#6095 TRANSGENIC INDUCTION OF EPITHELIAL SENESCENCE PROMOTES RENAL FIBROSIS INDEPENDENT OF INJURY
Authors: Docherty Marie-Helena , Campbell Ross , Denby Laura , Baird David , Mylonas Katie , Ferenbach David 
Published: June 2023

Abstract

Background and Aims
Chronic Kidney Disease (CKD) affects over 850 million people worldwide. Progressive renal fibrosis is a hallmark of CKD, irrespective of the initiating aetiology. Any episode of acute kidney injury (AKI) significantly increases the risk of development of CKD, even after apparent complete resolution of the initiating injury. This risk is exacerbated by age and pre-existing CKD. This implies that factors persist within damaged/aging kidneys which drive functional loss, impair complete repair and promote on-going fibrosis. Senescent cells (SCs) are metabolically active, permanently growth arrested cells produced in response to stress and DNA damage. SCs accumulate with age and persist at the sites of previous disease and injury. Their depletion in animal models is safe and extends organ function and healthspan. We have shown that pharmacological SC depletion in kidneys significantly improves kidney function and reduces fibrosis post injury. However, models of renal injury induce changes in multiple cell lineages, and pharmacological depletion is non-specific both in terms of cell lineage (i.e. epithelial vs mesenchymal vs leukocyte) and characteristics of senescent cell (i.e. acute vs chronic, primary vs secondary). We hypothesised that induction of epithelial senescence in the absence of other renal injuries is sufficient to initiate renal fibrosis. We developed a transgenic mouse allowing selective senescence induction in renal epithelia in the absence of injury, with tdTomato labelling of induced cells.

Method
By crossing existing strains, we produced a triple transgenic Pax8creERT2/mdm2flfl/tdTOMLSL mouse allowing conditional senescence induction by tamoxifen via mdm2 deletion in Pax8 expressing renal epithelia, alongside TdTomato expression. Levels of renal fibrosis, p21cip1 and TdTomato induction were quantified by picrosirius red staining, immunofluorescence and flow cytometry, with downstream image analysis on QuPath 0.3.2. CDKN1A gene expression was quantified by qPCR.

Results
Examination of kidneys and livers from young mice ± tamoxifen demonstrated that tdTomato induction was tissue specific and restricted to renal epithelia (Fig 1A-C). Administration of Tamoxifen resulted in increased expression of tdTomato and CDKN1A expression at day 7 in transgenic but not in WT mice. (Fig. 1C, 2 A-F). We observed a rapid induction of fibrosis in the first 7 days after tamoxifen induction in both young (Fig. 3A,C) and old mice (Fig 3C). This persists to day 42 (Fig. 3B). This demonstrated that epithelial senescence alone was sufficient to induce renal fibrosis in young mice, and exacerbate fibrosis in old mice. Further studies assessed the longer-term impact of acute senescence induction, with fibrosis stabilising in both young and old mice at 6 weeks post induction (Fig. 3D).

Conclusion
Using a novel transgenic mouse line, we demonstrate for the first time that induction of renal epithelial senescence in the absence of injury is sufficient to induce renal fibrosis in the early aftermath of SC induction. The evolution and/or clearance of senescent cells over time is the focus of on-going study and will be presented at the ERA.