#6679 ANALYSIS OF MACROPHAGE POPULATIONS IN THE KIDNEY WITH RENAL TUBULAR CELL-SPECIFIC SENESCENCE INDUCTION

Abstract

Abstract Background and Aims Ageing is a risk factor for multiple diseases, including kidney disease. To improve quality of life, pharmaceutical treatments are given to ageing patients, often with side-effects and cross-reactions. Research is needed into the effects and mechanisms of ageing in order to identify new druggable targets to improve quality of life. Cells can become senescent with age and are seen at sites of tissue injury. Senescent cells (SCs) undergo irreversible cell-cycle arrest, but remain metabolically active, producing a range of signalling molecules called the senescence associated secretory phenotype (SASP, made up of pro-inflammatory/fibrotic elements). Senescent cells within the kidney post-acute kidney injury (AKI) may be involved in ongoing damage, leading in some cases to chronic kidney disease (CKD), possibly due to interactions with macrophages. We hypothesise that the recruitment of monocytes/macrophages by senescent cells is due to the signalling components present in their SASP. Method Pax8 is renal tubule marker. We use Pax8-creERT2;mdm2 fl/fl mice to study kidney-specific senescence induction in absence of age/injury. Upon administration of tamoxifen, the mdm2 alleles are floxxed out by cre recombinase, allowing p53 to stabilise and activate the p21CIP1 cell-cycle inhibitor, inducing senescence in Pax8+ kidney-specific cells. The use of an endogenous tdTomato reporter allows visualisation of cells undergoing successful recombination upon the administration of tamoxifen (TAM). Results As expected, cell-cycle inhibitor/SC marker, Cdkn1a was upregulated by qPCR in TAM-treated young murine whole kidneys 10-fold compared to controls (p = 0.0173) (Figure 1). Ccl2 (monocyte chemoattractant) was up-regulated 20-fold in young (p = 0.0405) TAM-treated TG mice vs controls (Figure 1). In agreement with an increase of Ccl2 transcripts, there was a significant increase of renal macrophages as quantified by flow cytometry of kidney digests (p = 0.0141) in TAM treated young TG mice. Immunofluorescence staining of transgenic mice revealed an increase of Iba1 positive macrophages, correlated with staining of tdTomato tubules, suggesting that increase in macrophage numbers was in response to senescence induction (Figure 2). Iba1-p21 co-stains show a increases in p21 positive cells in TAM-treated transgenic mice, evidently a result of construct activation and appear in proximity to the macrophages (Figure 2). Conclusion Apparent increases in macrophages due to the presence of senescent cells may indicate one of the mechanisms by which AKI progresses to CKD, particularly if inflammatory macrophage phenotypes are present and persist. Macrophage and monocyte populations fluctuate with age and the use of this model allows analysis of macrophage recruitment/polarisation due to senescent cell burden in the absence of age/injury and possible confounding factors, in young mice.