2.1.3. Microvascular Disease

The coronary microvascular function is not well studied in CKD. Based on one old report around 30% of dialysis patients with clinical angina have only moderate epicardial coronary artery disease (CAD) [24], possibly explained with endothelial dysfunction associated with microvascular disease [11,25]. The presence of structural and coronary functional changes contributes to myocardiumcapillary mismatch which is not specific to uremia [9]. Under the condition of disbalance between high oxygen demand and a low oxygen supply microvascular coronary disease exposes cardiomyocytes to the risk of hypoxemia and beyond in possible ischemic myocardial injury at the microvascular level, which could be an explanation for persistently elevated serum troponin levels found in these patients [3,26].

Coronary artery calcification (CAC) as measured by computed tomography is noninvasive with excellent accuracy measurement of the burden of coronary atherosclerosis. CKD patients have higher CAC scores compared with age-matched controls without CKD, and those without baseline calcification present higher incidence rates of developing future de novo CAC [27]. Besides traditional factors, here, in particular, there are nontraditional risk factors such as hyperphosphatemia, calcium-phosphorus product, homocysteine, osteoprotegerin, and sclerostin which were independently related to the presence and high CAC scores [8,27,28].

### *2.2. Role of Phosphate in Uremic Cardiomyopathy*

Phosphate toxicity is a well described phenomenon in CKD [29,30]. Less than 1% of total phosphate is found in the blood and its balance was regulated by the interplay of bone, the parathyroid glands, intestines, and kidneys. The kidney is the principal organ regulating phosphate homeostasis. Following the loss of glomerular filtration rate (GFR), tubular phosphate reabsorption is significantly decreased by dual e ffect of compensatory increased concentration of two important hormones, the parathyroid hormone (PTH), and fibroblast growth factor 23 (FGF23). In addition, FGF23 suppresses the activation of vitamin D and acts to decrease parathyroid hormone synthesis and secretion being the major trigger in the path of CKD-MBD. FGF23 needs its cofactor klotho to ensure phosphate clearance [31]. Since the expression of Klotho declines in the kidney in the earlier stage CKD, FGF23 rises due to the resistance to FGF23 signaling in the kidney [31,32]. Although renal α-Klotho levels were significantly reduced and serum FGF23 levels were significantly elevated they can maintain serum phosphate within the normal range in early and intermediate stages of CKD. However, as CKD progresses, these defense mechanisms are ine ffective, so phosphate retention may occur, and hyperphosphatemia develops.

Elevated serum phosphate has revealed as a non-traditional risk factor for cardiovascular events in CKD and partially explains the increased mortality risk in CKD [29,31,32].

The role of phosphate in vascular calcification has been the focus of intense investigation in the past decades. In elevated phosphate conditions, the biology of the arterial tunica media is found greatly altered; there is vascular smooth muscle cell (VSMC) transition to bone phenotype, apoptosis inactivation of local anti-calcification factors, and elastin degradation [33,34].

The PiT-1 phosphate transporter seems to be a key mediator in phosphate-induced VSMC, activating bone formation-related gene expression, osteochondrogenic di fferentiation, and was recently shown to be relevant in cell proliferation and embryonic development, referring more functions for this protein than previously thought [33–35]. Vascular mineralization, especially a ffecting the coronary artery, is strongly related to mortality of CKD patients independently from the established atherogenic markers. The rate of coronary artery mineralization in CKD patients undergoing hemodialysis treatment was reported to be five times higher than in the non-dialysis CKD patients and is associated with features of valvular calcifications sharing the same changes [16,31,36]. Moreover, valvular heart disease is one of the most common complications observed in patients with CKD [37,38] and hyperphosphatemia directly a ffects progression of valvular calcification.

The progression of valvular calcification leads to obstruction of left ventricular outflow and inflow from the left atrium to the left ventricle associated with hemodynamic changes resulting in very di fficult clinical conditions [39].

Endothelial dysfunction is another early and crucial step in the development of cardiovascular disease apart from vascular calcification. Fewer reports have shown that phosphate level not in the physiologic range directly a ffects endothelial function and vascular remodeling [40,41]. Elevated phosphate level impairs endothelial function, hence diminishing microvascular function, angiogenic ability, and promoting endothelial sti ffness [42].

Endothelial sti ffness reflects changes in the structural and functional properties of the endothelium. These include cytoskeleton restructuring, successive mechano-signaling activity, intensified endothelial turnover (apoptosis), and diminished NO bioavailability [42].

High serum phosphate levels in HD patients were found to be independently associated with an increased number of endothelial microparticles (EMPs) and circulating (detached) endothelial cells [43]. These circulating submicron-sized membranous vesicles released by endothelium have a major biological role in the vascular injury; EMPs have been shown to act as primary and secondary messengers of vascular inflammation, thrombosis, vasomotor response, angiogenesis, and endothelial survival.

Phosphate is the major contributor to the level and biological activity of Calciprotein particles (CPPs) which are a new biological marker of CKD-MBD. Reports have shown that phosphate alone is not able to induce VSMCs mineralization, describing a synergistic action of both Ca and P in accelerated mineralization in vitro [16]. Insoluble CaP crystals generate when the concentration of calcium and phosphate exceeds the solubility limit. They can grow over time and finally precipitate as hydroxyapatite. The hepatic plasma protein fetuin-A (a natural calcification inhibitor) stabilizes colloidal protein–mineral complexes in the form of CPPs and mediates their clearance from the circulation. Primary CPPs, further, undergo topological rearrangemen<sup>t</sup> to find a more stable structure introduced as secondary CPPs [44]. The formation of CPP can be considered as a defense mechanism that prevents blood vessels from being occluded with insoluble CaP precipitates. The CPP level increases in the early stages of CKD, just before the rise of FGF23 and there are clinical findings that raise the hypothesis that CPPs might induce FGF23 [44,45]. In CKD patients, secondary CPPs have lower levels of calcification inhibitors including fetuin-A, and Gla-rich protein, readily taken up by the VSMCs inducing vascular calcification. While phosphate seems to be the driving force of CPP formation, his partner calcium seems to be a promoter of the inflammation-associated tissue damage forming a circle where increased mineralization triggers inflammation and vice-versa [44]. Recent studies have figured out the physiological and pathological significance of CPPs, its contributions to bone and mineral metabolism, and its role in tissue and organ impairments especially in cardiovascular damage and inflammatory responses [16,46,47] (Figure 1). Based on these findings secondary CPPs could be a

new biomarker for the pathological condition of CKD-MBD [47]. More studies are required to further clarify the role of CPPs as an essential mediator of CV damage and as a potential therapeutic target in CKD patients [47]. Recently, Ciceri et al. reported that ferric citrate prevents high Pi-induced calcium deposition by preventing apoptosis. Apoptosis has been proposed to be one of the mechanisms that initiate the calcification process by forming a nidus for the deposition of calcium and Pi crystals. Even in the status where VSMCs are already transformed with a procalcified stimulus being present, reverting apoptosis and inducing autophagy presumably contribute to stopping calcium deposition [48].

**Figure 1.** Role of Calciprotein particles in cardiovascular disease. In chronic kidney disease (CKD) patients, secondary Calciprotein particles (CPPs) have lower levels of calcification inhibitors including fetuin-A and were readily taken up by the vascular smooth muscle cells (VSMCs) inducing vascular calcification. Phosphate seems to be the driving force of CPP formation. Figure 1 shows the significance of CPPs, its contributions to bone and mineral metabolism, in an inflammatory response, and its role in the cardiovascular damage.

Animal experimental data sugges<sup>t</sup> that higher dietary phosphate engages multiple mechanisms involved in hypertension, including overactivation of the sympathetic nervous system, increased vascular stiffness, impaired endothelium-dependent vasodilation, together with an increased renal sodium absorption or renal injury [49].

On the other hand, there is limited evidence of a hyperphosphatemia-induced direct effect on cardiomyocytes. Dietary phosphate intake and hyperphosphatemia were frequently associated with abnormalities of the postcoronary arterial vessels in the myocardium and to interstitial fibrosis where hyperphosphatemia accelerate cardiac fibrosis as well as microvascular disease in experimental uremia [9,50]. In vitro studies showed that high Pi alone may not be able to generate cardiac hypertrophy but can initiate fibrosis [51]. Fibrosis, arising from non-myocytes and enhanced by cardiac myocytes, can promote increased wall stiffness and diastolic dysfunction. Moreover, fibrosis interrupts electrical signals, causing the tissue to be more arrhythmogenic [9,23]. Cardio markers and parameters of myocardial function, including Cardiac troponin T (cTnT), left ventricular max index (LVMi), left atrial dimensions (LAD), left ventricular end-systolic dimension (LVDs), left ventricular end-diastolic dimension (LVDd), interventricular septal thickness (IVST), and left ventricular posterior wall thickness (LVPWT), were reported consistently higher in a group of patients with higher serum phosphate (HSP) levels compared to those in the normal serum phosphate group (NSP) group, while left ventricular ejection fraction (LVEF) showed the opposite trend in a CKD cross-sectional study [52]. Furthermore, the lack of difference in mean arterial pressure (MAP) between the two groups suggested that cardiac remodeling including LVH and the declining LVEF might be associated with serum phosphate rather than hypertension and possibly this happens through triggering apoptosis of human cardiomyocytes.

With respect to CV mortality, it is reported that risk assessment varied from 1.09–1.13 for phosphorus (every 1 mg/dL increase) to 1.13–1.28 for calcium (every 1 mg/dL increase) [53].

In conclusion, enhanced phosphate has detrimental effects on the cardiovascular system seriously affecting patient outcomes (brief summary presented in Figure 2). Phosphate is toxic, impairs endothelial cells, promotes the formation of CPPs, induces VSMC transformation to osteogenic phenotype, and initiates cardiac fibrosis that leads to cardiac remodeling.

**Figure 2.** Pathophysiology of phosphate toxicity in the cardiovascular system. In CKD, higher serum phosphate levels are consistently linked with clinical and subclinical cardiovascular disease. Abbreviations: CPPs—Calciprotein particles; EMPs—endothelial microparticles; LVH—left ventricular hypertrophy; CHF—chronic heart failure; VSMCs—vascular smooth muscle cells; ↑ elevate; ↓ decrease.
