Molecular Changes in Slit Diaphragm Related Proteinuria

DESCRIPTION (provided by applicant): Proteinuria is a major manifestation of glomerular disease, and reducing proteinuria has been conclusively shown to retard the progression of kidney failure. Our research effort is directed towards investigating the pathogenesis of proteinuria, with the long term goal of developing specific anti-proteinuric therapies based disease mechanisms. The slit diaphragm plays a major role in maintaining glomerular permeability characteristics. We have recently shown that injecting a combination of anti-neph1 and anti-nephrin antibodies in individual sub-nephritogenic doses into rats results in proteinuria / albuminuria. In this proposal, we plan to use this new model to study changes in the expression of podocyte genes and proteins during selective slit diaphragm injury and proteinuria. Specific aim1: To characterize the full range of complement- and leukocyte-independent heterologous phase proteinuria induced in rats as a result of slit diaphragm injury using a combination of affinity purified anti-neph1 and anti-nephrin antibodies, and study its effect on the expression and phosphorylation of slit diaphragm proteins. Specific aim 2: To identify and characterize genes that are differentially expressed in the podocyte in this model using a combination of supression subtractive hybridization, real time PCR, in situ hybridization, Western blot and cultured cell transfection studies. This study will bring us one step closer to understanding the pathogenesis of proteinuria, and will also help to correlate our existing knowledge of the in vitro characteristics of slit diaphragm proteins with in vivo function.