Aged kidney exhibits exacerbated tubular injury and decreased interstitial fibrosis after ischemia

Elderly people have higher risks for acute kidney injury and its progression to chronic kidney disease. Age-related metabolic change has been proposed as a feature of kidney aging, however, assessing ATP dynamics in aged kidneys has been technically challenging. Here, we visualized age-related ATP changes in aged murine kidneys and revealed their association with pathological changes after ischemia reperfusion injury (IRI).

Intravital ATP imaging was performed using multiphoton microscopy in the kidneys of young (8-12 weeks old) and aged (>24 months old) mice that systemically express FRET-based ATP biosensor (GO-ATeam2 mice), both prior to and during IRI. To assess ATP producing pathways, ex-vivo ATP imaging was performed on kidney slices of GO-ATeam2 mice with inhibitors of glycolysis and oxidative phosphorylation (OXPHOS). Pathological changes and injury-associated genes in the kidneys were assessed on day1 and on day14 after IRI.

Intravital ATP imaging revealed significant increase in ATP levels in proximal tubules (PTs), distal tubules, and collecting ducts of aged kidneys in healthy condition. ATP levels in endothelial cells and fibroblasts were comparable between young and aged kidneys. The elevation in proximal tubular ATP was also observed in ex-vivo ATP imaging, suggesting this increase is independent of systemic regulation, such as blood components and blood pressure. In young kidney slices, ATP decreasing rates were comparable after OXPHOS inhibition and after dual OXPHOS-glycolysis inhibition. On the other hand, in aged kidney slices, the rate of ATP depletion tended to be faster after dual OXPHOS-glycolysis inhibition than after OXPHOS inhibition alone. Electron microscopy showed mitochondrial fragmentation in aged PTs in normal condition, suggesting mitochondrial dysfunction. We previously reported in young mice that ATP recovery in PTs after ischemia reperfusion (IR) predicts kidney prognosis. PTs of aged mice showed ATP recovery (recovery rate and recovery sufficiency) comparable to that of young mice. However, aged kidneys showed increased expression of Havcr1 (coding KIM1, a PT injury marker) and increased TUNEL positive cells on day 1 post-IRI compared to young kidneys, suggesting aggravated tubular injury. On day 14 after IRI, aged kidneys maintained a significantly higher proportion of injured tubules, while the expressions of fibrosis markers, Acta2 and Col1a1, were lower than in young kidneys. Interestingly, pre-ischemia ATP levels in PTs were significantly correlated with Havcr1 expression and the numbers of TUNEL positive cells on day 1, and were inversely correlated with the expressions of fibrotic markers, including Acta2, Col1a1, and Fn, on day 14.

Our results suggested that aged PTs acquire glycolytic capacity to compensate for mitochondrial dysfunction, potentially elevating basal ATP levels. Age-related ATP elevation in aged PTs predicts severer tubular injury in acute phase and less kidney fibrosis in chronic phase after IRI. Sustained tubular injury and reduced fibrosis in aged kidneys in chronic phase after IRI may indicate defects of recovery processes. Considering these, metabolic changes in aged PTs could alter processes of injury and recovery after IRI. This work was first presented at ASN Kidney Week 2025, and re-submission is permitted by ASN