Mechanisms of endocytic recycling in the renal proximal tubule

肾脏对保持健康、清除血液中的废物以及保持体内正确的水盐平衡极其重要。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。这发生在一个叫做近端小管(PT)的区域。PT可以回收许多类型的分子，包括内吞过程中摄取的蛋白质。过滤后的蛋白质与受体megalin结合，后者经历内化和循环的循环，不断将蛋白质吸收到PT细胞中。除了介导蛋白质回收外，megalin还可以吸收有毒药物和重金属，导致严重的肾脏损害。虽然我们知道巨蛋白回收对于维持其内吞能力非常重要，但我们对这一过程是如何发生的缺乏清楚的了解。这在一定程度上可能是由于研究巨蛋白在培养细胞系中运输的局限性，这些细胞系不能真实地概括PT中观察到的巨蛋白的高水平表达或内吞途径的特征组织。在我们的初步研究中，我们使用斑马鱼胚胎作为实验模型系统来研究巨蛋白在PT中的循环机制。斑马鱼胚胎的肾脏与哺乳动物的肾脏在组织和功能上都保存得很好。巨蛋白内吞作用在斑马鱼中也很保守。在我们的初步实验中，我们发现OCRL1蛋白是斑马鱼胚胎PT中巨蛋白循环所必需的，OCRL1蛋白是一种脂质代谢酶，它的结合伙伴IPIP27A是巨蛋白循环所必需的。我们还发现，IP27A可以将OCRL1与另一种名为Pacsin2的蛋白质联系起来。我们假设OCRL1、IPIP27A和Pacsin2共同作用于调节PT中megalin的循环。该提案将使用斑马鱼胚胎和各种既定的方法来验证这一假设。我们的初步结果还表明，OCRL1和IPIP27A的丢失可以影响一个重要的降解间隔，称为溶酶体，它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，并调查潜在的机制。我们还将研究OCRL1、IPIP27A和Pacsin2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整体功能的影响程度将是很重要的。综上所述，这些结果将告诉我们巨蛋白循环的生理相关过程是如何发生的，以及这如何对PT功能产生更广泛的影响。此外，这些发现将与我们对药物和重金属引起的肾脏毒性的理解有关。在未来，从这项建议中获得的知识可能会被用来开发更好的肾脏毒性测试，或者筛选可以减轻肾脏毒性的分子。

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.