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Pretransplant donor biopsy (PTDB)-based marginal donor allocation systems to single or dual renal transplantation could increase the use of organs with Kidney Donor Profile Index (KDPI) in the highest range (e.g. >80 or >90), whose discard rate approximates 50% in the United States. To test this hypothesis, we retrospectively calculated the KDPI and analyzed the outcomes of 442 marginal kidney transplants (340 single transplants: 278 with a PTDB Remuzzi score

Diabetic nephropathy (DN) is a complication associated with diabetes, leading to end-stage renal disease (ESRD). Despite significant progress in understanding DN, the cellular mechanisms leading to the renal damage are incompletely defined. In this study, with the aim to identify urine biomarkers for the early renal alterations in type 2 diabetes mellitus (T2D), we performed urinary proteomic analysis of 10 normoalbuminuric patients with T2D, 12 patients with type 2 DN (T2DN), and 12 healthy subjects. Proteins were separated by 2-DE and identified with ESI-Q-TOF MS/MS. Comparing the patients proteomic profiles with those of normal subjects, we identified 11 gradually differently changed proteins. The decreased proteins were the prostatic acid phosphatase precursor, the ribonuclease and the kallikrein-3. Eight proteins were progressively increased in both patients groups: transthyretin precursor, Ig κ chain C region, Ig κ chain V-II region Cum, Ig κ-chain V-III region SIE, carbonic anhydrase 1, plasma retinol-binding protein, β-2-microglobulin precursor, β-2-glycoprotein 1. The proteomic analysis allowed us to identify several increased urinary proteins, not only in T2DN but also in T2D patients in which the microalbuminuria was in the normal range. These patterns of urinary proteins might represent a potential tool for a better understanding of diabetic renal damage.

A multicentric study was carried out to investigate the safety and effectiveness of long-term treatment of chronic uremic patients by a regular combination of hemodialysis and charcoal hemoperfusion. 39 RDT patients from five dialysis centers were treated for up to 12 months. The combined treatment proved to be safe, well tolerated and effective in improving certain dialysis-resistant uremic signs, mainly severe peripheral neuropathy, asthenia, anorexia, nausea, lack of well-being and relapsing pericarditis. There was also a marked decrease, after several weeks of combined treatment, in plasma values of urea, uric acid and creatinine.

The results obtained by electrophysiological recording allow to discover the constant presence of alterations to the central and/or peripheral nervous system in the uremic syndrome. Thus, it becomes possible to demonstrate the existence of an actual 'uremic neuropathy'. Moreover, the results show the persistence and progression of uremic involvement in the course of dialytic treatment; only after kidney transplantation a return to normal takes place. Methodological and interpretative progress will allow us in the future to broaden our knowledge of uremia by providing a useful guide to therapeutic strategies.

Activation of coagulation during hemodialysis (HD) is a relevant clinical problem, especially when patients at risk of bleeding are treated. However, little is known about the relative contribution of the various components of the circuit to the thrombotic process. Thus, an experimental model was developed that is aimed at evaluating biochemical markers of coagulation activation at different times and sites throughout the HD circuit. A HD blood-tubing set with integrated arterial and venous chambers (cartridge-line set) was used, which was added with the following sampling points: at the beginning of the arterial line (P1), before the blood pump (P2), after the blood pump (P3), and at the end of the venous line (P4). A bypass system allowed us to circulate the blood only into the blood lines for the first 20 min of the extracorporeal circulation. The extracorporeal circuit was rinsed with 1.7 L of heparinized saline (2,500 IU/L) that was completely discarded before patient connection. A continuous administration of unfractionated heparin (500-800 IU/h) without a starting bolus was adopted as a low heparin extracorporeal treatment. Samples were collected before the start of the extracorporeal circulation from the fistula needle (T0P0), after 5 (T1), 10 (T2), and 20 min (T3) from P1, P2, P3, and P4. After 20 min, the blood was returned to the patient using only saline and HD was then started, circulating the blood through the dialyzer. Further samples were obtained from P1 and P4 after 5 (T4) and 210 min (T5). Plasma levels of coagulation activation markers-thrombin-antithrombin complex (TAT) and prothrombin fragment 1 + 2 (F1 + 2)-were evaluated in all the samples in 12 stable HD patients. In each patient, the activated partial thromboplastin time (APTT) was measured at T0P0 and T1-T5 from P1. No significant changes were found at any time as far as F1 + 2 is concerned. However, TAT levels increased over time only after the start of HD, suggesting that the latter test could be more useful in order to detect coagulation activation during HD. The same experiments performed with nonheparin-primed extracorporeal circuit showed similar results. The blood lines used did not significantly activate coagulation during the first 20 min, whereas only 5 min of blood circulation throughout the whole circuit increased TAT values, which still remained lower than previous reports, even after 210 min of treatment.

Benzoyl peroxide is a free-radical generating compound widely used in the polymer industry and also in pharmaceuticals as antimicrobial agent to treat acne. However, benzoyl peroxide causes irritation and contact dermatitis in about 1% of patients. Concern over the use of this compound is motivated by the demonstration that it can also act as skin tumor promoter in mice. In addition, benzoyl peroxide induces DNA strand breaks in many cells, including keratinocytes. Benzoyl peroxide toxicity is presumably mediated by the formation of reactive free radicals and by the consumption of intracellular antioxidants. In this work we investigated the effect of both the lipophilic antioxidant α-tocopherol and the hydrophilic thiol donor N-acetylcysteine (NAC) in human keratinocyte line HaCaT exposed to benzoyl peroxide. A protective effect against benzoyl peroxide cytotoxicity was achieved when cells were grown on a α-tocopherol layer. On the contrary, the addition of α-tocopherol dissolved in ethanol had a pro-oxidant effect, leading to an enhancement of benzoyl peroxide toxicity. Cytotoxicity was also reduced adding NAC to the culture medium; the presence of both NAC and α-tocopherol exerts a synergis- tic cytoprotection. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:107–114, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20008

Background andobjectivesThecalcimimeticcinacalcet reduced therisk of death or cardiovascular (CV) events in older, but not younger, patients with moderate to severe secondary hyperparathyroidism (HPT) who were receiving hemodialysis. To determine whether the lower risk in younger patients might be due to lower baseline CV risk and more frequent use of cointerventions that reduce parathyroid hormone (kidney transplantation, parathyroidectomy, and commercial cinacalcet use), this study examined the effects of cinacalcet in older ($65 years, n=1005) and younger (,65 years, n=2878) patients. Design, setting, participants, & measurements Evaluation of Cinacalcet HCl Therapy to Lower Cardiovascular Events (EVOLVE) was a global, multicenter, randomized placebo-controlled trial in 3883 prevalent patients on hemodialysis, whose outcomes included death, major CV events, and development of severe unremitting HPT. The age subgroup analysis was prespecified. ResultsOlderpatients hadhigher baselineprevalenceof diabetesmellitusandCV comorbidity. Annualizedrates of kidney transplantation and parathyroidectomy were .3-fold higher in younger relative to older patients and were more frequent in patients randomized to placebo. In older patients, the adjusted relative hazard (95% confidence interval) for the primary composite (CV) end point (cinacalcet versus placebo) was 0.70 (0.60 to 0.81); in younger patients, the relative hazard was 0.97 (0.86 to 1.09). Corresponding adjusted relative hazards for mortality were 0.68 (0.51 to 0.81) and 0.99 (0.86 to 1.13). Reduction in the risk of severe unremitting HPT was similar in both groups. Conclusions In the EVOLVE trial, cinacalcet decreased the risk of death and of major CV events in older, but not younger, patients with moderate to severe HPT who were receiving hemodialysis. Effect modification by age may be partly explained by differences in underlying CV risk and differential application of cointerventions that reduce parathyroid hormone. Clin J Am Soc Nephrol 10: ccc–ccc, 2015. doi: 10.2215/CJN.07730814

Changes in conjugated linoleic acid and its metabolites in patients with chronic renal failure. Background Conjugated linoleic acid (CLA) is a mixture of isomers of linoleic acid with conjugated double bonds that constitutes the most abundant fatty acid with conjugated dienes (CDs) in humans. CLA, erroneously considered in the past as a product of lipoperoxidation, has a dietary origin and has shown to possess anticarcinogenic and anti-atherogenic activity, mainly in animal studies. CLA can be metabolized to conjugated linolenic acid (CD18:3) and to conjugated eicosatrienoic acid (CD20:3) and these metabolites may be implicated in CLA activity. Because of the presence of dyslipidemia and the high incidence of cardiovascular and neoplastic diseases in uremic patients, we evaluated CLA and its metabolites in these patients in order to evaluate their metabolism and site distribution. Methods We measured CLA, CD18:3, CD20:3, CD fatty acid hydroperoxides (lipoperoxidation products), and linoleic acid in the plasma, adipose tissue, and red blood cell (RBC) membranes by using high-pressure liquid chromatography in the following groups: ( 1 ) 23 chronic renal failure (CRF) patients with creatine clearance (C Cr )> 10 mL/min (26.2 ± 16.7); ( 2 ) 21 end-stage CRF patients in conservative treatment with C Cr 3 ) 30 hemodialysis (HD) patients; and ( 4 ) 30 healthy controls. Results The incorporation of CLA, CD18:3, and CD20:3 in RBC membranes was significantly reduced in group 1 and was even more reduced in groups 2 and 3. CLA significantly increased both in the plasma and adipose tissue of end-stage CRF patients only. CD18:3 and CD20:3 did not change in the plasma and adipose tissue of any group. No significant changes in linoleic acid and CD fatty acid hydroperoxides were found. Conclusions The alterations of CD in CRF patients are not due to lipoperoxidation. The increased levels of CLA in plasma and adipose tissue of end-stage CRF patients may be due either to a reduced metabolization of CLA to CD18:3 and CD20:3, or to an altered site distribution with reduced incorporation in cellular membranes and accumulation in the plasma and adipose tissue. The clinical significance of these changes remains to be investigated.

Membranes made from synthetic polymers, in general, are considered as being biocompatible membranes and tend to be treated as a homogeneous group. However, all of these membranes have multiple and different characteristics that may contribute to interactions with blood components. As a consequence, the biocompatibility profile of synthetic membranes may vary. In the present cross-over study, we examined by flow cytometry the effects (expressed as % change from predialysis values) of three different synthetic polymers (polysulfone, PSF; polyacrylonitrile-co-sodium methallyl sulfonate, AN69; ethylenevinylalcohol, EVAL) on the expression of leukocyte adhesion molecules (CD11b/CD18, CD15s) and the interactions between leukocytes and platelets under conditions of routine clinical use. For neutrophils, a statistically significant difference was found in CD15s expression for EVAL as compared to AN69 (p Our data demonstrate that in terms of leukocyte adhesion receptors and platelet-leukocyte interactions, the biocompatibility profile of the synthetic membranes polysulphone, AN69 and EVAL shows many similarities but also several significant differences. Our results support the concept that biocompatibility evaluation of each membrane should be based exclusively on data generated by that membrane in order to avoid errors based on assumptions about group characteristics.

(i) Routine biochemistry: Plasma BUN, creatinine, electroThis paper deals with the results of clinical and laboratlytes, haematocrit, etc. ory investigations carried out on six chronic uraemic (ii) Erythropoiesis studies: 59Fe, 51Cr, transferrin, reticulopatients who had been on RDT for 1–3 years. They cytes, sternal puncture. presented evidence of dialysis resistant signs and (iii) Blood pressure studies: Plasma renin concentration, symptoms despite an increase in the total hours of serum aldosterone, 22 Na, cardiac index. dialysis/week from 22 to 30. These patients were put (iv) Nerve conduction velocity: peroneal and median nerves. on a new dialysis programme with short daily dialyses (v) Iliac crest percutaneous bone biopsies: using a trephine (3–4 h) for 5 days/week. of 5 mm internal diameter; calcium balance and 47Ca The overall results of this study indicate that a daily turnover. (vi) Metabolic studies: glucose (peripheral uptake, oral tolerdialysis programme may be considered as the most ance curves); lipid (triglycerides, NEFA, phospholipid physiological approach to dialysis treatment yielding and cholesterol ); serum aminoacids. Computer recordthe best clinical results. ing of all dialysis data was used. Results were recorded, memorized and then processed by punch cards.

Besides cell-bound adhesion molecules, which are of fundamental importance to a large number of physiological and pathological processes, soluble forms of adhesion molecules have been detected in the circulating blood in recent years. Circulating soluble adhesion molecules appear to be biologically active, and raised levels have been reported in a variety of disorders. In the present study, we used ELISA to measure the serum levels of four soluble adhesion molecules in 23 undialyzed patients with chronic renal failure (CRF), 13 patients on continuous ambulatory peritoneal dialysis (CAPD), 17 on chronic hemodialysis (HD) and 18 healthy controls having a similar mean and distribution of ages. The investigated soluble (s) molecules included intercellular adhesion molecule 1 (sICAM-1), vascular cell adhesion molecule 1 (sVCAM-1), sE-selectin and sP-selectin. sICAM-1 was found to be elevated in patients with CRF (p < 0.05), on CAPD (p < 0.02) and HD (p < 0.0001) compared with the controls but levels did not differ between the three patient groups. The higher sVCAM-1 values found in CRF (p < 0.02), CAPD (p < 0.05) and HD (p < 0.0001) as compared to controls again failed to differentiate the three groups of patients. Soluble E-selectin was also raised in the three groups (p < 0.0001) with no difference between them. Increased sP-selectin was found in CRF (p < 0.05), CAPD (p < 0.02) and in HD patients (p < 0.0001) compared to controls, and levels in HD were significantly higher (p < 0.02) than in CRF patients. Predialysis serum molecule levels did not differ between HD patients treated with cuprophan or with polyacrylonitrile dialyzers. HD sessions with both dialyzers had no effect on sICAM-1, while a decrease (p < 0.02) in sP-selectin was found after dialysis with cuprophan. In undialyzed patients with CRF, regression analysis showed a strong linear correlation between serum creatinine and serum levels of each soluble molecule. These results demonstrate that serum levels of soluble adhesion molecules ICAM-1, VCAM-1, E-selectin and P-selectin are elevated in both undialyzed patients with CRF and patients on CAPD or HD. The elevated serum levels of these proteins probably reflect inadequate clearance as well as enhanced synthesis/release.

The connection between lipids and the rate of progression of chronic renal disease was retrospectively examined in 70 patients who were divided into 2 groups according to their baseline creatinine clearance (CCr): Group 1 (Gp1) contained 30 patients with CCr 60-40 mL/min followed for 40.0 +/- 13.3 months; Group 2 (G2) contained 40 patients with CCr 39-15 mL/min followed for 39.0 +/- 18.2 months. The following parameters were considered: basal and final CCr proteinuria per unit of CCr (UProt/CCr); the difference between final and basal UProt/CCr (delta UProt/CCr); the change in CCr/month (delta CCr); baseline triglycerides (TG), total (TC), HDL (HDLC) and LDL (LDLC) cholesterol, Apo AI, Apo B, Lp(a). Besides in basal CCr the 2 groups significantly differed in the final CCr, final UProt/CCr, delta UProt/CCr, delta CCr. No differences were observed concerning lipid parameters except for Lp(a) (G1 14.8 +/- 13.6, G2 28.7 +/- 27.4 mg/dL; p0.05). Baseline TG (G1 184.1 +/- 61.3, G2 187.5 +/- 72.1 mg/dL) and Apo B (only G2 1.05 +/- 0.32 g/L) were significantly higher than normal subjects and the Apo AI/Apo B ratio (G1 1.42 +/- 0.43, G2 1.33 +/- 0.45) were significantly lower than in normal subjects. delta CCr, while inversely correlated in both groups with delta UProt/CCr (p0.01), only in G2 did it correlate directly with the Apo AI/Apo B ratio (p0.05) and inversely with Apo B and LDLC (p0.05). Although a correlation between Lp(a) and delta CCr was not found, 20/22 patients (3/5 G1, 17/17 G2) with a level30 mg% ran a progressive course. A natural progression of CRI, heralded by an increasing UProt, is highly frequent when baseline CCr is40 mL/min; only then lipids seem to add a burden to the renal damage.

Platelet interaction with leukocytes can occur to a significant degree during hemodialysis, but it remains to be determined what pathophysiological consequences stem from the intradialytic formation of platelet-leukocyte coaggregates. By the use of flow cytometry techniques, this study was set out to analyze intradialytic platelet-neutrophil coaggregate formation and neutrophil hydrogen peroxide production from 10 end-stage renal disease patients each dialyzed with cuprophane and polyacrylonitrile membranes. Platelet-neutrophil coaggregates increased during dialysis with cuprophane, whereas no changes occurred with polyacrylonitrile membranes. Dialysis with cuprophane, unlike that with polyacrylonitrile, also resulted in a significant increase in neutrophil hydrogen peroxide production 10 min after dialysis initiation which persisted at significantly higher levels than predialysis values through the first 20 min. We found that the increased hydrogen peroxide production by neutrophils essentially occurred in concomitance with neutrophil-platelet coaggregation. Intracellular fluorescence representing hydrogen peroxide formation significantly increased through the first 20 min of cuprophane dialysis in neutrophils aggregated to platelets. By contrast, no change occurred in neutrophils not aggregated to platelets. Neutrophils which had formed aggregates with platelets produced higher hydrogen peroxide levels, as assessed by significantly higher fluorescence values, than non-aggregate-forming neutrophils at all time points tested. The phenomenon was duplicated in vitro when ADP-activated normal platelets were incubated with neutrophil cells but was largely inhibited when ADP-activated platelets were treated with anti-P-selectin antibody before incubation with neutrophils. These results strongly suggest that platelet-neutrophil aggregates occurring during hemodialysis, representing cell-cell interactions with pathophysiological effects, may serve as a new parameter to assess biocompatibility.

It has been shown recently that Selenium (Se), an essential trace element for humans, is involved in the regulation of thyroid function, since the enzyme that catalyzes the liver conversion of the thyroid hormone T4 to the more active form T3 is a selenoenzyme. In chronic uremic patients, low blood Se levels as well as thyroid function abnormalities are often found. The present study was carried out to verify whether any correlation exists between Se levels and thyroid function, and to evaluate possible changes in hormonal pattern during Se supplementation in 10 chronic uremic patients on hemodialysis (HD) treatment. Se was supplemented orally as sodium selenite over six consecutive months. Basic plasma Se levels were significantly lower in patients than in normal controls. Right from the start of Se supplementation, plasma Se concentration promptly normalized and leveled off in the normal range throughout the study. Significant increase of FT3 and reduction of TSH levels were detected during Se supplementation. In Se-supplemented patients, a significant direct correlation was also found between reverse T3 (rT3) and TSH, and a significant inverse correlation was found between Se and TSH. Our results suggest that Se deficiency in chronic uremic patients represents a factor influencing the thyroid function and that the Se status should be determined in the evaluation of thyroid metabolism in these patients.

High molecular weight (MW) solutes are not removed during conventional hemodialysis (HD), and their accumulation is thought to play a role in some long-term HD complications (anemia, bone and joint pain, neuropathy, itching). The present trial was conducted to evaluate the removal capacity during in vivo HD of a new polymethylmethacrylate (PMMA) membrane (Filtryzer BK-F, 1.3 m2) compared to conventional PMMA (BK-P, 1.6 m2) and to cellulose acetate (CA, 1.3 m2). BK-F dialyzers, with a pore size of 100 A° and 62% porosity, are designed to remove high MW substances. Ten stable anuric RDT patients (53 ± 13 years) were treated for one week with each membrane in a randomized sequence. Plasma concentrations of creatinine, BUN and beta2-microglobulin (beta2-M) were measured before (b) and after (a) HD to determine the reduction rate for these substances (%). Beta2-M concentration after HD was corrected for changes in distribution volume. Samples of spent dialysate were collected after 3 minutes, 120 minutes and at the end of HD sessions, and appropriately treated and concentrated for HPLC analysis. The reduction rate for BUN and creatinine was similar for the 3 membranes. BK-F showed a higher beta2-M reduction rate than BK-P (p 10 kDa were infrequently detected. Peak profile during HD with BK-F was quite different, showing a predominant peak > 50 kDa which also included albumin. However, albumin loss significantly decreased after 120 minutes and at the end of dialysis compared with the 3-minute values, and was lower than that reported in CAPD patients. With BK-F a peak of MW > 500 kDa was also detected which previous studies indicated as a range characterized by the presence of erythropoiesis inhibitors. Use of the BK-F membrane in HD could afford satisfactory removal of high MW substances, thereby preventing or controlling some long-term HD complications such as anemia or beta2-M amyloid formation.

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A major role of CD62P/CD15s interaction in leukocyte margination during hemodialysis. We investigated expression of several antigens on neutrophils and monocytes, involved in cell adhesion, from patients hemodialyzed with cellulosic and polyacrylonitrile membranes. Among the antigens tested only the expression of CD15s and CD11b was significantly increased on neutrophils and monocytes in patients dialyzed with cellulosic membranes. No changes occurred with polyacrylonitrile membranes. Leukocyte counts from patients dialyzed with cuprophane membranes decreased at the same time as expression of cellular CD15s increased, resulting in a significant negative correlation at all time points tested. No correlation was found between the drop of monocytes and their expression of CD11b. When CD 15s expression increased on neutrophils and monocytes, we observed a concomitant increase of CD62P, a specific selectin of activated platelets. When whole blood cells were incubated with complement activated serum both antigens increased but not when cells were incubated with hrC5a. We also observed that CD61, a platelet phenotypic antigen, was present on leukocytes incubated with complement activated serum. At the time when platelet-leukocyte coaggregates decreased, CD62P expression remained stable on leukocytes, suggesting that both neutrophils and monocytes are able to trap either CD62P shed by activated platelets or soluble CD62P present in normal human serum. The present study documents a major role of P-selectin (CD62P)/sialyl-Lewis x (CD 15s) interaction in the transient leukocyte margination during hemodialysis.

Membranous Nephropathy (MN) represents a large amount of Nephrotic Syndromes in the adult population and its definitive diagnosis is currently carried out through biopsy. An autoimmune condition has been demonstrated in idiopathic MN (iMN) in which some kidney structures are targeted by patient autoantibodies. Some candidate antigens have been described and other likely involved target proteins responsible for the disease are not known yet. In this work our aim is to identify these proteins by screening a lambda-phage library with patients’ sera. We enrolled four groups of patients: two MN groups of 12 full iMN patients; one control group of 15 patients suffering from other renal diseases; one control group of 15 healthy individuals. A commercial cDNA phagemide library was screened using the above described sera, in order to detect positive signals due to antigen-antibody bond. We detected one phagemide clone expressing a protein which was shown to be targeted by the antibodies of the iMN sera only. Control sera were negative. The sequence analysis of cDNA matched the Synaptonemal Complex protein 65 (SC65) coding sequence. Further proteomic analyses were carried out to validate our results. We provide evidence of an involvement of SC65 protein as an autoimmune target in iMN. Considering the invasiveness and the resulting risk coming from renal biopsy, our ongoing aim is to set a procedure able to diagnose affected patients through a little- or non-invasive method such as blood sampling rather than biopsy.

Several studies have shown that, compared with standard therapy, cinacalcet reduces parathyroid hormone (PTH) as well as calcium and phosphorus levels in dialysis patients with secondary hyperparathyroidism (SHPT). The ECHO study evaluated cinacalcet use in actual clinical practice. ECHO enrolled 1865 patients treated with cinacalcet at 187 sites in 12 European countries. In Italy 263 patients were enrolled at 20 sites. The primary objective of the study was to evaluate K/DOQI target achievement in SHPT dialysis patients after the introduction of cinacalcet. Patients on cinacalcet treatment between July 2005 and October 2006 were enrolled and data were collected from 6 months prior to starting cinacalcet up to 12 months after. No treatment algorithm was provided to the investigators. Italian patients had suboptimally controlled SHPT at baseline according to K/DOQI targets (median PTH 760 pg/mL, P 5.4 mg/dL, Ca 9.7 mg/dL). After 1 year of cinacalcet treatment a reduction of PTH (-53.4%), Ca (-7.3%), P (-4.6%) and Ca x P (-14.4%) was observed. The proportion of patients that reached the K/DOQI targets after 1 year of cinacalcet treatment was higher compared to baseline for all parameters (PTH 32% vs 5%; Ca 48% vs 35%; P 59% vs 55%, Ca x P 82% vs 64%). The Italian ECHO data show that cinacalcet treatment increases the proportion of patients achieving the K/DOQI targets for PTH, Ca, P and Ca x P, confirming the effectiveness of cinacalcet in clinical practice in Italy. These findings are consistent with the phase III study results on cinacalcet use in dialysis patients in Europe.

Introduction: Development of renal biomarkers is required to improve on diagnostic accuracy, prognosis and prediction of response to therapy in renal disease. We describe a new method of obtaining from renal specimens a biologic fluid potentially enriched in secreted proteins. Methods: A renal biopsy specimen was centrifuged, and the interstitial fluid (IF) obtained was evaluated by SELDI-ToF, 1D and 2D gel electrophoresis. Twelve spots were extracted from the 2D gel and characterized by MALDI-TOF-MS. Results: The SELDI diagrams demonstrated abundant peptide peaks. One-dimensional gel electrophoresis demonstrated the presence of many bands indicating a diversity of proteins in the sample. Comparison of serum to IF demonstrated a number of bands that were not shared, suggesting that the IF is not a simple “replica” of plasma fluid. Employing 2D-PAGE, 418 spots were identified in the IF sample; 12 spots were selected and analyzed by mass spectrometry. Conclusions: We have described a novel technique to obtain a biologic fluid that contains a significant quantity and diversity of proteins from renal tissue. The procedure to obtain the fluid is simple and easily applicable to standard renal biopsy procedures. This fluid has the potential to identify informative proteins that are more concentrated than in any other renal biologic fluid previously analyzed and strictly related to renal pathophysiology. Future work includes the development of a clinical protocol to identify and validate informative biomarkers that have diagnostic and prognostic value.

Lipid disturbances have been linked to the progression of chronic renal disease. We examined 52 patients with a creatinine clearance (CCr) of 38.5 +/- 7.9 ml/min due to various nephropathies, on free diet. Bimonthly, over a 12-month period, we assessed: serum creatinine (Cr); CCr; daily urinary urea excretion; urinary protein excretion per unit of residual renal function (UProt/CCr); total, HDL, VLDL and LDL cholesterol; triglycerides; Apo A, Apo B. Chronic renal failure was progressive in 22 patients with a slope of 1/Cr-0.00358 +/- 0.00247, stable in 30 with a slope of 0.00420 +/- 0.00285. Lipid parameters did not differ significantly between the two groups but for the lower Apo A and Apo A/Apo B ratio values in the progressive group. Overall slope inversely correlated with basal CCr; in the progressive patients the slope correlated with the percentage variation of UProt/CCr and only partially with the altered Apo profile.

Introduction Lipoprotein glomerulopathy is a glomerulonephritis which was described for the first time by Saito in 1989 and is currently acknowledged as a separate nosological entity. It is histologically characterized by a marked dilatation of the glomerular capillaries and the presence of lipoprotein thrombi in the glomerular lumens. The dyslipidemic profile is similar to that of type III dyslipoproteinemia with Apolipoprotein E values that are often high; proteinuria and renal dysfunction are present. Proteinuria often does not respond to steroid and cytostatic treatments. The phenotypic expression of lipoprotein glomerulopathy is most probably correlated to a genetic alteration of the lipoprotein metabolism (mutation of the Apolipoprotein E coding gene). In literature, lipoprotein glomerulopathies have mainly been reported in Japanese and Chinese subjects, except for three cases in the Caucasian race, reported in France and the USA. Case presentation We describe the case of a 60-year-old female, Caucasian patient suffering from lipoprotein glomerulopathy, carrier of a new mutation on the Apolipoprotein E gene (Apolipoprotein EMODENA), and treated successfully with low density lipoprotein-apheresis with the Heparin induced extracorporeal lipoprotein precipitation system. After a first phase of therapeutic protocol with statins, the patient was admitted for nephrotic syndrome, renal failure and hypertension. Since conventional treatment alone was not able to control dyslipidemia, aphaeretic treatment with heparin-induced Extracorporeal Lipoprotein Precipitation - apheresis (HELP-apheresis) was started to maintain angiotensin converting enzyme inhibitor therapy for the treatment of hypertension. Treatment with HELP-apheresis led to a complete remission of the proteinuria in a very short time (four months), as well as control of hypercholesterolemia and renal function recovery. Conclusion According to this case of lipoprotein glomerulopathy, we believe that renal damage expressed by proteinuria correlates to the levels of lipids and, furthermore, the treatment with HELP-apheresis, by lowering low-density lipoprotein cholesterol and triglycerides, may be considered as a therapeutic option in synergy with pharmacological treatment in the treatment of lipoprotein glomerulopathy.

Nephrotic syndrome is a condition that is clinically associated with poor outcome. In this study, we compared different techniques of urine sample preparation in order to develop a robust analytical protocol to define the differential urinary proteome of urinary abnormalities compared to nephrotic proteinuria.We recruited 5 normal control subjects, 16 patients with urinary abnormalities and 16 patients with nephrotic syndrome. Proteins from normal urine were processed using three different protocols [acetone, ultrafiltration and trichloroacetic acid (TCA) precipitation], depletion of albumin and IgGs and then analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) gels and mass spectrometry.Comparing the three extraction methods by visual inspection of gels after 2D gel electrophoresis, the acetone precipitation and TCA methods yielded the best quality of protein extraction, while the acetone precipitation method was the most efficient. Furthermore, we tested three commercial kits for albumin and IgG depletion. We applied the optimized acetone extraction protocol to compare urinary samples from nephrotic patients (NP) to urinary samples obtained from patients presenting with urinary abnormalities (UAP). We observed a proteolytic activity directed against albumin. This observation was more prevalent in urinary samples from NP than from UAP. Within both groups, there was some inter-individual variability in the observed proteolytic activity. An increased concentration of alpha1 antitrypsin was also observed in urine of NP. We analysed albumin fragmentation by 1D and 2D western blots in the same samples skipping the albumin and IgG depletion steps to avoid the possible confound of albumin fragment removal. The analysis confirmed a stronger proteolytic activity in the nephrotic group.The proteolytic activity against albumin and the anti-proteolytic activity of alpha1 antitrypsin are likely linked and could play an important role in the nephrotic process. If replicated in larger samples, this methodology may lead to a better understanding of the underlying pathophysiological process of nephrotic syndrome.

Background Marginal organs not suitable for single kidney transplantation are considered for double kidney transplantation (DKT). Herein we have reviewed short and long-term outcomes of DKT over a 7-year experience. Patients and Methods Between 2001 and 2007, 80 DKT were performed in the transplant centers of Bologna, Parma, and Modena, Italy. Recipient mean age was 61 ± 5 years. The main indications were glomerular nephropathy (n = 33) and hypertensive nephroangiosclerosis (n = 14). Mean HLA A, B, and DR mismatches were 3.1 ± 1.2. Donor mean age was 69 ± 8 years and mean creatinine clearance was 75 ± 27 mL/min. Almost all kidneys were perfused with Celsior solution. Mean cold ischemia time was 17 ± 4 hours and mean warm ischemia time was 41 ± 17 minutes. Mean biopsy score was 4.4. Immunosuppression was based on tacrolimus (n = 52) or cyclosporine (n = 26). Results Fifty (62.5%) patients displayed good postoperative renal function. Thirty (37.5%) experienced acute tubular necrosis and required postoperative dialysis treatment; 8 acute rejections occurred. Urinary complications were 13.7% with 8/11 requiring surgical revision. There were 6 surgical reexplorations: intestinal perforation (n = 2), bleeding (n = 3), and lymphocele (n = 1). Two patients lost both grafts due to vascular and infectious complications at 7 or 58 days after transplantation. Two patients underwent intraoperative transplantectomy due to massive vascular thrombosis. Four (5%) patients underwent transplantectomy of a single graft due to vascular complications (n = 2), bleeding (n = 1), or infectious complications (n = 1). Graft and patient survivals were 95% and 100% versus 93% and 97% at 3 versus 36 months, respectively. Conclusions DKT is a safe approach for organ shortage. The score used in this study is useful to determine whether a kidney should be refused or accepted.

Haemodynamic instability is one of the most frequent problems occurring during dialysis treatment. Ten clinically stable patients (8 M and 2 F) undergoing chronic maintenance haemodialysis for at least 6 months were investigated. Two groups of five patients each, were selected on the basis of presence (IG) or absence (SG) of cardiovascular instability during dialysis. The cardiovascular function was assessed by computerised electrical bioimpedance performed during dialysis setting and by echocardiography immediately pre- and post-dialysis. In SG dialysis treatment did not change cardiac index (CI), stroke index (SI) and systemic vascular resistances index (SVRI). However CI, SI and SVRI, tended to decrease in IG patients; the reduction in CI was primarily due to a decrease in SI. Ejection velocity index increased significantly in SG but not in IG. Evaluation of cardiac function by Döppler echocardiography revealed a significant increment in fractional shortening, mean velocity of circumferential fiber shortening and Suga' index in SG with dialysis but not in IG. Stress index decreased significantly in both groups. Hormonal and biochemical parameters were not significantly different before and after dialysis in both groups. In IG the decrease in mean blood pressure, due to a reduction of SI, recognises in the inadequate response of myocardial contractility to volume subtraction, the genesis of its drop. Finally, impedance cardiography in uraemic patients helps to identify the factors that contribute to the impairment of cardiac performance and that should be studied before selecting new and advanced dialysis programmes.

This Phase II study aimed to determine the optimal dose and administration schedule of continuous erythropoietin receptor activator (C.E.R.A.) given subcutaneously (s.c.) in patients receiving dialysis converting directly from s.c. epoetin therapy 1-3 times/week. An extension phase examined long-term safety and efficacy.Patients were assigned to one of three C.E.R.A. dose groups determined by multiplying the previous weekly dose of epoetin by one of three ratios (0.4/150, 0.8/150, 1.2/150 for groups A, B and C, respectively). Within each group, patients were randomized to once weekly (QW), once every 3 weeks (Q3W) and once monthly (Q4W) schedules. Dose adjustments were not permitted for the first 6 weeks. The core study period was 19 weeks (21 weeks in the Q4W cohorts). Patients could enter a 12-month extension period at the same schedule, aiming to maintain haemoglobin (Hb) at 11-12 g/dL.137 patients entered the core period, and 62 continued into the extension period. A dose-dependent relationship was seen in the primary efficacy variable, change in Hb standardized to a 6 week period (p0.0001), but effect was independent of schedule. Hb levels were maintained throughout the study, with few dose changes. C.E.R.A. was generally well tolerated and the most frequent adverse event was hypotension.The results suggest that s.c. C.E.R.A. at up to once monthly intervals provides stable maintenance of Hb levels in dialysis patients converting directly from epoetin 1-3 times/week. Achieving tight Hb control with few dose adjustments at extended administration intervals may offer health benefits and improvements in resource management.

Water treatment system and dialysis monitors are susceptible to microbial contaminations and periodical disinfection procedures are mandatory to obtain results requested from international standards and guidelines. Several chemical germicides or some physical treatments are on the market validated by device manufacturer according to medical device directives. With time, interfering substances from dialysis device or water are able to modify disinfection efficiency. Simulating-use testing is not a common procedure to validate disinfectants and recent data document as biofilm represents the most important cause of disinfection inefficacy. Some international standards include tests in the presence of various interfering substances but their use is not widespread. When using a disinfectant, residue toxicity, material compatibility and potential risks for the staff also have to be considered. A quality assurance program has to be implemented to obtain adequate performances and to improve results on patients.

Background. Autosomal dominant polycystic kidney disease (ADPKD) is a renal disease characterized by an important variability in clinical course, which cannot be fully explained by the genetic heterogeneity of the disease. Although the role for the angiotensin I-converting enzyme (ACE) insertion/deletion (I/D) polymorphism as a modifier factor in ADPKD renal deterioration has been suggested, direct evidence from genetic association studies remain inconclusive. To provide a more robust estimate of the putative effect of the ACE I/D polymorphism on the renal progression in ADPKD, we performed a meta-analysis pooling data from all relevant studies in which the role of the ACE I/D variant in ADPKD clinical features was evaluated. Methods. We applied a random-effects model to combine odds ratio and 95% confidence intervals. Q-statistic was used to evaluate the homogeneity, and both Egger’s and Begg–Mazumdar tests were used to assess publication bias. Results. Altogether, three distinct meta-analyses were generated using data from 13 studies. Despite the absence of publication bias and the presence of homogeneity among study results, the DD genotype failed to show an influence on risk of end-stage renal disease (ESRD), mean age at ESRD or risk of hypertension in ADPKD patients when compared with I-allele carriers (DD vs ID þ II). Likewise, meta-analyses carried out separately for Caucasian and Asian studies showed no indication of an association between the DD genotype and a faster renal deterioration in ADPKD. Conclusion. These findings do not support the hypothesis that the enhanced ACE activity associated with the D allele might promote a significantly worse prognosis in patients with ADPKD.

To establish the accuracy of bioelectrical impedance analysis (BIA) for the assessment of total and appendicular body composition in peritoneal dialysis (PD) patients. Cross-sectional study. University Nephrology Clinic. In all, 20 PD patients and 77 healthy controls matched for gender, age and body mass index. Whole-body fat-free mass (FFM) and appendicular lean tissue mass (LTM) were measured by dual-energy X-ray absorptiometry. Resistance (R) of arms, trunk and legs was measured by eight-polar BIA at frequencies of 5, 50, 250 and 500 kHz. Whole-body resistance was calculated as the sum of R of arms, trunk and legs. The resistance index (RI) was calculated as the ratio between squared height and whole-body or segmental R. RI at 500 kHz was the best predictor of FFM, LTMarm and LTMleg in both PD patients and controls. Equations developed on controls overestimated FFM and LTMarm and underestimated LTMleg when applied to PD patients. Specific equations were thus developed for PD patients. Using these equations, the percent root mean-squared errors of the estimate for PD patients vs controls were 5 vs 6% for FFM, 8 vs 8% for LTMarm and 7 vs 8% for LTMleg. Eight-polar BIA offers accurate estimates of total and appendicular body composition in PD patients, provided that population-specific equations are used. University of Modena and Reggio Emilia.

An increase in conjugated linoleic acid (CLA), a natural fatty acid present in our diet, which possesses anticarcinogenic and antiatherogenic activities in experimental models, has been found in both the plasma and adipose tissue of end-stage chronic renal failure (ESCRF) patients. Increased levels of retinol have also been found in those patients, due to a reduced excretion of the retinol-binding protein. Since retinol is known to influence lipid metabolism, we evaluated whether changes in retinol, CLA, and other fatty acids are correlated in the plasma of CRF patients. We measured CLA, retinol, and unsaturated fatty acids in the plasma of the following groups: (A) 35 ESCRF patients; (B) 20 hemodialysis (HD) patients; (C) 20 healthy controls. Subjects with total cholesterol and/or triglycerides higher than 250 mg/dL were excluded. We found a significant increase in CLA, retinol, palmitoleic (16:1), and oleic (18:1) acids in ESCRF patients. In HD patients we found a similar pattern, however, CLA increase was not significant. No changes were observed in the other fatty acids measured. In the groups of ESCRF and HD patients, a positive correlation between the levels of plasma retinol and CLA, and between retinol and 16:1 was found. These correlations were not detected in controls. The abnormal levels of plasma retinol in CRF patients might partly explain the changes in CLA and 16:1. The influence of retinol levels on these fatty acids might be due to an induction of delta 9 desaturase. In fact, 16:1 is known to be produced, partly, by delta 9 desaturation of palmitic acid. Moreover, the formation of CLA from delta 9 desaturation of vaccenic acid-a trans-monounsaturated fatty acid present in our diet-has recently been demonstrated in humans. Nevertheless, our data do not represent direct evidence supporting an increased delta 9 desaturase activity in CRF patients. Another possible explanation might be a variation in the exogenous intake.

Water for dialysis represents an additive risk factors to the chronic inflammatory state documented in patients on ESRD. The possibility of sustaining proinflammatory cytokines through microbial derived products, coming from dialysate or infused solutions, is enhanced by biofilm presence on piping and on water treatment system or monitor components. Spread use of reverse osmosis, loop distribution system and pre-treatment components tailored to local raw water characteristics have greatly contributed to a general improvement in final water quality. Notwithstanding these contributions literature still reports fatal accidents or significant percentage of dialysis units not complying to the water quality standards. Technological improvement lowers chemical contamination but microbial quality relays more on quality assurance programs than on technology. Optimal water quality represents part of the anti-inflammatory strategies we need to assure to our dialysis patients to improve outcome.

An increased oxidative stress is now considered one of the major risk factors in chronic renal failure (CRF) patients that may be exacerbated by dialysis. It has been postulated that this increased oxidative stress might cause an augmented red blood cell (RBC) membrane lipid peroxidation with the consequent alteration in membrane deformability. The aim of this study was to evaluate RBC susceptibility to an in vitro induced oxidative stress and RBC antioxidant potential in different groups of CRF patients undergoing different substitutive treatment modalities. Fifteen end-stage CRF patients were evaluated in conservative treatment, 23 hemodialysis (HD) patients, 15 continuous ambulatory peritoneal dialysis (CAPD) patients, 15 kidney transplanted patients, and 16 controls. Their RBCs were incubated with the oxidative stress-inducing agent tert-butylhydroperoxide both in the presence and in the absence of the catalase inhibitor sodium azide, and the level of malondialdehyde (MDA) (a product of lipid peroxidation), was measured at 0, 5, 10, 15, and 30 min of incubation. In addition, the RBC content of reduced glutathione (GSH) was measured by HPLC. As opposed to the controls, RBCs from end-stage CRF patients exhibited an increased sensitivity to oxidative stress induced in vitro, both in the absence and presence of a catalase inhibitor, as demonstrated by a significantly higher level of MDA production at all the incubation times (P < 0.05). Different substitutive treatments had different impacts on this phenomenon; CAPD and kidney transplantation were able to normalize this alteration while HD was not. GSH appeared to be related to the increase in RBC susceptibility to oxidative stress; its content being significantly elevated in end-stage CRF and HD patients as compared with CAPD and transplanted patients and controls (P < 0.05). No significant changes were observed in the RBC glutathione content during the HD session. The increase of GSH in RBCs of end-stage CRF and HD patients seems to indicate the existence of an adaptive mechanism under increased oxidative stress occurring in vivo. Unlike HD, the beneficial effect of CAPD on the anemia of dialysis patients might partly be due to a condition of lower oxidative stress that might in addition counterbalance the cardiovascular negative effects of dislipidemia of CAPD patients.

Water for dialysis represents an additive risk factors to the chronic infammatory state documented in patients on ESRD. The possibility of sustaining proinflammatory cytokines trough microbial deriv

In this randomized trial renal transplant recipients were treated with basiliximab, everolimus 3 mg/day, low-dose CsA. At transplantation, patients were randomized to stop steroids at the seventh day (group A) or to continue oral steroids in low doses (group B). Of the 113 patients enrolled, 65 were randomized to group A and 68 to group B. All patients were followed for 2 years. During the study 28 (43%) group A patients required reintroduced corticosteroids. One patient died, in group B. The Graft survival rate was 97% in group A and 90% in group B. There were more biopsy-proven rejections in group A (32% vs 16%; P = .044). The mean creatinine clearance was 54 +/- 21 mL/min in group A vs 56 +/- 22 mL/min in group B. Mean levels of serum cholesterol tended to be lower in group A, but the difference was of borderline significance (191 +/- 91 vs 251 +/- 188 mg/dL; P = .07). Vascular thrombosis (0 vs 5) and pneumonia requiring hospitalization (2 vs 7) tended to be more frequent in group B. Only three cases of CMV infection (1 vs 2) occurred. An immunosuppressive therapy with everolimus and low-dose CsA allows one to obtain excellent renal graft survival and stable graft function at 2 years. Early interruption of steroids in patients treated with this regimen may increase the risk of acute rejection, but neither affects graft survival nor graft function, while possibly reducing the risk of hyperlipemia and vascular thrombosis. About 60% of patients given everolimus and low-dose CsA can definitively stop steroids after 1 week.

An increased free-radical production has been documented during hemodialysis (HD) particularly when bio-incompatible membranes are utilized. These highly reactive free radicals can cause damage through several pathways, one of the best known being lipid peroxidation. Malondialdehyde (MDA) is a product of lipid peroxidation, which can partly be removed by HD due to its low molecular weight and water solubility. Hydroperoxides are predominantly found in lipid substances, and therefore their removal by HD could be difficult. We evaluated the behavior of these two by-products of lipid peroxidation during HD, comparing their behavior in three different membranes, in order to study their reliability as markers of acute oxidative injury. Fifteen stable HD patients were dialyzed with each of the following membranes: cuprophan, polyamide, and polysulfone, three sessions for every membrane. MDA and hydroperoxides were measured pre-HD and then both from the arterial and venous line at 8, 15, 30, and 240 min. During HD with cuprophan membrane MDA decreased significantly in the venous line compared with the arterial line at 8, 15, and 30 min (P < 0.05). At the end of HD, MDA was significantly reduced compared with MDA pre-HD (P < 0.05). Plasma hydroperoxides increased significantly in the venous line compared with the arterial line at 8, 15, 30, and 240 min (P < 0.05). At the end of HD, hydroperoxides had increased significantly as compared with pre-HD (P < 0.05). When the polyamide and polysulfone membranes were used, the behavior of MDA was similar to that found with cuprophan. Hydroperoxides were unchanged during HD using both membranes. MDA is not a reliable marker of acute oxidative injury during HD as it is removed during HD. Hydroperoxide measurement is a better marker of acute oxidative injury during HD.

Aim Efficiency in removing middle molecules such as ß2-microglobulin (ß2-MG) is one of the main purposes of modern dialytic therapy. In order to achieve this, techniques requiring complex machines and substitution fluid have been developed over recent years. Alternatively, the internal filtration / back filtration phenomenon can be used. The recent development of a so-called “internal filtration enhanced dialyser” prompted us to compare the removal of ß2-MG together with other small molecules when the dialyser was used either in standard hemodiafiltration (HDF) or internal hemodiafiltration (iHDF). Methods Ten stable, anuric, hemodialysis (HD) patients treated by thrice weekly standard bicarbonate HD using low-flux synthetic membrane entered the study. A new high-flux polysulfone dialyser designed with the specific aim of enhancing internal filtration (BS-1.6 UL, 1.6 m2, Toray Industries) was used. Post dilution HDF (2.5 l/hour of substitution fluid, dialysate flow 500 ml/min) was compared with iHDF (dialysate flow 750 ml/min), with blood flow at 300 ml/min. Samples were obtained at the start and at the end of the session in order to measure the % removal of urea, creatinine, uric acid, phosphate and ß2-MG (corrected for total protein concentration). In addition, after 20 min of dialysis the clearances of the same molecules were measured. A mathematical model has been developed for the description of the hydrodynamic phenomena taking place within the dialyser and of fluid filtration across the membrane. Results No significant differences have been observed in removal rate switching from HDF to iHDF except for ß2-MG removal, which was slightly higher in HDF than in iHDF. Phosphate clearance is significantly higher than those obtained with creatinine in both HDF (pConclusions iHDF is a high flux dialysis method, which, if performed with a dialyser designed to enhance internal filtration, obtains a much higher removal rate in comparison with dialysers in traditional high flux dialysis, as previously reported in the literature. Provided that the dialyser is used on a dialysis machine working with ultra pure dialysate and UF control, this dialyser line can perform reliable internal HDF without the need for replacement solution. Considering the narrow difference in performance observed between iHDF and HDF, and the increasing number (and age) of patients leading to higher dialysis costs, iHDF represents a cost-effective alternative to other diffusive-convective techniques.