Andrzej Werynski

Simple models for description of small-solute transport in peritoneal dialysis.

The convective component in the general description of transport of solutes across the peritoneal membrane can be expressed as SQuc, where S is the sieving coefficient, Qu is the ultrafiltration flow rate, and c is the average concentration in the membrane (c = (1-F)cB + FcD, where cB and cD are blood plasma and dialysate solute concentration, respectively). F is a weighing function dependent on Qu, S, and the diffusive mass transport coefficient KBD. In this study a class of simple models of solute transport was considered in which S = 1 (justified for small solutes) was chosen, and F was selected as follows: F = 0 (as in the S = 1 (justified for small solutes) was chosen, and F was selected as follows: F = 0 (as in the widely used model of Garred and coworkers), F = 0.5 (theoretically justified model), F = 0.33 (theoretically justified for a high ultrafiltration period), and F = 1 (for convective transport from dialysate to blood). For all these models the estimation of KBD from clinical data can be performed with the aid of linear regression. The simple models were compared with the Pyle-Popovich model which takes into account the general expression for convective solute transport, for both the accuracy of the KBD determination (using linear regression) and the accuracy of theoretically calculated dialysate to plasma concentration ratios (D/P) to experimental D/P. Clinical evaluation of the new models was carried out in 28 6-hour dwell studies in 21 nondiabetic patients using 2 liters of hypertonic (glucose 3.86%) dialysis fluid. The differences between the simple models were small from the clinical point of view for urea, creatinine, glucose, and potassium, whereas for sodium the predictions were not satisfactory for any of the models. For urea and creatinine the model with F = 0.5 yielded the best fit of theoretical predictions to experimental data. For glucose and potassium small but systematic deviations of theoretical D/P from experimental D/P were observed for all simple models. The protein transport could be satisfactorily described by a model in which F = 1, as shown for total protein.

Diffusive mass transport coefficients are not constant during a single exchange in continuous ambulatory peritoneal dialysis.

Mass transport coefficients usually are assumed to be constant during single 6 hr exchanges of dialysis fluid in continuous ambulatory peritoneal dialysis (CAPD). To check this assumption, the authors estimated diffusive mass transport coefficients, KBD, for five low molecular weight solutes in 34 dwell studies with glucose 3.86% (20 studies), glucose 2.27% (nine studies), and glucose 1.36% (nine studies) dialysis fluids for time periods 3-30, 30-60, 60-90, 90-120, 120-180, 180-240, and 240-360 min. Dialysate volume and the rate of fluid reabsorption were measured using radiolabeled serum albumin (RISA) as a marker. Convective transport was described using a sieving coefficient of 0.55 for all solutes. The KBD values were constant for sodium, but higher at the beginning (3-30 min) than at the end (180-360 min) of the exchanges by an average of approximately 50% for urea, creatinine, and glucose, and by approximately 120% for potassium with all three dialysis fluids. This initial increment did not depend upon the concentration of glucose in dialysis fluid, except for urea. The steady state value of KBD was reached at 120 min for all solutes. The time patterns of KBD values for urea, creatinine, glucose, and potassium were well described by an exponential decay function, with the decay constant approximately 0.02 min-1. The patterns were similar for electrically neutral solutes, but different for electrolytes. The initial increments in KBD values mean that clearances during short dwell time (30-60 min) may be higher by 5-15% than clearances calculated from the steady state KBD values.

Lymphatic Absorption in CAPD Patients with Loss of Ultrafiltration Capacity

During continuous ambulatory peritoneal dialysis (CAPD) treatment, loss of ultrafiltration capacity (UFC) is a common complication that can be associated with increased peritoneal fluid absorption rate. The aim of the present study was to investigate the relative importance of lymphatic absorption for total peritoneal fluid absorption in patients with permanent loss of UFC associated with a high peritoneal absorption rate (KE, ml/min; high-KE group, n = 4). Clinically stable CAPD patients (n = 23) as well as patients with loss of UFC associated with increased diffusive mass transport coefficients (KBD, ml/min; high-KBD group, n = 8) served as control groups. The patients were investigated with a 6-hour dwell study with 3.86% glucose solution. The total fluid absorption rate was estimated by the disappearance rate (KE) of 131I-radioiodinated human serum albumin (RISA) from the peritoneal cavity, and the lymphatic absorption rate was estimated by the rate of RISA appearance in plasma (KPP, ml/min). The values of KE and KPP in the high-KE group (4.65 +/- 0.93 and 0.42 +/- 0.31 ml/min, respectively) were markedly higher than in the clinically stable CAPD patients (1.77 +/- 0.60 and 0.15 +/- 0.06 ml/min, respectively; both p < 0.001 vs. the high-KE group). In the high-KBD group, KE was lower (2.19 +/- 0.38 ml/min, p < 0.001) compared to the high-KE group, whereas KPP was similar (0.26 +/- 0.09 ml/min, NS). The fraction of KE which could be accounted for by KPP was on average only 9 +/- 5% in the high-KE group and did not differ from the fractions in the clinically stable patients or in the high-KBD group (9 +/- 5 and 12 +/- 4%, respectively). In 5 patients in whom plasma RISA activity was measured for 24 h from the beginning of the 6-hour dwell study, a continuous increase of the RISA level in plasma was observed during this time period. We conclude that although KPP was increased in patients with UFC loss associated with high KE, it accounted for only a minor part of KE. Furthermore, the relatively slow but prolonged appearance of RISA in plasma indicates that the interstitial compartment may serve as a reservoir of macromolecules which are slowly absorbed by local lymphatics. The present study supports previous findings that direct lymphatic absorption is only of relatively minor importance for the fluid absorption in peritoneal dialysis.

Simple models for fluid transport during peritoneal dialysis.

Peritoneal fluid transport can be predicted using different simplified formulas. To evaluate three such models, fluid transport was studied in 38 single six hour dwell studies using standard glucose 1.36% (n=9), 2.27% (n=9) and 3.86% (n=20) dialysis fluids as well as amino acid 2.70% fluid (n=8) in 33 patients on continuous ambulatory peritoneal dialysis (CAPD). Dialysate volume and the peritoneal absorption rate were measured using radioiodinated serum albumin (RISA) as a marker. The dialysate volume over dwell time curves were examined using three mathematical models of fluid transport for solutions with a crystalloid osmotic agent: Model P based on phenomenologically derived exponential function of time (Pyle, 1981), Model OS based on linear relationship between the rate of net volume change, Qv, to the difference of osmolality in dialysate and blood, and Model G based on linear relationship between Qv and the difference of glucose concentration in dialysate and blood. All these models provided a good description of the measured dialysate volume over time curves, however the descriptions with Models OS and G for glucose 3.86% fluid were slightly but significantly less precise. The coefficients of Model OS were stable in time, but the coefficients of Model G and P dependend in general on the time period used for their estimation, especially for glucose 3.86% dialysis fluid. The evaluation of dwell studies with solutions containing amino acid 2.70% (instead of glucose) as osmotic agent, using Model OS and P, showed that the transport coefficients were stable in time and both models provided equally precise descriptions. These results suggested that all three models can be used but models P and OS can be preferred for pratical applications such as predictions of fluid transport with alternative cristalloid osmotic agents. Furthermore, we found that the peritoneal barrier for fluid transport may change transiently during exchanges with the standard glucose - based dialysis fuid, whereas such changes were not observed with the amino acid-based fluid. This discrepancy may be due to a different composition of the dialysis fluids, including osmotic agent, buffer and pH.

Simple membrane models for peritoneal dialysis. Evaluation of diffusive and convective solute transport.

Currently used mathematical models to estimate parameters describing diffusive (diffusive mass transport coefficient, KBD) and convective (sieving coefficient, S) solute transport during peritoneal dialysis, as proposed by Pyle, Popovich, and Moncrief (PPM model) and Babb, Randerson, and Farrell (BRF model), require nonlinear regression and advanced numerical methods for parameter estimation. In this study, a simplified approach to the evaluation of KBD and S, using the same transport equation used in the PPM and BRF models but based on two-dimensional linear regression, is proposed. This new approach can be extended to generate a family of membrane models that differ in assumption concerning the average solute concentration (&OV0529;) inside the peritoneal membrane. In particular, &OV0529; was assumed to be equal to the arithmetic mean value of the dialysate and blood concentrations (PPM model), the blood concentration (BRF model), or the dialysate concentration (D model). The investigated family of models was used to study the transport of urea, creatinine, glucose, sodium, potassium, and total protein in 20 single, 6 hr dwell studies carried out in 20 nondiabetic patients in stable clinical condition using hypertonic (3.86%) glucose solution. For the PPM model, the linear and nonlinear regressions were able to provide almost identical values of KBD and S. The theoretical dialysate to plasma concentration ratio (D/P) was adequately fitted to experimental D/P for both the PPM and BRF models, but the fit was worse for the D model. However, unphysiologic (i.e., out of the 0–1 range) values of S were found for urea, potassium, and glucose independent of the version of the model used.

Effect of Alternative Osmotic Agents on Peritoneal Transport

To investigate the impact of osmotic agents on solute transport in continuous ambulatory peritoneal dialysis single 6-hour dwell studies were performed in nondiabetic patients using different osmotic agents: glucose 3.86%, amino acids 2.70, and glycerol 2.50%. Diffusive mass transport coefficient (KBD) and sieving coefficient (S) were assessed for urea, creatinine, glucose, glycerol, potassium, sodium, and total protein using the Babb-Randerson-Farrell model. The estimated KBD values for small solutes were higher in peritoneal dialysis fluid based on amino acids than in both glucose- and glycerol-based dialysis solutions. S values for small solutes were higher in glucose-based peritoneal dialysis fluid than in dialysis solutions based on amino acids and glycerol. Moreover, nonphysical, i.e., out of the range 0-1, S values were obtained for urea and potassium in glucose-based peritoneal dialysis fluid and for glucose and glycerol applied as osmotic agents. No difference in the transport parameters for total protein was found between the three investigated dialysis fluids. We conclude that the composition of dialysis fluid (osmotic agent, buffer solute, pH) can change the transport characteristics of the peritoneum. Furthermore, other physiological processes besides the diffusive and convective transport can contribute to the net peritoneal transport of some solutes.

ENCAPSULATION OF OKT3 CELLS IN HOLLOW FIBERS

Encapsulation of an OKT3 cell line in hollow fibers was evaluated in vitro and in vivo. The cell line is a mouse hybridoma producing immunoglobulin G2a (IgG2a) against CD3 human T lymphocytes and thus may function as a nonspecific activation system of a subpopulation of human T lymphocytes. For encapsulation purpose, hollow fibers of polypropylene K600 PP Accurel (Akzo, Germany) were selected. Hollow fibers were siliconized to improve membrane biocompatibility for in vivo experiments. The siliconized hollow fibers exhibited acceptable diffusive permeability (P) [ml/min/m2] for small solutes (for creatinine, p = 63.9 +/- 2.0, n = 3) and larger solutes (for albumin, p = 16.9 +/- 1.9, n = 3; for IgG, p = 1.0 +/- 0.2, n = 3). The 12 cm long hollow fibers were filled with a suspension of OKT3 cells of an average density of 10(6) cells/ ml, and both ends were sealed. The encapsulated cells were cultivated in RPMI 1640/10% CS medium at 37 degrees C, 5% CO2 for a period of 3 to 4 days. After the culture period, the medium was tested on human peripheral blood lymphocytes for the presence of anti-CD3 antibody and read in a flow FACS-trac cytometer (Becton Dickinson Immunocytochemistry Systems, San Diego, CA). The tightness of hollow fiber sealing was tested with a bubble point method. The number of cells increased after cultivation by four- to nine-fold on average (n = 11). Ten experiments were performed in vivo with OKT3 cells encapsulated in hollow fibers and implanted subcutaneously into mice for 3 days. In 50% of the experiments, some anti-CD3 antigens on human lymphocytes were found; however, the difference, in comparison with control, in percent of CD3+ was insignificant. In conclusion, the hollow fiber method for cultivation of hybridoma cells in vitro allows for separation of cells from the medium containing secreted anti-CD3 antibodies and is effective in maintaining cell viability. In vivo application needs additional study.

Fluid Transport in Peritoneal Dialysis

Standard peritoneal ultrafiltration characteristics in 18 patients undergoing continuous ambulatory peritoneal dialysis (CAPD) were investigated in a total of 21 single dwell studies of 6 h duration with 2 L of 3.86% glucose dialysis fluid. Intraperitoneal dialysate volumes were determined using radioiodinated serum albumin (RISA). Calculations were based on a novel mathematical method in which RISA elimination from the peritoneal cavity was taken into account. The RISA elimination rate, KE, was calculated as 2.1 ± 0.5 ml/min. The true dialysate volume after 360 min (2957±196 ml) was on the average 28% lower than the volume (3737 ± 260 ml) calculated without correction for the elimination of RISA. The mean maximum true volume plus sampling losses was 3255 ml at 240 min corresponding to a mean ultrafiltration of 762 ml between 3 min and 240 min. Our method of peritoneal volume determination proved to be useful for clinical investigations. The present study demonstrates that CAPD patients, without any major ultrafiltration problems, exhibit relatively small interpatient variations in their peritoneal volume over time curve.

Mathematical modeling of the glucose-insulin system during peritoneal dialysis with glucose-based fluids.

The purpose of this study was to analyze the effect of peritoneal dialysis with glucose-based solution on plasma glucose and insulin responses in patients on continuous ambulatory peritoneal dialysis (CAPD), describe the glucose-insulin system using a mathematical model, and identify abnormalities in this system. Six-hour dwell studies—using glucose 3.86% solution with a volume marker—were performed in 13 stable, fasting, nondiabetic CAPD patients. We used a mathematical model based on the previous works of Stolwijk and Hardy (1974) and Tolic et al (2000) to estimate the parameters of glucose-insulin system, insulin sensitivity index (Sl), and glucose effectiveness at basal (SG) and zero (GEZI) insulin. The individual peaks in plasma glucose and insulin concentrations occurred after 30–60 minutes of the dwell, with the average increase of 52% and 168% over the initial values, respectively. Increased insulin resistance was found in most of these patients. Both clinical and simulation results demonstrated a high interpatient variability in glucose and insulin kinetics and glucose-insulin system parameters in the patients. We demonstrated a successful control of increasing plasma glucose by insulin, despite an increased insulin resistance, during CAPD.

Spongy Polyethersulfone Membrane for Hepatocyte Cultivation: Studies on Human Hepatoma C3A Cells

There are different types of membranes used for hepatocyte cultivation. In our studies, spongy polyethersulfone (PES) membranes were examined as a support for hepatic cell cultivation in vitro. The extended surface of the membranes allows to introduce a high cell number especially in three-dimensional gel structure. Scanning electron microscopy analysis indicated that C3A cells used in our experiments grew well on PES membranes forming microvilli characteristic for normal hepatocytes. Analysis of cell viability proved that spongy PES membrane is well tolerated by J774 macrophages and did not stimulate nitric oxide synthesis. Bile canalicular structures were observed in fluorescence microscopy after F-actin staining with tetramethyl rhodamine iso-thiocyanate (TRITC)-phalloidin. The C3A cells showed high affinity to the PES membranes and adhered to almost 90% during the initial 24 h of incubation. Albumin production increased during static culture from the value of 805.2 +/- 284.4 (ng/24 h/initial 10(6) cells) during the first days, to 2017.6 +/- 505.9 (ng/24 h/initial 10(6) cells) after 10 days of culture. In conclusion, the spongy PES membranes can be used as scaffold for hepatocyte cultivation, especially for the creation of three-dimensional environments.