Kidney tubule polyploidization during physiologic ageing in mice

Abstract Background and Aims The aged population is constantly increasing, and kidney-aging is a risk factor for both acute kidney injury (AKI) and chronic kidney disease (CKD). Accordingly, CKD is a major global health problem with an increasing prevalence in older population. Therefore, there is an urgent need to understand the age-related mechanisms underpinning CKD development during ageing. Recent findings contributed to clarify the cellular and molecular mechanisms underlying CKD development and progression, posing tubular cells (TC) at the center of this process. In this regard, we have demonstrated that polyploidization of TC promotes fibrosis, CKD development and its progression in the long run. Here, we aimed to investigate TC polyploidization during physiologic ageing in mice. Method To investigate TC polyploidization during ageing, we took advantage of inducible Pax8/FUCCI2aR mice. In these mice, FUCCI2aR reporter is expressed by all TC after doxycycline hyclate induction. Mice were sacrificed and analyzed at 2, 5, 11 and 20 months of age. By combining DNA content with detection of FUCCI2aR fluorescent proteins, FACS analysis shows diploid (2C), tetraploid (4C) and octaploid or greater (≥8C) TC. Single cell RNA-sequencing (scRNA-seq) analysis was performed on mouse kidneys at 2 and 20 months of age to dissect the transcriptomic profile of polyploid TC and interrogate putative altered signaling pathways. Renal function parameters were assessed at 2, 5, 11 and 20 months before sacrifice. Kidneys were analyzed for senescence and interstitial fibrosis at 2, 5, 11 and 20 months of age. Results Genetic and single-cell transcriptomic approaches in mice were used to investigate TC polyploidization during ageing. Pax8/FUCCI2aR mice showed a progressive increase of polyploid TC during ageing starting from 5 months of age. ScRNA-seq analysis on kidneys of mice at 2 and 20 months of age revealed that polyploid TC were characterized by polyploidy regulators, p21 expression, accumulation of DNA damage and by acquiring senescent/fibrotic markers, demonstrating that polyploid TC rather than diploid TC accumulate DNA damage and acquire a senescent/pro-fibrotic state during physiologic ageing in vivo. In accordance with the increase of polyploid TC, mice developed interstitial fibrosis, showed a marked senescent phenotype and an age-related decline of renal function starting from 5 months of age. Collectively, these results suggest TC polyploidization is a potential trigger for CKD development and progression during physiologic kidney ageing in mice. Conclusion This study demonstrates the increase of TC polyploidization, which is associated with a progressive fibrosis, senescence and CKD development during physiologic kidney ageing in mice.