ИСТИНА

INTRODUCTION AND AIMS: Aging is accompanied by ischemia-associated pathologies and an important protective anti-ischemic measure is ischemic preconditioning (IPC) shown for a large number of organs, including the kidney. The studies of signaling pathways involved in IPC showed that mitochondria are an essential requisite for implementation of protective mechanisms and age-dependent changes of these organelles may affect the efficiency of IPC in the elderly. It stays unknown whether the protective effect of IPC remains in aged kidney. The goal of this study is to analyze possible changes in natural ischemic tolerance mechanisms in aged kidney with special attention to mitochondria quality control system. METHODS: Male outbred rats of age 4-6 and 20-23 months were used. Additional experiments were done with 6-month-old OXYS rats, demonstrating the signs of premature aging. The animals were subjected to IPC (4 cycles of 15-sec ischemia and 15-sec reperfusion each) of kidney followed by 40-min warm ischemia or to 40-min ischemia alone. Serum creatinine was measured 48 h after operation. Formaldehyde-fixed kidneys were stained for acetylated lysine. Autophagy and mitophagy related proteins were detected by Western blotting in whole kidney homogenates and isolated mitochondria. Lysosome abundance was assessed by Lysotracker Green. Flow cytometry was used to measure the transmembrane potential of isolated kidney mitochondria. Total antioxidative capacity of kidney homogenates was measured by the hemoglobin-hydrogen peroxide-luminol chemiluminescence protocol. RESULTS: In young rats IPC alleviated kidney injury caused by 40-min ischemia. However, in old rats IPC was not protecting. The same aged phenotype demonstrated 6-month-old OXYS rats. In the kidney of old and OXYS rats, the percent of tubules with acetylation-positive nuclei was higher than in young rats. In young rats the percent of such tubules after ischemia was increased and IPC prevented the ischemia-induced increment of protein acetylation. Vice versa, in old and OXYS rats IPC markedly augmented the percent of acetylation-positive tubules. In similar way in young rats ischemia increased and IPC decreased lysosome abundance and LC3-I content. In old and OXYS rats lysosome abundance was unchanged after ischemia as well as after IPC. Kidney LC3 levels were also lower in OXYS rats even under basal conditions, and mitochondrial PINK1 and ubiquitin levels were higher, suggesting impaired mitophagy. Moreover kidney mitochondria from old rats contained a population with diminished membrane potential and IPC expanded this fraction. Total antioxidative capacity of kidney homogenates was increased after IPC in young rats and decreased in old rats. CONCLUSIONS: Aging result in the accumulation of malfunctioning mitochondria in kidney due to low efficiency of autophagy. In such milieu IPC is no longer protective but rather damaging to the kidney.