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Ankle-Brachial Index and Bone Turnover in Patients on Dialysis Gérard M. London,*† Sylvain J. Marchais,* Alain P. Guérin,† and Marie-Christine de Vernejoul‡ *Department of Nephrology, Manhès Hospital, Fleury Mérogis, France; †Department of Pharmacology, Georges Pompidou European Hospital, National Institute of Health and Medical Research U970, Paris, France; and ‡ Lariboisiere Hospital, Vigot Petersen Centre, National Institute of Health and Medical Research U606, University Paris 7, Paris, France
ABSTRACT An association between atherosclerosis and osteoporosis has been reported in several studies. This association could result from local intraosseous atherosclerosis and ischemia, which is shown by limb osteoporosis in patients with peripheral artery disease (PAD), but also could result from bidirectional communication between the skeleton and blood vessels. Systemic bone disorders and PAD are frequent in ESRD. Here, we investigated the possible interaction of these disorders. For 65 prevalent nondiabetic patients on hemodialysis, we measured ankle-brachial pressure index (ABix) and evaluated mineral and bone disorders with bone histomorphometry. In prevalent patients on hemodialysis, PAD (ABix,0.9 or .1.4/incompressible) was associated with low bone turnover and pronounced osteoblast resistance to parathyroid hormone (PTH), which is indicated by decreased double-labeled surface and osteoblast surface (P,0.001). Higher osteoblast resistance to PTH in patients with PAD was characterized by weaker correlation coefficients (slopes) between serum PTH and double-labeled surface (P=0.02) or osteoblast surface (P=0.03). The correlations between osteoclast number or eroded surface and serum mineral parameters, including PTH, did not differ for subjects with normal ABix and PAD. Common vascular risk factors (dyslipidemia, smoking, and sex) were similar for normal, low, and incompressible ABix. Patients with PAD were older and had high C-reactive protein levels and longer hemodialysis vintage. These results indicate that, in prevalent nondiabetic patients with ESRD, PAD associates with low bone turnover and pronounced osteoblast resistance to PTH. J Am Soc Nephrol 26: ccc–ccc, 2014. doi: 10.1681/ASN.2014020169
The bone–vascular axis concept emerged from clinical and experimental findings linking bone and arterial remodeling and changes.1 Observations in diverse populations have revealed associations between atherosclerosis and vascular calcifications with decreased bone mineral density (BMD), risk for vertebral or hip fracture, low bone turnover, and cardiovascular disease.2–8 The biologic link between vascular disease and bone changes is certainly part of the aging process, but in many studies, these bone–vessel associations remained significant after adjustment for age, suggesting an age-independent causal relationship. Nevertheless, the factors or mechanisms underlying these associations are not well understood and could result from common J Am Soc Nephrol 26: ccc–ccc, 2014
mechanisms acting on both systems (e.g., inflammation, hyperlipidemia, diabetes, smoking,9–12 or generalized arterial disease per se). Blood vessels play a major role in osteogenesis, and factors affecting the blood supply to bones could have major effects on bone remodeling and structure.13–15
Received February 12, 2014. Accepted May 27, 2014. Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Gérard M. London, Manhes Hospital, 8, rue Roger-Clavier, 91712, Fleury Mérogis Cedex, France. Email: [email protected] Copyright © 2014 by the American Society of Nephrology
ISSN : 1046-6673/2602-ccc
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Ischemia resulting from intraosseous atherosclerosis and arterial calcifications could explain the association between osteoporosis and decreased BMD. The link between compromised bone circulation and osteoporosis was documented by the observation linking lower limb bone mineral content and abnormal bone turnover with increased risk of fractures in men with ischemic atherosclerotic peripheral artery disease (PAD).16–19 Lower limb PAD is a frequent complication of ESRD, and low ankle-brachial pressure index (ABix) or incompressible arteries are associated with all-cause and cardiovascular mortality.20–23 In general populations, associations between PAD and BMD are observed at the local (lower limb) level and frequently, in the absence of overt mineral metabolism disorders; in patients with CKD and ESRD, those interactions coexist frequently with mineral and bone disorders, including perturbed parathyroid hormone (PTH) and vitamin D metabolisms. In this study, we had the opportunity to analyze the relationships between ABix-assessed subclinical PAD and mineral and bone abnormalities, including systemic bone, on the basis of histomorphometry of iliac crest biopsies. These results indicate that, in prevalent nondiabetic patients with ESRD, PAD was associated with low bone turnover and pronounced osteoblast resistance to PTH.
RESULTS Patient Characteristics
We studied 65 patients whose clinical characteristics, brachial and lower limb systolic BP (SBP), and ABix are summarized in Table 1. ABix was normal in 30 of 65 (46.2%) patients, ABix was low in 11 (16.9%) patients, and ABix.1.4 or incompressible arteries were present in 24 (36.9%) patients. Patients with abnormal ABix were older. Peripheral artery calcifications were present in all subjects with pathologic ABix and 25.7% of patients whose ABix was normal. Hemodialysis vintage, the proportion of patients with previous parathyroidectomy (PTX), and the calcium phosphate binders prescribed dose
were higher in patients with low ABix or incompressible arteries. Blood chemistries are reported in Table 2 according to ABix. Patients with PAD had significantly higher serum high-sensitivity C-reactive protein (hCRP) and lower serum albumin. All others biochemical parameters, including PTH, were not significantly different among the three groups. Bone Histomorphometry
Bone histomorphometry parameters are given in Table 3. Osteoblast surface per bone surface (Ob.S/BS) and doublelabeled surface (dLS/BS) were significantly lower in patients with PAD. Patients with PAD had higher aluminum-stained surface but similar bone volume and osteoid volume. In multiple regression analyses, dLS/BS was positively associated with serum PTH (P,0.001), and it was negatively associated with serum hCRP (P,0.001) but not negatively associated with aluminum-stained surfaces (P=0.27). As shown in Figure 1 in pooled abnormal ABix, after adjustment for age and serum hCRP, the correlations between serum PTH, dLS/BS, and Ob.S/ BS were positive for normal and abnormal ABix (Figure 1). Upward shifts with significantly steeper slopes (b-coefficient) of the correlation between dLS/BS and PTH (mean b-coefficient6SD; 21624.4 versus 5.12630.3; T=2.34; P=0.03) and between Ob.S/BS and PTH (mean b-coefficient6SD; 17618.6 versus 8.4610.2; T=2.25; P=0.03) were observed for patients with normal ABix. Osteoclast number/per millimeter2 (N.Oc/T.A) and eroded surface (ES/BS) did not differ among the three groups (Figure 1). The correlations between circulating PTH, N.Oc/T.A, and ES/BS, were positive and similar for normal and abnormal ABix. Significant negative serum PTH-adjusted correlations were observed between serum hCRP, Ob.S/BS, and dLS/BS (Figure 2). Corrected univariate correlations showed that PAD was positively associated with serum CRP, age, dialysis vintage, and calcium carbonate dose and inversely related to dLS/BS, Ob.S/ BS, aluminum-stained surfaces, and PTX (Table 4). Because of multiple colinearities (for example, correlation between vintage and PTX: R=0.3615; P=0.004 or correlation between
Table 1. Clinical characteristics of patients with ESRD Characteristic Age (yr) Body mass index (kg/m2) Vintage (mo) Smoking (pack/yr) PTX (no.) CaCO3 (g/d) Brachial SBP (mmHg) Lower limb SBP (mmHg) ABix
ABix Normal (n=30)
Low (n=11)
>1.4 (n=24)
44 (29 to 50) 22.6 (21.3 to 24.1) 48 (29 to 65) 0 (0 to 3) 4/30 (13.3%) 1.2 (0.8 to 1.2) 147 (134 to 164) 175 (164 to 185) 1.14 (1.10 to 1.16)
59a (52 to 65) 20.9 (17.1 to 27.9) 85a (36 to 104) 0 (0 to 25) 5/11a (45.5%) 1.8 (1.4 to 2.4) 147 (131 to 156) 126a (104 to 139) 0.86a (0.65 to 0.89)
59a (52 to 64) 22.5 (20.3 to 23.5) 144a (96 to 173) 5.5 (0 to 16) 13/24a (54.2%) 2.4b (2 to 2.4) 156 (138 to 168) 300a (250 to 300) 1.76a (1.52 to 1.9)
ANOVA P Value ,0.001 NS ,0.001 NS 0.004 ,0.001 NS ,0.001 ,0.001
Values are medians (95% confidence intervals). Kruskal–Wallis one-way ANOVA on ranks. a Significantly different from normal ABix. b Significantly different from low ABix and ABix.1.4.
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Table 2. Blood chemistries of patients with ESRD according to ABix ABix
Parameter Cholesterol (mmol/L) Triglycerides (mmol/L) Uric acid (mmol/L) Glycemia (mmol/L) Serum Ca (mmol/L) Serum ionized Ca (mmol/L) Serum phosphate (mmol/L) Serum Mg (mmol/L) Serum PTH (pg/mL) Serum 25(OH) cholecalciferol (mg/L) Serum 1,25 (OH)2D3 (ng/L) Bone alkaline phosphatase (mg/L) Serum albumin (g/L) Hemoglobin (mmol/L) hCRP (mg/L)
Normal (n=30)
Low (n=11)
>1.4 (n=24)
5.03 (4.32 to 5.5) 1.52 (1.37 to 1.87) 309 (260 to 339) 4.88 (4.61 to 4.98) 2.45 (2.36 to 2.49) 1.23 (1.19 to 1.26) 1.84 (1.62 to 2.0) 1.15 (1.07 to 1.2) 322 (227 to 406) 21.35 (15.4 to 30.45) 18 (13 to 20) 25 (20 to 30) 39.9 (38.6 to 41) 10 (9.5 to 10.5) 3 (3 to 5)
5.8 (5.06 to 7.75) 1.7 (1.5 to 2.68) 313 (141 to 410) 5.03 (4.61 to 5.74) 2.43 (2.34 to 2.52) 1.23 (1.17 to 1.24) 1.79 (1.25 to 2.84) 1.19 (1 to 1.31) 215 (121 to 467) 17.85 (11.9 to 28.7) 12.8 (8 to 18) 17 (12 to 25) 37a (35 to 38.8) 9.7 (8.2 to 10.2) 10a (7 to 12)
4.97 (4.7 to 6) 1.63 (1.16 to 2.02) 340 (285 to 369) 5.02 (4.73 to 5.29) 2.46 (2.35 to 2.48) 1.22 (1.18 to 1.24) 1.98 (1.76 to 2.08) 1.17 (1.08 to 1.21) 212 (94 to 345) 19.8 (12.2 to 24.2) 16 (11.4 to 20.6) 18.5 (14 to 25) 37a (36 to 38.6) 10.9 (8.9 to 11.4) 8a (7 to 11)
ANOVA P Value NS NS NS NS NS NS NS NS NS NS NS (0.07) NS (0.06) ,0.001 NS ,0.001
Values are medians (95% confidence intervals). Kruskal–Wallis one-way ANOVA on ranks. a Statistically different from normal ABix.
Table 3. Bone histomorphometry Surfaces Ob.S/BS (%) dLS/BS (%) N.Oc/T.A (mm2) ES/BS (%) BV/TV (%) OV/BV (%) Aluminum-stained surface (%)
ABix Normal (n=30)
Low (n=11)
>1.4 (n=24)
11.81 (9.12 to 16.33) 10.52 (5.6 to 18.5) 1.42 (1 to 2.50) 2.58 (1.52 to 3.40) 21.01 (16 to 22.3) 8.76 (5.8 to 11.4) 5 (7.5 to 28.4)
2.42a (0.89 to 7.5) 1.33a (0 to 2.40) 0.40b (0.1 to 1) 0.48 (0 to 1.4) 19.23 (11.2 to 23.9) 7.80 (2.45 to 11.1) 48a (20 to 91)
2.66a (0.3 to 6.3) 1a (0 to 3.20) 0.80 (0.15 to 1.62) 1.38 (0.1 to 2.61) 15.8 (12.66 to 20.7) 6.15 (2.7 to 10.8) 48a (5 to 75)
ANOVA P Value 0.002 ,0.001 0.01 NS NS NS ,0.01
Values are medians (95% confidence intervals). Kruskal–Wallis one-way ANOVA on ranks with multiple comparison Z-value test. BV/TV, bone volume; OV/BV, osteoid volume. a Significantly different from normal ABix. b Significantly different from low ABix and ABix.1.4.
vintage and aluminum-stained surface: R=0.5365; P,0.001), multivariate stepwise correlation analysis identified only hCRP, vintage, and dLS/BS as significant cofactors associated with PAD (Table 5).
DISCUSSION
Our results indicated that, in patients with prevalent hemodialysis, subclinical PAD was associated with low bone turnover and more pronounced osteoblast resistance to PTH, which was assessed by decreased dLS/BS and Ob.S/BS and lower correlation b-coefficients (slopes) between serum PTH and dLS/BS or Ob.S/BS (Figure 1, A and B). The correlations between PAD and N.Oc/T.A or ES/BS did not differ from subjects with normal ABix. These bone–artery associations remained significant after adjustment for age, serum hCRP, and hemodialysis vintage. Although hemodialysis vintage and serum hCRP were independently associated with PAD, serum mineral parameters, J Am Soc Nephrol 26: ccc–ccc, 2014
including PTH, did not differ significantly between groups. Unlike the published association between the calcium phosphate binders doses and aortic calcifications,8 in multivariate analyses, this association was not observed in peripheral arteries with abnormal ABix. Decreased femoral neck or total hip BMD and increased risk of fractures were reported to be associated with PAD,16,18,19 and bone perfusion was reduced in osteoporotic patients compared with normal and osteopenic subjects.15 In a study on patients with unilateral PAD, Laroche et al.16 reported that BMD was significantly lower in the affected limb compared with the unaffected limb. Unlike the reported association between PAD and local femoral BMD, our results indicated an association between PAD and systemic bone changes in nonweight-bearing anterior iliac crest. Our study population did not include patients with diabetes, and the only common bone–artery risk factor was higher hCRP in patients with abnormal ABix (Tables 2 and 5). In an experimental in vivo study, Hjortnaes et al.24 showed Vascular and Skeletal Disorders in ESRD
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Figure 1. Correlations between serum PTH and bone histomorphometry parameters. (A) Ob.S/BS, (B) dLS/BS, (C) N.Oc/T.A, and (D) ES/BS for patients with normal ABix (white circles) and patients with abnormal ABix (black triangles). The correlations are adjusted to serum CRP.
the role of inflammation in the inverse relationship between osteoporotic bone remodeling and arterial and aortic valve calcifications. Inflammation is associated with atherosclerosis, a strong inductor of vascular calcifications,25,26 and a modulator of osteoblast activity.27 Independent of serum PTH, high serum hCRP was positively associated with abnormal ABix and inversely correlated with dLS/BS and Ob.S/BS (Figure 2). However, the correlations between PTH and bone turnover markers were independent (Figure 1) of hCRP, suggesting that the resistance to PTH could be associated with arterial changes. As shown in Figure 1 and Table 5, an abnormal ABix was associated with low bone turnover, principally affecting osteoblasts. In contrast, N.Oc/T.A and resorption activity did not differ significantly between patient groups. In a study on the general population, Pennisi et al.17 showed that peripheral vessels atherosclerosis was associated with biochemistry parameters suggestive of reduced bone formation and abnormal bone turnover. PTH resistance in patients with ESRD is well known28 and multifactorial, including abnormal PTH signaling. Downregulation of PTH/PTH-related protein-receptor type 1 (PTHrP1) gene expression and mRNA in osteoblasts,29–31 presence 4
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of antagonistic C-terminal PTH fragments,32 hyperlipidemiainduced oxidant stress,33 uremic milieu,34 uremic toxins,35 inflammation,36 and aging37 are all mechanisms accounting for skeletal resistance to PTH and adynamic bone. Could defective PTH signaling play a role in the development of bone–vascular axis abnormalities? Arterial calcifications are highly prevalent in subjects with low bone turnover8 and abnormal ABix.22 Experimental data showed that, by signaling through the PTH/PTHrP receptor (PTH1R), parathyroid PTH1–34 inhibited bovine vascular smooth muscle cell (VSMC) calcification38 and blocked the calcifying action of calcitriol.39 Shao et al.40 showed that teriparatide (human PTH1–34) inhibited osteogenic vascular calcification in diabetic LDL receptor-deficient mice. Cheng et al.41 showed that activation of VSMC PTH1R inhibited the Wnt/2-Catenin signaling pathway and prevented aortic calcifications and fibrosis in diabetic arteriosclerosis. The role of PTH1R signaling through PTH or PTHrP in arterial remodeling remains the subject of investigations, but experimental data indicate that PTHrP inhibited VSMC migration and proliferation42,43 and neointimal formation.44 PTH1R signaling plays an important J Am Soc Nephrol 26: ccc–ccc, 2014
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Figure 2. Correlations between serum hCRP and bone histomorphometry parameters. (A) Ob.S/BS, (B) dLS/BS, (C) N.Oc/T.A, and (D) ES/BS in patients with normal ABix (white circles) and patients with abnormal ABix (black triangles). The correlations are adjusted to serum PTH.
Table 4. Univariate correlations between PAD status (0, normal; 1, PAD) and clinical and biochemistry parameters Parameter
R Value
P Value
Age (yr) Vintage (mo) CaCO3 (g/d) hCRP (log mg/L) Serum albumin (g/L) dLS/BS (%) Ob.S/BS (%) N.Oc/T.A (mm2) Aluminum-stained surface (%) PTX (no, 0; yes, 1)
0.5936 0.4878 0.5230 0.7512 20.5920 20.5627 20.5054 20.3855 0.3539 0.3570
,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 0.002 0.004 0.004
role in the skeletal response to PTH through the control of osteocyte activation of SOST/sclerostin. In the presence of PTH resistance and impaired signaling, the transcriptional suppression of sclerostin production might be impaired.45,46 Resulting from experimental data, these hypotheses were not tested in this population, but serum sclerostin is increased in J Am Soc Nephrol 26: ccc–ccc, 2014
patients with ESRD47,48 and associated with cardiovascular calcifications.49 Our findings support, in a human ESRD population, the common role of inflammation in bone and cardiovascular remodeling, which was shown experimentally by Hjortnaes et al.,24 but the association between low bone turnover and arterial remodeling is not new.8,50 Hjortnaes et al.24 confirm the dissociation between serum PTH concentrations and bone histology and PTH concentration with clinical disease. Herein, the presence of subclinical PAD was not associated with any differences in serum PTH but was associated with a different clinical effect, which was characterized by skeletal resistance to PTH involving, more importantly, osteoblast function and number compared with osteoclast and bone erosion (Figure 3). Bone remodeling is maintained by a balance between bone erosion and bone formation and dependent on bidirectional communication between osteoblasts and osteoclasts through paracrine factors (receptor activator of NF-kB [RANK] ligand, osteoprotegerin, and ephrinB2 ligand), cell–cell contact, and cell–bone matrix interactions.50 Lesser coupling between bone erosion and bone Vascular and Skeletal Disorders in ESRD
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Table 5. Stepwise regression report for PAD status (0, normal; 1, PAD) Parameter
T Value
P Value
Vintage (mo) dLS/BS (%) Log hCRP (mg/L) Age (yr) CaCO3 (g/d) N.Oc/T.A (mm2) PTX (no, 0; yes, 1) Aluminum-stained surfaces (%)
4.040 22.877 6.087 0.850 1.1935 0.6961 0.5972 0.0509
,0.001 ,0.01 ,0.001 0.40 0.24 0.49 0.55 0.96
R2=0.6843.
the population, which included relatively young subjects with primary kidney disease, Partial R Adjusted for Rest excluding diabetes, treated in the 1990s. This 0.2111 population differs markedly from the pop0.1195 ulation treated today, which includes higher 0.3669 percentages of patients with diabetes and NA NA older patients with overt cardiovascular NA comorbidities and shorter vintage. The treatNA ment modalities have evolved with withNA drawal of aluminum-containing phosphate binders and the use of noncalcium-containing phosphate binders, the wider use of vitamin D receptor agonists and cholecalciferol supplementation, the prescription of calcimimetics, and the improvement of hemodialysis techniques. In summary, our results showed that, in prevalent nondiabetic patients with ESRD, the PAD was associated with low bone turnover and low bone formation with pronounced osteoblast resistance to PTH. 2
CONCISE METHODS Patients
Figure 3. Correlation between bone histomorphometry parameters. dLS/BS and ES/BS in patients with normal ABix (white circles) and patients with abnormal ABix (black triangles).
formation with decreased number and activity of osteoblasts was observed in several conditions, such as skeletal unloading during prolonged bed rest,51 aluminum-related osteodystrophy in patients on dialysis,52 and chronic inflammation.53,54 In this study, the PTH- and CRP-adjusted correlation between aluminumstained surfaces and dLS/BS was not significant, but patients with PAD had microinflammation and increased serum CRP. We can speculate that the microinflammation could play a role in bone erosioformation uncoupling. As shown on Figure 2, serum CRP was inversely correlated with dLS/BS and Ob.S/BS but less correlated or not correlated with N.Oc/T.A and ES/BS. Inflammation and proinflammatory cytokines, such as TNF-a and IL-1, could cause an imbalance in bone remodeling by downregulation of PTH/PTHrP136 and activation of osteoclasts through classic or alternative RANK/RANK ligand pathways favoring bone erosion.53,54 The possible role of abnormal PTH signaling in arterial remodeling and vascular pathology remains hypothetical, because observational studies on the basis of correlations studies and associations are not proof of causality. This limitation is an important limitation of this study. Another limitation concerns the limited number of patients and clinical characteristics of 6
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With the exception of 1 patient, the population of 65 prevalent nondiabetic patients on hemodialysis was previously detailed.55 Inclusion criteria were (1) hemodialysis vintage $12 months and (2) absence of clinical history of cardiovascular disease, including history of lower limb arteries angioplasty, stenting, amputation, or trophic disorders (ulcerations or gangrene). Dialysis duration was individually tailored (4–6 hours three times per week) to control body fluids and blood chemistries. The bicarbonate dialysate was prepared using double reverse osmosis-treated water with 1.5 or 1.75 mmol/L Ca according to the serum Ca-PO4 equilibrium and the need for active vitamin D3 (1a-OH-D3 prescribed in eight patients). Although CaCO3 was used exclusively as a PO4 binder at the time of the study, 28 patients had taken aluminum hydroxide in the past; 12 patients underwent subtotal PTX, and 10 patients underwent total PTX with heterotopic autotransplantation into the forearm. PTX had been performed 20–70 months before the study. Erythropoietin was administered to maintain hemoglobin$100 g/L when necessary. All subjects gave informed written consent to participate in the study, which was approved by our Institutional Review Board in accordance with the Declaration of Helsinki.
ABix Lower limb artery status was on the basis of ABix measurement and the ratio between SBP in the ankle (arteria tibialis posterior or dorsalis pedis) and the arm (arteria brachialis). SBPs were measured in a supine position after 15 minutes of rest. Brachial artery SBP was measured two times in the arm without arteriovenous shunt and averaged. Ankle SBP was measured two times in each foot and averaged. SBPs were measured with a Doppler SEGA M842 8-MHz Unit (Société Electronique Générale et Appliquée, Paris, France) during cuff deflation. Patients were divided in three groups according to their ABix values: normal ABix, 0.9–1.3; low ABix, ,0.9; .1.4 (i.e., incompressible). J Am Soc Nephrol 26: ccc–ccc, 2014
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Bone Histomorphometry Diagnostic anterior iliac crest bone biopsies were obtained after dLS/BS according to the schedule of 2 days on tetracycline, 10 days off tetracycline, and 2 days on tetracycline as previously detailed.55 The extent of the dLS/BS (percent) was measured on two unstained 10-mm-thick sections. On three Toluidine blue-stained 5-mm-thick sections, bone volume (percent), Ob.S/BS (percent), ES/BS (percent), and N.Oc/T.A were assessed. Bone Al staining was expressed as the percentage of the trabecular surface stained. All measurements were made using an eyepiece reticle (Carl Zeiss Integral Plate II, Oberkochen, Germany).
Blood Chemistries Blood chemistries, including serum Ca and PO4, blood lipids, blood glucose, uric acid, and serum albumin, are the mean values of all measurements in the 6 months preceding the ABix measurements. Routine biochemical parameters were determined by standard methods using autoanalyzers. Blood samples for measurement of bone alkaline phosphatase serum levels (Quidel MicroVue BAP EIA), 25 (OH) cholecalciferol, and 1,25(OH)2D3 were taken the day of the bone biopsy. Intact PTH (N-IRMA; Nichols Institute) was determined every 3 months, and the reported values are the average of two measurements done during the 6 months preceding the bone biopsy. Bone biopsy and measurement of ABix were done the same month.
Statistical Analyses Data are expressed as medians and 95% confidence intervals. The primary analysis concerned ABix subgroup normal, low, or .1.4 comparison with Kruskal–Wallis one-way ANOVA on ranks and Bonferroni multiple comparison Z-test value. For the univariate (Pearson or Spearman correlation coefficient), and multivariate regression studies, low and incompressible ABix groups were pooled and considered to have PAD. The correlations slopes are expressed as b-coefficients6SD and compared by t test. For these correlations, artery disease status was used as a dummy variable (0, normal ABix; 1, PAD). Multiple regression analyses were performed using the subset of univariate analysis-selected independent variables (after Bonferroni correction for the number of correlations studied). All analyses were calculated using NCSS 2000 (Gerry Hintze, Kaysville, UT).
ACKNOWLEDGMENTS We thank Ms. Janet Jacobson for editorial assistance. The study was supported by Groupe d’Étude de la Pathophysiologie de l’Insuffisance Rénale (GEPIR).
DISCLOSURES None.
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21. Liu J-H, Lin H-H, Yang Y-F, Liu Y-L, Kuo H-L, Wang I-K, Chou C-Y, Huang C-C: Subclinical peripheral artery disease in patients undergoing peritoneal dialysis: Risk factors and outcome. Perit Dial Int 29: 64–71, 2009 22. Adragao T, Pires A, Branco P, Castro R, Oliveira A, Nogueira C, Bordalo J, Curto JD, Prata MM: Ankle—brachial index, vascular calcifications and mortality in dialysis patients. Nephrol Dial Transplant 27: 318–325, 2012 23. Wang Y, Guo X, Li J, Hu D, Zhao D, Ma H, Mou Q, Liu J, Xu Y: Predictive value of ankle-brachial index to all-cause mortality and cardiovascular mortality in Chinese patients with chronic kidney disease. Vasa 41: 205– 213, 2012 24. Hjortnaes J, Butcher J, Figueiredo JL, Riccio M, Kohler RH, Kozloff KM, Weissleder R, Aikawa E: Arterial and aortic valve calcification inversely correlates with osteoporotic bone remodelling: A role for inflammation. Eur Heart J 31: 1975–1984, 2010 25. Aikawa E, Nahrendorf M, Figueiredo JL, Swirski FK, Shtatland T, Kohler RH, Jaffer FA, Aikawa M, Weissleder R: Osteogenesis associates with inflammation in early-stage atherosclerosis evaluated by molecular imaging in vivo. Circulation 116: 2841–2850, 2007 26. Al-Aly Z, Shao J-S, Lai C-F, Huang E, Cai J, Behrmann A, Cheng S-L, Towler DA: Aortic Msx2-Wnt calcification cascade is regulated by TNFa-dependent signals in diabetic Ldlr-/- mice. Arterioscler Thromb Vasc Biol 27: 2589–2596, 2007 27. Shapiro S, Tatakis DN, Dziak R: Effects of tumor necrosis factor alpha on parathyroid hormone-induced increases in osteoblastic cell cyclic AMP. Calcif Tissue Int 46: 60–62, 1990 28. Massry SG, Coburn JW, Lee DBM, Jowsey J, Kleeman CR: Skeletal resistance to parathyroid hormone in renal failure. Studies in 105 human subjects. Ann Intern Med 78: 357–364, 1973 29. Ureña P, Mannstadt M, Hruby M, Ferreira A, Schmitt F, Silve C, Ardaillou R, Lacour B, Abou-Samra AB, Segre GV, Drüeke T: Parathyroidectomy does not prevent the renal PTH/PTHrP receptor downregulation in uremic rats. Kidney Int 47: 1797–1805, 1995 30. Picton ML, Moore PR, Mawer EB, Houghton D, Freemont AJ, Hutchison AJ, Gokal R, Hoyland JA: Down-regulation of human osteoblast PTH/ PTHrP receptor mRNA in end-stage renal failure. Kidney Int 58: 1440– 1449, 2000 31. Iwasaki-Ishizuka Y, Yamato H, Nii-Kono T, Kurokawa K, Fukagawa M: Downregulation of parathyroid hormone receptor gene expression and osteoblastic dysfunction associated with skeletal resistance to parathyroid hormone in a rat model of renal failure with low turnover bone. Nephrol Dial Transplant 20: 1904–1911, 2005 32. Slatopolsky E, Finch J, Clay P, Martin D, Sicard G, Singer G, Gao P, Cantor T, Dusso A: A novel mechanism for skeletal resistance in uremia. Kidney Int 58: 753–761, 2000 33. Sage AP, Lu J, Atti E, Tetradis S, Ascenzi M-G, Adams DJ, Demer LL, Tintut Y: Hyperlipidemia induces resistance to PTH bone anabolism in mice via oxidized lipids. J Bone Miner Res 26: 1197–1206, 2011 34. Disthabanchong S, Hassan H, McConkey CL, Martin KJ, Gonzalez EA: Regulation of PTH1 receptor expression by uremic ultrafiltrate in UMR 106-01 osteoblast-like cells. Kidney Int 65: 897–903, 2004 35. Nii-Kono T, Iwasaki Y, Uchida M, Fujieda A, Hosokawa A, Motojima M, Yamato H, Kurokawa K, Fukagawa M: Indoxyl sulfate induces skeletal resistance to parathyroid hormone in cultured osteoblastic cells. Kidney Int 71: 738–743, 2007 36. Katz MS, Gutierrez GE, Mundy GR, Hymer TK, Caulfield MP, McKee RL: Tumor necrosis factor and interleukin 1 inhibit parathyroid hormoneresponsive adenylate cyclase in clonal osteoblast-like cells by downregulating parathyroid hormone receptors. J Cell Physiol 153: 206–213, 1992 37. Zhou S, Bueno EM, Kim SW, Amato I, Shen L, Hahne J, Bleiberg I, Morley P, Glowacki J: Effects of age on parathyroid hormone signaling in human marrow stromal cells. Aging Cell 10: 780–788, 2011 38. Jono S, Nishizawa Y, Shioi A, Morii H: Parathyroid hormone-related peptide as a local regulator of vascular calcification. Its inhibitory action
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on in vitro calcification by bovine vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 17: 1135–1142, 1997 Jono S, Nishizawa Y, Shioi A, Morii H: 1,25-Dihydroxyvitamin D3 increases in vitro vascular calcification by modulating secretion of endogenous parathyroid hormone-related peptide. Circulation 98: 1302–1306, 1998 Shao J-S, Cheng S-L, Charlton-Kachigian N, Loewy AP, Towler DA: Teriparatide (human parathyroid hormone (1-34)) inhibits osteogenic vascular calcification in diabetic low density lipoprotein receptordeficient mice. J Biol Chem 278: 50195–50202, 2003 Cheng S-L, Shao J-S, Halstead LR, Distelhorst K, Sierra O, Towler DA: Activation of vascular smooth muscle parathyroid hormone receptor inhibits Wnt/beta-catenin signaling and aortic fibrosis in diabetic arteriosclerosis. Circ Res 107: 271–282, 2010 Ishikawa M, Akishita M, Kozaki K, Toba K, Namiki A, Yamaguchi T, Orimo H, Ouchi Y: Amino-terminal fragment (1-34) of parathyroid hormone-related protein inhibits migration and proliferation of cultured vascular smooth muscle cells. Atherosclerosis 136: 59–66, 1998 Song GJ, Fiaschi-Taesch N, Bisello A: Endogenous parathyroid hormonerelated protein regulates the expression of PTH type 1 receptor and proliferation of vascular smooth muscle cells. Mol Endocrinol 23: 1681– 1690, 2009 Fiaschi-Taesch N, Sicari B, Ubriani K, Cozar-Castellano I, Takane KK, Stewart AF: Mutant parathyroid hormone-related protein, devoid of the nuclear localization signal, markedly inhibits arterial smooth muscle cell cycle and neointima formation by coordinate up-regulation of p15Ink4b and p27kip1. Endocrinology 150: 1429–1439, 2009 Saini V, Marengi DA, Barry KJ, Fulzele KS, Heiden E, Liu X, Dedic C, Maeda A, Lotinun S, Baron R, Pajevic PD: Parathyroid hormone (PTH)/ PTH-related peptide type 1 receptor (PPR) signaling in osteocytes regulates anabolic and catabolic skeletal responses to PTH. J Biol Chem 288: 20122–20134, 2013 Powell WF Jr., Barry KJ, Tulum I, Kobayashi T, Harris SE, Bringhurst FR, Pajevic PD: Targeted ablation of the PTH/PTHrP receptor in osteocytes impairs bone structure and homeostatic calcemic responses. J Endocrinol 209: 21–32, 2011 Cejka D, Herberth J, Branscum AJ, Fardo DW, Monier-Faugere M-C, Diarra D, Haas M, Malluche HH: Sclerostin and Dickkopf-1 in renal osteodystrophy. Clin J Am Soc Nephrol 6: 877–882, 2011 Pelletier S, Dubourg L, Carlier M-C, Hadj-Aissa A, Fouque D: The relation between renal function and serum sclerostin in adult patients with CKD. Clin J Am Soc Nephrol 8: 819–823, 2013 Brandenburg VM, Kramann R, Koos R, Krüger T, Schurgers L, Mühlenbruch G, Hübner S, Gladziwa U, Drechsler C, Ketteler M: Relationship between sclerostin and cardiovascular calcification in hemodialysis patients: A cross-sectional study. BMC Nephrol 14: 219–228, 2013 Irie N, Takada Y, Watanabe Y, Matsuzaki Y, Naruse C, Asano M, Iwakura Y, Suda T, Matsuo K: Bidirectional signaling through ephrinA2-EphA2 enhances osteoclastogenesis and suppresses osteoblastogenesis. J Biol Chem 284: 14637–14644, 2009 Zerwekh JE, Ruml LA, Gottschalk F, Pak CYC: The effects of twelve weeks of bed rest on bone histology, biochemical markers of bone turnover, and calcium homeostasis in eleven normal subjects. J Bone Miner Res 13: 1594–1601, 1998 Faugere MC, Arnala IO, Ritz E, Malluche HH: Loss of bone resulting from accumulation of aluminum in bone of patients undergoing dialysis. J Lab Clin Med 107: 481–487, 1986 Wei S, Siegal GP: Mechanisms modulating inflammatory osteolysis: A review with insights into therapeutic targets. Pathol Res Pract 204: 695– 706, 2008 Schett G: Effects of inflammatory and anti-inflammatory cytokines on the bone. Eur J Clin Invest 41: 1361–1366, 2011 London GM, Marchais SJ, Guérin AP, Boutouyrie P, Métivier F, de Vernejoul MC: Association of bone activity, calcium load, aortic stiffness, and calcifications in ESRD. J Am Soc Nephrol 19: 1827–1835, 2008
J Am Soc Nephrol 26: ccc–ccc, 2014
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Ankle-Brachial Index and Bone Turnover in Patients on Dialysis Gérard M. London,*† Sylvain J. Marchais,* Alain P. Guérin,† and Marie-Christine de Vernejoul‡ *Department of Nephrology, Manhès Hospital, Fleury Mérogis, France; †Department of Pharmacology, Georges Pompidou European Hospital, National Institute of Health and Medical Research U970, Paris, France; and ‡ Lariboisiere Hospital, Vigot Petersen Centre, National Institute of Health and Medical Research U606, University Paris 7, Paris, France
ABSTRACT An association between atherosclerosis and osteoporosis has been reported in several studies. This association could result from local intraosseous atherosclerosis and ischemia, which is shown by limb osteoporosis in patients with peripheral artery disease (PAD), but also could result from bidirectional communication between the skeleton and blood vessels. Systemic bone disorders and PAD are frequent in ESRD. Here, we investigated the possible interaction of these disorders. For 65 prevalent nondiabetic patients on hemodialysis, we measured ankle-brachial pressure index (ABix) and evaluated mineral and bone disorders with bone histomorphometry. In prevalent patients on hemodialysis, PAD (ABix,0.9 or .1.4/incompressible) was associated with low bone turnover and pronounced osteoblast resistance to parathyroid hormone (PTH), which is indicated by decreased double-labeled surface and osteoblast surface (P,0.001). Higher osteoblast resistance to PTH in patients with PAD was characterized by weaker correlation coefficients (slopes) between serum PTH and double-labeled surface (P=0.02) or osteoblast surface (P=0.03). The correlations between osteoclast number or eroded surface and serum mineral parameters, including PTH, did not differ for subjects with normal ABix and PAD. Common vascular risk factors (dyslipidemia, smoking, and sex) were similar for normal, low, and incompressible ABix. Patients with PAD were older and had high C-reactive protein levels and longer hemodialysis vintage. These results indicate that, in prevalent nondiabetic patients with ESRD, PAD associates with low bone turnover and pronounced osteoblast resistance to PTH. J Am Soc Nephrol 26: ccc–ccc, 2014. doi: 10.1681/ASN.2014020169
The bone–vascular axis concept emerged from clinical and experimental findings linking bone and arterial remodeling and changes.1 Observations in diverse populations have revealed associations between atherosclerosis and vascular calcifications with decreased bone mineral density (BMD), risk for vertebral or hip fracture, low bone turnover, and cardiovascular disease.2–8 The biologic link between vascular disease and bone changes is certainly part of the aging process, but in many studies, these bone–vessel associations remained significant after adjustment for age, suggesting an age-independent causal relationship. Nevertheless, the factors or mechanisms underlying these associations are not well understood and could result from common J Am Soc Nephrol 26: ccc–ccc, 2014
mechanisms acting on both systems (e.g., inflammation, hyperlipidemia, diabetes, smoking,9–12 or generalized arterial disease per se). Blood vessels play a major role in osteogenesis, and factors affecting the blood supply to bones could have major effects on bone remodeling and structure.13–15
Received February 12, 2014. Accepted May 27, 2014. Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Gérard M. London, Manhes Hospital, 8, rue Roger-Clavier, 91712, Fleury Mérogis Cedex, France. Email: [email protected] Copyright © 2014 by the American Society of Nephrology
ISSN : 1046-6673/2602-ccc
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Ischemia resulting from intraosseous atherosclerosis and arterial calcifications could explain the association between osteoporosis and decreased BMD. The link between compromised bone circulation and osteoporosis was documented by the observation linking lower limb bone mineral content and abnormal bone turnover with increased risk of fractures in men with ischemic atherosclerotic peripheral artery disease (PAD).16–19 Lower limb PAD is a frequent complication of ESRD, and low ankle-brachial pressure index (ABix) or incompressible arteries are associated with all-cause and cardiovascular mortality.20–23 In general populations, associations between PAD and BMD are observed at the local (lower limb) level and frequently, in the absence of overt mineral metabolism disorders; in patients with CKD and ESRD, those interactions coexist frequently with mineral and bone disorders, including perturbed parathyroid hormone (PTH) and vitamin D metabolisms. In this study, we had the opportunity to analyze the relationships between ABix-assessed subclinical PAD and mineral and bone abnormalities, including systemic bone, on the basis of histomorphometry of iliac crest biopsies. These results indicate that, in prevalent nondiabetic patients with ESRD, PAD was associated with low bone turnover and pronounced osteoblast resistance to PTH.
RESULTS Patient Characteristics
We studied 65 patients whose clinical characteristics, brachial and lower limb systolic BP (SBP), and ABix are summarized in Table 1. ABix was normal in 30 of 65 (46.2%) patients, ABix was low in 11 (16.9%) patients, and ABix.1.4 or incompressible arteries were present in 24 (36.9%) patients. Patients with abnormal ABix were older. Peripheral artery calcifications were present in all subjects with pathologic ABix and 25.7% of patients whose ABix was normal. Hemodialysis vintage, the proportion of patients with previous parathyroidectomy (PTX), and the calcium phosphate binders prescribed dose
were higher in patients with low ABix or incompressible arteries. Blood chemistries are reported in Table 2 according to ABix. Patients with PAD had significantly higher serum high-sensitivity C-reactive protein (hCRP) and lower serum albumin. All others biochemical parameters, including PTH, were not significantly different among the three groups. Bone Histomorphometry
Bone histomorphometry parameters are given in Table 3. Osteoblast surface per bone surface (Ob.S/BS) and doublelabeled surface (dLS/BS) were significantly lower in patients with PAD. Patients with PAD had higher aluminum-stained surface but similar bone volume and osteoid volume. In multiple regression analyses, dLS/BS was positively associated with serum PTH (P,0.001), and it was negatively associated with serum hCRP (P,0.001) but not negatively associated with aluminum-stained surfaces (P=0.27). As shown in Figure 1 in pooled abnormal ABix, after adjustment for age and serum hCRP, the correlations between serum PTH, dLS/BS, and Ob.S/ BS were positive for normal and abnormal ABix (Figure 1). Upward shifts with significantly steeper slopes (b-coefficient) of the correlation between dLS/BS and PTH (mean b-coefficient6SD; 21624.4 versus 5.12630.3; T=2.34; P=0.03) and between Ob.S/BS and PTH (mean b-coefficient6SD; 17618.6 versus 8.4610.2; T=2.25; P=0.03) were observed for patients with normal ABix. Osteoclast number/per millimeter2 (N.Oc/T.A) and eroded surface (ES/BS) did not differ among the three groups (Figure 1). The correlations between circulating PTH, N.Oc/T.A, and ES/BS, were positive and similar for normal and abnormal ABix. Significant negative serum PTH-adjusted correlations were observed between serum hCRP, Ob.S/BS, and dLS/BS (Figure 2). Corrected univariate correlations showed that PAD was positively associated with serum CRP, age, dialysis vintage, and calcium carbonate dose and inversely related to dLS/BS, Ob.S/ BS, aluminum-stained surfaces, and PTX (Table 4). Because of multiple colinearities (for example, correlation between vintage and PTX: R=0.3615; P=0.004 or correlation between
Table 1. Clinical characteristics of patients with ESRD Characteristic Age (yr) Body mass index (kg/m2) Vintage (mo) Smoking (pack/yr) PTX (no.) CaCO3 (g/d) Brachial SBP (mmHg) Lower limb SBP (mmHg) ABix
ABix Normal (n=30)
Low (n=11)
>1.4 (n=24)
44 (29 to 50) 22.6 (21.3 to 24.1) 48 (29 to 65) 0 (0 to 3) 4/30 (13.3%) 1.2 (0.8 to 1.2) 147 (134 to 164) 175 (164 to 185) 1.14 (1.10 to 1.16)
59a (52 to 65) 20.9 (17.1 to 27.9) 85a (36 to 104) 0 (0 to 25) 5/11a (45.5%) 1.8 (1.4 to 2.4) 147 (131 to 156) 126a (104 to 139) 0.86a (0.65 to 0.89)
59a (52 to 64) 22.5 (20.3 to 23.5) 144a (96 to 173) 5.5 (0 to 16) 13/24a (54.2%) 2.4b (2 to 2.4) 156 (138 to 168) 300a (250 to 300) 1.76a (1.52 to 1.9)
ANOVA P Value ,0.001 NS ,0.001 NS 0.004 ,0.001 NS ,0.001 ,0.001
Values are medians (95% confidence intervals). Kruskal–Wallis one-way ANOVA on ranks. a Significantly different from normal ABix. b Significantly different from low ABix and ABix.1.4.
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Table 2. Blood chemistries of patients with ESRD according to ABix ABix
Parameter Cholesterol (mmol/L) Triglycerides (mmol/L) Uric acid (mmol/L) Glycemia (mmol/L) Serum Ca (mmol/L) Serum ionized Ca (mmol/L) Serum phosphate (mmol/L) Serum Mg (mmol/L) Serum PTH (pg/mL) Serum 25(OH) cholecalciferol (mg/L) Serum 1,25 (OH)2D3 (ng/L) Bone alkaline phosphatase (mg/L) Serum albumin (g/L) Hemoglobin (mmol/L) hCRP (mg/L)
Normal (n=30)
Low (n=11)
>1.4 (n=24)
5.03 (4.32 to 5.5) 1.52 (1.37 to 1.87) 309 (260 to 339) 4.88 (4.61 to 4.98) 2.45 (2.36 to 2.49) 1.23 (1.19 to 1.26) 1.84 (1.62 to 2.0) 1.15 (1.07 to 1.2) 322 (227 to 406) 21.35 (15.4 to 30.45) 18 (13 to 20) 25 (20 to 30) 39.9 (38.6 to 41) 10 (9.5 to 10.5) 3 (3 to 5)
5.8 (5.06 to 7.75) 1.7 (1.5 to 2.68) 313 (141 to 410) 5.03 (4.61 to 5.74) 2.43 (2.34 to 2.52) 1.23 (1.17 to 1.24) 1.79 (1.25 to 2.84) 1.19 (1 to 1.31) 215 (121 to 467) 17.85 (11.9 to 28.7) 12.8 (8 to 18) 17 (12 to 25) 37a (35 to 38.8) 9.7 (8.2 to 10.2) 10a (7 to 12)
4.97 (4.7 to 6) 1.63 (1.16 to 2.02) 340 (285 to 369) 5.02 (4.73 to 5.29) 2.46 (2.35 to 2.48) 1.22 (1.18 to 1.24) 1.98 (1.76 to 2.08) 1.17 (1.08 to 1.21) 212 (94 to 345) 19.8 (12.2 to 24.2) 16 (11.4 to 20.6) 18.5 (14 to 25) 37a (36 to 38.6) 10.9 (8.9 to 11.4) 8a (7 to 11)
ANOVA P Value NS NS NS NS NS NS NS NS NS NS NS (0.07) NS (0.06) ,0.001 NS ,0.001
Values are medians (95% confidence intervals). Kruskal–Wallis one-way ANOVA on ranks. a Statistically different from normal ABix.
Table 3. Bone histomorphometry Surfaces Ob.S/BS (%) dLS/BS (%) N.Oc/T.A (mm2) ES/BS (%) BV/TV (%) OV/BV (%) Aluminum-stained surface (%)
ABix Normal (n=30)
Low (n=11)
>1.4 (n=24)
11.81 (9.12 to 16.33) 10.52 (5.6 to 18.5) 1.42 (1 to 2.50) 2.58 (1.52 to 3.40) 21.01 (16 to 22.3) 8.76 (5.8 to 11.4) 5 (7.5 to 28.4)
2.42a (0.89 to 7.5) 1.33a (0 to 2.40) 0.40b (0.1 to 1) 0.48 (0 to 1.4) 19.23 (11.2 to 23.9) 7.80 (2.45 to 11.1) 48a (20 to 91)
2.66a (0.3 to 6.3) 1a (0 to 3.20) 0.80 (0.15 to 1.62) 1.38 (0.1 to 2.61) 15.8 (12.66 to 20.7) 6.15 (2.7 to 10.8) 48a (5 to 75)
ANOVA P Value 0.002 ,0.001 0.01 NS NS NS ,0.01
Values are medians (95% confidence intervals). Kruskal–Wallis one-way ANOVA on ranks with multiple comparison Z-value test. BV/TV, bone volume; OV/BV, osteoid volume. a Significantly different from normal ABix. b Significantly different from low ABix and ABix.1.4.
vintage and aluminum-stained surface: R=0.5365; P,0.001), multivariate stepwise correlation analysis identified only hCRP, vintage, and dLS/BS as significant cofactors associated with PAD (Table 5).
DISCUSSION
Our results indicated that, in patients with prevalent hemodialysis, subclinical PAD was associated with low bone turnover and more pronounced osteoblast resistance to PTH, which was assessed by decreased dLS/BS and Ob.S/BS and lower correlation b-coefficients (slopes) between serum PTH and dLS/BS or Ob.S/BS (Figure 1, A and B). The correlations between PAD and N.Oc/T.A or ES/BS did not differ from subjects with normal ABix. These bone–artery associations remained significant after adjustment for age, serum hCRP, and hemodialysis vintage. Although hemodialysis vintage and serum hCRP were independently associated with PAD, serum mineral parameters, J Am Soc Nephrol 26: ccc–ccc, 2014
including PTH, did not differ significantly between groups. Unlike the published association between the calcium phosphate binders doses and aortic calcifications,8 in multivariate analyses, this association was not observed in peripheral arteries with abnormal ABix. Decreased femoral neck or total hip BMD and increased risk of fractures were reported to be associated with PAD,16,18,19 and bone perfusion was reduced in osteoporotic patients compared with normal and osteopenic subjects.15 In a study on patients with unilateral PAD, Laroche et al.16 reported that BMD was significantly lower in the affected limb compared with the unaffected limb. Unlike the reported association between PAD and local femoral BMD, our results indicated an association between PAD and systemic bone changes in nonweight-bearing anterior iliac crest. Our study population did not include patients with diabetes, and the only common bone–artery risk factor was higher hCRP in patients with abnormal ABix (Tables 2 and 5). In an experimental in vivo study, Hjortnaes et al.24 showed Vascular and Skeletal Disorders in ESRD
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Figure 1. Correlations between serum PTH and bone histomorphometry parameters. (A) Ob.S/BS, (B) dLS/BS, (C) N.Oc/T.A, and (D) ES/BS for patients with normal ABix (white circles) and patients with abnormal ABix (black triangles). The correlations are adjusted to serum CRP.
the role of inflammation in the inverse relationship between osteoporotic bone remodeling and arterial and aortic valve calcifications. Inflammation is associated with atherosclerosis, a strong inductor of vascular calcifications,25,26 and a modulator of osteoblast activity.27 Independent of serum PTH, high serum hCRP was positively associated with abnormal ABix and inversely correlated with dLS/BS and Ob.S/BS (Figure 2). However, the correlations between PTH and bone turnover markers were independent (Figure 1) of hCRP, suggesting that the resistance to PTH could be associated with arterial changes. As shown in Figure 1 and Table 5, an abnormal ABix was associated with low bone turnover, principally affecting osteoblasts. In contrast, N.Oc/T.A and resorption activity did not differ significantly between patient groups. In a study on the general population, Pennisi et al.17 showed that peripheral vessels atherosclerosis was associated with biochemistry parameters suggestive of reduced bone formation and abnormal bone turnover. PTH resistance in patients with ESRD is well known28 and multifactorial, including abnormal PTH signaling. Downregulation of PTH/PTH-related protein-receptor type 1 (PTHrP1) gene expression and mRNA in osteoblasts,29–31 presence 4
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of antagonistic C-terminal PTH fragments,32 hyperlipidemiainduced oxidant stress,33 uremic milieu,34 uremic toxins,35 inflammation,36 and aging37 are all mechanisms accounting for skeletal resistance to PTH and adynamic bone. Could defective PTH signaling play a role in the development of bone–vascular axis abnormalities? Arterial calcifications are highly prevalent in subjects with low bone turnover8 and abnormal ABix.22 Experimental data showed that, by signaling through the PTH/PTHrP receptor (PTH1R), parathyroid PTH1–34 inhibited bovine vascular smooth muscle cell (VSMC) calcification38 and blocked the calcifying action of calcitriol.39 Shao et al.40 showed that teriparatide (human PTH1–34) inhibited osteogenic vascular calcification in diabetic LDL receptor-deficient mice. Cheng et al.41 showed that activation of VSMC PTH1R inhibited the Wnt/2-Catenin signaling pathway and prevented aortic calcifications and fibrosis in diabetic arteriosclerosis. The role of PTH1R signaling through PTH or PTHrP in arterial remodeling remains the subject of investigations, but experimental data indicate that PTHrP inhibited VSMC migration and proliferation42,43 and neointimal formation.44 PTH1R signaling plays an important J Am Soc Nephrol 26: ccc–ccc, 2014
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Figure 2. Correlations between serum hCRP and bone histomorphometry parameters. (A) Ob.S/BS, (B) dLS/BS, (C) N.Oc/T.A, and (D) ES/BS in patients with normal ABix (white circles) and patients with abnormal ABix (black triangles). The correlations are adjusted to serum PTH.
Table 4. Univariate correlations between PAD status (0, normal; 1, PAD) and clinical and biochemistry parameters Parameter
R Value
P Value
Age (yr) Vintage (mo) CaCO3 (g/d) hCRP (log mg/L) Serum albumin (g/L) dLS/BS (%) Ob.S/BS (%) N.Oc/T.A (mm2) Aluminum-stained surface (%) PTX (no, 0; yes, 1)
0.5936 0.4878 0.5230 0.7512 20.5920 20.5627 20.5054 20.3855 0.3539 0.3570
,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 0.002 0.004 0.004
role in the skeletal response to PTH through the control of osteocyte activation of SOST/sclerostin. In the presence of PTH resistance and impaired signaling, the transcriptional suppression of sclerostin production might be impaired.45,46 Resulting from experimental data, these hypotheses were not tested in this population, but serum sclerostin is increased in J Am Soc Nephrol 26: ccc–ccc, 2014
patients with ESRD47,48 and associated with cardiovascular calcifications.49 Our findings support, in a human ESRD population, the common role of inflammation in bone and cardiovascular remodeling, which was shown experimentally by Hjortnaes et al.,24 but the association between low bone turnover and arterial remodeling is not new.8,50 Hjortnaes et al.24 confirm the dissociation between serum PTH concentrations and bone histology and PTH concentration with clinical disease. Herein, the presence of subclinical PAD was not associated with any differences in serum PTH but was associated with a different clinical effect, which was characterized by skeletal resistance to PTH involving, more importantly, osteoblast function and number compared with osteoclast and bone erosion (Figure 3). Bone remodeling is maintained by a balance between bone erosion and bone formation and dependent on bidirectional communication between osteoblasts and osteoclasts through paracrine factors (receptor activator of NF-kB [RANK] ligand, osteoprotegerin, and ephrinB2 ligand), cell–cell contact, and cell–bone matrix interactions.50 Lesser coupling between bone erosion and bone Vascular and Skeletal Disorders in ESRD
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Table 5. Stepwise regression report for PAD status (0, normal; 1, PAD) Parameter
T Value
P Value
Vintage (mo) dLS/BS (%) Log hCRP (mg/L) Age (yr) CaCO3 (g/d) N.Oc/T.A (mm2) PTX (no, 0; yes, 1) Aluminum-stained surfaces (%)
4.040 22.877 6.087 0.850 1.1935 0.6961 0.5972 0.0509
,0.001 ,0.01 ,0.001 0.40 0.24 0.49 0.55 0.96
R2=0.6843.
the population, which included relatively young subjects with primary kidney disease, Partial R Adjusted for Rest excluding diabetes, treated in the 1990s. This 0.2111 population differs markedly from the pop0.1195 ulation treated today, which includes higher 0.3669 percentages of patients with diabetes and NA NA older patients with overt cardiovascular NA comorbidities and shorter vintage. The treatNA ment modalities have evolved with withNA drawal of aluminum-containing phosphate binders and the use of noncalcium-containing phosphate binders, the wider use of vitamin D receptor agonists and cholecalciferol supplementation, the prescription of calcimimetics, and the improvement of hemodialysis techniques. In summary, our results showed that, in prevalent nondiabetic patients with ESRD, the PAD was associated with low bone turnover and low bone formation with pronounced osteoblast resistance to PTH. 2
CONCISE METHODS Patients
Figure 3. Correlation between bone histomorphometry parameters. dLS/BS and ES/BS in patients with normal ABix (white circles) and patients with abnormal ABix (black triangles).
formation with decreased number and activity of osteoblasts was observed in several conditions, such as skeletal unloading during prolonged bed rest,51 aluminum-related osteodystrophy in patients on dialysis,52 and chronic inflammation.53,54 In this study, the PTH- and CRP-adjusted correlation between aluminumstained surfaces and dLS/BS was not significant, but patients with PAD had microinflammation and increased serum CRP. We can speculate that the microinflammation could play a role in bone erosioformation uncoupling. As shown on Figure 2, serum CRP was inversely correlated with dLS/BS and Ob.S/BS but less correlated or not correlated with N.Oc/T.A and ES/BS. Inflammation and proinflammatory cytokines, such as TNF-a and IL-1, could cause an imbalance in bone remodeling by downregulation of PTH/PTHrP136 and activation of osteoclasts through classic or alternative RANK/RANK ligand pathways favoring bone erosion.53,54 The possible role of abnormal PTH signaling in arterial remodeling and vascular pathology remains hypothetical, because observational studies on the basis of correlations studies and associations are not proof of causality. This limitation is an important limitation of this study. Another limitation concerns the limited number of patients and clinical characteristics of 6
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With the exception of 1 patient, the population of 65 prevalent nondiabetic patients on hemodialysis was previously detailed.55 Inclusion criteria were (1) hemodialysis vintage $12 months and (2) absence of clinical history of cardiovascular disease, including history of lower limb arteries angioplasty, stenting, amputation, or trophic disorders (ulcerations or gangrene). Dialysis duration was individually tailored (4–6 hours three times per week) to control body fluids and blood chemistries. The bicarbonate dialysate was prepared using double reverse osmosis-treated water with 1.5 or 1.75 mmol/L Ca according to the serum Ca-PO4 equilibrium and the need for active vitamin D3 (1a-OH-D3 prescribed in eight patients). Although CaCO3 was used exclusively as a PO4 binder at the time of the study, 28 patients had taken aluminum hydroxide in the past; 12 patients underwent subtotal PTX, and 10 patients underwent total PTX with heterotopic autotransplantation into the forearm. PTX had been performed 20–70 months before the study. Erythropoietin was administered to maintain hemoglobin$100 g/L when necessary. All subjects gave informed written consent to participate in the study, which was approved by our Institutional Review Board in accordance with the Declaration of Helsinki.
ABix Lower limb artery status was on the basis of ABix measurement and the ratio between SBP in the ankle (arteria tibialis posterior or dorsalis pedis) and the arm (arteria brachialis). SBPs were measured in a supine position after 15 minutes of rest. Brachial artery SBP was measured two times in the arm without arteriovenous shunt and averaged. Ankle SBP was measured two times in each foot and averaged. SBPs were measured with a Doppler SEGA M842 8-MHz Unit (Société Electronique Générale et Appliquée, Paris, France) during cuff deflation. Patients were divided in three groups according to their ABix values: normal ABix, 0.9–1.3; low ABix, ,0.9; .1.4 (i.e., incompressible). J Am Soc Nephrol 26: ccc–ccc, 2014
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Bone Histomorphometry Diagnostic anterior iliac crest bone biopsies were obtained after dLS/BS according to the schedule of 2 days on tetracycline, 10 days off tetracycline, and 2 days on tetracycline as previously detailed.55 The extent of the dLS/BS (percent) was measured on two unstained 10-mm-thick sections. On three Toluidine blue-stained 5-mm-thick sections, bone volume (percent), Ob.S/BS (percent), ES/BS (percent), and N.Oc/T.A were assessed. Bone Al staining was expressed as the percentage of the trabecular surface stained. All measurements were made using an eyepiece reticle (Carl Zeiss Integral Plate II, Oberkochen, Germany).
Blood Chemistries Blood chemistries, including serum Ca and PO4, blood lipids, blood glucose, uric acid, and serum albumin, are the mean values of all measurements in the 6 months preceding the ABix measurements. Routine biochemical parameters were determined by standard methods using autoanalyzers. Blood samples for measurement of bone alkaline phosphatase serum levels (Quidel MicroVue BAP EIA), 25 (OH) cholecalciferol, and 1,25(OH)2D3 were taken the day of the bone biopsy. Intact PTH (N-IRMA; Nichols Institute) was determined every 3 months, and the reported values are the average of two measurements done during the 6 months preceding the bone biopsy. Bone biopsy and measurement of ABix were done the same month.
Statistical Analyses Data are expressed as medians and 95% confidence intervals. The primary analysis concerned ABix subgroup normal, low, or .1.4 comparison with Kruskal–Wallis one-way ANOVA on ranks and Bonferroni multiple comparison Z-test value. For the univariate (Pearson or Spearman correlation coefficient), and multivariate regression studies, low and incompressible ABix groups were pooled and considered to have PAD. The correlations slopes are expressed as b-coefficients6SD and compared by t test. For these correlations, artery disease status was used as a dummy variable (0, normal ABix; 1, PAD). Multiple regression analyses were performed using the subset of univariate analysis-selected independent variables (after Bonferroni correction for the number of correlations studied). All analyses were calculated using NCSS 2000 (Gerry Hintze, Kaysville, UT).
ACKNOWLEDGMENTS We thank Ms. Janet Jacobson for editorial assistance. The study was supported by Groupe d’Étude de la Pathophysiologie de l’Insuffisance Rénale (GEPIR).
DISCLOSURES None.
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