When man turns to stone: Extraosseous calcification in uremic patients

Kidney International, Vol. 65 (2004), pp. 2447–2462

NEPHROLOGY FORUM

When man turns to stone: Extraosseous calcification in uremic patients ¨ Principal discussant: JURGEN FLOEGE Division of Nephrology and Immunology, University of Aachen, Aachen, Germany

phosphate excesses and having skipped his phosphate binders (sevelamer plus calcium acetate), claiming that the pills were too big to swallow. Within 3 days he developed an abscess-like yet painless lesion on his thumb (Fig. 1A), which was identified as a calcification on radiograph (Fig. 1B). A bone scintigraphy (Fig. 2), chest (Fig. 3A), and cranial CT (Fig. 3B), as well as an ophthalmologic examination (Fig. 4) confirmed widespread extraosseous calcifications, involving but not limited to the lungs, pericardium, large arteries, abdominal wall, and conjunctiva. Serum fetuin-A was 0.33 g/L (normal range 0.5 to 1.0 g/L), and his IC 50 calcium-phosphate precipitation inhibitory activity in serum was 10.2 lL (normal IC 50 6.4 ± 2.6 lL). CASE PRESENTATIONS Patient 1

Patient 2

A 53-year-old man lost a kidney 35 years ago after an accident. Hypertension was detected 18 years ago, but he declined treatment. Last year he presented to the Aachen University Hospital with end-stage renal disease (ESRD), presumably due to hypertensive renal damage. Ambulatory peritoneal dialysis (PD) was started. In view of recurrent compliance problems, including skipping of PD exchanges, a transfer to hemodialysis (HD) was planned, which he refused. Finally, he was admitted again with uremic gastritis, pericardial effusion, polyneuropathy, and a urinary tract infection. The PD dose was increased, appropriate therapy for his medical problems was initiated, and his condition improved. Despite these measures, his serum phosphate rose from usual levels of 2.5 to 3.0 mmol/L to a maximum of 4.8 mmol/L. The serum calcium remained normal. He admitted dietary

A 64-year-old woman weighing 48 kg has undergone chronic HD in a private dialysis center for the past 17 years. She had ESRD due to tubulointerstitial nephropathy. She underwent surgery for tertiary hyperparathyroidism 7 years ago, and had reimplantation of one gland. Secondary hyperparathyroidism recurred; the iPTH serum levels ranged between 133 and 632 ng/L (mean 308 ng/L) over the past 6 years. Over the last 5 years, no episodes of hypercalcemia (that is, serum calcium >2.55 mmol/L) had been documented, and her serum phosphate had been well controlled with low doses of an aluminum-containing phosphate binder (mean serum phosphate over the previous 5 years was 1.36 ± 0.39 mmol/L; range, 0.7 to 2.57 mmol/L; N = 63). Last year, she was admitted to the Aachen University Hospital because of unstable angina. Angiography revealed diffuse coronary artery disease without options for interventional measures. Radiographic and CT evaluations documented extensive mitral valve ring calcifications, pericardial calcifications, and widespread vascular disease, including coronary calcification. C-reactive protein was chronically elevated, with a mean of 31 mg/L (range 28 to 38 mg/L). Serum fetuin-A was 0.38 g/L, and her IC 50 calcium-phosphate precipitation inhibitory activity in serum was 13.6 lL.

The Nephrology Forum is funded in part by grants from Amgen, Incorporated; Merck & Co., Incorporated; Dialysis Clinic, Incorporated; and Bristol-Myers Squibb Company. Key words: mediasclerosis, end-stage renal disease, hemodialysis, fetuin-A.
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2004 by the International Society of Nephrology 2447

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Fig. 1. Photograph of right thumb of Patient 1 (A). At first glance, the tumorous calcification resembles a small abscess. (B) Radiograph demonstrates the typical cloudy appearance of the extraosseous calcification (arrowheads) with interwoven fibrous tissue.

DISCUSSION ¨ DR. JURGEN FLOEGE (Professor of Medicine, Director, Renal Division, University Hospital, Aachen, Germany): These two patients provide important insights into the pathogenesis of uremic extraosseous calcification. Whereas Patient 1 is apparently a case of noncompliance and “metastatic” calcification, why does Patient 2 have such extensive calcification? She is not particularly old, not diabetic, ate little, and consequently had a low phosphate intake, was compliant with her medication, had not received calcium-containing phosphate binders over recent years, and had never had a markedly elevated calcium-phosphate product. Before embarking on this discussion, three points need to be clear: (1) I will attempt to avoid mixing data obtained in patients with ESRD with data from patients with normal renal function, as the pathogenetic principles overlap only in part. Also, insights into cardiovascular

disease gained in one group can be completely different in the other, a phenomenon termed “reverse epidemiology” [1]. (2) For didactic purposes, I will discuss vascular, valvular, organ, and soft tissue calcifications together, realizing that they may have different origins. However, in most cases, currently available information is too limited to distinguish different pathogenetic pathways. (3) Again, for didactic purposes and largely because of a lack of available data, I will use the global term “calcification” for extraosseous mineralization, realizing that important histologic and chemical subsets of calcification exist. Calcification in patients with ESRD can produce a range of pathologies, including calcific uremic arteriolopathy (formerly termed “calciphylaxis”; not covered in this review) [2], extraosseous soft tissue (Fig. 5) and solid organ calcification, corneal and conjunctival calcification, peritoneal calcification, and vascular and valvular calcification [3–7]. Nephrologists have been plagued by these complications from the early days of dialysis [3–5,

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Fig. 2. Bone scintigraphic image of Patient 1 following the injection of 570 MBq 99mTcHDP. Note extensive calcification of the lungs (arrows), right thumb (arrow), upper sternum, thyroid region, and base of the skull.

7, 8]. For example, data obtained in 120 uremic children between 1960 and 1983 revealed soft tissue calcification in 60%, most frequently involving blood vessels, lung, kidney, myocardium, central nervous system, and gastric mucosa [3]. Autopsy findings in the 1970s confirmed soft tissue, including vascular, calcification in 50% to 80% of HD patients [5, 6]. Thus, extraosseous calcification is neither a new nor a dramatically increasing problem in uremic patients. The latter impression may exist because new, non-invasive techniques have increased our ability to detect calcifications. Morphologic and biochemical features Vascular calcification involves large- and mediumsized arteries, whereas veins virtually never calcify unless placed into the arterial system. In rare cases, vascular

prostheses also calcify. Intimal calcification only occurs within inflammatory atherosclerotic plaques, whereas in mediasclerosis (Monckeberg ¨ sclerosis) muscular arteries undergo noninflammatory medial calcification. In mediasclerosis, elastic laminae chiefly in the inner two-thirds of the media calcify around fractured disorganized elastin fibers [9, 10], and formation of bone-like trabecular structures results. Glycoxidative modification of elastin in uremia might contribute to this process [11]. Mediasclerosis commonly occurs with normal aging, but it is markedly aggravated in uremia [6, 12] and diabetes mellitus [12], conditions associated with accelerated aging of various tissues [10]. The arterial tree has considerable variability in the susceptibility and the type of calcification. In older HD patients, calcification in the aortic wall and radial artery

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A

Fig. 3A. Chest CT image of Patient 1. Note the large pericardial calcifications (arrows) as well as calcification of the descending aorta (arrowhead).

consistently involves both the intima and media, whereas in younger dialysis patients, isolated mediasclerosis predominates [11, 13]. However, at least in the iliac artery, intimal lesions with microcalcifications have been described even in uremic children [14]. Vascular calcification is not a random process in chronic HD patients. For example, calcification of the internal thoracic or hepatic arteries is unusual [15, 16]. Valvular calcifications often start at the site of valve insertion, in particular the mitral annulus, and then extend in the direction of the cusps. Commissural fusion and stenosis, particularly of the aortic valve, develop in advanced stages [4, 7]. Compared with nonrenal disease populations, patients with ESRD are much more likely to have basal mitral leaflet calcification and papillary muscle calcification; also, calcification of intervalvular fibrosa and of the tricuspid annulus is noted only in ESRD patients [17]. In cases of uremic tumor-like calcinosis, the most common sites are the elbows, hips, hands, and wrists [18]. The calcium deposits are typically multiple, para-articular, labile, and have a fluid-viscous consistency [19]. “Active” lesions, that is, calcification centers surrounded by mononuclear or multinuclear macrophages, osteoclast-

like giant cells, fibroblasts, and chronic inflammatory elements, can be distinguished from “inactive” lesions, in which calcified material is surrounded by fibrous tissue (Fig. 5). Extraosseous calcifications in uremic patients can vary in chemical composition, even within the same site. Soft tissue and vascular calcifications in uremic patients can contain amorphous calcium phosphate or hydroxyapatite (Ca 5 (PO 4 ) 3 OH), that is, the same form of calcium crystals as in bone [8, 20, 21]. Another form of calcium (magnesium) phosphate, whitlockite (CaMg 3 )(PO 4 ) 2, has been reported in the elastic laminae in the media of non-uremic human aorta [9] as well as in uremic visceral organs [8]. Finally, brushites (CaHPO 4 ·2H 2 O) were found solely in stenotic arteriovenous fistulas of patients undergoing HD [22]. Calcium oxalate crystals, unless associated with primary oxalosis, are not detected in the uremic vasculature [20]. Detection and quantification The calcification pattern in large arteries as seen on conventional radiographs, patchy, irregular versus linear, “tramline”-like deposits, allows a crude distinction

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B

Fig. 3B. Cranial CT image of Patient 1 demonstrating massive vascular calcification of the medial and anterior cerebral arteries.

between atherosclerotic intimal and medial vascular calcification [23]. But ultrasound, intravascular ultrasound, and optical coherence tomography can provide better differentiation of vascular calcification types. Other techniques employed for detecting vascular or other calcifications include echocardiography, digital subtraction angiography, magnetic resonance tomography, and bone scintigraphy [7, 24] (Fig. 2). The two most widely used methods for detecting vascular, and particularly coronary, calcifications are electron-beam computed tomography (EBCT) and multi-row helical computerized tomography (CT) [25–27]. Multi-row helical CT offers better signal-to-noise ratio and higher spatial resolution, whereas EBCT offers better temporal resolution and

fewer cardiac motion artefacts [28]. Semi-quantitative assessments can be performed using the Agatston score (the product of plaque area and a density factor), total calcium volume, or a calcium mass score [28]. Neither CT method, however, can distinguish between calcifications of the vascular intima and media. Clinical consequences In contrast to atherosclerotic intimasclerosis, mediasclerosis is generally nonocclusive. Mediasclerosis reduces the dampening ability of blood vessels and thus induces pseudohypertension and increased pulse wave velocity [29]. Through these mechanisms waves return

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Fig. 4. Conjunctival calcifications in Patient 1.

more quickly from the periphery, further augmenting the systolic wave and lowering diastolic pressure. The resultant higher pulse pressure, like aortic stiffness and pulse wave velocity, is associated with a higher mortality rate in ESRD patients [29–31]. By increasing left-ventricular afterload, a high aortic pulse wave velocity is also associated with left-ventricular hypertrophy [29]. Similarly, valvular and cardiac calcifications are associated with the development of left-ventricular hypertrophy, heart failure, coronary ischemia, arrhythmias, valvular stenosis, increased risk of infective endocarditis, and, in rare cases, thromboses with emboli [4, 5, 7, 24, 32]. The degree of vascular or valvular calcification in HD patients correlates with the severity of cardiovascular disease as determined clinically, and predicts cardiovascular and all-cause mortality [23, 29, 33, 34]. Whether the presence of coronary calcifications in uremic patients has a similar high predictive value for future cardiac events, as in the general population, where it predicts total atherosclerotic plaque burden [35], is unknown. Soft tissue “metastatic” calcifications in dialysis patients are typically multiple, painless, large, and can become symptomatic with nerve compression and impairment of joint mobility [18] (Fig. 5). Rare consequences of

calcifications include vasculopathic proximal myopathy, penile necrosis, rupture of calcified tendons, respiratory failure, breast pain due to calcification of mammary vessels, headache due to dural calcification, and symptoms related to spinal compression. Risk factors Age and time on dialysis are central determinants of extraosseous and, in particular, vascular and valvular calcifications (Table 1), but even pediatric dialysis patients are not spared [3, 5, 14, 24, 25, 27, 34, 36, 37]. The development of calcifications can progress very rapidly, and calcification scores both in vessels and valves can increase by 20% to 30% within only one year [25, 38]. Various other risk factors for vascular and valvular calcifications have been identified (Table 1). Dialysis patients with a predominantly intimal calcification pattern in the pelvic vessels were older and characterized by the presence of “typical” cardiovascular risk factors (for example, smoking, dyslipidemia); patients with medial calcifications were younger and characterized by a longer duration of HD treatment and derangements of the calcium and phosphate balance [23]. A consistent risk

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A

¨ Fig. 5A. Teutschlander disease. Massive tumor-like calcification in the region of the left shoulder of a 50-year-old patient on chronic HD who refused phosphate binders.

factor is diabetes mellitus. This finding is not unexpected given that both atherosclerosis and mediasclerosis are associated with diabetes even in the absence of uremia. Furthermore, several studies noted that in patients with ESRD, the calcification risk increases in the presence of hypercalcemia, hyperphosphatemia, and/or with the use of calcium-containing phosphate binders [3, 7, 23, 27, 33, 34, 36, 38, 39]. Other studies found no such association [16, 25]. This contrast might relate to sample sizes, but in particular to the difficulty of relating a long-term event such as calcification to rapidly fluctuating serum parameters. Similarly, few studies [34, 36], but not the majority [5, 16, 25, 27], detected an association between calcification and parathyroid hormone (PTH) levels, or parathyroid or bone morphology. A direct role of PTH is also questionable, given that pronounced vascular calcification or even uremic calcific arteriolopathy can occur in the absence of hyperparathyroidism and that oversuppression of PTH, which results in adynamic bone disease, is associated with an increased risk of calcification [40, 41].

Tumorous soft-tissue calcifications (Teutschlander ¨ disease) usually develop in the context of long-standing pronounced hyperparathyroidism and/or excessive hyperphosphatemia (Figs. 1 and 5). Again, the role of PTH itself is questionable, since tumor-like calcifications can regress despite progressive hyperparathyroidism [42]. Several other risk factors for cardiovascular calcification are noteworthy. A number of studies noted an association between calcification and the malnutrition, inflammation, and atherosclerosis (MIA) syndrome, and with serum parameters related to it [27, 34, 36, 43]. Dyslipidemia was associated with more rapid progression of uremic vascular/valvular calcification in one small study [44] but not in others [33, 34], and another small study even noted lower total LDL and HDL cholesterol, as well as lower triglycerides in the presence of coronary calcifications [45]. The role of systemic acidosis or alkalosis in promoting uremic extraosseous calcification is not known.

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B

Fig. 5B, C. Intraoperative aspect, showing large nodules of whitish material (B). Histologic examination disclosed 2 calcified areas (C) of different morphology separated by a fibrous septum (F). Whereas the left calcified area shows larger crystals and more cellular reaction, including osteoclastlike cells (insert), the right calcified zone evokes relatively little cellular response (PAS stain, magnification ×50; with insert ×200; specimen kindly provided by R. Knuchel-Clarke, ¨ Dept. of Pathology, University Hospital Aachen) (C).

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Table 1. Factors promoting or inhibiting extraosseous calcification in vitro and in vivo Effect on calcification Promoting factors Age of patient Duration of dialysis Inorganic phosphate and/or Ca × PO 4 product Oral Ca2+ −containing PO 4 − binder dose Vitamin D 3 Diabetes, AGEs Increased serum fibrinogen or CRP or low albumin Inflammatory cytokines Modified low-density lipoprotein (LDL) Hyperhomocysteinemia Estrogen Dexamethasone Leptin Genetic factors Inhibitory factors Magnesium ions High-density lipoprotein (HDL) PTHrP Osteoprotegerin Osteopontin BMP-7 Inorganic pyrophosphate Matrix GLA protein Fetuin-A a

References

↑(epidemiologic studies) ↑(epidemiologic studies) ↑(VSMCa in vitro; klotho-deficient mice; epidemiologic studies) ↑(epidemiologic studies)

25, 27, 33, 34, 37, 43, 94 27, 33, 36, 37, 43 3, 7, 23, 27, 33, 34, 38, 39, 47–49, 58

↑(VSMC in vitro and epidemiologic data) ↑(microvascular pericytes in vitro; mouse model; in vivo association in HD patients; epidemiologic studies) ↑(epidemiologic studies)

37, 59, 95 33, 34, 60, 61, 94, 96

↑(VSMC in vitro) ↑(VSMC in vitro)

58 55, 95

↑(epidemiologic studies) ↑(VSMC in vitro) ↑(VSMC in vitro) ↑(VSMC in vitro) ↑(human in vivo data)

36, 43, 94 97 98 62 63, 64

↓(in vitro; in vivo association in PD patients) ↓(VSMC in vitro) ↓(VSMC in vitro) ↓(in vitro data, knockout mice, rat model, human in vivo data) ↓(in vitro and in vivo murine data) ↓(uremic mice) ↓(in vivo human data) ↓(knockout mice) ↓(in vitro data, knockout mice, human in vivo data)

21, 54 56 65 39, 66

27, 38

34, 36, 43

68, 69 67 74, 99 70 26, 79, 80, 82

VSMC, vascular smooth muscle cells.

Pathogenesis Although cardiovascular calcifications occur in the presence of normal renal function, even a moderate decrease in the glomerular filtration rate (GFR) dramatically augments their prevalence [12]. Also, atherosclerotic plaques in uremia calcify much earlier and more heavily [20]. Elevated serum phosphate levels are a potent predictor of cardiovascular events in patients with ESRD [46]. The most attractive explanation for these findings is a pathogenetic link among high serum phosphate levels, cardiovascular calcification, and increased mortality rates. In mice, hyperphosphatemia, even in the absence of renal failure, is associated with vascular calcifications [47]. Human aortic vascular smooth muscle cells (VSMC) cultured in a solution containing 2.0 mmol/L phosphate formed bioapatite but did not in a 1.4 mmol/L phosphate solution [48]. Bioapatite formation was inhibited when VSMC phosphate uptake was blocked with an antagonist of the sodium-phosphate co-transporter [48, 49]. This is perhaps the most convincing evidence that calcium and phosphate precipitation, at least in the vessel wall, is likely an active cellular rather than a passive “metastatic” process. In VSMC, increased intracellular phosphate down-

regulates typical VSMC genes and contributes to various osteoblast-like phenotype changes, including the formation of matrix vesicles, the expression of core binding factor 1 (Cbfa-1, a central transcription factor in osteogenic differentiation), the expression of alkaline phosphatase, the production of calcium-binding proteins such as osteocalcin and osteopontin, and the laying down of a collagen-rich extracellular matrix [48, 50]. However, the origin of these osteoblast-like, “calcifying vascular cells” in vivo is not fully clarified [51]. In vivo Cbfa-1, alkaline phosphatase, and osteopontin are present in uremic calcified arteries and calcific arteriolopathy [13, 40, 52]. In plasma of HD patients, osteopontin correlated with the extent of vascular calcifications [53]. It is currently unknown whether these processes are similarly operative in uremic intimal and medial calcification and how they relate to soft tissue calcifications. Also, none of this information excludes the existence of true “metastatic” calcification, that is, passive precipitation, in instances of an excessive calcium × phosphate product. In fact, today’s first patient is probably good evidence for the existence and pathogenetic relevance of this process. In light of today’s knowledge, it is certainly correct to place the main pathogenetic and

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therapeutic emphasis on phosphate, but other factors present in uremia also affect the development of calcifications [52]. While the role of calcium, in particular hypercalcemia, in promoting extraosseous calcifications is obvious and evidenced by several epidemiologic studies (Table 1), magnesium might inhibit calcification. In vitro and in vivo, the presence of Mg2+ ions reduces the crystallite size of apatite [21], and at least one study related higher serum magnesium levels in ESRD patients to a lower progress of arterial calcifications [54]. Blood lipids also can modulate calcification. In vitro, modified LDL increased, while HDL inhibited VSMC calcification [55, 56]. In non-ESRD patients, reducing total cholesterol slowed the progression of coronary artery calcification [57], but evidence for a role of lipids in uremic calcification is less clear. As I said a moment ago, epidemiologic studies on the association of dyslipidemia and uremic calcification are contradictory, and low cholesterol can even predict cardiovascular mortality in ESRD patients [1]. A role of inflammation in promoting uremic calcifications receives better support from epidemiologic studies (Table 1). In vitro, macrophage-derived TNF-a and oncostatin-M induce calcification in VSMC [58]. Furthermore, inflammation can contribute to the inverse correlation between calcification scores and bone mineral densities in dialysis patients [25]. Finally, links have been established in vitro and in vivo with a number of other potential modulators of calcification in ESRD (Table 1), including calcitriol [37, 59], HbA1c levels [60], advanced glycation end products [61], leptin [62], and some genetic factors [63, 64]. What agents inhibit calcification? Extracellular fluids are supersaturated with calcium and phosphate so extraosseous calcification must usually be prevented by inhibitors. Various factors decrease the rate of calcification or even reverse established calcifications, including parathyroid hormone–related peptide [65], osteoprotegerin [39, 66], bone morphogenic protein (BMP)-7 [67], and osteopontin [68, 69] (Table 1). The relevance of most of these various factors to human uremic calcification in vivo is currently unknown. The local inhibitor matrix GLA protein (MGP) is a gamma-carboxylated mineral-binding extracellular matrix protein, expressed by VSMC. MGP knockout mice develop severe cartilage and arterial medial calcifications [70]. Also, MGP has been found in human atherosclerotic plaques surrounding calcifications [71, 72]. In humans, mutations of MGP leading to a nonfunctional protein result in calcification of cartilage and vessels [73]. Vitamin K deficiency predisposes to undercarboxylated, inactive MGP, which might explain why warfarin therapy is a risk factor for calcific uremic arteriolopathy [2]. Another re-

cently identified inhibitor of vascular calcification is inorganic pyrophosphate, which is generated by a cell surface enzyme of VSMC. Inactivating mutations of the cell surface enzyme result in calcification of muscular arteries [74]. Studies in the mid-1970s linked increased bone pyrophosphate content to osteopenia and pulmonary calcifications in HD patients [75]. Systemic, circulating inhibitors also contribute to the prevention of calcium deposits. Fetuin-A, or alpha2-Heremans-Schmid glycoprotein, is synthesized by hepatocytes, exists in the extracellular space in high concentrations (0.5 to 1.0 g/L), and is downregulated during episodes of inflammation. Fetuin-A exhibits antiinflammatory activity [76] and increases phagocytosis of apoptotic bodies [77]. The latter activity might be important in preventing vascular calcification [78]. In vitro, fetuin-A is a highly potent inhibitor of hydroxyapatite formation [79]. Fetuin-A is responsible for about one-half the precipitation inhibitory effect of serum [80] by forming in serum a high-molecular-mass complex of calcium phosphate mineral, fetuin-A, as well as traces of MGP and secreted phosphoprotein 24 [81]. In fetuin-A knockout mice, most soft tissues became severely calcified [82]. In HD patients, serum fetuin-A levels correlate negatively with C-reactive protein (CRP) levels and are significantly lower when compared to controls with normal renal function [26]. Also, uremic sera were less efficient at inhibiting calcium phosphate crystal formation than were normal sera. That is, they exhibited higher IC 50 , and this effect was reversed by the addition of fetuin-A [26]. In particular, the second patient is a good example of the effects of chronic fetuin-A downregulation in the course of MIA syndrome. We observed extremely low serum levels in HD patients with calcific uremic arteriolopathy, thus offering evidence supporting the role of reduced fetuinA levels in inflammation [82]. Finally, in HD patients, low fetuin-A is an independent predictor of all-cause and cardiovascular mortality [26]. What determines the localization of calcifications? In atherosclerotic intimal vascular calcification as well as in mediasclerosis, it is assumed that membrane vesicles derived from apoptotic cells and/or spatially restricted calcium release serve as the nidus for calcium precipitation [78]. Such local cellular disturbances, probably combined with alterations of the extracellular matrix, likely are central determinants of the localization of calcium deposits. Therapeutic approaches Established calcifications are difficult to treat. For example, after valve surgery, calcifications can recur rapidly [4]. Even successful renal transplantation does not always halt the progress of vascular calcifications. However, in individual cases, marked clinical improvement and

Nephrology Forum: Extraosseous calcification in uremic patients

regression of extraosseous calcification have followed an increase in the dialysis dose, parathyroidectomy, optimization of the calcium × phosphate product, therapy with a bisphosphonate, therapy of aluminum overload, or successful renal transplantation [42, 83–86]. Large symptomatic tumorous calcinosis occasionally requires surgical intervention [18]. Existing options for preventing calcification include an optimization of dialysis dose, for example, initiating nightly HD or short-duration HD 6 days per week, parathyroidectomy if indicated, modifying calcium load, and the use of phosphate binders [87]. A single study also noted a slower progression or even regression of peripheral arterial calcifications, as detected on plain radiographs, in ESRD patients with high serum magnesium levels [54]. Sevelamer, a phosphate-binding polymer, suppresses ectopic calcifications in uremic rats [88]. In a clinical study, sevelamer slowed the progression of calcifications in coronary arteries and in the aorta, although whether these effects relate to sevelamer’s phosphatebinding or lipid-lowering properties remains unclear [38]. The possible etiologic mechanisms should lead to the development of novel approaches for treating and preventing calcification in dialysis patients. Vitamin D analogues [89], calcimimetics [90], and bisphosphonates [91] might provide alternative ways of modifying calcium and phosphate levels, that is, producing a lower calcium × phosphate product; control of lipid levels with statins or bile acid sequestrants as well as therapy with calcium antagonists also could have beneficial effects [57, 92]. Possible anti-inflammatory strategies could include using pyrogen-free dialysate, which can increase fetuin-A serum levels [abstract; Westenfeld R et al, Nephrol Dial Transplant 18(Suppl 4):701–702, 2003] and therapies that reduce cytokine levels. Finally, after we gain a better understanding of their regulatory elements, we could stimulate synthesis of inhibitors of calcification such as fetuin-A or use them as therapeutic agents. QUESTIONS AND ANSWERS DR. JOHN T. HARRINGTON (Division of Nephrology, Tufts-New England Medical Center, Boston, Massachusetts): You mentioned that inflammatory agents such as TNF-a might be involved in calcification. Has your precipitation inhibition test for calcification determined whether one of the newer anti-TNF agents, such as those used in rheumatoid arthritis, is effective? Second, given that calcification can occur in alkaline tissue, what is the role of pH? DR. FLOEGE: The efficacy of anti-TNF therapy is unproven but likely because in rheumatoid arthritis antiTNF therapy quickly lowers C-reactive protein levels and thereby presumably increases fetuin-A levels. We are just starting a study that will answer this latter question. I am

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not aware of anybody who has studied anti-TNF therapy in dialysis patients. It might make sense for us to treat, for example, our second patient, who has persistently high CRP levels, with anti-TNF agents. Now let me address your second question. Despite much speculation on the role of acid-base status in calcification, we have no data on it. In fact, pH is a potential confounder in many studies that have looked at calcification in dialysis patients. Looking at pH in dialysis patients is at best difficult because systemic pH is almost never stable and you would have to determine a weekly averaged pH to take into account bicarbonate loading, redistribution, dietary effects, etc. DR. MARKUS KETTELER (Assistant Professor, Division of Nephrology, University of Aachen, Germany): What would you recommend as treatment for uremic calcific arteriolopathy? DR. FLOEGE: If uremic calcific arteriolopathy is mediated, at least in part, through excessive lowering of fetuinA, it might make sense to administer fetuin. While it is difficult to conceive that this can be achieved using recombinant fetuin, given its high serum levels, plasma is an excellent source. One can speculate whether the administration of fresh frozen plasma or even plasma exchange might be helpful in uremic calcific arteriolopathy. Interestingly, there are now some first clinical observations that corticosteroids are helpful in this situation [2], and one of the effects of corticosteroids is an increase in fetuin-A synthesis (Jahnen-Dechent W, personal communication). Of course, in the situation of uremic calcific arteriolopathy, using corticosteroids can be problematic, as patients usually die from infection. The administration of plasma therefore might be a safer approach. To my knowledge, this has not been tested. ¨ DR. TILMAN DRUEKE (INSERM, Research Director and Associate Professor of Nephrology, Necker Hospital, Paris, France): With regard to John Harrington’s question on the role of acid-base status, it has been known for several years from studies by his former fellow David Bushinsky [93] and others that chronic acidosis increases the release of calcium and phosphate from bone, and this alteration might contribute to soft tissue calcification. On the other hand, we know that alkalosis favors the precipitation of phosphate. This could explain how, in the presence of systemic acidosis, local alkalotic conditions could favor the development of calcifications. DR. FLOEGE: Your comment raises the level of complexity even further but also increases uncertainty. Tissue damage—smooth muscle cell apoptosis or necrosis as well as local ischemia—is believed to favor calcification. However, such tissues usually are locally acidotic. I am not aware of many conditions in which tissue damage occurs during alkalosis. ¨ DR. DRUEKE : In many of the studies you discussed, PTH was not identified as a factor that increases the

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extent of calcifications. On the other hand, we know from a large number of observations that severe hyperparathyroidism promotes soft tissue and vascular calcification. Do you think that in recent years people have achieved better control of hyperparathyroidism so that PTH is not as numerically important as in the past? DR. FLOEGE: In most cases of secondary hyperparathyroidism, serum calcium does not increase, whereas its level does rise in severe tertiary hyperparathyroidism. Therefore, serum calcium remains normal over a very wide range of serum PTH levels. If hypercalcemia is the relevant pathogenetic mechanism, this lack of a linear relationship between PTH and calcium levels probably explains why calcium or the calcium × phosphate product, but usually not PTH levels, correlates with the presence of calcification. ¨ DR. DRUEKE : I think that phosphate is probably as important as calcium because very severe hyperparathyroidism is generally accompanied by very high phosphate levels. DR. FLOEGE: I agree. In fact, phosphate is probably even more important than calcium. In the study by Block et al [46], phosphate levels were usually much stronger predictors of mortality than were calcium levels. Again, however, this relationship might reflect a dampening of a statistical correlation; we do not determine on a routine basis the right parameter, namely, ionized calcium. Utilization of total serum calcium measurements clearly will weaken any correlation. DR. DAVID GOLDSMITH (Consultant Nephrologist, Guy’s Hospital, London, United Kingdom): The implications of vascular calcification depend on where the calcification is localized within the vessel wall. You showed us that the risk factors for intimal and medial calcification are quite different. Lipids, for example, are clearly associated with the progression of atherosclerosis, but not, as far as anybody knows, with the progression of mediasclerosis. One of our problems is that we have a lot of information on the quantification of calcification but much less on where it is in the vessel wall. How can we resolve that? DR. FLOEGE: The best methods we currently have for quantification of vascular calcification, namely, electronbeam computer scan (EBCT) and spiral CT, tell us whether calcification is present; they do not tell us where it is present within the vessel wall. One of the few, albeit crude, approaches for investigating this issue is analysis of plain radiographs, that is, determining whether the pattern reflects patchy atherosclerotic or tram-track-like medial calcifications. However, even this crude approach becomes difficult if both types of calcification co-exist. The techniques that most likely will be helpful in this context are intravascular ultrasound and optical coherence tomography. For example, we are involved in a research project in which our colleagues in the engineering

department of the Aachen University are attempting to improve optical coherence tomography to a resolution level that essentially will allow in vivo microscopy of any biologic surface including skin, mucosa, or vessel walls. Availability of such a technique in dialysis patients would be a major advance in our understanding of extraosseous calcification. DR. STEFAN HEIDENREICH (Associate Professor of ¨ Nephrology, University Hospital, Munster, Germany): What cells of the liver produce fetuin-A and what regulators of its production are known? DR. FLOEGE: As far as we know, fetuin-A is mostly produced by hepatocytes. As to regulators of its synthesis, I have already mentioned corticosteroids but there must be others. The promoter of fetuin-A was recently cloned and this should quickly advance our understanding of regulatory factors. DR. HARRINGTON: Has calcification of the native kidneys been invoked as one of the factors giving rise to renal neoplasias in dialysis patients? DR. FLOEGE: I cannot think of a direct mode by which intrarenal calcification might contribute to the sequence of persistent mitogenic stimulation of tubular epithelium that results in cyst formation, subsequent adenomatous transformation and, finally, carcinogenesis. Also, I am not aware of studies investigating at the histologic level whether carcinomas in acquired renal cystic disease are spatially related to calcification. DR. CATHY SHANAHAN (British Heart Foundation Lecturer, University of Cambridge, United Kingdom): Have you looked at fetuin-A levels in other chronic inflammatory diseases associated or not associated with calcification? DR. FLOEGE: We have not studied this on a systematic basis. We do have controls, of course, for example, patients with sepsis, who exhibit exceedingly low fetuin-A levels, but this is a short-term situation with no development of calcification. We are just starting a study in patients with rheumatoid arthritis to determine how their fetuin-A levels relate to calcification and mortality. These patients are obvious candidates for such a study given their increased cardiovascular risk and the fact that they do not suffer from hyperphosphatemia. On the other hand, such patients often receive vitamin D and other anti-osteoporotic therapy, so calcifications in these patients will be multifactorial in origin. ¨ DR. DRUEKE : In the Goodman study [27], an increase in calcification was demonstrated by EBCT only in adolescents. Do you think this is just because of the limitations of the method? I also would like to draw attention to a study by Milliner et al [3], which showed tremendous calcifications in uremic children much younger than age 20. She also showed in this post-mortem study that the most important contributory factor was vitamin D,

Nephrology Forum: Extraosseous calcification in uremic patients

not calcium or phosphate. Thus, we have more in vivo evidence for the role of vitamin D. DR. FLOEGE: I can only speculate that in the Goodman study no calcifications were detected in children because they usually receive a transplant relatively quickly. So one of the confounding factors might be the duration of dialysis; a second one is obviously the resolution of EBCT. DR. GERARD LONDON (Director, Nephrology Department, Manhes Hospital, Paris, France): I posed the same question to Bill Goodman, and his answer exactly confirmed your interpretation, that is, the absence of calcifications in these children was due to the short time between dialysis and transplantation. DR. FLOEGE: In this context I therefore believe that the Milliner study [3] is of particular importance because it clearly pointed out that children are not spared from this complication as opposed to other consequences of long-term dialysis, such as dialysis-related amyloidosis, which has never been observed in children. ¨ DR. DRUEKE : You showed the surgical approach in one of your patients with tumor-like calcinosis (Fig. 5) and reported that the tumor was largely solid. In our experience, such calcifications are mostly liquid and when you open them, a kind of liquid magma flows out. Also our experience has taught us that one should avoid surgery in patients with large calcifications as much as possible because these lesions often become infected. DR. FLOEGE: You raise two important points. The patient shown in Figure 5 might be unusual, because large solid areas were found in the tumor, but there also were some liquid parts. Maybe, and I am speculating here, liquid areas represent early stages of precipitation, that is, amorphous calcium phosphate, and these areas subsequently evolve into more crystalline, solid tumors. As to your second point, I fully agree, and I definitely do not advocate surgery in such patients. In fact, we refused surgery in a man with tumorous calcinosis that measured about 20 cm in diameter in the hip region. Instead we advised him to wait for a renal transplant, hoping that this will induce regression. The patient shown in Figure 5 actually had been refused for surgery as well in two other hospitals. She insisted that this tumor was disturbing her life to a degree that she almost became suicidal. We finally accepted her with great hesitation. Fortunately, no complications occurred after the surgery, but, again, this is clearly the exceptional approach. DR. KETTELER: What is your opinion on the role of calcimimetics, new calcium binders, and vitamin D analogues in the prevention of calcification? DR. FLOEGE: The evidence, based on the “treat-togoal” study [38], is good in the case of calcium-free phosphate binders. Limitations of this study include (1) the fact that vitamin D therapy was significantly different in the two groups of patients, (2) the lack of data on acidbase status in that study, which is important given that

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sevelamer will induce systemic acidosis through bicarbonate loss, and (3) its non-blinded nature. Despite these limitations, it is the only one that can serve for evidencebased recommendations. As far as calcimimetics are concerned, we do not know their role yet. There is a theoretical basis on which we can advocate their use for the prevention of calcification, namely, their ability to lower the calcium × phosphate product. Whether the effect of calcimimetics on PTH is also important in this context must remain open. Finally, in the case of vitamin D analogues, again, we lack data. As Tilman Drueke ¨ pointed out in his New England Journal of Medicine editorial [89], even the major recent studies comparing two vitamin D forms suffer from problems that render a head-tohead comparison of different vitamin D analogues almost impossible. DR. HARRINGTON: I’ve always been reluctant to accept data that demonstrate gene products such as bone proteins in unexpected locations. What were the controls in those studies? DR. FLOEGE: In the studies on this issue, mostly those by Sharon Moe’s group [13], the usual histochemical controls were of course included, but probably the best evidence for the validity of those findings is the complete absence of bone-related proteins in non-calcified arteries. DR. SHANAHAN: I agree. In our own studies in nonuremic, calcified vessels with diabetic or atherosclerotic injury, the expression of bone-related proteins also was strictly confined to calcified vessels and was never present in non-calcified arteries [72]. DR. LONDON: You mentioned that oxidized LDL could play a role in the induction of vascular calcification. Do we have any data on the effect of statins on intimal calcifications in dialysis patients? DR. FLOEGE: In contrast to non-ESRD patients, in whom statins clearly prevent the induction or progress of vascular calcifications [57], we have no such data in ESRD patients yet. The 4D- and CHORUS trials might provide an indirect answer in ESRD patients, as cardiovascular events are the primary end points of both studies. But these investigations don’t specifically address cardiovascular calcification in uremic diabetics. Given that the “treat-to-goal” study [38] also raised the question of whether the phosphate and/or lipid-lowering activity of sevelamer accounts for its beneficial effect on cardiovascular calcifications, we are clearly in need of studies specifically addressing this question.

ACKNOWLEDGMENTS The Principal Discussant gratefully acknowledges the help of Markus Ketteler, Willi Jahnen-Dechent, Ralf Westenfeld, and Phillip Bongartz in the preparation of this manuscript.

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Correspondence to Dr. J. Floege, Medizinische Klinik II, RWTH Aachen, 52057 Aachen, Germany. E-mail: juergen.floege@rwth-aachen. de. No reprints available.

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REFERENCES

25.

1. KALANTAR-ZADEH K, BLOCK G, HUMPHREYS MH, KOPPLE JD: Reverse epidemiology of cardiovascular risk factors in maintenance dialysis patients. Kidney Int 63:793–808, 2003 2. WILMER WA, MAGRO CM: Calciphylaxis: Emerging concepts in prevention, diagnosis, and treatment. Semin Dial 15:172–186, 2002 3. MILLINER DS, ZINSMEISTER AR, LIEBERMAN E, LANDING B: Soft tissue calcification in pediatric patients with end-stage renal disease. Kidney Int 38:931–936, 1990 4. LONDON GM, PANNIER B, MARCHAIS SJ, GUERIN AP: Calcification of the aortic valve in the dialyzed patient. J Am Soc Nephrol 11:778– 783, 2000 5. KUZELA DC, HUFFER WE, CONGER JD, WINTER SD, HAMMOND WS: Soft tissue calcification in chronic dialysis patients. Am J Pathol 86:403–424, 1977 6. IBELS LS, ALFREY AC, HUFFER WE, et al: Arterial calcification and pathology in uremic patients undergoing dialysis. Am J Med 66:790– 796, 1979 7. MAHER ER, YOUNG G, SMYTH-WALSH B, et al: Aortic and mitral valve calcification in patients with end-stage renal disease. Lancet 2:875–877, 1987 8. CONTIGUGLIA SR, ALFREY AC, MILLER NL, et al: Nature of soft tissue calcification in uremia. Kidney Int 4:229–235, 1973 9. REID JD, ANDERSEN ME: Medial calcification (whitlockite) in the aorta. Atherosclerosis 101:213–224, 1993 10. PROUDFOOT D, SHANAHAN CM: Biology of calcification in vascular cells: Intima versus media. Herz 26:245–251, 2001 11. SAKATA N, NOMA A, YAMAMOTO Y, et al: Modification of elastin by pentosidine is associated with the calcification of aortic media in patients with end-stage renal disease. Nephrol Dial Transplant 18:1601–1609, 2003 12. MERJANIAN R, BUDOFF M, ADLER S, et al: Coronary artery, aortic wall, and valvular calcification in nondialyzed individuals with type 2 diabetes and renal disease. Kidney Int 64:263–271, 2003 13. 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 61:638–647, 2002 14. NAYIR A, BILGE I, KILICASLAN I, et al: Arterial changes in paediatric haemodialysis patients undergoing renal transplantation. Nephrol Dial Transplant 16:2041–2047, 2001 15. URA M, SAKATA R, NAKAYAMA Y, et al: The impact of chronic renal failure on atherosclerosis of the internal thoracic arteries. Ann Thorac Surg 71:148–151, 2001 16. OKUDA K, KOBAYASHI S, HAYASHI H, et al: Case-control study of calcification of the hepatic artery in chronic hemodialysis patients: Comparison with the abdominal aorta and splenic artery. J Gastroenterol Hepatol 17:91–95, 2002 17. BITTRICK J, D’CRUZ IA, WALL BM, et al: Differences and similarities between patients with and without end-stage renal disease, with regard to location of intracardiac calcification. Echocardiography 19:1–6, 2002 18. COFAN F, GARCIA S, COMBALIA A, et al: Uremic tumoral calcinosis in patients receiving longterm hemodialysis therapy. J Rheumatol 26:379–385, 1999 19. STEINBACH LS, JOHNSTON JO, TEPPER EF, et al: Tumoral calcinosis: Radiologic-pathologic correlation. Skeletal Radiol 24:573–578, 1995 20. SCHWARZ U, BUZELLO M, RITZ E, et al: Morphology of coronary atherosclerotic lesions in patients with end-stage renal failure. Nephrol Dial Transplant 15:218–223, 2000 21. LEGEROS RZ: Formation and transformation of calcium phosphates: Relevance to vascular calcification. Z Kardiol 90(Suppl 3):116–124, 2001 22. OLSSON LF, ODSELIUS R, RIBBE E, HEGBRANT J: Evidence of calcium phosphate depositions in stenotic arteriovenous fistulas. Am J Kidney Dis 38:377–383, 2001 23. LONDON GM, GUERIN AP, MARCHAIS SJ, et al: Arterial media calcification in end-stage renal disease: Impact on all-cause and

26.

27. 28. 29. 30. 31. 32. 33. 34.

35.

36. 37. 38. 39. 40. 41. 42. 43.

44.

45. 46.

cardiovascular mortality. Nephrol Dial Transplant 18:1731–1740, 2003 GRUPPEN MP, GROOTHOFF JW, PRINS M, et al: Cardiac disease in young adult patients with end-stage renal disease since childhood: A Dutch cohort study. Kidney Int 63:1058–1065, 2003 BRAUN J, OLDENDORF M, MOSHAGE W, et al: Electron beam computed tomography in the evaluation of cardiac calcification in chronic dialysis patients. Am J Kidney Dis 27:394–401, 1996 KETTELER M, BONGARTZ P, WESTENFELD R, et al: Association of low fetuin-A (AHSG) concentrations in serum with cardiovascular mortality in patients on dialysis: A cross-sectional study. Lancet 361:827–833, 2003 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 342:1478–1483, 2000 HALLIBURTON SS, STILLMAN AE, WHITE RD: Noninvasive quantification of coronary artery calcification: methods and prognostic value. Cleve Clin J Med 69(Suppl 3):S6–11, 2002 LONDON GM: Cardiovascular disease in chronic renal failure: Pathophysiologic aspects. Semin Dial 16:85–94, 2003 SAFAR ME, BLACHER J, PANNIER B, et al: Central pulse pressure and mortality in end-stage renal disease. Hypertension 39:735–738, 2002 GUERIN AP, BLACHER J, PANNIER B, et al: Impact of aortic stiffness attenuation on survival of patients in end-stage renal failure. Circulation 103:987–992, 2001 DOULTON T, SABHARWAL N, CAIRNS HS, et al: Infective endocarditis in dialysis patients: New challenges and old. Kidney Int 64:720–727, 2003 RAGGI P, BOULAY A, CHASAN-TABER S, et al: Cardiac calcification in adult hemodialysis patients. A link between end-stage renal disease and cardiovascular disease? J Am Coll Cardiol 39:695–701, 2002 WANG AY, WANG M, WOO J, et al: Cardiac valve calcification as an important predictor for all-cause mortality and cardiovascular mortality in long-term peritoneal dialysis patients: A prospective study. J Am Soc Nephrol 14:159–168, 2003 TAYLOR AJ, FEUERSTEIN I, WONG H, et al: Do conventional risk factors predict subclinical coronary artery disease? Results from the Prospective Army Coronary Calcium Project. Am Heart J 141:463– 468, 2001 OH J, WUNSCH R, TURZER M, et al: Advanced coronary and carotid arteriopathy in young adults with childhood-onset chronic renal failure. Circulation 106:100–105, 2002 GOLDSMITH DJ, COVIC A, SAMBROOK PA, ACKRILL P: Vascular calcification in long-term haemodialysis patients in a single unit: A retrospective analysis. Nephron 77:37–43, 1997 CHERTOW GM, BURKE SK, RAGGI P: Sevelamer attenuates the progression of coronary and aortic calcification in hemodialysis patients. Kidney Int 62:245–252, 2002 NITTA K, AKIBA T, UCHIDA K, et al: The progression of vascular calcification and serum osteoprotegerin levels in patients on longterm hemodialysis. Am J Kidney Dis 42:303–309, 2003 AHMED S, O’NEILL KD, HOOD AF, et al: Calciphylaxis is associated with hyperphosphatemia and increased osteopontin expression by vascular smooth muscle cells. Am J Kidney Dis 37:1267–1276, 2001 CANAVESE C, CESARANI F, STRATTA C, et al: Dramatic worsening of vascular calcifications after kidney transplantation in spite of early parathyroidectomy. Clin Nephrol 54:487–491, 2000 GEFFRIAUD C, ALLINNE E, PAGE B, et al: Decrease of tumor-like calcification in uremia despite aggravation of secondary hyperparathyroidism: A case report. Clin Nephrol 38:158–161, 1992 STOMPOR T, PASOWICZ M, SULLOWICZ W, et al: An association between coronary artery calcification score, lipid profile, and selected markers of chronic inflammation in ESRD patients treated with peritoneal dialysis. Am J Kidney Dis 41:203–211, 2003 TAMASHIRO M, ISEKI K, SUNAGAWA O, et al: Significant association between the progression of coronary artery calcification and dyslipidemia in patients on chronic hemodialysis. Am J Kidney Dis 38:64–69, 2001 BONIFACIO DL, MALINENI K, KADAKIA RA, et al: Coronary calcification and cardiac events after percutaneous intervention in dialysis patients. J Cardiovasc Risk 8:133–137, 2001 BLOCK GA, PORT FK: Re-evaluation of risks associated with hyperphosphatemia and hyperparathyroidism in dialysis patients:

Nephrology Forum: Extraosseous calcification in uremic patients

47. 48. 49. 50. 51. 52. 53. 54. 55.

56. 57. 58.

59.

60.

61. 62. 63. 64. 65.

66. 67.

68. 69.

Recommendations for a change in management. Am J Kidney Dis 35:1226–1237, 2000 KURO-O M, MATSUMURA Y, AIZAWA H, et al: Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 390:45– 51, 1997 JONO S, MCKEE MD, MURRY CE, et al: Phosphate regulation of vascular smooth muscle cell calcification. Circ Res 87:E10–17, 2000 GIACHELLI CM, JONO S, SHIOI A, et al: Vascular calcification and inorganic phosphate. Am J Kidney Dis 38:S34–37, 2001 CHEN NX, O’NEILL KD, DUAN D, MOE SM: Phosphorus and uremic serum up-regulate osteopontin expression in vascular smooth muscle cells. Kidney Int 62:1724–1731, 2002 DEMER LL, TINTUT Y, PARHAMI F: Novel mechanisms in accelerated vascular calcification in renal disease patients. Curr Opin Nephrol Hypertens 11:437–443, 2002 MOE SM, DUAN D, DOEHLE BP, et al: Uremia induces the osteoblast differentiation factor Cbfa1 in human blood vessels. Kidney Int 63:1003–1011, 2003 NITTA K, ISHIZUKA T, HORITA S, et al: Soluble osteopontin and vascular calcification in hemodialysis patients. Nephron 89:455–458, 2001 MEEMA HE, OREOPOULOS DG, RAPOPORT A: Serum magnesium level and arterial calcification in end-stage renal disease. Kidney Int 32:388–394, 1987 PROUDFOOT D, DAVIES JD, SKEPPER JN, et al: Acetylated low-density lipoprotein stimulates human vascular smooth muscle cell calcification by promoting osteoblastic differentiation and inhibiting phagocytosis. Circulation 106:3044–3050, 2002 PARHAMI F, BASSERI B, HWANG J, et al: High-density lipoprotein regulates calcification of vascular cells. Circ Res 91:570–576, 2002 CALLISTER TQ, RAGGI P, COOIL B, et al: Effect of HMG-CoA reductase inhibitors on coronary artery disease as assessed by electronbeam computed tomography. N Engl J Med 339:1972–1978, 1998 SHIOI A, KATAGI M, OKUNO Y, et al: Induction of bone-type alkaline phosphatase in human vascular smooth muscle cells: roles of tumor necrosis factor-alpha and oncostatin M derived from macrophages. Circ Res 91:9–16, 2002 JONO S, NISHIZAWA Y, SHIOI A, MORII H: 1,25–Dihydroxyvitamin D 3 increases in vitro vascular calcification by modulating secretion of endogenous parathyroid horomone-related peptide. Circulation 98:1302–1306, 1998 ISHIMURA E, OKUNO S, KITATANI K, et al: Different risk factors for peripheral vascular calcification between diabetic and nondiabetic haemodialysis patients—Importance of glycaemic control. Diabetologia 45:1446–1448, 2002 YAMAGISHI S, FUJIMORI H, YONEKURA H, et al: Advanced glycation endproducts accelerate calcification in microvascular pericytes. Biochem Biophys Res Commun 258:353–357, 1999 PARHAMI F, TINTUT Y, BALLARD A, et al: Leptin enhances the calcification of vascular cells: Artery wall as a target of leptin. Circ Res 88:954–960, 2001 LANGE LA, LANGE EM, BIELAK LF, et al: Autosomal genome-wide scan for coronary artery calcification loci in sibships at high risk for hypertension. Arterioscler Thromb Vasc Biol 22:418–423, 2002 PEYSER PA, BIELAK LF, CHU JS, et al: Heritability of coronary artery calcium quantity measured by electron beam computed tomography in asymptomatic adults. Circulation 106:304–308, 2002 JONO S, NISHIZAWA Y, SHIOI A, MORII H: Parathyroid hormonerelated peptide as a local regulator of vascular calcification. Its inhibitory action on in vitro calcification by bovine vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 17:1135–1142, 1997 PRICE PA, JUNE HH, BUCKLEY JR, WILLIAMSON MK: Osteoprotegerin inhibits artery calcification induced by warfarin and by vitamin D. Arterioscler Thromb Vasc Biol 21:1610–1616, 2001 DAVIES MR, LUND RJ, HRUSKA KA: BMP-7 is an efficacious treatment of vascular calcification in a murine model of atherosclerosis and chronic renal failure. J Am Soc Nephrol 14:1559–1567, 2003 STEITZ SA, SPEER MY, MCKEE MD, et al: Osteopontin inhibits mineral deposition and promotes regression of ectopic calcification. Am J Pathol 161:2035–2046, 2002 SPEER MY, MCKEE MD, GULDBERG RE, et al: Inactivation of the osteopontin gene enhances vascular calcification of matrix Gla

70. 71. 72. 73. 74. 75. 76. 77. 78. 79.

80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90.

91.

92.

2461

protein-deficient mice: Evidence for osteopontin as an inducible inhibitor of vascular calcification in vivo. J Exp Med 196:1047–1055, 2002 LUO G, DUCY P, MCKEE MD, et al: Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature 386:78–81, 1997 DHORE CR, CLEUTJENS JP, LUTGENS E, et al: Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques. Arterioscler Thromb Vasc Biol 21:1998–2003, 2001 SHANAHAN CM, CARY NR, METCALFE JC, WEISSBERG PL: High expression of genes for calcification-regulating proteins in human atherosclerotic plaques. J Clin Invest 93:2393–2402, 1994 MUNROE PB, OLGUNTURK RO, FRYNS JP, et al: Mutations in the gene encoding the human matrix Gla protein cause Keutel syndrome. Nat Genet 21:142–144, 1999 RUTSCH F, RUF N, VAINGANKAR S, et al: Mutations in ENPP1 are associated with “idiopathic” infantile arterial calcification. Nat Genet 34:379–381, 2003 ALFREY AC, SOLOMONS CC: Bone pyrophosphate in uremia and its association with extraosseous calcification. J Clin Invest 57:700–705, 1976 OMBRELLINO M, WANG H, YANG H, et al: Fetuin, a negative acute phase protein, attenuates TNF synthesis and the innate inflammatory response to carrageenan. Shock 15:181–185, 2001 JERSMANN HP, DRANSFIELD I, HART SP: Fetuin-alpha2-HS glycoprotein enhances phagocytosis of apoptotic cells and macropinocytosis by human macrophages. Clin Sci (Lond) 105:273–278, 2003 PROUDFOOT D, SKEPPER JN, HEGYI L, et al: Apoptosis regulates human vascular calcification in vitro: Evidence for initiation of vascular calcification by apoptotic bodies. Circ Res 87:1055–1062, 2000 HEISS A, DUCHESNE A, DENECKE B, et al: Structural basis of calcification inhibition by alpha 2–HS glycoprotein/fetuin-A. Formation of colloidal calciprotein particles. J Biol Chem 278:13333–13341, 2003 JAHNEN-DECHENT W, SCHINKE T, TRINDL A, et al: Cloning and targeted deletion of the mouse fetuin gene. J Biol Chem 272:31496– 31503, 1997 PRICE PA, NGUYEN TM, WILLIAMSON MK: Biochemical characterization of the serum fetuin-mineral complex. J Biol Chem 278:22153– 22160, 2003 ¨ SCHAFER C, HEISS A, SCHWARZ A, et al: The serum protein alpha 2–Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification. J Clin Invest 112:357–366, 2003 KITOH C, MIYAGI K, ONOE T, et al: Reversible peritoneal calcification in a patient treated by CAPD. Clin Nephrol 52:329–332, 1999 CHAN CT, MARDIROSSIAN S, FARATRO R, RICHARDSON RM: Improvement in lower-extremity peripheral arterial disease by nocturnal hemodialysis. Am J Kidney Dis 41:225–229, 2003 DI LEO C, GALLIENI M, BESTETTI A, et al: Cardiac and pulmonary calcification in a hemodialysis patient: Partial regression 4 years after parathyroidectomy. Clin Nephrol 59:59–63, 2003 KURIYAMA S, TOMONARI H, NAKAYAMA M, et al: Successful treatment of tumoral calcinosis using CAPD combined with hemodialysis with low-calcium dialysate. Blood Purif 16:43–48, 1998 Clinical algorithms on renal osteodystrophy. Nephrol Dial Transplant 15(Suppl 5):39–57, 2000 KATSUMATA K, KUSANO K, HIRATA M, et al: Sevelamer hydrochloride prevents ectopic calcification and renal osteodystrophy in chronic renal failure rats. Kidney Int 64:441–450, 2003 DRUEKE TB, MCCARRON DA: Paricalcitol as compared with calcitriol in patients undergoing hemodialysis. N Engl J Med 349:496– 499, 2003 GOODMAN WG, HLADIK GA, TURNER SA, et al: The calcimimetic agent AMG 073 lowers plasma parathyroid hormone levels in hemodialysis patients with secondary hyperparathyroidism. J Am Soc Nephrol 13:1017–1024, 2002 PRICE PA, FAUS SA, WILLIAMSON MK: Bisphosphonates alendronate and ibandronate inhibit artery calcification at doses comparable to those that inhibit bone resorption. Arterioscler Thromb Vasc Biol 21:817–824, 2001 MOTRO M, SHEMESH J: Calcium channel blocker nifedipine slows down progression of coronary calcification in hypertensive patients compared with diuretics. Hypertension 37:1410–1413, 2001

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93. BUSHINSKY DA, SMITH SB, GAVRILOV KL, et al: Chronic acidosisinduced alteration in bone bicarbonate and phosphate. Am J Physiol (Renal Physiol) 285:F532–539, 2003 94. KRONENBERG F, MUNDLE M, LANGLE M, NEYER U: Prevalence and progression of peripheral arterial calcifications in patients with ESRD. Am J Kidney Dis 41:140–148, 2003 95. WATSON KE, BOSTROM K, RAVINDRANATH R, et al: TGF-beta 1 and 25-hydroxycholesterol stimulate osteoblast-like vascular cells to calcify. J Clin Invest 93:2106–2113, 1994 96. TOWLER DA, BIDDER M, LATIFI T, et al: Diet-induced diabetes activates an osteogenic gene regulatory program in the aortas of low

density lipoprotein receptor-deficient mice. J Biol Chem 273:30427– 30434, 1998 97. BALICA M, BOSTROM K, SHIN V, et al: Calcifying subpopulation of bovine aortic smooth muscle cells is responsive to 17 beta-estradiol. Circulation 95:1954–1960, 1997 98. MORI K, SHIOI A, JONO S, et al: Dexamethasone enhances in vitro vascular calcification by promoting osteoblastic differentiation of vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 19:2112– 2118, 1999 99. MASUDA I, HIROSE J: Animal models of pathologic calcification. Curr Opin Rheumatol 14:287–291, 2002