During the last 25 years our research has been directed toward understanding the cellular and molecular mechanisms of glomerular permeability and protein absorption as well as understanding the derangements in these processes that occur in glomerular diseases. Major findings of the previous renewal period were: 1) Demonstration that the adaptor protein ARH facilitates endocytosis and recycling of megalin;2) Discovery that ARH associates with centrosomal proteins and dynein;3) Identification of novel components of the nephrin multiprotein complex, including IQGAP, MAGI- 2, CASK, and spectrins;4) Demonstration that podocalyxin expression activates RhoA and induces actin reorganization;5) Demonstration that podocalyxin can bind directly to ezrin and is hyperphosphorylated in PAN nephrosis;6) Discovery of GIPN, a putative E3 ubiquitin ligase that interacts with RGS-GAIP and promotes downregulation of Gai3. The studies proposed in this application represent a direct continuation of our ongoing work on characterization of the slit diaphragm proteins, role of actin and actin binding proteins in regulating foot process organization, and the role of ARH and megalin in protein uptake.
Our specific aims are: 1) To isolate and characterize the glomerular junctional complexes from normal and PAN nephrotic rats;2) To investigate and regulation of the foot process organization through its interaction with actin. We will explore the possibility that GIV expression can serve as a marker for glomerular injury in proteinuria involving changes in the unique foot process organization of the podocyte;and 3) To further define the functions of the adaptor protein ARH in megalin's uptake of filtered proteins in the podocyte and proximal tubule cell as well as to explore its role in connecting endocytosis to centrosomal functions. In this work we will use a combination of morphological (EM, immunofluorescence), biochemical, molecular biological and proteomics approaches on glomeruli and cultured podocytes as well as live cell imaging on mouse podocytes and proximal tubule cells in culture. It is our hope and expectation that these studies will continue to provide new insights into our understanding of the cellular and molecular mechanisms of glomerular filtration, glomerular injury, protein absorption, and their alterations in glomerular diseases associated with proteinuria.

Public Health Relevance

Under this grant we have discovered and characterized a number of new proteins present on the podocyte that are important in normal glomerular permeability and in the pathogenesis of glomerular diseases associated with proteinuria, including minimal change disease, membranous glomerulonephritis and focal glomerulosclerosis. The studies planned can be expected to shed light not only on how normal podocytes function, but also they should provide key insights into the molecular basis of the signaling that directs changes in the actin cytoskeleton and organization of the foot processes of podocytes that occur in diseases associated with proteinuria and in the survival pathways for podocytes and proximal tubule cells in response to glomerular injury that lead to proteinuria.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Method to Extend Research in Time (MERIT) Award (R37)
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Pathobiology of Kidney Disease Study Section (PBKD)
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Ketchum, Christian J
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University of California San Diego
Other Basic Sciences
Schools of Medicine
La Jolla
United States
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Shah, Mehul; Baterina Jr, Oscar Y; Taupin, Vanessa et al. (2013) ARH directs megalin to the endocytic recycling compartment to regulate its proteolysis and gene expression. J Cell Biol 202:113-27
Fukasawa, Hirotaka; Obayashi, Hiroaki; Schmieder, Sandra et al. (2011) Phosphorylation of podocalyxin (Ser415) Prevents RhoA and ezrin activation and disrupts its interaction with the actin cytoskeleton. Am J Pathol 179:2254-65
Fukasawa, Hirotaka; Bornheimer, Scott; Kudlicka, Krystyna et al. (2009) Slit diaphragms contain tight junction proteins. J Am Soc Nephrol 20:1491-503
Lehtonen, Sanna; Shah, Mehul; Nielsen, Rikke et al. (2008) The endocytic adaptor protein ARH associates with motor and centrosomal proteins and is involved in centrosome assembly and cytokinesis. Mol Biol Cell 19:2949-61
Green, Ryan S; Stone, Erica L; Tenno, Mari et al. (2007) Mammalian N-glycan branching protects against innate immune self-recognition and inflammation in autoimmune disease pathogenesis. Immunity 27:308-20
Lehtonen, Sanna; Lehtonen, Eero; Kudlicka, Krystyna et al. (2004) Nephrin forms a complex with adherens junction proteins and CASK in podocytes and in Madin-Darby canine kidney cells expressing nephrin. Am J Pathol 165:923-36