INTRODUCTION

Renin (EC 3.4.23.15) is an aspartyl protease, which catalyzes the first step in the renin angiotensin system, the end product of which is the potent vasopressor peptide hormone, angiotensin II (AngII).¹ This octapeptide acts to increase peripheral vascular resistance and promote salt and fluid retention in concert with the hormone aldosterone. Renin is synthesized principally in the kidney juxtaglomerular (JG) cells, a group of modified smooth muscle cells located at the distal end of the renal afferent arteriole of the glomerulus (1). JG cells are in close contact with the macula densa, a specialized plaque of epithelial cells of the kidney distal tubule, which signal to the renal arterioles to regulate glomerular filtration rate and the secretion of renin, in response to ionic concentration and flow rate in the distal tubule (2, 3), the so-called tubuloglomerular feedback loop. Except for the submandibular gland (SMG) of the mouse, the JG cells are the only site where prorenin, the inactive zymogen, is known to be converted to the active form of renin. SMG renin does not, however, make its way into the plasma in large quantities and is thought not to play a significant role in blood pressure regulation under normal circumstances (reviewed in Bing et al. (4)). The release of renin from JG cells is mediated by two pathways: regulated release of the mature, active renin from modified lysosomal storage granules and constitutive release of the inactive zymogen. While the regulated pathway of renin secretion is responsive to baroreceptor, neurogenic, and macula densa signals (5), the physiological significance of the constitutive secretion of prorenin is not understood, nor are the molecular pathways that link secretory signals to renin maturation and release. Clarification of the mechanisms underlying these processes will be crucial to our understanding of the control of renin activity locally in the renal glomerulus, and in the plasma, and the regulation of fluid and ion homeostasis.

Human and rat genomes contain a single gene for renin, but mice display two alternative genotypes at the Ren locus. Thus some inbred mouse strains (e.g. strain C57BL/6) have only a single renin structural gene, termed Ren-1^c, while others (e.g. DBA/2 and 129) possess two renin genes, termed Ren-1^d and Ren-2. This probably results from the recent duplication of 21 kb of DNA containing a Ren-1^c-like ancestral gene (6, 7). All three mouse renin genes share the same overall genomic organization and encode highly homologous, but distinct, proteins, with approximately 97% similarity at the amino acid level, but having different glycosylation potentials (reviewed in Ref. 8). This arises because the renin-2 enzyme lacks putative consensus sites for asparagine-linked glycosylation, whereas renin-1^c and renin-1^d proteins can be glycosylated at three asparagine residues. The mouse renin genes are expressed in distinct, although overlapping, tissue-specific and developmental patterns (8). It has therefore been difficult to dissect the individual roles of each gene to date or to determine if renin-1^d and renin-2 play functionally equivalent roles in vivo. For example, Ren-1^d and Ren-2 are expressed at equivalent levels in JG cells, but Ren-2 is expressed at high levels (2% of total SMG protein) in the submandibular gland and is under the control of various hormones, including testosterone, whereas Ren-1^d is only detectable at trace levels in this organ (9). The evolution of non-identical, tightly regulated developmental expression profiles, and the biochemical differences between renin-1 and renin-2 proteins, suggest that the two genes may indeed possess functionally distinct properties.

The presence of two genetically distinct forms of renin, susceptible to manipulation by gene targeting, offers a unique opportunity to determine the importance of renin glycosylation for its role in vivo and to dissect the functions of renin, which in other animals are subserved by a single gene product. We have reported recently the targeted disruption of the Ren-2 gene in mice (10), which results in Ren-1^d being the only active renin gene present. Ren-2-null mice display elevated circulating active renin concentrations, and reduced circulating (inactive) prorenin; however, no abnormalities in the histomorphology of adult kidneys, adrenals, or submandibular glands, nor in resting blood pressures, have been found in adult animals to date (10). Here, we describe the generation of mice in which the Ren-1^d gene has been inactivated by homologous recombination. As part of a generalized construction strategy, the regions of DNA providing Ren-1^d gene homology in the targeting vector have been generated by long-range PCR amplification of isogenic substrate DNA, using a proofreading DNA polymerase. The phenotype of Ren-1^d/ mice shows that the Ren-1^d and Ren-2 genes are not functionally equivalent. First, female mice show a significant reduction in resting blood pressure. Furthermore, the level of plasma active renin is decreased while inactive prorenin is increased. Finally, the deletion of the glycosylated renin-1 results in the complete absence of dense secretory/storage granule formation in JG cells and altered morphology of the macula densa cells of the kidney distal tubule.

EXPERIMENTAL PROCEDURES

Regions of Ren-1^d gene homology for incorporation into the targeting vector were generated by long range PCR amplification using, as template, DNA from a bacteriophage P1 clone containing the entire mouse 129 Ren-1^d gene (P1-1249)² and the primer pairs, for the 5 arm, JJM 203 (5-CCGCTCGAGTCTGGACAGCCTACATGAC-3) and JJM 135 (5-AAGGTCTGGGGTGGGGTACC-3) and for the 3 arm, JJM 224 (5-GCCGCTCGAGGTACCAGCTACATGGAGAACGGGTC-3) and JJM 204 (5-GCAAGCTTGACAAAATGGCCCCCAGGAC-3). Natural (5 arm) and artificially introduced (3 arm) KpnI sites used in cloning are underlined. Reactions (100 µl) in 10 mM Tris-HCl, pH 8.8, 10 mM KCl, 0.002% (v/v) Tween 20®, 1.0 mM MgCl₂, 40 µM each dATP, dCTP, dGTP, dTTP, 5 µM each primer, 10-100 ng of template DNA, and 5 units of UlTma DNA polymerase (Perkin-Elmer ABI, Warrington, UK) were 40 cycles of 95 °C for 1 min, 66 °C (5 arm) or 68 °C (3 arm) for 1 min and 72 °C for 6.5 min, followed by one period of 10 min at 72 °C. Each PCR product was cloned into a plasmid vector and then manipulated to flank the PGKneo-selectable marker gene. The final targeting vector, pR1neoKO, contained 3.5- and 3.7-kb segments of the Ren-1^d gene flanking the selectable marker gene.

ES cells were grown in Glasgow modification of Eagle's medium + 10% fetal calf serum supplemented with mouse or human DIA/LIF on gelatin-coated plastic, as described (11). Targeting vector DNA (150 µg; pR1neoKO), was linearized by digestion with AscI and MluI and electroporated into 5 × 10⁹ E14Tg2a cells (12), a strain 129-derived embryonal stem cell line, with a discharge of 0.8 kV at 3 microfarads on a Bio-Rad gene pulser. Following G418 selection (175 µg/ml), drug-resistant colonies were expanded and genomic DNA prepared (13). Homologous recombination events were detected by Southern blotting of DNA digested with SacI and hybridized with an external 5 probe (a 297-bp PvuII/BamHI fragment, containing exon 1 of the Ren-1^d gene). Clones selected in this way were also hybridized with an external 3 probe after digestion with PvuII (a 746-bp HindIII/NcoI fragment, containing Ren-1^d exon 8 and part of exon 9; see Fig. 1D). One targeted clone was used to generate male chimaeras, which were crossed with strain 129 females to generate inbred 129 heterozygote offspring. These mice were intercrossed to produce an F2 generation with wild-type, heterozygous and homozygous inbred, Ren-1^d/ mice.

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Kidney RNA was amplified by reverse transcription-coupled PCR, using the Expand reverse transcription kit (Boehringer Mannheim, Lewes, UK), according to the manufacturer's instructions. cDNA was randomly primed with hexanucleotides, and renin sequences amplified using primers JJM 56 (5-CCAGCCCAGACCTTCAAAGTC-3) and JJM 141 (5-CCAGACAAATGGCCCCCAAG-3), specific for exons three and nine of the mouse renin genes, respectively. Amplification was for 10 cycles of 94 °C for 10 s, 62 °C for 20 s and 68 °C for 45 s, plus 25 cycles with a 5-s increment in each 68 °C extension phase. The resulting renin cDNA (999 bp) was digested with EarI, an enzyme that digests Ren-1^d cDNA twice and Ren-2 cDNA once.

Following sacrifice of animals by CO₂ anesthesia, tissues were immersion-fixed in 4% (w/v) formaldehyde, 0.9% (w/v) NaCl for 24 h and embedded in paraffin wax. Subsequently, 2-µm sections from kidneys, submandibular glands, adrenal glands, and testes were stained with hematoxylin and eosin and examined, in a blinded manner, by standard light microscopy.

Mean blood pressure (40% of systolic + 60% of diastolic pressures) was measured via direct cannulation of the abdominal aorta in adult mice (8-15 weeks old). Measurements were made over a 15-min period in conscious, resting animals housed in restraining tubes. All animals had undergone training (30 min/day) in restraining tubes for 5 consecutive days, beginning 7 days prior to the operation. Operations were performed as described (10). Briefly, a cannula made from drawn polyethylene tubing (Portex, Hythe, UK; Product Code 800/100/100) was inserted into the aorta (while the blood flow in the aorta and vena cava was occluded using a suture) and fixed in place using tissue glue. Cannulae were filled with heparin/saline and were flushed daily. Blood pressure was measured 24 h postoperation by connecting the cannula to a pressure transducer (Viggo-Spectralab, Oxnard, CA) and printing on a chart recorder. Statistical significance was assessed using a two-tailed Student's t-test.

Animals were sacrificed as above, and blood sampled immediately by cardiac puncture into 0.1 volumes of 125 mM EDTA, 25 mM -phenanthroline. Plasma was snap-frozen in liquid nitrogen in 100-µl aliquots. Plasma renin concentrations (PRC) and plasma prorenin concentrations (PPC) were calculated according to the method of Peters et al. (14). Total renin concentration was measured by activating 20 µl of plasma with 40 µl of trypsin (400 units/ml, dissolved in TES buffer; 0.1 M TES, pH 7.2, 0.01% neomycin, 10 mM EDTA). Samples were incubated on ice for 10 min and trypsin activation stopped by the addition of 40 µl of soybean trypsin inhibitor (600 units/ml, in TES buffer). Plasma active renin was measured by the addition of 80 µl of TES buffer (without trypsin) to 20 µl of plasma. Pretreated samples were incubated with lyophilized renin substrate, isolated from nephrectomized rat plasma (final concentration; 80 mg/ml, 0.11% 2,3-dimercapto-1-propanol, 1.15 mg/ml 8-hydroxyquinoline in TES buffer). Reactions proceeded for 1-3 h at 37 °C and were stopped with radioimmunoassay buffer (0.1 M Tris acetate, pH 7.4). The AngI generated by the plasma renin was measured by radioimmunoassay (15, 16). Plasma prorenin concentration is determined as the difference between total renin concentration and plasma active renin concentration. Statistical significance was assessed using the Wilcoxon rank test.

RESULTS

Initially, conditions were established for the efficient PCR amplification of DNA fragments in the size range 3-4 kb, using the thermostable proofreading UlTma DNA polymerase (described under "Experimental Procedures"). Subsequently, two regions of the Ren-1^d gene, extending from exon 1 to exon 3 and exon 4 to exon 9 (Fig. 1, A and B), were successfully amplified using Ren-1^d-specific primers, from bacteriophage P1 clone P1-1249, which contains the entire mouse 129 Ren-1^d gene. PCR products were then digested and used to assemble the final targeting vector, pR1neoKO, in which 3.5- and 3.7-kb segments of Ren-1^d gene homology flank the selectable marker (Fig. 1C). Following electroporation of the targeting construct into ES cells, Southern analysis using 5 and 3 external probes (Fig. 1D and not shown) identified 3 from 313 (1%) drug-resistant colonies that were correctly recombined in both the 5 and 3 arms. One clone was used to generate male chimeras, which were crossed with strain 129 females to generate inbred 129 heterozygote offspring. These mice were intercrossed to produce an F2 generation with wild-type, heterozygote and homozygote inbred Ren-1^d/ mice (Fig. 1E). The absence of recombination within the Ren-2 gene demonstrates the previously reported (17) highly specific recombination achievable, even when targeting closely related genes.

Amplification of total kidney RNA from Ren-1^d/ mice by reverse transcription-PCR, followed by restriction digestion of the product with EarI, confirmed that the disrupted Ren-1^d gene is unable to produce functional Ren-1^d mRNA and that Ren-2 mRNA is the only gene product present in these mice (Fig. 2). Primer extension analysis³ showed that Ren-2-derived mRNA is 2.8 ± 0.05- and 3.9 ± 0.23-fold more abundant in the kidneys of Ren-1^d/ males and females, respectively, compared with wild-type mice.

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Plasma renin concentration (PRC) and plasma prorenin concentration (PPC) were determined from mouse plasma samples (Fig. 3). PRC levels did not differ significantly between males of all three genotypes (+/+ = 240 ± 58; +/ = 170 ± 34; / = 243 ± 63 ng of AngI/ml/h; p > 0.05) (Fig. 3A, solid bars). However, PPC was significantly higher in Ren-1^d/ male mice (1341 ± 116 ng AngI/ml/h), as compared with both wild-type (717 ± 64 ng AngI/ml/h; p < 0.0003) and heterozygous males (566 ± 33 ng AngI/ml/h; p < 0.0003) (Fig. 3A, open bars). In females, PRC was reduced in Ren-1^d/ mice (123 ± 28 ng AngI/ml/h) compared with controls (229 ± 32 ng AngI/ml/h; p < 0.027), while heterozygous females had an intermediate level (164 ± 34 ng AngI/ml/h) (Fig. 3B, solid bars). Similar to male mice, PPC measurements revealed a significant increase in circulating prorenin in female Ren-1^d/ homozygotes (1632 ± 238 ng AngI/ml/h) compared with Ren-1^d+/ mice (528 ± 42 ng AngI/ml/h; p < 0.0003) and wild-type females (557 ± 56 ng AngI/ml/h; p < 0.0003) (Fig. 3B, open bars).

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Measurement of mean arterial blood pressures in males showed no significant (p > 0.05) difference between Ren-1^d genotypes (Table I). However, a significant decrease in blood pressure of 12.7 mmHg was seen in Ren-1^d/ females compared with wild-type controls (p < 0.01).

Table I. Resting blood pressure in Ren-1d gene knockout mice Blood pressure was determined in conscious, restrained wild-type (+/+), Ren-1d heterozygous (+/) and Ren-1d homozygous (/) mice by direct cannulation of the aorta as described (10). Values are mean ± S.E. (n). Mean arterial blood pressure +/+ +/  / mmHg Males 93.6  ± 5.2 (8) 93.0  ± 4.9 (11) 92.3  ± 3.8 (8) Females 93.6  ± 2.5 (8) 85.6  ± 2.8 (7) 80.9  ± 3.4 (8)a a p < 0.01 by Student's t test compared with +/+ females.

Kidneys, adrenal glands, submandibular gland, and testes or ovaries from Ren-1^d/ (n = 4), Ren-1^d+/ (n = 4), and wild-type animals (n = 2) from both sexes were studied. No differences were observed in adrenal glands, submandibular glands, testes, or ovaries from all three genotypes in both sexes. However, kidney sections showed two significant abnormalities in both Ren-1^d/ males and females (Fig. 4, A and B). The macula densa of Ren-1^d/ mice exhibited hypercellularity, and an altered epithelial morphology in which the cells showed a columnar appearance, which contrasts with the cuboidal morphology of the wild-type controls. The central three macula densa cells were measured in five JG regions from each of four individual mice (n = 20) of each genotype. Wild-type and Ren-1^d+/ mice had macula densa cells of 6.1 µm (range 5.6-6.3 µm) and 6.0 µm (range 5.8-6.2 µm) in height (basolateral to apical dimension), respectively, whereas the height of macula densa cells in Ren-1^d/ mice was 7.9 µm (range 7.6-8.1 µm). This represents a 30% increase in cell height in the Ren-1^d-deficient mice. Immunostaining of kidney sections with an antibody specific for renin showed that, in contrast to the granular appearance of the controls, Ren-1^d/ mice exhibited diffuse, uniform, low level cytoplasmic renin staining (approximately 5% of controls) consistent with constitutive secretion, and indicating that renin-2 is not stored in large quantities in the JG cells of these mice (Fig. 4, C and D). Kidney sections from homozygous mutant and control mice were examined by transmission electron microscopy, which demonstrated that the JG cells of the Ren-1^d/ mice were completely devoid of the storage/secretory granules typically present in wild-type controls (Fig. 4, E and F). Nevertheless, Ren-1^d/ JG cells contain an abundant rough endoplasmic reticulum (Fig. 4F).

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DISCUSSION

Disruption of the Ren-1^d gene, described here, provides the first demonstration that inactivation of a gene encoding renin affects blood pressure homeostasis, exemplified by the sexually dimorphic hypotension seen in Ren-1^d/ females. In addition, the Ren-1^d phenotype displays a decrease in the plasma concentration of active renin and an increase in plasma prorenin. Furthermore, a discrete and reproducible change was observed in the morphology of the macula densa cells of the kidney distal tubular epithelium. This small group of cells, which act as sensors in the tubular glomerular feedback loop, but do not express renin, show a 30% increase in basolateral to apical height. The physiological sequelae of this cellular change are presently unknown. Most striking is the complete absence of secretory/storage granules in the JG (modified smooth muscle) cells of the renal afferent arteriole. Thus, expression of the renin-1^d protein is a prerequisite for secretory granule formation and maturation, and the Ren-2 gene product is unable to act as substitute in this role.

A novel feature of the current study is the successful inactivation of the Ren-1^d gene by homologous recombination using a targeting construct in which the regions of DNA providing Ren-1^d gene homology were generated by long range PCR. Together with the use of a similar strategy to target the Ren-2 gene (10), these data demonstrate the feasibility of using homology regions generated entirely by PCR to target genes of interest. Optimized conditions for the efficient amplification of DNA fragments in the size range of 3-4 kb, using a cloned genomic DNA template and the thermostable proofreading UlTma DNA polymerase, included long (6.5 min) extension times, an increased number of cycles (40) and primers of 28-32 nucleotides in length. To facilitate molecular manipulations and/or screening for homologous recombinants, restriction enzyme recognition sequences can be usefully built into primers, but they should be situated at least five nucleotides from the end of the PCR product to permit efficient digestion. Although this experiment utilized a 130-kb bacteriophage P1 Ren-1^d genomic clone as the template for PCR, we have also demonstrated the efficient amplification of up to 10 kb fragments from genomic DNA, using a mixture of proofreading and Taq DNA polymerases (Expand kit (Boehringer Mannheim); data not shown). Thus, in principle, the regions of homology required to target any gene can be generated and tailored by direct PCR from genomic DNA.

Juxtaglomerular cells, the principal site of renin synthesis, represent a specialized population of the smooth muscle cells of the renal afferent arteriole. In particular they contain abundant modified lysosomal granules, where prorenin is activated and stored (1). It is thought that the release of active renin from these secretory/storage granules is by regulated exocytosis in response to specific physiological stimuli, whereas an additional distinct secretory pathway mediates the constitutive secretion of inactive prorenin, via clear secretory vesicles. This concept is based on previous work on cultured tumoral JG cells and human kidney slices (18) and AtT-20 cells, a mouse pituitary cell line that expresses both regulated and constitutive secretory pathways, and can process prorenin into active renin (19-22). It has also been suggested that the mouse enzymes renin-1^d and renin-2 can each be sorted through distinct secretory pathways in AtT-20 cells (23, 24), but whether renin-1^d and renin-2 are secreted separately via distinct pathways or coordinately through both pathways in vivo has yet to be determined. In the current study, the most striking consequences of ablating expression of the renin-1^d protein are a change in renin immunostaining from a punctate, abundant granular pattern in JG cells of wild-type mice, to diffuse, weak cytoplasmic staining (Fig. 4, C and D) and a complete lack of dense granule formation in Ren-1^d-null mice (Fig. 4, E and F). Thus signals required for sorting renin to the regulated secretory pathway of mouse JG cells in vivo reside exclusively in the renin-1^d protein and not in renin-2. This finding suggests that the trafficking and maturation of renin-2 protein within secretory granules in transfected AtT-20 cells (24, 25) may not mirror the situation in the intact mouse, especially given that this cell line displays a different range of prorenin processing activities compared with the JG cells of the kidney (see Ref. 26).

The reduced plasma concentrations of active renin and elevated prorenin seen in Ren-1^d/ mice (Fig. 3) are the converse of the situation in Ren-2^/ mice (10), The Ren-1^d/ phenotype might be explained by a compensatory stimulation of the constitutive secretory pathway, in the absence of regulated secretion of renin-1^d, leading to enhanced secretion of renin-2 in the inactive form. This supports the idea that renin-1^d secretion is predominantly via the regulated (granular) pathway and that renin-2 secretion is predominantly through the constitutive pathway. Higher rates of prorenin-2 secretion may be signaled by the deficit of active renin in the plasma, by the lack of JG secretory/storage granules, or directly by the absence of renin-1^d protein. The exact means by which one or other of these mechanisms stimulates Ren-2 gene expression (2.8-3.9-fold) is not yet clear. However, this resembles a case of human familial elevated plasma prorenin (26), where a mutation in exon 10 of one allele of the human renin gene introduces a premature termination codon. The elevated levels of plasma prorenin in this phenotype are postulated to result from a compensatory mechanism that enhances expression from the normal renin allele (26). The data in Fig. 3 clearly show that homozygous Ren-1^d-null mice display reduced, but nonetheless detectable, levels of active renin in the plasma. This active renin must be derived exclusively from the product of the Ren-2 gene, although the means by which prorenin-2 is converted to activate renin-2 is presently not clear. The complete absence of storage/secretory granules in Ren-1^d/ JG cells, the normal site of renin maturation activity, raises the possibility that prorenin-2 is activated in an extrarenal site in these mice.

The hypotension observed in female Ren-1^d/ mice demonstrates that the renin-2 protein cannot accomplish all the functions of renin-1^d in maintaining basal blood pressure. The fact that reduced blood pressure is seen only in female mice might well be a consequence of the sexually dimorphic expression of the Ren-2 gene (8). For example, male mice express much higher levels of renin-2 in the SMG than females, and this may compensate for a reduction in active renin concentration (Fig. 3) and the hypotension otherwise conferred by the Ren-1^d mutation. The altered macula densa cell morphology (Fig. 4) might reflect perturbations in the renin-angiotensin system in Ren-1^d/ mice, leading in turn to changes in chloride and fluid balance and altered signaling via the tubuloglomerular feedback loop. Studies of ion and fluid balance in Ren-1^d/ mice are presently underway to address these questions.

Recent gene-targeting experiments have shown that an intact renin-angiotensin system is fundamental to maintaining basal blood pressure, since mice lacking genes for angiotensinogen (Agt (27, 28), angiotensin converting enzyme (ACE (29)), and angiotensin type 1A receptor (AGTR1A (30, 31)) all share a reduction in blood pressure as a common phenotypic feature. Ablation of angiotensinogen, or of angiotensin converting enzyme, also results in renal vascular damage and defects in kidney morphology (27-29, 32). It is notable that while Ren-1^d/ mice also have altered renal morphology, these changes are much less severe, and more specific, than those seen in Agt and ACE knockout mice.

A critical feature of the present study is that all mouse stocks were maintained on the 129 strain inbred genetic background onto which the original Ren-1^d gene mutation was introduced. This eliminates the risk of introducing modifier loci, inherent in cross-breeding to other genetic strains, which may mask any phenotypic change caused solely by the introduced mutation (33). Strategies to account for modifier gene effects in gene targeting experiments exist (34), but these involve large breeding populations to ensure random segregation of loci, coupled with genotype assessment. Importantly, the maintenance of a pure genetic background also permits direct comparison with different knock-out animals on the same 129 strain background, for example, Ren-2^/ (10) and Ren-1^d/Ren-2^/ animals.⁴

In conclusion, these studies demonstrate that the mouse Ren-1^d and Ren-2 gene products fulfil distinct roles in renin secretory granule formation and blood pressure homeostasis and that the renin gene duplication in some strains of mice is not functionally redundant. The availability of both Ren-1^d/ (this study) and Ren-2^/ (10) mice presents additional opportunities to dissect renin gene function and the mechanisms underlying the trafficking, storage, maturation, and release of renin in vivo. Furthermore, Ren-1^d/ mice are likely to be especially useful in addressing the contribution of macula densa signaling in the tubuloglomerular feedback loop, particularly in response to perturbations in the renin-angiotensin system.