Kidney Hypothermic Perfusion with Mesenchymal Stromal Cells or Microvesicles Protects Rat Kidney from Ischemia By Upregulation of Cell Energy Metabolism and Ion Membrane Transport Key Genes

We evaluated in a rat model of Donor after Circulatory Death (DCD) the effects of pretransplant kidney conditioning with Mesenchymal Stromal Cells (MSC) or MSC derived microvescicles (MV) on ischemic injury. Fisher rats (F) were used as kidney donors, Transgenic Sprague-Dawley rats expressing Enhanced Green Fluorescence Protein (EGFP) as MSC donors. After 20 min of warm ischemia nephrectomy was performed and kidneys were perfused with Belzer solution (BS), BS supplemented with MSC (MSC) or with MV (MV) for 4 h, at 4°C. Renal damage was evaluated by histology, renal gene expression by microarray analysis and RT-PCR. Malondialdehyde, lactate, LDH, glucose and pyruvate were measured in effluent fluid. MSC/MV kidneys showed a significant minor global ischemic damage and up-regulation of three genes encoding proteins that improve cell energy metabolism (Isocitrate dehydrogenase 2, NADH dehydrogenase Fe-S protein 8, Pyruvate dehydrogenase beta) and three genes encoding proteins involved in ion membrane transport (Calbindin 1, Monocarboxylate transporter 1, Vacuolar H+-ATPase d2 subunit). Lactate, LDH and malondialdehyde in effluent fluid were significantly lower in MSC/MV kidneys than BS. Glucose was lower while pyruvate higher in MSC/MV effluent than BS, suggesting a larger use of energy substrates by MSC/MV kidneys. MSC/MV perfusion of kidneys in addition to BS through HMP protects from ischemia injury by preserving the enzymatic machinery essential for cell viability and prepares kidney to reperfusion damage. A rat model of DCD was reproduced. Rats were anesthetized using Isoflurane 2-5%. After 20 min of warm ischemia obtained by abdominal aorta clamping, nephrectomy was performed. Kidneys were then perfused with Belzer solution (BS), or BS supplemented with 3 millions of MSC (MSC), or BS supplemented with MV derived from 3 millions of MSC (MV). We studied 5 kidneys per group. Continuous perfusion was performed for 4 h at 4°C, than the effluent fluid was collected and stored at -20°C. Kidneys were fixed in 10% formalin for morphological studies, frozen in liquid nitrogen for RT-PCR. We studied also another group of non perfused kidneys (n 5) (NP) obtained after 20 min of warm ischemia and preserved in RNAlater to microarray studies. Malondialdehyde, lactate, LDH, glucose and pyruvate were measured in effluent fluid. All experiments were quadruplicated.