TRPC6 ameliorates renal ischemic reperfusion injury by inducing Zn2+ influx and activating autophagy to resist necrosis.

Original Article Page 1 of 15 TRPC6 ameliorates renal ischemic reperfusion injury by inducing Zn2+ influx and activating autophagy to resist necrosis Youmin Pu, Hongwen Zhao, Bingbing Shen, Qiang Zhou, Pan Xie, Xiongfei Wu Department of Nephrology, The First Affiliated Hospital of Army Medical University, Chongqing, China Contributions: (I) Conception and design: Y Pu, X Wu; (II) Administrative support: H Zhao; (III) Provision of study materials or patients: B Shen; (IV) Collection and assembly of data: Q Zhou, P Xie; (V) Data analysis and interpretation: Y Pu; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. Correspondence to: Xiongfei Wu. Department of Nephrology, The First Affiliated Hospital of Army Medical University, No. 30, Gaotanyan Street, Shapingba District, Chongqing 400038, China. Email: . 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 Zn2+ was tested with an Zn2+ 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, Zn2+ influx, and autophagy, and suppressed necrosis in OGD/R HK-2 cells. In addition, increase of Zn2+ 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 Zn2+ influx and autophagy of OGD/R HK-2 cells by inducing Zn2+ influx and autophagy. Moreover, TRPC6 could ameliorate kidney injury, block necrosis, and enhance autophagy in RIRI model rats by promoting Zn2+ influx and autophagy. Conclusions: TRPC6 could prevent necrosis and induce autophagy to alleviate RIRI by accelerating Zn2+ influx and autophagy. This shows TRPC6/Zn2+ influx/autophagy might be a novel therapeutic strategy for RIRI. Keywords: TRPC6; renal ischemic reperfusion injury (RIRI); Zn2+ influx; autophagy; necrosis Submitted Oct 08, 2021. Accepted for publication Jan 27, 2022. doi: 10.21037/atm-21-5837 View this article at: https://dx.doi.org/10.21037/atm-21-5837 Introduction Ischemia reperfusion injury (IRI) may occur following acute hemorrhage, severe trauma, toxic shock, or kidney transplantation (1,2). Due to the characteristics of high © Annals of Translational Medicine. All rights reserved. perfusion and high metabolism, the kidney is extremely sensitive to ischemia and hypoxia stimulation and prone to IRI (3), and research has shown renal IRI (RIRI) is one of the major factors affecting renal function recovery and longterm survival after renal ischemia or transplantation (4). Ann Transl Med 2022;10(5):249 | https://dx.doi.org/10.21037/atm-21-5837 Pu et al. TRPC6 in RIRI Page 2 of 15 Therefore, further exploration of the pathogenesis of RIRI and effective treatment strategies is of clinical significance. As a member of the transient receptor potential family of calcium channels, transient receptor potential channel 6 (TRPC6) is a nonselective cation channel and a key molecule of slit diaphragm (SD) (5,6). It was reported that TRPC6 could be involved in the intracellular flow of Ca2+, Fe2+, and Zn2+ (7,8), and Zn2+ had an antioxidant role and could prevent renal cell apoptosis in IRI (9). Thus, we speculated that TRPC6 could restrain programmed cell necrosis by mediating Zn 2+ influx and play a protective role in RIRI. However, the regulatory role of Zn 2+ on programmed cell necrosis in RIRI is not entirely clear. Role of Zn2+ in the activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway to protect against renal I/ R injury has been investigated in published research (10). It has been observed that I/R injury results in significant increase in the serum levels of blood urea nitrogen (BUN) and creatinine and increase in the fractional excretion of sodium (FENa) which is decreased by zinc chloride (ZnCl2) supplementation (11,12). Besides, Zn 2+ also improved glomerular filtration rate value, Cr-clearance, and urine flow rate beyond renal I/R induced renal injury (12,13). Furthermore, Major antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione (GSH) decreased after renal I/R-induced injury. It has been suggested that Zn2+ supplementation stimulates the activity of these enzyme (14). However, it is still unclear whether Zn2+ is the key regulator of TRPC6 in renal ischemic reperfusion injury. Autophagy is a highly conserved metabolic mechanism in the organic evolutionary process (15), and plays a fatal regulatory role in programmed cell necrosis (16). In addition, it has been recently disclosed that Zn 2+ can induce autophagy (17) and can protect RIRI by accelerating autophagy (18). However, it is still unclear whether Zn2+ can participate in the suppression of TRPC6 on cell necrosis by regulating cell autophagy. TRPC6 is a receptor-activated nonselective cation channel that is homogeneously expressed throughout the central nervous system and peripheral tissues, including the kidneys (19). It has been suggested to play a significant role in I/R injury of the lungs, retinas, and brain (20,21). Our previous study identified TRPC6 as an up-regulated differentially expressed gene in the pathogenesis of I/R injury, and protected against I/R injury, suggesting that it may be a potential novel target for therapy (22). However, the underlying mechanism of TRPC6 in renal I/R injury © Annals of Translational Medicine. All rights reserved. has not been investigated. In current study, we further verified the influences of TRPC6 on necrosis，Zn2+ influx, and autophagy in oxygen-glucose deprivation and reoxygenation (OGD/R) HK-2 cells and RIRI model rats. We present the following article in accordance with the ARRIVE reporting checklist (available at https://atm. amegroups.com/article/v (...truncated)