A Novel Role of NOD2 in Mediating Ca²⁺ Signaling:  Evidence from NOD2-regulated Podocyte TRPC6 Channels in Hyperhomocysteinemia

Huirong Han^1,2, Yupeng Wang¹, Xiang Li¹, Ping-An Wang¹, Xinbing Wei¹, Wei Liang³, Guohua Ding³, Xiao Yu⁴, Chanchan Bao⁵,Yan Zhang¹, Ziying Wang¹, and Fan Yi^1*

¹Department of Pharmacology, Shandong University School of Medicine, Jinan, China, 250012

²Department of Pharmacology, Weifang Medical University, Weifang, China, 261053

³Division of Nephrology, Renmin Hospital of Wuhan University, Wuhan, China, 430060, ⁴Department of Physiology, Shandong University School of Medicine, Jinan, China, 250012

⁵The Microscopy Characterization Platform, Shandong University, Jinan, China, 250012

Running title: NOD2 mediates TRPC6 expression and activity

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Reprint Requests to:   Fan Yi, Ph.D
                   Professor

Department of Pharmacology

Shandong University School of Medicine

                   44#, Wenhua Xi Road,

                   Jinan, Shandong, 250012, P.R. China

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E-mail: fanyi@sdu.edu.cn

Abstract

Although hyperhomocysteinemia (hHcys) has been recognized as an important independent risk factor in the progression of end-stage renal disease (ESRD) and in the development of cardiovascular complications related to ESRD, the mechanisms triggering the pathogenic actions of hHcys are not yet fully understood. The present study was designed to investigate the contribution of nucleotide-binding oligomerization domain containing 2 (NOD2), one intracellular innate immunity mediator, to the development of glomerulosclerosis in hHcys. Our results showed that NOD2 deficiency ameliorated renal injury in mice with hHcys. We further discovered the novel role of NOD2 in mediating Ca²⁺ signaling and found that homocysteine (Hcys)-induced NOD2 expression enhanced transient receptor potential cation channel 6 (TRPC6) expression and TRPC6-mediated calcium influx and currents, leading to intracellular Ca²⁺ release, ultimately results in podocyte cytoskeleton rearrangement and apoptosis. Moreover, we found that nephrin expression was dependently down-regulated by NOD2 and overexpression of nephrin attenuated Hcys-induced TRPC6 expression in podocytes. The results add evidence to support the essential role of nephrin in mediating NOD2-induced TRPC6 expression in hHcys. In conclusion, our results for the first time establish a previously unknown function of NOD2 for the regulation of TRPC6 channels, suggesting that TRPC6-dependent Ca²⁺ signaling is one of critical signal transduction pathways that links innate immunity mediator NOD2 to podocyte injury. Pharmacological targeting of NOD2 signaling pathways at multiple levels may help design a new approach to develop therapeutic strategies for treatment of hHcys-associated ESRD.