VITAMIN A AND RENAL PATTERNING

Vitamin A is required for formation of most organs and tissues including the kidney. Our studies demonstrate the requirement for 2 vitamin A receptors Rara and Rarb2 and Raldh2, a RA-synthesizing enzyme in stromal mesenchyme for normal renal development. In cortical stromal cells, Rara and Rarb2 are likely to be important for growth and branching of the renal collecting system, controlling expression of Ret at branching collecting duct tips where Ret is essential for branching morphogenesis. Definitive proof of this hypothesis is dependent on identification of vitamin A-inducible stromal factors that modulate Ret expression. We will identify these factors using differential hybridization and microarray analysis. Recent studies revealed medullary abnormalities in Rarab2- and Raldh2- mice at El8, suggesting a possible role for vitamin A signaling during tubule extension or collecting duct morphogenesis in the medulla. Consistent with this suggestion, we fmd a novel domain of RA synthesis in the immature loop of Henle, specifically in the hairpin turn, the site of tubule elongation, and we find that Rara and Rarb2 are co-localized in medullary stromal cells surrounding the large collecting ducts and loops of Henle, suggesting that vitamin A signaling in medullary stroma controls late stages of tubule elongation, collecting duct morphogenesis, or both. Alternately, impaired Vitamin A signaling at early stages may be the cause of medullary abnormalities. We will distinguish between these possibilities by generating mutants in which stromal cell signaling can be inducibly inactivated using the Tet-on system, then we will test the requirement for vitamin A in stromal cells at early and late stages. A new RA-synthesizing enzyme, Raldh3, has recently been identified. Raldh3 is localized exclusively in the ureteric bud and compound mutants lacking both Raldh2 and Raldh3 display more severe renal phenotypes than single Raldh2 or Raldh2 mutants, suggesting that RA-synthesis in the ureteric bud may be important directly, or indirectly activating vitamin A signaling in stromal cells. We will distinguish between these possibilities by generating mouse mutants in which the RA-response has been blocked either in the ureteric bud or in stroma by expression of a dominant negative Rar protein.