Elevated Blood Pressure and Heart Rate in Human Renin Receptor Transgenic Rats
Recently, a receptor for renin was described that may be important for vascular uptake and activation of (pro)renin, thus leading to local generation of angiotensin II. To assess the in vivo relevance of this protein, we generated transgenic rats overexpressing the human renin receptor gene in smooth muscle tissue, under the control of a 16-kb fragment of the mouse smooth muscle myosin heavy chain gene [TGR(SMMHC-HRR)]. Four lines of transgenic animals were obtained. The correct pattern of expression of the transgene was confirmed by RNase protection assay and in situ hybridization. TGR(SMMHC-HRR) rats are fertile and develop normally. After 6 months of age, transgenic rats develop a cardiovascular phenotype with an elevated systolic blood pressure (137.8±5 versus 118.9±3.7 mm Hg; P=0.008), and an augmentation in heart rate (349.1±7.7 versus 303.1±16.16 bpm; P=0.023) in TGR(SMMHC-HRR) and controls, respectively. These alterations are progressively increasing with aging. Although kidney function and plasma renin were normal in TGR(SMMHC-HRR), an increase in plasma aldosterone [TGR(SMMHC-HRR) 428±64.9 versus 207.3±73.24 pg/mL in control; P=0.02] and in aldosterone/renin ratio [TGR(SMMHC-HRR) 8.04±2.2 versus 2.8±0.55 in control; P=0.03] was observed. This suggests that renin receptor overexpression has resulted in increased intraadrenal angiotensin II, thereby provoking enhanced aldosterone generation in the absence of changes in plasma renin. The rise in aldosterone may underlie, at least in part, the observed cardiovascular phenotype of TGR(SMMHC-HRR).
The circulating renin angiotensin aldosterone system (RAAS) is an essential determinant for blood pressure control and hydroelectrolyte balance. Active renin (EC: 184.108.40.206), produced and secreted in a regulated manner by the myoepitheloid cells of the glomerular afferent arteriole, contributes to angiotensin generation in blood and at tissue sites. To allow tissue angiotensin generation, circulating renin, after its release from the kidney, needs to be sequestered by tissues, for example, through diffusion into the interstitial space1 or by binding to specific receptors.2 Two such receptors have been identified so far: the mannose-6-phosphate/insulin-like growth factor II receptor,3 and the “renin receptor.”4 The first occurs ubiquitously and appears to be a clearance receptor for renin and its inactive precursor, prorenin.5 The second binds both renin and prorenin to the cell surface, without resulting in internalization. Interestingly, binding to this receptor induces a conformational change, thereby increasing the catalytic activity of renin 5-fold and allowing prorenin to become fully enzymatically active.4 A similar concept for prorenin activation was recently proposed by Ichihara et al.6 The renin receptor is mainly expressed by vascular smooth muscle cells (VSMCs) and renal mesangial cells.4 Unexpectedly, (pro)renin binding to this receptor also induced phosphorylation and mitogen-activated protein kinase p42/p44 activation, thereby allowing renin and/or prorenin to exert angiotensin-independent effects. The latter finding might explain why prorenin transgenic rats develop a vascular and kidney phenotype mimicking pathological manifestations induced by hypertension, in the absence of high blood pressure and without activation of the circulating RAAS.7 It may also underlie the positive correlation between high prorenin levels and the increased risk of vascular damage observed in diabetic patients.8
To analyze the functional relevance of renin receptor in vivo we generated transgenic rats [TGR(SMMHC-HRR)] overexpressing the human renin receptor (HRR) in smooth muscle cells under the control of a 16-kb fragment of the mouse smooth muscle myosin heavy chain gene. TGR(SMMHC-HRR) showed strong transgene expression in the arterial wall, making the model valuable for studying the contribution of the renin receptor to circulating and tissue RAAS. TGR(SMMHC-HRR) spontaneously developed slow and progressive cardiovascular changes, marked by increased blood pressure and heart rate, accompanied by aldosterone elevation.
Generation of the Transgenic Rats TGR(SMMHC-HRR)
We used an HRR full-length cDNA (AF291814) cloned between the NotI and XbaI site of pcDNA 3.1+ vector (Invitrogen).4 A splicing acceptor site mimicking the splicing acceptor site of the second exon of the mouse myosin heavy chain gene was created using the following oligonucleotides: SMRR1=5′CGACTCGAGTTTACTCTGTCCCTGTCTTTTCACTCCACAGGGC-3′ and SMRR2=5′GGCCGCCCTGTGGAGTGAAAAGACAGGGACAGAGTAAAACTCGAGTCG-3′. The resulting double-stranded DNA fragment was cloned 5′ to the HRR sequence into the NruI and NotI sites. Others have defined9,10 a 16-kb fragment (−4229, +11600) of the mouse myosin heavy chain gene as sufficient to drive strong and specific expression of a reporter gene in smooth muscle cells. This smooth muscle myosin heavy chain (SMMHC) fragment was integrated 5′ to the HRR sequence using the SalI-compatible XhoI site of the splicing acceptor site. The resulting 17-kb NruI/NaeI fragment was used for production of transgenic rats as described previously.11 Genomic integration of the transgene was determined by PCR analysis of DNA obtained from offspring tail biopsies. The following primer set was used for genotyping (MHCHRR5: 5′-AGGAGAGGCAAGTAGATCCG-3′ and MHCHRR3: 5′-CTCCTGGTATAGGCCAATTTCCAT-3′). The expected transgene-specific 230-bp fragment, encompassing the last 3′ 100 bp of the SMMHC promoter, the splicing acceptor site, and the first 5′ 130 bp of the AF 291814 sequence, was subsequently cloned in to pGEM-T (Invitrogen). The resulting plasmid (SMMHC-HRR-230) was used for RNase protection assay (RPA). All of the procedures were performed according the guidelines of the American Physiological Society and were approved by local authorities (permit No. G0066/64).
Detection of HRR mRNA Expression
Tissue-specific mRNA expression of the transgene was verified by RPA from total tissue RNA. A 32P-labeled antisense probe was prepared with T7 RNA polymerase transcription of the plasmid SMMHC-HRR-230 after SpeI digestion. Total mRNA was isolated with TRIZOL from brain, lung, heart, aorta, adrenal gland, kidney, liver, intestine, bladder, uterus, skeletal muscle, and aortic smooth muscle cells, from both male and female rats at 8 weeks of age. RPA was performed according to the manufacturer’s protocol (AMBION, RPA kit II). For each hybridization reaction, 40 μg of RNA and 50 000 cpm of purified 32P-labeled transcript were incubated at 42°C overnight.
Integrity of the HRR mRNA
We checked the integrity of the transgenic mRNA by 5′ and 3′ rapid amplification of cDNA ends (RACE) PCR, using the RACE XLM PCR kit (Ambion). We followed the instructions of the manufacturer, starting from a uterus mRNA sample and using the following internal specific primers: HRR25, 5′-AACGAGTTTAGTATATTAAAT-3′, and HRR43, 5′-TTCCTCAGAAAAATAAGGAGTG-3′.
In Situ Hybridization
Correct expression pattern of the transgene was assessed by in situ hybridization, as described previously. Briefly a 35S-UTP–labeled mRNA probe was synthesized using a 600-bp fragment of the AF291814 as template.12
Prorenin Binding to Aortic Segments
Rat aorta segments (3 to 4 mm) were removed and incubated overnight in cold (4°C), oxygenated Krebs bicarbonate solution13 containing 100 U/L human recombinant prorenin. The next morning, the vessels were washed 5 times with cold Krebs bicarbonate buffer and homogenized. Prorenin was activated by acidification,2 and the acidified homogenates were incubated with excess sheep angiotensinogen to quantify the total amount of sequestered prorenin by enzyme-kinetic assay. Values were corrected for background angiotensin I–generating activity using acidified homogenized segments that had not been exposed to human prorenin.
Measurement of Blood Pressure and Heart Rate
Systolic blood pressure, diastolic blood pressure, and heart rate were recorded in conscious, freely moving animals by a telemetric pressure transducer implanted in the aorta as described.14
Measurement of Plasma RAS, Electrolytes, and Kidney Function
Rats were anesthetized briefly with ketamine and xylazine. Blood obtained from jugular vein puncture was collected into ice-cold microcentrifuge tubes containing EDTA and immediately centrifuged to isolate plasma. Plasma renin activity (PRA) and plasma renin concentration (PRC) were determined as described before.15 Plasma aldosterone was measured by radioimmunoassay. For the evaluation of kidney function, rats were placed for 3 days in metabolic cages, and urine was collected over a 24-hour period. Blood was collected in tubes containing heparin and centrifuged. K+, Na+, creatinine, albumin, and/or urea were measured in urine and blood plasma.
The results are expressed as mean±SEM. Hemodynamic parameters are presented as the mean of value of 3 days of sampling. Student t test was used for comparisons between groups. P<0.05 was considered significant.
Generation of TGR(SMMHC-HRR) Rats
The construct depicted in Figure 1A was used to generate the transgenic rat strain TGR(SMMHC-HRR). A 16-kb fragment of the SMMHC gene was cloned upstream the HRR cDNA, because this promoter sequence has been defined by others9,10 as sufficient to drive strong reporter gene expression in smooth muscle cells. We obtained 4 founder animals identified by PCR genotyping, 7324, 7328, 7329, and 7154 (Figure 1B). For each founder, lines were established, and heterozygous male and female animals were characterized with regard to the expression pattern of the transgene. The expression pattern was the same in the 4 lines, without sex variation, and was restricted to organs with a high smooth muscle content, such as aorta, bladder, uterus, and lung (Figure 1C). The expression was very high in VSMCs isolated from aorta as shown in Figure 1C and was not detectable in skeletal muscle and brain (data not shown). Kidney, heart, and adrenal gland showed a weak transgene mRNA expression (Figure 1C). The only notable expression in an organ with low smooth muscle content was detected in the testis (data not shown). Line 7154 and line 7329 showed the highest level of transgene mRNA expression, whereby expression in line 7329 was lower in all organs (Figure 1C). In situ hybridization confirmed a strong and specific VSMC layer expression of the transgene in aorta and kidney as shown in Figure 2. Transgene mRNA integrity was verified by 5′ and 3′ RACE PCR in lines 7154 and 7329 (data not shown).
After overnight incubation with human prorenin, homogenized aortic segments of TGR(SMMHC-HRR) contained 3 times as much prorenin (measured as angiotensin I–generating activity after prorenin activation by acidification) as aortic segments from control rats (n=5 for each; Figure 3; P=0.07).
Cardiovascular Phenotype in TGR(SMMHC-HRR)
Transgenic animals are fertile and develop normally. Male heterozygous animals of 2 lines (7154 and 7329) and control littermates were submitted to telemetry recording of blood pressure and heart rate in freely moving conscious animals for a time period covering 6 months to 1 year. Transgenic rats revealed a spontaneous progressive cardiovascular phenotype. Onset of the phenotype showed variability between the 2 lines, with earlier appearance in line 7154 presenting the highest expression level of the transgene. Before 3 months of age, line 7154 and control rats had comparable systolic blood pressure. After 6 months of age, a progressive increment in blood pressure, initially predominantly systolic, was observed (Figure 4A). At 8 months of age, the systolic blood pressure was 137.8 (±5) mm Hg, as compared with 118.7 (±3.7) mm Hg (P=0.008) in control rats. Simultaneously, a 20% increase in heart rate was observed in line 7154 (P=0.023; Figure 4B).
RAAS and Kidney Function in TGR(SMMHC-HRR)
PRA (3.6±1.1 versus 2.5±1.0 ng angiotensin I/mL per hour) and PRC (126±50 versus 104±69 ng angiotensin I/mL per hour) were identical in TGR(SMMHC-HRR) and control rats. No differences were observed in plasma or urinary electrolytes, creatinine, or albumin (Table). Plasma aldosterone was doubled in TGR(SMMHC-HRR) (Figure 5), and this correlated with a trend to lower plasma K+ in TGR(SMMHC-HRR) (Table). The aldosterone/renin ratio was significantly higher in TGR(SMMHC-HRR) (Figure 5).
There are conflicting data concerning the function of the recently described HRR protein/ATP6AP2.4,16,17 Sequence examination does not reveal any domain of homology with known receptors or proteins. However, the 70 C-terminal amino acids of the protein are highly conserved between species. In this particular region, a protein fragment of 8 to 9 kDa (M89) has been shown to interact with the integral domain of the vacuolar H+-ATP-ase.16 The mutant zebrafish for the homologous gene displayed a severe defect in early development (C. Burcklé, M. Bader, unpublished observation, 2005).17 In contrast, the large extracellular domain of this protein, which is less evolutionary conserved, has been shown to interact with renin and prorenin in vitro.4,6 Moreover, some distinct features, such as partial plasma membrane localization, VSMC and mesangial cell expression, high binding affinity for (pro)renin, and a prorenin unmasking enzymatic activity in cellular assays made this protein a relevant candidate for in vivo functional studies. Therefore, we have generated transgenic rats, TGR(SMMHC-HRR), expressing the HRR in smooth muscle cells.
In TGR(SMMHC-HRR), HRR mRNA expression is, as expected, restricted to organs with a high smooth muscle cell content and is very strong in smooth muscle cells of the aortic vascular wall. The smooth muscle cell layer location does not hinder (pro)renin accessibility as the enzyme diffuses freely by a paracellular pathway through the endothelial cell layer.1 Therefore, TGR(SMMHC-HRR) constitutes a valuable model for unraveling the impact of HRR overexpression on systemic and, particularly, on local RAS. We observed a clear tendency for increased uptake of prorenin by the TGR(SMMHC-HRR) aortic wall in vitro, hence confirming cell culture data, which showed increased binding of renin and prorenin at the cell surface after transfection of human or rat renin receptor cDNA.4,6
Interestingly, TGR(SMMHC-HRR) rats develop a delayed cardiovascular phenotype, defined by high blood pressure and heart rate. The increased blood pressure was accompanied by significant elevation of plasma aldosterone levels, a marked increase of the aldosterone/renin ratio, and a tendency to hypokalemia. Because PRC, PRA, and angiotensin I generation were comparable in transgenic and control rats, aldosterone hyperproduction possibly results from a primary adrenal cause. Consistently, there is no other strong stimulating systemic signal for aldosterone synthesis, such as hyperkalemia. Moreover, the likelihood of exaggerated corticotropin secretion seems to be very low, because the adrenal glands of the transgenic rats were macroscopically normal (data not shown).
In vitro, the renin receptor has been described as a stimulating cofactor for renin enzymatic activity.4 Because the HRR is expressed in the adrenal gland of TGR(SMMHC-HRR) with a vascular localization (data not shown), the elevated aldosterone level could be related to an intraadrenal RAS activation. However, it is not completely clear whether aldosterone elevation is the culprit for blood pressure increase. This will be additionally evaluated by administration of an aldosterone antagonist and adrenalectomy. Nevertheless, we cannot exclude a locally increased angiotensin II generation in resistance vessels as the cause for the hypertensive phenotype.
Another striking phenomenon in TGR(SMMHC-HRR) is the increase in heart rate. With a functioning baroreflex, one would expect normal or low heart rate in response to higher blood pressure. This may indicate a baroreflex dysfunction with sympathetic activation and may be caused by a stimulated central RAS.
A progressive cardiovascular phenotype is associated with VSMC renin receptor overexpression in TGR(SMMHC-HRR). In vitro, (pro)renin binding to the receptor increases its catalytic activity. In vivo, local activation of the intraadrenal RAS may contribute to aldosterone hyperproduction and to elevated blood pressure. Thus, inhibiting (pro)renin binding to the receptor may limit local RAS activation and may be considered as a potential new therapeutic target.
We acknowledge the expert help of Rosemarie Barnow, Reika Langanki, and Patrick Bruneval. C.A.B. was supported by fellowships of the Marie Curie training site “CVmodel” of the European Union and ACTELION Ltd.
- Received October 1, 2005.
- Revision received October 25, 2005.
- Accepted November 29, 2005.
Danser AHJ, van Kats JP, Admiraal PJJ, Derkx FHM, Lamers JMJ, Verdouw PD, Saxena PR, Schalekamp MADH. Cardiac renin and angiotensins. Uptake from plasma versus in situ synthesis. Hypertension. 1994; 24: 37–48.
Saris JJ, Derkx FHM, de Bruin RJA, Dekkers DHW, Lamers JMJ, Saxena PR, Schalekamp MADH, Danser AHJ. High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin. Am J Physiol. 2001; 280: H1706–H1715.
Saris JJ, van den Eijnden MMED, Lamers JMJ, Saxena PR, Schalekamp MADH, Danser AHJ. Prorenin-induced myocyte proliferation: no role for intracellular angiotensin II. Hypertension. 2002; 39: 573–577.
Ichihara A, Hayashi M, Kaneshiro Y, Suzuki F, Nakagawa T, Tada Y, Koura Y, Nishiyama A, Okada H, Uddin MN, Nabi AH, Ishida Y, Inagami T, Saruta T. Inhibition of diabetic nephropathy by a decoy peptide corresponding to the “handle” region for nonproteolytic activation of prorenin. J Clin Invest. 2004; 114: 1128–1135.
Regan CP, Manabe I, Owens GK. Development of a smooth muscle-targeted cre recombinase mouse reveals novel insights regarding smooth muscle myosin heavy chain promoter regulation. Circ Res. 2000; 87: 363–369.
Madsen CS, Regan CP, Hungerford JE, White SL, Manabe I, Owens GK. Smooth muscle-specific expression of the smooth muscle myosin heavy chain gene in transgenic mice requires 5′-flanking and first intronic DNA sequence. Circ Res. 1998; 82: 908–917.
Popova E, Bader M, Krivokharchenko A. Production of transgenic models in hypertension. Methods Mol Med. 2004; 108: 33–50.
Tom B, Garrelds IM, Scalbert E, Stegmann APA, Boomsma F, Saxena PR, Danser AHJ. ACE- versus chymase-dependent angiotensin II generation in human coronary arteries: a matter of efficiency? Arterioscler Thromb Vasc Biol. 2003; 23: 251–256.
Plehm R, Barbosa ME, Bader M. Animal models for hypertension/blood pressure recording. Methods Mol Med. In press.
Pilz B, Shagdarsuren E, Wellner M, Fiebeler A, Dechend R, Gratze P, Meiners S, Feldman DL, Webb RL, Garrelds IM, Danser AHJ, Luft FC, Muller DN. Aliskiren, a human renin inhibitor, ameliorates cardiac and renal damage in double-transgenic rats. Hypertension. 2005; 46: 569–576.
Ludwig J, Kerscher S, Brandt U, Pfeiffer K, Getlawi F, Apps D, Schägger H. Identification and characterization of a novel 9.2-kDa membrane sector-associated protein of vacuolar proton-ATPase from chromaffin granules. J Biol Chem. 1998; 273: 10939–10947.
Amsterdam A, Nissen RM, Sun Z, Swindell EC, Farrington S, Hopkins N. Identification of 315 genes essential for early zebra fish development. Proc Natl Acad Sci U S A. 2004; 101: 12792–12797.