#5741 POLYPLOID TUBULAR CELLS INITIATE A TGF-BETA CONTROLLED LOOP THAT SUSTAINS POLYPLOIDIZATION AND TUBULOINTERSTITIAL CROSSTALK DURING AKI-CKD TRANSITION

Abstract Background and Aims Acute Kidney Injury (AKI) is characterized by a sudden kidney failure accompanied by a transient decrease of kidney functionality. It is regarded as an important risk factor for chronic kidney disease (CKD) development, but the link is still elusive. Renal fibrosis, especially tubulointerstitial fibrosis, is the final manifestation of CKD and is characterized by an excessive synthesis and deposition of extracellular matrix associated with inflammatory infiltration, tubular epithelial (TC) cell damage and fibroblast activation. Although no targeted therapy yet exists to slow the progression of tubulointerstitial fibrosis, recent findings contributed to clarify the cellular and molecular mechanisms underlying its development and progression, posing TC at the center of this process. Accordingly, we have recently demonstrated that fibrosis and senescence are trade-offs of TC polyploidy occurring immediately after AKI to support fast kidney function recovery, but promoting consequent CKD. However, the mechanisms turning TC polyploidy to senescence and fibrosis still need to be elucidated. In this study, we propose that TC polyploidy is the primary driver of CKD progression after AKI. Methods Polyploid TC are characterized by an increased DNA content in the absence of cell division. To discriminate polyploid cells from actively proliferating cells, we employed a series of in vitro and in vivo transgenic models based on the Fluorescence Ubiquitin Cell Cycle Indicator (FUCCI) technology. AKI was triggered by unilateral ischemia reperfusion injury (IRI) or glycerol-induced rhabdomyolysis. This technology allows to follow the cell cycle phasing of living cells. Cell sorting and cytofluorimetric techniques were employed to isolate and characterize a subpopulation of polyploid TC that progressively accumulate DNA damage after AKI. These results were further corroborated by single cell RNA-sequencing (scRNA-seq) analyses in vitro and in vivo. Results In this study, we found that immediately after AKI, expression of cell cycle markers mostly identifies a population of DNA damaged polyploid TC. Employing transgenic mouse models and single cell RNA-sequencing we showed that after AKI, polyploid TC accumulate DNA damage and survive eventually resting in the G1 phase of cell cycle, while diploid cells do not survive DNA damage. This suggests that after AKI, polyploidization is a means to survive injury. Sorting of DNA-damaged polyploid TC showed that they express p21 and acquire a pro-fibrotic phenotype culminating in TGF-beta expression. in vitro analysis and single cell RNA-sequencing evidenced that TGF-beta directly promoted polyploidization, demonstrating that TC polyploidization is a self-sustained mechanism. Interactome analysis by single cell RNA-sequencing revealed that TGF-beta signaling fostered a reciprocal activation loop among polyploid TC, macrophages and fibroblasts to sustain kidney fibrosis and promote CKD progression. Conclusion Collectively, this study contributes to the ongoing revision of the paradigm of kidney tubule response to AKI supporting the existence of a tubulointerstitial crosstalk mediated by TGF-beta signaling produced by polyploid TC following DNA damage. Moreover, this study identifies polyploid TC as central actors during CKD progression with important implications for the development of novel targeted therapy to block CKD.