Study On The Mechanism Of EPO Improving Microvascular Injury In Renal Fibrosis Mice By HIF-1α/VEGF-A Pathway

Objective: To investigate the effects of erythropoietin(EPO)on microvascular injury related factors in renal fibrosis model mice,and to provide experimental basis for clinical treatment of chronic kidney disease.Methods: Will 90 male SPF C57BL/6 mice(body quality of 20±2g)using the random number table method were divided into Sham group,unilateral ureteral obstruction renal fibrosis model group(UUO)and EPO treatment group(EPO),30 in each group.Only free not ureteral ligation of Sham group.The left ureter was ligated and severed in UUO group to establish renal fibrosis model.Treatment group building daily intraperitoneal injection of EPO solution(1000IU/kg.d).Day 3,7,14 after surgery in each group were randomly kill 10 mice.Take 2ml blood from eyeball.Serum BUN content of the diacetyl oxime colorimetric method is used to detect,serum content of Scr sarcosine oxidase method is used to detect,El ISA method is used to detect serum Cys-C content.Choose obstruction side kidney tissues,for regular fixed,paraffin embedding,sectioning,and HE and Masson staining method to observe the renal tissue fibrosis degree.Using Western Blot method to detect microvascular injury in renal tissue related factor HIF-1α,VEGF,TSP-1 and renal fibrosis related factor TGF-β1,CTGF,ERK protein and ERK protein phosphorylation expression level.Results:1.Pathological examination resultsGross observation: There were no significant differences in kidneys on both sides of Sham group.The left kidney of mice in the group increased with the time of obstruction,turned white in color,increased in volume,edema of renal pelvis,and parenchyma thinning.The injury was relieved after EPO treatment.Microscopic observation: Compared with Sham group,renal fibrosis and microvascular injury were significantly observed in UUO group.With the extension of EPO treatment time,the degree of renal tubule dilation,inflammatory cell infiltration,interstitial collagen fiber accumulation and microvascular injury were significantly reduced.2.Renal function test resultsCompared with Sham group,the contents of BUN,Scr and Cys-C in UUO group were increased gradually(P<0.05),and all indexes were decreased after EPO treatment(P<0.05).On day 3,Scr and Cys-C in UUO group were higher than those in Sham group(P<0.05),and Cys-C content was decreased after EPO treatment(P<0.05),while Scr value was not changed(P>0.05).Day 7,14 UUO group of each index was significantly higher than in Sham group(P<0.01),EPO each index decreased significantly after treatment(P<0.01).3.Western Blot resultsRenal fibrosis related factors: The protein expression levels of TGF-β1,p-ERK1/2/ERK1/2 and CTGF in UUO group were higher than those in Sham group at the same time point(P<0.05).After EPO treatment,TGF-β1 protein expression was downregulated at different time points(P<0.05),and p-ERK1/2/ERK1/2 and CTGF protein expression were significantly down-regulated at day 7 and 14(P<0.01).Microvascular injury related factors: The expression of HIF-1α protein in UUO group was higher than that in Sham group on day 7 and 14(P<0.05),the expression of TSP-1 protein was higher than that in Sham group at different time points(P<0.05),and the expression of VEGF-A protein was significantly lower than that in Sham group at all time points(P<0.01).After EPO treatment,HIF-1α and TSP-1 protein expression were down-regulated at day 7 and 14(P<0.05),and VEGF-A protein expression was up-regulated at different time points(P<0.05).Conclusion:1.The EPO significantly reduce UUO model mice kidney damage.2.The EPO may regulate CTGF activity and delay the progression of renal interstitial fibrosis through TGF-β1/ERK signaling pathway.3.The EPO may regulate TSP-1 activity to reduce microvascular injury in renal fibrosis through HIF-1α/VEGF-A pathway.