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.

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.

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.

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.

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.

A single-oral bolus of 100,000 IU of cholecalciferol at hospital admission did not improve outcomes in the COVID-19 disease: the COVID-VIT-D-a randomised multicentre international clinical trial.

BACKGROUND: Vitamin D status has been implicated in COVID-19 disease. The objective of the COVID-VIT-D trial was to investigate if an oral bolus of cholecalciferol (100,000 IU) administered at hospital admission influences the outcomes of moderate-severe COVID-19 disease. In the same cohort, the association between baseline serum calcidiol levels with the same outcomes was also analysed. METHODS: The COVID-VIT-D is a multicentre, international, randomised, open label, clinical trial conducted throughout 1 year. Patients older than 18 years with moderate-severe COVID-19 disease requiring hospitalisation were included. At admission, patients were randomised 1:1 to receive a single oral bolus of cholecalciferol (n=274) or nothing (n=269). Patients were followed from admission to discharge or death. Length of hospitalisation, admission to intensive care unit (ICU) and mortality were assessed. RESULTS: In the randomised trial, comorbidities, biomarkers, symptoms and drugs used did not differ between groups. Median serum calcidiol in the cholecalciferol and control groups were 17.0 vs. 16.1 ng/mL at admission and 29.0 vs. 16.4 ng/mL at discharge, respectively. The median length of hospitalisation (10.0 [95%CI 9.0-10.5] vs. 9.5 [95%CI 9.0-10.5] days), admission to ICU (17.2% [95%CI 13.0-22.3] vs. 16.4% [95%CI 12.3-21.4]) and death rate (8.0% [95%CI 5.2-12.1] vs. 5.6% [95%CI 3.3-9.2]) did not differ between the cholecalciferol and control group. In the cohort analyses, the highest serum calcidiol category at admission (>25ng/mL) was associated with lower percentage of pulmonary involvement and better outcomes. CONCLUSIONS: The randomised clinical trial showed the administration of an oral bolus of 100,000 IU of cholecalciferol at hospital admission did not improve the outcomes of the COVID-19 disease. A cohort analysis showed that serum calcidiol at hospital admission was associated with outcomes. TRIAL REGISTRATION: COVID-VIT-D trial was authorised by the Spanish Agency for Medicines and Health products (AEMPS) and registered in European Union Drug Regulating Authorities Clinical Trials (EudraCT 2020-002274-28) and in ClinicalTrials.gov ( NCT04552951 ).

Role of the RANK/RANKL/OPG and Wnt/ß-Catenin Systems in CKD Bone and Cardiovascular Disorders.

In the course of chronic kidney disease (CKD), alterations in the bone-vascular axis augment the risk of bone loss, fractures, vascular and soft tissue calcification, left ventricular hypertrophy, renal and myocardial fibrosis, which markedly increase morbidity and mortality rates. A major challenge to improve skeletal and cardiovascular outcomes in CKD patients requires a better understanding of the increasing complex interactions among the main modulators of the bone-vascular axis. Serum parathyroid hormone (PTH), phosphorus (P), calcium (Ca), fibroblast growth factor 23 (FGF23), calcidiol, calcitriol and Klotho are involved in this axis interact with RANK/RANKL/OPG system and the Wnt/ß-catenin pathway. The RANK/RANKL/OPG system controls bone remodeling by inducing osteoblast synthesis of RANKL and downregulating OPG production and it is also implicated in vascular calcification. The complexity of this system has recently increased due the discovery of LGR4, a novel RANKL receptor involved in bone formation, but possibly also in vascular calcification. The Wnt/ß-catenin pathway plays a key role in bone formation: when this pathway is activated, bone is formed, but when it is inhibited, bone formation is stopped. In the progression of CKD, a downregulation of the Wnt/ß-catenin pathway has been described which occurs mainly through the not coincident elevations of sclerostin, Dickkopf1 (Dkk1) and the secreted Frizzled Related Proteins (sFRPs). This review analyzes the interactions of PTH, P, Ca, FGF23, calcidiol, calcitriol and Klotho with the RANKL/RANKL/OPG system and the Wnt/ß-catenin, pathway and their implications in bone and cardiovascular disorders 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.

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.

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.

Serum phosphate optimal timing and range associated with patients survival in haemodialysis: the COSMOS study.

BACKGROUND: Serum phosphate is a key parameter in the management of chronic kidney disease-mineral and bone disorder (CKD-MBD). The timing of phosphate measurement is not standardized in the current guidelines. Since the optimal range of these biomarkers may vary depending on the duration of the interdialytic interval, in this analysis of the Current management of secondary hyperparathyroidism: a multicentre observational study (COSMOS), we assessed the influence of a 2- (midweek) or 3-day (post-weekend) dialysis interval for blood withdrawal on serum levels of CKD-MBD biomarkers and their association with mortality risk. METHODS: The COSMOS cohort (6797 patients, CKD Stage 5D) was divided into two groups depending upon midweek or post-weekend blood collection. Univariate and multivariate Cox's models adjusted hazard ratios (HRs) by demographics and comorbidities, treatments and biochemical parameters from a patient/centre database collected at baseline and every 6 months for 3 years. RESULTS: There were no differences in serum calcium or parathyroid hormone levels between midweek and post-weekend patients. However, in post-weekend patients, the mean serum phosphate levels were higher compared with midweek patients (5.5 ± 1.4 versus 5.2 ± 1.4 mg/dL, P < 0.001). Also, the range of serum phosphate with the lowest mortality risk [HR ≤ 1.1; midweek: 3.5-4.9 mg/dL (95% confidence interval, CI: 2.9-5.2 mg/dL); post-weekend: 3.8-5.7 mg/dL (95% CI: 3.0-6.4 mg/dL)] showed significant differences in the upper limit (P = 0.021). CONCLUSION: Midweek and post-weekend serum phosphate levels and their target ranges associated with the lowest mortality risk differ. Thus, clinical guidelines should consider the timing of blood withdrawal when recommending optimal target ranges for serum phosphate and therapeutic strategies for phosphate control.

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.

Regulation of miR-29b and miR-30c by vitamin D receptor activators contributes to attenuate uraemia-induced cardiac fibrosis.

BACKGROUND: Uraemic cardiomyopathy, a process mainly associated with increased myocardial fibrosis, is the leading cause of death in chronic kidney disease patients and can be prevented by vitamin D receptor activators (VDRAs). Since some microRNAs (miRNAs) have emerged as regulators of the fibrotic process, we aimed to analyse the role of specific miRNAs in VDRA prevention of myocardial fibrosis as well as their potential use as biomarkers. METHODS: Wistar rats were nephrectomized and treated intraperitoneally with equivalent doses of two VDRAs: calcitriol and paricalcitol. Biochemical parameters, cardiac fibrosis, miRNA (miR-29b, miR-30c and miR-133b) levels in the heart and serum and expression of their target genes collagen I (COL1A1), matrix metalloproteinase 2 (MMP-2) and connective tissue growth factor (CTGF) in the heart were evaluated. RESULTS: Both VDRAs attenuated cardiac fibrosis, achieving a statistically significant difference in the paricalcitol-treated group. Increases in RNA and protein levels of COL1A1, MMP-2 and CTGF and reduced expression of miR-29b and miR-30c, known regulators of these pro-fibrotic genes, were observed in the heart of chronic renal failure (CRF) rats and were attenuated by both VDRAs. In serum, significant increases in miR-29b, miR-30c and miR-133b levels were observed in CRF rats, which were prevented by VDRA use. Moreover, vitamin D response elements were identified in the three miRNA promoters. CONCLUSIONS: VDRAs, particularly paricalcitol, attenuated cardiac fibrosis acting on COL1A1, MMP-2 and CTGF expression, partly through regulation of miR-29b and miR-30c. These miRNAs and miR-133b could be useful serum biomarkers for cardiac fibrosis and also potential new therapeutic targets.

MicroRNAs 29b, 133b, and 211 Regulate Vascular Smooth Muscle Calcification Mediated by High Phosphorus.

Vascular calcification is a frequent cause of morbidity and mortality in patients with CKD and the general population. The common association between vascular calcification and osteoporosis suggests a link between bone and vascular disorders. Because microRNAs (miRs) are involved in the transdifferentiation of vascular smooth muscle cells into osteoblast-like cells, we investigated whether miRs implicated in osteoblast differentiation and bone formation are involved in vascular calcification. Different levels of uremia, hyperphosphatemia, and aortic calcification were induced by feeding nephrectomized rats a normal or high-phosphorus diet for 12 or 20 weeks, at which times the levels of eight miRs (miR-29b, miR-125, miR-133b, miR-135, miR-141, miR-200a, miR-204, and miR-211) in the aorta were analyzed. Compared with controls and uremic rats fed a normal diet, uremic rats fed a high-phosphorous diet had lower levels of miR-133b and miR-211 and higher levels of miR-29b that correlated respectively with greater expression of osteogenic RUNX2 and with lower expression of several inhibitors of osteoblastic differentiation. Uremia per se mildly reduced miR-133b levels only. Similar results were obtained in two in vitro models of vascular calcification (uremic serum and high-calcium and -phosphorus medium), and experiments using antagomirs and mimics to modify miR-29b, miR-133b, and miR-211 expression levels in these models confirmed that these miRs regulate the calcification process. We conclude that miR-29b, miR-133b, and miR-211 have direct roles in the vascular smooth muscle calcification induced by high phosphorus and may be new therapeutic targets in the management of vascular calcification.

Direct inhibition of osteoblastic Wnt pathway by fibroblast growth factor 23 contributes to bone loss in chronic kidney disease.

Bone loss and increased fractures are common complications in chronic kidney disease. Because Wnt pathway activation is essential for normal bone mineralization, we assessed whether Wnt inhibition contributes to high-phosphorus-induced mineralization defects in uremic rats. By week 20 after 7/8 nephrectomy, rats fed a high-phosphorus diet had the expected high serum creatinine, phosphorus, parathyroid hormone, and fibroblast growth factor 23 (FGF23) levels and low serum calcium. There was a 15% reduction in tibial mineral density and a doubling of bone cortical porosity compared to uremic rats fed a normal-phosphorus diet. The decreases in tibial mineral density were preceded by time-dependent increments in gene expression of bone formation (Osteocalcin and Runx2) and resorption (Cathepsin K) markers, which paralleled elevations in gene expression of the Wnt inhibitors Sfrp1 and Dkk1 in bone. Similar elevations of Wnt inhibitors plus an increased phospho-ß-catenin/ß-catenin ratio occurred upon exposure of the osteoblast cell line UMR106-01 either to uremic serum or to the combination of parathyroid hormone, FGF23, and soluble Klotho, at levels present in uremic serum. Strikingly, while osteoblast exposure to parathyroid hormone suppressed the expression of Wnt inhibitors, FGF23 directly inhibited the osteoblastic Wnt pathway through a soluble Klotho/MAPK-mediated process that required Dkk1 induction. Thus, the induction of Dkk1 by FGF23/soluble Klotho in osteoblasts inactivates Wnt/ß-catenin signaling. This provides a novel autocrine/paracrine mechanism for the adverse impact of high FGF23 levels on bone in chronic kidney disease.

The challenge of controlling phosphorus in chronic kidney disease.

The pathogenesis and management of chronic kidney disease-mineral bone disorders (CKD-MBD) has experienced major changes, but the control of serum phosphorus at all stages of CKD still seems to be a key factor to improve clinical outcomes. High serum phosphorus is the most important uremia-related, non-traditional risk factor associated with vascular calcification in CKD patients and in the general population. Phosphorus may also be one of the key elements linking vascular calcification with low bone turnover. The main hormones and factors that contribute to the kidney regulation of phosphorus and calcium include parathyroid hormone, FGF-23, klotho and 1,25-dihydroxyvitamin D (1,25(OH)2D). Serum phosphorus did not start rising until CKD 3b in contrast with the earlier changes observed with fibroblast growth factor-23 (FGF-23), Klotho, calcitriol and parathyroid hormone (PTH). Despite FGF-23 and PTH having synergic effects regarding phosphorus removal, they have opposite effects on 1,25(OH)2D3. At the same stages of CKD in which phosphorus retention appears to occur, calcium retention also occurs. As phosphorus accumulation is associated with poor outcomes, an important question without a clear answer is at which level-range should serum phosphorus be maintained at different stages of CKD to improve clinical outcomes. There are four main strategies to manage phosphate homeostasis; phosphorus dietary intake, administration of phosphate binder agents, effective control of hyperparathyroidism and to ensure in the CKD 5D setting, an adequate scheme of dialysis. Despite all the available strategies, and the introduction of new phosphate binder agents in the market, controlling serum phosphorus remains challenging, and hyperphosphatemia continues to be extremely common in CKD 5 patients. Furthermore, despite phosphate binding agents having proved to be effective in reducing serum phosphorus, their ultimate effects on clinical outcomes remain controversial. Thus, we still need well-designed, large-scale, placebo-controlled studies to definitively prove that the reduction of serum phosphorus by phosphate binders improves clinical outcomes.

Improvement of mineral and bone metabolism markers is associated with better survival in haemodialysis patients: the COSMOS study.

BACKGROUND: Abnormalities in serum phosphorus, calcium and parathyroid hormone (PTH) have been associated with poor survival in haemodialysis patients. This COSMOS (Current management Of Secondary hyperparathyroidism: a Multicentre Observational Study) analysis assesses the association of high and low serum phosphorus, calcium and PTH with a relative risk of mortality. Furthermore, the impact of changes in these parameters on the relative risk of mortality throughout the 3-year follow-up has been investigated. METHODS: COSMOS is a 3-year, multicentre, open-cohort, prospective study carried out in 6797 adult chronic haemodialysis patients randomly selected from 20 European countries. RESULTS: Using Cox proportional hazard regression models and penalized splines analysis, it was found that both high and low serum phosphorus, calcium and PTH were associated with a higher risk of mortality. The serum values associated with the minimum relative risk of mortality were 4.4 mg/dL for serum phosphorus, 8.8 mg/dL for serum calcium and 398 pg/mL for serum PTH. The lowest mortality risk ranges obtained using as base the previous values were 3.6-5.2 mg/dL for serum phosphorus, 7.9-9.5 mg/dL for serum calcium and 168-674 pg/mL for serum PTH. Decreases in serum phosphorus and calcium and increases in serum PTH in patients with baseline values of >5.2 mg/dL (phosphorus), >9.5 mg/dL (calcium) and <168 pg/mL (PTH), respectively, were associated with improved survival. CONCLUSIONS: COSMOS provides evidence of the association of serum phosphorus, calcium and PTH and mortality, and suggests survival benefits of controlling chronic kidney disease-mineral and bone disorder biochemical parameters in CKD5D patients.