Introduction: Long-term graft failure remains a significant challenge in pediatric kidney transplantation, often leading to the need for repeated transplants and lifelong medical management. Cellular senescence plays a crucial role in the kidney’s response to transplant-related stress and injury, ultimately contributing to graft dysfunction. Targeting cells expressing the senescence protein p16^INK4a has been shown to improve kidney injury outcomes, but the specific cell populations implicated remain unclear. Given the key role of endothelial cell senescence in kidney injury and subsequent damage, we investigate the contribution of p16^INK4a-mediated endothelial senescence to injury-induced renal damage.
Methods: We developed a conditional knockout (KO) mouse model allowing the inducible deletion of Cdkn2a encoding p16^INK4a specifically in endothelial cells following tamoxifen injections. Young male mice, with or without endothelial-specific KO of p16^INK4a, were subjected to unilateral ischemia-reperfusion injury (IRI), bilateral IRI, or kidney transplantation. Kidney injury, inflammation, fibrosis, and senescence-related markers were analysed using histology, immunofluorescence, and RT-qPCR. Assessments were conducted at various time points: 6 hours, 1, 3, 7, and 30 days after unilateral IRI, 14 days after bilateral IRI, and 7 and 21 days after transplantation with each group including up to 17 mice.
Results: Acute kidney injury scoring showed reduced injury severity in kidneys from p16^INK4a KO mice at earlier time points after unilateral IRI or transplantation compared to control groups. Additionally, CD31 immunostaining indicated better preserved endothelial integrity in transplanted kidneys, as well as in bilateral and unilateral IRI with p16^INK4a KO starting day 3.  In parallel, SA-β-gal staining revealed lower cellular senescence and CD45 immunostaining showed reduced immune cell infiltration in p16^INK4a KO kidneys. At later time points, Picrosirius red staining additionally demonstrated less interstitial fibrosis. RT-qPCR analysis of genes related to kidney injury, endothelial function, senescence, inflammation, and fibrosis further supported these findings.
Conclusion: p16^INK4a KO in endothelial cells protected the kidneys from acute injury, maintained endothelial function, reduced inflammation and decreased cellular senescence and fibrosis in kidneys undergoing experimental IRI and transplantation, indicating the central role p16^INK4a-mediated endothelial senescence plays in driving kidney damage following injury. Senescent endothelial cells and endothelial p16^INK4a hence represents a promising therapeutic target for enhancing outcomes following kidney injury and transplantation.