Chronic kidney disease is a key risk factor for aortic stenosis progression.

BACKGROUND: Rapid progression of aortic stenosis (AS) has been observed in patients undergoing dialysis, but existing cross-sectional evidence is contradictory in non-dialysis-dependent chronic kidney disease (CKD). The present study sought to evaluate whether CKD is associated with the progression of AS over time in a large cohort of patients with AS. METHODS: We retrospectively studied all consecutive patients diagnosed with AS [peak aortic jet velocity (Vmax) ≥2.5 m/s] and left ventricular ejection fraction ≥50% in the echocardiography laboratories of two tertiary centers between 2000 and 2018. The estimated glomerular filtration rate (eGFR) (mL/min/1.73 m2) was calculated from serum creatinine values. Patients were divided into five CKD stages according to the baseline eGFR. Annual rates of change in the aortic valve area (AVA) were determined by a linear mixed-effects model. RESULTS: Among the 647 patients included, 261 (40%) had CKD. After a median follow-up of 2.9 (interquartile range 1.8-4.8) years, the mean overall rate of change in AVA was -0.077 (95% confidence interval -0.082; -0.073) cm2/year. There was an inverse relationship between the progression rate and kidney function. The more severe the CKD stage, the greater the AVA narrowing (P < .001). By multivariable linear regression analysis, the eGFR was also negatively associated (P < .001) with AS progression. An eGFR strata below 45 mL/min/1.73 m2 was associated with higher odds of rapid progression of AS than normal kidney function. During the clinical follow-up, event-free survival (patients free of aortic valve replacement or death) decreased as CKD progressed. Rapid progression of AS in patients with kidney dysfunction was associated with worse outcomes. CONCLUSIONS: Patients with CKD exhibit more rapid progression of AS over time and require close monitoring. The link between kidney dysfunction and rapid progression of AS is still unknown and requires further research.

Vasorin plays a critical role in vascular smooth muscle cells and arterial functions.

Within the cardiovascular system, the protein vasorin (Vasn) is predominantly expressed by vascular smooth muscle cells (VSMCs) in the coronary arteries and the aorta. Vasn knockout (Vasn-/- ) mice die within 3 weeks of birth. In the present study, we investigated the role of vascular Vasn expression on vascular function. We used inducible Vasn knockout mice (VasnCRE-ERT KO and VasnSMMHC-CRE-ERT2 KO , in which respectively all cells or SMCs only are targeted) to analyze the consequences of total or selective Vasn loss on vascular function. Furthermore, in vivo effects were investigated in vitro using human VSMCs. The death of VasnCRE-ERT KO mice 21 days after tamoxifen injection was concomitant with decreases in blood pressure, angiotensin II levels, and vessel contractibility to phenylephrine. The VasnSMMHC-CRE-ERT2 KO mice displayed concomitant changes in vessel contractibility in response to phenylephrine and angiotensin II levels. In vitro, VASN deficiency was associated with a shift toward the SMC contractile phenotype, an increase in basal intracellular Ca2+ levels, and a decrease in the SMCs' ability to generate a calcium signal in response to carbachol or phenylephrine. Additionally, impaired endothelium-dependent relaxation (due to changes in nitric oxide signaling) was observed in all Vasn knockout mice models. Our present findings highlight the role played by Vasn SMC expression in the maintenance of vascular functions. The mechanistic experiments suggested that these effects are mediated by SMC phenotype switching and changes in intracellular calcium homeostasis, angiotensin II levels, and NO signaling.

The Endothelium and COVID-19: An Increasingly Clear Link Brief Title: Endotheliopathy in COVID-19.

The endothelium has a fundamental role in the cardiovascular complications of coronavirus disease 2019 (COVID-19). Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particularly affects endothelial cells. The virus binds to the angiotensin-converting enzyme 2 (ACE-2) receptor (present on type 2 alveolar cells, bronchial epithelial cells, and endothelial cells), and induces a cytokine storm. The cytokines tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6 have particular effects on endothelial cells-leading to endothelial dysfunction, endothelial cell death, changes in tight junctions, and vascular hyperpermeability. Under normal conditions, apoptotic endothelial cells are removed into the bloodstream. During COVID-19, however, endothelial cells are detached more rapidly, and do not regenerate as effectively as usual. The loss of the endothelium on the luminal surface abolishes all of the vascular responses mediated by the endothelium and nitric oxide production in particular, which results in greater contractility. Moreover, circulating endothelial cells infected with SARS-CoV-2 act as vectors for viral dissemination by forming clusters that migrate into the circulation and reach distant organs. The cell clusters and the endothelial dysfunction might contribute to the various thromboembolic pathologies observed in COVID-19 by inducing the formation of intravascular microthrombi, as well as by triggering disseminated intravascular coagulation. Here, we review the contributions of endotheliopathy and endothelial-cell-derived extracellular vesicles to the pathogenesis of COVID-19, and discuss therapeutic strategies that target the endothelium in patients with COVID-19.

Decreased monocyte calcium sensing receptor expression in patients with chronic kidney disease is associated with impaired monocyte ability to reduce vascular calcification.

In chronic kidney disease (CKD), calcium-sensing receptor (CaSR) expression and function have been extensively studied in parathyroid tissue and vascular tissues. To examine whether similar changes occurred in other tissues, we measured total and surface CaSR expression in monocytes of patients with various stages of CKD and healthy volunteers respectively in cross-sectional studies. We further explored in vitro the impact of uremic serum on CaSR expression in monocytes (U937 and THP-1 cell lines), and whether human peripheral blood mononuclear cells or U937 and THP-1 monocytes might modify vascular calcium deposition in rat carotid arteries in vitro. CKD was associated with a decrease in peripheral blood mononuclear cell CaSR expression both in total and at the monocyte surface alone (43% and 34%, respectively in CKD stages 4-5). This decrease was associated with a reduction in the ability of monocytes to inhibit vascular calcification in vitro. Pretreatment with the calcimimetic NPSR568 of peripheral blood mononuclear cells isolated from patients with CKD significantly improved monocyte capacity to reduce carotid calcification in vitro. The fewer peripheral blood mononuclear cells expressing cell surface CaSR, the more calcimimetic treatment enhanced the decrease of carotid calcium content. Thus, we demonstrate that monocyte CaSR expression is decreased in patients with CKD and provide in vitro evidence for a potential role of this decrease in the promotion of vascular calcification. Hence, targeting this alteration or following monocyte CaSR expression as an accessible marker might represent a promising therapeutic strategy in CKD-associated arterial calcification.

Circulating Receptor Activator of Nuclear Factor kB Ligand and triglycerides are associated with progression of lower limb arterial calcification in type 2 diabetes: a prospective, observational cohort study.

BACKGROUND: Lower limb arterial calcification is a frequent, underestimated but serious complication of diabetes. The DIACART study is a prospective cohort study designed to evaluate the determinants of the progression of lower limb arterial calcification in 198 patients with type 2 diabetes. METHODS: Lower limb arterial calcification scores were determined by computed tomography at baseline and after a mean follow up of 31.20 ± 3.86 months. Serum RANKL (Receptor Activator of Nuclear factor kB Ligand) and bone remodeling, inflammatory and metabolic parameters were measured at baseline. The predictive effect of these markers on calcification progression was analyzed by a multivariate linear regression model. RESULTS: At baseline, mean ± SD and median lower limb arterial calcification scores were, 2364 ± 5613 and 527 respectively and at the end of the study, 3739 ± 6886 and 1355 respectively. Using multivariate analysis, the progression of lower limb arterial log calcification score was found to be associated with (ß coefficient [slope], 95% CI, p-value) baseline log(calcification score) (1.02, 1.00-1.04, p < 0.001), triglycerides (0.11, 0.03-0.20, p = 0.007), log(RANKL) (0.07, 0.02-0.11, p = 0.016), previous ischemic cardiomyopathy (0.36, 0.15-0.57, p = 0.001), statin use (0.39, 0.06-0.72, p = 0.023) and duration of follow up (0.04, 0.01-0.06, p = 0.004). CONCLUSION: In patients with type 2 diabetes, lower limb arterial calcification is frequent and can progress rapidly. Circulating RANKL and triglycerides are independently associated with this progression. These results open new therapeutic perspectives in peripheral diabetic calcifying arteriopathy. Trial registration NCT02431234.

The Metabolism of Epoxyeicosatrienoic Acids by Soluble Epoxide Hydrolase Is Protective against the Development of Vascular Calcification.

This study addressed the hypothesis that soluble epoxide hydrolase (sEH), which metabolizes endothelium-derived epoxyeicosatrienoic acids, plays a role in vascular calcification. The sEH inhibitor trans-4-(4-(3-adamantan-1-yl-ureido)-cyclohexyloxy)-benzoic acid (t-AUCB) potentiated the increase in calcium deposition of rat aortic rings cultured in high-phosphate conditions. This was associated with increased tissue-nonspecific alkaline phosphatase activity and mRNA expression level of the osteochondrogenic marker Runx2. The procalcifying effect of t-AUCB was prevented by mechanical aortic deendothelialization or inhibition of the production and action of epoxyeicosatrienoic acids using the cytochrome P450 inhibitor fluconazole and the antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE), respectively. Similarly, exogenous epoxyeicosatrienoic acids potentiated the calcification of rat aortic rings through a protein kinase A (PKA)-dependent mechanism and of human aortic vascular smooth muscle cells when sEH was inhibited by t-AUCB. Finally, a global gene expression profiling analysis revealed that the mRNA expression level of sEH was decreased in human carotid calcified plaques compared to adjacent lesion-free sites and was inversely correlated with Runx2 expression. These results show that sEH hydrolase plays a protective role against vascular calcification by reducing the bioavailability of epoxyeicosatrienoic acids.

Activation of the calcium-sensing receptor in human valvular interstitial cells promotes calcification.

INTRODUCTION AND AIMS: Calcific aortic valve disease (CAVD) is the most common heart valve disease in western countries. It has been reported that activation of the calcium-sensing receptor(CaSR) expressed by vascular smooth muscle cells prevents vascular calcification. However, to date, the CaSR's expression and function in cardiac valves have not been studied. The present study sought to evaluate the presence of the CaSR within human valvular interstitial cells (hVICs), assess the CaSR's functionality, and ascertain its involvement in hVIC calcification. METHODS AND RESULTS: Data from Western blot, flow cytometry and immunocytochemistry experiments demonstrated that primary hVICs express the CaSR. The receptor was functional, since the incubation of hVICs with the calcimimetic R-568 significantly increased Ca2+-induced ERK1/2 phosphorylation, and exposure to the calcilytic NPS2143 reduced ERK1/2 activation. A reduction in endogenous CaSR expression by hVICs (using siRNA) was associated with significantly lower levels of Ca2+-induced mineralization (quantified using Alizarin Red staining). Similar data were obtained after the pharmacological inhibition of CaSR activity by the calcilytic NPS2143. In contrast, overexpression of a functional CaSR amplified Ca2+-induced calcification. Pharmacological activation of the CaSR with the calcimimetic R-568 showed similar effects. CaSR's procalcific properties are associated with increased osteogenic transition (as characterized by elevated mRNA expression of bone morphogenetic protein 2 and osterix), and reduced the expression of the calcification inhibitor osteopontin. Histological analysis of 12 human aortic tricuspid valves showed that CaSR expression was greater in calcified areas than in non-calcified areas. These data were confirmed by Western blots. CONCLUSIONS: To the best of our knowledge, this study is the first to have demonstrated that hVICs express a functional CaSR. Taken as a whole, our data suggest that activation of the CaSR expressed by hVICs might be a key promoter of CAVD progression.

Differential Determinants of Tubular Phosphate Reabsorption: Insights on Renal Excretion of Phosphates in Kidney Disease.

We assessed the tubular reabsorption of phosphate (TRP) and maximal renal threshold for phosphate reabsorption to glomerular filtration rate (TmPi/GFR) and their determinants in 64 stages 2-4 chronic kidney disease (CKD) patients in order to define the early changes in phosphate metabolism in CKD. In multivariable analysis, TmPi/GFR correlates were estimated GFR (eGFR), intact parathyroid hormone (iPTH), and hemoglobin (R2 = 0.417), while TRP correlates were eGFR, iPTH, 24-h phosphaturia, and calcitriol (R2 = 0.72). This suggests that TmPi/GFR and TRP, respectively, assess hemoglobin-phosphate and bowel-kidney phosphate regulation axis. Iron supplementation based on TmPi/GFR or earlier phosphate restriction based on TRP should be investigated in view of modifying clinical outcomes in CKD.

Association between metformin use and below-the-knee arterial calcification score in type 2 diabetic patients.

BACKGROUND: Vascular calcification (VC) is common in type 2 diabetes, and is associated with cardiovascular complications. Recent preclinical data suggest that metformin inhibits VC both in vitro and in animal models. However, metformin's effects in patients with diabetic VC have not previously been characterized. The present study investigated the association between metformin use and lower-limb arterial calcification in patients with type 2 diabetes and high cardiovascular risk. METHODS: The DIACART cross-sectional cohort study included 198 patients with type 2 diabetes but without severe chronic kidney disease. Below-the-knee calcification scores were assessed by computed tomography and supplemented by colour duplex ultrasonography. Data on anti-diabetic drugs were carefully collected from the patients' medical records and during patient interviews. Biochemical and clinical data were studied as potential confounding factors. RESULTS: Metformin-treated patients had a significantly lower calcification score than metformin-free patients (mean ± standard deviation: 2033 ± 4514 and 4684 ± 9291, respectively; p = 0.01). A univariate analysis showed that metformin was associated with a significantly lower prevalence of severe below-the-knee arterial calcification (p = 0.02). VC was not significantly associated with the use of other antidiabetic drugs, including sulfonylureas, insulin, gliptin, and glucagon like peptide-1 analogues. A multivariate logistic regression analysis indicated that the association between metformin use and calcification score (odds ratio [95% confidence interval] = 0.33 [0.11-0.98]; p = 0.045) was independent of age, gender, tobacco use, renal function, previous cardiovascular disease, diabetes duration, neuropathy, retinopathy, HbA1c levels, and inflammation. CONCLUSIONS: In patients with type 2 diabetes, metformin use was independently associated with a lower below-the-knee arterial calcification score. This association may contribute to metformin's well-known vascular protective effect. Further prospective investigations of metformin's potential ability to inhibit VC in patients with and without type 2 diabetes are now needed to confirm these results.

Efficacy and safety of nicotinamide in haemodialysis patients: the NICOREN study.

BACKGROUND: Nicotinamide (NAM) has been proposed as an alternative treatment to phosphate binders for hyperphosphataemia in chronic kidney disease. METHODS: The NICOREN multicentre, open-label and randomized study was designed to examine non-inferiority and safety of NAM when compared with sevelamer (SEV) in chronic haemodialysis patients. One hundred patients were randomized to either NAM or SEV treatment for 24 weeks. Serum biochemistry and NAM's main metabolite, N -methyl-2-pyridone-5-carboxamide (2PY), were measured to assess compliance, efficacy and safety. RESULTS: After 24 weeks, we observed a comparable decrease in serum phosphorus in the NAM and SEV treatment arms, from 2.1 ± 0.4 to 1.8 ± 0.5 and 2.3 ± 0.5 to 1.7 ± 0.5 mM (P = not significant), respectively. The criterion for non-inferiority was, however, not met due to a more limited number of patients being included than planned. Treatment discontinuation due to adverse events was 1.6 times higher in the NAM than in the SEV group with only 55% of study completers in the NAM arm versus 90% in the SEV arm. Thrombocytopenia was observed in four NAM-treated patients. Serum 2PY levels were comparable at baseline, but increased markedly in the NAM group, but not in the SEV group, at 24 weeks (P < 0.0001). CONCLUSIONS: Thus, both drugs are equally effective in lowering serum phosphorus, but patients' tolerance of NAM was largely inferior to that of SEV. Extremely high 2PY levels may contribute to NAM's side effects.

The calcium-sensing receptor: A promising target for prevention of colorectal cancer.

The inverse correlation between dietary calcium intake and the risk of colorectal cancer (CRC) is well known, but poorly understood. Expression of the calcium-sensing receptor (CaSR), a calcium-binding G protein-coupled receptor is downregulated in CRC leading us to hypothesize that the CaSR has tumor suppressive roles in the colon. The aim of this study was to understand whether restoration of CaSR expression could reduce the malignant phenotype in CRC. In human colorectal tumors, expression of the CaSR negatively correlated with proliferation markers whereas loss of CaSR correlated with poor tumor differentiation and reduced apoptotic potential. In vivo, dearth of CaSR significantly increased expression of proliferation markers and decreased levels of differentiation and apoptotic markers in the colons of CaSR/PTH double knock-out mice confirming the tumor suppressive functions of CaSR. In vitro CRC cells stably overexpressing wild-type CaSR showed significant reduction in proliferation, as well as increased differentiation and apoptotic potential. The positive allosteric modulator of CaSR, NPS R-568 further enhanced these effects, whereas treatment with the negative allosteric modulator, NPS 2143 inhibited these functions. Interestingly, the dominant-negative mutant (R185Q) was able to abrogate these effects. Our results demonstrate a critical tumor suppressive role of CaSR in the colon. Restoration of CaSR expression and function is linked to regulation of the balance between proliferation, differentiation, and apoptosis and provides a rationale for novel strategies in CRC therapy.

Calcium sensing receptor signalling in physiology and cancer.

The calcium sensing receptor (CaSR) is a class C G-protein-coupled receptor that is crucial for the feedback regulation of extracellular free ionised calcium homeostasis. While extracellular calcium (Ca(2+)o) is considered the primary physiological ligand, the CaSR is activated physiologically by a plethora of molecules including polyamines and l-amino acids. Activation of the CaSR by different ligands has the ability to stabilise unique conformations of the receptor, which may lead to preferential coupling of different G proteins; a phenomenon termed 'ligand-biased signalling'. While mutations of the CaSR are currently not linked with any malignancies, altered CaSR expression and function are associated with cancer progression. Interestingly, the CaSR appears to act both as a tumour suppressor and an oncogene, depending on the pathophysiology involved. Reduced expression of the CaSR occurs in both parathyroid and colon cancers, leading to loss of the growth suppressing effect of high Ca(2+)o. On the other hand, activation of the CaSR might facilitate metastasis to bone in breast and prostate cancer. A deeper understanding of the mechanisms driving CaSR signalling in different tissues, aided by a systems biology approach, will be instrumental in developing novel drugs that target the CaSR or its ligands in cancer. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Vascular calcification in patients with type 2 diabetes: the involvement of matrix Gla protein.

BACKGROUND: Matrix Gla protein (MGP) is an important inhibitor of calcification. The objective of the present study of patients with type 2 diabetes and normal or slightly altered kidney function was to evaluate levels of inactive, dephospho-uncarboxylated MGP(dp-ucMGP) and total uncarboxylated MGP(t-ucMGP) and assess their links with biological and clinical parameters (including peripheral vascular calcification). METHODS: The DIACART study is a cross-sectional cohort study of 198 patients with type 2 diabetes and normal or slightly altered kidney function. Matrix Gla protein levels were measured with an ELISA and all patients underwent multislice spiral computed tomography scans to score below-knee arterial calcification. RESULTS: In the study population as a whole, the mean dp-ucMGP and t-ucMGP levels were 627 ± 451 pM and 4868 ± 1613 nM, respectively. Glomerular filtration rate, age and current vitamin K antagonist use were independently associated with dp-ucMGP levels. When the study population was divided according to the median peripheral arterial calcification score, patients with the higher score displayed significantly lower t-ucMGP and significantly higher dp-ucMGP levels. Furthermore, plasma dp-ucMGP was positively associated with the peripheral arterial calcification score (independently of age, gender, previous cardiovascular disease and t-ucMGP levels). CONCLUSIONS: High dp-ucMGP levels were independently associated with below-knee arterial calcification score in patients with type 2 diabetes and normal or slightly altered kidney function. The reversibility of the elevation of dp-ucMGP levels and the latter's relationship with clinical events merit further investigation.

Questions about Using the Induced Membrane Technique to Manage Cases of Congenital Tibial Pseudarthrosis.

The induced membrane technique is an innovative approach for repairing critical bone defects and has been applied recently in patients with congenital pseudarthrosis of the tibia (CPT). CPT is frequently associated with neurofibromatosis type 1 (NF1). Here, we briefly describe the clinical results of the induced membrane technique in NF1-deficient patients with CPT and in an animal model of CPT. Furthermore, we discuss the hypotheses used to explain inconsistent outcomes for the induced membrane technique in CPT-especially when associated with NF1.

Bone health in children with severe cerebral palsy.

Aim: To describe bone health and associated factors in children with severe cerebral palsy. Method: In a retrospective, single-centre study, we performed a comprehensive bone evaluation (including clinical, densitometric and bone biomarker assessments) of children with severe cerebral palsy. Results: None of the 19 included children had a normal BMCTBLH Z score, and only one had a BMDTBLH Z score greater than -2. Six children had a BMDLS Z score greater than -2. The bone biomarker data were suggestive of excessive bone remodelling. Levels of bone remodelling markers factors and densitometric variables were not significantly related. Age, weight and pubertal stage were significantly related to bone mass. Discussion: Our results highlights the insufficient increase in bone mass with age (probably due to excessive bone remodelling) and confirms the high prevalence of low bone mineral density in children with severe cerebral palsy. Possible preventive measures might include calcium + vitamin D supplementation and the systematic management of underweight and delayed puberty. Bone remodelling markers might be of value for follow-up.

Relationship between bone marrow adipose tissue and kidney function in postmenopausal women.

Introduction: Bone marrow adipose tissue (BMAT) is associated with aging, osteoporosis, and chronic kidney disease (CKD). To date, the association between BMAT and kidney function in postmenopausal women has not been thoroughly investigated. The main purpose of this study was to determine whether a relationship exists between proton density fat fraction (PDFF) and kidney function in postmenopausal women. Methods: We investigated the cross-sectional association between estimated glomerular filtration rate (eGFR) - calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation - and PDFF - measured at the lumbar spine and proximal femur using Water Fat Imaging (WFI) MRI - in 199 postmenopausal women from the ADIMOS cohort study. We also performed DXA scans and laboratory measurements of sclerostin and c-terminal Fibroblast Growth Factor 23 (cFGF23). Results: Participants' mean age was 67.5 (standard deviation, SD 10.0) years. Their median eGFR was 85.0 (interquartile range, IQR 72.2-95.0) ml/min/1.73 cm2, and their mean lumbar spine PDFF was 57.9 % (SD 9.6). When classified by eGFR-based CKD stages, 41.7 % of the cohort had an eGFR ≥ 90 (n = 83), 47.2 % had an eGFR of 60-89.9 (n = 94), and 11.1 % had an eGFR of 30-59.9 (n = 22). Participants with eGFR ≥ 90 had a lower lumbar spine PDFF than those with eGFR 60-89.9 (mean 55.8 % (9.8) vs. 58.9 % (9.0), p = 0.031) and those with eGFR 30-59.9 (55.8 % (9.8) vs. 60.8 % (9.8), p = 0.043). However, the differences did not remain significant after adjusting for predetermined confounders, including age, diabetes, Charlson comorbidity index, recent history of fragility fracture, appendicular lean mass, and lumbar spine BMD. The inclusion of sclerostin and/or cFGF23 as suspected mediators did not alter the findings. When proximal hip imaging-based PDFF was considered, no significant differences were found between the eGFR categories in the unadjusted and adjusted analyses. Conclusion: No evidence of an association between kidney function and bone marrow adiposity was found either in the lumbar spine or proximal femur in a cohort of postmenopausal women.

Indoxyl sulphate inhibits osteoclast differentiation and function.

BACKGROUND: Patients with chronic kidney disease (CKD) develop various bone abnormalities characterized by impaired bone remodelling. Recent data suggest that accumulation of the uraemic toxin indoxyl sulphate (IS) may be one of the factors involved in bone abnormalities in CKD patients. Indeed, it was recently reported that IS induces skeletal resistance to parathyroid hormone in cultured osteoblastic cells. However, it is not yet known whether IS also affects osteoclast cells. METHODS: In the present study, we assessed the direct effect of IS at uraemic concentrations and in the presence (to reach the 3 mM concentration) or absence of added inorganic phosphate (Pi) on osteoclast (OCL) differentiation and bone-resorbing activity in two well-established cellular models of monocyte/macrophage (peripheral blood mononuclear cells and the RAW 264.7 cell line). RESULTS: We found that IS inhibits both OCL differentiation and bone-resorbing activity in a dose-dependent manner and that these effects were enhanced in the presence of Pi at 3mM concentration. IS induced a gradual inhibition of JNK, Akt, p38, ERK1/2 phosphorylation and AP-1 DNA-binding activity. The effects of IS on OCL differentiation and AP-1 were prevented by probenecid, a competitive inhibitor of organic anion transporters, suggesting that IS's effects occur subsequently to its intake. CONCLUSION: Our findings strongly suggest that IS not only inhibits osteoblast function but also has an inhibitory effect on OCL function and thus could affect bone remodelling in CKD patients.

Strontium ranelate decreases receptor activator of nuclear factor-ΚB ligand-induced osteoclastic differentiation in vitro: involvement of the calcium-sensing receptor.

Strontium ranelate exerts both an anticatabolic and an anabolic effect on bone cells. To further investigate the mechanism by which strontium ranelate inhibits bone resorption, the effects of varying concentrations of Sr(o)(2+) on osteoclastic differentiation were studied using RAW 264.7 cells and peripheral blood monocytic cells (PBMCs). We report that increasing concentrations of Sr(o)(2+) down-regulate osteoclastic differentiation and tartrate-resistant acid phosphatase activity, leading to inhibition of bone resorption (-48% when PBMCs were cultured for 14 days in the presence of 2 mM Sr(o)(2+)). Using a dominant-negative form of the calcium-sensing receptor (CaR) and a small interfering RNA approach, we provide evidences that the inhibition of osteoclast differentiation by Sr(o)(2+) is mediated by stimulation of the CaR. Moreover, our results suggest that the effects of Sr(o)(2+) on osteoclasts are, at least in part, mediated by inhibition of the receptor activator of nuclear factor-κB ligand (RANKL)-induced nuclear translocation of nuclear factor-κB and activator protein-1 in the early stages of osteoclastic differentiation. In conclusion, our data indicate that Sr(2+) directly inhibits the formation of mature osteoclasts through down-regulation of RANKL-induced osteoclast differentiation and decreases osteoclast differentiation through the activation of the CaR.