Cx43 Promotes Angiotensin Ⅱ-induced Podocyte Injury By Mediating The Ca2+/calpain/ Autophagy Inhibition Pathway

Objective:Hypertension is the main cause of Chronic kidney disease（CKD）,second only to diabetes.In the early stage of chronic renal disease,proteinuria was the main clinical manifestation.Podocytes are absolutely differentiated cells which have wheels within wheels located on the lateral side of glomerular capillaries.Their foot processes interleave and attach to the basement membrane of the glomeruli,forming a crucial filtration barrier.Therefore,podocyte injury is an important link of proteinuria progression.It is very important to study the mechanism of podocyte injury and reduce podocyte injury for the treatment of glomerular diseases.Connexin43（Cx43）is a non-selective cationic channel widely present in renal podiatocytes in the form of gap junction（GJ）and hemichannel（HC）.Cx43 has been found to be closely related to glomerular disease in recent years.The purpose of this study was to investigate the mechanism of action of Cx43 and its relationship with autophagy in podocellular injury induced by angiotension Ⅱ,and to explore the protective effect of Cx43 targeted therapy on podocellular injury.Methods:In vivo experiments,C57 mice were divided into groups as follows:Control group and Ang Ⅱ group(10^-6mol/l)Ang Ⅱ treatment for 24h),Ang Ⅱ+losartan group,peptide5 group（5μmol/l）and Ang Ⅱ+peptide5 group were injected with angiotensin Ⅱ at the rate of 1000ng/kg/min with a micro-osmotic pump implanted subcutaneously into shoulder for 28 days,and tissue and blood samples were retained.Blood pressure was measured at 0,1,2,3 and 4 weeks after Ang Ⅱ administration.Serum biochemical indexes（BUN,Scr）were measured to detect renal function.HE staining and Masson staining were used to determine the pathological injury of podococyte.The protein expression levels of Cx43,Nephrin and Podocin were detected by Western blot;the expression levels of Cx43,Nephrin and Podocin markers were detected by tissue immunofluorescence;m RNA expression levels of Cx43,Nephrin and Podocin were detected by RT-qpcr.In vitro experiments,we cultured podocyocytes and used angiotensin Ⅱ(Ang Ⅱ,10^-6mol/L)to intervene podocyocytes to establish a model of hypertensive podocyocytes injury,while using si RNA to knock out Cx43 expression.The protein expression levels of Cx43,podocin,LC3,beclin-1,p62 and calpain1 were detected by Western blot;m RNA expression levels of Cx43 and podocin were detected by RT-q PCR;Nephrin expression was detected by cellular immunofluorescence.Intracellular Ca²⁺concentration was detected by Fluo3 am and apoptosis was detected by Annexin V-FITC/PI.Results:（1）To explore the effects of continuous Ang Ⅱ infusion on renal function in mice.After continuous Ang Ⅱ infusion,the blood pressure of mice in the Ang Ⅱ group was observed to be continuously increased,and the kidney function test showed that compared with the control group,creatinine and urea nitrogen in the Ang Ⅱ group were increased.Immunofluorescence indicated that the fluorescence intensity of Nephrin in Ang Ⅱ group was significantly higher than that in Control group.（2）To investigate the up-regulated expression of Cx43 in podocytes of Ang Ⅱ mouse model.The results of Western blot and RT-q PCR showed that compared with the control group,the expression levels of Cx43 protein and m RNA in Ang Ⅱ group were significantly increased.Tissue immunofluorescence showed that the fluorescence intensity of Cx43 in Ang Ⅱ group was stronger than that in Control group.The message Ang Ⅱ is related to Cx43.（3）To investigate the renal pathological manifestations of mice after blocking cx43 half-channel.HE staining showed that the morphology of Ang Ⅱ group was significantly worse than that of Control group.Masson staining showed interstitial fibrosis in Ang Ⅱ group,but no interstitial fibrosis or inflammatory cell infiltration occurred after losartan or peptide5treatment.The results of tissue immunofluorescence showed that,compared with the control group,the fluorescence intensity of Cx43 in Ang Ⅱ group was significantly increased,while the fluorescence intensity of Nephrin and WT1 was significantly decreased.After the application of losartan or peptide5,the fluorescence intensity of Cx43 was significantly decreased,while the fluorescence intensity of Nephrin and WT1was significantly increased.（4）To explore the effect of inhibiting Cx43 expression or blocking semi-channel on angiotensin Ⅱ-induced podocellular injury.Annexin V-FITC/PI results suggested that compared with the control group,the apoptosis of podiocytes in the Ang Ⅱ group was significantly increased,and the apoptosis of podiocytes was significantly decreased after si RNA Cx43 and peptide5.（5）Ang Ⅱ inhibited autophagy by activating calpain pathway,leading to podocyte injury.Increased expression levels of LC3Ⅱ/LC3I and p62 revealed impaired autophagy flow in podocyocytes.After blocking Cx43 half channel,the pathological manifestations of kidney in Ang Ⅱ group were improved significantly.（6）Cx43 inhibited autophagy by activating calpsin.The intracellular Ca2+concentration in each group was measured by fluo-3 AM calcium probe method.The Ca2+concentration in Ang Ⅱ group was significantly increased,and the Ca2+concentration in both groups decreased significantly after the administration of peptide5 or si RNA Cx43 silencing.In Ang Ⅱ group,calpain1 was significantly increased,while Atg5 and beclin-1 protein expression levels were significantly down-regulated.Conclusion:1.Continuous Ang Ⅱ infusion induced renal impairment and up-regulated Cx43 expression in mice.2.Inhibition of autophagy by Cx43 in podococyte injury induced by Ang Ⅱ.3.Inhibition of Cx43 can significantly reduce angiotensin Ⅱ-induced podococyte injury and renal pathological manifestations.4.Cx43 inhibited autophagy by activating calpsin.