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 withBelzer solution (BS), BS supplemented with MSC (MSC) or withMV (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.本研究在循环死亡后供体（Donor after Circulatory Death, DCD）大鼠模型中，评估了移植前采用间充质干细胞（Mesenchymal Stromal Cells, MSC）或MSC来源微囊泡（Microvescicles, MV）对肾脏进行预处理对缺血性损伤的影响。本研究以Fisher品系大鼠作为肾脏供体，以表达增强型绿色荧光蛋白（Enhanced Green Fluorescence Protein, EGFP）的转基因斯普拉-道来氏（Sprague-Dawley, SD）大鼠作为MSC供体。在20分钟热缺血后实施肾切除术，随后将肾脏置于4℃环境下，分别以贝尔泽灌注液（Belzer Solution, BS）、添加MSC的BS（MSC组）或添加MV的BS（MV组）进行4小时灌注。 肾脏损伤通过组织学方法进行评估，肾脏基因表达水平则通过微阵列分析（Microarray Analysis）与逆转录聚合酶链反应（Reverse Transcription Polymerase Chain Reaction, RT-PCR）检测；研究人员同时对灌注流出液中的丙二醛（Malondialdehyde, MDA）、乳酸、乳酸脱氢酶（Lactate Dehydrogenase, LDH）、葡萄糖及丙酮酸水平进行了检测。 结果显示，MSC/MV组肾脏的整体缺血损伤显著减轻，且有3个编码改善细胞能量代谢相关蛋白的基因表达上调，分别为异柠檬酸脱氢酶2、NADH脱氢酶铁硫蛋白8与丙酮酸脱氢酶β亚基；另有3个编码参与离子膜转运蛋白的基因表达上调，包括钙结合蛋白1、单羧酸转运蛋白1及空泡型H+-ATP酶d2亚基。 与BS组相比，MSC/MV组肾脏灌注流出液中的乳酸、LDH及丙二醛水平显著降低，而葡萄糖水平更低、丙酮酸水平更高，提示MSC/MV组肾脏对能量底物的利用更为充分。 综上，通过低温机械灌注（Hypothermic Machine Perfusion, HMP）在贝尔泽灌注液基础上联合MSC/MV进行肾脏灌注，可通过维持细胞存活所必需的酶系统以抵御缺血性损伤，并使肾脏能够更好地耐受再灌注损伤。