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Originally published In Press as doi:10.1074/jbc.M605720200 on August 8, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31753-31761, October 20, 2006
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Renin Enhancer Is Critical for Control of Renin Gene Expression and Cardiovascular Function*

David J. Adams{ddagger}12, Geoffrey A. Head§1, M. Andrea Markus{ddagger}, Frank J. Lovicu{ddagger}, Louise van der Weyden{ddagger}2, Frank Köntgen, Mark J. Arends||, Sathia Thiru||, Dmitry N. Mayorov§, and Brian J. Morris{ddagger}3

From the {ddagger}School of Medical Sciences and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia, §Baker Heart Research Institute, Melbourne, Victoria 8008, Australia, Ozgene Pty. Ltd., Perth 6102, Western Australia, and the ||Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom

Received for publication, June 15, 2006 , and in revised form, August 3, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The important cardiovascular regulator renin contains a strong in vitro enhancer 2.7 kb upstream of its gene. Here we tested the in vivo role of the mouse Ren-1c enhancer. In renin-expressing As4.1 cells stably transfected with Ren-1c promoter with or without enhancer, expression of linked beta-geo reporter, stable expression, and colony formation were dependent on the presence of the enhancer. We then generated mice carrying a targeted deletion of the enhancer (REKO mice) and found marked depletion of renin in renal juxtaglomerular and submandibular ductal cells, as well as hyperplasia of macula densa cells. Plasma creatinine was increased, but electrolytes were normal. Male REKO mice implanted with telemetry devices had 9 ± 1 mm Hg lower mean arterial pressure (p < 0.001), which was partly normalized by a high NaCl diet. Locomotor activity was lower, and baroreflex sensitivity was normal. Markedly reduced mean arterial pressure variability in the midfrequency band indicated a contribution of reduced sympathetic vasomotor tone to the hypotension. In conclusion, the renin enhancer is critical for renin gene expression and physiological sequelae, including response to alteration in salt intake. The REKO mouse may be useful as a low renin expression model.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Renin, produced in renal juxtaglomerular (JG)4 cells, is the rate-limiting enzyme of the renin-angiotensin system that generates angiotensin II. The renin-angiotensin system helps maintain fluid and electrolyte balance and blood pressure (BP) but also has important roles in development, vascular hypertrophy, angiogenesis, and clinical conditions, such as renal hypertension and heart failure (1). The JG cells are modified myoepithelial cells in the wall of the afferent arteriole and are in close contact with the macula densa segment of the distal tubule that signals the renal arterioles to regulate glomerular filtration rate and renin secretion (2).

Transcription of renin genes is regulated in both a tissue-specific and developmental manner (1, 3). In the mouse kidney, renin expression is first detected at embryonic day 14.5 in the developing arteries and renal arterial branches. Expression then becomes progressively restricted to smaller arteries and arterioles, eventually contracting to just the JG cells postpartum (4). In addition to the kidney, mice also express renin in the submandibular gland (SMG) and, at lower levels, in adrenal, brain, and various other tissues.

To ascertain the importance of renin, strains of mice have been generated with deletion of the entire renin gene (termed Ren-1c or Ren-1d, depending on mouse strain). Ren-1c null mice show neither detectable plasma renin activity nor plasma angiotensin I, a reduction in BP of 20-30 mm Hg, increased urine output and water intake, and altered renal morphology (5), as also seen in angiotensinogen-deficient mice (6, 7). Consistent with this, Ren-1d null mice show altered macula densa morphology, complete absence of JG cell granulation, and sexually dimorphic hypotension (8).

An understanding of the precise molecular mechanisms controlling renin gene expression and regulation in response to physiological cues remains, however, incomplete. Much progress has nevertheless been made using mouse As4.1 cells, a renin-expressing tumor cell line thought to be derived from JG cells (9). A potent enhancer located 2866-2625 bp upstream of Ren-1c in in vitro assays in As4.1 cells is critical for 100-fold activation of the proximal promoter (bp -197 to -50), doing so in a position- and orientation-independent manner (10). This important candidate element consists of at least 11 transcription factor-binding sites and is responsive to various signal transduction pathways that alter renin mRNA levels (11). An element 71 and 85% homologous to the mouse renin enhancer is present in the 5'-flanking DNA of humans (12) and rats (13), respectively.

Under pathophysiological conditions, such as stenosis of the ureter or renal artery, reactivation of renin expression occurs in renal arteriolar cells upstream of the JG region (14-17). A similar reactivation is produced by angiotensin-converting enzyme inhibition and low salt diet (18). The mechanism by which renin-expressing cells retract along the afferent arteriole during development or can be recruited back to express renin in adult animals is not known.

Here we address for the first time the contribution of the enhancer to renin gene expression and regulation in response to physiological cues in vivo.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Stable Transfection Experiment—For constructs, pGl3-Basic (Promega) was digested with HindIII and SalI, liberating the luciferase reporter gene, which was replaced with beta-geo cut as a HindIII and SalI fragment from pbeta-geo (19) to create pGl3-beta-geo.pbeta-geo was provided by Dr. P. Soriano (Fred Hutchinson Cancer Research Center, Seattle). Ren-1c promoter and promoter plus enhancer fragments were cloned 5' of the reporter into pGl3-beta-geo, to generate the plasmids PrompGl3-beta-geo and PromEnhpGl3-beta-geo, respectively. The cytomegalovirus-SEAP construct used has been described previously (20). These were then used for colony assays. As4.1 cells (1 x 106) were seeded into duplicate T75 flasks, so as to make them ~85% confluent at the time of transfection the next day. Transfection with 4 µg of PrompGl3-beta-geo or Prom+EpGl3-beta-geo and 1 µg of cytomegalovirus-SEAP was performed using Fugene 6 (Roche Applied Science) according to the manufacturer's instructions. Forty-eight hours post-transfection, 1 ml of medium was collected for a SEAP assay, and cells were fed with fresh Dulbecco's modified Eagle's medium supplemented with 200 µg/ml G418 (Invitrogen). Fresh medium with G418 was replenished every 48 h. Fourteen days after transfection, colonies >5 mm in size were counted. This experiment was performed three times using duplicate flasks, and the number of clones generated for each experiment was averaged to give n = 1. Results obtained for colony counts were normalized to SEAP values to account for differences in transfection efficiency. Statistics were generated using Student's t test.

Targeting Vector Construction for Renin Enhancer Knock-out (REKO) Mouse Production—The Ren-1c enhancer targeting construct was generated by amplification of a 2.2-kb HindIII-flanked 5' homology arm by PCR using the primers HindIIIREN5' (5'-CCC GAG CAG AAA GCT TGC CCA GAC ATC TGA CTC C-3') and HindIIIREN3' (5'-AAG GGA GAA GCT TGA CTA GTG ATT GAC-3'). The 3' homology arm was a 6.5-kb SalI/NotI-flanked PCR product generated using the primers SalIREN5' (5'-AAA AAA AAG TCG ACC GAG GCA CTG AGG CAT TCA CC-3') and NotIREN3' (5'-CGT GTC AAA GCG GCC GCC GAT GAC TTT GAA GGT CTG GGG-3'). The homology arms were cloned into the HindIII and SalI/NotI sites of pNATA-OzE, in which the neo/TK genes of pNATA (kindly provided by Dr. Hozumi Motohashi, University of Tsukuba, Japan) were removed by a HpaI/SalI digest and replaced with a HincII/XhoI-flanked PGK-neo selection cassette generated by PCR using primers P154 (5'-TAA GTT GGG TAA CGC CAG GG-3') and P144_01 (5'-AAG AGA GAA ACT CGA GCA TAT GTC TCT TGG ATC CGG AAC CCT TAA T-3') and pOzE plasmid as template (generously provided by Ozgene, Perth, Western Australia).

Generation of REKO Mice—C57BL/6J Bruce4 ES cells (21) were electroporated with NotI-linearized targeting vector and selected in 200 µg/ml G418. The resulting colonies were screened by Southern blot hybridization. The 5' probe (a 956-bp PCR fragment generated using the primers 5'-TCA CCT TCA CCA TCA GGG TCA G-3' and 5'-TGG GCT AGC CCG CTT TCT GCT C-3') hybridized on HindIII-digested genomic DNA gave a wild type (WT) band of 9.7 kb and a targeted band of 5.2 kb. The 3' probe (a 455-bp PCR fragment generated using the primers 5'-GGG TCC GAC TTC ACC ATC CAC TAC-3' and 5'-GTC TTT CTC TAC CTC TGG CAC AGC G-3') hybridized on BstEII-digested DNA gave a WT band of 8.5 kb and a targeted band of 10 kb. Two independent ES cell clones that had undergone homologous recombination, I_3A11 and I_5H8, were injected into BALB/c blastocysts, and the resulting chimeric males were mated to C57BL/6J females. Germ line transmission was confirmed by Southern blot hybridization using the probes described above. Founders were mated to C57BL/6J deleter mice (22) to remove the floxed PGK-neo cassette, which was confirmed by Southern blot analysis on HindIII-digested genomic DNA using first the 5' probe, followed by rehybridization with the Cre probe (to detect presence of the Cre transgene). The 884-bp Cre probe was generated by PCR using the pPCR_CreProbe plasmid (Ozgene) as template and the primers 5'-CGA GTG ATG AGG TTC GCA AG-3' and 5'-CAC CAG CTT GCA TGA TCT-3'). The offspring were subsequently back-crossed to C57BL/6J to remove the Cre transgene before being intercrossed to generate REKO mice. Production and experimentation with REKO mice had appropriate institutional animal ethics approval, and all mice were housed in accordance with specific pathogen-free regulations.

Genotyping—Genomic DNA was amplified using Taq polymerase (Qiagen) and 100 ng of each of three primer pairs: enhancer primers (5'-TGC TCC CTC TCC TCT AGG GCT TGG GAA GA-3' and 5'-AGT CAG TGA TAA ATG ACG GGC AGG ACC TAC-3'; 450 bp band), deletion primers (5'-GTG TCA AAG CAT GTT ATA GCC CAA TCA AG-3' and 5'-GAC CAG TAT GTG TCA GGG ACT CCC AGG-3'; 450 bp band for deleted enhancer allele and 800 bp for WT enhancer allele), and universal primers (5'-AGG CAA AAC CCA CAT TTC TTA CCG CAC AAC TAG-3' and 5'-CCT TTG CAG CCA AGT GAT GTC TGT GTC CAT G-3'; 450 bp band). The PCR cycle profile was as follows: 1 cycle at 94 °C for 30 s, followed by 30 cycles at 94 °C for 30 s, 65 °C for 30 s, and 72 °C for 30 s, with a final cycle of 72 °C for 10 min.

Histology and Immunohistochemistry—Tissues from WT and REKO mice were fixed for 48 h in 10% neutral buffered formalin before being dehydrated and embedded in paraffin, and 6-µm tissue sections were cut. For histology, the tissue sections were stained with hematoxylin and eosin and examined by light microscopy. For immunohistochemistry, sections were first incubated in 0.3% hydrogen peroxide (H2O2) in methanol for 20 min to block endogenous peroxidase activity, followed by three rinses in PBS supplemented with 0.1% BSA (PBS/BSA) and treatment with 2 mol/liter HCl for 20 min. After a further rinse with PBS/BSA (3 x 5 min), sections were incubated for 30 min with 3% normal rabbit serum diluted in PBS/BSA. Excess rabbit serum was removed, and sections were incubated overnight at 4 °C with an anti-renin goat polyclonal antibody (diluted 1:500 with PBS/BSA; Santa Cruz Biotechnology). The following day, sections were rinsed with PBS/BSA and incubated for 90 min at room temperature with a rabbit anti-goat IgG conjugated to horseradish peroxidase (diluted 1:250 with PBS; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Following two PBS/BSA washes and a third rinse with distilled water, 3'-diaminobenzidine tetrahydrochloride (0.5 mg/ml, diluted in 0.05 M Tris, 0.01% H2O2) was applied to sections, which were then rinsed with deionized H2O and counterstained with hematoxylin to visualize cell nuclei. Following a rinse with PBS, sections were mounted in 10% PBS/glycerol and visualized using bright field microscopy (Leica-DMLB). All photography was carried out using a digital camera (Leica DC-280).

Cardiovascular and Behavioral Measurements—In a preliminary operation under halothane open circuit anesthesia, male C57BL6 (WT) and REKO mice (n = 6 per group) were implanted with telemetry devices (TA11PA-C20; DataSciences International) to monitor mean arterial pressure (MAP), heart rate (HR), and locomotor activity in the unrestrained state as described previously (23). The catheter tip was placed in the aortic arch via the carotid artery, and the transmitter was placed under the skin at the flank. The radiotelemetry signals were collected by the receiver (model RPC-1; Data Sciences International) and were passed to an analogue converter (model RP11A; Data Sciences International). Ambient barometric pressure was also measured (APR-1; Data Sciences International) and subtracted from the telemetered pressure by the data collection system in order to compensate for changes in atmospheric pressure. The analogue voltage signal was then converted by a data acquisition card (NI PCI 6024E; National Instruments, Austin, TX) using software (Universal Acquisition) written in Labview (National Instruments). A special algorithm was used to detect systolic arterial pressure (SAP), diastolic arterial pressure (DAP), and pulse interval (24). MAP was calculated on a beat-to-beat basis, and instantaneous HR was calculated from the pulse interval. An index of locomotor activity was obtained by monitoring changes in the signal strength received, which occurred upon movement of the animal. Changes in signal strength of more than a predetermined threshold generated a digital pulse, which was counted by the acquisition system. For each heartbeat detected, SAP, MAP, and DAP, pulse interval and locomotor activity were stored in text format on an IBM-compatible computer. Lights in the holding room were set to automatically switch on at 6 a.m. and go off at 6 p.m. Being nocturnal, the mice were most active between 6 p.m. and 6 a.m.

Salt Diets—Mice were placed randomly on high (3.1%), normal (0.4%), and low (0.05%) NaCl ad libitum for 2 weeks, and the diet was rotated for a total of 6 weeks of study. At the end of each diet period, cardiovascular measurements (beat to beat) were recorded continuously for 72 h.


Figure 1
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  		FIGURE 1.
Ren-1c enhancer increases the likelihood that a stably integrated construct will express a linked reporter. The plasmids shown in A, PrompGl3-beta-geo (promoter alone) and Prom+EpGl3-beta-geo (promoter plus enhancer), were transfected into As4.1 cells with a SEAP internal control. After 48 h of recovery, cells were placed in medium containing 200 µg/ml G418. Following 14 days of selection, surviving colonies were counted, and results are shown in B.

 
Assessment of Baroreflex by Spectral Analysis and Sequences—While on a normal salt diet, beat to beat MAP and HR data between 11 a.m. and 12 p.m. (during the inactive period) were analyzed on 3 consecutive days. The auto- and cross-power spectra were calculated for multiple overlapping (by 50%) segments of MAP and HR using fast Fourier transform (25). The average value of the transfer gain in the frequency band between 0.3 and 0.5 Hz was used as the estimate of the baroreflex sensitivity (26). Other frequency bands included in the analysis were low frequency (0.08-0.3 Hz) and high frequency (0.5-1 Hz), and the total power was calculated between 0 and 1 Hz (27). During the same period, the spontaneous baroreflex slope was calculated as the slope of the linear regression lines between the SAP and HR, with a delay of 0-6 heartbeats, using the sequence technique (28, 29). The number of baroreflex sequences compared with nonbaroreflex sequences was highest with a four-beat delay in both strains and therefore used to calculate baroreflex gain. Sequences with at least three intervals, >0.5-mm Hg BP changes, and >5-ms heartbeat interval changes were analyzed only if the correlation coefficients were >0.85. Spontaneous baroreflex slope was calculated as the mean value of the significant slopes obtained.

Statistical Analyses of Physiological Data—Physiological data were analyzed by a multifactor, repeated measure, nested analysis of variance, where within-animal factors of diet and day/night effects and between animal factors (strain) were subtracted from the total sum of squares. The sum of the within-animal residuals was used for calculating F ratios for diet and day/night effects, whereas the animal x groups interaction plus within-animal residual was used for determining the between-strain effects, as described previously (30).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of the Ren-1c Enhancer in Single Cells—We first tested Ren-1c enhancer function in the context of a stably integrated transgene. Using constructs containing the Ren-1c promoter (PrompGl3-beta-geo) and the Ren-1c promoter plus enhancer (PromEnhpGl3-beta-geo) 5' of a beta-geo reporter gene, we were able to obtain colonies only when the enhancer was present (Fig. 1). That the result was not influenced by integration site was supported by fluorescence-activated cell sorting of As4.1 cells transiently transfected with constructs containing the renin promoter with or without enhancer, linked to enhanced green fluorescent protein reporter (data not shown).


Figure 2
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  		FIGURE 2.
Generation of Ren-1c enhancer null allele. A, a targeting vector containing a neomycin selection cassette (PGK-neo) flanked by loxP sites underwent homologous recombination with the Ren-1c locus and replaced the 241 bp core Ren-1c enhancer plus 349 bp upstream and 137 bp downstream. B, Southern blot analysis of the Ren-1c enhancer-targeted allele was performed using a 5' probe on HindIII-digested genomic DNA, and a 3' probe on BstEII-digested genomic DNA (data not shown), with restriction enzyme sites and fragment sizes as indicated (B, BstEII; H, HindIII). C, correctly targeted ES cell clones were sent through the germ line, and founder mice were bred with Cre deleter mice to remove the selection cassette. The latter was confirmed by Southern blot analysis on HindIII-digested genomic DNA using the 5' probe (C), followed by rehybridization with the Cre probe (D) to detect the presence of the Cre transgene. E, genotyping of Ren-1c enhancer knock-out (REKO) mice was performed by PCR using a combination of three primer pairs, enhancer primers (Enh-F/R), deletion primers (Del-F/R), and universal primers (Uni-F/R), which can distinguish between all three genotypes (+/+, +/-, and -/-) based on the presence and size of the product(s), resolved on a 2% agarose gel.

 
Generation of REKO Mice—To assess the physiological role of the Ren-1c enhancer, we generated a null allele of this enhancer by gene targeting in C57BL/6J ES cells. Correctly targeted clones containing a floxed neomycin selection cassette in place of the enhancer (Fig. 2A) were identified by Southern blotting using both 5' and 3' probes (Fig. 2B) (data not shown). These ES cell clones were used to generate chimeras. Male chimeric mice were crossed with C57BL/6J females, allowing us to transmit the targeted allele through the germ line on a pure C57BL/6J background. F1 offspring of this mutated allele were mated with a Cre deleter strain, maintained on a C57BL/6J background, to remove the floxed selection cassette, as confirmed by Southern blot analysis (Fig. 2, C and D). The resulting heterozygous mice were back-crossed to C57BL/6J to remove the Cre allele. Due to the breeding scheme described, we were able to perform all of our analysis on a pure C57BL/6J background. Heterozygous intercrosses genotyped by PCR (Fig. 2E) produced homozygous null (REKO) offspring at the expected Mendelian ratios and were indistinguishable from littermate controls in their growth and development.

REKO Mice Have Low Renin Expression—Typical renin immunostaining of JG cells was seen in WT mice on normal or low salt diet (Fig. 3, A and B). In contrast, across the entire renal cortex, no renin staining was detectable in REKO kidneys on either diet (Fig. 3, A and B). For SMG, the usual ductal renin immunostaining was seen in ductal cells of WT mice, but for REKO, staining was uniformly lower in all of these cells (Fig. 3C).

REKO Mice Have Elevated Plasma Creatinine—In WT and REKO mice, respectively, similar plasma concentrations (mmol/liter; ±S.D., each n = 6) were seen for sodium (135.7 ± 3.6 versus 136.4 ± 4.3), potassium (6.7 ± 1.5 versus 7.1 ± 1.5), chloride (101.0 ± 3.7 versus 101.1 ± 2.9), bicarbonate (13.6 ± 2.7 versus 14.4 ± 1.8), urea (7.4 ± 1.3 versus 7.3 ± 1.4), and glucose (15.8 ± 3.3 versus 15.3 ± 5.6). Plasma osmolarity (mosmol/liter) was also similar (298 ± 14.4 versus 300.0 ± 21.7), but plasma creatinine (25.4 ± 5.3 versus 35.3 ± 5.3 mmol/liter) was elevated by 39 ± 2% (p = 0.004), indicative of a decreased glomerular filtration rate.

Histological Analysis of REKO Mice—REKO kidneys exhibited macula densa hyperplasia (Fig. 3D). As well as an increase in number, the nuclei were piled up in a pseudostratified pattern. The hearts of REKO mice showed neither evidence of cardiac hypertrophy or atrophy nor differences in arterial wall thickness. No infarcts were seen, either watershed or lacunar, in the brain or elsewhere. The histomorphological appearance of the brain was similar between WT and REKO mice. In particular, in the hippocampus, the granular layer did not differ in thickness (8-11 nuclei with, at most, 10 or 11 nuclei at the thickest part), and density of granular cells was similar. Histological examination of all other organs showed no significant pathological abnormalities (data not shown).


Figure 3
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  		FIGURE 3.
Renin antibody staining in tissue sections of WT and REKO mice. A, immunohistochemistry of kidney sections from WT mice showed strong renin staining of JG cells, which, in mice fed a low NaCl diet for 1 week, extended up the afferent arteriole (B), whereas no immunostaining was seen in REKO mice on either a normal (A) or low salt (B) diet. C, SMG, showing that the ductal staining in WT was much reduced in REKO mice. D, macula densa hyperplasia, evident as an increase in nuclei and pseudostratification of cells (circled) in the distal tubule adjacent to a glomerulus of REKO mice, as opposed to a simple linear plaque of macula densa nuclei (circled) in WT. (n = 5 mice). Scale bar, 50 µM. AA, afferent arteriole; G, glomerulus.

 
Cardiovascular Measurements by Radiotelemetry—MAP, averaged over 24 h, was considerably lower in REKO versus WT mice (103 ± 1 versus 113 ± 0.8 mm Hg; p < 0.001, n = 6 per group), as were SAP and DAP (Table 1). Analysis of the hourly averages for SAP, HR, and activity showed that the lower BP in the REKO mice occurred over the entire 24-h period during times of high (dark) and low (light) activity (Fig. 4A). Both strains demonstrated similar circadian patterns of BP and HR, with highest values during the active period and lowest during the inactive period. The peak HR occurred in the hour after the lights went out. HR and body weight were similar in each group, but locomotor activity was 35% lower in REKO than in WT mice (p < 0.05) (Table 1). Mice were also assessed while on a low (0.05%) or high (3.1%) NaCl diet for 3 weeks. The REKO mice exhibited "salt sensitivity" in that all cardiovascular parameters showed a significant salt diet effect (F2,84 > 8, p < 0.01). The main effect on BP was seen for the high salt diet, which increased MAP by 5.7 mm Hg in REKO but only 1.9 mm Hg in WT. The difference in BP between WT and REKO mice persisted on the high salt diet (Table 1). On a normal diet, the HR of both strains was similar. The REKO mice were, however, particularly sensitive to either the low or the high salt diet, responding with a small but consistent increase in HR for both diets (Table 1). In the WT mice, the different salt diets had no effect on any variable except body weight, which showed a small increase on the high salt diet (Table 1). On the high salt diet the 24-h circadian patterns in BP, HR, and activity were similar to those observed on normal salt. The BP difference between REKO and WT was, however, considerably less (Fig. 4B).


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  		TABLE 1
Average SAP, DAP, MAP, HR, activity, and body weight in WT and REKO mice

In each column are shown absolute values and S.E. over the 24-h period. NS, not significant; p > 0.05.

 
Short Term Cardiovascular Variability and Baroreflex Sensitivity in REKO and WT Mice—Total BP and HR variability, as well as the variability in high frequency bands assessed by spectral analysis, were similar in the REKO and WT mice (Fig. 5). The midfrequency MAP and HR band corresponding to the autonomic frequencies and the low frequency HR band were, however, less in REKO compared with WT mice. Nevertheless the gain of the baroreceptor HR reflex calculated from the cross-spectral transfer function between MAP and HR in this midfrequency was similar in the two strains (Fig. 5). Furthermore, there was good coherence between BP and HR, being between 0.5 and 0.6 in both groups (Fig. 5). Using the average slope of spontaneous sequences of increasing and decreasing SAP over at least four beats, we confirmed that the HR baroreflex sensitivity was indeed similar in the REKO and WT mice (Fig. 6). Furthermore, high or low salt intake for 2 weeks did not alter the baroreflex gain in REKO or WT mice. Interestingly, the REKO mice displayed many fewer baroreflex sequences (56%) than did WT (Fig. 6) as well as a similarly lower number of nonbarosequences (60%, data not shown).


Figure 4
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  		FIGURE 4.
Telemetry of WT and REKO mice. Symbols show hourly averaged data ± S.E., indicating variation of SAP (circles) and DAP (triangles). Readings from WT mice are represented as open symbols, whereas readings from REKO mice are represented by the filled symbols. HR and behavioral activity during a 24-h period (black bar, dark period; white bar, light period) for REKO and WT mice fed a normal salt (A) or a high salt (B) diet are shown in the bottom panel.

 


Figure 5
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  		FIGURE 5.
Cardiovascular variability. This is shown for MAP (upper left), HR (lower left), baroreflex gain (upper right), and coherence (lower right) for REKO and WT mice on a normal salt diet. Measurements were made between 11 a.m. and 12 p.m. MAP is presented as the area under the curves (AUC) for low frequency (LF; 0.08-0.3 Hz), medium frequency (MF; 0.3-0.5 Hz), and high frequency (HF; 0.5-1 Hz) readings, *, p < 0.05; **, p < 0.01 for the between-groups comparison.

 


Figure 6
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  		FIGURE 6.
Average baroreflex gain. This is represented by the slope from down and up sequences (upper panels) and numbers of sequences (lower panels) in WT (open bars) and REKO mice (black bars) on a normal, high, or low salt diet. **, p < 0.01; ***, p < 0.001 for the between-groups comparison across diets.

 


Figure 7
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  		FIGURE 7.
Left panel, minute averages of MAP versus log of activity index from one WT (top) and one REKO (bottom) mouse measured over a 3-day period. The average regression is indicated by the solid line. The dashed lines represent the 95% predictive limits. Right panel, regression lines from 6 WT (top) and 6 REKO (bottom) mice. The average regression for each group is indicated by the bold line.

 
Relationship between Locomotor Activity and BP—Over a 24-h period the mice showed a positive relationship between MAP and locomotor activity, as determined using telemetry, such that a 10-fold increase in activity led to an ~8-mm Hg increase in MAP (Fig. 7). Our calculations in all 16 WT and REKO mice indicated no difference in the slope of the MAP-log activity relationship between strains (7.6 ± 2 mm Hg/log for both WT and REKO).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It is well established that enhancer elements play an important role in regulating the dynamic activation and inactivation of gene transcription (31, 32). They may function stochastically to allow expression by greatly influencing the likelihood that a randomly integrated transgene will express (31, 33) (i.e. suppress position effect variegation) (34). The native renin gene, like most genes (35), would be under the influence of silencing mechanisms that restrict ectopic expression of renin. We first examined the effect of the enhancer in single cells in vitro and show by colony assays comparing the Ren-1c promoter alone or the promoter linked to the Ren-1c enhancer that the enhancer exerts powerful control over the promoter. Several mechanisms could explain this result. These include the possibility that the enhancer influences the level of expression of the renin gene or that the enhancer suppresses silencing by repressive chromatin domains that would normally silence the gene in the absence of a physiological stimulus for renin expression and that the enhancer functions stochastically to allow expression. Our results are consistent with the possibility that, in the context tested, the Ren-1c enhancer might indeed be able to suppress silencing of renin gene expression. Since position effect variegation of randomly integrated transgenes can be influenced by their abnormal juxtaposition with heterochromatin (36), models based on such in vitro data should be reinforced by in vivo observations. Indeed, although the position effect variegation theory (34) is consistent with the binary (on/off) nature of renin expression observed in afferent arterioles in response to stimulation ("recruitment") (14-18, 37), beta-adrenoreceptor simulation causes a 4-fold increase in renal renin mRNA in the absence of recruitment (38). Moreover, we noted that in SMG of REKO mice, there was a uniform reduction, rather than a variegated pattern, of renin immunostaining in ductal cells. This argues against the on/off switching model, at least in SMG. Furthermore, the directional, rather than random, switching on of renin expression in afferent arterioles upstream of JG cells might suggest the involvement of intercellular signaling. The mechanisms operating in vivo in various renin-expressing tissues remain to be elucidated.

Consistent with the enhancer having a crucial role in renin expression, renin staining was undetectable in JG cells of REKO mice. A complete lack of renin granulation has been observed in JG cells of mice after deletion of the entire Ren-1d gene (39). In REKO mice, there was hyperplasia of the macula densa, presumably a compensatory response attempting to increase signaling to JG cells to increase renin secretion and gene expression (1). Macula densa hyperplasia and hypertrophy are also seen in mice lacking Ren-1c (5) or Ren-1d (39). In contrast to kidneys of Ren-1c null mice, however, REKO kidneys did not show hydronephrosis. The renal medullary hypoplasia and vascular thickening seen in Ren-1c null mice was not apparent in our REKO mice.

Targeted deletion of various key components of the renin-angiotensin system has produced severe phenotypes (5, 40, 41). For example, Ren-1c null mice show a decrease in density of granular cells in the hippocampus (5). In contrast, we saw no such change in REKO mice. This may be because renin is still present in sufficient quantities to avoid these pathologies in REKO mice.

Our physiological studies showed that REKO mice had significantly lower SAP and DAP than age-matched WT. This is consistent with the lower BP in mice with deletion of genes for renin (5), angiotensinogen (40, 42), angiotensin-converting enzyme (41), or AT1 receptors (43). The difference in BP in these was much greater than between REKO and WT, presumably because REKO mice still express renin, albeit at lower levels. Although most studies used tail-cuff to measure SAP, we employed direct telemetry, which is much more reliable and accurate, avoids effects from restraint stress, and gives data through active (night) and inactive (day) periods. We find that the renin enhancer is necessary to maintain BP and prevent a hypertensive response to the high salt diet, whose volume and sodium retention effects normally tend to increase BP. This is offset by reductions in renin secretion and sympathetic nerve activity. The inability to suppress renin (due to its low expression) could explain the pressor response to high salt in REKO but not WT mice and supports reduced renin expression being responsible for the lower BP. A contribution of nonrenin effects cannot, however, be excluded.

With the normal salt diet, we observed a markedly reduced midfrequency band of BP variability in the REKO mice but no difference in global BP or HR variability. Similarly, there was a lower number of short term sequences in REKO mice. Both of these observations can be explained by reduced sympathetic vasomotor activity. Since high frequency HR variability and baroreflex sensitivity were normal in REKO mice, vagal activity was probably normal. Lower short term variability but normal baroreflex sensitivity was also seen in AT1a receptor null mice (43). Thus, reduced renin expression may be responsible for the altered autonomic balance and reduced BP. The lack of effect on HR baroreflex suggests, however, that baroreflex autonomic pathways are not dependent on renin or angiotensin II. This is similar to conclusions from human renin overexpression studies in mice (44).

In mice, each log unit increase in activity produced an ~7-mm Hg higher BP. Although this relationship was similar in the two strains, REKO mice were half as active. This would account for only a portion (2 mm Hg) of the difference seen. The renin enhancer (via renin) thus influences central nervous system regulation of locomotor activity. This could involve release in the forebrain of dopamine, which is augmented by angiotensin II through AT1 receptors (45, 46). Although locomotor activity in AT1 receptor knock-out mice remains unreported, in AT2 receptor knock-out mice it is reduced (47, 48) or, after prolonged stimulation, increased (49). Similarly, angiotensinogen-deficient mice have either increased (50) or decreased (51) activity. Determination of how brain renin and angiotensin peptides regulate locomotor activity thus requires further investigation.

Taken together, our results demonstrate an important role for the renin enhancer in control of BP, HR, locomotor activity, and possibly sympathetic vasomotor tone. The in vitro and histological findings, as well as biochemical measurements, support this action being mediated by an effect on renin expression. Determination of the direct and indirect role of altered renin expression in the kidney, central nervous system, and other mechanisms involved in the cardiovascular alterations we observed will require further research.


   FOOTNOTES
 
* This work was supported by Australian Research Council Grants A10009169 (to B. J. M. and David I. K. Martin) and DP0664650 (to B. J. M. and Peter J. Leedman) and National Health and Medical Research Council of Australia Grant 225118 (to G. A. H. and D. N. M.). 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 These two authors contributed equally to this work. Back

2 Present address: The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambs, CB10 1SA, United Kingdom. Back

3 To whom correspondence should be addressed: Basic & Clinical Genomics Laboratory, School of Medical Sciences and Bosch Inst., Bldg. F13, University of Sydney, New South Wales 2006, Australia. Tel.: 61-2-93513688; Fax: 61-2-93512227; E-mail: brianm{at}medsci.usyd.edu.au.

4 The abbreviations used are: JG, juxtaglomerular; REKO, renin enhancer knock-out; WT, wild type; SMG, submandibular gland; BP, blood pressure; MAP, mean arterial pressure; SAP, systolic arterial pressure; DAP, diastolic arterial pressure; HR, heart rate; PBS, phosphate-buffered saline; BSA, bovine serum albumin; SEAP, secreted human alkaline phosphatase. Back


   ACKNOWLEDGMENTS
 
We thank Luisa La Greca and Kristy Jackson for able technical support in the cardiovascular telemetry studies and Jessica Boros for renin immunolabeling.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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