Altered vitamin K biodistribution and metabolism in experimental and human chronic kidney disease

Chronic kidney disease (CKD) is accompanied with extensive cardiovascular calcification, in part correlating with functional vitamin K deficiency. Here, we sought to determine causes for vitamin K deficiency beyond reduced dietary intake. Initially, vitamin K uptake and distribution into circulating lipoproteins after a single administration of vitamin K1 plus K2 (menaquinone 4 and menaquinone 7, respectively) was determined in patients on dialysis therapy and healthy individuals. The patients incorporated very little menaquinone 7 but more menaquinone 4 into high density lipoprotein (HDL) and low-density lipoprotein particles than did healthy individuals. In contrast to healthy persons, HDL particles from the patients could not be spiked with menaquinone 7 in vitro and HDL uptake was diminished in osteoblasts. A reduced carboxylation activity (low vitamin K activity) of uremic HDL particles spiked with menaquinone 7 vs. that of controls was confirmed in a bioassay using human primary vascular smooth muscle cells. Kidney menaquinone 4 tissue levels were reduced in 5/6-nephrectomized versus sham-operated C57BL/6 mice after four weeks of a vitamin K rich diet. From the analyzed enzymes involved in vitamin K metabolism, kidney HMG-CoA reductase protein was reduced in both rats and patients with CKD. In a trial on the efficacy and safety of atorvastatin in 1051 patients with type 2 diabetes receiving dialysis therapy, no pronounced vitamin K deficiency was noted. However, the highest levels of PIVKA-II (biomarker of subclinical vitamin K deficiency) were noted when a statin was combined with a proton pump inhibitor. Thus, profound disturbances in lipoprotein mediated vitamin K transport and metabolism in uremia suggest that menaquinone 7 supplementation to patients on dialysis therapy has reduced efficacy

Dissecting CD8+ T cell pathology of severe SARS-CoV-2 infection by single-cell immunoprofiling

Introduction: SARS-CoV-2 infection results in varying disease severity, ranging from asymptomatic infection to severe illness. A detailed understanding of the immune response to SARS-CoV-2 is critical to unravel the causative factors underlying differences in disease severity and to develop optimal vaccines against new SARS-CoV-2 variants. Methods: We combined single-cell RNA and T cell receptor sequencing with CITE-seq antibodies to characterize the CD8+ T cell response to SARS-CoV-2 infection at high resolution and compared responses between mild and severe COVID-19. Results: We observed increased CD8+ T cell exhaustion in severe SARS-CoV-2 infection and identified a population of NK-like, terminally differentiated CD8+ effector T cells characterized by expression of FCGR3A (encoding CD16). Further characterization of NK-like CD8+ T cells revealed heterogeneity among CD16+ NK-like CD8+ T cells and profound differences in cytotoxicity, exhaustion, and NK-like differentiation between mild and severe disease conditions. Discussion: We propose a model in which differences in the surrounding inflammatory milieu lead to crucial differences in NK-like differentiation of CD8+ effector T cells, ultimately resulting in the appearance of NK-like CD8+ T cell populations of different functionality and pathogenicity. Our in-depth characterization of the CD8+ T cell-mediated response to SARS-CoV-2 infection provides a basis for further investigation of the importance of NK-like CD8+ T cells in COVID-19 severity.</p

Dissecting CD8+ T cell pathology of severe SARS-CoV-2 infection by single-cell immunoprofiling

IntroductionSARS-CoV-2 infection results in varying disease severity, ranging from asymptomatic infection to severe illness. A detailed understanding of the immune response to SARS-CoV-2 is critical to unravel the causative factors underlying differences in disease severity and to develop optimal vaccines against new SARS-CoV-2 variants.MethodsWe combined single-cell RNA and T cell receptor sequencing with CITE-seq antibodies to characterize the CD8+ T cell response to SARS-CoV-2 infection at high resolution and compared responses between mild and severe COVID-19.ResultsWe observed increased CD8+ T cell exhaustion in severe SARS-CoV-2 infection and identified a population of NK-like, terminally differentiated CD8+ effector T cells characterized by expression of FCGR3A (encoding CD16). Further characterization of NK-like CD8+ T cells revealed heterogeneity among CD16+ NK-like CD8+ T cells and profound differences in cytotoxicity, exhaustion, and NK-like differentiation between mild and severe disease conditions.DiscussionWe propose a model in which differences in the surrounding inflammatory milieu lead to crucial differences in NK-like differentiation of CD8+ effector T cells, ultimately resulting in the appearance of NK-like CD8+ T cell populations of different functionality and pathogenicity. Our in-depth characterization of the CD8+ T cell-mediated response to SARS-CoV-2 infection provides a basis for further investigation of the importance of NK-like CD8+ T cells in COVID-19 severity

Vitamin K-Transport in urämisch veränderten Lipoproteinen

Patients receiving hemodialysis suffer from an increased cardiovascular risk compared to the general population. One of the most important causes of this increased cardiovascular risk is an accelerated process of vascular calcification in hemodialysis patients.The pathogenesis of an accelerated vascular calcification in the setting of chronic kidney disease and especially in the setting of hemodialysis therapy is not entirely understood. By now it is known that matrix gla-protein (MGP), a Gla-protein which is synthesized by the vascular endothelium and which is activated by vitamin K, is one of the most important inhibitors of vascular calcification.In the past it has been shown that patients on hemodialysis suffer from a subclinical vitamin K-deficiency. The aim of our study was to examine the lipoprotein-transport of three different isotypes of vitamin K (vitamin K1, MK-4 and MK-7) and to compare the patterns of vitamin K-transport between healthy subjects and hemodialysis patients to prove our hypothesis that in hemodialysis patients vitamin K-transport in lipoproteins is impaired. A disorder in vitamin K-transport in lipoproteins within the framework of uremia might lead to an impaired activation of MGP and by that contribute to an accelerated vascular calcification.For this, 7 healthy subjects and 10 hemodialysis patients took a vitamin K-preparation (Vitamin K-Komplex) for breakfast together with a croissant and a glass of orange juice after an initial blood withdrawal (0h). After one (1h), three (3h) and six (6h) hours another blood sample was taken. The different lipoprotein-fractions (triglyceride-rich lipoprotein fraction (TGRLP), LDL and HDL) were separated by ultracentrifugation of the serum and subsequently vitamin K-concentrations were determined by UV-detection after another purification by high performance liquid chromatography (HPLC).Our study could show that the MK-4 concentration in LDL and HDL of hemodialysis patients had significantly increased compared to the baseline-concentration, 3 and 6 hours after vitamin K-intake, while the concentration of MK-4 in the lipoproteins of the healthy subjects remained largely constant. Concerning the transport of MK-7 in lipoproteins, we could show that in our healthy subjects the HDL percentage of the total transport of MK-7 increased after vitamin K intake and after slightly more than one hour made up the bulk of the MK-7 transport. This increase failed to appear in the hemodialysis group; accordingly, LDL made up the bulk of MK-7 transport in the hemodialysis patients. Moreover, we could observe that in the healthy population the concentration of MK-7 in HDL increased after vitamin K-intake, while this increase could not be observed in HDL of hemodialysis patients.Concluding, our study showed that in the serum of hemodialysis patients there appears to be a disorder in the HDL-transport of MK-7. This finding is of great relevance as especially the menaquinones (vitamin K2), which include MK-7, are important for the activation of MGP and accordingly for the prevention of vascular calcification. A transport disorder in lipoprotein-transport of vitamin K in hemodialysis patients could make a large contribution to the understanding of the pathomechanisms of accelerated vascular calcification in the arteries of hemodialysis patients. As well, this finding brings up the question for a vitamin K-substitution. Before therapeutic conclusions can be drawn, in a next step our phenomenological observations have to be supported by explanations on a mechanical level

Vitamin K-Transport in urämisch veränderten Lipoproteinen

Patients receiving hemodialysis suffer from an increased cardiovascular risk compared to the general population. One of the most important causes of this increased cardiovascular risk is an accelerated process of vascular calcification in hemodialysis patients.The pathogenesis of an accelerated vascular calcification in the setting of chronic kidney disease and especially in the setting of hemodialysis therapy is not entirely understood. By now it is known that matrix gla-protein (MGP), a Gla-protein which is synthesized by the vascular endothelium and which is activated by vitamin K, is one of the most important inhibitors of vascular calcification.In the past it has been shown that patients on hemodialysis suffer from a subclinical vitamin K-deficiency. The aim of our study was to examine the lipoprotein-transport of three different isotypes of vitamin K (vitamin K1, MK-4 and MK-7) and to compare the patterns of vitamin K-transport between healthy subjects and hemodialysis patients to prove our hypothesis that in hemodialysis patients vitamin K-transport in lipoproteins is impaired. A disorder in vitamin K-transport in lipoproteins within the framework of uremia might lead to an impaired activation of MGP and by that contribute to an accelerated vascular calcification.For this, 7 healthy subjects and 10 hemodialysis patients took a vitamin K-preparation (Vitamin K-Komplex) for breakfast together with a croissant and a glass of orange juice after an initial blood withdrawal (0h). After one (1h), three (3h) and six (6h) hours another blood sample was taken. The different lipoprotein-fractions (triglyceride-rich lipoprotein fraction (TGRLP), LDL and HDL) were separated by ultracentrifugation of the serum and subsequently vitamin K-concentrations were determined by UV-detection after another purification by high performance liquid chromatography (HPLC).Our study could show that the MK-4 concentration in LDL and HDL of hemodialysis patients had significantly increased compared to the baseline-concentration, 3 and 6 hours after vitamin K-intake, while the concentration of MK-4 in the lipoproteins of the healthy subjects remained largely constant. Concerning the transport of MK-7 in lipoproteins, we could show that in our healthy subjects the HDL percentage of the total transport of MK-7 increased after vitamin K intake and after slightly more than one hour made up the bulk of the MK-7 transport. This increase failed to appear in the hemodialysis group; accordingly, LDL made up the bulk of MK-7 transport in the hemodialysis patients. Moreover, we could observe that in the healthy population the concentration of MK-7 in HDL increased after vitamin K-intake, while this increase could not be observed in HDL of hemodialysis patients.Concluding, our study showed that in the serum of hemodialysis patients there appears to be a disorder in the HDL-transport of MK-7. This finding is of great relevance as especially the menaquinones (vitamin K2), which include MK-7, are important for the activation of MGP and accordingly for the prevention of vascular calcification. A transport disorder in lipoprotein-transport of vitamin K in hemodialysis patients could make a large contribution to the understanding of the pathomechanisms of accelerated vascular calcification in the arteries of hemodialysis patients. As well, this finding brings up the question for a vitamin K-substitution. Before therapeutic conclusions can be drawn, in a next step our phenomenological observations have to be supported by explanations on a mechanical level

Mapping the human kidney using single-cell genomics

The field of single-cell genomics and spatial technologies is rapidly evolving and has already provided unprecedented insights into complex tissues. Major advances have been made in dissecting the cellular composition and spatiotemporal interactions that mediate developmental processes in the fetal kidney. Single-cell technologies have also provided detailed insights into the heterogeneity of cell types within the healthy adult and shed light on the complex cellular mechanisms that contribute to kidney disease. The in-depth characterization of specific cell types associated with acute kidney injury and glomerular diseases has potential for the development of prognostic biomarkers and new therapeutics. Analyses of pathway activity in clear-cell renal cell carcinoma can predict the sensitivity of tumour cells to specific inhibitors. The identification of the cell of origin of renal cell carcinoma and of new cell types within the tumour microenvironment also has implications for the development of targeted therapeutics. Similarly, single-cell sequencing has provided new insights into the mechanisms underlying kidney fibrosis, specifically our understanding of myofibroblast origins and the contribution of cell crosstalk within the fibrotic niche to disease progression. These and future studies will enable the creation of a map to aid our understanding of the cellular processes and interactions in the developing, healthy and diseased kidney

Fibrosis in Pathology of Heart and Kidney:From Deep RNA-Sequencing to Novel Molecular Targets

Diseases of the heart and the kidney, including heart failure and chronic kidney disease, can dramatically impair life expectancy and the quality of life of patients. The heart and kidney form a functional axis; therefore, functional impairment of 1 organ will inevitably affect the function of the other. Fibrosis represents the common final pathway of diseases of both organs, regardless of the disease entity. Thus, inhibition of fibrosis represents a promising therapeutic approach to treat diseases of both organs and to resolve functional impairment. However, despite the growing knowledge in this field, the exact pathomechanisms that drive fibrosis remain elusive. RNA-sequencing approaches, particularly single-cell RNA-sequencing, have revolutionized the investigation of pathomechanisms at a molecular level and facilitated the discovery of disease-associated cell types and mechanisms. In this review, we give a brief overview over the evolution of RNA-sequencing techniques, summarize most recent insights into the pathogenesis of heart and kidney fibrosis, and discuss how transcriptomic data can be used, to identify new drug targets and to develop novel therapeutic strategies.</p

Altered vitamin K biodistribution and metabolism in experimental and human chronic kidney disease

Chronic kidney disease (CKD) is accompanied with extensive cardiovascular calcification, in part correlating with functional vitamin K deficiency. Here, we sought to determine causes for vitamin K deficiency beyond reduced dietary intake. Initially, vitamin K uptake and distribution into circulating lipoproteins after a single administration of vitamin K1 plus K2 (menaquinone 4 and menaquinone 7, respectively) was determined in patients on dialysis therapy and healthy individuals. The patients incorporated very little menaquinone 7 but more menaquinone 4 into high density lipoprotein (HDL) and low-density lipoprotein particles than did healthy individuals. In contrast to healthy persons, HDL particles from the patients could not be spiked with menaquinone 7 in vitro and HDL uptake was diminished in osteoblasts. A reduced carboxylation activity (low vitamin K activity) of uremic HDL particles spiked with menaquinone 7 vs. that of controls was confirmed in a bioassay using human primary vascular smooth muscle cells. Kidney menaquinone 4 tissue levels were reduced in 5/6-nephrectomized versus sham-operated C57BL/6 mice after four weeks of a vitamin K rich diet. From the analyzed enzymes involved in vitamin K metabolism, kidney HMG-CoA reductase protein was reduced in both rats and patients with CKD. In a trial on the efficacy and safety of atorvastatin in 1051 patients with type 2 diabetes receiving dialysis therapy, no pronounced vitamin K deficiency was noted. However, the highest levels of PIVKA-II (biomarker of subclinical vitamin K deficiency) were noted when a statin was combined with a proton pump inhibitor. Thus, profound disturbances in lipoprotein mediated vitamin K transport and metabolism in uremia suggest that menaquinone 7 supplementation to patients on dialysis therapy has reduced efficacy

Altered vitamin K biodistribution and metabolism in experimental and human chronic kidney disease

Chronic kidney disease (CKD) is accompanied with extensive cardiovascular calcification, in part correlating with functional vitamin K deficiency. Here, we sought to determine causes for vitamin K deficiency beyond reduced dietary intake. Initially, vitamin K uptake and distribution into circulating lipoproteins after a single administration of vitamin K1 plus K2 (menaquinone 4 and menaquinone 7, respectively) was determined in patients on dialysis therapy and healthy individuals. The patients incorporated very little menaquinone 7 but more menaquinone 4 into high density lipoprotein (HDL) and low-density lipoprotein particles than did healthy individuals. In contrast to healthy persons, HDL particles from the patients could not be spiked with menaquinone 7 in vitro and HDL uptake was diminished in osteoblasts. A reduced carboxylation activity (low vitamin K activity) of uremic HDL particles spiked with menaquinone 7 vs. that of controls was confirmed in a bioassay using human primary vascular smooth muscle cells. Kidney menaquinone 4 tissue levels were reduced in 5/6-nephrectomized versus sham-operated C57BL/6 mice after four weeks of a vitamin K rich diet. From the analyzed enzymes involved in vitamin K metabolism, kidney HMG-CoA reductase protein was reduced in both rats and patients with CKD. In a trial on the efficacy and safety of atorvastatin in 1051 patients with type 2 diabetes receiving dialysis therapy, no pronounced vitamin K deficiency was noted. However, the highest levels of PIVKA-II (biomarker of subclinical vitamin K deficiency) were noted when a statin was combined with a proton pump inhibitor. Thus, profound disturbances in lipoprotein mediated vitamin K transport and metabolism in uremia suggest that menaquinone 7 supplementation to patients on dialysis therapy has reduced efficacy