Empagliflozin reduces kidney fibrosis and improves kidney function by alternative macrophage activation in rats with 5/6-nephrectomy

Background: Sodium glucose cotransporter 2 (SGLT2) inhibitors originally developed for the treatment of type 2 diabetes are clinically very effective drugs halting chronic kidney disease progression. The underlying mecha-nisms are, however, not fully understood.Methods: We generated single-cell transcriptomes of kidneys from rats with 5/6 nephrectomy before and after SGLT2 inhibitors treatment by single-cell RNA sequencing. Findings: Empagliflozin treatment decreased BUN, creatinine and urinary albumin excretion compared to placebo by 39.8%, 34.1%, and 55%, respectively (p < 0.01 in all cases). Renal interstitial fibrosis and glomerulosclerosis was likewise decreased by 51% and 66.8%; respectively (p < 0.05 in all cases). 14 distinct kidney cell clusters could be identified by scRNA-seq. The polarization of M2 macrophages from state 1 (CD206-CD68- M2 macro-phages) to state 5 (CD206+CD68+ M2 macrophages) was the main pro-fibrotic process, as CD206+CD68+ M2 macrophages highly expressed fibrosis-promoting genes and can convert into fibrocytes. Empagliflozin remarkably inhibited the expression of fibrosis-promoting (IFG1 and TREM2) and polarization-associated genes (GPNMB, LGALS3, PRDX5, and CTSB) in CD206+CD68+ M2 macrophages and attenuated inflammatory signals from CD8+ effector T cells. The inhibitory effect of empagliflozin on CD206+CD68+ M2 macrophages polari-zation was mainly achieved by affecting mitophagy and mTOR pathways.Interpretation: We propose that the beneficial effects of empagliflozin on kidney function and morphology in 5/6 nephrectomyiced rats with established CKD are at least partially due to an inhibition of CD206+CD68+ M2 macrophage polarization by targeting mTOR and mitophagy pathways and attenuating inflammatory signals from CD8+ effector T cells.Fundings: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.