Downregulation of TRPC6 regulates ERK1/2 to prevent sublytic C5b‑9 complement complex‑induced podocyte injury through activating autophagy

Li,Yuanyuan; Fang,Youfu; Liu,Jing

doi:10.3892/etm.2023.12275

Downregulation of TRPC6 regulates ERK1/2 to prevent sublytic C5b‑9 complement complex‑induced podocyte injury through activating autophagy

Authors:
- Yuanyuan Li
- Youfu Fang
- Jing Liu
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Affiliations: Department of Pediatrics, Weifang Yidu Central Hospital, Weifang, Shandong 262550, P.R. China
Published online on: October 26, 2023 https://doi.org/10.3892/etm.2023.12275
Article Number: 576
Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Idiopathic membranous nephropathy (IMN) is a common glomerular disease, in which 50‑60% of patients can progress to end‑stage renal disease within 10‑20 years, seriously endangering human health. Podocyte injury is the direct cause of IMN. Sublytic C5b‑9 complement complex induces damage in podocytes' structure and function. In sublytic C5b‑9 treated podocytes, the expression of canonical transient receptor potential 6 (TRPC6) is increased. However, the specific mechanism of TRPC6 in sublytic C5b‑9 treated podocytes is unclear. The present study aimed to reveal the effect and mechanism of TRPC6 on sublytic C5b‑9‑induced podocytes. Normal human serum was stimulated using zymosan to form C5b‑9. A lactate dehydrogenase release assay was used to examine C5b‑9 cytotoxicity in podocytes. The RNA and protein expression levels were analyzed using reverse transcription‑quantitative PCR, western blotting and immunofluorescent assay, respectively. Cell Counting Kit‑8 assay and flow cytometry were carried out to test the viability and apoptosis of podocytes, respectively. Transmission electron microscopy was used to observe autophagic vacuole. F‑actin was tested through phalloidin staining. Sublytic C5b‑9 was deposited and TRPC6 expression was boosted in podocytes stimulated through zymosan activation serum. Knockdown of TRPC6 raised the viability and reduced the apoptosis rate of sublytic C5b‑9‑induced podocytes. Meanwhile, transfection of small‑interfering (si)TRPC6 facilitated autophagy progression and enhanced the activation of cathepsin B/L in sublytic C5b‑9‑induced podocytes. The phosphorylation level of ERK1/2 was receded in siTRPC6 and sublytic C5b‑9 co‑treated podocytes. Moreover, the addition of the ERK1/2 activator partially reversed the effect of TRPC6 inhibition on sublytic C5b‑9‑induced podocytes. TRPC6 knockdown reduced the damage of sublytic C5b‑9 to podocytes by weakening the ERK1/2 phosphorylation level to activate autophagy. These results indicated that targeting TRPC6 reduced the injury of sublytic C5b‑9 on podocytes.

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1	Keri KC, Blumenthal S, Kulkarni V, Beck L and Chongkrairatanakul T: Primary membranous nephropathy: Comprehensive review and historical perspective. Postgrad Med J. 95:23–31. 2019.PubMed/NCBI View Article : Google Scholar
2	Safar-Boueri L, Piya A, Beck LH Jr and Ayalon R: Membranous nephropathy: Diagnosis, treatment, and monitoring in the post-PLA2R era. Pediatr Nephrol. 36:19–30. 2021.PubMed/NCBI View Article : Google Scholar
3	Liu W, Gao C, Liu Z, Dai H, Feng Z, Dong Z, Zheng Y, Gao Y, Tian X and Liu B: Idiopathic membranous nephropathy: Glomerular pathological pattern caused by extrarenal immunity activity. Front Immunol. 11(1846)2020.PubMed/NCBI View Article : Google Scholar
4	Ma H, Sandor DG and Beck LH Jr: The role of complement in membranous nephropathy. Semin Nephrol. 33:531–542. 2013.PubMed/NCBI View Article : Google Scholar
5	Nagata M: Podocyte injury and its consequences. Kidney Int. 89:1221–1230. 2016.PubMed/NCBI View Article : Google Scholar
6	Galindo-Izquierdo M and Pablos Alvarez JL: Complement as a therapeutic target in systemic autoimmune diseases. Cells. 10(148)2021.PubMed/NCBI View Article : Google Scholar
7	Yang P, Skiba NP, Tewkesbury GM, Treboschi VM, Baciu P and Jaffe GJ: Complement-mediated regulation of apolipoprotein E in cultured human RPE cells. Invest Ophthalmol Vis Sci. 58:3073–3085. 2017.PubMed/NCBI View Article : Google Scholar
8	Fishelson Z and Kirschfink M: Complement C5b-9 and Cancer: Mechanisms of cell damage, cancer counteractions, and approaches for intervention. Front Immunol. 10(752)2019.PubMed/NCBI View Article : Google Scholar
9	Takano T, Elimam H and Cybulsky AV: Complement-mediated cellular injury. Semin Nephrol. 33:586–601. 2013.PubMed/NCBI View Article : Google Scholar
10	Bateman RM, Sharpe MD, Jagger JE, Ellis CG, Solé-Violán J, López-Rodríguez M, Herrera-Ramos E, Ruíz-Hernández J, Borderías L, Horcajada J, et al: 36th International symposium on intensive care and emergency medicine: Brussels, Belgium. 15-18 March 2016. Crit Care. 20 (Suppl 2)(S94)2016.PubMed/NCBI View Article : Google Scholar
11	Tan Y and Zhao MH: Complement in glomerular diseases. Nephrology (Carlton). 23 (Suppl 4):S11–S15. 2018.PubMed/NCBI View Article : Google Scholar
12	Xie H, Yang L, Yang Y, Jiang W, Wang X, Huang M, Zhang J and Zhu Q: C5b-9 membrane attack complex activated NLRP3 inflammasome mediates renal tubular immune injury in trichloroethylene sensitized mice. Ecotoxicol Environ Saf. 208(111439)2021.PubMed/NCBI View Article : Google Scholar
13	Wang X, Liu J, Tian R, Zheng B, Li C, Huang L, Lu Z, Zhang J, Mao W, Liu B, et al: Sanqi oral solution mitigates proteinuria in rat passive heymann nephritis and blocks podocyte apoptosis via Nrf2/HO-1 Pathway. Front Pharmacol. 12(727874)2021.PubMed/NCBI View Article : Google Scholar
14	Zhou XJ, Klionsky DJ and Zhang H: Podocytes and autophagy: A potential therapeutic target in lupus nephritis. Autophagy. 15:908–912. 2019.PubMed/NCBI View Article : Google Scholar
15	Yoshibayashi M, Kume S, Yasuda-Yamahara M, Yamahara K, Takeda N, Osawa N, Chin-Kanasaki M, Nakae Y, Yokoi H, Mukoyama M, et al: Protective role of podocyte autophagy against glomerular endothelial dysfunction in diabetes. Biochem Biophys Res Commun. 525:319–325. 2020.PubMed/NCBI View Article : Google Scholar
16	Liu WJ, Li ZH, Chen XC, Zhao XL, Zhong Z, Yang C, Wu HL, An N, Li WY and Liu HF: Blockage of the lysosome-dependent autophagic pathway contributes to complement membrane attack complex-induced podocyte injury in idiopathic membranous nephropathy. Sci Rep. 7(8643)2017.PubMed/NCBI View Article : Google Scholar
17	Liu X, Yao X and Tsang SY: Post-Translational modification and natural mutation of TRPC Channels. Cells. 9(135)2020.PubMed/NCBI View Article : Google Scholar
18	Hou X, Huang M, Zeng X, Zhang Y, Sun A, Wu Q, Zhu L, Zhao H and Liao Y: The Role of TRPC6 in renal ischemia/reperfusion and cellular hypoxia/reoxygenation injuries. Front Mol Biosci. 8(698975)2021.PubMed/NCBI View Article : Google Scholar
19	Dryer SE, Roshanravan H and Kim EY: TRPC channels: Regulation, dysregulation and contributions to chronic kidney disease. Biochim Biophys Acta Mol Basis Dis. 1865:1041–1066. 2019.PubMed/NCBI View Article : Google Scholar
20	Moller CC, Wei C, Altintas MM, Li J, Greka A, Ohse T, Pippin JW, Rastaldi MP, Wawersik S, Schiavi S, et al: Induction of TRPC6 channel in acquired forms of proteinuric kidney disease. J Am Soc Nephrol. 18:29–36. 2007.PubMed/NCBI View Article : Google Scholar
21	Yu S and Yu L: Dexamethasone resisted podocyte injury via stabilizing TRPC6 expression and distribution. Evid Based Complement Alternat Med. 2012(652059)2012.PubMed/NCBI View Article : Google Scholar
22	Shengyou Y and Li Y: The effects of siRNA-silenced TRPC6 on podocyte autophagy and apoptosis induced by AngII. J Renin Angiotensin Aldosterone Syst. 16:1266–1273. 2015.PubMed/NCBI View Article : Google Scholar
23	Zhang MH, Fan JM, Xie XS, Deng YY, Chen YP, Zhen R, Li J, Cheng Y and Wen J: Ginsenoside-Rg1 protects podocytes from complement mediated injury. J Ethnopharmacol. 137:99–107. 2011.PubMed/NCBI View Article : Google Scholar
24	Liu Y, Zhang J, Wang Y and Zeng X: Apelin involved in progression of diabetic nephropathy by inhibiting autophagy in podocytes. Cell Death Dis. 8(e3006)2017.PubMed/NCBI View Article : Google Scholar
25	Ishikawa S, Tsukada H and Bhattacharya J: Soluble complex of complement increases hydraulic conductivity in single microvessels of rat lung. J Clin Invest. 91:103–109. 1993.PubMed/NCBI View Article : Google Scholar
26	Schmittgen TD and Livak KJ: Analyzing Real-time PCR data by the comparative C(T) method. Nat Protoc. 3:1101–1108. 2008.PubMed/NCBI View Article : Google Scholar
27	Pasquier B: Autophagy inhibitors. Cell Mol Life Sci. 73:985–1001. 2016.PubMed/NCBI View Article : Google Scholar
28	Jefferson JA, Pippin JW and Shankland SJ: Experimental models of membranous nephropathy. Drug Discov Today Dis Models. 7:27–33. 2010.PubMed/NCBI View Article : Google Scholar
29	Papagianni AA, Alexopoulos E, Leontsini M and Papadimitriou M: C5b-9 and adhesion molecules in human idiopathic membranous nephropathy. Nephrol Dial Transplant. 17:57–63. 2002.PubMed/NCBI View Article : Google Scholar
30	Zheng R, Deng Y, Chen Y, Fan J, Zhang M, Zhong Y, Zhu R and Wang L: Astragaloside IV attenuates complement membranous attack complex induced podocyte injury through the MAPK pathway. Phytother Res. 26:892–898. 2012.PubMed/NCBI View Article : Google Scholar
31	Wang L, Liang H, Sun B, Mi J, Tong X, Wang Y, Chen M, Yu L, Pan J, Liu S, et al: Role of TRPC6 in periodontal tissue reconstruction mediated by appropriate stress. Stem Cell Res Ther. 13(401)2022.PubMed/NCBI View Article : Google Scholar
32	Zhou LF, Chen QZ, Yang CT, Fu ZD, Zhao ST, Chen Y, Li SN, Liao L, Zhou YB, Huang JR and Li JH: TRPC6 contributes to LPS-induced inflammation through ERK1/2 and p38 pathways in bronchial epithelial cells. Am J Physiol Cell Physiol. 314:C278–C288. 2018.PubMed/NCBI View Article : Google Scholar
33	Putta P, Smith AH, Chaudhuri P, Guardia-Wolff R, Rosenbaum MA and Graham LM: Activation of the cytosolic calcium-independent phospholipase A₂ β isoform contributes to TRPC6 externalization via release of arachidonic acid. J Biol Chem. 297(101180)2021.PubMed/NCBI View Article : Google Scholar
34	Hartleben B, Godel M, Meyer-Schwesinger C, Liu S, Ulrich T, Kobler S, Wiech T, Grahammer F, Arnold SJ, Lindenmeyer MT, et al: Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice. J Clin Invest. 120:1084–1096. 2010.PubMed/NCBI View Article : Google Scholar
35	Fang L, Zhou Y, Cao H, Wen P, Jiang L, He W, Dai C and Yang J: Autophagy attenuates diabetic glomerular damage through protection of hyperglycemia-induced podocyte injury. PLoS One. 8(e60546)2013.PubMed/NCBI View Article : Google Scholar
36	Qi YY, Zhou XJ, Cheng FJ, Hou P, Ren YL, Wang SX, Zhao MH, Yang L, Martinez J and Zhang H: Increased autophagy is cytoprotective against podocyte injury induced by antibody and interferon-α in lupus nephritis. Ann Rheum Dis. 77:1799–1809. 2018.PubMed/NCBI View Article : Google Scholar
37	Lenoir O, Jasiek M, Henique C, Guyonnet L, Hartleben B, Bork T, Chipont A, Flosseau K, Bensaada I, Schmitt A, et al: Endothelial cell and podocyte autophagy synergistically protect from diabetes-induced glomerulosclerosis. Autophagy. 11:1130–1145. 2015.PubMed/NCBI View Article : Google Scholar
38	Hou X, Xiao H, Zhang Y, Zeng X, Huang M, Chen X, Birnbaumer L and Liao Y: Transient receptor potential channel 6 knockdown prevents apoptosis of renal tubular epithelial cells upon oxidative stress via autophagy activation. Cell Death Dis. 9(1015)2018.PubMed/NCBI View Article : Google Scholar
39	Farmer LK, Rollason R, Whitcomb DJ, Ni L, Goodliff A, Lay AC, Birnbaumer L, Heesom KJ, Xu SZ, Saleem MA and Welsh GI: TRPC6 binds to and activates calpain, independent of its channel activity, and regulates podocyte cytoskeleton, cell adhesion, and motility. J Am Soc Nephrol. 30:1910–1924. 2019.PubMed/NCBI View Article : Google Scholar