H(2)S inhibits hyperglycemia-induced intrarenal renin-angiotensin system activation via attenuation of reactive oxygen species generation

Abstract

Decrease in endogenous hydrogen sulfide (H2S) was reported to participate in the pathogenesis of diabetic nephropathy (DN). This study is aimed at exploring the relationship between the abnormalities in H2S metabolism, hyperglycemia-induced oxidative stress and the activation of intrarenal renin-angiotensin system (RAS). Cultured renal mesangial cells (MCs) and streptozotocin (STZ) induced diabetic rats were used for the studies. The expressions of angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin II (Ang II) type I receptor (AT1), transforming growth factor-β1 (TGF-β1) and collagen IV were measured by real time PCR and Western blot. Reactive oxygen species (ROS) production was assessed by fluorescent probe assays. Cell proliferation was analyzed by 5'-bromo-2'-deoxyuridine incorporation assay. Ang II concentration was measured by an enzyme immunoassay. AGT, ACE and AT1 receptor mRNA levels and Ang II concentration were increased in high glucose (HG) -treated MCs, the cell proliferation rate and the production of TGF-β1 and of collagen IV productions were also increased. The NADPH oxidase inhibitor diphenylenechloride iodonium (DPI) was able to reverse the HG-induced RAS activation and the changes in cell proliferation and collagen synthesis. Supplementation of H2S attenuated HG-induced elevations in ROS and RAS activation. Blockade on H2S biosynthesis from cystathione-γ-lyase (CSE) by DL-propargylglycine (PPG) resulted in effects similar to that of HG treatment. In STZ-induced diabetic rats, the changes in RAS were also reversed by H2S supplementation without affecting blood glucose concentration. These data suggested that the decrease in H2S under hyperglycemic condition leads to an imbalance between oxidative and reductive species. The increased oxidative species results in intrarenal RAS activation, which, in turn, contributes to the pathogenesis of renal dysfunction.

Figure 1. The changes in RAS components in the cultured renal MCs after HG treatment.

The mRNA levels of AGT (A), ACE (B) and AT1 receptor (C) were measured by real-time PCR. The Ang II concentrations in the medium were measured by enzyme immunoassay. The data were expressed as Mean±SE. *P<0.05 versus NG group; **P<0.01 versus NG group, n=5.

Figure 2. The effects of losartan on HG-induced changes in MCs proliferation, TGF-β1 and collagen IV expression.

(A) The cell proliferation was measured by BrdU incorporation assay at 48h after HG treatment with or without losartan (1 µM). TGF-β1 (B) and Collagen IV (C) mRNA levels were measured by real-time PCR at 24h after HG treatment. Collagen IV protein levels (D) were measured by Western blot. The data were expressed as Mean±SE. *P<0.05 versus NG group; **P<0.01 versus NG group; ^# P<0.05 versus HG group; ^{# #} P<0.01 versus HG group, n=5.

Figure 3. The effects of DPI on HG-induced changes in MCs proliferation, TGF-β1 and collagen IV expression.

(A) The cell proliferation was measured by BrdU incorporation assay at 48h after HG treatment with or without DPI (1 µM). The TGF-β1 (B) and Collagen IV (C) mRNA levels were measured by real-time PCR at 24h after HG treatment. The collagen IV protein levels (D) were measured by Western blot. The data were expressed as Mean±SE. *P<0.05 versus NG group; **P<0.01 versus NG group; ^# P<0.05 versus HG group; ^{# #} P<0.01 versus HG group, n=5.

Figure 4. The effects of NaHS, PPG, DPI or Apocynin on ROS production in the cultured MCs.

The ROS levels were measured by DCFH-DA fluorescent probe assay at 24 h after treatment. The data were expressed as Mean±SE. **P<0.01 versus NG group; ^{# #} P<0.01 versus HG group, n=8.

Figure 5. The effects of NaHS, PPG or DPI on HG-induced changes in AGT, ACE and AT1 receptor mRNA levels in the cultured MCs.

The mRNA levels were measured by real-time PCR at 24h after HG treatment. The data were expressed as Mean±SE. *P<0.05 versus NG group; **P<0.01 versus NG group; ^# P<0.05 versus HG group; ^{# #} P<0.01 versus HG group, n=5.

Figure 6. Measurement of blood glucose, AGT, ACE and AT1 receptor mRNA levels in animals of control (C), diabetic (D), diabetic treated with NaHS (D+NaHS) and control treated with NaHS (C+NaHS) rats.

The blood glucose (A) was measured by glucose detection kit, and the mRNA levels for AGT (B), ACE (C), and AT1 receptor (D) were measured by real-time PCR 3 weeks after STZ injection. The data were expressed as Mean±SE. *P<0.05 versus control rats; ^# P<0.05 versus diabetic rats, n=6.

Figure 7. The effects of Ang II on CSE or CBS expression in the cultured MCs.

The CSE and CBS mRNA levels were measured by real-time PCR at 24h after Ang II (10 nM) treatment. The data were expressed as Mean±SE, n=6.