TRPC6 ameliorates renal ischemic reperfusion injury by inducing Zn(2+) influx and activating autophagy to resist necrosis

BACKGROUND: Renal ischemic reperfusion injury (RIRI) is the most hackneyed cause of acute renal injury with high incidence. As a slit diaphragm (SD), TRPC6 (transient receptor potential channel 6) can maintain the structure and function of glomerular podocytes, and its activation has been reported to prominently alleviate ischemia reperfusion (I/R). However, the specific mechanism of TRPC6 in RIRI is uncertain. METHODS: The TRPC6 specific shRNA or overexpressing plasmid was used to decrease or increase TRPC6 level in HK-2 cells, respectively. Subsequently, the OGD/R (oxygen-glucose deprivation and re-oxygenation) HK-2 cells and RIRI model rats was established to examine the effect of TRPC6 in RIRI in vitro. After processing, viability was confirmed with MTT; cell necrosis was analyzed with flow cytometry; necrosis and autophagy-related proteins were verified with Western blot; free Zn(2+) was tested with an Zn(2+) fluorescent probe; and cell autophagy was monitored with MDC (monodansylcadaverine) method. Furthermore, TRPC6 agonist (OGA) or TRPC6 inhibitor (SKF96365) were introduced to increase or inhibit the activity of TRPC6 in RIRI model rats, and the kidney injury was assessed with H&E staining and RIP1 and PARP-1 expressions were examined with IHC (immunohistochemistry) staining. RESULTS: Our results verified TRPC6 could markedly enhance viability, Zn(2+) influx, and autophagy, and suppressed necrosis in OGD/R HK-2 cells. In addition, increase of Zn(2+) or autophagy activation produced similar results to TRPC6 overexpression in viability, autophagy, and necrosis of OGD/R HK-2 cells. Rescue experiment results also showed TRPC6 could prevent necrosis and facilitate Zn(2+) influx and autophagy of OGD/R HK-2 cells by inducing Zn(2+) influx and autophagy. Moreover, TRPC6 could ameliorate kidney injury, block necrosis, and enhance autophagy in RIRI model rats by promoting Zn(2+) influx and autophagy. CONCLUSIONS: TRPC6 could prevent necrosis and induce autophagy to alleviate RIRI by accelerating Zn(2+) influx and autophagy. This shows TRPC6/Zn(2+) influx/autophagy might be a novel therapeutic strategy for RIRI.