Connexin32 inhibits renal tubulointerstitial fibrosis in diabetic kidney disease by regulating NOX4/MRTF-A positive feedback loop.

Diabetic kidney disease (DKD) is typified by the epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells (TECs). EMT gives rise to renal tubulointerstitial fibrosis (TIF). Our previous study has shown that Connexin32 (Cx32) can improve renal TIF in DKD by inhibiting EMT. However, the specific mechanism of Cx32 in regulating EMT remains to be further elucidated. In the present study, we found that Cx32 protein expression was decreased in renal tubules of patients with DKD compared with controls. However, specific overexpression of Cx32 (tail vein injection of AAV9-Cx32) in renal tubular epithelial cells can effectively alleviate tubular mitochondrial damage, reduce reactive oxygen species (ROS) levels, inhibit EMT, and improve renal function and TIF in db/db mice. At the same time, overexpression of Cx32 inhibited HG-induced ROS overproduction and EMT in NRK-52E cells, and inhibited renal tubular epithelial cell fibrosis. Interestingly, there was a positive feedback regulation between NADPH oxidase 4 (NOX4) and Myocardin-related transcription factor A (MRTF-A) in NRK-52E cells induced by high glucose (HG). In addition, the treatment of AAV9-Cx32 inhibited the abnormal protein expression of NOX4 and MRTF-A, reduced the nuclear accumulation of MRTF-A in the kidney of db/db mice. Moreover, Cx32 overexpression reduced the colocalization of NOX4 and MRTF-A in HG-induced NRK-52E cells, thereby inhibiting the NOX4/MRTF-A positive feedback loop. Mechanistically, Cx32 regulates the NOX4/MRTF-A positive feedback loop through its C-terminal domain. Consequently, this study offers a more substantial experimental foundation for the utilization of Cx32 as a potential therapeutic target for DKD.