Luisa Sereni

OXIDATION OF ALBUMIN IS ENHANCED IN THE PRESENCE OF UREMIC TOXINS

Albumin has been considered a “sacrificial plasma antioxidant” due to the high reactivity of the protein sulfhydryl groups with oxidants such as hydrogen peroxide (H2O2) and hypochlorous acid (HOCl). Based on its large quantity and high turnover. It is considered as one of the most important plasma antioxidants for protecting key cellular and regulatory proteins. Since hemodialysis patients have lower overall levels of albumin and possible protein modifications due to uremic toxins, we investigated whether modifications by various uremic toxins would affect the susceptibility of albumin to an oxidative challenge. We incubated bovine serum albumin in the presence of carboxymethyllysine (CML) (10 μmol/L–1 mmol/L), methyl glyoxal (50 μmol/L–5 mmol/L), p-cresol (100 μmol/L–10 mmol/L) or hippuric acid (200 μmol/L–20 mmol/L) for 16 hours at 37°C and then subsequently added 0.5 mmol/L–1 mmol of H2O2/HOCl. We measured the extent of protein modification by the loss of protein sulfhydryl groups, dityrosine formation and the formation of advanced oxidation protein products (AOPP). Incubation of albumin with the uremic toxins caused a loss of protein sulfhydryl groups and an increase in dityrosines and AOPP. The presence of uremic toxins had no effect on the loss of protein sulfhydryl groups after addition of H2O2/HOCl; however, low levels of CML, p-cresol and methyl glyoxal inhibited the formation of AOPP and dityrosines. We suggest that uremic toxins may possibly play a role in mediating free radical initiated protein damage.

Hemodiafiltration With Online Regeneration of Ultrafiltrate: Effect on Heme‐Oxygenase‐1 and Inducible Subunit of Nitric Oxide Synthase and Implication for Oxidative Stress and Inflammation

Hemodiafiltration with regeneration of ultrafiltrate (HFR) has a positive impact on inflammation and oxidative stress (OxSt), risk factors for cardiovascular disease (CVD), the most common cause of excess morbidity and mortality for end-stage renal disease (ESRD) patients. However, studies have been of limited duration. This study extends our previous study of HFR effects by evaluating the effect on mononuclear cell protein expression of heme-oxygenase-1 (HO-1), induced by OxSt, and inducible subunit of nitric oxide synthase (iNOS), and plasma level of interleukin-1β (Il-1β) and oxidized low-density lipoproteins (OxLDL), marker of OxSt, for a 12-month period. Fourteen ESRD patients stable on hemodialysis over a period of at least 2 years and on conventional bicarbonate dialysis were switched to be treated with HFR. Blood samples were collected at baseline, after 3, 6, 9 and 12 months. HO-1 and iNOS protein expression were evaluated by Western blot, OxLDL by enzyme-linked immunosorbent assay (ELISA), and Il-1β by enzyme amplified sensitivity immumoassay assay. HFR significantly increased HO-1 at the 9 and 12 months (ANOVA = P < 0.00001): 0.17 ± 0.11 (baseline) versus 0.48 ± 0.20, P < 0.043 and 0.59 ± 0.32, P < 0.004, respectively. Il-1β declined (ANOVA = P < 0.0001) since the 3 months from 169.92 ± 92.39 pg/mL (baseline) to 39.03 ± 10.01 (12 months), P < 0.0001. HFR also reduced plasma OxLDL: 475.4 ± 110.8 ng/mL (baseline) versus 393.1 ± 101.9 ng/mL (12 months), P < 0.04. iNOS showed no changes upon HFR treatment. These results together with our previous results indicate that HFR improves OxSt and inflammation. Given the strong relationships between OxSt and inflammation with CVD, their reduction might provide a beneficial impact by reducing the risk of atherosclerotic CVD in dialysis patients.

Choosing new adsorbents for endogenous ultrapure infusion fluid: performances, safety and flow distribution.

Adsorption may notably contribute to the removal of uremic toxins and to the efficiency of hemodialysis. We examined different uncoated stationary matrixes, charcoals and synthetic resins to establish their adsorptive capacities in relation to low (urea, creatinine) and high molecular weight (β2-microglobulin, myoglobin) compounds in in vitro conditions (steady state and flow-through) using isotonic solutions or uremic ultrafiltrate. Trace metal, particle release analyses and scanning electron microscopy of different adsorbents were performed. Dynamic flow-distribution studies were made using 99Technetium and analysing the different regions of interest by single head γ-camera. We show that adsorbents may differ greatly as to their adsorptive capacity depending on flow rate, nature, and total mass of the compounds to be removed from the ultrafiltrate. These studies suggest a methodological approach for screening stationary matrixes for possible application in hemodialysis.

BLOOD TUBING AND CYTOKINE PRODUCTION: EFFECT OF STERILIZATION

Blood tubings commonly represent an integral component of hemodialysis circuits. Different factors may influence their biocompatibility, such as the type of material, the sterilization mode and the geometry. In vivo the final biocompatibility may be further complicated by the individual host response, the flow parameters, and the impact of mechanical trauma on bloods cellular components (i.e. erythrocytes). In this in vitro study we evaluated some commercially available blood tubings sterilized by different methods as to their interaction with normal leukocyte population and tested the response of these cells in terms of cytokines (IL-lβ, IL-1Ra, TNF-α). As a positive control, leukocytes were incubated with 0.5 ng/mL of bacterial lipopolysaccharide (LPS) or with Cuprophan of comparable surface. The results showed that cytokine production was markedly reduced, particularly in the case of γ-ray-sterilized tubings. Of interest, it was not always related to the adherence. However in some cases, particularly of γ-ray sterilization, adherence was none, despite the cytokine production.

Adsorption in Extracorporeal Blood Purification: How to Enhance Solutes Removal Beyond Diffusion and Convection

Uremic syndrome is linked to a plethora of uremic toxins circulating in the body in ESRD patients. Their overall spectrum is partly or entirely unexplored despite the need to urgently define the specimens and the patho-physiology beyond their high blood levels to address new or more selective removal strategies. It is generally accepted that convective hemodialysis is the best choice to remove large part of the molecular spectrum, even though it is not fully demonstrated its superiority in terms of clinical outcomes. Then, transport mechanisms can benefit from maximizing all the physi‐ co-chemical principles including diffusion for small solutes, convection for middle mole‐ cules and adsorption for large molecular size uremic toxins. The latter has not been fully adopted in hemodialysis and this transport mechanisms is limited to the intrinsic capability of dialysis membrane to adsorb macromolecules while transporting solutes by diffusion and/or convection. However, poorly has been explored about the use of sorbents to enhance the solute removal in hemodialysis. The purpose of this chapter is to summarize the main contributions of so far published clini‐ cal and technical experiences. The chapter will be structured as follow: first we introduced a summary of the basic princi‐ ples of solutes transport and relative contribution of the different mechanisms to the overall