Ren1d and Ren2 cooperate to preserve homeostasis: evidence from mice expressing GFP in place of Ren1d

ELLEN S. PENTZ1, MARIA LUISA S. SEQUEIRA LOPEZ1, HYUNG-SUK KIM2, OSCAR CARRETERO3, OLIVER SMITHIES2 and R. ARIEL GOMEZ1

1 Department of Pediatrics, University of Virginia, Charlottesville, Virginia 22908
2 Department of Pathology and Laboratory Medicine, University of North Carolina Chapel Hill, Chapel Hill, North Carolina 27959
3 Henry Ford Health Systems, Hypertension Research Division, Detroit, Michigan 48202


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To distinguish the contributions of Ren1d and Ren2 to kidney development and blood pressure homeostasis, we placed green fluorescent protein (GFP) under control of the Ren1d renin locus by homologous recombination in mice. Homozygous Ren1d-GFP animals make GFP mRNA in place of Ren1d mRNA in the kidney and maintain Ren2 synthesis in the juxtaglomerular (JG) cells. GFP expression provides an accurate marker of Ren1d expression during development. Kidneys from homozygous animals are histologically normal, although with fewer secretory granules in the JG cells. Blood pressure and circulating renin are reduced in Ren1d-GFP homozygotes. Acute administration of losartan decreases blood pressure further, suggesting a role for Ren2 protein in blood pressure homeostasis. These studies demonstrate that, in the absence of Ren1d, Ren2 preserves normal kidney development and prevents severe hypotension. Chronic losartan treatment results in compensation via recruitment of both Ren1d- and Ren2-expressing cells along the preglomerular vessels. This response is achieved by metaplastic transformation of arteriolar smooth muscle cells, a major mechanism to control renin bioavailability and blood pressure homeostasis.

renin; blood pressure; kidney; development; homologous recombination


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
RENIN IS THE KEY HORMONE-ENZYME in the renin-angiotensin system (RAS) which is responsible for the control of blood pressure and fluid homeostasis in the adult animal. In the RAS cascade, angiotensinogen is cleaved by renin to produce angiotensin I (ANG I). This is in turn cleaved by angiotensin converting enzyme (ACE) to produce angiotensin II (ANG II), the potent vasoconstrictor that ultimately exerts the effect on blood pressure. The major source of circulating renin in the adult is the kidney juxtaglomerular (JG) cells. In adult mammals, JG cells are located in the afferent arteriole at the entrance to the glomerulus. However, in the fetus, renin mRNA and protein are found along and throughout the developing preglomerular vasculature, including arcuate, interlobular arteries and afferent arterioles. As the kidney matures, renin expression becomes more restricted until in the adult it is found only in the JG cells (11). Treatments with agents that inhibit the response to ANG II [blockade of the ANG II type 1 receptor (AT1 receptor) or inhibition of ACE activity] or genetic manipulations that alter angiotensin availability result in an increase in renin synthesis and an accompanying increase in the number (recruitment) of renin-expressing cells along the afferent arteriole in a pattern resembling that of the fetus (9, 10, 17).

Humans, sheep, and rats have only one renin gene, whereas mice have two alternative genotypes at the renin locus: some strains have only one copy (Ren1c, as found in the C57Bl/6 strain), whereas others have two renin genes (Ren1d and Ren2, as in DBA/2 and 129J) (1, 6). The strains having two renin genes probably resulted from a duplication event of 21 kb of a Ren1c-like ancestral gene (1, 6). The mouse renin genes are expressed in distinct, but overlapping, tissue-specific and developmental patterns. Ren1c and Ren1d and Ren2 are all expressed in the kidney. In two renin gene mice, Ren1d expression is higher than Ren2 (70% of the renin transcripts in males and 80% in females) (17), whereas in the male submandibular gland Ren2 expression is predominant, reflecting the androgen responsiveness of Ren2 (8). In the developing adrenal gland, all three genes are expressed, but Ren1c expression is downregulated in the adult, whereas Ren1d and Ren2 expression continue into adulthood (14). These data come from in situ hybridization studies to localize renin mRNA and Northern analysis or primer extension to assess the different levels of each renin mRNA species. It is not known whether Ren1d and Ren2 are expressed in the same cells or in different cell populations during development or in the mature organ.

The three renin genes have the same overall genomic organization and encode highly homologous but distinct proteins. The Ren1 and Ren2 proteins share 97% amino acid similarity but differ in their potential glycosylation sites: Ren2 has no consensus sites for asparagine-linked glycosylation, whereas Ren1d and Ren1c have three sites (25). These potential glycosylation differences apparently affect the stability of the proteins, with the unglycosylated form being more labile. It is thought that the Ren1 protein is packaged in granules and released to the circulation in a regulated, stimulus-secretion coupling mechanism(s) in response to physiological demands such as hypotension, dehydration, etc. Of the two renins, Ren1 protein has thus been accepted as the major source of circulating renin [Ren1d being the majority transcript in the kidney (17)] and thus the major regulator of blood pressure homeostasis. However, the relative contribution of Ren2 to the pool of circulating renin and the potentially different functions of Ren1d and Ren2 proteins remain to be defined.

To assess the contribution of each allele to blood pressure homeostasis, each of the two renin genes (Ren1d and Ren2) has been individually deleted in two renin gene background mice (strain 129) by homologous recombination [Ren1d (2, 4); Ren2 (24)]. The Ren2 deletion mice are normal and have normal blood pressure. One Ren1d deletion strain was reported to have no change in blood pressure (2), whereas the other reported deletion strain has reduced blood pressure in females accompanied by degranulation of JG cells and alterations in the macula densa in both sexes (4). In contrast, when the single Ren1c gene is deleted in a one renin gene strain, there are profound effects on both kidney architecture and blood pressure (29). These deletion lines suggested that individually neither Ren1d nor Ren2 is absolutely essential for normal blood pressure homeostasis and kidney development. However, the interaction between Ren1d and Ren2 in the control of kidney development, blood pressure regulation, and the recruitment phenomenon has not been defined.

The present study was designed to define: 1) whether deletion of Ren1d in a two renin gene background resulted in alterations in kidney development and/or blood pressure regulation and 2) whether Ren2, in the absence of Ren1d, compensated Ren1d actions by contributing to the pool of circulating renin to regulate blood pressure and maintain normal kidney development. 3) In addition, because recruitment of renin-expressing cells has been identified as a major mechanism to regulate blood pressure homeostasis, we also investigated whether Ren2-expressing cells are involved in this phenomenon. 4) To be able to distinguish Ren1d-expressing cells from Ren2-expressing cells (and eventually to isolate and culture them), we labeled these cells with the vital marker green fluorescent protein (GFP) by replacing the Ren1d gene with GFP by homologous recombination.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Ren1d-GFP Targeting Vector
The targeting vector (Fig. 1C, below) was constructed from three fragments. A 1,242-bp 5' fragment was generated by PCR of 129J mouse genomic DNA using 5' primer 1 (5' GG C AGC TCT GCC CAG GCT TC) located at position 48 in the published upstream sequence of Ren1d (3) and 3' primer 2 (5' GGG TTC AGC CAA GGC TTT CTA A), which terminates at position 1284 adjacent to the ATG of the renin protein. The 5' primer contained additional bases at the 5' end (marked with underscore in the sequence) to introduce a SpeI restriction site for use in cloning. A proofreading DNA polymerase (Vent DNA polymerase; New England Biolabs, Beverly, MA) was used to ensure correct sequence. Two bases were altered adjacent to the ATG of renin (mouse atgg, PCR product atgg) to introduce a NcoI restriction site to facilitate cloning. The 5' DNA was cloned into the NcoI site (blunted with Klenow polymerase) at the ATG of GFP in the plasmid pEGFPN1 (Clontech, Palo Alto, CA). This GFP sequence includes also an SV40 poly-A addition signal. The DNA sequence of the PCR product and the sequence across the junction with GFP were confirmed by sequencing. The 3' targeting DNA was a 8.9-kb HindIII genomic fragment starting at position 9844 in renin intron 7 and containing the remaining 1.4 kb of the Ren1d gene plus 7.5 kb of flanking DNA. The 5' targeting fragment was cloned into the targeting vector osdupdel (available by request from O. Smithies) at a NheI site 5' to a MC1 NeoR gene-selectable marker and the 3' fragment into a HindIII site 3' to the marker.



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Fig. 1. Targeting GFP to the Ren1d gene. A: the strain 129 mouse renin locus showing the two renin genes. Relevant restriction sites are shown: BamHI (B), EcoRI (E), HindIII (H). Genomic DNA fragments detected by the renin 5' probe in Ren1d and Ren2 are shown above the map. Numbers indicate length in kb. B: expanded map of the Ren1d gene showing intron/exon structure and relevant restriction sites. Exons are the numbered black boxes. C: targeting construct showing 5' and 3' homology regions flanking GFP and the locations of the neo resistance cassette and thymidine kinase (TK) gene. Dashed lines show the regions in which recombination can take place. D: targeted gene. GFP replaces exons 1–7 and part of intron 7 of the Ren1d gene. Primers: The locations of the numbered PCR primers described in MATERIALS AND METHODS is indicated below the targeted construct. Probes: 5', 1.2-kb renin 5' DNA; GFP, 0.8-kb GFP DNA. E: Southern blot of DNA from the targeted embryonic stem (ES) cell line used to generate the Ren1d-GFP mouse. The additional bands present are those predicted for targeting GFP to Ren1d (8.5 kb in the BamHI digest and 2.6 kb in the EcoRI digest.) F: confirmation of targeting in the Ren1d-GFP mouse. Southern blot of BamHI digested tail DNA showing both the genomic Ren1d and Ren1d-GFP bands (7.0 and 8.5 kb, respectively) in the +/+ and +/- animals and only the Ren1d-GFP band (8.5 kb) in the -/- animal. GFP, green fluorescent protein.

 
Gene Targeting in Embryonic Stem Cells
Gene targeting was carried out using conventional procedures (16) in TC1 embryonic stem (ES) cells (a gift from Dr. Philip Leder). Targeted colonies were provisionally identified by PCR analysis (18) using primer 3 (5' GCC AGG GTC AGG TCA CTT C) located upstream of any sequences in the targeting vector and primer 4 (5' GTA GGT CAG GGT GGT TCA C) located in GFP. This PCR reaction generated a 1.4-kb product from correctly targeted DNA and no product from randomly inserted DNA. PCR-positive colonies were expanded and genomic DNA was extracted for Southern blotting to confirm the homologous recombination. DNA was digested with BamHI and EcoRI and hybridized with a 5' genomic DNA probe (the PCR product used as the 5' targeting DNA) and an internal probe (a 772-bp EcoRI-NotI DNA fragment) containing the GFP cDNA sequence.

Generation and Identification of Mutant Mice
ES cells from one targeted line were injected into C57Bl/6J blastocysts to produce chimeras, which were crossed to 129SvEv females (Taconic, Germantown, NY). Genotyping of the mice was by PCR of DNA from tail biopsies amplified with primers 3 and 4 as described above. PCR results were confirmed by Southern blots as for the targeted ES cells. To detect homozygous Ren1d-GFP animals, PCR was performed using a 5' primer (5' ATT AGG TTA ATA TGC AGG TCT CG) located at position 386 in the 5' flanking DNA and a 3' primer (5' TAG TAG AAG GGG GAA GTT GTG) located at position 1559 in the first intron of Ren1d. This PCR reaction generates a 1.2-kb fragment if the Ren1d gene is present. If the Ren1d gene is deleted (as in a homozygous Ren1d-GFP animal), then no product is detected.

RNA Extraction and RT-PCR Analysis
Kidneys were removed from -80°C storage, and total RNA was extracted using Tri-Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s directions. Contaminating DNA was removed using the DNA-Free kit (Ambion, Austin, TX). The cDNA was prepared from 2 µg of RNA using Moloney murine leukemia virus (MMLV) reverse transcriptase (Life Technologies, Grand Island, NY) and an oligo dT15 primer. PCR for renin mRNA used primers located in exons 1 and 5 of renin (5' ATG CCT CTC TGG GCA CTC TT and 5' GTC AAA CTT GGC CAG CAT GA) and exons 8 and 9 (primer 9, 5' TGT GAA CTG TAG CCA GGT G; and primer 10, 5' TCG GTT TTT CCT TAA AGC AG). The PCR products were purified using the Qiaquick PCR purification kit (Qiagen, Valencia, CA) and digested with restriction enzymes BstXI or AvaII for the exon 1–5 product or BsrI for the exon 8–9 product. The digestion products have different sizes from Ren1d and Ren2, and thus the two mRNAs can be distinguished. GFP mRNA was detected using primers internal to GFP [primer 5 (5' GTG AGC AAG GGC GAG GAG) and primer 6 (5' GCC GAT GGG GGT GTT CTG)] that amplify a 567-bp product. In addition, PCR was performed with primers from GFP (primer 5) and exon 8 (primer 7 5' AGT CCG TAC TGC TGA GTG TG) or exon 9 (primer 8 5' GCC AAG GCG AAT CCA ATG C).

Histological Analysis
Animals were anesthetized with pentobarbital. The left renal artery was clipped, and the kidney was removed and frozen immediately in liquid N2 for RNA extraction or fixed in Bouin’s. The animal was then perfused through the left cardiac ventricle with cold 4% paraformaldehyde (PFA) in PBS, pH 7.2–7.4. The right kidney was removed, and Vibratome sections (30–100 µm) were cut. The remainder of the kidney was cryoprotected in 30% sucrose in PBS at 4°C for 24–72 h and then frozen in OCT (Miles, Elkhart, IL) and stored at -20°C as previously described (11). Cryosections (10 µm) were cut with a Leica Cryocut 1800 cryostat, postfixed in 4% PFA in PBS, and examined microscopically for GFP fluorescence using standard fluorescein isothiocyanate filters. Bouin’s fixed tissue was dehydrated and embedded in paraffin, and 5-µm sections were cut. Tissues were stained with hematoxylin and eosin for morphological assessment or processed for immunohistochemistry. For electron microscopy, animals were perfused with 4% PFA and 2.5% glutaraldehyde. The kidneys were removed, and fixation continued overnight at 4°C. Tissue was postfixed in OsO4, embedded in epoxy resin by conventional methods, cut into 70- to 80-nm sections, and examined using a JEOL 100CX transmission electron microscope. For granule counts, 0.5-µm sections were stained with toluidine blue and examined at a magnification of x1,000. Only sections in which the JG apparatus (JGA) and the afferent vessel were visible together were selected for evaluation (9, 11).

Immunohistochemistry
Kidney tissue sections (5 µm thick) were deparaffinized in xylene and graded alcohols. Renin protein was detected using a goat anti-rat-renin polyclonal antibody (1:10,000 dilution) [gift of Dr. T. Inagami, Nashville, TN] and the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) as previously described (10).

Blood Pressure Measurements
Animals used for these measurements were 8–16 wk old.

Tail-cuff pressure measurements.
Systolic blood pressure in restrained, conscious mice was measured using a tail-cuff apparatus (Visitech Systems, Apex, NC) with the platform and tail cuff maintained at 38°C (19). Measurements were over a period of 4 days.

Intra-arterial pressure measurements.
Animals were anesthetized with isoflurane, placed on a thermostatically controlled heating table at 38°C and tracheotomized. A polyethylene catheter was threaded down the right carotid artery so that the catheter tip was at the aortic root. Mean arterial pressure was continuously recorded from this catheter by means of a Gould transducer coupled to a Biopac recorder. Measurements were taken over a 5-min period. Statistical significance was assessed by unpaired t-test.

Measurement of Renin Concentration
After measurement of intra-arterial blood pressure, blood was collected via the catheter into plasma separator tubes (Microtainer; Becton-Dickinson, Franklin Lakes, NJ) and stored on ice until separation by centrifugation for 5 min at 14,000 g at 4°C. Plasma samples were snap frozen in liquid N2 and stored at -80°C. Plasma renin concentration was measured by angiotensin I generation from added rat angiotensinogen substrate. A volume of 2 µl mouse plasma was incubated for 30 min at 37°C in incubation buffer (0.1 M sodium phosphate buffer, pH 6.5; 0.02 M disodium EDTA) with 0.5 mg/ml phenylmethylsulfonyl fluoride and 250 ng rat angiotensinogen substrate (2,396 ng ANG I/ml prepared from nephrectomized rat) in a volume of 200 µl. The reaction was stopped by boiling for 10 min. Angiotensin I generation was measured using the Clinical Assays Gamma Coat PRA [125I] RIA Kit. (DiaSorin, Stillwater, MN). The standard curve range was 5.0 ng to 0.02 ng. Statistical significance was assessed using Kruskal-Wallis analysis of variance on ranks.

Stimulation of the RAS
Adult male animals were injected daily for 7 days with 25 mg/kg of the angiotensin receptor antagonist losartan or vehicle (9). Newborn animals (8 days old) were treated with daily subcutaneous injections of 75 mg/kg losartan or vehicle for 6 days. At the end of the treatment period, kidneys and other organs were collected for assay of GFP and renin expression. For acute experiments, animals catheterized for blood pressure measurements were used. After the initial blood pressure recording, a bolus infusion of losartan (20 mg/kg) in no more than 50 µl, a dose previously shown (5) to give maximal response, was introduced through the catheter, and blood pressure was recorded for 5 min after drug infusion. Blood pressure levels were compared before and after losartan injection by paired t-test, and the magnitude of the changes was compared by ANOVA.

Isolation of Renin-Expressing Cells
Kidney microvessels were isolated by the iron oxide perfusion technique (7) and placed in culture. An enriched population of renin-expressing cells was isolated from Percoll gradients and placed into culture as described previously (12, 15).

Animals
Chimeric animals produced from the 129 strain ES cell injections were crossed to 129SvEv (Taconic) females to generate essentially inbred 129 offspring. Animals were maintained on this inbred two-renin gene 129SvEv background, so no Ren1c gene is present to affect assays. To establish timed pregnancies, animals were mated overnight, and the females were checked for vaginal plugs the following morning. The day of detection of a vaginal plug was regarded as embryonic day E0. Mice were fed regular mouse chow (Prolab 2000; PMI Feeds, St. Louis, MO) and tap water ad libitum and housed in a temperature-controlled (22 ± 2°C) environment with a 12:12-h light/dark cycle. All procedures were performed in accordance with the guidelines of the American Physiological Society and were approved by the University of Virginia Animal Care Committee


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Generation of the Ren1d-GFP Targeted Mouse
The 5' DNA sequences of both Ren1d and Ren2 are similar so the targeting construct was designed to have the larger region of Ren1d homology at its 3' end, to favor recombination with the Ren1d allele (Fig. 1). Following electroporation of the targeting construct into ES cells, PCR analysis identified 3/336 (0.9%) dual drug-resistant colonies. DNA from these three lines was examined on Southern blots to confirm that GFP had been incorporated intact and that the DNA upstream of GFP was that of the Ren1d promoter (two of the lines were confirmed, whereas the third had a rearrangement near the 3' end of GFP). In a BamHI digest of wild-type genomic DNA, the Ren 5' probe hybridizes to a 7.0-kb Ren1d band and to 0.2-kb and 4.8-kb Ren2 bands (Fig. 1, A and B) (1). If GFP is correctly targeted to Ren1d, then the size of the BamHI fragment will be 8.5 kb; if it is targeted to Ren2, then the band will be 2.6 kb (Fig. 1D). A Southern blot (Fig. 1E) shows the 8.5-kb band as predicted for targeting GFP to Ren1d, which is the same band hybridized with the GFP probe. In EcoRI digests, the 2.5-kb band hybridized as predicted for Ren1d targeting (3.5 kb would result from targeting to Ren2). One Ren1d-GFP targeted ES cell line was injected into mouse blastocysts to generate a targeted transgenic animal. Heterozygous transgenic animals (maintained on the 129/SvEv background) were intercrossed to produce isogenic wild-type, heterozygous, and homozygous Ren1d-GFP animals. [In the following, the genotypes will be designated as +/+ for wild-type (Ren2 Ren1d/Ren2 Ren1d), +/- (Ren2 Ren1d/Ren2 GFP) for a Ren1d-GFP heterozygote, and -/- (Ren2 GFP/Ren2 GFP) for homozygous Ren1d-GFP animals.] The Southern blot of BamHI digests of DNA from animals of the three genotypes (Fig. 1F) showed that the Ren1d 5' DNA probe hybridized only to the 8.5-kb Ren1d-GFP band in -/- animals, as expected, whereas the endogenous 7-kb Ren1d genomic band was present in both +/+ and +/- animals (Fig. 1F). These results confirm that the homozygous animals have only the correctly targeted Ren1d-GFP allele and no wild-type Ren1d gene.

Gene Expression Analysis
Renin mRNA expression in the Ren1d-GFP mice was examined by RT-PCR using primers located in renin exons 1 and 5. To verify there was no readthrough to the exons remaining in the targeted gene, RT-PCR for renin exons 8 and 9 was included. The exon 1–5 products were digested with BstXI and AvaII. The undigested product is 552 bp from Ren1d and 549 bp from Ren2. Digestion with BstXI cuts the Ren1d product into two fragments (392 and 159 bp) and Ren2 into three fragments (234, 161, and 153 bp). The 392- and 234-bp fragments are diagnostic for Ren1d and Ren2, respectively. The +/+ and +/- samples have both Ren1d and Ren2 bands, whereas in the -/- sample, the 392-bp Ren1d band is absent and only the 234-bp Ren2 band is present (Fig. 2A). Similarly, digestion with AvaII yields three fragments from Ren1d (207, 197, and 148 bp) and two from Ren2 (346 and 203 bp). The diagnostic Ren1d and Ren2 fragments (148 and 346 bp, respectively) are present in the +/+ and +/- samples; the 148-bp Ren1d fragment is absent from the -/- sample (Fig. 2A). The exon 8–9 product is 418 bp, which upon digestion with BsrI yields five fragments from Ren1d (128, 107, 79, 70, and 34 bp) and four from Ren2 (162, 107, 79, and 70 bp). The diagnostic Ren1d and Ren2 fragments (128 and 162 bp, respectively) are present in the +/+ sample; the 128-bp Ren1d fragment is absent from the -/- sample (Fig. 2B). In addition, no product was detected by RT-PCR using primers from GFP (primer 5) and either exon 8 (primer 7) or exon 9 (primer 8) (not shown). RT-PCR using primers for GFP yielded a 567-bp GFP fragment in +/- and -/- samples and no GFP fragment in +/+ (Fig. 2C). These results demonstrate that GFP mRNA is present in +/- and -/- kidney RNA, and Ren1d mRNA is absent from -/- kidneys, thus confirming that the homologous recombination that inserted GFP resulted in deletion of the Ren1d gene and abolished Ren1d mRNA expression.



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Fig. 2. Kidney renin and GFP mRNA expression. A: renin mRNA was amplified from total kidney RNA by RT-PCR of exons 1–5, and the products were digested with BstXI and AvaII to distinguish Ren1d and Ren2 transcripts. L, molecular weight marker; U, undigested PCR product containing both Ren1d (552 bp) and Ren2 (549 bp) products; +/+, wild type; -/-, homozygous Ren1d-GFP; +/-, heterozygous Ren1d-GFP. The diagnostic bands for Ren1d (BstXI, 392 bp; AvaII, 148 bp) and Ren2 (BstXI, 346 bp; and AvaII, 234 bp) are indicated. The diagnostic bands for Ren1d are absent in the -/- animals. B: the exon 8–9 products were digested with BsrI. U, undigested PCR product containing both Ren1d and Ren2 products (418 bp). The diagnostic 128-bp Ren1d and 162-bp Ren2 products are indicated. The 128-bp Ren1d band is absent in the -/- animals. C: RT-PCR for GFP demonstrated that GFP mRNA (567-bp product) is present only in +/- and -/- samples, but not +/+. GFP, control plasmid DNA used as PCR template.

 
Physiological Parameters
The body weights of both male and female Ren1d-GFP mice (+/- and -/-) are not different from the wild type (+/+) [Males: +/+, 27.8 ± 0.82 g (n = 8); +/-, 26.4 ± 0.92 g (n = 8); -/-, 26.2 ± 0.91 g (n = 9). Females: +/+, 19.9 ± 0.99 g (n = 8); +/-, 20.4 ± 0.64 g (n = 12); -/-, 20.6 ± 0.94 g (n = 9)]. The kidney weights in males and females (both +/- and -/-) are also not different from their respective wild type (+/+) [Males: +/+, 0.23 ± 0.02 g (n = 8); +/-, 0.19 ± 0.01 g (n = 8); -/-, 0.19 ± 0.01 g (n = 9). Females: +/+, 0.12 ± 0.01 g (n = 8); +/-, 0.14 ± 0.01 g (n = 12); -/-, 0.14 ± 0.01 g (n = 9)]. The three genotypes are present in the expected Mendelian 1:2:1 ratios from crosses of heterozygotes (44 +/+:92 +/-:40 -/-).

Tail-cuff blood pressure measurements on conscious, restrained animals showed no significant differences among the pressures of male mice from the three genotypes (+/+, +/-, and -/-), although there was a trend toward a lower pressure in the -/- males; the blood pressures in female -/- mice were significantly reduced compared with +/+ females (Table 1). In these same mice, intra-arterial pressures (measured under anesthesia) were significantly lower in both male and female -/- mice compared with +/+ (Table 1).


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Table 1. Blood pressure measurements in Ren1d-GFP mice

 
The plasma renin concentration in homozygotes was significantly reduced in both sexes; -/- males had 22% of +/+ levels, and -/- females had 7% of +/+ levels [Males: +/+, 22.3 ± 3.0 (n = 5); -/-, 4.9 ± 1.2 µg ANG I·ml-1·h-1 (n = 5), P = 0.001 vs. +/+. Females: +/+, 9.9 ± 1.0 (n = 5); -/-, 0.7 ± 0.1 µg ANG I·ml-1·h-1 (n = 6), P = 0.004 vs. +/+].

Histology and Immunostaining for Renin in Ren1d-GFP Kidneys
The kidneys of -/- animals are morphologically normal. There was no hypercellularity of the macula densa. Kidney sections from -/- and +/+ mice were examined for the presence of granules in the JG cells. The JG cells of +/+ animals have numerous secretory granules (Fig. 3A). [70% of the JGAs contained granules, and each positive JG area had 2–5 cells that had visible granules. These cells contained an average of 8 granules each (range 2 to >30).] The -/- animals have few granules in the JG cells (Fig. 3B) and fewer JG areas with granules. [Only 30% of JGAs have granules, and each positive JG area had only 1–2 granule-positive cells. The cells contained an average of 3 granules each (range 1 to 7).] The granules in -/- JG cells are smaller and more electron dense than in the wild-type animals. Immunostaining for renin in kidneys of adult -/- mice (Fig. 3D) showed renin protein in JG cells in the pattern previously described for +/+ animals (Fig. 3C), indicating that Ren2 protein is expressed in the same cells that normally express Ren1d when Ren1d protein is lacking.



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Fig. 3. Homozygous Ren1d-GFP JG cells retain some secretory granules and contain renin protein. Top (A and B): transmission electron micrographs of JG cells taken from control (+/+) and homozygous Ren1d-GFP (-/-) animals. A: +/+ animal showing abundant granules (arrows) in the JG cells. B: JG cell from -/- animal showing secretory granules (arrows). Magnification, x25,000. Bottom (C and D): immunostaining for renin in homozygous Ren1d-GFP kidneys. C: wild-type (+/+) adult kidney. D: homozygous Ren1d-GFP (-/-) adult kidney. Renin staining (brown) is present and localized to the JG areas in both the +/+ kidney (as expected) and the -/- kidney.

 
GFP is Expressed at the Appropriate Times and Locations During Development
The level of GFP expression at all stages was sufficiently high so that GFP fluorescence was readily detected in both +/- and -/-, and the pattern of expression was similar in both genotypes. In day E16 kidneys, GFP is clearly visible in the developing preglomerular vasculature in the pattern previously described by immunostaining for renin (10) (Fig. 4, AC). In the newborn kidney, GFP expression is evident along the afferent arterioles and in the JG cells (Fig. 4D), again corresponding with the renin protein pattern. In adult animals, GFP is restricted to the JG cells in the same pattern as renin (Fig. 3C, and Fig. 4, E and F). In addition, in day E15 adrenal glands (Fig. 4, G and H), there is a transient high level of expression of GFP that correlates with the previously reported renin mRNA expression at this developmental stage (14).



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Fig. 4. Developmental regulation of Ren1d-GFP expression. AC: fetal (day E16) kidney. A: GFP expression in Ren1d-GFP heterozygote. B: bright-field view of A showing GFP-positive vessel containing red cells. The vessels are outlined in white. C: wild-type renin immunostaining (brown). D: newborn (day N13) kidney, showing GFP expression in Ren1d-GFP homozygote. E and F: adult kidney. E: GFP expression in Ren1d-GFP heterozygote. F: bright-field view of F. Glomeruli with GFP-positive juxtaglomerular apparatus (JGA) areas are outlined. G, glomerulus. G and H: GFP expression in fetal (E15) adrenal gland. G: GFP in Ren1d-GFP heterozygote. H: bright-field view of G.

 
Response to Angiotensin Receptor Blockade
Blockade of the AT1 receptor with the specific antagonist losartan results in a decrease in blood pressure and recruitment of renin-expressing cells along the preglomerular vasculature in a pattern resembling that in fetal and newborn animals (9, 28). To determine whether GFP expression behaved as would be expected of endogenous renin production, i.e., could GFP serve as a reporter of promoter activation, and to address the question of whether the Ren2 protein present in homozygous Ren1d-GFP animals could compensate for the missing Ren1d protein, the response in both acute and chronic experiments was assessed.

In the acute experiments, the administration of losartan caused a significant decrease in the blood pressure in both +/+ and -/- males and females (Fig. 5). The mean change in females of both genotypes was the same (+/+, -5.7 ± 2.4 mmHg vs. -/-, -7.7 ± 2.2 mmHg; P = 0.554), whereas the male -/- animals had a smaller change than their +/+ controls (+/+, -9.7 ± 1.7 mmHg vs. -/-, -4 ± 1.2 mmHg; P = 0.012).



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Fig. 5. Change in blood pressure in response to acute losartan administration. A bolus injection of losartan (20 mg/kg) was administered via the carotid catheter in anesthetized animals. Blood pressure was recorded for 5 min after the drug administration. Graph displays the mean change in mean arterial pressure (MAP) during the 5 min after administration of losartan; n = 4 in each group.

 
Heterozygous Ren1d-GFP adult animals treated chronically with losartan showed recruitment of GFP expression along the preglomerular vasculature in a pattern similar to that previously found for renin immunostaining (9) (Fig. 6, AC). AT1A receptor blockade in young animals resulted in histological abnormalities in the treated animals (+/+ and -/-) regardless of genotype. The abnormalities included cyst formation and dilatation of arteries (Fig. 6, DG) as was previously found in treatment of newborn rats (28). There was a similar increase in the extent of renin immunostaining along the preglomerular vasculature in losartan-treated animals of both genotypes (Fig. 6, DG-). The extent of GFP expression in the vasculature was increased in a pattern similar to that of renin (Fig. 6, H and I).



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Fig. 6. Response to AT1 receptor blockade. A–C: recruitment of renin and GFP expression in adult animals (+/+ and +/-) treated with daily injections of losartan (25 mg/kg) for 7 days. A: renin immunostaining pattern in treated +/+ kidney. B: GFP expression in treated +/- Ren1d-GFP kidney. GFP expression extends along the afferent arteriole in the pattern of renin immunostaining. C: bright-field view of B. White outlines, glomeruli (G) and vessels. D–I: response to AT1 receptor blockade in young animals (day N14) given daily injections of losartan (75 mg/kg) for 6 days, showing immunostaining for renin in untreated +/+ kidney (D), losartan-treated +/+ kidney (E), untreated -/- kidney (F), and losartan-treated -/- kidney (G); and GFP expression in untreated -/- kidney (H) and losartan-treated -/- kidney (I). Note similar histological abnormalities (vascular dilatation and cysts indicated by asterisks), similar extension of renin immunostaining (brown) along the vasculature in both +/+ and -/- kidneys, and extension of GFP expression in losartan-treated -/- kidneys.

 
GFP Expression in Isolated Microvessels and JG Cells from Ren1d-GFP Kidneys
A major utility of labeling renin-expressing cells with GFP is that they can be identified by fluorescencewithout destroying the cells. The GFP marker could eventually be used to isolate a pure population of renin-expressing cells and enable manipulation of them in culture. To evaluate these potentials, two methods were used. First, microvessels isolated by the iron oxide perfusion technique were placed into culture. In these preparations, renin-expressing cells surrounding the microvessel are readily visible (Fig. 7, A and B). Second, an enriched population of renin-expressing cells isolated from Percoll gradients was placed into culture where initially there is a mixture of cell clumps and individual cells. GFP-expressing cells were easily identified in the cell clumps as small rounded cells (Fig. 7, C and D). After 2 days in culture, many of the cells had flattened out (Fig. 7E) but no longer expressed GFP and so consequently appeared to have ceased transcribing renin. However, after 3 h of exposure to 5 µg/ml forskolin, expression of GFP again became evident (Fig. 7F).



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Fig. 7. GFP expression in isolated microvessels and JG cells from Ren1d-GFP kidneys. Microvessels isolated by the iron oxide perfusion method were placed into culture and examined for GFP expression. A: GFP-expressing cells surrounding an isolated microvessel. B: bright-field view of A; the GFP-expressing cells are indicated by arrows. An enriched population of renin-expressing cells was isolated from Percoll gradients and placed into culture. C: GFP-expressing cells directly from the gradient. D: bright-field view of C; arrows point out the GFP-positive cells. E: cells flattened on dish after 2 days of culture; these cells do not express GFP. F: GFP expression in cells cultured for 2 days and then stimulated 3 h with 5 µg/ml forskolin; the cells now express GFP. Arrow in E indicates cell expressing GFP after forskolin stimulation.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study shows that GFP can replace the Ren1d gene and be appropriately expressed under control of the Ren1d promoter when introduced into the mouse genome by homologous recombination. The introduction of GFP had no obvious adverse effect on the mice. The body and kidney sizes of the +/- and -/- animals were not different from +/+, and the three genotypes were equally represented in the progeny from heterozygous parents. The kidneys of both +/- and -/- animals are normal. There is a small but significant reduction in the blood pressure of -/- animals. The fluorescence from GFP expression is sufficiently high to use the marker in heterozygous Ren1d-GFP animals. The expression of GFP in +/- and -/- animals follows the normal pattern for renin previously described by immunostaining and responds appropriately to stimulation of the RAS. In addition, GFP (renin)-expressing cells can be isolated, identified, and cultured in vitro.

GFP Is an Accurate Marker of Ren1d Gene Expression and Renin-Expressing Cells
The pattern of GFP expression during kidney development is identical to that previously described by immunostaining for renin. We could readily detect GFP-expressing kidney cells in blood vessels in day E16 embryonic kidneys and in a few isolated cells in E15 embryos. This is in agreement with previous work detecting renin by immunostaining as early as E14 in scattered renin cell precursors (23). In newborn and adult animals, GFP is localized to the vessels and JG cells in the pattern characteristic of renin mRNA and its protein as previously found by immunohistochemistry and in situ hybridization (10). These results validate the fidelity of the GFP expression pattern in the kidney and show that Ren1d-GFP expression and localization are appropriately developmentally regulated. Additionally, the expression of GFP in fetal adrenal glands in the pattern of renin further demonstrates the developmental fidelity of the GFP expression. Using a transgenic approach, Jones et al. (13) recently reported a 4.1-kb Ren1c promoter/GFP animal in which GFP expression appears to faithfully reflect renin expression during fetal development and in response to captopril stimulation.

Ren1d regulates Granulopoiesis and Blood Pressure and Participates in the Recruitment Response
Electron microscopy of the homozygous Ren1d-GFP kidneys shows the presence of a few granules in the JG cells in contrast to the abundant numbers in +/+ JG cells. In addition, there were fewer JG cells that contained any granules in -/- mice. The previously described Ren1d (4) and Ren1c (29) deletion mice were reported to have no granules in the JG cells, but, considering how sparse the population of granular cells is in -/- animals, the few granules in a few positive cells could have been missed in the previous reports. A recent report by Mullins et al. (21) showed that the presence of granules in the JG cells could be restored in a Ren1d -/- animal by introduction of an active Ren1d transgene. Taken together, the data indicate that the presence of granules in the JG cells mainly correlates with the presence of a Ren1 protein, which normally is 70–80% of the kidney renin in two gene animals. The few, electron-dense granules detected in Ren1d -/- JG cells may be either immature granules (27) that never fully develop in the absence of Ren1d protein or, alternatively, the JG cells may have some Ren2 protein stored and/or degraded in them.

Our results show that the blood pressure measured by tail cuff in conscious restrained mice was significantly reduced in female -/- animals and there was a trend to reduced pressure in males. Intra-arterial pressures measured under anesthesia in the same animals showed a significant reduction in blood pressure in homozygotes of both sexes. Blood pressure measurements in the two reported Ren1d knockout mouse lines gave conflicting results. Clark et al. (4) reported that Ren1d -/- female mice had significantly reduced blood pressures while the males were not different from normal. Bertaux et al. (2) reported that the blood pressures were normal in Ren1d -/- mice of both sexes. The different blood pressure effects in the three Ren1d deletion strains are most likely due to a combination of factors, including the different methods of measurement, subtle strain and environmental influences, and to the fact that the mutations are not identical.

The data suggest that renin expression due to the Ren2 gene partially, but not completely, compensates for the absence of Ren1d protein in regulating blood pressure. Nevertheless, Ren1d protein is a major source of active circulating renin. The active renin concentration in the plasma was significantly reduced in both male and female Ren1d-GFP homozygotes. Clark et al. (4) reported that the plasma renin concentration was reduced only in the female -/- animals and correlated this with their reduced blood pressure. In our mice both males and females have reduced blood pressures, and this correlates with the reduced circulating renin concentrations in both sexes. It appears that although the level of circulating renin was significantly reduced (to 10–20% of wild type), this reduction does not have a dramatic effect on the regulation of blood pressure. This is supported by the experiments in which losartan further reduced blood pressure in -/- mice, indicating that Ren2 was responsible for generating ANG II and preventing severe hypotension in these animals.

Our experiments show that the Ren1d gene participates in the response to AT1 receptor blockade with losartan. In adults there was recruitment of GFP expression extending along the afferent arteriole as described previously for renin (9). In newborn -/- animals, GFP expression (reflecting Ren1d transcription) was increased along the vessels in the previously described pattern of renin staining. Thus the absence of Ren1d does not alter the response to the apparent lack of angiotensin.

Compensation by Ren2 for the Lack of Ren1d
Although homozygous Ren1d-GFP animals have a marked reduction of renin granules and a clearcut reduction (but not complete absence) of circulating renin, these animals had immunostainable renin in JG cells. Clark et al. (4) showed only weak renin immunostaining in their wild-type and Ren1d deletion animals. Differences in fixation method, immunostaining procedure, and antibody titer may account for the different levels of renin detection in the two studies. In support of our finding of significant Ren2 staining in the -/- animals, Clark et al. (4) reported a three- to fourfold increase in Ren2 mRNA levels in -/- animals. Since normally Ren2 mRNA is 20–30% of the renin mRNA in the kidney (17), this increase correlates with the levels of Ren2 renin immunostaining we observed in -/- kidneys. These results indicate that the Ren2 gene compensates by expressing renin in the usual Ren1d location (the JG cells), and this results in circulating renin that maintains blood pressure near normal levels. In our homozygous Ren1d-GFP animals, there is a more marked decrease in blood pressure and plasma renin levels in the females than in the males, suggesting a sexually dimorphic compensation response in which males, via the androgen-responsive Ren2 gene, compensate more completely than the females. This interpretation is supported by data from studies of renin compensation in heterozygous angiotensinogen deletion mice. In these animals the Ren1d and Ren2 genes respond in a sexually dimorphic fashion, such that in females both renin genes respond in the same proportion as in the wild type, whereas in the males the greater proportion of the increase is mediated by the Ren2 gene (17).

The ability of Ren2 to compensate is evidenced also by the experiments in which an apparent lack of ANG II is produced by ACE inhibition (with captopril) or angiotensin receptor blockade (with losartan). Using captopril, Mullins et al. (21) showed that Ren1c, Ren1d, and Ren2 respond to lack of ANG II by increasing mRNA levels and thus potentially increasing the amount of renin protein. In the Ren1c/GFP transgenic, (13) captopril treatment resulted in expression of GFP along the adult vasculature in the pattern of renin recruitment. Our studies with Ren1d-GFP -/- mice treated with losartan allowed us to determine whether the increased Ren2 mRNA expression mentioned above (21) resulted in distribution of Ren2 protein in the typical recruitment pattern. We found that -/- mice responded with an increase in the number of renin (Ren2)-positive cells along the afferent arterioles, indicating that not only Ren1d but also Ren2 is involved in the increased number of cells that express renin to maintain renin availability and blood pressure homeostasis. In addition, the histological changes induced by losartan treatment were the same in +/+ animals which have both renin genes and the -/- animals which have only Ren2, indicating that in the unchallenged animal the action of Ren2 prevents the developmental abnormalities that occur when renin is absent (29).

This compensating effect was demonstrated also by the further drop in blood pressure in -/- animals when angiotensin actions were inhibited by an acute dose of losartan, suggesting that Ren2-derived renin had maintained (or was responsible for maintaining) ANG I generation and partially maintain blood pressure. This experiment demonstrates a complex regulation whereby Ren2 protein, in the absence of Ren1d protein, maintains blood pressure to about 85–90% of normal. Inhibition of angiotensin action further reduces the blood pressure by 5–16%, uncovering this unexpected action of Ren2. (Incidentally, this finding explains why in -/- animals there were no kidney developmental abnormalities, which, however, became obvious when angiotensin activity was further inhibited.) These findings suggest that there is a minimum requirement for angiotensin below which kidney abnormalities ensue. This finding agrees with previous experiments demonstrating that renin via ANG II generation is necessary for kidney development.

GFP (Ren1d)-Expressing Cells Can Be Identified in Vitro
The in vitro studies reported here demonstrate that the GFP (renin)-expressing cells can be readily visualized in microvessel preparations and in culture and the cells retain their ability to express GFP. Furthermore, the GFP expression responds appropriately to treatment with agents that stimulate renin expression, as shown by the upregulation of GFP expression after forskolin treatment. This forskolin response, which is mediated by cAMP, has both a transcriptional and posttranscriptional component, and the Ren1c, Ren1d, and Ren2 promoters all respond to cAMP by increased transcription in in vitro promoter-reporter studies (20, 21, 25). Our culture results show that GFP fluorescence in cells from the Ren1d-GFP animals allows visualization of stimulation of the Ren1d gene.

With the ability to now identify living renin-expressing cells, it will be possible to follow their movements during development. Preliminary studies of fetal kidneys cultured in vitro have demonstrated the appearance of GFP-expressing cells after 2–3 days in culture. Improvements in the detection optics should make these studies even more revealing and thereby facilitate the analysis of the roles of Ren1d and Ren2 in normal development and in response to challenges to homeostasis. In addition, the possibility of isolating reasonable numbers of renin-expressing cells by fluorescence-activated cell sorting will provide a source of cell-specific RNA for gene expression analysis with serial analysis of gene expression (SAGE) or microarray methods. Using the GFP fluorescence, it will also be possible to analyze the response of the renin gene to stimuli in individual cells in culture. These Ren1d-GFP animals thus provide mice that can serve as a means of isolating live renin-expressing cells for in vitro studies and cell culture, for in vivo analysis of the lineage of Ren1d-expressing cells, and for assessing the contribution of Ren1d and Ren2 to development and physiology.

In summary, the present studies demonstrate that both Ren1d and Ren2 participate in the maintenance of blood pressure homeostasis and normal kidney development. This work illustrates the utility of marking the activity of specific genes to unmask their homeostatic interactions and identify the cell population(s) involved in the response. Further studies with these animals will help elucidate the specific roles of Ren1d and Ren2 in the complex mechanisms involved in renin bioavailability for blood pressure homeostasis.


    ACKNOWLEDGMENTS
 
Special thanks are given to Kimberly Kluckman for teaching E. S. Pentz embryonic stem cell culture and gene targeting methods.

This work was supported by National Institutes of Health Grants DK-52612 and GM-20069. The use of the facilities of the Child Health Research Center core laboratories at the University of Virginia is gratefully acknowledged.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: R. A. Gomez, Univ. of Virginia, 300 Lane Rd., Bldg. MR-4, Rm. 2001, Charlottesville, VA 22908 (E-mail: rg{at}virginia.edu).


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