Luigi Amoroso

Mechanisms of uremic erythrocyte-induced adhesion of human monocytes to cultured endothelial cells†

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 pro‐inflammatory 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 mitogen‐activated 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‐αvβ3, anti‐thrombospondin‐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‐(αvβ3) 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. J. Cell. Physiol. 213:699–709.

Calcimimetic R-568 and Its Enantiomer S-568 Increase Nitric Oxide Release in Human Endothelial Cells

Background Calcimimetics, such as R-568, are thought to activate G protein-linked Ca2+-sensing receptor (CaSR) by allosterically increasing the affinity of the receptor for Ca2+ allowing for efficient control of uremic hyperparathyroidism. Several recent studies suggest they possess additional vascular actions. Although it has been postulated that calcimimetics may have a direct effect on CaSR in the blood vessels, further studies are needed to elucidate their vascular CaSR-dependent versus CaSR-independent effects. Methodology/Principal Findings Focusing on human umbilical vein endothelial cells (HUVECs), we studied the CaSR expression and distribution by Immunofluorescence and Western Blot analysis. CaSR function was evaluated by measuring the potential effect of calcimimetic R-568 and its enantiomer S-568 upon the modulation of intracellular Ca2+ levels (using a single cell approach and FURA-2AM), in the presence or absence of Calhex-231, a negative modulator of CaSR. To address their potential vascular functions, we also evaluated R- and S-568-stimulated enzymatic release of Nitric Oxide (NO) by DAF-2DA, by Nitric Oxide Synthase (NOS) radiometric assay (both in HUVECs and in Human Aortic Endothelial Cells) and by measuring eNOS-ser1177 phosphorylation levels (Immunoblotting). We show that, although the CaSR protein was expressed in HUVECs, it was mainly distributed in cytoplasm while the functional CaSR dimers, usually localized on the plasma membrane, were absent. In addition, regardless of the presence or absence of Calhex-231, both R- and S-568 significantly increased intracellular Ca2+ levels by mobilization of Ca2+ from intracellular stores, which in turn augmented NO release by a time- and Ca2+-dependent increase in eNOS-ser1177 phosphorylation levels. Conclusions/Significance Taken together, these data indicate that in human endothelium there is no stereoselectivity in the responses to calcimimetics and that CaSR is probably not involved in the action of R- and S-568. This suggests an additional mechanism in support of the CaSR-independent role of calcimimetics as vasculotrope agents.

Proteomic analysis of protein adsorption capacity of different haemodialysis membranes

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 membranes 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.

Leukocyte adhesion molecules and leukocyte-platelet interactions during hemodialysis: effects of different synthetic membranes.

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<0.05) and in CD11b/CD18 expression for PSF as compared to both EVAL (p<0.01) and AN69 (p<0.05). No difference between membranes was found on the expression of such adhesive molecules on monocytes. Significant differences in platelet-neutrophil (but not in platelet-monocyte) coaggregate formation were observed between PSF and both EVAL (p<0.001) and AN69 (p<0.01). Reactive oxygen species production by neutrophil population during hemodialysis was significantly different between each pair of synthetic polymers (PSF vs EVAL, p<0.001; PSF vs AN69, p<0.001; AN69 vs EVAL, p<0.05). 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.

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

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

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

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

Proteomic investigations on the effect of different membrane materials on blood protein adsorption during haemodialysis.

BACKGROUND During haemodialysis procedure, the contact of blood with the membrane material contained in the hemodialyser results in protein deposition and adsorption, and surface-adsorbed proteins may trigger a variety of biological pathways with potential pathophysiologic consequences. The present work was undertaken to examine for protein adsorption capacity of two membranes used for clinical haemodialysis, namely cellulose triacetate (a derivatized cellulosic membrane) and the synthetic polymer polysulfone-based helixone. MATERIALS AND METHODS We performed a prospective cross-over study in chronic haemodialysis patients, routinely treated with a cellulose triacetate dialyser (n=3) or with a helixone dialyser (n=3). Dialysers from each patient were obtained after dialysis session, and flushed with a litre of saline to remove residual blood. Adsorbed proteins were then eluted by a strong chaotropic buffer. Patients were next switched to the other membrane dialyser for four weeks, at the end of this period protein adsorption being evaluated again. After silver staining, expression profile protein of the two groups was analyzed by 2-DE gels, analyzed and identified by Peptide Mass-finger printing and MALDI-TOF-MS/MS sequency. Moreover nanoLC-MS/MS shotgun profiling was pursued using a semi-quantitative label free approach by emPAI data analysis. RESULTS A total of 54 differentially expressed proteins were identified: 22 proteins more concentrated in helixone membrane (predominantly low abundant plasma proteins) and 32 in cellulose triacetate (most represented by high abundant plasma proteins). The difference proved to be related to membrane material and not to patients characteristics. DISCUSSION Proteomic techniques represent a useful approach for the investigation of proteins surface-adsorbed onto a haemodialysis membrane, and can also be applied for critical assessment to compare efficiencies of different dialyser membrane materials in the adsorption of plasma proteins.

Platelet-leukocyte interactions in hemodialysis patients: culprit or bystander?

The formation of circulating platelet-leukocyte complexes has been observed in a variety of conditions and may be pathophysiologically significant. Platelet-leukocyte interactions in fact facilitate metabolic cooperation and mutual activation, which may be of relevance in many biological processes including inflammation, atherogenesis and hemostasis. During hemodialysis procedure, the series of reactions that can occur upon blood contact with the foreign membrane surface may involve a variety of changes affecting almost every cellular and plasmatic component of the blood. This article reviews the evidence for abnormal interactions between circulating platelets and leukocytes in uremic patients undergoing maintenance hemodialysis and the pathophysiologic implications which may stem from such interactions.

Proteomic Investigations into Hemodialysis Therapy

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

Phylogenetic relationships, biofilm formation, motility, antibiotic resistance and extended virulence genotypes among Escherichia coli strains from women with community-onset primitive acute pyelonephritis

The present work set out to search for a virulence repertoire distinctive for Escherichia coli causing primitive acute pyelonephritis (APN). To this end, the virulence potential of 18 E. coli APN strains was genotypically and phenotypically assessed, comparatively with 19 strains causing recurrent cystitis (RC), and 16 clinically not significant (control, CO) strains. Most of the strains belong to phylogenetic group B1 (69.8%; p<0.01), and APN strains showed unique features, which are the presence of phylogroup A, and the absence of phylogroup B2 and non-typeable strains. Overall, the most dominant virulence factor genes (VFGs) were ecpA and fyuA (92.4 and 86.7%, respectively; p<0.05), and the mean number of VFGs was significantly higher in uropathogenic strains. Particularly, papAH and malX were exclusive for uropathogenic strains. APN and RC strains showed a significantly higher prevalence of fyuA, usp, and malX than of CO strains. Compared to RC strains, APN ones showed a higher prevalence of iha, but a lower prevalence of iroN, cnf1, and kpsMT-II. Hierarchical cluster analysis showed a higher proportion of two gene clusters (malX and usp, and fyuA and ecpA) were detected in the APN and RC groups than in CO, whereas iutA and iha clusters were detected more frequently in APN strains. The motility level did not differ among the study-groups and phylogroups considered, although a higher proportion of swarming strains was observed in APN strains. Antibiotic-resistance rates were generally low except for ampicillin (37.7%), and were not associated with specific study- or phylogenetic groups. APN and RC strains produced more biofilm than CO strains. In APN strains, iha was associated with higher biofilm biomass formation, whereas iroN and KpSMT-K1 were associated with a lower amount of biofilm biomass. Further work is needed to grasp the virulence and fitness mechanisms adopted by E. coli causing APN, and hence develop new therapeutic and prophylactic approaches.