Fetuses deprived of the vasoactive peptide and potent growth factor Angiotensin II (Ang II) are born with renal dysplasia, and they require dialysis and transplantation for long term survival. Ang II binds at least two receptors, AT1, and AT2. AT1 mediates cell growth and division, vasoconstriction, and salt retention. Decreased AT1 activation likely mediates some of the fetotoxicity in Ang U -deprived babies. AT2, however, is the predominant Ang II receptor in the fetal kidney, and its expression declines at birth. In mediating cell death, vasodilation, and salt excretion, AT2 seems to oppose AT1 action, but its downstream signaling pathways have yet to be identified, and its role in fetal nephrogenesis has not been delineated. In preliminary studies using cultured rat metanephroi, isolated from confounding variables, we found that a) Ang II stimulated ureteric bud (UB) branching, b) AT1 blockade decreased UB branching, whereas c) AT2 antagonism increased UB branching. Normal nephrogenesis involves tightly controlled reciprocal interactions between the metanephric mesenchyme and the invading UB. Excessive UB branching results in abnormal and ectopic induction of mesenchyme, whereas insufficient UB branching causes renal hypoplasia. We hypothesize that AT2 activation inhibits UB branching, and we seek to identify changes in downstream gene expression associated with this process.
Specific Aim 1 : To further test the hypothesis that AT2 inhibits UB branching in cultured fetal rat kidneys. a. Using confocal microscopy and lectin staining we will examine UB branching under conditions that specifically activate or antagonize AT2.b. We will optimize culture conditions that maximally activate or suppress AT2 signaling for subsequent subtraction cloning studies.
Specific Aim 2 : To identify which genes are differentially expressed following AT2 activation or suppression.a. Subtraction cloning will be performed between kidneys at 2 time points following AT2 activation or antagonismb. Hybridization analysis will be used to confirm differentially expressed clones.c. Differentially expressed cDNAs will be sequenced to identify candidate genes. NCBI BLAST searches and bioinformatics will be used to sort differentially expressed genes into structural and functional categories.d. Quantitative RT-PCR will confirm the magnitude of change of selected clones.e. The spatial expression of differentially expressed clones will be assessed using in situ hybridization.The ability of candidate genes identified by this screen to influence ureteric bud branching will be tested in future studies.
