Single-cell analysis of proximal tubular cells with different DNA content reveals functional heterogeneity in the acute kidney injury to chronic kidney disease transition.

Abstract

INTRODUCTION Proximal tubular epithelial cells with different DNA contents emerge after acute kidney injury (AKI). However, their heterogeneity and roles in the acute kidney injury-to-chronic kidney disease (AKI-to-CKD) transition remain incompletely understood. METHODS Proximal tubular epithelial cells with different DNA contents were isolated at days 3 and 14 post-AKI following ischemia reperfusion injury for single-cell RNA sequencing. RESULTS Here, we found that proximal tubular epithelial cells with different DNA contents have existing and distinct bulk transcriptome profiles, especially those cells over 4N (polyploid cells with more than four chromosome sets) with upregulated profibrotic signatures. Heterogeneity existed within four distinct functional clusters. In particular, the polyploid cells demonstrated a preferential enrichment within specific proinflammatory and profibrotic clusters post-AKI. Polyploid cells within these specific clusters displayed the profibrotic trajectory, accompanied by increased fibrosis-driving regulon activity and very strong cell-cell interactions. This suggests polyploidy cells have an intrinsic role in promoting the AKI-to-CKD transition. Furthermore, we identified that secreted phosphoprotein 1 (SPP1) as the pivotal hub of polyploid cells and may be involved in various profibrotic signaling pathways. Genetic knockdown of SPP1 in the proximal tubule in vivo dramatically ameliorated kidney fibrosis. CONCLUSIONS Overall, our findings reveal the heterogeneity of proximal tubular epithelial cells with different DNA contents and identify intrinsic factors of polyploid cells such as SPP1 expression in promoting kidney fibrosis. Our study provides novel insights into potential therapeutic target of preventing the AKI-to-CKD transition.