#2440 Impaired renal progenitor regeneration and polyploid tubular cell progression underlie physiological kidney tubule aging

Abstract Background and Aims Aging is associated with a natural decline in nephron mass and number, leading to progressive kidney function loss and an increased risk of chronic kidney disease (CKD). With individuals over 65 years old being the fastest-growing demographic in developed nations, understanding the mechanisms driving CKD in this population is of critical clinical relevance. CKD often remains undiagnosed in up to 90% of patients due to the kidney tubule's remarkable compensatory capacity to maintain function despite nephron loss. However, these compensatory mechanisms are poorly understood. Aging in most organs is frequently linked to a loss of stem cell pools or impaired progenitor cell proliferation. In the kidney, we have revealed that a population of renal progenitor cells (RPC), interspersed among tubular cells (TC), are key to regenerating kidney tubules after acute kidney injury. Additionally, TC undergo polyploidization, acquiring extra genome copies, enabling them to sustain kidney function during stress. This is a key mechanism underlying kidney's ability to buffer against TC loss. This study investigates these two adaptive responses during physiological kidney aging. Method To trace RPC (Pax2⁺ cells), we used inducible Pax2/Confetti mice, where RPC are labeled with one of four fluorescent reporter genes. To study polyploid TC, we used inducible Pax8/FUCCI2aR mice, identifying polyploid TC by cell cycle fluorescent proteins and DNA content analysis. Mice aged 2, 5, 12, and 20 months were analyzed, and single-cell RNA sequencing (scRNA-seq) was performed at 2 and 20 months of age. Results Aging was associated with a natural decline in nephron number, progressive kidney function decline, fibrosis, and accumulation of senescent TC, culminating in CKD. In Pax2/Confetti mice, we observed a gradual loss of Pax2⁺ RPC and a decrease in their regenerative capacity starting from 12 months of age. scRNA-seq revealed an age-dependent signature in RPC characterized by DNA damage, TGF-β signaling, senescence markers, and enrichment of stress response pathways, which collectively underlie the impaired regenerative capacity of aged RPC. In Pax8/FUCCI2aR mice, tubular polyploidy progressively increased, accumulating additional chromosome sets as evinced by octaploid TC (8C DNA content), throughout aging. Polyploid TC showed elevated DNA damage and were detected in urine, suggesting ongoing loss. scRNA-seq of polyploid TC showed the increment of hypertrophy- and maladaptive-associated genes in aged mice and expression of fibrotic and senescent genes in polyploid TC expressing DNA damage markers. Additionally, we revealed the polyploid characteristic pathways, which were associated with tubulointerstitial fibrosis, including TGF-β signaling and epithelial-to-mesenchymal transition, TNF-α signaling, DNA damage responses, and mitochondrial dysfunction. These findings highlight the dual role of polyploid TC in acquiring a profibrotic/proinflammatory phenotype to buffer against DNA damage throughout aging, while driving fibrosis and CKD progression. Conclusion Aging-related kidney decline is characterized by: These age-related changes likely compromise the kidney tubule's capacity to compensate for nephron loss, increasing the susceptibility to CKD in the elderly population.