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. Although 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 using 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 receptor-negative cells. Analyses of cytokines, known to stimulate proliferation (PDGF-B) and senescence [transforming growth factor-β (TGF-β) and monocyte chemoattractant protein-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.NEW & NOTEWORTHY This study identifies the outer medulla as a critical spot where protein overload triggers a pathological cycle of proliferation and cellular senescence in proximal tubular epithelial cells (PTECs). By using a unique triple-knockout model, we demonstrate that protein endocytosis, rather than luminal protein presence alone, is the essential driver of this response through changes in the cytokine profile. We establish that TGF-β-dependent pathways are activated in response to protein overload, which may contribute to proliferation and senescence.