Effect of the phosphate binder sucroferric oxyhydroxide in dialysis patients on endogenous calciprotein particles, inflammation, and vascular cells.

BACKGROUND: Calciprotein particles (CPPs), colloidal mineral-protein nanoparticles, have emerged as potential mediators of phosphate toxicity in dialysis patients, with putative links to vascular calcification, endothelial dysfunction and inflammation. We hypothesized that phosphate binder therapy with sucroferric oxyhydroxide (SO) would reduce endogenous CPP levels and attenuate pro-calcific and pro-inflammatory effects of patient serum towards human vascular cells in vitro. METHODS: This secondary analysis of a randomised controlled crossover study compared the effect of 2-week phosphate binder washout with high-dose (2000 mg/day) and low-dose (250 mg/day) SO therapy in 28 haemodialysis patients on serum CPP levels, inflammatory cytokine/chemokine arrays and human aortic smooth muscle cell (HASMC) and coronary artery endothelial cell (HCAEC) bioassays. RESULTS: In our cohort (75% male, 62 ± 12 years) high-dose SO reduced primary (amorphous) and secondary (crystalline) CPP levels {-62% [95% confidence interval (CI) -76 to -44], P < .0001 and -38% [-62 to -0.14], P < .001, respectively} compared with washout. Nine of 14 plasma cytokines/chemokines significantly decreased with high-dose SO, with consistent reductions in interleukin-6 (IL-6) and IL-8. Exposure of HASMC and HCAEC cultures to serum of SO-treated patients reduced calcification and markers of activation (IL-6, IL-8 and vascular cell adhesion protein 1) compared with washout. Serum-induced HASMC calcification and HCAEC activation was ameliorated by removal of the CPP-containing fraction from patient sera. Effects of CPP removal were confirmed in an independent cohort of chronic kidney disease patients. CONCLUSIONS: High-dose SO reduced endogenous CPP formation in dialysis patients and yielded serum with attenuated pro-calcific and inflammatory effects in vitro.

Successful kidney transplantation in a patient with pre-existing chronic myeloid leukemia treated with imatinib.

Active malignancy is an absolute contraindication to kidney transplantation. As for chronic myeloid leukemia (CML), a Philadelphia chromosome-positive myeloproliferative neoplasm, the introduction of tyrosine kinase inhibitors has transformed CML from a lethal into a manageable chronic disease with a close-to-normal life expectancy. To date it is unknown whether kidney transplantation can be safely performed in patients with pre-existing CML. We describe the clinical course of a 57-year-old male patient with chronic kidney disease caused by reflux nephropathy. This patient had undergone first kidney transplantation 20 years earlier and had again been on chronic hemodialysis for 6 years when CML was diagnosed. First-line therapy with 400 mg imatinib daily was well tolerated and induced an optimal cytogenetic and molecular response 3 months after initiation. One and a half years after CML diagnosis, a second kidney transplantation from a deceased donor was performed. Immunosuppression included basiliximab, tacrolimus, mycophenolate mofetil, and corticosteroids. Currently, 2 years posttransplant, renal allograft function is stable (serum creatinine 1.09 mg/dL, estimated glomerular filtration rate 75 mL/min per 1.73 m2 ), and CML remains in deep molecular remission with imatinib. Imatinib-treated CML in deep molecular remission could be regarded as inactive malignancy and may therefore not be viewed as an absolute contraindication to kidney transplantation.

Overexpression of a functional calcium-sensing receptor dramatically increases osteolytic potential of MDA-MB-231 cells in a mouse model of bone metastasis through epiregulin-mediated osteoprotegerin downregulation.

INTRODUCTION AND AIMS: Osteolytic bone metastases are observed in advanced cases of breast cancer. In vitro data suggest that the activity of the calcium-sensing receptor (CaSR) expressed by metastatic cells could potentiate their osteolytic potential. This study aimed to demonstrate in vivo the involvement of the CaSR in breast cancer cells osteolytic potential and to identify potential targets linked to CaSR activity. METHODS AND RESULTS: MDA-MB-231 stably transfected with plasmids containing either a full-length wild-type CaSR (CaSR-WT), or a functionally inactive dominant negative mutant (CaSR-DN) or an empty vector (EV) were intratibially injected into Balb/c-Nude mice. X-ray analysis performed 19 days after injection showed a dramatic increase of osteolytic lesions in mice injected with CaSR-WT-transfected cells as compared to mice injected with EV- or CaSR-DN-transfected cells. This was associated with decreased BV/TV ratio and increased tumor burden. Epiregulin, an EGF-like ligand, was identified by a DNA microarray as a possible candidate involved in CaSR-mediated osteolysis. Indeed, in vitro, CaSR overexpression increased both epiregulin expression and secretion as compared to EV- or CaSR-DN-transfected cells. Increased epiregulin expression was also detected in osteolytic bone lesions from mice injected with CaSR-WT-transfected MDA-MB-231. In vitro, exposure of osteoblastic cells (HOB and SaOS2) to exogenous epiregulin significantly decreased OPG mRNA expression. Exposure of osteoblastic cells to conditioned media prepared from CaSR-WT-transfected cells also decreased OPG expression. This effect was partially blocked after addition of an anti-epiregulin antibody. CONCLUSIONS: Overexpression of a functional CaSR in metastatic breast cancer cells dramatically amplifies their osteolytic potential through epiregulin-mediated OPG downregulation.

Impact of CYP24A1 overexpression on growth of colorectal tumour xenografts in mice fed with vitamin D and soy.

Our previous studies showed that the 1,25-dihydroxyvitamin D (1,25-D3) catabolizing enzyme, 1,25-dihydoxyvitamin D 24 hydroxylase (CYP24A1) was overexpressed in colorectal tumours and its level correlated with increased proliferation. We hypothesised that cells overexpressing CYP24A1 have growth advantage and a diet rich in vitamin D and soy would restore sensitivity to the anti-tumourigenic effects of vitamin D. Soy contains genistein, a natural CYP24A1 inhibitor. To determine causality between CYP24A1 and tumour growth, we established xenografts in male SCID mice with HT29 cells stably overexpressing either GFP-tagged CYP24A1 or GFP. Mice were fed with either high (2500 IU D3/kg) or low vitamin D (100 IU D3/kg) diet in the presence or absence of soy (20% diet). In vitro, cells overexpressing CYP24A1 grew faster than controls. 1,25-D3, the active vitamin D metabolite, reduced cell number only in the presence of the CYP24A1 inhibitor VID400. Regardless of the amount of vitamin D in the diet, xenografts overexpressing CYP24A1 grew faster, were heavier and more aggressive. Soy reduced tumour volume only in the control xenografts, while the tumours overexpressing CYP24A1 were larger in the presence of dietary soy. In conclusion, we demonstrate that CYP24A1 overexpression results in increased aggressiveness and proliferative potential of colorectal tumours. Irrespective of the dietary vitamin D3, dietary soy is able to increase tumour volume when tumours overexpress CYP24A1, suggesting that combination of vitamin D3 and soy could have an anti-tumourigenic effect only if CYP24A1 levels are normal.

Low-dose calcium versus pentagastrin for stimulation of calcitonin in chronic hemodialysis patients: a pilot study.

CONTEXT: Elevated calcitonin levels occur in up to 46% of patients with chronic hemodialysis (CHD) and frequently reflect benign C-cell hyperplasia rather than medullary thyroid carcinoma. For the differential diagnosis of hypercalcitoninemia, the pentagastrin-stimulated calcitonin test was used until its availability became restricted. OBJECTIVE: This study sought to compare calcium and pentagastrin in terms of their ability to stimulate calcitonin secretion and their side effects in patients with CHD. SETTING AND DESIGN: This prospective pilot study was conducted at the chronic hemodialysis unit of the Medical University of Vienna between December 2012 and September 2013. PATIENTS: We studied six male patients with CHD with elevated basal calcitonin levels. INTERVENTION: The stimulation test was performed first with 0.5 µg/kg pentagastrin and then with 1 mg/kg calcium after a median washout period of 7 (6-9) months. MAIN OUTCOME MEASURES: We measured calcitonin, serum ionized calcium, intact PTH (iPTH), and C-terminal fibroblast growth factor 23 levels before and 2, 5, and 10 minutes after iv infusion of the stimulant and assessed the tolerability of the two substances by a questionnaire. RESULTS: Both pentagastrin and calcium significantly stimulated calcitonin secretion at 2 and 5 minutes. Partial correlation analysis revealed a strong association between calcium- and pentagastrin-stimulated calcitonin levels (r=0.875, P < .0001). Only after calcium infusion serum ionized calcium levels increased from 1.09 (0.91-1.16) mmol/l to 1.4 (1.14-1.65) mmol/l at 2 minutes (P < .01) but returned to baseline levels at 5 minutes. Moreover, calcium infusion led to a significant decrease in iPTH levels from 315 (203-723) pg/ml to 182 (121-415) pg/ml at 5 minutes (P < .05) and 171 (91-346) pg/ml at 10 minutes (P < .001). In general, calcium caused fewer and less severe side effects than pentagastrin. CONCLUSIONS: In patients with CHD, the response of calcitonin to calcium and pentagastrin was comparable, making calcium a potential substitute for pentagastrin in these patients.

Increased copy-number and not DNA hypomethylation causes overexpression of the candidate proto-oncogene CYP24A1 in colorectal cancer.

In colorectal cancer (CRC) the vitamin D catabolizing enzyme 1,25-dihydroxyvitamin D 24-hydroxylase (CYP24A1) is overexpressed with a potentially significant, positive impact on the catabolism of 1,25-dihydroxyvitamin D3 (1,25-D3 ). However, the underlying mechanism of CYP24A1 overexpression is poorly understood. In the present study, we investigated possible causes including hypomethylation of the CYP24A1 promoter, amplification of the CYP24A1 gene locus (20q13.2), and altered expression of CYP24A1-specific transcription factors. We quantified CYP24A1 gene copy-number, performed bisulfite sequencing of the CYP24A1 promoter to assess DNA methylation, and measured mRNA expression of CYP24A1, 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1), vitamin D receptor (VDR) and retinoid X receptor (RXR). We found that 77 (60%) out of 127 colorectal tumors showed increased CYP24A1 gene copy-number and that more than 6 copies of CYP24A1 correlated positively with CYP24A1 mRNA expression suggestive of a causal relationship. No differences in CYP24A1 promoter methylation were found between tumor tissue and adjacent mucosa from the same patient or between tissues with high or low mRNA expression, thus excluding DNA hypomethylation as a possible cause of CYP24A1 overexpression in CRC. Furthermore, mRNA expression of several factors involved in replication licensing positively correlated with CYP24A1 mRNA expression, raising the possibility that CYP24A1 overexpression might favor increased proliferation in tumors by suppressing local 1,25-D3 levels. We conclude that high copy-number gain is a key determinant of CYP24A1 overexpression in CRC. Other postulated causes of CYP24A1 overexpression including promoter hypomethylation and enhanced VDR and/or RXR expression do not appear to be involved.

Prevention of preneoplastic lesions by dietary vitamin D in a mouse model of colorectal carcinogenesis.

Colorectal cancer (CRC) is one of the leading causes of cancer morbidity and mortality in Western countries. One of the risk factors for colorectal tumorigenesis is vitamin D insufficiency. The aim of this study was to establish whether increasing dietary vitamin D intake can prevent or delay development of chemically induced preneoplastic lesions in the colon of mice. We fed six weeks old female C57BL/6J mice (n=28) with increasing vitamin D3 concentrations (100, 400, 1000, 2500, 5000IU/kg diet). To induce dysplasia, a preneoplastic lesion, we injected mice with the carcinogen azoxymethane (10mg/kg) intraperitoneally, followed by three cycles of 2% dextran sodium sulfate salt, a tumor promoter, in the drinking water. To test our hypothesis that high vitamin D intake prevents formation of preneoplastic lesions, we have investigated the effect of increasing dietary vitamin D on development of premalignant colorectal lesions, serum 25-hydroxyvitamin D3 (25-D3) levels, and expression of renal vitamin D system genes. Dietary vitamin D concentration correlated inversely with dysplasia score (Spearman's correlation coefficient, ρ: -0.579, p=0.002) and positively with serum 25-D3 levels (ρ: 0.752, p=0.001). Increasing dietary vitamin D concentration beyond 1000IU/kg led to no further increase in circulating 25-D3 levels, while the dysplasia score leveled out at ≥2500IU/kg vitamin D. High dietary vitamin D intake led to increased renal mRNA expression of the vitamin D catabolizing enzyme cyp24a1 (ρ: 0.518, p=0.005) and decreased expression of the vitamin D activating enzyme cyp27b1 (ρ: -0.452, p=0.016), protecting the body from toxic serum levels of the active vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25-D3). Our data showed that increasing dietary vitamin D intake is able to prevent chemically induced preneoplastic lesions. The maximum impact was achieved when the mice consumed more than 2500IU vitamin D/kg diet. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Calcium sensing receptor signalling in physiology and cancer.

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

Role of calcium, vitamin D, and the extrarenal vitamin D hydroxylases in carcinogenesis.

Vitamin D deficiency and low calcium intake are considered risk factors for several cancers. Vitamin D, synthesized in the skin or ingested through the diet, is transformed through two hydroxylation steps to the active metabolite, 1α,25-dihydroxyvitamin D3 (1,25-D3). 25-hydroxylases in the liver are responsible for the first hydroxylation step. The ultimate activation is performed by the renal 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1), while the 1,25-dihydroxyvitamin D 24-hydroxylase (CYP24A1) in the kidneys degrades the active metabolite. These two renal vitamin D hydroxylases control the endocrine serum 1,25-D3 levels, and are responsible for maintaining mineral homeostasis. In addition, the active vitamin D hormone 1,25-D3 regulates cellular proliferation, differentiation, and apoptosis in multiple tissues in a paracrine/autocrine manner. Interestingly, it is the low serum level of the precursor 25- hydroxyvitamin D3 (25-D3) that predisposes to numerous cancers and other chronic diseases, and not the serum concentration of the active vitamin D hormone. The extra-renal autocrine/paracrine vitamin D system is able to synthesize and degrade locally the active 1,25- D3 necessary to maintain normal cell growth and to counteract mitogenic stimuli. Thus, vitamin D hydroxylases play a prominent role in this process. The present review describes the role of the vitamin D hydroxylases in cancer pathogenesis and the cross-talk between the extra-renal autocrine/paracrine vitamin D system and calcium in cancer prevention.