THE DISCOVERY AND ANALYSIS OF A UNIQUE SUBPOPULATION OF PROXIMAL TUBULAR CELLS EXHIBITING PHENOTYPES INDICATIVE OF SENESCENCE

Cellular senescence, defined by cell cycle arrest and secretory phenotypes, has emerged as a potential therapeutic target for age-related diseases. While recent research indicates that cellular senescence may play a role in the pathogenesis of kidney diseases, the precise identification of senescent cells in vivo remains challenging, and its significance is not yet fully understood.

We utilized transgenic mice specifically engineered to express the G0 marker, mVenus-p27K-, in proximal tubular cells (PTCs) (PTC-G0 mice) and the FUCCI2aR system in PTCs (PTC-FUCCI mice) to induce kidney injury models through the administration of aristolochic acid and cisplatin. The G0 marker is conventionally used to identify cells in the quiescent phase (G0 phase), while FUCCI2aR comprises G1-FUCCI (mCherry positive) and S/G2/M-FUCCI (mVenus positive). Additionally, we conducted spatial transcriptomics on G0 marker-positive cells using a photo-isolation chemistry (PIC)-based system. We also performed primary culture of PTC-G0 mice and induced cellular senescence in these cells. Furthermore, we assessed the correlation between the accumulation of senescent cells in PTCs in kidney biopsy specimens and kidney dysfunction in humans.

In studies utilizing PTC-G0 mice, we identified a subpopulation of G0-positive proximal tubular cells (PTCs) characterized by robust Cyclin D1 expression, referred to as G0-double positive (G0DP) cells, within mouse models of kidney injury. These G0DP cells exhibited several characteristics of cellular senescence, including nuclear abnormalities and the accumulation of DNA damage foci. Notably, G0DP cells were exclusively located within failed-repair PTCs (FR-PTCs), which have recently garnered attention because of their proinflammatory phenotypes. These cells constituted 10-30% of FR-PTCs across various kidney injury models. Furthermore, using PTC-FUCCI mice, we determined that all Cyclin D1-positive PTCs were arrested in the G1 phase of the cell cycle. The prevalence of G0DP cells among FR-PTCs varied across kidney injury models, the time elapsed post-injury, and the severity of the injury. Spatial transcriptomic analysis based on PIC revealed that G0DP cells exhibited more pronounced senescent phenotypes than G0-positive cells lacking Cyclin D1 expression (G0-single positive cells: G0SP cells) within FR-PTCs, and ChIP-Atlas identified transcription factors such as Trp53, NF-κB, RUNX1, and STAT1 as activated in G0DP cells. Time-lapse imaging of primary PTCs from PTC-G0 mice treated with doxorubicin (DOXO) demonstrated sustained expression of the G0 marker, with permanent cell cycle arrest. DOXO-treated primary PTCs also exhibited Cyclin D1 positivity and senescent phenotypes. In human kidney biopsy specimens, Cyclin D1-positive FR-PTCs displayed similar senescent phenotypes, and the accumulation of Cyclin D1-positive FR-PTCs was significantly correlated with kidney dysfunction and poor kidney prognosis.

G0DP cells exhibit significant characteristics of cellular senescence both in vivo and in vitro, constituting a substantial subpopulation of FR-PTCs in mouse models of kidney injury. In human kidney diseases, Cyclin D1-positive FR-PTCs accumulate concurrently with kidney dysfunction, serving as a potential predictor of adverse kidney prognosis. This work was first presented at ASN Kidney Week 2023, and re-submission is permitted by ASN.