Medial vascular calcification has emerged as a putative key factor contributing to the excessive cardiovascular mortality of patients with chronic kidney disease (CKD). Hyperphosphatemia is ...considered a decisive determinant of vascular calcification in CKD. A critical role in initiation and progression of vascular calcification during elevated phosphate conditions is attributed to vascular smooth muscle cells (VSMCs), which are able to change their phenotype into osteo-/chondroblasts-like cells. These transdifferentiated VSMCs actively promote calcification in the medial layer of the arteries by producing a local pro-calcifying environment as well as nidus sites for precipitation of calcium and phosphate and growth of calcium phosphate crystals. Elevated extracellular phosphate induces osteo-/chondrogenic transdifferentiation of VSMCs through complex intracellular signaling pathways, which are still incompletely understood. The present review addresses critical intracellular pathways controlling osteo-/chondrogenic transdifferentiation of VSMCs and, thus, vascular calcification during hyperphosphatemia. Elucidating these pathways holds a significant promise to open novel therapeutic opportunities counteracting the progression of vascular calcification in CKD.
Expression of serum-and-glucocorticoid-inducible kinase-1 (SGK1) is low in most cells, but dramatically increases under certain pathophysiological conditions, such as glucocorticoid or ...mineralocorticoid excess, inflammation with TGFβ release, hyperglycemia, cell shrinkage and ischemia. SGK1 is activated by insulin and growth factors via phosphatidylinositide-3-kinase, 3-phosphoinositide-dependent kinase and mammalian target of rapamycin. SGK1 sensitive functions include activation of ion channels (including epithelial Na(+) channel ENaC, voltage gated Na(+) channel SCN5A transient receptor potential channels TRPV4 - 6, Ca(2+) release activated Ca(2+) channel Orai1/STIM1, renal outer medullary K(+) channel ROMK, voltage gated K(+) channels KCNE1/KCNQ1, kainate receptor GluR6, cystic fibrosis transmembrane regulator CFTR), carriers (including Na(+),Cl(-) symport NCC, Na(+),K(+),2Cl(-) symport NKCC, Na(+)/H(+) exchangers NHE1 and NHE3, Na(+), glucose symport SGLT1, several amino acid transporters), and Na(+)/K(+)-ATPase. SGK1 regulates several enzymes (e.g., glycogen synthase kinase-3, ubiquitin-ligase Nedd4-2) and transcription factors (e.g., forkhead transcription factor 3a, β-catenin, nuclear factor kappa B).
The phenotype of SGK1 knockout mice is mild and SGK1 is apparently dispensible for basic functions. Excessive SGK1 expression and activity, however, contributes to the pathophysiology of several disorders, including hypertension, obesity, diabetes, thrombosis, stroke, fibrosing disease, infertility and tumor growth. A SGK1 gene variant (prevalence ∼ 3 - 5% in Caucasians and ∼ 10% in Africans) is associated with hypertension, stroke, obesity and type 2 diabetes. SGK1 inhibitors have been developed and shown to reduce blood pressure of hyperinsulinemic mice and to counteract tumor cell survival.
Targeting SGK1 may be a therapeutic option in several clinical conditions, including metabolic syndrome and tumor growth.
A high intake of dietary salt (NaCl) has been implicated in the development of hypertension, chronic inflammation, and autoimmune diseases. We have recently shown that salt has a proinflammatory ...effect and boosts the activation of Th17 cells and the activation of classical, LPS-induced macrophages (M1). Here, we examined how the activation of alternative (M2) macrophages is affected by salt. In stark contrast to Th17 cells and M1 macrophages, high salt blunted the alternative activation of BM-derived mouse macrophages stimulated with IL-4 and IL-13, M(IL-4+IL-13) macrophages. Salt-induced reduction of M(IL-4+IL-13) activation was not associated with increased polarization toward a proinflammatory M1 phenotype. In vitro, high salt decreased the ability of M(IL-4+IL-13) macrophages to suppress effector T cell proliferation. Moreover, mice fed a high salt diet exhibited reduced M2 activation following chitin injection and delayed wound healing compared with control animals. We further identified a high salt-induced reduction in glycolysis and mitochondrial metabolic output, coupled with blunted AKT and mTOR signaling, which indicates a mechanism by which NaCl inhibits full M2 macrophage activation. Collectively, this study provides evidence that high salt reduces noninflammatory innate immune cell activation and may thus lead to an overall imbalance in immune homeostasis.
Background: Anemia is a major complication of end stage renal disease. The anemia is mainly the result of impaired formation of erythrocytes due to lack of erythropoietin and iron deficiency. ...Compelling evidence, however, points to the contribution of accelerated erythrocyte death, which decreases the life span of circulating erythrocytes. Erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine-exposure at the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+-activity (Ca2+i). Erythrocytes could be sensitized to cytosolic Ca2+ by ceramide. In end stage renal disease, eryptosis may possibly be stimulated by uremic toxins. The present study explored, whether the uremic toxin acrolein could trigger eryptosis. Methods: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, Ca2+i from Fluo3-fluorescence, and ceramide from fluorescent antibodies. Results: A 48 h exposure to acrolein (30 － 50 µM) did not significantly modify Ca2+i but significantly decreased forward scatter and increased annexin-V-binding. Acrolein further triggered slight, but significant hemolysis and increased ceramide formation in erythrocytes. Acrolein (50 µM) induced annexin-V-binding was significantly blunted in the nominal absence of extracellular Ca2+. Acrolein augmented the annexin-V-binding following treatment with Ca2+ ionophore ionomycin (1 µM). Conclusion: Acrolein stimulates suicidal erythrocyte death or eryptosis, an effect at least in part due to stimulation of ceramide formation with subsequent sensitisation of the erythrocytes to cytosolic Ca2+.
In chronic kidney disease (CKD), hyperphosphatemia promotes medial vascular calcification, a process augmented by osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs). VSMC ...function is regulated by sympathetic innervation, and these cells express α- and β-adrenergic receptors. The present study explored the effects of β2-adrenergic stimulation by isoproterenol on VSMC calcification. Experiments were performed in primary human aortic VSMCs treated with isoproterenol during control or high phosphate conditions. As a result, isoproterenol dose dependently up-regulated the expression of osteogenic markers core-binding factor α-1 (
CBFA1
) and tissue-nonspecific alkaline phosphatase (
ALPL
) in VSMCs. Furthermore, prolonged isoproterenol exposure augmented phosphate-induced calcification of VSMCs. Isoproterenol increased the activation of PKA and CREB, while knockdown of the PKA catalytic subunit α (
PRKACA
) or of
CREB1
genes was able to suppress the pro-calcific effects of isoproterenol in VSMCs. β2-adrenergic receptor silencing or inhibition with the selective antagonist ICI 118,551 blocked isoproterenol-induced osteogenic signalling in VSMCs. The present observations imply a pro-calcific effect of β2-adrenergic overstimulation in VSMCs, which is mediated, at least partly, by PKA/CREB signalling. These observations may support a link between sympathetic overactivity in CKD and vascular calcification.
Reduced homoarginine plasma levels are associated with unfavourable cardiovascular outcome in chronic kidney disease (CKD). Cardiovascular events in CKD are fostered by vascular calcification, an ...active process promoted by hyperphosphatemia and involving osteo-/chondrogenic transformation of vascular smooth muscle cells (VSMCs). The present study explored the effect of homoarginine on phosphate-induced osteo-/chondrogenic signalling and vascular calcification.
Experiments were performed in hyperphosphatemic klotho-hypomorphic mice (kl/kl), in subtotal nephrectomy and vitamin D3-overload mouse calcification models and in primary human aortic smooth muscle cells (HAoSMCs). As a result, plasma homoarginine levels were lower in kl/kl mice than in wild-type mice and in both genotypes significantly increased by lifelong treatment with homoarginine. Surprisingly, homoarginine treatment of kl/kl mice and of mice with renal failure after subtotal nephrectomy augmented vascular calcification and enhanced the transcript levels of plasminogen activator inhibitor 1 (Pai1) and of osteogenic markers Msx2, Cbfa1, and Alpl. Similarly, homoarginine treatment of HAoSMCs increased phosphate-induced calcium deposition, ALP activity, as well as PAI1, MSX2, CBFA1, and ALPL mRNA levels. Homoarginine alone up-regulated osteo-/chondrogenic signalling and indicators of oxidative stress in HAoSMCs. Furthermore, homoarginine reduced citrulline formation from arginine by nitric oxide (NO) synthase (NOS) isoforms. NO formation by NOS was reduced when using homoarginine as a substrate instead of arginine. The osteoinductive effects of homoarginine were mimicked by NOS inhibitor L-NAME and abolished by additional treatment with the NO donors DETA-NONOate and PAPA-NONOate or the antioxidants TEMPOL and TIRON. Furthermore, homoarginine augmented vascular calcification and aortic osteo-/chondrogenic signalling in mice after vitamin D3-overload, effects reversed by the NO donor molsidomine.
Homoarginine augments osteo-/chondrogenic transformation of VSMCs and vascular calcification, effects involving impaired NO formation from homoarginine.
In chronic kidney disease, hyperphosphatemia is a key pathological factor promoting medial vascular calcification, a common complication associated with cardiovascular events and mortality. This ...active pathophysiological process involves osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs) via complex intracellular mechanisms that are still incompletely understood. Little is known about the effects of phosphate on the bioenergetic profile of VSMCs during the onset of this process. Therefore, the present study explored the effects of the phosphate donor β-glycerophosphate on cellular bioenergetics of VSMCs. Mitochondrial and glycolytic functions were determined utilizing extracellular flux analysis in primary human aortic VSMCs following exposure to β-glycerophosphate. In VSMCs, β-glycerophosphate increased basal respiration, mitochondrial ATP production as well as proton leak and decreased spare respiratory capacity and coupling efficiency, but did not modify non-mitochondrial or maximal respiration. β-Glycerophosphate-treated VSMCs had higher ability to increase mitochondrial glutamine and long-chain fatty acid usage as oxidation substrates to meet their energy demand. β-Glycerophosphate did not modify glycolytic function or basal and glycolytic proton efflux rate. In contrast, β-glycerophosphate increased non-glycolytic acidification. β-Glycerophosphate-treated VSMCs had a more oxidative and less glycolytic phenotype, but a reduced ability to respond to stressed conditions via mitochondrial respiration. Moreover, compounds targeting components of mitochondrial respiration modulated β-glycerophosphate-induced oxidative stress, osteo-/chondrogenic signalling and mineralization of VSMCs. In conclusion, β-glycerophosphate modifies key parameters of mitochondrial function and cellular bioenergetics in VSMCs that may contribute to the onset of phenotypical transdifferentiation and calcification. These observations advance the understanding of the role of energy metabolism in VSMC physiology and pathophysiology of vascular calcification during hyperphosphatemia.
Key messages
β-Glycerophosphate modifies key parameters of mitochondrial respiration in VSMCs.
β-Glycerophosphate induces changes in mitochondrial fuel choice in VSMCs.
β-Glycerophosphate promotes a more oxidative and less glycolytic phenotype of VSMCs.
β-Glycerophosphate triggers mitochondrial-dependent oxidative stress in VSMCs.
Bioenergetics impact β-glycerophosphate-induced VSMC calcification.
Expression of the serum- and glucocorticoid-inducible kinase 1 (SGK1) is up-regulated by several types of cell stress, such as ischemia, radiation and hyperosmotic shock. The SGK1 protein is ...activated by a signaling cascade involving phosphatidylinositide-3-kinase (PI3K), 3-phosphoinositide-dependent kinase 1 (PDK1) and mammalian target of rapamycin (mTOR). SGK1 up-regulates Na
/K
-ATPase, a variety of carriers including Na
-,K
-,2Cl
- cotransporter (NKCC), NaCl cotransporter (NCC), Na
/H
exchangers, diverse amino acid transporters and several glucose carriers such as Na
-coupled glucose transporter SGLT1. SGK1 further up-regulates a large number of ion channels including epithelial Na
channel ENaC, voltagegated Na
channel SCN5A, Ca
release-activated Ca
channel (ORAI1) with its stimulator STIM1, epithelial Ca
channels TRPV5 and TRPV6 and diverse K
channels. Furthermore, SGK1 influences transcription factors such as nuclear factor kappa-B (NF-κB), p53 tumor suppressor protein, cAMP responsive element-binding protein (CREB), activator protein-1 (AP-1) and forkhead box O3 protein (FOXO3a). Thus, SGK1 supports cellular glucose uptake and glycolysis, angiogenesis, cell survival, cell migration, and wound healing. Presumably as last line of defense against tissue injury, SGK1 fosters tissue fibrosis and tissue calcification replacing energy consuming cells.
Background/Aims: Fibulin-3, an extracellular matrix glycoprotein, inhibits vascular oxidative stress and remodeling in hypertension. Oxidative stress is prevalent in chronic kidney disease (CKD) ...patients and is an important mediator of osteo-/chondrogenic transdifferentiation and calcification of vascular smooth muscle cells (VSMCs) during hyperphosphatemia. Therefore, the present study explored the effects of Fibulin-3 on phosphate-induced vascular calcification. Methods: Experiments were performed in primary human aortic smooth muscle cells (HAoSMCs) treated with control or with phosphate without or with additional treatment with recombinant human Fibulin-3 protein or with hydrogen peroxide as an exogenous source of oxidative stress. Results: Treatment with calcification medium significantly increased calcium deposition in HAoSMCs, an effect significantly blunted by additional treatment with Fibulin-3. Moreover, phosphate-induced alkaline phosphatase activity and mRNA expression of osteogenic and chondrogenic markers MSX2, CBFA1, SOX9 and ALPL were all significantly reduced by addition of Fibulin-3. These effects were paralleled by similar regulation of oxidative stress in HAoSMCs. Phosphate treatment significantly up-regulated mRNA expression of the oxidative stress markers NOX4 and CYBA, down-regulated total antioxidant capacity and increased the expression of downstream effectors of oxidative stress PAI-1, MMP2 and MMP9 as well as BAX/BLC2 ratio in HAoSMCs, all effects blocked by additional treatment with Fibulin-3. Furthermore, the protective effects of Fibulin-3 on phosphate-induced osteogenic and chondrogenic markers expression in HAoSMCs were reversed by additional treatment with hydrogen peroxide. Conclusions: Fibulin-3 attenuates phosphate-induced osteo-/ chondrogenic transdifferentiation and calcification of VSMCs, effects involving inhibition of oxidative stress. Up-regulation or supplementation of Fibulin-3 may be beneficial in reducing the progression of vascular calcification during hyperphosphatemic conditions such as CKD.
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