P1054COMBINING A HEPARIN-GRAFTED DIALYZER WITH A CITRATE ENRICHED DIALYSATE OFFERS ADEQUATE HEMODIALYSIS EFFICACY AVOIDING SYSTEMIC ANTICOAGULATION: RESULTS OF THE NON-INFERIORITY RANDOMIZED EVOCIT STUDY

Background and Aims The combined use of a heparin-grafted membrane with a citrate enriched dialysate is an effective hemodialysis strategy with low circuit clotting rates while avoiding systemic anticoagulation. Whether this technique results in dialysis efficacy that is non-inferior to regular hemodialysis using systemic anticoagulation has not been investigated up to now. Method Prevalent hemodialysis (n=26) patients were recruited for a randomized crossover non-inferiority trial powered at >90% to detect a prespecified non-inferiority threshold of 10% spKt/Vurea (NCT03887468). Hemodialysis using a heparin-grafted dialyzer in combination with a 1.0 mmol/L citrate enriched dialysate (“evocit”) was compared to hemodialysis using a heparin-grafted dialyzer, systemic unfractionated heparin and regular bicarbonate-based dialysate (“evohep”). Each treatment arm lasted 4 weeks with a 3x4hours weekly hemodialysis regimen. All sessions were standardized with fixed blood- and dialysate flow rates. Biological analyses were done during midweek sessions. The primary endpoint was spKt/Vurea. Secondary endpoints included alternative adequacy markers, premature treatment termination, retransfusion failure and loss of total cell volume of the dialyzer after dialysis. Results A total of 617 hemodialysis sessions were performed: 307 sessions according to evocit and 310 sessions according to evohep protocol. Mean spKt/Vurea was 1.45±0.25 for evocit sessions and 1.50±0.26 for evohep sessions. In a paired analysis, mean of the difference in spKt/Vurea between both study arms was 0.05 with a 95%CI of 0.012-0.098 (p=0.01), the upper bound of the estimate lying within the prespecified non-inferiority threshold (i.e. <0.15). Processed blood volume was 75.4±3L vs 75.8±1.5L and online Kt was 47.3±5L vs 48.3±4L for all evocit and evohep sessions respectively. Urea reduction rate (RR) was 71.3±5.7 vs 72.3±5.8, bèta2microglobulin RR 37.1±8 vs 37.9±8 and myoglobin RR 30.9±9.8 vs 34.5±12.5 for midweek evocit and evohep sessions respectively. Circuit thrombosis leading to premature treatment end occurred in 13/307 (4.23%) of evocit sessions in 6/26 patients but in none of the evohep sessions (p=0.03). Treatment time of evocit sessions complicated with circuit thrombosis (n=13) was reduced with 36 minutes (IQR 20-46 minutes) without impact on effective treatment times overall (236±12 vs 238±4 minutes for evocit and evohep sessions respectively). Retransfusion failure occurred in 3/307 (0.98%) of evocit sessions and none of the evohep sessions. Dialyzers’ total cell volume was reduced with 17% (IQR 11-33%) and 9% (IQR 6-17%) (p<0.0001) after evocit and evohep sessions respectively. Conclusion Hemodialysis avoiding systemic anticoagulation using a heparin-grafted dialyzer with a citrate enriched dialysate is an adequate technique for maintenance hemodialysis offering spKt/Vurea results within recommended dose, and is not inferior to standard hemodialysis using systemic anticoagulation with heparin in terms of spKt/Vurea. Circuit clotting complications occurred at low frequency during evocit sessions and did not have clinically significant repercussions on dialysis efficacy.

Water stress-induced changes in morphology and anatomy of flag leaf of spring wheat

Flag leaves of wheat (drought hardened and non-hardened) were examined by light microscopy to determine whether the differences in leaf anatomy could be related to the known differences in dehydration tolerance. Plants exposure to water stress during tissue differentiation of flag leaves resulted in an irreversible reduction of leaf area and thickness, increased frequencies of stomata and higher number of bulliform cells with simultaneous decrease in number of intermediate veins and an increase in the share of the cell walls in total cell volume. The smaller leaf thickness was due to a diminished number of mesophyll layers and a decreased size of mesophyll cells. Such altered leaf anatomy indicated development of leaf xerophily. It was found that the irreversible changes in anatomy of wheat flag leaves play a decisive role in acquiring drought tolerance during wheat acclimation to drought.

Differential response of vacuolar proton pumps to osmotica

The vacuole is a fundamental and dominant organelle and occupies a large part of the total cell volume in most mature plant cells. The higher-plant vacuole contains two types of proton-translocating pumps, H+-ATPase (EC 3.6.1.3) and H+-pyrophosphatase (EC 3.6.1.1), residing on the same membrane. These two enzymes generate roughly equal proton gradients across the vacuolar membrane for the secondary transport of ions and metabolites. However, the pumps respond differentially to stress in order to maintain critical functions of the vacuole. In this work, tonoplasts from etiolated mung bean seedlings (Vigna radiata L.) were used to investigate the function of these two enzymes under high osmotic pressure. At high concentrations of sucrose or sorbitol, the light scattering and volume of isolated vesicles were progressively changed. Concomitantly, enzymatic activities, proton translocation, and coupling efficiencies of these two proton-pumping enzymes were inhibited to various extents under high osmotic pressure. No significant change in enzymatic activities of purified vacuolar H+-PPase and H+-ATPase under similar conditions was observed. We thus believe that the membrane structure is an important determinant for proper function of proton pumping systems of plant vacuoles. Furthermore, kinetic analysis shows different variation in apparent Vmax but not in KM values of vacuolar H+-PPase and H+-ATPase at high osmolarity of sucrose and sorbitol, respectively, suggesting probable alterations in substrate hydrolysis reactions but not substrate-binding affinity of the enzymes. A working model is proposed to interpret supplemental roles of vacuolar H+-PPase and H+-ATPase to maintain appropriate functions of plant tonoplasts.

Quantification of Cytotoxicity by Cell Volume and Cell Proliferation

A fast and sensitive method for the quantification of cytotoxicity, using the cell counter and analyser system, CASY 1, was established. This system has a high resolution and a large dynamic range of volume determination, permitting the volume changes caused by cytotoxic effects to be measured in a reproducible and standardised manner. As a first approach, eight cytotoxic compounds with different modes of action were investigated. For seven of the compounds, changes in the mean cell volume could be demonstrated after three hours. All eight compounds showed a dramatic decrease in mean cell volume within 24 hours. Depending on the degree of membrane destruction caused by the direct or indirect actions of the cytotoxic compounds, values for the mean cell volume between the size of an undamaged cell and the size of the cell nucleus were determined. In addition, the number of living cells was substantially decreased by exposure to the chemicals. Both of the effects of cytotoxic compounds can be detected by following a single parameter, i.e. total cell volume. This parameter is highly sensitive for the detection and quantification of cytotoxicity. IC50 values were within the range obtained in testing by other methods. Cytotoxicity testing by CASY 1 is recommended when highly reproducible measurement is necessary, but the system is also valuable for cell counting and volume determination.

Cellular dimensions affecting the nucleocytoplasmic volume ratio.

Although it has long been appreciated that larger eukaryotic cells have larger nuclei, little is known about how this size relationship is maintained. Here we describe a method for measuring the aqueous volume ratio of nucleus to cytoplasm, two compartments which are interconnected via the pores in the nuclear envelope. We then use that method to identify proportional cellular dimensions in variously treated cells and in different cell types. Cells were scrape loaded with a mixture of fluorescent dextrans: Texas red dextran, average mol wt = 10,000 (TRDx10), and fluorescein isothiocyanate dextran, average mol wt = 70,000 (FDx70). After introduction into the cytoplasmic space, the TRDx10 distributed into both the nucleus and cytoplasm, whereas the FDx70 was restricted to cytoplasm, due to size exclusion by the nuclear pores. The aqueous nucleocytoplasmic volume ratio (RN/C) was determined by measuring, from fluorescence images of spread cells, total cellular fluorescence of each of the two probes and the fluorescence ratio of those probes in the cytoplasm. RN/C was unaffected by the measurement procedure or by varying temperatures between 23 degrees and 37 degrees C. Loading excess unlabeled dextrans had little effect on RN/C, with the single exception that high concentrations of large dextrans could lower RN/C in endothelial cells. Expanding intracellular membranous compartments of macrophages by phagocytosis of latex beads decreased RN/C. Expanding the same compartment by pinocytosis of sucrose, which nearly doubled total cell volume, had little effect on RN/C, indicating that nuclear volume was more closely linked to the cytoplasmic volume, exclusive of vesicular organelles, than to total cell volume. RN/C was the same in mononucleate and binucleate endothelial cells. Finally, measurements of RN/C in murine bone marrow-derived macrophages, bovine aortic endothelial cells, Swiss 3T3 fibroblasts, PtK2 cells, and CV-1 cells revealed that nuclear volume scaled allometrically with cell volume. The allometric relationship indicated that cell volume was proportional to nuclear surface area.

Early embryogenesis in Arabidopsis thaliana. II. The developing embryo

Embryo differentiation in Arabidopsis thaliana follows the classical Capsella variation of the Onagrad type. Fertilization occurs approximately 3 h after flowering, whereupon vacuolar organization in the zygote changes and the cell elongates rapidly to approximately three times its original length. Cytoplasmic polarization is maintained. During the first two division steps there is very little increase in total cell volume, and during subsequent divisions vacuole number increases, with a concomitant decrease in size. Plastids remain undifferentiated up to the late globular stage, after which grana begin to develop. Ribosomal concentration increases significantly after fertilization. Differences between embryo proper cells become evident by the heart stage; vacuole, plastid, and mitochondrial abundance, size, and complexity vary within the embryo. There are no plasmodesmatal connections with the endosperm or integuments. Suspensor development is complete by the early globular stage, when it consists of seven to nine highly vacuolate cells, each linked by end wall plasmodesmata. Ribosome and volume densities of plastids and mitochondria are significantly lower than in the embryo proper organelles, and dictyosomes are infrequent. Embryo sac wall projections proliferate throughout the micropylar chamber, especially adjacent to the filiform apparatus and zygote base, and ingrowths form on the basal cell proximal wall. Key words: Arabidopsis, embryogenesis, embryo differentiation, wall ingrowths.