Vittorio Sirolli

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.

Cell activation and cellular-cellular interactions during hemodialysis: Effect of dialyzer membrane

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.

New Treatment Approaches for the Anemia of CKD

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.

Platelet Activation Markers in Patients with Nephrotic Syndrome

Background/Aim: Enhanced platelet reactivity may play a significant role in the genesis of the hypercoagulable state of nephrotic syndrome. However, the role of platelet function testing in nephrosis is controversial, partly because the methods used to assess platelet function (platelet aggregation and immunoassays of plasma β-thromboglobulin and platelet factor 4) have such marked methodological problems. In the present study, we evaluated several tests assessing platelet function in 18 adult patients with idiopathic nephrotic syndrome and normal renal function. Methods: Platelet function was assessed by measurement of plasma β-thromboglobulin (enzyme-linked immunosorbent assay, ELISA), plasma P-selectin (ELISA), circulating platelets exposing the activation-dependent antigens P-selectin (CD62P) and lysosomal GP53 (CD63) (flow cytometry), and by aggregation response to agonists such as ADP and collagen. Results were compared to those obtained in a group of 16 age- and gender-matched healthy subjects. Results: Levels of plasma β-thromboglobulin (p = 0.001), plasma P-selectin (p < 0.001), and CD62P/CD63-positive platelets (p < 0.001 for both) were increased in nephrotic patients as compared to healthy controls. Platelet hyperaggregability in vitro was found in 13/18 patients. The reproducibility of platelet activation markers, as assessed by blood sample collection a week later from all patients, was found to be higher for plasma P-selectin (Spearman correlation coefficient, R = 0.99) and circulating activated platelets (CD62P: R = 0.97; CD63: R = 0.96) than for plasma β-thromboglobulin (R = 0.78). Conclusions: Pronounced platelet activation takes place in nephrotic syndrome and may contribute to the hypercoagulability of nephrosis. Whole blood flow cytometry assay of platelet activation and plasma P-selectin assay may represent useful tests to assess the hypercoagulable state in nephrotic patients.

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.

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.

Serum fetuin a in hemodialysis: a link between derangement of calcium-phosphorus homeostasis and progression of atherosclerosis?

BACKGROUND Fetuin A, a circulating inhibitor of ectopic calcification, is downregulated in hemodialysis and has been shown to predict cardiovascular mortality in this setting. The association of altered calcium-phosphorus with serum fetuin A levels is still a matter of debate. Although carotid intima-media thickness (cIMT) is a strong predictor of major cardiovascular events, its association with serum fetuin A levels is poorly defined. STUDY DESIGN Cohort study. PARTICIPANTS & SETTINGS 174 uremic patients on long-term hemodialysis therapy enrolled in 4 university hospitals. PREDICTORS Serum fetuin A levels at the beginning of the study (T0) and after 12 months (T12). OUTCOMES Progression of atherosclerosis assessed by means of cIMT measurements at 24 months (T24); cardiovascular morbidity and mortality at 36 months. RESULTS Serum fetuin A concentrations at T0 and T12 were 282.3 +/- 79.4 and 290.0 +/- 92.2 microg/mL, respectively. Mean T0 and T24 cIMT values were 1.02 +/- 0.2 and 1.06 +/- 0.2 mm, respectively (P < 0.001). Fatal and nonfatal cardiovascular disease occurred in 36 and 86 patients by 36 months, respectively. In multivariate logistic regression, higher calcium-phosphorus product was associated with lower serum fetuin A level (odds ratio, 0.96; 95% confidence interval [CI], 0.93 to 1.00; P = 0.02). Multiple regression analysis showed that T0 serum fetuin A level was associated with T24 cIMT (P = 0.01) after adjustments for age, cholesterol level, high-sensitivity C-reactive protein level, previous cardiovascular events, and T0 cIMT. In a multivariate Cox regression analysis, cardiovascular mortality was independently associated with a 1-tertile lower T0 serum fetuin A level, and a 1-tertile higher T0 cIMT value was independently associated with greater cardiovascular mortality (hazard ratio, 0.45; 95% CI, 0.15 to 0.65; P = 0.007 and hazard ratio, 10.00; 95% CI, 3.16 to 31.73; P < 0.001, respectively) after adjustment for age and previous cardiovascular events. LIMITATION Length of follow-up. CONCLUSION Calcium-phosphorus product in hemodialysis patients inversely correlated with serum fetuin A level, which, in turn, was associated inversely with progression of atherosclerotic lesions and cardiovascular mortality in this study population.

Calcium Sensing Receptor Expression in Ovine Amniotic Fluid Mesenchymal Stem Cells and the Potential Role of R-568 during Osteogenic Differentiation

Amniotic fluid-derived stem (AFS) cells have been identified as a promising source for cell therapy applications in bone traumatic and degenerative damage. Calcium Sensing Receptor (CaSR), a G protein-coupled receptor able to bind calcium ions, plays a physiological role in regulating bone metabolism. It is expressed in different kinds of cells, as well as in some stem cells. The bone CaSR could potentially be targeted by allosteric modulators, in particular by agonists such as calcimimetic R-568, which may potentially be helpful for the treatment of bone disease. The aim of our study was first to investigate the presence of CaSR in ovine Amniotic Fluid Mesenchymal Stem Cells (oAFMSCs) and then the potential role of calcimimetics in in vitro osteogenesis. oAFMSCs were isolated, characterized and analyzed to examine the possible presence of CaSR by western blotting and flow cytometry analysis. Once we had demonstrated CaSR expression, we worked out that 1 µM R-568 was the optimal and effective concentration by cell viability test (MTT), cell number, Alkaline Phosphatase (ALP) and Alizarin Red S (ARS) assays. Interestingly, we observed that basal diffuse CaSR expression in oAFMSCs increased at the membrane when cells were treated with R-568 (1 µM), potentially resulting in activation of the receptor. This was associated with significantly increased cell mineralization (ALP and ARS staining) and augmented intracellular calcium and Inositol trisphosphate (IP3) levels, thus demonstrating a potential role for calcimimetics during osteogenic differentiation. Calhex-231, a CaSR allosteric inhibitor, totally reversed R-568 induced mineralization. Taken together, our results demonstrate for the first time that CaSR is expressed in oAFMSCs and that calcimimetic R-568, possibly through CaSR activation, can significantly improve the osteogenic process. Hence, our study may provide useful information on the mechanisms regulating osteogenesis in oAFMSCs, perhaps prompting the use of calcimimetics in bone regenerative medicine.

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.