Influence of hemodialysis membrane permeability on serum levels of advanced glycation end products (AGEs) and homocysteine metabolites.

BACKGROUND: [corrected] Advanced glycation end products (AGEs), total homocysteine (tHcy) and the homocysteine metabolites cystathionine (Cysta) and dimethylglycine (DMG) are increased in serum of patients with end-stage renal disease. The aim of this prospective randomized study was to compare the efficacy of polysulfone high-flux vs. polysulfone low-flux hemodialysis (HD) treatment regarding removal of AGEs, tHcy, Cysta and DMG. PATIENTS AND METHODS: Twenty-nine patients on chronic HD treatment were randomly assigned to 2 groups in a 3-period 2-treatment design with low flux (A)--high flux (B)--low flux (A) for group I and B-A-B for group II, 6 weeks each period. The following parameters were measured in pre- and postdialytic serum samples at baseline and the end of each period: total serum fluorescence, Nepsilon-carboxymethyllysine (CML), free and protein-bound pentosidine, tHcy, Cysta and DMG. RESULTS: There was increased removal of free pentosidine during high-flux HD treatment compared to low-flux HD treatment, attaining significance between the second and third treatment periods (group 1: 86.0 +/- 4.7% vs. 79.2 +/- 8.8%, p = 0.007; group II: 84.0 +/- 6.3% vs. 79.8 +/- 9.8%, p = 0.049 for high vs. low flux). The intradialytic reduction rates for total serum fluorescence, tHcy, Cysta, DMG did not differ between high- and low-flux HD treatment. Protein-bound pentosidine and CML did not decrease during the dialysis sessions, neither with high-flux nor with low-flux HD membrane. Despite a strong decrease during single HD session, the predialytic levels of free pentosidine, tHcy, Cysta and DMG remained unchanged during the study period both for high- and low-flux HD treatment. CONCLUSION: The more pronounced effect of high-flux dialysis on the removal rate of free pentosidine, found in this randomized crossover study, could not translate into a significant difference in predialysis levels after a 6-week treatment period. We could not find any differences between polysulfone high- and low-flux membranes for lowering predialytic serum concentrations of the measured AGEs, which are mainly bound on albumin.

M-wave analysis and passive tilt in patients with different degrees of carotid artery disease.

BACKGROUND AND PURPOSE: Carotid artery disease (CAD) is able to critically impair cerebral autoregulation which increases the risk for stroke. As therapeutic strategy largely depends on the degree of CAD, we investigated whether this gradation is also related to significant changes in autoregulatory capacity. We applied cross-spectral analysis (CSA) of spontaneous Mayer-wave (M-wave) oscillations and passive tilting (PT) to test cerebral autoregulation. METHODS: Cerebral autoregulation was tested in 102 patients with carotid stenosis (> or =70%) or occlusion and 14 controls by comparison of continuous transcranial Doppler sonography of the middle cerebral artery and beat-to-beat arterial blood pressure (ABP) during PT to 80 degrees head-up position as well as by CSA of M-waves (3-9 cpm). RESULTS: The orthostatic decrease of cerebral blood flow velocity (CBFV) was not correlated with the degree of CAD and showed a lower sensitivity and specificity than phase angle shifts between M-waves in ABP and CBFV (sensitivity: 75-80%, specificity: 86%). Phase angles were gradually lowered in carotid stenoses > 70%, but apparently, they were only moderately correlated with the degree of CAD (r = -0.35, P < 0.01). An additional influencing factor seemed to be the sufficiency of collateralization. CONCLUSIONS: The results show that CSA of M-waves is more appropriate for testing autoregulation than PT. CSA suggests that the capacity to autoregulate depends to a certain extent on the degree of CAD but is also influenced by the sufficiency of collateral pathways and pre-existing strokes.

Determination of autofluorescence of red blood cells (RbCs) in uremic patients as a marker of oxidative damage.

AIM: It is suggested that the red blood cells (RBCs) of uremic patients have increased oxidative damage. The activities of different antioxidant enzymes and the levels of several antioxidants or lipid peroxidation products in RBCs are usually determined to estimate the oxidative stress in uremia. The autofluorescence of RBCs as measured by flow cytometry is caused by the formation of conjugated Schiff base compounds from aldehydes derived from lipid peroxidation and amino groups of phospholipids or cell proteins, and has been proposed as a marker of oxidative stress. The aim of this study was to evaluate if this method is suitable for estimation of oxidative stress in the RBCs of patients with different degrees of renal insufficiency. PATIENTS AND METHODS: To determine the oxidative damage in RBCs in uremia, the autofluorescence of RBCs was measured by flow cytometry in the following 3 groups of patients: group A: 16 patients with chronic renal failure (CRF); group B: 16 hemodialysis (HD) patients; group C: 16 patients with a well-functioning renal allograft. Twenty-four healthy volunteers served as controls. The basal value of RBC autofluorescence and the autofluorescence of RBCs after oxidative damage by treatment with 0.1 mM hydrogen peroxide (H2O2)/0.7 mM sodium azide were determined. RESULTS: In basal RBC autofluorescence values, no differences were found between the 3 groups and the controls. However, there was a significant correlation between the increase of serum creatinine and RBC autofluorescence in the group of patients with CRF (r = 0.521; p = 0.038). After H2O2 treatment, the RBC autofluorescence rose markedly in all individuals. This increase in RBC autofluorescence was significantly higher in the patients with CRF (p = 0.003) and in the HD patients (p = 0.001) compared to the controls. In contrast, there was no difference in RBC autofluorescence between the patients with renal allograft and the controls after H2O2 treatment. CONCLUSIONS: In conclusion, flow cytometry is a useful tool for determining oxidative damage in RBCs. The RBCs of uremic patients are more susceptible to oxidative damage induced by H2O2, likely caused by diminished antioxidant defense in the RBC membrane. Successful renal transplantation leads to a normal autofluorescence response in the RBCs after H2O2 treatment.

Cell-free DNA in urine: a marker for kidney graft rejection, but not for prenatal diagnosis?

Intrigued by the rapid clearance of free fetal DNA from the maternal circulation, we have investigated whether this fetal genetic material could be cleared via the kidney. For this purpose, we examined for the presence of Y chromosome-specific DNA sequences in urine samples obtained from 8 women pregnant with male fetuses. No male-specific sequences could be detected, despite the use of a very sensitive nested PCR assay nor a highly reproducible real-time PCR assay. We did, however, detect maternal DNA sequences. To determine if this cell-free DNA was derived from the kidney or another source, we next examined urine from female kidney transplant patients who had received male kidneys. Y chromosome-specific sequences were indeed detectable by both nested and real-time PCR in these samples, thereby confirming a recent report describing urinary DNA microchimerism. Quantitative analysis of serially obtained samples furthermore suggests that transplant-derived sequences are elevated during periods of graft rejection. These results imply that the measurement of graft-derived urinary DNA may serve as a new marker for kidney graft tolerance.

Determination of erythrocyte antioxidant capacity in haemodialysis patients using electron paramagnetic resonance.

BACKGROUND: The increased oxidative stress of uraemia is caused both by an increased generation of oxygen-free radicals and a decrease of antioxidative forces. There are, however, conflicting data concerning disturbances of the radical-scavenging power of red blood cells (RBCs) in uraemic patients. METHODS: The antioxidant capacities of the RBCs of 10 haemodialysis (HD) patients and 10 controls were examined after treatment with 0.324 mM tert-butylhydroperoxide (t-BOOH) in phosphate-buffered saline at 37 degrees C using electron paramagnetic resonance (EPR) with 5,5-dimethylpyrroline-N-oxide (DMPO) as a spin trap and glutathione (GSH) regeneration as an indicator of hexose monophosphate shunt (HMPS) activity. EPR investigations were also done after pre-incubation with N-ethylmaleimide (NEM) to inhibit the GSH system. Furthermore, we determined the RBC redox state in 15 HD patients and 15 controls. RESULTS: There was no difference between HD patients and controls in the elimination of t-BOOH-generated free radicals in the RBCs. A more than 20-fold increase in radical concentration was observed after GSH trapping with NEM. In this case, we found a delayed decrease of the relative radical concentration in HD patients compared with controls with a significant difference after 7 min (2.2+/-0.26 vs 1.60+/-0.21; P=0.005) and after 10 min (1.82+/-0.41 vs 0.83+/-0.44; P=0.001). GSH regeneration via HMPS did not differ between the RBCs of HD patients (99.5+/-13.5 nmol/min x ml RBC) and those of the controls (94.2+/-16.9 nmol/min x ml RBC). There were no differences in the RBC concentrations of GSH, GSSG, NADP, NADPH, and in the GSH/GSSG and NADP/NADPH ratios between HD patients and controls. CONCLUSIONS: These data suggest a strong antioxidant potential in the GSH system of erythrocytes without any evidence of a disturbance in HD patients. The HMPS pathway also appears not to be impaired in the RBCs of HD patients. However, the slower radical elimination in the RBCs of HD patients after inhibition of GSH-depending radical scavengers as compared with controls indicates a defect in the antioxidant forces outside the GSH system, and could be one reason for the reduced lifespan of RBCs in HD patients.

Flow, diffusion, and thermal convection in percolation clusters: NMR experiments and numerical FEM/FVM simulations.

Percolation objects were fabricated based on computer-generated, two- or three-dimensional templates. Random-site, semi-continuous swiss cheese, and semi-continuous inverse swiss-cheese percolation models above the percolation threshold were considered. The water-filled pore space was investigated by NMR imaging and, in the presence of a pressure gradient, NMR velocity mapping. The fractal dimension, the correlation length, and the percolation probability were evaluated both from the computer-generated templates and the corresponding NMR spin density maps. Based on velocity maps, the percolation backbones were determined. The fractal dimension of the backbones turned out to be smaller than that of the complete cluster. As a further relation of interest, the volume-averaged velocity was calculated as a function of the probe volume radius. In a certain scaling window, the resulting dependence can be represented by a power law the exponent of which was not yet considered in the theoretical literature. The experimental results favorably compare to computer simulations based on the finite-element method (FEM) or the finite-volume method (FVM). Percolation theory suggests a relationship between the anomalous diffusion exponent and the fractal dimension of the cluster, i.e., between a dynamic and a structural parameter. We examined interdiffusion between two compartments initially filled with H2O and D2O, respectively, by proton imaging. The results confirm the theoretical expectation. As a third transport mechanism, thermal convection in percolation clusters of different porosities was studied with the aid of NMR velocity mapping. The velocity distribution is related to the convection roll size distribution. Corresponding histograms consist of a power law part representing localized rolls, and a high-velocity cut-off for cluster-spanning rolls. The maximum velocity as a function of the porosity clearly visualizes the percolation transition.

Rayleigh-Bénard percolation transition study of thermal convection in porous media: numerical simulation and NMR experiments.

Thermal convection was studied as a function of the porosity in random-site percolation model objects in a Rayleigh-Bénard configuration. NMR velocity mapping experiments and numerical simulations using the finite-volume method are compared. Velocity histograms were evaluated and can be described by power laws in a wide range. The maximum velocity as a function of the porosity indicates a combined percolation/Rayleigh-Bénard transition.

Flow through percolation clusters: NMR velocity mapping and numerical simulation study.

Three- and (quasi-)two-dimensional percolation objects have been fabricated based on Monte Carlo generated templates. The object size was up to 12 cm (300 lattice sites) in each dimension. Random site, semicontinuous swiss-cheese, and semicontinuous inverse swiss-cheese percolation models above the percolation threshold were considered. The water-filled pore space was investigated by nuclear magnetic resonance (NMR) imaging and, after exerting a pressure gradient, by NMR velocity mapping. The spatial resolutions of the fabrication process and the NMR experiments were 400 microm and better than 300 microm, respectively. The experimental velocity resolution was 60 microm/s. The fractal dimension, the correlation length, and the percolation probability can be evaluated both from the computer generated templates and the corresponding NMR spin density maps. Based on velocity maps, the percolation backbones were determined. The fractal dimension of the backbones turned out to be smaller than that of the complete cluster. As a further relation of interest, the volume-averaged velocity was calculated as a function of the probe volume radius. In a certain scaling window, the resulting dependence can be represented by a power law, the exponent of which was not yet considered in the theoretical literature. The experimental results favorably compare to computer simulations based on the finite-element method (FEM) or the finite-volume method (FVM). This demonstrates that NMR microimaging as well as FEM/FVM simulations reliably reflect transport features in percolation clusters.

The nutation spin echo and its use for localized NMR

A suitably matched combination of unidirectional gradient pulses of the radio frequency amplitude B(1) and of the main magnetic field B(0) produces an unconventional type of spin echo, the nutation echo. The echo signal becomes volume selective if the gradients to be matched are inhomogeneously distributed in space. An example is a combination of a constant B(0) gradient and the inhomogeneous B(1) gradient of a surface coil. We suggest a method for localized NMR on this basis. Nutation echoes can also be used to map the spatial distribution of B(1) gradients of an arbitrary radio frequency coil geometry with the aid of a small probe sample. Copyright 2000 Academic Press.

Blockade of leucocyte function-associated antigen-1 (LFA-1) decreases lymphocyte trapping in the normal pulmonary vasculature: studies in the isolated buffer-perfused rat lung.

Adhesion molecules regulate the migration of lymphocytes in lymphoid and non-lymphoid organs. In the lung, little is known about lymphocyte sticking and migration through the pulmonary vascular endothelium in physiological or pathological situations. Therefore the isolated buffer-perfused rat lung was used to investigate the mobilization of lymphocytes out of the normal lung into the venous effluent and to the bronchoalveolar space. The lymphocyte subset composition was characterized in the venous effluent, the lung tissue and the bronchoalveolar lavage (BAL) using immunocytology. Lymphocytes continuously left the normal lung at a total of 5.0 +/- 0.7 x 106 cells within the first hour of perfusion. The injection of 200 x 106 lymphocytes via the pulmonary trunk increased the venous release of lymphocytes by 170%. To investigate the effect of LFA-1 and CD44 on the adhesion of lymphocytes to the pulmonary endothelium, lymphocytes preincubated with an anti-LFA-1 MoAb, which blocks the interaction of LFA-1 and intercellular adhesion molecule-1 (ICAM-1), or lymphocytes preincubated with an anti-CD44 MoAb, were injected. The injection of LFA-1-blocked lymphocytes led to an increase by 70% of injected cells recovered in the perfusate within the first hour, whereas anti-CD44 treatment of injected lymphocytes had no effect. The LFA-1-blocked lymphocytes showed higher numbers of T and B cells in the effluent. Thus, the present experiments demonstrate that LFA-1 influences the trapping of lymphocytes in the vasculature of the healthy rat lung.

31P-magnetic resonance spectroscopy (31P-MRS) of human allografts after renal transplantation.

BACKGROUND: 31P-Magnetic resonance spectroscopy (31P-MRS) can be used as a non-invasive tool for measuring the relative intracellular concentrations of several phosphorus metabolites in different organs. Various pathological conditions are characterized by different metabolic patterns. We studied the value of 31P-MRS after renal transplantation with both an uneventful and a clinically complicated course. METHODS: We determined the relative concentrations of phosphate-containing metabolites in renal allografts of humans with 31P-MRS (1.5 Tesla) in the first few weeks after transplantation; 18 patients with an uneventful clinical course and 10 patients who required dialysis after transplantation were examined. Six patients with a stable allograft function 2-3 months after transplantation served as controls. RESULTS: In patients with primary allograft function, we found a significant correlation between the phosphomonoester/phosphodiester-ratio (PME/PDE) (r = 0.66, r < 0.01) and the time after transplantation, but no correlation between the nucleoside triphosphate (beta-NTP)-concentration (r = -0.11) and the time course. In the patients with primary or early allograft dysfunction caused by histologically proven rejection (n=5), we found a low beta-NTP compared to patients with an uncomplicated clinical course (0.09+/-0.01 vs 0.15+/-0.03), but no differences in the PME/PDE ratio (0.73+/-0.21 vs 0.80+/-0.21). In contrast, the PME/PDE ratio was lowered in three patients with delayed graft function caused by acute tubular necrosis (0.45+/-0.07 vs 0.80+/-0.21), but the beta-NTP concentration was not reduced (0.15+/-0.003 vs 0.15+/-0.03). The 31P-MR spectrum of two patients with cyclosporin A damage was not altered compared to the controls. CONCLUSIONS: 31P-MRS can be used in patients in the early period after renal transplantation. A significant correlation between the PME/PDE ratio and the time course but no change in the beta-NTP concentration was found in patients with primary allograft function in the first 4 weeks after renal transplantation. Different patterns of 31P-MR spectra were observed depending on the different causes of primary and early transplant dysfunction.

Modulation of innate immune functions by intracerebroventricularly applied neuropeptide Y: dose and time dependent effects.

Centrally applied neuropeptide Y (NPY) interacts with the autonomic nervous system and the hypothalamo-pituitary-adrenal (HPA) axis activity. Since these physiological systems have been shown to modulate innate immune functions, the effects of intracerebroventricular (i.c.v.) NPY administration on leukocyte subsets in the blood, spleen and intravascular pool of the lung, blood granulocyte chemiluminescence response, and splenic natural killer (NK) cell-mediated lysis were studied in Lewis rats. Concentration-dependent NPY effects were tested at 15 min and 24 h post i.c.v. injection at dosages of 10(-6) M, 10(-9) M, and 10(-12) M. Time dependent effects were investigated at 15 min, 1 h and 24 h after i.c.v. administration of 10(-9) M NPY. Compared to saline controls, an increased number of granulocytes and NK cells in the blood, associated with a decreased granulocyte function and NK cytotoxicity was observed 15 min following NPY infusion. This initial immunosuppression was followed by long lasting stimulatory effects of NPY on the functional capacity of both cell populations when tested at 1 h and 24 h. The dosage of i.c.v. 10(-6) M NPY produced no changes, whilst 10(-9) M produced maximal, and 10(-12) M still significant effects. Results provide evidence that centrally applied NPY influences innate immunity in a dose and time dependent fashion. Cell mobilization from the vascular marginal pool is likely to be an underlying mechanism for the initial immunosuppression.

Lysosome membrane permeability to anions.

The permeability of rat liver lysosomes to some inorganic and aliphatic organic anions was investigated, using an osmotic-protection methodology. Lysosomes were incubated at 25 degreesC in 250 mOsm solutions of potassium salts of the anions, in the presence of valinomycin, and the latency of lysosomal hexosaminidase measured at intervals. Lysosomes suspended in 250 mM sucrose at 25 degreesC were stable for up to 4 h. When suspended in 250 mOsm solutions of potassium salts of inorganic acids, latency was lost at rates indicating anion permeance decreasing in the order thiocyanate, nitrate and iodide>bromide>chloride>sulfate. This rank order does not correspond with the anion selectivity of any known anion transporter, and is closer to that of the lyotropic series. Results with the potassium salts of aliphatic organic acids indicate little correlation between permeation and hydrocarbon chain length, although formate was more rapidly permeant than acetate and its higher homologs. By contrast, oxalate was less permeable than other dicarboxylic acids. The presence of one or more hydroxy groups decreased permeance. A correlation between permeance and the acid's lowest pKa suggested that penetration was due principally to the entry of the undissociated acid, but there is evidence that the (much more abundant) singly charged anionic form is also significantly permeant.

Effects of polyethyleneimine on endocytosis and lysosome stability.

Polyethyleneimine (PEI) is shown to destabilize isolated rat liver lysosomes, as indicated by a decrease in the latency of their acid N-acetyl-beta-glucosaminidase. PEI also inhibited the generation of radiolabeled digestion products from 125I-labeled bovine serum albumin endocytosed by rat visceral yolk sac in vitro. However, PEI did not greatly inhibit the endocytic uptake of a nondigestible fluid-phase substrate, fluorescein isothiocyanate (FITC)-dextran. It is hypothesized that PEI inhibits the adsorptive endocytosis of 125I-labeled bovine serum albumin, and thus its subsequent intralysosomal digestion, by competing with and displacing the labeled protein from its binding sites on the visceral yolk sac cell surface. This hypothesis suggests a plausible explanation for the ability of PEI to act as an efficient vector for gene and oligonucleotide transfer into mammalian cells. PEI present in the culture medium is carried into cells by adsorptive endocytosis. Concentrated thus on the endosome membrane, it permeabilizes this membrane and so affords DNA conjugated to the PEI an otherwise unavailable mode of access into the cytoplasm.

Lymphocyte subsets in distinct lung compartments show a different ability to produce interferon-gamma (IFN-gamma) during a pulmonary immune response.

Lymphocytes play an important immunoregulatory role in pulmonary immune responses. By releasing cytokines they can control the cell-cell communication of other participating cells. Although it is well established that the lung lymphocytes, localized in distinct compartments, differ in their subset composition, little is known about cytokine production in these compartments during immune responses. Lewis rats were immunized by intravenous administration of sheep erythrocytes on day 0 and day 7 and challenged intratracheally with sheep erythrocytes on day 10. Four days after intratracheal (i.t.) challenge the composition of lymphocyte subsets (CD2+, CD4+, CD8+, B cells, natural killer (NK) cells) in the spleen, blood, lung perfusate, lung tissue and bronchoalveolar lavage fluid (BALF) was characterized, and intracellular IFN-gamma was detected in these subsets by flow cytometry. Comparing control and immunized animals, no changes were found in lymphocyte numbers, subsets or the percentage of IFN-gamma-producing lymphocytes in the spleen, blood and lung perfusate. In lung tissue and BALF, however, the absolute number of all lymphocyte subsets and the percentage of IFN-gamma-producing lymphocytes were increased. When the lymphocyte subsets were analysed an increased percentage of IFN-gamma-producing T cells was found in lung tissue (4.5 +/- 0.6% versus 12.8 +/- 1.1%) and in BALF (7.8 +/- 1.4% versus 14.8 +/- 1.9%) of immunized animals opposed to controls, this increase being seen in both CD4+ and CD8+ cells. Thus, there is an accumulation of T cells with an increased potential to produce IFN-gamma in the lung interstitium and the bronchoalveolar space during pulmonary immune responses.

[Does treatment with thrombocyte aggregation inhibitors modify kidney transplantation surgery?]. / Beeinflusst die Einnahme von Thrombozytenaggregationshemmern die Durchführung einer Nierentransplantation?

The influence of aspirin treatment on haemostasis is still under debate. There are doubts in emergency operations, especially in transplant patients, concerning dosage and change in medication. Our results are based on an analysis of the therapeutic approaches in German transplantation centres. The question of transplant suitability, dosage and haemostatic effects are discussed with respect to the literature. Some 92.11% of the transplantation centres perform the operation even though the patient has been treated with aspirin. In these cases an increased bleeding tendency is tolerated and observed in 34.2% of the centres. An increased mortality has not been reported. Most of the transplantation centres accept a dosage of 100 mg aspirin daily. Only 7.89% of the transplantation centres refuse to operate on aspirin-treated patients. Correctly indicated aspirin treatment (for cardiac arrhythmia, embolism, or thrombosis, for example) does not contraindicate renal transplantation (daily dosage up to 100 mg). In elective surgery, however, a preoperative change in medication is recommended, e.g., the heparin instead of aspirin.

Lysosome membrane permeability to amines.

The permeability of rat liver lysosomes to xenobiotic organic compounds possessing nitrogen functions was investigated, using an osmotic-protection methodology. It was first shown that rat liver lysosomes are stable for at least one hour when incubated in 250 mM sucrose within the pH range 5 to 9. Primary and tertiary amines with pKa values within this pH range, and with differing numbers of aliphatic hydroxy or ether groups, were chosen for study and their permeability investigated at a range of pH values. The results indicate that uncharged amines can cross the lysosome membrane, and that the permeability of such molecules can be predicted from their total hydrogen-bonding capacity. The notional hydrogen-bonding capacity of an uncharged tri-substituted nitrogen with no attached hydrogen atom, as in pyridine or in a tertiary aliphatic amine, is deduced to be approximately 1, and that of an uncharged primary amine approximately 2. A hydrogen-bonding capacity of at least 11 is deduced for cationic nitrogen, implying that most if not all molecules containing a charged nitrogen atom cannot cross the lysosome membrane by passive diffusion. The implications for lysosome physiology and pharmacology are discussed.