Exogenous H2S Attenuates Hypertension by Regulating Renin Exocytosis under Hyperglycaemic and Hyperlipidaemic Conditions

Figures

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  Figure 1. Blood pressure was measured in patients with diabetes and db/db mice. (A–C) Blood glucose, SBP and DBP of normoglycemic patients and patients with diabetes and hypertension (n = 3–5). (D) SBP, MBP and DBP in 28-week db/db mice (n = 4). * p < 0.05, ** p < 0.01. SBP, systolic blood pressure. MBP, mean blood pressure. DBP, diastolic blood pressure.

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  Figure 2. CSE expression and H2S levels were analyzed in the kidney tissues of diabetic patients, db/db mice and JG cells. (A) CSE expression in the kidney tissues of patients obtained during renal tumor surgery detected by immunohistochemistry. Scale bar = 50 μm. H2S content in the kidney tissues of db/db mice (scale bar = 500 μm) (B) and JG cells (D) was detected by C-7Az fluorescence probe. (C,E) Western blot analysis of CSE expression in the kidney tissues of db/db mice (n = 4) and in JG cells (n = 5) treated with NaHS (100 μM) and PPG (10 μM). * p < 0.05, ** p < 0.01. PA, palmitic acid. OA, oleic acid. JG, juxtaglomerular. CSE, cystathionine-γ-lyase. PPG, DL-propargylglycine.

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  Figure 3. Effect of exogenous H2S on renin expression, renin activity and renin release. (A) Renin content in the serum of normoglycemic patients and patients with diabetes and hypertension (n = 4). (B) Renin expression level in kidney tissues of db/db mice was detected by Western blotting (n = 4). Renin activity and content in the serum and kidney tissues (C–F) and culture medium of JG cells (H,I) detected by ELISA kits (n = 4–5). (G) Western blot analysis of renin in JG cells (n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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  Figure 4. Exogenous H2S inhibited RAAS activation by modulating the PDE3B-cAMP pathway. (A,B) cAMP content in kidney tissues (n = 5) and serum of db/db mice (n = 4) assessed by an ELISA kit. (C,D) PDE3B expression levels in the kidney tissues (n = 5) and JG cells (n = 7) were detected by Western blotting. After treatment with cilostamide (10 μM) in JG cells, PDE3B expression level was detected. cAMP content in the intracellular (E) and cell culture medium (F), and renin content in cell culture medium (G) were assessed by ELISA kits (n = 4). (H) Western blot analysis of PDE3B expression level in JG cells treated with PPG (n = 5). * p < 0.05, ** p < 0.01. PDE3B, phosphodiesterases 3B.

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  Figure 5. Exogenous H2S attenuated renin release via SNARE proteins. (A) Immunohischemistry of the expression of VAMP2 in kidney tissues of patients. Scale bar = 50 μm. (B,C) VAMP2 and SNAP23 expression levels in JG cells were measured by Western blotting (n = 5). (D) The colocalization of renin with VAMP2 in JG cells was detected by immunofluorescence staining. Scale bar = 20 μm. * p < 0.05. VAMP2, vesicle-associated membrane protein 2. SNAP23, synaptosome-associated protein 23.

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  Figure 6. Exogenous H2S promoted renin consumption through activation of autophagy. (A–E) The expression levels of ATG7, LC3II/I and p62 were detected by Western blotting after treatment with bafilomycin A1 (100 nM) or wortmannin (1 μM) (n = 4–7). (F,G) The colocalization of renin-containing granules with autophagosomes in JG cells detected by immunofluorescence staining after transfection with EGFP-LC3 plasmid. Scale bar = 20 μm, (H) After treatment with an autophagy inhibitor, the renin content in the cell culture medium of JG cells was detected using ELISA kits. * p < 0.05, ** p < 0.01, *** p < 0.001. BafA1, bafilomycin A1. CQ, chloroquine. Wort, wortmannin.