Alleviation of Renal Fibrosis by PACS-2 Inhibition of Tubular Epithelial Cell G2/M Arrest Through CDKL1 Kinase

Background: Renal fibrosis is the final common pathway of chronic kidney diseases regardless of etiology. Studies have shown that tubular cell arrest in G2/M mediates renal fibrosis after injury. However, the underlying regulatory mechanisms are not well understood. Our previous studies revealed that phosphofurin acidic cluster sorting protein 2 (PACS-2) is mainly expressed in proximal tubular epithelial cells (PTECs) in the kidney and plays an important role in tubular injury of diabetic kidney disease. However, the role and molecular mechanism of PACS-2 in renal fibrosis remain unclear. Methods: PTEC-specific Pacs-2 knockout mice were generated by utilizing LoxP-Cre recombination system and subjected to unilateral ureteral obstruction (UUO)-D7 and aristolochic acid (AA)-D28 models to assess cell cycle and evaluate kidney fibrosis. Cultured human and mouse tubular epithelial cells were treated with TGF-β1 to analyze the underlying cellular mechanisms. Co-immunoprecipitation (CO-IP) coupled with mass spectrometry, molecular cloning and genetic manipulation were used to investigate PACS-2 interactions and specific binding domains. Results: PACS-2 expression is significantly decreased in the cortex of fibrotic kidney from UUO mouse. PACS-2 deficiency in PTECs exacerbates G2/M cell cycle arrest and renal fibrosis in UUO-D7 and AA-D28 mouse models. In vitro, overexpression of PACS-2 alleviates TGF-β1-induced fibrogenic responses in PTECs through inhibiting cell cycle arrest at G2/M phase. By mass spectrometry followed CO-IP, we find that cyclin-dependent kinase-like 1 (CDKL-1) may be the key molecule linking PACS-2 to cell cycle progression in PTECs. Knockdown of CDKL1 partially reversed the anti-fibrotic effects of PACS-2 by promoting G2/M cell cycle arrest in TGF-β1-stimulated HK-2 cells. Mechanistically, we demonstrate that PACS-2 can interact with kinase domain of CDKL1, possibly affecting its kinase activity to regulate cell cycle, rather than affecting its subcellular translocation and protein expression. Conclusion: Downregulation of PACS-2 promotes G2/M arrest in PTECs via interaction with CDKL1 kinase, thereby facilitating fibrotic maladaptive repair and kidney fibrosis. Our findings indicate that PACS-2 and CDKL1 may be promising therapeutic targets for kidney fibrosis. Funding: Government Support – Non-U.S.