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.

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.

Effect of Oat ß-Glucan Supplementation on Chronic Kidney Disease: A Feasibility Study.

OBJECTIVE: Dietary supplementation with grains containing high ß-glucan fiber has been shown to attenuate the progression of chronic kidney disease (CKD) and vascular calcification in animal models. The aim of this study was to investigate the feasibility of consuming an oat ß-glucan supplement and to assess its effects on certain uremic toxins and markers of mineral metabolism in patients with CKD. DESIGN: This is a 20-week, nonrandomized, single-center, pretest-posttest study. Twenty-eight subjects with CKD stages 3-4 were enrolled. The mean age was 67.6 ± 8.9 years, and the mean estimated glomerular filtration rate was 35 ± 14 mL/min/1.73 m2. Subjects received a dietary supplement containing 3 g of oat ß-glucan per day for 12 weeks. The 4-week period before the start of the intervention was used as a baseline comparison for each subject. The primary outcome was pre-post supplement changes in plasma levels of two uremic toxins: trimethylamine N-oxide (TMAO) and asymmetric dimethylarginine. Secondary outcomes were pre-post supplement changes in serum calcium, phosphorus, and Klotho levels. Repeated-measures analysis of variance was used to test the differences in outcomes over the three-month-long intervention. RESULTS: Serum levels of TMAO decreased by a median of -17% (interquartile range: -46%, 7%) at the end of the intervention. A nonstatistically significant change was observed for asymmetric dimethylarginine (median -0.6% [-12%, 20%]) and serum Klotho (median -3% [-8%, 7%]). There were no changes in serum levels of calcium and phosphorus. One month after discontinuation of ß-glucan therapy, TMAO levels increased by a median of 16% (-12%, 36%) but remained slightly below the pretreatment levels. Eight subjects experienced side effects and discontinued the treatment. CONCLUSION: A diet supplemented with ß-glucan is safe and potentially efficacious in lowering serum concentrations of TMAO in patients with CKD. Larger trials with longer follow-up times are needed to determine whether such reductions translate into clinical benefits.

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.

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.

Vitamin D deficiency and its association with fatigue and quality of life in advanced cancer patients under palliative care: A cross-sectional study.

BACKGROUND: A normal vitamin D status is required for bones and muscles to maintain their function and structure, but it also contributes to the functional integrity of other multiple physiologic systems in the body. AIM: To assess the relationship of Vitamin D deficiency with health-related quality-of-life issues, fatigue, and physical functioning in advanced cancer patients. DESIGN: This is a cross-sectional study. PATIENTS/SETTINGS: Adults under palliative care, having a locally advanced or metastatic or inoperable solid cancer. RESULTS: Among 30 patients in palliative care with advanced solid cancer, 90% were vitamin D deficient. Serum Vitamin D concentration was positively correlated with patient-reported absence of fatigue (s = 0.49), and physical and functional well-being (s = 0.44 and s = 0.41, respectively, p < 0.01). Fatigue was the symptom with the highest median impact on their lives and was the only one associated with serum vitamin D (p = 0.031), with lower fatigue in patients with vitamin D concentrations in the third tertile. There was no evidence of a direct association between health-related quality of life and vitamin D status. CONCLUSION: The 90% frequency of advanced cancer patients with vitamin D deficiency, together with the positive correlation of vitamin D status with the absence of fatigue and improved physical and functional well-being, points to vitamin D supplementation as a potential therapy to enhance the patient's quality of life.

A novel rat model of vitamin D deficiency: safe and rapid induction of vitamin D and calcitriol deficiency without hyperparathyroidism.

Vitamin D deficiency is associated with a range of clinical disorders. To study the mechanisms involved and improve treatments, animal models are tremendously useful. Current vitamin D deficient rat models have important practical limitations, including time requirements when using, exclusively, a vitamin D deficient diet. More importantly, induction of hypovitaminosis D causes significant fluctuations in parathyroid hormone (PTH) and mineral levels, complicating the interpretation of study results. To overcome these shortcomings, we report the successful induction of vitamin D deficiency within three weeks, with stable serum PTH and minerals levels, in Wistar rats. We incorporated two additional manoeuvres compared to a conventional diet. Firstly, the vitamin D depleted diet is calcium (Ca) enriched, to attenuate the development of secondary hyperparathyroidism. Secondly, six intraperitoneal injections of paricalcitol during the first two weeks are given to induce the rapid degradation of circulating vitamin D metabolites. After three weeks, serum 25-hydroxyvitamin D3 (25D) and 1,25-dihydroxyvitamin D3 (1,25D) levels had dropped below detection limits, with unchanged serum PTH, Ca, and phosphate (P) levels. Therefore, this model provides a useful tool to examine the sole effect of hypovitaminosis D, in a wide range of research settings, without confounding changes in PTH, Ca, and P.

The effect on quality of life of vitamin D administration for advanced cancer treatment (VIDAFACT study): protocol of a randomised controlled trial.

INTRODUCTION: Vitamin D is related to resistance to chronic diseases, physiological parameters and functional measures. All of these relationships underscore the potential benefits of cholecalciferol or D3 (nutritional vitamin D) in cancer. This is the first study designed to obtain conclusive evidence on the effect of cholecalciferol in advanced patients with cancer. The main goal is to assess its effects on the patient's perceived quality of life. Cholecalciferol's impact on fatigue and physical performance, as well as its cost utility, will also be assessed. METHODS AND ANALYSIS: A randomised triple-blind phase II/III placebo-controlled multicentre trial has been designed. Patients satisfying the inclusion and exclusion criteria will be randomly assigned to receive cholecalciferol or placebo. Eligible patients will be adults with a locally advanced or metastatic or inoperable solid cancer in palliative care, who have given signed informed consent and have matched inclusion and exclusion criteria. The randomisation will be based on a computer-generated procedure and centralised by the pharmacy service of the coordinating centre. The assigned treatment will be administered by the hospital's pharmacy to conceal group allocation for patients and healthcare providers. Cholecalciferol (4000 IU/day) or placebo, starting at day 15 and continuing up to day 42, will be added to palliative care treatment. Outpatient visits will be scheduled every 14 days. ETHICS AND DISSEMINATION: Ethical approval was received from the Medical Ethical Commitee of the HUAV (CEIC-1169). Participants and their families will receive the research findings which will also be disseminated on local and national media, presented at national and international meetings of the specialty, and published in peer-reviewed scientific journals. TRIAL REGISTRATION NUMBER: EudraCT: 2013-003478-29.

Vitamin D in chronic kidney disease.

In chronic kidney disease (CKD), abnormalities in vitamin D metabolism contribute to the development of mineral and skeletal disorders, elevations in parathyroid hormone (PTH), hypertension, systemic inflammation, renal and cardiovascular damage. CKD induces a progressive loss of the capacity of the kidney not only to convert 25-hydroxyvitamin D [25(OH)D] to circulating calcitriol, the vitamin D hormone, but also to maintain serum 25(OH)D levels for non-renal calcitriol synthesis. The resulting calcitriol and 25(OH)D deficiency associates directly with accelerated disease progression and death. This chapter presents our understanding of the pathophysiology behind 25(OH)D and calcitriol deficiency in CKD, of the adequacy of current recommendations for vitamin D supplementation and PTH suppression, and of potential markers of renal and cardiovascular lesions unrelated to PTH suppression, a knowledge required for the design of trials to obtain evidence-based recommendations for vitamin D and calcitriol replacement that improve outcomes at all stages of CKD.

Defective renal maintenance of the vitamin D endocrine system impairs vitamin D renoprotection: a downward spiral in kidney disease.

In kidney disease, the progressive loss of renal capacity to produce calcitriol, the vitamin D hormone, is a key contributor to elevations in parathyroid hormone (PTH) and mineral and skeletal disorders predisposing to renal and cardiovascular damage, ectopic calcifications, and high mortality rates. Thus, the safe correction of calcitriol deficiency to suppress PTH has been the treatment of choice for decades. However, recent epidemiological and experimental data suggest that calcitriol replacement may improve outcomes through renal and cardioprotective actions unrelated to PTH suppression. Furthermore, a striking incidence of vitamin D deficiency occurs in kidney disease and associates more strongly than calcitriol deficiency with a higher risk for kidney disease progression and death. Despite the translational relevance of these findings, no prospective trials are currently available in support of the efficacy of vitamin D supplementation and/or calcitriol replacement to safely halt/moderate renal disease progression. This review updates the pathophysiology behind the vicious cycle by which kidney injury impairs the maintenance of normal vitamin D and calcitriol levels, which in turn impedes vitamin D/calcitriol renoprotective actions, a requirement for the design of prospective trials to improve current recommendations for vitamin D interventions at all stages of kidney disease.

Kidney disease and vitamin D levels: 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, and VDR activation.

A normal vitamin D status is essential for human health. Vitamin D deficiency is a recognized risk factor for all-cause mortality in normal individuals and in chronic kidney disease (CKD) patients. The link between vitamin D deficiency and death is a defective activation of the vitamin D receptor (VDR) by 1,25-dihydroxyvitamin D (calcitriol, the vitamin D hormone) to induce/repress genes that maintain mineral homeostasis and skeletal integrity, and prevent secondary hyperparathyroidism, hypertension, immune disorders, and renal and cardiovascular (CV) damage. The kidney is the main site for the conversion of 25-hydroxyvitamin D (25D) to circulating calcitriol, and therefore essential for the health benefits of endocrine VDR activation. The kidney is also essential for the uptake of 25D from the glomerular ultrafiltrate for its recycling to the circulation to maintain serum 25D levels, extrarenal calcitriol synthesis, and the prosurvival benefits of autocrine/paracrine VDR activation. Indeed, both calcitriol and vitamin D deficiency increase progressively in the course of CKD, and associate directly with accelerated disease progression and death. Therefore, the safe correction of calcitriol and vitamin D deficiency/insufficiency is becoming a high priority among nephrologists. This review updates the pathophysiology behind 25D and calcitriol deficiency and impaired VDR activation in CKD, the adequacy of current recommendations for vitamin D supplementation, and potential markers of the efficacy of therapy to prevent or slow the development of renal and CV lesions unrelated to parathyroid hormone suppression, a knowledge required for the design of trials to obtain evidence-based recommendations for vitamin D and calcitriol replacement at all stages of CKD.

Calcium, phosphorus and vitamin D disorders in uremia.

BACKGROUND: Alterations in calcium, phosphate (P) and vitamin D metabolism play a critical role in the development of secondary hyperparathyroidism (SH), parathyroid hyperplasia and soft tissue and vascular calcification. METHODOLOGY: Studies were performed in uremic dogs and rats fed a low and high P diet over a period of 1-4 months. In addition, in vitro studies were performed in normal parathyroid glands incubated in culture media containing 0.2 mM P (low) or 2.0 mM P (high). RESULTS: Uremic rats maintained on a low P diet did not develop SH or parathyroid hyperplasia. There was an enhancement of p21, the suppressor of the cell cycle, in these parathyroid glands. Opposite results were obtained using a high P diet. There was an enhancement of transforming growth factor-alpha and epidermal growth factor receptor, known enhancers of cell proliferation. In vitro studies demonstrated the direct effect of P on parathyroid hormone secretion. CONCLUSIONS: Early dietary P restriction prevents the development of SH and parathyroid hyperplasia. If dietary P restriction is applied to rats with established SH, there is a significant amelioration of SH and parathyroid hyperplasia. In addition, control of serum P in uremic patients is crucial in the prevention of vascular calcification.

The effects of sevelamer hydrochloride and calcium carbonate on kidney calcification in uremic rats.

The control of serum phosphorus (P) and calcium-phosphate (Ca x P) product is critical to the prevention of ectopic calcification in chronic renal failure (CRF). Whereas calcium (Ca) salts, the most commonly used phosphate binders, markedly increase serum Ca and positive Ca balance, the new calcium- and aluminum-free phosphate binder, sevelamer hydrochloride (RenaGel), reduces serum P without altering serum Ca in hemodialysis patients. Using an experimental model of CRF, these studies compare sevelamer and calcium carbonate (CaCO(3)) in the control of serum P, secondary hyperparathyroidism (SH), and ectopic calcifications. 5/6 nephrectomized rats underwent one of the following treatments for 3 mo: uremic + high-P diet (U-HP); UHP + 3% CaCO(3) (U-HP+C); UHP + 3% sevelamer (U-HP+S). Sevelamer treatment controlled serum P independent of increases in serum Ca, thus reducing serum Ca x P product and further deterioration of renal function, as indicated by the highest creatinine clearances. Sevelamer was as effective as CaCO(3) in the control of high-P-induced SH, as shown by similar serum PTH levels, parathyroid (PT) gland weight, and markers of PT hyperplasia. Also, both P binders elicited similar efficacy in reducing the myocardial and hepatic calcifications induced by uremia. However, sevelamer caused a dramatic reduction of renal Ca deposition (29.8 +/- 8.6 micro g/g wet tissue) compared with both U-HP (175.5 +/- 45.7 micro g/g wet tissue, P < 0.01) and the U-HP+C (58.9 +/- 13.7 micro g/g wet tissue, P < 0.04). Histochemical analyses using Von Kossa and Alizarin red S staining of kidney sections confirmed these findings. The high number of foci of calcification in the kidney of uremic controls (108 +/- 25) was reduced to 33.0 +/- 11.3 by CaCO(3) and decreased even further with sevelamer (16.4 +/- 8.9, P < 0.02 versus CaCO(3)). Importantly, the degree of tubulointerstitial fibrosis was also markedly lower in U-HP+S (5%) compared with either U-HP+C (30%) or U-HP (50%). It is concluded that in experimental CRF in rats, despite a similar control of serum P and SH, sevelamer is more effective than CaCO(3) in preventing renal Ca deposition and tubulointerstitial fibrosis, including better preservation of renal function. These findings cannot be extrapolated to human disease, and further studies in patients are necessary to determine the benefits of either P binder.