KCNQ1 is an essential mediator of the sex-dependent perception of moderate cold temperatures.

Low temperatures and cooling agents like menthol induce cold sensation by activating the peripheral cold receptors TRPM8 and TRPA1, cation channels belonging to the TRP channel family, while the reduction of potassium currents provides an additional and/or synergistic mechanism of cold sensation. Despite extensive studies over the past decades to identify the molecular receptors that mediate thermosensation, cold sensation is still not fully understood and many cold-sensitive peripheral neurons do not express the well-established cold sensor TRPM8. We found that the voltage-gated potassium channel KCNQ1 (Kv7.1), which is defective in cardiac LQT1 syndrome, is, in addition to its known function in the heart, a highly relevant and sex-specific sensor of moderately cold temperatures. We found that KCNQ1 is expressed in skin and dorsal root ganglion neurons, is sensitive to menthol and cooling agents, and is highly sensitive to moderately cold temperatures, in a temperature range at which TRPM8 is not thermosensitive. C-fiber recordings from KCNQ1-/- mice displayed altered action potential firing properties. Strikingly, only male KCNQ1-/- mice showed substantial deficits in cold avoidance at moderately cold temperatures, with a strength of the phenotype similar to that observed in TRPM8-/- animals. While sex-dependent differences in thermal sensitivity have been well documented in humans and mice, KCNQ1 is the first gene reported to play a role in sex-specific temperature sensation. Moreover, we propose that KCNQ1, together with TRPM8, is a key instrumentalist that orchestrates the range and intensity of cold sensation.

GWAS meta-analysis followed by Mendelian randomization revealed potential control mechanisms for circulating α-Klotho levels.

The protein α-Klotho acts as transmembrane co-receptor for fibroblast growth factor 23 (FGF23) and is a key regulator of phosphate homeostasis. However, α-Klotho also exists in a circulating form, with pleiotropic, but incompletely understood functions and regulation. Therefore, we undertook a genome-wide association study (GWAS) meta-analysis followed by Mendelian randomization (MR) of circulating α-Klotho levels. Plasma α-Klotho levels were measured by enzyme-linked immunosorbent assay (ELISA) in the Ludwigshafen Risk and Cardiovascular Health and Avon Longitudinal Study of Parents and Children (mothers) cohorts, followed by a GWAS meta-analysis in 4376 individuals across the two cohorts. Six signals at five loci were associated with circulating α-Klotho levels at genome-wide significance (P < 5 × 10-8), namely ABO, KL, FGFR1, and two post-translational modification genes, B4GALNT3 and CHST9. Together, these loci explained >9% of the variation in circulating α-Klotho levels. MR analyses revealed no causal relationships between α-Klotho and renal function, FGF23-dependent factors such as vitamin D and phosphate levels, or bone mineral density. The screening for genetic correlations with other phenotypes followed by targeted MR suggested causal effects of liability of Crohn's disease risk [Inverse variance weighted (IVW) beta = 0.059 (95% confidence interval 0.026, 0.093)] and low-density lipoprotein cholesterol levels [-0.198 (-0.332, -0.063)] on α-Klotho. Our GWAS findings suggest that two enzymes involved in post-translational modification, B4GALNT3 and CHST9, contribute to genetic influences on α-Klotho levels, presumably by affecting protein turnover and stability. Subsequent evidence from MR analyses on α-Klotho levels suggest regulation by mechanisms besides phosphate-homeostasis and raise the possibility of cross-talk with FGF19- and FGF21-dependent pathways, respectively. Significance statement: α-Klotho as a transmembrane protein is well investigated along the endocrine FGF23-α-Klotho pathway. However, the role of the circulating form of α-Klotho, which is generated by cleavage of transmembrane α-Klotho, remains incompletely understood. Genetic analyses might help to elucidate novel regulatory and functional mechanisms. The identification of genetic factors related to circulating α-Klotho further enables MR to examine causal relationships with other factors. The findings from the first GWAS meta-analysis of circulating α-Klotho levels identified six genome-wide significant signals across five genes. Given the function of two of the genes identified, B4GALNT3 and CHST9, it is tempting to speculate that post-translational modification significantly contributes to genetic influences on α-Klotho levels, presumably by affecting protein turnover and stability.

Increased ß-adrenergic stimulation augments vascular smooth muscle cell calcification via PKA/CREB signalling.

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 ß-adrenergic receptors. The present study explored the effects of ß2-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. ß2-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 ß2-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.

Protective effects of spironolactone on vascular calcification in chronic kidney disease.

BACKGROUND: Vascular calcification is common in chronic kidney disease (CKD) and associated with increased cardiovascular mortality. Aldosterone has been implicated as an augmenting factor in the progression of vascular calcification. The present study further explored putative beneficial effects of aldosterone inhibition by the mineralocorticoid receptor antagonist spironolactone on vascular calcification in CKD. METHODS: Serum calcification propensity was determined in serum samples from the MiREnDa trial, a prospective, randomized controlled clinical trial to investigate efficacy and safety of spironolactone in maintenance hemodialysis patients. Experiments were conducted in mice with subtotal nephrectomy and cholecalciferol treatment, and in calcifying primary human aortic smooth muscle cells (HAoSMCs). RESULTS: Serum calcification propensity was improved by spironolactone treatment in patients on hemodialysis from the MiREnDa trial. In mouse models and HAoSMCs, spironolactone treatment ameliorated vascular calcification and expression of osteogenic markers. CONCLUSIONS: These observations support a putative benefit of spironolactone treatment in CKD-associated vascular calcification. Further research is required to investigate possible improvements in cardiovascular outcomes by spironolactone and whether the benefits outweigh the risks in patients with CKD.

Inflammation: a putative link between phosphate metabolism and cardiovascular disease.

Dietary habits in the western world lead to increasing phosphate intake. Under physiological conditions, extraosseous precipitation of phosphate with calcium is prevented by a mineral buffering system composed of calcification inhibitors and tight control of serum phosphate levels. The coordinated hormonal regulation of serum phosphate involves fibroblast growth factor 23 (FGF23), αKlotho, parathyroid hormone (PTH) and calcitriol. A severe derangement of phosphate homeostasis is observed in patients with chronic kidney disease (CKD), a patient collective with extremely high risk of cardiovascular morbidity and mortality. Higher phosphate levels in serum have been associated with increased risk for cardiovascular disease (CVD) in CKD patients, but also in the general population. The causal connections between phosphate and CVD are currently incompletely understood. An assumed link between phosphate and cardiovascular risk is the development of medial vascular calcification, a process actively promoted and regulated by a complex mechanistic interplay involving activation of pro-inflammatory signalling. Emerging evidence indicates a link between disturbances in phosphate homeostasis and inflammation. The present review focuses on critical interactions of phosphate homeostasis, inflammation, vascular calcification and CVD. Especially, pro-inflammatory responses mediating hyperphosphatemia-related development of vascular calcification as well as FGF23 as a critical factor in the interplay between inflammation and cardiovascular alterations, beyond its phosphaturic effects, are addressed.

Acid sphingomyelinase promotes SGK1-dependent vascular calcification.

In chronic kidney disease (CKD), hyperphosphatemia is a key factor promoting medial vascular calcification, a common complication associated with cardiovascular events and high mortality. Vascular calcification involves osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs), but the complex signaling events inducing pro-calcific pathways are incompletely understood. The present study investigated the role of acid sphingomyelinase (ASM)/ceramide as regulator of VSMC calcification. In vitro, both, bacterial sphingomyelinase and phosphate increased ceramide levels in VSMCs. Bacterial sphingomyelinase as well as ceramide supplementation stimulated osteo-/chondrogenic transdifferentiation during control and high phosphate conditions and augmented phosphate-induced calcification of VSMCs. Silencing of serum- and glucocorticoid-inducible kinase 1 (SGK1) blunted the pro-calcific effects of bacterial sphingomyelinase or ceramide. Asm deficiency blunted vascular calcification in a cholecalciferol-overload mouse model and ex vivo isolated-perfused arteries. In addition, Asm deficiency suppressed phosphate-induced osteo-/chondrogenic signaling and calcification of cultured VSMCs. Treatment with the functional ASM inhibitors amitriptyline or fendiline strongly blunted pro-calcific signaling pathways in vitro and in vivo. In conclusion, ASM/ceramide is a critical upstream regulator of vascular calcification, at least partly, through SGK1-dependent signaling. Thus, ASM inhibition by repurposing functional ASM inhibitors to reduce the progression of vascular calcification during CKD warrants further study.

Stimulation of ORAI1 expression, store-operated Ca2+ entry, and osteogenic signaling by high glucose exposure of human aortic smooth muscle cells.

Diabetes and chronic kidney disease (CKD) both trigger vascular osteogenic signaling and calcification leading to early death by cardiovascular events. Osteogenic signaling involves upregulation of the transcription factors CBFA1, MSX2, and SOX9, as well as alkaline phosphatase (ALP), an enzyme fostering calcification by degrading the calcification inhibitor pyrophosphate. In CKD, osteogenic signaling is triggered by hyperphosphatemia, which upregulates the serum and glucocorticoid-inducible kinase SGK1, a strong stimulator of the Ca2+-channel ORAI1. The channel is activated by STIM1 and accomplishes store-operated Ca2+-entry (SOCE). The present study explored whether exposure of human aortic smooth muscle cells (HAoSMCs) to high extracellular glucose concentrations similarly upregulates ORAI1 and/or STIM1 expression, SOCE, and osteogenic signaling. To this end, HAoSMCs were exposed to high extracellular glucose concentrations (15 mM, 24 h) without or with additional exposure to the phosphate donor ß-glycerophosphate. Transcript levels were estimated using qRT-PCR, protein abundance using Western blotting, ALP activity using a colorimetric assay kit, calcium deposits utilizing Alizarin red staining, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). As a result, glucose enhanced the transcript levels of SGK1 and ORAI1, ORAI2, and STIM2, protein abundance of ORAI1, SOCE, the transcript levels of CBFA1, MSX2, SOX9, and ALPL, as well as calcium deposits. Moreover, glucose significantly augmented the stimulating effect of ß-glycerophosphate on transcript levels of SGK1 and ORAI1, SOCE, the transcript levels of osteogenic markers, as well as calcium deposits. ORAI1 inhibitor MRS1845 (10 µM) significantly blunted the glucose-induced upregulation of the CBFA1 and MSX2 transcript levels. In conclusion, the hyperglycemia of diabetes stimulates expression of SGK1 and ORAI1, thus, augmenting store-operated Ca2+-entry and osteogenic signaling in HAoSMCs.

Reversal of phosphate-induced ORAI1 expression, store-operated Ca2+ entry and osteogenic signaling by MgCl2 in human aortic smooth muscle cells.

In chronic kidney disease, renal phosphate retention leads to hyperphosphatemia with subsequent vascular osteogenic signaling and calcification. Osteogenic signaling involves up-regulation of the transcription factors CBFA1, MSX2, and SOX9, as well as alkaline phosphatase (ALP), an enzyme stimulating calcification by degrading the calcification inhibitor pyrophosphate. Stimulation of osteogenic signaling and calcification by phosphate donor ß-glycerophosphate in human aortic smooth muscle cells (HAoSMCs) is attenuated by MgCl2, an effect mimicked by Ca2+-sensing receptor agonist GdCl3. Most recent observations revealed that the effect of ß-glycerophosphate on osteogenic signaling requires ORAI1, a Ca2+-channel accomplishing store-operated Ca2+-entry (SOCE), which is stimulated by Ca2+-sensor STIM1. The present study explored whether ORAI1 and/or STIM1 expression and, thus, SOCE and osteogenic signaling in HAoSMCs are sensitive to MgCl2 and/or GdCl3. To this end, transcript levels were estimated using q-RT-PCR, protein abundance with western blotting, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1  µM). As a result, 24 h exposure to ß-glycerophosphate (2 mM) significantly enhanced transcript levels of ORAI1 and STIM1 as well as SOCE, effects significantly blunted or virtually abrogated by 1.5 mM MgCl2 and by 50  µM GdCl3. In conclusion, MgCl2 and GdCl3 are powerful inhibitors of ORAI1 and STIM1 expression and store-operated Ca2+-entry, effects affecting osteogenic signalling in vascular smooth muscle cells.

Klotho Deficiency Induces Arteriolar Hyalinosis in a Trade-Off with Vascular Calcification.

Hyalinosis is a vascular lesion affecting the renal vasculature and contributing to aging-related renal function decline. We assessed whether arteriolar hyalinosis is caused by Klotho deficiency, a state known to induce both renal and vascular phenotypes associated with aging. Histochemistry was used to assess hyalinosis in Klotho-/- kidneys, compared with Klotho+/- and wild-type littermates. Immunohistochemistry was used to investigate vascular lesion composition and the different layers of the vascular wall. Finally, spironolactone was used to inhibit calcification in kl/kl mice, and vascular lesions were characterized in the kidney. Arteriolar hyalinosis was detected in Klotho-/- mice, which was present up to the afferent arterioles. Hyalinosis was accompanied by loss of α-smooth muscle actin expression, whereas the endothelial lining was mostly intact. Hyalinous lesions were positive for IgM and iC3b/c/d, indicating subendothelial leakage of plasma proteins. The presence of extracellular matrix proteins suggested increased production by smooth muscle cells (SMCs). Finally, in Klotho-/- mice with marked vascular calcification, treatment with spironolactone allowed for replacement of calcification by hyalinosis. Klotho deficiency potentiates both endothelial hyperpermeability and SMC dedifferentiation. In the absence of a calcification-inducing stimulus, SMCs assume a synthetic phenotype in response to subendothelial leakage of plasma proteins. In the kidney, this results in arteriolar hyalinosis, which contributes to the decline in renal function. Klotho may play a role in preventing aging-related arteriolar hyalinosis.

Associations of Thyroid Hormones and Resting Heart Rate in Patients Referred to Coronary Angiography.

Resting heart rate (RHR) is associated with increased risk of cardiovascular morbidity and mortality. Thyroid hormones exert several effects on the cardiovascular system, but the relation between thyroid function and RHR remains to be further established. We evaluated whether measures of thyroid hormone status are associated with RHR in patients referred to coronary angiography. Thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), free thyroxin (FT4), and RHR were determined in 2795 participants of the Ludwigshafen Risk and Cardiovascular Health (LURIC) Study. Median (25th to 75th percentile) serum concentrations were 1.25 (0.76-1.92) mU/l for TSH, 4.8 (4.2-5.3) pmol/l for FT3 and 17.1 (15.4-19.0) pmol/l for FT4, and mean (±standard deviation) RHR was 68.8 (±11.7) beats/min. Comparing the highest versus the lowest quartile, RHR (beats/min) was significantly higher in the fourth FT4 quartile [3.48, 95% confidence interval (CI): 2.23-4.73; p <0.001] and in the fourth FT3 quartile (2.30, 95% CI: 1.06-3.55; p <0.001), but there was no significant difference for TSH quartiles. In multiple linear regression analyses adjusting for various potential confounders, FT3 and FT4 were significant predictors of RHR (p <0.001 for both). In subgroups restricted to TSH, FT3, and FT4 values within the reference range, both FT3 and FT4 remained significant predictors of RHR (p <0.001 for all). In conclusion, in patients referred to coronary angiography, FT3 and FT4 but not TSH were positively associated with RHR. The relationship between free thyroid hormones and RHR warrants further investigations regarding its diagnostic and therapeutic implications.

Signaling pathways involved in vascular smooth muscle cell calcification during hyperphosphatemia.

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.

Role of SGK1 in the Osteogenic Transdifferentiation and Calcification of Vascular Smooth Muscle Cells Promoted by Hyperglycemic Conditions.

In diabetes mellitus, hyperglycemia promotes the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) to enhance medial vascular calcification, a common complication strongly associated with cardiovascular disease and mortality. The mechanisms involved are, however, still poorly understood. Therefore, the present study explored the potential role of serum- and glucocorticoid-inducible kinase 1 (SGK1) during vascular calcification promoted by hyperglycemic conditions. Exposure to high-glucose conditions up-regulated the SGK1 expression in primary human aortic VSMCs. High glucose increased osteogenic marker expression and activity and, thus, promoted the osteogenic transdifferentiation of VSMCs, effects significantly suppressed by additional treatment with the SGK1 inhibitor EMD638683. Moreover, high glucose augmented the mineralization of VSMCs in the presence of calcification medium, effects again significantly reduced by SGK1 inhibition. Similarly, SGK1 knockdown blunted the high glucose-induced osteogenic transdifferentiation of VSMCs. The osteoinductive signaling promoted by high glucose required SGK1-dependent NF-kB activation. In addition, advanced glycation end products (AGEs) increased the SGK1 expression in VSMCs, and SGK1 inhibition was able to interfere with AGEs-induced osteogenic signaling. In conclusion, SGK1 is up-regulated and mediates, at least partly, the osteogenic transdifferentiation and calcification of VSMCs during hyperglycemic conditions. Thus, SGK1 inhibition may reduce the development of vascular calcification promoted by hyperglycemia in diabetes.

SGK1-dependent stimulation of vascular smooth muscle cell osteo-/chondrogenic transdifferentiation by interleukin-18.

The serum- and glucocorticoid-inducible kinase 1 (SGK1) is a key regulator of osteo-/chondrogenic transdifferentiation and subsequent calcification of vascular smooth muscle cells (VSMCs). The phenotypical transdifferentiation of VSMCs is associated with increased interleukin-18 (IL-18) levels and generalized inflammation. Therefore, the present study investigated the possible involvement of SGK1 in IL-18-induced vascular calcification. Experiments were performed in primary human aortic smooth muscle cells (HAoSMCs) treated with recombinant human IL-18 protein in control or high phosphate conditions and following SGK1 knockdown by siRNA or pharmacological inhibition of SGK1, PI3K, and PDK1. As a result, IL-18 treatment increased SGK1 mRNA and protein expression in HAoSMCs. IL-18 upregulated SGK1 mRNA expression in a dose-dependent manner. This effect was paralleled by upregulation of the mRNA expression of MSX2 and CBFA1, osteogenic transcription factors, and of tissue-nonspecific alkaline phosphatase (ALPL), an osteogenic enzyme, as markers of increased osteo-/chondrogenic transdifferentiation. Phosphate treatment increased SGK1 and osteogenic markers mRNA expression as well as ALPL activity and induced calcification of HAoSMCs, all effects significantly augmented by additional treatment with IL-18. Conversely, silencing of SGK1 or cotreatment with the SGK1 inhibitor EMD638683 blunted the effects of IL-18 on osteo-/chondrogenic transdifferentiation and calcification of HAoSMCs. The procalcific effects of IL-18 were similarly suppressed in the presence of PI3K or PDK1 inhibitors. In conclusion, SGK1 expression is upregulated by IL-18 in VSMCs and SGK1 participates in the intracellular signaling of IL-18-induced osteo-/chondrogenic transdifferentiation of VSMCs. Thus, SGK1 may serve as therapeutic target to limit the progression of medial vascular calcification during vascular inflammation.

An overview of the mechanisms in vascular calcification during chronic kidney disease.

PURPOSE OF REVIEW: Chronic kidney disease (CKD) facilitates a unique environment to strongly accelerate vascular calcification - the pathological deposition of calcium-phosphate in the vasculature. These calcifications are associated with the excessive cardiovascular mortality of CKD patients. RECENT FINDINGS: Vascular calcification is a multifaceted active process, mediated, at least partly, by vascular smooth muscle cells. These cells are able to transdifferentiate into cells with osteo/chondrogenic properties, which exert multiple effects to facilitate vascular tissue mineralization. As the understanding of the underlying pathophysiology increases, first therapeutic concepts begin to emerge. SUMMARY: This brief review provides an overview on the so far known mechanisms involved in the initiation and progression of vascular calcification in CKD.

Effects of vitamin D supplementation on FGF23: a randomized-controlled trial.

PURPOSE: Fibroblast growth factor-23 (FGF23) is critical for phosphate homeostasis. Considering the high prevalence of vitamin D deficiency and the association of FGF23 with adverse outcomes, we investigated effects of vitamin D3 supplementation on FGF23 concentrations. METHODS: This is a post-hoc analysis of the Styrian Vitamin D Hypertension trial, a single-center, double-blind, randomized, placebo-controlled trial, conducted from 2011 to 2014 at the Medical University of Graz, Austria. Two hundred subjects with 25(OH)D concentrations < 30 ng/mL and arterial hypertension were randomized to receive either 2800 IU of vitamin D3 daily or placebo over 8 weeks. Primary outcome was the between-group difference in FGF23 levels at study end while adjusting for baseline values. RESULTS: Overall, 181 participants (mean ± standard deviation age, 60.1 ± 11.3; 48% women) with available c-term FGF23 concentrations were considered for the present analysis. Mean treatment duration was 54 ± 10 days in the vitamin D3 group and 54 ± 9 days in the placebo group. At baseline, FGF23 was significantly correlated with serum phosphate (r = 0.135; p = 0.002). Vitamin D3 supplementation had no significant effect on FGF23 in the entire cohort (mean treatment effect 0.374 pmol/L; 95% confidence interval - 0.024 to 0.772 pmol/L; p = 0.065), but increased FGF23 concentrations in subgroups with baseline 25(OH)D concentrations below 20 ng/mL (n = 70; mean treatment effect 0.973 pmol/L; 95% confidence interval - 0.032 to 1.979 pmol/L; p = 0.019) and 16 ng/mL (n = 40; mean treatment effect 0.593 pmol/L; 95% confidence interval 0.076 to 1.109; p = 0.022). CONCLUSIONS: Vitamin D3 supplementation had no significant effect on FGF23 in the entire study cohort. We did, however, observe an increase of FGF23 concentrations in subgroups with low baseline 25(OH)D.

Zinc Inhibits Phosphate-Induced Vascular Calcification through TNFAIP3-Mediated Suppression of NF-κB.

Background The high cardiovascular morbidity and mortality of patients with CKD may result in large part from medial vascular calcification, a process promoted by hyperphosphatemia and involving osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). Reduced serum zinc levels have frequently been observed in patients with CKD, but the functional relevance of this remains unclear.Methods We performed experiments in primary human aortic VSMCs; klotho-hypomorphic (kl/kl), subtotal nephrectomy, and cholecalciferol-overload mouse calcification models; and serum samples from patients with CKD.Results In cultured VSMCs, treatment with zinc sulfate (ZnSO4) blunted phosphate-induced calcification, osteo-/chondrogenic signaling, and NF-κB activation. ZnSO4 increased the abundance of zinc-finger protein TNF-α-induced protein 3 (TNFAIP3, also known as A20), a suppressor of the NF-κB pathway, by zinc-sensing receptor ZnR/GPR39-dependent upregulation of TNFAIP3 gene expression. Silencing of TNFAIP3 in VSMCs blunted the anticalcific effects of ZnSO4 under high phosphate conditions. kl/kl mice showed reduced plasma zinc levels, and ZnSO4 supplementation strongly blunted vascular calcification and aortic osteoinduction and upregulated aortic Tnfaip3 expression. ZnSO4 ameliorated vascular calcification in mice with chronic renal failure and mice with cholecalciferol overload. In patients with CKD, serum zinc concentrations inversely correlated with serum calcification propensity. Finally, ZnSO4 ameliorated the osteoinductive effects of uremic serum in VSMCs.Conclusions Zinc supplementation ameliorates phosphate-induced osteo-/chondrogenic transdifferentiation of VSMCs and vascular calcification through an active cellular mechanism resulting from GPR39-dependent induction of TNFAIP3 and subsequent suppression of the NF-κB pathway. Zinc supplementation may be a simple treatment to reduce the burden of vascular calcification in CKD.

Fibulin-3 Attenuates Phosphate-Induced Vascular Smooth Muscle Cell Calcification by Inhibition of Oxidative Stress.

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.

Role of PKB/SGK-dependent phosphorylation of GSK-3α/ß in vascular calcification during cholecalciferol overload in mice.

Medial vascular calcification is a highly regulated process involving osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells. Both, protein kinase B (PKB) and serum- and glucocorticoid-inducible kinase 1 (SGK1) are involved in the intracellular signaling of vascular calcification and both phosphorylate and inactivate glycogen synthase kinase 3 (GSK-3). The present study explored whether PKB/SGK-dependent phosphorylation of GSK-3α/ß is involved in vascular calcification. Experiments were performed in Gsk-3α/ß double knockin mice lacking functional PKB/SGK phosphorylation sites (gsk-3KI) and corresponding wild-type mice (gsk-3WT) following high-dosed cholecalciferol treatment as well as ex vivo in aortic ring explants from gsk-3KI and gsk-3WT mice treated without and with phosphate. In gsk-3WT mice, high-dosed cholecalciferol induced vascular calcification and aortic osteo-/chondrogenic signaling, shown by increased expression of osteogenic markers Msx2, Cbfa1 and tissue-nonspecific alkaline phosphatase (Alpl). All these effects were suppressed in aortic tissue from gsk-3KI mice. Cholecalciferol decreased aortic Gsk-3α/ß phosphorylation (Ser21/9) in gsk-3WT mice, while no phosphorylation was observed in gsk-3KI mice. Moreover, the mRNA expression of type III sodium-dependent phosphate transporter (Pit1) and plasminogen activator inhibitor 1 (Pai1) was increased following cholecalciferol treatment in aortic tissue of gsk-3WT mice, effects again blunted in gsk-3KI mice. In addition, phosphate treatment induced mineral deposition and osteogenic markers expression in aortic ring explants from gsk-3WT mice, effects reduced in aortic ring explants from gsk-3KI mice. In conclusion, vascular PKB/SGK-dependent phosphorylation of GSK-3α/ß contributes to the osteoinductive signaling leading to vascular calcification.

Adenylyl cyclase 6 in acid-base balance - adding complexity.

Systemic acid-base balance is tightly controlled within a narrow range of pH. Disturbances in systemic acid-base homeostasis are associated with diverse detrimental effects. The kidney is a key regulator of acid-base balance, capable of excreting HCO3- or H+, and chronic kidney disease invariably leads to acidosis. However, the regulatory pathways underlying the fine-tuned acid-base sensing and regulatory mechanisms are still incompletely understood. In the article published recently in Clinical Science (vol 132 (16) 1779-1796), Poulson and colleagues investigated the role of adenylyl cyclase 6 (AC6) in acid-base homeostasis. They uncovered a complex role of AC6, specifically affecting acid-base balance during HCO3- load, which causes pronounced alkalosis in AC6-deficient mice. However, the phenotype of AC6-deficient mice appears much more complex, involving systemic effects associated with increased energy expenditure. These observations remind us that there is much to be learned about the intricate signaling pathways involved in renal control of acid-base balance and the complex ramifications of acid-base regulation.

Mineralocorticoid Receptor Blockers and Aldosterone to Renin Ratio: A Randomized Controlled Trial and Observational Data.

Current guidelines recommend to withdraw mineralocorticoid receptor (MR) blocker treatment for at least 4 weeks when measuring the aldosterone to renin ratio (ARR) as a screening test for primary aldosteronism (PA). We aimed to evaluate the effect of MR blocker treatment on ARR and its components, plasma aldosterone concentration (PAC), and direct renin concentration (DRC). First, we performed a post-hoc analysis of the effect of eplerenone on parathyroid hormone levels in primary hyperparathyroidism (EPATH) study, a randomized controlled trial (RCT) in 110 patients with primary hyperparathyroidism (pHPT). Patients were 1:1 randomly assigned to receive either 25 mg eplerenone once daily (up-titration after 4 weeks to 50 mg/day) or placebo for 8 weeks. Second, we measured the ARR in 4 PA patients from the Graz Endocrine Causes of Hypertension Study (GECOH) before and after MR blocker treatment. Ninety-seven participants completed the EPATH trial, and the mean treatment effect (95% confidence interval) for log(e)ARR was 0.08 (-0.32 to 0.48) ng/dl/µU/ml (p=0.694). The treatment effect was 0.71 (0.47 to 0.96; p<0.001) ng/dl for log(e)PAC and 0.64 (0.19 to 1.10; p=0.006) µU/ml for log(e)DRC, respectively. In the 4 PA patients, the ARR decreased from 11.24±3.58 at baseline to 2.70±1.03 (p=0.013) ng/dl/µU/ml after MR blocker treatment. In this study with limited sample size, MR blocker treatment did not significantly alter the ARR in pHPT patients but significantly reduced the ARR in PA patients. Diagnostic utility of ARR and its components for PA diagnostics under MR blocker treatment warrants further study.