Modulation of PKCα/ETS1 by klotho restores CYB5R4-dependent mitochondrial function in proximal tubular epithelial cells to attenuate the progression of diabetic kidney disease

Objective: Diabetic kidney disease (DKD) progression involves early proximal tubular injury, which precedes podocyte injury. The protective role of the protein Klotho in DKD is well-documented, but its impact on early tubular injury and mitochondrial dysfunction in proximal tubule epithelial cells (PTECs) remains underexplored. This study aimed to determine whether Klotho alleviates DKD by targeting mitochondrial dysfunction in PTECs and to uncover the molecular mechanisms involved. Methods: The role of Klotho was investigated using human kidney biopsies from patients at different DKD stages and a diabetic mouse model (induced by high-fat diet and streptozotocin). In vivo and in vitro techniques, including immunofluorescence, Western blot, transmission electron microscopy, and single-cell RNA sequencing, were used to assess tubular injury, mitochondrial integrity, and key protein interactions. The function of a newly identified protein, CYB5R4, was validated using knockdown and overexpression approaches in mouse models and human kidney (HK-2) cells. Results: Our results reveal a novel molecular pathway where Klotho alleviates early tubular injury in DKD by targeting the mitochondrial protein CYB5R4. We demonstrate that CYB5R4 is critically downregulated in DKD and that its restoration is both necessary and sufficient for Klotho's protective effect on mitochondrial function in PTECs. This regulation follows a defined signaling cascade where Klotho suppresses PKCα, which in turn inhibits the transcription factor ETS1. This inhibition of ETS1 leads to the de-repression of the CYB5R4 promoter, ultimately reducing tubular apoptosis and injury. This CYB5R4-dependent mechanism positions CYB5R4 as a key therapeutic target. Conclusion: Our findings uncover a novel Klotho/PKCα/ETS1/CYB5R4 signaling axis in PTECs that restores mitochondrial function and mitigates DKD progression, offering a promising therapeutic target for managing DKD. Supplementary Information: The online version contains supplementary material available at 10.1186/s12933-026-03150-y.