Glucose-induced Decrease Of Cystathionine ?-synthase Mediates Renal Injuries

Background:DKD(diabetic kidney disease)is a common complication of diabetic microvascular and also one of the important causes of ESRD(end stage renal disease),with high morbidity and mortality.The pathogenesis of diabetic nephropathy is complex and diverse,including renal hemodynamic changes,ischemia,RAAS system activation,oxidative stress,inflammation,and so on.The main H₂S synthetases in vivo are CBS(Cystathionine-?-synthase)?CSE(Cystathionine-?-lyase)?3-MST(3-mercaptopyruvate sulfurtransferase)?CAT(Cysteine aminotransferase).At present,there are many studies on the role of CSE and 3-MST in diabetes and diabetic nephropathy,but the exact role of CBS in diabetic nephropathy is still unclear.Objective:To investigate the effect of high glucose on the production of H₂S enzyme CBS,to further clarify the mechanism of high glucose induced decrease of CBS,to explore the role of renal CBS/H₂S in diabetic nephropathy,and the protective effect of exogenous supplement of H₂S on kidney.Methods:We detected the level of H₂S in blood of diabetic patients and normal control group.Established diabetic mice model by intraperitoneal injection of STZ,and divided them into normal control group,diabetic nephropathy group and hydrogen sulfide(GYY4137)treatment group The levels of CBS and ubiquitinated CBS protein were detected by western blot,and the ubiquitination of CBS was evaluated by immunoprecipitation.The kidneys of diabetic mice were stained by histology and immunohistochemistry,and examined by electron microscope.The levels of CBS,CSE,3-MST and NT in renal homogenate of diabetic mice were detected by western blot,and compared among the three groups.The renal tissues were cultured with immortalized proximal tubular cells(m RPTCs),CBS and Na⁺/H⁺exchange isomer 3(NHE3)were detected by confocal microscopy immune-fluorescence.The early CBS of high glucose treated mrptcs were co stained with proteasome marker PMSC6 or lysosome marker LAMP2,They were observed by confocal microscope.Real time PCR was used to detect CBS level and si RNA transfected CBS level.Results:1.We found that the level of plasma hydrogen sulfide(H₂S)of diabetic nephropathy patients was decreased,the level of H₂S in plasma and kidney of diabetic mice was also decreased,CBS in diabetic mice was decreased,and ubiquitinated CBS was increased.2.High glucose induced the increase of protein oxidative stress product NT in diabetic mice.Exogenous supplement of H₂S donor GYY4137 could restore NT,improve urinary albumin and renal pathology(increased mesangial matrix,thickened glomerular basement membrane and foot process fusion).3.The expression of CBS in renal tubules of diabetic mice was increased by immunohistochemical staining.CBS was co localized with the proximal tubule marker Na⁺/H⁺exchange isomer 3(NHE3)in renal tubular epithelial cells;4.High glucose decreased the expression of CBS protein and increased the expression of ubiquitinated CBS in the early stage of mrptcs,and the degradation of CBS was via proteasome pathway rather than lysosomal pathway;5.High glucose treated mrptcs reduced CBS protein expression by 60-70%in the late stage by inhibiting m RNA synthesis,which could be reversed by treating with exogenous H₂S.Conclusion:In vivo and in vitro,high glucose can induce the decrease of CBS protein and CBS in proximal tubular cells.The mechanism is to increase UPS degradation pathway in early stage and inhibit its m RNA expression in late stage The loss of CBS caused by si RNA deletion mediates the renal injury induced by oxidative/nitrification stress.Exogenous H₂S corrects the renal abnormalities related to the decrease of CBS/H₂S in diabetic mice.