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Intravenous iron supplementation is essential in hemodialysis (HD) patients to recover blood loss and to meet the requirements for erythropoiesis and, in patients receiving erythropoietin, to avert the development of iron deficiency. In a recent real-world study, Hofman et al. showed that a therapeutic shift from iron sucrose (IS) to ferric carboxymaltose (FCM) in HD patients improves iron parameters while reducing use of iron and erythropoietin. The objective of this economic analysis is to compare the weekly cost of treatment of FCM vs IS in hemodialysis patients in Italy. The consumption of drugs (iron and erythropoietin) was derived from Hofman’s data, while the value was calculated at Italian ex-factory prices. The analysis was carried on the total patient sample and in two subgroups: patients with iron deficiency and patients anemic at baseline. In addition, specific sensitivity analyses considered prices currently applied at the regional level, simulating the use of IS vs iron gluconate (FG) and epoetin beta vs epoetin alfa. In the base-case analysis, the switch to FCM generates savings of -€12.47 per patient/week (-21%) in all patients, and even greater savings in the subgroups with iron deficiency -€17.28 (-27%) and in anemic patients -€23.08 (-32%). Sensitivity analyses were always favorable to FCM and confirmed the robustness of the analysis. FCM may represent a cost-saving option for the NHS, and Italian real-world studies are needed to quantify the real consumption of resources in dialysis patients., (Copyright by Società Italiana di Nefrologia SIN, Rome, Italy.)

Osteoarthritis (OA) is a disease associated to age or conditions that precipitate aging of articular cartilage, a post-mitotic tissue that remains functional until the failure of major homeostatic mechanisms. OA severely impacts the national health system costs and patients' quality of life because of pain and disability. It is a whole-joint disease sustained by inflammatory and oxidative signaling pathways and marked epigenetic changes responsible for catabolism of the cartilage extracellular matrix. OA usually progresses until its severity requires joint arthroplasty. To delay this progression and to improve symptoms, a wide range of naturally derived compounds have been proposed and are summarized in this review. Preclinical in vitro and in vivo studies have provided proof of principle that many of these nutraceuticals are able to exert pleiotropic and synergistic effects and effectively counteract OA pathogenesis by exerting both anti-inflammatory and antioxidant activities and by tuning major OA-related signaling pathways. The latter are the basis for the nutrigenomic role played by some of these compounds, given the marked changes in the transcriptome, miRNome, and methylome. Ongoing and future clinical trials will hopefully confirm the disease-modifying ability of these bioactive molecules in OA patients.

While high levels of saturated fatty acids are associated with impairment of cardiovascular functions, n-3 polyunsaturated fatty acids (PUFAs) have been shown to exert protective effects. However the molecular mechanisms underlying this evidence are not completely understood. In the present study we have used rat H9c2 ventricular cardiomyoblasts as a cellular model of lipotoxicity to highlight the effects of palmitate, a saturated fatty acid, on genetic and epigenetic modulation of fatty acid metabolism and fate, and the ability of PUFAs, eicosapentaenoic acid, and docosahexaenoic acid, to contrast the actions that may contribute to cardiac dysfunction and remodeling. Treatment with a high dose of palmitate provoked mitochondrial depolarization, apoptosis, and hypertrophy of cardiomyoblasts. Palmitate also enhanced the mRNA levels of sterol regulatory element-binding proteins (SREBPs), a family of master transcription factors for lipogenesis, and it favored the expression of genes encoding key enzymes that metabolically activate palmitate and commit it to biosynthetic pathways. Moreover, miR-33a, a highly conserved microRNA embedded in an intronic sequence of the SREBP2 gene, was co-expressed with the SREBP2 messenger, while its target carnitine palmitoyltransferase-1b was down-regulated. Manipulation of the levels of miR-33a and SREBPs allowed us to understand their involvement in cell death and hypertrophy. The simultaneous addition of PUFAs prevented the effects of palmitate and protected H9c2 cells. These results may have implications for the control of cardiac metabolism and dysfunction, particularly in relation to dietary habits and the quality of fatty acid intake., Competing Interests: The authors declare no conflict of interest.

Systemic inflammation and uremic toxins (UT) determine the increased cardiovascular mortality observed in chronic hemodialysis (HD) patients. Among UT, the adipokine Chemerin induces vascular dysfunction by targeting both endothelial and vascular smooth muscular cells (EC and VSMC). As Citrate anion modulates oxidative metabolism, systemic inflammation and vascular function, we evaluated whether citrate-buffered dialysis improves HD efficiency, inflammatory parameters and chemerin-mediated microvascular injury. 45 patients were treated in sequence with acetate, citrate and, again, acetate-buffered dialysis solution (3 months per interval). At study admission and after each treatment switch, we evaluated dialysis efficacy and circulating levels of chemerin and different inflammatory biomarkers. In vitro, we stimulated EC and VSMC with patients' plasma and we investigated the role of chemerin as UT. Citrate dialysis increased HD efficacy and reduced plasma levels of CRP, fibrinogen, IL6 and chemerin. In vitro, patients' plasma induced EC and VSMC dysfunction. These effects were reduced by citrate-buffered solutions and paralleled by the decrease of chemerin levels. Consistently, chemerin receptor knockdown reduced EC and VSMC dysfunction. In conclusion, Switching from acetate to citrate improved dialysis efficacy and inflammatory parameters; in vitro, chemerin-induced EC and VSMC injury were decreased by using citrate as dialysis buffer.

Objective: The arteriovenous fistula (AVF) is recommended as the preferred hemodialysis access. However, placing an AVF in all patients may result in poor access outcomes and increased central venous catheter (CVC) use because of increased comorbid conditions, age, and suboptimal vessels. In patients with inadequate superficial veins for AVFs, the use of the brachial veins for creation of forearm arteriovenous grafts (AVGs) has received limited attention. This retrospective study aimed to evaluate outcomes of forearm brachial-brachial AVGs (BB-AVGs) placed in patients with poor superficial veins., Methods: We identified 111 BB-AVGs created in 111 consecutive patients, using standard-walled polytetrafluoroethylene grafts, between January 2010 and December 2015. After excluding 6 patients (non-dialysis initiation, missing information, and death within 1 month), we included 105 patients from 21 dialysis centers. We analyzed primary failures, time to cannulation, patency, complications, and revisions. Patency rates were calculated by the Kaplan-Meier method. The incidence of complications and revisions was expressed as number of events per person-year., Results: A total of 105 patients (median age, 69 years) were followed up for a median time of 21.2 months (interquartile range, 9.2-36.5 months). Of the patients, 72.4% were on chronic hemodialysis and had previously undergone one or more access procedures. At the time of BB-AVG placement, prior accesses were 39 AVFs, 20 tunneled CVCs, and 17 AVGs. BB-AVG rates of primary failure and revision before cannulation were 7.6% and 5.7%, respectively. BB-AVGs were cannulated after a median time of 3.4 weeks (interquartile range, 2.8-4.1 weeks). Primary patency rates at 12, 24, and 36 months were 49.5%, 29.5%, and 19.5%. Secondary patency rates at 12, 24, and 36 months were 76.3%, 62.7%, and 54.6%. After cannulation, the incidence of complications and revisions was 1.054 and 0.649 per person-year, respectively. Most complications and interventions were due to thrombosis (0.527 per person-year) or stenosis (0.381 per person-year) and related interventions (0.490 per person-year). A minority of patients experienced AVG infections (0.052 per person-year), with only two requiring access removal., Conclusions: In patients with poor superficial veins, the forearm BB-AVG is a reliable access because of low access-related morbidity and considerable long-term access survival. BB-AVG placement has the advantage of preserving proximal vessels. In these patients, such an approach can delay both rapid exhaustion of vascular sites and early recourse to CVC permanent use., (Copyright © 2018 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Decreased inflammation and cardiovascular mortality are evident in patients with end-stage chronic kidney disease treated by online hemodiafiltration. Extracellular vesicles (EV) are mediators of cell-to-cell communication and contain different RNA types. This study investigated whether mixed online hemodiafiltration (mOL-HDF) beneficial effects associate with changes in the RNA content of plasma EV in chronic kidney disease patients. Thirty bicarbonate hemodialysis (BHD) patients were randomized 1:1 to continue BHD or switch to mOL-HDF. Concentration, size, and microRNA content of plasma EV were evaluated for 9 mo; we then studied EV effects on inflammation, angiogenesis, and apoptosis of endothelial cells (HUVEC) and on osteoblast mineralization of vascular smooth muscle cells (VSMC). mOL-HDF treatment reduced different inflammatory markers, including circulating CRP, IL-6, and NGAL. All hemodialysis patients showed higher plasma levels of endothelial-derived EV than healthy subjects, with no significant differences between BHD and mOL-HDF. However, BHD-derived EV had an increased expression of the proatherogenic miR-223 with respect to healthy subjects or mOL-HDF. Compared with EV from healthy subjects, those from hemodialysis patients reduced angiogenesis and increased HUVEC apoptosis and VSMC calcification; however, all these detrimental effects were reduced with mOL-HDF with respect to BHD. Cell transfection with miR-223 mimic or antagomiR proved the role of this microRNA in EV-induced HUVEC and VSMC dysfunction. The switch from BHD to mOL-HDF significantly reduced systemic inflammation and miR-223 expression in plasma EV, thus improving HUVEC angiogenesis and reducing VSMC calcification., (Copyright © 2019 by The American Association of Immunologists, Inc.)

CD200, a transmembrane type Ia glycoprotein belonging to the immunoglobulin superfamily, has been shown to have a differential expression in B-cell neoplasms. Here, we retrospectively assessed the diagnostic relevance of CD200 on 427 patients with B-cell chronic neoplasms in leukemic phase (median age, 69 y; range, 35-97 y). The final diagnosis based on the investigator's assessment was chronic lymphocytic leukaemia (CLL) in 75% of cases and non-CLL in 25% of cases. Sensitivity and specificity for the diagnosis of CLL (vs non-CLL) were calculated for the following markers: CD200, CD5, CD22, CD23, CD79b, FMC7, and SmIg. CD23 was the only marker without a statistically significant difference between the investigator assessment and the flowcytometric analysis. The other markers were unable-when individually evaluated-to discriminate between CLL and non-CLL, requiring the integration into a scoring system. The modified score no. 1 (addition of CD200) showed superimposable sensitivity and specificity compared with the Matutes score. The substitution of CD79b (modified score no. 2), surface membrane immunoglobulins (SmIg) (modified score no. 3), and CD79b and FMC7 (modified score no. 4) with CD200 showed that only the modified score no. 4 had both higher sensitivity and higher specificity compared with standard Matutes score. In conclusion, this work defines a simplified score, compared with the classical Matutes score, for the differential diagnosis of chronic B-cell leukaemia-which only requires 4 markers instead of 5 (CD5, CD23, CD200, and SmIg)., (Copyright © 2018 John Wiley & Sons, Ltd.)

Hyporesponsiveness to erythropoietin stimulating agents (ESAs) is a condition associated with increased mortality. Even after identifying the condition, the causes are difficult to treat and only partially reversible in end-stage renal disease patients. Thus, the role of more recent hemodialysis (HD) techniques in improving such conditions is an emerging issue. However, major randomized clinical trials have not confirmed the results of smaller observational studies in which online hemodiafiltration has shown some efficacy in improving patients' response to ESAs. In our interpretation, these findings are not in contrast, but they can be explained by a better understanding of the interactions between HD and ESAs on iron mobilization, first of all through the role of hepcidin. The kinetics of hepcidin removal through HD combined with the action of selected ESAs may help the clinician in prescribing the best association between HD treatment and ESAs to overcome hyporesponsiveness., (© 2018 S. Karger AG, Basel.)

Purpose of the Study: Little information have been provided till now regarding the effect of high volume HDF (hv-OL-HDF) in respect to standard bicarbonate dialysis (BHD) in medium-long term protein-bound toxins removal., Procedures: A randomised cross-over multicentre study (REDERT study) was designed to compare the effects of hv-OL-HDF and low-flux BHD on uremic toxins serum levels in 36 chronic dialysis patients followed for 13 months. Group 1 patients were treated with BHD (Treatment A) for 6 months, and afterwards, they were transferred to hv-OL-HDF for a further 6 months (Treatment B). Group 2 patients were treated with Treatment B for 6 months, and afterwards, they were transferred to Treatment A for a further 6 months. Total and free pre-dialysis indoxyl-sulfate (IS) and p-cresyl-sulfate (pCS) were determined starting a midweek dialysis session at baseline and after six months of hv-OL-HDF or BHD. IS and pCS, were simultaneously measured, by liquid chromatography/electrospray ionization-tandem mass spectrometry, Kt/v and pre and post-dialysis b-2microglobulin (b2MG) levels were measured every three months., Results: Kt/V was significantly increased in hv-OL-HDF (from 1.47 ± 0.24 to 1.49 ± 0.16; p < 0.01) and was reduced in BHD (from 1.51 ± 0.2 to 1.36 ± 0.21; p < 0.001). The mean infusion volume in HDF was 20.9 ± 2.1 L with a mean total convective volume of 23.8 ± 2.3 L and a significant removal of b2MG was obtained in hv-OL-HDF at month 3 and month 6. Both free and total levels of IS and pCS were significantly reduced in hv-OL-HDF at month 6 in respect to BHD., Conclusions: In the present study we confirm the assumption that post-HDF is an effective technique in small and protein-bound uremic toxins removal.

Patients affected by cardiovascular disease (CVD) are treated with antiplatelet agents (AA) and/or anticoagulant drugs, which are fundamental in the management of stroke, coronary atherosclerotic disease, peripheral vascular disease and atrial fibrillation. CVD is the most important cause of death in chronic renal failure (CRF). Death rates from myocardial infarction (MI) and from all other cardiac causes exceed those for the general population. Incidence of MI in CRF is triple that in the general population. Moreover, mortality is seven- to eight-fold higher in patients requiring chronic hemodialysis compared to the general population, and approximately 40% of deaths in this population are attributable to coronary artery disease (CAD). For these reasons, AA are widely used in patients affected by CRF. Current data do not support a protective effect of antiplatelet administration in hemodialytic patients as primary prevention of cardiovascular mortality. Different results have been obtained concerning secondary prevention of CVD. The Cooperative Cardiovascular Project demonstrated that dialysis patients treated with aspirin following MI had a 43% lower mortality. Another study reported that the use of aspirin and beta-blockers following MI was associated with lower mortality in CRF patients. However, aspirin plus clopidogrel seems to increase the hemorrhagic risk without a significant reduction in cardiovascular mortality and there are insufficient data to support the use of new AA drugs in hemodialytic patients. In conclusion, since CRF patients are one of the groups at highest risk for atherosclerotic events, it could be reasonable to use aspirin in HD patients. However, the bleeding risk in HD patients needs to be strongly evaluated, especially before starting dual AA treatment.

Within the framework of the LUST trial (LUng water by Ultra-Sound guided Treatment to prevent death and cardiovascular events in high-risk end-stage renal disease patients), the European Renal and Cardiovascular Medicine (EURECA-m) working group of the European Renal Association-European Dialysis Transplant Association established a central core lab aimed at training and certifying nephrologists and cardiologists participating in this trial. All participants were trained by an expert trainer with an entirely web-based programme. Thirty nephrologists and 14 cardiologists successfully completed the training. At the end of training, a set of 47 lung ultrasound (US) videos was provided to trainees who were asked to estimate the number of B-lines in each video. The intraclass correlation coefficient (ICC) for the whole series of 47 videos between each trainee and the expert trainer was high (average 0.81 ± 0.21) and >0.70 in all but five cases. After further training, the five underperforming trainees achieved satisfactory agreement with the expert trainer (average post-retraining ICC 0.74 ± 0.14). The Bland-Altman plot showed virtually no bias (difference between the mean 0.03) and strict 95% limits of agreement lines (-1.52 and 1.45 US B-lines). Only four cases overlapped but did not exceed the same limits. Likewise, the Spearman correlation coefficient applied to the same data series was very high (r = 0.979, P < 0.0001). Nephrologists and cardiologists can be effectively trained to measure lung congestion by an entirely web-based programme. This web-based training programme ensures high-quality standardization of US B-line measurements and represents a simple, costless and effective preparatory step for clinical trials targeting lung congestion., (© The Author 2016. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Background and Objectives: Accumulation of fluid in the lung is the most concerning sequela of volume expansion in patients with ESRD. Lung auscultation is recommended to detect and monitor pulmonary congestion, but its reliability in ESRD is unknown., Design, Setting, Participants, & Measurements: In a subproject of the ongoing Lung Water by Ultra-Sound Guided Treatment to Prevent Death and Cardiovascular Complications in High Risk ESRD Patients with Cardiomyopathy Trial, we compared a lung ultrasound-guided ultrafiltration prescription policy versus standard care in high-risk patients on hemodialysis. The reliability of peripheral edema was tested as well. This study was on the basis of 1106 pre- and postdialysis lung ultrasound studies (in 79 patients) simultaneous with standardized lung auscultation (crackles at the lung bases) and quantification of peripheral edema., Results: Lung congestion by crackles, edema, or a combination thereof poorly reflected the severity of congestion as detected by ultrasound B lines in various analyses, including standard regression analysis weighting for repeated measures in individual patients (shared variance of 12% and 4% for crackles and edema, respectively) and κ-statistics (κ ranging from 0.00 to 0.16). In general, auscultation had very low discriminatory power for the diagnosis of mild (area under the receiver operating curve =0.61), moderate (area under the receiver operating curve =0.65), and severe (area under the receiver operating curve =0.68) lung congestion, and the same was true for peripheral edema (receiver operating curve =0.56 or lower) and the combination of the two physical signs., Conclusions: Lung crackles, either alone or combined with peripheral edema, very poorly reflect interstitial lung edema in patients with ESRD. These findings reinforce the rationale underlying the Lung Water by Ultra-Sound Guided Treatment to Prevent Death and Cardiovascular Complications in High Risk ESRD Patients with Cardiomyopathy Trial, a trial adopting ultrasound B lines as an instrument to guide interventions aimed at mitigating lung congestion in high-risk patients on hemodialysis., (Copyright © 2016 by the American Society of Nephrology.)

Pure red cell aplasia (PRCA) may develop in patients with chronic kidney disease receiving erythropoiesis-stimulating agents (ESA). We report on a 72-year-old patient who developed hypo-proliferative anaemia unresponsive to ESA following the administration of epoetin zeta subcutaneously for 7 months. On the basis of severe isolated hypoplasia of the erythroid line in the bone marrow and high-titre neutralizing anti-erythropoietin antibodies (Ab), a diagnosis of Ab-mediated PRCA was made. Epoetin zeta was discontinued and the patient was given steroids. This was associated with anaemia recovery. To our knowledge this is the first PRCA case related to epoetin zeta.

Background: Optimizing vascular access outcomes is still a challenge, since 30-60% of arteriovenous fistulas fail or do not mature and catheters are widely used in contemporary patients., Methods: This study reports on strategies and outcomes in a single center in which access planning, surgery and maintenance are managed by a team of nephrologists. We retrospectively analyzed 305 fistulas and 61 grafts created in 270 consecutive patients between 2002 and 2013., Results: The percentage of patients receiving a fistula or graft who initiated hemodialysis with a mature access was 68.6%. Among prevalent patients, 71.7% used a fistula, 15.7% a graft and 12.6% a catheter. Rates of primary failure and revision before cannulation were 14.4 and 1.6% for fistulas vs. 4.9 and 3.3% for grafts. After maturation, complications (1.040 vs. 0.188 per patient-year (py)) and interventions (0.743 vs. 0.066 per py) were greater for grafts than for fistulas (p < 0.001). Secondary patency did not significantly differ between grafts and fistulas (median survival 34.8 vs. 57.3 months, p = 0.36), unless primary failures were excluded from Kaplan-Meier analysis (median survival 34.9 vs. 70.9 months, p = 0.03)., Conclusions: High fistula prevalence, low access-related morbidity and catheter dependence were achieved using individualized strategies, including mid-forearm or perforating vein fistula creation and selective graft placement in high risk patients. Direct involvement of nephrologists throughout all steps of access care can improve access outcomes, by promoting a patient-centered approach., (© 2016 S. Karger AG, Basel.)

Introduction: Several studies demonstrated that endothelium dependent vasodilatation is impaired in cardiovascular and chronic kidney diseases because of oxidant stress-induced nitric oxide availability reduction. The Mediterranean diet, which is characterized by food containing phenols, was correlated with a reduced incidence of cardiovascular diseases and delayed progression toward end stage chronic renal failure. Previous studies demonstrated that both red and white wine exert cardioprotective effects. In particular, wine contains Caffeic acid (CAF), an active component with known antioxidant activities., Aim of the Study: The aim of the present study was to investigate the protective effect of low doses of CAF on oxidative stress-induced endothelial injury., Results: CAF increased basal as well as acetylcholine-induced NO release by a mechanism independent from eNOS expression and phosphorylation. In addition, low doses of CAF (100 nM and 1 μM) increased proliferation and angiogenesis and inhibited leukocyte adhesion and endothelial cell apoptosis induced by hypoxia or by the uremic toxins ADMA, p-cresyl sulfate and indoxyl sulfate. The biological effects exerted by CAF on endothelial cells may be at least in part ascribed to modulation of NO release and by decreased ROS production. In an experimental model of kidney ischemia-reperfusion injury in mice, CAF significantly decreased tubular cell apoptosis, intraluminal cast deposition and leukocyte infiltration., Conclusion: The results of the present study suggest that CAF, at very low dosages similar to those observed after moderate white wine consumption, may exert a protective effect on endothelial cell function by modulating NO release independently from eNOS expression and phosphorylation. CAF-induced NO modulation may limit cardiovascular and kidney disease progression associated with oxidative stress-mediated endothelial injury.

Background: In haemodialysis (HD) patients, anaemia is associated with reduced survival. Despite treatment with erythropoiesis-stimulating agents (ESAs), a large number of patients with chronic kidney disease show resistance to this therapy and require much higher than usual doses of ESAs in order to maintain the recommended haemoglobin (Hb) target, and recent studies suggest that hepcidin (HEP) may mediate the ESA resistance index (ERI). High-volume online haemodiafiltration (HV-OL-HDF) has been shown to improve anaemia and to reduce the need for ESAs in HD patients; this effect is associated with a reduced inflammatory state in these patients. The aim of the REDERT study (role of haemodiafiltration on ERI) was to investigate the effect of different dialysis techniques on ERI and HEP levels in chronic dialysis patients., Methods: A single cross-over, randomized, multicentre study (A-B or B-A) was designed. Forty stable HD patients from seven different dialysis units (male 65%, mean age 67.6 ± 14.7 years and mean dialytic age 48 ± 10 months) were enrolled. Patients were randomized to the standard bicarbonate dialysis (BHD) with low-flux polysulfone (PS) membrane group or to the HV-OL-HDF group with high-flux PS membranes and exchange volume of >20 L/session. After 6 months, patients were shifted to the other dialytic group for a further 6 months. Clinical data, Hb, ESA doses and iron metabolism were recorded every month. HEP, beta2-microglobulin (b2MG) and C-reactive protein (CRP) were determined every 3 months, and ERI was calculated monthly as the weekly ESA dose per kilogram of body weight divided by Hb level. Data were analysed using paired-samples t-test, Wilcoxon signed-rank test and Spearman's correlation coefficient., Results: Dialysis efficiency for small molecules assessed as Kt/V was significantly increased in HV-OL-HDF from 1.47 ± 0.24 to 1.49 ± 0.16; P < 0.01. A significant reduction of b2MG was obtained in HV-OL-HDF from month 3 whereas CRP values were not significantly changed during the study period either in BHD or HV-OL-HDF.ERI was significantly reduced in HV-OL-HDF at month 3 and 6 (from 9.1 ± 6.4 UI/weekly/Kg/Hb to 6.7 ± 5.3 UI/weekly/Kg/Hb; P < 0.05) due to a higher ESA consumption in BHD in spite of similar Hb levels. HEP levels were reduced in HV-OL-HDF with respect to BHD after 3 and 6 months. Iron consumption was not significantly different during BHD or HV-OL-HDF treatment as well as transferrin, ferritin and TSAT levels. A significant positive linear correlation between HEP and ERI (r(2) = 0.258, P < 0.001) was observed., Conclusions: In a uraemic patient population with low-grade inflammation treated with HV-OL-HDF, we observed a significant reduction of ERI values as well as HEP levels. The positive correlation between these two parameters supports a role for HEP in the development of ERI in the dialytic population. Moreover, the lower b2MG and the higher Kt/V achieved in HV-OL-HDF confirms the better depurative effect of this technique in comparison with BHD with respect to middle molecules and small-molecular-weight molecules., (© The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Background: Endothelial progenitor cells (EPCs) are known to induce tissue repair by paracrine mechanisms including the release of growth factors and extracellular vesicles (EVs), nanoparticles able to carry proteins and genetic information to target cells. The aim of this study was to evaluate whether EVs derived from EPCs may protect from complement-mediated mesangial injury in experimental anti-Thy1.1 glomerulonephritis., Methods: EVs were isolated by serial ultracentrifugation from supernatants of cultured human EPCs and characterized for their protein and RNA content. In vivo, EVs were injected i.v. in the experimental rat model of mesangiolytic anti-Thy1.1 glomerulonephritis evaluating renal function, proteinuria, complement activity and histological lesions. In vitro, the biological effects of EPC-derived EVs were studied in cultured rat mesangial cells incubated with anti-Thy1.1 antibody and rat or human serum as complement source., Results: After i.v. injection in Thy1.1-treated rats, EVs localized within injured glomeruli and inhibited mesangial cell activation, leucocyte infiltration and apoptosis, decreased proteinuria, increased serum complement haemolytic activity (CH50) and ameliorated renal function. EV treatment decreased intraglomerular deposition of the membrane attack complex (MAC or C5b-9) and expression of smooth muscle cell actin and preserved the endothelial antigen RECA-1 and the podocyte marker synaptopodin. The protective effect of EVs was significantly reduced by pre-treatment with a high dose of RNase (1 U/mL), suggesting a key role for EV-carried RNAs in these mechanisms. Indeed, EPC-derived EVs contained different mRNAs coding for several anti-apoptotic molecules and for the complement inhibitors Factor H, CD55 and CD59 and the related proteins. The in vitro experiments aimed to investigate the mechanisms of EV protection indicated that EVs transferred to mesangial cell mRNAs coding for Factor H, CD55 and CD59 and inhibited anti-Thy1.1 antibody/complement-induced apoptosis and C5b-9/C3 mesangial cell deposition., Conclusions: EVs derived from EPCs exert a protective effect in Thy1.1 glomerulonephritis by inhibition of antibody- and complement-mediated injury of mesangial cells., (© The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Background: Mediterranean-style diet has been considered for its important beneficial effects on the progression of CV disease. Wine is an important component of the Mediterranean diet, and moderate wine drinkers have lower mortality rates than nondrinkers and heavy drinkers in epidemiologic studies. The beneficial effects of red wine are thought to be dependent on the polyphenol compounds such as resveratrol that exhibit potent antioxidant activity. However, white wine, although lacking polyphenols, contains simple phenols, such as tyrosol (Tyr) and hydroxytyrosol (OH-Tyr), characteristic also of extra-virgin olive oil, which may share similar antioxidant and inflammatory properties., Patients and Methods: The effect of white wine and extra-virgin olive oil on inflammatory markers was evaluated in 10 healthy volunteers and in 10 patients with CKD (chronic kidney disease) K-DOQI stage III-IV in a prospective, single blind, randomized, cross-over trial. After two weeks of wash-out from alcoholic beverages, subjects were randomized to a cross-over design A-B or B-A of a 2-week treatment with white wine (4 ml/kg body weight, 0.48 g/kg of alcohol 12%, corresponding to 2-3 glasses/daily) and extra-virgin olive oil (treatment A) or extra-virgin olive oil alone (treatment B). The two study periods were separated by a two-week wash-out period. At baseline and at the end of each treatment, plasma levels of inflammatory markers C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and interleukin-8 (IL-8) concentration were determined. Urinary levels of Tyr, OH-Tyr, and their metabolites were measured at the same time., Results: During combined consumption of white wine and extra-virgin olive oil (treatment A), plasma levels of CRP and IL-6 decreased from 4.1 ± 1.8 to 2.4 ± 1.9 mg/l (p < 0.05) and from 5.3 ± 3.2 to 3.4 ± 2.3 mg/l (p < 0.05) in CKD patients. CRP decreased from 2.6 ± 1.2 to 1.9 ± 0.9 mg/l (p < 0.05), and IL-6 decreased from 2.2 ± 1.8 to 1.7 ± 1.3 mg/l (p = ns) in healthy volunteers. No significant variation versus baseline was observed during treatment B. A significant increase in urinary Tyr and OH-Tyr was observed during treatment A (white wine and extra-virgin olive oil)., Conclusions: Plasma markers of chronic inflammation were significantly reduced in CKD patients during the combined consumption of white wine and olive oil, suggesting a possible anti-inflammatory effect of this nutritional intervention., (© 2015 S. Karger AG, Basel.)

Background: A pharmacoeconomic analysis of the RISCAVID database aimed at assessing the cost effectiveness of phosphate binders in preventing CV mortality and morbidity over 7 years was performed., Methods: Morbid or fatal events occurring in 750 chronic HD patients were recorded. Statistical analysis evaluated the distribution of variables and the effect of sevelamer on survival. A cost-effectiveness evaluation was performed using a probabilistic model based on a Markov chain., Results: Multivariate analysis showed that treatment with sevelamer was associated with a reduced stroke incidence by 52% (p = 0.04) and reduced levels of C-reactive protein (p < 0.01). Cost-effectiveness evaluation evidenced a 33% decrease in hospital-days for patients treated with sevelamer, with and without comorbidities compared to patients undergoing calcium binders treatment., Conclusion: Treatment with sevelamer was associated with a reduced risk of stroke in HD patients, with a clear saving on disease-related costs for the Italian National Healthcare System., (© 2015 S. Karger AG, Basel)

Introduction: Malnutrition is a common complication in hemodialysis (HD) patients and it is related to morbidity and mortality. Although a gold standard method for diagnosis of malnutrition is not available, serum albumin, body weight and height are commonly used and are included in the calculation of the Geriatric nutritional risk index (GNRI). Recently the association between GNRI and mortality in chronic HD patients has been documented in Asian populations. Our aim was to examine the relative reliability of the GNRI as a mortality and morbidity predictor in an Italian HD cohort., Methods: We prospectively examined the GNRI of 753 maintenance HD patients aged 65.7 ± 14.1 years, 457 males, included in the Riscavid cohort, and followed them up for 84 months. Predictors for all-cause death were examined using Kaplan-Meier and Cox proportional-hazards analyses., Results: Low GNRI was significantly associated with signs of wasting, i.e. low Body mass index, hypoalbuminemia, low normalized protein catabolic rate. Patients within the lowest GNRI quartile had a significantly lower survival rate than those in the 2nd to 4th quartile (p < 0.001). Multivariate Cox proportional-hazards analysis demonstrated that the lowest quartile of GNRI was a significant predictor of case mix adjusted all-cause mortality (HR 1.72; CI 1.35-2.18, p < 0.001)., Conclusions: These results demonstrate that low GNRI (<92) is associated with malnutrition and is a strong predictor of overall mortality in HD patients. However, cardiovascular events did not differ among the GNRI quartiles. A low GNRI score can be considered a simple and reliable marker of malnutrition and predictor for mortality risk in Caucasian HD patients.

Background: Citrate has anticoagulative properties and favorable effects on inflammation, but it has the potential hazards of inducing hypocalcemia. Bicarbonate dialysate (BHD) replacing citrate for acetate is now used in chronic haemodialysis but has never been tested in postdilution online haemodiafiltration (OL-HDF)., Methods: Thirteen chronic stable dialysis patients were enrolled in a pilot, short-term study. Patients underwent one week (3 dialysis sessions) of BHD with 0.8 mmol/L citrate dialysate, followed by one week of postdilution high volume OL-HDF with standard bicarbonate dialysate, and one week of high volume OL-HDF with 0.8 mmol/L citrate dialysate., Results: In citrate OL-HDF pretreatment plasma levels of C-reactive protein and β 2-microglobulin were significantly reduced; intra-treatment plasma acetate levels increased in the former technique and decreased in the latter. During both citrate techniques (OL-HDF and HD) ionized calcium levels remained stable within the normal range., Conclusions: Should our promising results be confirmed in a long-term study on a wider population, then OL-HDF with citrate dialysate may represent a further step in improving dialysis biocompatibility.

Objective: To assess the knowledge of adult hemodialysis patients and nurses working in dialysis units, specifically with regard to knowledge of phosphorus and other nutrients related to dietary management of end-stage renal disease., Design: Cross-sectional cohort study., Setting: Hemodialysis unit., Subjects: One hundred ninety-one hemodialysis patients and 105 dialysis nurses, as well as 86 control hospital employees who are not health professionals., Intervention: Nutritional knowledge was assessed by a 25-item chronic kidney disease knowledge assessment tool for nutrition, which includes 15 questions on phosphorus and 10 questions on protein, sodium, and potassium knowledge., Results: The scores obtained by patients were much lower than those of nurses (11.6 ± 3.9 vs. 16.0 ± 2.2, P < .001) but slightly higher than those of controls (10.6 ± 3.2, P < .05). Patients with phosphorus serum level >5.5 mg/dL showed chronic kidney disease knowledge assessment tool for nutrition scores similar to those of patients with a serum phosphorus level <5.5 mg/dL. The prevalence of right answers to questions regarding knowledge of phosphorus was lower than that regarding knowledge of the other nutrients, both for patients (38.4% ± 17.8% vs. 57.3% ± 19.9%, P < .001) and nurses (55.6% ± 11.1% vs. 74.8% ± 11.7%, P < .001) as well as for controls (30.7% ± 14.5% vs. 60.1% ± 17.4%, P < .001)., Conclusions: Our study suggests that nutritional knowledge of hemodialysis patients, although higher than the general population, is lower for phosphorus with respect to the other nutrients, such as protein, sodium, and potassium. This occurs even in patients with hyperphosphatemia or those taking phosphate binder medications. Nurses showed the best scores; however, improvement is necessary, especially with regard to knowledge of phosphorus. Training programs on nutrition for nurses and on information for patients should be implemented. They can contribute to achievement of a more effective control of phosphate balance, reduction of costs, and improvement of the quality of care for hemodialysis patients., (Copyright © 2012 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

Background: Mixed diffusive-convective dialysis therapies offer greater removal capabilities than conventional dialysis. The aim of this study was to compare two different on-line, post-dilution hemodiafiltration (HDF) treatments with regard to achieved convective volume and middle-molecule dialysis efficiency: standard volume control (sOL-HDF) and automated control of the transmembrane pressure (TMP) (UC-HDF)., Methods: We enrolled 30 ESRD patients (55.9 ± 14.0 years, 20/10 M/F) in a randomized, prospective, cross-over study. The patients received a 3-month period of sOL-HDF followed by UC-HDF for a further 3 months, or vice versa, using the same dialysis machine. In sOL-HDF, fixed exchange volumes were set according to a filtration fraction greater than or equal to 25%. In UC-HDF therapy, the exchanged volume was driven by a biofeedback system controlling the TMP and its set point in a double loop. Patients maintained their treatment time, dialyzer, blood flow rate, and anticoagulant regimen unchanged throughout the study., Results: Greater convective volumes were achieved in UC-HDF than in sOL-HDF (23.8 ± 3.9 vs.19.8 ± 4.8 L; p<0.001) with high pre-dialysis Ht value (sOL-HDF 34.0 ± 4.5% and UC-HDF 34.0 ± 4.4%; p = 0.91). The average clearance values of ß2m and P were higher in UC-HDF than in sOL-HDF (respectively 123 ± 24 vs. 111 ± 22 ml/min, p<0.002 and 158 ± 26 vs. 152 ± 25 ml/min, p<0.05). Moreover, the UC-HDF mode led to a significantly increased rate of call-free sessions from 88% to 97% (p<0.0001)., Conclusions: This study showed that the biofeedback module, applied to the automatic control of TMP in on-line HDF, results in higher convective volumes and correspondingly higher ß2m and P clearances. By making the HDF treatment more automated and less complex to perform, it significantly reduced the staff workload.

Cardiovascular disease is the leading cause of death in the general population; traditional risk factors seem inadequate to explain completely the remarkable prevalence of cardiovascular mortality and morbidity observed in the uremic population. A role for chronic inflammation has been well established in the development of atherosclerotic disease, and, on the basis of these observations, atherosclerosis might be considered an inflammatory disease. Inflammation has been implicated in the etiology of coronary artery disease in the general population, and traditional inflammatory biomarkers such as C-reactive protein (CRP) and interleukin-6 (IL-6) have been shown to predict cardiovascular events in both symptomatic and asymptomatic individuals as well as those in the uremic population. Later on, new nontraditional markers were related to the risk of cardiovascular morbidity and mortality in general and in uremic population. As a consequence of the expanding research base and availability of assays, the number of inflammatory marker tests ordered by clinicians for cardiovascular disease (CVD) risk prediction has grown rapidly and several commercial assays have become available. So, up to now we can consider that several new nontraditional markers as CD40-CD40 ligand system and pentraxin-3 seem to be significant features of cardiovascular disease in general and in ESRD population.

Background: Resistance to erythropoiesis-stimulating agents (ESAs) is often associated with chronic inflammation. Here, we investigated how anaemia, ESA resistance and the plasma levels of biological markers of inflammation could influence all-cause and cardiovascular disease morbidity and mortality., Methods: Seven hundred and fifty-three haemodialysis (HD) patients (mean age 66 ± 14.2 years, mean dialytic age 70 ± 77 months and diabetes 18.8%) were enrolled and followed-up for 36 months. Demographic, clinical and laboratory data, co-morbidity conditions, administered drugs, all-cause mortality and fatal/non-fatal cardiovascular (CV) events were recorded. We measured ESA resistance index, C-reactive protein (CRP) and interleukin-6 (IL-6)., Results: Six hundred and fifty-one patients (86.4%) received ESAs. Patients with haemoglobin level <11 g/dL (n = 225) showed increased risk of CV [relative risk (RR) 1.415, 95% confidence interval (CI) 1.046-1.914] and overall mortality (RR 1.897, 95% CI 1.423-2.530) versus patients with haemoglobin levels >11 g/dL. ESA resistance values categorized into quartiles (Quartile I <5.6, Quartile II 5.7-9.6, Quartile III 9.7-15.4 and Quartile IV >15.4) correlated with all-cause mortality and fatal/non-fatal CV events (RR 1.97, 95% CI 1.392-2.786; RR 1.619, 95% CI 1.123-2.332, respectively). Furthermore, albumin was significantly reduced versus reference patients and correlated with all-cause mortality and CV events; CRP levels were higher in hyporesponders (Quartile IV) (P < 0.001) and predicted all-cause mortality and CV events. IL-6 but not CRP was a strong predictor of ESA resistance., Conclusions: ESA responsiveness can be considered a strong prognostic factor in HD patients and seems to be tightly related to protein-energy wasting and inflammation.

The biological effects in vitro and in vivo of acetate as the main dialysate buffer have been well documented since the introduction of bicarbonate dialysis in the late 1970s. Bicarbonate dialysis solutions have become the standard but still contain 3-5 mmol/L of acetate for chemical stability. This is an important controversy given the recent introduction in numerous hemodialysis techniques of totally acetate-free solutions in an attempt to improve the hemodynamic efficiency and prevent inflammatory and cardiovascular damage. The issue is particularly relevant with the use of on-line techniques, in which, by summing the quota diffused from the dialysate and the quota infused directly into the blood, elevated quantities of acetate can be transferred to the patient's blood. In spite of the relatively small concentration of acetate in the bicarbonate dialysate, the acetate mass transfer is underestimated, leading to various and serious side effects especially in malnourished patients and/ or patients with a low muscle mass or with hepatocellular dysfunction. These patients do not have the capacity to effectively and rapidly metabolize the acetate, leading inevitably to acetate intoxication and accumulation. As the acetate cannot be transformed, it will follow alternative metabolic pathways, with worsening of the acidosis, and will induce the production of nitric oxide with negative hemodynamic effects such as vasodilation. The hemodynamic consequences are myocardial disease with contractile dysfunction and hemodynamic instability. Other problems triggered by acetate include cytokine production with higher microinflammation, dyslipidemia, arterial hypotension, and myocardial dysfunction with a rise in troponin. The use of acetate is hard to defend. Bicarbonate dialysate with a low acetate concentration can be used in patients with a good acetate tolerance. In such patients acidosis adjustment can be continued in the post-dialysis period. Some broad scientific documentation on the benefits of convective-diffusive treatments, almost all of which performed with non-acetate-free techniques, seems to contradict the negative effects of acetate but several studies have shown better hemodynamic stability during dialysis sessions with totally acetate- free dialysate. In conclusion, the future of hemodialysis is likely to be without acetate because acetate-free solutions are easy to produce and their cost will diminish.

Background: Hemodialysis is complicated by a high incidence of intradialytic hypotension and disequilibrium symptoms caused by hypovolemia and a decrease in extracellular osmolarity. Automatic adaptive system dialysis (AASD) is a proprietary dialysis system that provides automated elaboration of dialysate and ultrafiltration profiles based on the prescribed decrease in body weight and sodium content., Study Design: A noncontrolled (single arm), multicenter, prospective, clinical trial., Setting & Participants: 55 patients with intradialytic hypotension or disequilibrium syndrome in 15 dialysis units were studied over a 1-month interval using standard treatment (642 sessions) followed by 6 months using AASD (2,376 sessions)., Intervention: AASD (bicarbonate dialysis with dialysate sodium concentration and ultrafiltration rate profiles determined by the automated procedure)., Outcomes: Primary and major secondary outcomes were the frequency of intradialytic hypotension and symptoms (hypotensive events, headache, nausea, vomiting, and cramps), respectively., Results: More stable intradialytic systolic and diastolic blood pressures with lower heart rate were found using AASD compared with standard treatment. Sessions complicated by hypotension decreased from 58.7% ± 7.3% to 0.9% ± 0.6% (P < 0.001). The incidence of other disequilibrium syndrome symptoms was lower in patients receiving AASD. There were no differences in end-session body weight, interdialytic weight gain, or presession natremia between the standard and AASD treatment periods., Limitations: A noncontrolled (single arm) study, no crossover from AASD to standard treatment., Conclusions: This study shows the long-term clinical efficacy of AASD for intradialytic hypotension and disequilibrium symptoms in a large number of patients and dialysis sessions., (Copyright © 2011 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

Background: Oxidative stress is prevalent in dialysis patients and has been implicated in the pathogenesis of cardiovascular disease and anemia. Vitamin E is a fat-soluble antioxidant that plays a central role in reducing lipid peroxidation and inhibiting the generation of reactive oxygen species. The aim of this cross-over randomized study was to compare the effects of a vitamin E-coated polysulfone (Vit E PS) membrane and a non-vitamin E-coated polysulfone (PS) membrane on inflammatory markers and resistance to erythropoietin-stimulating agents (ESAs)., Methods: After a 1-month run-in period of standard bicarbonate dialysis with a synthetic membrane, 62 patients of both genders, and older than 18 years, dialysis vintage 48 ± 27 months, BMI 22 ± 3 (from 13 different dialysis units) were randomized (A-B or B-A) in a cross-over design to Vit E PS (treatment A) and to PS (treatment B) both for 6 months. C-reactive protein (CRP) and interleukin-6 (IL-6) concentrations were determined by a sandwich enzyme immunoassay at baseline and every 2 months; red blood cell count, ESA dose and ESA resistance index (ERI) were assessed monthly., Results: Hemoglobin (Hb) levels significantly increased in the Vit E PS group from 11.1 ± 0.6 g/dl at baseline to 11.5 ± 0.7 at 6 months (p < 0.001) and remained unchanged in the PS group. Although ESA dosage remained stable during the observation periods in both groups, ERI was significantly reduced in the Vit E PS group from 10.3 ± 2.2 IU-dl/kg/g Hb week at baseline to 9.2 ± 1.7 at 6 months (p < 0.001). No significant variation of ERI was observed in the PS group. A significant reduction in plasma CRP and IL-6 levels was observed in the Vit E PS group: CRP from 6.7 ± 4.8 to 4.8 ± 2.2 mg/l (p < 0.001) and IL-6 from 12.1 ± 1.4 to 7.5 ± 0.4 pg/ml (p < 0.05). In the PS group, CRP varied from 6.2 ± 4.0 to 6.4 ± 3.7, and IL-6 from 10.6 ± 2.1 to 9.6 ± 3.5 (p = n.s.)., Conclusions: Treatment with Vit E PS membranes seems to lead to a reduction in ESA dosage in HD patients; in addition, a low chronic inflammatory response may contribute to a sparing effect on exogenous ESA requirements., (Copyright © 2011 S. Karger AG, Basel.)

Cardiovascular disease is a significant complication in chronic kidney disease (CKD) and a major cause of death in dialysis patients. Clinical studies have shown that anemia is associated with reduced survival in patients undergoing chronic hemodialysis. Furthermore, an association between anemia and adverse cardiovascular outcomes has also been observed in patients with earlier stages of CKD not yet requiring dialysis. Although this fact still remains controversial, high-efficiency on-line hemodiafiltration (HDF) has been shown to improve anemia and to reduce the need for erythropoietin-stimulating agents in hemodialysis (HD) patients. This positive effect has been attributed to the fact that the convective methods might remove some protein-bound erythropoietic inhibitor substances. Moreover, in HD patients, renal anemia is linked to the inflammatory state of uremic syndrome. It is also worth nothing that the improvement in anemia is associated with a reduced inflammatory state in patients undergoing on-line HDF. Here, we have reviewed the current knowledge of the effect of dialysis technique on renal anemia., (Copyright © 2011 S. Karger AG, Basel.)

Despite significant advances in hemodialysis (HD) and medical therapy, mortality rates in patients with chronic kidney disease stage 5 (CKD-5D) remain unacceptably high, with cardiovascular (CV) mortality risk in dialysis patients being several times higher than that of a general population. The CV mortality risk of a 25-to 34-year-old dialysis patient is approximately the same as the one of an otherwise healthy person aged over 85 in the general population. HD patients are not only exposed to the various traditional risk factors valid for the general population but in addition also to the non-traditional risk factors resulting either from uremia per se or from the dialysis treatment. Our understanding of chronic inflammation in CKD-5D and of its influence in the accelerated atherosclerotic process has greatly evolved. Dialysis therapy, performed efficiently, has the potential to provide CKD-5D patients with benefits beyond the life-saving function of removal accumulated waste products and excess fluid. There is sufficient evidence to indicate that on-line hemodiafiltration has the potential to improve chronic inflammation, fluid overload, left ventricular hypertrophy, anemia and quality of life of CKD-5D patients. In the search for more effective, safer and less expensive approaches to the management of the above conditions, an innovative concept of biocompatibility should be broadened to the HD system/patient evaluation. Here, we intend to present new insights obtained from a prospective observational study (RISCAVID, 'RISchio CArdiovascolare nei pazienti afferenti all'Area Vasta In Dialisi') performed on a large HD population in the northwestern region of Tuscany, Italy, on the assessment of the different parameters of chronic inflammation and gross/CV mortality and anemia management., (Copyright © 2011 S. Karger AG, Basel.)

Background: Despite substantial progress in medical care, the mortality rate remains unacceptably high in dialysis patients. Evidence suggests that bone mineral dismetabolism (CKD-MBD) might contribute to this burden of death. However, to date only a few papers have investigated the clinical relevance of serum mineral derangements and the impact of different therapeutic strategies on mortality in a homogeneous cohort of south European dialysis patients., Methods: The RISCAVID study was a prospective, observational study in which all patients receiving hemodialysis (HD) in the north-western region of Toscany in June 2004 were enrolled (N=757) and followed up for 24 months., Results: At study entry, only 71 (9%) patients of the entire study cohort exhibited an optimal control of serum phosphorous (Pi), calcium (Ca), calciumX-phosphorous product (CAXPi) and intact parathyroidhormone (iPTH) according to the Kidney Disease Outcomes Quality Initiative (K/DOQI) clinical guidelines. Despite a similar prevalence, the severity of CKD-MBD appeared different to the results reported in the USA. Interestingly, none of the serum biomarkers or number of serum biomarkers within KDOQI targets was independently associated with all-cause and cardiovascular (CV) mortality. Among treatments, Sevelamer was the only drug independently associated with lower all-cause and cardiovascular mortality (p<0.001)., Conclusion: The RISCAVID study highlights the difficulty of controlling bone mineral metabolism in HD patients and lends support to the hypothesis that a carefully chosen phosphate binder might impact survival in HD patients.

Background: Asymmetric dimethylarginine (ADMA)and symmetric dimethylarginine (SDMA) originate from hydrolysis of methylated proteins, including dietary proteins, and are retained in end-stage renal disease(ESRD). This study aimed to detect the correlation of ADMA and SDMA to nutritional parameters in dialysis patients., Methods: Before and after a single dialysis session, larginine, ADMA and SDMA plasma levels were measured in 38 hemodialysis patients by HPLC-tandem mass spectrometry. Biochemistry, protein intake, anthropometry and bioelectric impedance analysis were evaluated., Results: Predialysis plasma levels of ADMA were higher than in normal controls (n=20) (1.14 +/- 0.27 mumol/Lvs. 0.56 +/- 0.09 mumol/L, p<0.001), as were SDMA levels(3.49 +/- 1.00 mumol/L vs. 0.44 +/- 0.13 mumol/L, p<0.001).On univariate analysis, predialysis ADMA levels were inversely related to BMI and albumin levels, whereas SDMA was directly related to nPNA, phase angle, prealbumin and creatinine serum levels. ADMA/SDMA ratio was inversely related to prealbumin and albumin, creatinine, urea and phosphorus serum levels, as wellas nPNA, but positively to C-reactive protein. On multiple regression analysis, serum albumin and BMI were the stronger predictors of ADMA, whereas prealbumin serum levels followed by dietary protein intake were the stronger predictors of SDMA. Prealbumin followed by C-reactive protein was predictive of the ADMA/SDMA molar ratio., Conclusions: Our results confirm that ADMA and SDMA levels are increased in ESRD patients and suggest that a link may exist with inflammation and nutritional status. High ADMA levels associated with reduced SDMA may be a predictive marker of malnutrition-inflammation-atherosclerosis syndrome.

Background: The 'RISchio CArdiovascolare nei pazienti afferenti all' Area Vasta In Dialisi' (RISCAVID) study is an observational and prospective trial including the whole chronic haemodialysis (HD) population in the northwest part of Tuscany (1.235 million people). The aim of the study was to elucidate the relevance of traditional and non-traditional risk factors of mortality and morbidity in HD patients as well as the impact of different HD modalities., Methods: A total of 757 HD patients (mean age 66 +/- 14 years, mean dialytic age 70 +/- 76 months, diabetes 19%) were prospectively followed up for 30 months and all-cause mortality, cardiovascular (CV) mortality and non-fatal CV events (acute myocardial infarction and stroke) were registered. At the time of the enrolment, demographic, clinical and laboratory data of the whole population were entered into a centralized database. Serum albumin, high-sensitive C-reactive protein (CRP), interleukin-6 (IL-6) and interleukin-8 (IL-8) were centrally determined at the start of the study. Patients were stratified into three groups according to the HD modality: standard bicarbonate HD (BHD) (n = 424), haemodiafiltration (HDF) with sterile bags (n = 204) and online HDF (n = 129). The Cox proportional hazards regression assessed adjusted differences in CV morbidity and mortality risk; a multivariate analysis was also performed., Results: All-cause and CV mortality was 12.9%/year and 5.9%/year, respectively. Patients with combined high levels of CRP and pro-inflammatory cytokines showed an increased risk for CV (RR 1.9, P < 0.001) and all-cause mortality (RR 2.57, P < 0.001). Multivariate analysis adjusted for comorbidity and demographic showed CRP as the most powerful mortality predictor (P < 0.001) followed by IL-6. The Cox proportional hazards regression assessed that online HDF and HDF patients had a significantly increased adjusted cumulative survival than BHD (P < 0.01)., Conclusions: Data at 30 months from this study showed the synergic effect of CRP and pro-inflammatory cytokines as the strong predictors of all-cause and CV mortality. HDF was associated with an improved cumulative survival independent of the dialysis dose.

Background: The endogenous inhibitor of nitric oxide (NO) synthase, asymmetric dimethylarginine (ADMA), is a strong cardiovascular (CV) risk marker in patients with chronic renal insufficiency. Statins have pleiotropic effects and are currently considered as potential ADMA-lowering agents., Methods: We investigated the effect of simvastatin on plasma ADMA levels in 35 patients with chronic kidney disease (CKD) by performing a secondary analysis of a randomized double-blind placebo-controlled trial where patients were randomized to receive simvastatin or placebo for 6 months., Results: Plasma ADMA was higher in CKD patients (0.84 +/- 0.14 micromol/L) than in healthy subjects (0.69 +/- 0.10 micromol/L) (p<0.001). In CKD patients, ADMA at baseline was related directly with triglycerides (r=0.42, p=0.01) and inversely with HDL cholesterol (r=-0.37, p=0.03) and creatinine clearance (p=0.03). As expected, simvastatin caused significant reductions in total cholesterol, LDL cholesterol and triglycerides, as well as in C-reactive protein (CRP; -28%, p=0.001) and IL-6 (-20%, p=0.05) but failed to decrease plasma ADMA both in crude and adjusted analyses., Conclusions: Simvastatin does not modify plasma ADMA. Because raised ADMA is known to prevent the favorable effect of statins on myocardial blood flow, cointerventions aimed at lowering or antagonizing ADMA may either prompt or potentiate the cardiovascular protective effect of simvastatin.

Background: Statins reduce lipid levels, inflammation and cardiovascular events in patients with coronary artery disease; CKD patients show increased risk of cardiovascular and increased plasma levels of IL-6 and IL-8., Aim: To evaluate the in vitro effect of simvastatin (S) or fluvastatin (F) on the lipopolysaccharide (LPS) stimulated secretion of IL-6 and IL-8 from monocytes of chronic kidney disease patients (CKD) in K-DOQI stages 3-5., Methods and Subjects: Monocytes enriched peripheral blood (PBMC) from 28 CKD (15 in K-DOQI stages 3-4, Group I, and 13 in K-DOQI stage 5 on hemodialysis, Group II) and 10 healthy subjects (HS), were isolated by Ficoll-gradient centrifugation. Cells were incubated with LPS 100 ng/ml or with LPS plus increasing doses of statins (from 10(-6) to 10(-8) M ) for 24 h. Surnatant IL-6 and IL-8 concentrations were determined by EIA., Results: Basally the mean concentration of IL-6 and IL-8 was higher in patients than in HS and in Group II than in Group I (IL6: HS 285 +/- 77 pg/ml, Group I 365 +/- 178 pg/ml, Group II 520 +/- 139 pg/ml- IL8 HS 180 +/- 75 pg/ml, Group I 1722 +/- 582 pg/ml, Group II 4400 +/- 1935 pg/ml). After addition of LPS the mean concentration of IL-6 and IL-8 increased in all groups (IL6: HS 1740 +/- 178 pg/ml, Group I 3754 +/- 672 pg/ml, Group II 4800 +/- 967 pg/ml; IL8: HS 450+/-132 pg/ml, Group I 9700+/-2837 pg/ml, Group II 11608 +/- 2316 pg/ml). After the addition of LPS plus increasing doses of S or F from 10(-10) to 10(-6) M, a significantly lower cytokine concentration compared to the data after LPS alone was observed (IL6: HS 45%, Group I 75%, Group II 50%; IL8: HS 100%, Group I 65%, Group II 35%)., Conclusions: These data confirm that cytokine release is increased in CKD patients and that is highest in the most severe patients. Furthermore they suggest that fluvastatin or simvastatin can be used in order to reduce the high cardiovascular risk.

Macrophage-stimulating protein (MSP) is a scatter factor that causes cell proliferation and migration, and receptor origin nantaise (RON) is its receptor. RON is expressed in macrophages and mesangial cells, and MSP is produced by renal tubular cells. This study investigated whether MSP/RON participate in the pathogenesis of anti-Thy 1 nephritis, a glomerular disease that is characterized by invasion of circulating monocytes into glomeruli and migration and proliferation of mesangial cells. In vivo, renal function and histopathology were studied in rats that had anti-Thy 1 disease and were untreated and treated with a neutralizing anti-MSP antibody. In vitro, whether monocytes express RON and whether MSP has a chemotactic effect on monocytes were studied. In vivo, in anti-Thy 1 disease, MSP was expressed de novo in glomeruli, and neutralization of MSP attenuated the rise in serum creatinine and proteinuria, stopped glomerular neutrophil and monocyte influx, protected from glomerular injury, and lessened mesangial cell overgrowth. In vitro, unstimulated monocytes did not express RON, but the stimulation with LPS induced de novo RON expression. LPS-stimulated monocytes were attracted by MSP. These results demonstrate a pathogenic role of the MSP/RON system in anti-Thy 1 nephritis.

Introduction: Although several registries collecting data of patients with kidney diseases exist, only a few specifically collect data relating to renal biopsy. Kidney biopsy has been performed routinely in Pisa since 1977; the aim of this study was to report the relative frequency of nephropathies according to gender, age at time of biopsy, clinical presentation and renal function, based on histological diagnoses during the years 1977 through 2005. During this time, 3,810 kidney biopsies were performed, of which 89.3% were from native (n=3,446) and 10.7% from transplant kidneys. Throughout this period, 5% of renal biopsies were not diagnostic, so in this paper we report data regarding 3,269 native kidney nephropathies., Methods: During the years 1977 through 2005, data for renal biopsies were collected on specific registers filled out by clinicians. Information collected in the database included a variety of indicators, such as clinical anamnesis, creatinine clearance, daily proteinuria, hemoglobin levels, blood pressure, height and weight, clinical presentation, and current medications. Clinical presentation was defined as urinary abnormalities (UA), nephrotic syndrome (NS) and acute nephritic syndrome (ANS). Renal diseases were divided into 4 major categories: primary glomerulonephritis (GN), secondary GN, tubulointerstitial nephropathies (TIN) and vascular nephropathies (VN)., Results: From 1977 up to 1987, a mean of 95 +/- 18 renal biopsies/year were performed; this number significantly increased to 185 +/- 22 renal biopsies/year (range 138-200) (p<0.001) in the following period (1988-2005). Renal biopsy was more frequently performed in males (59%) compared with females (41%). Of all diseases of the native kidney, primary GN was the most frequent (66%), followed by secondary GN (25.6%), TIN (4.2%) and VN (4.2%). The type of primary GN with the highest frequency was mesangial GN (both IgA and non-IgA) (45.7%), followed by membranous GN (23%), focal segmental glomerulosclerosis (19.8%), minimal change disease (5.3%), crescentic GN (4.2%) and postinfectious GN (2%). In terms of age, renal biopsy was more frequently performed in patients aged 20 to 60 years, and nearly 60% of patients presented a glomerular filtration rate (GFR) >60 ml/min at the time of biopsy. The main clinical reason for performing renal biopsy was UA, in all the types of nephropathies., Conclusions: We confirm data that renal diseases are more frequent in men, with the exception of secondary GN. The mean age at diagnosis was 42 years resulting from the tendency not to perform renal biopsies in children and in elderly patients. Renal biopsy was mainly performed in patients with GFR >60 ml/min and asymptomatic urinary abnormalities suggesting concern on the part of clinicians regarding glomerular diseases. The tendency to perform renal biopsies has been significantly increasing throughout our follow-up period.

Background: Since it has been demonstrated that soy diet can improve endothelial function, in the present study we evaluated the effect of dietary substitution of 25 g of animal proteins with soy proteins on endothelial dysfunction in renal transplant patients., Methods: In 20 renal transplant patients (55 +/- 11 years, serum creatinine 1.7 +/- 0.6 mg/dl), brachial artery flow mediated dilation (FMD) and endothelium-independent vasodilation (sublingual nitroglycerine, 25 microg) were measured at baseline, after 5 weeks of a soy diet and finally after 5 weeks of soy wash-out. Changes in plasma lipids, markers of oxidative stress (lipid peroxides, LOOH) and inflammation (C-reactive protein), isoflavones (genistein and daidzein), asymmetric dimethyl arginine (ADMA) and L-arginine were also evaluated., Results: At baseline, patients showed a significantly lower FMD as compared with age-matched healthy subjects (3.2 +/- 1.8 vs 6.3 +/- 1.9, respectively; P < 0.001), while response to nitroglycerine was similar. After soy diet, actual protein intake was not changed, cholesterol and lipid peroxides were significantly reduced, and isoflavones were detectable in plasma. Soy diet was associated with a significant improvement in FMD (4.4 +/- 2.0; P = 0.003 vs baseline), while response to nitroglycerine was unchanged. Improvement in FMD was related to L-arginine/ADMA ratio changes, but no significant relation was found to changes in cholesterol, lipid peroxides or genistein and daidzein plasma concentrations. After 5 weeks of soy diet discontinuation, FMD (3.3 +/- 1.7%) returned to baseline values and isoflavones were no longer detectable in plasma., Conclusions: A soy protein diet for 5 weeks improves endothelial function in renal transplant patients. This effect seems to be strictly dependent on soy intake as it disappears after soy withdrawal and is mediated by an increase in the L-arginine/ADMA ratio, independently of change in lipid profile, oxidative stress or isoflavones.

Background: HFR [double chamber haemodiafiltration (HDF) with reinfusion of regenerated ultrafiltrate] is a novel dialytic method which combines the processes of diffusion, convection and adsorbance. In this technique an adsorbent cartridge of resin and charcoal may regenerate the ultrafiltrate suggesting its use as an endogenous substitution fluid. The aim of this multicentre randomized cross-over study was to compare HFR to online HDF in terms of inflammatory and nutritional parameters., Methods: After a 1 month run-in period of standard bicarbonate dialysis (HD) with a synthetic membrane, 25 chronic dialytic patients were randomized (A-B or B-A) to be treated by HFR (A) with a two-chamber filter (SG 8 Plus - high permeability Polysulphone HF 0.7 m2 + SMC 1.95 sqm; Bellco, Mirandola, Italy) or by online sterile bicarbonate HDF. Each study period of 4 months was separated by 1 month of HD and the entire length of the study was 10 months. CRP levels were measured by a highly sensitive nephelometric assay (Dade, Behring) with a sensitivity of 0.1 microg/ml. Cytokine concentrations were determined by EIA [Interleukin (IL) 6, Biosource, USA and IL-10 Bender MED-Systems, Vienna]. The sensitivity thresholds were < 5 pg/ml for IL-6 and < 8 pg/ml for IL-10. Serum leptin was determined with a ELISA method (Biosource, USA). All parameters were determined monthly in patients starting a midweek dialytic session., Results: Plasma CRP and IL-6 were significantly reduced during the 4 months of HFR and HDF: CRP from 8.0 +/- 3.2 to 5.6 +/- 3.4 mg/l with HFR (P < 0.05) and from 9.4 +/- 4.3 to 5.9 +/- 3.9 mg/l with HDF (P < 0.05). IL-6 decreased from 14.8 +/- 6.3 to 10.1 +/- 3.2 with HFR (P < 0.02) and from 12.1 +/- 4.2 to 9.6 +/- 3.7 with HDF (P = ns) with a percentage decrease after 4 months of 32% with HFR vs 21% with HDF. During the 1 month wash-out period with HD, CRP increased from 5.7 +/- 3.6 to 8.7 +/- 3.9 mg/l (P < 0.01) and IL-6 from 10 +/- 3.4 to 13.5 +/- 5.2 pg/ml (P < 0.01). A significant increase in IL-10 was detected either in HFR (from 4.8 +/- 2.1 to 6.89 +/- 1.7 pg/ml) and in HDF (from 3.3 +/- 1.7 to 8.95 +/- 4.3 pg/ml; P < 0.05) after 4 months. No significant variation in serum leptin levels were observed during the study. CRP and IL-6 were highly correlated (r = 0.54; P < 0.001) as was serum albumin and prealbumin (r = 0.39; P < 0.001). Serum albumin was negatively correlated with CRP (r = -0.26; P < 0.01) and IL-6 (r = -0.19; P < 0.05); serum prealbumin was correlated with IL-6 (r = 0.37; P < 0.001) and with CRP (r = 0.24; P < 0.01)., Conclusions: Haemodiafiltration with online regeneration of ultrafiltrate and online HDF are highly biocompatible techniques and no significant difference between HFR and online HDF was observed in terms of reduction of inflammatory markers. Further studies with a longer follow-up are needed to evaluate the clinical relevance of the online endogenous reinfusion to counteract the chronic inflammatory state of the uraemic patient.

Background: The beneficial effects of statins in reducing cardiovascular events have been attributed predominantly to their lipid-lowering effects, recent studies suggest that these effects might be due to their anti-inflammatory properties. We here investigate the in vivo and in vitro effects of simvastatin on cytokine production in pre-dialysis chronic renal failure patients., Methods: Our clinical study has been designed as a randomized double-blind placebo controlled study. A total of 55 chronic kidney disease (CKD) patients at stages 3 and 4 (mean creatinine clearance 45 ml/min, range 15-60) were randomly assigned to receive simvastatin 40 mg/day or placebo, added to their ongoing treatment, for 6 months. Blood samples were obtained at baseline, and after 3 and 6 months of observation for the determination of lipids, inflammatory markers and renal function. For the in vitro studies, the effect of increasing doses of simvastatin on cytokine production [namely interleukin (IL)-6 and IL-8] in human cultured monocytes from 10 healthy subjects (HS) and 15 CKD patients stimulated by lipopolysaccharide (LPS) was investigated., Results: A significant reduction in total cholesterol from 221+/-44 mg/dl to 184+/-41 mg/dl (3 months) and to 186+/-39 mg/dl (6 months) (P<0.02) and low-density lipoprotein cholesterol from 139+/-40 mg/dl to 104+/-29 mg/dl (3 months) and to 100+/-31 mg/dl (6 months) (P<0.001) was observed in the 28 patients treated with simvastatin. In this group, C-reactive protein (CRP) levels significantly decreased from 2.6 mg/l [interquartile range (IQR 4.9)] to 2.0 mg/l (IQR 1.9) (P = 0.03) at 6 months (P<0.05). A parallel reduction of IL-6 levels from 5.1 pg/ml (IQR 3.8) to 3.5 pg/ml (IQR 3.1) (P = 0.001) at 6 months was also observed. No significant reduction in inflammatory markers [CRP from 5.1 mg/l (IQR 1.9) to 5.4 mg/l (IQR 1.3) (P = NS) at 6 months] or plasma lipids [LDL-cholesterol from 127+/-32 mg/dl to 131+/-21 mg/dl (6 months)] was observed in the 27 patients of the placebo group. In the in vitro studies, the average value for cell-associated IL-6 and IL-8 was higher in CKD (155+/-95 pg/ml monocytes for IL-6 and 722+/-921 pg/ml monocytes for IL-8) vs HS (137+/-87 pg/ml monocytes and 186+/-125 pg/ml monocytes) (P<0.01) and was not affected by simvastatin alone. LPS resulted in a significant increase in cytokine production (IL-6: 1954+/-321 pg/ml monocytes for CKD and 1451+/-237 pg/ml monocytes for HS; P<0.001); the simultaneous addition of increasing doses of simvastatin to these cultures induced a dose-dependent inhibition of IL-6 and IL-8 production in stimulated peripheral blood mononuclear cells in all groups., Conclusions: These results indicate that simvastatin in commonly used doses has an in vitro and in vivo anti-inflammatory effect in CKD patients, and may play an important role in counteracting the mechanisms involved on the pathogenesis of cardiovascular disease.

Cyclosporine (CyA) is an immunosuppressive agent used after solid organ transplantation, but its clinical use is limited by side effects, the most important of which is nephrotoxicity. In a previous work we demonstrated that L-propionylcarnitine (L-PC), a propionyl ester of L-carnitine, is able to prevent CyA-induced acute nephrotoxicity reducing lipid peroxidation in the isolated and perfused rat kidney. CyA administration was associated with a dose dependent increase in renovascular resistance prevented by a pretreatment with L-PC. The aim of the present study was to confirm L-PC protective effect, previously described in vitro, in an in vivo rat model. Chronic nephrotoxicity study was carried out for 28 days. L-PC was administered (i.p. 25 mg/kg b.w.) since the first day, while CyA treatment was performed for the last 21 days (by oral administration 25 mg/kg b.w.). We demonstrate that L-PC was able to significantly lower blood pressure in CyA treated animals and to prevent CyA induced decrease in creatinine clearance. Moreover renal tissue analysis revealed that L-PC was able to reduce lipid hydroperoxide content and morphological abnormalities associated to chronic CyA administration. In conclusion our study demonstrated for the first time in vivo that L-PC protects against functional and tissue damage associated to chronic CyA administration.

Online hemodiafiltration appears to be the most effective technique of renal replacement therapy in many respects. Removal of small and high-molecular weight substances is enhanced. Modern technology ensures a safe, online production of reinfusion fluids. Nonetheless, stringent maintenance rules are required for the production of sterile and nonpyrogenic-dialysate solutions. In this review, we will critically review the state of the art of the clinical effects derived from the use of ultrapure dialysate and the online production of dialysate fluids in high-flux hemodiafiltration.

Background: Cohort studies have demonstrated an association between C-reactive protein (CRP) and interleukin-6 (IL-6) and all-cause and cardiovascular mortality in end-stage renal disease (ESRD) patients. Interleukin-8 (IL-8) appears to be not only the plasma expression of the acute-phase response but also a direct pathogenetic mediator of the atherosclerotic process., Methods: To evaluate the role of IL-8 in predicting outcome, 76 chronic dialytic patients were prospectively followed for 18 months. At baseline, blood samples were taken for analysis of high-sensitivity CRP, IL-6, IL-8 and other standard laboratory analyses., Results: Median IL-8 was 5.2 mg/l, therefore near half of the patients had IL-8 values within the range of 'normal limits'. IL-6 and CRP were significantly correlated (r = 0.45, p < 0.001) and a positive correlation was also found between IL-6 and IL-8 (r = 0.39, p < 0.001). The correlation coefficient between IL-6 and CRP was 0.43 (p < 0.001) and 0.50 (p < 0.001) in patients without and with history and/or clinical signs of cardiovascular disease, respectively. After a follow-up of 1.5 years, 8 patients had died from cardiovascular causes and another 7 patients for other reasons; furthermore 9 major nonfatal cardiovascular events were recorded. Stepwise regression analysis showed IL-8 as the strongest independent predictor of all-cause and cardiovascular events (p = 0.0025) even after adjustment for age and dialytic age, followed by IL-6 and CRP (p < 0.01)., Conclusion: Despite a small population and a relatively short follow-up period, this study firstly demonstrated that IL-8 is a powerful independent predictive factor for cardiovascular and overall mortality cause in ESRD patients., (2006 S. Karger AG, Basel.)

In this study, we investigated the effect of 1,25(OH)2D3 on proteinuria and on the alteration of slit diaphragm-associated proteins induced by anti-Thy 1.1 in Wistar rats. Four groups of animals were studied: group I, anti-Thy 1.1 treated rats; group II, anti-Thy1.1 treated group that at day 2, after the onset of overt proteinuria, started the treatment with 1,25(OH)2D3; group III, normal control rats injected with vehicle alone; group IV, rats that received only 1,25(OH)2D3. At day 2, in group I and II, before the administration of 1,25(OH)2D3, protein excretion was significantly increased when compared to controls. Overt proteinuria was maintained until day 14 in group I whereas in group II protein excretion was significantly reduced from day 3 to day 14. Moreover, treatment with 1,25(OH)2D3 abrogated podocytes injury, detected as desmin expression and loss of nephrin and zonula occludens-1 (ZO-1), two slit diaphragm-associated proteins, and glomerular polyanion staining, that were observed in group I. In conclusion, these results suggest that 1,25(OH)2D3 administrated with a therapeutic regiment may revert proteinuria, counteracting glomerular podocyte injury.

Ochratoxin A (OTA), is a nephrotoxic mycotoxin present in wine, which is nephrotoxic in humans. Our working hypothesis is that natural substances in wine may counteract OTA toxicity. Thirty-six rats were randomized to OTA dissolved in saline, red wine, or 13.5% ethanol or to OTA-free wine, ethanol, or saline. OTA (289 microg/kg of body weight/48 h) was administered by gastric gavage for 2 weeks. Serum creatinine, tubular enzymuria, renal lipohydroperoxides (LOOH), reduced (GSH) and oxidized (GSSG) glutathione, and renal superoxide dismutase activity (SOD) were determined in renal tissue. OTA alone produced significant increases in renal lipoperoxides and significant decreases in SOD and GSH/GSSG ratio. In red wine or ethanol, OTA was less nephrotoxic, reducing oxidative damage as revealed by LOOH. In OTA-wine and OTA-ethanol groups, SOD activity was higher than in the OTA-treated one, suggesting that both ethanol and nonalcoholic fractions may preserve antioxidant reserve. GSH/GSSG ratio was significantly preserved only in the OTA-wine group and not in OTA-ethanol. Red wine may exert a protective effect against OTA nephrotoxicity by limiting oxidative damage. The ostensible protection afforded by ethanol deserves further investigation.

Chronic inflammation has been repeatedly reported in individuals undergoing hemodialysis. C-reactive protein (CRP) is considered a marker of chronic inflammation, as well as a mediator of the atherosclerotic process. Clinical and epidemiologic studies are based on plasma values obtained with the use of various automated methods. Our aim was to test 3 commercially available methods and compare the values obtained with the use of these tests in a population of individuals undergoing hemodialysis. We compared the following methods: immunoturbidimetry (AU2700 biochemistry analyzer; Olympus, Rungis, France) laser nephelometry (Behring Diagnostics, Marburg, Germany), and nephelometry (Beckman Instruments, Fullerton, Calif. The 3 methods were used in 3 different centers: Montpellier, France; and Pisa and Turin, Italy, respectively. We prepared samples for the estimation of imprecision values (ie, coefficient of variation [CV]) from the plasma of normal patients by adding purified C-reactive protein at concentrations ranging from 2.6 to 180 mg/L for intraassay variation and concentrations of 0, 1, 2, 3, 5, 10, 20, 50, 100, 150, and 180 mg/L for interassay variation. Intraassay imprecision was determined with the use of 10 replicate analyses on the same sample of the same day. We assessed interassay imprecision using the same sample, divided into aliquots and measured on 5 consecutive days. Agreement between methods was assessed on predialysis serum samples collected from patients with stable chronic kidney disease who were undergoing long-term hemodialysis at the 3 different centers (Montpellier,192; Pisa, 56; Turin,98). Serum was separated from the red cells and stored in 3 aliquots at -70 degrees C until it could be analyzed. Samples were thawed only once, circulated among the 3 centers, and each tested with all 3 of the methods. The Beckman method yielded the most precise results, with intraassay CVs ranging from 1 to 2 and interassay CVs ranging from 1 to 4. The Behring method was the least precise, with intraassay and interassay CVs ranging from 12 to 15 and 7 to 16, respectively. The results of the Olympus method fell between those of the other 2 methods. Agreement between the results of the Olympus and Behring methods was satisfactorily. The Beckman and Olympus methods yielded, on average, similar results over the entire range of CRP values. We detected significant disagreement between the Beckman method and the other 2 methods, obtaining results 10 to 100 times lower with the Beckman method. This became evident in terms of kappa-statistics. Our findings emphasize the need for careful assessment of the methods used to detect CRP in serum samples. Failure to do so may ultimately have a negative impact on the real relevance of CRP as a marker and on the role of chronic implication particularly in epidemiologic studies.