Excessive protein uptake by endocytic receptors causes elevated proliferation and senescence in proximal tubules primarily in outer medulla of proteinuric mice

Proteinuria contributes to the progression of chronic kidney disease and causes protein overload in proximal tubular epithelial cells (PTECs), fibrosis and inflammation. While protein overload is suggested to stimulate PTEC proliferation, its role in inducing cellular senescence remains unclear. This study investigated the effects of protein overload on PTEC proliferation and senescence using both in vivo and in vitro models. Experiments were performed using podocin-knockout (KO) (proteinuric model) and megalin-cubilin-podocin KO mice (proteinuric mice without endocytosis, triple KO). Human immortalized proximal tubular cells (RPTEC/TERT1) were incubated with human serum albumin (HSA) with and without fatty acids. Mouse kidneys and cells were analyzed by western blotting, quantitative RT-PCR, and immunofluorescence. In RPTEC/TERT1 cells, treatment with HSA carrying fatty acids induced proliferation and senescence. In vivo, proteinuric podocin-KO mice exhibited a significant increase in proliferation and senescence markers, particularly within the outer medulla. This response was markedly attenuated in triple-KO mice. Proliferation and senescence occurred in both receptor positive and negative cells. Analyses of cytokines, known to stimulate proliferation (PDGF-B) and senescence (TGFβ and MCP-1), were increased in podocin KO, but diminished in triple KO mice. Mechanistically, TGF-β receptor inhibition prevented HSA-induced senescence, whereas recombinant TGF-β sufficed to induce the phenotype in RPTEC/TERT1. In conclusion, excessive endocytosis of filtered proteins induces a changed cytokine environment with impact for PTEC proliferation and cellular senescence, particularly within the outer medulla.