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Am J Physiol Renal Physiol (February 14, 2006). doi:10.1152/ajprenal.00413.2005
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Submitted on October 20, 2005
Accepted on February 3, 2006

Effects of water restriction on gene expression in mouse renal medulla: Identification of 3{beta}HSD4 as a collecting duct protein

Qi Cai1, Maggie Keck1, Matthew R. McReynolds1, Janet D. Klein2, Kevin Greer3, Kumar Sharma4, James B. Hoying3, Jeff M. Sands2, and Heddwen L. Brooks1*

1 Department of Physiology, University of Arizona, College of Medicine, Tucson, AZ, USA
2 Renal Division, Emory University School of Medicine, Atlanta, GA, USA
3 Biomedical Engineering Program, Genomics Research Laboratory, University of Arizona, Tucson, AZ, USA
4 Division of Nephrology, Thomas Jefferson, Philadelphia, PA, USA

* To whom correspondence should be addressed. E-mail: brooksh{at}email.arizona.edu.

In order to identify novel gene targets of vasopressin regulation in the renal medulla, we performed a cDNA microarray study on the inner medullary tissue of mice following a 48 hour water restriction protocol. In this study 4625 genes of the possible approximately 12,000 genes on the array were included in the analysis, and of these 157 transcripts were increased and 63 transcripts were decreased by 1.5-fold or more. Quantitative, real-time PCR measurements confirmed the increases seen for 12 selected transcripts and the decreases were confirmed for 7 transcripts. In addition we measured transcript abundance for many renal collecting duct proteins that were not represented on the array; AQP2, AQP3, Pax-8 and alpha and beta NaKATPase subunits were all significantly increased in abundance; the beta and gamma subunits of ENaC and the vasopressin type 1A receptor were significantly decreased. To correlate changes in mRNA expression with changes in protein expression, we carried out quantitative immunoblotting. For most of the genes examined, changes in mRNA abundances were not associated with concomitant protein abundance changes; however aquaporin-2 transcript abundance and protein abundance did correlate. Surprisingly, Aldolase B transcript abundance was increased but protein abundance was decreased following 48 hours of water restriction. Several transcripts identified by microarray were novel with respect to their expression in mouse renal medullary tissues. The steroid hormone enzyme 3-beta hydroxysteroid dehydrogenase 4 (3{beta}-HSD4) was identified as a novel target of vasopressin regulation and via dual labeling immunofluorescence we co-localized the expression of this protein to AQP2 expressing collecting ducts of the kidney. These studies have identified several transcripts whose abundances are regulated in mouse inner medulla in response to an increase in endogenous vasopressin levels and could play roles in the regulation of salt and water excretion.




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