MOLECULAR MECHANISMS IN NEPHROTIC SYNDROME

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Nephrotic syndrome (NS), a common kidney disease in children, is characterized by retraction (effacement) of the distal "foot" processes of glomerular epithelial cells (GEC) that surround glomerular capillaries. Foot processes are an essential part of the kidney's filtration barrier, and their structure is regulated primarily by actin microfilaments which are abundant in foot processes. Actin and the GEC itself are thought to be anchored the glomerular basement membrane by a complex of actin-associated proteins and beta1 integrin adhesion molecules. Effacement of GEC foot processes has been linked to altered distributions of GEC actin microfilaments and increased glomerular expression and phosphorylation of hsp27, a heat shock protein reported to regulate actin polymerization. We hypothesize that: 1) Specific changes in the expression and/or interaction of actin microfilaments, the actin-associated proteins vinculin, talin, and alpha-actin, and beta1 integrin adhesion molecules are critical for the development of GEC foot process effacement during nephrotic syndrome, and 2) These changes are mediated by altered expression of GEC hsp27. To test these hypotheses we plan to determine; 1) If specific changes in the expression or interaction of actin, the actin-associated proteins vinculin, talin and alpha-actinin, and beta1 integrins in GECs are necessary for the development of foot process effacement and NS, and to determine the relationship between these alterations and variable degrees of GEC foot process effacement, and 2) If induced changes in the expression of glomerular hsp27 result in development of, or protection from, GEC foot process effacement and NS. Using both in vivo (PAN nephrosis in rats) and in vitro (protamine treatment of isolated glomeruli) models of GEC foot process effacement, we will analyze changes in F- and G-actin, actin-associated proteins (vinculin, talin, alpha- actinin), and beta1 integrins in glomeruli using semiquantitative confocal microscopy, Northern, and Western blots, and immunoprecipitation studies. The relationship between changes in the expression and interaction of these molecules and the extent of GEC foot process effacement at multiple stages of disease will be determined using electron microscopy. The effect of hsp27 induction on foot process effacement will be studied both in vitro (heat shock and hsp27 transfections of glomeruli) and in vivo (whole body hyperthermia and inducible sense and antisense hsp27 cDNA transgenic mice). Identification of specific molecular alterations in the regulation of GEC structure which are critical for the development of GEC foot process effacement and NS would permit the development of more specific and less toxic therapies for NS.