Dose Effects of Human Renin in Rats Transgenic for Human Angiotensinogen
Abstract We examined the effect of chronic human renin infusion and human renin inhibition on blood pressure in a unique transgenic rat model. We infused incremental doses of human renin (1 to 500 ng/h) with minipumps for 10 days into rats harboring the human angiotensinogen gene [TGR(hAOGEN)1623]. We measured blood pressure and heart rate continuously by telemetry. We found that human renin at 5 ng/h was necessary to increase blood pressure, whereas 10 ng/h caused systolic blood pressure to increase to 215±13 mm Hg. Heart rate decreased initially but then increased by 100 beats per minute compared with basal values. Drinking behavior also increased. Doses as high as 500 ng/h did not increase blood pressure further. A linear relationship was found between the log of plasma renin activity and systolic blood pressure that increased in slope from days 2 to 9. Rat angiotensinogen levels were low and not influenced by human renin infusion. Human angiotensinogen levels remained stable until 500 ng/h human renin was infused, at which time they decreased by 50% at 9 days. Rat renin gene expression (RNase protection assay) was decreased by human renin infusion, whereas rat and human angiotensinogen gene expressions in liver and kidney as well as angiotensin-converting enzyme gene expression in kidney were not affected. The human renin inhibitor Ro 42-5892 was given by gavage repeatedly to rats receiving human renin at 40 ng/h. Ro 42-5892 lowered blood pressure promptly to basal values. High human renin hypertension in this model is dose dependent, features a steeper relationship between blood pressure and plasma renin activity over time, and is associated with tachycardia and increased drinking. We conclude that the human angiotensinogen transgenic rat offers new perspectives in the study of human renin–induced hypertension.
Blocking the RAS is presently one of the most important therapeutic options for reducing the global morbidity and mortality in a variety of cardiovascular diseases, including hypertension, heart failure, and myocardial infarction. ACE inhibitors and type 1 Ang II receptor antagonists can both effectively block the RAS in vivo and in vitro after the initial generation of Ang I by renin. However, cleavage of AOGEN by renin remains the key step in the system. This step is controlled by renal renin secretion and provides for the rapid adjustment of the overall activity of the system.1 ACE inhibition interferes with the metabolism of other vasoactive hormones, such as bradykinin and substance P, whereas Ang II receptor antagonists are confined to pharmacologically distinct receptor subtypes.2 Experimental renin inhibition is necessary for interpreting the consequences of inhibiting the RAS specifically. However, human renin inhibitors cannot be validly tested in the usual laboratory animals because of the species specificity of the renin-AOGEN reaction. For this reason, an effective preclinical screening of human renin inhibitors, including their pharmacokinetic and pharmacodynamic characterization, is not easily achieved. Relatively little is known about the cardiovascular and renal effects of inhibiting renin, the rate-limiting enzyme of the system, which has no known physiological substrate other than AOGEN.
Human renin cleaves rAOGEN poorly and at a very slow rate. Similarly, hAOGEN is converted to Ang I by human but not by rat renin.3 A TGR that harbors the hAOGEN gene permits the study of human renin in an animal model.4 5 We showed previously that a short infusion of human renin into these TGR acutely increases their BP compared with controls.4 We then developed assay systems to specifically and simultaneously measure components of the rat and human RAS in plasma samples from these TGR. We could further demonstrate that human renin–induced hypertension can be maintained over several days.3 The longer-term effects of human renin infusion and dose-response relationships between doses of human renin or renin plasma concentrations and BP have not been explored in detail. In the current study, we therefore examined the effect of a 10-day chronic human renin infusion on PRA, PRC, Ang II concentrations, hAOGEN, and rAOGEN as well as telemetrically monitored BP and heart rate. We also used an RNase protection assay to examine rAOGEN, hAOGEN, and ACE expressions in these rats. The dose-response relationships between human renin and BP as well as between PRA and BP were successfully defined. Human renin infusion suppressed endogenous (rat) renin gene expression and rPRC. We were able to document the efficacy of a human renin inhibitor in this model. We conclude that this novel rat model permits short- and long-term studies for characterization of the hemodynamic effects of human renin and its inhibition in a species that is one of the most convenient for the study of drug pharmacokinetics, pharmacodynamics, and toxicology.
Male Sprague-Dawley rats heterozygous for the complete human genomic AOGEN gene [TGR(hAOGEN)1623] and male nontransgenic Sprague-Dawley rats weighing 320 to 430 g were used for all experiments. The TGR line and its characteristics have been described elsewhere.3 4 Briefly, TGR show high hAOGEN gene expression in the liver, kidney, heart, aorta, and adrenal glands. Their plasma hAOGEN concentrations exceed endogenous AOGEN concentrations by 50- to 100-fold. TGR were kept under standard conditions at 24±2°C and were fed a commercial rat chow (No. C-1000, Altromin) containing 0.2% sodium by weight, with free access to tap water. All experiments were performed according to American Physiological Society guidelines and were approved by local authorities.
Dose Dependency of Chronic Human Renin–Induced Hypertension
We performed this protocol to determine the relationship between the intravenous human renin dose and arterial BP, plasma RAS parameters, and tissue mRNA expressions of renin, endogenous rat and transgenic human AOGEN, and ACE in various organs. The procedures have been detailed elsewhere.3 Briefly, TGR were anesthetized with ketamine and xylazine (15 and 5 mg/kg body wt IP, respectively). A polyethylene catheter was inserted into the right jugular vein and exteriorized at the nape. A minipump (No. 2002, Alzet Corp) filled with human recombinant renin (generous gift of Drs W. Fischli and S. Mathews, Hoffmann–La Roche AG, Basel, Switzerland) was connected to the tubing and placed into a subcutaneous pouch. The human renin was diluted in sterile water containing 5 mg/L bovine serum albumin (vehicle). BP, heart rate, and ambulatory activity were continuously recorded with a radiotelemetric system (Data Sciences International) implanted into the infrarenal aorta and harbored in the abdominal cavity. Data were averaged over 10 minutes and then stored on a personal computer using the manufacturer’s DataQuest IV software.
We previously showed3 that 50 ng/h human renin increases systolic BP chronically to values greater than 200 mm Hg. We now infused human renin for a total of 10 days at rates of 1, 2.5, 5, and 10 ng/h into groups of six TGR (0.5 μL/h discharge rate) to determine the minimum effective renin dose. Six TGR received vehicle alone. Blood samples (0.6 mL) were drawn from the jugular venous catheter or by jugular venous puncture during surgery before the minipumps were placed (day 0), 2 days thereafter (day 2), and on the last infusion day (day 9). The rats were anesthetized with ketamine. On day 9, we also collected blood for Ang II determinations. A catheter was placed in the carotid artery, and blood was allowed to flow into prechilled tubes containing Na2EDTA (6.25×10−6 mol/L), human renin inhibitor (Ro 42-5892, 10−5 mol/L), and phenanthroline (final concentrations, 2×10−3 mol/L). Two milliliters of plasma was required for the assay. hAOGEN and rAOGEN concentrations, total PRA, and human- and rat-specific PRC were determined by enzyme-kinetic assay. At the end of the experiment, rats were killed. The kidneys, hearts, and livers were removed, snap-frozen in liquid nitrogen, and stored at −70°C for subsequent mRNA extraction and analysis of gene expression. Hematocrit values, drinking volumes (daily), and body weights (every 3 days) were measured longitudinally.
To examine the effects of maximal human renin values, we gave six TGR human renin at a dose of 500 ng/h by minipump. In three rats, the minipumps were removed on day 7 while BP and heart rate were observed for another 3 days. Only plasma RAS parameters were determined in these three rats.
Effects of Human Renin Inhibition
We performed this protocol to investigate the utility of this model in testing human renin inhibitors. TGR were given 40 ng/h human renin intravenously as described to achieve a rapid rise in BP. Four days after the renin infusion was started (day 0), rats received 2 mL water by gavage (day 4). On the following 2 days (days 5 and 6), the human renin inhibitor Ro 42-58926 (remikiren, Hoffmann–La Roche; 30 mg/kg body wt) was given by gavage. BP was observed until day 9 when the rats were killed by an overdose of intraperitoneal ketamine. BP and heart rate were monitored by telemetry. Venous blood samples were obtained by jugular vein puncture before the human renin infusion (control), 3 days after the infusion had started, 6 hours after the first Ro 42-5892 dose (day 5), and at the end of the experiment.
Enzyme-Kinetic Determinations and Radioimmunoassay
The human- and rat-specific components of the plasma RAS were determined by an in vitro enzyme-kinetic assay specifically developed and validated by us for this purpose.3 These assays distinguish human- from rat-specific Ang I–generating pathways by using the human renin inhibitor Ro 42-5892 and by relying on the selective substrate specificities of human and rodent renins. The Ang I generated during the various in vitro incubations was measured by a direct radioimmunoassay.5 All radioimmunoassay determinations were done in triplicate, and measurements were made at two different concentrations. Ro 42-5892 did not interfere with measurements. The sensitivity of the radioimmunoassay as defined by the Ang I concentration capable of displacing the 125I-labeled Ang I tracer by 20% was 3 to 4 pg per assay tube. A displacement of 50% of the tracer was achieved at a concentration of 21±4 pg per tube (n=11). The mean intra-assay and interassay variabilities were 9% and 13%, respectively. Our methods for measuring plasma Ang II concentrations are outlined elsewhere.7 The method included an extraction procedure, high-performance liquid chromatographic (HPLC) separation of the Ang II fraction, and subsequent radioimmunoassay of Ang II. The immunoassay5 showed 20% displacement of 125I-labeled Ang II tracer at about 1 to 2 pg Ang II per assay tube and 50% displacement at 12±3 pg (n=6) per tube. Assay variabilities were similar to those for the Ang I assay. Serial dilutions of HPLC-purified Ang II fractions were tested with an upper limit of detection of 700 pg Ang II per mL.
For determination of hAOGEN concentrations, 50 μL of prediluted plasma (1:5000 in buffer) was incubated together with 445 μL of 0.15 mol/L citrate phosphate buffer (pH 5.7) containing 0.05 mol/L Na2EDTA, 1.0 g/L bovine serum albumin (citrate phosphate buffer), and an excess of 1.0 pmol human recombinant renin as well as with 5 μL PMSF (50 g/L ethanol) for 1 hour at 37°C. For determination of rAOGEN concentrations, 50 μL of prediluted (1:50) plasma was incubated together with 0.2 mol/L Tris-HCl buffer (pH 7.4) containing 0.05 mol/L Na2EDTA and 1.0 mg/mL bovine serum albumin (Tris buffer) and an excess of purified mouse submaxillary gland renin equivalent to a concentration that would generate 50 μg Ang I/mL per hour under conditions of substrate abundance (tested separately), as well as with 5 μL PMSF for 1 hour at 37°C. Mouse renin cleaves rAOGEN but not hAOGEN, whereas human renin is specific for hAOGEN. The AOGEN concentrations were expressed as micrograms Ang I per milliliter based on an equimolar production of Ang I from cleaved rAOGEN or hAOGEN. We also used a direct radioimmunoassay to measure hAOGEN.8 The assay determines total hAOGEN protein concentrations including human [des-Ang I]-AOGEN.
For determination of global PRA, 50 μL plasma was incubated together with 47 μL Tris buffer (pH 7.4) and 3 μL PMSF for 1 hour at 37°C. For measurement of hPRC, 25 μL of plasma was incubated together with an excess of hAOGEN contained in 75 μL renin-free plasma from TGR (hAOGEN), 95 μL of 0.15 mol/L citrate phosphate buffer (pH 5.7), and 5 μL of PMSF for 1 hour at 37°C. Because of higher dilution factors, the hPRC assay was about five times less sensitive in detecting human renin than the PRA assay. However, the PRC assay has the advantage that it controls for the presence of excess homologous substrate.3 Renin-free plasma from TGR was obtained 48 hours after bilateral nephrectomy and contained 120 nmol/mL hAOGEN. To control for species specificity, we repeated all incubations in the presence of the specific human renin inhibitor Ro 42-5892, which does not inhibit rat renin at a concentration of 3×10−7 mol/L in the assay. For determination of rPRC, 25 μL plasma was incubated with 200 μL renin-free nontransgenic rat plasma obtained 48 hours after bilateral nephrectomy (4.3 nmol/mL rAOGEN) and 200 μL Tris buffer (pH 7.4) containing Ro 42-5892 as well as with 5 μL PMSF for 2 hours at 37°C. Assays for hPRC and rPRC had similar sensitivities in detecting human or rat renin. Postnephrectomy plasma pools were checked before use for the absence of any residual Ang I generation. PRA and PRC were expressed as nanograms Ang I per milliliter per hour.
RNase Protection Assay
Total RNA was isolated from snap-frozen tissues by a lithium chloride/urea precipitation technique.9 mRNAs specific for hAOGEN, rAOGEN, rat renin, ACE, and β-actin were then identified by RNase protection assay with the use of an Ambion RPA III kit (ITC Biotechnology GmbH) and according to protocols provided by the manufacturer. Antisense RNA probes were prepared by T7 polymerase transcription with the use of cDNA fragments specific for hAOGEN and rAOGEN subcloned into pGEM5 and pGEM4 vectors4 10 and cDNA fragments specific for rat renin and rat β-actin subcloned into pGEM4 and pBluescript SK II+ vectors,11 respectively. The ACE-specific antisense probe was obtained by T3 polymerase transcription of an endothelial ACE cDNA fragment subcloned into pBluescript KS+ II vector.12 The protected sequences of the various probes were 132, 290, 297, 150, and 375 nucleotides in the given order. All probes were labeled with [32P]UTP to a specific activity greater than 2×108 cpm/μg RNA. One to 20 μg of total RNA, depending on tissue type and investigated gene, was hybridized together with a minimum of 1.5×105 cpm of specific antisense probe and cohybridized with a similar excess of β-actin–specific antisense RNA serving as an internal control. Probe fragments subsequently protected from combined RNase A/T1 digestion were separated by electrophoresis on a 5% denaturing polyacrylamide gel and visualized with a FUJIX BAS 2000 Phospho-Imager system after 3 to 36 hours of autoradiography. Signal intensities were quantified by computer-aided densitometry, and ratios for the investigated gene with β-actin were calculated.
Mean values with SD and linear regression parameters were calculated. Differences between contrasting groups were tested by either one-way ANOVA or Student’s t test with the use of StatView software on a Macintosh computer. A value of P<.05 was regarded as significant. The terms “increased” and “decreased” are used only when significant.
Six rats each received vehicle or incremental doses of human renin (1, 2.5, 5, 10, and 500 ng/h) chronically via minipumps. These rats were monitored continuously with telemetry. Hemodynamics and RAS parameters were not significantly different between groups before the infusions started (data not shown). Telemetric recordings of BP and heart rate of a representative TGR receiving the maximum 500 ng/h human renin dose are shown in Fig 1A⇓ to exemplify BP characteristics. Twenty-four hours after implantation, the infusion began to be effective (tubing dead space) and immediately increased systolic BP to more than 200 mm Hg. BP increased further over the following days. Heart rate initially decreased from 400 to 300 beats per minute (bpm) and after 3 days of infusion began to increase again to 500 bpm until the minipumps were removed. Thereafter, BP decreased progressively within hours to subnormal levels, whereas heart rate gradually returned to normal over the following days. All three rats tested in this way showed the same hemodynamic responses to high human renin administration and discontinuation. Diastolic BPs exhibited a parallel pattern; pulse pressure remained constant; the long-term variability of systolic or diastolic BPs as defined by the difference of maximum and minimum values observed during a 24-hour period also remained unchanged (data not shown).
Fig 2⇓ shows mean 24-hour systolic BP as a function of human renin infusion dose. Systolic BP at days 2 and 9 are displayed. Human renin at 5 ng/h was the minimum effective dose that increased BP by day 9 in 3 of 6 rats. At human renin doses less than 5 ng/h, only 1 rat of 12 developed a moderate BP elevation by day 9. At 10 ng/h, human renin increased BP by day 2 to 170 mm Hg and to 210 mm Hg by day 9. As described above, the human renin dose of 500 ng/h did not increase BP significantly further.
Fig 3⇓ shows the relationship between PRA and mean 24-hour systolic BP in these rats on days 2 and 9. Only rats receiving human renin were included. PRA measurements in rats receiving human renin infusions correlated closely (r=.9, P<.05) with determinations for hPRC, indicating that excess amounts of hAOGEN were present in TGR plasma and that PRA and hPRC were qualitatively interchangeable. Since our PRA assay was more sensitive at low hPRC values than the hPRC assay and since rat renin was undetectable in plasma, the analysis in Fig 3⇓ was based on PRA. The relationship on day 9 was significantly steeper than the relationship on day 2, implying a greater effect of renin on BP at day 9 than day 2.
The Table⇓ presents PRA, rPRC, hPRC, rAOGEN, hAOGEN, and plasma Ang II concentrations in these rats as a function of human renin dose on day 9. The endogenous PRC values were suppressed in all TGR receiving human renin infusions, including the lowest infusion dose of 1 ng/h, although that dose did not increase BP. From the lowest infusion dose on, PRA specifically depended on the presence of human renin in the plasma, as shown by control in vitro determinations in the presence of Ro 42-5892. The changes in PRA and hPRC between control rats and the 1 ng/h infusion dose demonstrates the shift from rat to human renin in controlling PRA. At low doses, our methods were possibly not sensitive enough to detect significant changes in hPRC. The 10 ng/h infusion dose, which effectively increased BP in all rats, was also the dose that effectively increased global PRA and hPRC. Combining the results from our previous study, in which 50 ng/h human renin was infused,3 PRA increased progressively as a function of dose to extremely high levels. Doubling of the human renin dose from 5 to 10 ng/h increased global PRA by a factor of 10, whereas the rise in hPRC was even steeper, clearly indicating saturation of human renin metabolism by rat liver and kidneys. hPRC was also a function of infused human renin. rAOGEN was stable and remained at normal levels across the human renin infusions. On the other hand, hAOGEN was not significantly influenced at human renin doses between 0 and 10 ng/h. At the 500 ng/h dose, hAOGEN was half the usual value at day 9. Direct radioimmunoassay determinations of total hAOGEN protein confirmed these low plasma concentrations and thereby excluded possible underestimation of concentrations by our indirect measurement methods based on the release of Ang I from AOGEN. Ang II concentrations increased above baseline with the 5 ng/h renin dose. They increased further with the 10 ng/h dose and were above the limits for our assay with the 500 ng/h dose. No Ang II concentrations are available for the 2.5 ng/h renin infusion.
Significant tachycardia was present only in rats receiving 10 and 500 ng/h human renin and after more than 7 days of infusion. The mean increases in heart rate compared with pretreatment values were 47±34 and 80±27 bpm in these groups, respectively. These rats also showed a significant increase in hematocrit on day 9 of infusion, paralleled by an increase in daily drinking volume by approximately 50% (n=6) and 200% (n=3) in the two groups (data not shown). Rats receiving 500 ng/h human renin exhibited a significant loss of body weight by 26±9% and reduced ambulatory activity (n=6).
Fig 4⇓ shows the results of our gene expression studies performed by RNase protection assay in various organs of TGR. Fig 4A⇓ shows rat renin gene expression in kidneys of TGR with or without human renin infusion. Gene expression of rat renin in kidney decreased to barely detectable levels with human renin infusion. This decrease in rat renin mRNA levels in the kidney was predictive of hypertension in all cases. Fig 4B⇓ shows hAOGEN gene expression in TGR liver, kidney, and heart with (2.5, 5, and 500 ng/h) and without (C) human renin infusion. Human renin infusion did not influence hAOGEN gene expression in these organs. Fig 4C⇓ shows rAOGEN in liver and ACE mRNA in kidney and heart. No effects on these gene expressions were observed with renin infusion. However, we observed an approximately threefold increase of ACE mRNA expression in hearts from rats treated with 500 ng/h human renin. Measurements of optical density, which allowed for statistical analysis, confirmed these observations.
We also infused three TGR with human renin at a dose of 40 ng/h to test the effect of the human renin inhibitor Ro 42-5892. The inhibitor (30 mg/kg) was given by gavage on days 5 and 6, after which BP was observed until day 10. A representative example is shown in Fig 1B⇑. Water gavage had no effect. Systolic and diastolic BPs decreased promptly to control values with Ro 42-5892 on both days the drug was given. Thereafter, BP again increased to levels observed before the renin inhibitor was given. During the experiment hPRC and rPRC showed a mirror-like behavior. hPRC increased from 0 to 1159±424 ng Ang I/mL per hour with human renin infusion and then decreased to 15±13 ng Ang I/mL per hour 6 hours after Ro 42-5892 gavage, indicating an almost complete inhibition of human renin–dependent Ang I generation in the enzyme kinetic assay. Interestingly, rPRC, which had decreased under human renin infusion from 37±47 to 0 ng Ang I/mL per hour, returned to 31±9 ng Ang I/mL per hour 6 hours after Ro 42-5892 gavage. At the end of the experiment, rPRC was suppressed again (1±1 ng Ang I/mL per hour), and hPRC was 305±153 ng Ang I/mL per hour.
Since the initial observations of Goldblatt et al,13 Wilson and Byrom,14 and Volhard,15 clinicians and researchers in the field of hypertension have been aware of the BP-raising effects of renin and the resultant Ang II–induced vasculopathy associated with consistently high circulating renin concentrations. Because of the specificity of human renin for hAOGEN, valid animal studies involving human renin have not been possible.3 The hAOGEN TGR was specifically developed to enable such studies. Our observations are the first to investigate in depth the effects of human renin in this model. We found that human renin increased BP in a dose-dependent manner; however, the effective range was narrow. A dose of 5 ng/h was necessary to effectively elevate BP chronically in three of six rats, whereas a dose of 10 ng/h markedly elevated BP in all tested animals. Thereafter, no further increases in BP were observed with doses as high as 500 ng/h. We found that Ang II concentrations determined on day 9 paralleled the increasing levels of renin infusion. We found increased values with the 5 ng/h dose, which increased further with the higher doses. We were unable to determine an increase in hPRC at low doses; however, the sensitivity of our renin assays was not sufficient to show an effect at this level.
In the past few decades, infusions of exogenous Ang I and Ang II into experimental animals have been used in an attempt to mimic high renin–induced hypertension or the physiological effects associated with the cleavage of AOGEN by renin. The number of studies involving the infusion of renin itself is relatively small and almost exclusively comprises short-term experiments over a few hours.16 The only experiment similar to ours was one by Blackett et al,17 who investigated the BP effects of long-term homologous renin infusions in the rabbit. They were able to influence BP level by the amount of renin they infused; however, they were not able to produce sustained hypertension at very high renin doses. The paucity of renin infusion studies is explained by the fact that renal extracts and early preparations of renin were likely to contain contaminating materials that may have their own vasoactive effects. The amounts of purified renin available for study were not adequate for such experiments to be conducted. The use of recombinant human renin obviates these problems; however, the material can be applied only to models harboring hAOGEN or its cleavable homologues. Thus far, only humans or primate models could be investigated.16 The transgenic hAOGEN rat now allows for such studies in the species generally preferred and usually selected for pharmacological characterization and toxicological testing of new drugs.
Ang II infusion studies have revealed that distinct direct and slow pressor effects are observed. Slow pressor responses require days to become established and involve an upward shift of the dose-response curve to Ang II toward higher BP.18 These characteristics are highly germane to human renovascular hypertension, in which PRA and Ang II levels may revert to normal values. For instance, Bianchi et al19 demonstrated that after renal arterial constriction in dogs, the dose-response curve to acute Ang II infusions shifted in parallel with the progressively increasing BP during the 9 weeks after surgery. Bean et al20 generated a slowly developing pressure response by chronic low-dose Ang II infusion also in the dog. They reported a similar and characteristic parallel upward shift of the acute dose-response curve to Ang II in dogs. These studies suggested that the progressive long-term increase in BP after acute renal artery constriction was a slowly developing Ang II pressure response. However, neither these nor other21 studies could clearly show that the dose-response curve to acute Ang II infusion itself became steeper with time. Ames et al22 infused Ang II chronically into human volunteers. Decreasing amounts of Ang II were needed to maintain elevated BP at constant levels in their study. Other investigators demonstrated that individuals with renovascular hypertension had a steeper relationship between BP and peripheral plasma Ang II concentrations than normotensive subjects, whose BP was titrated by acute injections of the peptide.23 Chronic and acute effects were compared; thus, there is no conclusive evidence from acute infusion experiments to show that the slow pressure response observed in models of Ang II–dependent and renovascular hypertension is directly related to increased sensitivity to Ang II. Instead, the BP responses may be independent of Ang II and related to other mechanisms.
We describe a comparable, albeit different, phenomenon in our TGR. For instance, renin infusion at 5 ng/h evoked no change in BP by day 2; however, by day 9, BP was increased in some of the rats. We speculate that given enough time, even the lowest renin infusion groups would develop hypertension. Interestingly, the very low renin dose groups had a reduced global PRA compared with vehicle, as if the Ang II generated from infused human renin were able to suppress rat renin secretion before being able to increase BP. In rats receiving 2.5 and 5 ng/h, the infused human renin was apparently still in equilibrium with the Ang II–mediated rat renin suppression and then massively overrode the equilibrium when human renin was infused at 10 ng/h. The relationship between BP and global PRA was undoubtedly exponential and significantly steeper on day 9 than on day 2. Although rats were grouped according to renin infusion, we believe a combined analysis based on PRA was permissible because PRA indicated the actual Ang I–generating capacity of each individual plasma. This value was variable and showed a roughly even distribution over the entire range. The data suggest a greater pressure response for a given renin level on day 9 than day 2 in the same rats. Accumulation of renin in the plasma cannot be the only possible explanation for this observation, because the slope of the relationship would then have remained constant. If BP sensitivity to Ang II is defined by the change in BP observed with a given increase in PRA, our findings argue strongly for the idea that Ang II indeed chronically increases the sensitivity to its own action over time. The mechanisms involved are not within the scope of the present experiments; however, our conclusion is supported by the fact that BP returned to normal immediately in the presence of Ro 42-5892 or when renin infusions were discontinued.
We attempted to define the minimal effective renin dose. In retrospect, the doses we selected were not ideal in terms of establishing dose-response relationships; a conventional logarithmic relationship may have been better. Nevertheless, we did find a sequential increase in Ang II concentrations at the infusion doses at which this measurement was available. Furthermore, we found a high correlation between mean 24-hour systolic BP and log PRA on both days 2 and 9. We feel that these observations support the notion that a dose-response relationship is present.
We were puzzled by the persistent tachycardia observed with chronic renin infusions that was present even long after BP had returned to normal. Baroreceptor-mediated changes in heart rate were expected and may have played a role during the initial phase of renin infusion. However, heart rate remained elevated even when BP had returned to pretreatment values. In the dog, chronic low-dose Ang II infusions resulted in an initial decrease in cardiac output followed by gradual increases in cardiac output over the following 5 days.24 No significant long-term changes in heart rate were observed. When Ang II was discontinued, the dogs exhibited an increase in heart rate of 1.3 bpm per 1 mm Hg of decrease in BP, which was attributed to a resetting of the baroreceptor reflex. Our rats initially had no change in or slightly decreased their heart rate while BP increased. Thereafter, heart rate proceeded to increase by up to 100 bpm by day 9 of infusion. Ang II is known to interact with sympathetic nervous system activity at various levels and may have played a role. There is extensive evidence that Ang II stimulates sympathetic outflow from the sympathetic nervous system and facilitates sympathetic neurotransmission in the periphery.25 Under conditions of chronic low-dose Ang II infusions, potentiation of the adrenergic response has been reported.26 A negative sodium balance with hypovolemia could provide another explanation.
We observed markedly stimulated drinking behavior in our rats, which occurred only at doses in which BP also increased. Hematocrit showed a parallel increase, indicating significant contraction of plasma volume. Cowley and DeClue24 did not report such changes in their experiment of chronic Ang II infusion. Detailed balance studies have not yet been performed in our model, and we did not measure serum sodium or osmolality; however, the drinking response resembles the increased drinking observed in some individuals with malignant hypertension.27 In TGR the abnormalities disappeared after renin infusions were stopped.
Human renin infusion resulted in prompt decreases in circulating rat renin in TGR. We were able to confirm that rat renin gene expression was shut off by infusion of human renin at higher doses when hypertension occurred in the individual animal. At very low doses, no significant suppression of rat renin mRNA was noted. Possibly, feedback regulation of rat renin at the gene level begins when counterregulation at the renin secretory level is no longer possible. Furthermore, the suppression of renin mRNA at the gene level appears to be a crucial step and a very sensitive indicator of the onset of hypertension. Interestingly, human renin infusion had no effect on either rAOGEN or hAOGEN gene expression. Earlier studies have suggested a regulatory effect of Ang II on AOGEN production28 29 ; however, rAOGEN values were not significantly affected by any dose of human renin, and rAOGEN gene expression in the liver was also not influenced. These results are at variance with earlier reports suggesting that Ang II actually stimulates hepatic AOGEN production.30 31 32 The existence and role of such a positive feedback loop by the RAS is not clear. Our results correspond to those of Clauser et al,33 who found no evidence for tonic stimulation of AOGEN by Ang II in rats. The decreasing plasma hAOGEN in rats receiving the maximum human renin dose was most likely due to illness, with decreased hepatic protein secretion or globally enhanced protein metabolism. Furthermore, we found no evidence on the basis of our RNase protection assays that renin infusion and the resulting Ang II–induced hypertension had any effect on ACE gene expression in the kidney or heart, except for an extremely high renin dose in the heart. These results were obtained at day 9, and we cannot state for certain that longer-term effects would not have occurred, for example, in association with cardiac remodeling. ACE gene expression and ACE production are increased in the heart and vessels of rats with renovascular hypertension, although the cellular localizations and mechanisms of such increases are not entirely clear.34
The main objective of the model was fulfilled by testing the effect of the orally administered human renin inhibitor Ro 42-5892. This demonstration is unique for an experimental animal model. We performed only acute experiments with a renin inhibitor. Moreover, the bioavailability of the tested compound is suboptimal for clinical purposes.35 In TGR, very high doses of human renin inhibitor resulted in a prompt and inhibitor-specific decrease in BP to basal values. The TGR(hAOGEN) rat provided for steady-state experimental conditions. Since exogenous human renin infusion remained constant, counterregulatory renal mechanisms such as changes in renal renin secretion were of no importance for the degree of human renin inhibition. However, the endogenous RAS was reactivated when the “exogenous” human system became blocked. This feature may have influenced to some extent the duration and magnitude of the hypotensive effect of the tested renin inhibitor. Chronic experiments of renin inhibition in this model with appropriate new renin inhibitors remain to be performed. Such experiments will allow future comparison of renin inhibition with ACE inhibitors or Ang II receptor blockers in the most widely used species in hypertension research.
In summary, we have demonstrated the utility of hAOGEN TGR as a model for chronic, high human renin hypertension. Our data suggest a chronic increase in sensitivity to Ang II with time as well as potential sympathetic nervous system activation. The renin-dependent increase in BP was accompanied by negative fluid balance. We have shown that the model is associated with suppression of endogenous rat renin release and synthesis, with no significant effect on hepatic AOGEN synthesis or ACE and AOGEN expressions in various other organs. We have demonstrated that a single administration of an orally active renin inhibitor is efficacious in lowering BP to basal values. We suggest that this model will have utility in the study of human RAS physiology and pharmacology.
Selected Abbreviations and Acronyms
|Ang I, II||=||angiotensin I, II|
|PRA||=||plasma renin activity|
|PRC||=||plasma renin concentration|
This study was supported by a grant-in-aid from Hoffmann–La Roche Inc, Basel, Switzerland. J. Ménard is a Humboldt Foundation Distinguished Research Scholar. We wish to thank I. Strauss, G. Born, Ch. Lipka, M. Somnitz, A. Müller, and M.-F. Gonzales for their skillful technical help. We are grateful to Dr U. Ganten for her expert advice. We also thank Drs H.-W. Fischli and S. Mathews, Hoffmann–La Roche, for their support and for providing us with human recombinant renin. J.-P. Clozel, Hoffmann–La Roche, gave helpful comments.
- Received August 2, 1996.
- Revision received September 5, 1996.
- Accepted October 23, 1996.
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