Role of Nef in Pathogenesis of HIV Associated Nephropathy

We have previously demonstrated that HIV Nef stimulates distinct downstream signaling pathways to induce podocyte proliferation, dedifferentiation, and loss of normal cytoskeletal organization. However, the linkage of this cell signaling network to downstream transcription factor and gene regulatory networks remains unknown. To address this, we have performed both DNA/protein arrays and gene chip arrays in kidneys of Tg26 HIV transgenic mice compared to littermates, demonstrating that 10 transcription factors are consistently up-regulated and 14 are down-regulated in kidneys of Tg26 mice. By in silico promoter analysis of our DNA/protein arrays and gene arrays, we identified Stat3, NFkB, p53, SPIC, and Myc as important mediators of gene activation in the kidneys of Tg26 mice. Therefore, we hypothesize that these transcription factors contribute to the pathology of HIVAN through regulation of their target genes. We will computationally analyze the transcription factor-gene networks in kidneys of Tg26 mice using a systems biology approach. We will perform promoter analysis using the Chip-on-Chip database and match these to our microarray database. We will also identify the co-regulatory factors and other intermediary factors that are involved in regulation of these transcription factors, using literature-based protein-protein interaction networks and graph analysis. Similar analyses will be performed using data from DNA/protein arrays and gene arrays in glomeruli isolated from Tg26 mice and their littermates. We plan to experimentally validate the transcription factor-gene networks identified by computational analysis in the kidneys of Tg26 mice by EMSA, real-time PCR or western blot. Finally, we will determine the relevance of the identified transcription factor-gene networks in the pathogenesis of HIVAN, both in vitro in cultured kidney cells and in vivo in Tg26 mice. Since Stat3 and NFkB are the key transcription factors activated in the kidneys of Tg26 mice, we will first study the role of Stat3 by using a knockout mouse model and the role of NFkB using a specific inhibitor.