Original Report: Laboratory Investigation American
Journal of
Nephrology
Am J Nephrol 2014;39:400–408 DOI: 10.1159/000362507
Received: October 4, 2013 Accepted: March 26, 2014 Published online: May 6, 2014
Phosphate Binding Reduces Aortic Angiotensin-Converting Enzyme and Enhances Nitric Oxide Bioactivity in Experimental Renal Insufficiency Arttu Eräranta a Suvi Törmänen a Peeter Kööbi a, b Tuija I. Vehmas a Päivi Lakkisto d, e Ilkka Tikkanen d, e Eeva Moilanen a Onni Niemelä f Jukka Mustonen a, c Ilkka Pörsti a, c a
School of Medicine, University of Tampere, and Departments of b Ophthalmology and c Internal Medicine, Tampere University Hospital, Tampere, d Minerva Institute for Medical Research, and e Department of Medicine, Helsinki University Central Hospital, Helsinki, and f Department of Clinical Chemistry, Etelä-Pohjanmaa Central Hospital Laboratory, Seinäjoki, Finland
Abstract Background: Disturbed calcium-phosphorus metabolism is associated with increased kidney angiotensin-converting enzyme (ACE) in experimental chronic renal insufficiency (CRI). However, information about the effects of phosphate binding and loading on vascular ACE is lacking. Methods: Fifteen weeks after 5/6 nephrectomy (NX), rats were placed on a phosphate-binding (NX+Ca, 3.0% Ca), phosphate-loading (NX+Pi, 1.5% Pi), or control diet for 12 weeks (NX and sham). Results: Aortic ACE, blood pressure, plasma phosphate, and parathyroid hormone were increased in the NX and NX+Pi groups, but were reduced with phosphate binding. Endothelium-mediated relaxations of isolated mesenteric conduit artery rings to acetylcholine were impaired in the NX and NX+Pi groups, but did not differ from sham in NX+Ca rats. Experiments with nitric oxide (NO) synthase inhibition in vitro suggested that the NO-mediated component of acetylcholine
© 2014 S. Karger AG, Basel 0250–8095/14/0395–0400$39.50/0 E-Mail [email protected] www.karger.com/ajn
response was lower in the NX and NX+Pi groups, but did not differ from sham in NX+Ca rats. In all NX groups, aortic endothelial NO synthase (eNOS) was reduced, while plasma and urine concentrations of NO metabolites were increased. Aortic nitrated proteins and calcification were increased in the NX and NX+Pi groups when compared with the NX+Ca and sham groups. Conclusion: Hypertension in the NX model of CRI was associated with reduced vasorelaxation, decreased eNOS, and increased ACE and nitrated proteins in the aorta. Phosphate binding with calcium carbonate enhanced vasorelaxation via endogenous NO and suppressed elevation of ACE and nitrated proteins, suggesting reduced vascular oxidative stress. Our findings support the view that correction of the calcium-phosphorus balance prevents CRI-induced vascular pathophysiology. © 2014 S. Karger AG, Basel
Introduction
In chronic kidney disease, cardiovascular complications are aggravated by increased serum phosphate and parathyroid hormone (PTH) concentrations [1]. ReIlkka Pörsti, MD School of Medicine/Internal Medicine University of Tampere FI–33014 Tampere (Finland) E-Mail ilkka.porsti @ uta.fi
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Key Words Angiotensin-converting enzyme · Calcium · Chronic renal insufficiency · Nitric oxide · Phosphate · Renin-angiotensin system
Methods Animals and Experimental Design We utilized samples from rats previously used for reports elucidating resistance artery function [4] and kidney ACE [5] in CRI. The NX groups underwent removal of the right kidney and surgical resection of the upper and lower poles (2/3) of the left kidney, whereas the sham operation was kidney decapsulation [4, 5, 14, 15]. Diets, anesthesia, antibiotics, postoperative pain relief, and tail-cuff measurement of systolic blood pressure (BP) were as previously described [4, 14, 15].
Phosphate, Aortic ACE, and NO in Renal Insufficiency
A preliminary 12-week study examined whether a calcium carbonate diet influences aortic or cardiac ACE. Four weeks after NX (rat age: 12 weeks), four groups with corresponding systolic BPs and body weights in the sham versus sham+Ca groups and NX versus NX+Ca groups were formed (n = 10–11) [4]. Then for 8 weeks, sham and NX rats ingested 0.3% calcium, while sham+Ca and NX+Ca rats ingested 3% calcium chow (carbonate supplement; AnalyCen, Lidköping, Sweden) [4]. Tissue samples were harvested after 12 study weeks. For the 27-week study, 15 weeks after NX, three groups (n = 13–14) of remnant kidney rats, having equal mean systolic BP, body weight, and plasma creatinine, and sham rats (n = 11) of equal age were put on chow containing 0.3% Ca and 0.5% Pi (NX and sham), 3.0% Ca and 0.5% Pi (NX+Ca), and 0.3% Ca and 1.5% Pi (NX+Pi) (AnalyCen) [5]. These diets represent intakes of low Ca/moderate Pi, high Ca/moderate Pi, and low Ca/high Pi, respectively. The calcium diets were based on our previous experiences of 0.3 versus 3.0% calcium intake in experimental CRI [4, 5, 15]. Then for 12 weeks, body weight and systolic BP were monitored fortnightly. The 24-hour water intake and urine output were measured in metabolic cages and urine samples stored at –80 ° C. From the NX, NX+Pi, and NX+Ca groups, 7, 6, and 1 rats were lost, and the final numbers of animals in these groups were 7, 7, and 12, respectively [5]. At the close of the study, the rats were anesthetized (urethane 1.3 g/kg), and blood samples were drawn from carotid artery with EDTA and heparin as anticoagulants [4, 5, 15]. The main branches of the mesenteric arteries were excised, and the hearts were removed. Tissue samples for ACE, eNOS, and nitrated protein measurements were frozen in isopentane at –40 ° C and stored at –80 ° C. Aortic samples were fixed in 4% formaldehyde for 24 h and embedded in paraffin. 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 Arterial Responses in vitro The mesenteric artery was cleaned and three successive 3-mmlong rings were cut. In proximal rings the endothelium was left intact, while it was denuded from the distal sample [16]. The rings were suspended between hooks in organ bath chambers (20 ml) with physiological salt solution (pH 7.4) containing (in mmol/l): NaCl 119, NaHCO3 25, glucose 11.1, CaCl2 1.6, KCl 4.7, KH2PO4 1.2, and MgSO4 1.2, and aerated with 95% O2/5% CO2. The rings were equilibrated for 2 h at +37 ° C with a preload of 4.905 mN/mm (FT-03 transducer, 7E Polygraph; Grass Instrument Co., Quincy, Mass., USA) [17]. Responses to acetylcholine (ACh) in the absence and presence of 0.1 mmol/l NG-nitro-L-arginine methyl ester (L-NAME) or 1 mmol/l L-arginine, norepinephrine (NE), and Ang II were studied in endothelium-intact rings, and to KCl and nitroprusside in endothelium-denuded rings. All vasorelaxations were studied after precontractions induced by 1 μmol/l NE. Stock solutions of all compounds were freshly prepared and protected from light.
Biochemical Measurements NOx was measured by conversion of nitrite and nitrate to NO [18]. Proteins were precipitated with ethanol (+20 ° C, 2 h), and a 20-μl sample was injected into a cylinder containing vanadium
Am J Nephrol 2014;39:400–408 DOI: 10.1159/000362507
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duced renal function is associated with large arterial stiffening, increased arterial intima-media thickness, vascular smooth muscle hyperplasia, and medial calcification [2, 3]. In addition, functional and structural changes in resistance arteries have been described in both clinical [3] and experimental chronic renal insufficiency (CRI) [4]. Phosphate retention, hypocalcemia, increased fibroblast growth factor 23, and reduced vitamin D levels lead to the development of secondary hyperparathyroidism already at stages 2 and 3 of CRI [5, 6], and these factors also influence the remodeling of the vascular wall [7]. In order to manage CRI-induced hyperphosphatemia and hyperparathyroidism, oral phosphate binders are frequently needed [8]. Endothelial cells synthesize nitric oxide (NO) from Larginine by constitutive endothelial NO synthase (eNOS) [9]. When compared with small vessels, the contribution of eNOS to vasomotion seems greater in conduit-sized arteries [10, 11]. The interaction between activated reninangiotensin system and bioavailability of endotheliumderived NO is considered an important regulator of vascular tone [12]. A key target for the pharmacological inhibition of the renin-angiotensin system is angiotensinconverting enzyme (ACE), which converts angiotensin I (Ang I) to Ang II and degrades kinins [13]. We recently found that dietary phosphate loading was associated with increased kidney ACE expression and tissue damage in the 5/6 nephrectomy (NX) rat model of CRI [5]. In contrast, phosphate binding with 3.0% calcium carbonate reduced kidney ACE and tissue damage [5]. The present study explored whether phosphate binding and loading influences ACE content in the abdominal aorta and endothelium-dependent vasodilatation in the mesenteric artery. In order to evaluate oxidative stress, nitrated protein and eNOS contents in the abdominal aorta, and NO metabolites (NOx) in plasma and urine were determined. Rats were subjected to NX, followed for 15 weeks, and divided into groups given a control, phosphate-lowering, or high-phosphate diet for 12 weeks.
(III) chloride (VCl3) in HCl (0.8 g VCl3/100 ml of 1 mol/l HCl) at 95 ° C, and the NO formed was measured (NOA 280 analyzer; Sievers Instruments, Boulder, Colo., USA) [19]. Measurements of plasma phosphate, PTH, and ionized calcium were as described [5]. Autoradiography of Tissue ACE Quantitative autoradiography of ACE in vitro was performed on tissue sections (20 μm thick) with the radioligand [125I]MK351A (tyrosyl residue of lisinopril), as described earlier [20, 21]. The optical densities were quantified by an image analysis system (AIDA 2D) coupled to the FUJIFILM BAS-5000 phosphorimager (Tamro, Vantaa, Finland), for a total of 24 analyses per sample. Western Blotting of Aortic NOS and Nitrated Proteins Frozen tissues were homogenized (Ultra-Turrax T25, IKA®Labortechnik, Staufen, Germany) in 400 μl of distilled H2O containing protease inhibitors (CompleteTM Mini EDTA-free, Roche Diagnostics GmbH, Mannheim, Germany). After centrifugation (12,000 g, 15 min, 4 ° C), supernatant protein concentrations were determined (Coomassie PlusTM Protein Assay Kit; Pierce, Rockford, Ill., USA). SDS-PAGE was run on 8% resolving gel and 4% stacking gel, and proteins were transferred to Hybond-ECL nitrocellulose membrane (Amersham Biosciences Ltd., Amersham, UK). The primary antibodies were: mouse eNOS 1: 2,500 dilution (BD Biosciences-Pharmingen, San Diego, Calif., USA), polyclonal rabbit NOS2 1:4,000 dilution (Santa Cruz Biotechnology, Dallas, Tex., USA), and monoclonal mouse nitrotyrosine 1: 3,333 dilution (Cayman Chemical, Ann Arbor, Mich., USA). Antibody binding was detected using SuperSignal® West Pico chemiluminescent substrate (Pierce), and the signal was analyzed using FluorChem 8800 (Alpha Innotech Corporation, San Leandro, Calif., USA).
Aortic Calcification Calcifications were determined from von Kossa-stained samples [5]. The index of calcification for each rat was expressed as a percentage of the calcified area related to the total area of aortic cross-section. Drugs The drugs were: ketamine (Parke-Davis Scandinavia AB, Solna, Sweden), ACh chloride, L-arginine, L-NAME hydrochloride, NE bitartrate, Ang II (Sigma Chemical Co., St. Louis, Mo., USA), and sodium nitroprusside (Fluka Chemie AG, Buchs, Switzerland). Data Presentation and Analysis of Results Contractions were expressed in mN/mm, and EC50 concentrations as the negative logarithm (pD2) values. Vasorelaxation was presented as a percentage of the precontraction. Area under concentration response curve (AUC) analysis was applied to evaluate the contribution of NO to vasorelaxation. Statistical analyses were by one-way analysis of variance (ANOVA), supported by post-hoc Tukey’s test or ANOVA for repeated measurements, as appropriate. If skewed variable distribution was observed, Kruskal-Wallis and post-hoc Mann-Whitney U tests were used. Spearman’s correlations were calculated, as appropriate. Results were presented as means ± SEM, and p < 0.05 was considered significant.
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Results
Am J Nephrol 2014;39:400–408 DOI: 10.1159/000362507
12-Week Pilot Study Quantitative in vitro autoradiography showed a decrease in aortic and cardiac ACE protein content after 8 weeks of the 3.0% calcium carbonate diet (online suppl. fig. S1; see www.karger.com/doi/10.1159/000362507 for all online suppl. material). The analyses were performed on 8–10 rats randomly selected in each group. 27-Week Study: Animal Data, Plasma, and Urine Determinations At week 15, systolic BP was elevated in all NX groups (table 1). At study week 27, BP was higher in all NX groups than in the sham rats, but BP in the NX-Ca group was lower than in NX and NX+Pi rats. There were no differences in body weight at week 15, but body weights at week 27 were lower in the NX+Ca and NX+Pi groups than in the sham rats, while no significant differences were detected between the NX groups. The heart/body weight ratios were increased, and creatinine clearance was decreased by about 45–55%, corresponding to stage 3 chronic kidney disease, in all NX rats versus sham rats (table 1). Plasma phosphate and PTH were increased in the NX group, and further elevated in the NX+Pi group, while both phosphate and PTH were suppressed to values below those in sham rats in the NX+Ca group. Plasma ionized calcium did not differ from sham in the NX group, but was increased in the NX+Ca, and decreased in the NX+Pi group. Both plasma and urine NOx were increased in all NX groups when compared with sham (table 1). Aortic ACE, eNOS, Nitrated Proteins, and Calcification After 27 study weeks, aortic ACE protein content was higher in the NX and NX+Pi groups than in the sham rats. Notably, aortic ACE content was clearly lower in the NX+Ca group when compared with the NX and NX+Pi groups (fig. 1a, b). Aortic eNOS protein content was similarly decreased in all NX groups when compared with sham rats (fig. 2a). However, no difference was detected in aortic inducible NOS protein between the study groups, with average values ranging ±18% of the level observed in sham rats (not shown). Aortic nitrated protein content was higher in the NX and NX+Pi groups versus sham, but was lower in the NX+Ca than in NX and NX+Pi rats (fig. 2b). The index of aortic calcification was increased in the NX and NX+Pi groups when compared with the NX+Ca and sham groups (fig. 2c, online suppl. fig. S2). Eräranta et al.
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1.75
*
Sham
NX
Aortic ACE vs. sham
1.50
*
1.25
†, ‡
1.00
0.75
a
NX+Ca
NX+Pi
b
Sham
NX
NX+Ca
NX+Pi
Fig. 1. Representative original tracings (a) and mean ± SEM plots (b) of aortic ACE content determined using
autoradiography; n = 8–10 in each group. * p < 0.05 vs. sham; † p < 0.05 vs. NX; ‡ p < 0.05 vs. NX+Pi.
Table 1. Experimental animal data and laboratory findings in the experimental groups
Sham Systolic BP At week 15, mm Hg At week 27, mm Hg Body weight at week 15, g Body weight at week 27, g Heart weight/body weight, g/kg Creatinine clearance week 27, μmol/ml/100 g Plasma Phosphate, mmol/l PTH, pg/ml Ionized calcium, mmol/l NOx, μmol/l Urine NOx, μmol/24 h
NX
NX+Ca
133 ± 2 129 ± 2 499 ± 8 557 ± 7 3.21 ± 0.03 327 ± 19
154 ± 6* 173 ± 4* 476 ± 9 507 ± 38 4.19 ± 0.43* 167 ± 34*
153 ± 4* 145 ± 3*, † 478 ± 9 488 ± 13* 4.03 ± 0.13* 177 ± 16*
1.16 ± 0.06 88 ± 12 1.36 ± 0.06 8.9 ± 0.4
2.52 ± 0.46* 1,172 ± 343* 1.34 ± 0.03 11.4 ± 0.9*
0.81 ± 0.08*, † 3.7 ± 0.5*, † 1.55 ± 0.03*, † 14.2 ± 5.1*
0.03 ± 0.01
1.36 ± 0.66*
1.49 ± 0.84*
NX+Pi 152 ± 4* 161 ± 4* 484 ± 11 431 ± 35* 4.46 ± 0.40* 145 ± 29* 5.47 ± 1.12*, †, ‡ 3,620 ± 236*, †, ‡ 0.93 ± 0.09*, †, ‡ 18.4 ± 5.1* 0.80 ± 0.36*
Endothelium-Dependent and -Independent Vasorelaxation The relaxations induced by ACh in endothelium-intact mesenteric artery rings were impaired in the NX and NX+Pi groups, but did not differ from sham rats in the NX+Ca group (fig. 3a). The NOS inhibitor L-NAME reduced the relaxations to ACh in all groups, but the effect was more pronounced in the NX+Ca than in the NX and
NX+Pi groups, as the ACh response did not differ between the NX groups in the presence of L-NAME (fig. 3b). The change in the AUC of the ACh-response induced by L-NAME suggested that the contribution of NO to the relaxation was reduced in the NX and NX+Pi groups, but did not differ from sham rats in the NX+Ca group (fig. 3c). Both aortic nitrated protein content and index of calcification correlated inversely with maximal ACh-induced
Phosphate, Aortic ACE, and NO in Renal Insufficiency
Am J Nephrol 2014;39:400–408 DOI: 10.1159/000362507
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Values are means ± SEM; n = 7–14 for all groups. * p < 0.05 vs. sham; † p < 0.05 vs. NX; ‡ p < 0.05 vs. NX+Ca.
1.25
Aortic eNOS vs. sham
1.00
0.75
*
0.50
*
*
0.25
0
Sham
NX
NX+Ca
Aortic nitrated proteins vs. sham
6
*
4 3 2 †, ‡
1 0
NX+Pi
*
5
Sham
NX
NX+Ca
NX+Pi
b
a
and index of aortic calcification (percent of cross-section) determined using a von Kossa stain (c). Means ± SEM; n = 7–11 in each group; intensity of white bands represents binding to specific antibody (a, b). * p < 0.05 vs. sham; † p < 0.05 vs. NX; ‡ p < 0.05 vs. NX+Pi.
relaxation (r = –0.438, p = 0.002; r = –0.637, p < 0.001, respectively). The addition of exogenous L-arginine (1 mmol/l) had no significant effects on the ACh-induced relaxations (data not shown). Vasorelaxation to the NO donor nitroprusside in endothelium-denuded rings was slightly decreased in the NX+Pi group when compared with sham and NX+Ca rats, but did not differ between the sham, NX, and NX+Ca groups (fig. 3d). Vasoconstrictor Responses Vasoconstrictor sensitivity and maximal responses of mesenteric arterial rings to NE did not differ between the sham, NX, and NX+Ca groups, while sensitivity to NE was slightly higher in the NX+Pi than the sham and NX+Ca groups, and maximal response was lower in the 404
Am J Nephrol 2014;39:400–408 DOI: 10.1159/000362507
c
*
25 20 15 10
*
5 0
†, ‡ Sham
NX
NX+Ca
NX+Pi
NX+Pi than sham group (online suppl. table S1). The sensitivity and maximal responses to KCl did not differ between the study groups. Vasoconstrictor sensitivity to Ang II was lower in the NX+Ca than sham group, while maximal wall tension in response to Ang II was higher in the NX versus sham group, and lower in the NX+Ca versus NX group (online suppl. table S1).
Discussion
This study investigated the effects of dietary phosphate binding and loading on aortic ACE protein, eNOS content, and calcification, and vascular tone in vitro of isolated mesenteric arterial rings in experimental CRI. In the Eräranta et al.
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Fig. 2. Bar graphs and representative bands of aortic eNOS protein (a), and nitrated proteins (b), determined using Western blotting,
Calcification (% of aortic cross-section)
30
0
NX
20
20
NX+Ca NX+Pi
Relaxation (%)
Relaxation (%)
0
Sham
40 60
*
80
a
5
9
b
250
8 7 6 Acetylcholine (–log mol/l)
5
0 †, ‡
200 150
20
*
*
100
Relaxation (%)
Change in AUC by L-NAME (arbitrary units)
With 0.1 mmol/l L-NAME
60
100 8 7 6 Acetylcholine (–log mol/l)
9
*
40 60 80
50 0
40
80
100
c
*
100 Sham
NX
NX+Ca
NX+Pi
d
9
8 7 6 Nitroprusside (–log mol/l)
5
27-week study, elevated systolic BP, decreased creatinine clearance, and increased heart/body weight were seen in all NX groups (table 1). However, the phosphate-lowering diet blunted the increase in BP, while phosphate loading did not influence BP. In experimental studies, the lowering of BP after a high-calcium diet has been attributed to increased natriuresis, reduced sympathetic activity, suppressed PTH levels, and enhanced vasodilatation [4, 15, 22–25]. The present results suggest that reduction of tissue ACE might also play a role in the lowering of BP. Abnormal calcium, phosphate, and PTH metabolism are known causes for increased cardiovascular calcification in CRI [1, 3]. Effective phosphate control is a culprit
in the prevention of cardiovascular problems in CRI, and several calcium-based and noncalcium-containing phosphate binders are available for this purpose. Concern has been raised that high intake of calcium salts may predispose to calcifications in CRI, but the superiority of noncalcium-containing phosphate binders over calciumbased binders remains unclear [8]. The present experimental results apply to calcium-based phosphate binding, and show that suppression of PTH and phosphate to levels below those in sham rats was associated with reduction in aortic calcifications. Due to reduced survival in the NX and NX+Pi groups [5], samples from the most uremic rats were not available for laboratory analyses, and the
Phosphate, Aortic ACE, and NO in Renal Insufficiency
Am J Nephrol 2014;39:400–408 DOI: 10.1159/000362507
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Fig. 3. Relaxations to ACh in endothelium-intact mesenteric arterial rings in the absence (a) and presence (b) of 0.1 mmol/l L-NAME. c Bar graph showing the change induced by NOS inhibition with L-NAME in the AUC (arbitrary units) of the ACh response. d Relaxation to nitroprusside in endothelium-denuded mesenteric arterial rings. Means ± SEM; n = 7–11 in each group. * p < 0.05, ANOVA for repeated measurements (a, b, d); * p < 0.05 vs. sham; † p < 0.05 vs. NX; ‡ p < 0.05 vs. NX+Pi (c), one-way ANOVA.
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increases superoxide release [34]. Increased oxidative stress is considered as one of the culprits leading to the loss of NO-mediated vasodilatation [34]. When superoxide reacts with NO, peroxynitrite is formed at a reaction rate that exceeds the scavenging of superoxide by superoxide dismutase [31, 35, 36]. Peroxynitrite can modify tyrosine residues in proteins creating nitrotyrosine, thus leaving a footprint of oxidative stress in tissues [36]. Here we found that the aortic nitrated protein levels were increased in the NX and NX+Pi groups, but were reduced in the NX+Ca group, suggesting that the calcium carbonate diet reduced oxidative stress. One mechanism leading to reduced Ang II and superoxide generation could be reduced arterial tissue ACE content, and this could also increase the bioactivity of endothelium-derived NO. NO has a short half-life, and NOx determination was used to evaluate NO production in vivo. Plasma NOx concentration was increased in NX rats, in agreement with previous results [30, 31]. Urine NOx was also increased in all NX groups. Elevated NOx is considered to reflect oxidative stress and inflammation in CRI [29, 31]. The source of NOx remains unknown, as we detected reduced eNOS and no differences in abdominal aortic inducible NOS content after 27 weeks of CRI. Previously, studies examining NO release have shown increased production, whereas those examining NO bioactivity have shown attenuated responses in CRI, and even endothelial cells cultured with uremic plasma in vitro have shown increased NO release [31]. Thus, elevated plasma NOx could result from the effects of a uremic milieu on NOS activity in vivo, while reduced NO bioactivity in uremia has been related to excess NO consumption due to oxidative stress [31]. Elevated NOx could also result from the increased oxidative stress in CRI since peroxynitrite can break down to form NOx [29]. The arterial contractions were examined to uncover possible differences in vasoconstrictor sensitivity that would interfere with the interpretation of relaxation experiments. No differences were found in responses to KCl, and contractions elicited by NE were corresponding in the sham, NX, and NX+Ca groups. Therefore, alterations in vasoconstrictor sensitivity did not explain enhanced vasorelaxation in NX+Ca rats when compared with NX rats. The NX+Pi group exhibited higher sensitivity to NE when compared with sham rats, and increased vasoconstrictor sensitivity may partially explain impaired vasodilator response to exogenous NO after phosphate loading. Finally, mesenteric arterial rings in the NX group exhibited increased maximal wall tension to Ang II, while the response in the NX+Ca group did not differ from the Eräranta et al.
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results may have underestimated the extent of tissue damage and decline of renal function in these groups. Previously, we found that the calcium-phosphorus balance influences ACE content in kidney tissue [5, 15], and the results of the 12-week study showed that the calcium diet reduced ACE in the heart and aorta. In the 27week study, aortic ACE protein was decreased after 12 weeks of an oral calcium carbonate diet. In our earlier study, the correlation between ACE protein and ACE mRNA content in kidney tissue was good (r = 0.83) [5], suggesting that changes in tissue ACE are explained by alterations at the level of gene expression. In contrast to our previous results on kidney ACE, the content of ACE in the aorta was not increased after phosphate loading [5]. This may be attributed to the calcifications in the aortas of NX+Pi rats, as calcified tissue sections would not contain ACE to bind the autoradiography tracer. Deficient endothelium-mediated vasodilatation is common in CRI [23, 26–28]. Previously, we observed that the correction of calcium, phosphate, and PTH levels in NX rats improved endothelium-mediated vasorelaxation via K+-channels and NO in small 2nd- and 3rd-order mesenteric arterial branches [4]. In the present study, we found that phosphate binding normalized impaired relaxation to ACh in a conduit-sized artery. Lower BP in the NX+Ca than in other NX groups probably contributed to the improved vasorelaxation. As the improved response was inhibited by the NOS inhibitor L-NAME, and the change in AUC induced by L-NAME was more pronounced in the NX+Ca than NX and NX+Pi groups, the beneficial effect could be attributed to enhanced NO-mediated response. The contribution of endothelium-derived NO to vasorelaxation seemed corresponding in the NX and NX+Pi groups. Enhanced vasorelaxation after calcium carbonate was not explained by changes in NO sensitivity in arterial smooth muscle, as the response to nitroprusside did not differ in the sham, NX, and NX+Ca groups. Many studies have examined the effect of CRI on Larginine metabolism and the functional role of the NOS enzymes with contradictory results [29–32]. Here we found that eNOS content in the abdominal aorta was equally reduced in all NX groups. Thus, enhanced endothelium-mediated vasorelaxation in the Ca+NX group was not explained by changes in the quantity of eNOS protein. The major effector peptide of the renin-angiotensin system, Ang II, can reduce NO bioactivity through increased production of superoxide radicals, which rapidly inactivate NO [13, 33]. At the molecular level, Ang II upregulates and activates vascular NADPH oxidases, and
sham rats, and contractile sensitivity to Ang II was even lower in NX+Ca than sham rats. This implies that the calcium carbonate diet reduced Ang II-mediated responses in arterial tissue even at the level of the AT1 receptor or its signal transduction cascade. In conclusion, elevated BP in experimental stage 3 chronic kidney disease was associated with impaired vasorelaxation via endothelium-derived NO, and decreased eNOS and increased ACE and nitrated proteins in the aorta, and elevated levels of plasma and urine NOx. Phosphate binding with 3.0% calcium carbonate reduced aortic ACE and nitrated proteins, and enhanced vasorelaxation via increased endothelium-derived NO bioactivity. Thus, reduction of plasma phosphate to levels below those in sham-operated controls with oral calcium carbonate beneficially influenced vascular pathophysiology in experimental CRI.
Acknowledgments The excellent technical assistance of Marja-Leena Koskinen, Riina Hatakka, and Jarkko Lakkisto is sincerely acknowledged. This study was supported by Finnish Kidney Foundation, Finnish Foundation for Cardiovascular Research, Competitive State Research Financing of the Expert Responsibility Area of Tampere University Hospital, Paavo Nurmi Foundation, Emil Aaltonen Foundation, Pirkanmaa Regional Fund of the Finnish Cultural Foundation, Sigrid Jusélius Foundation, and Competitive Research Funding of the Hospital District of Helsinki and Uusimaa.
Disclosure Statement The authors declare no conflicts of interest.
References
Phosphate, Aortic ACE, and NO in Renal Insufficiency
9 Moncada S, Higgs A: The L-arginine-nitric oxide pathway. N Engl J Med 1993;329:2002– 2012. 10 Geiger M, Stone A, Mason SN, Oldham KT, Guice KS: Differential nitric oxide production by microvascular and macrovascular endothelial cells. Am J Physiol 1997;273:L275–L281. 11 Garland CJ, Plane F, Kemp BK, Cocks TM: Endothelium-dependent hyperpolarization: a role in the control of vascular tone. Trends Pharmacol Sci 1995;16:23–30. 12 Raij L: Workshop: hypertension and cardiovascular risk factors: role of the angiotensin II-nitric oxide interaction. Hypertension 2001;37:767–773. 13 Enseleit F, Hurlimann D, Luscher TF: Vascular protective effects of angiotensin converting enzyme inhibitors and their relation to clinical events. J Cardiovasc Pharmacol 2001; 37(Suppl 1):S21–S30. 14 Kööbi P, Kalliovalkama J, Jolma P, Rysä J, Ruskoaho H, Vuolteenaho O, Kähönen M, Tikkanen I, Fan M, Ylitalo P, Pörsti I: AT1 receptor blockade improves vasorelaxation in experimental renal failure. Hypertension 2003;41:1364–1371. 15 Pörsti I, Fan M, Kööbi P, Jolma P, Kalliovalkama J, Vehmas TI, Helin H, Holthöfer H, Mervaala E, Nyman T, Tikkanen I: High calcium diet down-regulates kidney angiotensin-converting enzyme in experimental renal failure. Kidney Int 2004;66:2155–2166. 16 Arvola P, Pörsti I, Vuorinen P, Pekki A, Vapaatalo H: Contractions induced by potassium-free solution and potassium relaxation in vascular smooth muscle of hypertensive and normotensive rats. Br J Pharmacol 1992; 106: 157–165.
17 Kööbi P, Jolma P, Kalliovalkama J, Tikkanen I, Fan M, Kähönen M, Moilanen E, Pörsti I: Effect of angiotensin II type 1 receptor blockade on conduit artery tone in subtotally nephrectomized rats. Nephron Physiol 2004; 96:p91–p98. 18 Kalliovalkama J, Jolma P, Tolvanen JP, Kähönen M, Hutri-Kähönen N, Saha H, Tuorila S, Moilanen E, Pörsti I: Potassium channel-mediated vasorelaxation is impaired in experimental renal failure. Am J Physiol 1999; 277:H1622–H1629. 19 Braman RS, Hendrix SA: Nanogram nitrite and nitrate determination in environmental and biological materials by vanadium (III) reduction with chemiluminescence detection. Anal Chem 1989;61:2715–2718. 20 Kohzuki M, Johnston CI, Chai SY, Jackson B, Perich R, Paxton D, Mendelsohn FA: Measurement of angiotensin converting enzyme induction and inhibition using quantitative in vitro autoradiography: tissue selective induction after chronic lisinopril treatment. J Hypertens 1991;9:579–587. 21 Bäcklund T, Palojoki E, Grönholm T, Eriksson A, Vuolteenaho O, Laine M, Tikkanen I: Dual inhibition of angiotensin converting enzyme and neutral endopeptidase by omapatrilat in rat in vivo. Pharmacol Res 2001; 44: 411–418. 22 Hatton DC, McCarron DA: Dietary calcium and blood pressure in experimental models of hypertension. A review. Hypertension 1994; 23:513–530.
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1 Marco MP, Craver L, Betriu A, Belart M, Fibla J, Fernandez E: Higher impact of mineral metabolism on cardiovascular mortality in a European hemodialysis population. Kidney Int Suppl 2003:S111–S114. 2 Safar ME, London GM, Plante GE: Arterial stiffness and kidney function. Hypertension 2004;43:163–168. 3 Tyralla K, Amann K: Morphology of the heart and arteries in renal failure. Kidney Int Suppl 2003:S80–S83. 4 Jolma P, Kööbi P, Kalliovalkama J, Saha H, Fan M, Jokihaara J, Moilanen E, Tikkanen I, Pörsti I: Treatment of secondary hyperparathyroidism by high calcium diet is associated with enhanced resistance artery relaxation in experimental renal failure. Nephrol Dial Transplant 2003;18:2560–2569. 5 Eräranta A, Riutta A, Fan M, Koskela J, Tikkanen I, Lakkisto P, Niemelä O, Parkkinen J, Mustonen J, Pörsti I: Dietary phosphate binding and loading alter kidney angiotensin-converting enzyme mRNA and protein content in 5/6 nephrectomized rats. Am J Nephrol 2012;35:401–408. 6 Slatopolsky E, Brown A, Dusso A: Role of phosphorus in the pathogenesis of secondary hyperparathyroidism. Am J Kidney Dis 2001; 37:S54–S57. 7 Amann K, Tyralla K, Gross ML, Eifert T, Adamczak M, Ritz E: Special characteristics of atherosclerosis in chronic renal failure. Clin Nephrol 2003;60(Suppl 1):S13–S21. 8 Navaneethan SD, Palmer SC, Vecchio M, Craig JC, Elder GJ, Strippoli GF: Phosphate binders for preventing and treating bone disease in chronic kidney disease patients. Cochrane Database Syst Rev 2011;2:CD006023.
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28 Rabelink TJ, Koomans HA: Endothelial function and the kidney. An emerging target for cardiovascular therapy. Drugs 1997;53(Suppl 1):11–19. 29 Thuraisingham RC, Roberts NB, Wilkes M, New DI, Mendes-Ribeiro AC, Dodd SM, Yaqoob MM: Altered L-arginine metabolism results in increased nitric oxide release from uraemic endothelial cells. Clin Sci (Lond) 2002;103:31–41. 30 Aiello S, Noris M, Todeschini M, Zappella S, Foglieni C, Benigni A, Corna D, Zoja C, Cavallotti D, Remuzzi G: Renal and systemic nitric oxide synthesis in rats with renal mass reduction. Kidney Int 1997;52:171–181. 31 Thuraisingham RC, Yaqoob MM: Oxidative consumption of nitric oxide: a potential mediator of uremic vascular disease. Kidney Int Suppl 2003;84:S29–S32.
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32 Vaziri ND, Ni Z, Wang XQ, Oveisi F, Zhou XJ: Downregulation of nitric oxide synthase in chronic renal insufficiency: role of excess PTH. Am J Physiol 1998;274:F642–F649. 33 Chabrashvili T, Kitiyakara C, Blau J, Karber A, Aslam S, Welch WJ, Wilcox CS: Effects of ANG II type 1 and 2 receptors on oxidative stress, renal NADPH oxidase, and SOD expression. Am J Physiol Regul Integr Comp Physiol 2003;285:R117–R124. 34 Förstermann U: Nitric oxide and oxidative stress in vascular disease. Pflügers Arch 2010; 459:923–939. 35 Bouloumie A, Bauersachs J, Linz W, Scholkens BA, Wiemer G, Fleming I, Busse R: Endothelial dysfunction coincides with an enhanced nitric oxide synthase expression and superoxide anion production. Hypertension 1997;30: 934–941. 36 Beckman JS, Koppenol WH: Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol 1996;271:C1424– C1437.
Eräranta et al.
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23 Kööbi P, Vehmas TI, Jolma P, Kalliovalkama J, Fan M, Niemelä O, Saha H, Kähönen M, Ylitalo P, Rysä J, Ruskoaho H, Pörsti I: Highcalcium vs high-phosphate intake and small artery tone in advanced experimental renal insufficiency. Nephrol Dial Transplant 2006; 21:2754–2761. 24 Bukoski RD, Kremer D: Calcium-regulating hormones in hypertension: vascular actions. Am J Clin Nutr 1991;54:220S–226S. 25 Bukoski RD: Dietary Ca2+ and blood pressure: evidence that Ca2+-sensing receptor activated, sensory nerve dilator activity couples changes in interstitial Ca2+ with vascular tone. Nephrol Dial Transplant 2001;16:218–221. 26 Joannides R, Richard V, Haefeli WE, Benoist A, Linder L, Luscher TF, Thuillez C: Role of nitric oxide in the regulation of the mechanical properties of peripheral conduit arteries in humans. Hypertension 1997;30:1465–1470. 27 van Guldener C, Lambert J, Janssen MJ, Donker AJ, Stehouwer CD: Endothelium-dependent vasodilatation and distensibility of large arteries in chronic haemodialysis patients. Nephrol Dial Transplant 1997; 12 (Suppl 2):14–18.
Journal of
Nephrology
Am J Nephrol 2014;39:400–408 DOI: 10.1159/000362507
Received: October 4, 2013 Accepted: March 26, 2014 Published online: May 6, 2014
Phosphate Binding Reduces Aortic Angiotensin-Converting Enzyme and Enhances Nitric Oxide Bioactivity in Experimental Renal Insufficiency Arttu Eräranta a Suvi Törmänen a Peeter Kööbi a, b Tuija I. Vehmas a Päivi Lakkisto d, e Ilkka Tikkanen d, e Eeva Moilanen a Onni Niemelä f Jukka Mustonen a, c Ilkka Pörsti a, c a
School of Medicine, University of Tampere, and Departments of b Ophthalmology and c Internal Medicine, Tampere University Hospital, Tampere, d Minerva Institute for Medical Research, and e Department of Medicine, Helsinki University Central Hospital, Helsinki, and f Department of Clinical Chemistry, Etelä-Pohjanmaa Central Hospital Laboratory, Seinäjoki, Finland
Abstract Background: Disturbed calcium-phosphorus metabolism is associated with increased kidney angiotensin-converting enzyme (ACE) in experimental chronic renal insufficiency (CRI). However, information about the effects of phosphate binding and loading on vascular ACE is lacking. Methods: Fifteen weeks after 5/6 nephrectomy (NX), rats were placed on a phosphate-binding (NX+Ca, 3.0% Ca), phosphate-loading (NX+Pi, 1.5% Pi), or control diet for 12 weeks (NX and sham). Results: Aortic ACE, blood pressure, plasma phosphate, and parathyroid hormone were increased in the NX and NX+Pi groups, but were reduced with phosphate binding. Endothelium-mediated relaxations of isolated mesenteric conduit artery rings to acetylcholine were impaired in the NX and NX+Pi groups, but did not differ from sham in NX+Ca rats. Experiments with nitric oxide (NO) synthase inhibition in vitro suggested that the NO-mediated component of acetylcholine
© 2014 S. Karger AG, Basel 0250–8095/14/0395–0400$39.50/0 E-Mail [email protected] www.karger.com/ajn
response was lower in the NX and NX+Pi groups, but did not differ from sham in NX+Ca rats. In all NX groups, aortic endothelial NO synthase (eNOS) was reduced, while plasma and urine concentrations of NO metabolites were increased. Aortic nitrated proteins and calcification were increased in the NX and NX+Pi groups when compared with the NX+Ca and sham groups. Conclusion: Hypertension in the NX model of CRI was associated with reduced vasorelaxation, decreased eNOS, and increased ACE and nitrated proteins in the aorta. Phosphate binding with calcium carbonate enhanced vasorelaxation via endogenous NO and suppressed elevation of ACE and nitrated proteins, suggesting reduced vascular oxidative stress. Our findings support the view that correction of the calcium-phosphorus balance prevents CRI-induced vascular pathophysiology. © 2014 S. Karger AG, Basel
Introduction
In chronic kidney disease, cardiovascular complications are aggravated by increased serum phosphate and parathyroid hormone (PTH) concentrations [1]. ReIlkka Pörsti, MD School of Medicine/Internal Medicine University of Tampere FI–33014 Tampere (Finland) E-Mail ilkka.porsti @ uta.fi
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Key Words Angiotensin-converting enzyme · Calcium · Chronic renal insufficiency · Nitric oxide · Phosphate · Renin-angiotensin system
Methods Animals and Experimental Design We utilized samples from rats previously used for reports elucidating resistance artery function [4] and kidney ACE [5] in CRI. The NX groups underwent removal of the right kidney and surgical resection of the upper and lower poles (2/3) of the left kidney, whereas the sham operation was kidney decapsulation [4, 5, 14, 15]. Diets, anesthesia, antibiotics, postoperative pain relief, and tail-cuff measurement of systolic blood pressure (BP) were as previously described [4, 14, 15].
Phosphate, Aortic ACE, and NO in Renal Insufficiency
A preliminary 12-week study examined whether a calcium carbonate diet influences aortic or cardiac ACE. Four weeks after NX (rat age: 12 weeks), four groups with corresponding systolic BPs and body weights in the sham versus sham+Ca groups and NX versus NX+Ca groups were formed (n = 10–11) [4]. Then for 8 weeks, sham and NX rats ingested 0.3% calcium, while sham+Ca and NX+Ca rats ingested 3% calcium chow (carbonate supplement; AnalyCen, Lidköping, Sweden) [4]. Tissue samples were harvested after 12 study weeks. For the 27-week study, 15 weeks after NX, three groups (n = 13–14) of remnant kidney rats, having equal mean systolic BP, body weight, and plasma creatinine, and sham rats (n = 11) of equal age were put on chow containing 0.3% Ca and 0.5% Pi (NX and sham), 3.0% Ca and 0.5% Pi (NX+Ca), and 0.3% Ca and 1.5% Pi (NX+Pi) (AnalyCen) [5]. These diets represent intakes of low Ca/moderate Pi, high Ca/moderate Pi, and low Ca/high Pi, respectively. The calcium diets were based on our previous experiences of 0.3 versus 3.0% calcium intake in experimental CRI [4, 5, 15]. Then for 12 weeks, body weight and systolic BP were monitored fortnightly. The 24-hour water intake and urine output were measured in metabolic cages and urine samples stored at –80 ° C. From the NX, NX+Pi, and NX+Ca groups, 7, 6, and 1 rats were lost, and the final numbers of animals in these groups were 7, 7, and 12, respectively [5]. At the close of the study, the rats were anesthetized (urethane 1.3 g/kg), and blood samples were drawn from carotid artery with EDTA and heparin as anticoagulants [4, 5, 15]. The main branches of the mesenteric arteries were excised, and the hearts were removed. Tissue samples for ACE, eNOS, and nitrated protein measurements were frozen in isopentane at –40 ° C and stored at –80 ° C. Aortic samples were fixed in 4% formaldehyde for 24 h and embedded in paraffin. 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 Arterial Responses in vitro The mesenteric artery was cleaned and three successive 3-mmlong rings were cut. In proximal rings the endothelium was left intact, while it was denuded from the distal sample [16]. The rings were suspended between hooks in organ bath chambers (20 ml) with physiological salt solution (pH 7.4) containing (in mmol/l): NaCl 119, NaHCO3 25, glucose 11.1, CaCl2 1.6, KCl 4.7, KH2PO4 1.2, and MgSO4 1.2, and aerated with 95% O2/5% CO2. The rings were equilibrated for 2 h at +37 ° C with a preload of 4.905 mN/mm (FT-03 transducer, 7E Polygraph; Grass Instrument Co., Quincy, Mass., USA) [17]. Responses to acetylcholine (ACh) in the absence and presence of 0.1 mmol/l NG-nitro-L-arginine methyl ester (L-NAME) or 1 mmol/l L-arginine, norepinephrine (NE), and Ang II were studied in endothelium-intact rings, and to KCl and nitroprusside in endothelium-denuded rings. All vasorelaxations were studied after precontractions induced by 1 μmol/l NE. Stock solutions of all compounds were freshly prepared and protected from light.
Biochemical Measurements NOx was measured by conversion of nitrite and nitrate to NO [18]. Proteins were precipitated with ethanol (+20 ° C, 2 h), and a 20-μl sample was injected into a cylinder containing vanadium
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duced renal function is associated with large arterial stiffening, increased arterial intima-media thickness, vascular smooth muscle hyperplasia, and medial calcification [2, 3]. In addition, functional and structural changes in resistance arteries have been described in both clinical [3] and experimental chronic renal insufficiency (CRI) [4]. Phosphate retention, hypocalcemia, increased fibroblast growth factor 23, and reduced vitamin D levels lead to the development of secondary hyperparathyroidism already at stages 2 and 3 of CRI [5, 6], and these factors also influence the remodeling of the vascular wall [7]. In order to manage CRI-induced hyperphosphatemia and hyperparathyroidism, oral phosphate binders are frequently needed [8]. Endothelial cells synthesize nitric oxide (NO) from Larginine by constitutive endothelial NO synthase (eNOS) [9]. When compared with small vessels, the contribution of eNOS to vasomotion seems greater in conduit-sized arteries [10, 11]. The interaction between activated reninangiotensin system and bioavailability of endotheliumderived NO is considered an important regulator of vascular tone [12]. A key target for the pharmacological inhibition of the renin-angiotensin system is angiotensinconverting enzyme (ACE), which converts angiotensin I (Ang I) to Ang II and degrades kinins [13]. We recently found that dietary phosphate loading was associated with increased kidney ACE expression and tissue damage in the 5/6 nephrectomy (NX) rat model of CRI [5]. In contrast, phosphate binding with 3.0% calcium carbonate reduced kidney ACE and tissue damage [5]. The present study explored whether phosphate binding and loading influences ACE content in the abdominal aorta and endothelium-dependent vasodilatation in the mesenteric artery. In order to evaluate oxidative stress, nitrated protein and eNOS contents in the abdominal aorta, and NO metabolites (NOx) in plasma and urine were determined. Rats were subjected to NX, followed for 15 weeks, and divided into groups given a control, phosphate-lowering, or high-phosphate diet for 12 weeks.
(III) chloride (VCl3) in HCl (0.8 g VCl3/100 ml of 1 mol/l HCl) at 95 ° C, and the NO formed was measured (NOA 280 analyzer; Sievers Instruments, Boulder, Colo., USA) [19]. Measurements of plasma phosphate, PTH, and ionized calcium were as described [5]. Autoradiography of Tissue ACE Quantitative autoradiography of ACE in vitro was performed on tissue sections (20 μm thick) with the radioligand [125I]MK351A (tyrosyl residue of lisinopril), as described earlier [20, 21]. The optical densities were quantified by an image analysis system (AIDA 2D) coupled to the FUJIFILM BAS-5000 phosphorimager (Tamro, Vantaa, Finland), for a total of 24 analyses per sample. Western Blotting of Aortic NOS and Nitrated Proteins Frozen tissues were homogenized (Ultra-Turrax T25, IKA®Labortechnik, Staufen, Germany) in 400 μl of distilled H2O containing protease inhibitors (CompleteTM Mini EDTA-free, Roche Diagnostics GmbH, Mannheim, Germany). After centrifugation (12,000 g, 15 min, 4 ° C), supernatant protein concentrations were determined (Coomassie PlusTM Protein Assay Kit; Pierce, Rockford, Ill., USA). SDS-PAGE was run on 8% resolving gel and 4% stacking gel, and proteins were transferred to Hybond-ECL nitrocellulose membrane (Amersham Biosciences Ltd., Amersham, UK). The primary antibodies were: mouse eNOS 1: 2,500 dilution (BD Biosciences-Pharmingen, San Diego, Calif., USA), polyclonal rabbit NOS2 1:4,000 dilution (Santa Cruz Biotechnology, Dallas, Tex., USA), and monoclonal mouse nitrotyrosine 1: 3,333 dilution (Cayman Chemical, Ann Arbor, Mich., USA). Antibody binding was detected using SuperSignal® West Pico chemiluminescent substrate (Pierce), and the signal was analyzed using FluorChem 8800 (Alpha Innotech Corporation, San Leandro, Calif., USA).
Aortic Calcification Calcifications were determined from von Kossa-stained samples [5]. The index of calcification for each rat was expressed as a percentage of the calcified area related to the total area of aortic cross-section. Drugs The drugs were: ketamine (Parke-Davis Scandinavia AB, Solna, Sweden), ACh chloride, L-arginine, L-NAME hydrochloride, NE bitartrate, Ang II (Sigma Chemical Co., St. Louis, Mo., USA), and sodium nitroprusside (Fluka Chemie AG, Buchs, Switzerland). Data Presentation and Analysis of Results Contractions were expressed in mN/mm, and EC50 concentrations as the negative logarithm (pD2) values. Vasorelaxation was presented as a percentage of the precontraction. Area under concentration response curve (AUC) analysis was applied to evaluate the contribution of NO to vasorelaxation. Statistical analyses were by one-way analysis of variance (ANOVA), supported by post-hoc Tukey’s test or ANOVA for repeated measurements, as appropriate. If skewed variable distribution was observed, Kruskal-Wallis and post-hoc Mann-Whitney U tests were used. Spearman’s correlations were calculated, as appropriate. Results were presented as means ± SEM, and p < 0.05 was considered significant.
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12-Week Pilot Study Quantitative in vitro autoradiography showed a decrease in aortic and cardiac ACE protein content after 8 weeks of the 3.0% calcium carbonate diet (online suppl. fig. S1; see www.karger.com/doi/10.1159/000362507 for all online suppl. material). The analyses were performed on 8–10 rats randomly selected in each group. 27-Week Study: Animal Data, Plasma, and Urine Determinations At week 15, systolic BP was elevated in all NX groups (table 1). At study week 27, BP was higher in all NX groups than in the sham rats, but BP in the NX-Ca group was lower than in NX and NX+Pi rats. There were no differences in body weight at week 15, but body weights at week 27 were lower in the NX+Ca and NX+Pi groups than in the sham rats, while no significant differences were detected between the NX groups. The heart/body weight ratios were increased, and creatinine clearance was decreased by about 45–55%, corresponding to stage 3 chronic kidney disease, in all NX rats versus sham rats (table 1). Plasma phosphate and PTH were increased in the NX group, and further elevated in the NX+Pi group, while both phosphate and PTH were suppressed to values below those in sham rats in the NX+Ca group. Plasma ionized calcium did not differ from sham in the NX group, but was increased in the NX+Ca, and decreased in the NX+Pi group. Both plasma and urine NOx were increased in all NX groups when compared with sham (table 1). Aortic ACE, eNOS, Nitrated Proteins, and Calcification After 27 study weeks, aortic ACE protein content was higher in the NX and NX+Pi groups than in the sham rats. Notably, aortic ACE content was clearly lower in the NX+Ca group when compared with the NX and NX+Pi groups (fig. 1a, b). Aortic eNOS protein content was similarly decreased in all NX groups when compared with sham rats (fig. 2a). However, no difference was detected in aortic inducible NOS protein between the study groups, with average values ranging ±18% of the level observed in sham rats (not shown). Aortic nitrated protein content was higher in the NX and NX+Pi groups versus sham, but was lower in the NX+Ca than in NX and NX+Pi rats (fig. 2b). The index of aortic calcification was increased in the NX and NX+Pi groups when compared with the NX+Ca and sham groups (fig. 2c, online suppl. fig. S2). Eräranta et al.
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Fig. 1. Representative original tracings (a) and mean ± SEM plots (b) of aortic ACE content determined using
autoradiography; n = 8–10 in each group. * p < 0.05 vs. sham; † p < 0.05 vs. NX; ‡ p < 0.05 vs. NX+Pi.
Table 1. Experimental animal data and laboratory findings in the experimental groups
Sham Systolic BP At week 15, mm Hg At week 27, mm Hg Body weight at week 15, g Body weight at week 27, g Heart weight/body weight, g/kg Creatinine clearance week 27, μmol/ml/100 g Plasma Phosphate, mmol/l PTH, pg/ml Ionized calcium, mmol/l NOx, μmol/l Urine NOx, μmol/24 h
NX
NX+Ca
133 ± 2 129 ± 2 499 ± 8 557 ± 7 3.21 ± 0.03 327 ± 19
154 ± 6* 173 ± 4* 476 ± 9 507 ± 38 4.19 ± 0.43* 167 ± 34*
153 ± 4* 145 ± 3*, † 478 ± 9 488 ± 13* 4.03 ± 0.13* 177 ± 16*
1.16 ± 0.06 88 ± 12 1.36 ± 0.06 8.9 ± 0.4
2.52 ± 0.46* 1,172 ± 343* 1.34 ± 0.03 11.4 ± 0.9*
0.81 ± 0.08*, † 3.7 ± 0.5*, † 1.55 ± 0.03*, † 14.2 ± 5.1*
0.03 ± 0.01
1.36 ± 0.66*
1.49 ± 0.84*
NX+Pi 152 ± 4* 161 ± 4* 484 ± 11 431 ± 35* 4.46 ± 0.40* 145 ± 29* 5.47 ± 1.12*, †, ‡ 3,620 ± 236*, †, ‡ 0.93 ± 0.09*, †, ‡ 18.4 ± 5.1* 0.80 ± 0.36*
Endothelium-Dependent and -Independent Vasorelaxation The relaxations induced by ACh in endothelium-intact mesenteric artery rings were impaired in the NX and NX+Pi groups, but did not differ from sham rats in the NX+Ca group (fig. 3a). The NOS inhibitor L-NAME reduced the relaxations to ACh in all groups, but the effect was more pronounced in the NX+Ca than in the NX and
NX+Pi groups, as the ACh response did not differ between the NX groups in the presence of L-NAME (fig. 3b). The change in the AUC of the ACh-response induced by L-NAME suggested that the contribution of NO to the relaxation was reduced in the NX and NX+Pi groups, but did not differ from sham rats in the NX+Ca group (fig. 3c). Both aortic nitrated protein content and index of calcification correlated inversely with maximal ACh-induced
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Values are means ± SEM; n = 7–14 for all groups. * p < 0.05 vs. sham; † p < 0.05 vs. NX; ‡ p < 0.05 vs. NX+Ca.
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and index of aortic calcification (percent of cross-section) determined using a von Kossa stain (c). Means ± SEM; n = 7–11 in each group; intensity of white bands represents binding to specific antibody (a, b). * p < 0.05 vs. sham; † p < 0.05 vs. NX; ‡ p < 0.05 vs. NX+Pi.
relaxation (r = –0.438, p = 0.002; r = –0.637, p < 0.001, respectively). The addition of exogenous L-arginine (1 mmol/l) had no significant effects on the ACh-induced relaxations (data not shown). Vasorelaxation to the NO donor nitroprusside in endothelium-denuded rings was slightly decreased in the NX+Pi group when compared with sham and NX+Ca rats, but did not differ between the sham, NX, and NX+Ca groups (fig. 3d). Vasoconstrictor Responses Vasoconstrictor sensitivity and maximal responses of mesenteric arterial rings to NE did not differ between the sham, NX, and NX+Ca groups, while sensitivity to NE was slightly higher in the NX+Pi than the sham and NX+Ca groups, and maximal response was lower in the 404
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c
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NX+Pi than sham group (online suppl. table S1). The sensitivity and maximal responses to KCl did not differ between the study groups. Vasoconstrictor sensitivity to Ang II was lower in the NX+Ca than sham group, while maximal wall tension in response to Ang II was higher in the NX versus sham group, and lower in the NX+Ca versus NX group (online suppl. table S1).
Discussion
This study investigated the effects of dietary phosphate binding and loading on aortic ACE protein, eNOS content, and calcification, and vascular tone in vitro of isolated mesenteric arterial rings in experimental CRI. In the Eräranta et al.
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Fig. 2. Bar graphs and representative bands of aortic eNOS protein (a), and nitrated proteins (b), determined using Western blotting,
Calcification (% of aortic cross-section)
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27-week study, elevated systolic BP, decreased creatinine clearance, and increased heart/body weight were seen in all NX groups (table 1). However, the phosphate-lowering diet blunted the increase in BP, while phosphate loading did not influence BP. In experimental studies, the lowering of BP after a high-calcium diet has been attributed to increased natriuresis, reduced sympathetic activity, suppressed PTH levels, and enhanced vasodilatation [4, 15, 22–25]. The present results suggest that reduction of tissue ACE might also play a role in the lowering of BP. Abnormal calcium, phosphate, and PTH metabolism are known causes for increased cardiovascular calcification in CRI [1, 3]. Effective phosphate control is a culprit
in the prevention of cardiovascular problems in CRI, and several calcium-based and noncalcium-containing phosphate binders are available for this purpose. Concern has been raised that high intake of calcium salts may predispose to calcifications in CRI, but the superiority of noncalcium-containing phosphate binders over calciumbased binders remains unclear [8]. The present experimental results apply to calcium-based phosphate binding, and show that suppression of PTH and phosphate to levels below those in sham rats was associated with reduction in aortic calcifications. Due to reduced survival in the NX and NX+Pi groups [5], samples from the most uremic rats were not available for laboratory analyses, and the
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Fig. 3. Relaxations to ACh in endothelium-intact mesenteric arterial rings in the absence (a) and presence (b) of 0.1 mmol/l L-NAME. c Bar graph showing the change induced by NOS inhibition with L-NAME in the AUC (arbitrary units) of the ACh response. d Relaxation to nitroprusside in endothelium-denuded mesenteric arterial rings. Means ± SEM; n = 7–11 in each group. * p < 0.05, ANOVA for repeated measurements (a, b, d); * p < 0.05 vs. sham; † p < 0.05 vs. NX; ‡ p < 0.05 vs. NX+Pi (c), one-way ANOVA.
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increases superoxide release [34]. Increased oxidative stress is considered as one of the culprits leading to the loss of NO-mediated vasodilatation [34]. When superoxide reacts with NO, peroxynitrite is formed at a reaction rate that exceeds the scavenging of superoxide by superoxide dismutase [31, 35, 36]. Peroxynitrite can modify tyrosine residues in proteins creating nitrotyrosine, thus leaving a footprint of oxidative stress in tissues [36]. Here we found that the aortic nitrated protein levels were increased in the NX and NX+Pi groups, but were reduced in the NX+Ca group, suggesting that the calcium carbonate diet reduced oxidative stress. One mechanism leading to reduced Ang II and superoxide generation could be reduced arterial tissue ACE content, and this could also increase the bioactivity of endothelium-derived NO. NO has a short half-life, and NOx determination was used to evaluate NO production in vivo. Plasma NOx concentration was increased in NX rats, in agreement with previous results [30, 31]. Urine NOx was also increased in all NX groups. Elevated NOx is considered to reflect oxidative stress and inflammation in CRI [29, 31]. The source of NOx remains unknown, as we detected reduced eNOS and no differences in abdominal aortic inducible NOS content after 27 weeks of CRI. Previously, studies examining NO release have shown increased production, whereas those examining NO bioactivity have shown attenuated responses in CRI, and even endothelial cells cultured with uremic plasma in vitro have shown increased NO release [31]. Thus, elevated plasma NOx could result from the effects of a uremic milieu on NOS activity in vivo, while reduced NO bioactivity in uremia has been related to excess NO consumption due to oxidative stress [31]. Elevated NOx could also result from the increased oxidative stress in CRI since peroxynitrite can break down to form NOx [29]. The arterial contractions were examined to uncover possible differences in vasoconstrictor sensitivity that would interfere with the interpretation of relaxation experiments. No differences were found in responses to KCl, and contractions elicited by NE were corresponding in the sham, NX, and NX+Ca groups. Therefore, alterations in vasoconstrictor sensitivity did not explain enhanced vasorelaxation in NX+Ca rats when compared with NX rats. The NX+Pi group exhibited higher sensitivity to NE when compared with sham rats, and increased vasoconstrictor sensitivity may partially explain impaired vasodilator response to exogenous NO after phosphate loading. Finally, mesenteric arterial rings in the NX group exhibited increased maximal wall tension to Ang II, while the response in the NX+Ca group did not differ from the Eräranta et al.
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results may have underestimated the extent of tissue damage and decline of renal function in these groups. Previously, we found that the calcium-phosphorus balance influences ACE content in kidney tissue [5, 15], and the results of the 12-week study showed that the calcium diet reduced ACE in the heart and aorta. In the 27week study, aortic ACE protein was decreased after 12 weeks of an oral calcium carbonate diet. In our earlier study, the correlation between ACE protein and ACE mRNA content in kidney tissue was good (r = 0.83) [5], suggesting that changes in tissue ACE are explained by alterations at the level of gene expression. In contrast to our previous results on kidney ACE, the content of ACE in the aorta was not increased after phosphate loading [5]. This may be attributed to the calcifications in the aortas of NX+Pi rats, as calcified tissue sections would not contain ACE to bind the autoradiography tracer. Deficient endothelium-mediated vasodilatation is common in CRI [23, 26–28]. Previously, we observed that the correction of calcium, phosphate, and PTH levels in NX rats improved endothelium-mediated vasorelaxation via K+-channels and NO in small 2nd- and 3rd-order mesenteric arterial branches [4]. In the present study, we found that phosphate binding normalized impaired relaxation to ACh in a conduit-sized artery. Lower BP in the NX+Ca than in other NX groups probably contributed to the improved vasorelaxation. As the improved response was inhibited by the NOS inhibitor L-NAME, and the change in AUC induced by L-NAME was more pronounced in the NX+Ca than NX and NX+Pi groups, the beneficial effect could be attributed to enhanced NO-mediated response. The contribution of endothelium-derived NO to vasorelaxation seemed corresponding in the NX and NX+Pi groups. Enhanced vasorelaxation after calcium carbonate was not explained by changes in NO sensitivity in arterial smooth muscle, as the response to nitroprusside did not differ in the sham, NX, and NX+Ca groups. Many studies have examined the effect of CRI on Larginine metabolism and the functional role of the NOS enzymes with contradictory results [29–32]. Here we found that eNOS content in the abdominal aorta was equally reduced in all NX groups. Thus, enhanced endothelium-mediated vasorelaxation in the Ca+NX group was not explained by changes in the quantity of eNOS protein. The major effector peptide of the renin-angiotensin system, Ang II, can reduce NO bioactivity through increased production of superoxide radicals, which rapidly inactivate NO [13, 33]. At the molecular level, Ang II upregulates and activates vascular NADPH oxidases, and
sham rats, and contractile sensitivity to Ang II was even lower in NX+Ca than sham rats. This implies that the calcium carbonate diet reduced Ang II-mediated responses in arterial tissue even at the level of the AT1 receptor or its signal transduction cascade. In conclusion, elevated BP in experimental stage 3 chronic kidney disease was associated with impaired vasorelaxation via endothelium-derived NO, and decreased eNOS and increased ACE and nitrated proteins in the aorta, and elevated levels of plasma and urine NOx. Phosphate binding with 3.0% calcium carbonate reduced aortic ACE and nitrated proteins, and enhanced vasorelaxation via increased endothelium-derived NO bioactivity. Thus, reduction of plasma phosphate to levels below those in sham-operated controls with oral calcium carbonate beneficially influenced vascular pathophysiology in experimental CRI.
Acknowledgments The excellent technical assistance of Marja-Leena Koskinen, Riina Hatakka, and Jarkko Lakkisto is sincerely acknowledged. This study was supported by Finnish Kidney Foundation, Finnish Foundation for Cardiovascular Research, Competitive State Research Financing of the Expert Responsibility Area of Tampere University Hospital, Paavo Nurmi Foundation, Emil Aaltonen Foundation, Pirkanmaa Regional Fund of the Finnish Cultural Foundation, Sigrid Jusélius Foundation, and Competitive Research Funding of the Hospital District of Helsinki and Uusimaa.
Disclosure Statement The authors declare no conflicts of interest.
References
Phosphate, Aortic ACE, and NO in Renal Insufficiency
9 Moncada S, Higgs A: The L-arginine-nitric oxide pathway. N Engl J Med 1993;329:2002– 2012. 10 Geiger M, Stone A, Mason SN, Oldham KT, Guice KS: Differential nitric oxide production by microvascular and macrovascular endothelial cells. Am J Physiol 1997;273:L275–L281. 11 Garland CJ, Plane F, Kemp BK, Cocks TM: Endothelium-dependent hyperpolarization: a role in the control of vascular tone. Trends Pharmacol Sci 1995;16:23–30. 12 Raij L: Workshop: hypertension and cardiovascular risk factors: role of the angiotensin II-nitric oxide interaction. Hypertension 2001;37:767–773. 13 Enseleit F, Hurlimann D, Luscher TF: Vascular protective effects of angiotensin converting enzyme inhibitors and their relation to clinical events. J Cardiovasc Pharmacol 2001; 37(Suppl 1):S21–S30. 14 Kööbi P, Kalliovalkama J, Jolma P, Rysä J, Ruskoaho H, Vuolteenaho O, Kähönen M, Tikkanen I, Fan M, Ylitalo P, Pörsti I: AT1 receptor blockade improves vasorelaxation in experimental renal failure. Hypertension 2003;41:1364–1371. 15 Pörsti I, Fan M, Kööbi P, Jolma P, Kalliovalkama J, Vehmas TI, Helin H, Holthöfer H, Mervaala E, Nyman T, Tikkanen I: High calcium diet down-regulates kidney angiotensin-converting enzyme in experimental renal failure. Kidney Int 2004;66:2155–2166. 16 Arvola P, Pörsti I, Vuorinen P, Pekki A, Vapaatalo H: Contractions induced by potassium-free solution and potassium relaxation in vascular smooth muscle of hypertensive and normotensive rats. Br J Pharmacol 1992; 106: 157–165.
17 Kööbi P, Jolma P, Kalliovalkama J, Tikkanen I, Fan M, Kähönen M, Moilanen E, Pörsti I: Effect of angiotensin II type 1 receptor blockade on conduit artery tone in subtotally nephrectomized rats. Nephron Physiol 2004; 96:p91–p98. 18 Kalliovalkama J, Jolma P, Tolvanen JP, Kähönen M, Hutri-Kähönen N, Saha H, Tuorila S, Moilanen E, Pörsti I: Potassium channel-mediated vasorelaxation is impaired in experimental renal failure. Am J Physiol 1999; 277:H1622–H1629. 19 Braman RS, Hendrix SA: Nanogram nitrite and nitrate determination in environmental and biological materials by vanadium (III) reduction with chemiluminescence detection. Anal Chem 1989;61:2715–2718. 20 Kohzuki M, Johnston CI, Chai SY, Jackson B, Perich R, Paxton D, Mendelsohn FA: Measurement of angiotensin converting enzyme induction and inhibition using quantitative in vitro autoradiography: tissue selective induction after chronic lisinopril treatment. J Hypertens 1991;9:579–587. 21 Bäcklund T, Palojoki E, Grönholm T, Eriksson A, Vuolteenaho O, Laine M, Tikkanen I: Dual inhibition of angiotensin converting enzyme and neutral endopeptidase by omapatrilat in rat in vivo. Pharmacol Res 2001; 44: 411–418. 22 Hatton DC, McCarron DA: Dietary calcium and blood pressure in experimental models of hypertension. A review. Hypertension 1994; 23:513–530.
Am J Nephrol 2014;39:400–408 DOI: 10.1159/000362507
407
Downloaded by: Duke Medical Center Library 152.3.102.254 - 11/28/2017 7:52:54 AM
1 Marco MP, Craver L, Betriu A, Belart M, Fibla J, Fernandez E: Higher impact of mineral metabolism on cardiovascular mortality in a European hemodialysis population. Kidney Int Suppl 2003:S111–S114. 2 Safar ME, London GM, Plante GE: Arterial stiffness and kidney function. Hypertension 2004;43:163–168. 3 Tyralla K, Amann K: Morphology of the heart and arteries in renal failure. Kidney Int Suppl 2003:S80–S83. 4 Jolma P, Kööbi P, Kalliovalkama J, Saha H, Fan M, Jokihaara J, Moilanen E, Tikkanen I, Pörsti I: Treatment of secondary hyperparathyroidism by high calcium diet is associated with enhanced resistance artery relaxation in experimental renal failure. Nephrol Dial Transplant 2003;18:2560–2569. 5 Eräranta A, Riutta A, Fan M, Koskela J, Tikkanen I, Lakkisto P, Niemelä O, Parkkinen J, Mustonen J, Pörsti I: Dietary phosphate binding and loading alter kidney angiotensin-converting enzyme mRNA and protein content in 5/6 nephrectomized rats. Am J Nephrol 2012;35:401–408. 6 Slatopolsky E, Brown A, Dusso A: Role of phosphorus in the pathogenesis of secondary hyperparathyroidism. Am J Kidney Dis 2001; 37:S54–S57. 7 Amann K, Tyralla K, Gross ML, Eifert T, Adamczak M, Ritz E: Special characteristics of atherosclerosis in chronic renal failure. Clin Nephrol 2003;60(Suppl 1):S13–S21. 8 Navaneethan SD, Palmer SC, Vecchio M, Craig JC, Elder GJ, Strippoli GF: Phosphate binders for preventing and treating bone disease in chronic kidney disease patients. Cochrane Database Syst Rev 2011;2:CD006023.
408
28 Rabelink TJ, Koomans HA: Endothelial function and the kidney. An emerging target for cardiovascular therapy. Drugs 1997;53(Suppl 1):11–19. 29 Thuraisingham RC, Roberts NB, Wilkes M, New DI, Mendes-Ribeiro AC, Dodd SM, Yaqoob MM: Altered L-arginine metabolism results in increased nitric oxide release from uraemic endothelial cells. Clin Sci (Lond) 2002;103:31–41. 30 Aiello S, Noris M, Todeschini M, Zappella S, Foglieni C, Benigni A, Corna D, Zoja C, Cavallotti D, Remuzzi G: Renal and systemic nitric oxide synthesis in rats with renal mass reduction. Kidney Int 1997;52:171–181. 31 Thuraisingham RC, Yaqoob MM: Oxidative consumption of nitric oxide: a potential mediator of uremic vascular disease. Kidney Int Suppl 2003;84:S29–S32.
Am J Nephrol 2014;39:400–408 DOI: 10.1159/000362507
32 Vaziri ND, Ni Z, Wang XQ, Oveisi F, Zhou XJ: Downregulation of nitric oxide synthase in chronic renal insufficiency: role of excess PTH. Am J Physiol 1998;274:F642–F649. 33 Chabrashvili T, Kitiyakara C, Blau J, Karber A, Aslam S, Welch WJ, Wilcox CS: Effects of ANG II type 1 and 2 receptors on oxidative stress, renal NADPH oxidase, and SOD expression. Am J Physiol Regul Integr Comp Physiol 2003;285:R117–R124. 34 Förstermann U: Nitric oxide and oxidative stress in vascular disease. Pflügers Arch 2010; 459:923–939. 35 Bouloumie A, Bauersachs J, Linz W, Scholkens BA, Wiemer G, Fleming I, Busse R: Endothelial dysfunction coincides with an enhanced nitric oxide synthase expression and superoxide anion production. Hypertension 1997;30: 934–941. 36 Beckman JS, Koppenol WH: Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol 1996;271:C1424– C1437.
Eräranta et al.
Downloaded by: Duke Medical Center Library 152.3.102.254 - 11/28/2017 7:52:54 AM
23 Kööbi P, Vehmas TI, Jolma P, Kalliovalkama J, Fan M, Niemelä O, Saha H, Kähönen M, Ylitalo P, Rysä J, Ruskoaho H, Pörsti I: Highcalcium vs high-phosphate intake and small artery tone in advanced experimental renal insufficiency. Nephrol Dial Transplant 2006; 21:2754–2761. 24 Bukoski RD, Kremer D: Calcium-regulating hormones in hypertension: vascular actions. Am J Clin Nutr 1991;54:220S–226S. 25 Bukoski RD: Dietary Ca2+ and blood pressure: evidence that Ca2+-sensing receptor activated, sensory nerve dilator activity couples changes in interstitial Ca2+ with vascular tone. Nephrol Dial Transplant 2001;16:218–221. 26 Joannides R, Richard V, Haefeli WE, Benoist A, Linder L, Luscher TF, Thuillez C: Role of nitric oxide in the regulation of the mechanical properties of peripheral conduit arteries in humans. Hypertension 1997;30:1465–1470. 27 van Guldener C, Lambert J, Janssen MJ, Donker AJ, Stehouwer CD: Endothelium-dependent vasodilatation and distensibility of large arteries in chronic haemodialysis patients. Nephrol Dial Transplant 1997; 12 (Suppl 2):14–18.
