Patterns of Proteins Removed with High-Flux Membranes on High-Volume Hemodiafiltration Detected with a MultiDimensional LC-MS/MS Strategy.

Background: Aim of this prospective crossover study was to identify the nature of the middle-molecular weight solutes removed during high-volume post-dilution HDF. Methods: The efficiency in removing small molecules, protein-bound and middle-molecular proteins was evaluated in 16 chronic dialysis patients on post-dilution HDF with two high-flux dialyzer membranes (Amembris and Polyamix). Multidimensional Protein Identification Technology (MudPIT) was employed to identify middle-molecular weight solutes in spent dialysate. Results: Efficiency of post-dilution HDF in removing solutes of different MW was high with both membranes, but higher with Amembris than with Polyamix. With MudPIT analysis, 277 proteins were identified in the dialysate fluids. Although the protein-removal pattern was similar among patients and tested membranes, the total and protein-specific peptide spectral count (mass spectrometric quantitation criteria) of most proteins were higher using the Amembris membrane. Conclusions: The MudPIT approach showed to be a powerful tool to identify a broad molecular weight spectrum of proteins removed with post-dilution HDF. Short Summary: Aim of this prospective crossover study was to analyze the hydraulic properties of two high-flux dialyzer membranes (Amembris and Polyamix) during high-volume, post-dilution HDF and to evaluate the influence of these properties on the removal of proteins and peptides using an in-depth analysis of the spent dialysate. For this analysis, a liquid chromatography tandem mass spectrometry approach called MudPIT (Multidimensional Protein Identification Technology) was used to identify the middle molecular weight solutes present in the spent dialysate of patients. The capability of post-dilution HDF in removing solutes of different MW was very high with both dialyzers, but higher with the Amembris membrane. The proteomic MudPIT approach showed to be a powerful tool to identify a wide molecular spectrum of proteins removed from blood during post-dilution HDF. These results may contribute to address research toward a better knowledge of uremic toxins and the balance between the intended and unintended removal of undesired and beneficial proteins next to identification of new target proteins as potential candidates for uremic toxicity. © 2014 S. Karger AG, Basel.

Efficiency of post-dilution hemodiafiltration with a high-flux α-polysulfone dialyzer.

PURPOSE: Intra-individual comparison of technical and clinical characteristics of two hemodiafiltration (HDF) strategies, namely, post-dilution HDF (post-HDF) with a high-flux α-polysulfone hemodiafilter and reverse mid-dilution HDF (MD-HDF). METHODS: Fifteen patients who were stable on RRT were randomly submitted to both HDF techniques under matched operational conditions. Removal of small and middle molecular compounds was compared. The pressure regimen within the dialyzers and the hydraulic and solute permeability indexes of the membrane were monitored on-line during the sessions. RESULTS: Urea removed was not statistically different between post-HDF and MD-HDF (41.7±10.2 vs. 39.9±8.2 g/session). High and comparable removal of phosphate (KDQ,132±30 vs. 138±21 ml/min) and middle molecules (ß2-m KDQ, 79.1±6.1 vs. 74.1±13.5 ml/min) was shown in post-HDF and MD-HDF. Albumin leakage tended to be lower after post-HDF (914±370 vs. 1313±603 mg/session, p=0.075). There were no cases of blood circuit clotting, hypotensive episodes, or other clinical or technical problems. In post-HDF, a very high ultrafiltration rate (QUF, 7.4 l/h) and filtration fraction of 59% were maintained through the sessions with safe trans-membrane pressure (TMP) values strictly retained within the planned range (280-350 mmHg). Larger volume exchange (10 l/h) was obtained in MD-HDF, but the very high QUF established high and risky TMP in the post-dilution section of the MD 220 dialyzer. CONCLUSIONS: The hemodiafilter tested in this study proved its high efficiency when used in post-dilution HDF with the application of an automatic ultrafiltration/pressure feedback, which guaranteed maximal convection within controlled hydraulic conditions.

Gene silencing in adult rat cardiac myocytes in vitro by adenovirus-mediated RNA interference.

RNA interference (RNAi) by short double stranded RNA (siRNA) represents an efficient and frequently used tool for gene silencing to study gene function. Whereas efficient ablation of genes has been demonstrated in neonatal cardiac myocytes, thus far information on successful application of this technique in adult cardiac myocytes (ACM), a standard experimental model in cardiac physiology and pathophysiology, is sparse. Here we demonstrate efficient ablation of a transgene encoding for enhanced green fluorescent protein (EGFP) and a cell specific endogenous gene encoding for an inward-rectifier channel subunit (Kir2.1) in ACM in vitro using adenovirus driven transcription of siRNA hairpins. EGFP fluorescence and density of background inward rectifier current (IK1) were reduced by > 90% within about 6-8 days after transformation with the corresponding virus. In Kir2.1-silenced myocytes resting membrane potential was significantly reduced. Survival of these cells in culture was compromised, presumably due to Ca2+ -overload caused by the depolarization. The sequence-specific knockdowns of EGFP and Kir2.1 were confirmed on the RNA level using real-time RT-PCR. In Kir2.1-silenced myocytes density of transient outward current, carried predominantly by Kv4.x subunits remained unaffected. This communication for the first time demonstrates proof of principle of efficient RNA interference using adenovirus-based vectors and demonstrates its large potential in phenotyping of ACM.