Luteolin Alleviates Renal Fibrosis in Diabetic Kidney Disease via Activation of the Cx32‐Nrf2 Signaling Axis

ABSTRACT Luteolin (Lut) is a flavonoid found in many medicinal plants, and previous studies have shown that it can slow the progression of diabetic kidney disease (DKD), although the mechanism is incompletely defined. This study asked whether Lut protects against DKD by acting on the connexin32 (Cx32)‐Nrf2 signaling axis to suppress ferroptosis and partial epithelial‐mesenchymal transition (EMT). Renal fibrosis, oxidative stress, EMT, and ferroptosis related markers, as well as Cx32/Nrf2 expression were assessed in db/db mice and high glucose (HG)‐induced NRK‐52E cells. Microdissected human kidney transcriptomes were obtained from GEO and analyzed in R. Molecular docking and cellular thermal shift assay (CETSA) were used to test whether Lut interacts with Cx32. siRNA knockdown was used to test whether Cx32 is required for its effects. Lut improved renal function, reduced fibrosis, lowered ROS, EMT, and ferroptosis in db/db mice and HG‐induced NRK‐52E cells. Additionally, Cx32 and Nrf2 protein levels were reduced, which was restored by Lut treatment. And overexpression of Cx32 promoted Nrf2 nuclear translocation. In human diabetic kidney transcriptomes, Cx32 and Nrf2 transcripts changed reciprocally and were inversely correlated across two independent cohorts, supporting Cx32 as a candidate upstream node. Molecular docking indicated that Lut binds to the ARG22 and THR18 residues of Cx32, and CETSA showed that Lut increased the thermal stability of Cx32. Knockdown of Cx32 abolished Nrf2 activation by Lut. Therefore, we concluded that Lut attenuates renal fibrosis in DKD by activating the Cx32‐Nrf2 signaling axis.