The relationship between hydrogen sulfide (H2S), microRNAs (miRs), matrix metalloproteinases (MMPs) and poly-ADP-ribose-polymerase-1 (PARP-1) in diabetic kidney remodeling remains mostly obscured. We aimed at investigating whether alteration of miR-194-dependent MMPs and PARP-1 causes renal fibrosis in diabetes kidney, and whether H2S ameliorates fibrosis. Wild type, diabetic Akita mice as well as mouse glomerular endothelial cells (MGECs) were used as experimental models, and GYY4137 as H2S donor. In diabetic mice, plasma H2S levels were decreased while ROS and expression of its modulator (ROMO1) were increased. In addition, alteration of MMPs-9, −13 and −14 expression, PARP-1, HIF1α, and increased collagen biosynthesis as well as collagen cross-linking protein, P4HA1 and PLOD2 were observed along with diminished vascular density in diabetic kidney. These changes were ameliorated by GYY4137. Further, downregulated miRNA-194 was normalized by GYY4137 in diabetic kidney. Similar results were obtained in in vitro condition. Interestingly, miR-194 mimic also diminished ROS production, and normalized ROMO1, MMPs-9, −13 and −14, and PARP-1 along with collagen biosynthesis and cross-linking protein in HG condition. We conclude that decrease H2S diminishes miR-194, induces collagen deposition and realignment leading to fibrosis and renovascular constriction in diabetes. GYY4137 mitigates renal fibrosis in diabetes through miR-194-dependent pathway.

Hydrogen sulfide (H2S) attenuates N-methyl-d-aspartate receptor-R1 (NMDA-R1) and mitigates diabetic renal damage; however, the molecular mechanism is not well known. Whereas NMDA-R1 facilitates Ca2+permeability, H2S is known to inhibit L-type Ca2+channel. High Ca2+activates cyclophilin D (CypD), a gatekeeper protein of mitochondrial permeability transition pore (MPTP), thus facilitating molecular exchange between matrix and cytoplasm causing oxidative outburst and cell death. We tested the hypothesis of whether NMDA-R1 mediates Ca2+influx causing CypD activation and MPTP opening leading to oxidative stress and renal injury in diabetes. We also tested whether H2S treatment blocks Ca2+channel and thus inhibits CypD and MPTP opening to prevent renal damage. C57BL/6J and Akita (C57BL/6J-Ins2Akita) mice were treated without or with H2S donor GYY4137 (0.25 mg·kg−1·day−1ip) for 8 wk. In vitro studies were performed using mouse glomerular endothelial cells. Results indicated that low levels of H2S and increased expression of NMDA-R1 in diabetes induced Ca2+permeability, which was ameliorated by H2S treatment. We observed cytosolic Ca2+influx in hyperglycemic (HG) condition along with mitochondrial-CypD activation, increased MPTP opening, and oxidative outburst, which were mitigated with H2S treatment. Renal injury biomarker KIM-1 was upregulated in HG conditions and normalized following H2S treatment. Inhibition of NMDA-R1 by pharmacological blocker MK-801 revealed similar results. We conclude that NMDA-R1-mediated Ca2+influx in diabetes induces MPTP opening via CypD activation leading to increased oxidative stress and renal injury, and H2S protects diabetic kidney from injury by blocking mitochondrial Ca2+permeability through NMDA-R1 pathway.

IntroductionElevated plasma levels of homocysteine, also known as hyperhomocysteinemia (HHcy), is associated with chronic kidney disease and end‐stage renal failure. HHcy is also a vascular risk factor that causes glomerular remodeling by inducing and accumulating extracellular matrix (ECM) in the glomerular and peri‐glomerular spaces. This remodeling is, in part, due to Hcy‐mediated imbalance between matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) and disruption of cell‐to‐cell gap junction protein, connexins (Cxs). HHcy also contribute to renal microvascular impairment and vasoconstriction due to decreased endogenous hydrogen sulfide (H2S) production and homocysteinylation of endothelial nitric‐oxide synthase (eNOS). However, the molecular events targeting Caveolin, eNOS, MMPs/TIMPs, Cxs and the role of H2S in HHcy associated ECM turnover and renovascular remodeling remain elusive.HypothesisWe investigated whether GYY4137, slow releasing H2S donor ameliorates HHcy effect and renal remodeling.MethodsCaveolin protein 1 knockout B6.Cg‐Cav1 (Cav1−/ −), C57BL6/J (WT) and hyperhomocysteinemic CBS+/− mice were supplemented without or with Hcy (2% in diet) and treated without or with GYY4137 (0.5 mg/Kg/d, IP) for a period of 8 weeks.ResultsHHcy decreased plasma H2S levels, upregulated Cav1 and decreased eNOS activity in the kidney. HHcy also resulted in the upregulation of mRNA and protein levels of MMPs −1, 2, −9, −8, −13 as well as connexins – 40 and −43 and decreased the activities of (TIMPs) −1, −2, −3, −4. Immunohistochemistry results indicated increased collagen IV deposition and expression of connexins in the glomerular and interstitial areas. GYY4137 treatment mitigated these changes in HHcy. In conclusion, our data suggests that H2S alleviates renovascular remodeling by modulating Cav‐1, eNOS, MMPs/TIMPs and Cxs in HHcy.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

IntroductionHyperhomocysteinemia (HHcy), an increase in plasma homocysteine levels induces collagen deposition by causing imbalance between MMPs and TIMPs leading to fibrosis, and predisposing hypertension. HHcy also decreases the endogenous production of hydrogen sulfide (H2S) and homocysteinylates endothelial nitric‐oxide synthase (eNOS) resulting in further increase of vascular resistance. In the contrary, deletion of a membrane protein Caveolin‐1 (Cav‐1) is known to mitigate hypertension. However, the interplay between Caveolin, eNOS and MMPs/TIMPs and the role of H2S in HHcy associated renovascular remodeling and hypertension remains largely unknown. We tested the hypothesis that GYY4137, a H2S donor mitigates hyperhomocysteinemic effect in renal microvasculature and thus normalizes vascular tone.MethodsB6.Cg‐Cav1(Cav‐1) mice representing caveolae protein 1 knock‐out and C57BL6/J (WT) mice were treated without or with GYY4137 (0.5 mg/Kg/d, IP) and supplemented without or with Hcy (2% in diet) for a period of 8 weeks.ResultsHHcy decreased plasma H2S levels, upregulated Cav1 and decreased eNOS activity in kidney. HHcy resulted in the upregulation of mRNA and protein levels of matrix metalloproteinases (MMPs) ‐2,‐9,‐1,‐8,‐13 and decreased the activities of tissue inhibitor of metalloproteinases (TIMPs) ‐1,‐2,‐3,‐4. Immunohistochemistry showed increased collagen IV deposition in the glomerular and interstitial areas. GYY4137 treatment reversed these changes in HHcy. In conclusion, our data suggests that H2S mitigates renovascular remodeling by modulating Cav‐1, eNOS and MMPs/TIMPs in HHcy.Support or Funding InformationThis study was supported by National Institute of Health Grant ‐ DK104653 (to U.S.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.