The functional kidney filter is likely created by the capillary endothelium, the glomerular basement membrane, and the "slit diaphragm" or specialized intercellular junction that lies between the interdigitating foot processes of the visceral epithelial cells (podocyte). Glomerular diseases including diabetic nephropathy present with protein leak (proteinuria) in the urine. Several podocyte specific gene mutations including NPHS1 and NPHS2 that result in proteinuric renal diseases have placed the emphasis on podocyte as primary component of the filtration barrier and important for normal glomerular function. The most widely accepted model is that the glomerular filtration barrier functions as a series of sieves with increasing size selectivity. It is not clear, however, why the filter does no routinely clog with large proteins that enter the glomerular basement membrane. Recent investigations have suggested that the podocytes are very active in the process of endocytosis that routinely remove proteins like immunoglobulin that accumulate at the filtrations barrier. Podocytes express FcRn receptor which is reported as an albumin and immunoglobulin transporter. Mice lacking the FcRn receptor accumulated IgG as they aged and showed delayed clearance of IgG in tracer studies. In a more recent study, podocyte uptake of Evans Blue labeled albumin was observed in puromycin aminonucleoside injury model. Podocyte vesicles have often been observed in TEM studies on kidney biopsies from patients with nephrotic syndrome. These studies support the idea that podocytes play an active role in removing proteins from the GBM and suggest that genetic or acquired impairment (overwhelmed by the amount of protein leak) of the clearance machinery is likely to be a common mechanism promoting glomerular diseases. To better understand the role of endocytosis in maintaining the filtration barrier we generated a mouse model to disrupt the endocytic activity in the podocytes. Vps34 (PI3K class III) is member of the phosphoinositides 3-kinsae (PI3K) family of lipid kinases and is important in regulation of vesicular trafficking in the endosomal/lysosomal system. As anticipated, mice deleted of vps34 in a podocyte specific manner develop early proteinuria and large vacuoles are visible in the podocytes. Using this model we would like to further investigate the role of endocytosis in podocyte homeostasis. Based on the preliminary work this project we will focus on several aspects of this mechanism. Specifically we will: (1) Analyze the effect of podocyte specific deletion of vsp34 in vivo. We will analyze the characteristics of the vacuoles observed in the podocytes. We will also analyze the effect of vps34 deletion in an inducible model. (2) Using this model system we will investigate the role of podocyte in endocytosis of proteins that accumulate at the slit diaphragm. We anticipate by disrupting the cellular endocytosis/vesicular trafficking processes will enable us to visualize basal levels of ongoing endocytosis that enable the podocytes to maintain a clean fitler.
Glomerular diseases are a major cause of morbidity, mortality, and public health expenditure, accounting for over 55% of patients starting hemodialysis in the United States. Our treatments for patients with glomerular disease to slow or prevent progression of disease are not sufficiently effective, and do not precisely target the causes of glomerular disease. A major component of the glomeruli, the podocyte are essential for the proper function of the kidney filter. Injury to the podocyte result in protein leak which i a major clinical sign of the presence of glomerular disease and predicts outcome based on its severity. This proposal seeks to understand the mechanisms by which podocyte health is affected by proteinuria. Recent investigations have suggested that podocytes take up the leaked proteins, a process that might be detrimental to their overall health. This proposal is relevant to the goals of the NIDDK to promote research on basic kidney biology, including applications within Cell and Molecular Biology of the Kidney (protein trafficking and cell polarity) and within the Pathobiology of Kidney Disease (normal biology of glomerular cells, and proteinuria).
|Venkatareddy, Madhusudan; Verma, Rakesh; Kalinowski, Anne et al. (2016) Distinct Requirements for Vacuolar Protein Sorting 34 Downstream Effector Phosphatidylinositol 3-Phosphate 5-Kinase in Podocytes Versus Proximal Tubular Cells. J Am Soc Nephrol 27:2702-19|
|Verma, Rakesh; Venkatareddy, Madhusudan; Kalinowski, Anne et al. (2016) Shp2 Associates with and Enhances Nephrin Tyrosine Phosphorylation and Is Necessary for Foot Process Spreading in Mouse Models of Podocyte Injury. Mol Cell Biol 36:596-614|
|Verma, Rakesh; Venkatareddy, Madhusudan; Kalinowski, Anne et al. (2016) Integrin Ligation Results in Nephrin Tyrosine Phosphorylation In Vitro. PLoS One 11:e0148906|
|Ikonomov, Ognian C; Sbrissa, Diego; Venkatareddy, Madhusudan et al. (2015) Class III PI 3-kinase is the main source of PtdIns3P substrate and membrane recruitment signal for PIKfyve constitutive function in podocyte endomembrane homeostasis. Biochim Biophys Acta 1853:1240-50|