The Protective Effect Of MiR-138 SiRNA On Podocyte Injury In Diabetic Nephropathy Model Mouse

Diabetic Nephropathy(DN)has become the most common cause of End-Stage Renal Disease(ESRD).The pathogenesis of DN is complicated.Various factors,such as abnormal glucose metabolism,renal hemodynamic changes,oxidative stress,inflammatory response and genetic susceptibility,are involved in the pathogenesis of DN.Currently the leading view is that DN is podocyte disease.More than 20%of podocyte loss will result in irreversible glomerular damage,characterized by progressive proteinuria,glomerulosclerosis,tubulointerstitial fibrosis,and eventually progressing to ESRD.With the exploration of the pathogenesis of DN,more and more researches demonstrated the key role of inflammation.More importantly,inflammatory factors are harmful for renal structure and function through different mechanisms.The phenomenon that podocytes are damaged by inflammatory factors is receiving increasing attention.MiRNA is non-coding single-stranded microRNA(miRNA)with approximately 22 nucleotides in length.MiRNA regulates gene expression at the level of post-transcriptional.The key role of miRNA in the development of DN has been widely recognized and mi RNA provides a new target for the diagnosis and prevention of DN.Altering the expression of specific mi RNAs that involved in DN may be an effective method to prevent the development of DN.Our previous experiments found that the expression of miR-138 in patients with DN was increased,while miR-138 hasn't been reported to be involved in the pathogenesis of DN.Objective:1.To investigate the expression of miR-138 in renal tissue of db/db mice and its relationship with urine protein.2.To determine that mi R-138 induces podocyte injury by regulating the expression of inflammatory factors in renal tissue of db/db mice.3.To explore the protective effect of miR-138 siRNA on podocyte injury in renal tissue of db/db mice.Method:Experimental mice were randomly divided into the following four groups:(1)db/m group,12 db/m mice,without any intervention;(2)db/db group,20 db/db mice,tail intravenous injection of normal saline after mouse sacrificed at 10 weeks and 14weeks respectively;(3)vehicle group,20 db/db mice,tail intravenous injection of miR-138 irrelevant sequences lentivirus after mouse sacrificed at 10 weeks and 14weeks respectively;(4)si-miR group,20 db/db mice,tail intravenous injection of miR-138 siRNA lentivirus after mouse sacrificed at 10 weeks and 14 weeks respectively.Blood glucose and body weight were measured regularly from 6 weeks to 22 weeks,and blood and urine samples were collected for the detection of serum creatinine and urine protein.Mice were sacrificed at 10 weeks,14 weeks,18 weeks and 22 weeks respectively,and samples were collected.The expression of miR-138was detected by real-time quantitative PCR(qRT-PCR).Western blotting was performed to detect the expression of nephrin,podocin,desmin,IL-6 and IL-18.The expression of podocin was aslo demonstrated by immunofluorescence staining.The renal tissue structure was observed by PAS staining and electron microscopic.Results:1.The levels of blood glucose,body weight,serum creatinine and urinary albumin in db/db mice increased with time,and were higher than those in db/m mice at the same age(p<0.05).After injection of miR-138 siRNA,there was no significant change in the levels of body weight and blood glucose(p>0.05),while the levels of serum creatinine and urinary albumin decreased in db/db mice(p<0.05).2.Compared with db/m mice,the expression of miR-138 in renal tissue of db/db mice was increased(p<0.05),and the level of miR-138 was positively correlated with the level of urine albumin/creatinine ratio(R~2=0.64,p<0.01).3.Compared with db/m mice,the expression of IL-6 and IL-18 in renal tissue of db/db mice was increased.After injection of miR-138 siRNA,the expression of IL-6and IL-18 was down-regulated in db/db mice.4.Compared with db/m mice,the expression of nephrin and podocin in renal tissue of db/db mice was decreased,while the level of desmin was increased.After injection of miR-138 siRNA,the expression of nephrin and podocin was up-regulated and the expression of desmin was down-regulated in db/db mice.5.The result of PAS staining and electron microscopy showed that these pathological changes,including glomerular hypertrophy,mesangial expansion,sclerosis,glomerular basement membrane thickening and podocyte foot process fusion were appeared in renal tissue of db/db mice.After injection of miR-138 siRNA,the above pathological changes were alleviated in db/db mice.Conclusion:1.The expression of mi R-138 in renal tissue of db/db mice is increased and the level of miR-138 is positively correlated with the level of urine albumin/creatinine ratio,suggesting that miR-138 is a potential biomarker and therapeutic target in DN.2.MiR-138 may induce podocyte injury by upregulating the expression of IL-6and IL-18 in renal tissue of db/db mice.3.In renal tissue of db/db mice,miR-138 siRNA may alleviate the podocyte injury by down-regulating the expression of IL-6 and IL-18,which may provide new target for the treatment of DN.