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【摘要】 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 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; 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.
【关键词】 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 osmotic pressure crucial for inducing water reabsorption ( 11 ). V2Rs are expressed on the basolateral membrane of CD cells and activation mediates both rapid and long-term regulation of cell water, urea, and sodium permeability. Many studies have documented the long-term regulation of aquaporin-2 (AQP2) expression by vasopressin action on V2Rs in renal CD cells. An increase in vasopressin increases the water permeability of CDs in vasopressin-deficient Brattleboro rats, and this effect correlates well with increased levels of AQP2 mRNA and protein ( 6, 12 ). Vasopressin-regulated AQP2 expression is also increased in many animal models of clinical disorders such as diabetes ( 19 ) and cardiac failure ( 23, 32 ). Transcription factors AP1 and cAMP-responsive elements have been identified in the 5'-flanking region of the AQP2 gene ( 17, 33 ), adding additional evidence that vasopressin increases the transcription rate of the AQP2 gene.
In this study, we performed a 48-h water restriction protocol in mice to increase endogenous levels of circulating vasopressin. cDNA analysis of renal medullary gene expression was performed with the aim of identifying new gene targets of potential vasopressin action (the water restriction protocol could also potentially increase the renin-angiotensin-aldosterone system and the sympathetic nervous system) and to characterize the response to increased vasopressin in mouse medullary tissues. We present here our microarray data, real-time PCR data, and protein expression data from these studies. Although many genes were significantly altered at the mRNA level, protein expression levels were variable and often only small changes were detected using this protocol. However, both AQP2 protein and mRNA abundance were significantly increased in mouse renal medullas following water restriction for 2 days. We identified the steroid-inactivating enzyme 3 HSD4 as a CD protein and report its colocalization to APQ2-expressing cells.
METHODS
Animals. Mice were maintained in the animal facility of the University of Arizona Health Sciences Center, or in Emory University under National Institutes of Health guidelines. We used mice CD1 (ICR) that were 10 wk old. Control mice received regular food and water ad libitum, water-restricted mice received a gel diet, as previously described ( 14 ), which contained 1.5 ml of water per 20 g body wt per day (1.5 ml·20 g body wt -1 ·day -1 ). Three animals were used in each group, i.e., control and water-restricted, for microarray analysis; for protein abundance, five animals were used in each group.
RNA isolation, amplification, and cDNA purification. Full methodology for the RNA purification, amplification, and cDNA production has been previously published ( 21 ). Briefly, RNA samples isolated from individual mouse inner medullas were labeled C1-C3 for control mice and E1-E3 for water-restricted mice. RNA was amplified using the MessageAmp kit (Ambion cat. no. 1750) according to the manufacturer?s protocols. Three micrograms of total RNA from each individual mouse were used as a template for each amplification reaction, and this gave a yield of 50 µg of amplified RNA. Amplified RNA was then reverse transcribed to cDNA using the EndoFree RT kit (Ambion cat. no. 1740) according to the manufacturer?s protocol. Amino allyl modified cDNA was purified using PCR purification columns (Qiagen cat. no 28104) according to the manufacturer?s protocol. The modified cDNA was labeled with Alexa dyes via the free amine modification (A-20002-546 Alexa Fluor and A-20006-647 Alexa Fluor, Molecular Probes, Eugene, OR).
Microarray slide preparation and hybridization. Microarrays for our study were prepared within the Genomic Research Laboratory (GRL) at the University of Arizona using the NIA mouse 15K clone set http://lgsun.grc.nia.nih.gov/cDNA/15k.html. Full methodology for the production of the microarrays and the hybridization protocols has been published previously ( 21 ). Labeled modified cDNA in hybridization buffer was loaded onto a slide and set to hybridize at 47°C for a minimum of 16 h. After completion, a short wash was run in the hybridization station after which the slide was removed and dipped in 0.05 x SSC to remove any residual nonhybridized cDNA. The slide was dried and analyzed using the arrayWORx e CCD-based microarray scanner from Applied Precision, capable of multichannel fluorescence scanning.
Microarray analysis. Microarray data were reduced and evaluated by CARMA as previously described ( 21 ); the results were submitted to the Gene Expression Omnibus (GEO) and can be found under GEO reference number GSE3498 .
Real-time quantitative PCR. Real-time quantitative PCR was carried out using the Rotor-Gene RG-3000 (Corbett Research) sequence detection system and SYBR Green reagents from Qiagen (Quantitect Sybr Green PCR Kit, cat. no. 20414). Primers were designed using Primer3 software ( 25 ) and are listed in supplemental data B along with the gene accession number for the target gene. (Supplemental data for this article are available online at the AJP-Renal Physiology Web site.) Three micrograms of total or amplified RNA were reverse transcribed with the Endofree RT kit, according to the manufacturer?s protocol (Ambion). The cDNA was diluted to 8 ng/µl and the PCR reaction mixture contained 5 µl of Sybr master mix, 0.4 µl 25 mM MgCl 2, 0.6 µl RNAse free water, 100 pmol of forward and reverse primers, and 16 ng cDNA, in a volume of 10 µl. Each reaction was performed in triplicate at 95°C, 15 min; then 95°C, 15 s, and 58°C, 15 s, and 20 s at 72°C for 40 cycles. This was followed by a melt cycle which consisted of stepwise increase in temperature from 72 to 99°C. A single predominant peak was observed in the dissociation curve of each gene, supporting the specificity of the PCR product. Ct numbers (threshold values) were set within the exponential phase of the PCR and were used to calculate the expression levels of the genes of interest and were normalized to endogenous cellular actin RNA. The level of actin RNA was measured in parallel samples using actin specific primers (see supplemental data B for primer sequences).
Sample preparation, SDS-PAGE, and immunoblotting. Full details of sample preparation, SDS-PAGE, and immunoblotting procedures have been published previously ( 6 ). Briefly, kidneys were dissected into inner medulla (IM) and outer medulla (OM) and were homogenized in 300 µl of ice-cold isolation solution (250 mmol/l sucrose and 10 mmol/l triethanolamine, pH 7.6, containing 1 µg/ml leupeptin and 0.1 mg/ml phenylmethylsulfonyl fluoride) using a tissue homogenizer (Omni 1000 fitted with a micro-sawtooth generator) at maximum speed for three 15-s intervals. Total protein concentrations were measured (BCA kit, Pierce Chemical), and the samples were solubilized in Laemmli sample buffer at 60°C for 15 min. Semiquantitative immunoblotting was carried out as described previously ( 7 ) to assess the relative abundances of the proteins of interest between control and water-restricted samples. To confirm that protein loading of the gels was equal, preliminary 12% polyacrylamide gels were stained with Coomassie blue. Densitometry (Personal Densitometer SI, Molecular Dynamics) was performed on representative bands to ensure equal loading (generally, <5% variation relative to the mean). Proteins were separated on 7.5, 10, or 12% polyacrylamide gels by SDS-PAGE, and the proteins were transferred to nitrocellulose membranes electrophoretically (Bio-Rad Mini Trans-Blot Cell). Membranes were blocked for 1 h at room temperature with 5% nonfat dry milk and probed overnight at 4°C with the respective primary antibodies. Membranes were washed and exposed to secondary antibodies (goat anti-rabbit IgG conjugated to horseradish peroxidase, Cell Signaling; rabbit anti-mouse IgG conjugated to horseradish peroxidase, Cell Signaling; both diluted to 1:2,000) for 1 h at room temperature. Bands were visualized by using a luminol-based enhanced chemiluminescence substrate (Amersham). Band densities were determined by laser densitometry. To facilitate comparisons, we normalized the densitometry values such that the mean for the control group is defined as 100%. Values from water-restricted animals were compared with controls using an unpaired t -test. P < 0.05 was considered statistically significant.
Immunohistochemistry and immunofluorescence labeling. Mouse kidneys were fixed in 4% paraformaldehyde overnight. Kidneys were embedded in paraffin and sectioned (4 µm) by the University of Arizona Pathology Laboratory. Sections were deparaffinized with xylene and rehydrated in graded ethanol. For peroxidase staining, endogenous peroxidase activity was quenched with 0.3% (vol/vol) hydrogen peroxide in absolute methanol for 30 min. Sections were heated in citrate buffer (pH 6.0) in a microwave oven for 10 min for antigen retrieval. Nonspecific binding was blocked in 2% BSA.
For immunohistochemistry, the sections were incubated overnight at 4°C with primary antibody, followed by a biotinylated secondary antibody (anti-rabbit IgG, Zymed) for 30 min at 37°C and horseradish peroxidase-conjugated streptavidin (Zymed) for 30 min at 37°C. Labeling was visualized with chromogen diaminobenzidine (Zymed). Finally, the slides were counterstained with hematotoxylin.
For immunofluorescence staining, the sections were incubated with a goat anti-AQP2 antibody (1:200 dilution, Santa Cruz Biotechnology) and rabbit anti-3 HSD antibody (1:500 dilution) ( 28 ) overnight at 4°C, followed by an incubation of Texas red-conjugated donkey anti-goat secondary antibody (1:200 dilution) and FITC-conjugated donkey anti-rabbit (1:200 dilution, Jackson ImmunoResearch) for 1 h at room temperature. Finally, the slides were mounted with antifade medium, Vectashield (Vector Lab).
RESULTS
Urinary osmolality was significantly increased following the 48-h water restriction protocol, control mice urine osmolality was 1,536 ± 211 mosmol/kgH 2 O; water-restricted values were 2,570 ± 200 mosmol/kgH 2 O, P < 0.05, n = 4. This was reflected in the increased weight loss observed in the water-restricted mice, 3.7 ± 0.25 g, compared with a change of 1.36 ± 0.38 g in control mice, P < 0.05, n = 5.
Microarray analysis. Total RNA was isolated from the inner medulla of control (samples C1, C2, C3) and water-restricted mice (samples E1, E2, and E3) for microarray analysis of gene expression using our previously published methods ( 21 ). Total RNA from each sample was then amplified before being labeled in the cDNA reaction and each sample from an individual mouse was hybridized to an array four times (see MIAME document, supplemental data). Data were analyzed and transformed as previously described ( 21 ). The ANOVA was performed on a gene by gene basis and was limited to genes that were measured confidently on a minimum of three of the four hybridizations, for at least one sample. In this study, 4,625 genes out of the possible 12,000 genes on the array were included in the ANOVA. We considered genes to be significantly differentially expressed if the ANOVA P value was 0.05. Genes that exhibited small changes in gene expression, but were identified as significant because of unusually small variance, were excluded based on a cut-off of 1.5-fold up- or downregulation between the control and water-restricted mice; 220 genes were in this selected group, 157 were significantly increased, and 63 were significantly decreased following water restriction. An output file showing individual measurements, gene name (if known), and links to the NIA and GenBank databases was generated (see supplemental data A).
Validation of array results via real-time quantitative PCR. We selected several genes from the microarray analysis that were significantly increased or decreased in response to water restriction and used quantitative real-time PCR to confirm the mRNA expression levels. We confirmed our microarray results for a randomly selected number of genes that were either up- or downregulated in the water-restricted mice, compared with control mice using the same amplified RNA samples that were used for the microarrays. We previously demonstrated that amplified RNA is a good measure of the relative gene expression in total RNA samples ( 21 ). We used actin as an internal standard for the real-time analysis as it was unchanged on the microarray data. Figure 1, A and B, presents the real-time PCR data for 12 genes that were identified by microarray analysis as significantly increased in mRNA abundance in water-restricted mice compared with control mice. All genes were confirmed by real-time PCR as significantly increased. Figure 1 C presents the real-time PCR data for seven genes that were identified via microarray analysis as significantly decreased in mRNA abundance in water-restricted mice compared with control mice; real-time PCR data presented in Fig. 1 C confirms the microarray data for each of the seven genes selected. Figure 2 presents the changes in mRNA abundance measured for several renal CD expressed genes that were not represented on the microarrays. AQP2 and AQP3 mRNA abundance were increased following 48 h of water restriction. There was a significant decrease in the mRNA abundance of the vasopressin type 1A receptor; however, we saw no change in V2R mRNA abundance after 48 h of water restriction (not shown). We observed a significant decrease in the mRNA abundance for two of the epithelial sodium channel (ENaC) subunits, (0.35 ± 0.24, compared with 1.00 ± 0.17 in control IM, P < 0.05) and (0.29 ± 0.18 compared with 1.00 ± 0.44 in control IM, P < 0.05), there was no change in the abundance of ENaC- subunit mRNA (not shown). We previously demonstrated that high NaCl concentration increased the mRNA abundance of the paired-box genes 2 and 8 (Pax-2 and -8) in cultured inner medullary cells ( 8 ). In this in vivo study, Pax-8 mRNA was significantly increased in abundance following water restriction (2.25 ± 0.3 compared with 1.00 ± 0.17 in control IM, P < 0.05), in contrast, Pax-2 mRNA abundance was unchanged (not shown).
Fig. 1. SYBR Green I real-time PCR assay validation for a subset of differentially expressed genes selected from genes shown to increase ( A and B ) or decrease ( C ) in the microarray data. The data shown are the mean relative fold-change in the renal medullas of control vs. water-restricted (WR) mice; *significant difference between control and WR mice ( t -test; P < 0.05). Assays for actin were run in parallel on each sample for subsequent normalization of the data. A : 3 HSD4, 3 -hydroxysteroid-dehydrogenase 4; AR6, aldehyde reductase 6, renal specific oxidoreductase; CA4, carbonic anhydrase 4, CES3, carboxylesterase 3; P8, nuclear protein 1. B : AK2, adenylate kinase 2; GRP78, glucose-regulated protein 78; mATF4, activating transcription factor 4; tPA, tissue plasminogen activator; ALDH7, aldehyde dehydrogenase family 7. C : GSTm2, glutathione s transferase mu 2; COLa2, procollagen, type I, alpha 2; CNN2, Calponin 2; IREG1, ferroportin 1; CHIF, corticosteroid hormone-induced factor; IGF-1, insulin-like growth factor 1.
Fig. 2. SYBR Green I real-time PCR assay validation of collecting duct genes. The data shown are the mean relative fold-change in the renal medullas of control vs. WR mice. *Significant difference between control and WR mice ( t -test; P < 0.05). Assays for actin were run in parallel on each sample for subsequent normalization of the data. Pax-8, paired box domain-8; AQP2, aquaporin-2; AQP3, aquaporin-3; -NKATP, Na-K-ATPase, -subunit; -NKATP, sodium potassium ATPase, -subunit; V1a, vasopressin receptor subtype 1a; ENaC-, epithelial sodium channel- subunit; ENaC-, epithelial sodium channel- subunit.
Protein expression studies for selected genes. Changes in mRNA abundance do not necessarily correlate to changes in protein abundance, particularly over short physiological time frames such as 48 h. Therefore, we selected a few genes for analysis of protein expression in the renal medullas of water-restricted mice. We analyzed protein expression using semiquantitative immunoblotting, immunohistochemistry, and immunofluorescence.
Localization of 3 HSD4 to CDs in the renal medulla. In mice, 3 HSD4 is a 3-ketosteroid reductase which uses NADPH as the preferred cofactor. It is an isoform of the 3- HSD steroid enzyme family and has been proposed to function to metabolize steroid hormones, including progesterone and dihydrotestosterone. Previous studies identified 3 HSD4 as expressed in glomeruli of the kidney ( 28 ), but its expression in renal medulla has not been demonstrated. Using immunocytochemistry, we show in Fig. 3 A that 3 HSD4 protein is expressed in CD-like tubules of the renal medulla. To specifically localize the expression of 3 HSD4 to CDs of the medulla, we performed dual labeling with AQP2, a renal CD marker. A fluorescein-labeled secondary antibody was used to identify cells that expressed 3 HSD4 as shown in Fig. 3 B (shown in green), whereas a secondary antibody labeled with Texas Red was used to label cells that expressed AQP2 as shown in Fig. 3 C (shown in red). Figure 3 D shows the overlay demonstrating coexpression of AQP2 and 3 HSD4 in CDs of the renal medulla. Semiquantitative immunoblotting indicated that there was no significant increase in 3 HSD4 protein abundance following water restriction (data not shown).
Fig. 3. Immunolocalization of 3 HSD4 to collecting duct tubules of the kidney. A : 3 HSD4 immunperoxidase labeling in paraffin-embedded mouse kidney sections, positive 3 HSD4 immunostaining is observed in collecting tubule cells. Immunofluorescent localization of 3 HSD4 in green ( B ) and AQP2 in red ( C ) to collecting duct tubules in the mouse medulla ( D ) merged picture. Magnification: x 400.
Corticosteroid hormone-induced factor. Corticosteroid hormone-induced factor (CHIF) is a small epithelial-specific protein known to be regulated by aldosterone and potassium intake. In the kidney it is specifically expressed in the basolateral membrane of kidney CDs ( 10, 27 ). Recent studies suggest that CHIF along with other members of the transmembrane protein FXYD family interact with the - and -subunits of the Na-K-ATPase pump and can alter pump kinetics ( 5, 15 ). In this study, CHIF mRNA was significantly decreased in abundance in the renal medullary cells of water-restricted mice (0.24 ± 0.03 compared with control 1.00 ± 0.17, P < 0.05); however, protein abundance as shown in Fig. 4 was not significantly altered (108 ± 9 compared with 100 ± 10% in control IM). CHIF mRNA and protein have previously been reported as responding differently; CHIF mRNA and protein is elevated by aldosterone in the colon ( 1, 27 ); however, in the kidney CD CHIF protein increases with aldosterone, but CHIF mRNA does not ( 30, 31 ). The mRNA for the -subunit of Na-K-ATPase was significantly increased in abundance (1.53 ± 0.2 compared with control 1.00 ± 0.14, P < 0.05) as was the mRNA for the -subunit of Na-K-ATPase (2.43 ± 0.13 compared with control 1.00 ± 0.44, P < 0.05). We also detected a small but significant increase in -Na-K-ATPase protein expression as shown in Fig. 4.
Fig. 4. Immunoblots examining transporter abundance in inner medulla homogenates of control and WR mice. Each lane represents a homogenate from an individual mouse. Densitometry values are normalized to a control value of 100 to facilitate comparison. * P < 0.05. -NKATP, sodium potassium ATPase -subunit; -ENaC, epithelial sodium channel -subunit.
Aldolase B. Aldolase B catalyzes the reversible conversion of fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxy-acetate phosphate and is a key player in both gluconeogenic and glycolytic pathways ( 22 ). Aldolase B has previously been localized to proximal tubules in rat kidney ( 26 ) and data suggest that aldolase B, as part of the glycolytic pathway, regulates V-ATPase assembly by direct protein-protein interactions. This would provide a mechanism for coupling V-ATPase assembly directly to glucose metabolism ( 20 ). V-ATPases are present at high densities on the plasma membrane of proximal tubule cells and intercalated cells of the CD and have a central role in maintaining the acid-base balance of the organism ( 16 ). However, no expression of aldolase B has previously been reported in the renal medulla. Data shown in Fig. 4 demonstrate that aldolase B protein was significantly decreased in expression in the renal medulla, following 2 days of water restriction (44 ± 11 compared with 100 ± 22% in control IM, P < 0.05). In contrast, aldolase B mRNA was significantly increased ( Fig. 1 A ).
Renal CD proteins. Water restriction for 48 h significantly increased the abundance of AQP2 mRNA in renal medullary tissue (1.81 ± 0.07 compared with control 1.00 ± 0.04 P < 0.05). This increase in mRNA abundance was confirmed at the protein level in the inner medulla (256 ± 23 compared with 100 ± 11% in control IM) as shown in Fig. 4. Immunocytochemistry demonstrated the expression of AQP2 in collecting tubules of the mouse kidney, and following 48 h of water restriction, an increase in expression was observed in the apical membrane of the tubules as expected, shown in Fig. 5. In contrast, there was no change in the protein expression of AQP3 in the inner medulla of water-restricted mice ( Fig. 4 ), despite a significant increase in AQP3 mRNA abundance. Pax-2 mRNA was not changed in abundance following 48-h water restriction, and the protein expression of Pax-2 was also unchanged ( Fig. 4 ). Water restriction for 48 h significantly decreased the mRNA abundance of -ENaC in renal medullary tissue and this decrease was confirmed at the protein level in the inner medulla (37 ± 9 compared with 100 ± 6% in control IM, P < 0.05), shown in Fig. 4.
Fig. 5. Immunohistochemical labeling of AQP2 in mouse kidney medulla. AQP2 labeling was seen dispersed within the cytoplasm in collecting duct cells of control mice ( A ) and there was an increase in labeling intensity in collecting duct cells in response to water restriction ( B ). Magnification: x 400.
DISCUSSION
Vasopressin is known to increase the water permeability of CD, thus allowing osmotically driven water reabsorption to occur both in the cortex and in the medulla, leading to concentration of the urine. As expected, our water restriction protocol (48 h) significantly increased urine osmolarity in mice and increased AQP2 mRNA expression and protein abundance. Vasopressin?s action to increase renal water absorption is well documented, in contrast, vasopressin?s role in regulating renal sodium absorption is less clear. In vivo, vasopressin has been reported to have both antinatriuretic and natriuretic effects, with recent data suggesting that vasopressin activation of V1aR causes natriuresis, but V2R activation contributes to the antinatiuretic effect ( 3, 4 ).
We observed a significant decrease in the mRNA abundance of the epithelial sodium channel - and -subunits, with no change in the -subunit. We confirmed the decrease in mRNA expression at the protein level with Western blot analysis; both - and -subunit protein abundance was decreased in water-restricted mice. Previous studies using the oocyte expression system have shown that the - and -subunits of ENaC facilitate the surface expression of functional ENaC channels ( 9 ); thus a decrease in the mRNA and protein abundance of these subunits suggests that sodium transport may be reduced in the inner medullary CDs of water-restricted mice. It is well documented that long-term activation of the V2 receptor, and not the V1a receptor, such as via dDAVP infusion for 5 days, increases sodium retention, with a corresponding increase in the abundance of the - and -subunit of ENaC. However, long-term (7 days) water restriction, i.e., increases in AVP which would activate both V1aR and V2R, demonstrated differential effects on ENaC subunit abundance in rats; -ENaC abundance was significantly increased by 7-day dDAVP infusion but not by 7-day water restriction; in contrast, both long-term water restriction and dDAVP infusion increased the expression of the - and -subunits ( 13 ).
Both AQP2 and AQP3 protein expression is known to be increased by a 48-h thirsting protocol in rat inner medullary tissue ( 29 ). As expected, our data demonstrated that a 48-h water restriction protocol in mice significantly increased both AQP2 protein and mRNA abundance in the renal medulla. In contrast, AQP3 mRNA was increased after 48-h water restriction, but protein abundance was not changed. We also saw a small but significant increase in protein expression of the -subunit of the Na-K-ATPase, and a significant increase in the mRNA abundance for the - and -subunit of Na-K-ATPase following water restriction. Previously published data demonstrated that the protein abundance of the -subunit of Na-K-ATPase was increased by 7 days of water restriction, but not by dDAVP infusion, suggesting again that V1A and V2 receptor activation has differential effects on protein abundance in the renal medulla.
Our microarray data identified 3 HSD4 mRNA as increased in expression following water restriction in mice, and we localized the expression of 3 HSD4 protein to the CDs of the renal medulla. There are multiple isoforms of the family of 3 HSD steroid converting enzymes which can be separated into two categories based on their enzyme activities. Mouse isoforms 1-3 confer 3 -hydroxysteriod dehydrogenase/ 5 - 4 isomerase activity using NAD + as a cofactor. These isoforms catalyze an essential step in the biosynthesis of all steroid hormones, the conversion of pregnenalone to progesterone. Expression of the isoforms exhibiting this activity is typically found in steroidogenic organs (adrenals and gonads). The other category of activity of this enzyme family is a 3-ketosteroid reductase which uses NADPH as the preferred cofactor. These isoforms (3 HSD4 and 5 in mice) are typically expressed in the kidneys and liver ( 24 ) and function to metabolize steroid hormones, including progesterone and dihydrotestosterone. A potential role for 3 HSD4 in renal tissue may be its ability to inactivate androgens. Previous molecular profiling studies of diabetic mouse kidneys identified 3 HSD4 as a marker gene that strongly correlated with the development of diabetic nephropathy. Glomerular staining of 3 HSD4 was decreased in human and murine diabetic samples with advanced glomerulosclerosis ( 28 ). A potential role for 3 HSD4 in renal tissue may be its ability to inactivate dihydrotestosterone (DHT) ( 28 ). High levels of dihydrotestosterone in the glomerulus are known to contribute to glomerular dysfunction, albuminuria and glomerulosclerosis. No data yet exist on the possible functional role of 3 HSD4 in the renal medulla.
Our microarray data demonstrated that a large number of genes were increased in the renal medulla of the water-restricted mice; however, this increase in mRNA abundance did not translate into a corresponding increase in protein abundance for several proteins that we tested. Aldolase B catalyzes the reversible conversion of fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxy-acetate phosphate and is a key player in both gluconeogenic and glycolytic pathways ( 22 ). Microarray data identified the presence of aldolase B mRNA in the renal medulla and the mRNA abundance of aldolase B was significantly increased following water restriction. In contrast, protein expression of aldolase B was significantly decreased following 48 h of water restriction. Aldolase B has not previously been demonstrated as a CD protein, but immunostaining in the IM of control mice revealed that it colocalized with AQP2 protein (data not shown). Staining in the cortical region confirmed previous observation ( 26 ) with very intense aldolase B expression in the proximal tubules with no staining in distal tubules (not shown). Aldolase B mRNA was previously identified by us as significantly decreased in the inner medulla of aquaporin-1 null mice compared with control wild-type mice.
In summary, water restriction for 48 h in mice increased the mRNA and protein expression of AQP2 in the renal medulla and decreased the expression of the - and -subunits of ENaC. Using microarray technology, we identified several mRNAs whose expression was either significantly increased or decreased in the renal medulla following water restriction and identified the steroid hormone enzyme 3 HSD4 as a renal CD protein.
GRANTS
This work was funded by a grant from the University of Arizona Foundation (H. L. Brooks) and by National Institutes of Health Grant DK-064706 (H. L. Brooks).
ACKNOWLEDGMENTS
We are grateful to Dr. H. Garty for providing the CHIF antibody.
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作者单位：1 Department of Physiology, College of Medicine, and 3 Biomedical Engineering Program, Genomics Research Laboratory, University of Arizona, Tucson, Arizona; 2 Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and 4 Division of Nephrology, Thomas Jefferson