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【关键词】 cells
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung
Blood Institute, National Institutes of Health, Bethesda, Maryland
ABSTRACT
The urea transporters, UT-A1 and UT-A3, two members of the UT-A gene family, are localized to the terminal portion of the inner medullary collecting duct (IMCD). In this manuscript, we demonstrate that 4.2 kb of the 5'-flanking region of the UT-A gene (UT-A promoter) is sufficient to drive the IMCD-specific expression of a heterologous reporter gene, -galactosidase (-Gal), in transgenic mice. RT-PCR, immunoblotting, and immunohistochemistry demonstrate that within the kidney, transgene expression is confined to the terminal portion of the IMCD. Colocalization studies with aquaporin-2 show that expression is localized to the principal cells of the IMCD2 and IMCD3 regions. Utilizing -Gal activity assays, we further show that within the kidney, the -Gal transgene can be regulated by both water restriction and glucocorticoids, similar to the regulation of the endogenous UT-A gene. These results demonstrate that 4.2 kb of the UT-A promoter is sufficient to drive expression of a heterologous reporter gene in a tissue-specific and cell-specific fashion in transgenic mice.
transgenic; kidney; -galactosidase
THE STUDY OF GENE FUNCTION in vivo has been enhanced by the use of genetically modified animals. Numerous mouse models of renal function have been developed that use "classical" gene-targeting techniques to disrupt a particular gene in the germline of an animal, and the subsequent study of the "knockout" model can be used to assess the renal phenotype of the ablated gene (9, 1821, 36, 37). However, gene deletion throughout the body often results in difficulties assessing the role of a gene in renal function or a particular nephron segment or cell type. In its most extreme scenario, deletion of a gene results in mortality of the mice, rendering the model unsuitable for characterization (2, 14, 38). In these cases, there is a need to conditionally regulate gene targeting.
As a first step to achieving cell-specific transgene expression, it has been necessary to experimentally determine whether a given promoter region can target expression in a cell- or tissue-specific manner. This has been achieved by the generation of transgenic animals, where a promoter region of interest is linked to a reporter gene, such as -galactosidase (-Gal), green fluorescent protein (GFP), or firefly luciferase, and the localization and expression of the reporter gene are examined. Using this approach, several promoters have been identified that are capable of targeting genes to particular nephron segments. For example, the visceral epithelial cells of the glomerulus can be targeted with the promoter for the nephrin gene (Nphs1) (23, 35); proximal tubule cells can be targeted with the type II promoter for -glutamyl transpeptidase (29) or the androgen-regulated protein (KAP) (5, 6), whereas proximal tubule cells of the outer medulla can be targeted with the human plasminogen activator inhibitor type I (PAI-1) gene promoter (7); the epithelial cells of the thick ascending limb of the loop of Henle and early distal convoluted tubule can be targeted with the promoter regions of either Tamm-Horsfall protein (13, 34, 40) or the dopamine- and adenosine cAMP-regulated inhibitor of protein phosphatase-1 (DARPP-32) (4); both the human CLC-KB promoter (17) and the B1 subunit of vacuolar H+-ATPase (V-ATPase) promoter (22) can localize transgene expression to, among other regions, the connecting tubule; and the principal cells of the collecting duct can be targeted with the aquaporin-2 promoter (27, 39).
In the series of experiments detailed in this study, we generated a transgenic line where the expression of a reporter gene, -Gal, is under the control of the mouse UT-A urea transporter promoter, UT-A (10). This promoter region, situated at the 5'-end of the gene, is believed to drive the expression of the inner medulla collecting duct (IMCD)-specific UT-A isoforms, UT-A1 and UT-A3. Characterization of this line, termed UT-A-Gal, shows for the first time that this promoter region is sufficient to drive reporter gene expression in only the principal cells of the IMCD, thus making the promoter a useful tool for achieving cell-specific gene expression or ablation in this segment of the nephron.
METHODS
Construction of targeting vector and generation of transgenic mice. Using standard PCR methodologies and mouse genomic DNA, a 4.2-kb fragment of the 5'-flanking region and part of the first noncoding exon of the mouse UT-A gene were amplified. This PCR fragment was directionally cloned into the SbfI/XhoI sites of the pWHERE plasmid (Invivogen), upstream of the insulated -Gal reporter gene (Fig. 1). The entire targeting vector was sequenced for confirmation (Lark Technologies). The plasmid was linearized, and Escherichia coli DNA was removed by restriction digest with PacI and separated by gel electrophoresis. The DNA was gel extracted (Qiagen), phenol/chloroform extracted, ethanol precipitated and purified with an Elutip-D column (Schleicher & Schuell). The National Heart, Lung, and Blood Institute Transgenic Core facility generated transgenic founder mice by standard methods. Briefly, the purified transgene was used for microinjection into the male pronucleus of blastocysts derived from C57BL/6 mice and transferred to pseudopregnant females. Transgene incorporation was confirmed in the resultant offspring by Southern blot analysis after an NcoI/NheI restriction enzyme digest of 10 μg of genomic DNA. Blots were probed with a 2.5-kb -Gal probe. Transgenic mice showed a 3.1-kb fragment resulting from the NcoI/NheI digestion within the transgene. Subsequent to the proven intergration of the transgene by Southern blot analysis, further genotyping of offspring was performed using PCR and the sense primer 5'-CACACTCTGCTCCTCACATATCACTTG (spanning the 3'-end of the UT-A promoter, UT-A-F1) and the antisense primer 5'-GCTGTGAGGAAGGCAGAGAGGTCA (reporter gene specific, -Gal-R1). Transgenic positive animals were subsequently bred with C57BL/6 mice and generations F1-F3 were analyzed.
Determination of copy number. Real-time PCR was performed on an ABI Prism 7900HT system, using 50 ng of genomic DNA, 20 pmol (each) of gene-specific primers UT-A-F1 and -Gal-R1 and a Quantitect SYBR green PCR kit (Qiagen) according to the manufacturer’s protocol. Specificity of the amplified product was determined using melting curve analysis software, gel electrophoresis, and sequencing. The copy number of founder transgenic mice was determined using a serial dilution of mouse genomic DNA containing 0100,000 copies per cell equivalent of transgene DNA (original purified transgene). A standard curve of threshold cycle (Ct) vs. copy number was generated, and unknowns were determined. Equal loading and integrity of genomic DNA were determined using actin-specific primers (forward, 5'-GTGCTGTCTGGCGGCACCACCAT and reverse, 5'-CCTGTAACAACGCATCTCATAT).
RT-PCR analysis of transgene expression. Total RNA was extracted from transgenic or nontransgenic control male mice using a Ribopure kit (Ambion) according to the manufacturer’s guidelines. Potential contaminating DNA was removed from the RNA preparations by 30-min incubation with DNase I (Ambion). Total RNA (1 μg) was reverse transcribed using oligo-dT and Superscript II reverse transcriptase (Invitrogen) following the manufacturer’s recommended protocol. RT-negative controls were performed to assess the presence of possible genomic contamination of RNA samples. PCR amplification was performed using 0.2 μl of cDNA, 10 pmol of gene-specific primers and HotStarTaq (Qiagen). The primers used were 1) -Gal specific, designed to amplify a 541-bp product (forward, 5'-GTTGTGCTGCAAAGGAGAGACTGGGAG and reverse, 5'-GCTCTGAGGAAGGCAGAGAGGTCA); and 2) actin specific (see above). After an initial denaturation step at 95°C for 15 min, PCR cycling conditions were 35 cycles of denaturation at 94°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 60 s, and a final extension at 72°C for 8 min. All PCR products were sequenced for verification.
-Gal activity assays. Tissues from male transgenic or nontransgenic mice were homogenized in 0.5 ml of 100 mM potassium phosphate (pH 7.8), 0.2% Triton X-100 in the presence of the protease inhibitors leupeptin (5 μg/ml) and AEBSF (0.2 mM). The samples were centrifuged at 4,000 g for 2 min to pellet cell debris. Total protein concentrations were determined in the supernatant (BCA kit; Pierce Chemical). -Gal activity was measured in triplicate 10-μl samples using the Galacto-Light Plus chemiluminescence reporter gene assay system (Applied Biosystems) according to the manufacturer’s protocol and a Victor-3 1420 Multi-label Counter (Wallac).
-Gal staining. Male transgenic mice were anesthetized with isoflurane and perfused via the heart with cold PBS (pH 7.4) for 2 min. Multiple tissues were harvested and cut into small (<1 cm2) sections. The kidney was dissected into inner medulla, outer medulla, and cortex. Tissues were immersion fixed in cold 4% paraformaldehyde on ice for 30 min and then subsequently washed in PBS several times. Samples were stained in the dark overnight at 37°C in a solution containing 50 mM Tris?HCl (pH 7.5), 15 mM NaCl, 1 mM MgCl2, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 0.5 mg/ml X-gal. Samples were washed multiple times with PBS, and kidney images were captured using a Leica MZ FL III Fluorescence Stereomicroscope. For the vas deferens, the sections were embedded in paraffin; 10-μm longitudinal sections were cut, mounted on glass slides, dewaxed with xylene, counterstained with Nuclear Fast Red (Sigma), and viewed with a Nikon E800 Upright microscope.
Immunoblotting. Mice were euthanized by decapitation and the kidney was dissected into inner medulla, outer medulla, and cortex. Protein preparation and immunoblotting were performed as previously described using an anti--Gal-purified antibody (Promega), diluted 1:1,000.
Immunocytochemistry. Kidneys were fixed by perfusion via the heart with cold PBS for 60 s, followed by 4% paraformaldehyde in PBS (pH 7.4) for 3 min. The kidneys were removed and the midregion was sectioned into 5-μm transverse sections and postfixed for 1 h in cold 4% paraformaldehyde/PBS, followed by 3 x 10-min washes with PBS. The tissue was dehydrated in a series of ethanol concentrations and left overnight in xylene. The tissue was embedded in paraffin, and 5-μm sections were cut (Histopath) and mounted on Superfrost Plus slides (VWR International). Staining was performed as described previously (16) using the following primary antibodies, mouse anti--Gal (Promega, diluted 1:1,000) or chicken anti-aquaporin-2 (diluted 1:1,000), and the following secondary antibodies, goat anti-mouse IgG, Alexa Fluor 680 and goat anti-chicken IgG Alexa Fluor 488 (Molecular Probes). After 1-h incubation at room temperature, coverslips were mounted with Vectashield mounting medium containing DAPI (Vector labs). Microscopy was performed with a Nikon E800 Upright microscope.
Effect of water restriction on UT-A promoter activity in vivo. All animal studies in this paper were done under National Institutes of Health Animal Care and Use Committee-approved animal protocols. Male transgenic mice were used for all studies. Animals were maintained in mouse metabolic cages (Hatteras Instruments) for the duration of the study, under controlled temperature and light conditions (12:12-h light-dark cycles). Mice received a fixed daily ration of gelled diet per day containing both food and water. The gelled diet was made up of 4 g of special low-NaCl synthetic food (0.001% Na wt/wt, Research Diets), 0.2 mmol NaCl, 25 mg agar, and either 1.7 ml (water restricted) or 5 ml (control) of deionized water. Five mice were used for each group. Mice did not have access to supplemental drinking water during this period. After 3 days of adaptation to the cages, urine was collected under mineral oil in preweighed collection vials for a 24-h period. Urine volume was measured gravimetrically assuming a density of one. Urine osmolality was determined using a vapor pressure osmometer (Wescor). Kidney inner medullas were dissected, one inner medulla was processed for real-time RT-PCR and the other for -Gal activity.
Real-time RT-PCR. Total RNA was extracted using a Ribopure kit (Ambion) according to the manufacturer’s guidelines. Real-time RT-PCR was performed as detailed in Ref. 8 using UT-A1 or UT-A3 isoform-selective primers (24).
Effect of glucocorticoids on UT-A promoter activity in vivo. Male transgenic mice were anesthetized with isoflurane for the subcutaneous implantation of osmotic minipumps (model 2001, Alzet) containing either dexamethasone (Sigma) in 20% DMSO/isotonic saline (5 mice) or control minipumps containing 20% DMSO/isotonic saline (5 mice). Dexamethasone was delivered at 2 μg?g body wt1?day1. For 6 days, mice received a fixed daily ration of gelled diet containing 1.7 ml of deionized water but also had free access to supplemental drinking water. Kidney inner medullas were dissected out and processed as described in -Gal activity assays. In an identical experiment, the kidney inner medullas were dissected and processed for real-time RT-PCR.
Statistical analysis. All values are quoted as means ± SE. Analysis was performed by t-tests or one-way ANOVA as appropriate, with significance assumed at the 5% level. If ANOVA indicated a significant difference, comparison between groups was performed with the Student-Newman-Keuls method.
RESULTS
Creation of transgenic mice. A 4.2-kb region upstream of the first coding exon of the UT-A gene, but containing the transcriptional start site of UT-A1 and UT-A3, was used to generate the UT-A-Gal transgene. A similar size region has been characterized previously and was shown to be sufficient for promoter activity in Madin-Darby canine kidney cells (10). The UT-A-Gal transgene was used to create transgenic mice by pronuclear microinjection using standard methodologies. Initially, 51 pups were analyzed for transgene expression by Southern analysis using a -Gal-specific probe (not shown). Five founder animals were identified and UT-A-Gal expression was confirmed by PCR analysis using transgene-specific primers. Real-time PCR and specific primers were used to estimate the transgene copy number in the founder animals, by comparing the threshold cycle (Ct) of the founder DNA to a standard curve generated from nontransgenic mouse DNA spiked with 0100,000 copies per cell equivalent of the transgene (Fig. 2). Founders 2470, 2471, and 2472 had 2, 6, and 10 copies per cell equivalent, respectively, of the transgene, founder 2473 had 110 copies, and line 2474 had 50 copies. Founder animals were crossed with C57BL6/J mice, and the offspring were analyzed. Both male and female offspring were analyzed for transgene expression from each of the five founder animals, but ultimately only two of the lines, 2473 and 2474, were found to functionally express the transgene. These animals transmitted the transgene to greater than 50% of their offspring and were used to propagate the transgenic line of mice for further experiments. All data shown are from the line 2473.
RT-PCR analysis of transgene expression. Multiple tissues were analyzed for transgene expression using primers specific for the -Gal transcript (Fig. 3). -Gal was only expressed in the kidney inner medulla (IM) of the tissues analyzed (Fig. 3A) and transgene expression was not evident in nontransgenic control mice (Fig. 3B). An identical reaction performed in the absence of reverse transcriptase confirmed that the IM-specific product did not result from genomic DNA contamination. Analysis of actin served as a positive control for the RT-PCR reaction.
Localization of -Gal. Of the tissues analyzed, X-Gal staining was only apparent in the kidney and vas deferens, whereas no staining was observed in testis, heart, lung, liver, and brain. In the kidney, X-Gal staining was localized to the papillary tip and was strongest in the terminal portion of this region (Fig. 4). To further characterize the localization of UT-A-Gal in the kidney, immunohistochemistry was performed using an antibody against E. coli -gal. Initially, the specificity of the antibody was determined by Western blotting of different kidney regions. A single 114-kDa band representing E. coli -gal was only detected in the kidney IM of transgenic positive animals and was not observed in any other kidney region or in nontransgenic control mice (Fig. 5). Staining of kidney sections using the same antibody localized -Gal immunoreactivity to the IMCD cells, whereas no staining was observed in the thin descending limb of the loop of henle (tDL) (representative images shown in Fig. 6, A and B). The staining of the IMCD was strongest in the terminal portion of the papillary tip, IMCD2 and IMCD3, and little staining was apparent in the base region, IMCD1 (Fig. 6C). Colocalization studies using an aquaporin-2 (AQP2)-specific antibody showed that -Gal staining was localized to AQP2 expressing cells in the IMCD (Fig. 6, D-F). Of the sections analyzed, from four different animals, there were zero -Gal-positive cells that did not contain AQP2, and virtually all AQP2-containing cells in the IMCD were also positive for -Gal, suggesting that -Gal expression is almost complete and localized to the IMCD principal cells.
In the vas deferens, staining was observed in cells surrounding the tubule lumen (Fig. 7, left). At higher magnification, staining was clearly localized to the columnar epithelial principal cells; the cells that form the apical membrane of the vas deferens tubule (Fig. 7, right).
-Gal activity. To further characterize transgene expression and as a means of examining the regulation of the UT-A-Gal transgene, -Gal enzyme activity was assayed in various tissues. A representative experiment is shown in Fig. 8. In UT-A-Gal mice, only kidney IM (20-fold) and vas deferens (15-fold) showed enzyme activity that was greater than nontransgenic control mice.
Effect of water restriction on UT-A promoter activity in vivo. UT-A--Gal mice were kept in metabolic cages and received either a water-restricted (1.7 ml water/day) or a control (5 ml water/day) diet. After 3 days, urine osmolality was significantly greater in the water-restricted group (3,317 ± 80 mosmol/kgH2O) compared with the control group (1,061 ± 22 mosmol/kgH2O). Kidney IMs were dissected and -Gal activity was measured in one IM, whereas the second IM was processed for real-time RT-PCR. -Gal activity was significantly greater in the water-restricted animals, 428 ± 62 relative luminescence units (RLU)/μg protein, compared with the control animals, 241 ± 59 RLU/μg, suggesting that the UT-A-Gal transgene could be regulated by osmolality or vasopressin (Fig. 9). Real-time RT-PCR using isoform-specific primers showed that the normalized mRNA levels of the UT-A3 isoform were significantly increased under water-restricted conditions (2.1 ± 0.22) compared with control conditions (1.0 ± 0.14), whereas the normalized mRNA levels of the UT-A1 isoform were not changed by water restriction (Fig. 10).
Effect of glucocorticoids on UT-A promoter activity in vivo. Glucocorticoids increase fractional urea excretion and decrease urea permeability and UT-A1 protein abundance in the IMCD (26). Further studies have shown that dexamethasone administration significantly decreases the activity of the rat UT-A promoter in LLC-PK(1)-GR101 cells and also decreases UT-A1 and UT-A3 mRNA expression (28). To determine whether the same effects of glucocortcoids could be observed in vivo, UT-A-Gal mice were administered dexamethasone for 6 days. -Gal activity in the IM of dexamethasone-treated mice was significantly lower than in control mice, 115 ± 29 RLU/μg compared with 273 ± 58 (Fig. 11), suggesting that the UT-A-Gal transgene was regulated similarly to the endogenous UT-A promoter. In an identical experiment, real-time RT-PCR using isoform-specific primers showed that the normalized mRNA levels of both UT-A1 and UT-A3 were significantly decreased after 6 days of dexamethasone administration (Fig. 12).
DISCUSSION
The kidney maintains body fluid homeostasis by a series of specific transport processes that occur throughout the highly differentiated nephron. Due to the multiple different cell types and functional heterogeneity of the nephron, standard gene-deletion methods are often unable to provide precise information about a protein’s specific role in any particular nephron segment. However, powerful new techniques to analyze gene function in a nephron-specific manner are now being used (reviewed in Refs. 3, 15, 33). The first requisite to generate these cell- or tissue-specific knockouts is to characterize the promoter of a particular gene to determine whether this region contains all the necessary elements that confer expression in a cell-specific manner.
The urea transporters, UT-A1 and UT-A3, are two members of the UT-A gene family. They are splice products of a single gene and the UT-A promoter region, situated at the 5'-end of the gene, drives their transcription (1, 10, 25). In the mouse, immunohistochemistry has localized both UT-A1 and UT-A3 to the terminal portion of the IMCD. Specifically, UT-A1 is expressed both in the apical region and cytoplasm, whereas UT-A3 is both intracellular and in the basolateral membrane of IMCD principal cells (12, 32). Together, they mediate rapid transepithelial urea transport across the IMCD and thus prevent a urea-induced osmotic diuresis (9). In the studies outlined in this manuscript, we used the mouse UT-A promoter to drive the expression of the reporter gene -Gal. The aim of the study was to determine whether the promoter fragment used contained the elements necessary to confer IMCD-specific gene targeting.
Transgenic mice were created with 4.2 kb of the mouse UT-A promoter driving the expression of a -Gal reporter gene. This fragment of the UT-A promoter was found to be sufficient to drive -Gal expression in both the kidney and, surprisingly, the vas deferens. In the kidney, -Gal expression was localized to the terminal portion of the papillary tip. Colocalization studies with AQP2 determined that -Gal was expressed in the principal cells of the IMCD, and no expression was observed in cells of the tDL. Within the IMCD, in the line 2473, virtually all AQP2-containing principal cells were also positive for -Gal, suggesting that the expression of the UT-A-Gal transgene was complete in this mouse line. However, importantly, -Gal expression was only evident in the IMCD2 and IMCD3 segments, and in contrast to AQP2, there was no staining of IMCD1, the OMCD and the CCD. This pattern of -Gal expression is consistent with the localization of UT-A1 and UT-A3 in the mouse kidney. In addition to IMCD-specific expression, the UT-A promoter region was sufficient to confer increased expression of -Gal in response to water restriction. The promoter fragment used in these studies contains a tonicity-enhancer element and previous studies in cell culture have shown that the promoter can be upregulated in response to either hypertonicity or increased cAMP levels (10). However, the use of this transgenic model has shown in vivo that the UT-A promoter can be regulated by prolonged antidiuresis. Interestingly, real-time RT-PCR studies revealed that, although their transcription is driven by the same promoter, only the mRNA transcript levels of the UT-A3 isoform were increased in response to water restriction. These results are similar to previous observations in mouse and suggest that there may be a difference in mRNA stability between the UT-A1 and UT-A3 mRNA transcripts; whether this is conferred by the different untranslated regions at the 3'-end of the mRNA transcripts remains to be determined.
The administration of dexamethasone to UT-A-Gal mice significantly reduced -Gal, UT-A1, and UT-A3 expression in the IMCD. These results are similar to those that have been observed previously in cell culture (28), indicating that the UT-A promoter is downregulated by glucocorticoids. However, it is unlikely that this atypical regulation is via a glucocorticoid response element (GRE) (28).
A novel finding from this study was that the UT-A promoter also targeted expression of -Gal to the columnar epithelial principal cells within the vas deferens. The vas deferens, along with the epydidymis, is part of a series of ducts responsible for the transport and maturation of sperm after they leave the rete testis. Within this ductal system, sperm is concentrated by water extraction and the composition of the luminal fluid is modified. Although further studies are required to determine the exact UT-A isoform expressed within the vas deferens, it is possible that the role of a urea transporter in this segment is to create an osmotic gradient between the vas deferens lumen and the interstitium and, with the aid of AQP2 also localized in this region (27, 30), provide a mechanism for water extraction and sperm concentration.
In the present study, two unexplained findings were that the UT-A-Gal transgene was not expressed in either the testis, where UT-A5 is expressed (11), or in the colon, where UT-A proteins have been localized using various UT-A-specific antibodies (31). Two possible explanations for the lack of -Gal activity in these tissues are that the UT-A transgene did not contain all the necessary "testis-specific" or "colon-specific" enhancer elements or that, in the case of UT-A5, a completely different internal promoter drives its transcription. Further studies are required to determine the mechanism of both colon- and testis-specific expression.
In summary, we show here that 4.2 kb of the 5'-flanking region of the UT-A gene directs expression to the IMCD principal cells of the kidney and that the transcriptional activity of the transgene can be regulated by either glucocorticoids or prolonged antidiuresis, similarly to the endogenous promoter. This transgenic model should facilitate future studies aimed at targeted gene deletion within the IMCD.
GRANTS
This study was funded by the Intramural Budget of the National Heart, Lung, and Blood Institute (National Institutes of Health to M. A. Knepper).
ACKNOWLEDGMENTS
The authors are grateful for the assistance of C. Liu, manager of the National Heart, Lung, and Blood Institute (NHLBI) Transgenic Core Facility at the National Institutes of Health (NIH), and C. A. Combs, manager of the NHLBI Light Microscopy Imaging Facility at the NIH. The authors also thank E. Johns for expert technical assistance.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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