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Nephrol Dial Transplant (2003) 18: III86-III89
© 2003 European Renal Association-European Dialysis and Transplant Association

Original Article

Phosphate overload accelerates vascular calcium deposition in end-stage renal disease patients

Takashi Shigematsu1,, Takashi Kono2, Kenichi Satoh2, Keitaro Yokoyama1, Toyohiko Yoshida2, Tatsuo Hosoya1 and Kohji Shirai3

1 Division of Nephrology and Hypertension, Jikei University School of Medicine, 2 Division of Urology and Blood Purification, Mihama Hospital, Chiba and 3 Center of Diabetes, Endocrinology and Metabolism, Sakura Hospital, Toho University School of Medicine, Japan



   Abstract
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 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Cardiovascular disease is a major problem in end-stage renal disease (ESRD) patients, with calcification being one of the conspicuous features of arteriosclerotic vessels. In the present study, clinical analysis and in vitro cell culture were used to investigate factors promoting vascular calcification in ESRD patients. The aortic arch calcification score (AACS) was the method used to estimate vascular calcification by evaluation of the simple posterior–anterior view chest X-rays. Factors that relate significantly to vascular calcification and the AACS are the CaxPi, age, dialysis period, blood pressure, smoking and diabetes mellitus, but not total cholesterol or triglyceride. The CaxPi, which depends on the serum phosphate concentration, is the only specific factor with the possibility for correction in ESRD patients, and so control of serum phosphate concentration is an important factor for reducing vascular calcification. The effects of phosphate overload on calcium deposition in human vascular smooth muscle cells (hVSMCs) using a primary cell culture system were also investigated. hVSMCs were harvested from the radial artery in ESRD patients and it was found that they could secrete extracellular matrix with a high affinity for calcium in a high phosphate medium (Pi=5.4 mg/dl). Therefore, phosphate overload might stimulate the hVSMCs to accelerate the calcium deposition in ESRD patients. These results suggest that the control of phosphate excess is important for prevention of calcium deposition on arteriole walls in ESRD patients.

Keywords: cardiovascular disease; end-stage renal disease (ESRD); hyperphosphataemia; vascular calcification; vascular smooth muscle cells (VSMCs)



   Introduction
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 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 References
 
The number of patients with end-stage renal disease (ESRD) undergoing chronic renal replacement therapy is increasing worldwide, which is a major problem not only in the medical field but also socially and economically, so cost-effective care is important in this field. One of the unfortunate medical problems that many ESRD patients have is vascular disease, which is a major cause of mortality from ischaemic heart disease, cerebral vascular disease and arteriosclerosis obliterans [1]. Vascular calcification is often observed in ESRD, even in young patients [2], and is related to the mortality risk in ESRD patients. Identification of the factors that cause vascular calcification is therefore important.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 References
 
Clinical study of factors modulating vascular calcification
We analysed 286 ESRD patients (F/M=194/92) aged 59.6±11.5 years (mean±SD) undergoing long-term haemodialysis for a period of 9.7±7.6 years. We used the aortic arch calcification score (AACS) method to estimate vascular calcification by examination of simple, posterior–anterior view chest X-ray films. We scored the area of calcification from grade 0 (no calcification) to 5 (severe calcification) by detailed measurement of the calcification in the aortic arch (Figure 1Go). We checked the clinical significance of age, dialysis period, serum calcium–phosphate product (CaxPi), blood pressure, smoking, original kidney disease (e.g. diabetes mellitus, hypercholesterolaemia, hyperuricaemia), intact parathyroid hormone (i-PTH) concentration and gender.



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  		Fig. 1.  Vascular calcification was estimated by the aortic arch calcification score (AACS) by posterior–anterior view on simple chest X-ray film. We scored the size of the area from grade 0 (no calcification) to 5 (severe calcification), by detailed measurement of the area of calcification in the aortic arch.

 
The PTH concentration was measured by i-PTH assay using the Allegro i-PTH IRMA Kit (Sumitomo-Metaphysics Pham, Osaka, Japan). Other assays were performed by standard methods with an enzyme technique as the basic procedure. All data are shown as mean±SD. Statistical analysis was performed by analysis of covariance (ANOVA) with multiple comparisons, with a significance level set at 5%. Multiple factorial regression analysis with the Cox-Hazard model was also used.

Binding of 45Ca to the extracellular matrix produced by hVSMCs
We harvested human vascular smooth muscle cells (hVSMCs) from the radial artery of nine ESRD patients by an explant method. The cells were cultured for 7 days in 2.7 mg/dl phosphate Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS) after confluent growth. In the tests using excess phosphate, the phosphate concentration was increased 2-fold to 5.4 mg/dl using glycerophosphate. After 7 days, hVSMCs were removed using KOH (0.01 mol/l). 45Ca (1.0 µCi) used as a tracer was added to the remaining extracellular matrix. After 3 h incubation with 45Ca, the conditioned medium was washed, and the radioactivity of the extracellular matrix was counted in the calcium precipitate.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 References
 
Clinical study of the modulating factors of vascular calcification
The multiple factorial analysis was adjusted for age and also for other aspects of the procedure. Factors that can affect the AACS are age, dialysis period, CaxPi, blood pressure, smoking and diabetes mellitus. Age, dialysis period and diabetes are impossible to modify, but hypertension and smoking are now controllable risk factors, even in the normal population. The control of CaxPi is specific for the minimization of vascular calcification in ESRD patients (Figure 2Go). High serum cholesterol, hyperuricaemia, i-PTH and gender are not significant factors in the estimation of the AACS.



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  		Fig. 2.  Relationship between aortic arch calcification score (AACS) and serum CaxPi. The CaxPi >65 mg/dl2, was significantly (P<0.01) related to the AACS. This effect on the abdominal aorta with ECG disorder (ischaemic change shown as ST–T change) was also closely related to the serum CaxPi (data not shown).

 
We found that the powerful determinant for CaxPi is the serum phosphate concentration (Figure 3Go).



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  		Fig. 3.  Correlation between the CaxPi and serum calcium or phosphate concentration. The CaxPi is determined almost only by the Pi concentration and is closely correlated with serum phosphate (Y=10.476–5.155, r2=0.914), but not serum calcium (r2=0.229).

 

Binding of 45Ca to the extracellular matrix produced by hVSMCs
The results of the in vitro study are shown in Figure 4Go. The extracellular matrix secreted from the conditioned hVSMCs in the high phosphate medium had a high affinity for 45Ca. The effect of phosphate excess was almost 2-fold.



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  		Fig. 4.  Impact of phosphorus on calcium precipitation in primary cultured human vascular smooth muscle cells (hVSMCs) from dialysis patients, which can secrete extracellular matrix with a high affinity for calcium in a high phosphate medium.

 



   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
References
 
Cardiovascular diseases have a great deal of influence on the long-term survival of ESRD patients [1]. A major problem in ESRD patients is vascular calcification, which is known to occur more in the intima (where the VSMCs are located) than in the endothelium [3]. In the present study, we investigated the factors affecting vascular calcification by clinical studies, and then evaluated the impact of elevated phosphate on calcification by in vitro cell culture.

Typical vascular calcification seems to be similar to that of bone in ESRD patients, which strongly suggests that vascular calcification is related to bone mineral metabolism. We analysed vascular calcification according to the classical method of AACS (Figure 1Go). An advantage of this method is that it can be carried out in any hospital or haemodialysis centre, as opposed to some more recent procedures such as electron beam CT analysis. The AACS is related positively to serum CaxPi (Figure 2Go), which is recognized as a significant factor controlling vascular calcification in ESRD patients [4]. It has been reported that the CaxPi is ~65 mg2/dl2, and our results corresponded to that figure. We also demonstrated that phosphate was the significant factor in determining the CaxPi (Figure 3Go); so maintaining it at <65 mg2/dl2 controls hyperphosphataemia in much the same way as keeping the serum phosphate concentration at <6.0–6.5 mg/dl. Block et al. have reported that control of serum phosphorus and CaxPi has a major impact in reducing the mortality risk in chronic haemodialysis patients [5]. They concluded that patients with a serum phosphorus concentration >6.5 mg/dl have an increased mortality risk, and our results are in agreement with their conclusion.

In addition, we evaluated the effect of raised phosphate on vascular calcification in vitro. hVSMCs harvested by an explant method from the radial artery of ESRD patients and cultured in a 2-fold more concentrated phosphate medium (5.4 mg/dl) than controls can secrete extracellular matrix that has a high affinity for calcium. This effect of phosphate excess was almost 2-fold greater than in the normal phosphate controls. The progression of calcium deposition in vessels appears to be dependent on phosphate overload. The composition of the extracellular matrix is still obscure, but some factors, such as Gla protein, osteocalcin and osteopontin, that can cause calcium deposition, may be present in the matrix [6,7]. The mechanism whereby hVSMCs recognize the high phosphate signal is unknown, but there is some information available about the relationship between VSMCs and osteoblasts [8]. It has been reported that osteoblasts can act as human Glvr-1 phosphate transporting agents [9], and hVSMCs may possess the phosphate transporting system that responds to the high phosphate signal.

In conclusion, phosphate overload may accelerate vascular calcium deposition, and control of phosphate excess is important in the treatment of this condition in ESRD patients.



   Notes
 
Correspondence and offprint requests to: Takashi Shigematsu, MD, PhD, Assistant Professor, Division of Nephrology and Dialysis Unit, Department of Internal Medicine, Jikei University School of Medicine, Aoto-General Hospital, 6-41-2 Aoto, Katsushika-Ku, Tokyo 125-8506, Japan. Email: aotaki{at}jikei.ac.jp Back



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Muntner P, Coresh J, Klag MJ et al. History of myocardial infarction and stroke among incident end-stage renal disease cases and population-based controls: an analysis of shared risk factors. Am J Kidney Dis 2002; 40:23–30
  2. Goodman WG, Goldin J, Kuizon BD et al. Coronary-artery calcification in young adults with end-stage renal disease who are undergoing dialysis. N Engl J Med 2000; 18:1478–1483
  3. Davies MR, Hruska KA. Pathophysiological mechanisms of vascular calcification in end-stage renal disease. Kidney Int 2001; 60:472–479[CrossRef][Web of Science][Medline]
  4. Cozzolino M, Dusso AS, Slatopolsky E. Role of calcium–phosphate product and bone-associated proteins on vascular calcification in renal failure. J Am Soc Nephrol 2001; 12:2511–2516[Free Full Text]
  5. Block GA, Hulbert-Shearon TE, Levin NW et al. Association of serum phosphorus and calciumxphosphate product with mortality risk in chronic hemodialysis patients: a national study. Am J Kidney Dis 1998; 31:607–617[Web of Science][Medline]
  6. Moe SM, O'Neill KD, Duan D et al. Medial artery calcification in ESRD patients is associated with deposition of bone matrix proteins. Kidney Int 2002; 61:638–647[CrossRef][Web of Science][Medline]
  7. Shanahan CM, Cary NR, Salisbury JR et al. Medial localization of mineralization-regulating proteins in association with Monckeberg's sclerosis: evidence for smooth muscle cell-mediated vascular calcification. Circulation 1999; 100:2168–2176[Abstract/Free Full Text]
  8. Hofbauer LC, Shui C, Riggs BL et al. Effects of immunosuppressants on receptor activator of NF-kappaB ligand and osteoprotegerin production by human osteoblastic and coronary artery smooth muscle cells. Biochem Biophys Res Commun 2001; 280:334–339[CrossRef][Web of Science][Medline]
  9. Palmer G, Bonjour JP, Caverzasio J. Expression of a newly identified phosphate transporter/retrovirus receptor in human SaOS-2 osteoblast-like cells and its regulation by insulin-like growth factor I. Endocrinology 1997; 138:5202–5209[Abstract/Free Full Text]

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