Proteinuric kidney diseases, including diabetic nephropathy are responsible for majority of the patients that develop end stage renal disease. Proteinuria is not only important for progression of kidney disease but is also an important cardiovascular risk factor. Podocytes have been the main focus of investigations in glomerular diseases that present with proteinuria. Recent studies suggest that podocyte foot process effacement is a form of podocyte mobility that is a result of increase in lamellipodia formation. Nephrin ability to regulate podocyte actin dynamics, lamellipodia formation and focal adhesion dynamics in a phosphorylation dependent manner suggests its role in foot process effacement. Hypothesis: Hypothesis: Inhibition of signaling events that increase podocyte mobility or prevent lamellipodia formation will prevent podocyte foot process spreading and loss following injury. Rationale: Increase in Nephrin phosphorylation has been observed following podocyte injury. Our preliminary data suggests that Src kinase dependent Nephrin phosphorylation is regulated by non- receptor tyrosine phosphatase shp2. Furthermore, inhibition of Shp2 prevents podocyte foot process effacement in protamine sulfate model of podocyte injury.
Specific Aims : We will test our hypothesis by pursuing the following two specific aims. 1) Define regulation of Nephrin phosphorylation by Shp2. Using biochemical and cell biology techniques we will expand on our preliminary observations and define the protein-protein interaction and functional consequences of Shp2 dependent Nephrin phosphorylation. 2) Characterize signaling mechanism by which integrin activation regulates Nephrin phosphorylation. We will examine the molecular mechanism that result in Nephrin phosphorylation. Our preliminary experiments suggest that Nephrin phosphorylation occurs as a result of integrin activation. 2) Determine the role of Shp2 and Nephrin phosphorylation on podocyte homeostasis in vivo. We will generate an inducible and a non-inducible podocyte specific conditional shp2 knockout mouse. This mouse will enable us to study the role of shp2 during development and following injury. We will also be able to examine the benefit of preventing foot process effacement in podocyte injury models like protamine sulfate specifically in regards to podocyte loss and proteinuria. Using these mice we will also be able to test other models of podocyte injury.
Title: Shp2 a non-receptor tyrosine phosphatase regulates Nephrin phosphorylation and podocyte mobility. Relevance: The proposed research will identify novel potential therapeutic targets that will help in maintaining podocyte homeostasis following injury. Use of Shp2 inhibitors is presently being pursued for cancer treatment, and can be applied to proteinuric kidney diseases as well. Successful completion of the proposal will generate new treatment modalities that will help in slowing down or preventing the progression of proteinuric kidney diseases.
|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|
|Compton, Lauren M; Ikonomov, Ognian C; Sbrissa, Diego et al. (2016) Active vacuolar H+ ATPase and functional cycle of Rab5 are required for the vacuolation defect triggered by PtdIns(3,5)P2 loss under PIKfyve or Vps34 deficiency. Am J Physiol Cell Physiol 311:C366-77|
|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) 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|