HIF orchestrated metabolic shift confers protection against Acute Kidney Injury (AKI)

Renal hypoxia is widespread in acute kidney injury (AKI) of various aetiologies. Hypoxia adaptation, conferred through the hypoxia-inducible factor (HIF), appears to be insufficient. Here we show that HIF activation in renal tubules through Pax8-rtTA-based inducible knockout of von Hippel-Lindau protein (VHL-KO) protects from rhabdomyolysis-induced AKI. In this model, histological observations indicate that injury mainly affects proximal convoluted tubules, with 5% necrosis at d1 and 40% necrosis at d2. HIF-1alpha up-regulation in distal tubules reflects renal hypoxia. However, lack of HIF in proximal tubules suggests insufficient adaptation by HIF. AKI in VHL-KO mice leads to prominent HIF activation in all nephron segments, as well as to reduced serum creatinine, serum urea, tubular necrosis, and apoptosis marker caspase-3 protein. At d1 after rhabdomyolysis, when tubular injury is potentially reversible, HIF mediated protection in AKI is associated with activated glycolysis, cellular glucose uptake and utilization, autophagy, vasodilation, and proton removal as demonstrated by qPCR, pathway enrichment analysis and immunohistochemistry. Together, our data provide evidence for a HIF-orchestrated multi-level shift towards glycolysis as a major mechanism for protection against acute tubular injury. All experiments were carried out in transgenic mice in which selective renal tubular VHL knockout (VHL-KO) was inducible by doxycycline (Reference: Mathia S, Paliege A, Koesters R, Peters H, Neumayer HH, Bachmann S, Rosenberger C. Action of hypoxia-inducible factor in liver and kidney from mice with Pax8-rtTA-based deletion of von Hippel-Lindau protein. Acta Physiol (Oxf). 2013; 207(3):565-76.). Four groups of animals were used: 1) controls: untreated mice; 2) VHL-KO: injected with doxycycline (0.1 mg per 10 g body weight SC), 4 days prior to sacrifice; 3) AKI: rhabdomyolysis; 4) VHL-KO/AKI: doxycycline plus rhabdomyolysis. To induce AKI, 50% glycerol (0.05 ml per 10 g body weight) was injected IM into the left hind limb under isoflurane narcosis. Drinking water was withdrawn between 20 h prior and 24 h after glycerol injection.

多种病因所致急性肾损伤（acute kidney injury, AKI）中，肾脏缺氧普遍存在。由缺氧诱导因子（hypoxia-inducible factor, HIF）介导的缺氧适应似乎并不充分。本研究证实，通过基于Pax8-rtTA的诱导性敲除希佩尔-林道蛋白（von Hippel-Lindau protein, VHL）以激活肾小管内的HIF，可对横纹肌溶解（rhabdomyolysis）诱导的AKI起到保护作用。
在该模型中，组织学观察显示损伤主要累及近端曲小管（proximal convoluted tubules），造模后第1天坏死率为5%，第2天坏死率达40%。远端小管（distal tubules）内HIF-1α上调反映了肾脏缺氧状态，但近端小管内HIF的缺失则提示HIF介导的适应不足。
VHL敲除（VHL-KO）小鼠的AKI可导致所有肾单位节段出现显著的HIF激活，同时可降低血清肌酐（serum creatinine）、血清尿素（serum urea）水平，减轻肾小管坏死及凋亡标志物半胱天冬酶-3（caspase-3）的蛋白表达。
在横纹肌溶解造模后第1天，此时肾小管损伤尚处于可逆阶段，HIF介导的AKI保护作用与糖酵解（glycolysis）激活、细胞葡萄糖摄取与利用、自噬（autophagy）、血管舒张（vasodilation）以及质子清除相关，该结论通过定量聚合酶链反应（quantitative polymerase chain reaction, qPCR）、通路富集分析及免疫组织化学（immunohistochemistry）得以证实。
综上，本研究数据证实，HIF统筹下的多层面糖酵解转向是对抗急性肾小管损伤的核心保护机制。所有实验均在转基因小鼠（transgenic mice）中开展，该小鼠可通过多西环素（doxycycline）诱导实现肾小管特异性VHL敲除（VHL-KO）（参考文献：Mathia S, Paliege A, Koesters R, Peters H, Neumayer HH, Bachmann S, Rosenberger C. 缺氧诱导因子在Pax8-rtTA介导的希佩尔-林道蛋白缺失小鼠肝肾中的作用. 牛津生理学学报. 2013; 207(3):565-76.）
本研究共设置4组实验动物：1）对照组：未作任何处理的正常小鼠；2）VHL-KO组：于处死前4天经皮下注射（subcutaneous, SC）多西环素（0.1 mg/10 g体重）；3）AKI组：构建横纹肌溶解模型；4）VHL-KO/AKI组：给予多西环素干预联合横纹肌溶解造模。
构建AKI模型时，于异氟烷（isoflurane）麻醉下向小鼠左侧后肢肌内注射（intramuscular, IM）50%甘油（0.05 ml/10 g体重），并于甘油注射前20小时至注射后24小时期间禁水。