Prabal K. Chatterjee a , Nimesh S.A. Patel a , Espen O. Kvale a , Paul A.J. Brown b , Keith N. Stewart c , Helder Mota-Filipe d , Martyn A. Sharpe e , Rosanna Di Paola f , Salvatore Cuzzocrea f , Christoph Thiemermann a
08 April 2004
Background/Aims: Oxidative and nitrosative stress plays important roles in the pathogenesis of renal ischemia/reperfusion (I/R) injury. Here we investigate the effect of EUK-134, a synthetic superoxide dismutase and catalase mimetic, (i) on renal dysfunction and injury caused by I/R in vivo and (ii) on proximal tubular cell (PTC) injury and death caused by oxidative and nitrosative stress. Methods: Rats, subjected to bilateral renal ischemia (45 min) followed by reperfusion (6 h), were administered EUK-134 (0.3 and 3 mg/kg, i.v.) prior to and during reperfusion, after which biochemical and histological indicators of renal dysfunction and injury were measured. The expression of poly(ADP-ribose) (PAR) and inducible nitric oxide (NO) synthase (iNOS) and nitrotyrosine formation were determined immunohistochemically and used as indicators of oxidative and nitrosative stress. Primary cultures of rat PTCs, isolated and cultured from the kidney cortex, were incubated with hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>; 1 m M for 2 h) in the presence of increasing concentrations of EUK-134 (1–100 µ M) after which PTC injury and death were measured. The effects of EUK-134 on serum levels of NO in rats subjected to renal I/R or on NO production by PTCs incubated with interferon-γ (IFN-γ, 100 IU/ml) and bacterial lipopolysaccharide (LPS, 10 µg/ml) in combination for 24 h were also measured. Results: EUK-134 produced a significant reduction in renal dysfunction and injury caused by I/R. Specifically, serum creatinine levels, an indicator of renal dysfunction, were reduced from 227 ± 11 (n = 12, I/R only) to 146 ± 9 µ M (n = 12, I/R +3 mg/kg EUK-134). Urinary N-acetyl-β- D-glucosaminidase activity, an indicator of tubular damage, was reduced from 42 ± 5 (n = 12, I/R only) to 22 ± 3 IU/l (n = 12, I/R +3 mg/kg EUK-134). EUK-134 significantly reduced renal injury caused by oxidative stress in vivo (reduction in PAR formation), and in vitro EUK-134 reduced PTC injury and death caused by H<sub>2</sub>O<sub>2</sub>. However, EUK-134 also reduced nitrosative stress caused by I/R in vivo (reduction of iNOS expression and nitrotyrosine formation), which was reflected by a significant reduction in serum NO levels in rats subjected to renal I/R. Specifically, serum NO levels were reduced from 57 ± 12 (n = 12, I/R only) to 23 ± 3 m M (n = 12, I/R +3 mg/kg EUK-134). In vitro, EUK-134 significantly reduced NO production by PTCs incubated with IFN-γ/LPS. Conclusion: We propose that EUK-134 reduces renal I/R injury not only via reduction of oxidative stress, but also by reducing nitrosative stress caused by renal I/R.