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

MUC1/HIF-1a 复合物在缺血再灌注损伤期间肾近曲小管中的作用。

基本信息

批准号：
10541885
负责人：
Mohammad Al-bataineh
金额：
$ 11.74万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-12-15 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10541885
关键词：
2-oxoglutarate 3-dioxygenase proline ARNT protein Acute Renal Failure with Renal Papillary Necrosis Adult Affect Apical Award Binding Biological Assay Cell Line Cell Nucleus Cell surface Cells Cessation of life Chimera organism Chronic Kidney Failure Clinical Co-Immunoprecipitations Complex Data Dimerization Distal Elements Epithelial Cells Epithelium Excess Mortality Financial Hardship Functional disorder Genes Genetic Genetic Transcription Glucose Goals Grant Human Hydroxylation Hypotension Hypoxia Immunofluorescence Microscopy In Vitro Incidence Injury Injury to Kidney Ischemia Kidney Knock-out Knockout Mice Limb structure Location Mediator Membrane Glycoproteins Mercuric chloride Metabolic dysfunction Metabolism Morbidity - disease rate Morphology Mucin 1 protein Mus Names Nephrons Nuclear Oxygen Pathway interactions Patients Phosphorylation Play Proteins Proximal Kidney Tubules Publishing Pyruvate Kinase Recovery Regulation Renal function Reperfusion Injury Reporting Research Research Personnel Resistance Role Sepsis Severities Signal Transduction Surface Testing Therapeutic Intervention Thick Tissues Transactivation Transcriptional Activation Transfection Tubular formation Work beta catenin career congenic coping experimental study hypoxia inducible factor 1 inhibitor ischemic injury kidney cell mortality mouse model mutant novel novel therapeutics pancreatic cancer cells prevent renal damage repaired response therapy design trafficking transcription factor urinary

项目摘要

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.

项目摘要。急性肾损伤（AKI）是一种常见且具有破坏性的临床问题。尽管有发病率，死亡率和财务流失相关，除了透析治疗之外没有成熟的治疗方法。最近的工作已经确定了肾小管上皮细胞应对缺血再灌注损伤（IRI）的适应性反应。迫切需要确定 AKI 中适应性反应的调节方式，以便治疗干预措施预防和治疗 AKI 的发生。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缺氧损伤的适应性反应的最重要的介质。有新的证据肾上皮细胞顶端表面表达的跨膜糖蛋白粘蛋白 1 (MUC1) 发挥着重要作用在增强 HIF-1 活性方面具有新颖且重要的作用。我们有强有力的初步数据表明遗传从小鼠肾小管上皮细胞中删除 Muc1 会加剧损伤，尤其是近端肾小管 (PT) 来自 IRI，限制适应性 HIF-1 反应，改变肾小管代谢，并抑制恢复。这本申请的中心假设是 MUC1 在 IRI 期间通过 HIF-1 的反式激活来保护肾脏特别是在恢复中的近端小管中的适应性反应。具体目标是： 1. 确定 IRI 期间 MUC1 对肾脏的保护是否需要 MUC1 依赖性 HIF-1a 核 HIF-1 通路的靶向和随后的转录激活，特别是在恢复过程中 PT。我将检验以下假设：MUC1 在 IRI 期间通过 HIF-1 适应性反式激活来保护肾脏恢复中的 PT 中的反应。建议进行实验以稳定 Muc1 近端小管中的 HIF-1a 通过引入 VHL 的 PT KO，并用载体或特定 MUC1 抑制剂治疗，在 IRI 期间 KO 小鼠阻断 MUC1 内化和核递送。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 途径的转导是否至关重要。 2. 表征培养肾细胞中的 MUC1-HIF-1a 复合物。我将检验 MUC1 的假设通过直接结合稳定 HIF-1a。这里建议进行实验来表征 MUC1-HIF-1a 复合物在原代 PT 细胞和人肾细胞系中使用体外 Pull-down 测定和 co-IP 研究类型和突变体构建体，并确定 MUC1 与 HIF-1a 的结合是否会导致其转运至细胞核。我的研究目标是全面表征 MUC1-HIF-1a 相互作用并确定其机制 IRI 期间其对肾脏 PT 的保护作用。该信息可用于设计诱导 MUC1 的疗法并限制 AKI 的严重程度。该奖项将为我提供额外的支持来生成初步数据获得 R01 级别的资助，以进一步研究肾脏 MUC1 水平的增加是否可以提供对 IRI 的抵抗力。