The slit diaphragm is essential for normal glomerular filtration. Mutations in the slit diaphragm protein podocin are associated with steroid-resistant nephrotic syndrome which is characterized by early childhood onset of proteinuria, rapid progression to end-stage renal disease and focal segmental glomerulosclerosis [1-3]. Although it is known that podocin localizes to the slit diaphragm and that it is crucial for the filtration process, our understanding of the actual role of this protein remains poor. Podocin is thought to be a scaffolding protein, essential for the correct recruitment of other proteins, including nephrin, to the slit diaphragm. In disease and in the presence of nephrotic plasma, podocin localizes incorrectly resulting in defective signalling from the slit diaphragm [4-6]. Increasing our knowledge of the regulation, trafficking and interactions of podocin and how these can be impaired is essential for our understanding of the disease state.
We have a number of unique and well characterised podocyte cell lines from both normal glomeruli and glomeruli from kidneys removed because of congenital nephrotic syndrome. These, coupled with state-of-the-art cell imaging and proteomic facilities available within the university, will be used to identify the role podocin plays within the podocyte and to understand how disease-causing mutations affect this. This will include: (1) using super resolution (STED) and total internal reflection (TIRF) microscopy in living single cells to study (a) the dynamics of podocin trafficking (using GFP) in both normal and diseased states and (b) podocin interactions with known partners e.g. nephrin (using two colour imaging), and (2) using proteomic approaches to identify and characterise novel interacting partners. The resulting information will lead to improved understanding of glomerular filtration barrier function and pathophysiology.
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1 Boute, N., Gribouval, O., Roselli, S., Benessy, F., Lee, H., Fuchshuber, A., Dahan, K., Gubler, M. C., Niaudet, P. and Antignac, C. (2000) NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome. Nature genetics. 24, 349-354
2 Tsukaguchi, H., Sudhakar, A., Le, T. C., Nguyen, T., Yao, J., Schwimmer, J. A., Schachter, A. D., Poch, E., Abreu, P. F., Appel, G. B., Pereira, A. B., Kalluri, R. and Pollak, M. R. (2002) NPHS2 mutations in late-onset focal segmental glomerulosclerosis: R229Q is a common disease-associated allele. The Journal of clinical investigation. 110, 1659-1666
3 Pereira, A. C., Pereira, A. B., Mota, G. F., Cunha, R. S., Herkenhoff, F. L., Pollak, M. R., Mill, J. G. and Krieger, J. E. (2004) NPHS2 R229Q functional variant is associated with microalbuminuria in the general population. Kidney international. 65, 1026-1030
4 Huber, T. B., Schermer, B. and Benzing, T. (2007) Podocin organizes ion channel-lipid supercomplexes: implications for mechanosensation at the slit diaphragm. Nephron. Experimental nephrology. 106, e27-31
5 Schermer, B. and Benzing, T. (2009) Lipid-protein interactions along the slit diaphragm of podocytes. Journal of the American Society of Nephrology : JASN. 20, 473-478
6 Huber, T. B., Simons, M., Hartleben, B., Sernetz, L., Schmidts, M., Gundlach, E., Saleem, M. A., Walz, G. and Benzing, T. (2003) Molecular basis of the functional podocin-nephrin complex: mutations in the NPHS2 gene disrupt nephrin targeting to lipid raft microdomains. Human molecular genetics. 12, 3397-3405