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，清除血液中的废物以及维持体内正确的水和盐平衡非常重要。血液在肾脏中被过滤，通过过滤器的小分子不是废物，需要被肾脏重新吸收。这发生在称为近端小管（PT）的区域。PT可以回收许多类型的分子，包括通过内吞过程吸收的蛋白质。过滤后的蛋白质与受体巨蛋白结合，巨蛋白经历内化和再循环的循环，不断将蛋白质吸收到PT细胞中。除了介导蛋白质修复外，megalin还可以吸收有毒药物和重金属，导致严重的肾脏损伤。虽然我们知道megalin循环对于维持其内吞能力非常重要，但我们对这一过程如何发生缺乏清楚的了解。这可能部分是由于在培养的细胞系中研究巨蛋白运输的局限性，其不能忠实地概括在PT中观察到的巨蛋白表达的高水平或内吞途径的特征性组织。在我们的初步研究中，我们已经使用斑马鱼胚胎作为实验模型系统，以探讨在PT的megalin回收的机制。斑马鱼胚胎的肾脏与哺乳动物的肾脏在组织和功能方面都很保守。巨蛋白内吞作用在斑马鱼中也是很保守的。在我们的初步实验中，我们已经发现，OCRL 1蛋白，这是一种脂质代谢酶，和它的结合伴侣，IPIP 27 A，是所需的巨蛋白循环在PT的斑马鱼胚胎。我们还发现IPIP27A可以将OCRL 1与另一种名为Pacsin 2的蛋白质连接起来。我们假设OCRL 1，IPIP27A和Pacsin 2共同作用，介导PT中megalin的再循环。该提案将使用斑马鱼胚胎和各种已建立的方法来验证这一假设。我们的初步研究结果还表明，OCRL 1和IPIP 27 A的丢失可以影响称为溶酶体的重要降解区室，溶酶体是内吞途径的终点站，这表明再循环可以影响PT中溶酶体的形成。因此，我们将测试这种可能性，并研究其潜在的机制。我们还将研究OCRL 1，IPIP27A和Pacsin 2丢失后受损的再循环如何在基因表达水平上影响PT的整体功能。这对于确定回收对PT功能整体影响的程度非常重要。总之，这些结果将告诉我们巨蛋白再循环的生理相关过程是如何发生的，以及这如何更普遍地影响PT功能。此外，研究结果将有助于我们了解药物和重金属引起的肾毒性。将来，从这一提议中获得的知识可能会被用来开发更好的肾毒性测试或筛选可以减轻肾毒性的分子。

项目成果

Genetic Renal Diseases: The Emerging Role of Zebrafish Models.

遗传肾脏疾病：斑马鱼模型的新兴作用。

• 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