEKSANDROWICZ2,
WIESLAWA LYSIAK-SZYDLOWSKA2 & BOLESLAW RUTKOWSKI1
Department of 1Nephrology, Transplantology and Internal Medicine, and 2Clinical Nutrition and Laboratory Diagnostics,
Medical University of Gdańsk, Gdańsk, Poland
Abstract
Objective. Despite the proven effectiveness of combination therapy with an angiotensin I-converting enzyme inhibitor
(ACEI) and angiotensin II-receptor blockers (ARBs) for the prevention and treatment of kidney disease, it has not proved
possible to inhibit the progress of chronic nephropathies completely. To improve renal outcome one may consider using
increased dosages of ACEI above those usually recommended for hypertension. Material and methods. A randomized, open,
controlled study was conducted to evaluate the influence of two combination therapies on proteinuria, markers of tubular
injury and renal fibrosis. A total of 18 patients with a creatinine level of 109936 mmol/l and proteinuria of 0.9790.76 g/24 h
were enrolled in the study. In the 8-week run-in period, an ACEI (cilazapril 5 mg once-daily) and an ARB (telmisartan
80 mg once-daily) were administered to achieve the target blood pressure of 5130/80 mmHg. Next, the patients were
randomly assigned to either an increased dose of cilazapril (10 mg) or the previous dose (5 mg) in two active-treatment
periods, each lasting 8 weeks. Results. A significant increase in renin activity was observed after administration of cilazapril
10 mg (6.4691.12 vs 4.6790.7 ng/ml/h; p0.028). Proteinuria, urine excretion of N-acetyl-b-D-glucosaminidase, and
a1-microglobulin and amino-terminal propeptide of type III procollagen were unchanged. Conclusion. An increased dosage
of cilazapril (twice the maximum recommended dose) in addition to combination therapy with telmisartan was associated
with increased blockade of the reninangiotensinaldosterone system, with no additional effect on proteinuria, markers of
tubular injury or renal fibrosis.
Key Words: Angiotensin-converting enzyme inhibitor, angiotensin-receptor blocker, combination therapy, high dose,
proteinuria, tubules, fibrosis
Introduction
Intervention in the reninangiotensinaldosterone
system (RAAS) is currently the most effective
strategy for combined blood pressure-lowering and
renoprotection. Agents that inhibit the RAAS, such
as angiotensin I-converting enzyme inhibitors
(ACEIs) and angiotensin II-receptor blockers
(ARBs), prevent and retard the progression of both
diabetic and non-diabetic native kidney disease [1].
It has been suggested [2,3] that they also exert a
renoprotective effect in patients after kidney transplantation. It was shown [1] that the protective renal
effects of the RAAS-inhibiting drugs are partly
independent of the changes in glomerular circulation
and also involve limiting the non-haemodynamic
effects of angiotensin II and aldosterone, such as
mitogenesis, inflammation and fibrosis. Combination therapy involving concomitant use of an ACEI
and an ARB has been studied extensively for some
years with respect to renal protection [4,5]. Published in 2003 [6], (COOPERATE) trial findings
gave, for the first time, evidence that combined
therapy can provide additional renoprotection beyond blood-pressure control in non-diabetic renal
diseases.
Correspondence: Leszek Tylicki, MD, PhD, Department of Nephrology, Transplantology and Internal Medicine, Medical University of Gdańsk, De˛binki 7 St.,
Gdańsk 80-211, Poland. Tel: 48 58 349 25 05. Fax: 48 58 346 11 86. E-mail: leszek.tylicki@amg.gda.pl
(Received 25 July 2007; accepted 19 December 2007)
ISSN 0036-5599 print/ISSN 1651-2065 online # 2008 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)
DOI: 10.1080/00365590801905943
L. Tylicki et al.
Despite great progress in conservative therapy for
chronic nephropathies and a decrease in the rate of
progression of chronic nephropathies by accurate
RAAS blockade, it has not proved possible to inhibit
their progress completely. The question arises as to
whether we can really go beyond slowing the rate of
progression of chronic nephropathies and stop the
decline in kidney function or even achieve regression
of existing renal scarring [7]. It has been suggested in
some experimental studies [8] that resolution of
established kidney sclerosis is possible by using an
ACEI or an ARB at doses well above those usually
recommended to lower blood pressure. To shed
more light on this issue, we performed a randomized, open, controlled study to evaluate the influence of up-titration with the ACEI cilazapril to twice
the maximum dose recommended for hypertension
in combination with the ARB telmisartan on surrogate markers of kidney injury, i.e. proteinuria, and
markers of tubular involvement and kidney fibrosis.
General protocol
The study was a randomized, open, 2 2 crossover
trial in which the renal effects of two different dual
pharmacological blockades of the RAAS were compared. It consisted of 8 weeks of dual treatment with
telmisartan (Micardis; Boehringer Ingelheim Polska,
Warsaw, Poland) 80 mg once-daily (o.d.) and cilazapril (Inhibace; Roche Polska, Warsaw, Poland) 5 mg
o.d. (Period 1), and 8 weeks of an alternative
combined therapy with telmisartan 80 mg o.d. and
cilazapril 10 mg o.d. (Period 2) (Figure 1). At the
beginning of the study, subjects who met the inclusion
criteria entered an 8-week run-in period, during
which any other previously used ACEIs or ARBs
were stopped. Cilazapril and telmisartan were either
continued or newly administered to those patients
who had not received these agents previously. The
maximal recommended doses of these agents for
hypertension were determined. Patients were also
administered hydrochlorothiazide at a dose of
12.5 mg o.d. In addition, adjuvant therapy with
doxazosin was added and titrated up, if necessary, to
achieve the target office trough blood pressure (BP) of
5130/80 mmHg. There was no wash-out period
between administration of the antihypertensive
agents used previously and the study treatment.
When the target BP was achieved, the patients
received this adjusted therapy until the end of the
run-in period, but not for B6 weeks. Patients were
recommended not to change their usual daily intakes
of protein and sodium during the study period.
At the end of the run-in period, patients were
randomly allocated to one of the two treatment
sequences: cilazapril 5 mgcilazapril 10 mg (Sequence 1); or cilazapril 10 mgcilazapril 5 mg
(Sequence 2) (Figure 1). Afterwards, the same 8week therapy as used during the run-in period was
administered during a control period to stabilize
background proteinuria. The dosages of telmisartan
Material and methods
Patients
Patients were selected from a cohort that consecutively attended our renal outpatient department. The
inclusion criteria were established as follows: age
1865 years; chronic non-diabetic proteinuric
nephropathy; normal or slightly impaired stable
renal function expressed as serum creatinine level
B155 mmol/l [estimated glomerular filtration rate
(eGFR) 0.75 ml/s]; stable proteinuria 0.3 g/
24 h at the randomization point; hypertension; and
no steroids or other immunosuppressive treatment
for a minimum of 6 months before the study.
Patients with nephrotic syndrome were excluded.
The study was approved by the local ethical committee, and the investigated patients all gave their
written consent to participate in the study.
C5
C10
Sequence 1 (C5-C10)
Run-in
Randomization
382
End-period
C10
C5
Sequence 2 (C10-C5)
*
*
*
*
Figure 1. Study scheme. The asterisks denote time points at which urine and blood were taken for analyses. C5cilazapril 5 mg; C10
cilazapril 10 mg.
High-dose dual RAAS blockade
and hydrochlorothiazide, once adjusted during
the run-in period, were left unchanged throughout
the study. Drug compliance was assessed by
means of tablet counts. At the end of each of the
two treatment periods, office trough BP, 24-h
ambulatory BP, serum creatinine, potassium, haemoglobin, plasma renin activity (PRA) as well as
urine excretion of protein (UPE), N-acetyl-b-Dglucosaminidase (NAG), a1-microglobulin (a1-m),
amino-terminal propeptide of type III procollagen
(PIIINP), creatinine, sodium (NaEX) and urea were
determined.
Procedures and laboratory methods
Office trough BP was measured using a mercury
sphygmomanometer in a sitting position after 10 min
of rest and expressed as the mean value of two
consecutive measurements taken 2-min apart. Ambulatory BP was measured continuously for 24 h
using the Mobil-o-graph (version 12; I.E.M. GmbH,
Stolberg, Germany) monitoring system. BP was
measured every 15 min during the day (07.00 to
22.00) and every 30 min during the night (22.00 to
07.00). Regarding the office BP measurements,
systolic (SBP) and diastolic (DBP) values were
analysed; for the ambulatory BP measurements,
24-h SBP and 24-h DBP were analysed. UPE, NaEX
and urea excretion were evaluated on the basis of
24-h urine collection. All of the patients were
equipped with a scaled container and strictly informed how to collect urine. They collected two 24-h
urine samples, from which the mean values of
UPE were calculated for data evaluation. Patients
were asked not to perform heavy physical activity on
the urine collection days. The excretion of urea was
used to calculate the protein intake according to
Maroni equation: protein intake normalized to
weight (g/kg/day) 6.25{[urea-N-excretion urine
24h (g/day)][0.0031body weight (kg)]}/body
weight (kg) [9]. eGFR was calculated according to
the CockcroftGault equation [10].
Blood samples for determination of PRA were
taken after overnight fasting and 30 min of rest with
the patient lying down and before administration of
the study drugs. The samples were stored at 758C
until assayed. PRA was measured by means of a
radioimmunoassay (RIA; RENCTK; DiaSorin,
Stillwater, MN), which estimates the amount of
angiotensin I generated by the action of renin on
angiotensinogen. Haemoglobin, urea, potassium,
sodium, protein and creatinine levels were measured
by means of standard laboratory techniques. Adverse
effects were recorded at each visit using questionnaires or as observed by the investigators.
383
The first morning urine sample was collected for
the determination of PIIINP. The samples were
stored at 758C until assayed. Urinary PIIINP
was measured using an RIA kit obtained from Orion
Diagnostica (Espoo, Finland). The intra- and interassay coefficients of variation were 3.0% and 6.5%
for concentrations of 2.8 and 2.7 mg/l, respectively.
The measurement range of the assay is 1.050 mg/l,
and the detection limit is 0.3 mg/l.
NAG and a1-m were analysed in the second
morning spot urine sample. NAG was determined
by means of a spectrophotometric method according
to Maruhn [11]. The incubation medium contained,
in a final volume of 0.4 ml, 5 mmol/l p-nitrophenyl2-acetamido-b-D-glucopyranoside as a substrate in
50 mmol/l citrate buffer (pH 4.14). The reaction was
started by the addition of 0.2 ml of undialysed urine,
continued for 15 min at 378C, and then terminated
with 1 ml of glycine buffer (pH 10.5). Absorbance
was measured at 405 nm against a sample terminated at time zero. The calculation of the NAG level
was based on the molar extinction coefficient of the
product of the reaction, p-nitrophenol, which is
equal to 18.5 cm2/mmol. From preliminary experiments it was clear that dialysis of urine did not affect
the NAG level in urine. An immunoturbidimetric
test (Tina-quant a1-microglobulin; Roche, Basle,
Switzerland) was used for the quantitative determination of a1-m in urine. The detection limit of the
method was 2 mg/l. Urinary NAG, a1-m and
PIIINP were reported per milligramme or gramme
of urine creatinine to correct for variation in the
urine concentration.
Statistics
Data from our preliminary studies were used for
the sample size calculation. The primary endpoint of
the study was a difference in 24-h UPE from
measurements available for each patient after treatment with standard and high doses of cilazapril.
Secondary endpoints included urine NAG, and
a1-m and PIIINP urine excretions. A sample size
of 18 patients adequately allowed a power of 80% to
detect a difference in means across the levels
of repeated-measures factors equal to the withinpatient SD, i.e. a standardized effect size of 1.0 at a
significance level of 0.05 (two-tailed). The normality
and homogeneity of the variances were verified
by means of the ShapiroWilk and Levene tests,
respectively. Because of their skewed distributions,
eGFR, UPE, excretion of NAG and PIIINP and
NaEX were logarithmically transformed before statistical analysis and expressed as geometric means
and 95% CIs. Other results are expressed as mean9
SEM. Differences in variables were assessed using
384
L. Tylicki et al.
Student’s t-test. p B0.05 (two-tailed) was considered
statistically significant. Data were evaluated using
the Statistica (version 6.0; StatSoft Inc, Tulsa, OK)
software package.
To prevent or limit the possibility of a period
effect, we introduced a degree of balance into the
study design, with a randomization scheme allowing
every treatment to be represented in every period
with the same frequency. Overall, we had two
different therapy sequences during the two treatment
periods (Figure 1). An equal number of patients
(n 9) per sequence was randomly assigned. Because all 18 patients completed the protocol, this
balance was fully respected at the end of the study.
To check for the presence of a period effect, the
differences between variables at the randomization
point and at the end of the study were also
examined. To prevent or limit the risk of a carryover
effect, we planned each treatment period to last 8
weeks. Previous studies [12] have shown that the
effects of ACEIs and ARBs on proteinuria and
glomerular permselectivity are fully reversible within
4 weeks. Thus, prolonging each treatment period for
8 weeks allowed us to rule out a residual effect of
previous treatment at the end of Week 8, when
proteinuria was measured.
Results
Table I. Characteristics of patients.
n
Mean (9SD) age (years)
No. of females/males
18
42.4491.89
7/11
Primary non-diabetic nephropathy (n)
Chronic glomerulonephritis
Chronic interstitial nephritis
Amyloidosis
Unknown
12
1
1
4
BMI (kg/m2)
Serum creatinine (mmol/l)
eGFR (ml/s)
Urinary protein excretion (g/24 h)
Office SBP (mmHg) before run-in period
Office DBP (mmHg) before run-in period
29.9191.17
113910
1.35 (1.191.75)
0.75 (0.61.35)
128.492.3
79.391.6
Treatment with ACEI/ARB before the study (n)
Cilazapril 5 mg
3
Cilazapril 2.5 mg/telmisartan 40 mg
4
Cilazapril 5 mg/telmisartan 80 mg
1
Benazepril 20 mg/losartan 50 mg
1
Benazepril 10 mg/losartan 50 mg
2
Benazepril 20 mg/telmisartan 80 mg
1
Benazepril 20 mg/telmisartan 40 mg
1
Benazepril 40 mg
1
Lisinopril 20 mg/losartan 50 mg
1
Enalapril 40 mg
3
to the standard dosage (6.4691.12 vs 4.6790.7
ng/ml/h; p 0.028) (Figure 2).
Of the 18 patients who entered the study, all of them
completed the protocol. Their characteristics are
presented in Table I. To achieve the target BP,
doxazosin had to be supplemented in 2/18 patients
in all the treatment periods (average dose 3.09
0.53 mg o.d.). Before data analysis, the carryover
effect was tested for and found not to be significant.
To exclude the presence of the period effect, the
differences between levels of UPE, urine excretion of
NAG, a1-m and PIIINP after the run-in period (at
the randomization point) and at the end of the study
were compared and found not to be significant.
There were no significant differences between urinary NAG (p 0.49) and a1-m (p 0.63) excretions at the end of the two different combination
treatments (Table III).
BP
PIIINP
Control of BP was adequate in all study periods,
with all patients achieving the target office trough BP
of 5130/80 mmHg. There were no differences in
office trough SBP and DBP between the treatments.
There were also no significant differences in ambulatory SBP (p0.29) and DBP (p 0.13) measurements between the treatments (Table II).
There were no significant differences between urinary PIIINP excretion at the end of the two different
combination treatments (p0.89; Table III).
PRA
A significant increase in PRA was observed after
treatment with the higher dose of cilazapril compared
UPE
There were no significant differences in UPE level at
the end of the two different combination treatments
(p 0.56; Table III).
Urinary NAG and a1-m excretion
Renal function, sodium and protein intake
Renal function assessed by means of serum creatinine and eGFR remained stable during the study
periods (p0.37 and 0.42, respectively). There
were no differences in sodium and protein intakes
between treatment periods (p 0.35 and p0.72,
respectively; Table II).
385
Table II. Results of BP, protein intake, NaEX, potassium and haemoglobin level.
Parameter
Randomization point
24-h SBP (mmHg)
24-h DBP (mmHg)
NaEX (mmol/24 h)
Daily protein intake (g/24 h)
Potassium (mmol/l)
Haemoglobin (g/l)
a
118.491.67
74.1792.24
255 (227305)
85.8693.8
4.4990.12
141.993.1
Cilazapril 5 mg
Cilazapril 10 mg
Study end
117.4491.81
73.591.8
248 (94575)
78.4492.06
4.5590.11
141.393.9
116.0691.81a
71.4491.92a
228 (203274)a
78.2494.62a
4.5590.14a
138.393.7b
116.7891.93
73.7891.76
263 (235307)
79.1894.32
4.3690.14
141.593.4
Non-significant difference between treatment with cilazapril 5 mg and cilazapril 10 mg.
Significant difference (p 0.01) between treatment with cilazapril 5 mg and cilazapril 10 mg.
b
Adverse effects
There were no significant differences in potassium
concentration between the treatments (p 0.9). In
one patient, the potassium level increased to 6.2
mmol/l during combination treatment with the
higher dose of cilazapril. The haemoglobin level
decreased significantly after combination treatment
with the higher dose of cilazapril (p 0.01; Table II).
This, however, was not associated with symptoms of
anaemia and none of the patients needed treatment
for anaemia. The study treatments were well tolerated by all the patients and no adverse effects were
reported in the questionnaires.
Discussion
Several clinical studies [6,13] have investigated dual
RAAS blockade in non-diabetic or mixed renal
diseases and documented a greater antiproteinuric
effect of combined therapy with an ACEI and an
ARB than with monotherapy. Given these results, it
now seems reasonable to consider dual RAAS
blockade as the new gold standard of treatment for
chronic proteinuric kidney disease, at least in those
patients with a non-diabetic origin of nephropathy
[14]. Following this recommendation, all patients in
the present study were administered a dual RAAS
blockade. Furthermore, for ethical reasons there was
no wash-out of the pharmacological blockade of the
RAAS during the run-in period. The study agents
were introduced into the therapeutic regimen instead
of the previously used antihypertensive drugs.
Despite the proven effectiveness of standard doses
of ACEIs and ARBs in the prevention and treatment
of renal complications, it has not proved possible to
inhibit the progress of chronic nephropathies completely [1]. The reasons for this may reflect the fact
Plasma renin activity ng/ml/h
8,0
7,5
7,0
6,5
6,0
5,5
5,0
4,5
p=0.028
4,0
Mean
Mean ± SEM
3,5
cilazapril 5
cilazapril 10
Figure 2. PRA after treatment with standard and high doses of cilazapril.
386
L. Tylicki et al.
Table III. Results of serum creatinine, proteinuria, urine excretion of NAG, a1-m and PIIINP.
Parameter
Serum creatinine (mmol/l)
eGFR (ml/s)
UPE (g/24 h)
NAG excretion (IU/g creat.)
a1-m excretion (mg/g creat.)
PIIINP excretion (mg/g creat.)
a
Randomization point
113910
1.35 (1.191.75)
0.75 (0.61.35)
3.08 (2.525.01)
8.8991.54
1.7 (1.382.56)
Cilazapril 5 mg
10298
1.49 (1.31.87)
0.61 (0.471.27)
3.02 (2.315.22)
7.5691.83
1.57 (1.112.83)
Cilazapril 10 mg
Study end
10699a
1.45 (1.271.85)a
0.66 (0.451.51)a
2.92 (1.47.45)a
8.8992.18a
1.49 (0.93.12)a
10398
1.46 (1.31.82)
0.62 (0.441.49)
2.91 (2.494.36)
8.2791.66
1.84 (1.512.53)
Non-significant difference between treatment with cilazapril 5 mg and cilazapril 10 mg.
that even combined treatment does not provide
complete, persistent blockade of the RAAS [15].
Therefore, it is necessary to optimize this modality
and to search for alternative therapeutic strategies
which can further improve renal outcome.
The aim of this study was to find out whether or
not an increased dose of ACEI well above the
maximum recommended limit makes sense given
the renoprotective aspect. The rationale comes
from the experimental observations that up-titration
of the dose of an ACEI or ARB to levels above those
usually recommended for lowering BP may provide
an additional nephroprotective effect, i.e. by reversing the destructive processes within the kidney [16].
In a doseresponse study in proteinuric Sprague
Dawley rats with non-diabetic kidney disease, Peters
et al. [17] demonstrated a BP-independent reduction in renal expression of transforming growth
factor (TGF)-b1, the most potent profibrotic cytokine during high-dose therapy with losartan and
enalapril. Moreover, other disease markers, including glomerular matrix accumulation, and glomerular
production and mRNA expression of the matrix
protein fibronectin and the protease inhibitor plasminogen activator inhibitor type 1, closely followed
TGF-b1 expression. Similarly, in another study [18],
a high dose of the ACEI enalapril, far in excess of
that used for BP control, was reported to induce a
partial regression of glomerulosclerosis as well as
sclerotic changes in interstitial and vascular compartments. Other authors [19,20] have demonstrated regression of glomerulosclerosis during
therapy with high doses of ARBs in animal models
of hypertensive nephroangiosclerosis and age-related
glomerulosclerosis. The mechanisms appear to involve both the inhibition of collagen synthesis and
the enhancement of matrix degradation due to
activation of metalloproteinases. In addition, segments of glomeruli regenerate both by capillary
lengthening and branching, whereas the sclerosed
segments are largely reabsorbed. Clinical data on
this point are extremely limited.
In the present study, the renal effects of doubling
the dosage of cilazapril on top of combination
therapy were assessed using measurement of surrogate markers of kidney injury, i.e. proteinuria (a
marker of glomerular injury), NAG and a1-m
(tubular involvement markers) and PIIINP (an
indirect marker of kidney fibrosis). Doubling the
dosage of cilazapril was shown to be associated with
an increase in blockade of the RAAS. A significant
increase in PRA was observed during treatment with
the higher dose of cilazapril. Previously, additional,
albeit non-significant, increases in PRA and angiotensin I levels were reported by Haas et al. [21]
during therapy with the ACEI spiramil at twice the
maximal antihypertensive dose. Both these findings
are in agreement with another clinical observation
[15] suggesting that increasing the dose of the ARB
losartan above the recommended limit improves and
prolongs RAAS blockade. We demonstrated that,
although the higher dose of cilazapril significantly
increased RAAS blockade, it had no significant
additional effect on proteinuria reduction. In accordance with this observation, Haas et al. [21]
reported that increasing the spiramil dose to twice
its maximal antihypertensive dosage also does not
alter proteinuria. Surprisingly, the administration of
very high doses of the ARBs telmisartan, losartan
and irbesartan seems to be more effective at reducing
proteinuria, as well as slowing the progression of
chronic proteinuric nephropathies, than standard
doses of these agents [2225]. The reasons for these
discrepancies are not clear. It is unlikely that confounding factors would have influenced the present
study outcomes. The two treatment periods did not
differ with respect to BP, the sodium and protein
intakes of the patients or renal function.
Considering the fact that the extent of tubulointerstitial damage is a fundamental predictor of
kidney outcome, tubular cells have become a renal
site of particular interest [26]. To evaluate the
tubular effects of our interventions, two different
markers of tubular injury were analysed in the
present study. NAG is an enzyme of the hydrolase
class which is abundant in the kidney, predominantly
in the lysosomes of the proximal tubular cells. It is
physiologically excreted in low amounts in urine as a
387
consequence of the normal exocytosis process. The
increased excretion of NAG is thought to be a
specific marker of tubular injury in many renal
pathologies [27]. Increased urinary excretion of a1m, a low-molecular-weight protein physiologically
filtered and reabsorbed by tubular cells, may indicate
a reduced capacity of a1-m reabsorption by such
cells and thus a1-m may represent a marker for
established tubular damage, with greater urinary
concentrations pointing to a greater severity of
damage [28]. In the present study, up-titration of
cilazapril on top of dual RAAS blockade resulted in
no improvement in tubular status.
Given the previous assumption that an incremental dosage of an ACEI may affect angiotensin IImediated, non-haemodynamic processes, such as
inflammation and fibrosis, the authors analysed the
influence of the study treatments on urine excretion
of PIIINP, a non-invasive marker of interstitial
fibrosis [29]. A close association between urinary
PIIINP excretion and the degree of interstitial
kidney fibrosis was previously evidenced [30]. During the synthesis and deposition of type III collagen,
PIIINP is degraded from the collagen and secreted
into the surroundings. In the present study, no
significant differences in PIIINP excretion were
observed during the two treatment options. Given
the fact that urinary PIIINP was previously found to
originate from the kidney [30], one may assume that
an increased dose of cilazapril will not affect fibrotic
processes in the kidney. However, it should be taken
into consideration that the lack of effect on kidney
fibrosis markers may be a result of the relatively short
study period as well as adequate treatment with
ACEIs and ARBs before entering the study.
A potential limitation of the study is the relatively
small sample size, which was sufficiently powered to
detect a significant difference equal to the SD value
between the treatment periods. We realize that the
effect size of 1.0 in such a case is relatively large and
only allows a conclusion of a preliminary nature. In
addition, one should realize that the potential
benefits for tubules and interstitium were extrapolated from presumptive early surrogates, tubular
origin enzymes and collagen degradation product
excretions. Evidence may be provided only by
histological examinations.
One should realize that the study results do not
call into question the proven effectiveness of dual
RAAS blockade in the field of nephroprotection.
This was evidenced in many previous studies,
including those performed by the authors [4,5].
For ethical reasons this aspect of nephroprotection
was not evaluated in the present study. Considering
the prognostic impact of proteinuria and the extent
of tubular injury on renal outcome, however, the
question arises as to whether increasing ACEI doses
to very high in addition to dual pharmacological
RAAS blockade is really useless. Long-term controlled studies involving histological assessment
would seem to be necessary to give a conclusive
answer to this question.
Conclusions
In an open, controlled, cross-over fashion the
authors demonstrated that a twofold increase in the
dosage of the ACEI cilazapril in addition to dual
ACEI and ARB therapy was associated with an
increase in RAAS blockade, with no additional
effects on proteinuria or markers of tubular injury
and kidney fibrosis. Such an intervention seems to
be safe; nevertheless, a slight but clinically irrelevant
decrease in haemoglobin level was observed.
Acknowledgements
The study was supported by a grant (No. ST-4) from
the Polish Committee for Scientific Research (KBN)
via the Medical University of Gdansk. The authors
thank Roche Polska and Boehringer Ingelheim
Polska for providing drugs. The authors are indebted
to mgr Joanna Wierzchowska for expert technical
assistance with the radioimmunological analyses. The
study results were partially presented in abstract form
during the XLIIIth European Renal Association
European Dialysis and Transplant Association Congress in Glasgow, UK in 2006.
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NDT Advance Access published December 2, 2008
Nephrol Dial Transplant (2008) 1 of 2
Letter
High-dose angiotensin-converting enzyme inhibitor
attenuates oxidative stress in patients with chronic
kidney disease
Sir,
A pharmacological blockade of the renin–angiotensin–
aldosterone system (RAAS) constitutes a cornerstone strategy for inhibiting progression of chronic nephropathies.
In a recent NDT issue, Tomas Berl [1] discussed the improvements in renal outcome associated with maximal
RAAS inhibition achieved using combined therapies that
included angiotensin-converting enzyme inhibitors (ACEI),
angiotensin receptor blockers (ARB), renin inhibitors and
mineralocorticoid receptor antagonists. Alternatively, patients were administered ACEI or ARB at doses highly
exceeding those approved for blood pressure control. Here,
we elaborate on this very interesting discussion by reporting
that a double RAAS blockade with high-dose ACEI attenuates oxidative stress phenomena in patients with chronic
kidney disease (CKD).
In an open, randomized, cross-over study, 18 white adult
patients (11 men and 7 women; mean age: 42 years) with
nondiabetic proteinuric CKD were evaluated to test the hypothesis that high-dose ACEI (10 mg cilazapril) combined
with standard ARB (telmisartan) therapy increases nephroprotection by lowering the level of potentially nephrotoxic,
oxidative stress-dependent products. The trial treatment was
based on 80-mg telmisartan therapy combined with either
5 mg cilazapril or 10 mg cilazapril for 2 months of the study;
the alternative being used for the next 2 months. A commercial ELISA kit (Cayman Chemical Co., USA) was then
used to measure the urinary excretion of 15-F2t -isoprostane
in the treated patients. 15-F2t -isoprostane is accepted as a reliable and sensitive marker of oxidative stress in the human
body [2.]
It was found that the higher dose cilazapril treatment
significantly reduced urinary levels of 15-F2t -isoprostane
relative to the control group (ANOVA P = 0.008; post hoc
P = 0.044) with no changes observed in systemic blood
pressure, serum creatinine or potassium levels (Table 1).
This finding may be of clinical relevance, as 15-F2t isoprostane has biological activity as a potent renal vasoconstrictor [3] and has been implicated as a causative mediator
in hepatorenal syndrome [4].
Interestingly enough, we have previously demonstrated
that a combined therapy with telmisartan and high-dose
cilazapril (doubling the dose recommended for antihypertensive treatment) has no additional effect on proteinuria
[5], a finding in accordance with the observations of Berl
[1]. However, our present data suggest that high-dose administration of ACEI may attenuate oxidative stress, as indicated by reduced generation of potentially nephrotoxic
isoprostanes, thus providing additional renal protection for
patients with CKD.
Conflict of interest statement. None declared.
Editorial Note: Dr Berl had no further comments on this letter.
1
Department of Nephrology
Transplantology & Internal
Medicine
2
Department of Medical Chemistry
Medical University of Gdansk
7 Debinki St. 80-211 Gdansk
Poland
3
Blood Brain Barrier and
Neuro-Oncology Program
Oregon Health & Science
University, 3181 SW San Jackson
Park Rd, Portland, OR 97239, USA
E-mail: mrenke@amg.gda.pl
Marcin Renke1
Leszek Tylicki1
Narcyz Knap2
Przemyslaw
Rutkowski1
Alexander
Neuwelt3
Andriy Petranyuk2
Wojciech
Larczynski1
Michał Wozniak2
Boleslaw
Rutkowski1
1. Berl T. Maximizing inhibition of the renin-angiotensin system with
high doses of converting enzyme inhibitors or angiotensin receptor
blockers. Nephrol Dial Transplant 2008; 23: 2443–2447
2. Fam SS, Morrow JD. The isoprostanes: unique products of arachidonic
acid oxidation—a review. Curr Med Chem 2003; 10: 1723–1740
3. Takahashi K, Nammour TM, Fukunaga M et al. Glomerular actions
of a free radical-generated novel prostaglandin, 8-epi-prostaglandin
F2 alpha, in the rat. Evidence for interaction with thromboxane A2
receptors. J Clin Invest 1992; 90: 136–141
4. Morrow JD, Moore KP, Awad JA et al. Marked overproduction of
non-cyclooxygenase derived prostanoids (F2-isoprostanes) in the hepatorenal syndrome. J Lipid Mediat 1993; 6: 417–420
5. Tylicki L, Renke M, Rutkowski P et al. Dual blockade of the reninangiotensin-aldosterone system with high-dose angiotensin-converting
enzyme inhibitor for nephroprotection: an open, controlled, randomized
study. Scand J Urol Nephrol 2008; 13: 1–8
doi: 10.1093/ndt/gfn665
C The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
For Permissions, please e-mail: journals.permissions@oxfordjournals.org
2
Nephrol Dial Transplant (2008)
Table 1. Serum creatinine, potassium and urinary excretion of 15-F2t -isoprostane
Parameter
Randomization
Cilazapril 5 mg
Cilazapril 10 mg
End of study
Serum creatinine, mean ± SEM (mg/dL)
Serum potassium, mean ± SEM (mmol/L)
Urinary 15-F2t -isoprostane, geometric mean
(95% CI) (ng/mg of creatinine)
1.28 ± 0.11
4.49 ± 0.12
0.63 (0.47–1.29)
1.15 ± 0.09
4.55 ± 0.11
0.69 (0.56–1.24)
1.20 ± 0.10
4.55 ± 0.14
0.39 (0.30–0.89)
1.17 ± 0.09
4.36 ± 0.14
0.67 (0.53–1.27)
Triple Pharmacological Blockade of the Renin-Angiotensin-Aldosterone
System in Nondiabetic CKD: An Open-Label Crossover Randomized
Controlled Trial
Leszek Tylicki, MD, PhD,1 Przemysław Rutkowski, MD, PhD,1 Marcin Renke, MD, PhD,1
Wojciech Larczyński, MD,1 Ewa Aleksandrowicz, PhD,2 Wiesława Lysiak-Szydlowska, MD, PhD,2
and Bolesław Rutkowski, MD, PhD1
Background: Agents inhibiting the renin-angiotensin-aldosterone (RAAS) system have an important
role in slowing the progression of chronic kidney disease. We evaluated the hypothesis that the addition
of an aldosterone receptor antagonist to an angiotensin-converting enzyme (ACE) inhibitor and
angiotensin II type 1 (AT-1) receptor blocker (ARB) (triple RAAS blockade) may provide an additional
benefit compared with an ACE inhibitor and ARB (double RAAS blockade).
Design: Randomized open controlled crossover study.
Setting & Participants: 18 whites (7 women, 11 men) from the Outpatient Department of Nephrology
with chronic nondiabetic proteinuric kidney diseases, mean age 42.4 ⫾ 1.9 years (SEM).
Interventions: In the 8-week run-in period, all participants received the ACE inhibitor cilazapril (5
mg), the ARB telmisartan (80 mg), and the diuretic hydrochlorothiazide (12.5 mg) as double RAAS
blockade to achieve the target blood pressure of less than 130/80 mm Hg. Participants were then
randomly assigned to 2 treatment sequences, either the addition of spironolactone (25 mg) (triple RAAS
blockade) through 8 weeks followed by double RAAS blockade through 8 weeks (sequence 1) or double
RAAS blockade followed by triple RAAS blockade (sequence 2).
Main Outcome Measures: 24-hour urine protein excretion (primary end point) and markers of tubular
injury and fibrosis (secondary end points). Analysis was performed using analysis of variance for
repeated measurements.
Results: At baseline, mean serum creatinine level was 1.16 ⫾ 0.09 mg/dL (103 ⫾ 8 ␮mol/L), estimated
glomerular filtration rate was 107.8 mL/min (95% confidence interval, 93 to 140.9 [1.8 mL/s; 95% confidence
interval, 1.55 to 2.35; Cockcroft-Gault formula), and 24-hour mean proteinuria was 0.97 ⫾ 0.18 g. Mean
urine protein excretion was 0.7 g/24 h (95% confidence interval, 0.48 to 0.92) less after triple RAAS blockade
than after double RAAS blockade (P ⫽ 0.01), without change in blood pressure. Urine excretion of
N-acetyl-␤-D-glucosaminidase (P ⫽ 0.02) and amino-terminal propeptide of type III procollagen (P ⫽ 0.05)
also significantly decreased. Potassium levels increased significantly after triple therapy (P ⫽ 0.02).
However, no patient was withdrawn because of adverse effects.
Limitations: Absence of blinding, small sample size, short treatment period, absence of histological
assessment.
Conclusions: Administration of an aldosterone receptor antagonist in addition to double RAAS
blockade with an ACE inhibitor and ARB may slow the progression of chronic kidney disease. Additional
studies are necessary to confirm this result.
Am J Kidney Dis 52:486-493. © 2008 by the National Kidney Foundation, Inc.
INDEX WORDS: Proteinuria; renin-angiotensin-aldosterone system; ACE inhibitor; angiotensin receptor blocker; aldosterone receptor blocker; spironolactone.
he renin-angiotensin-aldosterone system
(RAAS) has an important role in the progression of chronic kidney diseases (CKDs), and
inhibition of the RAAS with angiotensin-converting enzyme (ACE) inhibitors and angiotensin II
type 1 receptor blockers (ARBs) may retard
T
CKD progression.1,2 Double pharmacological
blockade of the RAAS with an ACE inhibitor
and ARB is recommended as standard renoprotective management, at least in patients with nondiabetic proteinuric CKD.3,4 In our previous studies, we confirmed the validity of this therapy in
From the Departments of 1Nephrology Transplantology
and Internal Medicine and 2Clinical Nutrition and Laboratory
Diagnostic, Medical University of Gdansk, Gdansk, Poland.
Received September 26, 2007. Accepted in revised form
February 14, 2008. Originally published online as doi:
10.1053/j.ajkd.2008.02.297 on April 17, 2008.
Trial registration: clinicaltrials.gov, study number:
NCT00528385.
Address correspondence to Leszek Tylicki, MD, PhD,
Department of Nephrology, Transplantology and Internal
Medicine, Medical University of Gdansk, De˛binki 7 St.
Gdańsk 80-211, Poland. E-mail: leszek.tylicki@amg.gda.pl
© 2008 by the National Kidney Foundation, Inc.
0272-6386/08/5203-0014$34.00/0
doi:10.1053/j.ajkd.2008.02.297
486
American Journal of Kidney Diseases, Vol 52, No 3 (September), 2008: pp 486-493
Triple RAAS Blockade as a Nephroprotection
such patients and also in subjects after renal
transplantation.5,6 However, neither ACE inhibitors nor ARBs, even in high doses or concomitant use, abrogate the progression of CKD completely. Innovative approaches are needed to keep
patients with CKD off dialysis therapy. Additional blockade of the aldosterone pathway may
prove to be such a beneficial therapeutic concept.
Aldosterone, a final effector of the RAAS, has
a significant role in the pathogenesis of CKD
independently of angiotensin II through direct
cellular action. This includes promoting inflammatory response, endothelial dysfunction, and
fibrosis by increasing plasminogen activator inhibitor 1 and transforming growth factor ␤1
expression and reactive oxygen species stimulation.7 Other mechanisms include the ability of
aldosterone to potentiate the deleterious effects
of angiotensin II as a result of upregulation of
angiotensin II receptor type AT-1.8 A number of
observations suggested that the nongenomic vasoconstrictor action of aldosterone led to increased arterial and glomerular capillary pressure.9
Both ACE inhibitors and ARBs, even at currently approved doses, fail to suppress aldosterone synthesis effectively. This may occur, at
least in part, because aldosterone synthesis is
strongly regulated out of the renin-angiotensin
axis via potassium levels, and adrenocorticotropic hormone.7 Moreover, the initial suppression of aldosterone is not sustained long term. In
40% to 50% of patients, an aldosterone escape
phenomenon was observed; that is, circulating
aldosterone levels increased after some months
of treatment with an ACE inhibitor or ARB.10
Given these facts, additional administration of an
aldosterone receptor antagonist to combination
treatment with an ACE inhibitor and ARB, socalled triple RAAS blockade, may provide additional renal protection. To shed more light on this
issue, we performed a randomized open controlled study to evaluate the influence of triple
RAAS therapy on surrogate markers of kidney
injury, ie, proteinuria, markers of tubular involvement, and kidney fibrosis.
487
our renal outpatient department. Inclusion criteria were age
of 18 to 65 years, nondiabetic proteinuric CKD, normal or
slightly impaired stable renal function expressed as serum
creatinine level less than 1.7 mg/dL (⬍150 ␮mol/L; estimated glomerular filtration rate [eGFR] ⬎ 45 mL/min [⬎0.75
mL/s]), stable proteinuria greater than 0.3 g/24 h at the
randomization point, hypertension, and no steroids or other
immunosuppressive treatment a minimum of 6 months before the study. Patients with nephrotic syndrome were excluded. The study was approved by the local ethical committee, and patients gave their written consent for participation
in this study.
General Protocol
The study was a randomized open 2 ⫻ 2 crossover trial in
which renal effects of double and triple RAAS blockade
were compared. At the beginning, participants who met the
inclusion criteria entered the 8-week run-in period, during
which any previously used ACE inhibitors or ARBs were
stopped. Cilazapril (Inhibace; Hoffman La-Roche, Basel,
Switzerland) was continued or newly administered to patients who had not received this agent previously. A maximal
recommended dose of 5 mg once daily in the morning was
set. Patients also were administered hydrochlorothiazide in a
dose of 12.5 mg once daily and telmisartan, 80 mg, once
daily in the morning (Micardis; Boehringer Ingelheim, Ingelheim, Germany). There was no washout period between
antihypertensive agents used previously and the study treatment involving cilazapril and telmisartan. To achieve the
target office trough blood pressure (BP) of 130/80 mm Hg or
less, adjuvant antihypertensive treatment with doxazosin
was used, if necessary. When the target BP was achieved,
patients received such adjusted therapy (background therapy)
until the end of the run-in period, but not less than 6 weeks.
Patients were recommended not to change their usual daily
protein and sodium intake during the study periods. At the
end of the run-in period, they were randomly assigned to 1 of
2 treatment sequences: 8-week background therapy with the
addition of 25 mg of spironolactone/8-week background
therapy without spironolactone (sequence 1) or 8-week
background therapy without spironolactone/8-week background therapy with the addition of 25 mg of spironolactone
(sequence 2; Fig 1). There was no washout between the 2
treatments in each sequence. Afterward, the same 8-week
background therapy as in the run-in period was administered
as a control period for stability of background proteinuria.
METHODS
Patients
Patients of white race were recruited from March 2005 to
February 2006 from the cohort that consecutively attended
Figure 1. Study scheme. *Urine and blood were obtained for analyses.
488
Spironolactone, the aldosterone receptor antagonist (Spironol; Polfa Grodzisk, Grodzisk Mazowiecki, Poland), was
administered once daily in the morning. Allocation was
performed independent of the research team person according to a computer-generated randomization list. Dosages of
cilazapril, telmisartan, and hydrochlorothiazide, once established in the run-in period, were unchanged throughout the
study. Target BP was an office trough BP of 130/80 mm Hg
or less. Drug adherence was assessed by means of tablet
counts. At the end of each of the 2 treatment periods, values
for office trough BP, 24-hour ambulatory BP, serum creatinine, potassium, hemoglobin, and plasma renin activity
(PRA) and 24-hour urine excretion of protein (UPE),
N-acetyl-␤-D-glucosaminidase (NAG), ␣1-microglobulin
(␣1m), amino-terminal propeptide of type III procollagen
(PIIINP), creatinine, sodium, and urea were determined.
Procedures and Laboratory Methods
Office trough BP was measured using a mercury sphygmomanometer with the patient in a sitting position after 10
minutes of rest and expressed as a mean value of 2 consecutive measurements obtained 2 minutes apart. Ambulatory BP
was measured continuously for 24 hours using the Mobil-ograph (version 12) monitoring system (I.E.M. GmbH, Stolberg, Germany). BP was measured every 15 minutes during
the day (7:00 AM to 10:00 PM) and every 30 minutes during
the night (10:00 PM to 7:00 AM). All patients were equipped
with a scaled container and were strictly informed how to
collect urine. On 2 different days, they collected two 24-hour
urine samples. From those, mean values of UPE, sodium
excretion, and urea excretion were calculated for data evaluation. Patients were asked not to perform heavy physical
activity on urine collection days. Urea excretion was used to
calculate protein intake according to the equation of Maroni
et al11: protein intake normalized to weight (g/kg/d) ⫽ 6.25
⫻ ([urea nitrogen excretion in urine (g/d)] ⫹ [0.0031 ⫻
body weight (kg)])/body weight (kg). eGFR was calculated
according to the Cockroft-Gault equation.12
Blood samples for PRA were obtained after overnight
fasting and 30 minutes at rest lying down before study drug
administration. Samples were stored at ⫺75°C until assayed. PRA was measured by means of radioimmunoassay
(RENCTK; DiaSorin, Stillwater, MN) that estimates the
amount of angiotensin I generated by the action of renin on
angiotensinogen. Hemoglobin, urea, potassium, sodium, protein, and creatinine were measured by using standard laboratory techniques. Adverse effects were recorded at each visit
in response to questionnaires or as observed by the investigators.
The first morning urine sample was collected for determination of PIIINP. Samples were stored at ⫺75°C until
assayed. Urinary PIIINP was measured by using a radioimmunoassay kit obtained from Orion Diagnostica (Espoo,
Finland). Intra-assay and interassay coefficients of variation
were 3.0% and 6.5% for 2.8 and 2.7 ␮g/L, respectively. The
measuring range of the assay is 1.0 to 50 ␮g/L, and the
detection limit is 0.3 ␮g/L.
NAG and ␣1m were analyzed in the second morning
spot-urine sample. NAG was determined by using the spectrophotometric method according to Maruhn13 and was
described in detail elsewhere.6 Immunoturbidimetric test
Tylicki et al
(Tina-quant ␣1-microglobulin; Roche, Basel, Switzerland)
was used for quantitative determination of ␣1m in urine. The
detection limit of the method was 2 mg/L. Urinary NAG,
␣1m, and PIIINP were reported per milligram or gram of
urine creatinine to correct the variation in urine concentration.
Statistics
Data from previous studies were used for sample size
calculation.14,15 The primary end point of this study was a
difference in 24-hour UPE in measurements available for
each patient after treatment with double and triple RAAS
blockade. Baseline 24-hour proteinuria of patients from this
population of 0.75 ⫾ 0.5 g/24 h was predicted. Assuming a
30% decrease in proteinuria after adding spironolactone to
double RAAS blockade, we predicted a decrease in proteinuria from 0.75 to 0.50 g/24 h with triple RAAS therapy. To
give the study 80% power to detect such a difference as
statistically significant (P ⬍ 0.05, 2 tailed) with an expected
SD of 0.25 g/24 h, 18 patients had to complete the study.
Secondary end points included urine NAG, ␣1m, and PIIINP excretion. Normality and homogeneity of variances
were verified by means of Shapiro-Wilk test and Levene test,
respectively. Because of their skewed distribution, diastolic
BP, eGFR, PRA, and PIIINP urine excretion were logarithmically transformed before statistical analysis and expressed
as geometric means and 95% confidence intervals. Other
results are expressed as mean ⫾ SEM. Differences in variables measured more than twice were assessed by using
analysis of variance (ANOVA) for repeated measurements
with Bonferroni correction for paired comparisons, otherwise paired t-test. P less than 0.05 (2 tailed) is considered
statistically significant. Data were evaluated using the Statistica (version 7.1; StatSoft Inc, Tulsa, OK) software package.
To prevent or limit the possibility of a period effect, we
introduced a degree of balance into the study design with a
scheme of randomization allowing every treatment to be
represented in every period with the same frequency (Fig 1).
To check for the presence of period effect, values for
variables at the randomization point and end of the study
were compared. To prevent or limit the risk of carryover
effect, we planned each treatment period for 8 weeks.
Previous studies showed that effects of aldosterone receptor
blockade on proteinuria were fully reversible within 4
weeks.14 Thus, prolonging each treatment period for 8
weeks allowed us to rule out a residual effect of previous
treatment at the end of week 8, when proteinuria was
measured. The Grizzle approach was used to investigate the
possibility of a carryover effect from the first to the second
treatment periods of the study.16 An analysis restricted to the
first treatment period was planned if a carryover effect could
not be excluded by using this approach. The test by Grubbs17
was used for detecting outliers.
RESULTS
Of 18 patients who entered the study, 18
(100%) completed the protocol (Fig 2). Baseline
clinical data at the randomization point are listed
in Table 1. To achieve the target BP, doxazosin (4
Figure 2. Patient flow through recruitment, randomization, and follow-up. Abbreviation: RAAS, renin-angiotensinaldosterone system.
mg once daily) had to be supplemented in 1 of 18
patients in all treatment periods. Grizzle analysis
excluded the possibility of a carryover effect
between 2 treatment periods for UPE, PRA,
urine NAG, ␣1m, and PIIINP excretion. To check
for the presence of period effect, differences
between main variables at the randomization
point and the end of the study were compared
and found not to be significant.
Control of BP was adequate in all study periods; 17 of 18 patients reached the target office
trough BP of less than 130/80 mm Hg. There also
were no differences in ambulatory systolic and
diastolic BP and daily protein and sodium intake
between treatments (P ⫽ 0.9; P ⫽ 0.1; P ⫽ 0.2;
and P ⫽ 0.3, respectively). Renal function assessed by means of serum creatinine and eGFR
remained stable during the study periods (P ⫽
0.6 and P ⫽ 0.9, respectively; Table 2). A significant increase in PRA was observed after treatment with triple RAAS blockade compared with
double RAAS blockade (P ⫽ 0.02; Table 2). The
increment in PRA was shown in 16 of 18 patients.
Triple therapy provided an additional 55.37%
decrease in proteinuria; ie, 0.7 g/24 h (95%
confidence interval, 0.48 to 0.92) compared with
double RAAS blockade (ANOVA P ⫽ 0.01; post
489
hoc P ⫽ 0.01; Table 2). The decrease in proteinuria was shown in 16 of 18 patients. Changes in
proteinuria did not correlate with changes in
systolic BP, diastolic BP, or PRA. The addition
of spironolactone resulted in a significant decrease in urine NAG (ANOVA P ⫽ 0.006; post
hoc P ⫽ 0.02) and PIIINP (ANOVA P ⫽ 0.03;
post hoc P ⫽ 0.05) excretion compared with
double RAAS blockade (Table 2). It was shown
in 15 and 16 of 18 patients, respectively. ␣1m
excretion decreased numerically after triple
RAAS blockade (P ⫽ 0.9).
The study treatments were well tolerated by
all patients. Adverse effects were not reported in
questionnaires. There was a significant increase
in potassium levels after triple RAAS blockade
compared with baseline (ANOVA P ⫽ 0.02; post
hoc P ⫽ 0.02; Table 2). The increase in potassium levels after triple RAAS blockade was
shown in 10 of 18 patients. In 2 patients, potassium levels significantly increased to 5.7 and 5.9
mmol/L, respectively.
DISCUSSION
In the present study, we show that administration of the aldosterone receptor antagonist spironolactone in addition to combination treatment
with an ACE inhibitor and ARB leads to a further
increase in RAAS blockade. A significant increase in PRA was observed after triple RAAS
blockade, similar to other reports.18,19
The addition of spironolactone to double
RAAS blockade with cilazapril and telmisartan
Table 1. Patient Characteristics (at the
randomization point)
No. of patients
Age (y)
Women/men
Primary nondiabetic nephropathy
Minimal change disease
Mesangial proliferative
glomerulonephritis
Immunoglobulin A nephropathy
Chronic membranous
glomerulonephritis
Chronic membranoproliferative
glomerulonephritis
Focal sclerosing glomerulonephritis
Chronic interstitial nephritis
Amyloidosis
Unknown
Body mass index (kg/m2)
18
42.44 ⫾ 1.89
7/11
1
3
4
1
2
1
1
1
4
26.91 ⫾ 1.17
490
Tylicki et al
Table 2. Results for BP, Protein Intake, Sodium Excretion, Potassium, Hemoglobin, Serum Creatinine, PRA,
Proteinuria, and Urine Excretion of NAG, ␣1m, and PIIINP
Randomization Point
End of Triple RAAS
Blockade
End of Double RAAS
Blockade
Study End
No. of patients
18
18
18
18
24-h systolic BP (mm Hg)
115.28 ⫾ 2.79
114.72 ⫾ 2.51
116.11 ⫾ 2.12
115.28 ⫾ 2.27
24-h diastolic BP (mm Hg)
73.83 (70.62-77.71) 70.17 (66.75-74.36) 71.63 (68.52-75.37) 70.99 (68.09-74.47)
Sodium urine excretion (mmol/24 h)
276.33 ⫾ 16.29
252.41 ⫾ 24.94
238.76 ⫾ 23.11
258.94 ⫾ 22.26
Daily protein intake (g/kg/24 h)
1.09 ⫾ 0.08
1.05 ⫾ 0.09
1.04 ⫾ 0.07
0.91 ⫾ 0.06
Potassium (mEq/L)
4.5 ⫾ 0.12
4.81 ⫾ 0.12*
4.66 ⫾ 0.09
4.55 ⫾ 0.09
Hemoglobin (g/dL)
14.19 ⫾ 0.34
13.79 ⫾ 0.33
14.16 ⫾ 0.36
13.9 ⫾ 0.34
Serum creatinine (mg/dL)
1.16 ⫾ 0.09
1.18 ⫾ 0.09
1.15 ⫾ 0.1
1.17 ⫾ 0.1
eGFR (mL/min/1.73 m2)
107.8 (93-140.9)
106.7 (89.2-146.2) 103.5 (83.9-147)
99,6 (81.6-137.9)
0.97 ⫾ 0.18
0.51 ⫾ 0.1†
1.21 ⫾ 0.2
0.99 ⫾ 0.26
NAG excretion (IU/g creatinine)
3.76 ⫾ 0.59
2.19 ⫾ 0.21†
3.44 ⫾ 0.44
3.16 ⫾ 0.48
␣1-Microglobulin excretion (mg/g
8.89 ⫾ 1.54
6.25 ⫾ 1.18
8.68 ⫾ 1.6
8.89 ⫾ 3.35
creatinine)
1.7 (1.38-2.56)
1.32 (1.14-2.0)†
2.8 (2.0-4.68)
1.35 (1.04-2.38)
PIIINP excretion (␮g/g creatinine)
PRA (ng/mL/h)
—
3.78 (2.79-6.85)†
2.56 (1.94-4.78)
—
Note: To convert serum creatinine in mg/dL to ␮mol/L, multiply by 88.4; eGFR in mL/min/1.73 m2 to mL/s/1.73 m2, multiply
by 0.01667; hemoglobin in g/dL to g/L, multiply by 10. Potassium levels expressed in mEq/L and mmol/L are equivalent.
Abbreviations: BP, blood pressure; NAG, N-acetyl-␤-D-glucosaminidase; PIIINP, amino-terminal propeptide of type III
procollagen; eGFR, estimated glomerular filtration rate; PRA, plasma renin activity.
*Significant difference compared with randomization point (P ⬍ 0.05).
†Significant difference compared with double renin-angiotensin-aldosterone system blockade (P ⬍ 0.05).
resulted in a significant decrease in UPE of
55.4%. Of note, the decrease in UPE was not
significantly associated with a decrease in BP.
No confounding changes in renal function or
protein and sodium intake were observed. UPE
decreased in 16 of 18 patients who completed the
study (Fig 3), suggesting that the antiproteinuric
effect is not restricted to patients with aldosterone escape phenomenon, occurring in approximately 40% of patients receiving long-term pharIndividual patients data on UPE
2,6
2,4
2,2
2,0
UPE g/24 hours
1,8
1,6
1,4
1,2
1,0
0,8
0,6
0,4
0,2
0,0
Triple RAAS blockade
Double RAAS blockade
Figure 3. Individual patient data for 24-hour proteinuria
(urinary protein excretion [UPE]) after double and triple
renin-angiotensin-aldosterone system (RAAS) blockade.
macological RAAS blockade,10 or patients with
a particular type of nephropathy. The antiproteinuric effect of spironolactone seen in our study
confirms and extends findings of previous clinical studies in patients with nondiabetic CKD.15,20
In a double-blind placebo-controlled study performed in 41 patients with various CKDs, Chrysostomou and Becker15 showed that the addition
of the aldosterone receptor antagonist to an ACE
inhibitor resulted in a 42% decrease in proteinuria that was sustained up to 12 months. Of
interest, they showed for the first time that triple
therapy offered an advantage to double therapy
with an ACE inhibitor and ARB, causing a greater
proteinuria decrease by 32.5%. However, they
observed no differences between triple RAAS
blockade and a combination of ACE inhibitor
and spironolactone. In a prospective randomized
open-label study, Bianchi et al evaluated the
effects of spironolactone (25 mg/d for 1 y) on
proteinuria and eGFR in 83 patients with CKD
already treated with an ACE inhibitor and/or
ARB.14 After 1 year of therapy, proteinuria and
aldosterone levels decreased significantly. Their
subgroup analysis performed in 43 individuals
treated with a combination of an ACE inhibitor
and ARB showed that the addition of an aldoste-
rone receptor antagonist to double RAAS blockade may provide an additional significant decrease in proteinuria by even 43%.
Unfortunately, the study of Chrysostomou and
Becker,15 although very interesting, did not use
an ACE inhibitor and ARB in their maximal
recommended doses; therefore, it did not answer
the crucial question about whether triple RAAS
blockade provided additional renoprotection in
comparison to combination ACE-inhibitor and
ARB therapy. In the report by Bianchi et al, there
was no information about doses of the ACE
inhibitor and ARB used in the study.14 The
particular strength of our study is to observe a
decrease in proteinuria when spironolactone was
added to double RAAS blockade using an ACE
inhibitor and ARB in maximal recommended
hypotensive doses. These findings point toward a
potential benefit of blockade of all components
of the RAAS with the use of an aldosterone
receptor antagonist, ACE inhibitor, and ARB as a
new promising nephroprotective strategy to effectively decrease the deleterious actions of both
angiotensin II and aldosterone in patients with
CKD. One may speculate about whether increasing doses of an ACE inhibitor and/or ARB to
greater than commonly recommended levels may
provide additional nephroprotection. Aranda et al21
showed that a high dose of telmisartan (ie, 160
mg) produced a greater decrease in proteinuria
than the standard dose of 80 mg. These findings
indicate that maximal recommended hypotensive doses of an ARB may be not optimal in the
aspect of nephroprotection. Of interest also may
be the issue of whether divided or bedtime administration of RAAS blockers may provide a better
antiproteinuric response in a comparison with a
1-time morning administration of the same daily
doses of these agents.22
Several mechanisms may be involved in the
antiproteinuric effect of aldosterone blockade.
First, as a result of differential effects on the
afferent and efferent glomerular arterioles,9 aldosterone could increase intraglomerular pressure,
an action independent of that of angiotensin II.
Using isolated afferent and efferent arterioles
from rabbit kidneys, Arima et al9 showed that
aldosterone exerted a vasoconstriction action on
both the afferent and efferent arterioles, but the
sensitivity of the efferent arterioles to aldosterone was greater than that of the afferent arte-
491
rioles. Another potential mechanism incorporates the ability of aldosterone to potentiate the
pressor effects of angiotensin II as a result of
upregulation of angiotensin II receptors in vascular smooth muscle cells.8
Worthy of analysis is the issue of whether
the renal benefit of additional aldosterone blockade lies in its diuretic effect or its ability to
modulate the non–volume-mediated effects of
aldosterone. One should realize that the addition
of a diuretic to pharmacological blockade of the
RAAS may decrease proteinuria further.23 To
reduce the influence of sodium-dependent mechanisms on study results, all subjects received an
unchanged dose of diuretic, hydrochlorothiazide,
during all periods of the study. In addition, patients were instructed not to change their daily
sodium intake during study periods. Sodium excretion was monitored and found not to change
during all study phases, which may suggest that
the renal effect of spironolactone was not caused
by diuretic effects of the drug. Of course, we
cannot completely exclude such a mechanism.
To solve the problem, a study design involving
direct comparison of renal effects of spironolactone and a diuretic without aldosterone antagonism properties in addition to double pharmacological RAAS blockade should be adopted.
No clinical evidence is available about whether
the addition of an aldosterone receptor antagonist can inhibit the long-term progression of
CKD to end-stage failure. Promising pilot data
recently were published by Bianchi et al, who
showed that the monthly rate of decrease in
eGFR from baseline was lower in patients treated
with combined RAAS blockade including spironolactone than in controls without an aldosterone receptor antagonist.14 In a proteinuriainduced renal damage model, a combination of
ACE inhibitor and spironolactone recently was
shown to decrease tubular damage and prevent
kidney fibrosis,24 fundamental predictors of kidney outcome.25 To evaluate tubulointerstitial effects of our interventions, the tubular involvement markers NAG and ␣1m and an indirect
marker of kidney fibrosis, PIIINP, were analyzed.
A close association between urinary PIIINP excretion and degree of interstitial fibrosis previously was evidenced.26 During the synthesis and
deposition of type III collagen, PIIINP is degraded from the collagen and secreted into the
492
surroundings. The increased excretion of NAG is
believed to be a specific marker of tubular injury
in many renal pathological states, including nondiabetic CKD.27 Increased urinary excretion of
␣1m, a low-molecular-weight protein physiologically filtered and reabsorbed by tubular cells,
might indicate the decreased capacity of its reabsorption by such cells and might be the marker of
established tubular damage, with greater urinary
concentrations pointing to greater severity of
damage.28 In the present study, triple RAAS
blockade was shown to decrease both NAG and
PIIINP compared with double RAAS blockade,
and these effects were completely independent
of BP changes. Decreased ␣1m levels also were
observed; however, the difference did not reach
the significance level. These beneficial renal effects may contribute to the decrease in tubulotoxic proteinuria, as well as direct nonhemodynamic consequences of aldosterone antagonism,
including decreased production of such prosclerotic cytokines as transforming growth factor ␤1,
plasminogen activator inhibitor type 1, and decreased macrophage infiltration, processes involved in the development of fibrosis.29 Thus,
we expanded the evidence of the beneficial renal
impact of triple RAAS blockade beyond looking
at proteinuria.
A potential limitation of the study is the relatively small sample size, which is sufficiently
powered to detect a significant difference equal
to the SD value between treatment periods.
Twenty-four–hour urine collections used to assess proteinuria may be associated with significant collection errors, largely because of improper timing and missed samples, leading to
overcollection and undercollection. Another limitation may be the relatively short treatment periods, during which beneficial tubulointerstitial
effects may not yet fully develop. In addition,
one should realize that the potential benefits for
tubules and interstitium were extrapolated from
presumptive early surrogates. Evidence may be
provided by only histological examination.
Side effects of triple RAAS blockade also
need to be considered. There is potential risk of
life-threatening hyperkalemia during aldosterone
receptor blockade, particular when patients are
treated with other agents that increase potassium
levels, such as ACE inhibitors and ARBs.30 Because the risk of hyperkalemia is clearly dose
Tylicki et al
dependent, we used a low dose of spironolactone. In addition, only patients with normal or
slightly impaired kidney function were included
in the study. Within this regimen, triple RAAS
blockade induced only moderate hyperkalemia.
Although hyperkalemia was not a clinical problem in our study, we do not advocate the use of
triple RAAS blockade without careful screening
and ongoing monitoring of candidate patients.
There were no reports of antiandrogen side effects in our study, but it is necessary to note that
exposure time was short.
In summary, we found that an aldosterone
receptor antagonist in addition to recommended
renoprotective treatment using combination
therapy with an ACE inhibitor and ARB, socalled triple RAAS blockade, decreased proteinuria and urine excretion of proteins associated
with tubular damage and interstitial renal fibrosis
and thus may have a beneficial effect on the
outcome of patients with proteinuric nondiabetic
CKD.
ACKNOWLEDGEMENTS
Results of this study were presented in abstract form
during the 44th European Renal Association-European Dialysis and Tansplant Association Congress, Barcelona, Spain,
on June 21-24, 2007.
Support: The study was supported by a grant from the
Polish Committee for Scientific Research (KBN) through
the Medical University of Gdansk (ST-4). The authors thank
Roche Polska and Boehringer Ingelheim Polska for providing the drugs. The drug providers and sponsors had no
involvement in the study design, patient recruitment, analysis, interpretation of data, writing of the report, or the
decision to submit the report for publication.
Financial Disclosure: None.
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Spironolactone Attenuates Oxidative Stress in Patients With Chronic Kidney
Disease
Marcin Renke, Leszek Tylicki, Narcyz Knap, Przemyslaw Rutkowski, Alexander
Neuwelt, Wojciech Larczynski, Michal Wozniak and Boleslaw Rutkowski
Hypertension 2008;52;e132-e133; originally published online Sep 29, 2008;
DOI: 10.1161/HYPERTENSIONAHA.108.120568
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Letter to the Editor
Letters to the Editor will be published, if suitable, as space permits. They should not exceed 1000 words
(typed, double-spaced) in length and may be subject to editing or abridgment.
Spironolactone Attenuates Oxidative Stress in
Patients With Chronic Kidney Disease
To the Editor:
In one of the latest issues of Hypertension, Michea et al1
reported that the mineralocorticoid receptor antagonist spironolactone attenuates cardiac hypertrophy and oxidative stress of the
heart in uremic rats. The results of our recent clinical study
indicate that spironolactone acts to decrease the amount of
oxidative stress in patients being treated for chronic kidney
disease. In an open, randomized, crossover study, 16 white adult
patients (10 men and 6 women; mean age: 41 years) with
nondiabetic proteinuric chronic kidney disease were evaluated to
test the hypothesis that spironolactone combined with standard
nephroprotective therapy may act as a clinically beneficial
antioxidant.
All of the study participants, during a preliminary period of 8
weeks, received the angiotensin-converting enzyme inhibitor
cilazapril (5 mg), angiotensin II type 1 receptor blocker telmisartan (80 mg), and diuretic hydrochlorothiazide (12.5 mg),
reducing the blood pressure to ⬍130/80 mm Hg. The trial
treatment was either based solely on the unchanged double
blockade of the renin-angiotensin system or combined with 25
mg of spironolactone, thus providing triple renin-angiotensin
system blockade during the first 2 months of the study, with the
alternative being used for the next 2 months. A commercial
ELISA kit (Cayman Chemical Co) was then used to measure the
urinary excretion of 15-F2t-isoprostane, widely accepted as a
reliable and sensitive marker of oxidative stress in the human
body.2
It was found that spironolactone significantly reduced urinary
levels of 15-F2t-isoprostane relative to the control group
(ANOVA P⫽0.035; posthoc P⫽0.041), with no change observed in systemic blood pressure or serum creatinine levels
(Table). This finding may be of clinical relevance, because
15-F2t-isoprostane isoprostane has biological activity as a potent
renal vasoconstrictor3 and has been implicated as a causative
mediator in hepatorenal syndrome.4
Interestingly, Furumatsu et al5 recently observed a beneficial
effect from the incorporation of spironolactone into a combined
treatment regimen consisting of angiotensin-converting enzyme
inhibitor and angiotensin II type 1 receptor blocker for use
against chronic kidney disease; Furumatsu et al5 specifically
noted improved intrarenal hemodynamics, as well as decreased
proteinuria levels, in patients receiving spironolactone. Thus,
taken together with the findings of previous studies, our results
indicate that spironolactone may be a useful addition to standard
nephroprotective therapy, playing a beneficial role as a clinically
effective antioxidant.
Source of Funding
The study was fully supported by Medical University of Gdansk
via ST-U grant.
Disclosures
None.
Marcin Renke
Leszek Tylicki
Department of Nephrology, Transplantology
and Internal Medicine
Gdansk, Poland
Narcyz Knap
Gdansk, Poland
Przemysław Rutkowski
Gdansk, Poland
Alexander Neuwelt
Blood Brain Barrier and Neuro-Oncology Program
Oregon Health and Science University
Portland, Ore
Wojciech Larczyński
Gdansk, Poland
Michał Woźniak
Gdansk, Poland
Bolesław Rutkowski
Gdansk, Poland
Table. Serum Creatinine and Urinary Excretion of 15-F2t-Isoprostane
Parameter
Randomization
Spironolactone
Control
End of Study
Serum creatinine, mean⫾SEM, mg/dL
1.12⫾0.08
1.16⫾0.10
1.13⫾0.11
1.09⫾0.10
Urinary 15-F2t-isoprostane, geometric
mean (95% CI), ng/mg of creatinine
0.76 (0.48 to 2.48)
0.65 (0.51 to 0.98)
0.94 (0.67 to 2.55)
0.91 (0.55 to 2.51)
(Hypertension. 2008;52:e132-e133.)
© 2008 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.108.120568
e132 INSTHEALTH LIB on November 13, 2008
Downloaded from hyper.ahajournals.org at NATIONAL
1. Michea L, Villagrán A, Urzúa A, Kuntsmann S, Venegas P, Carrasco L,
Gonzalez M, Marusic ET. Mineralocorticoid receptor antagonism
attenuates cardiac hypertrophy and prevents oxidative stress in uremic
rats. Hypertension. 2008;52:1– 6.
2. Fam SS, Morrow JD. The isoprostanes: unique products of arachidonic
acid oxidation-a review. Curr Med Chem. 2003;10:1723–1740.
3. Takahashi K, Nammour TM, Fukunaga M, Ebert J, Morrow JD, Roberts LJ,
Hoover RL, Badr KF. Glomerular actions of a free radical-generated novel
prostaglandin, 8-epi-prostaglandin F2 alpha, in the rat. Evidence for interaction with thromboxane A2 receptors. J Clin Invest. 1992;90:136 –141.
e133
4. Morrow JD, Moore KP, Awad JA, Ravenscraft MD, Marini G, Badr KF,
Williams R, Roberts LJ. Marked overproduction of non-cyclooxygenase
derived prostanoids (F2-isoprostanes) in the hepatorenal syndrome.
J Lipid Mediat. 1993;6:417– 420.
5. Furumatsu Y, Nagasawa Y, Tomida K, Mikami S, Kaneko T, Okada N,
Tsubakihara Y, Imai E, Shoji T. Effect of renin-angiotensin-aldosterone
system triple blockade on non-diabetic renal disease: addition of an
aldosterone blocker, spironolactone, to combination treatment with an
angiotensin-converting enzyme inhibitor and angiotensin II receptor
blocker. Hypertens Res. 2008;31:59 – 67.
Downloaded from hyper.ahajournals.org at NATIONAL INSTHEALTH LIB on November 13, 2008
Original Paper
Kidney Blood Press Res 2008;31:404–410
DOI: 10.1159/000185828
Received: March 27, 2008
Accepted: October 24, 2008
Published online: December 18, 2008
The Effect of N-Acetylcysteine on Proteinuria and
Markers of Tubular Injury in Non-Diabetic Patients
with Chronic Kidney Disease
A Placebo-Controlled, Randomized, Open, Cross-Over Study
Marcin Renke a Leszek Tylicki a Przemysław Rutkowski a Wojciech Larczyński a
Ewa Aleksandrowicz b Wiesława Łysiak-Szydłowska b Bolesław Rutkowski a
Departments of a Nephrology, Transplantology and Internal Medicine and b Clinical Nutrition and
Laboratory Diagnostics, Medical University of Gdansk, Gdansk , Poland
Key Words
N-Acetylcysteine ⴢ Chronic kidney disease ⴢ Proteinuria ⴢ
Tubular injury
Abstract
Background: Inhibition of the renin-angiotensin-aldosterone system with angiotensin-converting enzyme inhibitors
(ACEI) and/or angiotensin II subtype 1 receptor antagonists
(ARB) constitutes a strategy in the management of patients
with chronic kidney disease. There is still no optimal therapy
which can stop the progression of chronic kidney disease.
Antioxidants such as N-acetylcysteine (NAC) have been reported as a promising strategy in this field. Methods: In a
placebo-controlled, randomized, open, 2-period cross-over
study, we evaluated the influence of NAC (1,200 mg/day)
added to renin-angiotensin-aldosterone system blockade
on proteinuria and surrogate markers of tubular injury and
renal fibrosis in 20 non-diabetic patients with proteinuria
(0.4–6.36 g/24 h) with normal or decreased kidney function
(estimated glomerular filtration rate 61–163 ml/min). Subjects entered the 8-week run-in period during which the
therapy using ACEI and/or ARB was established with blood
pressure below 130/80 mm Hg. Next, patients were randomly assigned to 1 of 2 treatment sequences: NAC/washout/
© 2008 S. Karger AG, Basel
1420–4096/08/0316–0404$24.50/0
Fax +41 61 306 12 34
E-Mail karger@karger.ch
www.karger.com
Accessible online at:
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placebo or placebo/washout/NAC. Clinical evaluation and
laboratory tests were performed at the randomization point
and after each period of the study. Results: No significant
changes in laboratory tests were observed. Conclusion: NAC
had no effect on proteinuria, surrogate markers of tubular
injury or renal fibrosis in non-diabetic patients with chronic
kidney disease.
Copyright © 2008 S. Karger AG, Basel
Introduction
Pharmacological inhibition of the renin-angiotensinaldosterone system constitutes a cornerstone strategy in
the management of patients with chronic nephropathies
with proteinuria and with chronic renal failure [1]. Angiotensin-converting enzyme inhibitors (ACEI) as well as
angiotensin II subtype 1 receptor antagonists (ARB) have
been shown to decrease proteinuria, reduce the local renal inflammatory processes and slow the progression of
renal insufficiency [2–6]. Despite recent progress, there is
still no optimal therapy which stops the progression of
renal disease. Therefore, it is necessary to search for alternative therapeutic strategies which can further improve renal outcome. The administration of various anMarcin Renke, MD, PhD
Department of Nephrology, Transplantology and Internal Medicine
Medical University of Gdansk, Debinki 7
PL–80-211 Gdansk (Poland)
Tel. +48 58 349 25 05, Fax +48 58 346 11 86, E-Mail mrenke@amedec.amg.gda.pl
Table 1. Patient characteristics at baseline
Sequence 1
NAC
1,200 mg/day
Washout
Placebo
Parameter
Random
End
point
Run-in
period
Sequence 2
Placebo
Washout
NAC
1,200 mg/day
8 weeks
8 weeks
8 weeks
8 weeks
Fig. 1. Study scheme.
tioxidants has been reported to exhibit beneficial effects
in a number of experimental models of chronic kidney
diseases, suggesting that reactive species (RS), their
sources and the signalling pathways modified by RS may
represent important therapeutic targets to halt or attenuate renal injury [7].
N-Acetylcysteine (NAC), a synthetic precursor of reduced glutathione (GSH), is a thiol-containing compound which stimulates the intracellular synthesis of
GSH, enhances glutathione-S-transferase activity and
acts solely as an RS scavenger [8]. NAC has been shown
to prevent renal function in animal studies [9–12], but
clinical data concerning the renal effects of NAC are still
very limited [13–15]. Consequently, in the present study,
we evaluated the effects of the addition of NAC to background nephroprotective therapy with ACEI and/or ARB
on proteinuria and renal function as well as surrogate
markers of tubular injury and renal fibrosis in non-diabetic patients with chronic kidney disease.
Patients and Methods
Patients
Patients were selected from the cohort that attended our renal
outpatients department. The inclusion criteria were as follows:
age 18–65 years, chronic non-diabetic proteinuric nephropathy,
normal or slightly impaired stable renal function, expressed as an
estimated glomerular filtration rate (eGFR) above 60 ml/min, stable proteinuria above 300 mg/24 h and no steroids or other immunosuppressive treatment for a minimum of 6 months before
the study. Stable renal function and proteinuria were defined as a
variability of serum creatinine and proteinuria of less than 20%
during the 6 months before the patients started the study.
A Placebo-Controlled, Randomized,
Open, Cross-Over Study
Females/males
Age, years (mean 8 SEM)
Systolic blood pressure, mm Hg
(mean 8 SEM)
Mean diastolic blood pressure, mm Hg
Urinary protein excretion, g/24 h
Serum creatinine, mg/dl (mean 8 SEM)
eGFR, ml/min (mean 8 SEM)
BMI
Histopathological diagnosis
Mesangial glomerulonephritis
Mesangiocapillary glomerulonephritis
Membranous glomerulonephritis
Focal sclerosing glomerulonephritis
Other primary chronic glomerulonephritis
Unknown non-diabetic proteinuric chronic
kidney diseases
Background hypotensive therapy
ACEI and ARB
ACEI
ARB
Diuretic (hydrochlorothiazide 12.5 mg)
Doxazosin (8 mg)
8/12
39.3582.6
118.1582.8
75.9 (71.5–80.3)
1.41 (0.75–2.08)
1.0380.05
100.985.9
25.33 (23.65–27.0)
13
8
2
1
1
1
7
10
9
1
13
1
Values are numbers of patients, except where indicated otherwise. Figures shown in parentheses are ranges.
General Protocol
The study was a prospective, placebo-controlled, randomized, open, 2-period cross-over trial in which the effects on the
kidney of the addition of NAC to background nephroprotective
therapy with ACEI and/or ARB were evaluated. At the beginning, subjects entered the 8-week run-in period during which the
background nephroprotective therapy using pharmacological
blockade of the renin-angiotensin-aldosterone system was established with the target blood pressure (BP) below 130/80 mm Hg
(table 1). At the end of the run-in period, patients were randomly assigned to 1 of 2 treatment sequences: 8-week NAC (1,200
mg/day)/8-week washout-background therapy/8-week placebo
(sequence 1) or 8-week placebo/8-week washout-background
therapy/8-week NAC (1,200 mg/day) (sequence 2) (fig. 1). Allocation was performed by a person who was independent of the research team according to a computer-generated randomization
list. The patients received 600 mg of NAC as effervescent tablets
(ACC 600; Hexal AG) twice a day. The target BP during the whole
study was an office trough BP of 130/80 mm Hg or less. To achieve
a comparable BP level during all of the treatment periods, adjuvant antihypertensive treatment with doxazosin was allowed.
The dosages of ACEI, ARB and diuretics, once established in the
run-in period, were left unchanged throughout the study, including in the washout period. At the randomization point and after
405
Table 2. Changes in parameters after NAC and placebo
Proteinuria (DPE), g/24 h
Serum creatinine, mg/dl
eGFR, ml/min
PIIINP excretion, ␮g/g creatinine
NAG excretion, IU/creatinine
␣1m excretion, mg/g creatinine
Baseline-NAC
NAC (+)
0.99 (0.68–2.06)
1.02 (0.94–1.15)
101.386.5
0.94 (0.81–1.28)
1.63 (1.36–3.23)
8.682.4
0.96 (0.74–1.72)
0.98 (0.91–1.08)
105.787.1
1.19 (0.99–1.65)
1.35 (0.86–2.73)
7.8684.53
⌬
Baseline-placebo Placebo
–0.1380.23
–0.0580.02
4.3881.9
0.1580.22
–0.6780.29
–3.8282.75
0.80 (0.54–1.86)
1.02 (0.93–1.15)
95.187.7
1.00 (0.86–1.37)
1.67 (1.36–3.36)
9.8983.08
0.89 (0.69–1.71)
1.02 (0.94–1.12)
101.786.4
0.95 (0.81–1.28)
1.27 (0.97–2.60)
6.1782.27
⌬
–0.00180.13
–0.0180.03
6.683.5
–0.0880.12
–0.5780.37
–1.5382.28
p
0.6
0.31
0.59
0.34
0.83
0.52
Figures shown in parentheses are ranges.
the end of each treatment period, office trough BP, serum creatinine, potassium, proteinuria measured as 24-hour urine protein excretion (DPE), sodium excretion, urea excretion, surrogate markers of tubular injury, i.e. urine excretion of N-acetyl␤-D -glucosaminidase (NAG) and ␣-1 microglobulin (␣1m), and
an indirect marker of renal fibrosis, i.e. amino-terminal propeptide of type III procollagen (PIIINP), were determined. The study
was approved by the local ethical committee and the investigated
patients all gave their informed consent. The study was registered in www.clinicaltrials.gov and received a positive opinion
(NCT00572663).
Procedures and Laboratory Analyses
The office trough BP was measured by a Speidel and Keller
sphyngomanometer with the patient in a sitting position after
10 min of rest and expressed as the mean value of 2 consecutive
measurements taken 2 min apart. DPE, Na excretion and urea
excretion were evaluated on the basis of 24-hour urine collection. All of the patients were supplied with a scaled container
and were strictly informed how to collect 24-hour urine. They
collected two 24-hour urines, from which the mean value of
DPE was calculated for data evaluation. Patients were asked not
to perform heavy physical activity on the urine collection days
and were recommended not to change their usual daily protein
and sodium intake during the study period. The excretion of
urea was used to calculate the protein intake according to the
Maroni equation: protein intake normalized to weight (g/kg/
day) = 6.25 ! ([urea-N-excretion urine 24 h (g/day)] + [0.0031
! body weight (kg)])/body weight (kg) [16]. eGFR was calculated according to the Cockcroft-Gault formula. The first-morning urine sample was collected for the determination of PIIINP.
The samples were stored at –75 ° C until assayed. Urinary PIIINP
was measured using a radioimmunoassay kit obtained from Orion Diagnostica (Espoo, Finland). The intra- and interassay coefficients of variation were 3.0 and 6.5% for 2.8 and 2.7 ␮g/l, respectively. The measuring range of the assay is from 1.0 to 50
␮g/l, and the detection limit is 0.3 ␮g/l. NAG and ␣1m were
analysed in the second-morning spot urine sample. NAG was
determined by the spectrophotometric method according to
Maruhn [17]. Incubation medium contained, in a final volume of
0.4 ml, 5 nmol/l p-nitrophenyl-2-acetamido-␤-D -glucopyranoside as a substrate in 50 mmol/l citrate buffer (pH 4.14). The reaction was started by the addition of 0.2 ml of undialysed urine,
carried out for 15 min at 37 ° C, and then terminated with 1 ml of
glycine buffer, pH 10.5. Absorbance was measured at 405 nm
406
against a sample terminated at time zero. The calculation of the
NAG level was performed from the molar extinction coefficient
of the product of the reaction, p-nitrophenol, equal to 18.5 cm 2/
␮mol. From the preliminary experiments it was clear that the
dialysis of urine did not affect the NAG level in urine. An immunoturbidimetric test (Tina-quant ␣1-microglobulin; Roche, Basel, Switzerland) was used for the quantification of ␣1m in urine.
The detection limit of the method was 2 mg/l. Urinary NAG,
␣1m and PIIINP were reported per milligram or gram of urine
creatinine to correct the variation in urine concentration. Potassium, sodium, urea, protein and creatinine levels were measured
by standard laboratory techniques. Adverse effects were recorded at each visit in response to questionnaires or as observed by
the investigators.
Statistics
The primary end point of this study was a change in DPE in
measurements available for each patient after treatment with
NAC and placebo. A sample size of 18 patients adequately allowed
a power of 80% to detect a difference in variables equal to withinpatient standard deviation, that is a standardized effect size of 1.0
at a significance level of 0.05 (2-tailed). Secondary end points included urine NAG, ␣1m and PIIINP excretion. Normality and
homogeneity of the variances were verified by means of the Shapiro-Wilk and Levene tests, respectively. Because of their skewed
distribution, diastolic BP, DPE, NAG excretion, PIIINP, serum
creatinine and daily protein intake were logarithmically transformed before statistical analysis and expressed as geometric
means and 95% confidence intervals. Other results are presented
as means 8 SEM. Differences in variable changes between treatment with NAC and placebo were assessed using Student’s t test
(table 2). Differences in variables measured more than twice (table 3) were assessed using ANOVA. A p value less than 0.05 (2tailed) was considered statistically significant. Data were evaluated using the Statistica (version 7.1, StatSoft Inc., Tulsa, Okla.,
USA) software package.
Results
Of the 20 patients who entered the study, 19 (95%)
completed the protocol. One patient dropped out because
of the withdrawal of informed consent, but not due to a
Renke et al.
Table 3. Changes in parameters during the study
Parameter
Randomization point
After NAC
After placebo
p
Na urine excretion, mmol/24 h
Daily protein intake, g/24 h
Systolic BP, mm Hg
Diastolic BP, mm Hg
242.8821.6
1.02 (0.93–1.14)
118.282.8
75.4 (71.5–80.3)
243.3813.5
1.0 (0.94–1.09)
121.182.13
77.5 (74.0–81.7)
221.3824.98
0.93 (0.83–1.09)
123.782.53
78.6 (74.8–83.2)
0.56
0.36
0.14
0.41
Figures shown in parentheses are ranges.
side effect of the therapy. Clinical characteristics of the
patients are listed in table 1.
24-Hour Urine Protein Excretion
There were no significant changes in DPE level after
NAC as compared to placebo (table 2).
Urinary NAG and ␣1m Excretion
There were no significant changes in urinary NAG
and ␣1m excretion levels after adding NAC as compared
to placebo (table 2).
PIIINP Excretion
There were no significant changes in urinary PIIINP
after adding NAC as compared to placebo (table 2).
BP, Renal Function, Sodium and Protein Intake
The control of BP was adequate during all study periods; all patients achieved a target office trough BP of below 130/80 mm Hg. There were no differences in office
trough systolic and diastolic BP between the treatment
periods. Renal function assessed by means of serum creatinine and eGFR remained stable throughout the study
periods. There were no differences in sodium and protein
intake between treatment periods (table 3).
Adverse Effects
NAC therapy was well tolerated by all patients. Adverse effects were not reported.
Discussion
Evidence is available that oxidative stress contributes
to the pathophysiology of kidney injury [7]. First, RS are
suggested to induce processes known to be involved in
chronic renal scarring, such as cell proliferation, apoptosis, inflammation and vascular injury [18–21]. RS also
participate in vascular smooth muscle cell growth and
migration [22], impairment of endothelial-dependent
vascular relaxation [23] and the development of atherosclerosis [24], processes closely related to vascular injury.
Second, induction of oxidative stress at the kidney level
may cause pathological changes resembling those seen in
chronic kidney diseases. Rats subjected to sustained impairment of antioxidant defence displayed enhanced expression of genes for interstitial and basement membrane
collagens, increased interstitial infiltration, proteinuria
and decreased glomerular filtration. More than a 3-fold
increase in transforming growth factor-␤1 mRNA expression was also found, suggesting a critical role of this
cytokine in oxidative kidney damage [25]. Third, increased generation of RS has been shown to occur in glomerulonephritis [26], interstitial fibrosis [27], hypertensive nephroangiosclerosis [28] and obstructive nephropathy [29]. Human studies support this concept as well.
Increased glomerular expression of antioxidant enzymes
was found in IgA nephropathy and lupus nephritis [30].
Neutrophils taken from patients with glomerulonephritis show higher RS generation than in those taken from
healthy individuals [31]. Finally, the administration of
various natural or synthetic antioxidants has been shown
to be of benefit in the prevention and attenuation of renal
scarring in numerous animal models of kidney diseases.
These include vitamins, ␣-lipoic acid, melatonin, dietary
flavonoids, phytoestrogens and many others [reviewed in
7]. Given these facts one may consider the concept that
antioxidants might be applied therapeutically as a nephroprotective strategy.
Antioxidant strategies are based on 2 main mechanisms: the inhibition of RS generation and the enhancement of RS elimination. NAC, a synthetic precursor of
GSH, is a thiol-containing compound which stimulates
the intracellular synthesis of GSH, enhances glutathioneS-transferase activity and acts solely as an RS scavenger
[8, 32]. The rationale for the use of this antioxidant in
407
kidney diseases stems also from findings that NAC suppresses plasma and tissue angiotensin-converting enzyme activity [33], attenuates the cytotoxic properties of
advanced glycation end products [34] and decreases the
homocysteine plasma level [35]. NAC was also found to
decrease systemic BP [36]. Some reports [37], but not all
[38], have shown that it can inhibit NF-␬B activation in
renal mesangial and epithelial cells.
Interventional animal studies have confirmed the
nephroprotective properties of NAC in cyclosporine- and
mercury-induced nephrotoxicity as well as in ischemia/
reperfusion injury [39–42]. The attenuation of histological abnormalities involving tubular injury as well as preservation of renal function were observed after NAC. In
contrast, benefits of NAC administration were not found
in a model of interstitial inflammation [38]. Clinical data
on this point are very limited and exclusively associated
with the prevention of contrast-induced nephropathy. In
a randomized placebo-controlled study of 83 patients,
Tepel et al. [43] showed that oral administration of NAC
in combination with hydration significantly reduces the
incidence of radiocontrast nephropathy. The results of
subsequent studies are not unequivocal [44]. Some of
them confirmed the preventive effect of NAC against
contrast injury [45], while some did not [46, 47]. Nor were
there shown to be any protective effects of NAC on the
kidney in patients undergoing cardiac surgery and elective aortic aneurysm repair [14, 15].
To the best of our knowledge, the present study is the
first to evaluate the influence of NAC on the progression
of chronic kidney disease. We analysed the effects of
NAC on proteinuria, the fundamental marker of glomerular injury and impaired glomerular permselectivity as
well as a marker of poor long-term renal outcome. Considering that tubular epithelial cell injury may initiate the
fibrotic process in kidneys and the fact that the extent of
tubulointerstitial damage is a crucial predictor of renal
outcome [48], tubular cells have become a site of particular interest in the kidney. To evaluate the tubulointerstitial effects of our interventions, the tubular involvement
markers NAG and ␣1m, as well as an indirect marker of
kidney fibrosis, PIIINP, were analysed. A close association between urinary PIIINP excretion and the degree of
interstitial fibrosis was previously evidenced [49]. During
the synthesis and deposition of type III collagen, PIIINP
is degraded from the collagen and secreted into the surroundings. The increased excretion of NAG is thought to
be a specific marker of tubular injury in many renal pathologies including non-diabetic chronic kidney disease
[50]. Increased urinary excretion of ␣1m, a low-weight
408
protein physiologically filtered and reabsorbed by tubular cells, might indicate the reduced capacity of its reabsorption by such cells, and thus it might be a marker of
established tubular damage, with greater urinary concentrations pointing to greater severity of damage [51].
In the present study, the administration of NAC induced no change in proteinuria levels in patients with
non-diabetic chronic kidney disease. The therapy did not
affect the urine excretion of tubular enzymes, suggesting
no improvement in tubular status either. NAC did not
change urine excretion of PIIINP. Given the fact that urinary PIIINP was previously found to originate from the
kidney [49], one may assume that the addition of NAC to
standard therapy with ACEI and ARB did not beneficially affect the fibrotic processes in the kidney.
We do not know why this was so, but at least 3 explanations may be considered. First, one should take into
account the relatively short treatment period, during
which beneficial renal effects may not yet fully develop.
Second, one should realize that the potential benefits for
tubules and the interstitium were extrapolated from presumptive early surrogates. Evidence may be provided
only by histological examinations. Finally, we should also
consider that NAC may not actually reveal nephroprotective properties. Incidentally, NAC was suggested to increase the expression of collagen I and IV and transforming growth factor-␤ [32]. Other authors reported that
NAC has some value as an antioxidant, but only in oxidative stress conditions [52]. Moreover, at doses as low as
1,200 mg daily, it may even exert pro-oxidative properties
in persons with normal intracellular GSH levels [53]. It is
unlikely that confounding factors influenced the present
study outcomes. There were no differences during the
treatment periods with respect to BP, patients’ sodium
and protein intake or renal function. The authors think
that the nephroprotective properties of NAC need to
be further addressed in future controlled long-term
studies.
A potential limitation of the present study is the relatively small sample size, which is sufficiently powered to
detect a significant difference equal to the standard deviation value between treatment periods. Twenty-fourhour urine collections used to assess proteinuria may be
associated with significant collection errors, largely because of improper timing and missed samples, leading to
over- and undercollection. Another limitation may be the
relatively short treatment periods, during which beneficial tubulointerstitial effects may not yet fully develop. In
addition, as mentioned above, one should realize that the
potential benefits for tubules and the interstitium were
Renke et al.
extrapolated from presumptive early surrogates. Evidence may be provided only by histological examination.
In conclusion, the results of the present study suggest
that NAC neither influences proteinuria level nor exerts
a beneficial effect against tubular injury and kidney fibrosis.
Acknowledgements
This study was supported by a grant from the Polish Committee for Scientific Research via the Medical University of Gdansk
(ST-4). The authors thank Hexal Polska and Adamed Polska for
providing drugs.
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Renke et al.
WWW. M ED S CI M ONIT .COM
Product Investigation
The effect of N-acetylcysteine on blood pressure and
markers of cardiovascular risk in non-diabetic patients
with chronic kidney disease: A placebo-cotrolled,
randomized, cross-over study
Authors’ Contribution:
A Study Design
B Data Collection
C Statistical Analysis
D Data Interpretation
E Manuscript Preparation
F Literature Search
G Funds Collection
Marcin Renke ABCDEF, Leszek Tylicki ABCDE, Przemysław Rutkowski ABCDE,
Wojciech Larczynski1 B, Alexander Neuwelt2 EF, Ewa Aleksandrowicz3 B,
Wiesława Łysiak-Szydłowska3 AB, Bolesław Rutkowski1 ADE
1
PI
1
R
SO
O N
N A
LY L
U
1
SE
Received: 2009.09.07
Accepted: 2010.02.08
Published: 2010.07.01
Department of Nephrology, Transplantology and Internal Medicine, Medical University of Gdansk, Gdansk, Poland
Blood Brain Barrier and Neuro-Oncology Program, Oregon Health & Science University, Portland, OR, U.S.A.
3
Department of Clinical Nutrition and Laboratory Diagnostics, Medical University of Gdansk, Gdansk, Poland
1
2
Source of support: The study was supported by a grant from the Polish Committee for Scientific Research via the
Medical University of Gdansk (ST-4). The authors thank Hexal Polska and Adamed Polska for providing drugs
Summary
Background:
Material/Methods:
Cardiovascular complications in patients with chronic kidney disease (CKD) are frequent. They
show increased cardiovascular mortality and morbidity attributable to accumulation of several risk
factors; e.g., hypertension, oxidative stress and elevated plasma homocysteine concentration. Despite
recent progress in their management, there is still no optimal therapy that can stop progression of
CKD and decrease cardiovascular outcome in these patients. Antioxidants, e.g., N-acetylcysteine
(NAC), have been suggested as a promising medicament in this field.
In a placebo-controlled, randomized, two-period cross-over study we evaluated the influence of
eight weeks of NAC therapy (1200 mg/day) added to pharmacological renin-angiotensin system
blockade on ambulatory blood pressure and surrogate markers of cardiovascular risk and injury in
20 non-diabetic patients with albuminuria [30–915 mg per creatinine mg] and normal or slightly
decreased kidney function [eGFR 61–163 ml/min]. After eight weeks run-in period during which
the therapy using angiotensin converting enzyme inhibitors and/or angiotensin receptor blockers was settled, patients were randomly assigned to one of two treatment sequences: NAC/washout/placebo or placebo/washout/NAC.
PE
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© Med Sci Monit, 2010; 16(7): PI13-18
PMID: 20581787
Results:
Conclusions:
key words:
Full-text PDF:
Word count:
Tables:
Figures:
References:
Author’s address:
No significant changes in blood pressure, albuminuria and homocysteine plasma level were observed.
NAC had no effect on blood pressure and surrogate markers of cardiovascular injury in non-diabetic patients with CKD.
N-acetylcysteine • blood pressure • chronic kidney disease • albuminuria • homocyteine
http://www.medscimonit.com/fulltxt.php?ICID=880910
2715
3
1
45
Marcin Renke, Department of Nephrology, Transplantology and Internal Medicine, Medical University of Gdansk,
Debinki 7 Str., 80-211 Gdansk, Poland, e-mail: mrenke@gumed.edu.pl
Current Contents/Clinical Medicine • IF(2009)=1.543 • Index Medicus/MEDLINE • EMBASE/Excerpta Medica • Chemical Abstracts • Index Copernicus
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Med Sci Monit, 2010; 16(7): PI13-18
Background
Table 1. Patient characteristics at baseline.
Parameter
Gender: female/male (n)
8/12
39.35±2.6
Mean systolic blood pressure mmHg
(±SEM)
118.15±2.8
Mean diastolic blood pressure mmHg
75.9 (71.5–80.3)
SE
Mean age years (±SEM)
Albuminuria mg/g creatinine
Risk factors for CVD in CKD may be divided into two broad
categories: traditional and nontraditional [9]. The traditional cardiovascular risk factors, such as hypertension,
left ventricular hypertrophy, diabetes mellitus, smoking,
dyslipidemia and older age, are common in these patients
but are not adequate to fully explain the high prevalence
of CVD. It has been documented that patients with CKD
also are exposed to other nontraditional uremia-related
risk factors, such as anemia, altered calcium-phosphorus
metabolism, inflammation, malnutrition, and oxidative
stress (OS) [10,11].
OS occurs when reactive oxygen species (ROS) production
exceeds local antioxidant capacity, resulting in increased oxidation of important macromolecules, including proteins,
lipids, carbohydrates, and damage to DNA structure [12].
There is increasing evidence associating the role of ROS with
ischemia-reperfusion injury in the heart and in the pathogenesis of atherosclerosis, hypertension, and heart failure
[13]. Evidence shows that OS contributes to the pathophysiology of cardiovascular complications. OS is the central
mechanism by which risk factors, such as hyperlipidemia,
hypertension, diabetes mellitus, and smoking, lead to vascular damage and the clinical sequelae of atherosclerosis.
Given these facts, OS may be a potentially important treatment target, and the administration of antioxidants may be
a promising supplement to RAAS in reducing cardiovascular complications.
N-acetylcysteine (NAC), a synthetic precursor of reduced
glutathione (GSH), is a thiol-containing compound that
stimulates the intracellular synthesis of GSH, enhances
glutathione-S-transferase activity, and acts as an ROS scavenger [14]. In addition to its antioxidant properties, NAC
has other biological actions that may be helpful in cardiovascular protection. By stabilizing nitric oxide (NO), NAC
may have a vasodilatory effect in certain situations [15]. In
addition, NAC’s sulfhydryl group may inhibit angiotensinconverting enzyme, reducing production of the vasoconstrictor angiotensin II. NAC has also been shown to reduce
systemic blood pressure in animals [15,16].
In the present study we evaluated the effects of addition of
NAC to background RAAS treatment on blood pressure and
important cardiovascular risk factors, i.e., albuminuria and
plasma homocysteine levels.
181.7 (160–365)
Serum creatinine mg/dl (±SEM)
eGFR ml/min (±SEM)
BMI kg/m2
R
SO
O N
N A
LY L
U
Cardiovascular disease (CVD) in patients with chronic kidney disease (CKD) is frequent and accounts for approximately 40% of all deaths in these patients. Cardiovascular
mortality is considerably higher in CKD patients than in
the general population [1–3]. Pharmacological inhibition of the renin-angiotensin-aldosterone system (RAAS)
constitutes a cornerstone strategy in the management of
patients with CKD, slowing the progression of renal insufficiency [4–7], as well as reducing cardiovascular complications [8]. Despite recent progress, there is still no optimal therapy that can halt progression of renal disease
and completely protect patients with CKD against cardiovascular problems. Therefore, it is necessary to search for
alternative therapeutic strategies to further improve patient outcomes.
PE
1.03±0.05
100.9±5.9
25.33 (23.65–27.0)
Background hypotensive therapy: (n)
ACEI and AT1RA
10
ACEI
9
AT1RA
1
Diuretic (Hydrochlorotiazide 12.5 mg)
13
Material and Methods
Individuals
Patients were selected from the cohort that attended our renal outpatient department. The inclusion criteria were established as follows: age 18–65 years, chronic non-diabetic
proteinuric nephropathy, normal or slightly impaired stable renal function expressed as estimated glomerular filtration rate (eGFR) above 60 ml/min, stable albuminuria
above 300 mg/ 24 hours, and no steroids or other immunosuppressive treatment for a minimum of six months before the study.
General protocol
The study was a prospective, placebo-controlled, randomized, two-period cross-over trial in which the renal effects of
adding NAC to background nephroprotective therapy with
angiotensin-converting enzyme inhibitors (ACEI) and/or
angiotensin II receptor antagonists (AT1RA) were evaluated. At the beginning, subjects entered the eight weeks
run-in period during which the background nephroprotective therapy using pharmacological blockade of RAAS was
settled with the target blood pressure (BP) below 130/80
mmHg (Table 1). At the end of the run-in period, patients
were randomly assigned to one of two treatment sequences: 8-week NAC (1200 mg/day)/ 8-week washout – background therapy/ 8-week placebo (sequence 1), or 8-week
placebo/ 8-week washout – background therapy/ 8-week
NAC (1200 mg/day) (sequence 2) (Figure 1). Allocation
was performed by a person that was independent of the research team person according to a computer generated randomization list. The patients received 1200 mg of NAC as
effervescent tablets (ACC 600, Hexal AG) divided into two
doses a day. The target BP throughout the study was a BP
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Renke M et al – NAC and cardiovascular risk factors
Figure 1. Study scheme.
Sequence 1
Random
Run in period
I
NAC-1200 mg/day
I
Washout
Placebo
End-point
I
I
Washout
8-weeks
NAC-1200 mg/day
8-weeks
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PLacebo
8-weeks
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I
Sequence 2
8-weeks
of 130/80 mmHg or less. The dosages of ACEI, AT1RA and
diuretics, once established in the run-in period, were left
unchanged throughout the study, including in the washout period. At the randomization point and after the end
of each of the treatment periods ambulatory BP (ABP), serum creatinine and the surrogate markers of cardiovascular injury homocysteine and albuminuria were determined.
Nondipper status was defined as a night-day (N/D) ratio
of mean ABP greater than 0.9. The study was approved by
the local ethics committee (NKEBN/153/2004) and registered in www.clinicaltrials.gov (NCT00572663). The investigated patients all gave their informed consent.
Procedures and laboratory analyses
The office trough BP was measured by Speidel+Keller sphyngomanometer in a sitting position after 10 minutes of rest
and expressed as a mean value of two consecutive measurements taken two minutes apart. Ambulatory BP was measured continuously for 24-h using the Mobil-o-graph (version
12) monitoring system. BP was measured every 15 minutes
during the day (7:00 a.m. to 10:00 p.m.) and every 30 minutes during the night (10:00 p.m. to 7:00 a.m.). Results of
office BP measurements were analysed for systolic (SBP)
and diastolic (DBP); those of ABP measurements for 24-h
SBP, 24-h DBP as well as day-time (D) and night-time (N)
values. A night-day (N/D) ratio of mean ABP was calculated. Albuminuria excretion was measured in a first morning spot urine sample. A first morning urine specimen is
preferable because it correlates best with 24-hour protein
excretion and is required to avoid postural albuminuria.
The authors calculated the ratio of albumin to creatinine
to correct for the variations in urinary concentration due
to hydration. The concentration of albumin was measured
by enzyme-linked immunosorbent assay (ELISA) using an
Albumin (Immunodiagnostic AG, Bensheim, Germany) kit
in accordance with manufacturer’s recommendations. The
intra-assay and inter-assay coefficients of variations for this
assay were 5.0% and 8.0%, respectively. The measurements
of two samples collected within one week were averaged.
Sodium (Na) and urea excretion were evaluated on the basis of 24-hour urine collection. All patients were equipped
with a scaled container and were strictly informed how to
collect 24-hour urine. They collected two 24-hour urines,
and of those the mean value of Na excretion were calculated for data evaluation. Patients were asked not to perform
heavy physical activity on the urine collection days and were
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Med Sci Monit, 2010; 16(7): PI13-18
instructed not to change their usual daily protein and sodium intake during the study period. eGFR was calculated
according to the Cockroft-Gault equation. The serum sample was collected for the quantitative determination of total L-homocysteine level. The samples are stored at –75°C
until assayed. Homocysteine was measured via Enzyme
Immunoassay (EIA) using a kit obtained from Axis-Shield
Diagnostic Ltd. (United Kingdom). The homocysteine reference range was established based on 95% confidence limits as 5–15 µmol/L. The calibration range was from 2 to
50 µmol/L. The Axis Homocysteine Enzyme Immunoassay
was compared to the University of Bergen HPLC method.
Creatinine levels were measured by standard laboratory
techniques. Adverse effects were recorded at each visit in response to questionnaires or as observed by the investigators.
Statistics
The primary end point of this study was a change in ABP
in measurements available for each patient after treatment
with NAC and placebo. A sample size of 18 patients adequately allowed a power of 80% to detect a difference in a
standardised effect size of 1.0 at a significance level of 0.05
(two-tailed). Data from our pilot measurements and previous studies were used to sample size calculations. A baseline mean blood pressure of 130/80 mmHg was predicted,
consistent with target blood pressure of the run-in period.
According to previous studies we assumed a reduction in
systolic and diastolic blood pressure of approximately 5%,
i.e. from 130 to 123.5 mmHg and from 80 to 76 mmHg,
respectively, with NAC and no changes with placebo. To
give the study an 80% power to detect such a difference
as statistically significant (p<0.05; two-tailed) with a standardised effect size of 1.0 (difference between blood pressure changes in NAC and control group equal to one
standard deviation), 18 patients had to complete the
study. Secondary end points included albuminuria and homocysteine levels. Normality and homogeneity of the variances were verified by means of the Shapiro-Wilk test and
Levene test, respectively. Because of their skewed distribution, diastolic and systolic ABP, albuminuria and serum
creatinine were logarithmically transformed before statistical analysis and expressed as geometric means and 95%
confidence intervals. Other results are presented as means
±SEM. Differences in variables’ changes between treatment
with NAC and placebo were assessed using Student’s t-test
(Table 2). Differences in variables measured more than
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Med Sci Monit, 2010; 16(7): PI13-18
Table 2. Changes of parameters after NAC and placebo.
Baseline–NAC
NAC (+)
Δ
Baseline–Placebo
Placebo
Δ
P
Albuminuria
152.4 (129–315.5) 202.8 (106–299) –14.6±45.6
mg/g creatinine
135.8(115.7–264.7) 107.4 (88.5–230.2) –30.9±18.4
0.3
Serum creatinine
1.02 (0.94–1.15)
mg/dl
0.98 (0.91–1.08) –0.05±0.02
1.02 (0.93–1.15) 1.02 (0.94–1.12) –0.01±0.03
0.31
eGFR ml/min. 101.3±6.5
105.7±7.1
4.38±1.9
95.1±7.7
101.7±6.4
0.59
Homocysteine
12.2±0.84
µmol/L
13.9±1.23
1.52±1.55
13.3±0.72
13.24±0.94
6.6±3.5
SE
–0.05±1.0
0.63
To convert serum creatinine in mg/dL to µmol/L, multiply by 88.4; eGFR in mL/min/1.73 m2 to mL/s/1.73 m2, multiply by 0.01667.
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Table 3. Changes of parameters during the study.
Parameter
Randomisation point
242.8±21.6
After NAC
Na urine excretion mmol/24h
Daily protein intake g/kg/24h
1.02 (0.93–1.14)
Systolic BP mmHg
114.8 (110.2–120.4)
243.3±13.5
1.0
(0.94–1.09) 115.8 (111.5–120.7)
After placebo
p
221.3±24.98
0.56
0.93 (0.83–1.09)
0.36
117.7 (112.2–124.2)
0.68
Systolic Daytime BP mmHg
118.8±2.5
121.6±1.3
122.3±2.6
0.77
Systolic Nighttime BP mmHg
104.2±2.5
108.5±2.4
106.8±3.2
0.63
Diastolic BP mmHg
72.3
(69–76.5)
74.1
(71.2–77.5) 74.9
(70.7–80.2)
0.39
Diastolic Daytime BP mmHg
75.9±1.6
77.1±1.3
79.0±2.2
0.41
Diastolic Nighttime BP mmHg
64.5±2.2
65.6±2.7
65.7±2.7
0.69
N/D ratio Systolic BP mmHg
0.88±0.01
0.89±0.02
0.87±0.01
0.7
N/D ratio Diastolic BP mmHg
0.85±0.02
0.85±0.01
0.83±0.02
0.9
twice (Table 3) were assessed using ANOVA. P values less
than 0.05 (2-tailed) were considered statistically significant.
Data were evaluated using Statistica (version 7.1; StatSoft
Inc, Tulsa, OK) software package.
Albuminuria
Results
Renal function, sodium and protein intake
Of the 20 patients who entered the study, 19 (95%) completed the protocol. One subject dropped out because of
the withdrawal of informed consent non-dependent on a
side effect of therapy. Clinical characteristics of patients
are listed in Table 1.
Renal function assessed by means of serum creatinine and
eGFR remained stable during the study periods. There were
no differences in sodium and protein intake between treatment periods (Tables 2, 3).
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Blood pressure
The control of BP was adequate in all study periods;
all patients reached the target office trough BP below
130/80 mmHg. There were no differences in systolic and
diastolic ABP between the treatment periods. N/D ratio was
unchanged during the study period (Table 3).
Homocysteine
There were no significant differences between homocysteine plasma levels at the end of the two treatment periods (Table 2).
There were no significant changes in albuminuria after NAC
as compared to placebo (Table 2).
Adverse effects
NAC therapy was well tolerated by all patients. Adverse effects were not reported.
Discussion
Evidence is available that OS contributes to the pathophysiology of cardiovascular complications. The overproduction of ROS leads to the loss of NO bioavailability, endothelial dysfunction, lipid peroxidation, atherosclerosis and
plaque instability [11]. OS presents in early stages of CKD
and appears to increase significantly during the progression of nephropathies [17–19]. These facts suggest that
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in dialyzed patients. NAC improved coronary and peripheral vascular function in atherosclerosis and attenuated pulmonary vascular endothelial damage induced by tumor necrosis factor-alfa [33,34]. Tepel et al. [35] reported that use
of 600 mg NAC twice daily in 134 patients for two years reduced composite cardiovascular endpoints in HD patients.
In many studies NAC has been reported to reduce albuminuria in acute states of endothelial injury, including contrastinduced glomerular injury [36]; however, other studies have
failed to confirm such beneficial effects of NAC. The results of the Sharif study showed that low concentrations of
NAC do not offer better endothelial protection than does
heparinized saline. At the maximum concentration NAC
even impaired endothelial relaxation in human saphenous
veins [37]. Miner et al. reported that endothelial function
was unaffected by NAC in cardiac transplant patients [38].
Similarly, NAC did not affect the albumin/creatinine ratio
in severe sepsis, indicating that NAC might not abrogate
sepsis-induced endothelial dysfunction [39].
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Caballos-Picot et al. investigated the glutathione antioxidant
system in patients with CKD and demonstrated diminished
plasma glutathione levels and a profound drop in glutathione peroxidise function [20]. Others have demonstrated
a generalized increase in thiol oxidation and a concomitant decrease in both protein-associated and low molecular weight reduced plasma thiols [11]. Of particular note,
extracellular thiols constitute an important component in
antioxidant defence relevant to cardiovascular disease [21].
These facts strongly support the choice of NAC, a thiol containing antioxidant, as a cardioprotective agent. The rationale for the use of NAC stems also from findings that NAC
suppresses plasma and tissue angiotensin-converting enzyme
activity [22,23] and attenuates cytotoxic properties of advanced glycation end-products (AGEs) [24].
Renke M et al – NAC and cardiovascular risk factors
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antioxidants might be applied therapeutically as a cardioprotective strategy in CKD.
We thus analyzed the influence of NAC administration on
important cardiovascular risk factors (systemic BP, albuminuria and homocysteine plasma levels) in CKD patients. In
experimental studies NAC was found to decrease BP in rats
via enhancing NO-dependent vasodilation [15,16,25]. We
believe the present study is the first to clinically evaluate the
influence of NAC on BP in CKD patients. Ambulatory BP
was analyzed, a technique with improved prognostic value
over office BP as a predictor of cardiac outcome in a CKD
population [26]. ABP monitoring also provides valuable information about the circadian rhythm of BP. Lack of a BP
decrease of at least 10% at night (nondipping phenomenon) is suggested to be associated with enhanced cardiovascular mortality and morbidity in the CKD and general population [26]. In the present study we didn’t find changes
in systolic and diastolic BP after NAC administration, and
no changes in circadian rhythm of BP were observed. This
finding is in contrast to a recently published report on hypertensive patients with type 2 diabetes [27], which found
that NAC and L-arginine administration for six months reduced systolic, diastolic and mean BP. NAC was demonstrated to enhance the bioavailability of NO by forming S-nitrosoNacetylcysteine and S-nitrocysteine and neutralizing ROS.
NO is the most important endothelium-derived vasorelaxant, producing baseline vasodilatation and thus contributing to the maintenance of normal blood flow [28].
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Med Sci Monit, 2010; 16(7): PI13-18
In the present study the influence of NAC on endothelial
function was analyzed. Albuminuria is thought to be a marker
of extensive endothelial dysfunction or generalised vasculopathy, which may lead to heightened atherogenic states. One
possible explanation is that endothelial dysfunction might
promote increased penetration of atherogenic lipoprotein
particles through the arterial wall [29]. Albuminuria is also
an established risk factor for cardiovascular morbidity and
mortality [30]. In this study no beneficial effects of NAC on
albuminuria were observed. In many previous reports, NAC
was shown to improve endothelial function. For instance,
NAC has been demonstrated to improve brachial artery endothelial function as assessed using high-resolution ultrasound in patients with coronary artery diseases [31]. Sahin
et al. [32] proved that oral administration of 600 mg NAC
twice daily could improve endothelial function by preventing the reduction of flow-mediated dilatation as measured
in high-resolution Doppler ultrasound of the brachial artery
We don’t know exactly why NAC was ineffective in reducing BP and albuminuria in the present study, but at least
three explanations may be considered. First, one should
take into account the relatively short treatment period
used, during which beneficial endothelial effects may not
have had time to fully develop. Second, NAC was previously suggested to protect against development of hypertension and decrease elevated BP, but it may not affect BP in
a normal range. Third, the authors have taken as an axiom
that NAC attenuates OS, but they did not document real
antioxidant effects in the studied patients. We should also
consider that NAC may actually have no protective effects.
Other authors reported that NAC has some value as an antioxidant, but only in certain conditions [40]. Moreover, at
doses as low as 1200 mg daily, NAC may even exert pro-oxidative properties in persons with the normal intracellular
GSH level [41]. It is also possible that NAC may elicit some
toxic effects; for instance, systemic toxicity of NAC after intravenous infusion was previously documented [42].
There is strong evidence that the reduction of elevated
plasma homocysteine concentrations in patients with CKD
is of clinical importance. A recent study showed an association of quartiles of plasma homocysteine concentration with mortality in patients with end-stage renal failure
[43], and prospective study demonstrated that the relative
risk for cardiovascular events, including death, increased
1% per micromolar increase in plasma homocysteine concentration [44]. In some previous reports [31,45] suggested NAC alters the protein-binding of thiol components in
plasma, displaces circumlating thiols from their protein
binding sites, increases homocysteine urine excretion and
decreases homocysteine plasma levels. The authors failed
to confirm such beneficial effects in their population with
CKD. Previously, Miner and colleagues reported no changes in homocysteine plasma levels after NAC supplementation in cardiac transplant recipients [38]. The explanation
for these negative results may be that homocysteine plasma
levels were in the normal range in all participants.
Conclusions
In conclusion, the results of the present study suggest that
NAC used over a short time period neither influences
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Med Sci Monit, 2010; 16(7): PI13-18
homocysteine plasma levels nor exerts beneficial effects
against albuminuria and blood pressure elevation in patients with CKD stage 1 and 2.
23. Tylicki L, Renke M, Rutkowski P et al: Effects of N-Acetylcysteine on
Angiotensin-Converting Enzyme Plasma Activity in Patients with Chronic
Kidney Diseases. Blood Purif, 2008; 26(4): 354
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4. Tylicki L, Rutkowski P, Renke M et al: Triple Pharmacological Blockade
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risk factor for cardiovascular disease and all-cause mortality: a pooled
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5. Renke M, Tylicki L, Rutkowski P et al: Low-dose dual blockade of the
renin-angiotensin system improves tubular status in non-diabetic proteinuric patients. Scand J Urol Nephrol, 2005; 39(6): 511–17
6. Tylicki L, Larczynski W, Rutkowski B: Renal protective effects of the renin-angiotensin-aldosterone system blockade: from evidence-based approach to perspectives. Kidney Blood Press Res, 2005; 28: 230–42
7. Rutkowski P, Tylicki L, Renke M et al: Low-dose dual blockade of the
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8. Mann JF, Gerstein HC, Pogue J et al: Renal insufficiency as a predictor
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9. Sarnak MJ, Levey AS: Cardiovascular disease and chronic renal disease:
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14. Aruoma OI, Halliwell B, Hoey BM, Butler J: The antioxidant action of
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21. Mills BJ, Weiss MM, Lang CA et al: Blood glutathione and cysteine changes in cardiovascular disease. J Lab Clin Med, 2000; 135(5): 396–401
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Published online: May 19, 2008
Blood Purif 2008;26:354
DOI: 10.1159/000133431
Effects of N-Acetylcysteine on AngiotensinConverting Enzyme Plasma Activity in Patients
with Chronic Kidney Diseases
Leszek Tylicki a Marcin Renke a Przemyslaw Rutkowski a Wojciech Larczynski a
Ewa Aleksandrowicz b Wieslawa Lysiak-Szydlowska b Boleslaw Rutkowski a
a
Department of Nephrology, Transplantology and Internal Medicine, and a Department of Clinical Nutrition,
Medical University of Gdansk , Poland
In one of the previous issues of Blood
Purification Sahin et al. [1] reported that
N-acetylcysteine (NAC) could improve
endothelial dysfunction in patients with
chronic kidney disease (CKD). The authors suggested that this effect is related to
the action of NAC as an antioxidant, i.e.
free-radical scavenger, or as a reactive sulfhydryl compound that increases the reducing capacity of the cell. Here, we would
like to extend these observations and present the results of our recent clinical study
which indicate that NAC could also interfere with the renin-angiotensin system reducing the concentration of circulating
angiotensin-converting enzyme (ACE). In
a placebo-controlled, randomized, open
two-period cross-over study, we evaluated
the influence of NAC on plasma concentration of ACE in 20 nondiabetic patients
with persistent proteinuria (0.4–6.36 g per
24 h) with normal or slightly lowered kidney function (eGFR 61–163 ml/min). Subjects entered the 8-week run-in period
during which antihypertensive therapy
was settled with a target blood pressure below 130/80 mm Hg. Next, patients were
randomly assigned to one of two treatment
sequences: 8 weeks NAC (1,200 mg/day)/8
weeks washout/ 8 weeks placebo (sequence
1) or 8 weeks placebo/8 weeks washout/8
weeks NAC (1,200 mg/day) (sequence 2).
The dosages of any drugs once established
in the run-in period, were left unchanged
throughout the study, as well as in the
washout period. Circulating ACE concentration was determined in plasma with a
commercial ELISA system (Human ACE
Immunoassay Quantikine, R&D Systems,
Minneapolis, Minn., USA). NAC significantly reduced ACE concentration (⌬
mean 8 SEM) relative to placebo (–20.13
8 8.96 vs. –7.59 8 7.43 ng/ml; p = 0.036).
A similar conclusion was reached previously in experimental conditions showing
that different antioxidants including NAC
may decrease local endothelial ACE activity [2]. The molecular and cellular mechanisms by which oxidative stress may mediate ACE activity are not clear so far. Given
that increased endothelial expression of
ACE plays an important role in cardiovascular remodeling [3], one may indicate
that NAC not only improves endothelial
© 2008 S. Karger AG, Basel
0253–5068/08/0264–0354$24.50/0
Fax +41 61 306 12 34
E-Mail karger@karger.ch
www.karger.com
Accessible online at:
www.karger.com/bpu
dysfunction but also prevents against vascular and myocardial structural changes.
It may have a great clinical implication given that the population with CKD is at very
high risk of cardiovascular complications.
References
1 Sahin G, Yalcin AU, Akcar N: Effect of Nacetylcysteine on endothelial dysfunction in
dialysis patients. Blood Purif 2007; 25: 309–
315.
2 Usui M, Egashira K, Kitamoto S, Koyanagi
M, Katoh M, Kataoka C, Shimokawa H,
Takeshita A: Pathogenic role of oxidative
stress in vascular angiotensin-converting
enzyme activation in long-term blockade of
nitric oxide synthesis in rats. Hypertension
1999;34:546–551.
3 Takemoto M, Egashira K, Usui M, Numaguchi K, Tomita H, Tsutsui H, Shimokawa H,
Sueishi K, Takeshita A: Important role of tissue angiotensin-converting enzyme activity
in the pathogenesis of coronary vascular and
myocardial structural changes induced by
long-term blockade of nitric oxide synthesis
in rats. J Clin Invest 1997;99:278–287.
Dr. Leszek Tylicki
Department of Nephrology, Transplantology and Internal Medicine
PL–80211 Gdansk (Poland)
Tel./Fax +48 58 346 1186, E-Mail leszek.tylicki@amg.gda.pl
Vol. 57, No 4/2010
547–552
on-line at: www.actabp.pl
Regular paper
Atorvastatin improves tubular status in non-diabetic patients
with chronic kidney disease — placebo controlled, randomized,
cross-over study
Marcin Renke1*, Leszek Tylicki1, Przemysław Rutkowski1, Alexander Neuwelt2, Wojciech
Larczyński1, Marcin Ziętkiewicz3, Ewa Aleksandrowicz4, Wiesława Łysiak-Szydłowska4 and
Bolesław Rutkowski1
Department of Nephrology, Transplantology and Internal Medicine, Medical University of Gdansk, Poland; 2Blood Brain Barrier and NeuroOncology Program, Oregon Health & Science University, Portland, Oregon, USA; 3Department of Internal Medicine, Connective Tissue Diseases
and Geriatrics, and 4Department of Clinical Nutrition and Laboratory Diagnostics, Medical University of Gdansk, Poland
1
Background. There is evidence that dyslipidemia is associated with chronic kidney disease (CKD) and it has
been implicated in the progression of renal damage.
Optimal management of dyslipidemia should therefore
lead to renal benefits. A number of experimental models demonstrate a beneficial effect of statins in ameliorating renal damage. However, the exact mechanism
by which statins protect against renal damage remains
unclear. Methods. In a placebo-controlled, randomized,
cross-over study we evaluated the influence of atorvastatin (ATO) 40 mg/day added to the renin-angiotensinaldosterone systeme (RAAS) blockade on proteinuria
and surrogate biomarkers of tubular damage or injury
in 14 non-diabetic patients with proteinuria (0.4–1.8 g
per 24 h) with normal or declined kidney function (eGFR
55–153 ml/min). In the eight-week run-in period, therapy
using angiotensin converting enzyme inhibitors (ACEI)
and/or angiotensin II subtype 1 receptor antagonists
(ARB) was adjusted to achieve a blood pressure below
130/80 mm Hg. Next, patients were randomly assigned
to one of two treatment sequences: ATO/washout/placebo or placebo/washout/ATO. Clinical evaluation and laboratory tests were performed at the randomization point
and after each period of the study. The primary end
point of this study was a change in proteinuria measured as 24-h urine protein excretion (DPE). Secondary
end points included urine N-acetyl-β-d-glucosaminidase
(NAG) and α1-microglobulin (α1m) excretion. Results.
The ATO therapy significantly reduced urine excretion of
α1m (p=0.033) and NAG (p=0.038) as compared to placebo. There were no differences in proteinuria, blood pressure, eGFR and serum creatinine between the ATO and
placebo groups. Conclusion. Atorvastatin treatment is
safe and improves biomarkers of tubular damage or injury in non-diabetic patients with CKD.
Keywords: Atorvastatin, kidney, chronic kidney disease, proteinuria,
tubular injury
Received: 21 May, 2010; revised: 16 October, 2010; accepted:
06 November, 2010; available on-line: 16 November, 2010
INTRODUCTION
Despite recent progress, there is still no optimal
therapy that stops progression of renal disease. Therefore, it is necessary to search for alternative therapeu-
tic strategies which can further improve renal outcome
(Renke et al., 2010). There is evidence that dyslipidemia
is associated with chronic kidney disease (CKD) (Guijarro & Keane, 1993; Samuelsson et al., 1997). Experimental studies have established that lipids are damaging to the kidney (Keane et al., 1988; Rutkowski et al.,
2003). The administration of various statins has been
reported to exhibit beneficial effects in a number of
experimental models of chronic kidney diseases suggesting that lipids may represent important therapeutic
targets to halt or attenuate renal injury (Tylicki et al.,
2003). The benefits of statins can be explained not only
by their lipid-lowering potential but also by non-lipid
related mechanisms, the so called “pleiotropic effects”.
Several studies have evaluated the effects of statins
on the progression of CKD in human subjects but
the results are controversial (Chan et al., 1992; Fuiano
et al., 1996; Bianchi et al., 2003; Strippoli et al., 2008;
Banach et al., 2009). Considering the prognostic impact
of proteinuria reduction on long-term renal outcome,
in the present study we evaluated the effects of addition of atorvastatin (ATO), a 3-hydroxy-3-methyglutaryl
coenzyme A (HMG-CoA) reductase inhibitor, to background nephroprotective therapy consisting of angiotensin converting enzyme inhibitors (ACEI) and/or
angiotensin II subtype 1 receptor antagonists (ARB).
ATO, in contrast to many other statins, does not require dosage modification at any level of renal function
(K/DOQI 2003). The patients were then evaluated for
proteinuria, inflammation, renal function, and surrogate
biomarkers of tubular injury. The primary end point
of this study was a change in proteinuria measured as
24-h urine protein excretion (DPE), in measurements
available for each patient. Secondary end points included urine N-acetyl-β-d-glucosaminidase (NAG) and α1microglobulin (a1m) excretion.
*
e-mail: mrenke@gumed.edu.pl
Abbreviations: α1m, α1-microglobulin; ACEI, angiotensin converting enzyme inhibitors; ALAT, alanine aminotransferase; ARB, angiotensin II subtype 1 receptor antagonists; ASAT, aspartate aminotransferase; ATO, atorvastatin; BMI, body mass index; BP, blood
pressure; CK, creatine phosphokinase; CKD, chronic kidney disease;
CVD, cardiovascular diseases; DPE, 24-h urinary protein excretion;
eGFR, estimated glomerular filtration rate; hsCRP, high sensitive Creactive protein; NAG, N-acetyl-β-d-glucosaminidase; RAAS-rennin,
angiotensin-aldosterone systeme
548
M. Renke and others
METHODS
Patients were selected from a cohort that attended our renal outpatients’ department. The inclusion
criteria were as follows: age 18–65 years, chronic
non-diabetic proteinuric nephropathy without dyslipidemia, normal or slightly impaired stable renal function expressed as estimated glomerular filtration rate
(eGFR) above 45 ml/min, stable proteinuria above
300 mg/24 h, and no steroids or other immunosuppressive treatment for a minimum of six months before the study. Stable renal function and proteinuria
were defined as a variability of serum creatinine and
proteinuria less than 25 % during six months before
the start of the study. Patients with total cholesterol
less than 200 mg/dl, low-density lipoprotein (LDL)
cholesterol < 130 mg/dl, and triglycerides < 150 mg/
dl were included. Exclusion criteria were as follows:
nephritic syndrome, diabetes mellitus, cardiovascular
disease (CVD), potassium serum level > 5.1 mmol/l,
history of malignancy including leukemia and lymphoma, fertile women who were not taking oral contraceptives, pregnant or lactating women, patients
with active liver disease, i.e., aspartate aminotransferase (ASAT) or alanine aminotransferase (ALAT)
values more than three times the upper reference
values, and known or suspected contraindications to
the study medications, including a history of adverse
reactions to statins, ACEI or ARB.
General protocol. The study was a prospective,
placebo-controlled, randomized, two-period cross-over
trial in which the renal effects of adding ATO (Sortis; Parke-Davis, Pfizer Polska) to background nephroprotective therapy with ACEI and/or ARB (Xartan;
Adamed Polska) were evaluated. At the beginning,
subjects entered an eight week run-in period during
which the background nephroprotective therapy using pharmacological blockade of RAAS was adjusted
to give a target blood pressure (BP) below 130/80
mm Hg (Table 1). At the end of the run-in period,
patients were randomly assigned to one of two treatment sequences: twelve-week ATO (40 mg/day)/12week washout — background therapy/12-week placebo (sequence 1) or 12-week placebo/12-week washout
— background therapy/12-week ATO (40 mg/day)
(sequence 2) (Fig. 1). Allocation was performed by a
Figure 1.
2010
person that was independent of the research team according to a computer generated randomized list. The
patients received 40 mg of ATO as tablets (Sortis 40,
Pfizer) once a day. The target BP during the whole
study was an office visit BP of 130/80 mm Hg or
less. The dosages of ACEI, ARB and diuretics, once
established in the run-in period, were left unchanged
throughout the study and in the washout period. At
the randomization point and after the end of each
treatment periods, office trough BP, serum creatinine,
potassium, proteinuria measured as 24-h urine protein
excretion (DPE), sodium excretion (Na ex), urea excretion, and surrogate markers of tubular injury (urine
excretion of N-acetyl-β-d-glucosaminidase (NAG),
α-1-microglobulin (α1m)) were determined. The study
was approved by the local ethical committee (NKEBN/749/2003) and all the patients gave informed
consent. The study was registered at www.clinicaltrials.
gov and received a positive opinion (NCT00572312).
Procedures and laboratory analyses. The office trough BP was measured with Speidel+Keller
sphyngomanometer in a sitting position after 10 min
of rest and expressed as a mean value of two consecutive measurements taken 2 min apart. DPE, Na
ex and urea excretion were evaluated on the basis of
24-h urine collection. All patients were equipped with
a scaled container and were strictly informed how to
collect 24-h urine. They collected two 24-h urines —
of those the mean value of DPE was calculated for
data evaluation. Patients were asked not to perform
heavy physical activity on the urine collection days
and were recommended not to change their usual daily protein and sodium intake during the study period.
The excretion of urea was used to calculate the protein intake according to Maroni equation: protein intake normalized to weight (g/kg per day)=6.25×([ureaN-excretion urine 24 h (g/day)]+[0.0031×body weight
(kg)])/ body weight (kg) (Maroni et al., 1985). eGFR
was calculated according to Cockcroft-Gault formula (Cockcroft & Gault, 1976). NAG and α1m were
analyzed in the second morning spot urine sample.
NAG was determined by the spectrophotometric
method according to Maruhn (1976). Incubation medium contained in a final volume of 0.4 ml, 5 nmol/l
P-nitrophenyl-2-acetamido-β-d-glucopyranoside as a
substrate in 50 mmol/l citrate buffer (pH 4.14). The
reaction was started by the
addition of 0.2 ml of undialysed urine, carried out
for 15 min. at 37 °C, and
then terminated with 1 ml
of glycine buffer, pH 10.5.
Absorbance was measured
at 405 nm against a sample terminated at time zero.
The calculation of the NAG
level was made from the molar absorbance coefficient of
the product of the reaction,
P-nitrophenol, equal to 18.5
cm2/μmol. From preliminary
experiments it was clear that
the dialysis of urine did not
affect NAG level in urine.
Immunoturbidimetric
test
(Tina-quant α1-microglobulin,
Roche, Basel, Switzerland)
was used for quantification of
Vol. 57 Atorvastatin in chronic kidney diseases
Parameter
7/7
Mean age (years)
34.2 ± 6.94
Mean systolic blood pressure (mm Hg)
111.5 ± 7.8
Mean diastolic blood pressure (mm Hg)
71.2 (66.4–75.7)
0.85 (0.35–1.8)
Serum creatinine (mg/dl)
1.05 ± 0.27
eGFR (ml/min)
104.7 ± 33.3
Total cholesterol (mg/dl)
191.9 ± 21
hsCRP (mg/l)
0.91 (0.33–2.22)
BMI (kg/m2)
25.97 (23.3–29.3)
Histopatological diagnosis: (n)
8
1
3
1
IgA nephropathy
3
Unknown non-diabetic proteinuric chronic kidney diseases
6
ACEI and ARB
10
ACEI
3
ARB
1
Note: To convert serum creatinine in mg/dl to µmol/l, multiply by 88.4; eGFR in ml/min/1.73 m2
to ml/s/1.73 m2, multiply by 0.01667; Abbreviations: BMI, Body mass index; hsCRP, high sensitive
C-reactive protein; eGFR, estimated glomerular filtration rate
α1m in urine. The detection limit of the method was
2 mg/l. Urinary NAG and α1m were reported per mg
or g of urine creatinine to correct for the variation
in urine concentration. We measured high sensitive
C-reactive protein (hsCRP) with a commercial ELISA
Kit (DRG, EIA-3954) and reported it in mg/l. Total
cholesterol, LDL cholesterol, HDL cholesterol, serum
triglyceride, ASAT, ALAT, creatine phosphokinase
(CK), potassium, sodium, urea, protein and creatinine
levels were measured in fresh blood samples drawn
after fasting overnight for at least 12 h. These parameters were measured by standard laboratory techniques. Body mass index (BMI) was calculated asmass
(kilograms) divided by height (meters) squared. Adverse effects were recorded at each visit in response
to questionnaires or as observed by the investigators.
549
Statistics. The primary end
point of this study was a change
in DPE in measurements available for each patient. A sample
size of 12 patients adequately allowed a power of 80 % to detect
a difference in variables equal to
within patient standard deviation,
that is a standardized effect size of
1.0 at a significance level of 0.05
(two-tailed). Secondary end points
included urine NAG and a1m excretion. Normality and homogeneity of the variances were verified
by means of the Shapiro-Wilk test
and Levene test, respectively. Because of their skewed distribution,
diastolic BP, DPE, NAG excretion, hsCRP, serum creatinine and
daily protein intake were logarithmically transformed before statistical analysis and expressed as geometric means and 95 % confidence
intervals. Other results are presented as means ± S.E.M. Differences in variable changes between
treatment with ATO and placebo
were assessed using Student’s t-test
(Table 2). Differences in variables
measured more than twice (Table
3) were assessed using ANOVA. P
values less than 0.05 (2-tailed) were
considered statistically significant.
Data were evaluated using Statistica (version 7.1; StatSoft Inc., Tulsa, OK) software package.
RESULTS
Of the 14 patients who entered the study, 12 (86 %)
completed the protocol. Two of them were dropped out
because of withdrawal of informed consent. This decision was not due a side effect of therapy. Clinical characteristics of patients are listed in Table 1.
Twenty-four-hour urine protein excretion (DPE)
There was no significant change in DPE after ATO as
compared to placebo (Table 2).
Table 2. Changes of parameters after ATO and placebo
Baseline — ATO Δ
Baseline — Placebo Δ
p
DPE (g/24 h)
–0.23 ± 0.08
–0.001 ± 0.13
0.98
α1m excretion (mg/g creat.)
–8.18 ± 3.39
–0.17 ± 0.68
0.033
NAG excretion (IU/creatinine)
–0.92 ± 0.29
–0.16 ± 0.18
0.038
Total cholesterol (mg/dl)
–68.88 ± 7.52
–0.11 ± 8.44
0.001
LDL-C (mg/dl)
–49.0 ± 4.25
–3.22 ± 6.02
0.001
HDL-C (mg/dl)
–5.88 ± 2.85
–1.22 ± 1.21
0.14
Triglycerides (mg/dl)
–21.88 ± 14.2
15.4 ± 16.16
0.12
Note: To convert total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) in mg/dl to mmol/l multiply by 0.02586. To convert triglycerides in mg/dl to mmol/l multiply by 0.01129. Abbreviations: DPE, urinary protein excretion; α1m, α1-microglobulin;
NAG, N-acetyl-β-d-glucosaminidase
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M. Renke and others
2010
Table 3. Changes of parameters during the study
Randomization point
After ATO
After Placebo
p
223.9 ± 28.8
208.8 ± 27.3
215.1 ± 28.4
0.68
1.1 (0.93–1.14)
1.0 (0.94–1.12)
1.12 (0.83–1.39)
0.64
1.05 ± 0.1
1.07 ± 0.1
1.12 ± 0.1
0.08
hsCRP (mg/l)
0.91 (0.3–2.21)
0.47 (0.29–0.77)
0.77 (0.4–1.49)
0.47
ALAT (IU/l)
22.3 (18.6–27.8)
29.6 (19.3–48.2)
22.2 (17.1–30.5)
0.049
ASAT (IU/l)
20.4 (17.8–23.8)
25.2 (16.8–38.6)
19.9 (17.4–23.0)
0.089
97 (18–252)
111.8 (85.7–153)
93.5 (18–240.6)
0.14
Parameter
Sodium urine excretion (mmol/24 h)
Daily protein intake (g/24 h)
Serum creatinine (mg/dl)
Creatine phosphokinase (IU/l)
Systolic blood pressure (mmHg)
111.5 ± 2.5
114.4 ± 2.12
115.0 ± 1.97
0.27
Diastolic blood pressure (mmHg)
71.2 (66.4–76.6)
72.1 (68.8–75.6)
69.5 (67.5–71.5)
0.67
Note: To convert serum creatinine in mg/dl to µmol/l, multiply by 88.4. Abbreviations: hsCRP, high sensitive C-reactive protein; ASAT, aspartate
aminotransferase; ALAT, alanine aminotransferase
Urinary NAG and α1m excretion
Urinary NAG (p=0.038) and α1m excretion (p=0.033)
decreased significantly after adding of ATO as compared
to placebo (Table 2).
Serum lipid levels
Total cholesterol (p=0.001) and LDL cholesterol
(p=0.001) decreased significantly after ATO as compared
to placebo. There were no significant changes in triglyceride and HDL cholesterol during the study (Table 2).
Blood pressure, renal function, hsCRP, sodium and
protein intake
The control of BP was adequate in all study periods; all patients reached the target office trough BP
below 130/80 mm Hg. There were no differences in
office trough systolic and diastolic BP between the
treatment periods. Renal function assessed by means of
serum creatinine and eGFR remained stable throughout
the study. hsCRP levels had a tendency to decrease in
ATO treatment but it was not statistically significant
(p=0.47). There were no differences in sodium and
protein intake between treatment periods (Table 3).
Safety
ATO therapy was well tolerated by all patients. Adverse effects were not reported. ASAT and CK were
unchanged during the study period. ALAT statistically
increased after ATO (p=0.049) but it was still in the
normal range.
DISCUSSION
To the best of our knowledge, the present study was
the first to evaluate the influence of atorvastatin, an
HMG-CoA reductase inhibitor, on the markers of renal outcome in proteinuric CKD patients without dyslipidemia and CVD. We analysed the effects of ATO
(40 mg/day) on proteinuria, the fundamental marker
of glomerular injury and impaired glomerular permselectivity. Proteinuria is also a marker of long-term renal
outcome. In the present study, the administration of
ATO provided no change in proteinuria level (p=0.98)
in non-diabetic CKD patients. Only a few randomized
controlled trials directly addressing the effect of statins
on renal function and proteinuria have been reported.
Most of those studies were of small size or short duration, used a variety of statins, and many did not include a placebo arm. Some of them suggest that statins
reduce proteinuria and the rate of decline of GFR (Bianchi et al., 2003; Tonelli, 2006). These positive effects
have been summarized in published meta-analyses (Fried
et al., 2001; Sandhu et al., 2006; Strippoli et al., 2008). Interestingly, there are also studies suggesting that statins,
particularly at high doses, may increase proteinuria (Deslypere et al., 1990; Verhulst et al., 2004). Finally, the National Lipid Association Statin Safety Task Force recently
reported that statin-induced proteinuria is not associated
with renal impairment or renal failure (McKenney et al.,
2006).
Considering that tubular epithelial cell injury may initiate the fibrosis process in kidneys and the fact that the
extent of tubulointerstitial damage is a crucial predictor
of renal outcome, tubular cells have become a renal site
of particular interest. To evaluate tubulointerstitial effects
of our interventions, the tubular involvement markers
NAG and α1m were analysed (Bazzi et al., 2002).
An increased excretion of NAG is thought to be a
specific marker of tubular injury in many renal pathologies including non-diabetic CKD (Bazzi et al., 2002). Increased urinary excretion of α1m, a low-weight protein
physiologically filtered and reabsorbed by tubular cells,
might indicate a reduced capacity of its reabsorption by
such cells and it might be a marker of established tubular damage, with greater urinary concentrations pointing to greater severity of damage (Holdt-Lehmann et al.,
2000). Our results show that treatment with ATO reduces markers of tubular injury. Similar results (although
in experimental models) were described by Tsujihata and
co-workers (2008). That group reported that ATO had
inhibitory effects on renal tubular cell injury. In human
subjects Nakamura and co-workers (2006) presented data
suggesting that pitavastatin ameliorated tubulointerstitial
damage in CKD patients. That effect was independent
of the lipid-lowering effect (Nakamura et al., 2006).
The pleiotropic effects of statins have important clinical implications, independent of their lipid-lowering effects (Fathi et al., 2004; Tonelli et al., 2004; Epstein and
Campese, 2005; Nissen et al., 2005; Ridker et al., 2005;
Goicoechea et al., 2006; Panichi et al., 2006; Renke et al.,
2010). In our previous pilot study we confirmed that
ATO therapy attenuated oxidative stress in patients with
CKD (Renke et al., 2010). They are at an increased risk
Vol. 57 Atorvastatin in chronic kidney diseases
for CVD, and recent reviews suggested that inflammation and oxidative stress could be the primary mediators
explaining the burden of CVD in CKD patients (Arici &
Walls, 2001). Moreover, inflammation plays a central role
in the progression of CKD (Tonelli et al., 2005; Zoja et
al., 2006). Our study used hsCRP, a protein found in
the blood, as a marker of inflammation. Interestingly,
patients with elevated basal levels of CRP are at an increased risk of diabetes, hypertension and cardiovascular
disease (Pradhan et al., 2001; Dehghan et al., 2007). In
our study this parameter had a tendency to decrease with
ATO treatment, but the result was not statistically significant (p=0.47). The fact that most of the patients had
serum hsCRP levels in the normal range at the beginning of the study is probably the main reason why our
results are different from those of some other authors
(Chang et al., 2002; Ichihara et al., 2002; Vernaglione et
al., 2004). Our study confirms the findings of others
(Newman et al., 2006; Shurraw & Tonelli 2006; Newman et al., 2008) that ATO therapy is well tolerated by
CKD patients. Adverse effects were not reported during the study period. It is unlikely that confounding factors might have influenced the outcome of the present
study. The treatment periods did not differ with respect
to blood pressure, patients` sodium and protein intake as
well as renal function. We believe that the nephroprotective properties of ATO need to be addressed further in
future controlled long term studies.
small sample size, although it was sufficiently powered
to detect a significant difference equal to the S.D. value
between treatment periods. A further limitation would
be the fact that the participants were selected on the
basis of their stability. The 24-h urine collections used
to assess proteinuria may be associated with significant
collection errors, largely because of improper timing and
missed samples, leading to over- and under-collection. In
addition, one should realize that the potential benefits
for tubules and interstitium were extrapolated from presumptive early surrogates. Such evidence should be confirmed by histological examination.
In conclusion, the study results suggest that treatment
with ATO (40 mg/day) for 12 weeks in nondialysis patients with CKD induced, in addition to its lipid-lowering effect, a significant decrease in biomarkers of tubular
injury and damage without change in proteinuria. The
treatment was safe and well tolerated by patients.
Acknowledgements
The study was supported by grant from the Committee for Scientific Research through the Medical University of Gdansk (ST-4 and W-80). The authors thank Pfizer
Polska and Adamed for providing drugs. The drug providers and sponsors had no involvement in the study design, patient recruitment, analysis, interpretation of data,
writing of the report, or the decision to submit the report for publication.
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Letter to Editor
Dear Editor,
R
SO
O N
N A
LY L
U
The pharmacological blockade of the renin-angiotensin-aldosterone system (RAAS) is a cornerstone strategy for inhibiting progression of chronic nephropathies. In a recent
Medical Science Monitor paper, Banach et al. [1] discussed
the role of statins in patients with chronic kidney disease
(CKD). Here we elaborate on this very interesting discussion by reporting that atorvastatin attenuates oxidative stress
phenomena in patients with CKD.
tion has no additional effect on proteinuria [5], a finding
in opposition with the observations of Bianchi et al. [6].
However, our present data suggest that atorvastatin may attenuate oxidative stress, as indicated by reduced generation
of potentially nephrotoxic isoprostanes, thus providing additional renal protection for patients with CKD.
SE
Atorvastatin attenuates oxidative stress in patients with
chronic kidney disease
In a randomized, placebo controlled, cross-over study, 14
white adult patients (7 men and 7 women; mean age: 34
years) with nondiabetic proteinuric CKD were evaluated
to test the hypothesis that atorvastatin 40 mg (ATO) combined with standard angiotensin converting enzyme inhibitors (ACEI) and/or angiotensin receptor blockers (ARB)
therapy increases nephroprotection by lowering the level
of potentially nephrotoxic oxidative stress-dependent products. Subjects entered the 8 weeks run-in period when the
therapy using ACEI and/or ARB. Next, patients were randomly assigned to one of two treatment sequences: ATO/
washout/placebo or placebo/washout/ATO. Clinical evaluation and ambulatory blood pressure with laboratory tests
were performed at the randomization point and after each
of 3 periods in the study (every 12 weeks). A commercial
ELISA kit (Cayman Chemical Co) was then used to measure
the urinary excretion of 15-F2t-isoprostane in the treated patients. 15-F2t-isoprostane is accepted as a reliable and sensitive marker of oxidative stress in the human pathologies [2].
PE
WWW. M ED S CI M ONIT.COM
It was found that the ATO treatment significantly reduced
urinary levels of 15-F2t-isoprostane relative to the placebo
group (ANOVA P=0.019) with no changes observed in systemic blood pressure, eGFR and serum creatinine (Table 1).
This finding may be of clinical relevance, as 15-F2t-isoprostane
has biological activity as a potent renal vasoconstrictor [3]
and has been implicated as a causative mediator in hepatorenal syndrome [4].
Interestingly enough, we have previously demonstrated that
a combined therapy with ATO and standard nephroprotec-
References:
1. Banach M, Mikhalidis DP, Kjedsen SE, Rysz J: Time for new indications
for statins? Med Sci Monit, 2009; 15(12): MS1–5
LE
2. Fam SS, Morrow JD: The isoprostanes: unique products of arachidonic acid oxidation-a review. Curr Med Chem, 2003, 10: 1723–40
3. Takahashi K, Nammour TM, Fukunaga M et al: Glomerular actions of
a free radical-generated novel prostaglandin, 8-epi-prostaglandin F2 alpha, in the rat. Evidence for interaction with thromboxane A2 receptors. J Clin Invest, 1992; 90: 136–41
4. Morrow JD, Moore KP, Awad JA et al: Marked overproduction of noncyclooxygenase derived prostanoids (F2-isoprostanes) in the hepatorenal syndrome. J Lipid Mediat, 1993; 6: 417–20
5. Renke M, Rutkowski P, Tylicki L et al: The effect of atorvastatin on proteinuria and markers of tubular injury In non-diabetic patients with
chronic kidney disease – placebo controlled, randomized, cross-over
study. Int Urol Nephrol, 2009; in press
6. Bianchi S, Bigazzi R, Caiazza A, Campese VM: A controlled, prospective study of effects of atorvastatin on proteinuria and progression of
kidney disease. Am J Kidney Dis, 2003; 41: 565–70
Sincerelly,
Marcin Renke1, Narcyz Knap2, Leszek Tylicki1, Przemyslaw
Rutkowski1, Alexander Neuwelt3, Wojciech Larczynski1,
Michal Wozniak2 Boleslaw Rutkowski1
1
Department of Nephrology, Transplantology & Internal
Medicine, Medical University of Gdańsk, Gdańsk, Poland
2
Department of of Medical Chemistry, Medical University
of Gdańsk, Gdańsk, Poland
3
Blood Brain Barrier and Neuro-Oncology Program,
Oregon Health & Science University, Portland, OR,
U.S.A.
Marcin Renke, Department of Nephrology,
Transplantology & Internal Medicine, Medical University
of Gdańsk, Debinki 7 Str., 80-211 Gdańsk, Poland, e-mail:
mrenke@gumed.edu.pl
Received: 2010.01.02
Table 1. Changes of eGFR and Urine Excretion of iPF2α after atorvastatin-ATO and placebo.
Baseline – ATO Δ
Baseline – Placebo Δ
P
eGFR (ml/min.)
–3.37±2.68
1.18±0.09
0.420
Urine Excretion of iPF2α (ng/mg creatinine)
–1.41±0.69
0.53±0.29
0.019
Current Contents/Clinical Medicine • IF(2008)=1.514 • Index Medicus/MEDLINE • EMBASE/Excerpta Medica • Chemical Abstracts • Index Copernicus
LE3
PRACE POGL¥DOWE
Marcin RENKE
Przemys³aw RUTKOWSKI
Leszek TYLICKI
Marcin ZIÊTKIEWICZ
Wojciech LARCZYÑSKI
Boles³aw RUTKOWSKI
Pentoksyfilina stary lek czy nowa nadzieja
nefrologii?
Katedra i Klinika Nefrologii Transplantologii
i Chorób Wewnêtrznych
Akademia Medyczna, Gdañsk
Kierownik:
Prof. dr hab. med. Boles³aw Rutkowski
Farmakologiczna blokada uk³adu
renina-angiotensyna-aldosteron (RAA)
stanowi obecnie podstawow¹ strategiê leczenia przewlek³ych nerfopatii
przebiegaj¹cych z bia³komoczem lub
w fazie przewlek³ej niewydolnoci nerek. Wprowadzenie leków hamuj¹cych
uk³ad RAA do terapii pacjentów z
uszkodzeniem nerek doprowadzi³o do
zwolnienia tempa progresji niewydolnoci nerek. Nie uda³o siê jednak jak
do tej pory ca³kowicie zahamowaæ jej
postêpu. Sk³oni³o to do poszukiwañ
uzupe³niaj¹cych strategii terapeutycznych. Byæ mo¿e pentoksyfilina (PTF)
lek znany i stosowany od wielu lat m.in.
w przewlek³ym niedokrwieniu koñczyn
dolnych i zaburzeniach kr¹¿enia mózgowego bêdzie cennym uzupe³nieniem terapii blokuj¹cej uk³ad RAA i
umo¿liwi pe³niejsz¹ ochronê funkcji
nerek. Dane z badañ dowiadczalnych
oraz pierwsze doniesienia kliniczne
pozwalaj¹ mieæ nadziejê, ¿e znaleziono kolejny orê¿ w walce z chorobami
nerek. Lek ten ma cenne w³aciwoci
antycytokinowe, co pozwala na prze³amanie opornoci na dzia³anie erytropoetyny, zmniejsza nasilenie stanu zapalnego i byæ mo¿e spowalnia tempo
powstawania mia¿d¿ycy miêdzy innymi przez ograniczenie produkcji reaktywnych form tlenu. Prawdopodobnie
najbli¿sze lata przynios¹ odpowied na
pytanie, czy faktycznie PTF jest now¹
nadziej¹ nefrologii.
Pharmacological inhibition of the
renin-angiotensin-aldosteron system
(RAAS) constitutes a cornerstone
strategy in the management of patients
with chronic nephropathies with proteinuria and with chronic renal failure.
Angiotensin converting enzyme inhibitors (ACEI) as well as angiotensin II
subtype 1 receptor antagonists have
been shown to decrease proteinuria,
reduce the local renal inflammatory
processes and slow the progression
of renal insufficiency. Despite recent
progress, there is still no optimal
therapy that would stop progression
of renal disease. May be pentoxifilline
(PTF) - the old medication which is still
used to treat peripheral vascular disease and brain ischemia will be the
new adjunct to RAAS blockade. In addition, PTF has been shown to decrease the production of pro-inflammatory cytokines and reactive oxygen
species. PTF therapy may improve the
hemoglobin response in patients with
previously rh-EPO resistant anemia in
renal failure. This may occur due to
inhibition of proinflammatory cytokine
production. Probably in the next few
years we will get answer to the question of PTF role in nephrology.
Wstêp
Uk³ad renina-angiotensyna-aldosteron
(RAA) odgrywa kluczow¹ rolê w regulacji
cinienia têtniczego, utrzymaniu homeostazy wodno-elektrolitowej oraz procesach
zwi¹zanych ze wzrostem i proliferacj¹ komórek. Podstawowym zadaniem uk³adu
RAA jest utrzymanie sta³ej objêtoci p³ynu
wewn¹trznaczyniowego oraz systemowego
cinienia krwi. Angiotensyna II (Ang II) jest
g³ównym efektorem uk³adu RAA i w trakcie
jego aktywacji indukuje obkurczenie naczyñ
krwiononych oraz nasilenie reabsorbcji
sodu w cewkach nerkowych bezporednio
lub poprzez pobudzenie uk³adu adrenergicznego oraz zwiêkszenie produkcji aldosteronu i wazopresyny. Mimo roli, jak¹ odgrywa
uk³ad RAA w utrzymaniu homeostazy, jego
przewlek³e pobudzenie prowadziæ mo¿e do
niekorzystnych nastêpstw w uk³adzie ser-
cowo-naczyniowym. Wykazano, ¿e Ang II
odgrywa kluczow¹ rolê w procesach zwi¹zanych z uszkodzeniem nerek oraz rozwojem i progresj¹ przewlek³ej ich niewydolnoci. Udzia³ uk³adu RAA w procesach chorobowych w obrêbie nerek nie ogranicza siê
tylko do dzia³ania Ang II. Wykazano, ¿e aldosteron mo¿e równie¿ bezporednio stymulowaæ procesy w³óknienia w nerkach na
drodze aktywacji TGF-ß1. Do patologicznych zmian w obrêbie nerek dochodzi te¿
wskutek dzia³añ katecholamin uwalnianych
z zakoñczeñ nerwowych obwodowego uk³adu adrenergicznego, który stymulowany jest
przez Ang II centralnie oraz obwodowo [38].
Dysponujemy dwiema podstawowymi
grupami leków zmniejszaj¹cymi efekty dzia³ania uk³adu RAA. Ograniczaj¹ one efekty
biologiczne zwi¹zane z aktywacj¹ receptorów AT-1. Inhibitory konwertazy angioten-
Dodatkowe s³owa kluczowe:
pentoksyfilina
nefroprotekcja
niedokrwistoæ
k³êbuszkowe zapalenie nerek
nefropatia cukrzycowa
Additional key words:
pentoxyfilline
kidney protection
anemia
glomerulonephritis
diabetic nephropathy
Adres do korespondencji:
Dr med. Marcin Renke
Katedra i Klinika Nefrologii, Transplantologii
i Chorób Wewnêtrznych AM
Gdañsk 80-211, ul. Dêbinki 7
Tel./Fax: +48 58 3461186
e-mail: mrenke@amg.gda.pl
358
Pentoxifylline old drug or new hope for nephrology?
Przegl¹d Lekarski 2008 / 65 / 7-8
M. Renke i wsp.
syny II (IKA) realizuj¹ to poprzez hamowanie aktywnoci podstawowego enzymu uk³adu RAA, konwertuj¹cego angiotensynê (KA)
i zmniejszenie syntezy Ang II. Blokada ta
nie jest jednak zupe³na z powodu syntezy
Ang II szlakami enzymatycznymi niezale¿nymi od KA, jak równie¿ zjawiska okrelanego jako ucieczka od IKA, to jest opornoci na dzia³anie IKA, rozwijaj¹cej siê u
czêci pacjentów po pewnym czasie skutecznego leczenia tymi lekami. Antagonici
receptora AT-1 (ARA) blokuj¹ wi¹zanie Ang
II z ich najwa¿niejszym receptorem, przy
zachowanej syntezie peptydu [37].
Wprowadzenie leków hamuj¹cych uk³ad
RAA do terapii pacjentów z uszkodzeniem
nerek doprowadzi³o do zwolnienia tempa
progresji niewydolnoci nerek [13,3234,36,39]. Nie uda³o siê jednak jak do tej
pory ca³kowicie zahamowaæ jej postêpu.
Sk³ania to do poszukiwañ uzupe³niaj¹cych
strategii terapeutycznych. Jednym z leków,
który od wielu lat pojawia siê w krêgu zainteresowania nefrologów jest pentoksyfilina
(PTF). Pierwsze doniesienia na temat farmakokinetyki tego preparatu wród chorych
z przewlek³¹ niewydolnoci¹ nerek pochodz¹ sprzed 30 lat [35]. Jednak dopiero wyniki badañ z ostatnich lat pozwalaj¹ mieæ
nadziejê, ¿e posiadamy cenny lek, który
mo¿e mieæ wp³yw na rokowanie pacjentów
z chorobami nerek.
Pentoksyfilina miejsce w nefrologii
Ochrona funkcji nerki przeszczepionej
oraz nerek po transplantacji innych narz¹dów.
Historycznie rzecz bior¹c pocz¹tkowo
zwrócono uwagê na potencjalnie korzystne
dzia³anie PTF wród chorych po transplantacji narz¹dów. Istnieje wiele badañ dowiadczalnych potwierdzaj¹cych tezê, ¿e
PTF chroni funkcjê nerek po transplantacji
narz¹dów [1,2]. Lek ten mia³ miêdzy innymi
ograniczaæ dzia³anie nefrotoksyczne cyklosporyny rutynowo stosowanej wród chorych po transplantacji. Niestety, wiêkszoæ
przeprowadzonych randomizowanych badañ klinicznych nie potwierdzi³a nefroprotekcyjnego dzia³ania PTF w tej grupie pacjentów [6,29,30].
Leczenie wspomagaj¹ce pierwotnych
i wtórnych glomerulopatii
Kilka badañ eksperymentalnych podkrela korzystn¹ rolê PTF w leczeniu k³êbuszkowych zapaleñ nerek. Badania dotycz¹
miêdzy innymi modelu glomerulopatii mezangialnej [7] oraz glomerulopatii z przeciwcia³ami przeciw b³onie podstawnej k³êbuszka (anty GBM) [9]. Niestety, dane kliniczne
s¹ sk¹pe, ale istnieje kilka ciekawych doniesieñ na ten temat. W 2001 roku Doucloux i wsp. [14] przedstawili wyniki badañ przeprowadzonych w grupie 10 chorych z potwierdzonym biopsyjnie b³oniastym k³êbuszkowym zapaleniem nerek, którzy nie odpowiedzieli na typowe leczenie sterydami i do
standartowej terapii otrzymali PTF w dawce 1200 mg/dziennie przez okres 6 miesiêcy. Stwierdzono istotne statystycznie zmniejszenie dobowej utraty bia³ka (p=0,001), obni¿enie poziomu czynnika martwicy nowotworu (TNF=tumor necrosis factor) w surowicy (p=0,001) i moczu (p=0,02) oraz zwiêkPrzegl¹d Lekarski 2008 / 65 / 7-8
szenie poziomu albumin w surowicy krwi
(p=0,0004) przy niezmienionym stê¿eniu
kreatyniny w surowicy krwi. W innym badaniu, które objê³o 11 chorych z opornym na
typowe leczenie immunosupresyjne zespo³em nerczycowym w przebiegu nefropatii
toczniowej, stwierdzono istotne statystycznie zmniejszenie dobowej utraty bia³ka
(p=0,003) pod wp³ywem stosowanej PTF w
dawce od 800 do 1600 mg dziennie przez 6
miesiêcy. Leczenie by³o dobrze tolerowane,
¿aden chory nie przerwa³ terapii z powodu
objawów ubocznych stosowanego leczenia
[16]. W innym badaniu klinicznym, którego
wyniki opublikowano ostatnio, 17 chorych z
pierwotnym k³êbuszkowym zapaleniem nerek i funkcj¹ nerek okrelan¹ jako szacunkowy wskanik filtracji k³êbuszkowej
(eGFR=estimated Glomerular Filtration
Rate) pomiêdzy 25 a 115 ml/min./1,73 m2
otrzymywa³o 800 mg dziennie PTF przez 6
miesiêcy. Leczenie by³o dobrze tolerowane
i pozwoli³o na zmniejszenie dobowej utraty
bia³ka [8].
Zapobieganie rozwojowi i leczenie
nefropatii cukrzycowej
Istnieje szereg badañ eksperymentalnych podkrelaj¹cych ochronn¹ rolê PTF w
wywo³ywanej dowiadczalnie cukrzycy,
szczególnie w zapobieganiu rozwojowi nefropatii cukrzycowej [12,17]. Istniej¹ te¿ nieliczne niestety doniesienia kliniczne podkrelaj¹ce rolê PTF w ochronie funkcji nerek
wród chorych z cukrzyc¹ typu 2. Navarro i
wsp. [28] opublikowali w 1999 roku wyniki
prospektywnego badania przeprowadzonego w grupie 24 chorych na cukrzycê z niewydolnoci¹ nerek (klirens kreatyniny < 35
ml/min.). 14 chorych otrzymywa³o doustnie
400 mg PTF dziennie przez okres 6 miesiêcy, natomiast 10 pacjentów stanowi³o grupê kontroln¹. W grupie badanej stwierdzono statystycznie znamienne zmniejszenie
dobowej utraty bia³ka i zmniejszenie w surowicy TNF-a w porównaniu do grupy kontrolnej (p<0,001) po 6 miesi¹cach badania.
Stê¿enie kreatyniny w surowicy krwi i klirens
kreatyniny pozosta³y niezmienione w obu
grupach podczas badania. Znaleziono równie¿ korelacjê pomiêdzy redukcj¹ dobowej
utraty bia³ka i zmniejszeniem TNF-a (r=0,72,
p<0,01). Wed³ug autorów wiadczy to o
istotnej roli cytokin w progresji nefropatii
cukrzycowej oraz znaczeniu PTF jako leku
antycytokinowego w ochronie funkcji nerek
w tym typie nefropatii. W innym badaniu tej
grupy autorów z 2003 roku udowodniono,
¿e podawanie PTF wród chorych z cukrzyc¹ typu 2 nie tylko ogranicza bia³komocz,
ale równie¿ zmniejsza wydalanie N-acetylbeta-glucosaminidazy (NAG), który jest
markerem uszkodzenia cewek nerkowych
[3]. Badanie by³o prospektywne i przeprowadzone na grupie 45 chorych z cukrzyc¹
typu 2 (30 chorych otrzymywa³o 1200 mg
PTF na dobê przez 4 miesi¹ce, a 15 chorych stanowi³o grupê kontroln¹). Wyniki porównywano z badaniami 15 zdrowych osób
w podobnym wieku i podobnym rozk³adzie
p³ci. Uzyskane dane pozwoli³y wyci¹gn¹æ
wnioski, które podkrelaj¹ ochronn¹ rolê
PTF w populacji chorych z nefropati¹ cukrzycow¹ w przebiegu cukrzycy typu 2 [27].
W innym stosunkowo niedawno opubliko-
wanym randomizowanym, kontrolowanym
badaniu ten sam autor ocenia³ efekt podawanego doustnie PTF w dawce 1200 mg w
grupie 30 chorych z cukrzyc¹ typu 2 w porównaniu do 31 pacjentów w grupie kontrolnej. Wszyscy pacjenci byli leczeni standardow¹ terapi¹ nefroprotekcyjn¹ z u¿yciem
blokera dla receptora AT 1 dla angiotensyny II (ARA) przez okres co najmniej jednego roku. Stwierdzono w grupie otrzymuj¹cej dodatkowo PTF istotne statystycznie (p
<0,001) zmniejszenie albuminurii oraz
zmniejszenie wydalania z moczem TNF-a.
Efekt ten by³ niezale¿ny od zmian cinienia
têtniczego i kontroli metabolicznej [26].
Rola PTF w leczeniu wspomagaj¹cym
niedokrwistoci
Obecnie nie ma w¹tpliwoci, ¿e stosowanie erytropoetyny (EPO) u chorych z
przewlek³¹ chorob¹ nerek (PChN) niesie ze
sob¹ wiele korzyci [18,21]. Pierwsze obserwacje kliniczne dotycz¹ce leczenia
PTF w ma³ych grupach chorych z PChN i
niedokrwistoci¹ s¹ bardzo obiecuj¹ce. W
badaniu prospektywnym Navarro i wsp. z
1999 roku [25] przedstawiono wyniki 7 pacjentów z klirensem kreatyniny < 30 ml/min.
leczonych PTF w dawce 400 mg doustnie
przez okres 6 miesiêcy. Obserwowano znamienny statystycznie wzrost wartoci hemoglobiny (9,9 ± 0,5 g/dl vs 10,6 ± 0,6 g/dl)
przy niezmiennym poziomie EPO w surowicy. Jednoczenie poziom TNF-a w surowicy pacjentów otrzymuj¹cych PTF obni¿y³ siê
znamiennie, przy braku zmian w grupie kontrolnej. W innym opublikowanym w 2004
roku badaniu [11] przeprowadzonym wród
16 chorych z PChN (11 leczonych hemodializ¹, 4 leczonych dializ¹ otrzewnow¹ i 1
z niewydolnoci¹ nerki przeszczepionej)
oraz z³¹ odpowiedzi¹ na stosowane leczenie EPO, które definiowano jako poziom
hemoglobiny < 10,7 g/dl na 6 miesiêcy przed
w³¹czeniem do badania przy dawce EPO a
12 000 IU/tydzieñ, wykazano korzystne dzia³anie PTF. Chorzy otrzymali lek doustnie 400
mg/dobê przez 4 miesi¹ce. 12 pacjentów
ukoñczy³o badanie, wród nich poziom hemoglobiny wzrós³ z 9,5 ± 0,9 g/dl przed terapi¹ do 11,7 ± 1,0 g/dl (p=0,0001) po 4 miesi¹cach stosowania PTF. Efekt ten t³umaczono dzia³aniem przeciwzapalnym, u pod³o¿a którego le¿y hamowanie dzia³ania cytokin prozapalnych TNF-a i IFN-g. Obecnie
mo¿na spodziewaæ siê opublikowania wyników badania przeprowadzonego wród
160 chorych z PChN leczonych EPO lub
darbaerytropoetyn¹ a oraz PTF. Zosta³o ono
zaprojektowane jako badanie randomizowane podwójnie lepe, kontrolowane placebo z grup¹ kontroln¹ st¹d jego wyniki mog¹
odpowiedzieæ na wiele nurtuj¹cych pytañ
dotycz¹cych roli PTF w leczeniu wspomagaj¹cym chorych z PChN i niedokrwistoci¹ [21].
Inne potencjalnie korzystne dzia³ania
PTF w nefrologii
W badaniach eksperymentalnych
stwierdzono, ¿e PTF ma hamuj¹cy wp³yw
na syntezê kolagenu przez komórki b³ony
otrzewnej u ludzi, co mog³oby potencjalnie
znaleæ zastosowanie w leczeniu b¹d zapobieganiu wyst¹pienia bardzo gronego
359
sji PChN przedstawiono na rycinie 1. Istniej¹
równie¿ doniesienia podkrelaj¹ce potencjaln¹ rolê PTF w zapobieganiu rozwojowi
ostrej niewydolnoci nerek (ONN). Mo¿e do
tego dochodziæ poprzez dzia³anie stymuluj¹ce produkcjê prostaglandyn prowadz¹cych
do rozszerzenia naczyñ nerkowych [40] lub
na drodze zmniejszenia efektów endotoksemii towarzysz¹cej ONN [41]. Warto dodaæ, ¿e dzia³anie antycytokinowe PTF mog¹
znaleæ zastosowanie nie tylko w nefrologii, ale równie¿ w reumatologii, diabetologii
i kardiologii.
Rycina 1
Schemat potencjalnego udzia³u pentoksyfiliny (PTF) w hamowaniu progresji PChN.
Scheme of potential pentoxifylline role in prevention of chronic renal failure progression.
powik³ania przewlek³ego leczenia dializ¹
otrzewnow¹ jakim jest stwardniaj¹ce zapalenie otrzewnej (EPS) [15]. Stwierdzono
równie¿, ¿e PTF w badaniach eksperymentalnych hamuje u szczurów p³ytkowy czynnik wzrostu (PDGF) i TGF-ß, co mog³oby
mieæ znaczenie w hamowaniu progresji
mia¿d¿ycy naczyñ, tak powszechnej wród
chorych z PChN [10]. PTF poprzez wp³yw
na receptory dla TNF-a ma te¿ mieæ wp³yw
na zwolnienie katabolizmu bia³ek wród chorych jeszcze nie dializowanych, ale z zaawansowan¹ PChN [4]. Mo¿e mieæ to znaczeniu w zapobieganiu rozwojowi niedo¿ywienia w tej grupie chorych, na które jest
szczególnie nara¿ona ta populacja. Jak wiemy istnieje cis³y zwi¹zek niedo¿ywienia ze
miertelnoci¹ wród chorych z PChN [20].
Inne ciekawe doniesienia podkrelaj¹ rolê
PTF w ochronie funkcji nerek chorych poddawanych radioterapii i chemioterapii
[22,31]. Istnieje te¿ pilota¿owe badanie, w
którym stosowano profilaktycznie PTF do¿ylnie u chorych poddawanych zabiegom
kardiochirurgicznym. Wyniki badañ 20 pacjentów powy¿ej 80 roku ¿ycia, którzy otrzymywali PTF w trakcie i w dwóch kolejnych
dobach po zabiegu przês³owania aortalnowieñcowego porównano z wynikami 20 chorych z grupy kontrolnej, którzy otrzymywali
placebo. By³o to prospektywne, randomizowane, kontrolowane placebo badanie, które pozwoli³o na wykazanie korzyci p³yn¹cych z tego typu postêpowania, które polega³y na ochronie funkcji nerek, ale równie¿
360
w¹troby i ródb³onka naczyñ [5]. Korzystne
dzia³anie nefroprotekcyjne PTF w po³¹czeniu z indometacyn¹ i alfa-tokoferolem, wykazano równie¿ w badaniu klinicznym przeprowadzonym wród 54 chorych poddawanych zabiegom urologicznym lub ESWL z
powodu kamicy dróg moczowych [19].
Domniemane mechanizmy dzia³ania
nefroprotekcyjnego PTF
Na podstawie przeprowadzonych badañ
wiadomo, ¿e PTF poza znanym od wielu lat
dzia³aniem przeciwzakrzepowym m.in. poprzez wp³yw na funkcjê p³ytek krwi, posiada
jeszcze szereg innych potencjalnie korzystnych dzia³añ. Stwierdzono wp³yw hamuj¹cy
PTF na produkcjê cytokin pozapalnych, takich jak TNF-a, przez monocyty i limfocyty
T, czy te¿ interferonu a przez wspomniane
limfocyty. Wydaje siê, ¿e to dzia³anie antycytokinowe mo¿e mieæ znaczenie zarówno
w hamowaniu progresji PChN [27], jak i poprawie wartoci hemoglobiny wród chorych
z zaawansowan¹ PChN leczonych EPO z
powodu niedokrwistoci [21,24]. Wiele
wskazuje na to, ¿e wygaszanie stanu zapalnego mo¿e te¿ pe³niæ istotn¹ rolê w zapobieganiu rozwojowi niedo¿ywienia wród
pacjentów z PChN [4]. Ponadto, PTF poprzez dzia³anie zwalniaj¹ce podzia³y komórkowe oraz hamowanie produkcji kolagenu
[10] mo¿e spowalniaæ postêp ró¿nych nefropatii, które mog¹ prowadziæ do rozwoju
PChN [8,42]. Uproszczony schemat potencjalnego udzia³u PTF w hamowaniu progrePrzegl¹d Lekarski 2008 / 65 / 7-8
Podsumowanie
Obecnie powszechnie uwa¿a siê, ¿e
hamowanie uk³adu RAA pozwala na zwolnienie tempa utraty funkcji nerek wród chorych z PChN. Od kilku lat terapia oparta na
IKA i ARA jest preferowanym leczeniem
nadcinienia i standartowym postêpowaniem nefroprotekcyjnym wród chorych z
ró¿nymi typami nefropatii [13,23,36,37]. Nie
uda³o siê jednak ca³kowicie zahamowaæ jej
postêpu. Sk³ania to do poszukiwañ uzupe³niaj¹cych strategii terapeutycznych. Jednym
z leków, który od wielu lat pojawia siê w krêgu zainteresowania nefrologów jest pentoksyfilina. Wydaje siê, ¿e PTF mo¿e byæ stosowana w terapii uzupe³niaj¹cej u chorych
z PChN przebiegaj¹c¹ z bia³komoczem.
Wymaga to jednak potwierdzenia w du¿ych,
kontrolowanych badaniach klinicznych. Byæ
mo¿e PTF znajdzie sta³e miejsce w terapii
chorych leczonych nerkozastêpczo, zarówno metod¹ hemodializy, jak i dializy otrzewnowej. Wstêpne doniesienia na temat poprawy wartoci hematologicznych w tej grupie chorych, czy zapobieganiu niedo¿ywienia, ograniczaniu stanu zapalnego, czy
wreszcie zapobieganiu wyst¹pienia gronych powik³añ jak np. stwardniaj¹cego zapalenia otrzewnej s¹ interesuj¹ce i wymagaj¹ wyjanienia. Równie¿ nie do koñca
poznana jest rola PTF w profilaktyce uszkodzeñ nerek w okresie oko³ooperacyjnym,
podczas radio czy chemioterapii oraz w leczeniu urologicznym. Wstêpne doniesienia
nakazuj¹ prowadzenie dalszych badañ
szczególnie, ¿e dotychczasowe wyniki s¹
zachêcaj¹ce, a badana substancja jest tania i stosunkowo dobrze tolerowana przez
chorych.
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Vol. 57, No 1/2010
119–123
on-line at: www.actabp.pl
Regular paper
Effect of pentoxifylline on proteinuria, markers of tubular injury
and oxidative stress in non-diabetic patients with
chronic kidney disease — placebo controlled, randomized,
cross-over study
Marcin Renke1, Leszek Tylicki1, Przemysław Rutkowski1, Narcyz Knap2, Marcin Ziętkiewicz3,
Alexander Neuwelt4, Ewa Aleksandrowicz5, Wiesława Łysiak-Szydłowska5, Michał Woźniak2
and Bolesław Rutkowski1
Department of Nephrology, Transplantology and Internal Medicine, 2Department of Medical Chemistry, 3Department of Internal Medicine,
Connective Tissue Diseases and Geriatrics, Medical University of Gdańsk, Gdańsk, Poland; 4Blood Brain Barrier and Neuro-Oncology Program,
Oregon Health & Science University, Portland, Oregon, USA; 5Department of Clinical Nutrition and Laboratory Diagnostics. Medical University
of Gdańsk, Gdańsk, Poland
1
Background: Inhibition of the renin-angiotensin-aldosterone system (RAAS) with angiotensin converting enzyme
inhibitors (ACEI) and/or angiotensin II subtype 1 receptor antagonists (ARB) is a common strategy used in the
management of patients with chronic kidney disease
(CKD). However, there is no universal therapy that can
stop progression of CKD. Pentoxifylline (PTE) is a nonspecific phosphodiesterase inhibitor with anti-inflammatory properties. It has been reported to have promising
effects in CKD treatment. Methods: In a placebo-controlled, randomized, cross-over study we evaluated the influence of PTE (1200 mg/day) added to RAAS blockade
on proteinuria, surrogate markers of tubular injury and
oxidative stress-dependent products in 22 non-diabetic
patients with proteinuria (0.4–4.3 g per 24 h) with normal or declined kidney function [eGFR 37–178 mL/min].
In an eight-week run-in period, therapy using ACEI and/
or ARB was adjusted to achieve a blood pressure below
130/80 mm Hg. Next, patients were randomly assigned
to one of two treatment sequences: PTE/washout/placebo or placebo/washout/PTE. Clinical evaluation and laboratory tests were performed at the randomization point
and after each period of the study. Results: The PTE
therapy reduced proteinuria (by 26 %) as compared to
placebo. There were no differences in α1-microglobulin,
urine excretion of N-acetyl-β-d-glucosaminidase (NAG),
hsCRP, the urinary excretion of 15-F2t-isoprostane, blood
pressure (BP), eGFR and serum creatinine between the
PTE and placebo groups. Conclusion: Pentoxifylline may
decrease proteinuria in non-diabetic patients with CKD.
enzyme inhibitors (ACEI) and angiotensin II subtype
1 receptor antagonists (ARB) have been shown to decrease proteinuria, reduce local renal inflammatory processes and slow down the progression of renal insufficiency (Renke et al., 2004; Renke et al., 2005; Rutkowski
et al., 2004; Tylicki et al., 2007a; 2007b). Despite recent
progress, there is still no optimal therapy that stops progression of CKD. Therefore, it is necessary to search for
alternative therapeutic strategies which can further improve renal outcome.
Considering the prognostic impact of proteinuria reduction on long-term renal outcome, in the present
study we evaluated the effects of pentoxifylline (PTE)
addition to background nephroprotective therapy consisting of ACEI and/or ARB. PTE, a methyl-xanthine
derivative, is a non-selective phosphodiesterase inhibitor
with anti-inflammatory and immunomodulatory effects.
PTE is also widely used to treat peripheral vascular disorders because of its potent hemorrheological properties (Frampton & Brogden, 1995). Moreover, PTE potently inhibits cell proliferation and extracellular matrix
accumulation, factors that play important roles in CKD
progression. The PTE’s benefit when administered in
conjunction with RAAS blockade in patients with CKD
is not clear. Our study evaluated the effects of this treatment on proteinuria, inflammation, oxidative stress, renal
function and surrogate markers of tubular injury.
Keywords: pentoxifylline, oxidative stress, kidney, chronic kidney disease, proteinuria, tubular injury
Individuals. Patients were selected from the cohort
that attended our renal outpatient department. The inclusion criteria were as follows: age 18–65 years, chronic
non-diabetic proteinuric nephropathy without dyslipidemia, normal or slightly impaired stable renal function
expressed as estimated glomerular filtration rate (eGFR)
Received: 04 January, 2010; revised: 11 March, 20201; accepted:
19 March, 2010; available on-line: 22 March, 2010
INTRODUCTION
The incidence and prevalence of chronic kidney disease (CKD) is increasing worldwide. Pharmacological
inhibition of the renin-angiotensin-aldosterone system
(RAAS) constitutes a cornerstone strategy in the management of patients with chronic nephropathies with
proteinuria (Tylicki et al., 2005). Angiotensin converting
MATERIAL AND METHODS
e-mail: mrenke@gumed.edu.pl
Abbreviations: ACEI, angiotensin converting enzyme inhibitors;
ARB, angiotensin II subtype 1 receptor antagonists; BP, blood pressure; CKD, chronic kidney disease; CVD, cardiovascular diseases;
DPE, 24-h urinary protein excretion; eGFR, estiamted glomerular
filtration rate; PTE, pentoxifylline; RAAS, renin-angiotensin-aldosterone system.
120
M. Renke and others
2010
Parameter
7/15
Mean age years (±S.E.M.)
38.6 ± 10.3
Mean systolic blood pressure
mm Hg (± S.E.M.)
123.8 ± 12.6
Mean diastolic blood pressure mm Hg
75.3 (70.6-81.0 )
Urinary protein excretion g/24 h
1.2 (0.4-4.3 )
Serum creatinine mg/dL
1.0 (0.9-1.3 )
eGFR mL/min (± S.E.M.)
121.8 ± 50.2
hsCRP mg/L
2.36 (0.29–10.4 )
BMI kg/m2
27.7 (19.3-36.1)
Histopathological diagnosis: (n)
14
4
1
2
Focal segmental glomerulosclerosis
(FSGS)
2
IgA nephropathy
5
Unknown non-diabetic proteinuric
chronic kidney diseases
8
ACEI and ARB
14
ACEI
7
ARB
1
above 30 mL/min, stable proteinuria above 300 mg/
24 h, and no steroids or other immunosuppressive treatment for a minimum of six months before the study.
Stable renal function and proteinuria were defined as a
variability of serum creatinine and proteinuria less than
20 % during 6 months before the start of the study.
Exclusion criteria were as follows: fertile women who
were not taking oral contraceptives, pregnant or lactating
women, and a history of adverse reactions to PTE.
General protocol. The study was a prospective, placebo-controlled, randomized, two-period cross-over trial in
which the renal effects of adding PTE to a background
nephroprotective therapy with ACEI and/or ARB were
evaluated. Subjects entered an eight-week run-in period
during which background nephroprotective therapy using pharmacological blockade of RAAS was adjusted to
keep target blood pressure (BP) below 130/80 mm Hg
(Table 1). At the end of the run-in period, patients were
randomly assigned to one of two treatment sequences:
8-week PTE (1200 mg/day)/8-week washout — background therapy/8-week placebo (sequence 1) or 8-week
placebo/8-week washout — background therapy/8-week
PTE (1200 mg/day) (sequence 2) (Fig. 1). The allocation
was performed according to a computer-generated randomization list by a person that was independent of the
research team. The patients received 1200 mg of PTE,
in tablet form (Polfilin 400, Polpharma), once a day. The
dosages of ACEI, ARB and diuretics, once established
in the run-in period, were left unchanged throughout the
study. At the randomization point, and after the end of
each treatment period, office through BP, serum creatinine, potassium, hsCRP, proteinuria (measured as 24-h
Figure 1. Study scheme
urinary protein excretion (DPE)), sodium excretion (Na
ex), and urea excretion were measured. Further, surrogate markers of tubular injury were analyzed, namely
urine excretion of N-acetyl-β-d-glucosaminidase (NAG),
α-1microglobulin (α1m) and 15-F2t-isoprostane. 15-F2tisoprostane is accepted as a reliable and sensitive marker
of oxidative stress in human pathologies (Fam & Morrow, 2003). The study was approved by the local ethical committee and the investigated patients all gave informed consent.
Procedures and laboratory analyses. The office through BP was measured with a Speidel+Keller
sphyngomanometer in a sitting position after 10 min
of rest and expressed as a mean value of two consecutive measurements taken 2 min apart. DPE, Na
ex and urea excretion were evaluated on the basis
of 24-h urine collection. All patients were equipped
with a graded container and were informed how to
collect 24-h urine. They collected two 24-h urines
— of those the mean value of DPE was calculated
for data evaluation. Patients were asked not to perform heavy physical activity on the urine collection days and were recommended not to change
their usual daily protein and sodium intake during
the study period. The excretion of urea was used to
calculate the protein intake according to the Maroni
equation: protein intake normalized to weight (g/
kg per day) = 6.25×([urea-N-excretion urine 24 h (g/
day)] + [0.0031 × body weight (kg)])/body weight (kg)
(Maroni et al., 1985). eGFR was calculated according
to the Cockcroft-Gault formula (Cockcroft & Gault,
1976). NAG and α1m were analyzed in the second
morning spot urine sample. NAG was determined by
the spectrophotometric method according to Maruhn
(1976). Incubation medium had a final volume of 0.4
mL, containing 5 nmol/L p-nitrophenyl-2-acetamidoβ-d-glucopyranoside as a substrate in 50 mmol/L citrate buffer (pH 4.14). The reaction was started by the
addition of 0.2 mL of undialysed urine, carried out
for 15 min at 37 °C, and then terminated with 1 mL
of glycine buffer, pH 10.5. Absorbance was measured
at 405 nm against a sample terminated at time zero.
The calculation of the NAG level was made from the
molar absorption coefficient of the product of the reaction, p-nitrophenol, which is 18.5 cm2/μmol. From
preliminary experiments it was clear that the dialysis
did not affect NAG levels in the urine. Immunoturbidimetric test (Tina-quant α1-microglobulin, Roche,
Basel, Switzerland) was used for the quantification
of α1m in urine. The detection limit of the method
was 2 mg/L. Urinary NAG, and α1m were reported
per mass of urine creatinine to correct for the variation in urine concentration. High sensitivity C-reac-
Vol. 57 Pentoxifylline in chronic kidney diseases
121
Table 2. Changes of parameters after PTE and placebo
PTE — Baseline (Δ)
Placebo — Baseline (Δ)
P
–0.41 ± 0.48
0.01 ± 0.62
0.11
α1m excretion mg/g creatinine
0.61 (–8.4–9.6)
0.41 (–5.7–6.5)
0.96
NAG excretion IU/ g creatinine
1.0 ± 1.96
1.16 ± 4.9
0.91
–1.66 ± 1.77
–0.89 ± 4.69
0.63
0.08 (–0.01–0.18)
–0.04 (–0.12–0.03)
0.8
Proteinuria (DPE) g/24 h
hsCRP mg/L
Urine excretion of iPF2α ng/mg creatinine
tive protein (hsCRP) was measured with a commercial
ELISA kit (DRG, EIA-3954) and reported as mg/L.
A commercial ELISA kit (Cayman Chemical Co.) was
then used to measure the urinary excretion of 15-F 2tisoprostane in the treated patients. Potassium, sodium,
urea, protein and creatinine levels were measured in
fresh blood samples drawn after fasting overnight for
at least 12 h. These parameters were measured using standard laboratory techniques. Body mass index
(BMI) was calculated as weight (kilograms) divided by
height (meters) squared. Adverse effects were recorded at each visit in response to questionnaires or as
observed by the investigators.
Statistics. The primary end point of this study was
a change in DPE in measurements available for each
patient after treatment with PTE and placebo. The
sample size of 16 patients adequately allowed a power of 80 % to detect a difference in variables equal
to within one standard deviation, that is a standardized effect size of 1.0 at a significance level of 0.05
(two-tailed). Secondary end points included urine
NAG, α1m, and 15-F2t-isoprostane excretions. Normality and homogeneity of the variances were verified
by means of the Shapiro-Wilk test and Levene test,
respectively. Because of their skewed distribution, diastolic BP, DPE, NAG excretion, 15-F2t-isoprostane,
hsCRP, serum creatinine and daily protein intake were
logarithmically transformed before statistical analysis,
and expressed as geometric means and 95 % confidence intervals. Other results are presented as means
± S.E.M. Differences in the variables’ changes between treatment with PTE and placebo were assessed
using Student’s t-test (Table 2). The differences in the
variables measured more than twice (Table 3) were assessed using ANOVA. P less than 0.05 (2-tailed) was
considered statistically significant. Data were evaluated
using Statistica (version 7.1; StatSoft Inc, Tulsa, OK)
software package.
RESULTS
Of the 22 patients who entered the study, 14 (64 %)
completed the protocol. Five of the patients dropped
out because of the withdrawal of informed consent
due to a side effect of therapy (gastrointestinal symptoms — 23 %). The other patients resigned from participation in the study for personal reasons. Clinical
characteristics of the patients are listed in Table 1.
24-h urinary protein excretion (DPE)
The PTE therapy reduced proteinuria (by 26 %) as
compared to placebo, but the result was not significant
(P = 0.11) (Table 2). The exact change of DPE in single patients before and after PTE is shown separately
(Fig. 2).
Urinary NAG and α1m excretion
There were no significant changes in urinary NAG
(P = 0.91) and α1m excretion level (P = 0.96) using PTE
as compared to placebo (Table 2).
15-F2t-isoprostane excretions and hsCRP
There were no significant changes in15-F2t-isoprostane
excretions and hsCRP during the study (Table 2).
Blood pressure, renal function, sodium and protein
intake
The control of BP was adequate in all study periods; all patients reached the target office trough BP
below 130/80 mm Hg. There were no differences in
the office through systolic and diastolic BP between
the treatment periods. Renal function assessed by
means of serum creatinine and eGFR remained stable
during the study periods. There were no differences in
sodium and protein intake between treatment periods
(Table 3).
Safety
Interestingly, the PTE therapy was not well tolerated
in this study. Adverse effects were reported in five patients (22.7 %) who suffered from gastrointestinal symptoms — nausea, dyspepsia and diarrhea.
Table 3. Changes of parameters during study
Parameter
Randomization point
After PTE
After Placebo
P
Na urinary excretion mmol/24 h
295 ± 30.2
247 ± 34.5
268 ± 35.5
0.64
Daily protein intake g/24 h
1.1 ± 0.3
1.1 ± 0.3
1.0 ± 0.3
0.45
Serum creatinine mg/dL
1.0 (0.9–1.3 )
1.1 (0.9–1.4 )
1.1 (0.9–1.5 )
0.86
Systolic BP mm Hg
123.8 ± 12.6
122.9 ± 11.2
123.8 ± 10
0.55
Diastolic BP mm Hg
75.3 (70.6–81.0 )
74.3 (70.2–79.0 )
77.6 (73.8–82.1 )
0.64
122
M. Renke and others
2010
Figure 2. Daily protein excretion (DPE) before and after the therapy with pentoxifylline.
DISCUSSION
To the best of our knowledge the present study was
the first to evaluate tubulointerstitial effects of pentoxifylline in proteinuric non-diabetic CKD patients. PTE
has potential value as an antiproliferative and antifibrogenic agent, an effect documented in animal research
(Chen et al., 1999a; 1999b; Lin et al., 2005) and in patients with diabetic kidney disease (Navarro et al., 2003).
Considering that tubular epithelial cell injury may initiate the fibrotic process in kidneys and that the extent
of tubulointerstitial damage is a crucial predictor of renal
outcome, tubular cells have become a site of particular
interest. To evaluate the tubulointerstitial effects of the
described interventions, the tubular involvement markers NAG and α1m were analyzed (Bazzi et al., 2002). An
increased excretion of NAG is thought to be a specific
marker of tubular injury in many renal pathologies including non-diabetic CKD (Bazzi et al., 2002). Increased
urinary excretion of α1m, a low-molecular weight protein
physiologically filtered and reabsorbed by tubular cells,
may indicate a reduced capacity of reabsorption by tubular cells, and thus can act as a marker of established
tubular damage, with greater urinary concentrations suggesting greater severity of damage (Holdt-Lehmann et al.,
2000). Our results show that treatment with PTE had no
influence on these markers of tubular injury.
The effects of PTE (1200 mg/day) on proteinuria
were also analyzed. Proteinuria is considered a marker
of long-term renal outcome. In the present study, the
administration of PTE decreased the proteinuria levels
in non-diabetic CKD patients, but this was not significant (P = 0.11). Only a few randomized controlled trials
directly addressing the effect of PTE on renal function
and proteinuria have been reported. Most of those studies were of small size or short duration, used a variety of
doses, and many did not include a placebo arm. Some
of these studies suggest that PTE reduces proteinuria
(Ducloux et al., 2001; Galindo-Rodriguez et al., 2003;
Lin et al., 2008) and the rate of GFR decline (Perkins
et al., 2009). These positive effects were summarized in
published meta-analyses (McCormick et al., 2008) and review articles (Lin et al., 2004; Lin et al., 2005; Renke et
al., 2008; Vilayur & Harris, 2009). The pleiotropic effects
of PTE have important clinical implications, as it displays anti-tumor necrosis factor alpha (TNF-α) (Mandell,
1995) and anti-interferon gamma (IFN-γ) (Benbernou et
al., 1995; Bienvenu et al., 1995) action, as well as antioxidant (Freitas & Filipe, 1995) and antiapoptotic effects
(Belloc et al., 1995). Patients with CKD are at increased
risk for cardiovascular disease (CVD), and recent reviews
suggest that inflammation and oxidative stress could be
the primary mediators of CVD in CKD patients (Arici &
Walls, 2001). Moreover, inflammation plays a central role
in the progression of CKD (Tonelli et al., 2005; Zoja et
al., 2006). In our study we used hsCRP, a protein found
in the blood, as a marker of inflammatory process. Interestingly, patients with elevated basal levels of CRP are
at an increased risk of diabetes, hypertension and cardiovascular disease (Pradhan et al., 2001; Dehghan et al.,
2007). In our study this parameter had a tendency to
decrease with PTE treatment (70 %), but the result was
not statistically significant (P = 0.63). The facts that most
of the patients had serum hsCRP levels within the normal range at the beginning of the study and the small
number of participants are probably the main reasons
why our results differ from those of other studies. The
urinary excretion of 15-F2t-isoprostane, a reliable and
sensitive marker of oxidative stress, was also measured.
Urinary excretion of 15-F2t-isoprostane was not found to
change with treatment (P = 0.8). Interestingly, the PTE
therapy was not well tolerated in this study, a finding in
contrast to the perception that PTE has few side effects
in CKD patients (Ward & Clissold, 1987; McCormick et
al., 2008). Adverse effects, namely gastrointestinal symptoms, were reported in 5 patients (23 %) during the study
period. This finding is perhaps attributable to accumulation of PTE metabolites, a known mechanism of toxicity
in patients with chronic renal failure (Paap et al., 1996).
In the present study, the PTE doses were unchanged in
patients with moderate renal dysfunction (Navarro et al.,
2003).
small sample size, which was unsufficiently powered to
detect a significant difference equal to the S.D. value
between treatment periods. Further, 24-h urine collections used to assess proteinuria may be associated with
significant collection errors, largely because of improper
timing and missed samples, leading to overcollection and
undercollection.
In conclusion, the study results suggest that treatment
with PTE (1200 mg/day) for 8 weeks in nondialysed
patients with CKD induced the reduction of DPE (by
26 %), without affecting markers of tubular injury and
Vol. 57 Pentoxifylline in chronic kidney diseases
oxidative stress. However, the potential nephroprotective
properties of PTE need to be addressed further in future
controlled long term studies.
Acknowledgements
The study was supported by grant from the State
Committee for Scientific Research via the Medical University of Gdańsk (ST-4).
The authors thank Polpharma for providing drugs.
The drug providers and sponsors had no involvement in
the study design, patient recruitment, analysis, interpretation of data, writing of the report, or the decision to
submit the report for publication.
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Marcin Renke

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