Genomic reprograming analysis of the Mesothelial to Mesenchymal Transition identifies biomarkers in peritoneal dialysis patients.

Peritoneal dialysis (PD) is an effective renal replacement therapy, but a significant proportion of patients suffer PD-related complications, which limit the treatment duration. Mesothelial-to-mesenchymal transition (MMT) contributes to the PD-related peritoneal dysfunction. We analyzed the genetic reprograming of MMT to identify new biomarkers that may be tested in PD-patients. Microarray analysis revealed a partial overlapping between MMT induced in vitro and ex vivo in effluent-derived mesothelial cells, and that MMT is mainly a repression process being higher the number of genes that are down-regulated than those that are induced. Cellular morphology and number of altered genes showed that MMT ex vivo could be subdivided into two stages: early/epithelioid and advanced/non-epithelioid. RT-PCR array analysis demonstrated that a number of genes differentially expressed in effluent-derived non-epithelioid cells also showed significant differential expression when comparing standard versus low-GDP PD fluids. Thrombospondin-1 (TSP1), collagen-13 (COL13), vascular endothelial growth factor A (VEGFA), and gremlin-1 (GREM1) were measured in PD effluents, and except GREM1, showed significant differences between early and advanced stages of MMT, and their expression was associated with a high peritoneal transport status. The results establish a proof of concept about the feasibility of measuring MMT-associated secreted protein levels as potential biomarkers in PD.

Longitudinal Trend in Lipid Profile of Incident Peritoneal Dialysis Patients is Not Influenced by the Use of Biocompatible Solutions.

UNLABELLED: ♦ BACKGROUND: The longitudinal trends of lipid parameters and the impact of biocompatible peritoneal dialysis (PD) solutions on these levels remain to be fully defined. The present study aimed to a) evaluate the influence of neutral pH, low glucose degradation product (GDP) PD solutions on serum lipid parameters, and b) explore the capacity of lipid parameters (total cholesterol [TC], triglyceride [TG], high density lipoprotein [HDL], TC/HDL, low density lipoprotein [LDL], very low density lipoprotein [VLDL]) to predict cardiovascular events (CVE) and mortality in PD patients. ♦ METHODS: The study included 175 incident participants from the balANZ trial with at least 1 stored serum sample. A composite CVE score was used as a primary clinical outcome measure. Multilevel linear regression and Poisson regression models were fitted to describe the trend of lipid parameters over time and its ability to predict composite CVE, respectively. ♦ RESULTS: Small but statistically significant increases in serum TG (coefficient 0.006, p < 0.001), TC/HDL (coefficient 0.004, p = 0.001), and VLDL cholesterol (coefficient 0.005, p = 0.001) levels and a decrease in the serum HDL cholesterol levels (coefficient -0.004, p = 0.009) were observed with longer time on PD, whilst the type of PD solution (biocompatible vs standard) received had no significant effect on these levels. Peritoneal dialysis glucose exposure was significantly associated with trends in TG, TC/HDL, HDL and VLDL levels. Baseline lipid parameter levels were not predictive of composite CVEs or all-cause mortality. ♦ CONCLUSION: Serum TG, TC/HDL, and VLDL levels increased and the serum HDL levels decreased with increasing PD duration. None of the lipid parameters were significantly modified by biocompatible PD solution use over the time period studied or predictive of composite CVE or mortality.

Laboratory and dialysis characteristics in hemodialysis patients suffering from chronic itch--results from a representative cross-sectional study.

BACKGROUND: A representative cross-sectional study showed that chronic itch (lasting for a minimum of 6 weeks) affects 25.2 % (point prevalence) of hemodialysis (HD) patients. Pathophysiology and etiology of chronic itch (CI) in HD are still unclear. METHODS: We investigated 860 HD patients from a representative randomly selected cluster-sample considering the regional distributions of dialysis units in Germany. The current analyses report comorbidities, laboratory values and dialysis characteristics of HD patients in relation to CI. RESULTS: Diabetes was the only comorbidity that was associated with the occurrence of itch but interestingly with less CI. Except for creatinine, phosphorus, and parathormone, there were no significant associations between the occurrence and characteristics of CI and any laboratory value. Kt/V was not associated with the presence of CI. Patients dialyzed with polyarylethersulfone-membrane showed significantly more CI in all prevalence estimates and those dialyzed with polysulfone-membrane were significantly less affected by CI. CONCLUSIONS: Long-term follow-up studies will show if the type of dialysis membrane influences the development of CI in HD patients. It is most likely that several factors e.g. elevated parathormone, origin of end stage renal disease (ESRD), type of dialysis membrane, and a neuropathic component all contribute to the occurrence of CI in HD patients. Future research should consider a multifactorial origin of itch in HD.

Serum Magnesium and Mortality in Hemodialysis Patients in the United States: A Cohort Study.

BACKGROUND: Low serum magnesium levels in patients with kidney disease have been linked to increased mortality. This study investigated whether similar associations existed in maintenance hemodialysis (HD) patients. STUDY DESIGN: Cohort study. SETTING & PARTICIPANTS: All Fresenius Medical Care North America in-center HD patients with available serum magnesium measurements were studied. The initial exploratory study in 21,534 HD patients evaluated associations among serum magnesium level, dialysate magnesium concentration, and mortality from April 2007 through June 2008. The follow-up study in 27,544 HD patients evaluated associations between serum magnesium levels and mortality over 1 year (January through December 2008). PREDICTORS: The primary predictor was serum magnesium level, with adjustment for case-mix (age, sex, race, diabetes, and dialysis vintage and additionally for follow-up study: body surface area and vascular access) and laboratory variables (albumin, hemoglobin, phosphorus, equilibrated Kt/V, potassium, calcium, and intact parathyroid hormone values). OUTCOME: Primary outcome variable was 1-year mortality risk, evaluated using Cox proportional hazards models. RESULTS: Among 21,534 HD patients in the exploratory study, there were 3,682 deaths. Higher dialysate magnesium level was associated with higher serum magnesium level (R=0.22; P<0.001). Patients with the lowest serum magnesium levels (<1.30 mEq/L) were at highest risk for death (HR, 1.63; 95% CI, 1.30-1.96; reference serum magnesium, 1.60-<1.90 mEq/L). Among 27,544 HD patients in the follow-up study, there were 4,531 deaths. In Cox proportional hazards models, there was a linear decline in death risk from the lowest to the highest serum magnesium category, with the best survival at serum magnesium levels ≥ 2.50 mEq/L (HR, 0.68; 95% CI, 0.56-0.82). However, risk estimates were attenuated with case-mix and lab adjustment. This pattern was consistent within diabetes subgroups and for cardiovascular or noncardiovascular causes of death. LIMITATIONS: Observational study with cross-sectional serum magnesium measurements and no information for oral magnesium intake. CONCLUSIONS: Elevated serum magnesium levels > 2.10 mEq/L were associated with better survival than low serum magnesium levels < 1.30 mEq/L in HD patients. Prospective studies may determine whether manipulation of low serum magnesium levels affects survival.

Prevalence of chronic itch and associated factors in haemodialysis patients: a representative cross-sectional study.

Chronic itch is a common symptom in haemodialysis (HD) patients, which is often underestimated. The aim of this cross-sectional study was to investigate the prevalence and factors associated with chronic itch in HD patients. A total of 860 HD patients from a randomly selected cluster-sample of patients attending dialysis units in Germany were included. The patients' mean?±?SD age was 67.2?±?13.5 years, 57.2% were male. The point prevalence of chronic itch was 25.2% (95% CI 22.4-28.1), 12-month prevalence was 27.2% (95% CI 24.1-30.3) and lifetime prevalence was 35.2% (95% CI 31.9-38.3). Chronic itch was significantly less prevalent in patients with secondary glomerulonephritis as primary renal disease. A history of dry skin, eczema, and age

Characterisation of calcium phosphate crystals on calcified human aortic vascular smooth muscle cells and potential role of magnesium.

BACKGROUND: Cardiovascular disease including vascular calcification (VC) remains the leading cause of death in patients suffering from chronic kidney disease (CKD). The process of VC seems likely to be a tightly regulated process where vascular smooth muscle cells are playing a key role rather than just a mere passive precipitation of calcium phosphate. Characterisation of the chemical and crystalline structure of VC was mainly led in patients or animal models with CKD. Likewise, Mg2+ was found to be protective in living cells although a potential role for Mg2+ could not be excluded on crystal formation and precipitation. In this study, the crystal formation and the role of Mg2+ were investigated in an in vitro model of primary human aortic vascular smooth muscle cells (HAVSMC) with physical techniques. METHODOLOGY/PRINCIPAL FINDINGS: In HAVSMC incubated with increased Ca x Pi medium, only calcium phosphate apatite crystals (CPA) were detected by Micro-Fourier Transform InfraRed spectroscopy (µFTIR) and Field Effect Scanning Electron Microscope (FE-SEM) and Energy Dispersive X-ray spectrometry (EDX) at the cell layer level. Supplementation with Mg2+ did not alter the crystal composition or structure. The crystal deposition was preferentially positioned near or directly on cells as pictured by FE-SEM observations and EDX measurements. Large µFTIR maps revealed spots of CPA crystals that were associated to the cellular layout. This qualitative analysis suggests a potential beneficial effect of Mg2+ at 5 mM in noticeably reducing the number and intensities of CPA µFTIR spots. CONCLUSIONS/SIGNIFICANCE: For the first time in a model of HAVSMC, induced calcification led to the formation of the sole CPA crystals. Our data seems to exclude a physicochemical role of Mg2+ in altering the CPA crystal growth, composition or structure. Furthermore, Mg2+ beneficial role in attenuating VC should be linked to an active cellular role.

Interference of peritoneal dialysis fluids with cell cycle mechanisms.

INTRODUCTION: Peritoneal dialysis fluids (PDF) differ with respect to osmotic and buffer compound, and pH and glucose degradation products (GDP) content. The impact on peritoneal membrane integrity is still insufficiently described. We assessed global genomic effects of PDF in primary human peritoneal mesothelial cells (PMC) by whole genome analyses, quantitative real-time polymerase chain reaction (RT-PCR) and functional measurements. METHODS: PMC isolated from omentum of non-uremic patients were incubated with conventional single chamber PDF (CPDF), lactate- (LPDF), bicarbonate- (BPDF) and bicarbonate/lactate-buffered double-chamber PDF (BLPDF), icodextrin (IPDF) and amino acid PDF (APDF), diluted 1:1 with medium. Affymetrix GeneChip U133Plus2.0 (Affymetrix, CA, USA) and quantitative RT-PCR were applied; cell viability was assessed by proliferation assays. RESULTS: The number of differentially expressed genes compared to medium was 464 with APDF, 208 with CPDF, 169 with IPDF, 71 with LPDF, 45 with BPDF and 42 with BLPDF. Out of these genes 74%, 73%, 79%, 72%, 47% and 57% were downregulated. Gene Ontology (GO) term annotations mainly revealed associations with cell cycle (p = 10(-35)), cell division, mitosis, and DNA replication. One hundred and eighteen out of 249 probe sets detecting genes involved in cell cycle/division were suppressed, with APDF-treated PMC being affected the most regarding absolute number and degree, followed by CPDF and IPDF. Bicarbonate-containing PDF and BLPDF-treated PMC were affected the least. Quantitative RT-PCR measurements confirmed microarray findings for key cell cycle genes (CDK1/CCNB1/CCNE2/AURKA/KIF11/KIF14). Suppression was lowest for BPDF and BLPDF, they upregulated CCNE2 and SMC4. All PDF upregulated 3 out of 4 assessed cell cycle repressors (p53/BAX/p21). Cell viability scores confirmed gene expression results, being 79% of medium for LPDF, 101% for BLPDF, 51% for CPDF and 23% for IPDF. Amino acid-containing PDF (84%) incubated cells were as viable as BPDF (86%). CONCLUSION: In conclusion, PD solutions substantially differ with regard to their gene regulating profile and impact on vital functions of PMC, i.e. on cells known to be essential for peritoneal membrane homeostasis.

A magnesium based phosphate binder reduces vascular calcification without affecting bone in chronic renal failure rats.

The alternative phosphate binder calcium acetate/magnesium carbonate (CaMg) effectively reduces hyperphosphatemia, the most important inducer of vascular calcification, in chronic renal failure (CRF). In this study, the effect of low dose CaMg on vascular calcification and possible effects of CaMg on bone turnover, a persistent clinical controversy, were evaluated in chronic renal failure rats. Adenine-induced CRF rats were treated daily with 185 mg/kg CaMg or vehicle for 5 weeks. The aortic calcium content and area% calcification were measured to evaluate the effect of CaMg. To study the effect of CaMg on bone remodeling, rats underwent 5/6th nephrectomy combined with either a normal phosphorus diet or a high phosphorus diet to differentiate between possible bone effects resulting from either CaMg-induced phosphate deficiency or a direct effect of Mg. Vehicle or CaMg was administered at doses of 185 and 375 mg/kg/day for 8 weeks. Bone histomorphometry was performed. Aortic calcium content was significantly reduced by 185 mg/kg/day CaMg. CaMg ameliorated features of hyperparathyroid bone disease. In CRF rats on a normal phosphorus diet, the highest CaMg dose caused an increase in osteoid area due to phosphate depletion. The high phosphorus diet combined with the highest CaMg dose prevented the phosphate depletion and thus the rise in osteoid area. CaMg had no effect on osteoblast/osteoclast or dynamic bone parameters, and did not alter bone Mg levels. CaMg at doses that reduce vascular calcification did not show any harmful effect on bone turnover.

Mineral bone disorder in chronic kidney disease: head-to-head comparison of the 5/6 nephrectomy and adenine models.

BACKGROUND: Experimental models are important to the understanding of the pathophysiology of, as well as the effects of therapy on, certain diseases. In the case of chronic kidney disease-mineral bone disorder, there are currently two models that are used in evaluating the disease: 5/6 nephrectomy (Nx) and adenine-induced renal failure (AIRF). However, the two models have never been compared in studies using animals maintained under similar conditions. Therefore, we compared these two models, focusing on the biochemical, bone histomorphometry, and vascular calcification aspects. METHODS: Wistar rats, initially fed identical diets, were divided into two groups: those undergoing 5/6 Nx (5/6Nx group) and those that were switched to an adenine-enriched diet (AIRF group). After 9 weeks, animals were sacrificed, and we conducted biochemical and bone histomorphometry analyses, as well as assessing vascular calcification. RESULTS: At sacrifice, the mean body weight was higher in the 5/6Nx group than in the AIRF group, as was the mean blood pressure. No differences were seen regarding serum phosphate, ionized calcium, intact parathyroid hormone (PTH), or fibroblast growth factor 23 (FGF23). However, creatinine clearance was lower and fractional excretion of phosphate (FeP) was higher in the AIRF group rats, which also had a more severe form of high-turnover bone disease. Vascular calcification, as evaluated through von Kossa staining, was not observed in any of the animals. CONCLUSIONS: Overt vascular calcification was not seen in either model as applied in this study. Under similar conditions of diet and housing, the AIRF model produces a more severe form of bone disease than does 5/6 Nx. This should be taken into account when the choice is made between these models for use in preclinical studies.

Magnesium inhibits Wnt/ß-catenin activity and reverses the osteogenic transformation of vascular smooth muscle cells.

Magnesium reduces vascular smooth muscle cell (VSMC) calcification in vitro but the mechanism has not been revealed so far. This work used only slightly increased magnesium levels and aimed at determining: a) whether inhibition of magnesium transport into the cell influences VSMC calcification, b) whether Wnt/ß-catenin signaling, a key mediator of osteogenic differentiation, is modified by magnesium and c) whether magnesium can influence already established vascular calcification. Human VSMC incubated with high phosphate (3.3 mM) and moderately elevated magnesium (1.4 mM) significantly reduced VSMC calcification and expression of the osteogenic transcription factors Cbfa-1 and osterix, and up-regulated expression of the natural calcification inhibitors matrix Gla protein (MGP) and osteoprotegerin (OPG). The protective effects of magnesium on calcification and expression of osteogenic markers were no longer observed in VSMC cultured with an inhibitor of cellular magnesium transport (2-aminoethoxy-diphenylborate [2-APB]). High phosphate induced activation of Wnt/ß-catenin pathway as demonstrated by the translocation of ß-catenin into the nucleus, increased expression of the frizzled-3 gene, and downregulation of Dkk-1 gene, a specific antagonist of the Wnt/ß-catenin signaling pathway. The addition of magnesium however inhibited phosphate-induced activation of Wnt/ß-catenin signaling pathway. Furthermore, TRPM7 silencing using siRNA resulted in activation of Wnt/ß-catenin signaling pathway. Additional experiments were performed to test the ability of magnesium to halt the progression of already established VSMC calcification in vitro. The delayed addition of magnesium decreased calcium content, down-regulated Cbfa-1 and osterix and up-regulated MGP and OPG, when compared with a control group. This effect was not observed when 2-APB was added. In conclusion, magnesium transport through the cell membrane is important to inhibit VSMC calcification in vitro. Inhibition of Wnt/ß-catenin by magnesium is one potential intracellular mechanism by which this anti-calcifying effect is achieved.

Magnesium modulates parathyroid hormone secretion and upregulates parathyroid receptor expression at moderately low calcium concentration.

BACKGROUND: The interest on magnesium (Mg) has grown since clinical studies have shown the efficacy of Mg-containing phosphate binders. However, some concern has arisen for the potential effect of increased serum Mg on parathyroid hormone (PTH) secretion. Our objective was to evaluate the direct effect of Mg in the regulation of the parathyroid function; specifically, PTH secretion and the expression of parathyroid cell receptors: CaR, the vitamin D receptor (VDR) and FGFR1/Klotho. METHODS: The work was performed in vitro by incubating intact rat parathyroid glands in different calcium (Ca) and Mg concentrations. RESULTS: Increasing Mg concentrations from 0.5 to 2 mM produced a left shift of PTH-Ca curves. With Mg 5 mM, the secretory response was practically abolished. Mg was able to reduce PTH only if parathyroid glands were exposed to moderately low Ca concentrations; with normal-high Ca concentrations, the effect of Mg on PTH inhibition was minor or absent. After 6-h incubation at a Ca concentration of 1.0 mM, the expression of parathyroid CaR, VDR, FGFR1 and Klotho (at mRNA and protein levels) was increased with a Mg concentration of 2.0 when compared with 0.5 mM. CONCLUSIONS: Mg reduces PTH secretion mainly when a moderate low calcium concentration is present; Mg also modulates parathyroid glands function through upregulation of the key cellular receptors CaR, VDR and FGF23/Klotho system.

A comparison of calcium acetate/magnesium carbonate and sevelamer-hydrochloride effects on fibroblast growth factor-23 and bone markers: post hoc evaluation from a controlled, randomized study.

BACKGROUND: Different phosphate binders exert differing effects on bone mineral metabolism and levels of regulating hormones. The objective of this post hoc evaluation of the CALcium acetate MAGnesium carbonate (CALMAG) study was to compare the effects of calcium acetate/magnesium carbonate (CaMg) and a calcium-free phosphate binder, sevelamer-hydrochloride (HCl), on serum levels of fibroblast growth factor-23 (FGF-23) and markers of bone turnover. METHODS: This secondary analysis of the controlled, randomized CALMAG study, comparing the effect of CaMg and sevelamer-HCl on serum phosphorus (P), aimed to investigate the parameters described above. The analysis included 204 patients who completed the initial study per protocol (CaMg, n = 105; sevelamer-HCl, n = 99). RESULTS: The study showed that serum levels of FGF-23 were significantly reduced with CaMg and sevelamer-HCl, with no difference between groups at Week 25 [analysis of covariance (ANCOVA); log-intact FGF-23 (iFGF-23), P = 0.1573]. FGF-23 levels strongly correlated with serum P levels at all time points in both groups. The bone turnover parameters alkaline phosphatase (AP), bone AP (BAP), procollagen type 1 amino-terminal propeptide 1 (P1NP), osteoprotegerin (OPG), beta-crosslaps (ß-CTX) and tartrate-resistant acid phosphatase 5b (TRAP 5b) increased significantly in the sevelamer-HCl group; they remained almost unchanged in the CaMg group, after the initial phase of P lowering (ANCOVA, P < 0.0001 for all except OPG, P = 0.1718). CONCLUSIONS: CaMg and sevelamer-HCl comparably lower serum levels of iFGF-23. Changes in bone parameters were dependent on characteristics of the phosphate binder; in contrast with sevelamer-HCl, CaMg had no influence on bone turnover markers.

Effect of a magnesium-based phosphate binder on medial calcification in a rat model of uremia.

Calcium-based phosphate binders are used to control hyperphosphatemia; however, they promote hypercalcemia and may accelerate aortic calcification. Here we compared the effect of a phosphate binder containing calcium acetate and magnesium carbonate (CaMg) to that of sevelamer carbonate on the development of medial calcification in rats with chronic renal failure induced by an adenine diet for 4 weeks. After 1 week, rats with chronic renal failure were treated with vehicle, 375 or 750 mg/kg CaMg, or 750 mg/kg sevelamer by daily gavage for 5 weeks. Renal function was significantly impaired in all groups. Vehicle-treated rats with chronic renal failure developed severe hyperphosphatemia, but this was controlled in treated groups, particularly by CaMg. Neither CaMg nor sevelamer increased serum calcium ion levels. Induction of chronic renal failure significantly increased serum PTH, dose-dependently prevented by CaMg but not sevelamer. The aortic calcium content was significantly reduced by CaMg but not by sevelamer. The percent calcified area of the aorta was significantly lower than vehicle-treated animals for all three groups. The presence of aortic calcification was associated with increased sox9, bmp-2, and matrix gla protein expression, but this did not differ in the treatment groups. Calcium content in the carotid artery was lower with sevelamer than with CaMg but that in the femoral artery did not differ between groups. Thus, treatment with either CaMg or sevelamer effectively controlled serum phosphate levels in CRF rats and reduced aortic calcification.

Removal of protein-bound, hydrophobic uremic toxins by a combined fractionated plasma separation and adsorption technique.

Protein-bound uremic toxins, such as phenylacetic acid, indoxyl sulfate, and p-cresyl sulfate, contribute substantially to the progression of chronic kidney disease (CKD) and cardiovascular disease (CVD). However, based on their protein binding, these hydrophobic uremic toxins are poorly cleared during conventional dialysis and thus accumulate in CKD-5D patients. Therefore, we investigated whether hydrophobic and cationic adsorbers are more effective for removal of protein-bound, hydrophobic uremic toxins than conventional high-flux hemodialyzer. Five CKD-5D patients were treated using the fractionated plasma separation, adsorption, and dialysis (FPAD) system for 5 h. A control group of five CKD patients was treated with conventional high-flux hemodialysis. Plasma concentrations of phenylacetic acid, indoxyl sulfate, and p-cresyl sulfate were measured. Removal rates of FPAD treatment in comparison to conventional high-flux hemodialysis were increased by 130% for phenylacetic acid, 187% for indoxyl sulfate, and 127% for p-cresol. FPAD treatment was tolerated well in terms of clinically relevant biochemical parameters. However, patients suffered from mild nausea 2 h after the start of the treatment, which persisted until the end of treatment. Due to the high impact of protein-bound, hydrophobic uremic toxins on progression of CKD and CVD in CKD-5D patients, the use of an adsorber in combination with dialysis membranes may be a new therapeutic option to increase the removal rate of these uremic toxins. However, larger, long-term prospective clinical trials are needed to demonstrate the impact on clinical outcome.

Magnesium prevents phosphate-induced calcification in human aortic vascular smooth muscle cells.

BACKGROUND: Vascular calcification (VC) is prevalent in patients suffering from chronic kidney disease. Factors promoting calcification include abnormalities in mineral metabolism, particularly high phosphate levels. Inorganic phosphate (Pi) is a classical inducer of in vitro VC. Recently, an inverse relationship between serum magnesium concentrations and VC has been reported. The present study aimed to investigate the effects of magnesium on Pi-induced VC at the cellular level using primary HAVSMC. METHODS: Alive and fixed HAVSMC were assessed during 14 days in the presence of Pi with increasing concentrations of magnesium (Mg(2+)) chloride. Mineralization was measured using quantification of calcium, von Kossa and alizarin red stainings. Cell viability and secretion of classical VC markers were also assessed using adequate tests. Involvement of transient receptor potential melastatin (TRPM) 7 was assessed using 2-aminoethoxy-diphenylborate (2-APB) inhibitor. RESULTS: Co-incubation with Mg(2+) significantly decreased Pi-induced VC in live HAVSMC, no effect was found in fixed cells. At potent concentrations in Pi-induced HAVSMC, Mg(2+) significantly improved cell viability and restored to basal level increased secretions of osteocalcin and matrix gla protein, whereas a decrease in osteopontin secretion was partially restored. The block of TRPM7 with 2-APB at 10(-4) M led to the inefficiency of Mg(2+) to prevent VC. CONCLUSIONS: Increasing Mg(2+) concentrations significantly reduced VC, improved cell viability and modulated secretion of VC markers during cell-mediated matrix mineralization clearly pointing to a cellular role for Mg(2+) and 2-APB further involved TRPM7 and a potential Mg(2+) entry to exert its effects. Further investigations are needed to shed light on additional cellular mechanism(s) by which Mg(2+) is able to prevent VC.

Influence of bicarbonate/low-GDP peritoneal dialysis fluid (BicaVera) on in vitro and ex vivo epithelial-to-mesenchymal transition of mesothelial cells.

BACKGROUND: Peritoneal membrane damage induced by peritoneal dialysis (PD) is largely associated with epithelial-to-mesenchymal transition (EMT) of mesothelial cells (MCs), which is believed to be a result mainly of the glucose degradation products (GDPs) present in PD solutions. OBJECTIVES: This study investigated the impact of bicarbonate-buffered, low-GDP PD solution (BicaVera: Fresenius Medical Care, Bad Homburg, Germany) on EMT of MCs in vitro and ex vivo. IN VITRO STUDIES: Omentum-derived MCs were incubated with lactate-buffered standard PD fluid or BicaVera fluid diluted 1:1 with culture medium. Ex vivo studies: From 31 patients randomly distributed to either standard or BicaVera solution and followed for 24 months, effluents were collected every 6 months for determination of EMT markers in effluent MCs. RESULTS: Culturing of MCs with standard fluid in vitro resulted in morphology change to a non-epithelioid shape, with downregulation of E-cadherin (indicative of EMT) and strong induction of vascular endothelial growth factor (VEGF) expression. By contrast, in vitro exposure of MCs to bicarbonate/low-GDP solution had less impact on both EMT parameters. Ex vivo studies partially confirmed the foregoing results. The BicaVera group, with a higher prevalence of the non-epithelioid MC phenotype at baseline (for unknown reasons), showed a clear and significant trend to gain and maintain an epithelioid phenotype at medium- and longer-term and to show fewer fibrogenic characteristics. By contrast, the standard solution group demonstrated a progressive and significantly higher presence of the non-epithelioid phenotype. Compared with effluent MCs having an epithelioid phenotype, MCs with non-epithelioid morphology showed significantly lower levels of E-cadherin and greater levels of fibronectin and VEGF. In comparing the BicaVera and standard solution groups, MCs from the standard solution group showed significantly higher secretion of interleukin 8 and lower secretion of collagen I, but no differences in the levels of other EMT-associated molecules, including fibronectin, VEGF, E-cadherin, and transforming growth factor ß1. Peritonitis incidence was similar in both groups. Functionally, the use of BicaVera fluid was associated with higher transport of small molecules and lower ultrafiltration capacity. CONCLUSIONS: Effluent MCs grown ex vivo from patients treated with bicarbonate/low-GDP BicaVera fluid showed a trend to acquire an epithelial phenotype, with lower production of proinflammatory cytokines and chemokines (such as interleukin 8) than was seen with MCs from patients treated with a lactate-buffered standard PD solution.

Relationship between magnesium and clinical biomarkers on inhibition of vascular calcification.

BACKGROUND: Arteriosclerosis and cardiovascular disease are strongly associated with vascular calcification. Hyperphosphatemia is an essential risk factor for increased vascular calcification. End-stage renal disease (ESRD) patients could serve as an in vivo model for accelerated calcification. This study focuses on the most likely protective effects of magnesium ion (Mg(2+)) on phosphate-induced vascular calcification ex vivo/in vitro. Furthermore, plasma Mg(2+) concentrations of ESRD and healthy controls were investigated for association with surrogate parameters of vascular calcification in vivo. METHODS: Aortic segments of male Wistar-Kyoto rats were incubated and the phosphate concentration of the medium was elevated. The aortic segments were incubated in the absence and presence of MgCl(2); tissue calcification was quantified by different methods. Serum Mg(2+) concentrations of patients with chronic kidney disease (CKD stage 5; ESRD) and patients without CKD (controls) were associated with carotid intima media thickness (IMT) and aortic pulse wave velocity (PWV) as surrogate parameter for arteriosclerosis and arterial stiffening. RESULTS: Incubation of aortic segments in the presence of ß-glycerophosphate and NaH(2)PO(4) caused an increased tissue Ca(2+) deposition compared to control conditions. This increased amount of Ca(2+) in the aortic rings was significantly decreased in the presence of Mg(2+). In CKD patients, but not in controls, magnesium serum concentration was associated with the IMT of the carotid arteries. In addition, CKD patients with higher magnesium serum concentration had a significantly lower PWV. DISCUSSION AND CONCLUSION: Elevated phosphate concentrations in the culture media induce ex vivo/in vitro medial calcification in intact rat aortic rings in the presence of alkaline phosphatase. Mg(2+) ions reduced ex vivo/in vitro vascular calcification despite increased phosphate concentration. This hypothesis is additionally based on the fact that CKD patients with high Mg(2) serum levels had significantly lower IMT and PWV values, which may result in a lower risk for cardiovascular events and mortality in these patients. Therefore, Mg(2+) supplementation may be an option for treatment and prevention of vascular calcification resulting in a reduction of cardiovascular events in CKD patients.

The impact of dialysis solution biocompatibility on ultrafiltration and on free water transport in rats.

This study compares different peritoneal dialysis fluids (PDF) in rats over a short contact time. For greater accuracy, net ultrafiltration (UF) and peritoneal transport indices, mass transfer area coefficient (MTAC) were scaled for the in vivo peritoneal surface area recruited (ivPSA) measured by microcomputerized tomography. Wistar rats underwent nephrectomy (5/6ths), were randomized into two groups and given 1.5% glucose PDF, either conventional acidic lactate (n = 14) or pH neutral bicarbonate (BicaVera) (n = 13); MTAC and UF were measured using a 90-min peritoneal equilibrium test (PET), fill volume (IPV) of 10 ml/100 g; small pore fluid transport was determined from sodium balance and used to calculate free water transport (FWT). Each ivPSA value was significantly correlated with the actual IPV, which varied from one rat to another. At 90 min of contact, there was no difference in recruited ivPSA in relation to PDFs. There was a difference (p < 0.01) in net UF/ivPSA 0.45 vs. 1.41 cm(2)/ml for bicarbonate versus lactate, as there was in the proportion of FWT with bicarbonate (42 ± 5% of net UF) compared to lactate (29 ± 4% of net UF). Net UF for individual values of ivPSA differs between conventional PDF and more biocompatible solutions, such as bicarbonate PDF. This observed change in UF cannot be fully explained by differences in glucose transport. The changes in FWT may be explained by the impact of the PDF biocompatibility on aquaporin function.

Oral supplementation with sulodexide inhibits neo-angiogenesis in a rat model of peritoneal perfusion.

BACKGROUND: Peritoneal dialysis (PD) is associated with functional and morphological alterations of the peritoneal membrane (PM). It is hypothesized that vascular endothelial growth factor (VEGF) plays a role in this process. Sulodexide is a glycosaminoglycan with effects on vascular biology. Therefore, the impact of oral sulodexide on PM function and morphology in a rat model of peritoneal perfusion was evaluated. METHODS: Rats received 10 mL peritoneal dialysate fluid (PDF) twice daily via a tunnelled PD catheter. The test-PD group (Sul) received 15 mg/kg/day oral sulodexide versus none in the control-PD group (Con). A third group received no PDF (Sham). After 12 weeks, a peritoneal equilibration test was performed and the PM was sampled. Neo-angiogenesis was evaluated using immunostaining with von Willebrand, and epithelial-to-mesenchymal transition (EMT) using co-localization of cytokeratin and α-smooth muscle actin. VEGF was determined in the dialysate by enzyme-linked immunosorbent assay. RESULTS: PD induced loss of ultrafiltration, also in the sulodexide group. Creatinine and glucose transport were better preserved, and sodium dip was more pronounced in the sulodexide group versus control. Submesothelial thickness, neo-angiogenesis and EMT were more pronounced in the Con versus Sul versus Sham group. VEGF in the dialysate, corrected for diffusion was higher in Con and Sul versus Sham. CONCLUSION: Oral sulodexide administration diminishes neo-vascularization, submesothelial thickening and EMT induced by exposure to PDF in a rat model. As there was no difference in VEGF at the protein level in the dialysate, we hypothesize that oral sulodexide inhibits VEGF locally by binding.

Magnesium reduces calcification in bovine vascular smooth muscle cells in a dose-dependent manner.

BACKGROUND: Vascular calcification (VC), mainly due to elevated phosphate levels, is one major problem in patients suffering from chronic kidney disease. In clinical studies, an inverse relationship between serum magnesium and VC has been reported. However, there is only few information about the influence of magnesium on calcification on a cellular level available. Therefore, we investigated the effect of magnesium on calcification induced by ß-glycerophosphate (BGP) in bovine vascular smooth muscle cells (BVSMCs). METHODS: BVSMCs were incubated with calcification media for 14 days while simultaneously increasing the magnesium concentration. Calcium deposition, transdifferentiation of cells and apoptosis were measured applying quantification of calcium, von Kossa and Alizarin red staining, real-time reverse transcription-polymerase chain reaction and annexin V staining, respectively. RESULTS: Calcium deposition in the cells dramatically increased with addition of BGP and could be mostly prevented by co-incubation with magnesium. Higher magnesium levels led to inhibition of BGP-induced alkaline phosphatase activity as well as to a decreased expression of genes associated with the process of transdifferentiation of BVSMCs into osteoblast-like cells. Furthermore, estimated calcium entry into the cells decreased with increasing magnesium concentrations in the media. In addition, higher magnesium concentrations prevented cell damage (apoptosis) induced by BGP as well as progression of already established calcification. CONCLUSIONS: Higher magnesium levels prevented BVSMC calcification, inhibited expression of osteogenic proteins, apoptosis and further progression of already established calcification. Thus, magnesium is influencing molecular processes associated with VC and may have the potential to play a role for VC also in clinical situations.