1Department of Pharmacological Sciences, and 4The Immunopharmacological Research Group, Medical School, University of Tampere, Tampere, 2Department of Anaesthesia and Intensive Care, 3Department of Internal Medicine, and 5Department of Clinical Chemistry, Tampere University Hospital, Tampere, 6Department of Medicine, Helsinki University Central Hospital, Helsinki and 7Minerva Institute for Medical Research, Helsinki, Finland
Correspondence and offprint requests to: Ilkka Pörsti, MD, PhD, Medical School, Department of Internal Medicine, FIN-33014 University of Tampere, Finland. Email: ilkka.porsti{at}uta.fi P. Jolma and P. K\|[ouml ]\|\|[ouml ]\|bi contributed equally to the manuscript.
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Abstract |
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Methods. We investigated the influence of an 8-week high calcium diet (0.3 vs 3.0%) on resistance artery tone in 5/6 nephrectomized (NTX) rats. Calcium was supplemented as carbonate salt, blood pressure measured by tail-cuff, urine collected in metabolic cages, and samples taken for blood chemistry and parathyroid hormone (PTH). Functional studies of isolated third-order branches of the mesenteric artery in vitro were performed using the Mulvany multimyograph.
Results. Plasma urea was elevated 1.6-fold and systolic blood pressure by 10 mmHg after NTX, while increased calcium intake was without effect on these variables. Plasma PTH and phosphate were raised following NTX, and suppressed by high calcium diet. Vasorelaxations induced by K+ channel agonists 11,12-epoxyeicosatrienoic acid and levcromakalim were impaired after NTX. Vasorelaxation induced by acetylcholine was also reduced following NTX, and experiments with NG-nitro-L-arginine methyl ester, diclofenac and charybdotoxin + apamin suggested that the K+ channel-mediated component of endothelium-dependent relaxation was deficient after NTX. Increased calcium intake corrected all impairments of vasodilatation in NTX rats.
Conclusions. Deficient vasorelaxation via K+ channels was normalized by high calcium diet in experimental RF. This effect was independent of the degree of renal impairment and blood pressure, but was associated with improved calcium metabolism: plasma levels of PTH and phosphate were decreased and ionized calcium was increased.
Keywords: arterial smooth muscle; calcium diet; endothelium; phosphate binding; renal failure
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Introduction |
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Phosphate excretion is decreased in RF, and elevated phosphate together with reduced synthesis of active vitamin D [1,25(OH)2D3] lead to the development of secondary hyperparathyroidism [2,7]. High parathyroid hormone (PTH) and phosphate levels predispose to ectopic calcifications, and disturbed calcium-phosphate balance significantly contributes to the cardiovascular pathology in RF [2,7]. Excess of PTH is also associated with elevated blood pressure, and it may directly influence the function of arterial smooth muscle [2,8]. It is of note that in primary hyperparathyroidism, endothelial vasodilatory function is impaired, and this deficit can be normalized by parathyroidectomy [9].
Oral calcium carbonate and other phosphate-binding agents, which reduce intestinal phosphate absorption, are used in RF to reduce circulating levels of phosphate and PTH [2]. In experimental hypertension, high calcium intake has consistently reduced blood pressure [10], and this effect is associated with improved vascular relaxation, supporting the view that calcium supplementation reduces blood pressure by decreasing peripheral arterial resistance [10].
The effect of high calcium diet on vascular function has not been studied in RF. Here we examined the influence of increased calcium intake on arterial tone in rats subjected to 5/6 nephrectomy (NTX). The focus of the study was especially on the phase before these animals developed overt hypertension. We tested the hypothesis that treatment of hyperphosphataemia and secondary hyperparathyroidism by high calcium diet will improve vasorelaxation in experimental RF. The roles of different endothelium-derived mediators in the vasodilator responses of resistance vessels from NTX rats were elucidated, and possible functional changes in arterial smooth muscle were addressed.
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Subjects and methods |
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High calcium diet continued for 8 weeks, and 24-h fluid consumption and urine output were measured during study week 20. The rats were weighed and anaesthetized (urethane 1.3 g/kg), and blood samples from cannulated carotid artery for plasma electrolyte, creatinine, urea, phosphate, 1,25 (OH)2D3, PTH and haemoglobin measurements were drawn into chilled tubes, and for ionized Ca2+ into glass capillaries, with heparin as anticoagulant. The hearts and kidneys were removed and weighed, and third order branches from the mesenteric arterial bed were excised under a dissecting microscope (Nikon, Japan).
The Mulvany multimyograph Model 610A (J.P. Trading, Aarhus, Denmark) was employed in functional studies in four separate vascular ring preparations. In this isometric system the force transducer is directly linked to a computer (Myodaq software, J.P. Trading). Vessels were suspended as rings (length 1.9 mm) on two 40-µm stainless steel wires, each of which was attached to a myograph jaw. The physiological salt solution (PSS; pH 7.4) contained (mM): 119.0 NaCl, 25.0 NaHCO3, 11.1 glucose, 1.6 CaCl2, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, and was aerated with 95% O2 and 5% CO2. Normalization of the preparations was performed so that the vessel internal diameter was set at 90% of that obtained when exposed to an intraluminal pressure of 100 mmHg in the relaxed state [12]. Morphology of small arteries was examined with a pressure myograph (Living Systems Instrumentation Inc., Burlington, VT, USA) as reported previously, and the development of myogenic tone was inhibited by Ca2+-free solution containing 30 mmol/l EDTA [13]. The experimental design of the study was approved by the Animal Experimentation Committee of the University of Tampere, and the Provincial Government of Western Finland Department of Social Affairs and Health, Finland. The investigation conforms to the Guiding Principles for Research Involving Animals.
Mesenteric resistance arterial responses in vitro
Presence of intact endothelium was confirmed by a clear relaxation to 1 µM acetylcholine (ACh) in 5 µM noradrenaline (NA)-pre-contracted rings, and the absence of endothelium by the lack of this response. The endothelium was removed by perfusing air through the lumen. A 30 min period in PSS was allowed between each cumulative response described below.
Ring preparation 1: contraction to NA; role of nitric oxide (NO), prostanoids and K+ channels in endothelium-mediated relaxations (endothelium-intact rings). Contractions to NA were first determined. Relaxations to ACh were examined in arteries pre-contracted with 5 µM NA, which induced 75% of maximal contraction in all groups. Responses to ACh were also elicited in the presence of 0.1 mM NG-nitro-L-arginine methyl ester (L-NAME; NO synthase inhibitor); L-NAME and 3 µM diclofenac (cyclooxygenase inhibitor); L-NAME, diclofenac and 50 nM apamin plus 0.1 µM charybdotoxin (blockers of small and large conductance Ca2+-activated K+ channels; KCa, respectively) [14]. Without the introduction of the above inhibitors the response to ACh was highly reproducible: the pD2 (log mol/l) of the first vs fourth response (n = 8) was 7.64 ± 0.06 vs 7.47 ± 0.07, and maximal relaxation (%) 89.1 ± 2.9 vs 88.0 ± 1.8, respectively.
Ring preparation 2: contractions to KCl and endothelin-1 (ET-1) (endothelium-denuded rings). Contractions to KCl were elicited, and in solutions containing high concentrations of K+, NaCl was replaced with KCl on an equimolar basis. After 30 min, contractions to ET-1 were recorded.
Ring preparation 3: relaxations to activation of ß-adrenoceptors and exogenous NO (endothelium-denuded rings). Responses to isoprenaline (ß-adrenoceptor agonist) and nitroprusside (NO donor) were examined after pre-contraction with 5 µM NA.
Ring preparation 4: relaxations to opening of Ca2+-activated and ATP-sensitive K+ channels (endothelium-denuded rings). Responses to 11,12-epoxyeicosatrienoic acid (EET; opener of KCa) [14], and levcromakalim (opener of ATP-sensitive K+ channels; KATP) were examined after pre-contraction with 5 µM NA.
Plasma concentrations of electrolytes, phosphate, creatinine, urea, protein, haemoglobin, 1,25(OH)2D3 and PTH
Potassium and sodium were measured by potentiometric direct dry chemistry, urea by colorimetric enzymatic dry chemistry, and phosphate by colorimetric dry chemistry (Vitros 950 analyzer, Johnson & Johnson Clinical Diagnostics, Rochester, NY, USA). Creatinine was determined by colorimetric assay according to Jaffe, and proteins by colorimetric measurement according to Biuret (Cobas Integra analyzer, F. Hoffman-La Roche Ltd, Basel, Switzerland). Ionized calcium was measured by ion selective electrode (result adjusted for pH 7.4, Ciba Corning 634 Ca2+/pH Analyzer, Ciba Corning Diagnostics, Sudbury, UK), and haemoglobin photometrically (Technicon H*2TM, Technicon Instruments Corporation, Tarrytown, NY). PTH levels were measured by an immunoradiometric assay specific for intact rat PTH (Catalog #50-2000, Immunotopics, San Clemente, CA, USA), and vitamin D by radioassay designed for the quantitative determination of 1,25(OH)2D3 (competitive protein-binding assay, Catalog #40-6041, Nichols Institute Diagnostics, San Juan Capistrano, CA, USA).
Data presentation and analysis of results
Wall tensions were expressed in mN/mm, and the EC50 in each ring was calculated as a percentage of maximal response and presented as the negative logarithm (pD2). The relaxations were presented as percentage of pre-existing contraction. In addition, the area under curve (AUC) for each individual relaxation response was determined and expressed in arbitrary units. In the two NTX groups, correlation analyses were performed between plasma levels of creatinine, urea and the pD2 values for vasorelaxation. The sham-operated animals were excluded from these analyses, as increased calcium intake had no effect on arterial tone in these animals.
Statistical analysis was carried out by one-way and two-way analyses of variance (ANOVA) supported by two-tailed t-test when carrying out pair-wise comparisons between the test groups. ANOVA for repeated measurements was applied for data consisting of repeated observations at successive points. Spearmans correlation coefficient (r; two-tailed) was used in the correlation analyses. Unless otherwise indicated, the P-values in the text refer to ANOVA for repeated measurements. Results are expressed as mean ± SEM, and differences were considered significant when P < 0.05.
Drugs
The drugs were: ketamine (Parke-Davis Scandinavia AB, Solna, Sweden), cefuroxim, diazepam (Orion Pharma Ltd., Espoo, Finland), metronidazole (B. Braun AG, Melsungen, Germany), buprenorphine (Reckitt & Colman, Hull, UK), levcromakalim (SmithKline Beecham AB, West Sussex, UK), ACh chloride, apamin, charybdotoxin, EET, ET-1, isoprenaline hydrochloride, NA bitartrate, L-NAME hydrochloride (Sigma Chemical Co, St Louis, MO), sodium nitroprusside (Fluka Chemie AG, Buchs SG, Switzerland) and diclofenac (Voltaren® injection solution, Ciba-Geigy, Basle, Switzerland). Stock solutions of the compounds were made by dissolving them in distilled water; levcromakalim was dissolved in 50% ethanol, and EET in 99% ethanol and kept on ice. The final contents of ethanol in PSS were 0.14 and 2.38% when levcromakalim and EET were used, respectively. The 0.14% ethanol was without effect on arterial tone, while the minor effect of 2.38% ethanol is depicted in Figure 2. Solutions were freshly prepared before use and protected from light.
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Results |
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Arterial preparations from all groups showed similar contractile sensitivity (i.e. pD2 values) and maximal wall tensions in response to NA, KCl and ET-1 (Table 4). Therefore, no changes in vasoconstrictor responses were observed in the NTX + calcium rats that could explain the enhanced vasorelaxation in the arterial rings of these animals.
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Calcium metabolism, renal function and vasorelaxation in small mesenteric arteries
Increased calcium intake, which reduced plasma phosphate and PTH, and elevated ionized calcium in NTX rats, was associated with enhanced vasorelaxation to ACh, levcromakalim and EET. The scatter plots depicting the variables of calcium metabolism in relation to endothelium-dependent vasorelaxation in resistance arteries, i.e. AUC values for ACh, showed clear clustering of data points in separate subgroups in the NTX and NTX + calcium groups (Figure 3AC), whereas such clustering was not observed when the relation of plasma urea to the AUC for ACh was illustrated (Figure 3D). The variables reflecting renal function were not different in the two NTX groups, and plasma creatinine and urea showed no correlation to the sensitivity (r = 0.255 and 0.186; P = 0.307 and P = 0.445, respectively) or maximal relaxation to ACh (r = 0.269 and 0.323; P = 0.296 and P = 0.206, respectively) in the NTX and NTX + calcium groups. The scatter plot analyses depicting variables of calcium metabolism in relation to the AUC of vasorelaxations elicited by levcromakalim and EET in the two NTX groups showed similar clustering of data points (data not shown).
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Discussion |
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Blood pressure in the NTX rats was only moderately elevated, and was not affected by increased calcium intake. The development of hypertension after subtotal nephrectomy depends on rat strain, renal ablation procedure and especially dietary sodium load [6,11]. We were interested in the vascular effects of increased calcium intake that would not be related to changes in blood pressure, whereby the sodium content of the chow (0.27%) was not increased. This amount of dietary sodium has not previously elevated blood pressure in NTX rats [6,11].
Secondary hyperparathyroidism is a universal complication in chronic RF. The main up-regulators of PTH secretion are high plasma phosphate, low ionized calcium and low calcitriol [7]. Both secondary hyperparathyroidism and hyperphosphataemia contribute to vascular pathology during impaired kidney function, while treatment of high phosphate can ameliorate the progression of secondary hyperparathyroidism and reduce cardiovascular complications [2,7]. In the present study, plasma PTH and phosphate were elevated in experimental RF, and effectively reduced by the high calcium diet. The lowering of PTH by increased calcium intake in the NTX rats can be attributed to reduced plasma phosphate and elevated calcium levels.
Plasma 1,25(OH)2D3 levels were reduced after NTX, but were not affected by increased calcium intake. The main regulators of renal 1,25(OH)2D3 synthesis are phosphate, PTH, calcium and 1,25(OH)2D3 [7,15]. Already high-normal concentration of phosphate inhibits and low-normal stimulates 1,25(OH)2D3 synthesis. In addition, high PTH increases, while hypercalcaemia decreases the production. 1,25(OH)2D3 also regulates its own synthesis by a negative feedback mechanism [7,15]. In this study, the net effect of reduced plasma PTH and phosphate, together with elevated calcium, can explain why active vitamin D levels were not reduced by high calcium diet. In RF, the altered vitamin D status has been suggested as a possible contributor to the cardiovascular pathology, and vitamin D may also directly influence the function of various tissues including vascular smooth muscle [2,15]. As the plasma 1,25(OH)2D3 levels were not changed by calcium supplementation in the present study, the observed changes in vasorelaxation after increased calcium intake probably did not result from changes in the levels of active vitamin D.
In spite of the crucial role of small arteries in the regulation of peripheral vascular resistance, the present knowledge about resistance vessel function in chronic RF is scarce. Isolated subcutaneous resistance arteries from uraemic patients with systolic hypertension have shown impaired endothelium-mediated relaxation [4], but no such impairment has been found in the skin microcirculation of normotensive patients with RF [16]. However, several in vivo studies suggest that endothelial function is impaired in RF [5]. In this study, we found that endothelium-dependent relaxation was clearly impaired 12 weeks after subtotal nephrectomy, while high calcium diet for 8 weeks normalized the response to ACh in NTX rats.
Vasodilatation induced by ACh is mediated via NO, prostacyclin and endothelium-derived hyperpolarizing factor (EDHF), which plays a major role in the relaxation of resistance arteries, probably via opening of smooth muscle KCa [14,19]. The contribution of these components to vasodilatation was addressed by NOS inhibition, cyclooxygenase inhibition and K+ channel blockade, respectively. NOS inhibition reduced the relaxations to ACh, but the responses were still impaired in the NTX group when compared with other groups. Cyclooxygenase inhibition with diclofenac was without effect on the responses to ACh. In contrast, KCa blockade with apamin and charybdotoxin reduced the L-NAME and diclofenac-resistant relaxation to ACh [14], and this effect was less marked in the NTX group than others. As the impaired response to ACh in the NTX group was observed in the presence of NOS and cyclooxygenase inhibition, but not in the presence of KCa blockade, these results imply that endothelium-dependent vasodilatation in experimental RF was reduced via a mechanism, which involved activation of K+ channels in arterial smooth muscle. This impairment was normalized by high calcium diet.
Reduced endothelium-dependent relaxation via K+ channels could result from decreased endothelial release of EDHF, or reduced sensitivity of smooth muscle to EDHF. We found that the endothelium-independent relaxations to the KATP opener levcromakalim and the KCa opener EET were impaired in NTX rats, while these responses in NTX + calcium rats did not differ from the sham-operated controls. Therefore, deficient vasodilatation at the level of smooth muscle K+ channels was corrected by increased calcium intake in RF. Moreover, the reduced response to EET, a putative EDHF [14,19], indicates that smooth muscle sensitivity to EDHF was also decreased in the NTX rats. The attenuated endothelium-mediated relaxations in the NTX rats may well be explained by deficient vasorelaxation at the level of smooth muscle K+ channels.
Changes in the plasma concentration of phosphate, calcium and PTH could affect blood vessels in many ways. Elevated phosphate may influence the metabolism of vascular smooth muscle, and induce phenotypic changes that predispose the vessel wall to calcification [17]. A link between extracellular calcium and arterial tone is the calcium receptor located in perivascular nerves, the activation of which causes vasorelaxation via the release of a hyperpolarizing mediator [18]. The link between PTH and vascular tone is complex. PTH excess may increase cytoplasmic Ca2+ or alter the production of endothelium-derived vasoactive factors [2]. Acutely PTH causes vasodilatation, but subacute infusion of physiological doses of PTH raises blood pressure, as vascular desensitization to PTH takes place rapidly (for a review see [2]). Interestingly, at the cellular level PTH is linked to increased production of 20-hydroxyeicosatetraenoic acid (20-HETE), an endogenous vasoconstrictor that acts in part by inhibiting the opening of KCa in smooth muscle [19]. Thus, a putative explanation for the enhanced vasorelaxation following high calcium diet in NTX rats would be reduced synthesis of the KCa blocker 20-HETE in arterial smooth muscle.
The effect of high calcium intake on resistance artery morphology in RF has not been characterized. We found that small mesenteric arteries of NTX rats showed increased wall to lumen ratio, and this change was not affected by increased calcium intake. As the cross-sectional area of arterial wall was not increased, the observed change in vascular morphology in NTX rats is compatible with eutrophic inward remodelling [20]. The vascular wall to lumen ratio gives information about the ability of the vessel to contract against intravascular pressure, while the cross-sectional area indicates the amount of material within the vascular wall, and provides information about vascular growth [20]. The present results showed that high calcium diet improved vasorelaxation in RF, although resistance vessel structure was not corrected.
In clinical RF, the importance of effective treatment of hyperphosphataemia in the prevention of cardiovascular complications is well recognized [2,7]. The treatment of secondary hyperparathyroidism remains slightly controversial, as a 23-fold elevation in plasma PTH has been considered beneficial to the bones of patients with impaired kidney function. However, the cardiovascular actions of moderate chronic elevations in PTH are not known in RF. Therefore, our experimental results warrant such clinical studies, where the vascular actions of long-term treatments of secondary hyperparathyroidism are elucidated in patients with moderate kidney impairment. Different therapeutics approaches (calcium salts, other phosphate binders, calcimimetic agents), possibly at different levels of PTH control, should be compared with respect to their influences on arterial structure and tone.
In conclusion, renal insufficiency induced by subtotal nephrectomy resulted in modest elevation of blood pressure, secondary hyperparathyroidism and impaired vasorelaxation via K+ channels in resistance vessels. High calcium diet significantly lowered plasma PTH and phosphate levels without influencing blood pressure or plasma creatinine, and normalized the deficient resistance artery vasorelaxation in experimental RF. These findings suggest that correction of the disturbances of calcium-phosphate metabolism by increased calcium intake is beneficial to the vasorelaxant properties of resistance arteries in RF.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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