PRESENTED BY
MARKUS KETTELER

Presentation Summary

Written by Jasna Trbojevic-Stankovic
Reviewed by Markus Ketteler

Calcification is a rather complex and active pathological process involving several interrelated mechanisms. Patients with chronic kidney disease (CKD) exhibit high risk for the development of calcifications, especially vascular, which are associated with significant morbidity and mortality in this population. However, unlike vascular calcifications related to older age as a feature of atherosclerosis, which typically occur in tunica intima, vascular calcifications due to CKD-related metabolic changes are restricted to the smooth muscle cell layer and especially to the elastic laminae in tunica media (1-4). Thus, CKD patients, and especially those on dialysis, may exhibit either or both types of calcifications – intimal and/or medial. While intimal calcifications may lead to adverse cardiovascular and cerebrovascular events due to acute occlusion, medial calcifications cause non-occlusive vascular stiffness promoting quicker return of the systolic pulse wave from the periphery, increasing left ventricular afterload and, consequently, left ventricular mass (1).

In the recent years, several mechanisms have been proposed to explain vascular calcification including loss of inhibitors, induction of bone formation, circulating nucleational complexes and cell death (3). Various imaging techniques are essential in evaluating and monitoring of vascular calcifications. These include plain and lateral thoracic X rays, echocardiography, and lateral abdominal X rays, which correspond well with findings on CT scan. Abdominal aortic calcification, diagnosed with plain lateral abdominal x-ray, and central arterial stiffness are independent predictors of mortality and nonfatal cardiovascular events in dialysis patients (5). Carotid-femoral pulse wave velocity is also good predictor of mortality risk in dialysis patients (5). Thus, both the intimal and the medial calcification are associated with increased cardiovascular mortality in this population.

The ultrastructure of vascular calcification
The underlying pathological mechanisms initiating microcalcification of the media have only been studied in the last decade. It was previously assumed that vascular calcifications result from passive precipitation of calcium and phosphate due to disturbed mineral metabolism in CKD patients. However, the finding of different mineral phases in medial calcification led to belief that the process is a result of different pathomechanisms, involving active cellular processes (6). A systematic analysis of ultrastructural properties of medial calcifications in iliac artery segments collected from end-stage renal disease patients prior to transplantation confirmed that microcalcifications originate from nanocrystals, are chemically diverse and intimately associate with proteinaceous inhibitors of calcification (6). A core-shell layered structure of the microcalcifications has been confirmed with transmission electron microscopy, suggesting that apoptotic bodies or matrix vesicles may serve as a calcification nidus (Figure 1).

Figure 1. Comparison of the recent randomized controlled trials on the timing of RRT initiation in AKI (6, 7, 8, 9, 10)

The role of calciprotein particles
Prevention of calcification is also a rather complex process involving Fetuin-A, a liver-derived blood protein that acts as a potent inhibitor of ectopic mineralization. Fetuin-A stabilizes supersaturated mineral solutions by forming soluble colloidal nanospheres termed calciprotein particles (CPP), in analogy to lipoprotein particles (8). They were first observed in haemodialysis patients a decade ago in a study which revealed that free Fetuin-A circulates free and does not precipitate, while under extra-osseous calcification stress Fetuin-A forms particles to inhibit mineral precipitation (9). These particles occur substantially where bone resorption releases high levels of calcium and phosphate, or when mineral homeostasis is severely disturbed, as in dialysis patients (9). Calciprotein particles in the interstitial spaces are cleared by monocytes, macrophages or other endocytosing cells. However, it has been hypothesized that overly abundant particles may overcome the clearing capacity and result in apoptosis of macrophages, endothelial cells and smooth muscle cells, and deposition of calcified apoptotic fragments (10).

Pasch et al. recently developed an assay for measuring extra-osseous calcification propensity by detecting spontaneous transition of spherical colloidal primary CPP to secondary elongate crystalline ones in the presence of artificially elevated calcium and phosphate concentrations (11). The test was named T50 in reference to the half-time needed for the maturation from primary to secondary CPP. The assay was validated in vivo using the sera from heavily calcified Fetuin-A–deficient, and age-matched noncalcified heterozygous, and wild-type mice, as well as on blood samples from haemodialysis patients and healthy volunteers. It showed excellent ability to discriminate the calcification-prone from the noncalcification-prone individuals (11). In transplant patients, reduced serum T50 was associated with increased risk of all-cause mortality, cardiovascular mortality, and graft failure (12). Furthermore, serum magnesium, albumin and parathyroid hormone levels were positively associated, and phosphate, haemoglobin, and the use of calcineurin inhibitors or vitamin K antagonists were inversely associated with serum T50 in transplanted patients (12). In a randomized, double-blind, placebo-controlled EVOLVE clinical trial, which recruited nearly four thousand haemodialysis patients with moderate to severe secondary hyperparathyroidism, lower T50 was associated with higher probability of cardiovascular events and mortality (13, 14).

The role of magnesium
In the recent years, several studies have demonstrated association between low magnesium levels and increased risk of vascular calcification, leading to the hypothesis that magnesium might counteract the development of calcifications. To test this assumption, a group of authors treated vascular smooth muscle cells with high phosphate or secondary CPP and supplemented with magnesium. The experiment confirmed that secondary CPP mediate phosphate-induced vascular calcifications, but also revealed that magnesium delays CPP maturation and therefore prevents calcification in a dose dependent manner (15). These in vitro results only confirmed previous observation that increasing dialysate magnesium increases T50 and hence, decreases calcification propensity in haemodialysis patients (16).

Calciphylaxis and novel treatments
Calciphylaxis is another important issue related to vascular calcification. It is a rare and serious condition caused by calcific occlusion of micro vessels in the subcutaneous adipose tissue and dermis that results in ischemic skin lesions (17). It is associated with severe pain sensation and poor survival. Even though multiple risk factors have been identified, the pathogenesis of calciphylaxis remains elusive. Multiple factors seem to be involved in this process, including calcification promoters and inhibitors (e.g., carboxylated matrix Gla protein, Fetuin-A, pyrophosphate and phytate) (17).

Figure 2. Pathogenetic mechanisms involved in calciphylaxis (17, 7)

Phytate (myo-inositol hexaphosphate) is a naturally-occurring substance found in cereals and other high-fibre foods, and also present in mammalian cells and tissues in micro concentrations. Phytate has been shown to prevent formation of renal stones and vascular calcifications. The intravenous formulation of hexasodium phytate, SNF472, is a novel experimental drug for the treatment of calciphylaxis and decelerating progression of cardiovascular calcification in haemodialysis patients. It inhibits the development and progression of ectopic calcifications by binding to the growth sites of the hydroxyapatite crystal, the main component of cardiovascular deposits, irrespective of the aetiology of the calcifications and plasma calcium and phosphate levels (18). The effects of SNF472 have been evaluated in three small studies in rat models. The first focused on prevention of vitaminD3-induced cardiovascular calcifications with an intravenous SNF472 bolus; the second investigated inhibition of progression of vitamin D3-induced cardiovascular calcification with a subcutaneous SNF472 bolus; while the third inspected the effects of SNF472 infusion on cardiovascular calcifications in adenine-induced uremic rats. All experiments rendered very encouraging results, reporting that SNF472 treatment inhibited cardiovascular calcifications by 60-70% in non-uremic and by up to 80% in uremic rats (18).

The first clinical trial of SNF472 in patients with calciphylaxis was announced four years ago and recently presented the initial results. The double-blind, placebo-controlled phase 2b trial recruited 274 haemodialysis patients randomized to receive SNF472 300mg or 600mg, or placebo infusions thrice weekly during 52 weeks. It concluded that compared with placebo, SNF472 significantly attenuated the progression of coronary artery and aortic valve calcification, but called for further studies to determine the effects of experimental therapeutic on cardiovascular morbidity and mortality (19).

References

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