The Effect Of Mitochondrial Double-stranded RNA In Tubular Injury In Diabetic Nephropathy

Diabetic nephropathy(DN)is a chronic kidney disease caused by diabetes mellitus,and tubular injury is one of the important pathological features of DN.The pathogenesis of DN is complex,including increased reactive oxygen species(ROS)and mitochondrial damage due to high glucose.The mitochondrial ROS(mt ROS)production in tubular cells is increased,which is involved in DN tubular injury,but the detailed mechanism remains to be identified.In recent years,it has been found that mitochondria-encoded double-stranded RNA(mt-ds RNA)plays an important role in cellular physiological and pathological states.The release of mt-ds RNA can lead to protein translation arrest and apoptosis through the protein kinase R(PKR)/eukaryotic translation initiation factor 2subunit alpha(e IF2α)pathway,and the production and release of mtds RNA is closely related to mt ROS.Therefore,we speculate that mt ROSmediated release of mt-ds RNA may be involved in DN tubular injury.Objective: To clarify the role of mt-ds RNA in DN tubular injury,and to investigate whether mt ROS is involved in DN tubular injury by promoting mt-ds RNA release and regulating the mt-ds RNA/PKR/e IF2αaxis.Methods: 1.To observe the effect of high glucose on mt-ds RNA expression and release in tubular epithelial cells,human proximal tubular epithelial cells(HK-2 cells)were divided into five groups:(1)Normal glucose(NG,5 m M glucose),(2)High glucose(HG,30 m M glucose),(3)HG+PNPase(mt-ds RNA degradase)si RNA,(4)HG+PKR phosphorylation inhibitor(C16),(5)HG+PNPase si RNA+C16.Immunofluorescence and Western blot were used to detect the localization and expression changes of mt-ds RNA in HK-2 cells,as well as the expression of p-PKR,p-e IF2αand apoptosis indicators.2.To observe the effect of mt ROS on mt-ds RNA expression and release in HK-2 cells,HK-2 cells were divided into 6 groups:(1)NG,(2)HG,(3)HG+Mito-tempo(antioxidants),(4)HG+Mito-tempo+PNPase si RNA,(5)HG+Mito-tempo+C16,(6)HG+Mito-tempo+PNPase si RNA+C16.Immunofluorescence and Western blot were used to detect the localization and expression changes of mt-ds RNA in HK-2 cells,as well as the expression of p-PKR,p-e IF2α and apoptosis indicators.3.To investigate the renal protective effect and mechanism of Mitotempo on DN mice,two animal models were used for animal experiments:(1)Type 2 DN model,divided into 3 groups:(1)db/m,(2)db/db,(3)db/db+Mito-tempo;(2)Streptozotocin(STZ)-induced type 1 DN model,divided into 3 groups:(1)Control,(2)STZ,(3)STZ+Mito-tempo.Mice in db/db+Mito-tempo and STZ+Mito-tempo groups were given Mito-tempo(1 mg/kg)intraperitoneally daily,and the other groups were given saline.After 12 weeks,urine albumin and serum creatinine were measured,and histopathological changes in kidney were observed.Mitochondrial damage and oxidative stress indexes,localization and expression of mt-ds RNA in kidney tissues,p-PKR,p-e IF2α and apoptosis indicators were detected by immunofluorescence,immunohistochemistry and Western blot.Results: 1.Compared with NG,mt-ds RNA expression was significantly increased,mt-ds RNA co-localization with mitochondria was significantly reduced,p-PKR and p-e IF2α expression were significantly increased,and apoptosis was increased in HG-treated HK-2 cells.However,this phenomenon was partially blocked by C16.2.Compared with normal controls,mt ROS production was increased,mt-ds RNA expression was increased and co-localization with mitochondria was reduced in HK-2 cells treated with HG,accompanied by increased expression of p-PKR and p-e IF2α.However,this phenomenon was partially reversed by Mito-tempo treatment.3.Compared with the control group,the kidneys of DN mice were more pathologically damaged,with increased tubular atrophy and interstitial fibrosis.At the same time,DN mice showed a significant increase in renal tubular mt-ds RNA expression,accompanied by enhanced expression of p-PKR and p-e IF2α and increased apoptosis.In addition,KIM-1,NGAL,Fibronectin and a-SMA were elevated,ATP5a-1,UQCRC1,NDUFS8 and TOM20,key molecules of mitochondrial function were reduced,and the expression levels of oxidative stress indicators 8-OHd G and 4-HNE were also increased in DN mice,but these changes were partially alleviated by Mito-tempo treatment.Conclusions: In DN,increased mt ROS in renal tubular epithelial cells promotes tubular injury through the release of mt-ds RNA and subsequent activation of the PKR/e IF2α pathway.