Mechanisms of Vascular Calcification in Chronic Kidney Disease

The Pathophysiology of Vascular Calcification

Although calcification can occur in the arterial intima adjacent to plaques and in the medial layers, it is not clear if these forms of calcification are identical, or if they have different inciting factors leading to a common pathogenic mechanism that parallels bone formation. Transcription factors such as Cbfa1/RUNX2 and MSX-2, critical for normal bone development, have been identified in cells surrounding human arterial calcification in both the general population and in chronic kidney disease patients, in animal models, and in vitro.⁷ The bone proteins osteonectin, osteopontin, bone sialoprotein, type I collagen, and alkaline phosphatase have also been identified in multiple sites of extraskeletal calcification. In cell culture, vascular smooth muscle cells and vascular pericytes are capable of producing these same bone-forming transcription factors and proteins, and can be induced to do so with high concentrations of phosphorus, uremic serum, high glucose, oxidized lipids, cytokines, and several other factors.

Vascular smooth muscle cells that express these proteins are capable of forming mineralized nodules in cell culture experiments in the presence of phosphorus, either sodium phosphate or as a phosphate donor β-glycerophosphate that is cleaved to phosphorus by membrane-bound alkaline phosphatase. Thus, in addition to calcium, phosphorus is also a critical element of calcification both in bone¹³ and in blood vessels, and the two are additive in their effects on in vitro vascular calcification.¹⁴ For mineralization to occur in vitro, there is a need for cellular transformation and access to mineral. If one raises the mineral concentration high enough in culture media it will spontaneously precipitate, even in the absence of cells—the so-called ‘physicochemical’ deposition that is highly pH dependent. Various proteins can inhibit this physicochemical component of vascular calcification (see below). As recently reviewed, these data question the usefulness of the long argued concept of a calcium x phosphorus (Ca x P) product in blood as predictive of extraskeletal mineralization in patients with chronic kidney disease.¹⁵ Indeed, this concept of a ‘safe Ca x P’ product, has given physicians a convenient way to analyze their patient's monthly labs, but is not based on scientific data, and the risk associated with a given Ca x P product depends on the patient's existing arterial disease, abnormal mineral homeostasis, and the availability of inhibitors of mineralization.

Uremic animal models of arterial calcification complement our clinical and in vitro work and have helped to characterize the types of abnormalities that are important. These animal models can be broadly classified into five groups supporting the complex pathogenesis outlined in Figure 1: animals with hyperphosphatemia due to chronic kidney disease and high phosphorus diet, or those having genetic defects that impair renal excretion of phosphorus (Klotho or FGF-23, for example), animals made hypercalcemic with toxic doses of vitamin D, animals with atherosclerosis made uremic (ApoE and LDL receptor null mice), animals with abnormal bone remodeling (osteoprotegerin null mice), and animals with defects in inhibitors such as matrix Gla protein. Importantly, in these same animal models, arterial calcification can be prevented or reduced by therapies that normalize serum phosphorus (phosphate binders or low phosphate diet), correct secondary hyperparathyroidism (calcimimetics, and in some studies, vitamin D analogues), and by therapies that inhibit bone turnover, (bisphosphonates, osteoprotegerin, a vacuolar ATPase osteoclast inhibitor, and bone morphogenic protein 7). These findings provide strong evidence that hyperphosphatemia and calcium load are key risk factors, and that impaired bone remodeling leads to vascular calcification, confirming the link between abnormal bone remodeling and arterial calcification that exists in humans in the general population and in patients with chronic kidney disease.⁹ It appears in patients with chronic kidney disease that both extremes of bone remodeling, low turnover (adynamic bone) and hyperparathyroid bone, may accelerate vascular calcification by not allowing calcium or phosphorus into bone, or resorbing it out of bone, respectively.

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Figure 1.

Normally, mesenchymal stem cells differentiate to adipocytes, osteoblasts, chondrocytes, and vascular smooth muscle cells (VSMC). In the setting of chronic kidney disease, diabetes, aging, inflammation, and multiple other toxins, these VSMC can dedifferentiate or transform into osteo/chondrocytic-like cells by upregulation of transcription factors such as RUNX-2 and MSX2. These transcription factors are critical for normal bone development and thus their upregulation in VSMC is indicative of a phenotypic switch. These osteo/chondrocytic-like VSMC then become calcified in a process similar to bone formation. These cells lay down collagen and noncollagenous proteins in the intima or media and incorporate calcium and phosphorus into matrix vesicles to initiate mineralization and further mineralize into hydroxyapatite. The overall positive calcium and phosphorus balance of most dialysis patients feeds both the cellular transformation and the generation of matrix vesicles. In addition, the extremes of bone turnover in chronic kidney disease (low and high or adynamic and hyperparathyroid bone, respectively) will increase the available calcium and phosphorus by altering the bone content of these minerals. Ultimately, whether an artery calcifies or not depends on the strength of the army of inhibitors (I) standing by in the circulation (fetuin-A) and in the arteries (PPI = pyrophosphate, MGP = matrix Gla protein, and OP = osteopontin as examples).

Interestingly, not all dialysis patients develop arterial calcifications, despite similar exposure to these risk factors, and importantly, do not develop calcification with increased duration of dialysis.^5,7 These findings imply there are protective factors, either in blood vessels or in the circulation, or both. If human serum is added to a solution containing high calcium and phosphorus, with or without cells, calcification is inhibited. Thus, serum contains numerous inhibitors of calcification. The most abundant of these is fetuin-A, a reverse acute-phase reactant that acts as a ‘vacuum cleaner’ to rid plasma of excess calcium and phosphorus molecules. Levels of fetuin-A go down during inflammation, and low levels in dialysis patients are associated with vascular and valvular calcification and death.¹⁶ Matrix Gla protein, pyrophosphate, and osteopontin are also local inhibitors of calcification. It is certainly likely other inhibitors exist as well. The importance of calcification inhibitors is demonstrated by the profound phenotype and site specificity of vascular calcification that occur in mice with null mutations, suggesting that, similar to bone,¹³ calcification would proceed unabated if not regulated by these inhibitors. Different anatomic sites may have a unique profile for these modulators.

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