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Hemodynamics and Vascular Regulation |
* Departments of Pediatrics and Internal Medicine, University of Heidelberg, Heidelberg; Institute of Nuclear Chemistry, University of Mainz; and Institute of Pharmacology, University of Kiel, Germany
Address correspondence to: Dr. Claus Peter Schmitt, Division of Pediatric Nephrology, University Hospital for Pediatric and Adolescent Medicine, Im Neuenheimer Feld 153, D-69120 Heidelberg, Germany. Phone: +49-6221-5639313; Fax: +49-6221-564203; E-mail: claus.peter.schmitt{at}med.uni-heidelberg.de
Received for publication September 7, 2005. Accepted for publication December 3, 2005.
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Abstract |
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Introduction |
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So far, no consistent effect of calcimimetics on casual BP has been reported in controlled prospective trials in patients who are on dialysis (2–5). However, these studies were not designed to evaluate an impact of the calcimimetic on BP. We believed that it was important to characterize the effects of continuous and intermittent administration of R-568 on BP and heart rate (HR) by measuring the circadian BP profile as well as several determinants that have an impact on BP. To this end, we compared SNX and sham-operated Sprague-Dawley rats. They received R-568 by continuous infusion in a first period and by daily intermittent subcutaneous injection in a subsequent second period.
Gain-of-function mutations in the CaR reduce NaCl reabsorption in the cortical ascending limb and result in a negative sodium balance (6). Accordingly, continuous administration of the calcimimetic R-568 may induce net sodium losses and thus exert BP-lowering effects. To this end, we determined total body sodium content after R-568 treatment in healthy rats using neutron activation analysis.
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Materials and Methods |
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Experiment I: Effect of R-568 on BP in Sham-Operated and SNX Rats
For subtotal nephrectomy, the right kidney was removed in a first session under anesthesia with ketamine (100 mg/kg body wt) and xylazine (2 mg/kg body wt). At the time of the first operation, a BP sensor (model PA-C40; Data Science International, Arden Hills, MN) was inserted into the aorta below the level of the renal arteries and the radiofrequency transmitter was fixed intraperitoneally. Signals were sent to a telemetry receiver that was placed under the cage and transmitted to the LabPRO Acquisition System, Version 3.10. BP was measured every 5 min (approximately 12,000 readings/rat). Seven days after uninephrectomy, we selectively removed cortical tissue from the hypertrophied left kidney corresponding to 75% of the weight of the previously excised right kidney. We kept the amount constant between rats, thus achieving similar removal of number of glomeruli. Particular care was taken to preserve adrenals without damage. In sham-operated rats, the kidneys were mobilized in two consecutive sessions.
Five days after the second operation, treatment was started. Sham-operated rats were treated with solvent and R-568 for 2 wk (n = 4 per group) and for 6 wk (n = 4 per group), respectively, and compared with SNX rats that were treated with solvent and R-568 for 6 wk (n = 7 per group). R-568 (Amgen, Thousand Oaks, CA) was dissolved in 45% aqueous cyclodextrin (2-hydroxypropyl-
-cyclodextrin; Sigma-Aldrich Chemie, Steinheim, Germany) and administered via osmotic minipumps (Alzet 2002; Alza Corp., Palo Alto, CA). After 2 wk, minipumps were exchanged for a subsequent 2-wk treatment period. This was followed by once-daily subcutaneous injections of the same total amount of R-568 for 2 wk. Food intake and body weight were measured every other day, blood sampling (100 µl) was performed weekly to measure ionized calcium. Forty-eight hours before rats were killed and 2 h after the last R-568 injection, blood was sampled for hormone analysis; 24 h before the rats were killed, they were transferred into metabolic cages.
Experiment II: Effect of R-568 on Total Body Sodium in Intact Rats
Intact rats were kept either on standard or on high-phosphate diet (Altromin C1049; 1.65% phosphate, 0.24% sodium, 0.95% calcium, 0.07% magnesium, and 0.7% potassium) to induce secondary hyperparathyroidism. They received either solvent (45% cyclodextrin) or R-568 (20 mg/kg per day) via osmotic minipumps for 2 wk (n = 9 per group). A total of 100 µl of blood was sampled on day 7. At the end of the study, the minipumps and the gastrointestinal tract were removed and body sodium was determined in the carcass. Meticulous attention was paid to avoid any loss of blood or extracellular fluid to prevent artificial changes of the body sodium content.
Biochemical Measurements
Blood ionized calcium was measured within 30 s after sampling using an ion selective electrode system (Ionometer EH-F; Fresenius, Oberursel, Germany). The results were corrected for pH 7.4; the mean intra- and interassay coefficient of variation was 2.5%. PTH was measured using a rat intact PTH ELISA (Immuntopics Inc., San Clemente, CA), which is specific for rat PTH1 to 84. It has no cross-reactivity with N-terminal 1 to 34 or mid- and C-terminal 39 to 84 fragments. Serum and urine electrolytes, creatinine, and urea were measured using standard laboratory techniques.
Plasma ACE Activity.
Angiotensin-converting enzyme (ACE) activity was assayed by a modified fluorometric method using Z-Phe-His-Leu as substrate (7). Plasma (50 µl) was diluted with phosphate buffer to a volume of 450 µl. The enzyme reaction was started by adding 50 µl of a 10-mM substrate solution to the samples and incubated for 30 min at 37°C. The reaction was terminated by transferring 100-µl aliquots into 1 ml of 0.1 N NaOH. All subsequent steps were continued in the dark. A total of 25 µl of 2% ortho-phthaldialdehyde solution in DMSO was added to the samples. After 30 min, the reaction was terminated by addition of 1 ml of 0.8 M HCl, and precipitates were spun down by 3000 x g centrifugation for 3 min. Fluorescence was measured within 60 min. Zero time blank values were subtracted from the corresponding test values. All assays were performed in duplicate. The results are expressed as nmol His-Leu/ml per min.
Plasma Aldosterone.
Aldosterone was measured by a specific in-house RIA, using tritiated aldosterone (1,2,4,6, 3H aldosterone; Amersham Biosciences, Freiburg, Germany) and an antibody, developed as described elsewhere (8). Before RIA, extraction and chromatographic purification were performed to remove cross-reacting steroids. The chromatographic separation of aldosterone was modified from the method previously described (9) using Celite (Celite 545 AW; Sigma Aldrich, Taufkirchen, Germany) as an inert support for partition chromatography. The stationary phase consisted of 30% formamide in water, and the mobile phase consisted of a mixture of ethyl acetate in n-hexane with increasing polarity, eluting aldosterone with 50% ethyl acetate in n-hexane. The standard curve ranged from 1 to 200 pg; sensitivity was 2 pg per tube (1 ng/100 ml). The recovery rate was 104 ± 8.2% (n = 12; mean ± SD), and intra- and interassay coefficients of variation were 3.5 to 8.5 and 9.6 to 12.2%, respectively.
Neutron Activation Analysis
Body sodium content was measured by neutron activation analysis using the research reactor TRIGA of the Institute of Nuclear Chemistry (Mainz, Germany). The sodium content was determined via the 23Na(n,
)24Na neutron capture process. Rats were irradiated in special polymer capsules for 1 h at 100 kW reactor power in a carousel position, corresponding to a flux of thermal neutrons of 7 x 109 neutrons/cm2. The contribution of 24Na activation resulting from those capsules was considered. To simulate the radioactivity resulting from those irradiations, a calibration standard was prepared, according to the reported rat tissue content: 1.64 g/L NaCl, 1.29 g/L NaHCO3, 1.98 g/L FeSO4 x 7H2O, 4.24 g/L K2CO3, and 10.66 g/L NH4H2PO4 (10). Four aliquots of 200 ml of this stock solution were irradiated under identical conditions, yielding a mean experimental error of 24Na activities of 1.68%. This reflects the accuracy of the results of the 24Na determinations in the irradiated rats. After a cooling period of 5 h, rats were positioned at a 100-cm distance to an HPGe detector; an additional 50-mm lead shielding was placed in between. Gamma spectra were recorded for 1 h, and the areas of the two most relevant photo peaks of 24Na (t1/2 = 14.659 h), namely 1369 keV (100% abundance) and 2754 keV (99.944% abundance), were obtained using the Intertechniq Intergamma software. Both peaks produced similar results. Using the stock solution as calibration source, final data from the 2754 keV peak are given as mmol sodium/g body wt.
Statistical Analyses
The t test was used for group comparison. Even though BP recordings were obtained every 5 min, only the 24-h mean BP was used for calculations of long-term BP effects, resulting in very conservative estimates of P values and reducing false-positive results. Data from days of minor interventions (blood sampling) and 48 h after major interventions (implantation, exchange, and removal of minipumps) were excluded from the analysis. Graphs that displayed BP over 6 wk were smoothed with a uniformly weighted moving average spanning 36 data points (equivalent to 3 h). For significance testing of acute BP effects, only one value per rat (averaged from 7 d in sham-operated and 9 d in SNX rats) was used to compare groups.
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Results |
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Long-Term Effects of R-568 on BP and HR.
Controls.
Infusion of R-568 in sham-operated rats induced only small changes in SBP and diastolic BP (DBP) within the first 14 d of treatment (Figure 1). On day 7, SBP/DBP was 5.9 ± 14/3.3 ± 8.5 and on day 11 was 5.1 ± 11/1.2 ± 7 mmHg lower than in solvent-treated sham-operated rats (both NS). In the subgroup that was followed for an additional 4 wk, a similar BP profile was observed initially, and no difference between the groups was observed from the third week onward. HR was similar in R-568–and solvent-treated rats throughout the study period.
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In sham-operated R-568–treated rats there was no day/night difference during the first 2 wk (days 8 to 10: daytime MAP 100 ± 8 mmHg; nighttime MAP 101 ± 10 mmHg), whereas in solvent-treated controls, daytime dipping persisted (days 8 to 10: daytime MAP 103 ± 7 mmHg; nighttime MAP 108 ± 6 mmHg; P = 0.017).
Experiment II
Effect of R-568 on Total Body Sodium Content.
To assess a potential effect of R-568 on sodium balance, we measured total body sodium in healthy rats that were fed a normal or a high-phosphate diet to induce secondary hyperparathyroidism. Because initial food intake was less in R-568–treated rats (68 ± 8 versus 81 ± 9 g/kg per d in rats on normal phosphate diet and 50 ± 8 versus 72 ± 8 g/kg per d in rats on a high-phosphate diet; P < 0.05), pair feeding was performed from the fourth day onward, resulting in identical food intake subsequently. Water intake was similar in rats that were on a normal phosphate diet, significantly increased in rats that were on high-phosphate diet, and partially reduced by R-568 treatment (Table 2). On day 7, sodium, potassium, and pH were similar between groups, whereas ionized calcium was strongly reduced by R-568. Tail-cuff plethysmography revealed a lower SBP in R-568–treated rats that were on a normal phosphate diet after 7 d but similar BP after 14 d. In rats that were on high-phosphate diet, however, the difference in BP persisted after 14 d. Total body sodium content was not lower in R-568–treated rats that were on normal phosphate diet but was even 10% higher compared with solvent-treated controls. In rats that were on high-phosphate diet, body sodium content was not changed by R-568 treatment. Dietary sodium intake did not correlate with total body sodium content in rats that were fed a normal and a high-phosphate diet, respectively.
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Discussion |
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An antihypertensive effect of calcimimetic agents in rats was first suggested by Ogata et al. (1), who reported a reduction in SBP by tail plethysmography in uremic rats that were treated with intermittent R-568. Our study confirms this finding using the gold standard of telemetry and adds a number of important details.
Measurement of BP by means of tail plethysmography is difficult to standardize and may easily result in imprecise data. Accordingly, in nonuremic R-568–treated rats, we measured greater BP differences with tail-cuff plethysmography as compared with telemetric BP measurements, suggesting a transient BP-lowering effect of R-568 after 1 wk but no longer after 2 wk. In intact rats that were fed a high-phosphate diet to induce hyperparathyroidism, the antihypertensive effect persisted after 2 wk. This is in line with the persistent antihypertensive effect of R-568 observed in uremic hyperparathyroid rats. Whether the transient effect in nonuremic rats reflects the intervention of compensatory mechanisms or less parathyroid activity compared with uremic rats is unclear.
Neutron activation analysis allows for a highly precise determination of total body sodium. In contrast, measurement of urinary sodium output at the end of the study does not necessarily reflect cumulative renal sodium losses, because sodium depletion may already occur within the first days of R-568 treatment and thus escape later analysis, when a new steady state may already have been obtained. Repeated 24-h urine collections in metabolic cages are not well tolerated by the rats and may vitiate the sodium balance by unmeasured sodium losses, e.g., from bleeding food pads. Analysis of total body sodium by neutron activation analysis circumvents these potential artifacts. Surprising, it did not reveal a negative sodium balance in healthy rats that were fed a standard diet and were treated with R-568, as one may have expected given that gain-of-function mutations in the CaR reduce NaCl reabsorption in the cortical ascending limb and induce natriuresis (6). To the contrary, body sodium content was significantly increased within 14 d of R-568 infusion, although food intake and thus sodium intake was, if anything, lower with R-568. A compensatory activation of the renin-angiotensin and aldosterone system in response to the BP decline may have escaped the single-point measurements of plasma ACE activity and aldosterone levels.
In SNX rats, R-568 persistently reduced the progressive increase in BP that was observed in the solvent-treated SNX controls. HR declined within the first 10 d in both groups but remained higher in R-568 rats during the subsequent 2 wk and even increased further in R-568–treated rats after that. This is compatible with a compensatory increase in HR in response to a decrease of BP induced by vasodilation of resistance arteries. The CaR is expressed in a wide variety of rat small arteries, including renal, mesenteric, cerebral, and subcutaneous vessels (11–13). It has been localized in adventitial perivascular nerves. Activation of the CaR by a progressive increase in ambient calcium results in a U-shaped response with constriction at medium calcium concentrations and marked vasodilation at high and low calcium concentrations. The known agonists magnesium and aminoglycosides induce dose-dependent vascular relaxation (11); the neuronally released vasodilator substance is a cannabinoid receptor agonist (14). Accordingly, R-568 may result in reduced peripheral vascular resistance in the rat model, either directly via activation of the CaR or indirectly by the decrease of ionized calcium to subnormal levels.
Alternatively, the antihypertensive effect of R-568 may be mediated via suppression of PTH plasma levels. Infusion of PTH significantly increases BP in rats (15) and humans (16,17). Conversely, in normotensive and spontaneously hypertensive eucalcemic rats, parathyroidectomy reduces BP to a similar degree as the calcimimetic agent (1,18). The acute injection of R-568 reduces BP within 50 min in spontaneously hypertensive but not in normotensive rats (19). Clinical studies with calcimimetic agents have not been designed to evaluate effects on BP, and none of the published trials has reported any impact on BP (2–5).
Meanwhile, >600 ESRD patients have been treated with the calcimimetic cinacalcet HCl in phase 3 studies. All together, lower SBP and DBP values were recorded with cinacalcet (144.4 ± 2.4 and 78.6 ± 1.5 versus 138.6 ± 2.4 and 76.9 ± 1.5 mmHg after 1 yr; personal communication, U. Fraass, Amgen, March 2005). Still, these findings may be confounded by factors that influence BP, including fluid and salt intake, antihypertensive medication, and dialysis regimen.
The striking acute increase in BP after subcutaneous injection of R-568 is more difficult to explain. Presumably, it is not an artifact of differences in pain perception, because the HR increased to the same degree in sham-operated and to a smaller degree in SNX rats that were treated with R-568. The brisk and transient increase in BP was twice as high with R-568 compared with solvent injection. In view of the expression of the CaR in different central nervous system structures (20,21), central effects cannot be excluded. The parallel striking increase in BP and HR argues in favor of central effects mediated by R-568, whereas the subsequent reduction in HR below baseline that was observed in uremic rats would be consistent with a hypothetical overshooting baroreceptor-mediated counterregulation. Whether the acute R-568 effects are also related to differences in PTH and ionized calcium concentration between groups is unknown. It is interesting that some patients do not experience an immediate decline in PTH plasma levels but a transient increase before a sustained reduction, suggesting an initial antagonistic effect of calcimimetics (2,22).
So far, no data have been published on circadian BP rhythm in uremic rats. Analogous to the findings in uremic patients (23), circadian BP rhythm was absent in SNX rats. It could be restored neither by R-568 infusion nor by subcutaneous administration during the daytime.
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Conclusion |
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Acknowledgments |
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We are grateful to M. Jennewein and S. Zauner for assistance with neutron activation analysis. We are thankful to H. Ehmke for constructive comments.
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Footnotes |
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T.O. and K.N. contributed equally to this work.
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) on days with no other interventions (7 d in sham-operated and 9 d in SNX rats). Mean values ± SE for 30-min intervals are given.