The DR1‑CSE/H2S system inhibits renal fibrosis by downregulating the ERK1/2 signaling pathway in diabetic mice

Figures

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  Figure 1. Activation of DR1 upregulates the CSE/H2S pathway in the kidneys of T1D mice. (A and C) The expression of DR1 and CSE in the kidneys was determined by western blotting (n=3). The intensity of each band was quantified by densitometry and data was normalized to the β-actin signal. (B and D) H2S levels were detected by ultraviolet spectrophotometer in the kidneys (n=6). The results were expressed as the mean ± standard error of the mean. *P<0.05, **P<0.01 vs. control group; ##P<0.01 vs. T1D-12 W group. DR1, dopamine 1 receptors; CSE, cystathionine-γ-lyase; T1D, diabetes group; W, week.

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  Figure 2. DR1 activation inhibits cell proliferation and collagen deposition by upregulates the CSE/H2S pathway in the kidneys of T1D mice. Morphology of kidney by hematoxylin and eosin staining (magnification, ×400; scale bars, 100 µm), collagen deposition by Masson staining (magnification, ×400; scale bars, 100 µm, the blue stain by the arrow indicated is collagens) and thickness of renal basement membrane by transmission electron microscope (magnification, ×20,000; scale bars, 2 µm). Renal injury was aggravated, including glomerular hypertrophy, collagen deposition in interstitial kidney tissue and the thickness of the glomerular basement membrane were increased in the T1D-4 W, T1D-8 W and T1D-12 W groups. These changes were most significant in the T1D-12 W group. Pretreatment with SKF38393 or NaHS significantly reduced these pathological changes in the T1D-12 W group. All data were from at least 4 independent experiments. DR1, dopamine 1 receptors; CSE, cystathionine-γ-lyase; T1D, diabetes group; W, week.

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  Figure 3. DR1 activation increases the CSE/H2S pathway in the HG-induced MCs. (A) The expression of DR1 and CSE in the HG-induced MCs was determined by western blotting (n=3). The intensity of each band was quantified by densitometry and data was normalized to the β-actin signal. (B) H2S levels were detected by fluorescence microscopy in the HG-induced MCs (n=6, magnification, ×200; scale bars, 200 µm). The results were expressed as the mean ± standard error of the mean. *P<0.05, **P<0.01 vs. control group; #P<0.05, ##P<0.01 vs. HG group. DR1, dopamine 1 receptors; CSE, cystathionine-γ-lyase; HG, high glucose; MC, mesangial cell.

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  Figure 4. Activation of the DR1-CSE/H2S pathway attenuates HG-stimulated MC proliferation. (A) Cell viability was measured by CCK-8 assay (n=8). (B) Cell proliferation rate was measured by EdU assay (n=6, magnification, ×200; scale bars, 200 µm). (C) Quantify the results of (B) with a histogram (n=6). (D) The expression of cyclin D1, PCNA and P21 in the HG-induced MCs was determined by western blotting (n=3). The results were expressed as the mean ± standard error of the mean. *P<0.05, **P<0.01 vs. control group; #P<0.05, ##P<0.01 vs. HG group; &P<0.05, &&P<0.01 vs. HG + SKF38393 group. DR1, dopamine 1 receptors; CSE, cystathionine-γ-lyase; HG, high glucose; MC, mesangial cell.

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  Figure 5. Activation of the DR1-CSE/H2S pathway inhibits HG-induced collagen deposition in MCs. The expression of COL1, α-SMA and MMP9 in the HG-induced MCs was determined by western blotting. The experiments were repeated at least three times. The results were expressed as the mean ± standard error of the mean. *P<0.05, **P<0.01 vs. control group; #P<0.05, ##P<0.01 vs. HG group; &P<0.05, &&P<0.01 vs. HG + SKF38393 group. DR1, dopamine 1 receptors; CSE, cystathionine-γ-lyase; HG, high glucose; MC, mesangial cell; COL1, collagen 1; α-SMA, α-smooth muscle actin.

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  Figure 6. Activation of the DR1-CSE/H2S pathway downregulates ERK1/2 pathway in HG-induced MCs. The activity of p-ERK1/2 and t-ERK1/2 was detected through western blotting from different groups in HG-induced MCs. PD98059 is an ERK1/2 inhibitor. The intensity of each phosphorylated band was quantified by densitometry and data was normalized to the corresponding total band signal. The experiments were repeated at least three times. The results were expressed as the mean ± standard error of the mean. *P<0.05 vs. control group; #P<0.05, ##P<0.01 vs. HG group; &P<0.05 vs. HG + SKF38393 group. DR1, dopamine 1 receptors; CSE, cystathionine-γ-lyase; HG, high glucose; MC, mesangial cell; p-, phosphorylated; t-, total;

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  Figure 7. The mechanism of DR1 activation inhibiting ECM deposition and MC proliferation. DR1 activation increases (Ca2+)i and then enhances interaction of CaM and CSE and promotes generation of endogenous H2S, which inhibits MC proliferation and ECM deposition by downregulating ERK1/2 signaling pathway. DR1, dopamine 1 receptors; ECM, extracellular matrix; MC, mesangial cell; CaM, calmodulin; CSE, cystathionine-γ-lyase; p-, phosphorylated.