Angiotensin II type 1 receptor blocker ameliorates uncoupled endothelial nitric oxide synthase in rats with experimental diabetic nephropathy

Abstract

Background: Recent studies showed that angiotensin II type 1 receptor blocker (ARB) slows progression of chronic renal disease in patients with type 2 diabetes, regardless of changes in blood pressure. We showed that the imbalance of nitric oxide (NO) and reactive oxygen species (ROS) due to endothelial NO synthase (eNOS) uncoupling contributed to renal dysfunction in the diabetic nephropathy. The aim of this study was to determine the effects of ARB on uncoupled eNOS in rat diabetic nephropathy.

Methods: Diabetes was induced in Sprague-Dawley rats with streptozotocin (65 mg/ kg body weight). After 6 weeks, rats were divided into saline (DM; n = 11) and ARB, losartan groups (DM+Los; n = 11). After 2-week treatment, glomerular ROS production was assessed by 2',7'-dichlorofluorescin diacetate (DCFH-DA)-derived chemiluminescence. Renal NO and ROS production were imaged by confocal laser microscopy after renal perfusion with DCFH-DA and diaminorhodamine-4M acetoxymethyl ester with L-arginine. The dimeric form of eNOS was measured by low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Serum tetrahydrobiopterin (BH4) concentrations were determined by high-performance liquid chromatography. Protein and mRNA expression of GTP cyclohydrolase 1 (GTPCH1), key enzyme of BH4 synthesis, were examined.

Results: Losartan attenuated glomerular ROS production in DM. Accelerated ROS production and diminished bioavailable NO caused by NOS uncoupling were noted in DM glomeruli. Losartan reversed the decreased GTPCH1 and decreased dimeric form of eNOS and glomerular NO production by increased BH4 bioavailability.

Conclusions: ARB improved the NOS uncoupling in diabetic nephropathy by increasing BH4 bioavailability.

Fig. 1

Superoxide production and pathway in diabetic glomeruli. (A) Isolated glomeruli of control with no treatment rats (Cont), diabetic rats (DM) and losartan-treated diabetic rats (DM+Los) were incubated with 2′,7′-dichlorofluorescein diacetate (DCFH) and superoxide production was measured (Bar = 100 μm). Relative intensity was quantified. Data are shown relative to the Cont. Data are mean ± SD of 100 glomeruli from six rats in each group (total 600 glomeruli). Los; losartan treatment. ^*P < 0.05 versus Cont, ^†P < 0.05 versus DM + no treatment. (B) Glomeruli of DM were separately incubated with substrates of various enzymes and superoxide production was measured by DCFH staining (Bar = 100 μm). Relative intensity was quantified. Data are shown relative to the Cont. Data are mean ± SD of 100 glomeruli from six Cont or DM rats in each group (total 600 glomeruli). DPI, diphenylene iodonium; L-NAME, N(G)-nitro-l-arginine methyl ester. ^*P < 0.05 versus Cont, ^†P < 0.05 versus DM (−). (C) The superoxide production in glomeruli was measured by lucigenin chemiluminescence. Extracted protein from Cont, Cont+Los, DM and DM+Los glomeruli were used. Data are mean ± SD of six rats in each group. ^*P < 0.05 versus Cont, ^†P < 0.05 versus DM + no treatment.

Fig. 2

In situ detection of nitrate oxide (NO) and reactive oxygen species (ROS). Representative images of NO (top) and ROS (bottom) in renal cortex of control rats (Cont), diabetic rats (DM) and DM rats treated with losartan (DM+Los). Upper NO and lower ROS images are the same region of renal cortex. Arrowhead indicates glomerulus. Generation of ROS and NO was evaluated by the fluorescent intensity of DCF and DAR-4M, respectively (Bar = 100 μm). Data are mean ± SD of 20 glomeruli from five rats in each group (total 100 glomeruli). ^*P < 0.05 versus Cont, ^†P < 0.05 versus DM + no treatment.

Fig. 3

Western blot analysis for eNOS in the glomeruli. Representative blots of eNOS after low-temperature sodium dodecyl sulfate–polyacrylamide gel electrophoresis and phosphorylated (phospho)-eNOS. The intensity of dimers, monomers eNOS, phospho-eNOS and total eNOS was determined by NIH Image software, and the results are expressed as the dimer/monomer ratio and phospho-eNOS/total eNOS ratio. Data are mean ± SD of six rats in each group. ^*P < 0.05 versus Cont, ^†P < 0.05 versus DM.

Fig. 4

Nitrotyrosine formation in the glomeruli. (A) Immunohistochemistry of nitrotyrosine in control rats (Cont), diabetic rats (DM) and DM rats treated with losartan (DM+Los) (Bar = 100 μm). (B) Representative blots of nitrotyrosine. The intensity of the 55 kDa band was determined by NIH Image software, and the results are expressed as relative to the Cont. Data are mean ± SD of six rats in each group. ^*P < 0.05 versus Cont, ^†P < 0.05 versus DM.

Fig. 5

Serum biopterin concentration and glomerular GTPCH1 expression. (A) Serum levels of tetrahydrobiopterin (BH4) and dihydrobiopterin (BH2) in control rats (Cont), diabetic rats (DM) and DM rats treated with losartan (DM+Los) were determined using HPLC. Data are mean ± SD of six rats in each group. ^*P < 0.05 versus Cont, ^†P < 0.05 versus DM. (B) Glomerular mRNA expression of GTPCH1 in Cont, DM and DM+Los. (C) Glomerular protein expression of GTPCH1 in Cont, DM and DM+Los. Representative blots of GTPCH1 are shown. Data are mean ± SD of six rats in each group. ^*P < 0.05 versus Cont, ^†P < 0.05 versus DM.