Youthful Systemic Milieu Impacts On Renal Ischemia Reperfusion Injury In Elderly Mice

Background and objective:The incidence of acute kidney injury(AKI)is high in elderly people.AKI is difficult to prevent and treat.One of its major causes is renal ischemia reperfusion injury(IRI).A young systemic environment may prevent the senescence of old organs;however,it is unknown whether a young milieu may reduce renal IRI in the elderly.This study applied a parabiosis model to examine the therapeutic effect of the youthful systemic internal environment on AKI in the old mice,and to explore the mechanism.Methods:(1)At first,male C57 BL/6 mice and C57/BL6-TgN(ACTbEGFP)mice were established parabiosis model.Peripheral blood was observed and analyzed by flow cytometry to verity the shared blood circulation between two mice.(2).The mice were divided into four groups at random,and each group included 6-8 single mice or pairs of animals.Group 1 was the old sham group(O:sham)and included wild-type old mice that underwent surgery to expose the bilateral renal pedicles,but the pedicles were not clamped.Group 2 was the old IRI group(O:IRI)and included old mice that underwent a procedure to clamp the bilateral renal pedicles.Group 3 was the old-old parabiosis IRI group(O-O:IRI).In this group,the old-old parabiosis model was established first.Then 3 weeks after the parabiosis operation,the bilateral renal pedicles were clamped in the old recipient mouse.Group 4 was the young-old parabiosis IRI group(Y-O:IRI).In this group,the young-old parabiosis model was established first.Then 3 weeks after the parabiosis operation,the bilateral renal pedicles were clamped in the old recipient mouse..(3)At 24hour and 72hour after IRI,blood,kidney samples were harvested for further processing.The renal tissues were PAS stained.Quantitative analysis of histopathology score and Tunel-positive cell,CD3-positive cell,ED1-positive cell,EDU-positive cell,PCNA-positive cell counts was done.The renal tissue protein expression was measured by western blot,included that oxidative stress index MDA,PC,SOD,GSH-PX;inflammatory index IL1?,IL6,NF?B;apoptotic index bax,bcl2,cytochronic C,cleaved caspase-3,autophagy indicators Atg5,beclinl,LC3,p62,proliferation index cyclinD1 cyclinEl,dedifferentiation index Pax2,vimentin,ERK1/2.Quantibody mouse cytokine antibody array was used to filter out significant different cytokines..Results:(1)We Successfully established parabiosis mouse model and veritied the cross circulation between two mice by flow cytometry.(2)At 24 hour after IRI,the serum Cr and BUN level of old IRI mice in young-old parabiosis model were better than the old IRI mice in old-old parabiosis.The similar changes were observed in other index which included tubular injury score,expression of marker protein related to oxidative stress injury,inflammory injury,apoptosis.Autophagy was increased in old IRI mice of young-old parabiosis model compared to old-old parabiosis model.Our results suggested that the youth environment can significantly reduce damage extent of the aged mice with acute ischemia/reperfusion renal injury.(3)At 72 hour after IRI,the serum Cr and BUN level of old IRI mice in young-old parabiosis model were not significantly different from the old IRI mice in old-old parabiosis.The similar changes were observed in other index.These index included tubular injury score,percent of EDU-positive cells and PCNA-positive cells in renal tubular,expression of cyclinDl,cyclinEl,Pax2,vimentin,ERK1/2 protein.At 72 hours after IRI,compared to old sham group,the old IRI mice had significantly poor percent survival,which was 21.4%.However,old parabiosis IRI mice showed 100%percent survival.Compared to old IRI mice,old parabiosis IRI mice exhibited decreased renal histology,significantly increased cell proliferation,dedifferentiation.less serum Cr,BUN level.(4).We also used a high-throughput screening method to measure 120 kinds of cytokines of blood.compared to old IRI mice,old parabiosis IRI mice exhibited up-regulated expression which included promoted proliferation factors,many inflammatory factors.Conclusions:(1)Parabiosis mouse model was successfully established and veritied the cross circulation between two mice.(2)At 24 hour after IRI,compared to old-old parabiosis mouse model,the youthful milieu provided by young-old parabiosis model can significantly reduce damage extent of the aged mice with acute renal ischemic reperfusion injury.The machnisms were related to decrease oxidative stress injury,inflammory injury,apoptosis and increase autophagy.(3)At 72 hour after IRI,compared to old-old parabiosis mouse model,the youthful internal environment provided by young-old parabiosis model did not show significantly different in the tubular cell proliferation and dedifferentiation.Exogenous biological renal support promoted renad cell proliferation and dedifferentiation,improve prognosis and pathology in the elderly IRI mice.The regulated blood cytokines probably produced important effects.