IN VIVO EVIDENCE LINKING CELL CYCLE ARREST, CELLULAR SENESCENCE AND FIBROSIS IN THE KIDNEY

In recent years, attention has focused on the accumulation of senescent cells following kidney injury; however, the mechanisms inducing these cells and their specific roles remain insufficiently understood. Phosphorylated p38 (p-p38) is induced in injured kidneys in vivo, and in vitro studies have demonstrated that p-p38 promotes cellular senescence in cultured cells. However, the role of p-p38 in cellular senescence induction in vivo and in the progression of chronic kidney disease (CKD) has not been fully elucidated.

We established a mouse model (PT-MKK6EE mice) and a cell line (MKK6EE HK-2) that can induce constitutive p38 phosphorylation in proximal tubular cells (PTCs) in response to doxycycline (Dox) administration both in vitro and in vivo. We analyzed the role of p-p38 in PTCs in cellular senescence in vivo and CKD progression.

When Dox was administered to PT-MKK6EE mice to induce p-p38 expression in PTCs, Kim-1, a marker of injured PTCs, was observed in nearly all p-p38-positive PTCs, despite the absence of kidney injury. Additionally, VCAM1, a marker of failed repair PTCs (FR-PTCs), was also detected in some p-p38 positive PTCs. Furthermore, macrophage infiltration and fibrosis were observed around p-p38-positive PTCs, indicating that sustained p38 phosphorylation in PTCs alone may contributes to CKD progression.

p-p38-positive PTCs in these mice exhibited senescence hallmarks including lysosomal protein accumulation and nuclear enlargement, along with significant upregulation of senescence-associated genes. This suggests that p38 activation in PTCs also induces cellular senescence in vivo. Notably, these senescent cells were positive for VCAM1.

To verify cell cycle arrest, another critical feature of cellular senescence, PT-MKK6EE mice were subjected to ischemia-reperfusion injury (IRI) to promote cell cycle entry of PTCs. The expression of Ki67, a marker of cell proliferation, was significantly reduced in p-p38 positive PTCs, indicating that p38 phosphorylation induces cell cycle arrest. Indeed, in the wild-type mouse subjected to IRI, p-p38 and Ki67 were present in a mutually exclusive spatiotemporal pattern, supporting the findings in PT-MKK6EE mice.

To elucidate the mechanism underlying p38-mediated cell cycle arrest and the induction of cellular senescence, MKK6EE HK-2 cells were employed. Induction of p-p38 decreased cyclin D1 protein levels and increased p21 and p27 protein levels, indicating that the cells were arrested in the G0 phase of the cell cycle. Furthermore, Skp2, which is involved in the ubiquitination of p21 and p27, was reduced, confirming that the accumulation of p21 and p27 occurs via Skp2. Additionally, time-course RNA-seq analysis revealed time-dependent increase in the expression of cellular senescence-related genes, demonstrating that sustained p38 phosphorylation facilitates cell cycle arrest and subsequent stepwise induction of cellular senescence.

We demonstrated that p-p38 in PTCs initiates a sequential process of cell cycle arrest and cellular senescence. Moreover, senescent cells induced by p-p38 are present in VCAM1 positive "failed repair PTCs", and contribute to CKD progression.

This work was first presented at ASN Kidney Week 2024, and re-submission is permitted by ASN.