Jakob Voelkl

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.

Therapeutic Interference With Vascular Calcification—Lessons From Klotho-Hypomorphic Mice and Beyond

Medial vascular calcification, a major pathophysiological process associated with cardiovascular disease and mortality, involves osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). In chronic kidney disease (CKD), osteo-/chondrogenic transdifferentiation of VSMCs and, thus, vascular calcification is mainly driven by hyperphosphatemia, resulting from impaired elimination of phosphate by the diseased kidneys. Hyperphosphatemia with subsequent vascular calcification is a hallmark of klotho-hypomorphic mice, which are characterized by rapid development of multiple age-related disorders and early death. In those animals, hyperphosphatemia results from unrestrained formation of 1,25(OH)2D3 with subsequent retention of calcium and phosphate. Analysis of klotho-hypomorphic mice and mice with vitamin D3 overload uncovered several pathophysiological mechanisms participating in the orchestration of vascular calcification and several therapeutic opportunities to delay or even halt vascular calcification. The present brief review addresses the beneficial effects of bicarbonate, carbonic anhydrase inhibition, magnesium supplementation, mineralocorticoid receptor (MR) blockage, and ammonium salts. The case is made that bicarbonate is mainly effective by decreasing intestinal phosphate absorption, and that carbonic anhydrase inhibition leads to metabolic acidosis, which counteracts calcium-phosphate precipitation and VSMC transdifferentiation. Magnesium supplementation, MR blockage and ammonium salts are mainly effective by interference with osteo-/chondrogenic signaling in VSMCs. It should be pointed out that the, by far, most efficient substances are ammonium salts, which may virtually prevent vascular calcification. Future research will probably uncover further therapeutic options and, most importantly, reveal whether these observations in mice can be translated into treatment of patients suffering from vascular calcification, such as patients with CKD.

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

PurposeFibroblast 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.MethodsThis 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 2014xa0at the Medical University of Graz, Austria. Two hundred subjects with 25(OH)D concentrationsu2009<u200930xa0ng/mL and arterial hypertension were randomized to receive either 2800xa0IU of vitamin D3 daily or placebo over 8xa0weeks. Primary outcome was the between-group difference in FGF23 levels at study end while adjusting for baseline values.ResultsOverall, 181 participants (meanu2009±u2009standard deviation age, 60.1u2009±u200911.3; 48% women) with available c-term FGF23 concentrations were considered for the present analysis. Mean treatment duration was 54u2009±u200910 days in the vitamin D3 group and 54u2009±u20099 days in the placebo group. At baseline, FGF23 was significantly correlated with serum phosphate (ru2009=u20090.135; pu2009=u20090.002). Vitamin D3 supplementation had no significant effect on FGF23 in the entire cohort (mean treatment effect 0.374xa0pmol/L; 95% confidence interval −u20090.024 to 0.772xa0pmol/L; pu2009=u20090.065), but increased FGF23 concentrations in subgroups with baseline 25(OH)D concentrations below 20xa0ng/mL (nu2009=u200970; mean treatment effect 0.973xa0pmol/L; 95% confidence interval −u20090.032 to 1.979xa0pmol/L; pu2009=u20090.019) and 16xa0ng/mL (nu2009=u200940; mean treatment effect 0.593xa0pmol/L; 95% confidence interval 0.076 to 1.109; pu2009=u20090.022).ConclusionsVitamin 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.

Effect of eplerenone on markers of bone turnover in patients with primary hyperparathyroidism – The randomized, placebo-controlled EPATH trial

Mineralocorticoid receptor (MR) antagonism may affect bone turnover via direct and indirect pathways involving parathyroid hormone, but randomized controlled trials are lacking. In a pre-specified analysis of the Eplerenone in primary hyperparathyroidism placebo-controlled, randomized trial (ISRCTN 33941607), effects of eight weeks MR-blockade with eplerenone on bone turnover markers in 97 patients with primary hyperparathyroidism were tested. Mean age was 67.5±9.5years, and 76 (78.4%) were females. In analysis of covariance with adjustment for baseline values, eplerenone had no significant effect on isoform 5b of the tartrate-resistant acid phosphatase (TRAP), beta-crosslaps, N-terminal propeptide of procollagen type 1 (P1NP), osteocalcin and bone-specific alkaline phosphatase. There was no significant cross-sectional correlation between plasma aldosterone concentration or the aldosterone-to-renin ratio and markers of bone turnover in multivariate linear regression models at baseline. These data provide first evidence from a randomized and placebo-controlled trial that short-term MR antagonism may not affect bone turnover, at least in patients with primary hyperparathyroidism.

Heterotrimeric G‐protein subunit Gαi2 contributes to agonist‐sensitive apoptosis and degranulation in murine platelets

Gαi2, a heterotrimeric G‐protein subunit, regulates various cell functions including ion channel activity, cell differentiation, proliferation and apoptosis. Platelet‐expressed Gαi2 is decisive for the extent of tissue injury following ischemia/reperfusion. However, it is not known whether Gαi2 plays a role in the regulation of platelet apoptosis, which is characterized by caspase activation, cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) translocation to the platelet surface. Stimulators of platelet apoptosis include thrombin and collagen‐related peptide (CoRP), which are further known to enhance degranulation and activation of αIIbβ3‐integrin and caspases. Using FACS analysis, we examined the impact of agonist treatment on activation and apoptosis in platelets drawn from mice lacking Gαi2 and their wild‐type (WT) littermates. As a result, treatment with either thrombin (0.01 U/mL) or CoRP (2 μg/mL or 5 μg/mL) significantly upregulated PS‐exposure and significantly decreased forward scatter, reflecting cell size, in both genotypes. Exposure to CoRP triggered a significant increase in active caspase 3, ceramide formation, surface P‐selectin, and αIIbβ3‐integrin activation. These molecular alterations were significantly less pronounced in Gαi2‐deficient platelets as compared to WT platelets. In conclusion, our data highlight a previously unreported role of Gαi2 signaling in governing platelet activation and apoptosis.

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 25u2009mg eplerenone once daily (up-titration after 4 weeks to 50u2009mg/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)u2009μU/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.

Impact of Altered Mineral Metabolism on Pathological Cardiac Remodeling in Elevated Fibroblast Growth Factor 23

Clinical and experimental studies indicate a possible link between high serum levels of fibroblast growth factor 23 (FGF23), phosphate, and parathyroid hormone (PTH), deficiency of active vitamin D (1,25D) and klotho with the development of pathological cardiac remodeling, i.e., left ventricular hypertrophy and myocardial fibrosis, but a causal link has not been established so far. Here, we investigated the cardiac phenotype in klotho hypomorphic (kl/kl) mice and Hyp mice, two mouse models of elevated FGF23 levels and klotho deficiency, but differing in parameters of mineral metabolism, by using histology, quantitative real-time PCR, immunoblot analysis, and serum and urine biochemistry. Additionally, the specific impact of calcium, phosphate, PTH, and 1,25D on hypertrophic growth of isolated neonatal rat cardiac myocytes was investigated in vitro. Kl/kl mice displayed high serum Fgf23 levels, increased relative heart weight, enhanced cross-sectional area of individual cardiac myocytes, activated cardiac Fgf23/Fgf receptor (Fgfr) 4/calcineurin/nuclear factor of activated T cell (NFAT) signaling, and induction of pro-hypertrophic NFAT target genes including Rcan1, bMHC, brain natriuretic peptide (BNP), and atrial natriuretic peptide (ANP) as compared to corresponding wild-type (WT) mice. Investigation of fibrosis-related molecules characteristic for pathological cardiac remodeling processes demonstrated ERK1/2 activation and enhanced expression of Tgf-β1, collagen I, and Mmp2 in kl/kl mice than in WT mice. In contrast, despite significantly elevation of serum and cardiac Fgf23, and reduced renal klotho expression, Hyp mice showed no signs of pathological cardiac remodeling. Kl/kl mice showed enhanced serum calcium and phosphate levels, while Hyp mice showed unchanged serum calcium levels, lower serum phosphate, and elevated serum iPTH concentrations compared to corresponding WT mice. In cultured cardiac myocytes, treatment with both calcium or phosphate significantly upregulated endogenous Fgf23 mRNA expression and stimulated hypertrophic cell growth and expression of pro-hypertrophic genes. The treatment with PTH induced hypertrophic cell growth only, and stimulation with 1,25D had no significant effects. In conclusion, our data indicate that Hyp mice, in contrast to kl/kl mice appear to be protected from pathological cardiac remodeling during conditions of high FGF23 levels and klotho deficiency, which may be due, at least in part, to differences in mineral metabolism alterations, i.e., hypophosphatemia and lack of hypercalcemia.

Role of PKB/SGK-dependent phosphorylation of GSK-3α/β 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/β is involved in vascular calcification. Experiments were performed in Gsk-3α/β 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 exxa0vivo 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/β 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/β contributes to the osteoinductive signaling leading to vascular calcification.

Ldlr and ApoE mice better mimic the human metabolite signature of increased carotid intima media thickness compared to other animal models of cardiovascular disease

BACKGROUND AND AIMSnPreclinical experiments on animal models are essential to understand the mechanisms of cardiovascular disease (CVD). Metabolomics allows access to the metabolic perturbations associated with CVD in heart and vessels. Here we assessed which potential animal CVD model most closely mimics the serum metabolite signature of increased carotid intima-media thickness (cIMT) in humans, a clinical parameter widely accepted as a surrogate of CVD.nnnMETHODSnA targeted mass spectrometry assay was used to quantify and compare a series of blood metabolites between 1362 individuals (KORA F4 cohort) and 5 animal CVD models: ApoE-/-, Ldlr-/-, and klotho-hypomorphic mice (kl/kl) and SHRSP rats with or without salt feeding. The metabolite signatures were obtained using linear regressions adjusted for various co-variates.nnnRESULTSnIn human, increased cIMT [quartile Q4 vs. Q1] was associated with 26 metabolites (9 acylcarnitines, 2 lysophosphatidylcholines, 9 phosphatidylcholines and 6 sphingomyelins). Acylcarnitines correlated preferentially with serum glucose and creatinine. Phospholipids correlated preferentially with cholesterol (total and LDL). The human signature correlated positively and significantly with Ldlr-/- and ApoE-/- mice, while correlation with kl/kl mice and SHRP rats was either negative and non-significant. Human and Ldlr-/- mice shared 11 significant metabolites displaying the same direction of regulation: 5 phosphatidylcholines, 1 lysophosphatidylcholines, 5 sphingomyelins; ApoE-/- mice shared 10.nnnCONCLUSIONSnThe human cIMT signature was partially mimicked by Ldlr-/- and ApoE-/- mice. These animal models might help better understand the biochemical and molecular mechanisms involved in the vessel metabolic perturbations associated with, and contributing to metabolic disorders in CVD.