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Original Research
29 April 2021
Live Imaging of Calciprotein Particle Clearance and Receptor Mediated Uptake: Role of Calciprotein Monomers
Sina Koeppert
6 more and 
Willi Jahnen-Dechent
CPM-derived fetuin-A is reabsorbed in proximal renal tubules. Mice received intravenous injections of CPM prepared with Alexa488-labeled bovine fetuin-A (green). Nuclei were visualized by Hoechst 33258 (blue). Videos were recorded using a two-photon microscope and a video camera. Fluorescence intensity was recorded using a GaAsP detector. A renal filtration unit was continuously monitored up to 60 min. Representative micrographs demonstrating a typical sequence of fetuin-A tubular reabsorption. Yellow autofluorescence in distal tubules (yellow arrows, t = 0.0 min) was already visible before CPM injection. Within seconds after injection, green signal was detectable in glomerular and peritubular capillaries (white arrows, t = 0.3 min). At 2.5 min, proximal tubular (PCT) epithelial cells (filled white arrowheads) became green fluorescence positive showing a steadily increasing signal until 10 min, whereas distal tubular (DCT) cells showed no green fluorescent signal (white empty arrowheads), suggesting cellular uptake of green CPM-associated fetuin-A protein by proximal tubular epithelial cells from the tubule lumen. Fluorescence remained strong at the origin of the proximal tubuli where they connect to the glomeruli, while fluorescence continuously decreased in glomerular and peritubular capillaries until 60 min. Photographs illustrating proximal tubule re-uptake were taken from Supplementary Movie 2.

Background: The 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.

Methods: We 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.

Results: Live 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.

Conclusions: Fetuin-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.

6,866 views
39 citations
Mini Review
29 April 2021
Keutel Syndrome, a Review of 50 Years of Literature
M. Leonor Cancela
3 more and 
Monzur Murshed
Literature timeline for Keutel syndrome (KS) and matrix Gla protein (MGP) (A). MGP gene and protein structure mapping the eight mutations associated with KS (B). Most common traits observed in KS patients genotyped for MGP (C).

Keutel syndrome (KS) is a rare autosomal recessive genetic disorder that was first identified in the beginning of the 1970s and nearly 30 years later attributed to loss-of-function mutations in the gene coding for the matrix Gla protein (MGP). Patients with KS are usually diagnosed during childhood (early onset of the disease), and the major traits include abnormal calcification of cartilaginous tissues resulting in or associated with malformations of skeletal tissues (e.g., midface hypoplasia and brachytelephalangism) and cardiovascular defects (e.g., congenital heart defect, peripheral pulmonary artery stenosis, and, in some cases, arterial calcification), and also hearing loss and mild developmental delay. While studies on Mgp–/– mouse, a faithful model of KS, show that pathologic mineral deposition (ectopic calcification) in cartilaginous and vascular tissues is the primary cause underlying many of these abnormalities, the mechanisms explaining how MGP prevents abnormal calcification remain poorly understood. This has negative implication for the development of a cure for KS. Indeed, at present, only symptomatic treatments are available to treat hypertension and respiratory complications occurring in the KS patients. In this review, we summarize the results published in the last 50 years on Keutel syndrome and present the current status of the knowledge on this rare pathology.

6,682 views
21 citations
Hypothetical molecular processes in calcium phosphate medial vascular mineralization. At pH up to 7.40 Ca2+ ions are hydrated, and the majority of the phosphate molecules are in the form of HPO4-2- ions. The precipitation is triggered by a local increase of pH to above 7.90, associated with a marked increase in PO43– ions; the main component in ACP and HAP. Ca2+ and PO43– ions form complexes of increasing coordination number, eventually forming multinuclear clusters, which contain nine Ca2+ ions and six PO43– ions. Subsequently, promoter macromolecules with charged Ca2+-ligand groups (phosphate, carboxylate, and/or sulfate) produce a local accumulation of Posner clusters and the appearance of ACP precipitates. Finally, as the process progresses, the precipitate clusters rearrange into dense crystalline HAP nanoparticles.
Review
14 April 2021
The Thermodynamics of Medial Vascular Calcification
Ángel Millán
1 more and 
Víctor Sorribas

Medial vascular calcification (MVC) is a degenerative process that involves the deposition of calcium in the arteries, with a high prevalence in chronic kidney disease (CKD), diabetes, and aging. Calcification is the process of precipitation largely of calcium phosphate, governed by the laws of thermodynamics that should be acknowledged in studies of this disease. Amorphous calcium phosphate (ACP) is the key constituent of early calcifications, mainly composed of Ca2+ and PO43– ions, which over time transform into hydroxyapatite (HAP) crystals. The supersaturation of ACP related to Ca2+ and PO43– activities establishes the risk of MVC, which can be modulated by the presence of promoter and inhibitor biomolecules. According to the thermodynamic parameters, the process of MVC implies: (i) an increase in Ca2+ and PO43– activities (rather than concentrations) exceeding the solubility product at the precipitating sites in the media; (ii) focally impaired equilibrium between promoter and inhibitor biomolecules; and (iii) the progression of HAP crystallization associated with nominal irreversibility of the process, even when the levels of Ca2+ and PO43– ions return to normal. Thus, physical-chemical processes in the media are fundamental to understanding MVC and represent the most critical factor for treatments’ considerations. Any pathogenetical proposal must therefore comply with the laws of thermodynamics and their expression within the medial layer.

6,728 views
19 citations
Review
16 March 2021

Mitochondria are crucial bioenergetics powerhouses and biosynthetic hubs within cells, which can generate and sequester toxic reactive oxygen species (ROS) in response to oxidative stress. Oxidative stress-stimulated ROS production results in ATP depletion and the opening of mitochondrial permeability transition pores, leading to mitochondria dysfunction and cellular apoptosis. Mitochondrial loss of function is also a key driver in the acquisition of a senescence-associated secretory phenotype that drives senescent cells into a pro-inflammatory state. Maintaining mitochondrial homeostasis is crucial for retaining the contractile phenotype of the vascular smooth muscle cells (VSMCs), the most prominent cells of the vasculature. Loss of this contractile phenotype is associated with the loss of mitochondrial function and a metabolic shift to glycolysis. Emerging evidence suggests that mitochondrial dysfunction may play a direct role in vascular calcification and the underlying pathologies including (1) impairment of mitochondrial function by mineral dysregulation i.e., calcium and phosphate overload in patients with end-stage renal disease and (2) presence of increased ROS in patients with calcific aortic valve disease, atherosclerosis, type-II diabetes and chronic kidney disease. In this review, we discuss the cause and consequence of mitochondrial dysfunction in vascular calcification and underlying pathologies; the role of autophagy and mitophagy pathways in preventing mitochondrial dysfunction during vascular calcification and finally we discuss mitochondrial ROS, DRP1, and HIF-1 as potential novel markers and therapeutic targets for maintaining mitochondrial homeostasis in vascular calcification.

13,872 views
59 citations
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