Cellular response and clearance mechanisms triggered by protein-mineral complexes

Koeppert, Sina; Jahnen-Dechent, Wilhelm (Thesis advisor); Elling, Lothar (Thesis advisor)

Aachen : RWTH Aachen University (2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021

Abstract

The liver-derived plasma protein fetuin-A is a systemic inhibitor of ectopic calcification. Fetuin-A stabilizes saturated mineral solutions initially as ion clusters to form calciprotein monomers (CPM) and then as larger multimeric consolidations containing amorphous calcium phosphate referred to as primary calciprotein particles (CPP). Time-, temperature-, pH-, and mineral saturation-dependently, primary CPP spontaneously convert into secondary CPP which are larger, oblongated, more crystalline, and less soluble. CPM and CPP mediate excess mineral stabilization, transport and clearance from circulation. The main objective of this work was to determine the clearance mechanisms of CPM and CPP and investigate resulting cellular responses. The majority of experiments was performed with synthetic CPM/CPP prepared with purified bovine fetuin-A. Synthetic CPP proved a good model for CPP research as results were transferable to CPP prepared with species-specific plasma. Clearance of primary versus secondary CPP and CPM was investigated in vivo and in vitro identifying liver sinusoidal endothelial cells (LSEC) as the major clearing cell type of primary CPP, whereas the majority of secondary CPP was cleared by liver Kupffer cells (macrophages). CPP endocytosis by LSEC and macrophages was receptor-mediated. CPM were cleared through the kidneys and CPM-associated fetuin-A protein was reabsorbed from the primary urine by proximal epithelial cells. CPP triggered TLR4-dependent TNFα and IL-1β secretion in cultured macrophages, which was associated with increased calcium-dependent inflammasome activation, suggesting that inflammation and calcification may be considered consequences of prolonged CPP circulation and clearance. Further, cytotoxicity analysis revealed increased CPP cytotoxicity. Unlike CPP, CPM did not trigger a formation of the inflammasome, showed no cytotoxic effect, and were readily cleared in the kidneys, suggesting CPM as a physiological transporter of excess calcium and phosphate and confirming the role of fetuin-A as a mineral chaperone. Upon prolonged circulation, e.g. in chronic kidney disease, CPM will coalesce and form CPP, which cannot enter the fast clearance pathway through the kidneys, yet still can be cleared by the liver. Chronic clearance deficiency may cause calcification starting by CPP deposition in blood vessels and soft tissues, which in turn will trigger local inflammation.