Regulation of Podocyte Structure by the Binding Protein

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DESCRIPTION (provided by applicant): Nephrotic syndrome (NS) is a common kidney disease, but the molecular mechanisms underlying the disease remain unclear for most cases. We previously reported that the small heat shock protein, hsp27, a known regulator of actin polymerization, is highly expressed in glomerular podocytes (the cells most affected in NS), that its expression and phosphorylation are induced in podocytes in experimental NS, and that hsp27 overexpression can protect cultured podocytes from injury. To clarify the mechanism of this protection, we screened a glomerular yeast two-hybrid library for hsp27-binding proteins, and found hic-5, a known focal adhesion and steroid receptor co-activator protein. We confirmed hic-5 as a true hsp27 binding protein by coimmunoprecipitation, partially mapped the interaction domains, and demonstrated that interaction of hic-5 with hsp27 can alter hsp27 function. Recently, we also found that glucocorticoids, the primary treatment for NS, can act directly on podocytes to protect and enhance recovery from PAN-induced injury, in striking contrast to the dominant paradigm that glucocorticoids exert their therapeutic effect in NS by suppressing production of a soluble disease mediator by circulating lymphocytes. We therefore hypothesize that the interaction between hsp27 and the multifunctional binding protein, hic-5, plays a critical role in regulating both podocyte injury during NS and podocyte recovery following glucocorticoid therapy, via hic-5's known roles in: 1) Focal adhesion dynamics, and 2) Glucocorticoid receptor coactivation. To test these hypotheses we will determine the molecular states of hic-5 and hsp27 required for their protein-protein interaction, and the effects of alterations in these states on their intracellular localization in podocytes. We will also determine if induced alterations in the expression, phosphorylation, or hsp27- binding of hic-5 can regulate: 1) Podocyte focal adhesion protein composition, rate and extent of formation, and function before and after podocyte injury, and 2) Glucocorticoid-induced activation of podocyte gene transcription, as well as protection and enhanced recovery of podocytes from injury. Podocytes will be infected with adenovirus containing full-length and phosphorylation- and/or truncation-mutant hic-5 and hsp27 constructs, and detailed analyses performed of the effects of altered hic-5-hsp27 interaction on the: 1) Intracellular localization of hsp27 and hic-5, 2) Focal adhesion composition, formation rate, and function, and 3) Glucocorticoid-induced protection and enhanced recovery from PAN induced injury and actin filament disruption by latrunculin A and cytochalasin D. Correlative in vivo studies will include analyses of hic-5 and hsp27 co-localization in podocytes, as well as protection against disease by dexamethasone, in PAN-induced nephrotic syndrome in rats. Identification of a biologically important role for the interaction between hic-5 and hsp27 in regulating podocyte structure and the therapeutic effect of glucocorticoids in NS would improve our understanding of the molecular mechanism(s) underlying the development of NS, and permit the development of more highly targeted and less toxic therapies for this very common kidney disease.