Hydrogen sulfide reduced renal tissue fibrosis by regulating autophagy in diabetic rats

Abstract

The present study aimed to explore the effect of hydrogen sulfide (H2S) on renal tissue fibrosis and its mechanism in diabetic rats. Rats were randomly divided into four groups (n=13/group): Control group; induced diabetes mellitus group (STZ); induced diabetes mellitus treated with H2S group (STZ + H2S); normal rats treated with H2S group (H2S). The diabetic model was induced by intraperitoneal (i.p.) injections of 40 mg/kg body weight streptozotocin (STZ); the control group was treated with saline every day (i.p); NaHS (100 µmol/kg i.p.) was administered to rats of STZ + H2S group and H2S group. After 8 weeks, rat body weight and 24 h proteinuria levels were determined in each group, renal pathological morphology was analyzed by Masson's trichrome staining, collagen IV content was detected by immunohistochemistry, and periodic acid‑Schiff (PAS) staining was performed on renal glomerular and tubular basement membranes. The expression levels of matrix metalloproteinase 9 (MMP9), MMP7, tissue inhibitor of metalloproteinase 1 (TIMP1), superoxide dismutase (SOD), serine/threonine kinase AKT, transforming growth factor (TGF)‑β1, nuclear factor (NF)‑κB and several autophagy related proteins were assessed by western blot analysis. Compared with the control group, renal tissue fibrosis was observed, collagen IV expression and the 24 h proteinuria quantity was markedly increased and the amount of PAS positive material in renal glomerular and tubular basement membranes was notably increased in STZ‑treated rats. Furthermore, the expression levels of MMP9, MMP7, TIMP1, autophagy‑associated proteins, AKT, TGF‑β1 and NF‑κB protein were significantly increased, and SOD expression levels were significantly decreased in the STZ group compared with the control (P<0.05). In the H2S+STZ group, renal tissue fibrosis and the expression of collagen IV were improved, 24 h proteinuria was decreased, the amount of PAS positive material in renal glomerular and tubular basement membranes was decreased, the expression levels MMP9, MMP7, TIMP1, autophagy‑associated proteins, AKT, TGF‑β1 and NF‑κB protein were significantly decreased, and the expression levels of SOD were significantly increased compared with the STZ group (P<0.05). In conclusion, H2S may improve renal tissue fibrosis by inhibiting autophagy, upregulating SOD and downregulating AKT, TGF‑β1 and NF-κB.

Figure 1.

Masson's trichrome staining in STZ-diabetic rats. Pathologic morphology and collagen deposition was assessed using Masson's trichrome staining. Magnification, ×400. STZ, streptozotocin; H₂S, hydrogen sulfide.

Figure 2.

Immunohistochemical analysis of collagen IV in STZ-diabetic rats. Kidney tissues were stained for collagen IV and counterstained with hematoxylin. Magnification, ×400. STZ, streptozotocin; H₂S, hydrogen sulfide.

Figure 3.

Periodic acid-Schiff staining in renal tissue from STZ-diabetic rats. Magnification, ×400. STZ, streptozotocin; H₂S, hydrogen sulfide.

Figure 4.

Expression of renal CBS in STZ-diabetic rats. CBS levels were assessed by western blot analysis. Data are presented as the mean ± standard deviation (n=3). ^##P<0.05 vs. the control group, **P<0.05 vs. the STZ group. STZ, streptozotocin; H₂S, hydrogen sulfide; CBS, cystathionine β synthase.

Figure 5.

Expression of MMP9, MMP7, TIMP1 and SOD in STZ-diabetic rats. Data are presented as the mean ± standard deviation (n=3). ^##P<0.05 vs. the control group, **P<0.05 vs. the STZ group. MMP, matrix metalloproteinase; TIMP1, tissue inhibitor of metalloproteinase; SOD, superoxide dismutase; STZ, streptozotocin; H₂S, hydrogen sulfide.

Figure 6.

Expression of LC3, Atg3, Atg5, Atg7, Atg12 and Atg16 in STZ-diabetic rats. Data are presented as the mean ± standard deviation (n=3). ^##P<0.05 vs. the control group, **P<0.05 vs. the STZ group. LC3, microtubule associated protein light chain 3; Atg, autophagy related; STZ, streptozotocin; H₂S, hydrogen sulfide.

Figure 7.

Expression of TGF-β1, NF-κB, AKT protein in STZ-diabetic rats. Data are presented as the mean ± standard deviation (n=3). ^##P<0.05 vs. the control group, **P<0.05 vs. the STZ group. TGF-β1, transforming growth factor β1; NF-κB, nuclear factor-κB; STZ, streptozotocin; H₂S, hydrogen sulfide.