Roles Of TRPC6 Channel In Renal Ischemia/Rreperfusion Injury And Renal Tubular Epithelial Cells Hypoxia/Reoxygenation Injury

Background:Renal Ischemia Reperfusion Injury?IRI?is a phenomenon that renal tissue injury worsens instead of being repaired after the recovery of blood supply based on ischemia,and is one of the major causes of clinical Acute Kidney Injury?AKI?.AKI is a critical clinical disease that leads to Chronic Kidney Disease?CKD?and End-Stage Renal Disease?ESRD?.Therefore,it is of great significance to alleviate the damage caused by renal IRI.At present,the diagnostic criteria for AKI are as follows:within 48 hours,Serum creatinine?Scr?is increased by absolute value greater than or equal to 26.5 umol/L,or the Scr is increased by 50%compared with the previous first7 days,or 6 hours of urine?0.5 ml/kg/h.Common clinical factors leading to renal IRI include kidney transplantation,acute ischemic renal failure,shockwave lithotripsy,and extracorporeal nephrolithotomy.Ischemia reperfusion?I/R?involves a series of complex pathophysiological processes,including oxidative stress,lipid peroxidation,intracellular calcium accumulation,mitochondrial dysfunction,inflammatory mediators and cytokines,etc.Calcium overload is one of the important mechanisms of renal IRI.Increasing evidence shows that Transient Receptor Potential Canonical?TRPC?channel 6 may be involved in cell damage after I/R in target organs.In the kidney,TRPC6 is concentrated in the podocyte and is an important component of the silt diaphragm of podocytes.The abnormal expression of TRPC6 is closely related to focal segmental glomerulosclerosis?FSGS?and diabetic nephropathy.The expression of TRPC6 was increased after renal I/R,and was involved in the process of podocytes after ischemic injury.Renal Tubular Epithelial Cell?TEC?is vulnerable to ischemia and hypoxia due to its high metabolic physiology characteristic.TEC damage occupies the key position in the process of I/R.Preliminary results of our laboratory confirmed that the knockout or drug inhibition of TRPC6 may play a protective role in TEC injury caused by hydrogen peroxide?H₂O₂?.In this study,I/R in vivo model and the Hypoxia/Reoxygenation?H/R?model was further used to study the role of TRPC6 in TEC injury.Objective:In this study,wild type?WT?and TRPC6 gene knockout(Trpc6^-/-)mice were used to establish the kidney I/R model?in vivo?,and primary TEC was used to establish H/R model?in vitro?,to explore the role and mechanism of TRPC6 in I/R injury.Methods:WT and Trpc6^-/-mice were used to establish the kidney I/R model?in vivo?by bilateral renal pedicle clamp for 40 min and reperfusion for 24 h after ischemia.The H/R model?in vitro?was established to simulate the I/R injury in vivo by using the primary TEC hypoxia for 24 h and reoxygenation for 24 h.Biochemical indexes of Scr and Blood Urea Nitrogen?BUN?were detected by automatic Nitrogen analyzer.Hematoxylin Eosin?HE?and periodic acid-schiff stain?PAS?in renal histopathology were observed.Western blot,immunofluorescence and mitochondrial membrane potential detection kit?JC-1?were used to detect the changes of TEC apoptosis.Results:1.After I/R,Scr and BUN were increased.2.Western blot and immunofluorescence showed increased expression of TRPC6 protein after I/R and H/R.3.Western blot showed that,after I/R and H/R,the expression of Cleaved Caspase3?CC3?,Bax/Bcl2 increased.4.Western blot and immunofluorescence showed that,after I/R and H/R,the CC3 and Bax/Bcl-2 were reduced in the Trpc6^-/-group compared with the WT group.5.JC-1 staining showed that the reduction of mitochondrial membrane potential of primary TEC in Trpc6^-/-group was lower than that in WT group after H/R.6.After I/R,the Scr and BUN of Trpc6^-/-group were lower than those of WT group.7.HE and PAS staining showed that WT-SHAM and Trpc6^-/--SHAM group had clear renal tissue structure and tight TEC arrangement,and no abnormalities were observed.In the WT-I/R group,TEC arrangement was disordered,and a large number of cells showed obvious swelling,vacuolar degeneration,cell brush-like margin shedding,obvious tubular type in the lumen,and interstitial hyperemia.Trpc6^-/--I/R group showed the degree of cell swelling,cell destruction and degeneration were significantly slighter than WT-I/R.8.The increased level of p-AKT and p-ERK1/2 proteins in Trpc6^-/--I/R group were lower than those in WT group.The expression of apoptotic proteins decreased after the addition of inhibitors U0126 and MK2206,respectively.Conclusion:The results of this experiment showed that I/R induced calcium influx through activating TRPC6,and then up-regulated the PI3K/AKT and ERK signaling pathways to promote apoptosis.However,Trpc6^-/-can down-regulate PI3K/AKT and ERK pathway activity,reduce TEC calcium overload,thus reduce reduce TEC apoptotic rates and renal IRI.