AXL Disrupts the Integrity of the Slit Diaphragm by Inhibiting ZO-1 Phase Separation and Aggravates Podocyte Injury in Diabetic Nephropathy

Background: SD injury of podocyte hiatal septum is an important cause of podocyte lesions and proteinuria in diabetic nephropathy. ZO tight junction protein (Zonula occludens - 1, 1) is an important link protein, SD area in maintaining normal cell structure and biological function plays a key role. However, how ZO-1 is regulated and its mechanism remain unclear. Methods: The expression level and localization of AXL in renal tissues from DKD patients and diabetic mice were examined by analyzing the database, single-cell sequencing and molecular biology techniques. The potential binding partners of AXL were screened and confirmed by mass spectrometry and Co-IP analysis. FRAP was used to detect the effect of AXL on ZO-1 phase separation. The role of AXL on the phenotype of diabetic nephropathy was investigated using podocyte-specific AXL-KO mice. Results: We found that AXL expression level was upregulated in podocytes of DKD patients and mice, causing podocyte injuries and proteinuria. AXL up-regulation in renal tissues of DKD patients was associated with increased albumiuria and deteriorated renal function. In db/db diabetic mice in vivo, overexpression of AXL worsened podocyte injury, whereas AXL inhibitor R428 significantly alleviated this effect. Podocyte-specific AXL-KO diabetic mice showed aggravated albuminuria and podocyte injury. AXL was expressed in podocytes and localized at the slit diaphragm. In cultured podocytes in vitro, AXL and ZO-1 were co-localized in a bead-like pattern along the cell membrane. High glucose up-regulated the expression level of AXL and reduced ZO-1 phase separation and membrane expression level. Knocking down AXL, however, restored ZO-1 membrane localization and alleviated podocyte injury. ZO-1 protein formed phase separation droplets, and AXL disrupted its phase separation by binding to the SH3 domain of ZO-1. Conclusion: Our results suggest that AXL disrupts the integrity of the slit diaphragm by inhibiting the ZO-1 phase separation, causing the loosening of tight junctions, thereby disrupting the SD integrity and exacerbating podocyte injury and proteinuria. AXL may serve as a potentially new target for podocyte-specific therapeutics in regulating the slit diaphragm, providing a novel direction for the treatment of diabetic nephropathy.