Independent Prognostic and Predictive Role of Interstitial Macrophages in Kidney Biopsies of IgA Nephropathy Patients.

A relevant percentage of IgAN patients experience a progressive decline in kidney function. According to the KDIGO guidelines, proteinuria and eGFR are the only validated prognostic markers. The role of interstitial macrophages in kidney biopsies of IgAN patients and the outcome of patients treated with renin-angiotensin system inhibitors (RASBs) alone or combined with glucocorticoids were evaluated. Clinical and laboratory records (age, gender, hypertension, hematuria, proteinuria, eGFR, serum creatinine, and therapy), MEST-C parameters of the Oxford classification, C4d deposition, peritubular capillaries, and glomerular and interstitial macrophages in 47 IgAN patients undergoing kidney biopsy consecutively between 2003 and 2016 were examined. A high number of interstitial macrophages significantly correlated with peritubular capillary rarefaction and impairment of kidney function. Cox's multivariable regression analysis revealed that a value > 19.5 macrophages/HPF behaved as an independent marker of an unfavorable outcome. Patients exhibiting > 19.5 macrophages/HPF treated at the time of diagnosis with RASBs combined with methylprednisolone had an estimated probability of a favorable outcome higher than patients treated with RASBs alone. Thus, a value > 19.5 macrophages/HPF in IgAN biopsies can predict an unfavorable outcome and endorse a well-timed administration of glucocorticoids. Studies evaluating urine biomarkers associated with peritubular capillary rarefaction in patients with marked macrophage infiltration may help personalized treatment decisions.

Erythropoietin induces miRNA-210 by JAK2/STAT5 signaling in PBMCs of End-stage Renal Disease patients.

Anemia of chronic kidney disease is associated with blunted response/resistance to erythropoietin-stimulating agents (ESAs) in hemodialysis (HD) patients. Several molecules have been successfully associated with ESA responsiveness; however, none of them is now considered a valid therapeutic biomarker of erythropoietin resistance in these patients. We performed an evaluation of the level of specific plasma circulating miRNAs in blood samples of HD patients, in relation to ESA treatment, with a follow-up of 1 year (T0-T3). We found significantly lower circulating levels of all miRNAs analyzed at baseline (T0) in HD patients vs. healthy control (HC). The plasmatic levels of miRNA-210 resulted significantly and negatively associated with Erythropoietin Resistance Index (ERI), and the variance of ΔmiRNA-210 (miRNA-210T3 minus miRNA-210T0 ) explained significant percentage of ΔERI (ERIT3 minus ERIT0 ) variance. The receiver operating characteristic analysis at T0 showed that the plasmatic level of miRNA-210 could distinguish HD patients with positive or negative trend in ERI at T3. In vitro, recombinant human erythropoietin (EPO) induced significant release of miRNA-210 from cultured peripheral blood mononuclear cells, through the activation of Janus kinase 2 (JAK2)/ signal transducer and activator of transcription 5 (STAT5) signaling, but not by the activation of the MAPK protein 38α and extracellular signal-regulated kinase ½. Accordingly, HD patients with negative ΔERI showed higher level of phosphor-Janus kinase 2 and nuclear translocation of phosphor-signal transducer and activator of transcription 5. vs. patients with positive ΔERI or HC. Our data highlighted that chronic HD significantly reduces the circulating level of the miRNAs evaluated; within the targets analyzed, the miRNA-210 could be considered as a prognostic indicator of ESA responsiveness and index for anemia management.

Dietary Patterns and Mortality in a Multinational Cohort of Adults Receiving Hemodialysis.

RATIONALE & OBJECTIVE: Clinical practice guidelines for dietary intake in hemodialysis focus on individual nutrients. Little is known about associations of dietary patterns with survival. We evaluated the associations of dietary patterns with cardiovascular and all-cause mortality among adults treated by hemodialysis. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: 8,110 of 9,757 consecutive adults on hemodialysis (January 2014 to June 2017) treated in a multinational private dialysis network and with analyzable dietary data. EXPOSURES: Data-driven dietary patterns based on the GA2LEN food frequency questionnaire. Participants received a score for each identified pattern, with higher scores indicating closer resemblance of their diet to the identified pattern. Quartiles of standardized pattern scores were used as primary exposures. OUTCOMES: Cardiovascular and all-cause mortality. ANALYTICAL APPROACH: Principal components analysis with varimax rotation to identify common dietary patterns. Adjusted proportional hazards regression analyses with country as a random effect to estimate the associations between dietary pattern scores and mortality. Associations were expressed as adjusted HRs with 95% CIs, using the lowest quartile score as reference. RESULTS: During a median follow-up of 2.7 years (18,666 person-years), there were 2,087 deaths (958 cardiovascular). 2 dietary patterns, "fruit and vegetable" and "Western," were identified. For the fruit and vegetable dietary pattern score, adjusted HRs, in ascending quartiles, were 0.94 (95% CI, 0.76-1.15), 0.83 (95% CI, 0.66-1.06), and 0.91 (95% CI, 0.69-1.21) for cardiovascular mortality and 0.95 (95% CI, 0.83-1.09), 0.84 (95% CI, 0.71-0.99), and 0.87 (95% CI, 0.72-1.05) for all-cause mortality. For the Western dietary pattern score, the corresponding estimates were 1.10 (95% CI, 0.90-1.35), 1.11 (95% CI, 0.87-1.41), and 1.09 (95% CI, 0.80-1.49) for cardiovascular mortality and 1.01 (95% CI, 0.88-1.16), 1.00 (95% CI, 0.85-1.18), and 1.14 (95% CI, 0.93-1.41) for all-cause mortality. LIMITATIONS: Self-reported food frequency questionnaire, data-driven approach. CONCLUSIONS: These findings did not confirm an association between mortality among patients receiving long-term hemodialysis and the extent to which dietary patterns were either high in fruit and vegetables or consistent with a Western diet.

Fruit and Vegetable Intake and Mortality in Adults undergoing Maintenance Hemodialysis.

BACKGROUND AND OBJECTIVES: Higher fruit and vegetable intake is associated with lower cardiovascular and all-cause mortality in the general population. It is unclear whether this association occurs in patients on hemodialysis, in whom high fruit and vegetable intake is generally discouraged because of a potential risk of hyperkalemia. We aimed to evaluate the association between fruit and vegetable intake and mortality in hemodialysis. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Fruit and vegetable intake was ascertained by the Global Allergy and Asthma European Network food frequency questionnaire within the Dietary Intake, Death and Hospitalization in Adults with ESKD Treated with Hemodialysis study, a multinational cohort study of 9757 adults on hemodialysis, of whom 8078 (83%) had analyzable dietary data. Adjusted Cox regression analyses clustered by country were conducted to evaluate the association between tertiles of fruit and vegetable intake with all-cause, cardiovascular, and noncardiovascular mortality. Estimates were calculated as hazard ratios with 95% confidence intervals (95% CIs). RESULTS: During a median follow up of 2.7 years (18,586 person-years), there were 2082 deaths (954 cardiovascular). The median (interquartile range) number of servings of fruit and vegetables was 8 (4-14) per week; only 4% of the study population consumed at least four servings per day as recommended in the general population. Compared with the lowest tertile of servings per week (0-5.5, median 2), the adjusted hazard ratios for the middle (5.6-10, median 8) and highest (>10, median 17) tertiles were 0.90 (95% CI, 0.81 to 1.00) and 0.80 (95% CI, 0.71 to 0.91) for all-cause mortality, 0.88 (95% CI, 0.76 to 1.02) and 0.77 (95% CI, 0.66 to 0.91) for noncardiovascular mortality and 0.95 (95% CI, 0.81 to 1.11) and 0.84 (95% CI, 0.70 to 1.00) for cardiovascular mortality, respectively. CONCLUSIONS: Fruit and vegetable intake in the hemodialysis population is low and a higher consumption is associated with lower all-cause and noncardiovascular death.

Proteomic Characterization of a New asymmetric Cellulose Triacetate Membrane for Hemodialysis.

PURPOSE: The artificial membrane inside the haemodialyzer is the main determinant of the quality and success of haemodialysis therapy. The performances of haemodialysis membranes are highly influenced by the interactions with plasma proteins, which in turn are related to the physical and chemical characteristics of the membrane material. The present cross-over study is aimed to analyse the haemodialysis performance of a newly developed asymmetric cellulose triacetate membrane (ATA) in comparison to the conventional parent symmetric polymer (CTA). EXPERIMENTAL DESIGN: In four chronic non diabetic haemodialysis patients, the protein constituents of the adsorbed material from the filters after the haemodialysis session, and the proteins recovered in the ultrafiltrate during the session, are identified using a bottom-up shotgun proteomics approach. RESULTS: The ATA membrane shows a lower protein adsorption rate and a lower mass distribution pattern of the proteinaceous material. CONCLUSIONS AND CLINICAL RELEVANCE: By highlighting the differences between the two haemodialysis filters in terms of adsorbed proteins and flow through, it is demonstrated the higher biocompatibility of the novel ATA membrane, that fulfils the indications for the development of more performant membranes and may represent a step forward for the treatment of patients on chronic haemodialysis.

The Association of Mediterranean and DASH Diets with Mortality in Adults on Hemodialysis: The DIET-HD Multinational Cohort Study.

Background Mediterranean and Dietary Approaches to Stop Hypertension (DASH) diets associate with lower cardiovascular and all-cause mortality in the general population, but the benefits for patients on hemodialysis are uncertain.Methods Mediterranean and DASH diet scores were derived from the GA2LEN Food Frequency Questionnaire within the DIET-HD Study, a multinational cohort study of 9757 adults on hemodialysis. We conducted adjusted Cox regression analyses clustered by country to evaluate the association between diet score tertiles and all-cause and cardiovascular mortality (the lowest tertile was the reference category).Results During the median 2.7-year follow-up, 2087 deaths (829 cardiovascular deaths) occurred. The adjusted hazard ratios (95% confidence intervals) for the middle and highest Mediterranean diet score tertiles were 1.20 (1.01 to 1.41) and 1.14 (0.90 to 1.43), respectively, for cardiovascular mortality and 1.10 (0.99 to 1.22) and 1.01 (0.88 to 1.17), respectively, for all-cause mortality. Corresponding estimates for the same DASH diet score tertiles were 1.01 (0.85 to 1.21) and 1.19 (0.99 to 1.43), respectively, for cardiovascular mortality and 1.03 (0.92 to 1.15) and 1.00 (0.89 to 1.12), respectively, for all-cause mortality. The association between DASH diet score and all-cause death was modified by age (P=0.03); adjusted hazard ratios for the middle and highest DASH diet score tertiles were 1.02 (0.81 to 1.29) and 0.70 (0.53 to 0.94), respectively, for younger patients (≤60 years old) and 1.05 (0.93 to 1.19) and 1.08 (0.95 to 1.23), respectively, for older patients.Conclusions Mediterranean and DASH diets did not associate with cardiovascular or total mortality in hemodialysis.

Proteomic Investigations into Hemodialysis Therapy.

The retention of a number of solutes that may cause adverse biochemical/biological effects, called uremic toxins, characterizes uremic syndrome. Uremia therapy is based on renal replacement therapy, hemodialysis being the most commonly used modality. The membrane contained in the hemodialyzer represents the ultimate determinant of the success and quality of hemodialysis therapy. Membrane's performance can be evaluated in terms of removal efficiency for unwanted solutes and excess fluid, and minimization of negative interactions between the membrane material and blood components that define the membrane's bio(in)compatibility. Given the high concentration of plasma proteins and the complexity of structural functional relationships of this class of molecules, the performance of a membrane is highly influenced by its interaction with the plasma protein repertoire. Proteomic investigations have been increasingly applied to describe the protein uremic milieu, to compare the blood purification efficiency of different dialyzer membranes or different extracorporeal techniques, and to evaluate the adsorption of plasma proteins onto hemodialysis membranes. In this article, we aim to highlight investigations in the hemodialysis setting making use of recent developments in proteomic technologies. Examples are presented of why proteomics may be helpful to nephrology and may possibly affect future directions in renal research.

Biocompatibility assessment of haemodialysis membrane materials by proteomic investigations.

The exposure of blood to an artificial surface such as the haemodialysis membrane results in the nearly instantaneous deposition of a layer of plasma proteins. The composition of the protein layer profoundly influences all subsequent events, and to a large extent determines the biocompatibility of the biomaterial. In the present study, we examine the protein adsorption capacity and coagulation profiles of the polysulfone-based helixone material in comparison to cellulose triacetate. A differential profiling investigation using shotgun proteomics data-independent analysis was applied to eluates obtained with each membrane after a dialysis session, in order to assess the function of desorbed proteins. Functional classification and network analysis performed using bioinformatics tools shed light on the involvement of adsorbed proteins into important molecular processes, such as lipid transport and metabolism, cell growth differentiation and communication, and the coagulation cascade. The collected evidence was further validated by targeted mass spectrometry using selected reaction monitoring on proteotypic transitions of key protein effectors, confirming the different panels of adsorbed protein on each membrane. The coagulation profile during haemodialysis of patients under polysulfone-based helixone filter cartridges was also assessed showing a slightly higher platelet activation profile after the dialysis session. The overall collected evidence highlights a modulation of the coagulation biological pathway during haemodialysis, which is largely influenced by the biomaterial used.

Effect of erythropoietin on primed leucocyte expression profile.

Resistance to erythropoietin (EPO) affects a significant number of anaemic patients with end-stage renal disease. Previous reports suggest that inflammation is one of the major independent predictors of EPO resistance, and the effects of EPO treatment on inflammatory mediators are not well established. The aim of this study was to investigate EPO-induced modification to gene expression in primary cultured leucocytes. Microarray experiments were performed on primed ex vivo peripheral blood mononuclear cells (PBMCs) and treated with human EPO-α. Data suggested that EPO-α modulated genes involved in cell movement and interaction in primed PBMCs. Of note, EPO-α exerts anti-inflammatory effects inhibiting the expression of pro-inflammatory cytokine IL-8 and its receptor CXCR2; by contrast, EPO-α increases expression of genes relating to promotion of inflammation encoding for IL-1ß and CCL8, and induces de novo synthesis of IL-1α, CXCL1 and CXCL5 in primed cells. The reduction in MAPK p38-α activity is involved in modulating both IL-1ß and IL-8 expression. Unlike the induction of MAPK, Erk1/2 activity leads to upregulation of IL-1ß, but does not affect IL-8 expression and release. Furthermore, EPO-α treatment of primed cells induces the activation of caspase-1 upstream higher secretion of IL-1ß, and this process is not dependent on caspase-8 activation. In conclusion, our findings highlight new potential molecules involved in EPO resistance and confirm the anti-inflammatory role for EPO, but also suggest a plausible in vivo scenario in which the positive correlation found between EPO resistance and elevated levels of some pro-inflammatory mediators is due to treatment with EPO itself.

Toward personalized hemodialysis by low molecular weight amino-containing compounds: future perspective of patient metabolic fingerprint.

BACKGROUND: L-carnitine deficiency is commonly observed in chronic hemodialysis patients, and this depletion may cause clinical symptoms like muscle weakness, anaemia, and hypotension. MATERIALS AND METHODS: We pursued a targeted metabonomics investigation in 28 hemodialysis patients (13 non diabetics and 15 diabetics) and in 10 age-matched healthy controls, on plasma levels of all carnitine esters and of several amino acids. Samples were taken before and after the first hemodialysis treatment of the week. Multiplexed data were collected in LCMRM (Multiple Reaction Monitoring) and analysed by unsupervised multivariate analysis. RESULTS: In diabetic uremic patients, we observed lower values of propionylcarnitine than in other groups, while acylcarnitine concentration was higher in uremics compared to controls. The hemodialysis session induced a decline in free, short-chain, medium-chain and dicarboxylic acylcarnitines, whereas the long chain acylcarnitines remained unaffected. Plasma levels of amino acid proline, ornithine, citrulline and serine were significantly elevated in uremic patients before dialysis compared to controls. For most tested plasma amino acids, a significant reduction after hemodialysis session was found. DISCUSSION: Our study is the first that investigated on possible modifications of the system of carnitine in diabetic patients in hemodialysis not only in relation to the condition of deficiency but also compared to lipid and glucose homeostasis alteration typical of diabetics. We proposed the application of targeted metabolic fingerprint in the management of the hemodialysis patients.

Beta2-microglobulin causes abnormal phosphatidylserine exposure in human red blood cells.

The exposure of the aminophospholipid phosphatidylserine on the external leaflet of red blood cell plasma membrane can have several pathophysiological consequences with particular regard to the processes of cell phagocytosis, haemostasis and cell-cell interaction. A significant increase in phosphatidylserine-exposing erythrocytes has been reported in chronic haemodialysis patients and found to be strongly influenced by the uraemic milieu. To identify uraemic compound(s) enhancing phosphatidylserine externalization in erythrocytes, we fractionated by chromatographic methods the ultrafiltrate obtained during dialysis, and examined by flow cytometry the effect of the resulting fractions on phosphatidylserine exposure in human red cells. Chromatographic procedures disclosed a homogeneous fraction able to increase erythrocyte phosphatidylserine exposure. The inducer of such externalization was identified by monodimensional gel electrophoresis and mass spectrometry investigations as beta2-microglobulin. To confirm the beta2-microglobulin effect and to examine the influence of protein glycation (as it occurs in uraemia) on phosphatidylserine erythrocyte exposure, erythrocytes from normal subjects were incubated with recombinant beta2-microglobulin (showing no glycation sites at mass analysis), commercial beta2-microglobulin (8 glycation sites), or with in vitro glycated recombinant beta2-microglobulin (showing multiple glycation sites). Elevated concentrations of beta2-microglobulin (corresponding to plasma levels reached in dialysis patients) increased slightly but significantly the protein's ability to externalize phosphatidylserine on human erythrocytes. Such an effect was markedly enhanced by glycated forms of the protein. Beta2-microglobulin is recognized as a surrogate marker of middle-molecule uraemic toxins and represents a key component of dialysis-associated amyloidosis. Our study adds further evidence to the potential pathophysiologic consequences of beta2-microglobulin accumulation in chronic uraemic patients.

Adsorption and carbonylation of plasma proteins by dialyser membrane material: in vitro and in vivo proteomics investigations.

BACKGROUND: Protein carbonylation is an irreversible and not reparable reaction which is caused by the introduction into proteins of carbonyl derivatives such as ketones and aldehydes, generated from direct oxidation processes or from secondary protein reaction with reactive carbonyl compounds. Several studies have demonstrated significantly increased levels of reactive carbonyl compounds, a general increase in plasma protein carbonyls and carbonyl formation on major plasma proteins in blood from uremic patients, particularly those undergoing chronic haemodialysis. MATERIALS AND METHODS: In the present preliminary study, we first assessed by an in vitro filtration apparatus the possible effects of different materials used for haemodialysis membranes on protein retention and carbonylation. We employed hollow fiber minidialyzers of identical structural characteristics composed of either polymethylmethacrylate, ethylenevinyl alcohol, or cellulose diacetate materials. Protein Western Blot and SDS-PAGE coupled to mass spectrometry analysis were applied to highlight the carbonylated protein-binding characteristics of the different materials. We also investigated in vivo protein carbonylation and carboxy methyl lisine-modification in plasma obtained before and after a haemodialysis session. RESULTS: Our data underline a different capability on protein adsorption associated with the different properties of the filter materials, highlighting the central buffering and protective role of serum albumin. In particular, polymethylmethacrylate and cellulose diacetate showed, in vitro, the highest capacity of binding plasma proteins on the surface of the hollow fiber minidialyzers. CONCLUSIONS: The present study suggests that biomaterials used for fabrication of haemodialysis membrane may affect the carbonyl balance in chronic uremic patients.