Role of the MUC1/HIF-1a Complex in the kidney proximal tubule during ischemia-reperfusion injury.

项目摘要。急性肾损伤(AKI)是一种常见且严重的临床问题。尽管发病率很高， 死亡率，以及相关的经济损失，除了透析治疗之外，还没有既定的治疗方法。近期 研究发现了肾小管上皮细胞对缺血再灌注损伤(IRI)的适应性反应。 迫切需要确定AKI中适应性反应是如何调节的，以便治疗性干预 预防和治疗AKI是可以开发的。缺氧诱导因子-1(HIF-1)是一种被认为是转录因子 作为细胞对低氧侮辱适应性反应的最重要的介体。有新的证据表明 肾上皮细胞顶面表达的跨膜糖蛋白粘蛋白1在肾小管上皮细胞中的作用 在增强HIF-1活性方面的新的重要作用。我们有强有力的初步数据表明基因 小鼠肾小管上皮细胞MUC1缺失加重损伤，尤其是近曲小管 通过IRI，限制适应性HIF-1反应，改变肾小管代谢，抑制恢复。这个 这一应用的中心假设是MUC1在IRI期间通过反式激活HIF-1来保护肾脏 适应性反应特别是在恢复中的近端小管。具体目标是： 1.确定在IRI期间MUC1对肾脏的保护是否需要依赖MUC1的HIF-1a核 HIF-1通路在恢复期的靶向和随后的转录激活 PT.我将测试MUC1在IRI期间通过反式激活HIF-1适应性信号来保护肾脏的假设 在恢复的PT中的反应。建议进行实验以稳定MUC1近端小管中的HIF-1a KO小鼠在IRI期间通过引入VHL的PT KO，并用赋形剂或特异性MUC1抑制剂治疗 阻断MUC1内化和核传递。这些研究的结果将揭示MUC1是否简单地稳定下来 HIF-1a水平，或者MUC1核靶向对于保护性HIF-1途径的转导是必不可少的。 2.鉴定MUC1-HIF-1a复合体在培养肾细胞中的表达。我将测试MUC1的假设 通过直接结合稳定HIF-1a。这里提出了实验来表征MUC1-HIF-1a络合物 用体外下拉试验和共IP研究原代PT细胞和人肾细胞系 类型和突变结构，并确定MUC1与HIF-1a结合是否导致其转运到细胞核。 本研究的目的是充分表征MUC1-HIF-1a的相互作用，并确定MUC1-HIF-1a的机制 它在IRI期间对肾脏PT的保护作用。这一信息可用于设计诱发MUC1的治疗方法 并限制AKI的严重程度。这一奖项将为我提供额外的支持，以生成初步数据 R01水平的拨款，以进一步调查肾脏MUC1水平的增加是否会对IRI产生抵抗力。

Project Abstract. Acute kidney injury (AKI) is a common and devastating clinical problem. Despite the morbidity, mortality, and financial drain associated, there are no established therapies outside of dialytic treatment. Recent work has identified adaptive responses in the tubular epithelium to cope with ischemia-reperfusion injury (IRI). There is a critical need to identify how adaptive responses are regulated in AKI so that therapeutic interventions to prevent and treat AKI can be developed. Hypoxia inducible factor-1 (HIF-1) is a transcription factor regarded as the most significant mediator of cellular adaptive responses to hypoxic insult. There is emerging evidence that the transmembrane glycoprotein mucin 1 (MUC1) expressed on the apical surface of kidney epithelia plays a novel and important role in enhancing HIF-1 activity. We have strong preliminary data suggesting that genetic deletion of Muc1 from mouse tubular epithelial cells exacerbates damage especially in the proximal tubule (PT) from IRI, limits adaptive HIF-1 responses, alters tubular metabolism, and inhibits recovery. The central hypothesis of this application is that MUC1 protects the kidney during IRI by transactivation of the HIF-1 adaptive response specifically in the recovering proximal tubule. The specific aims are: 1. To determine if MUC1 protection of the kidney during IRI requires MUC1-dependent HIF-1a nuclear targeting and subsequently transcriptional activation of the HIF-1 pathway specifically in the recovering PT. I will test the hypothesis that MUC1 protects the kidney during IRI by transactivation of the HIF-1 adaptive response in the recovering PT. Experiments are proposed to stabilize HIF-1a in the proximal tubule in the Muc1 KO mice during IRI by introducing a PT KO of VHL, and treatment with vehicle or a specific MUC1 inhibitor to block MUC1 internalization and nuclear delivery. Results of these studies will reveal if MUC1 simply stabilizes HIF-1a levels, or if MUC1 nuclear targeting is essential for transduction of the protective HIF-1 pathway. 2. To characterize the MUC1-HIF-1a complex in cultured kidney cells. I will test the hypothesis that MUC1 stabilizes HIF-1a by direct binding. Experiments are proposed here to characterize the MUC1-HIF-1a complex using both in vitro pull-down assays and co-IP studies in primary PT cells and human kidney cell lines with wild type and mutant constructs, and to determine if MUC1 binding to HIF-1a results in its trafficking to the nucleus. The goal of my research is to fully characterize the MUC1-HIF-1a interaction and determine the mechanism of its protective role in the kidney PT during IRI. This information can be used to design therapies to induce MUC1 and limit the severity of AKI. This award will provide me with additional support to generate preliminary data for an R01-level grant to further investigate whether increased levels of renal MUC1 provide resistance to IRI.