Glomerular visceral epithelial cells play a central role in maintaining the filtration barrier of the renal glomerulus. These cells are also called podocytes to describe the foot-like appearance of numerous interdigitating processes that arise from their cell bodies and surround glomerular capillary walls. In response to glomerular injury, podocytes undergo a dramatic change in morphology termed "foot process effacement", a process which is invariably associated with proteinuria, and which results from retraction and spreading of foot processes due to an alteration in podocyte cytoskeletal and intercellular junctional architecture. The goal of the project proposed herein is to contribute to understanding the cellular and molecular mechanisms that govern the dynamics of podocyte morphology and filter integrity with the expectation that providing a mechanistic understanding of these processes will expose potential disease mechanisms and therapeutic targets. Several unique protein complexes have been identified that are targeted to the foot process intercellular junction: among these is the Nephrin-Neph1-Podocin receptor complex, which appears to function as a transmembrane receptor. Loss of any one of the Nephrin-Neph1-Podocin receptor components, either in patients carrying an inherited genetic mutation or in experimental gene-targeted mice, causes proteinuria and effacement of podocyte foot processes. For this reason, we propose that the Nephrin-Neph1-Podocin complex functions to integrate podocyte cytoskeletal dynamics and intercellular junction dynamics. Our preliminary work suggests that the Nephrin complex transducers outside-in signals that participate in regulating actin dynamics. In work that first demonstrated this function of Nephrin, we showed that upon engagement of Nephrin's extracellular domain, the protein kinase Fyn catalyzes Nephrin phosphorylation during foot process formation and following induction of podocyte effacement. Nephrin phosphorylation results in recruitment of the Nck to the junction where it assembles an actin polymerization complex capable of nucleating and elongating actin filaments. In unpublished work described herein, we find that Neph1 behaves in a similar fashion, becoming tyrosine phosphorylated by Fyn and recruiting the adaptor protein Grb2, an event that is necessary for Neph1-induced actin polymerization. Moreover, Nephrin and Neph1 function cooperatively in regulating actin polymerization. While these results support our hypothesis, direct evidence that the Nephrin-Neph1 complex regulates actin dynamics in the podocyte in situ is lacking and the mechanisms by which the Nephrin-Neph1 complex regulates actin dynamics remain obscure. Therefore, this application represents a new proposal aimed at addressing these deficiencies by (1) Investigating cellular mechanisms by which Nephrin-Nck and Neph1-Grb2 cooperate to induce actin nucleation, (2) testing in vivo the relevance of the interaction between Neph1 and Grb2 by creating and characterizing a new mouse model in which the Neph1-Grb2 interaction is interrupted;(3) examining the relationship between the Nephrin and Crk-dependent regulation of actin dynamics. Project Narrative: Relevance. Diseases resulting in the nephrotic syndrome such as diabetes mellitus and other diseases of the glomerulus frequently cause progressive kidney damage resulting in irreversible loss of renal function and account for nearly 60% of end-stage renal disease (ESRD) in the United States (X). While dialysis and kidney transplantation are effective treatments for ESRD, they are expensive and imperfect. Indeed, ESRD is associated with a mortality rate of 60% at five years. Moreover, the economic burden of ESRD treatment in the United States is approaching $30 billion dollars per year. Because little is understood about the biology of diseases of the glomerulus effective therapies are lacking. Therefore, prevention of ESRD by understanding the molecular pathogenesis of these diseases is a critical problem requiring medical research. Diseases of the renal glomerulus that result in the nephrotic syndrome are important causes of morbidity and mortality. Unfortunately, the molecular mechanisms governing development of the nephrotic syndrome remain poorly understood. By continuing to study in detail the cellular and molecular biology of the junctional protein complex of the podocyte, it is anticipated that this project will contribute to a better understanding of the biology of the response of the podocyte to injury or disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK080751-06
Application #
8230613
Study Section
Special Emphasis Panel (ZRG1-RUS-B (02))
Program Officer
Rys-Sikora, Krystyna E
Project Start
2009-11-03
Project End
2013-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
6
Fiscal Year
2012
Total Cost
$333,234
Indirect Cost
$124,963
Name
University of Pennsylvania
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Arif, Ehtesham; Rathore, Yogendra S; Kumari, Babita et al. (2014) Slit diaphragm protein Neph1 and its signaling: a novel therapeutic target for protection of podocytes against glomerular injury. J Biol Chem 289:9502-18
George, Britta; Fan, Qingfeng; Dlugos, Christopher P et al. (2014) Crk1/2 and CrkL form a hetero-oligomer and functionally complement each other during podocyte morphogenesis. Kidney Int 85:1382-94
George, Britta; Verma, Rakesh; Soofi, Abdulsalam A et al. (2012) Crk1/2-dependent signaling is necessary for podocyte foot process spreading in mouse models of glomerular disease. J Clin Invest 122:674-92
Mallik, Leena; Arif, Ehtesham; Sharma, Pankaj et al. (2012) Solution structure analysis of cytoplasmic domain of podocyte protein Neph1 using small/wide angle x-ray scattering (SWAXS). J Biol Chem 287:9441-53
Garg, Puneet; Holzman, Lawrence B (2012) Podocytes: gaining a foothold. Exp Cell Res 318:955-63
Holzman, Lawrence B; Garg, Puneet (2009) Initial insight on the determinants of podocyte polarity. J Am Soc Nephrol 20:683-5