Mechanisms of endocytic recycling in the renal proximal tubule

肾近曲小管内吞再循环机制

• 批准号: BB/N000641/1
• 负责人: Martin Lowe
• 金额: $ 49.23万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FN000641%2F1
• 关键词: Mechanisms; endocytic; recycling; renal; proximal

The kidney is extremely important for maintaining health, removing waste products from the blood and maintaining correct water and salt balance in the body. Blood is filtered in the kidney and small molecules that pass through the filter which are not waste need to be reabsorbed by the kidney. This occurs in a region called the proximal tubule (PT). The PT can retrieve many types of molecules, including proteins that are taken up by the process of endocytosis. Filtered proteins bind to the receptor megalin, which undergoes cycles of internalization and recycling to continually take up proteins into the PT cells. In addition to mediating protein retrieval, megalin can also take up toxic drugs and heavy metals, resulting in severe kidney damage. Although we know megalin recycling is very important for maintaining its endocytic capability, we lack a clear understanding of how this process occurs. This may in part be due to the limitations of studying megalin trafficking in cultured cell lines, which do not faithfully recapitulate the high levels of megalin expression or the characteristic organization of the endocytic pathway that are observed in the PT. In our preliminary studies, we have used the zebrafish embryo as an experimental model system to investigate the mechanisms of megalin recycling in the PT. The kidney of zebrafish embryos is well conserved with that of mammals, both in terms of its organization and functions. Megalin endocytosis is also well conserved in zebrafish. In our preliminary experiments we have found that the OCRL1 protein, which is a lipid-metabolizing enzyme, and its binding partner, IPIP27A, are required for megalin recycling in the PT of zebrafish embryos. We have also found that IPIP27A can link OCRL1 with another protein called Pacsin2. We hypothesize that OCRL1, IPIP27A and Pacsin2 act together to mediate recycling of megalin in the PT. The proposal will test this hypothesis, using zebrafish embryos and a variety of established methods. Our preliminary results have also shown that loss of OCRL1 and IPIP27A can affect an important degradative compartment called the lysosome, which is the terminal station of the endocytic pathway, suggesting that recycling can influence lysosome formation in the PT. We will therefore test this possibility, and investigate the underlying mechanisms. We will also investigate how impaired recycling upon loss of OCRL1, IPIP27A and Pacsin2 influences the global functions of the PT at the level of gene expression. This will be important for determining the extent to which recycling can impact PT function as a whole. In summary, the results will inform us of how the physiologically relevant process of megalin recycling occurs, and how this can impact upon PT function more generally. Moreover, the findings will be relevant to our understanding of drug and heavy metal induced kidney toxicity. In the future the knowledge gained from this proposal may be exploited to develop better tests for kidney toxicity or to screen for molecules that can alleviate kidney toxicity.

肾脏对于维持健康，从血液中去除废物并保持体内的水和盐平衡非常重要。在肾脏中过滤血液，通过过滤器的小分子需要被肾脏重新吸收。这发生在称为近端小管（PT）的区域中。 PT可以检索许多类型的分子，包括由内吞作用过程所吸收的蛋白质。过滤后的蛋白质与受体梅加林结合，该受体经历了内在化的周期，并回收以不断将蛋白质吸收到PT细胞中。除了介导蛋白质检索外，梅加林还可以吸收有毒药物和重金属，从而导致严重的肾脏损伤。尽管我们知道Megalin回收对于保持其内吞能力非常重要，但我们对这一过程的发生缺乏明确的了解。这可能部分是由于在培养的细胞系中研究巨林运输的局限性，这些细胞系中没有忠实地概括高水平的梅加林表达或在PT中观察到的内吞途径的特征组织。在我们的初步研究中，我们使用斑马鱼胚作为实验模型系统来研究PT中Megalin回收的机制。斑马鱼胚胎的肾脏在其组织和功能方面都具有哺乳动物的肾脏。斑马鱼内吞作用在斑马鱼中也有充分的保守。在我们的初步实验中，我们发现脂质代谢酶的OCRL1蛋白及其结合伴侣IPIP27A需要在斑马鱼胚胎的PT中进行梅加林回收。我们还发现，IPIP27A可以将OCRL1与另一种称为PACSIN2的蛋白联系起来。我们假设OCRL1，IPIP27A和PACSIN2一起起作用以介导PT中的Megalin的回收利用。该提案将使用斑马鱼胚胎和各种既定方法检验该假设。我们的初步结果还表明，OCRL1和IPIP27A的丧失会影响一个称为溶酶体的重要降解室，这是内吞途径的末端站，这表明回收可以影响PT中的溶酶体形成。因此，我们将测试这种可能性，并研究潜在的机制。我们还将研究OCRL1，IPIP27A和PACSIN2损失后，回收的损害如何影响PT在基因表达水平上的全局功能。这对于确定回收利用会影响PT整体功能的程度很重要。总而言之，结果将为我们告知我们如何发生梅加林回收的生理相关过程，以及这如何更普遍地影响PT功能。此外，这些发现将与我们对药物和重金属诱导肾脏毒性的理解有关。将来，可以利用从该提案中获得的知识来开发更好的肾脏毒性测试或筛选可以减轻肾脏毒性的分子。

项目成果

Genetic Renal Diseases: The Emerging Role of Zebrafish Models.

• DOI: 10.3390/cells7090130
• 发表时间: 2018-09-01
• 期刊: Cells
• 影响因子: 6
• 作者: Elmonem MA;Berlingerio SP;van den Heuvel LP;de Witte PA;Lowe M;Levtchenko EN
• 通讯作者: Levtchenko EN

Cystinosis (ctns) zebrafish mutant shows pronephric glomerular and tubular dysfunction.

• DOI: 10.1038/srep42583
• 发表时间: 2017-02-15
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Elmonem MA;Khalil R;Khodaparast L;Khodaparast L;Arcolino FO;Morgan J;Pastore A;Tylzanowski P;Ny A;Lowe M;de Witte PA;Baelde HJ;van den Heuvel LP;Levtchenko E
• 通讯作者: Levtchenko E

Cell-Based Phenotypic Drug Screening Identifies Luteolin as Candidate Therapeutic for Nephropathic Cystinosis

• DOI: 10.1681/asn.2019090956
• 发表时间: 2020-07-01
• 期刊: JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY
• 影响因子: 13.6
• 作者: De Leo, Ester;Elmonem, Mohamed A.;Rega, Laura Rita
• 通讯作者: Rega, Laura Rita