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Frontiers in Cardiovascular Medicine 9, 1-18 (2022). doi:10.3389/fcvm.2022.959457, Published by Frontiers Media, Lausanne

Background and Aims Chronic hypokalemia causes kidney fibrosis with cystic lesions and arterial hypertension. In contrast, potassium-rich diet lowers blood pressure. The acute effects of hypo- and hyperkalemia on heart and kidney are not well understood. Method Wild-type mice were fed with low (LK), normal (NK) and high (HK) potassium diet for 4 and 20 days. Kidneys were examined for site of acute injury, inflammation and fibrosis. Blood analysis of electrolytes and kidney parameters were analyzed. Echocardiography and ECG were used to assess heart function. Further, KCNJ10 knockout mice were used to investigate kidney damage in a genetically induced hypokalemia model. Results Proximal tubule injury as detected by KIM-1+ staining and yH2AX+ DNA-damage was observed after 4 and 20 days of LK diet. Injury was associated with strong Ki-67+ proliferation of proximal tubule cells. No injury was detected in mice on NK and HK diet. After 20 days of LK diet, F4/80+ inflammation and aSMA+ extracellular matrix accumulation, typical for fibrosis, were observed. LK mice developed polyurie, volume depletion, loss of body weight and high BUN. Lower cardiac output and signs of myocardial stress was seen in echocardiography and ECG. Consistent with WT mice on LK diet, KCNJ10 knockout mice developed same pattern of kidney injury. Nine months after deletion of KCNJ10, cysts were observed in the proximal tubule in outer medzulla. Conclusion Acute hypokalemia causes kidney injury and myocardial stress. Cystic lesions originate from late proximal tubule. Hypokalemia should be corrected rapidly to stop progression into kidney fibrosis.

Toxins 12(3), 161 (2020). doi:10.3390/toxins12030161 special issue: "Special Issue "Comorbidities in Chronic Kidney Disease (CKD)" / Special Issue Editors: Prof. Dr. Joachim Jankowski, Guest Editor; Dr. Heidi Noels, Guest Editor", Published by MDPI, Basel

ObjectiveThe plasma protein fetuin-A mediates the formation of protein-mineral colloids known as calciprotein particles (CPP) – rapid clearance of these CPP by the reticuloendothelial system prevents errant mineral precipitation and therefore ectopic mineralization (calcification). The mutant mouse strain D2,Ahsg-/- combines fetuin-A deficiency with the mineralization-prone DBA/2 genetic background, having a particularly severe compound phenotype of microvascular and soft tissue mineralization. Here we studied mechanisms leading to soft tissue mineralization, organ damage and premature aging in these mice.Approach and ResultsWe analyzed mice longitudinally by echocardiography, X-ray-computed tomography, analytical electron microscopy, histology, mass spectrometry proteomics, and genome-wide microarray-based expression analyses of D2 wildtype and Ahsg-/- mice.Fetuin-A deficient mice had calcified lesions in myocardium, lung, brown adipose tissue, reproductive organs, spleen, pancreas, kidney and the skin, associated with reduced growth, cardiac output and premature aging. Importantly, early stage calcified lesions presented in the lumen of the microvasculature suggesting precipitation of mineral containing complexes from the fluid phase of blood. Genome-wide expression analysis of calcified lesions and surrounding (not calcified) tissue, together with morphological observations, indicated that the ectopic mineralization was not associated with osteochondrogenic cell differentiation, but rather with thrombosis and fibrosis.ConclusionsCollectively, these results demonstrate that pathological mineralization can start by intravascular mineral deposition causing microvasculopathy, which impacts on growth, organ function and survival. Our study underscores the importance of fetuin-A and related systemic regulators of mineralized matrix metabolism to prevent cardiovascular disease, especially in dysregulated mineral homeostasis.

ObjectiveCalcifications can disrupt organ function in the cardiovascular system and the kidney, and are particularly common in patients with chronic kidney disease (CKD). Fetuin-A deficient mice maintained against the genetic background DBA/2 exhibit particularly severe soft tissue calcifications, while fetuin-A deficient C57BL/6 mice remain healthy. We employed molecular genetic analysis to identify risk factors of calcification in fetuin-A deficient mice. We sought to identify pharmaceutical therapeutic target that could be influenced by dietary of parenteral supplementation.Approach and ResultsWe studied the progeny of an intercross of fetuin-A deficient DBA/2 and C57BL/6 mice to identify candidate risk genes involved in calcification. We determined that a hypomorphic mutation of the Abcc6 gene, a liver ATP transporter supplying systemic pyrophosphate, and failure to regulate the TRPM6 magnesium transporter in kidney were associated with severity of calcification. Calcification prone fetuin-A deficient mice were alternatively treated with dietary phosphate restriction, magnesium supplementation, or by parenteral administration of fetuin-A or pyrophosphate. All treatments markedly reduced soft tissue calcification, demonstrated by computed tomography, histology and tissue calcium measurement.ConclusionsWe show that pathological ectopic calcification in fetuin-A deficient DBA/2 mice is caused by a compound deficiency of three major extracellular and systemic inhibitors of calcification, namely fetuin-A, pyrophosphate, and magnesium. All three of these are individually known to contribute to stabilize protein-mineral complexes and thus inhibit mineral precipitation from extracellular fluid. We show for the first time a compound triple deficiency that can be treated by simple dietary or parenteral supplementation. This is of special importance in patients with advanced CKD, who commonly exhibit reduced serum fetuin-A, pyrophosphate and magnesium levels.Subject CodesAnimal Models of Human Disease, Fibrosis, Inflammation, Proteomics, Peripheral Vascular Disease

Calcifications can disrupt organ function in the cardiovascular system and the kidney, and are particularly common in patients with chronic kidney disease (CKD). Fetuin-A deficient mice maintained against the genetic background DBA/2 exhibit particularly severe soft tissue calcifications, while fetuin-A deficient C57BL/6 mice remain healthy. We employed molecular genetic analysis to identify risk factors of calcification in fetuin-A deficient mice. We sought to identify pharmaceutical therapeutic targets that could be influenced by dietary of parenteral supplementation. We studied the progeny of an intercross of fetuin-A deficient DBA/2 and C57BL/6 mice to identify candidate risk genes involved in calcification. We determined that a hypomorphic mutation of the Abcc6 gene, a liver ATP transporter supplying systemic pyrophosphate, and failure to regulate the Trpm6 magnesium transporter in kidney were associated with severity of calcification. Calcification prone fetuin-A deficient mice were alternatively treated with parenteral administration of fetuin-A dietary magnesium supplementation, phosphate restriction, or by or parenteral pyrophosphate. All treatments markedly reduced soft tissue calcification, demonstrated by computed tomography, histology and tissue calcium measurement. We show that pathological ectopic calcification in fetuin-A deficient DBA/2 mice is caused by a compound deficiency of three major extracellular and systemic inhibitors of calcification, namely fetuin-A, magnesium, and pyrophosphate. All three of these are individually known to contribute to stabilize protein-mineral complexes and thus inhibit mineral precipitation from extracellular fluid. We show for the first time a compound triple deficiency that can be treated by simple dietary or parenteral supplementation. This is of special importance in patients with advanced CKD, who commonly exhibit reduced serum fetuin-A, magnesium and pyrophosphate levels.

The journal of infectious diseases 212(suppl 2), S247-S257 (2015). doi:10.1093/infdis/jiv140, Published by Oxford Univ. Press, Oxford [u.a.]

Background The liver-derived plasma protein fetuin-A is strongly expressed during fetal life, hence its name. Fetuin-A protein is normally present in most fetal organs and tissues, including brain tissue. Fetuin-A was neuroprotective in animal models of cerebral ischemia and lethal chronic inflammation, suggesting a role beyond the neonatal period. Little is known, however, on the presence of fetuin-A in mature human brain tissue under different physiological and pathological conditions. Methods We studied by immunohistochemistry (IHC) the distribution of fetuin-A protein in mature human brain autopsy tissues from patients without neurological disease, patients with inflammatory brain disorders, and patients with ischemic brain lesions. To identify fetuin-A-positive cells in these tissues we co-localized fetuin-A with GFAP (astrocytes) and CD68 (macrophages, activated microglia). Results and discussion Unlike previous reports, we detected fetuin-A protein also in mature human brain as would be expected from an abundant plasma protein also present in cerebrospinal fluid. Fetuin-A immunoreactivity was increased in ischemic white matter and decreased in inflamed cerebellar tissue. Fetuin-A immunostaining was predominantly associated with neurons and astrocytes. Unlike the developing brain, the adult brain lacked fetuin-A immunostaining in CD68-positive microglia. Our findings suggest a role for fetuin-A in tissue remodeling of neonatal brain, which becomes obsolete in the adult brain, but is re-activated in damaged brain tissue. To further assess the role of fetuin-A in the mature brain, animal models involving ischemia and inflammation need to be studied.

Frontiers in immunology 9, 1991 (2018). doi:10.3389/fimmu.2018.01991, Published by Frontiers Media, Lausanne

Sclerostin is a soluble antagonist of canonical Wnt signaling and a strong inhibitor of bone formation. We present experimental data on the role of sclerostin in chronic kidney disease - bone mineral disorder (CKD-MBD). Methods We performed 5/6 nephrectomies in 36-week-old sclerostin-deficient (SOST−/−) B6-mice and in C57BL/6J wildtype (WT) mice. Animals received a high phosphate diet for 11 weeks. The bones were analyzed by high-resolution micro-computed tomography (μCT) and quantitative bone histomorphometry. Aortic tissue was analyzed regarding the extent of vascular calcification. Results All nephrectomized mice had severe renal failure, and parathyroid hormone was highly increased compared to corresponding sham animals. All SOST−/− animals revealed the expected high bone mass phenotype. Overall, the bone compartment in WT and SOST−/− mice responded similarly to nephrectomy. In uremic WT animals, μCT data at both the distal femur and lumbar spine revealed significantly increased trabecular volume compared to non-uremic WTs. In SOST−/− mice, the differences between trabecular bone volume were less pronounced when comparing uremic with sham animals. Cortical thickness and cortical bone density at the distal femur decreased significantly and comparably in both genotypes after 5/6 nephrectomy compared to sham animals (cortical bone density − 18% and cortical thickness − 32%). Overall, 5/6 nephrectomy and concomitant hyperparathyroidism led to a genotype-independent loss of cortical bone volume and density. Overt vascular calcification was not detectable in either of the genotypes. Conclusion Renal osteodystrophy changes were more pronounced in WT mice than in SOST−/− mice. The high bone mass phenotype of sclerostin deficiency was detectable also in the setting of chronic renal failure with severe secondary hyperparathyroidism. Abbreviations α-SMA, alpha smooth muscle actin; AjAR, adjusted apposition rate; B.Ar, bone area; BFR, bone formation rate; cbfa1, core binding factor 1; CKD-MBD, chronic kidney disease and mineral bone disorder; EPm, eroded perimeter; Ntx, nephrectomy; O.Ar, osteoid area; Ob, osteoblast perimeter; Oc, osteoclast perimeter; O.Wi, osteoid width; PTH, parathyroid hormone; s.c., subcutaneous; VSMCs, vascular smooth muscle cells; WT, wildype

PLoS one 12(10), e0187030 (2017). doi:10.1371/journal.pone.0187030, Published by PLoS, Lawrence, Kan.

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (µCT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal µCT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (µCT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal µCT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Matriptase-2 is a type II transmembrane serine protease controlling the expression of hepcidin, the key regulator of iron homeostasis. By cleaving hemojuvelin, matriptase-2 suppresses bone morphogenetic protein/sons of mothers against decapentaplegic signaling. So far, the only known putative substrates of matriptase-2 are hemojuvelin and matriptase-2 itself. In this study, fetuin-A (α2-Heremans-Schmid glycoprotein) was identified in vitro as a substrate of matriptase-2. The protease–substrate interaction was validated by isolating matriptase-2 via the affinity to fetuin-A. Fetuin-A is a liver-derived plasma protein with multiple functions, which is proteolytically processed to yield a disulfide-linked two-chain form. In co-transfected cells, a matriptase-2-dependent conversion of unprocessed fetuin-A into a two-chain form was detected. Conversely, downregulation of endogenously expressed matriptase-2 stabilized fetuin-A. Arg and Lys residues located within the 40 residue spanning connecting peptide of fetuin-A were identified as cleavage sites for matriptase-2. Analysis of hepcidin expression revealed an inductive effect of fetuin-A, which was abolished by matriptase-2. Fetuin-A deficiency in mice resulted in decreased hepcidin mRNA levels. These findings implicate a role of fetuin-A in iron homeostasis and provide new insights into the mechanism of how matriptase-2 might modulate hepcidin expression.

The purpose of this study was to determine if chest tubes that are not milked or stripped occlude more frequently than milked or stripped tubes, and if the amount of drainage varies according to the treatment of the tubes. Following coronary artery bypass graft procedures, 49 male subjects had their chest tubes milked every 2 hours, had them stripped every 2 hours, or served as controls (i.e., their tubes were neither milked nor stripped). An analysis of variance was applied to the results. There was no significant difference in total drainage volume, hourly zero drainage, heart rate, or occurrence of arrhythmias among the three groups of subjects. Four to 16 hours postoperatively, a significantly higher volume of drainage occurred in the subjects whose chest tubes had been stripped. Stripping is particularly discouraged during this interval. The chest tubes remained patent with or without milking or stripping. We conclude that neither milking nor stripping is necessary for the proper care of chest tubes. We recommend that tubes be positioned such that they promote continuous drainage.