this is a RO1 research grant with the long-term objectives to identify new molecular pathomechanisms and their underlying genetic programs by which TGF-beta/Smad signaling pathways my mediate chronic progression of renal diseases. The overall hypothesis is that depletion of podocytes is a pathomechanism of chronic progression in glomerular diseases. Toxic/genetic insults in podocytes may activate autocrine TGF-beta and/or Smad7 to trigger polocyte apoptosis. TGF-beta secreted by injured podocytes may also provide a paracrine signal to promote mesangial matrix expansion. In contrast, primary endocapillary/mesangial injuries may activate TGF-B providing an autocrine signal for matrix expansion and a paracrine signal for secondary podocyte apoptosis. The first Specific Aim will test whether podocyte toxins induce injury/apoptosis by activation TGF-beta- and/or Smad7-dependent genetic programs. We will examine phenotypic features in control and Smad7-deficient podocytes after exposure to TGF-beta and the podocyte-toxins protamine sulfate and puromycin aminonucleoside, and we will identify the underlying genetic programs (gene expression profiles using cDNA microarrays. The second Specific Aim will test whether Smad7 mediates podocyte depletion associated with glomerulosclerosis in TGF-beta1 transgenic mice. Compound genetic mice carrying the TGF-beta1 transgene and Smad7 null alleles will be analyzed for the effect of Smad7 gene dosage on the development of TGF-beta1-induced glomerulosclerosis and interstitial fibrosis, focusing on in vivo podocyte damage and apoptosis indices. We will profile gene expression to identify Smad7-dependent genetic programs that may underlie Smad7-dependent phenotypic manifestations. The third Specific Aim will test whether podocyte defects and/or apoptosis are mediated by TGF-beta- and/or Smad7-dependent genetic reprogramming and these parameters correlate with progression of glomerulosclerosis in CD2AP knockout mice. We will quantitate podocyte damage and apoptosis indices, mesangial matrix expansion glomerulosclerosis indices and correlate these parameter with TGF-beta and Smad7 expression, and activation of genetic programs as determined by microarray analysis in CD2AP knockout mice at different stages of disease. All gene expression datasets (see Aim 1, 2 and 3) will be cross-referenced in a relational database. Genetic programs with i) putative roles in podocyte injury in vitro (Aim 1), ii) differential regulation according to genotype in compound TGF-beta1/Smad7 genetic mice (Aim 2), and iii) during stages of the progressive glomerulosclerosis in CD2AP knockout mice (Aim 3), will be queried to delineate genetic programs that may underlie structural defects and apoptosis in podocytes induced by epigenetic (protamine sulfate and puromycin aminonucleoside) or genetic (CD2Ap minus/minus) insults.
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