Supplementary Material for: Intrarenal Anti-Leptin Treatment Attenuates Ischemia and Reperfusion Injury

Introduction: Renal ischemia and reperfusion (IR) injury introduces cellular stress and is the main cause of acute kidney damage. Renal cells exposed to noxious stress induce the expression of the pleiotropic hormone leptin. As we have previously revealed a deleterious stress-related role for leptin expression, these results suggested that leptin is also involved in pathological renal remodeling. The systemic functions of leptin preclude the study of its local effects using conventional approaches. We have therefore designed a method to locally perturb leptin activity in specific tissues without affecting its systemic levels. This study explores whether local anti-leptin strategy is reno-protection in a post-IR porcine kidney model.
Methods: We induced renal IR injury in pigs by exposing kidneys to ischemia and revascularization. Upon reperfusion, kidneys instantly received an intraarterial bolus of either a leptin antagonist (LepA) or saline solution. Peripheral blood was sampled to assess systemic leptin, IL-6, creatinine, and BUN levels, and post-operative tissue samples were analyzed by H&E histochemistry and immunohistochemistry.
Results: Histology of IR/saline kidneys exhibited extensive necrosis of proximal tubular epithelial cells, as well as elevated levels of apoptosis markers and inflammation. In contrast, IR/LepA kidneys showed no signs of necrosis or inflammation, with normal IL-6 and TLR4 levels. LepA treatment led to upregulation in mRNA levels of leptin, leptin receptor, ERK1/2, STAT3, and transport molecule NHE3.
Conclusions: Local, intrarenal post-ischemic LepA treatment at reperfusion prevented apoptosis and inflammation and was reno-protective. Selective intrarenal administration of LepA at reperfusion may provide a viable option for clinical implementation.

引言：肾缺血再灌注（IR）损伤引发细胞应激，是急性肾脏损伤的主要原因。暴露于有害应激的肾细胞会诱导多效性激素脂联素的表达。鉴于此，我们先前揭示了脂联素表达在应激相关的不良作用，这些结果暗示脂联素亦参与肾脏病理重塑。脂联素的全身性功能阻碍了通过传统方法研究其局部效应。因此，我们设计了一种方法，在不影响其全身水平的情况下，局部干扰特定组织中的脂联素活性。本研究旨在探讨局部抗脂联素策略在IR损伤后的猪肾脏模型中是否具有肾脏保护作用。
方法：我们通过暴露猪肾脏于缺血和再血管化来诱导肾IR损伤。再灌注后，肾脏立即接受动脉内注射脂联素拮抗剂（LepA）或盐水溶液。采集外周血样以评估全身脂联素、IL-6、肌酐和BUN水平，并通过H&E组织化学和免疫组织化学分析术后组织样本。
结果：IR/盐水肾脏的病理学检查显示近端肾小管上皮细胞广泛坏死，以及凋亡标志物和炎症水平升高。相比之下，IR/LepA肾脏没有坏死或炎症的迹象，IL-6和TLR4水平正常。LepA治疗导致脂联素、脂联素受体、ERK1/2、STAT3和转运分子NHE3的mRNA水平上调。
结论：局部、肾脏缺血后再灌注时的LepA治疗可以防止细胞凋亡和炎症，具有肾脏保护作用。在再灌注时选择性肾脏内注射LepA可能为临床应用提供一种可行的方案。