Prorenin Contributes to Angiotensin Peptide Formation in Transgenic Rats With Rat Prorenin Expression Targeted to the Liver
We reported previously that targeted expression of rat prorenin to the liver under the control of the human α1-antitrypsin promoter increased plasma prorenin levels by several-hundred–fold in male transgenic rats and caused cardiac hypertrophy, severe renal lesions, and myocardial fibrosis by 20 weeks of age, despite normal blood pressure. We examined the evolution of the phenotype of male transgenic rats over 12 months and the effects of binephrectomy on the renin-angiotensin (Ang) system. Plasma prorenin levels were >1000-fold higher than in wild type littermates, whereas plasma and renal Ang II levels were no different from wild-type (WT) levels, and kidney renin levels were suppressed in transgenic rats. In contrast to our earlier report, transgenic rats had increased systolic blood pressure at 3 to 12 months of age, and only modest renal lesions and myocardial fibrosis were evident after 6 months of age. Binephrectomy reduced plasma renin activity and concentration and prorenin levels by 50% to 80% and Ang II levels by 90% in WT rats. By contrast, binephrectomy increased plasma renin activity and concentration and prorenin levels by 52.0-, 13.0-, and 5.8-fold, respectively, without change in Ang II levels in transgenic rats. We conclude that, in the animals studied in this report, elevated prorenin levels did not cause renal lesions or myocardial fibrosis during the first 6 months of age. Ang peptide formation consequent to the increased prorenin levels prevented reduction of Ang II levels after binephrectomy and was likely to have contributed to hypertension, cardiac hypertrophy, and suppression of kidney renin levels in these transgenic rats.
Prorenin, the biosynthetic precursor of renin, contains a prosegment that masks the active site, thereby preventing access by the renin substrate, angiotensinogen.1,2 Renal juxtaglomerular cells are the only known site of production of renin, and the kidney produces both renin and prorenin, whereas a number of extrarenal tissues produce prorenin.1,2 Plasma prorenin concentrations are 10- to 20-fold higher than renin concentrations in humans.3,4 Whether plasma prorenin has biological activity in vivo is a matter of controversy.1,2 Partial conversion to renin and a low degree of intrinsic activity because of transitory unfolding of the prosegment may contribute to angiotensin (Ang) formation by prorenin. In addition, prorenin binding to the (pro)renin receptor may initiate signal transduction by mechanisms independent of Ang peptide formation, and the (pro)renin receptor may activate prorenin by promoting unfolding of the prosegment.1,2,5
We reported previously the development of a transgenic rat model with high plasma prorenin levels that suggested a direct pathogenic effect of prorenin.6 These transgenic rats, designated TGR(hAT-rpR), had rat prorenin expression targeted to the liver by a human α1-antitrypsin promoter and exhibited sexual dimorphism, with plasma prorenin levels increased ≈600-fold in males but only 2- to 3-fold in females.6 Despite the absence of elevation of plasma renin activity (PRA) or systolic blood pressure (SBP) by 20 weeks of age, male transgenic (hAT-rpR) rats exhibited cardiac hypertrophy, and histological analysis revealed severe renal lesions, hypertrophic cardiomyocytes, interstitial and perivascular fibrosis in the heart, and increased aortic wall thickness.6 To examine further the mechanism of the phenotype of these (hAT-rpR) rats we studied these animals ≤12 months of age, measured Ang II levels in plasma and kidney, and examined the effects of removal of renal renin by binephrectomy on the renin-Ang system. Because expression of the rat prorenin transgene in (hAT-rpR) rats showed sexual dimorphism, and because only male rats showed renal, cardiac, and aortic abnormalities,6 our study was confined to male rats. In contrast to our initial report,6 we found that male (hAT-rpR) rats were hypertensive by 3 months of age, developed only modest renal lesions and cardiac fibrosis after 6 months of age, and had normal aortic wall thickness. Plasma and kidney Ang II levels of male (hAT-rpR) rats were similar to the levels in WT littermates, and binephrectomy experiments showed that prorenin was the major contributor to plasma Ang II levels in (hAT-rpR) rats.
Materials and Methods
The generation of (hAT-rpR) rats by the Centre for Genome Research, Edinburgh, has been described.6 This transgenic rat line was created by inserting a rat prorenin cDNA fused to a human α1-antitrypsin promoter into the genome of the Fischer F344 rat. In December 1996, 9 male and 12 female heterozygous (hAT-rpR) rats, strain 85-26, and 8 WT male Fischer F344 rats were received at Institut National de la Santé et de la Recherche Médicale U367. The colony was maintained by mating heterozygous male and female (hAT-rpR) rats. Heterozygous male (hAT-rpR) rats and their WT male littermates were used for all of the experiments, carried out in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes. The rat experiments were completed in 2000. For detailed Materials and Methods, please see the online Data Supplement, available at http://hyper.ahajournals.org.
SBP, Body Weight, and Left Ventricular and Kidney Weights
Male (hAT-rpR) rats had higher SBP than male WT rats from 3 to 12 months of age (Table 1). This difference in SBP between male WT and (hAT-rpR) rats was also seen in the original breeding stock received from Edinburgh: 142±2 versus 207±13 mm Hg (mean±SEM; n=8 to 9; P=0.0003) at 6 months of age and 147±2 versus 218±7 mm Hg (n=6; P<0.0001) at 12 months of age. By contrast, female (hAT-rpR) rats received from Edinburgh did not show increased SBP at either 4 months (129±3 mm Hg; n=12) or 10 months of age (138±5 mm Hg; n=6).
Body weights were similar for male WT and (hAT-rpR) rats (Table 1). Left ventricular weight was higher in (hAT-rpR) than in WT rats at 6 and 12 months of age, and the left ventricular weight:body weight ratio was higher in (hAT-rpR) than in WT rats at 3, 6, and 12 months of age (Table 1). Although there was a slight difference in kidney weight, there was no difference in the kidney weight:body weight ratio between WT and (hAT-rpR) rats (Table 1).
Plasma Renin, Prorenin, Angiotensinogen, and Ang II and Kidney Renin and Ang II
PRA and plasma renin concentration (PRC) values of (hAT-rpR) rats were 1.6- to 3.0-fold higher, and plasma prorenin levels were >1000-fold higher than in WT rats (Table 2). Plasma angiotensinogen levels of (hAT-rpR) rats were 8% to 17% lower than in WT rats (Table 2). By contrast, there were no differences between WT and (hAT-rpR) rats in plasma Ang II levels. Kidney renin levels in (hAT-rpR) rats were suppressed to 13% to 35% of the levels in WT rats (Table 2); these kidney renin levels were similar to those of WT rats maintained from 3 to 12 months of age on a diet with 2% sodium chloride (0.6±0.2 μg of Ang I/mg of protein per hour; n=7) and were not further suppressed when (hAT-rpR) rats were maintained on a diet with 2% sodium chloride for the same period (0.4±0.1 μg of Ang I/mg of protein per hour; n=8). Renal Ang II levels of (hAT-rpR) rats (113±16 fmol/g of wet weight, mean±SEM; n=6) were not different from the levels in WT rats (164±17 fmol/g of wet weight).
Effects of Binephrectomy
Measurements were made in both sham-nephrectomized and binephrectomized WT and (hAT-rpR) rats (Figure 1). PRA, PRC, plasma prorenin, and angiotensinogen were measured on both the day of surgery and 24 hours after surgery, whereas plasma Ang II was measured only 24 hours after surgery. Sham nephrectomy caused similar changes in PRA, PRC, and prorenin levels in WT and (hAT-rpR) rats. Sham nephrectomy increased PRA ≈2-fold and did not change PRC or prorenin levels in WT rats, whereas sham nephrectomy increased PRA by ≈6.0-fold and PRC by ≈1.7-fold and did not change prorenin levels in (hAT-rpR) rats. There were, however, marked differences between WT and (hAT-rpR) rats in the effects of binephrectomy. In comparison with the values before surgery, binephrectomy reduced PRA by ≈80%, PRC by ≈60%, and prorenin by ≈50% in WT rats. By contrast, binephrectomy increased PRA by ≈52.0-fold, PRC by ≈13.0-fold, and prorenin by ≈5.8-fold in (hAT-rpR) rats, reaching an average prorenin level of 201 638 ng of Ang I/mL per hour. The changes in plasma angiotensinogen levels were similar for WT and (hAT-rpR) rats. Sham surgery increased angiotensinogen levels by ≈3-fold, and nephrectomy increased angiotensinogen levels by ≈7-fold in both WT and (hAT-rpR) rats. In comparison with sham nephrectomy, binephrectomy reduced plasma Ang II levels by ≈90% in WT rats; by contrast, there was a nonsignificant increase in Ang II levels of (hAT-rpR) rats to ≈124 fmol/mL after binephrectomy (Figure 1).
Renal and Cardiac Histology
There were no differences between WT and (hAT-rpR) rats in renal histology at 3 and 6 months of age, although the glomerular, tubulointerstitial, and vascular injury scores of (hAT-rpR) rats were higher than in WT rats at 9 and 12 months of age (Figures 2 and S1, available in the online Data Supplement). There were no differences between WT and (hAT-rpR) rats in myocardial collagen density at 3, 6, and 9 months of age, although collagen density (both interstitial and perivascular) was increased above the level in WT rats at 12 months of age (Figures 3 and S2). There were no differences between WT and (hAT-rpR) rats in aortic medial area or thickness at any age (Figure 3).
This study confirmed our original report of elevated prorenin levels, cardiac hypertrophy, and suppressed kidney renin levels in (hAT-rpR) rats.6 In contrast to our original report, however, we found that (hAT-rpR) rats in the present study were hypertensive with mildly elevated PRA and PRC, and the markedly elevated prorenin levels were not associated with histological lesions during the first 6 months of age; only modest renal and cardiac lesions were evident after 6 months of age, and (hAT-rpR) rats had no aortic medial hypertrophy. Plasma and renal Ang II levels in (hAT-rpR) rats were similar to WT levels, but, contrary to WT rats, plasma Ang II levels did not fall after binephrectomy in (hAT-rpR) rats. These data demonstrate in vivo Ang peptide formation consequent to the increased prorenin levels, which likely contributed to the hypertension and suppression of kidney renin levels in (hAT-rpR) rats. Increased blood pressure may have contributed to the cardiac hypertrophy and renal and cardiac lesions in (hAT-rpR) rats. We were unable to test the role of Ang II by treating (hAT-rpR) rats with an Ang receptor blocker, because this strain of rats is no longer extant; new transgenic strains would need to be established to perform additional experiments, and, although a close approximation could be generated, it is not possible to remake the identical strain.
We studied the same transgenic rat line 85-26 described in our original report, and we are unable to explain why the phenotype of (hAT-rpR) rats in the present study differed from that described in our original report.6 We previously showed a critical influence of genetic background on the phenotype of mouse prorenin transgenic (mRen-2)27 rats,7 but we have no information regarding whether the genetic background of (hAT-rpR) rats in this study was different from that of (hAT-rpR) rats in our original report, because they are no longer extant in either center. Differences in diet and animal housing may have also contributed to the differences in phenotype. The altered phenotype of (hAT-rpR) rats was evident in the breeding stock received from Edinburgh in that their blood pressure was elevated when first measured after arrival in Paris and remained elevated, and their renal and cardiac lesions were modest when examined at 12 months of age. There was a trend for a higher SBP in (hAT-rpR) than in WT rats at 15 and 20 weeks of age in our original study, which may have failed to achieve statistical significance because the number of rats provided insufficient statistical power. Moreover, the measurement of SBP by tail-cuff plethysmography in rats briefly anesthetized with 2% halothane in our original study may have made hypertension more difficult to detect than SBP measurement in conscious rats, as performed in the present study.
Other transgenic models have failed to provide evidence for a direct pathogenic role of prorenin. Peters et al8 concluded that ≈180-fold elevation of prorenin levels, per se, did not cause glomerulosclerosis in rats transgenic for the Ren-2 gene under the transcriptional control of the cytochrome P450 Cyp1a1 promoter. Moreover, mice with human prorenin expression targeted to the liver and human angiotensinogen expression targeted to the heart had no abnormality of heart size or fibrosis, despite plasma prorenin levels 20-fold higher than in control mice.9 Furthermore, mice with mouse prorenin expression targeted to the liver showed no increase in cardiac fibrosis or renal glomerulosclerosis despite hypertension and 13- to 28-fold elevation of plasma prorenin levels, and captopril treatment normalized blood pressure in these mice.10
Although PRA and plasma angiotensinogen levels were similar for (hAT-rpR) and WT rats in our initial study,6 PRA and PRC were 1.6- to 3.0-fold higher, with an 8% to 17% reduction in angiotensinogen levels in (hAT-rpR) rats in the present study. PRC levels were ≤0.1% of the prorenin levels in (hAT-rpR) rats, and a critical issue in the interpretation of the PRA and PRC values for these rats was the possible inadvertent activation of prorenin during handling of plasma and performance of the assays, despite precautions. The similar Ang II levels in WT and (hAT-rpR) rats suggest that the higher PRA and PRC levels in (hAT-rpR) rats were consequent to inadvertent prorenin activation, although the modest decrease in plasma angiotensinogen levels was consistent with higher PRA and PRC in (hAT-rpR) rats.
The marked suppression of kidney renin levels in (hAT-rpR) rats was confirmation of our earlier study in which we also found >90% suppression of renin mRNA detected by hybridization in situ of the juxtaglomerular apparatus of the (hAT-rpR) kidney.6 This evidence for markedly reduced renin secretion suggests that the (hAT-rpR) kidney made little contribution to the elevated PRA and PRC in these rats. The failure of a high-sodium diet to reduce kidney renin levels in (hAT-rpR) rats was consistent with their kidney renin levels being predominantly attributed to plasma prorenin trapped in the tissue that was subsequently activated during tissue processing before renin assay. These data, therefore, suggest that the major part of renin activity and Ang II detected in the plasma of intact (hAT-rpR) rats was a consequence of the elevated prorenin levels of hepatic origin. This interpretation is strongly supported by the failure of plasma Ang II levels to fall after binephrectomy in these animals. Hepatic prorenin production was the predominant source of prorenin in (hAT-rpR) rats and was not subject to the same regulatory influences as kidney renin production in WT rats. For example, the increase in prorenin levels after binephrectomy was probably related to the acute-phase response of the α1-antitrypsin promoter of the prorenin transgene. The different regulation of hepatic prorenin production may, therefore, explain why Ang II levels were not suppressed by the increased blood pressure of (hAT-rpR) rats, as would be expected when renin is secreted from juxtaglomerular cells in similarly hypertensive WT rats. The failure of Ang II levels to increase in parallel with the increases in PRA and PRC after binephrectomy suggests that much of the increases in PRA and PRC were attributable to in vitro activation of the increased prorenin levels during PRA and PRC measurement.
The present data enable estimation of the contribution of prorenin to Ang formation in (hAT-rpR) rats in vivo by comparing the plasma Ang II and prorenin levels of anephric and intact rats and adjusting for differences in the angiotensinogen level, given that essentially all of the plasma Ang II in anephric (hAT-rpR) rats was produced by prorenin activated in vivo. Anephric (hAT-rpR) rats had plasma Ang II levels of ≈124 fmol/mL; adjusting for the 6-fold elevation in angiotensinogen levels, which would approximately double Ang I formation by a constant renin concentration,11 this corresponded with an Ang II level of ≈60 fmol/mL for an animal with normal angiotensinogen levels, which corresponded with a PRC of ≈60 ng of Ang I/mL per hour in a WT rat. Given the prorenin level of anephric (hAT-rpR) rats (201 638 ng of Ang I/mL per hour), a PRC level of 60 ng of Ang I/mL per hour corresponded with ≈0.03% activity of prorenin in vivo. It also suggests that prorenin levels of intact (hAT-rpR) rats aged 9 to 12 months (≈50 000 ng of Ang I/mL per hour) were sufficient to produce all of the Ang II measured in plasma. We acknowledge that these calculations have limitations, but the estimated contribution of prorenin to Ang II levels in the (hAT-rpR) rat is consistent with their hypertension and suppressed renal renin levels.
Our study does not provide information about the mechanism by which prorenin contributed to Ang formation, whether by a low degree of intrinsic activity of prorenin because of unfolding of the prosegment or by cleavage of the prosegment to produce renin. The (pro)renin receptor was proposed to bind and activate prorenin,5 thereby facilitating Ang peptide formation in tissues. One tissue where this might occur is the kidney, because the (pro)renin receptor is expressed in glomeruli, tubules, and vessels of the rat kidney.5,12,13 Moreover, a soluble form of the (pro)renin receptor is reported to be present in plasma.14 However, our finding that renal Ang II levels of (hAT-rpR) rats were no different from the levels in WT rats provides no support for this or any other mechanism of local prorenin activation in kidney, and our estimate of ≈0.03% activity of rat prorenin, on the basis of plasma Ang II levels, suggests very little systemic activation of prorenin in vivo.
In summary, we found that the phenotype of (hAT-rpR) rats studied in this report was similar to that described in our initial report with respect to the elevated prorenin levels, cardiac hypertrophy, and suppressed kidney renin levels but differed from our original report in that (hAT-rpR) rats in the present study were hypertensive with mildly elevated PRA and PRC, and the markedly elevated prorenin levels were not associated with histological lesions during the first 6 months of age. Genetic or environmental factors may be responsible for the different pathological and blood pressure responses to the elevated plasma prorenin levels seen in (hAT-rpR) rats described in this study and in our initial report. The failure of Ang II levels to fall after binephrectomy demonstrated that prorenin contributed to Ang II levels in (hAT-rpR) rats. We propose that Ang peptide formation consequent to the increased prorenin levels contributed to the hypertension, cardiac hypertrophy, and suppression of kidney renin levels in (hAT-rpR) rats. Furthermore, the similar renal Ang II levels of WT and (hAT-rpR) rats did not support a role for the (pro)renin receptor in the activation of prorenin in the kidney.
The 10- to 20-fold higher concentration of prorenin than renin in human plasma raises many questions about the role that prorenin may play in health and disease states. Our studies of the (hAT-rpR) rat indicate that, although prorenin may contribute to Ang peptide formation in vivo, the contribution of physiological levels of prorenin to Ang formation is much less than that of renin. Additional studies are required to define the contribution of prorenin to Ang peptide formation in specific tissues, such as those that produce prorenin, and in conditions associated with elevated prorenin levels, such as diabetes mellitus and pregnancy.
We thank Marie-Francoise Gonzalez and Thanh-Tam Guyene for assistance with these experiments and Didier Heudes for assistance with histological analysis of cardiac and aortic tissues.
Sources of Funding
This work was supported by grants from Institut National de la Santé et de la Recherche Médicale, the Association Claude Bernard (Paris, France), a Kidney Research United Kingdom (formerly National Kidney Research Fund) award (to J.J.M.), a Wellcome Trust Programme Grant WTO53646, the Wellcome Trust Cardiovascular Research and Functional Genomics initiatives, and the EURATools consortium. D.J.C. is recipient of a senior research fellowship from the National Health and Medical Research Council of Australia (grant 395508). J.J.M. is recipient of the Wellcome Trust Principal Fellowship.
D.J.C. has had research contracts with Solvay Pharmaceutical Company and Novartis and has been a member of an advisory board for Novartis. H.K. is an employee of Novartis. J.M. is a consultant for Actelion and for Novartis.
- Received June 29, 2009.
- Revision received July 23, 2009.
- Accepted September 22, 2009.
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