#2352 Polyploid tubular cells drive functional recovery after nephron mass loss and can be employed to determine likelyhood of CKD progression

Abstract Background and Aims Tubular epithelial cells (TC) represent the metabolic unit of the nephrons. Nephrons - the kidney functional unit - are established early during development and cannot increase. Therefore, their loss can promote the development of chronic kidney disease (CKD). CKD represents a growing global health challenge with more than 850 million people living with some form of kidney disease - 20 times the prevalence of cancer. Timely CKD identification is complicated by the remarkable capacity of the kidney tubule to buffer against nephron loss to maintain kidney function. In contrast to the general belief that partial regeneration promoted by cell dedifferentiation and proliferation is at the base of these buffering mechanisms, we identified TC polyploidy as a novel mechanism of tubule hypertrophy. Polyploid cells, which acquire additional genome copies, are metabolically more active than diploid cells, providing a rapid response to stress. Throughout life, nephron loss can occur naturally with aging or triggered by injury, kidney donation or partial nephrectomy due to cancer. These conditions can predispose to CKD over time, but the link is still elusive. Here, we hypothesize that polyploid TC are the main determinant of compensatory hypertrophy to preserve kidney function after nephron mass reduction. In addition, determining their accumulation and incidence may give important clues to determine the risk of developing CKD. Method To discriminate polyploid cells from actively proliferating cells, we employed in vivo transgenic models based on the Fluorescence Ubiquitin Cell Cycle Indicator (FUCCI2aR) technology (Pax8/WT). An additional transgenic model in which YAP1 is knocked-out specifically in TC while expressing Fucci2aR reporter resulting in a reduced capacity of undergoing polyploidy was employed (Pax8/YAP1ko.) To determine the contribution of progenitor cells, an in vivo model based on the Confetti reporter under the control of the Pax2 promoter was employed (Pax2/Confetti). Nephrectomy was performed by removing the left kidney and mice were sacrificed at different days after uni- Nephrectomy (Nx). Cytofluorimetric techniques were employed to characterize polyploid TC and histology investigation was employed to analyze fibrosis development. γH2AX straining was used to quantify DNA damage. These results were further corroborated by single cell RNA-sequencing (scRNA-seq) analyses in vitro and in vivo. Results Pax2/Confetti mice proved that progenitor proliferation at different days after uni-Nx is negligible. Accordingly, YAP1 continuous nuclear expression is observed 90 days after uni-Nx suggesting that polyploidization response is activated in compensatory hypertrophy. Quantification of polyploid TC at different days after uni-Nx as well as kidney weight 4 days after uni-Nx in Pax8/WT and Pax8/YAP1ko mice confirm that YAP1-activated TC polyploidy mediates compensatory hypertrophy. Interestingly, γH2AX staining showed no significant DNA damage accumulation in polyploid TC of Pax8/WT mice in comparison to healthy kidney while Pax8/YAP1ko accumulate progressive instability in the absence of a direct injury. This suggests that in the absence of YAP1, polyploid TC are genetically unstable accumulating DNA damage. Binucleated polyploid TC are found in the urine at different days after uni-Nx indicating that polyploid TC are continuously accumulated and shed therefore they can be used to monitor kidney adaptation to a reduced kidney mass. In line with the role of YAP1 in mediating compensatory hypertrophy via TC polyploidy, late YAP1 recombination in Pax8/YAP1ko mice 4 days after uni-Nx (i.e., when tubular hypertrophy has already occurred) preserved kidney function. Finally, scRNA-seq analysis performed at different days after uni-Nx was used to investigate polyploid TC signature in the absence of a direct injury. Conclusion Polyploid TC drive compensatory hypertrophy following nephron mass reduction to maintain kidney function. As they are continuously shed in the urine they can be employed to monitor residual kidney function reserve.