Molecular charge influences transperitoneal macromolecule transport

Kidney International, Vol. 43 (1993), pp. 837—844

Molecular charge influences transperitoneal macromolecule transport JOHN K. LEYPOLDT and LEE W. HENDERSON' Medical and Research Services, Veterans Affairs Medical Center, San Diego, and Department of Medicine, and Department of Applied Mechanics and Engineering Sciences, University of California, San Diego, La Jolla, California, USA

Molecular charge influences transperitoneal macromolecule transport. The influence of molecular charge on macromolecule transport during peritoneal dialysis was assessed by determining transperitoneal transport rates for fluorescent-labeled macromolecules (molecular radii from 15 to 40 A) that differed only in molecular charge: neutral dextran, anionic dextran sulfate and cationic diethylaminoethyl (DEAE) dextran. The test macromolecules were infused into the bloodstream of

ity [5]. The effects of molecular charge and configuration have

unanesthetized New Zealand White rabbits at a constant rate, and

mesothelial cells [6—8].

isotonic dialysis solution (40 mI/kg) was instilled into the peritoneal cavity. Blood and dialysate samples were taken hourly over a four hour dwell. Transpentoneal transport rates were assessed by calculating both the dialysate to plasma concentration ratio at four hours and the permeability-area product for the peritoneum, the latter parameter determined from the increase in the dialysate concentration with time. Transport rates for DEAE dextran were less (P < 0.05) than those for both neutral dextran and dextran sulfate; transport rates for neutral dextran and dextran sulfate were not different. Moreover, transperitoneal transport rates for fluorescent-labeled DEAE dextran were not

not, however, been extensively studied. Molecular charge might be expected to significantly influence transperitoneal solute transport since recent anatomical studies have identified an abundance of negative surface charge on mesenteric endo-

thelial cells, adjacent basement membranes and peritoneal The present studies were designed to examine the role of molecular charge on transperitoneal transport of macromolecules in a rabbit model of peritoneal dialysis. The test macromolecules employed in these studies were fluorescent-labeled, polydisperse dextran fractions that differed only in molecular charge. Methods

affected by adding unlabeled DEAE dextran to the intravenous infusion solution, an observation suggesting that low transport rates for DEAE dextran were not due to its binding to plasma protein. We conclude that

molecular charge is a determinant of transpentoneal macromolecule transport.

Fluorescent-labeled dextran preparation

Neutral dextran fractions Tb, weight-averaged molecular weight ——10,000 (Pharmacia Fine Chemicals, Piscataway, New Jersey, USA), and T2000, average molecular weight —2,000,000 (Sigma Chemical, St. Louis, Missouri, USA), were labeled with

Peritoneal dialysis is increasingly employed in the treatment 5-fluorescein isothiocyanate (FITC; Eastman Kodak, Rochesof end-stage renal disease [1]. A major concern with the chronic ter, New York, USA) using the method of de Belder and use of peritoneal dialysis is the significant loss of protein into Granath [9]. High molecular weight fractions of dextran sulfate, —500,000 the dialysis solution that routinely occurs [2, 3]. Proteins that are lost during peritoneal dialysis originate primarily from the (Pharmacia), and DEAE dextran, —500,000 (Pharmacia), were vascular pool [3]; thus, mechanisms governing peritoneal trans- first hydrolyzed by heating in 1 N HC1 at 100°C for 10 to 25

port of macromolecules likely reflect general physiological principles of transport within the microcirculation. Previous studies have demonstrated that the major factors influencing macromolecule transport within the microcirculation are molecular size, hemodynamic conditions, molecular charge, and

minutes. Hydrolyzed fractions of dextran sulfate and DEAE

dextran were employed in these studies since they had a comparable molecular weight distribution to the neutral dextran

fraction Tb. Since dextran sulfate and DEAE dextran were essentially insoluble in dimethyl sulfoxide, the alternative labeling procedure of de Belder and Granath [9] using the

molecular configuration [4]. Previous work has demonstrated that transperitoneal solute fluorescein analog 5-(4,6-dichlorotriazin-2-yl)-amino fluorescein transport rates decrease with increasing molecular size and can hydrochloride (DTAF; Eastman Kodak) was employed. be altered when employing dialysis solutions of different tonic- Briefly, 200 mg of DTAF were reacted in water with I g of either

hydrolyzed dextran sulfate or hydrolyzed DEAE dextran for Present Address: Baxter Healthcare Corporation, Renal Division, 1620 Waukegan Road, McGaw Park, IL 60085-6730, USA. Received for publication December 26, 1991 and in revised form November 19, 1992 Accepted for publication November 23, 1992

two hours at pH 12. The unreacted label was separated from the fluorescent-labeled macromolecules by precipitation with cold ethanol followed by membrane dialysis using Spectralpor tubing (molecular wt cutoff for protein of 12,000 to 14,000; Spectrum Medical, Los Angeles, California, USA) for more than 48 hours. The fluorescent yield of DTAF-labeled dexti-an sulfate

© 1993 by the International Society of Nephrology

and DEAE dextran was comparable to that of FITC-labeled 837

838

Leypoldt and Henderson: Charge influences peritoneal transport

neutral dextran. The degree of substitution of the fluorescent- change. This solution was employed instead of commercial labeled macromolecules was not characterized nor was it isotonic peritoneal dialysis solutions since the latter are hypercritical since only relative fluorescence values were employed tonic to rabbit plasma. A 1 ml dialysate sample was taken five in subsequent calculations. It has been previously shown that minutes after dialysate infusion was completed and then hourly the fluorescent properties of FITC, DTAF and their conjugated over a four hour dwell. A 2 ml arterial blood sample was obtained concomitantly with each dialysate collection. After products are virtually identical [10]. The net charge on the fluorescent-labeled dextran fractions the four hour samples had been taken, the peritoneal cavity was was characterized by observing their binding to ion exchange drained as completely as possible. Following drainage, 30 ml of columns prepared from DEAE-Sephadex (anion exchanger Normosol R solution were instilled into the peritoneal cavity. A25; Pharmacia) and SP-Sephadex (cationic exchanger C25; The contents of the peritoneal cavity were mixed for three Pharrnacia). DTAF-labeled dextran sulfate was found to bind to minutes and an aliquot of this solution was collected for DEAE-Sephadex but not to SP-Sephadex. DTAF-labeled calculation of the residual volume of dialysis solution remaining DEAE dextran was found to bind to SP-Sephadex; moreover, in the peritoneal cavity. Urine output was collected and monitrace quantities were also observed to bind to DEAE-Sepha- tored throughout each experiment; output in excess of the dex. DTAF-labeled DEAE dextran that had been filtered infusion volume was replaced volume for volume with a soluthrough one DEAE-Sephadex column showed no significant tion containing 0.45% NaC1 and 2.5% glucose. The experiment binding upon second passage through a DEAE-Sephadex col- was terminated by a barbiturate overdose. Twenty-seven experiments were performed in total. The umn; this filtered preparation was used in certain experiments to examine the influence of residual anionic components on the protocol for each experiment was identical except for the experimental results (see below). FITC-labeled neutral dextran dextran fractions infused into the bloodstream. Six experiments were performed using FITC-labeled neutral dextran, five were was not observed to bind to either ion exchanger. performed using DTAF-labeled dextran sulfate, and six were In vivo experiments performed using DTAF-labeled DEAE dextran. Seven addiMale New Zealand White rabbits, body weight ranging from tional experiments were performed using DTAF-labeled DEAE 2.2 to 3.6 kg, were fasted overnight and anesthetized with dextran; three of these used the preparation of DTAF-labeled halothane during the surgical procedure. The animals were DEAE dextran that had been filtered through an anionic explaced supine on a heating pad and an indwelling Foley catheter change column to remove residual anionic components (see was passed into the bladder. Following a ventral neck incision, above) and the remaining four added unlabeled DEAE dextran the carotid artery and jugular vein were cannulated for blood at a concentration approximately sevenfold that of DTAFsampling and the infusion of solutions, respectively. These labeled DEAE dextran to the intravenous infusion solution. cannulas were exteriorized and taped to the skin to allow access Three additional experiments were also performed with FITCto them during the experiment. A one inch incision in the skin labeled neutral dextran in the presence of unlabeled dextran and through the first layer of muscle of the right lumbar region Tb. The concentration of unlabeled dextran Tl0 employed in was required for insertion of the peritoneal catheter (Sil-Med these experiments was comparable to that used previously [11]. Corporation, Taunton, Massachusetts, USA) using a trocar. In vitro experiments The peritoneal catheter was secured into position with a purseTo assess the validity of fluorescent-labeled dextrans as test string suture. After catheter placement, the anesthetic was turned off and the rabbit was given pure oxygen until conscious- macromolecules for investigating transperitoneal transport, in vitro membrane transport studies using fluorescent-labeled dcxness was regained. After surgery was completed, a 90-minute washout exchange trans were performed and were compared with those using of the peritoneal cavity using Normosol R (Abbott Laborato- unlabeled dextrans. In vitro experiments were performed on ries, North Chicago, Illinois, USA) was performed (40 mIkg). commercial hemodialyzers containing polysulfone membranes Normosol R solutions had a pH of approximately 6.8 and of high hydraulic permeability (F60 hemodialyzers, Fresenius, contained 140 mEq/liter sodium, 5 mEq/liter potassium, 3 Bad Homburg, Germany). The device was studied in a postdimEq/liter magnesium, 98 mEq/liter chloride, 27 mEq/liter ace- lutional hemofiltration circuit described previously [12]. Sieving tate and 23 mEq/liter gluconate. At the beginning of this coefficients were determined at perfusate flow rates of 150 and washout exchange a baseline arterial blood sample was taken 400 ml/min with an ultrafiltration rate of 60 mllmin. The

and a priming dose of test solutes, consisting of 90 mg of perfusate solution was prepared by dilution of commercial creatinine (Sigma) and 20 mg of a fluorescent-labeled dextran dialysate concentrate (Renalab RL5O, Irvine, California, USA). fraction dissolved in 20 ml of a 0.9% NaCl solution, was then The solution reservoir was continuously maintained at 37°C infused into the animal over a five minute interval. This was throughout the experiment. Sieving coefficients were calculated to assess transmembrane followed by a constant intravenous infusion of a solution containing the corresponding solutes to maintain their plasma transport and were determined by concentrations in the input, concentrations relatively constant. After 90 minutes the perito- output and ultrafiltrate streams as described previously [12]. neal cavity was drained by gravity as completely as possible Sieving coefficients were relatively independent of perfusate flow rate; the mean value of these determinations was therefore and the experiment was initiated. Isotonic dialysis solution (Normosol R plus 0.5% glucose) used. Nine in vitro experiments were performed in total. Three

containing 10 sg/ml of FITC-labeled dextran T2000 was replicates were performed using the following test solutes: warmed to 37°C and instilled into the peritoneal cavity (40 unlabeled neutral dextran, unlabeled DEAE dextran, and mllkg) immediately following drainage of the washout cx- DTAF-labeled DEAE dextran.

Leypoldt and Henderson: Charge influences peritoneal transport

Analytical techniques A molecular weight characterization of unlabeled dextrans was performed on each relevant sample using high performance size exclusion (gel permeation) chromatography [12]. Size

exclusion chromatography was performed using a TSKG4000PW column with a column buffer containing 0.15 M ammonium acetate and 0.05 M sodium phosphate, pH 6.5. A Waters Model R401 differential refractometer was employed for monitoring concentration changes in the column effluent. Column calibration was performed with dextran standards of very

839

Calculations

The dialysate concentration for all test solutes increased continuously with time during an exchange; the increase in dialysate concentration relative to that in plasma was more rapid for smaller solutes. Transperitoneal solute transport was assessed using two different approaches. First, total overall transport was assessed simply by calculating the dialysate to plasma concentration ratio at the end of the exchange (at 4 hours). During an isotonic exchange, this measure of solute

low polydispersity [13], and the concentration of unlabeled transport is independent of dialysate volume measurements as a dextran was determined at appropriate retention volumes as function of time but depends on both the initial volume of determined from column calibration, isotonic dialysis solution and the length of the dwell. The No sample preparation was necessary for the analysis of second approach was to calculate the permeability-area product unlabeled dextran concentrations in the in vitro experiments; (PA) from the change in the dialysate concentration and volume samples were injected directly onto the column. For in vivo with time during an exchange [11]. During an isotonic exexperiments using unlabeled DEAE dextran, no attempt was change, where convective solute transport may be assumed made to analyze their concentrations. For in vivo experiments negligible, the change in the dialysate concentration between using unlabeled neutral dextran, plasma and dialysate samples any two times points, t1 and t2, is described by the following were prepared for chromatographic analysis by procedures equation [11] described previously [11]. A molecular weight characterization of fluorescent-labeled dextrans in each sample was also performed using previously

reported methods [14]. Size exclusion chromatography was

Cd(t2) = [Cd(tl) —

PAC PAC 1I[V(t2)/V(ti)V' + PAIq) + PA + q qj

PA +

(2)

performed using a TSK-G4000PW column with a column buffer containing 1.0 M ammonium acetate and 0.05 M sodium phos- where Cd denotes the dialysate concentration at the correphate, pH 9.0. A Waters Model 420-E fluorescence detector sponding time points and C, denotes the plasma concentration

was employed for monitoring changes in fluorescence in the (assumed constant). The value of PA has units of volume per

column effluent, with excitation at 450 nm (bandpass filter) and time (comparable to clearance) and is a measure of the fluorescence at 500 nm (cutoff filter) being monitored. Column transperitoneal solute transport rate that is independent of calibration was performed with FITC-labeled dextran standards dwell time. Values of PA were estimated for each experiment

of very low polydispersity [13]. FITC-labeled dextran T2000 by comparing the experimental data with equation (2) using concentration was taken as the relative height of the chromato- nonlinear regression [11]. The actual or true volume of dialysis solution in the peritogram at the column void volume, and the concentration of dextran at all other molecular weights was determined at neal cavity as a function of time is difficult to estimate accuappropriate retention volumes as determined from column rately. It has been previously shown that the single estimate of dialysate volume V to employ when using equation (2) to obtain calibration. Samples analyzed for fluorescent-labeled dextrans were in- the most accurate estimate of PA is that obtained using indicajected directly onto the column with no sample preparation. In tor dilution methods [17]. Therefore, changes in dialysate the experiments employing FITC-labeled and unlabeled neutral volume with time were calculated from changes in the concendextran, isolation of FITC-labeled neutral dextran from each tration of FITC-labeled dextran T2000 in the dialysis solution sample was performed as described previously [11]. The plasma [11, 18]. For these calculations, no correction was made for loss concentration of dextran was determined as the fluorescence in of the marker macromolecule from the peritoneal cavity. It the sample minus the background fluorescence measured in the should be emphasized that this calculated volume is not an accurate estimate of the actual volume of dialysis solution baseline arterial plasma sample. The molecular radius (R) of both unlabeled and fluorescent- within the peritoneal cavity. The value of q in equation (2) is the labeled neutral dextrans in A was computed from previously transperitoneal ultrafiltration rate and was calculated from sequential indicator dilution estimates of dialysate volume [11, reported data [15, 16] by the following equation

R=

0.305M°47

where M denotes neutral dextran molecular weight. Molecular radii for charged and neutral dextrans were assumed to be equal if they had the same column retention volume. Creatinine concentrations were measured by an automated Jaffè rate methodology (Creatinine Analyzer 2, Beckman In-

18].

The residual volume remaining after gravity drainage of the peritoneal cavity was determined from the decrease in FITClabeled dextran T2000 concentration following instillation of 30 ml of Normosol R solution at the end of the four-hour experimental exchange. The final true volume of dialysis solution remaining in the peritoneal cavity at the end of the exchange

struments, Fullerton, California, USA). Glucose concentra- was calculated as the sum of the volume drained and the tions were measured by a YSI Model 27 Industrial Analyzer calculated residual volume. Fluid absorption rates from the (Yellow Springs Instrument, Yellow Springs, Ohio, USA). peritoneal cavity were calculated as the difference between Each concentration of creatinine was corrected for the concen- the indicator dilution volume at the end of the exchange minus tration of glucose. the final true volume divided by dwell time.

840

Leypoldt and Henderson: Charge influences peritoneal transport

1.4

Table 1. Dialysate volume measurements (mean SEM) Dwell time

hr

Method

Volume

1.2

0

Indicator dilution Indicator dilution Indicator dilution Indicator dilution Indicator dilution Drain + residual

109 4 125 5 127 5 130 7 132 7

1.0

1

2 3

4 4

61 7

0 0.8 0.6

Table 2. Dialysate to plasma concentration ratios for creatinine

(mean SEM)

0.4

Dwell time

hr 0 1

2 3

4

Neutral dextran 0.08 0.62 0.89

0.01

1.04 1.04

0.05

0.04 0.04 0.02

DEAE dextran 0.05 0.55 0.75 0.92 0.99

0.02 0.06 0.07 0.05 0.05

Dextran sulfate

0.05 0.62 0.78

0.01 0.04

0.86 1.04

0.10 0.10

0.08

0.2 0 15

20

25

30

35

40

Molecular radius, A Fig. 1. The dialysate to plasma concentration ratio (C/C) at the end

Statistics All experimental values are expressed as the mean the standard error of the mean (sEM). Statistical significance of differences between study groups was assessed using single

of a four hour dwell plotted as afunction of molecular size for dextrans

with different charge. Symbols are: (— —) neutral dextran; (- - -) dextran sulfate; (—) DEAE dextran. Coded lines connect each data point, and error bars are shown only at selected data points.

classification analysis of variance [19]. Individual group differ-

The dependence of plasma and dialysate concentrations of ences were further analyzed by Student's t-test for unpaired samples with modified confidence limits computed by the fluorescent-labeled dextrans on time paralleled those of creatiDunn-Sidák method [19]. Differences were considered statistically significant at the 0.05 level.

nine except that dialysate concentrations relative to their plasma values were considerably smaller. Figure 1 shows the

dependence of the dialysate to plasma concentration ratio

Results (Cd/CP) at the end of the exchange as a function of molecular Dialysate volume measurements were independent of the test radius for dextrans with different charge. The results shown in macromolecules infused into the bloodstream; all experimental this figure were from experiments that did not employ unlabeled

results were therefore combined together (Table 1). Results from three experiments were excluded, however, because of catheters that were difficult to drain at the end of the experiment. Indicator dilution estimates of dialysate volume in-

dextrans. The dialysate to plasma concentration ratio at four hours was not different for neutral dextran and dextran sulfate at any molecular radius. The dialysate to plasma concentration ratio at the end of the exchange was lower for DEAE dextran

creased gradually with time throughout the four hour exchange. than for neutral dextran with molecular radii between 19 and 22 The volume of dialysis solution determined by indicator dilution A (P < 0.05) and anionic dextrans with molecular radii between at the end of the exchange was approximately twice the final 15 and 34 A (P < 0.05). Calculated values of PA are displayed true volume. These results are similar to previous observations in Figure 2 as a function of molecular radius. Values of PA for from this laboratory [11, 20], and this discrepancy in volume neutral dextran and DEAE dextran were different for molecular measurements is best explained by fluid absorption from the radii between 27 and 40 A (P < 0.05). Moreover, calculated PA peritoneal cavity at an average rate of 0.30 0.03 mI/mm (N = values for dextran sulfate and DEAE dextran were different for molecular radii between 20 and 29 A (P < 0.05). These results 24). Plasma creatinine concentrations were similar when using demonstrate that transperitoneal transport of cationic dextran is different test macromolecules and remained relatively constant less than that for both neutral and anionic dextran. For macromolecules of the size considered in this study, during each experimental exchange. The dialy sate creatinine

concentration increased with time during the four hour ex- transport rates during peritoneal dialysis are thought to be change, and the dialysate to plasma concentration ratio as a governed largely by diffusion [11, 21]. (For macromolecules function of time was independent of the test macromolecule with radii greater than approximately 40 A, however, convecemployed (Table 2). Moreover, the calculated values of PA for tion has been proposed as the predominant transport mechacreatinine when using neutral dextran, dextran sulfate and nism [22].) Figure 3 displays the calculated PA values shown in 0.38 the previous figure divided by the diffusion coefficient in free DEAE dextran as test macromolecules were 2.49 ml/min, 2.09 0.34 mI/mm and 1.86 0.31 mLlmin, respectively; these differences were not statistically significant. These results demonstrate that transperitoneal transport of creatinine was independent of the test macromolecule employed.

solution (PAID) plotted as a function of molecular radius. The diffusion coefficient in free solution for each test macromolecule was calculated from its molecular radius calculated in equation (1) [11]. This plot demonstrates that the dependence of PA/D on

841

Leypoldt and Henderson: Charge influences peritoneal transport

0.5

1.4 1.2

0.4 U)

0

0.3

0.8 ci)

E a.

1.0

0.6 0.2 0.4 0.1

0.2 0

0

15 15

20

25

30

35

40

Molecular radius, A Fig. 2. The calculated value of peritoneal permeability-area product (PA) plotted as a function of molecular size for dextrans with different charge. Symbols are: (— . —) neutral dextran; (- - -) dextran sulfate; (—) DEAE dextran. Coded lines connect each data point, and error bars are shown only at selected data points.

20

25

30

35

40

Molecular radius, A Fig. 4. The dialysate to plasma concentration ratio (CIC) at the end

of a four hour dwell plotted as a function of molecular size for neutral dextran assessed with or without a fluorescent label. Symbols are: (—) FITC labeled; (- --) FITC labeled (I); (—• —) unlabeled. The symbol (I) indicates that the FITC-labeled neutral dextran was first isolated before being separated by chromatography. Coded lines connect each data point, and error bars are shown only at selected data points.

10,000

decreased with increasing molecular size, an indication of hindered transperitoneal diffusion. These results therefore sug-

gest that the major peritoneal transport barrier to cationic macromolecules may differ from that for neutral and anionic macromolecules. The results of additional in vivo experiments employing different combinations of fluorescent-labeled and unlabeled dextrans are shown in Figures 4 and 5. The results shown in 1,000 Figure 4 demonstrate that the dialysate to plasma concentration ratio at the end of the exchange is the same for both FITClabeled and unlabeled forms of neutral dextran. Moreover, the results for FITC-labeled neutral dextran were not dependent on whether dextran was first isolated from the plasma and dialysate samples before being separated by chromatography. Figure 5 displays PA values plotted as a function of molecular radius for DTAF-labeled DEAE dextran. These results demonstrate that transperitoneal transport of DTAF-labeled DEAE dextran 100 did not depend on prior filtration through an anionic exchange 15 20 25 30 35 40 column nor whether unlabeled DEAE dextran was simultaMolecular radius, A neously infused into the bloodstream. The observations shown Fig. 3. The value of peritoneal permeability-area product divided by in Figures 4 and 5 suggest that the addition of the fluorescent the diffusion coefficient in free solution (PAID) plotted as a function of label to neutral and cationic dextrans does not significantly molecular size for dextrans with different charge. Symbols are: (— —) influence their transport rate across the peritoneum. Moreover, neutral dextran; (- - -) dextran sulfate; (—) DEAE dextran. Coded lines infusion of excess unlabeled DEAE dextran into the bloodconnect each data point, and error bars are shown only at selected data stream does not influence the transperitoneal transport rate for points. DTAF-labeled DEAE dextran. Further confirmation of the validity of the present chromatomolecular size for neutral and anionic dextrans was similar to graphic methods was obtained by the results of the in vitro that for these macromolecules in free solution. Stated in other experiments. Sieving coefficients for F60 hemodialyzers are words, transperitoneal diffusion of these macromolecules was shown in Figure 6 as a function of molecular radius where unhindered. In contrast, the values of PA/D for DEAE dextran results for unlabeled neutral dextran, unlabeled DEAE dextran

842

Leypoldt and Henderson: Charge influences peritoneal transport

0.5

0.4 C a)

0

0.3

0 0

E

.( a-

0) C

> a)

0.2

Cl)

0.1

15

0 15

20

25

30

35

40

Molecular radius, A Fig. 5. The calculated value of peritoneal permeability-area product

(PA) plotted as a function of molecular size for DEAE dextran whether the DTAF-labeled inacromolecule was infused into the bloodstream either alone (—), after being filtered through an anionic exchange column (— • —), or in the presence of excess unlabeled DEAE dextran (- - -). Coded lines connect each data point, and error bars are shown only at selected data points.

20

25

30

35

40

Molecular radius, A Fig. 6. Sieving coefficients plotted as a function of molecular size for neutral dextran and DEAE dextran for the F60 hemodialyzer. Symbols are: (—) unlabeled; (- - -) DTAF-labeled. Coded lines connect each data point, and error bars are shown only at selected data points.

tance of molecular charge on glomerular transport of macromolecules [24, 25]. Within the glomerulus, anionic macromolecules were filtered less rapidly and cationic macromolecules more rapidly than neutral macromolecules of identical molecuand DTAF-labeled DEAE dextran are compared. As reported lar size. These observations have been explained by postulating previously [23], addition of positive molecular charge does not that the fixed negative charges on the glomerular capillary wall influence macromolecule sieving coefficients across this syn- inhibit the transport of anionic macromolecules and accelerate thetic membrane. Sieving coefficients for unlabeled and DTAF- the transport of cationic macromolecules by classic electrolabeled DEAE dextran are indistinguishable. These results static interactions [26]. The effect of molecular charge on macromolecule transport in provide important evidence confirming the validity of the chromatographic methods for fluorescent-labeled dextrans per- other capillary beds has not, however, been studied as extensively as in glomerular capillaries. Indeed, the results from such formed in the present study. studies are sometimes difficult to interpret. For example, cerDiscussion

Since the capillary wall is generally thought to be the primary

barrier governing macromolecule transport during peritoneal dialysis, the major determinants of transcapillary and transperitoneal protein transport are likely similar. Indeed, previous work has demonstrated that molecular size and transcapillary (or transperitoneal) fluid movement are determinants of solute transport both across the capillary wall and during peritoneal dialysis [4, 5]. This similarity between transcapillary and transperitoneal solute transport has recently been emphasized by Rippe and Stelin [22], who have suggested that a three-pore model of the capillary wall can adequately describe all relevant aspects of transperitoneal solute transport. The present study demonstrates that molecular charge is a determinant of transperitoneal macromolecule transport and should therefore be included in the list of common factors governing both transcapillary and transperitoneal solute transport. Molecular charge has previously been demonstrated to be an important determinant of transcapillary macromolecule exchange in several different microcirculatory beds. Studies in rat glomerular capillaries have clearly demonstrated the impor-

tain studies have reported that reflection coefficients for anionic macromolecules were greater (or capillary diffusive permeabilities were lower) than for either neutral or cationic macromol-

ecules, observations that suggest more hindrance to transcap-

illary transport of anionic macromolecules. These results, which are similar to those reported for the glomerular microcirculation, have been observed in tissues such as skin [27], muscle [28, 29] and mesentery [30]. On the contrary, macromolecule transport studies in intestinal [31] and pulmonary [32] capillary beds have found that lymph to plasma concentration ratios (primarily a measure of capillary permeability) for cationic macromolecules were lower than for neutral or anionic

ones, observations that are opposite to those reported for glomerular capillaries. These observations were not dependent on the structure of the test macromolecules since both polymers such as dextrans as well as proteins such as isozymes of lactate dehydrogenase bearing different charges gave similar results.

These disparate observations were tentatively resolved by suggesting that the capillary wall can be negatively charged in

some capillary beds but positive in others [31]. However, ultrastructural studies using electron dense macromolecules to

Leypoldt and Henderson: Charge influences peritoneal transport

locate and identify charged moieties on vascular endothelium have observed a net negative charge on virtually all tissues that have been examined [33, 34]. Parker and colleagues [32, 35] have extensively studied the effect of molecular charge on macromolecule transport within the pulmonary microcirculation and have suggested that the electrochemical properties of the interstitium may be as important as those of the capillary wall. They studied the transport of two isozymes of lactate dehydrogenase with different molecular

843

responsible for the present observations. A more complete description of transperitoneal transport of charged macromole-

cules will require additional studies. The use of dextrans of identical size but with different molecular charge will provide a useful tool for these future investigations. Acknowledgments Preliminary reports of these studies were presented at the FASEB 72nd Annual Meeting, Las Vegas, May 1—5, 1988, and the 21st Annual

charge by determining initial tissue clearance, steady state Meeting of The American Society of Nephrology, San Antonio, Delymph to plasma concentration ratios, and interstitial distribution volumes. Their data was most consistent with the hypothesis that the negatively-charged interstitial tissue behaves as a cation exchanger, facilitating the initial clearance of and creating a larger interstitial distribution volume for cationic macromolecules. They have hypothesized that the cation exchange properties of interstitial tissue result from the binding of cationic macromolecules to the negatively-charged ground sub-

cember 11—14, 1988 and have appeared in abstract form (FASEB J 2:Al523, 1988 and Kidney mt 35:273, 1989). This work was supported by DVA Medical Research Funds. The authors thank Elana Varnum and Sharon Okamoto for technical assistance.

Reprint requests to John K. Leypoldt, Ph.D., Renal Section (IJH), VA Medical Center, 500 Foothill Blvd., Salt Lake City, Utah 84148, USA.

References

stance of the interstitial matrix. Their hypothesis appears consistent with most previous work and provides an interesting unifying hypothesis that would explain how a microcirculatory

bed could act as a positively-charged barrier yet still have a negatively-charged vascular endothelium. The results of the present study demonstrate that the peritoneum exhibits macromolecule transport properties with respect to molecular charge similar to those of the pulmonary microcirculation; that is, transport rates for cationic macromolecules

are less than those for either neutral or anionic macromolecules. The present observations are also consistent with the general hypothesis of Parker, Gilchrist and Cartledge [32] since the low transport rate for cationic macromolecules may be due to their binding to interstitial tissue. Moreover, it is likely that

1. U.S. Renal Data System: USRDS 1991 Annual Data Report, The National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, August 1991 2. BLTJMENKRANTZ MJ, GAHL GM, K0PPLE JD, KAMDAR AV, JONES

MR. KESSEL M, COBURN JW: Protein losses during peritoneal dialysis. Kidney mt 19:593—602, 1981

3. DULANEY JT, HATCH FE Ja: Peritoneal dialysis and loss of proteins: A review. Kidney mt 26:253—262, 1984 4. TAYLOR AE, GRANOER DN: Exchange of macromolecules across

the microcirculation, in Handbook of Physiology. Section 2: The Cardiovascular System. Volume IV. Microcirculation, edited by RENKIN EM, MICHEL CC, Bethesda, American Physiological Society, 1984, pp. 467—520 5. POPOVICH RP, MONCRIEF JW, PYLE WK: Transport kinetics, in

Peritoneal Dialysis, edited by NOLPH KD, Dordrecht, Kluwer Academic, 1989, pp. 96—116

transperitoneal transport rates for neutral and anionic macro-

6. G0TL0IB L, BAR SELLA P, JAICHENKO J, SHUSTACK A: Ruthenium-

molecules are not different since neither can bind to the

red-stained polyanionic fixed charges in peritoneal microvessels.

negatively-charged components of interstitial tissue. Therefore, the influence of molecular charge on macromolecule transport

7. Goioi L, SHOSTACK A, JAICHENKO J: Ruthenium-red-stained anionic charges of rat and mice mesothelial cells and basal lamina:

during peritoneal dialysis is likely determined more by the properties of peritoneal interstitial tissue than those of the peritoneal capillary wall.

It should be emphasized that the present observations are

limited and do not permit a unique explanation for low transperitoneal transport rates for cationic macromolecules

Nephron 47:22—28, 1987

The peritoneum is a negatively charged dialyzing membrane. Nephron 48:65—70, 1988

8. LEAK LV: Distribution of cell surface charges on mesothelium and lymphatic endothelium. Microvasc Res 31:18—30, 1986

9. DE BELDER AN, GRANATH K: Preparation and properties of fluorescein-labelled dextrans. Carbohydr Res 30:375—378, 1973 10. BLAKESLEE D, BAINES MG: Immunofluorescence using dichiorotriazinylaminofluorescein (DTAF). I. Preparation and fractionation of labelled IgG. J Immunol Methods 13:305—320, 1976

when compared with those for neutral and anionic macromolecules of the same molecular size. Indeed, direct extrapolation of these observations in a rabbit model using dextrans to protein loss rates during peritoneal dialysis may not be valid. Certain in

11. LEYPOLDT JK, PARKER HR, FRIGON RP, HENDERSON LW: Molec-

vitro experiments have shown that neutral dextrans do not always behave as ideal solid spheres. For example, recent

12. LEYPOLDT JK, FRIGON RP, HENDERSON LW: Macromolecular charge affects hemofilter solute sieving. Trans ASAIO 32:384—387,

studies have shown that neutral dextrans diffuse more rapidly across polycarbonate track-etch membranes than theoretical

predictions for solid spheres [36]. It is important to note,

ular size dependence of peritoneal transport. J Lab Clin Med 110:207—216, 1987

1986 13. FRIGON RP, LEYPOLDT JK, UYEJI S. HENDERSON LW: Disparity

between Stokes radii of dextrans and proteins as determined by retention volume in gel permeation chromatography. Anal Chem

55:1349—1354, 1983 however, that such nonideal behavior is not because the polymers unravel and reptate through pores smaller than the Stokes 14. BAYLIS C, FREDERICKS M, LEYPOLDT J, FRIGON R, WILSON C, HENDERSON L: The mechanisms of proteinuria in aging rats. Mech radius of the molecule itself [36]. Indeed, there is no evidence Ageing Dev 45:111—126, 1988 that neutral dextrans can reptate through pores of the size range 15. GRANATH KA: Solution properties of branched dextrans. J Co/bid Sci 13:308—328, 1951 studied here [37]. Furthermore, the present in vitro experiments

have demonstrated that the transport rates of neutral and cationic dextrans across a neutral synthetic membrane are

16. GRANATH KA, KvIST BE: Molecular weight distribution analysis by gel chromatography on Sephadex. J Chromatogr 28:69—81, 1967

equal. These observations suggest that differences in molecular

volume measurements required for determining peritoneal solute

configuration between neutral and DEAE dextran are not

17. LEYPOLDT JK, PUST AH, FRIGON RP, HENDERSON LW: Dialysate

transport. Kidney mt 34:254—261. 1988

844

Leypoldt and Henderson: Charge influences peritoneal transport

18. PUST AH, LEYPOLDT 1K, FiuGON RP, HENDERSON LW: Peritoneal

sulphate-substituted and neutral dextran fractions in the rat hind-

dialysate volume determined by indicator dilution measurements.

quarter vascular bed. Acta Physiol Scand 115:397—404, 1982 29. HARALDSSON B, EKHOLM C, RIPPE B: Importance of molecular

Kidney tnt 33:64—70, 1988

19. SOKAL RH, ROHLF FJ: Biometry (2nd ed). San Francisco, W.H. Freeman, 1981 20. LEYPOLDT JK, CHIU AS, FRIGON RP, HENDERSON LW: Dialysate

to blood transport of macromolecules during peritoneal dialysis. Am J Physiol 257:H1851—H 1859, 1989 21. KREDIET RT, KOOMEN 0CM, KOOPMAN MG, HOEK Fl, STRUUK DG, BOESCHOTEN EW, ARIsz L: The peritoneal transport of serum proteins and neutral dextran in CAPD patients. Kidney mt 35:1064— 1072, 1989

22. RIPPE B, STELIN G: Simulations of peritoneal solute transport during CAPD. Application of two-pore formalism. Kidney Int 35:1234—1244, 1989

23. LEYPOLDT JK, FRIGON RP, HENDERSON LW: Macrosolute charge

independent of sign decreases sieving coefficient. (abstract) Abs ASAJO 16:48, 1987 24. CHANG RLS, DEEN WM, ROBERTSON CR, BRENNER BM: Permse-

lectivity of the glomerular capillary wall: III. Restricted transport of polyanions. Kidney tnt 8:212—218, 1975 25. BOHRER MP, BAYLI5 C, HUMES HD, GLASSOCK RJ, ROBERTSON

CR, BRENNER BM: Permselectivity of the glomerular capillary wall. Facilitated filtration of circulating polycations. J Gun Invest 61:72—78, 1978

26. DEEN WM, SATVAT B, JAMIESON JM: Theoretical model for glomerular filtration of charged solutes. Am J Physiol 238:Fl26—

charge for the passage of endogenous macromolecules across continuous capillary walls, studied by serum clearance of lactate dehydrogenase (LDH) isoenzymes. Ada Physiol Scand 117:123— 130, 1983 30. ADAMSON RH, HUXLEY VH, CURRY FE: Single capillary perme-

ability to proteins having similar size but different charge. Am .1 Physiol 254:H304—H312, 1988 31. PERRY MA, BENOIT JN, KVIETYS PR, GRANGER DN: Restricted

transport of cationic macromolecules across intestinal capillaries. Am J Physiol 245:G568—G572, 1983 32. PARKER IC, GILcHRI5'r 5, CARTLEDGE JT: Plasma-lymph exchange

and interstitial distribution volumes of charged macromolecules in the lung. JAppiPhysiol 59:1128—1136, 1985 33. SiMioNascu N, SIMI0NEScu M, PALADE GE: Differentiated micro-

domains on the luminal surface of the capillary endothelium. I. Preferential distribution of anionic sites. J Cell Biol 90:605—613, 1981

34. Miiici AJ, L'HERNAULT N, PALADE GE: Surface densities of diaphragmed fenestrae and transendothelial channels in different murine capillary beds, Circ Res 56:709—717, 1985 35. GILCHRIST SA, PARKER JC: Exclusion of charged macromolecules in the pulmonary interstitium. Microvasc Res 30:88—98, 1985 36. DAVIDSON MG, DEEN WM: Hindered diffusion of water-soluble

F139, 1980 27. AREEKUL 5: Reflection coefficients of neutral and sulphate-substi-

macromolecules in membranes. Macromolecules 21:3474-3481,

tuted dextran molecules in the isolated perfused rabbit ear. Acta

37. Mociiizuiu 5, ZYDNEY AL: Dextran transport through asymmetric ultrafiltration membranes: Comparison with hydrodynamic models. J Membr Sd 68:21—41, 1992

Soc Med Upsal 74:129—138, 1969 28. HARALDSSON B, MOXHAM BJ, RIPPE B: Capillary permeability to

1988