Study On The Role And Mechanism Of QDPR And KRAS Interaction In Renal Fibrosis

Objective To investigate whether overexpression of quinoid dihydropteridine reductase(QDPR)in unilateral ureteral obstruction(UUO)rat model affects fibrosis by affecting KRAS and its downstream pathway,and investigating the effect and mechanism of QDPR affects KRAS and its downstream pathway to affect fibrosis development.Methods Selected 24 SPF male SD rats aged 3 weeks and divided into 8 groups randomly:(1)7 days of sham group(7d Sham,7S),(2)7 days of UUO group(7d UUO,7U),(3)7 days of control group(UUO + overexpression lentivirus control 7d,7C),(4)7 days QDPR overexpression model group(UUO + lentivirus overexpression QDPR 7d,7Q),(5)14 days of sham group(14d Sham,14S),(6)14 days UUO group(14d UUO,14U),(7)14 days control group(UUO + overexpression lentivirus control 14 d,14C),(8)14 days QDPR overexpression model group(UUO + lentivirus overexpression QDPR 14 d,14Q).Constructed UUO model by unilateral ureteral ligation,and sacrificed euthanized for 7 and 14 days after operation.Masson staining,immunohistochemical staining of type I collagen,immunohistochemical staining of QDPR,immunohistochemical staining of KRAS,and western blot detected QDPR and KRAS proteins in UUO model to detecte the fibrosis and the expression of QDPR and KRAS in the UUO model.Immunohistochemical staining,colloidal gold technology,information biology,organelle extraction and Co-immunoprecipitation(Co-IP)methods detected the interaction between QDPR and KRAS in normal physiological kidney.On the basis of UUO model,constructed the animal models of over-expressing QDPR and its control by retrograde injection of lentivirus.Sacrificed rats for anle 7 and 14 days after operation,masson staining and immunohistochemical staining of type I collagen detected the effect of overexpressing QDPR on renal interstitial fibrosis,and immunohistochemical and western blot detected the expression and localization of KRAS and QDPR,and western blot detected the expression of ECa,Erk,P-Erk,Akt and P-Akt.Results 1 Construct rat models of renal interstitial fibrosis successfully.2 The protein content of QDPR and KRAS in 7U group are lower than that in 7S group,the protein content of QDPR and KRAS in 14 U group are lower than that in 14 S group,and QDPR and KRAS mainly express in the renal tubules.3 In humans and rats kidneys,both QDPR and KRAS are highly express in the renal tubules,and a small amount are also express in the glomeruli.Colloidal gold electron microscopy find that QDPR express in the rat kidney podocyte membrane,it is highly likely that QDPR enter the mitochondria,QDPR and KRAS both express in mitochondria and cytoplasm by subcellular localization.The analysis of Hex software suggests that QDPR can affect the function of KRAS.At this time,KRAS can be detected by western blot when Co-IP experiments pulled down QDPR,and QDPR can be detected by western blot when Co-IP experiments pulled down KRAS.4 Construct the UUO models of over-expressed QDPR and its control successfully.The QDPR is not significantly increased in 7Q model than that of the control group,and the QDPR content of 14 Q is significantly higher than that of the control group(P<0.05).5 The content of ECa protein in 14 Q group is higher than that in 14 C group(P<0.05).6 The content of KRAS protein in 14 Q group is higher than that in 14 C group(P<0.05),and Akt,P-Akt,and P-Erk do not change significantly,while the Erk protein content in 14 Q group is significantly higher than that in 14 C group(P<0.05),and the P-Erk/Erk is significantly lower than 14 C group(P<0.05).Conclusions 1 In the UUO model,QDPR and KRAS expression are significantly down-regulated,suggesting that QDPR and KRAS may have an important effect on rat renal interstitial fibrosis.2 QDPR and KRAS have similar locations,both QDPR and KRAS highly express in the renal tubules and less express in the glomeruli,and both QDPR and KRAS express in the mitochondria and cytoplasm,and there is an interaction between QDPR and KRAS in a normal physiological kidney.3 Overexpression of QDPR can improve renal fibrosis,and overexpression of QDPR can improve renal fibrosis in the UUO model by regulating KRAS downstream Erk pathway.Figure7;Table2;Reference 128...