#5715 IMMUNE LANDSCAPING IN RHABDOMYOLYSIS INDUCED ACUTE KIDNEY INJURY IDENTIFIES SENESCENCE AS A POTENTIAL TARGET
Authors: Rao Snigdha N , Zahm Margot , Buleon Marie , Casemayou Audrey , Faguer Stanislas , Feuillet Guylene , Iacovoni Jason , Joffre Olivier , Gonzalez-Fuentes Ignacio , Martins Frédéric , Riant Elodie , Zakaroff-Girard Alexia , Schanstra Joost , Blache Jean Sébastien Saulnier , Belliere Julie 
Published: June 2023

Abstract

Background and Aims
Worldwide, acute kidney injury (AKI) has an incidence of 13.3 million cases per year leading to 1.7 million deaths. Rhabdomyolysis (RM) accounts for 10% of the AKI cases, and frequently occurs due to falling (often in elderly), crush syndrome or exposure to drugs. By causing skeletal muscle damage, RM leads to a release of muscle contents such as myoglobin into the circulatory system and trigger renal sterile inflammation. The role of macrophages in driving inflammatory phase of RM-AKI lesions and fibrosis-related prognosis has been reported but both the role of other immune cells and the macrophage heterogeneity in RM-AKI still lack detail. The aim of this study was to describe renal immune cell recruitment and more specifically the mononuclear phagocytic cell (MPC) population (macrophages, monocytes and dendritic cells) during RM-AKI through single cell RNA sequencing (scRNA-seq) in mice.

Method
Two-month-old C57BL/6J male mice were administered intramuscularly saline (control) or 50% glycerol for induction of RM. Forty-eight hours later mice were sacrificed, kidneys were collected and dissociated and CD45 live cells were sorted. Single cell cDNA libraries were prepared, and sequencing was performed according to the 10X Genomics protocol. Creatinine phosphokinase and blood urea nitrogen were quantified to validate muscle injury and renal function respectively. A combination treatment of dasatinib and quercetin (DQ) as senolytic drugs was performed. Intraperitoneal injections of dasatinib (10mg/kg) and quercetin (50mg/kg) were conducted with and without RM-AKI.

Results
scRNA-seq of CD45+ renal cells revealed the existence of 7 immune cell clusters with macrophages and monocytes as the most highly recruited cells in RM-AKI condition, indicating a major change in the MPC population. Specific clustering of MPCs divulged 10 clusters (8 macrophages, 1 monocyte and 1 dendritic cell cluster) revealing a higher heterogeneity of this population compared to what was previously observed by flow cytometry. Trajectory, pseudotime and KEGG pathway enrichment analyses disclosed a unique cluster highly expressing Stathmin 1 present in both control and RM-AKI condition where 164 cells were observed in control condition which expanded to 333 cells in RM-AKI condition. This unique cluster was characterized by a bimodal expression of MHCII markers and a downregulation after RM-AKI suggesting a transcriptional reprogramming. Cellular senescence was observed to be a predominant pathway in this unique cluster. Senolytics were identified as a possible pharmacological target based on pathway analysis. DQ protocol accompanied with an improvement of kidney function as well as an attenuation of MPC phenotypic modifications.

Conclusion
This study revealed the MPC diversity in RM-AKI through immune landscaping, trajectory and pathways in RM-AKI in mice. A unique cluster associated with possible reprogramming of macrophages was identified. Senolytics were established as a potential nephroprotective strategy in RM-AKI.