Mechanisms of endocytic recycling in the renal proximal tubule

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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.