Reappraisal of profibrotic phenotype and cell-cycle state of renal tubular epithelium after ischemia–reperfusion injury

Abstract

Chronic kidney disease progression involves phenotypic changes in tubular epithelial cells, and recent studies have highlighted the relationship between such pro-fibrotic phenotypes and cell cycle arrest in injured tubular epithelial cells undergoing repair. We investigated these processes using a mouse unilateral ischemia–reperfusion injury model with γGT.Fucci2aR mice, which express fluorescent cell cycle markers, and in vitro experiments with human kidney-2 cells and public single-cell RNA sequencing data. In the unilateral ischemia–reperfusion injury model, mVenus-positive cells (S/G2/M phases) in the γGT.Fucci2aR mice peaked on Day 3 post-injury, then rapidly declined. In kidneys with progressive tubular atrophy and interstitial fibrosis between 7–12 days post-injury, S/G2/M phase cells were limited. These findings were corroborated by analysis using public single-cell RNA sequencing data from the same mouse models, which confirmed dynamic cell cycle changes in the acute phase post-injury but no significant G2/M phase cells in the chronic phase. In vitro experiments with human kidney-2 cells demonstrated that cellular communication network factor 2 and transforming growth factor-β expression increased significantly as proliferating cells reached confluence and cell cycle progression slowed. Pro-fibrotic phenotypes in tubular epithelial cells were not exclusively acquired by G2/M-arrested cells, as reported in previous studies, but can also be acquired by cells in the quiescent G0/G1 phase during normal cell cycling. To develop novel therapeutics for chronic kidney disease, regulating pro-fibrotic gene expression in injured tubular epithelial cells, independent of specific cell cycle phases, appears to be crucial.