Live Imaging of Calciprotein Particle Clearance and Receptor Mediated Uptake: Role of Calciprotein Monomers

Abstract

BackgroundThe liver-derived plasma protein fetuin A is a systemic inhibitor of ectopic calcification. Fetuin-A stabilizes calcium phosphate mineral initially as ion clusters to form calciprotein monomers (CPM), and then as larger multimeric consolidations containing amorphous calcium phosphate (primary CPP, CPP 1) or more crystalline phases (secondary CPP, CPP 2). CPM and CPP mediate excess mineral stabilization, transport and clearance from circulation.MethodsWe injected i.v. synthetic fluorescent CPM and studied their clearance by live two-photon microscopy. We analyzed organ sections by fluorescence microscopy to assess CPM distribution. We studied cellular clearance and cytotoxicity by flow cytometry and live/dead staining, respectively, in cultured macrophages, liver sinusoidal endothelial cells (LSEC), and human proximal tubule epithelial HK-2 cells. Inflammasome activation was scored in macrophages. Fetuin A monomer and CPM charge were analyzed by ion exchange chromatography.ResultsLive mice cleared CPP in the liver as published previously. In contrast, CPM were filtered by kidney glomeruli into the Bowman space and the proximal tubules, suggesting tubular excretion of CPM-bound calcium phosphate and reabsorption of fetuin A. Fetuin-A monomer clearance was negligible in liver and low in kidney. Anion exchange chromatography revealed that fetuin A monomer was negatively charged, whereas CPM appeared neutral, suggesting electrochemical selectivity of CPM versus fetuin A. CPM were non-toxic in any of the investigated cell types, whereas CPP 1 were cytotoxic. Unlike CPP, CPM also did not activate the inflammasome.ConclusionsFetuin-A prevents calcium phosphate precipitation by forming CPM, which transform into CPP. Unlike CPP, CPM do not trigger inflammation. CPM are readily cleared in the kidneys, suggesting CPM as a physiological transporter of excess calcium and phosphate. Upon prolonged circulation, e.g., in chronic kidney disease, CPM will coalesce and form CPP, which cannot be cleared by the kidney, but will be endocytosed by liver sinusoidal endothelial cells and macrophages. Large amounts of CPP trigger inflammation. Chronic CPM and CPP clearance deficiency thus cause calcification by CPP deposition in blood vessels and soft tissues, as well as inflammation.

Spatio-Temporal Multiscale Analysis of Western Diet-Fed Mice Reveals a Translationally Relevant Sequence of Events during NAFLD Progression

“…Intravital imaging of lipid droplets and macrophages in mouse livers was performed using an inverted two-photon microscope LSM MP7 (Zeiss, Germany), as previously described [19][20][21]. Briefly, after induction of anesthesia in mice [21], bolus tail vein injections of Hoechst 33258 (nucleus marker), TMRE/Rhodamine123 (markers of lobular zonation and mitochondrial membrane potential), Bodipy (lipid marker), and/or F4/80 antibody (macrophage marker) were performed approximately 30 min before the start of recording (Table 4).…”

Matrix-GLA-Protein and Vascular Calcification: Can Diet Influence the Consequences of Matrix GLA Protein Inactivation? A Review

“…Several experimental studies have demonstrated the potent cytotoxic and procalcific Ca-phosphate crystals in the absence of fetuin-A [156,157] . A decrease in systemic fetuin-A increases systemic calciprotein particles and procalcification of calciprotein particles, thus promoting vascular calcification [158][159][160][161][162] .…”

Colchicine overdose impairs the capacity of Kupffer cells to clear foreign particles and endotoxins

“…To visualize bile canaliculi, a bolus of the bile acid analogue cholyl-lysyl-fluorescein (CLF; #451041, Corning) was intravenously injected via a tail vein catheter approximately 15 min before the end of the recording (Vartak et al 2021 ). Image analysis was done in defined regions of interest using Zen software (Zeiss, Germany), as previously described (Ghallab et al 2021a ; Koeppert et al 2021 ). Representative video/image of at least 4 mice per condition was analyzed and presented in the result section.…”