Novel Biomarkers of Bone Metabolism.

Bone represents a metabolically active tissue subject to continuous remodeling orchestrated by the dynamic interplay between osteoblasts and osteoclasts. These cellular processes are modulated by a complex interplay of biochemical and mechanical factors, which are instrumental in assessing bone remodeling. This comprehensive evaluation aids in detecting disorders arising from imbalances between bone formation and reabsorption. Osteoporosis, characterized by a reduction in bone mass and strength leading to heightened bone fragility and susceptibility to fractures, is one of the more prevalent chronic diseases. Some epidemiological studies, especially in patients with chronic kidney disease (CKD), have identified an association between osteoporosis and vascular calcification. Notably, low bone mineral density has been linked to an increased incidence of aortic calcification, with shared molecules, mechanisms, and pathways between the two processes. Certain molecules emerging from these shared pathways can serve as biomarkers for bone and mineral metabolism. Detecting and evaluating these alterations early is crucial, requiring the identification of biomarkers that are reliable for early intervention. While traditional biomarkers for bone remodeling and vascular calcification exist, they suffer from limitations such as low specificity, low sensitivity, and conflicting results across studies. In response, efforts are underway to explore new, more specific biomarkers that can detect alterations at earlier stages. The aim of this review is to comprehensively examine some of the emerging biomarkers in mineral metabolism and their correlation with bone mineral density, fracture risk, and vascular calcification as well as their potential use in clinical practice.

Soluble Klotho, a Potential Biomarker of Chronic Kidney Disease-Mineral Bone Disorders Involved in Healthy Ageing: Lights and Shadows.

Shortly after the discovery of Klotho, interest grew in its potential role in chronic kidney disease (CKD). There are three isoforms of the Klotho protein: αKlotho, ßKlotho and γKlotho. This review will focus on αKlotho due to its relevance as a biomarker in CKD. αKlotho is synthesized mainly in the kidneys, but it can be released into the bloodstream and urine as soluble Klotho (sKlotho), which undertakes systemic actions, independently or in combination with FGF23. It is usually accepted that sKlotho levels are reduced early in CKD and that lower levels of sKlotho might be associated with the main chronic kidney disease-mineral bone disorders (CKD-MBDs): cardiovascular and bone disease. However, as results are inconsistent, the applicability of sKlotho as a CKD-MBD biomarker is still a matter of controversy. Much of the inconsistency can be explained due to low sample numbers, the low quality of clinical studies, the lack of standardized assays to assess sKlotho and a lack of consensus on sample processing, especially in urine. In recent decades, because of our longer life expectancies, the prevalence of accelerated-ageing diseases, such as CKD, has increased. Exercise, social interaction and caloric restriction are considered key factors for healthy ageing. While exercise and social interaction seem to be related to higher serum sKlotho levels, it is not clear whether serum sKlotho might be influenced by caloric restriction. This review focuses on the possible role of sKlotho as a biomarker in CKD-MBD, highlighting the difference between solid knowledge and areas requiring further research, including the role of sKlotho in healthy ageing.

Redox Metabolism and Vascular Calcification in Chronic Kidney Disease.

Vascular calcification (VC) is a common complication in patients with chronic kidney disease which increases their mortality. Although oxidative stress is involved in the onset and progression of this disorder, the specific role of some of the main redox regulators, such as catalase, the main scavenger of H2O2, remains unclear. In the present study, epigastric arteries of kidney transplant recipients, a rat model of VC, and an in vitro model of VC exhibiting catalase (Cts) overexpression were analysed. Pericalcified areas of human epigastric arteries had increased levels of catalase and cytoplasmic, rather than nuclear runt-related transcription factor 2 (RUNX2). In the rat model, advanced aortic VC concurred with lower levels of the H2O2-scavenger glutathione peroxidase 3 compared to controls. In an early model of calcification using vascular smooth muscle cells (VSMCs), Cts VSMCs showed the expected increase in total levels of RUNX2. However, Cts VMSCs also exhibited a lower percentage of the nucleus stained for RUNX2 in response to calcifying media. In this early model of VC, we did not observe a dysregulation of the mitochondrial redox state; instead, an increase in the general redox state was observed in the cytoplasm. These results highlight the complex role of antioxidant enzymes as catalase by regulation of RUNX2 subcellular location delaying the onset of VC.

Serum phosphate is associated with increased risk of bone fragility fractures in hemodialysis patients.

BACKGROUND: Bone fragility fractures are associated with high morbidity and mortality. This study analysed the association between the current biochemical parameters of CKD-MBD and bone fragility fractures in the COSMOS project. METHODS: COSMOS is a 3-year, multicentre, open cohort, prospective, observational study carried out in 6797 hemodialysis patients (227 centres from 20 European countries). The association of bone fragility fractures (outcome) with serum calcium, phosphate and PTH (exposure), was assessed using Standard Cox proportional hazards regression and Cox proportional hazards regression for recurrent events. Additional analyses were performed considering all-cause mortality as a competitive event for bone fragility fracture occurrence. Multivariable models were used in all strategies, with the fully adjusted model including a total of 24 variables. RESULTS: During a median follow-up of 24 months 252 (4%) patients experienced at least one bone fragility fracture (incident bone fragility fracture rate 28.5 per 1000 patient-years). In the fractured and non-fractured patients, the percentage of men was 43.7% and 61.4%, mean age 68.1 and 63.8 years and a haemodialysis vintage of 55.9 and 38.3 months respectively. Baseline serum phosphate > 6.1 mg/dL (reference value 4.3-6.1 mg/dL) was significantly associated with a higher bone fragility fracture risk in both regression models (HR: 1.53[95%CI: 1.10-2.13] and HR: 1.44[95%CI: 1.02-2.05]. The significant association persisted after competitive risk analysis (subHR: 1.42[95%CI: 1.02-1.98]) but the finding was not confirmed when serum phosphate was considered as a continuous variable. Baseline serum calcium showed no association with bone fragility fracture risk in any regression model. Baseline serum PTH > 800 pg/mL was significantly associated with a higher bone fragility fracture risk in both regression models, but the association disappeared after a competitive risk analysis. CONCLUSIONS: Hyperphosphatemia was independently and consistently associated with an increased bone fracture risk, suggesting serum phosphate could be a novel risk factor for bone fractures in hemodialysis patients.

Phosphorus May Induce Phenotypic Transdifferentiation of Vascular Smooth Muscle Cells through the Reduction of microRNA-145.

Phosphorus is a vital element for life found in most foods as a natural component, but it is also one of the most used preservatives added during food processing. High serum phosphorus contributes to develop vascular calcification in chronic kidney disease; however, it is not clear its effect in a population without kidney damage. The objective of this in vivo and in vitro study was to investigate the effect of high phosphorus exposure on the aortic and serum levels of miR-145 and its effect on vascular smooth muscle cell (VSMCs) changes towards less contractile phenotypes. The study was performed in aortas and serum from rats fed standard and high-phosphorus diets, and in VSMCs exposed to different concentrations of phosphorus. In addition, miR-145 silencing and overexpression experiments were carried out. In vivo results showed that in rats with normal renal function fed a high P diet, a significant increase in serum phosphorus was observed which was associated to a significant decrease in the aortic α-actin expression which paralleled the decrease in aortic and serum miR-145 levels, with no changes in the osteogenic markers. In vitro results using VSMCs corroborated the in vivo findings. High phosphorus first reduced miR-145, and afterwards α-actin expression. The miR-145 overexpression significantly increased α-actin expression and partially prevented the increase in calcium content. These results suggest that miR-145 could be an early biomarker of vascular calcification, which could give information about the initiation of the transdifferentiation process in VSMCs.

Experimental Models to Study Diabetes Mellitus and Its Complications: Limitations and New Opportunities.

Preclinical biomedical models are a fundamental tool to improve the knowledge and management of diseases, particularly in diabetes mellitus (DM) since, currently, the pathophysiological and molecular mechanisms involved in its development are not fully clarified, and there is no treatment to cure DM. This review will focus on the features, advantages and limitations of some of the most used DM models in rats, such as the spontaneous models: Bio-Breeding Diabetes-Prone (BB-DP) and LEW.1AR1-iddm, as representative models of type 1 DM (DM-1); the Zucker diabetic fatty (ZDF) and Goto-kakizaki (GK) rats, as representative models of type 2 DM (DM-2); and other models induced by surgical, dietary and pharmacological-alloxan and streptozotocin-procedures. Given the variety of DM models in rats, as well as the non-uniformity in the protocols and the absence of all the manifestation of the long-term multifactorial complications of DM in humans, the researchers must choose the one that best suits the final objectives of the study. These circumstances, added to the fact that most of the experimental research in the literature is focused on the study of the early phase of DM, makes it necessary to develop long-term studies closer to DM in humans. In this review, a recently published rat DM model induced by streptozotocin injection with chronic exogenous administration of insulin to reduce hyperglycaemia has also been included in an attempt to mimic the chronic phase of DM in humans.

Vitamin D Receptor From VSMCs Regulates Vascular Calcification During CKD: A Potential Role for miR-145a.

BACKGROUND: Vascular calcification (VC) is a highly prevalent complication of chronic kidney disease (CKD) and is associated with the higher morbidity-mortality of patients with CKD. VDR (vitamin D receptor) has been proposed to play a role in the osteoblastic differentiation of vascular smooth muscle cells (VSMCs), but the involvement of vitamin D in VC associated to CKD is controversial. Our aim was to determine the role of local vitamin D signaling in VSMCs during CKD-induced VC. METHODS: We used epigastric arteries from CKD-affected patients and individuals with normal renal function, alongside an experimental model of CKD-induced VC in mice with conditional deletion of VDR in VSMC. In vitro, experiments in VSMC with or without VDR incubated in calcification media were also used. RESULTS: CKD-affected patients and mice with CKD showed an increase in VC, together with increased arterial expression of VDR compared with controls with normal renal function. Conditional gene silencing of VDR in VSMCs led to a significant decrease of VC in the mouse model of CKD, despite similar levels of renal impairment and serum calcium and phosphate levels. This was accompanied by lower arterial expression of OPN (osteopontin) and lamin A and higher expression of SOST (sclerostin). Furthermore, CKD-affected mice showed a reduction of miR-145a expression in calcified arteries, which was significantly recovered in animals with deletion of VDR in VSMC. In vitro, the absence of VDR prevented VC, inhibited the increase of OPN, and reestablished the expression of miR-145a. Forced expression of miR-145a in vitro in VDRwt VSMCs blunted VC and decreased OPN levels. CONCLUSIONS: Our study provides evidence proving that inhibition of local VDR signaling in VSMCs could prevent VC in CKD and indicates a possible role for miR-145a in this process.

Mineral and bone metabolism markers and mortality in diabetic patients on haemodialysis.

BACKGROUND: Diabetic patients on haemodialysis have a higher risk of mortality than non-diabetic patients. The aim of this COSMOS (Current management of secondary hyperparathyroidism: a multicentre observational study) analysis was to assess whether bone and mineral laboratory values [calcium, phosphorus and parathyroid hormone (PTH)] contribute to this risk. METHODS: COSMOS is a multicentre, open-cohort, 3-year prospective study, which includes 6797 patients from 227 randomly selected dialysis centres in 20 European countries. The association between mortality and calcium, phosphate or PTH was assessed using Cox proportional hazard regression models using both penalized splines smoothing and categorization according to KDIGO guidelines. The effect modification of the association between the relative risk of mortality and serum calcium, phosphate or PTH by diabetes was assessed. RESULTS: There was a statistically significant effect modification of the association between the relative risk of mortality and serum PTH by diabetes (P = .011). The slope of the curve of the association between increasing values of PTH and relative risk of mortality was steeper for diabetic compared with non-diabetic patients, mainly for high levels of PTH. In addition, high serum PTH (>9 times the normal values) was significantly associated with a higher relative risk of mortality in diabetic patients but not in non-diabetic patients [1.53 (95% confidence interval 1.07-2.19) and 1.17 (95% confidence interval 0.91-1.52)]. No significant effect modification of the association between the relative risk of mortality and serum calcium or phosphate by diabetes was found (P = .2 and P = .059, respectively). CONCLUSION: The results show a different association of PTH with the relative risk of mortality in diabetic and non-diabetic patients. These findings could have relevant implications for the diagnosis and treatment of chronic kidney disease-mineral and bone disorders.

Effects of a Losartan-Antioxidant Hybrid (GGN1231) on Vascular and Cardiac Health in an Experimental Model of Chronic Renal Failure.

Drugs providing antihypertensive and protective cardiovascular actions are of clinical interest in controlling cardiovascular events and slowing the progression of kidney disease. We studied the effect of a hybrid compound, GGN1231 (derived from losartan in which a powerful antioxidant was attached), on the prevention of cardiovascular damage, cardiac hypertrophy, and fibrosis in a rat model of severe chronic renal failure (CRF). CRF by a 7/8 nephrectomy was carried out in male Wistar rats fed with a diet rich in phosphorous (0.9%) and normal calcium (0.6%) for a period of 12 weeks until sacrifice. In week 8, rats were randomized in five groups receiving different drugs including dihydrocaffeic acid as antioxidant (Aox), losartan (Los), dihydrocaffeic acid+losartan (Aox+Los) and GGN1231 as follows: Group 1 (CRF+vehicle group), Group 2 (CRF+Aox group), Group 3 (CRF+Los group), Group 4 (CRF+Aox+Los group), and Group 5 (CRF+GGN1231 group). Group 5, the CRF+GGN1231 group, displayed reduced proteinuria, aortic TNF-α, blood pressure, LV wall thickness, diameter of the cardiomyocytes, ATR1, cardiac TNF-α and fibrosis, cardiac collagen I, and TGF-ß1 expression. A non-significant 20% reduction in the mortality was also observed. This study showed the possible advantages of GGN1231, which could help in the management of cardiovascular and inflammatory processes. Further research is needed to confirm and even expand the positive aspects of this compound.

Role of Klotho and AGE/RAGE-Wnt/ß-Catenin Signalling Pathway on the Development of Cardiac and Renal Fibrosis in Diabetes.

Fibrosis plays an important role in the pathogenesis of long-term diabetic complications and contributes to the development of cardiac and renal dysfunction. The aim of this experimental study, performed in a long-term rat model, which resembles type 1 diabetes mellitus, was to investigate the role of soluble Klotho (sKlotho), advanced glycation end products (AGEs)/receptor for AGEs (RAGE), fibrotic Wnt/ß-catenin pathway, and pro-fibrotic pathways in kidney and heart. Diabetes was induced by streptozotocin. Glycaemia was maintained by insulin administration for 24 weeks. Serum and urine sKlotho, AGEs, soluble RAGE (sRAGE) and biochemical markers were studied. The levels of Klotho, RAGEs, ADAM10, markers of fibrosis (collagen deposition, fibronectin, TGF-ß1, and Wnt/ß-catenin pathway), hypertrophy of the kidney and/or heart were analysed. At the end of study, diabetic rats showed higher levels of urinary sKlotho, AGEs and sRAGE and lower serum sKlotho compared with controls without differences in the renal Klotho expression. A significant positive correlation was found between urinary sKlotho and AGEs and urinary albumin/creatinine ratio (uACR). Fibrosis and RAGE levels were significantly higher in the heart without differences in the kidney of diabetic rats compared to controls. The results also suggest the increase in sKlotho and sRAGE excretion may be due to polyuria in the diabetic rats.

Serum and Urinary Soluble α-Klotho as Markers of Kidney and Vascular Impairment.

This study was designed to investigate the controversy on the potential role of sKlotho as an early biomarker in Chronic Kidney Disease-Mineral Bone Disorder (CKD-MBD), to assess whether sKlotho is a reliable marker of kidney α-Klotho, to deepen the effects of sKlotho on vascular smooth muscle cells (VSMCs) osteogenic differentiation and to evaluate the role of autophagy in this process. Experimental studies were conducted in CKD mice fed a normal phosphorus (CKD+NP) or high phosphorus (CKD+HP) diet for 14 weeks. The patients' study was performed in CKD stages 2-5 and in vitro studies which used VSMCs exposed to non-calcifying medium or calcifying medium with or without sKlotho. The CKD experimental model showed that the CKD+HP group reached the highest serum PTH, P and FGF23 levels, but the lowest serum and urinary sKlotho levels. In addition, a positive correlation between serum sKlotho and kidney α-Klotho was found. CKD mice showed aortic osteogenic differentiation, together with increased autophagy. The human CKD study showed that the decline in serum sKlotho is previous to the rise in FGF23. In addition, both serum sKlotho and FGF23 levels correlated with kidney function. Finally, in VSMCs, the addition of sKlotho prevented osteogenic differentiation and induced autophagy. It can be concluded that serum sKlotho was the earliest CKD-MBD biomarker, a reliable indicator of kidney α-Klotho and that might protect against osteogenic differentiation by increasing autophagy. Nevertheless, further studies are needed to investigate the mechanisms of this possible protective effect.

MicroRNA-145 and microRNA-486 are potential serum biomarkers for vascular calcification.

INTRODUCTION: MicroRNAs (miRs) regulate vascular calcification (VC), and their quantification may contribute to suspicion of the presence of VC. METHODS: The study was performed in four phases. Phase 1: miRs sequencing of rat calcified and non-calcified aortas. Phase 2: miRs with the highest rate of change, plus miR-145 [the most abundant miR in vascular smooth muscle cells (VSMCs)], were validated in aortas and serum from rats with and without VC. Phase 3: the selected miRs were analyzed in epigastric arteries from kidney donors and recipients, and serum samples from general population. Phase 4: VSMCs were exposed to different phosphorus concentrations, and miR-145 and miR-486 were overexpressed to investigate their role in VC. RESULTS: miR-145, miR-122-5p, miR-486 and miR-598-3p decreased in the rat calcified aortas, but only miR-145 and miR-486 were detected in serum. In human epigastric arteries, miR-145 and miR-486 were lower in kidney transplant recipients compared with donors. Both miRs inversely correlated with arterial calcium content and with VC (Kauppila index). In the general population, the severe VC was associated with the lowest serum levels of both miRs. The receiver operating characteristic curve showed that serum miR-145 was a good biomarker of VC. In VSMCs exposed to high phosphorus, calcium content, osteogenic markers (Runx2 and Osterix) increased, and the contractile marker (α-actin), miR-145 and miR-486 decreased. Overexpression of miR-145, and to a lesser extent miR-486, prevented the increase in calcium content induced by high phosphorus, the osteogenic differentiation and the loss of the contractile phenotype. CONCLUSION: miR-145 and miR-486 regulate the osteogenic differentiation of VSMCs, and their quantification in serum could serve as a marker of VC.

Effects of calcitriol and paricalcitol on renal fibrosis in CKD.

BACKGROUND: In chronic kidney disease, the activation of the renin-angiotensin-aldosterone system (RAAS) and renal inflammation stimulates renal fibrosis and the progression to end-stage renal disease. The low levels of vitamin D receptor (VDR) and its activators (VDRAs) contribute to worsen secondary hyperparathyroidism and renal fibrosis. METHODS: The 7/8 nephrectomy model of experimental chronic renal failure (CRF) was used to examine the anti-fibrotic effects of treatment with two VDRAs, paricalcitol and calcitriol, at equivalent doses (3/1 dose ratio) during 4 weeks. RESULTS: CRF increased the activation of the RAAS, renal inflammation and interstitial fibrosis. Paricalcitol treatment reduced renal collagen I and renal interstitial fibrosis by decreasing the activation of the RAAS through renal changes in renin, angiotensin receptor 1 (ATR1) and ATR2 mRNAs levels and renal inflammation by decreasing renal inflammatory leucocytes (CD45), a desintegrin and metaloproteinase mRNA, transforming growth factor beta mRNA and protein, and maintaining E-cadherin mRNA levels. Calcitriol showed similar trends without significant changes in most of these biomarkers. CONCLUSIONS: Paricalcitol effectively attenuated the renal interstitial fibrosis induced by CRF through a combination of inhibitory actions on the RAAS, inflammation and epithelial/mesenchymal transition.

Fibrosis in Chronic Kidney Disease: Pathogenesis and Consequences.

Fibrosis is a process characterized by an excessive accumulation of the extracellular matrix as a response to different types of tissue injuries, which leads to organ dysfunction. The process can be initiated by multiple and different stimuli and pathogenic factors which trigger the cascade of reparation converging in molecular signals responsible of initiating and driving fibrosis. Though fibrosis can play a defensive role, in several circumstances at a certain stage, it can progressively become an uncontrolled irreversible and self-maintained process, named pathological fibrosis. Several systems, molecules and responses involved in the pathogenesis of the pathological fibrosis of chronic kidney disease (CKD) will be discussed in this review, putting special attention on inflammation, renin-angiotensin system (RAS), parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), Klotho, microRNAs (miRs), and the vitamin D hormonal system. All of them are key factors of the core and regulatory pathways which drive fibrosis, having a great negative kidney and cardiac impact in CKD.

Chronic Kidney Disease-Mineral and Bone Disorders: Pathogenesis and Management.

The key players of the chronic kidney disease-mineral and bone disorders (CKD-MBD) are calcium, phosphate, PTH, FGF23, and the vitamin D hormonal system. The progressive reduction of kidney function greatly modifies the tightly interrelated mechanisms that control these parameters. As a result, important changes occur in the bone and mineral hormonal axis, leading to changes in bone turnover with relevant consequences in clinical outcomes, such as decrease in bone mass with increased bone fragility and bone fractures and increased vascular and valvular calcification, also with great impact in the cardiovascular outcomes. So far, the knowledge of the mineral and bone disorders in CKD and the increased variety of efficacious therapies should lead to a better prevention and management of CKD-MBD.

The receptor activator of nuclear factor κΒ ligand receptor leucine-rich repeat-containing G-protein-coupled receptor 4 contributes to parathyroid hormone-induced vascular calcification.

BACKGROUND: In chronic kidney disease, serum phosphorus (P) elevations stimulate parathyroid hormone (PTH) production, causing severe alterations in the bone-vasculature axis. PTH is the main regulator of the receptor activator of nuclear factor κB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system, which is essential for bone maintenance and also plays an important role in vascular smooth muscle cell (VSMC) calcification. The discovery of a new RANKL receptor, leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4), which is important for osteoblast differentiation but with an unknown role in vascular calcification (VC), led us to examine the contribution of LGR4 in high P/high PTH-driven VC. METHODS: In vivo studies were conducted in subtotally nephrectomized rats fed a normal or high P diet, with and without parathyroidectomy (PTX). PTX rats were supplemented with PTH(1-34) to achieve physiological serum PTH levels. In vitro studies were performed in rat aortic VSMCs cultured in control medium, calcifying medium (CM) or CM plus 10-7 versus 10-9 M PTH. RESULTS: Rats fed a high P diet had a significantly increased aortic calcium (Ca) content. Similarly, Ca deposition was higher in VSMCs exposed to CM. Both conditions were associated with increased RANKL and LGR4 and decreased OPG aorta expression and were exacerbated by high PTH. Silencing of LGR4 or parathyroid hormone receptor 1 (PTH1R) attenuated the high PTH-driven increases in Ca deposition. Furthermore, PTH1R silencing and pharmacological inhibition of protein kinase A (PKA), but not protein kinase C, prevented the increases in RANKL and LGR4 and decreased OPG. Treatment with PKA agonist corroborated that LGR4 regulation is a PTH/PKA-driven process. CONCLUSIONS: High PTH increases LGR4 and RANKL and decreases OPG expression in the aorta, thereby favouring VC. The hormone's direct pro-calcifying actions involve PTH1R binding and PKA activation.

High-serum phosphate and parathyroid hormone distinctly regulate bone loss and vascular calcification in experimental chronic kidney disease.

BACKGROUND: In chronic kidney disease (CKD), increases in serum phosphate and parathyroid hormone (PTH) aggravate vascular calcification (VC) and bone loss. This study was designed to discriminate high phosphorus (HP) and PTH contribution to VC and bone loss. METHODS: Nephrectomized rats fed a HP diet underwent either sham operation or parathyroidectomy and PTH 1-34 supplementation to normalize serum PTH. RESULTS: In uraemic rats fed a HP diet, parathyroidectomy with serum PTH 1-34 supplementation resulted in (i) reduced aortic calcium (80%) by attenuating osteogenic differentiation (higher α-actin; reduced Runx2 and BMP2) and increasing the Wnt inhibitor Sclerostin, despite a similar degree of hyperphosphataemia, renal damage and serum Klotho; (ii) prevention of bone loss mostly by attenuating bone resorption and increases in Wnt inhibitors; and (iii) a 70% decrease in serum calcitriol levels despite significantly reduced serum Fgf23, calcium and renal 24-hydroxylase, which questions that Fgf23 is the main regulator of renal calcitriol production. Significantly, when vascular smooth muscle cells (VSMCs) were exposed exclusively to high phosphate and calcium, high PTH enhanced while low PTH attenuated calcium deposition through parathyroid hormone 1 receptor (PTH1R) signalling. CONCLUSIONS: In hyperphosphataemic CKD, a defective suppression of high PTH exacerbates HP-mediated osteogenic VSMC differentiation and reduces vascular levels of anti-calcifying sclerostin.

Variants in cardiac GATA genes associated with bicuspid aortic valve.

BACKGROUND: Bicuspid aortic valve (BAV) is a heterogeneous and still not fully understood condition, with diverse genetic aetiology and associated phenotypes ranging from aortic stenosis or regurgitation to aneurysm and dissection. Several genes have been associated with the presence of BAV, notably some members of the GATA family of transcription factors that play important roles in cardiac embryogenesis. METHODS: A case-control study with 122 unrelated and ethnically matched patients with bicuspid and 154 with tricuspid aortic valve was performed. All exons of GATA4, GATA5, and GATA6 genes were sequenced searching for variants. Frequencies were compared and functional effects of rare variants were analysed by informatic prediction tools. RESULTS: Four rare and potentially pathogenic variants were identified in only five sporadic cases: a missense p.Arg202Gln (rs782614097) in GATA5 and three synonymous variants, p.Cys274= (rs55980825) and p.His302= (rs201516339) in GATA4, and p.Asn458= (rs143026087) in GATA6. Minor alleles of p.His302=, p.Arg202Gln and rs3764962 are enriched in BAV patients compared to ExAC database (P = 0.01, 0.03, and 0.01 respectively). In addition, a common polymorphism in GATA5 (p.Asp203=, rs41305803) is associated with BAV showing a protective effect in carriers of the minor allele (OR [95%CI] = 0.45[0.25-0.81]; P = 0.004). CONCLUSION: This study associates additional genetic variants in GATA4 and GATA5 with BAV, supporting the implication of these genes in the development of this valvulopathy. The discovery of all the genetic factors involved will contribute to a better understanding of the process and, therefore, to detect a genetic predisposition and even to the identification of therapeutic targets.

Rare genetic variants in GATA transcription factors in patients with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a very heterogeneous disease. Although primarily caused by mutations in genes encoding sarcomeric proteins, other genes might explain that heterogeneity. Potential candidate genes are GATA transcription factors that regulate the expression of proteins associated with HCM. Exons of GATA2, GATA4, and GATA6 genes were sequenced in 212 patients with unrelated HCM previously analyzed for genes encoding the most frequently mutated sarcomeric proteins. Functional effects of variants were predicted by in silico analyses. 3 potentially pathogenic variants were identified: c.-77G>A in GATA2, p.Ala343Thr (rs370588269) in GATA4, and p.Pro555Ala (rs146243018) in GATA6 Multivariate analyses showed that angina was more frequent in patients carrying sarcomeric and GATA rare variants (55% vs 23.2% in non-carriers of GATA rare variants, OR (95% CI) 7.12 (1.23 to 41.27), p=0.029). Among patients without a known causal mutation, GATA rare variants were associated with a greater maximum posterior wall thickness (16.4±4.4 vs 14.0±3.1 mm in non-carriers, p=0.021). Thus, variants having a putative effect on GATA genes would alter the expression of their target genes and could modify the hypertrophic response. Therefore, although relatively infrequent in patients with HCM, they may represent a novel insight into the molecular mechanisms related to the pathogenesis of HCM.

Regeneration of mandibular defects using adipose tissue mesenchymal stromal cells in combination with human serum-derived scaffolds.

Bone regeneration is a challenging issue. Traditional solutions bring risks, potential complications, and morbidity. The aim of the present study was to regenerate critical-sized mandible defects in athymic rats with adipose tissue mesenchymal stromal cells (AT-MSCs) in combination with human serum-derived scaffolds. Two approaches to treatment were performed. The first approach used differentiated stromal cells that became osteogenic cell lines. The second approach used no pre-differentiation. Follow-up periods were 45 days and 90 days. Both cell types were combined with human serum-derived scaffolds. Afterward, histological (haematoxylin-eosin and Masson's Trichrome stain modified by Goldner), immunohistochemical (human vimentin and Stro-1), and radiological (microCT) studies were performed. The level of calcification between the groups was compared by analysis of variance, and statistical significance was set at p < 0.05. The results demonstrate that bone regeneration can be achieved with both undifferentiated and pre-differentiated cells, but that the structure and level of calcification were better achieved with pre-differentiated cells (p < 0.05). The scaffold is suitable for this cell type, is osteoconductive and simple to perform. This article highlights the possible application of adipose tissue mesenchymal stromal cells in combination with a non-mineralized scaffold in bone regeneration.