TRPC6 Inhibition Attenuates Renal Tubulointerstitial Fibrosis via the Reactive Oxygen Species/TXNIP/NLRP3 Signaling Pathway

Abstract – Introduction: Chronic kidney disease is a worldwide public health issue primarily characterized by glomerulosclerosis and the renal tubulointerstitial fibrosis. Recent studies have shown that TRPC6 is essential in renal interstitial fibrosis, although the precise mechanisms involved are not yet fully understood. Methods: UUO model was established using C57BL/6 male mice in which HK-2 cells were stimulated with TGF-β1. H&E and Masson staining were used to observe pathological changes. IHC staining was also conducted to measure the α-SMA, fibronectin (Fn), TRPC6, reactive oxygen species (ROS), and NLRP3 expressions. Scanning electron microscopy was used to observe morphological changes in the tubular cell membrane, and flow cytometry was utilized to measure ROS levels. In addition, Western blotting was performed to detect Fn, α-SMA, TRPC6, TXNIP, NLRP3, and the downstream pyroptosis-related molecule levels. Results: TRPC6 protein levels were enhanced in UUO mice and HK-2 cells upon TGF-β1 stimulation, which coincided with noticeable morphological changes associated with pyroptosis. Treatment with the TRPC6 inhibitor SAR7334 effectively reduced renal fibrosis markers and diminished levels of ROS, TXNIP, and proteins related to NLRP3-mediated pyroptosis (including NLRP3, cGSDMD, and IL-1β). Furthermore, application of the NLRP3 inhibitor MCC950 in HK-2 cells reinforced our findings, as it attenuated renal fibrosis-related proteins and counteracted the elevated levels of Fn, α-SMA, and NLRP3-mediated pyroptosis proteins observed in TGF-β1-stimulated HK-2 cells. Additionally, inhibiting TRPC6 appeared to dampen the activity of the ROS/TXNIP/NLRP3 pathway. Conclusion: TRPC6 may represent a promising target for mitigating renal interstitial fibrosis, potentially through its effects on the ROS/TXNIP regulatory pathway involving NLRP3-mediated pyroptosis.