Preservation of Intracellular Renin Expression Is Insufficient to Compensate for Genetic Loss of Secreted Renin
The primary product of the renin gene is preprorenin. A signal peptide sorts renin to the secretory pathway in juxtaglomerular cells where it is released into the circulation to initiate the renin-angiotensin system cascade. In the brain, transcription of renin occurs from an alternative promoter encoding an mRNA starting with a new first exon (exon 1b). Exon 1b initiating transcripts skip over the classical first exon (exon 1a) containing the initiation codon for preprorenin. Exon 1b transcripts are predicted to use a highly conserved initiation codon within exon 2, producing renin, which should remain intracellular, because it lacks the signal peptide. To evaluate the roles of secreted and intracellular renin, we took advantage of the organization of the renin locus to generate a secreted renin (sRen)-specific knockout, which preserves intracellular renin expression. Expression of sRen mRNA was ablated in the brain and kidney, whereas intracellular renin mRNA expression was preserved in fetal and adult brains. We noted a developmental shift from the expression of sRen mRNA in the fetal brain to intracellular renin mRNA in the adult brain. Homozygous sRen knockout mice exhibited very poor survival at weaning. The survivors exhibited renal lesions, low hematocrit, an inability to generate a concentrated urine, decreased arterial pressure, and impaired aortic contraction. These results suggest that preservation of intracellular renin expression in the brain is not sufficient to compensate for a loss of sRen, and sRen plays a pivotal role in renal development and function, survival, and the regulation of arterial pressure.
Renin is the first and rate-limiting enzyme in the renin-angiotensin (Ang) system (RAS). Renin processes angiotensinogen into Ang-I, which is further proteolytically cleaved by Ang-converting enzyme into Ang-II. In the canonical pathway, Ang-II derived from the circulation binds to Ang II type 1 and Ang II type 2 receptors in target tissues to exert its function to regulate cardiovascular and water/electrolyte homeostasis. In addition to Ang-II, ample evidence now supports the hypothesis that alternative Ang peptides, such as Ang-(1-7), may act as effector peptides in cardiovascular regulation.1,2 The relative contributions of Ang-II and Ang-(1-7) remain a source of debate and have added significant complexity to the RAS. A second level of complexity stems from the wealth of data showing that many, if not all, components of the RAS are expressed in many tissues. The concept that tissue-specific RAS pathways exist in tissues, including the kidney, brain, heart, vasculature, and adrenal gland, resulting in local production and action of Ang peptides, continues to gain experimental support.3 In the kidney, local Ang-II is thought to regulate blood flow and sodium reabsorption, whereas in the brain it stimulates thirst and sympathetic activity. That antihypertensive agents targeting the RAS are effective in hypertensive patients with normal or even low plasma renin activity have contributed to the argument that tissue RASs are physiologically relevant and important.
The classical renin protein is composed of a signal peptide that leads to its secretion and release into the systemic circulation, a prosegment that protects the active site of the enzyme from interacting with angiotensinogen, and mature active renin. The canonical sites of renin synthesis, storage, and release are the juxtaglomerular cells of the kidney. It is thought that other sites of renin synthesis release primarily prorenin. The first exon of renin (termed “exon 1a”) transcribed in renal juxtaglomerular cells harbors the transcription start site and encodes the initiation codon for translation. We and others identified an alternative isoform of renin mRNA in the brain of the mouse, rat, and human.4,5 This isoform is derived from a different transcription start site and transcribes a unique first exon (termed “exon 1b”). Transcripts initiating at exon 1b skip over exon 1a and splice directly to exon 2, which contains an evolutionarily conserved in-frame ATG, which may act as an alternative translation initiation codon.6 Exon 1b transcripts are predicted to encode a truncated form of renin that lacks the signal peptide, and, thus, remains intracellular, and the first third of the prosegment, and, thus, is constitutively active. Previous studies have shown that this form of renin, which we have termed “intracellular renin” (icRen), is enzymatically active and functional both in vitro and in vivo.5–7 We hypothesize that secreted renin (sRen) and icRen isoforms play differential roles in cardiovascular regulation and fluid homeostasis. Functional studies examining the importance of renin in the brain have been very challenging, because the level of renin protein is extremely low. Moreover, because there are no unique peptides in icRen (the sequence of icRen is a subset of sRen), there is no clear way to differentiate the products. We, therefore, used a unique gene targeting strategy to generate knockouts (both null and conditional) of one isoform while retaining expression of the other. Herein we report that we have established a null model of sRen with the preservation of icRen expression.
Generation of sRen Knockout Mice
Gene targeting was performed in mouse embryonic stem cells derived from a tyrosinase mutant line of C57BL/6J-Tyr(c-2J). Germ-line transmission was screened in the offspring from the chimeras bred with C57BL/6J. Details of vector construction are in the Supplemental Methods (available at http://hyper.ahajournals.org). All of the experimental procedures on mice were approved by the University of Iowa Animal Care and Use Committee and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
RNA was extracted from mouse tissues using TriReagent (Invitrogen). Mouse renin mRNA was measured with an RNase protection assay, as described previously.4 Specific primers were used to amplify the sRen and icRen mRNA. The forward primer for sRen and icRen mRNA anchors to exon 1a and exon 1b, respectively. A common reverse primer located in exon 3 was used for both. A primer set in exon 4 and exon 5 was used to detect total renin mRNA. Taqman probes were used for real-time PCRs for sRen and total renin mRNA. Cyber green with Takara Taq polymerase was used to measure icRen mRNA. Primers sequences are listed in Table S1 (available in the online Data Supplement at http://hyper.ahajournals.org).
Renal Histology and Function
Kidneys were harvested and incubated in Pen-Fix for 3 days, paraffin embedded, and subjected to hematoxylin/eosin staining. After a 2-day acclimation period, urine was collected for 24 hours in metabolism cages designed for mice (Nalgene). Urine osmolarity was determined by freezing-point analysis (Fiske 2400). Urine sodium and potassium were determined by flame photometry (Instrumentation Laboratory 943). Animals were killed by CO2 asphyxia and decapitated, and blood was transferred into three 75-mm heparin-coated capillary tubes for hematocrit. Tubes were centrifuged at 12 600g for 3 minutes (BD Triac) before reading. Remaining blood (400 μL) was collected into 50 μL of 0.5-mol/L EDTA, mixed, and placed on ice for 5 minutes. Plasma was stored at −80°C until analysis. Plasma aldosterone was determined by ELISA (Cayman Chemical, No. 10004377) using the manufacturer’s instructions.
Under ketamine and xylazine anesthesia (85.5:12.5 mg/kg), by means of an anterior neck incision, left common carotid artery was implanted with radiotelemetry catheters (PA-C10; Data Sciences International). The radiotelemeter transmitter was kept subcutaneously in the right flank. After 10 days of recovery, direct arterial pressure and heart rate were recorded continuously during 10 days (sampling every 5 minutes with 10-second intervals). After the day/night recordings, the sampling frequency was increased to 2000 Hz, and a 2-hour recording was made for analysis of the spontaneous baroreflex using the sequence method.8 The sympathetic and vagal effects to the heart were assessed by autonomic blockade with propranolol (5 mg/kg) and methylatropine (2 mg/kg), respectively. The intrinsic heart rate was calculated after simultaneous β-adrenergic and muscarinic blockade. Aortic function was measured ex vivo. Aortas were dissected and incubated in an organ bath, while constrictor or dilator agents were added into the chamber at increasing doses, as described.9
All of the comparisons were between sex- and age-matched wild-type and knockout mice. Data are plotted as mean±SEM and were analyzed with ANOVA. P<0.05 is considered statistically significant.
We used a unique gene-targeting approach to preserve expression of icRen while deleting sRen. To accomplish this, it was necessary to retain the common portions of the gene encoding renin (exons 2 to 9). Therefore, we specifically ablated exon 1a of the mouse renin gene along with the classical promoter while preserving exon 1b, as well as its discrete transcriptional regulatory elements in the genome of C57BL/6J (Figure 1). The use of C57BL/6 embryonic stem cells allowed us to work in a genetic background containing only a single allele of the renin gene (Ren-1c). Recall that the 129 strain carries 2 alleles of renin (Ren-2 and Ren-1d). The introduction of the Cre-LoxP system confers in this model the capability to cripple sRen expression in specific tissues or cell types. Embryonic stem cell clones were screened via PCR genotyping, and chimeric mice were generated from embryonic stem clone FV115. Founder mice harboring the floxed exon 1a allele along with the neomycin cassette were bred with mice expressing FLPase in the early embryo. The resulting mice containing the floxed exon 1a allele (lacking the neomycin gene) were then bred with E2A-Cre mice expressing Cre-recombinase in the early embryo to generate a null allele of sRen. A series of PCR assays was sequentially performed to verify the fidelity of gene targeting and to screen for germline transmission, the floxed allele, and ultimately the null allele (data not shown). Southern blotting using probes outside the homology arms in the targeting construct confirmed the generation and transmission of the sRen null allele in +/− and −/− mice (Figure 2).
Intercrosses of +/− mice were performed to obtain −/− homozygotes. An analysis of the first 105 offspring from these crosses at 3 weeks of age revealed significant preweaning lethality, because only 2 −/− mice (of 26 expected) survived to 3 weeks of age (Figure 3A). On the contrary, live sRen−/− mice were obtained at birth (Figure S1), and an analysis of fetuses collected at prenatal day 18.5 revealed a normal ratio of +/+, +/−, and −/− offspring (Figure 3B). This implicates a defect in survival between birth and weaning. Consistent with other models of RAS gene deficiency, sRen−/− mice exhibited a significantly reduced hematocrit (Figure 3C).10 Although kidneys from sRen−/− newborns appeared structurally normal (Figure 4A through 4D), kidneys from surviving adult sRen−/− mice exhibited severe renal atrophy compared with sRen+/+ controls (Figure 4E through 4H). Also consistent with previous models, sRen−/− mice exhibited increased urine output and an inability to generate a concentrated urine (Table 1).11 Probably as a compensatory mechanism, plasma aldosterone was elevated by 35% (Table 1). We conclude that sRen is required for survival, and its loss causes severe defects in the structure and function of the kidney after birth.
We next sought to obtain evidence for the preservation of icRen mRNA expression in the sRen-null mice. We used primer sets capable of individually detecting sRen versus icRen mRNA and total renin (sRen+icRen mRNA). There was a marked reduction in total renin mRNA in the kidney and brain from 18.5-day gestation fetuses from sRen−/− as compared with +/+ and +/− littermates (Figure 5A). This was attributed to a loss of sRen mRNA, because there was clear preservation of icRen mRNA in sRen−/− mice. These data were confirmed by real-time quantitative PCR (Figure 5B). It is notable that, at this stage of fetal development, renin mRNA in the brain is mainly attributable to sRen mRNA, because the decrease in total renin mRNA completely paralleled the decrease in sRen mRNA in sRen−/− mice. On the contrary, icRen mRNA was preserved and even increased in the brain of sRen−/− fetuses (Figure 5B). Similar results for total renin and sRen mRNA were observed in the kidney, consistent with the highly predominant transcription from the classic promoter in renal juxtaglomerular cells.
In adult survivors, total renin mRNA was completely ablated in the submandibular gland and kidney of sRen−/− mice (Figure 6A). As expected, there was no expression of renin mRNA in the liver. Quantitative PCR revealed that the levels of sRen mRNA were completely ablated, whereas the levels of icRen mRNA were increased in the brain of adult sRen−/− mice (Figure 6B). Interestingly, however, there was no apparent change in the level of total renin mRNA in the brain from adult sRen−/− mice, suggesting a developmental shift in the use of renin mRNA isoforms. Our data suggest that, during late embryonic development, sRen mRNA is the major isoform expressed in the brain, whereas, in adults, icRen mRNA is the predominant form expressed in the brain.
We next measured mean arterial blood pressure and heart rate via radiotelemetry. sRen null mice that survived to adulthood displayed a normal circadian rhythm (Figure 7A) but exhibited a significant decrease in mean arterial blood pressure compared with age- and sex-matched wild-type littermates during both the daytime and nighttime hours (Figure 7B). Baseline heart rate was unchanged in both groups (Figure 7C). Baroreflex function in sRen−/− was essentially normal except that there was a decrease in the number of baroreflex sequences (Table 2). This may reflect a decrease in the blood pressure variability. The autonomic regulation of heart rate was also normal in sRen−/−, although there was a trend toward an increase in the vagal tone to the heart, as observed after muscarinic blockade with methyl-atropine. The intrinsic heart rate was similar in both groups.
Aorta from sRen−/− mice exhibited normal responses to acetylcholine and nitroprusside after preconstriction with prostaglandin F2α and exhibited normal contractile responses to prostaglandin F2α, phenylephrine, and KCl (Figure S2). However, an impaired constrictor response to 5-hydroxytryptamine was noted in the aorta from sRen−/− mice (Figure 7D).
This is the first study designed to examine the differential roles, if any, of sRen and icRen in the regulation of cardiovascular function. The long-term goal of this project is test the provocative hypothesis that the unique expression of icRen in the brain plays a role in blood pressure and water and electrolyte homeostasis.12 As a first step toward this goal, we used a novel gene-targeting strategy to fully ablate sRen while preserving icRen mRNA. The novel findings from our study are that complete loss of sRen, even with the preservation of icRen, causes lethality, and, in adult survivors, severe renal abnormalities, anemia, an inability to concentrate urine, and hypotension. This suggests that sRen is an essential component required during early neonatal life and is a critical regulator of arterial pressure in adults. Our data also suggest that preservation of icRen is not sufficient to rescue defects caused by the loss of sRen.
RAS Deficiency, Lethality, and Renal Defects
The increased lethality and renal structural abnormalities observed in adult sRen-null mice are consistent with other models of RAS gene ablation, including renin-null mutants lacking the entire renin gene.11,13–15 That the mice are born in normal numbers and exhibit normal renal histology at birth but then succumb within a few weeks is also consistent with other RAS deficiencies. Similarly, anemia has been reported in mice lacking Ang-converting enzyme.10 This implies that Ang II generated in the extracellular spaces in tissues or in the systemic circulation is required for continued development of the kidney after birth and is a major regulator of arterial pressure in adults. Mice lacking sRen were impaired in their ability to generate concentrated urine, although their total 24-hour sodium and potassium excretion was essentially normal. The modest elevation in aldosterone is likely a consequence of these renal defects. Although icRen mRNA is the predominant form of renin mRNA in the brain of adult mice, it represents only a very small fraction (estimated <1%) of the total renin mRNA in the kidney. Consequently, it is not surprising that preservation of this small fraction of icRen mRNA in the kidney was insufficient to rescue the defects caused by loss of sRen. Indeed, it remains unclear what minimum level of renal renin is required to retain viability and renal structure and function and whether this renin needs to be secreted.
Complementation studies have been used to identify the important sites of RAS expression to rescue defects observed in RAS knockout mice. For example, we reported previously that the human renin and human angiotensinogen genes could fully complement lethality and renal defects in angiotensinogen-deficient mice.16 Because the transgenes were expressed in many tissues, and plasma Ang II was elevated, it provided a proof-of-principle that genetic means could be used to replace endogenous Ang II with transgenic Ang II. Other genetic data from our laboratory showing that exclusive targeting of renal angiotensinogen and renal Ang II is insufficient to rescue lethality in angiotensinogen-deficient mice implicated the importance of circulating Ang II for the maintenance of renal structure.17 This is supported by complementation studies from other investigators, where overexpression of RAS components in adipose tissue or liver resulted in Ang II release in the circulation, which rescued renal defects.18,19 More contradictory to this hypothesis were data from Lochard et al20 who reported that brain-specific overproduction of Ang II normalized blood pressure and corrected some renal defects in angiotensinogen-deficient mice. The Ang II level in the brain was elevated by ≈6-fold and was presumably generated in the extracellular space, because the construct was cleverly designed to secrete Ang II. Interestingly, the increase in brain Ang II did not translate into an increase in circulating Ang II in their transgenic mice. It should be noted, however, that they did not repeat these measurements when their transgene was bred on the angiotensinogen-deficient genetic background. Consequently, the mechanism by which brain-specific Ang II improved renal structure and function remains unclear. In our study, preservation, or even an increase in icRen expression in the brain, was clearly ineffective at preventing renal abnormalities.
Evidence for an icRen in the Brain
Our data showing preservation of icRen mRNA expression in the sRen-null mice provide additional support for the concept that an independent renin mRNA is transcribed at the renin locus. The increase in icRen mRNA in the brain of sRen-null mice and the developmental shift in the expression from sRen in the fetal brain to icRen in the adult brain suggest that independent regulatory elements and a novel promoter mediate expression of icRen mRNA. This conclusion is strengthened when one considers that the ablation of sRen was generated by deletion of exon 1a and 500 bp of surrounding DNA, including the classical renin promoter. Sequence analysis of the region directly upstream of mouse exon 1b reveals the absence of a classic TATA box but the presence of a potential CCAAT box at approximately −50. A promoter prediction program also identified a potential promoter sequence ≈700 bp upstream of exon 1b (within intron 1 of the renin locus).21 The same algorithm predicted a promoter upstream of exon 1a but did not identify a promoter in the second intron. Understanding the transcriptional elements and physiological signals regulating expression of icRen will require additional investigation. It may be important to consider whether the low levels of icRen mRNA in the brain reflect the activity of a very weak promoter or are attributable to other factors. For example, Mercure et al22 showed previously that, whereas truncation of the renin prosegment resulted in increased renin activity, it also caused a marked decrease in secretion and expression of renin. Therefore, it is unclear whether the very truncation that eliminates the signal peptide and part of the prosegment also causes decreased expression.
Lee-Kirsch et al5 first identified transcripts initiating at exon 1b in the brain of the mouse, rat, and human. AtT-20 cells transfected with exon 1b cDNAs resulted in renin activity in cell lysates, but not medium, and in vitro–translated icRen was not processed in microsomal membranes. These results were consistent with the production of active prorenin and retention of the protein intracellularly. We then identified the presence of the exon 1b mRNA in the brain of transgenic mice carrying a highly regulated genomic construct encoding the human renin gene and validated its presence in human fetal brain RNA.4 The exon 1b form of renin mRNA was the predominant form in the brain of these mice, and the protein was localized throughout the hypothalamus.23 The hypertension in double-transgenic mice carrying this highly regulated human renin gene was reduced after intracerebroventricular losartan, providing indirect evidence supporting the function of icRen. More direct evidence for the function of icRen is derived from studies specifically overexpressing this form of the protein. We reported that brain-specific expression of either sRen or icRen in transgenic mice resulted in a similar increase in blood pressure.6 Peters et al7 reported that expression of icRen in transgenic rats is retained in the cytoplasm and results in increased aldosterone production. Clausmeyer et al24,25 reported the presence of icRen mRNA in the adrenal gland, and its synthesis in the heart was stimulated after myocardial infarction. Therefore, icRen may not be exclusive to the brain.
There are many lingering questions surrounding the intracellular RAS concept (reviewed in Reference12). Is icRen localized in a cellular compartment where it can generate Ang peptides? What happens to these peptides if and when they are generated? Of obvious importance would be to determine whether this occurs in neurons, and, if so, what the function of intracellular Ang II is in neurons. Although our data clearly suggest that icRen cannot substitute for loss of sRen, they do not rule out a physiological function for icRen. By taking advantage of the fact that sRen and icRen mRNAs are derived from independent transcription start sites, we have generated the first of a series of models designed to dissect the physiological significance of sRen and icRen. Unfortunately, because the sRen and icRen proteins are essentially identical in sequence (icRen is a subset of secreted prorenin), antibodies specific to each isoform cannot be generated. This, coupled with the low level of renin expression in the brain, has hampered studies addressing its function. Consequently, the generation of the complementary model described herein, that is, ablation of icRen with preservation of sRen, should provide a unique experimental platform from which to assess the function of this protein. Because icRen is the primary isoform of renin mRNA expressed in the adult brain and its expression is largely, but not exclusively, brain specific, an icRen null should function, essentially, as a brain-specific renin knockout. The generation of this model is currently in progress.
We thank Drs Nobuyuki Takahashi and Feng Li for their technical advice in performing subcutaneous saline injections in newborn mice and Henry Keen for bioinformatics.
Sources of Funding
C.D.S. is the recipient of National Institutes of Health grants HL048058, HL061446, and HL084207 and the Roy J. Carver Trust. D.X. is a recipient of an American Heart Association Predoctoral Fellowship (0910035G). J.L.G. is the recipient of an American Physiological Society Physiological Genomics Fellowship.
- Received July 1, 2009.
- Revision received July 27, 2009.
- Accepted September 10, 2009.
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