In end-stage renal disease (ESRD) endothelium may represent a key target for the action of circulating elements, such as modified erythrocytes (RBC) and/or plasmatic factors, that may facilitate inflammation and the vasculopathy associated with uremia. We have previously demonstrated that phosphatidylserine (PS) exposure on the surface of RBC from ESRD patients increases RBC-human umbilical vein endothelial cell (HUVEC) interactions and causes decreased nitric oxide (NO) production. We postulated that, besides the pro-inflammatory effects due to decreased NO bio-availability, enhanced ESRD-RBC-HUVEC interactions might directly stimulate proinflammatory pathways leading to increased vascular adhesion molecule expression. ESRD-RBC-endothelial cell interactions induced a time-dependent up-regulation of VCAM-1 and ICAM-1 (measured by Western blot (WB) and real-time PCR), associated with mitogenactivated protein kinase (MAPK) activation and impairment of the Akt/endothelial nitric oxide synthase (eNOS) signaling cascade, measured by WB. In reconstitution experiments, normal RBC incubated with uremic plasma showed increased PS exposure and significantly increased VCAM-1 and ICAM-1 mRNA levels when incubated on HUVEC. Interestingly, ESRD-RBC induced increased expression of adhesion molecules was prevented by Annexin-V (AnV, able to mask PS on RBC surface), anti-integrin-avb3, antithrombospondin-1 (TSP-1), and PD98059 (a selective inhibitor of MAPK phosphorylation). Moreover, AnV reversed the ESRD-RBC effects on MAPK and Akt/eNOS signaling pathways. Our data demonstrate that, possibly via a direct interaction with the endothelial thrombospondin-(avb3) integrin complex, ESRD-RBC-HUVEC adhesion induces a vascular inflammatory phenotype. Thus, intervention targeting ESRD-RBC increased adhesion to endothelium and/or MAPK and Akt/eNOS pathways may have the potential to prevent vascular lesions under uremic conditions.

Normocytic normochromic anemia is a common complication in chronic kidney disease and is associated with many adverse clinical consequences. Erythropoiesis-stimulating agents (ESAs) and adjuvant iron therapy represent the primary treatment for anemia in chronic kidney disease. The introduction of ESAs into clinical practice was a success story, mediating an increase in hemoglobin concentrations without the risk for recurrent blood transfusions and improving quality of life substantially. However, recombinant ESAs are still expensive and require a parenteral route of administration. Moreover, concern has arisen following randomized clinical trials showing that higher hemoglobin targets and/or high ESA doses may cause significant harm. This, together with changes in ESA reimbursement policy in some countries, has resulted in a significant reduction in ESA prescribing and the hemoglobin level targeted during therapy. Several attempts are being made to develop new drugs with improved characteristics and/or easier manufacturing processes compared with currently available ESAs, including new treatment approaches that may indirectly improve erythropoiesis. We give an update on the new investigational strategies for increasing erythropoiesis, examining in depth their characteristics and possible advantages in the clinical setting and the caveats to be aware of at the present stage of development.

Summary. Platelet activation is associated with exposure of the aminophospholipid phosphatidylserine (PS) to the outer hemileaflet of the plasma membrane bilayer, which seems to be involved in the coagulation process. Because platelet activation may occur in patients suffering from chronic uremia, which is frequently associated with a thrombophilic tendency, we studied whether uremic platelets show an increased propensity to expose PS on the outer membrane leaflet and whether this process is linked with important functional and molecular changes. Flow cytometric percentage of annexin V-positive platelets, a measure of PS externalization, was significantly elevated (P < 0.001) in uremic patients when compared to normal controls under both unstimulated and agonist-stimulated conditions. Uremic platelet procoagulant activity, as measured by thrombin generation, was more than twice as high (4.13 ± 0.3 l mL ). Two independent assays showed that the enzymatic activity of caspase-3, a protease involved in the loss of membrane PS asymmetry, was significantly greater in the platelets of uremic subjects than in those of healthy controls. PS exposure in agonist-stimulated platelets was markedly reduced by inhibition of caspase-3 activity but was not affected by inhibition of calpain activity. These results support the view that the thrombophilic susceptibility of uremic patients may be partly ascribed to increased PS exposure to the outer membrane leaflet of platelets. This process seems to be causally linked to an increase in caspase-3 activity, particularly during platelet activation.

During hemodialysis (HD), circulating blood cells can be activated and also engage in dynamic interplay. These phenomena may be important factors behind dialysis membrane bio(in)compatibility. In the present prospective cross-over study, we have used flow cytometry to evaluate the influence of different dialysis membranes on the activation of circulating blood cells (leukocytes, platelets) and their dynamic interactions (formation of circulating platelet-leukocyte and platelet-erythrocyte aggregates) during in vivo HD. Each patient (n = 10) was treated with dialyzers containing membranes of cellulose diacetate, polysulfone and ethylenevinylalcohol (EVAL) in a randomized order. Upregulation of adhesion receptor expression (CD15s, CD11b/CD18) occurred mainly with the cellulosic membrane, though an increase in CD11b/CD18 circulating on neutrophils was also found with both synthetic membranes. Circulating activated platelets (P-selectin/CD63-positive platelets) increased during HD sessions with cellulose diacetate and polysulfone. An increased formation of platelet-neutrophil aggregates was found at 15 and 30 min during dialysis with cellulose diacetate and polysulfone but not with EVAL. Platelet-erythrocyte aggregates also increased with cellulose diacetate and at 15 min with polysulfone as well. Generally in concomitance with the increase in platelet-neutrophil coaggregates, there was an increased hydrogen peroxide production by neutrophils. The results of this study indicate that cellular mechanisms can be activated during HD largely depending on the membrane material, EVAL causing less reactivity than the other two membranes. It appears that each dialysis membrane has multiple and different characteristics that may contribute to interactions with blood components. Our results also indicate that derivatizing cellulose (cellulose diacetate) may be a useful way to improve the biocompatibility of the cellulose polymer and that there may be great variability in the biocompatibility profile of synthetic membranes, dialysis with polysulfone being in general associated with a higher degree of cell activation than EVAL membrane.

Protein-adsorptive properties are a key feature of membranes used for haemodialysis treatment. Protein adsorption is vital to the biocompatibility of a membrane material and influences membrane's performance. The object of the present study is to investigate membrane biocompatibility by correlating the adsorbed proteome repertoire with chemical feature of the membrane surfaces. Dialyzers composed of either cellulose triacetate (Sureflux 50 L, effective surface area 0.5 m(2); Nipro Corporation, Japan) or the polysulfone-based helixone (FX40, effective surface area 0.4 m(2); Fresenius Medical Care AG, Germany) materials were employed to develop an ex vivo apparatus to study protein adsorption. Adsorbed proteins were eluted by a strong chaotropic buffer condition and investigated by a proteomic approach. The profiling strategy was based on 2D-electrophoresis separation of desorbed protein coupled to MALDI-TOF/TOF analysis. The total protein adsorption was not significantly different between the two materials. An average of 179 protein spots was visualised for helixone membranes while a map of retained proteins of cellulose triacetate membranes was made up of 239 protein spots. The cellulose triacetate material showed a higher binding capacity for albumin and apolipoprotein. In fact, a number of different protein spots belonging to the gene transcript of albumin were visible in the cellulose triacetate map. In contrast, helixone bound only a small proportion of albumin, while proved to be particularly active in retaining protein associated with the coagulation cascade, such as the fibrinogen isoforms. Our data indicate that proteomic techniques are a useful approach for the investigation of proteins surface-adsorbed onto haemodialysis membranes, and may provide a molecular base for the interpretation of the efficacy and safety of anticoagulation treatment during renal replacement therapy.

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

Protein-adsorptive properties are a key feature of membranes used for hemodialysis treatment. Protein adsorption is vital to the biocompatibility of a membrane material and influences membrane's performance. The object of the present study is to investigate membrane biocompatibility by correlating the adsorbed proteome repertoire with structural feature of the membrane surfaces. Minidialyzers of identical structural characteristics composed of either cellulose diacetate or ethylenevinyl alcohol materials were employed to develop an ex vivo apparatus to investigate protein adsorption. Adsorbed proteins were eluted by a strong chaotropic buffer condition and investigated by 2-DE coupled to both MALDI-TOF mass spectrometry (MS) mass fingerprinting and fragmentation analysis on a nanoLC-MS/MS hybrid instrument. Membrane surface characterization included evaluation of roughness (atomic force microscopy), elemental chemical composition (X-ray-photoelectron-spectroscopy), and hydrophilicity (pulsed nuclear magnetic resonance). The present study identifies a number of different proteins as common or characteristic of filter material interaction, showing that proteomic techniques are a promising approach for the investigation of proteins surface-adsorbed onto hemodialysis membrane. Proteomic analysis enables the characterization of protein layers of unknown composition.

These findings suggest that PS externalization may promote increased uremic erythrocyte adhesion to endothelium, possibly via a direct interaction with matrix thrombospondin.