Fetuin-a-containing calciprotein particles in inflammation, premature vascular ageing and chronic kidney disease

Age-related arterial remodelling is accelerated in patients with Chronic Kidney Disease (CKD). This vascular phenotype is characterised by increased arterial calcification and stiffening and is associated with greatly increased risk of adverse cardiovascular outcomes. Chronic exposure of the vasculature to dysregulated mineral metabolism and an increasingly pro-inflammatory environment, characteristic of the uraemic milieu, drives phenotypic changes in multiple vascular cell types leading to mineralisation, senescence and attrition within the arterial wall. Recent studies highlight the importance of potently proinflammatory mineral nanocrystals, rather than soluble mineral ions, in mediating many of these cellular effects, potentiating the vicious cycle of vascular inflammation and mineralisation. Various systemic and local inhibitory networks have evolved to protect against the deposition of mineral nuclei and prevent extraosseous calcification. Fetuin-A is a liver-derived phosphorylated glycoprotein that coats mineral nanocrystals to form calciprotein particles (CPP), soluble colloidal high molecular weight complexes that inhibit further crystal growth and aggregation and which may facilitate clearance of mineral debris from the extracellular fluid. We hypothesised that disturbances in this humoral homeostatic system and resultant accumulation of these nanoparticles in uraemia may be directly pathogenic and predictive of vascular dysfunction and poor patient outcome. This thesis presents a number of co-ordinated clinical and pre-clinical analyses undertaken to examine the potential significance of CPP in uraemic vascular pathology. Using carefully validated methodology, serum CPP were undetectable in healthy adult controls but elevated in a cohort of patients with pre-dialysis CKD, where levels were strongly associated with systemic inflammatory markers and aortic stiffness (Chapter 2). Serum CPP were also found to be elevated in a group of patients with chronic rheumatologic inflammatory disease but normal renal function and, markedly so in patients with calcific uraemic arteriolopathy where levels tracked closely with serial changes in inflammatory cytokine concentrations (Chapter 3). Elevated CPP levels may reflect increased genesis of CPP from bone or calcifying tissues and/or reduced clearance due to impairment of renal elimination pathways or inadequate buffering by bone. In prospective analysis, higher serum CPP levels emerged as a strong inflammation-dependent predictor of an increased risk of all-cause mortality in the pre-dialysis CKD setting, while the transformation time of primary amorphous mineral containing-CPP to secondary crystalline-mineral containing CPP (calcification propensity) was associated with the risk of death, independent of conventional renal and cardiovascular risk factors (Chapter 7). In vitro, CPP were found to be rapidly internalised by murine macrophages, principally by the class A scavenger receptor, wherein they induced secretion of interleukin-1β and tumour necrosis factor-α, and increased intracellular oxidative stress, but only at very high levels (Chapter 4). Notably, fetuin-A-containing CPP were markedly less pro-inflammatory than naked hydroxyapatite (HAP) crystals of equivalent calcium load and size. Moreover, only CPP containing phosphorylated fetuin-A had significant effects on cytokine expression and cell viability. The effect of CPP on interleukin-1β secretion in human macrophage appeared predominantly mediated by activation of the NLRP3 inflammasome, an intracellular danger sensor, following internalisation and disruption of the endolysosomal compartment when CPP were present in gross excess (Chapter 5). In this context, fetuin-A appeared to dampen activation of the NLRP3 platform through inhibition of lysosomal cathepsin B. A failure to clear CPP by SR-A-dependent pathways in vascular smooth muscle cells (VSMC) resulted in the passive deposition of mineral without reprogramming to a more bone-like phenotype in a normophosphataemic environment (Chapter 6). However, in co-culture with macrophage, CPP augmented mineralisation via inflammatory and direct cell-to-cell signalling. Importantly, these cellular effects were found to be critically dependent on CPP ripening and dose, with only large amoutns of secondary crystalline CPP eliciting pro-inflammatory and pro-calcific cascades. Taken together these results underscore the intimate relationship between systemic inflammation and pro-calcific factors in patients with CKD. Both clinical and pre-clinical studies presented here attest to a consistent association between CPP and inflammatory processes. However, although secondary CPP appear to induce an inflammatory response and mineralisation in the macrophage and VSMC in vitro, these effects were only observed at levels 10 to 100-fold higher than those found in patient serum and hence the relevance of these findings to in vivo vascular pathology is questionable, although chronic exposure or appreciably higher tissue levels may elicit such a response. Consequently the epidemiological relationships observed may be purely associative rather than causal. Rather the data highlights a protective role for fetuin-A in limiting unwanted immune activation and potentially deleterious effects by mineral debris. The accumulation of CPP in CKD and other chronic inflammatory diseases may therefore be seen as a marker of extraosseous mineral stress rather than directly pathogenic. That aspects of vascular risk associatied with serum CPP levels and calcification propensity scores are not captured by traditional mineral and cardiovascular factors, suggests, regardless of their direct pathogenicity, that ascertainment of these novel mineral measures may be clinically useful. Indeed, irrespective of expiermental readout, the transformation of primary to secondary CPP remained a strikingly consistent determinant of cellular effect in vitro. Thus interventions aimed at retarding this transformation phenomenon may have merit. Further studies are needed to investigate the source of circulating CPP and to evaluate whether CPP excess has pathological effects on the vasculature in vivo. Moreover, it remains to be demonstrated whether targeting CPP excess may be of benefit to the patient.