Knowledge about the molecular mechanisms that control development and differentiation of the renal tubule is incomplete. Ontogeny of the collecting duct is of particular importance because the two major cell types, intercalated and principal cells, are critical to maintenance of acid-base, sodium and water balance; and dysfunction of these cells likely accounts for urinary acidification, edema forming and concentrating disorders, developmental disorders of the kidney, and malignancies. We plan to study renal principal cell specific regulation of the aquaporin-2 gene, because it is specific for and essential to the function of principal cells within the collecting duct.
Our specific aims are: 1) Develop a transgenic mouse model for the In vivo study of renal principal cell specific regulatory regions within the aquaporin-2 gene. 2) Develop a cell culture model for the in vitro study of the principal cell specific regulatory regions within the aquaporin-2 gene. 3) Identify the binding sites for nuclear transcription factors within the aquaporin-2 gene that are responsible for principal cell specific expression of aquaporin-2. 4) Identify regulatory regions within the aquaporin-2 gene that collectively confer principal cell specific expression by using a combination of the in vitro cell culture model and in vivo transgenic mouse model. 5) Isolate cDNAs encoding transcription factors that are responsible for renal principal cell specific expression of the aquaporin-2 gene. These studies will utilize a combination of reporter gene studies in transgenic mice and either transfected or microinjected kidney cells, Dnase I hypersensitivity analysis, in vivo footprinting, and electrophoretic mobility shift assays to define the specific regulatory regions and nuclear protein binding sites within the aquaporin-2 gene that are responsible for renal principal cell specific expression. cDNAs encoding transcription factors that interact with these regulatory regions will be isolated using one- hybrid cloning system, or a combination of affinity purification, microsequencing and degenerate RT-PCR cloning techniques. The identification of existing or novel transcription factors involved in kidney and collecting duct specific gene expression will open the door to understanding the molecular basis for renal tubule differentiation, and will provide insight into potential disease mechanisms in the kidney.
|Kishore, B K; Carlson, N G; Ecelbarger, C M et al. (2015) Targeting renal purinergic signalling for the treatment of lithium-induced nephrogenic diabetes insipidus. Acta Physiol (Oxf) 214:176-88|
|Yu, Ming-Jiun; Miller, R Lance; Uawithya, Panapat et al. (2009) Systems-level analysis of cell-specific AQP2 gene expression in renal collecting duct. Proc Natl Acad Sci U S A 106:2441-6|
|Miller, R Lance; Lucero, Olivia M; Riemondy, Kent A et al. (2009) The V-ATPase B1-subunit promoter drives expression of Cre recombinase in intercalated cells of the kidney. Kidney Int 75:435-9|
|Miller, R Lance; Zhang, Ping; Chen, Tong et al. (2006) Automated method for the isolation of collecting ducts. Am J Physiol Renal Physiol 291:F236-45|
|Sun, Rujia; Miller, R Lance; Hemmert, Andrew C et al. (2005) Chronic dDAVP infusion in rats decreases the expression of P2Y2 receptor in inner medulla and P2Y2 receptor-mediated PGE2 release by IMCD. Am J Physiol Renal Physiol 289:F768-76|
|Miller, R Lance; Zhang, Ping; Smith, Maren et al. (2005) V-ATPase B1-subunit promoter drives expression of EGFP in intercalated cells of kidney, clear cells of epididymis and airway cells of lung in transgenic mice. Am J Physiol Cell Physiol 288:C1134-44|
|Kishore, Bellamkonda K; Krane, Carissa M; Miller, R Lance et al. (2005) P2Y2 receptor mRNA and protein expression is altered in inner medullas of hydrated and dehydrated rats: relevance to AVP-independent regulation of IMCD function. Am J Physiol Renal Physiol 288:F1164-72|
|Garcia-Villalba, Pilar; Denkers, Nathaniel D; Wittwer, Carl T et al. (2003) Real-time PCR quantification of AT1 and AT2 angiotensin receptor mRNA expression in the developing rat kidney. Nephron Exp Nephrol 94:e154-9|
|Zharkikh, Ludmilla; Zhu, Xiaohong; Stricklett, Peter K et al. (2002) Renal principal cell-specific expression of green fluorescent protein in transgenic mice. Am J Physiol Renal Physiol 283:F1351-64|