A porcine model of hemodialyzer reactions: roles of complement activation and rinsing back of extracorporeal blood.

Hemodialysis reactions (HDRs) resemble complement-activation-related pseudoallergy (CARPA) to certain i.v. drugs, for which pigs provide a sensitive model. On this basis, to better understand the mechanism of human HDRs, we subjected pigs to hemodialysis using polysulfone (FX CorDiax 40, Fresenius) or cellulose triacetate (SureFlux-15UX, Nipro) dialyzers, or Dialysis exchange-set without membranes, as control. Experimental endpoints included typical biomarkers of porcine CARPA; pulmonary arterial pressure (PAP), blood cell counts, plasma sC5b-9 and thromboxane-B2 levels. Hemodialysis (60 min) was followed by reinfusion of extracorporeal blood into the circulation, and finally, an intravenous bolus injection of the complement activator zymosan. The data indicated low-extent steady rise of sC5b-9 along with transient leukopenia, secondary leukocytosis and thrombocytopenia in the two dialyzer groups, consistent with moderate complement activation. Surprisingly, small changes in baseline PAP and plasma thromboxane-B2 levels during hemodialysis switched into 30%-70% sharp rises in all three groups resulting in synchronous spikes within minutes after blood reinfusion. These observations suggest limited complement activation by dialyzer membranes, on which a membrane-independent second immune stimulus was superimposed, and caused pathophysiological changes also characteristic of HDRs. Thus, the porcine CARPA model raises the hypothesis that a second "hit" on anaphylatoxin-sensitized immune cells may be a key contributor to HDRs.

Dietary magnesium supplementation prevents and reverses vascular and soft tissue calcifications in uremic rats.

Although magnesium has been shown to prevent vascular calcification in vitro, controlled in vivo studies in uremic animal models are limited. To determine whether dietary magnesium supplementation protects against the development of vascular calcification, 5/6 nephrectomized Wistar rats were fed diets with different magnesium content increasing from 0.1 to 1.1%. In one study we analyzed bone specimens from rats fed 0.1%, 0.3%, and 0.6% magnesium diets, and in another study we evaluated the effect of intraperitoneal magnesium on vascular calcification in 5/6 nephrectomized rats. The effects of magnesium on established vascular calcification were also evaluated in uremic rats fed on diets with either normal (0.1%) or moderately increased magnesium (0.6%) content. The increase in dietary magnesium resulted in a marked reduction in vascular calcification, together with improved mineral metabolism and renal function. Moderately elevated dietary magnesium (0.3%), but not high dietary magnesium (0.6%), improved bone homeostasis as compared to basal dietary magnesium (0.1%). Results of our study also suggested that the protective effect of magnesium on vascular calcification was not limited to its action as an intestinal phosphate binder since magnesium administered intraperitoneally also decreased vascular calcification. Oral magnesium supplementation also reduced blood pressure in uremic rats, and in vitro medium magnesium decreased BMP-2 and p65-NF-κB in TNF-α-treated human umbilical vein endothelial cells. Finally, in uremic rats with established vascular calcification, increasing dietary magnesium from 0.1% magnesium to 0.6% reduced the mortality rate from 52% to 28%, which was associated with reduced vascular calcification. Thus, increasing dietary magnesium reduced both vascular calcification and mortality in uremic rats.

Metabolism Regulates Cellular Functions of Bone Marrow-Derived Cells used for Cardiac Therapy.

Administration of bone marrow-derived mononuclear cells (BMC) may increase cardiac function after myocardial ischemia. However, the functional capacity of BMC derived from chronic heart failure (CHF) patients is significantly impaired. As modulation of the energy metabolism allows cells to match the divergent demands of the environment, we examined the regulation of energy metabolism in BMC from patients and healthy controls (HC). The glycolytic capacity of CHF-derived BMC is reduced compared to HC, whereas BMC of metabolically activated bone marrow after acute myocardial infarction reveal increased metabolism. The correlation of metabolic pathways with the functional activity of cells indicates an influence of metabolism on cell function. Reducing glycolysis without profoundly affecting ATP-production reversibly reduces invasion as well as colony forming capacity and abolishes proliferation of CD34(+) CD38(-) lin(-) hematopoietic stem and progenitor cells (HSPC). Ex vivo inhibition of glycolysis further reduced the pro-angiogenic activity of transplanted cells in a hind limb ischemia model in vivo. In contrast, inhibition of respiration, without affecting total ATP production, leads to a compensatory increase in glycolytic capacity correlating with increased colony forming capacity. Isolated CD34(+) , CXCR4(+) , and CD14(+) cells showed higher glycolytic activity compared to their negative counterparts. Metabolic activity was profoundly modulated by the composition of media used to store or culture BMC. This study provides first evidence that metabolic alterations influence the functional activity of human HSPC and BMC independent of ATP production. Changing the balance between respiration and glycolysis might be useful to improve patient-derived cells for clinical cardiac cell therapy. Stem Cells 2016;34:2236-2248.

Nalfurafine suppresses pruritogen- and touch-evoked scratching behavior in models of acute and chronic itch in mice.

The kappa-opioid agonist, nalfurafine, has been approved in Japan for treatment of itch in patients with chronic kidney disease. We presently investigated if systemic administration of nalfurafine inhibited ongoing or touch-evoked scratching behavior (alloknesis) following acute intradermal injection of histamine or the non-histaminergic itch mediator, chloroquine, in mice. We also investigated if nalfurafine suppressed spontaneous or touch-evoked scratching in an experimental model of chronic dry skin itch. Nalfurafine reduced scratching evoked by histamine and chloroquine. Following acute histamine, but not chloroquine, low-threshold mechanical stimuli reliably elicited directed hindlimb scratching behavior, which was significantly attenuated by nalfurafine. In mice with experimental dry skin, nalfurafine abolished spontaneous scratching but had no effect on alloknesis. Nalfurafine thus appears to be a promising treatment for acute itch as well as ongoing itch of dry skin.

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.

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.

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.

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 Improves Cardiac Function in Experimental Uremia by Altering Cardiac Elastin Protein Content.

Cardiovascular complications are accompanied by life-threatening complications and represent the major cause of death in patients with chronic kidney disease (CKD). Magnesium is important for the physiology of cardiac function, and its deficiency is common in CKD. In the present study, we investigated the impact of oral magnesium carbonate supplementation on cardiac function in an experimental model of CKD induced in Wistar rats by an adenine diet. Echocardiographic analyses revealed restoration of impaired left ventricular cardiac function in animals with CKD. Cardiac histology and real-time PCR confirmed a high amount of elastin protein and increased collagen III expression in CKD rats supplemented with dietary magnesium as compared with CKD controls. Both structural proteins are crucial in maintaining cardiac health and physiology. Aortic calcium content increased in CKD as compared with tissue from control animals. Magnesium supplementation numerically lowered the increases in aortic calcium content as it remained statistically unchanged, compared with controls. In summary, the present study provides evidence for an improvement in cardiovascular function and aortic wall integrity in a rat model of CKD by magnesium, as evidenced by echocardiography and histology.

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.

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.

Steviol glycosides as an alternative osmotic agent for peritoneal dialysis fluid.

Background: Peritoneal dialysis (PD) is a renal replacement technique that requires repeated exposure of the peritoneum to hyperosmolar PD fluids (PDFs). Unfortunately, it promotes alterations of the peritoneal membrane (PM) that affects its functionality, including mesothelial-mesenchymal transition (MMT) of mesothelial cells (MCs), inflammation, angiogenesis, and fibrosis. Glucose is the most used osmotic agent, but it is known to be at least partially responsible, together with its degradation products (GDP), for those changes. Therefore, there is a need for more biocompatible osmotic agents to better maintain the PM. Herein we evaluated the biocompatibility of Steviol glycosides (SG)-based fluids. Methods: The ultrafiltration and transport capacities of SG-containing and glucose-based fluids were analyzed using artificial membranes and an in vivo mouse model, respectively. To investigate the biocompatibility of the fluids, Met-5A and human omental peritoneal MCs (HOMCs) were exposed in vitro to different types of glucose-based PDFs (conventional 4.25% glucose solution with high-GDP level and biocompatible 2.3% glucose solution with low-GDP level), SG-based fluids or treated with TGF-ß1. Mice submitted to surgery of intraperitoneal catheter insertion were treated for 40 days with SG- or glucose-based fluids. Peritoneal tissues were collected to determine thickness, MMT, angiogenesis, as well as peritoneal washings to analyze inflammation. Results: Dialysis membrane experiments demonstrated that SG-based fluids at 1.5%, 1%, and 0.75% had a similar trend in weight gain, based on curve slope, as glucose-based fluids. Analyzing transport capacity in vivo, 1% and 0.75% SG-based fluid-exposed nephrectomized mice extracted a similar amount of urea as the glucose 2.3% group. In vitro, PDF with high-glucose (4.25%) and high-GDP content induced mesenchymal markers and angiogenic factors (Snail1, Fibronectin, VEGF-A, FGF-2) and downregulates the epithelial marker E-Cadherin. In contrast, exposition to low-glucose-based fluids with low-GDP content or SG-based fluids showed higher viability and had less MMT. In vivo, SG-based fluids preserved MC monolayer, induced less PM thickness, angiogenesis, leukocyte infiltration, inflammatory cytokines release, and MMT compared with glucose-based fluids. Conclusion: SG showed better biocompatibility as an osmotic agent than glucose in vitro and in vivo, therefore, it could alternatively substitute glucose in PDF.

Validation of a Novel Predictive Algorithm for Kidney Failure in Patients Suffering from Chronic Kidney Disease: The Prognostic Reasoning System for Chronic Kidney Disease (PROGRES-CKD).

Current equation-based risk stratification algorithms for kidney failure (KF) may have limited applicability in real world settings, where missing information may impede their computation for a large share of patients, hampering one from taking full advantage of the wealth of information collected in electronic health records. To overcome such limitations, we trained and validated the Prognostic Reasoning System for Chronic Kidney Disease (PROGRES-CKD), a novel algorithm predicting end-stage kidney disease (ESKD). PROGRES-CKD is a naïve Bayes classifier predicting ESKD onset within 6 and 24 months in adult, stage 3-to-5 CKD patients. PROGRES-CKD trained on 17,775 CKD patients treated in the Fresenius Medical Care (FMC) NephroCare network. The algorithm was validated in a second independent FMC cohort (n = 6760) and in the German Chronic Kidney Disease (GCKD) study cohort (n = 4058). We contrasted PROGRES-CKD accuracy against the performance of the Kidney Failure Risk Equation (KFRE). Discrimination accuracy in the validation cohorts was excellent for both short-term (stage 4-5 CKD, FMC: AUC = 0.90, 95%CI 0.88-0.91; GCKD: AUC = 0.91, 95% CI 0.86-0.97) and long-term (stage 3-5 CKD, FMC: AUC = 0.85, 95%CI 0.83-0.88; GCKD: AUC = 0.85, 95%CI 0.83-0.88) forecasting horizons. The performance of PROGRES-CKD was non-inferior to KFRE for the 24-month horizon and proved more accurate for the 6-month horizon forecast in both validation cohorts. In the real world setting captured in the FMC validation cohort, PROGRES-CKD was computable for all patients, whereas KFRE could be computed for complete cases only (i.e., 30% and 16% of the cohort in 6- and 24-month horizons). PROGRES-CKD accurately predicts KF onset among CKD patients. Contrary to equation-based scores, PROGRES-CKD extends to patients with incomplete data and allows explicit assessment of prediction robustness in case of missing values. PROGRES-CKD may efficiently assist physicians' prognostic reasoning in real-life applications.

Global gene expression analysis in nonfailing and failing myocardium pre- and postpulsatile and nonpulsatile ventricular assist device support.

Mechanical unloading by ventricular assist devices (VAD) leads to significant gene expression changes often summarized as reverse remodeling. However, little is known on individual transcriptome changes during VAD support and its relationship to nonfailing hearts (NF). In addition no data are available for the transcriptome regulation during nonpulsatile VAD support. Therefore we analyzed the gene expression patterns of 30 paired samples from VAD-supported (including 8 nonpulsatile VADs) and 8 nonfailing control hearts (NF) using the first total human genome array available. Transmural myocardial samples were collected for RNA isolation. RNA was isolated by commercial methods and processed according to chip-manufacturer recommendations. cRNA were hybridized on Affymetrix HG-U133 Plus 2.0 arrays, providing coverage of the whole human genome Array. Data were analyzed using Microarray Analysis Suite 5.0 (Affymetrix) and clustered by Expressionist software (Genedata). We found 352 transcripts were differentially regulated between samples from VAD implantation and NF, whereas 510 were significantly regulated between VAD transplantation and NF (paired t-test P < 0.001, fold change >or=1.6). Remarkably, only a minor fraction of 111 transcripts was regulated in heart failure (HF) and during VAD support. Unsupervised hierarchical clustering of paired VAD and NF samples revealed separation of HF and NF samples; however, individual differentiation of VAD implantation and VAD transplantation was not accomplished. Clustering of pulsatile and nonpulsatile VAD did not lead to robust separation of gene expression patterns. During VAD support myocardial gene expression changes do not indicate reversal of the HF phenotype but reveal a distinct HF-related pattern. Transcriptome analysis of pulsatile and nonpulsatile VAD-supported hearts did not provide evidence for a pump mode-specific transcriptome pattern.

Salt intake induces epithelial-to-mesenchymal transition of the peritoneal membrane in rats.

BACKGROUND: Dietary salt intake has been linked to hypertension and cardiovascular disease through volume-mediated effects. Accumulating evidence points to direct negative influence of salt intake independent of volume overload, such as cardiac and renal fibrosis, mediated through transforming growth factor beta (TGF-beta). Epithelial-to-mesenchymal transition (EMT) has been implicated as a key process in chronic fibrotic diseases, such as chronic kidney disease or heart failure. The potential role of dietary salt intake on cell transdifferentiation has never been investigated. This study analysed the effect of dietary salt intake on EMT and fibrosis in the peritoneal membrane (PM) in a rat model. METHODS: Twenty-eight Wistar rats were randomized to a normal salt (NS) or a high salt (HS) intake. NS and HS rats had free access to tap water or NaCl 2% as drinking water, respectively. After 2 weeks, samples of peritoneum were taken, and TGF-beta(1), Interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) mRNA expression were quantified with qRT-PCR. Fibrosis and submesothelial PM thickness were scored. EMT was evaluated using fluorescence staining with cytokeratin and alpha smooth muscle actin (alpha-SMA). RESULTS: Dietary salt intake caused peritoneal fibrosis and thickening of the submesothelial layer and induced EMT as identified by colocalization of cytokeratin and alpha-SMA in cells present in the submesothelial layer. Peritoneal TGF-beta(1) and IL-6 mRNA expression were upregulated in the HS group. CONCLUSION: High dietary salt intake induces EMT and peritoneal fibrosis, a process coinciding with upregulation of TGF-beta1.

Elimination of glutamate using CRRT for 72 h in patients with post-cardiac arrest syndrome: A randomized clinical pilot trial.

AIM: Glutamine and glutamate are major mediators of secondary brain cell death during post-cardiac arrest syndrome. As there is an equilibrium between brain tissue and plasma concentrations of glutamine and glutamate, their elimination from systemic circulation by extracorporeal blood purification may ultimately lead to reduced secondary cell death in the brain. We hypothesized that systemic glutamine and glutamate can be significantly reduced by continuous venovenous hemodiafiltration (CVVHDF). METHODS: This was a prospective, randomized clinical trial in post cardiac-arrest survivors evaluating standard of care or additional CVVHDF over 72 h immediately after admission. Glutamine and glutamate plasma concentrations were analyzed at eight time points in both groups. Primary endpoint was reduction of glutamine and glutamate plasma concentrations. The trial has been registered at clinical trial.gov (NCT02963298). RESULTS: In total, 41 patients were randomized over a period of 12 months (control n = 21, CVVHDF n = 20). The primary aim reduction of glutamine and glutamate plasma concentrations by CVVHDF, was not achieved; both groups-maintained concentrations within a normal range over the study period (glutamate: 4.7-11.1 mg/dL; glutamine: 0.2-3.7 mg/dL). However, post-filter concentrations of glutamine and glutamate in CRRT patients were significantly decreased as compared to pre-filter concentrations (glutamate: pre-filter median 8.85 mg/dL IQR 7.1-9.6; post-filter 0.95 mg/dL IQR 0.5-2; p < 0.001; glutamine: pre-filter 0.7 mg/dL IQR 0.6-1; post-filter 0.2 mg/dL IQR 0-0.2; p < 0.001). CONCLUSION: In this trial, CVVHDF was not able to statistically significantly lower systemic plasma glutamine and glutamate levels. Post-cardiac arrest patients had plasma glutamine and glutamate levels within the normal range.

A Bifunctional Adsorber Particle for the Removal of Hydrophobic Uremic Toxins from Whole Blood of Renal Failure Patients.

Hydrophobic uremic toxins accumulate in patients with chronic kidney disease, contributing to a highly increased cardiovascular risk. The clearance of these uremic toxins using current hemodialysis techniques is limited due to their hydrophobicity and their high binding affinity to plasma proteins. Adsorber techniques may be an appropriate alternative to increase hydrophobic uremic toxin removal. We developed an extracorporeal, whole-blood bifunctional adsorber particle consisting of a porous, activated charcoal core with a hydrophilic polyvinylpyrrolidone surface coating. The adsorption capacity was quantified using analytical chromatography after perfusion of the particles with an albumin solution or blood, each containing mixtures of hydrophobic uremic toxins. A time-dependent increase in hydrophobic uremic toxin adsorption was depicted and all toxins showed a high binding affinity to the adsorber particles. Further, the particle showed a sufficient hemocompatibility without significant effects on complement component 5a, thrombin-antithrombin III complex, or thrombocyte concentration in blood in vitro, although leukocyte counts were slightly reduced. In conclusion, the bifunctional adsorber particle with cross-linked polyvinylpyrrolidone coating showed a high adsorption capacity without adverse effects on hemocompatibility in vitro. Thus, it may be an interesting candidate for further in vivo studies with the aim to increase the efficiency of conventional dialysis techniques.

Magnesium Chloride promotes Osteogenesis through Notch signaling activation and expansion of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSC) are osteoblasts progenitors and a variety of studies suggest that they may play an important role for the health in the field of bone regeneration. Magnesium supplementation is gaining importance as adjuvant treatment to improve osteogenesis, although the mechanisms involving this process are not well understood. The objective of this study was to investigate the effects of magnesium on MSC differentiation. Here we show that in rat bone marrow MSC, magnesium chloride increases MSC proliferation in a dose-dependent manner promoting osteogenic differentiation and mineralization. These effects are reduced by 2-APB administration, an inhibitor of magnesium channel TRPM7. Of note, magnesium supplementation did not increase the canonical Wnt/ß-catenin pathway, although it promoted the activation of Notch1 signaling, which was also decreased by addition of 2-APB. Electron microscopy showed higher proliferation, organization and maturation of osteoblasts in bone decellularized scaffolds after magnesium addition. In summary, our results demonstrate that magnesium chloride enhances MSC proliferation by Notch1 signaling activation and induces osteogenic differentiation, shedding light on the understanding of the role of magnesium during bone regeneration.

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.