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Originally published In Press as doi:10.1074/jbc.M608055200 on September 21, 2006

J. Biol. Chem., Vol. 281, Issue 46, 35296-35304, November 17, 2006
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The Human Renin Kidney Enhancer Is Required to Maintain Base-line Renin Expression but Is Dispensable for Tissue-specific, Cell-specific, and Regulated Expression*

Xiyou Zhoux{ddagger}, Deborah R. Davis§, and Curt D. Sigmund{ddagger}§1

From the {ddagger}Molecular and Cellular Biology Graduate Program, Departments of §Internal Medicine and Molecular Physiology & Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242

Received for publication, August 22, 2006 , and in revised form, September 20, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Renin is the rate-limiting enzyme in the renin-angiotensin system and thus dictates the level of the pressor hormone angiotensin-II. The classical site of renin expression and secretion is the renal juxtaglomerular cell, where its expression is tightly regulated by physiological cues. An evolutionarily conserved transcriptional enhancer located 11 kb upstream of the human RENIN gene has been reported to markedly enhance transcription in renin expressing cells in vitro. However, its importance in vivo remains unclear. We tested whether this enhancer is required for appropriate tissue- and cell-specific expression, or for physiological regulation of the human RENIN gene. To accomplish this, we used a retrofitting technique employing homologous recombination in bacteria to delete the enhancer from a 160-kb P1-artificial chromosome containing human RENIN, two upstream genes and one downstream gene, and then generated two lines of transgenic mice. We previously showed that human renin expression in transgenic mice containing the wild type construct is tightly regulated as is expression of the linked genes. Deletion of the enhancer had no effect on tissue-specific expression of human RENIN, but using the downstream gene as an internal control, found that human RENIN mRNA levels were 3-10-fold decreased compared with constructs containing the enhancer. Despite this decrease in expression, renin protein remained localized to renal juxtaglomerular cells and was appropriately regulated by cues that either increase or decrease expression of renin. Our results suggest that sequences other than the enhancer may be necessary for tissue-specific, cell-specific, and regulated expression of human RENIN.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Renin is the first and rate-limiting aspartyl protease in the renin-angiotensin system (RAS)2 cascade that leads to the production of angiotensin-II (Ang-II) by the consecutive cleavage of angiotensinogen by renin and angiotensin-I by angiotensin converting enzyme. Ang-II is an important regulator of cardiovascular function, stimulating vasoconstriction, cardiac output, aldosterone synthesis, and sympathetic activity, thereby directly and indirectly enhancing peripheral vascular resistance and renal tubular water and salt reabsorption. The critical importance of the RAS is evidenced by gene targeted ablation of the RAS genes in mice. The elimination of any RAS gene in mice causes cardiovascular consequences that include hypotension and renal insufficiency, and developmental consequences including postnatal lethality (1-4). The cardiovascular and developmental defects can be complemented in mice by substitution of the mouse RAS with their human orthologs (5) or by targeted expression of Ang-II (6). In humans, genetic defects in the RAS were reported to cause renal tubular dysgenesis, hypotension, and early stillbirth (7). Consequently, both the mouse and human genetic data illustrate the importance of the system both developmentally and in adults.

Renin is primarily expressed in renal juxtaglomerular (JG) cells in the wall of the afferent arteriole proximal to the glomerulus. Its expression is tightly regulated by renal baroreceptor, macula densa, beta-adrenergic receptor, and Ang-II receptor-mediated mechanisms. For example, Ang-II elicits negative feedback on renin synthesis and secretion, whereas inhibition of Ang-II synthesis caused by angiotensin converting enzyme inhibition increases renin synthesis. Despite our descriptive understanding of physiological cues that regulate renin expression, the molecular mechanisms operant in vivo remain unclear. Recent advances in the development of sophisticated mouse models is beginning to provide clues to understand the mechanisms regulating the RENIN gene. For example, using cell-specific cre-LoxP-mediated gene ablation, Chen et al. (8) recently reported that the G-protein G{alpha}s is an essential component of the signal transduction machinery regulating Renin expression and release (8). However, exactly what DNA sequences control the tissue-specific, cell-specific, and regulated expression of the RENIN gene in vivo remain unclear.

Previous studies by us and others have identified a potent transcriptional enhancer 2.6 kb upstream of the mouse Renin (mRen) gene (9). A sequence with 75-85% homology was also identified ~6 kb upstream of the rat and 11-kb upstream of the human RENIN (hREN) gene (10, 11). Detailed analysis of the enhancer using an immortalized renin-expressing JG cell line in vitro has led many to conclude that a minimum of 10 different transcription factors exhibiting either stimulatory and inhibitory effects on renin expression may bind to the enhancer (12-16). Sequences in the enhancer also appear necessary to mediate the inhibitory effect of cytokines (17-21). Consequently, this sequence becomes an important candidate as a major regulator of Renin expression. Recently, the Morris laboratory (22), using a gene targeted deletion of the mouse Renin enhancer, reported diminished levels of juxtaglomerular renin protein. Their conclusion that the enhancer is a critical regulator of Renin gene expression was made without defining the tissue-specific expression of renin or measuring the level of renal Renin mRNA. Moreover, their deletion, whereas encompassing the enhancer, extended 349 bp upstream and 137 bp downstream of the enhancer. The function of these additional 5'-flanking sequences remains unclear.

We previously reported that transgenic mice carrying a 160-kb P1 artificial chromosome (PAC) encoding the hREN gene including 75 kb of upstream and 70 kb of downstream sequences exhibited appropriate tissue-specific, cell-specific, copy number proportional, and exquisitely regulated expression in response to cues that either stimulate or inhibit renin expression (23, 24). Moreover, expression of neighboring upstream (GOLT1A and KISS1) and downstream (ETNK2) genes also encoded on PAC160 were appropriately expressed (25). Therefore to develop a robust experimental platform to accurately and completely assess the importance of the hREN enhancer in vivo, we used a PAC/BAC retrofitting technique to delete the kidney enhancer (termed KE) (26). We then characterized the expression and regulation of hREN in PAC160{Delta}KE transgenic mice using the neighboring genes as internal controls. This has major advantages over traditional promoter-reporter gene approaches that often suffer from ectopic expression due to position artifacts, exhibit variegated expression, highly variable expression not in proportion to copy number, and generally lack internal controls to normalize the level of transgene expression. We report herein a retention of appropriate tissue- and cell-specific expression and appropriate regulation of hREN even after deletion of the kidney enhancer. However, loss of the enhancer causes a 3-10-fold decrease in base-line hREN expression. We conclude that the enhancer is not required for appropriate spatial hREN expression but is required to maintain the normal level of base-line expression.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of the Kidney Enhancer-deficient PAC160 Construct—The PAC160 (Genome Systems control number 4917) was used as the template for deletion of the human kidney enhancer (KE) as previously reported (23, 26). Briefly, ~500 bp upstream and downstream of the KE (KE-L and KE-R) were PCR amplified and cloned between two clusters of three adjacent {chi}-site sequences in the vector pRM4-N (27). Chi sequences are known to facilitate homologous recombination in Escherichia coli. The flanking sequences were designed to specifically delete the 241-bp KE (GenBankTM accession number AF140238 [GenBank] ) segment without altering adjacent sequences. Another PCR fragment was cloned containing a LoxP511-flanked chloramphenicol resistance (CAM) marker used for selection in bacteria. LoxP511 is a cre-recombinase recognizable heterotopic form of LoxP that does not recombine with the wild type LoxP site in the PAC160 parent vector. The LoxP511-CAM-LoxP511 was then inserted between KE-L and KE-R. The targeting construct was linearized and transformed to E. coli strain MC1061 containing the PAC160 with selection for kanamycin (on the PAC vector) and CAM resistance. Colonies were screened to ensure they were ampicillin sensitive. The PAC160{Delta}KE+CAM vector was isolated and subjected to battery of PCR and Southern blots to ensure faithful recombination. This vector was then transformed into E. coli strain BS591 (a gift of Brian Sauer), which constitutively expresses cre-recombinase, to excise the CAM gene. Kanamycin-resistant, CAM-sensitive colonies were selected for further analysis. The PAC160{Delta}KE vector was isolated and purified using the large construct DNA isolation kit from Qiagen. Again, this PAC was subjected to a number of PCR and Southern blot analysis to ensure fidelity of the KE deletion. Sequence analysis confirmed the precise replacement of the KE with a single LoxP511 site.

PCR and Southern Blot Analysis of PAC160{Delta}KE Mice—Microinjection of PAC160{Delta}KE was performed by the University of Iowa Transgenic Animal Facility as described previously (23). Two lines of founder mice, 17853/2 (PAC160{Delta}KE2) and 26913/1 (PAC160{Delta}KE4), were initially identified by hREN-specific PCR. Spleen DNA from PAC160{Delta}KE and PAC160 control mice was isolated as described previously. 200 ng of genomic DNA was used for PCR. 10 or 20 µg of genomic DNA digested with HindIII or MscI was used for standard Southern blot hybridization. The hREN cDNA, KE, and KE-L probes were labeled with Rediprime II Random Primer Labeling System (Amersham Biosciences).

RNase Protection Assay (RPA)—Mouse RNA was isolated using Tri-Reagent (Molecular Research Center Inc., Cincinnati, OH) as described by the manufacturer. The RPAIII Kit (Ambion, Austin, TX) was used to analyze the expression of hREN, mRen, ETNK2, and GOLT1A genes. The size of the protected fragments are 300, 326, 225, and 412 bases, respectively. Three different loading controls (Ambion) were used, 28S (115 nucleotides), beta-actin (245 nucleotides), or cyclophilin (105 nucleotides). Total RNA (50 µg) was hybridized with 2 x 105 dpm of hREN, ETNK2, and GOLT1A probes, and 5 x 104 dpm of loading controls (28S, beta-actin, and cyclophilin). RNase digestion was performed with RNase A and RNase T1 at a dilution of 1:75 for 45 min. Quantifications of RPA results were performed with PhosphorImager and ImageQuant software (GE Healthcare).

Treatment of Transgenic Mice—Captopril was dissolved in the drinking water (0.5 mg/ml) and administered to mice for 10 days, whereas control mice were given only drinking water as the vehicle. For Ang-II treatment, all mice were first trained for 7 days for tail-cuff measurement of blood pressure using the BP-2000 apparatus (Visitech System Apex, NC). Systolic blood pressure was then measured for 4 consecutive days. Microosmotic mini-pumps (Alzet; model 1007D) were implanted subcutaneously in mice under ketamine/xylazine anesthesia. The pumps contained either saline (vehicle) or a pressor dose (1000 ng/kg/min) of Ang-II (Sigma). Blood pressure was then measured for the next 5 days before sacrificing the mice. For each day of measurement, 10 practice cuff inflations were followed by 20-30 test inflations. Systolic blood pressure data were transferred to Excel, and any blood pressure datum outside the range of mean ± 2 S.D. was discarded before further statistics analysis. Kidneys were collected on dry ice and stored at -80 °C.


Figure 1
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  		FIGURE 1.
Generation of the PAC160{Delta}KE transgene. A, a schematic representation of the hREN gene, the kidney enhancer (KE, solid square), chorionic enhancer (CE, checkerboard square), and neighboring genes (gray arrows) in PAC 160 are shown. The relative direction of transcription is indicated by the direction of the arrows. The two terminal genes PEPP3 (also called PLEKHA6) and Sox 13 are truncated by the end of the PAC160 clone as indicated by open boxes. The GOLT1A and KISS1 genes lie upstream of hREN, whereas ETNK2 lies directly downstream. The LoxP site present in the PAC parent vector is indicated by the solid arrowhead. B, flow chart for generation of the {Delta}KE knock-out. Homologous sequences (~500 bp each) upstream (KE-L) and downstream (KE-R) of KE were used to flank a CAM surrounded by heterotopic LoxP511 sites (crosshatched arrowheads). The entire construct was flanked by 3 repeats of {chi}-sites (GGTGGTCG) to facilitate homologous recombination. After selection for homologous recombinants, the LoxP511 flanked CAM gene was removed by propagation in E. coli constitutively expressing cre-recombinase. C, PAC160{Delta}KE is identical to PAC160 except the KE is replaced by a single LoxP511 site.

 
All mice were fed with standard mouse chow (LM-485; Teklad Premier Laboratory Dies) and water ad libitum. Care and use of mice met the standard set forth by the National Institutes of Health and all procedures were approved by the University Animal Care and Use Committee at the University of Iowa.

Immunofluorescence and Confocal Microscopy—Antibodies against hREN or mREN were previously demonstrated for their specificity (23, 28, 29). They were kindly provided by Dr. Walter Fischli, (Hoffmann-La Roche, Basel, Switzerland) and Dr. Kenneth W. Gross (Roswell Park Cancer Institute, Buffalo, NY), respectively. Two to seven mice for each line were perfused transcardially and fixed with 4% paraformaldehyde. The kidneys were frozen in OCT compound and sectioned 5-8-µm thick using a Cryocut 1800 cryostat (Leica). Frozen kidney sections were incubated with hREN antibody (x1:250 dilution), then detected with fluorescein isothiocyanate-labeled goat anti-rabbit secondary antibody (x1:100 dilution, Pierce). To study the co-localization of hREN and endogenous mREN, antibodies to both renins were used with fluorescein isothiocyanate-labeled donkey anti-rabbit secondary antibody (x1:40 dilution, Santa Cruz) and rhodamine-labeled donkey anti-mouse secondary antibody (x1:100 dilution, Santa Cruz). The images were observed with a fluorescence microscope (Nikon Eclipse E600) equipped with Spot advanced digital camera (Diagnostic Instruments, Inc.) or Bio-Rad MRC1024 laser confocal microscope. All photos in each experiment were taken with the same exposure. Image J (National Institutes of Health) and Adobe Photoshop were used to minimally process all figures. In most cases, brightness and contrast were increased until the biological tissue and background fluorescence could be clearly observed. The same setting was used on control and experimental sections.

Statistical Analysis—All results were compared by Student's t test using the SigmaStat software (SPSS Scientific). Values are shown as mean ± S.E. A value of p < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
To assess the contribution of the KE in the regulation of hREN gene expression we utilized homologous recombination in bacteria to accurately delete the KE sequence. This retrofitting strategy involved: 1) cloning short homologous segments (500 bp) upstream (KE-L) and downstream (KE-R) of the target deletion; 2) the use of an easily selectable marker (CAM) for recombination in bacteria; 3) heterotopic LoxP511 sites that can facilitate cre-mediated recombination with each other but not with the wild type LoxP present in the PAC parent vector; and 4) {chi}-sites to facilitate homologous recombination in E. coli. Bacterial cells containing the PAC160 vector were transformed with the targeting vector, recombinants were selected, and the CAM gene was then removed by propagation in bacteria expressing cre-recombinase (Fig. 1). This strategy deleted the KE, leaving a single LoxP511 site as a footprint of the deletion (Fig. 1C). PAC160{Delta}KE recombinant clones shown to faithfully delete the KE were identified and characterized by a series of PCR, Southern blot, sequencing, and fingerprint experiments (26).

Four transgenic lines containing PAC160{Delta}KE were initially identified by PCR using primers within hREN. Southern blot analysis of HindIII-digested genomic DNA using an hREN cDNA as probe indicated that all four lines contained the entire hREN gene (Fig. 2), but only two ({Delta}KE2 and {Delta}KE4) were shown to be fully intact when the Southern blot was probed either with the entire PAC160, or a probe derived from GOLT1A (data not shown). PAC160{Delta}KE2 and PAC160{Delta}KE4 were also found to contain an intact copy of the chorionic enhancer, a sequence located ~5 kb upstream of the transcription start site that was reported to be active in choriodecidual cells (30), but has little homology with sequences upstream of the mouse or rat renin genes. To verify the absence of the KE in transgenic mice carrying PAC160{Delta}KE2 and PAC160{Delta}KE4, we first performed PCR using primers inside the KE-L and KE-R homology and analyzed the sequence. Sequence analysis clearly showed a faithful deletion of the KE and replacement with a single LoxP511 site. We next performed Southern blot analysis using DNA flanking the KE (KE-L) as a probe. As expected, a higher molecular weight band was identified in both PAC160{Delta}KE transgenic lines consistent with loss of a MscI site in the enhancer (Fig. 2C, top). When the blot was stripped and reprobed with a KE probe, bands were detected in the wild type PAC160 mice, but not the PAC160{Delta}KE mice, nor the non-transgenic controls (Fig. 2C, bottom).

We next examined the tissue-specific expression of hREN in PAC160 transgenic mice retaining an intact KE and in both lines of mice lacking the KE. To accomplish this, we performed multiplex RNase protection assays using probes to hREN, 28S, and two neighboring genes ethanolamine kinase 2 (ETNK2) and Golgi transport 1 homolog A (GOLT1A) that exhibit overlapping but distinct tissue-specific expression profiles (Fig. 3). This provided a mechanism to not only assess the effect of the KE on hREN, but also on its neighboring genes, as well as to provide an internal control in the occurrence that loss of the KE dramatically affected the level of hREN expression. In wild type PAC160 mice, hREN expression was evident in kidney and to a much lower content in lung. Consistent with our previous report, ETNK2 was abundantly expressed in the kidney, liver, and testes (25), whereas GOLT1A was primarily expressed in the liver and at lower levels in kidney, brain, and lung. Deletion of the KE did not dramatically alter the tissue specificity of any of the three genes examined. Expression of renal hREN was lowest in PAC160{Delta}KE2 that exists in 1-2 copies per genome, and higher in PAC160{Delta}KE4, which exists in several copies per genome.


Figure 2
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  		FIGURE 2.
Southern blot of PAC160{Delta}KE transgenic mice. A, a schematic representation of the region surrounding the hREN gene showing the approximate location of HindIII and MscI restriction sites. Probe locations are indicated by the solid boxes. B, the hREN cDNA probe was used to hybridize to HindIII-digested genomic DNA purified from PAC160 transgenic mice. The cDNA detects three hREN-specific (h) and two endogenous mRen-specific (m) bands. P160 DNA is purified PAC160 DNA grown in bacteria. C, the KE probe is derived from the KE sequence deleted in PAC160{Delta}KE, whereas the KE-L probe is located directly upstream of the KE. The KE-L (top) and KE (bottom) probes were used to hybridize to MscI-digested genomic DNA purified from PAC160 transgenic mice. The KE probe only detects bands in WT PAC160 mice, whereas the KE-L probe detects bands of difference sizes in all mice. The larger band in {Delta}KE mice is due to loss of one MscI site in the KE. WT1 and WT2, two lines of wild type PAC160 mice. WT3, one line of PAC140 mice. NT, non-transgenic. {Delta}2, PAC160{Delta}KE line 2; {Delta}4, PAC160{Delta}KE line 4; {Delta}17 is a line of PAC160{Delta}KE that also lacks the chorionic enhancer (CE). Both gels are derived from the same Southern blot first probed with KE-L, stripped, and then reprobed with KE. The gels are representative of at least 4 different Southern blots.

 


Figure 3
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  		FIGURE 3.
Tissue-specific expression of the PAC160{Delta}KE transgenes. Representative multiplex RNase protection assays of hREN, GOLT1A, ETNK2, and 28S expression from total tissue RNA (50 µg) from one line of WT and two lines of PAC160{Delta}KE mice are shown. The location of the protected fragments are indicated. The RPAs are representation of n = 2-3 mice per construct. Tissues labels are: brain (B), heart (H), kidney (K), liver (Lv), lung (Lg), skeletal muscle (Sk), submandibular gland (Sg), and testis (T).

 
Although the KE appears dispensable for appropriate tissue-specific expression of hREN (and its closely linked genes), a comparison of the ratio of renal hREN to ETNK2 expression in the PAC160{Delta}KE2 and PAC160{Delta}KE4 lines suggested diminished expression of hREN compared with wild type PAC160 mice. To formally test the possibility that loss of the enhancer diminishes the level of base-line hREN mRNA, we evaluated hREN expression in kidney samples from a line of PAC160 (line 2, n = 10) and PAC160{Delta}KE (line 4, n = 9) that exhibit similar levels of ETNK2 (one of two internal controls). RPA analysis clearly shows equivalent levels of ETNK2 and cyclophillin, but lower levels of hREN in kidney RNA from PAC160{Delta}KE4 mice (Fig. 4, A and B). Quantification of the results shows a 2.8-fold decrease in hREN mRNA when compared with either cyclophillin or ETNK2 (Fig. 4, C and D). A comparison of the hREN/ETNK2 ratio of PAC160{Delta}KE4 with another line of wild type PAC160 mice similarly revealed a 2.7-fold decrease in hREN expression (Fig. 4E). Because the copy number PAC160{Delta}KE2 is significantly lower than that in either line of PAC160 wild type mice, we compared the ratio of hREN/ETNK2 with a line of PAC140 wild type mice that has a similar copy number. The PAC140 construct is similar to PAC160, exhibits copy number dependent expression, but differs in the extent of flanking sequences (75 kb upstream and 70 kb downstream in PAC160 versus 35 kb upstream and 90 kb downstream in PAC140) (23). In this comparison, loss of the KE caused an 11-fold decrease in base-line hREN expression suggesting that the loss of the KE may be greater in mice with fewer copies of the transgene (Fig. 4F).


Figure 4
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  		FIGURE 4.
Influence of KE deletion on hREN expression. A, a representative RPA was performed with total kidney RNA (50 µg) from independent kidney samples from one line of wild type (WT) PAC160 and one line of PAC160{Delta}KE mice. The positions of the hREN, ETNK2, and Cyclophilin protected products are indicated. B-D, RPAs were quantified using the phosphorimager software and ratio of ETNK2/Cyclophillin (B), hREN/Cyclophillin (C), and hREN/ETNK2 (D) are shown. Filled bars, WT2 (n = 10); crosshatched bars, PAC160{Delta}KE4 (n = 9). E, the hREN/ETNK2 ratio comparing PAC160{Delta}KE4 (n = 10) with a different line of WT mice (n = 6) is shown. F, the hREN/ETNK2 ratio comparing PAC160{Delta}KE2 (n = 9) with a line of PAC140 WT mice with similar copy number (n = 9) is shown. *, p < 0.001 versus control by Student's unpaired t test.

 


Figure 5
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  		FIGURE 5.
Immunostaining of mouse and human renin in kidney. Representative confocal immunofluorescent images of frozen kidney sections from PAC160-WT (A-C) and PAC160{Delta}KE4 (D-F) transgenic mice immunostained with mouse renin antisera (A and D), human renin antisera (B and E), and merged (C and F). Scale bars represent 20 µm.

 
To address whether the KE is required for JG cell-specific expression of hREN we performed immunohistochemistry for mouse and human renin using antisera specific to each (23, 28, 29). JG cell-specific co-expression of both mouse and human renin was clearly demonstrated in kidney from wild type PAC160 (Fig. 5, A-C) and in the higher copy number PAC160{Delta}KE4 (Fig. 5, D-F). Because the expression of renal hREN mRNA was lower in PAC160{Delta}KE2 we performed immunohistochemistry for human renin alone to avoid misinterpreting a carry over signal from the higher mouse renin. A low level of human renin, likely in a single JG cell per positive JG apparatus was detected in PAC160{Delta}KE2 mice (Fig. 6, B, and C, arrows), but not in kidney sections from non-transgenic littermate controls (Fig. 6A). Interestingly, the level of human renin, the number of positive JG cells per apparatus, and the number of positively labeled JG apparatuses was increased in PAC160{Delta}KE2 mice treated with the angiotensin converting enzyme inhibitor Captopril (Fig. 6, E and F), but not in Captopril-treated controls (Fig. 6D). Similarly, there was an increase in the level and number of JG cells labeled by mouse and human renin in PAC160 wild type (Fig. 7, D-F) and PAC160{Delta}KE4 mice treated with Captopril (Fig. 7, G-I), whereas no human renin signal was detected in Captopril-treated non-transgenic mice (Fig. 7B). We quantified the induction of renal mRen and hREN mRNA of vehicle and Captopril-treated mice (Fig. 8). Captopril caused a significant induction of both mRen (3.75-, 6.2-, and 3.5-fold) and hREN (5.0-, 7.4-, and 9.9-fold) mRNA in PAC160, PAC160{Delta}KE4, and PAC160{Delta}KE2, respectively.

Provided evidence for stimulation of hREN mRNA in response to inhibition of Ang-II synthesis, we sought to determine whether the KE is required to mediate a repression of hREN in the presence of exogenously administered Ang-II. PAC160 and PAC160{Delta}KE mice were treated with Ang-II by osmotic mini-pumps for 5 days after which renin expression was measured by RPA (Fig. 9). Control mice were treated with saline in the mini-pumps. Saline caused a non-significant 6.6 ± 5.0 mm Hg rise in blood pressure, whereas Ang-II elevated blood pressure by 29.5 ± 4.7 mm Hg. Ang-II treatment reduced the level of hREN mRNA irrespective of the presence of the KE (3.1-fold in PAC160 and 3.4-fold in PAC160{Delta}KE4). These data clearly show, that whereas the kidney enhancer is necessary for maintaining base-line renin expression, it is dispensable for tissue-specific, cell-specific, and regulated expression of the hREN gene.


Figure 6
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  		FIGURE 6.
Immunostaining of human renin in kidney. Representative immunofluorescent images of human renin staining in the kidney of non-transgenic (A and D) and PAC160{Delta}KE2 (B, C, E, and F) that were either vehicle (VEH, A-C) or Captopril-treated (CAP, D-F). The arrows point to human renin-stained cells.

 


Figure 7
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  		FIGURE 7.
Immunostaining of mouse and human renin in kidney from Captopril-treated mice. Representative confocal immunofluorescent images of frozen kidney sections from non-transgenic (Non-Tg, A-C), PAC160-WT (D-F), and PAC160{Delta}KE4 (G-I) transgenic mice immunostained with mouse renin antisera (A, D, and G) and human renin antisera (B, E, and H), or merged (C, F, and I). Scale bars represent 20 µm. Note the complete absence of immunostaining for human renin in non-transgenic controls (B).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We tested the hypothesis that the KE mediates tissue- and cell-specific expression of hREN, and is required for regulated expression in renal JG cells in response to physiological cues. Our results show that the KE is dispensable for these functions, but is required for the maintenance of base-line levels of hREN mRNA. The KE was originally identified as a sequence that strongly stimulated activity of the renin promoter in As4.1 cells, a renin expressing cell line derived from the kidney with many properties of JG cells (9). Mutational and biochemical analysis revealed the enhancer to contain the binding sites for CREB, CREM, RAR{alpha}/RXR, USF1/2, NFI, and SP1/3, all of which are required to maximally stimulate transcriptional activity of the renin promoter (12, 14, 16). However, in addition to this clustering of stimulatory transcription factors, binding sites for inhibitory factors were also identified. An NF-Y binding site overlaps with the binding site for RAR{alpha}/RXR and may thus prevent the binding of factors to the RARE (13). Ear2, an orphan member of the steroid hormone superfamily is a potent inhibitor of thyroid hormone receptor, luteinizing hormone receptor, and renin expression (15, 31, 32). In the renin enhancer, Ear2 shares the same binding site as RAR{alpha}/RXR. The enhancer has also been implicated as a target of inhibitory cytokines (17, 19, 21, 33), and NF{kappa}B was reported to inhibit renin transcription in response to tumor necrosis factor-{alpha} via interference with CREB binding to the CRE in the enhancer (18). Based on all the in vitro studies, we considered the enhancer to be a complex regulatory element capable of both stimulating and inhibiting the renin promoter depending on the physiological status of the renin expressing cell and the complement of transcription factors binding at any given time. Perhaps, given the results of the current study, its original definition as a transcriptional enhancer, a sequence that can stimulate transcriptional activity in an orientation and position independent manner remains the most accurate.

The in vivo definition of the KE as an element required to maintain base-line levels of renin is consistent with the recent results of Adams et al. (22). They generated a germ line deletion of the KE and an additional 486-bp flanking the enhancer by gene targeting the endogenous Ren-1c locus in C57BL/6J mice and showed a loss of renin protein-containing JG cells under base-line and low salt diet conditions. This loss of renin staining is consistent with the 3-10-fold decrease in renal hREN mRNA measured in our PAC160{Delta}KE mice. However, it becomes necessary to ask whether their conclusion that the renin enhancer is critical for control of renin gene expression is justified. Whereas our results of lower base-line hREN expression are consistent with this conclusion, our results demonstrating that the hREN KE is not necessary for normal tissue specificity or regulation by angiotensin converting enzyme inhibition and Ang-II are in opposition to that conclusion. Consequently, a determination of the residual level of renin mRNA in the kidney and elsewhere in their KE-null mice is necessary. Arguing that there must be residual renin expression even after deletion of the mRen KE and its flanking sequence is their genetic data reporting no lethality and their cardiovascular data reporting a blood pressure reduction of 9 mm Hg. On the contrary, renin null mice generated by directly targeting the renin locus in the same genetic background exhibit 80% lethality within a few days of birth, hydrone-phrosis, thickening of renal arterial walls, and fibrosis (4). Moreover, renin null mice exhibit a 32 mm Hg decrease in blood pressure. The renin null phenotype is consistent with the null phenotype of other RAS genes (1-3, 34). Therefore it is likely that the KE-null is a renin-hypomorph and not a renin-null thus supporting our conclusion.


Figure 8
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  		FIGURE 8.
Induction of renal hREN and mRen mRNA in response to Captopril. Total kidney mRNA was isolated from the indicated lines and analyzed by RPA using probes specific for either mRen or hREN and normalized for expression of beta-actin. The qualification of the RPAs was performed as described under "Experimental Procedures." Levels of gene expression were expressed as a ratio of renin/actin. Crosshatched bars, vehicle treated; closed bars, Captopril treated. PAC160-WT2 (n = 5), PAC160{Delta}KE4 (n = 10), PAC160{Delta}KE2 (vehicle n = 4, Captopril n = 5). *, p < 0.001 versus vehicle-treated mice.

 


Figure 9
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  		FIGURE 9.
Repression of renal hREN and mRen mRNA in response to Ang-II infusion. Total kidney mRNA was isolated from the indicated lines and analyzed by RPA using probes specific for either mRen or hREN and normalized for expression of beta-actin. The qualification of the RPAs was performed as described under "Experimental Procedures." Levels of gene expression were expressed as a ratio of renin/actin. Crosshatched bars, saline treated; closed bars, Ang-II treated. Saline treatment caused an increase in blood pressure by 6.6 ± 5.0 mm Hg, whereas pressor Ang-II (1000 ng/kg/min) caused a 29.5 ± 4.7 mm Hg increase in blood pressure (p = 0.003). PAC160-WT1, vehicle n = 5, Ang-II n = 7; PAC160{Delta}KE4, vehicle n = 5, Ang-II n = 7. *, p < 0.001 versus saline-infused mice. {dagger}, p < 0.03 versus saline-infused mice.

 
If the KE enhancer is indeed dispensable, what other sequences control renin expression in vivo? Pinet and colleagues (30) identified a second enhancer 5 kb upstream of the hREN promoter that was highly active in choriodecidual cells and less active in Calu-6 and As4.1 cells. We feel that this sequence is an unlikely candidate as it is not conserved in any other renin gene. However, other sequences upstream of the KE enhancer between KISS1 and REN exhibit a high degree of sequence conservation across species. In particular, two sequence blocks located at approximately -15 and -17 kb exhibit strong conservation in many mammals and therefore could potentially be considered candidates for the type of analysis described in this report.

The other obvious candidate region is the proximal promoter itself. Early studies of the proximal promoter implicated both Pit-1 and CREB binding sites as important elements regulating renin expression (35-37). More recently, the liver X receptor has been implicated as both a cAMP responsive transcription factor regulating renin expression, and a factor responsible for repression of renin expression in non-renin expressing tissues such as the liver (38, 39). Perhaps the most intriguing data implicating the proximal promoter comes from a developmental analysis of renin expression. During development, renin expressing cells in the metanephric kidney are first identified surrounding growing and branching blood vessels. Renin expression becomes progressively more restricted as the arterial tree branches, until only mature JG cells express the gene (reviewed in Ref. 40). The elegant gene targeting and lineage mapping studies of Sequeira Lopez et al. (41) suggest that renin-expressing cells identified early in renal development are precursors of cells that ultimately differentiate into cell types that do not normally express renin in the adult kidney, but retain the potential to re-synthesize renin when homeostasis is threatened. In establishing a link between the developmental profile of renin expression and transcriptional regulation, Gross and colleagues (42) reported that the Abd-B class of Hox transcription factors (HoxD10 and its binding partner PBX1b) binds to a sequence at -60 in the proximal promoter and is necessary for maximal renin promoter activity. They also reported that CBF1, a transcriptional repressor converted to an activator by the intracellular domain of notch, and Ets-1, a factor implicated in cellular proliferation and differentiation, both have conserved binding sites in the proximal promoter and regulate renin expression (43). Clearly, future studies will be necessary to directly link these developmentally regulated transcriptional pathways with the developmental and pluripotential nature of the renin expressing phenotype.

Another important clue implicating the proximal promoter region comes from our previous studies (44, 45) examining hREN expression in transgenic mice containing much shorter genomic constructs. Genomic constructs consisting of all exons and introns, but extending only 140 or 896 bp upstream of the gene exhibit JG cell-specific expression in kidney (44, 45). Despite this renal cell specificity, ectopic expression in other tissues was evident and the transgenes were inappropriately regulated (46). Ectopic expression was eliminated and appropriate regulation restored in transgenic models employing substantially more 5'-flanking sequences (23, 47). Similarly, transgenic mice containing constructs consisting only of short hREN 5'-flanking regions fused to reporter genes generally failed to exhibit correct expression (48, 49). One recent study fusing 12.2 kb of the hREN promoter including the KE to cre-recombinase exhibited cre activity in JG cells but also the renal medulla suggesting either some ectopic expression or the identification of a renal medullary lineage of renin expressing cells (50). In aggregate, these data suggest the proximal promoter may be necessary and sufficient to target renal JG cells, but that a combination of other sequences either upstream of the gene (e.g. KE and other conserved non-coding sequences), within the gene itself (e.g. intronic sequences), or downstream of the gene may be required to confer appropriate tissue specificity and regulation. Evidence supporting the regulation of renin mRNA stability has also been reported suggesting that post-transcriptional mechanisms may also play an important but indeterminate role in vivo (51-53).

It is widely accepted that transcriptional enhancers can operate over large distances both upstream and downstream of genes. Therefore, it is useful to consider that a genetic locus is a continuum of DNA that stretches upstream and downstream and perhaps beyond neighboring genes. Boundary elements or insulators separate loci from each other providing protection from nearby regulatory elements. Position effects in transgenic mice are thought to occur due to the juxtaposition of the transgene near regulatory elements controlling a gene at or near the insertion. In this light, there are no studies that have examined the region downstream of the renin gene, between renin and ETNK2. This may be of particular relevance considering that the genes are only 3 kb apart and exhibit overlapping yet distinct expression profiles. Indeed, ETNK2 is highly expressed in the liver, whereas renin is not. Whether these sequences act as insulators and are mechanistically involved in renin expression remains unknown.

Finally, we need to determine whether the KE has species-specific effects. In other words, does loss of the mRen KE have more dramatic effects on renin expression than loss of the hREN KE? There is circumstantial evidence that this may indeed be the case. First, it is well documented that renin levels in mice are much higher than in humans suggesting the enhancer may be more active in mice. Second, although the physiological significance remains unclear, the position of the enhancer is much closer to the mRen promoter (-2.6 kb) than the hREN promoter (-11 kb). Third, although the enhancer is conserved among mammalian renin genes, there are some critical differences that markedly effect the level of enhancer activity. For example, although the sequence of the mouse and human renin enhancers are identical with respect to the CRE (element-d), E-box (element-e), and one half-site of the RARE (element-c), the other half-site (element-b) of the RARE is different rendering the hREN enhancer much less active than the mRen enhancer (10, 11). Given this, it is surprising that the hREN enhancer lacks the NF-Y binding site (element-a) that, in the mRen enhancer, overlaps the RARE and inhibits its activity (13). Perhaps the reduced activity of the hREN enhancer caused by its distance from the promoter, or by alterations in the RARE sequence eliminated the selective pressure needed to retain a sequence with inhibitory properties (the NF-Y binding site). Therefore, future studies must focus on both the mouse and human renin genes to precisely determine which alternative sequences are physiologically important in the control of renin expression.


   FOOTNOTES
 
* This work was supported National Institutes of Health Grants HL48058, HL61446, and HL55006. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: 3181B Medical Education and Biomedical Research Facility, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242. Tel.: 319-335-7604; Fax: 319-353-5350; E-mail: curt-sigmund{at}uiowa.edu.

2 The abbreviations used are: RAS, renin-angiotensin system; Ang-II, angiotensin-II; JG, juxtaglomerular; mRen, mouse renin; hREN, human renin; PAC, P1 artificial chromosome; GOLT1A, Golgi transport 1 homolog A; ETNK2, ethanolamine kinase 2; KE, kidney enhancer; CAM, chloramphenicol resistance; CREB, cAMP-response element-binding protein; RPA, RNA protection assay. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Henry Keen for bioinformatic support for this project, Norma Sinclair, Trish Yarolem, and JoAnn Schwarting in the University of Iowa Transgenic Animal Facility and the University of Iowa Central Microscopy Facility. We gratefully acknowledge the generous research support of the Roy J. Carver Trust.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kim, H. S., Krege, J. H., Kluckman, K. D., Hagaman, J. R., Hodgin, J. B., Best, C. F., Jennette, J. C., Coffman, T. M., Maeda, N., and Smithies, O. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2735-2739[Abstract/Free Full Text]
  2. Krege, J. H., John, S. W., Langenbach, L. L., Hodgin, J. B., Hagaman, J. R., Bachman, E. S., Jennette, J. C., O'Brien, D. A., and Smithies, O. (1995) Nature 375, 146-148[CrossRef][Medline] [Order article via Infotrieve]
  3. Oliverio, M. I., Madsen, K., Best, C. F., Ito, M., Maeda, N., Smithies, O., and Coffman, T. M. (1998) Am. J. Physiol. 274, F43-F50
  4. Takahashi, N., Lopez, M. L., Cowhig, J. E., Jr., Taylor, M. A., Hatada, T., Riggs, E., Lee, G., Gomez, R. A., Kim, H. S., and Smithies, O. (2005) J. Am. Soc. Nephrol. 16, 125-132[Abstract/Free Full Text]
  5. Davisson, R. L., Kim, H. S., Krege, J. H., Lager, D. J., Smithies, O., and Sigmund, C. D. (1997) J. Clin. Investig. 99, 1258-1264[Medline] [Order article via Infotrieve]
  6. Lochard, N., Silversides, D. W., Van Kats, J. P., Mercure, C., and Reudelhuber, T. L. (2003) J. Biol. Chem. 278, 2184-2189[Abstract/Free Full Text]
  7. Gribouval, O., Gonzales, M., Neuhaus, T., Aziza, J., Bieth, E., Laurent, N., Bouton, J. M., Feuillet, F., Makni, S., Ben Amar, H., Laube, G., Delezoide, A. L., Bouvier, R., Dijoud, F., Ollagnon-Roman, E., Roume, J., Joubert, M., Antignac, C., and Gubler, M. C. (2005) Nat. Genet. 37, 964-968[CrossRef][Medline] [Order article via Infotrieve]
  8. Chen, L., Kim, S. M., Oppermann, M., Faulhaber-Walter, R., Huang, Y. G., Mizel, D., Chen, M., Sequeira Lopez, M. L., Weinstein, L. S., Gomez, R. A., Briggs, J. P., and Schnermann, J. B. (2006) Am. J. Physiol. Renal Physiol., in press
  9. Petrovic, N., Black, T. A., Fabian, J. R., Kane, C. M., Jones, C. A., Loudon, J. A., Abonia, J. P., Sigmund, C. D., and Gross, K. W. (1996) J. Biol. Chem. 271, 22499-22505[Abstract/Free Full Text]
  10. Yan, Y., Jones, C. A., Sigmund, C. D., Gross, K. W., and Catanzaro, D. F. (1997) Circ. Res. 81, 558-566[Abstract/Free Full Text]
  11. Shi, Q., Black, T. A., Gross, K. W., and Sigmund, C. D. (1999) Circ. Res. 85, 479-488[Abstract/Free Full Text]
  12. Shi, Q., Gross, K. W., and Sigmund, C. D. (2001) J. Biol. Chem. 276, 3597-3603[Abstract/Free Full Text]
  13. Shi, Q., Gross, K. W., and Sigmund, C. D. (2001) Hypertension 38, 332-336[Abstract/Free Full Text]
  14. Pan, L., Black, T. A., Shi, Q., Jones, C. A., Petrovic, N., Loudon, J., Kane, C., Sigmund, C. D., and Gross, K. W. (2001) J. Biol. Chem. 276, 45530-45538[Abstract/Free Full Text]
  15. Liu, X., Huang, X., and Sigmund, C. D. (2003) Circ. Res. 92, 1033-1040[Abstract/Free Full Text]
  16. Pan, L., Glenn, S. T., Jones, C. A., Gronostajski, R. M., and Gross, K. W. (2003) Biochim. Biophys. Acta 1625, 280-290[Medline] [Order article via Infotrieve]
  17. Pan, L., Wang, Y., Jones, C. A., Glenn, S. T., Baumann, H., and Gross, K. W. (2004) Am. J. Physiol. 288, F117-F124
  18. Todorov, V. T., Volkl, S., Muller, M., Bohla, A., Klar, J., Kunz-Schughart, L. A., Hehlgans, T., and Kurtz, A. (2004) J. Biol. Chem. 279, 1458-1467[Abstract/Free Full Text]
  19. Todorov, V. T., Volkl, S., Friedrich, J., Kunz-Schughart, L. A., Hehlgans, T., Vermeulen, L., Haegeman, G., Schmitz, M. L., and Kurtz, A. (2005) J. Biol. Chem. 280, 24356-24362[Abstract/Free Full Text]
  20. Klar, J., Sigl, M., Obermayer, B., Schweda, F., Kramer, B. K., and Kurtz, A. (2005) Hypertension 46, 1340-1346[Abstract/Free Full Text]
  21. Liu, X., Shi, Q., and Sigmund, C. D. (2006) Endocrinology, in press
  22. Adams, D. J., Head, G. A., Markus, M. A., Lovicu, F. J., van der Weyden, L., Kontgen, F., Arends, M. J., Thiru, S., Mayorov, D. N., and Morris, B. J. (2006) J. Biol. Chem. 281, 31753-31761[Abstract/Free Full Text]
  23. Sinn, P. L., Davis, D. R., and Sigmund, C. D. (1999) J. Biol. Chem. 274, 35785-35793[Abstract/Free Full Text]
  24. Sinn, P. L., and Sigmund, C. D. (2000) Physiol. Genomics 3, 25-31[Abstract/Free Full Text]
  25. Nistala, R., Zhang, X., and Sigmund, C. D. (2004) Physiol. Genomics 17, 4-10[Abstract/Free Full Text]
  26. Nistala, R., and Sigmund, C. D. (2002) Nucleic Acids Res. 30, e41[Abstract/Free Full Text]
  27. Jessen, J. R., Meng, A., McFarlane, R. J., Paw, B. H., Zon, L. I., Smith, G. R., and Lin, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5121-5126[Abstract/Free Full Text]
  28. Lavoie, J. L., Lake-Bruse, K. D., and Sigmund, C. D. (2004) Am. J. Physiol. 286, F965-F971
  29. Nguyen, G., Delarue, F., Burckle, C., Bouzhir, L., Giller, T., and Sraer, J. D. (2002) J. Clin. Investig. 109, 1417-1427[CrossRef][Medline] [Order article via Infotrieve]
  30. Germain, S., Bonnet, F., Philippe, J., Fuchs, S., Corvol, P., and Pinet, F. (1998) J. Biol. Chem. 273, 25292-25300[Abstract/Free Full Text]
  31. Zhu, X. G., Park, K. S., Kaneshige, M., Bhat, M. K., Zhu, Q., Mariash, C. N., McPhie, P., and Cheng, S. Y. (2000) Mol. Cell. Biol. 20, 2604-2618[Abstract/Free Full Text]
  32. Zhang, Y., and Dufau, M. L. (2001) Mol. Endocrinol. 15, 1891-1905[Abstract/Free Full Text]
  33. Baumann, H., Wang, Y., Richards, C. D., Jones, C. A., Black, T. A., and Gross, K. W. (2000) J. Biol. Chem. 275, 22014-22019[Abstract/Free Full Text]
  34. Oliverio, M. I., Best, C. F., Kim, H.-S., Arendshorst, W. J., Smithies, O., and Coffman, T. M. (1997) Am. J. Physiol. 272, F515-F520
  35. Sun, J., Oddoux, C., Gilbert, M. T., Yan, Y., Lazarus, A., Campbell, W. G., and Catanzaro, D. F. (1994) Circ. Res. 75, 624-629[Abstract/Free Full Text]
  36. Gilbert, M. T., Sun, J., Yan, Y., Oddoux, C., Lazarus, A., Tansey, W. P., Lavin, T. N., and Catanzaro, D. F. (1994) J. Biol. Chem. 269, 1-6[Free Full Text]
  37. Nakamura, N., Burt, D. W., Paul, M., and Dzau, V. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 56-69[Abstract/Free Full Text]
  38. Tamura, K., Chen, Y. E., Horiuchi, M., Chen, Q., Daviet, L., Yang, Z., Lopez-Ilasaca, M., Mu, H., Pratt, R. E., and Dzau, V. J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8513-8518[Abstract/Free Full Text]
  39. Tomita, S., Tomita, N., Yamada, T., Zhang, L., Kaneda, Y., Morishita, R., Ogihara, T., Dzau, V. J., and Horiuchi, M. (1999) Circ. Res. 84, 1059-1066[Abstract/Free Full Text]
  40. Harris, J. M., and Gomez, R. A. (1997) Microsc. Res. Tech. 39, 211-221[CrossRef][Medline] [Order article via Infotrieve]
  41. Sequeira Lopez, M. L., Pentz, E. S., Nomasa, T., Smithies, O., and Gomez, R. A. (2004) Dev. Cell 6, 719-728[CrossRef][Medline] [Order article via Infotrieve]
  42. Pan, L., Xie, Y., Black, T. A., Jones, C. A., Pruitt, S. C., and Gross, K. W. (2001) J. Biol. Chem. 276, 32489-32494[Abstract/Free Full Text]
  43. Pan, L., Glenn, S. T., Jones, C. A., and Gross, K. W. (2005) J. Biol. Chem. 280, 20860-20866[Abstract/Free Full Text]
  44. Sigmund, C. D., Jones, C. A., Kane, C. M., Wu, C., Lang, J. A., and Gross, K. W. (1992) Circ. Res. 70, 1070-1079[Abstract/Free Full Text]
  45. Sinn, P. L., Zhang, X., and Sigmund, C. D. (1999) Am. J. Physiol. 277, F634-F642
  46. Keen, H. L., and Sigmund, C. D. (2001) Hypertension 37, 403-407[Abstract/Free Full Text]
  47. Yan, Y., Hu, L. F., Chen, R. P., Sealey, J. E., Laragh, J. H., and Catanzaro, D. F. (1998) Hypertension 32, 205-214[Abstract/Free Full Text]
  48. Sinn, P. L., and Sigmund, C. D. (2000) in Drugs, Enzymes and Receptors of the Renin-Angiotensin System, Celebrating a Century of Discovery (Husain, A., and Graham, R. M., eds) pp. 259-278, Harwood Academic Publishers, Amsterdam
  49. Sinn, P. L., and Sigmund, C. D. (2000) Regul. Pept. 86, 77-82[CrossRef][Medline] [Order article via Infotrieve]
  50. Castrop, H., Oppermann, M., Weiss, Y., Huang, Y., Mizel, D., Lu, H., Germain, S., Schweda, F., Theilig, F., Bachmann, S., Briggs, J., Kurtz, A., and Schnermann, J. (2006) Physiol. Genomics 25, 277-285[Abstract/Free Full Text]
  51. Sinn, P. L., and Sigmund, C. D. (1999) Hypertension 33, 900-905[Abstract/Free Full Text]
  52. Adams, D. J., Beveridge, D. J., van der Weyden, L., Mangs, H., Leedman, P. J., and Morris, B. J. (2003) J. Biol. Chem. 278, 44894-44903[Abstract/Free Full Text]
  53. Skalweit, A., Doller, A., Huth, A., Kahne, T., Persson, P. B., and Thiele, B. J. (2003) Circ. Res. 92, 419-427[Abstract/Free Full Text]

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