Hypothermic Machine Perfusion Decreases Renal Cell Apoptosis During Ischemia/Reperfusion Injury via the Ezrin/AKT Pathway.

Abstract

This study aimed to explore the potential mechanisms of hypothermic machine perfusion (HMP)-a more efficient way to preserve kidneys from donors after cardiac death than static cold storage (CS), then to provide the basis for further improving donor quality. Twelve healthy male New Zealand rabbits (12 weeks old, weighing 3.0 ± 0.3 kg) were randomly divided into two groups: the HMP group and CS group (n = 6). Rabbits' left kidney was subjected to 35 min of warm ischemic time by clamping the left renal pedicle and 1 h of reperfusion. The kidneys were then hypothermically (4-8°C) preserved in vivo for 4 h with HCA-II solution using HMP or CS methods. Then rabbits underwent a right nephrectomy and the kidney tissues were collected after 24 h of reperfusion. TUNEL staining was performed on paraffin sections to detect apoptosis, and the expressions of cleaved caspase-3, ezrin, AKT, and p-AKT in frozen kidney tissues were detected by Western blotting. The ezrin expression was further confirmed by immunohistochemistry analysis. The apoptosis rate and expression of cleaved caspase-3 in the HMP group were significantly lower than the CS group (P < 0.001 and P = 0.002), meanwhile the expression of cleaved caspase-3 in the HMP and CS groups was significantly increased compared with the normal group (P = 0.035 and P < 0.001), and the expression of ezrin and p-AKT in the HMP group was significantly higher than the CS group (P = 0.005, 0.014). HMP decreased the renal cell apoptosis rate during ischemia/reperfusion injury via the ezrin/AKT pathway.