Mechanisms of endocytic recycling in the renal proximal tubule

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.

An important function of the proximal tubule (PT) is the retrieval of low molecular weight proteins from the renal filtrate. Consequently, the PT has an extremely high rate of endocytosis. The major endocytic receptor in the PT is megalin. Megalin-dependent endocytosis is important for normal physiology and human health, and is also a major route for uptake of nephrotoxic drugs and heavy metals. The molecular mechanisms of megalin internalization are well described, but how the receptor undergoes recycling is very poorly understood. We have used zebrafish, which is an excellent model for investigating kidney function and renal endocytosis, to investigate the process of megalin recycling in vivo. Our preliminary results indicate that the inositol 5-phosphatase OCRL1 and its binding partner IPIP27A are required for efficient megalin recycling in the PT of zebrafish embryos. We have also found that IPIP27A can link OCRL1 to the F-BAR protein Pacsin2, leading to the hypothesis that OCRL1, IPIP27A and Pacsin2 act together to facilitate megalin recycling in the PT. This proposal will test this hypothesis using zebrafish embryos and established experimental approaches. We aim to decipher the mechanisms by which these proteins function in recycling, and to identify additional associated machinery that also functions in this process. Our preliminary results also indicate that loss of OCRL1 and IPIP27A can impact upon lysosome abundance. We will therefore investigate the mechanisms by which loss of OCRL1 and IPIP27A affect lysosome biogenesis and turnover. Finally, using transcriptomics, we will investigate how impairment of endocytic recycling affects PT function as a whole. Together, the results will provide new insight into how megalin recycling occurs, and how this process impacts upon the PT homeostasis and function more generally. The findings will be relevant to our understanding of the endocytic pathway, renal physiology and nephrotoxicity.

Wherever possible we will try to maximise the impact of our research. We will adopt several strategies to achieve this, as indicated below.

Beneficiaries of the research and potential exploitation
The scientific data obtained will increase the knowledge of UK and international scientists in the academic, clinical and commercial sectors. This contribution to knowledge will lead to improved understanding of renal physiology and pathology that in turn may be exploited by a number of interested parties. The development of robust zebrafish based methods for measuring renal endocytosis and proximal tubular function is likely to be of interest to the pharmaceutical industry, for whom drug-induced nephrotoxicity is major problem. Zebrafish reporter strains may be used to screen for drug toxicity, or alternatively, may be used to screen for compounds that can restore renal tubular function in the disease state, or that can ameliorate drug-induced toxicity. Similarly, zebrafish reporter strains may be used to screen for heavy metal toxicity or compounds that can ameliorate such toxicity. The findings therefore have potential application in a number of areas including in the pharmaceutical and aquaculture industries. Ways to exploit our findings will be explored with UMIP (University of Manchester Intellectual Property).

NC3Rs
Given the suitability of zebrafish to study kidney function it may be used as a replacement for mice or rats, which are typically used in academia and the pharmaceutical industry to study the physiology and pathology of this organ. In the current proposal we will use zebrafish as our model of choice, which combine a high degree of conservation of renal function with mammals with increased tractability for the types of experiments we propose. The replacement of mice or rats with zebrafish can be considered a refinement as fish have lower neurological complexity than mice or rats.

Public engagement
We will communicate our findings to the public via the open access University website and through the Faculty Research brochure. We will also notify the dedicated Faculty press officer of our findings at around the point of publication in academic journals. The press officer will then contact local and national news agencies, and prepare press releases that these agencies can use. We will also contact appropriate charities including the Lowe Syndrome Trust in the UK and the Lowe Syndrome Association in the USA. ML has existing links with the LST, and findings arising from the work will be communicated to the Charity through personal contact. The Lowe laboratory has participated in several public engagement initiatives and we plan for this to continue. The lab has hosted two A-level students at different times as part of the Nuffield bursary scheme. The Faculty of Life Sciences continues to participate in the Nuffield scheme and we envisage hosting another A-level student should the current application be successful. Members of the Lowe lab have participated in several public engagement exercises involving visiting schoolchildren, hosted at the Manchester Museum with whom our Faculty has close links. Members of the Lowe lab will continue to participate in these exercises in the future.

Training
The technical and intellectual knowledge acquired by the post-doctoral researcher during the course of the proposed work will equip these people to pursue a career in science or science-related discipline. The researcher will also learn transferable skills that will increase their employability. Such a highly trained individual will contribute to the UK knowledge and skill base and ultimately the UK economy.