Effects of water restriction on gene expression in mouse renal medulla: identification of 3βHSD4 as a collecting duct protein

Abstract

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-h water restriction protocol. In this study, 4,625 genes of the possible ϳ12,000 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; aquaporin-2 (AQP2), AQP3, Pax-8, and ␣-and ␤-Na-K-ATPase subunits were all significantly increased in abundance; the ␤-and ␥-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, AQP2 transcript abundance and protein abundance did correlate. Surprisingly, aldolase B transcript abundance was increased but protein abundance was decreased following 48 h 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␤-hydroxysteroid dehydrogenase 4 (3␤HSD4) was identified as a novel target of vasopressin regulation, and via dual labeling immunofluorescence we colocalized 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.vasopressin; microarray VASOPRESSIN, THE PEPTIDE hormone, mediates its physiological effects by interactions with G protein-coupled receptors that are expressed along the nephron and in the microvasculature (vasa recta) of the renal medulla. Vasopressin (AVP) actions on the kidney are crucial for the maintenance of sodium and water balance and for the control of blood volume and consequently blood pressure. There are two main subtypes of vasopressin receptors: the vasopressin type 1A receptor (V1aR) which is linked to the phospholipase C (PLC) pathway and increases intracellular calcium via an IP3 dependent mechanism and the vasopressin type 2 receptors (V2R) which couple to G s proteins to stimulate intracellular cAMP formation and activate PKA. V1aRs are found in collecting duct (CD) cells and in the medullary vasa recta (2, 18). Stimulation of the V1aR causes vasoconstriction of the vasa recta, thus decreases medullary blood flow. During antidiuresis, this action minimizes the escape of solutes from the medullary interstitium via ascending vasa recta, thus favoring the maintenance of the high...

Sugar, Sex, and TGF-β in Diabetic Nephropathy

“…39 In male mice, 48-hour water restriction increased expression of 3 β -HSD4 in the renal medulla, suggesting it is regulated by vasopressin, which is increased in diabetes. 66 Further data regarding the regulation and function of this important protein are largely absent. Given that expression levels of 3 β -HSD4 are predictive of the degree of the development of glomerulosclerosis, 64 further research into the interaction of 3 β -HSD4 with TGF- β signaling is warranted.…”

Vasopressin increases expression of UT-A1, UT-A3, and ER chaperone GRP78 in the renal medulla of mice with a urinary concentrating defect

“…Our previously published gene array studies identified GRP78 as a gene that was upregulated in the renal medullas of AQP1KO mice (6) following long-term dDAVP infusion (7 days) and in wild-type mice following a water-restriction protocol (5).…”

“…Studies in vitro have demonstrated that hyperosmolality can directly activate heat shock proteins and transcription factors (23,36). Our previous gene array studies identified the endoplasmic reticulum (ER) stress protein GRP78 as significantly increased in the renal medulla following vasopressin-receptor activation (5,6). ER stress responses can be stimulated by changes in the external environment of a cell, including, potentially, the changes in interstitial osmolality experienced by cells in the renal medulla.…”

Increased Renal Responsiveness to Vasopressin and Enhanced V2 Receptor Signaling in RGS2 −/− Mice

“…The exact molecular targets of RGS2 in vasopressin-sensitive parts of the nephron remain unknown. However, the V2R itself or adenylyl cyclases ACIII, ACV, and ACVI, which are abundantly expressed in the principal cell of the collecting duct, are likely candidates for future studies (7,18). Ϫ8 M) or phenylephrine (PE; 2 ϫ 10 Ϫ4 M).…”