Mechanisms of Crystalloid Versus Colloid Osmosis Across the Peritoneal Membrane

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Mechanisms of Crystalloid versus Colloid Osmosis across the Peritoneal Membrane

Johann Morelle ,1,2 Amadou Sow,2 Charles-André Fustin,3 Catherine Fillée,4 Elvia Garcia-Lopez,5 Bengt Lindholm,5 Eric Gofﬁn,1,2 Fréderic Vandemaele,6 Bengt Rippe,7,a Carl M. Öberg ,7 and Olivier Devuyst 1,2,8

Due to the number of contributing authors, the afﬁliations are listed at the end of this article.

ABSTRACT Background Osmosis drives transcapillary ultrafiltration and water removal in patients treated with peritoneal dialysis. Crystalloid osmosis, typically induced by glucose, relies on dialysate tonicity and occurs through endothelial aquaporin-1 water channels and interendothelial clefts. In contrast, the mechanisms mediating water flow driven by colloidal agents, such as icodextrin, and combinations of osmotic agents have not been evaluated. Methods We used experimental models of peritoneal dialysis in mouse and biophysical studies combined with mathematical modeling to evaluate the mechanisms of colloid versus crystalloid osmosis across the peritoneal membrane and to investigate the pathways mediating water flow generated by the glucose polymer icodextrin. Results In silico modeling and in vivo studies showed that deletion of aquaporin-1 did not influence osmotic water transport induced by icodextrin but did affect that induced by crystalloid agents. Water flow induced by icodextrin was dependent upon the presence of large, colloidal fractions, with a reflection coefficient close to unity, a low diffusion capacity, and a minimal effect on dialysate osmolality. Combining crystalloid and colloid osmotic agents in the same dialysis solution strikingly enhanced water and sodium transport across the peritoneal membrane, improving ultrafiltration efficiency over that obtained with either type of agent alone.

Conclusions These data cast light on the molecular mechanisms involved in colloid versus crystalloid osmosis and characterize novel osmotic agents. Dialysis solutions combining crystalloid and colloid particles may help restore ﬂuid balance in patients treated with peritoneal dialysis.

J Am Soc Nephrol 29: 1875–1886, 2018. doi: https://doi.org/10.1681/ASN.2017080828

Peritoneal dialysis (PD) is a renal replacement modality that applies the principle of osmosis through the peritoneal membrane to restore water balance in patients Received August 3, 2017. Accepted April 25, 2018. with ESRD. Fluid overload is associated with poor C.M.O. and O.D. are cosenior authors. 1 outcome in patients on dialysis, and achieving ade- aDeceased. quate ultraﬁltration (UF) is key to successful PD, especially in patients with low residual renal function.2–6 Published online ahead of print. Publication date available at www.jasn.org. Osmosis refers to the movement of water across a barrier that is permeable to water but restricts the Correspondence: Prof. Johann Morelle, Division of Nephrology, — Cliniques universitaires Saint-Luc, Avenue Hippocrate 10, B-1200 transport of solutes which therefore act as os- Brussels, Belgium, or Prof. Olivier Devuyst, Institute of Physiology, motic agents. In 1861, Graham was the ﬁrst to University of Zurich, Zurich, Switzerland. E-mail: johann.morelle@ introduce a distinction between osmotic agents uclouvain.be or [email protected] on the basis of their physical properties: small-sized Copyright © 2018 by the American Society of Nephrology

J Am Soc Nephrol 29: 1875–1886, 2018 ISSN : 1046-6673/2907-1875 1875 BASIC RESEARCH www.jasn.org osmotic agents prone to crystallization and that easily diffuse Significance Statement across membranes were termed crystalloid, whereas noncrystal- line substances retained by membranes because of their larger size Osmosis sustains the efficiency of peritoneal dialysis to restore fluid were referred to as colloid (from the Ancient Greek kοlla, balance in patients with ESRD. The water channel aquaporin-1 glue).7 Although glucose has been used as the prototypic crystal- (AQP1) plays a critical role in glucose-driven (crystalloid) osmosis across the peritoneal membrane, but it is not known whether it loid osmotic agent to drive water removal in PD for decades, its contributes to ultrafiltration induced by colloid osmotic agents such use carries major limitations. Because of their small size, glucose as icodextrin. On the basis of studies in Aqp1 mice, biophysical molecules easily diffuse across the peritoneal membrane, thereby experiments, and computer simulations, these data re-examine limiting UF during long dwells. Furthermore, glucose leads to mechanisms of crystalloid versus colloid osmosis and demonstrate fl adverse metabolic effects,8 and its long-term use is associated that colloidal fractions of icodextrin induce an osmotic ow that is independent of AQP1. They also show the role of large icodextrin with structural and functional changes of the peritoneal mem- fractions to generate colloid osmosis and provide a rationale for – brane that contribute to loss of UF capacity.9 11 These drawbacks using combinations of osmotic agents to improve fluid volume have led to the development of alternative osmotic agents in- control in patients treated with peritoneal dialysis. cluding icodextrin, a mixture of glucose polymers. Thanks to their larger hydrodynamic radius (R ), polymers of icodextrin H Mouse TPM are absorbed from the peritoneal cavity at a much slower rate The initial parameters of the TPM were adapted on the basis of than glucose, via the lymphatic circulation, making them par- osmotic conductance for glucose and peritoneal solute trans- ticularly suitable for long dwells and sustained UF.12,13 +/+ port rate measured in independent experiments in Aqp1 The transport of water and solutes across the endothelium 2/2 and Aqp1 mice; the aC parameter was adjusted from 0.02 lining peritoneal capillaries during PD can be functionally de- +/+ to 0.04, to match both the difference between Aqp1 and scribed in terms of a three-pore model (TPM).14,15 On the basis 2/2 Aqp1 mice (using 3.86% glucose as osmotic agent) and of the TPM, numerous small pores located at interendothelial the osmotic water transport for icodextrin (Supplemental clefts mediate solute-coupled fluid transport, ultrasmall pores Table 1). All relevant exchange parameters were rescaled by located in endothelial cells facilitate transcellular water trans- 0.75 (body weight) , corresponding to a scaling factor of ap- port, and a relatively small number of large pores account for proximately 336 from man to mouse, similar to that previ- transport of macromolecules but have a negligible role in water ously described.20 transport.14–17 The identification of aquaporin-1 (AQP1) water channels in the endothelium of peritoneal capillaries and studies Aqp1 Animals of peritoneal transport in mouse models established AQP1 Aqp1 as the molecular counterpart of ultrasmall pores, playing a crit- Sex-matched mouse littermates generated as previously described,27 aged 8–12 weeks, were used to assess the ical role in water removal during PD with glucose-based dialysis fl 18–23 in uence of the water channel AQP1. All animals had solutions. In contrast with crystalloid osmosis, the colloidal ad libitum size of some glucose polymers and the absence of sodium sieving access to standard diet and tap water .Experi- suggested that icodextrin induces transperitoneal UF by different ments were conducted in accordance with the National Re- mechanisms.12,24 In addition, computer-based predictions and search Council Guide for the Care and Use of Laboratory scarce observations in patients undergoing PD suggested the Animals and the Animal Ethics Committee of the UCL Med- potential advantage of combining icodextrin and glucose in ical School. the same dwell to potentiate UF.25,26 However, the pathways and mechanisms of colloid versus crystalloid osmosis across Peritoneal Transport Studies the peritoneal membrane, and the mechanism of action of com- Transport across the mouse peritoneum was investigated binations of osmotic agents, have not been investigated. using a modified peritoneal equilibration test with different In this study, we combined experimental studies in mouse PD solutions (Baxter Healthcare, Belgium) (Table 1), as de- models of PD, biophysical studies, and advanced computer scribed previously.20,21,28,29 Baseline characteristics of the an- simulations to characterize the basic principles of colloid ver- imals are presented in Supplemental Table 2. Intraperitoneal sus crystalloid osmosis across the peritoneal membrane and to volume (IPV) versus time curves were performed using a fluo- investigate the pathways mediating water flow generated by the rescent BSA conjugate as an indicator-dilution technique, as glucose polymer icodextrin. We also demonstrate how dialysis previously described,21 or by direct volumetry. Both tech- solutions combining crystalloid and colloid particles may im- niques yielded values that were closely correlated, including prove the control of fluid volume in PD. when icodextrin was used as osmotic agent (Pearson r=0.98, P=0.003). Dialysate sodium was measured using the indirect ion selective electrode method. METHODS Dynamic Light Scattering Full details of the methods can be found in the Supplemental Dynamic light scattering was used to determine the apparent Material. RH and size distribution profile of the glucose polymer

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Table 1. Conventional and bimodal PD solutions: composition and osmolarity Variable 1.36% Gluc. 3.86% Gluc. 1.1% A.A. 7.5% Icod. CIG 1.36% CIG 3.86% Sodium, mmol/L 132 132 132 133 132 132 Calcium, mmol/L 1.25 1.25 1.25 1.75 1.75 1.75 Magnesium, mmol/L 0.25 0.25 0.25 0.25 0.25 0.25 Lactate, mmol/L 40 40 40 40 40 40 Chloride, mmol/L 95 95 105 96 96 96 Osmotic agent Glucose, mmol/L 75.49 214.25 ——75.49 214.25 Glucose monohydrate, g/dl 1.5 4.25 ——1.5 4.25 Amino acids, mmol/L ——87 ——— Icodextrin, g/dl ———7.5 7.5 7.5 Osmolarity (mOsm/L) 344 483 365 284 358 497 pH 5.2 5.2 6.7 5.0–6.0 5.0–6.0 5.0–6.0 Gluc., glucose; A.A., amino acids; icod., icodextrin; —, not applicable. icodextrin in solution (Extraneal 7.5%). The measurements The original version of the TPM was adapted to the intrinsic were performed on a Malvern CGS3 equipped with a HeNe permeability characteristics of the mouse peritoneal mem- laser (l=632.8 nm) at a temperature of 37.3°C and an angle of brane (Supplemental Table 1), to allow crossvalidation be- 90°. The size distributions were obtained by a CONTIN anal- tween computer simulations and experimental data obtained ysis of the data. in vivo and provide mechanistic insights into the physiology of water transport. We applied this mouse-adapted version of the Determination of Total Icodextrin, Icodextrin TPM to simulate changes in IPV, dialysate sodium kinetics, Metabolites, and Absorption of Carbohydrates and peritoneal solute transport over time, using hypertonic Total icodextrin in the dialysate was measured by enzymatic glucose and icodextrin as prototypic osmotic agents. We next hydrolysis with amyloglucosidase (Sigma-Aldrich, Saint-Louis, performed the same procedure in vivo, using experimental PD MO), icodextrin metabolites were determined in the dialysate of on Aqp1 mice (Figure 1). mice exposed to 7.5% icodextrin using a weak anion-exchange In both models, hypertonic glucose-based dialysate column (BioBasic AX; 15034.6 mm, 5 mm; Thermo Electron, induced a rapid increase in IPVand a dialysate sodium sieving Woburn, MA) and a refractive index detector, and total HMW during the first part of the dwell in the presence of ultrasmall fractions were estimated as the difference between total icodex- pores (water channels) (Figure 1, A–D). The absence of water trin and LMW G2-G7 fractions, as previously described.30–32 channels was associated with a significant reduction in the The amount of carbohydrates absorbed from the peritoneal cav- osmotic water permeability of the peritoneal membrane, the ity was calculated as the difference between the mass of carbo- abolition of sodium sieving, and an approximately 50% de- hydrates (glucose and icodextrin) infused into the peritoneal crease in total net UF (Figure 1, A–D, Table 2), in line with cavity and the mass removed. previous studies.20,21 To the contrary, solutions containing icodextrin induced a slow and sustained UF, with no sodium Statistical Methods sieving (Figure 1, G–J). The latter kinetics were essentially Data are presented as mean6SEM. Comparisons between re- unchanged in absence of water channels (modified TPM or sults from different groups were performed using a t test, one-, deletion of AQP1) (Figure 1, G–J). Peritoneal solute transport or two-way ANOVA, as appropriate. Significance level was in- rate—expressed as the dialysate-to-plasma ratio (D/P) of urea dicated in the legend of each figure (*P,0.05, **P,0.01, —was not affected by the absence of water channels (Figure 1, ***P,0.001). E, F, K, and L). The osmotic effect of glucose was transient, with a decreased UF rate that paralleled dissipation of the osmotic gradient. In contrast, the water flow induced by ico- RESULTS dextrin was constant over time, independent of the osmotic gradient, occurring despite the lower tonicity of the dialysate Osmosis Induced by Icodextrin Occurs Independently compared with that of plasma (Supplemental Figure 1). In of Water Channels and Tonicity these experiments, measured changes in IPV closely correlated We first assessed the relative contribution of transcellular and with predictions from the TPM (Pearson r=0.98, P,0.001) paracellular routes to osmotic water transport induced by var- and both techniques were in excellent agreement (Supplemen- ious osmotic agents, using TPM-based modeling and a well tal Figure 2), thereby validating the mouse-adapted version of established mouse model of PD with commercially available the TPM. Longer dwell times (up to 4 hours) using 7.5% ico- PD solutions (Table 1). dextrin in our model and in the adapted version of the TPM

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Figure 1. Osmosis induced by icodextrin occurs independently of water channels and tonicity. Results from computer simulations on the basis of the TPM and from an experimental model of PD in Aqp1 mice showing intraperitoneal (IP) volume versus time curves (A, B, G, and H), kinetics of dialysate sodium (C, D, I, and J), and dialysate-to-plasma ratio (D/P) of urea (E, F, K, and L). The TPM applied a +/+ fractional Kf for ultrasmall pores (ac) of either 0.04 (solid line) or 0.00 (dotted line), and in vivo experiments were performed in Aqp1 2 2 (open circles, solid line) and Aqp1 / (close circles, dotted lines) mice during 2-hour dwells, using 3.86% glucose (blue) or 7.5% icodextrin (orange). Data in mice are mean6SEM; n=4–6/group. Na, sodium. were associated with a progressive and linear increase in net trend ,0.01). To the contrary, using hypertonic glucose as UF (from 10.162.2 to 18.161.9 and 24.263.3 ml/g body wt osmotic agent, IPV reached a plateau after 90–100 minutes, for 2, 3, and 4-hour dwells in mice, respectively; P for linear followed by a decrease.

Table 2. Transport parameters in Aqp1 mice using various osmotic agents Net UF Solution Genotype MTAC Urea Garred (ml/min) MTAC Urea Waniewski (ml/min) (ml/g body wt) 1.36% glucose Aqp1+/+ 0.461.5 37.963.2 37.763.1 2 2 Aqp1 / 25.861.4a 42.562.5 43.762.6 3.86% glucose Aqp1+/+ 34.061.6 31.561.4 29.861.3 2 2 Aqp1 / 11.661.2b 40.561.8 39.261.7 1.1% aminoacids Aqp1+/+ 6.060.7 32.461.9 31.961.8 2 2 Aqp1 / 21.761.0b 35.461.8 35.661.9 7.5% icodextrin Aqp1+/+ 5.461.3 31.461.1 31.061.1 2 2 Aqp1 / 5.960.7 35.061.4 34.461.4 MTAC urea calculated using an f value of 0.33. n=4–6 mice per group. MTAC, mass transfer area coefﬁcient. aP,0.05 versus Aqp1+/+. bP,0.001 versus Aqp1+/+. cP,0.01 versus Aqp1+/+.

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correlation between net UF and solute transport during glucose-based dwells (Pearson correlation coefficient, r=20.85, P,0.001, n=15), whereas both parameters positively correlated when icodextrin was used (r=0.71, P=0.003, n=15) (Supplemental Figure 3C). We next tested the influence of AQP1 on the osmotic water transport generated by other crystalloid dialysis solutions (Tables 1 and 2), by comparing the net UF obtained in Aqp1 mice after 2-hour dwells using 1.36% glucose, 1.1% amino acids, 3.86% glucose, or 7.5% icodextrin. As shown in Figure 2A, glucose and amino acids, but not icodextrin, required the presence of water channels to induce transperitoneal osmosis. Solute transport, evaluated by the mass transfer area coefficient for urea, was similar in all groups (Table 2). The amount of UF generated by crystalloid agents closely correlated with the osmotic gradient between plasma and dialysis solution at baseline (Pearson r=0.98, P,0.0001) (Figure 2B). These findings are in agreement with simulations from the TPM, highlighting the importance of transcellular ultrasmall pores for crystalloid osmotic agents (i.e.,lowRH)(Figure2C). The modelized data, validated in vivo, substantiated predictions that icodextrin induces a slow but sustained transperitoneal water flow exclusively via a paracellu- Figure 2. Critical role of water channels and tonicity for crystalloid osmosis. (A) Net UF lar route, independently of the crystalloid generated at the end of 2-hour dwells with 2.5 ml of dialysate containing 1.36% osmotic gradient. Conversely, glucose and glucose, 3.86% glucose, 1.1% aminoacids, or 7.5% icodextrin, in Aqp1+/+ (black amino acids provide a fast but transient 2 2 boxes) and Aqp1 / (red boxes) mice. Boxes and whiskers represent minimum to water transport both via paracellular and maximum values; n=6/group. (B) Correlation between net UF obtained in the mouse transcellular pathways; they require the model and the transperitoneal osmotic gradient at baseline (dialysate-to-plasma ratio presence of an osmotic gradient and endo- of osmolality, D/Posm) for crystalloid osmotic agents (glucose and aminoacids, black thelial water channels to generate UF. circles, red line). Values for icodextrin are provided (gray circles) for comparison; each circle represents a mouse. (C) Predictions from the TPM showing the relationship Colloid Osmosis Is Induced by Large between osmotic pressure and R for fractional UF coefficients for ultrasmall pores (a ) H c Icodextrin Fractions of 0.04 (red line), 0.02 (black), and 0.00 (gray). BW, body weight. Because the ability of solutes to permeate the peritoneal membrane is primarily deter- Toinvestigatehowvariationsinmouseperitonealsolutetrans- mined by their RH and the functional radius of small pores 33 port influence water removal induced by different osmotic (approximately 4.0 nm), we assessed the RH of glucose poly- agents, we used intraperitoneal exposure to LPS (10 mg/kg) to mers by dynamic light scattering (Figure 3A). Mean RH of modulate solute transport. Twenty-four hours after exposure to icodextrin was 4.9 nm, with fractions ranging from 1.0 to 23.0 LPS, the D/P urea increased by 22% and 18% in 3.86% glucose- nm, versus 3.65 nm for albumin (Figure 3B). The presence of and 7.5% icodextrin-based solutions, respectively (P=0.92) icodextrin fractions larger than albumin—the prototypic colloid (Supplemental Figure 3A). Whereas the approximately 20% in- osmotic agent—substantiated the fact that some icodextrin crease in solute transport was associated with a 47% decrease in fractions induce colloid osmosis.12 net UF generated by glucose, water removal achieved by icodex- To confirm the role of large fractions of glucose polymers in trin-based solutions increased by +114% from the basal condi- osmosis, we used UF membranes with different molecular mass tion (P,0.001 between glucose and icodextrin) (Supplemental cut-offs to selectively remove the largest particles. The use of a 30 Figure 3B). When analyzing all experiments, there was a negative kD cut-off membrane was predicted to remove polysaccharides

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Figure 3. Colloid osmosis is induced by large icodextrin fractions. (A) Principle of dynamic light scattering (DLS). The sample is illu- minated by a laser beam (l=632.8 nm) and the ﬂuctuations of the scattered light are detected at a known scattering angle by a fast photon detector. The particles in solution scatter the light, providing information about their Brownian motion, size, and distribution. (B) CONTIN size distribution of polymers in a 7.5% icodextrin solution (red line, mean of ﬁve independent measures) compared with

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14 R with an RH.4.0 nm (i.e., the functional radius of small pores) Table 3. Effect of removing large icodextrin fractions on H, and to abrogate transperitoneal water flow. UF of icodextrin stock solution dispersity, osmotic gradient, and water transport in solutionwitha30kDmolecularmass cut-off membrane effec- vivo tively decreased the concentration of total icodextrin from 75.0 to Icodextrin Polymers Variable 24.8 mg/ml along with an approximately 60% reduction in high- All <30 kD <10 kD molecular-mass metabolites. It also resulted in a shift toward R ,nm 4.960.02 3.860.03a 3.460.00a R H lower H values, reduced the size dispersity of glucose polymers Relative peak width 0.5860.01 0.5060.02b 0.4660.00a 6 6 P, (relative peak width 0.50 0.02 versus 0.58 0.01, 0.01), and AUC D/Posm 125.561.4 122.660.4 119.960.8 abrogated icodextrin-induced osmosis in vivo (net UF 25.261.4 Net UF (ml/g BW) 8.061.8 25.261.4a 29.962.3a 6 m P, – versus 8.0 1.8 l/g, 0.001) (Figure 3, C I). Using a 10 kD AUC, area under the curve; D/Posm, dialysate-to-plasma ratio of osmolality; molecular mass cut-off membrane further reduced the concen- BW, body weight. aP,0.001. tration of total icodextrin (14.4 mg/ml), high-molecular-mass bP,0.01. icodextrin fractions (280%), size of polymers in solution R (mean H 3.4 nm), dispersity (relative peak width 0.46), and water A–F). These predictions were verified in the mouse model, 2 6 m P, transport(netUF 9.9 2.3 l/g, 0.001 versus stock 7.5% where combinations of glucose and icodextrin yielded a net – icodextrin) (Figure 3, C I). To the contrary, removing the largest UF that exceeded the sum of UF generated by each of the os- fi fi icodextrin fractions had no signi cant effect on the pro le of low- motic agents used separately (Figure 4, G–I, Table 4). The effect molecular-mass metabolites (negligible amounts of G2, approxi- on water removal was accompanied by a similar increase in mately 0.01 mg/ml; G3, G4, and G5 approximately 0.1 mg/ml; transperitoneal sodium removal (Figure 4J), indicating en- – and G6 and G7 approximately 0.2 0.3 mg/ml), the concentration hanced water transport at the level of interendothelial junctions. of electrolytes, and the transperitoneal osmotic gradient (Table 3). In both models, the kinetics of IPV showed a fast movement of Using molecular mass distribution (derived from dynamic light water across the peritoneum during the first part of the dwell, scattering and concentrations of icodextrin metabolites) of un- followed by a sustained osmosis over time (Figure 4, A, B, G, and fractionated versus fractionated icodextrin, predictions from the H). As expected, water channels had a critical role in the crystal- in vivo TPM closely correlated with values obtained (Pearson loid osmotic water flow generated during the initial part of the r P =0.998, =0.03). dwell (Supplemental Figure 4), whereas colloidal icodextrin frac- fi Altogether, these data veri ed the hypothesis that transca- tions enhanced and prolonged small pore–mediated water trans- fl pillary, paracellular water ow induced by icodextrin relies on port (Figures 4, C–F and 5). It is noteworthy that the use of R the presence of colloidal particles in solution, with an H ex- combined solutions was associated with a reduced absorption ceeding that of the functional radius of interendothelial clefts of carbohydrates, as compared with the sum of osmotic agents fl fi and a high re ection coef cient. used alone (219% and 211.5% of carbohydrates absorbed for CIG 3.86% and CIG 1.36%, respectively) (Table 4). Predictions Combinations of Crystalloid and Colloid Osmotic from the TPM were in agreement with these observations, Agents Enhance Water Removal during PD showing a 10%–15% reduction of carbohydrates absorbed during On the basis of the different mechanisms of osmotic water CIG dwells. As a result, combinations of glucose and icodextrin transport, we next tested the effect of combining glucose significantly increased UF efficiency—i.e., the amount of UF gen- and icodextrin mixed in the same PD solution (Table 1) on erated per amount of carbohydrate absorbed (Table 4). Alto- transperitoneal water removal. gether, these data validated predictions that crystalloid and colloid PredictionsfromtheTPMsuggestedthatcombining1.36%or osmotic agents mixed in a same solution enhance UF. This strik- 3.86% glucose with icodextrin (CIG) in the same dwell would ing effect is mediated by an increase in solute-coupled water trans- enhance water transport via the paracellular route (Figure 4, port across the paracellular route.

human serum albumin (HSA) (gray line). The average apparent RH (Stokes–Einstein radius) of icodextrin is 4.960.04 nm—exceeding that of HSA (3.7 nm)—with fractions ranging between 1.0 and 23.0 nm. In comparison, RH of glucose (molecular mass, 180 D) is 0.37 nm. (C–E) (C) Correlation curves, (D) apparent RH, and (E) relative peak width obtained by DLS of icodextrin stock solution (red), and after UF using 30 kD molecular mass (blue) or 10 kD molecular mass (green) cut-off membranes. n=3–6 measures/solution. P,0.001 between curves. (F and G) (F) Osmolality and (G) mass spectra of unfractionated and fractionated icodextrin, with high-molecular-mass fractions directly derived from concentrations of icodextrin metabolites and from DLS. Although high-molecular-mass icodextrin fractions have an important contribution to the total mass of particles in solution, they only have a minimal effect on osmolality. (H) Intraperitoneal (IP) volume versus time curves predicted by the TPM using either unfractionated (red) or fractionated (,30 kD, blue; ,10 kD, green) icodextrin. (I) Net UF measured at the end of 2-hour dwells performed with unfractionated (red) or fractionated (,30 kD, blue; ,10 kD, green) icodextrin. Dots represent individual measures; bars are mean6SEM. Inlet, the relationship between mean values of net UF generated in vivo versus those predicted by the TPM. A.U., arbitrary units; BW, body weight; DLS, dynamic light scattering; Unfract., unfractionated.

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Figure 4. Combinations of crystalloid and colloid osmotic agents enhance water and sodium removal. (A and B) Predictions from the TPM of the changes in intraperitoneal (IP) volume induced by (A) 1.36% or (B) 3.86% glucose and 7.5% icodextrin, used alone or in combination. (C and D) Contribution of transcellular and paracellular routes to transcapillary ﬂow rate derived from TPM simulations. Simulations were performed for (C) 1.36% glucose, (D) 3.86% glucose, and combinations of icodextrin with (E) 1.36% glucose or with (F) 3.86% glucose. JvC denotes transcellular water transport across “ultrasmall” pores (blue line); JvS, paracellular water transport across “small pores” (green line); and the sum of JvC and JvS is represented in red. (G and H) Changes in IP volume over time in mice using (G) 1.36% or (H) 3.86% glucose alone or in combination with icodextrin. (I) Net UF generated in mice at the end of 2-hour dwells using 1.36% glucose (light blue bars), 3.86% glucose (dark blue), 7.5% icodextrin (orange), or a combination of osmotic agents (light and dark green, for CIG 1.36% and CIG 3.86%, respectively). (J) Sodium removal across the peritoneal membrane of mice using the same dialysis solutions. Data are mean6SEM; n=3–6/group. BW, body weight.

Metabolism of Icodextrin in the Mouse Peritoneal measured a-amylase activity and icodextrin metabolites in Cavity plasma and dialysate in mice. Both Aqp1 wild-type and knock- Once in the peritoneal cavity, icodextrin undergoes some me- out mice showed elevated but similar circulating levels of the tabolization due to a-amylase.34,35 Because rodents, contrary enzyme in basal conditions (2965641 versus 3147695 IU/L, +/+ 2/2 to humans, have high plasma levels of a-amylase that may in Aqp1 and Aqp1 animals, respectively, P=0.26). Mean diffuse to the cavity and interfere with that metabolism, we dialysate a-amylase activity was 176616 UI/L at the end of

Table 4. Mouse transport parameters using crystalloid and colloid osmotic agents, used alone or in combination

Solution Net UF (ml/g body wt) D120/D0 Glucose UF Efﬁciency (ml/g BW per mg CHO absorbed) 1.36% glucose 0.461.5 0.6260.01 0.0360.30 CIG 1.36% 29.261.3a 0.6060.04 2.6561.13a 3.86% glucose 34.061.6 0.3560.04 0.8060.44 CIG 3.86% 51.261.3a 0.3860.02 2.0660.95b 7.5% icodextrin 5.461.3 NA 0.3260.20

Results are mean6SEM values; n=4–6 mice per group. D120/D0 glucose, dialysate glucose concentration at 120 min of the dwell/baseline dialysate glucose concentration; BW, body weight; CHO, carbohydrate; CIG, combination 7.5% icodextrin-glucose; NA, not applicable. aP,0.001 versus the sum of net UF provided by each osmotic agent used alone. bP,0.05.

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Figure 5. Schematic representation of the mechanisms and pathways of crystalloid, colloid, and combined osmosis across the peritoneal membrane. Using crystalloid osmotic agents such as glucose or aminoacids (left panels), transcapillary UF occurs across small and ultrasmall (AQP1) pores and is directly related to the tonicity of the dialysis solution. Once the osmotic gradient has dissipated because of systemic absorption of the osmotic agent, reabsorption of fluid (backfiltration) occurs, mainly across the small pores. Large icodextrin subfractions (middle panels) generate an osmotic water transport independently of AQP1 water channels and tonicity, exclusively through the small pore system. The peritoneal coefficient of reflection for these large molecules is close to unity, indicating that they are not able to cross the membrane and induce a colloid osmosis. The slow absorption—via peritoneal lymphatics—and intraperitoneal metabolism of icodextrin molecules provide a sustained transcapillary UF making it suitable for long dwells, especially in patients with fast peritoneal solute rate. Combining glucose and icodextrin in the same dwell (right panels) enhances transcapillary UF, thanks to the added effects of initial AQP1- dependent free-water transport generated by glucose, and the prevention of backfiltration by large icodextrin molecules.

2-hour dwells in mice (corresponding to 5–7-hour dwells in that of patients at the end of their first icodextrin exchange.34 patients36). The dialysate levels of a-amylase were similar for These data support the validity of the mouse model of PD to 3.86% glucose and 7.5% icodextrin (190610 versus 140635 assess osmotic water transport induced by icodextrin. IU/L, respectively, P=0.37), and they were not influenced by longer dwell duration (103610 and 11869 IU/L, for 3- and 4- DISCUSSION hour dwells, respectively). The remaining concentration of total icodextrin (sum of high and low-molecular mass-fractions, Our modelization, biophysical, and experimental studies in 42.460.9 mg/ml) and the profile and relative concentration of transgenic mouse models validate, for the first time, the fun- low-molecular-mass icodextrin metabolites (G2–G7, Supple- damental differences between crystalloid and colloid osmosis mental Figure 5) in the drained dialysate were similar to what across the peritoneal membrane. Whereas crystalloid agents has been observed in the effluent of patients receiving PD.34,35 such as glucose and amino acids require hypertonicity and the The sum of G2–G4 metabolites represented 18% of the total presence of AQP1, large icodextrin fractions generate a water glucose polymers in the dialysate, a proportion identical to flow that predominantlyoccurs across smallporesof peritoneal

J Am Soc Nephrol 29: 1875–1886, 2018 Osmosis across the Peritoneal Membrane 1883 BASIC RESEARCH www.jasn.org capillaries and is independent of tonicity. Combinations of endothelial water channels (AQP1) to promote osmotic water crystalloid and colloid osmotic agents enhance water transport transport across the peritoneal membrane. In addition, we via the paracellular route, potentiating sodium and water re- confirm in this model that faster peritoneal solute transport moval during PD. increases water removal induced by icodextrin whereas it The osmotic flow through an ideal semipermeable mem- decreases glucose-driven UF, in line with clinical observa- brane (permeable to water but not to solutes) is mainly de- tions.46–48 These studies substantiate the different and poten- termined by the total number of particles in solution (i.e., tially complementary pathways of water transport during osmolality), independently of their size—each 1 mM of solute crystalloid versus colloid osmosis. exerting an osmotic pressure of 19.3 mm Hg according to van’t Our investigations of the potential benefit from combining Hoff’s law. Conversely, the flow across a solute-permeable crystalloid and colloid agents reveal that these combinations membrane—like most biologic membranes, including the during the same dwell enhance osmotic water transport, sodium peritoneum—depends upon the relatively nonpermeable sol- removal, and UF efficiency during PD. These results are in line utes (Supplemental Figure 6). In continuous capillaries like with mathematical modeling both in mice and in patients,25 and those lining the peritoneal membrane, the protein-reflecting data from clinical studies.49–53 The use of combined solutions element, or small pore, has a functional radius of approxi- led to a fast, glucose-induced, and AQP1-dependent osmotic mately 4.0 nm, and is located at the interendothelial clefts, cov- water transport during the first part of the dwell, and a sustained ered by the glycocalyx.14,37–39 Because peritoneal capillaries—in UF thanks to the presence of large, colloidal fractions of icodex- contrast to the glomerular barrier—show no significant charge- trin. Importantly, these large fractions enhanced solute-coupled 40–42 selectivity, particles with an RH similar to or larger than the water removal and prevented the backflow of water from dialy- functional radius of small pores are expected to generate colloid sate to peritoneal capillaries after the dissipation of crystalloid osmosis during PD. osmotic gradient. In turn, the enhanced UF achieved using com- On the basis of albumin and its oncotic effect in the micro- binations of osmotic agents diluted the carbohydrates in the vasculature, the glucose polymer icodextrin was developed to dialysis solution, thereby reducing their transperitoneal diffu- achieve UF during long dwells in patients receiving PD, while sion and systemic absorption. At the same time, fluid absorption reducing the deleterious effects of glucose.12 Despite its global use rate from the cavity remained constant using either combined or as a dialysis solution for .20 years, the mechanism by which separate solutions. Combining crystalloid and colloid osmosis icodextrin induces water flow was never formally demonstrated. offers the potential to enhance UF and sodium removal, and Our results demonstrate that large fractions of icodextrin are achieve euvolemia, especially in patients with fast peritoneal sol- colloidal in nature and induce colloid osmosis. First, a significant ute transport rate causing loss of UF capacity. proportion of the icodextrin glucose polymers have an RH larger The translational relevance of our findings is supported by than the functional radius of small pores (approximately 4 nm). severalpoints.Regardingtheissueofscaling,2-hourdwellsin Second, the selective removal of polymers with a RH.4nmcom- mice can be compared with approximately 5–7hoursdwells pletely abrogates osmotic water flow across the mouse perito- in humans, as a result of higher area-to-volume ratio and neum, whereas it has no effect on the transperitoneal osmotic more rapid solute (approximately 3–4 times faster) and fluid gradient. Third, biophysical and in vivo data are matched by (approximately 2.5 times) transport across the peritoneal computer simulations suggesting the effective osmolality mainly membrane of small animals.36,54 Such dwell duration is depends on high-molecular-mass fractions.25 We confirmed that closetotheoptimaldurationofanicodextrindwelltoin- large fractions of icodextrin have an important contribution to duce UF in patients receiving PD, which has been shown to the total mass of particles in solution, but only a minimal effect be approximately 8 hours.12 The relatively low net UF on osmolality, whereas low-molecular-mass fractions signifi- achieved using 7.5% icodextrin as compared with 3.86% cantly contribute to the tonicity of the solution. Our data validate glucose-based dialysis solutions in our mouse model may predictions that solutes with a reflection coefficient close to unity be explained by time-scaling issues and potential additional induce colloid osmosis,43–45 and that approximately 25% of the factors, including differences in plasma osmolality, the ab- particles in solution of icodextrin act as colloid.22,25 These specific sence of ESRD, intrinsic physiologic properties of the mouse properties provide a rationale supporting the effectiveness of ico- peritoneal membrane (i.e.,higheraC), and transperitoneal dextrin to achieve sustained UF during long dwells and restore diffusion of a-amylase. Studies in rat models of PD have fluid and sodium balance in patients receiving PD.46–48 raised the issue of high intraperitoneal levels of a-amylase, Our studies also demonstrate that the pathways and funda- potentially influencing the breakdown and metabolism of mental mechanisms of osmosis differ between crystalloid and icodextrin.30–32,55–57 The a-amylase activity measured in colloid agents. For the first time, we provide direct evidence the peritoneal effluent of our mouse models yielded values that the water flow generated by colloid particles occurs across closer to those in patients receiving PD than in rats. In interendothelial clefts and is independent of the presence of addition, the remaining icodextrin concentration in the water channels and tonicity (i.e., iso- or even hypotonic os- dialysate (approximately 40 mg/ml) and the profile of low- mosis). Conversely, crystalloid agents such as glucose and molecular-mass icodextrin metabolites (with smaller poly- amino acids require an osmotic gradient and the presence of mers [G2 to G4] predominating over the larger ones [G5 to

1884 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1875–1886, 2018 www.jasn.org BASIC RESEARCH

G7]) observed in mice at the end of the dwells were all Increased peritoneal membrane transport is associated with decreased similar to what is reported in patients receiving PD.34,56 It patient and technique survival for continuous peritoneal dialysis – should be noted that the inclusion of the amylase activity in patients. JAmSocNephrol9: 1285 1292, 1998 3. Brown EA, Davies SJ, Rutherford P, Meeus F, Borras M, Riegel W, et al.; the TPM could potentially optimize predictions of both net EAPOS Group: Survival of functionally anuric patients on automated UF and carbohydrate absorption in models using icodex- peritoneal dialysis: The European APD Outcome Study. JAmSoc trin.58 Altogether, these data support the usefulness and Nephrol 14: 2948–2957, 2003 reliability of the mouse model of PD to assess icodextrin- 4. Davies SJ: Longitudinal relationship between solute transport and ultrafiltra- induced water transport during PD. tion capacity in peritoneal dialysis patients. Kidney Int 66: 2437–2445, 2004 These studies, on the basis of a multilevel approach, unravel 5. Brimble KS, Walker M, Margetts PJ, Kundhal KK, Rabbat CG: Meta- analysis: Peritoneal membrane transport, mortality, and technique essential features of crystalloid and colloid osmosis across bi- failure in peritoneal dialysis. JAmSocNephrol17: 2591–2598, 2006 ologic membranes. They formally demonstrate that large frac- 6. Lin X, Lin A, Ni Z, Yao Q, Zhang W, Yan Y, et al.: Daily peritoneal ul- tions of icodextrin induce colloid osmosis and elucidate how trafiltration predicts patient and technique survival in anuric peritoneal combining crystalloid and colloid particles enhances water and dialysis patients. Nephrol Dial Transplant 25: 2322–2327, 2010 sodium transport across the peritoneal membrane. These in- 7. Graham T: Liquid diffusion applied to analysis. Philos Trans R Soc Lond 151: 183–224, 1861 sights provide a rationale for using combinations of osmotic – fl 8. Holmes CJ: Glucotoxicity in peritoneal dialysis solutions for the solu- agents to improve uid balance in patients treated with PD. tion! Adv Chronic Kidney Dis 14: 269–278, 2007 9. Williams JD, Craig KJ, Topley N, Von Ruhland C, Fallon M, Newman GR, et al.; Peritoneal Biopsy Study Group: Morphologic changes in the ACKNOWLEDGMENTS peritoneal membrane of patients with renal disease. J Am Soc Nephrol 13: 470–479, 2002 10. Davies SJ, Phillips L, Naish PF, Russell GI: Peritoneal glucose exposure We dedicate this work to the memory of Professor Bengt Rippe (Goth- and changes in membrane solute transport with time on peritoneal enburg, 1950–Lund, 2016), whose seminal work conceptualized the basis dialysis. J Am Soc Nephrol 12: 1046–1051, 2001 of fluid and solute transport across capillaries and, by extension, across 11. Devuyst O, Margetts PJ, Topley N: The pathophysiology of the peri- – the peritoneal membrane during peritoneal dialysis. The engaging and toneal membrane. JAmSocNephrol21: 1077 1085, 2010 12. Mistry CD, Mallick NP, Gokal R: Ultrafiltration with an isosmotic solution enthusiast personality of B.R. inspired a whole generation of physician- during long peritoneal dialysis exchanges. Lancet 2: 178–182, 1987 scientists and contributed to the diffusion of peritoneal dialysis world- 13. Mistry CD, Gokal R, Peers E: A randomized multicenter clinical trial wide. We thank Yvette Cnops, Ann-Christin Bragfors-Helin, Monica comparing isosmolar icodextrin with hyperosmolar glucose solutions in Eriksson, and Sebastien Druart for expert technical assistance. CAPD. MIDAS Study Group. Multicenter Investigation of Icodextrin in This work was supported in part by Baxter Healthcare (extramural Ambulatory Peritoneal Dialysis. Kidney Int 46: 496–503, 1994 grant to J.M. and O.D.), the Fondation Saint-Luc (J.M.), the Fondation 14. Rippe B, Stelin G, Haraldsson B: Computer simulations of peritoneal fluid transport in CAPD. Kidney Int 40: 315–325, 1991 Horlait-Dapsens (J.M.), the Société Francophone de Dialyse (J.M.), 15. Rippe B, Rosengren BI, Venturoli D: The peritoneal microcirculation in the National Fund for Scientific Research (to J.M. and O.D.), and the peritoneal dialysis. Microcirculation 8: 303–320, 2001 Concerted Research Action (ARC16/21-074). A.S. was supported by the 16. Krediet RT, Lindholm B, Rippe B: Pathophysiology of peritoneal Special Research Fund of the Université Catholique de Louvain Medical membrane failure. Perit Dial Int 20[Suppl 4]: S22–S42, 2000 School (Brussels, Belgium). Baxter Novum is the result of a grant from 17. Krediet RT: The physiology of peritoneal solute, water, and lymphatic transport. In: Nolph and Gokal’s Textbook of Peritoneal Dialysis,3rd Baxter Healthcare to the Karolinska Institutet. C.-A.F. is a Senior Re- Ed., edited by Khanna R, Krediet RT, New York, Springer, 2009, pp search Associate of the National Fund for Scientific Research. 137–172 J.M., B.R., C.M.O., and O.D. designed the study; J.M., A.S., C.-A.F., 18. Devuyst O, Nielsen S, Cosyns JP, Smith BL, Agre P, Squifflet JP, et al.: C.F., E.G.-L., B.L., E.G., and F.V. carried out experiments; B.R. and C. Aquaporin-1 and endothelial nitric oxide synthase expression in capillary – M.O. performed computer simulations; J.M., C.-A.F., B.L., E.G., C.M. endothelia of human peritoneum. Am J Physiol 275: H234 H242, 1998 19. Yang B, Folkesson HG, Yang J, Matthay MA, Ma T, Verkman AS: Re- O., and O.D. analyzed the data; J.M., C.M.O., and O.D. made the duced osmotic water permeability of the peritoneal barrier in aqua- fi gures; J.M., C.M.O., and O.D. drafted and revised the paper; all porin-1 knockout mice. Am J Physiol 276: C76–C81, 1999 authors approved the final version of the manuscript. 20. 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Krediet RT, Koomen GC, Koopman MG, Hoek FJ, Struijk DG, tion of icodextrin in rats and humans including the effect of a-amylase Boeschoten EW, et al.: The peritoneal transport of serum proteins and activity in dialysate. Perit Dial Int 35: 288–296, 2015 neutral dextran in CAPD patients. Kidney Int 35: 1064–1072, 1989 41. Buis B, Koomen GC, Imholz AL, Struijk DG, Reddingius RE, Arisz L, et al.: Effect of electric charge on the transperitoneal transport of plasma proteins during CAPD. Nephrol Dial Transplant 11: 1113–1120, 1996 42. Asgeirsson D, Axelsson J, Rippe C, Rippe B: Similarity of permeabil- This article contains supplemental material online at http://jasn.asnjournals. ities for Ficoll, pullulan, charge-modified albumin and native albumin org/lookup/suppl/doi:10.1681/ASN.2017080828/-/DCSupplemental.

AFFILIATIONS

1Division of Nephrology and 4Department of Clinical Biochemistry, Cliniques Universitaires Saint-Luc, Brussels, Belgium; 2Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Brussels, Belgium; 3Bio and Soft Matter Division (BSMA), Institute of Condensed Mater and Nanosciences, Université Catholique de Louvain, Louvain-la-Neuve, Belgium; 5Division of Renal Medicine and Baxter Novum, Department of Clinical Science, Intervention and Technology, Karolinska Institute, Karolinska University Hospital Huddinge, Stockholm, Sweden; 6Baxter Healthcare, Braine-l’Alleud, Belgium; 7Department of Nephrology, Lund University, Skane University Hospital, Lund, Sweden; and 8Institute of Physiology, University of Zurich, Zurich, Switzerland

1886 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1875–1886, 2018 JASN-2017-08-0828-R2

Mechanisms of Crystalloid versus Colloid Osmosis across the Peritoneal Membrane

Johann Morelle, Amadou Sow, Charles-André Fustin, Catherine Fillée, Elvia Garcia-Lopez, Bengt Lindholm, Eric Goffin, Frederic Vandemaele, Bengt Rippe, Carl M. Öberg*, and Olivier Devuyst*

Supplementary material

TABLE OF CONTENT

Detailed material and methods ...... P2 Suppl. Fig. 1: Dynamic changes in UF rate and osmotic gradient using glucose or icodextrin in the mouse model of PD ...... P8 Suppl. Fig. 2: Correlation and agreement between IPV measured in vivo and predicted by the three-pore model (TPM) ...... P9 Suppl. Fig. 3: Effect of lipopolysaccharide-induced modulation of peritoneal solute transport on water removal achieved by hypertonic glucose vs. icodextrin in the mouse model of PD ...... P10 Suppl. Fig. 4: Role of AQP1 in osmotic water transport induced by combinations of icodextrin and glucose ...... P11 Suppl. Fig. 5: Concentration of low molecular weight icodextrin metabolites (G2-G7) in the peritoneal effluent of Aqp1 mice ...... P12 Suppl. Fig. 6: Molecular mechanisms of osmosis ...... P13 Suppl. Table 1: Parameters of the modified, mouse-adapted three-pore model ...... P14 Suppl. Table 2: Clinical and biological characteristics of Aqp1 mice at baseline ...... P15 References ...... P16

1 DETAILED MATERIAL AND METHODS

Animals. Gender-matched Aqp1 mouse littermates generated as previously described1, aged 8–12 weeks, were used to assess the influence of the water channel AQP1. All animals had access to standard diet and tap water ad libitum. The experiments were conducted in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals and the Animal Ethics Committee of the UCL Medical School.

Peritoneal transport studies. Transport across the mouse peritoneum was investigated using a modified peritoneal equilibration test with different PD solutions (Baxter Healthcare, Belgium) (Table 1), as described previously2-6. The combined glucose-icodextrin PD solutions were prepared by Baxter on laboratory scale using the same raw materials as the current glucose and icodextrin PD solutions. These PD solutions were filtered through a 0.2 µm filter, filled into bags, overpouched, heat sterilized, and their final electrolyte composition was verified. Although no endotoxin screening was performed, the lack of impact of these solutions on solute transport - as compared with commercial solutions – rules out any significant inflammation. Briefly, following anesthesia with ketamine (100 mg/kg SC, Merial, Brussels, Belgium) and xylazine (10 mg/kg subcutaneously, Bayer, Belgium), mice were laid in a supine position on a thermopad at 37°C. The right common carotid artery was cannulated for blood sampling. The fur over the abdominal wall of the mice was shaved after meticulous disinfection with 70% alcohol. A silicon catheter (Venflon 22 G, 0.9 mm diameter, Terumo, Belgium) was inserted percutaneously into the right lower quadrant of the peritoneal cavity. This catheter was used for dialysate fluid infusion and sampling. Dialysate microsamples were taken from the bag at 0 minute, and from the Venflon catheter at 30 and 60 minutes, and at the end of the dwell (120 minutes). Prior to each sampling, 50 to 100 µL of the dialysate was flushed back and forth, and the abdomen was gently agitated to facilitate fluid mixing. Dialysate recovery was performed at the end of the dwell, using syringes and pre-weighed safe-lock tubes; the remaining dialysate in the peritoneal cavity was collected using gauze tissue (~5% of the recovered volume). Instilled and recovered dialysate volumes were weighed on a precision balance (AG35, Mettler Toledo; margin of error during experiments is 0.1 mg). Provided a dialysate density of 1.020 ± 0.001 g/cm³ in experimental conditions (3.86% glucose), the volume margin of error can be calculated as 0.1 µl. Values rounded of to 1 µl were used for both instilled and recovered volumes. These parameters were used to measure net UF, as previously described5-6. Previous experiments have demonstrated that a low intraperitoneal pressure in our mouse model of PD (<2 mmHg for IPV up to 10 ml) is

2 associated with a negligible absorption of fluid and fluorescently-labeled or radioiodinated albumin4-6; as a result, fluid absorption in basal conditions in our model can be neglected and net UF roughly represents transcapillary UF. Blood microsamples were taken from carotid artery at time 0, 30 and 60 minutes, and at the end of the dwell (exsanguination, 120 minutes). Hematocrit was measured before PD exchange. Plasma and dialysate urea and glucose, and plasma sodium were assayed using a Kodak Ektachem DT60 II and DTE II analyser (Eastman Kodak Company, USA). Dialysate sodium was determined using an indirect ion-selective electrode on a Roche Diagnostics Cobas 8000 Module ISE. Plasma and dialysate osmolality was measured using a Fiske Osmometer (Needham Heights, MA). Baseline characteristics of the animals are presented in Supplementary table 2. In some experiments, a single dose of lipopolysaccharide (10 mg/kg, E. coli serotype O111: B4; Sigma-Aldrich, Buchs, Switzerland) was instilled into the peritoneal cavity 24-h before assessing peritoneal transport.

Calculations. The transport of small solute was evaluated by the D/D0 glucose ratio and the mass transfer area coefficient (MTAC) of urea, using (1) the Garred two-sample model7 and (2) the Henderson and Nolph equation corrected for convective transport according to Waniewski8:

Vav V0 P  D0  MTAC urea (µL/min) (Garred) =  ln  (1) t 120 Vt P  Dt 

1 f Vt  V0 (P  D0 )  MTAC urea (µL/min) (Waniewski) = ln 1 f  (2) t120 Vt ln(P  Dt  in which Vav is the average of the initial and final volumes; V0, the instilled volume; Vt, the drained volume; f, a weighing factor between diffusion and convection (set at 0.33 for a large degree of convective transport, and at 0.5 for negligible convection); P, the plasma 9 concentration of urea corrected for aqueous plasma water ; Dt, the dialysate concentration of urea at the end of the dwell; D0, the initial concentration of urea in dialysate, which is set at 0. UF efficiency was calculated as the net UF (µL/g of body weight) divided by the amount of carbohydrates absorbed, as previously described10.

Intraperitoneal volume vs. time curves. Intraperitoneal volume versus time curves were performed using a fluorescent bovine serum albumin conjugate as an indicator-dilution technique, as previously described6, or by direct volumetry. Both techniques closely correlated, including when icodextrin was used as osmotic agent (Pearson r, 0.98, P=0.003). Dialysate sodium was measured using indirect ion selective electrode method.

3 Dynamic light scattering. Dynamic light scattering was used to determine the apparent hydrodynamic radius and size distribution profile of the glucose polymer icodextrin in solution (Extraneal 7.5%). The polydispersity index measurements were performed on a Malvern CGS3 equiped with a HeNe laser (λ=632.8 nm) at a temperature of 37.3°C and an angle of 90°. The size distributions were obtained by a CONTIN analysis of the data.

Determination of total icodextrin, icodextrin metabolites, and absorption of carbohydrates. Total icodextrin in the dialysate was measured by enzymatic hydrolysis with amyloglucosidase (Sigma-Aldrich, Saint-Louis, MO, USA); icodextrin metabolites were determined in the dialysate of mice exposed to 7.5% icodextrin using a weak anion-exchange column (BioBasic AX; 150 x 4.6 mm, 5 µm; Thermo Electron, Woburn, MA, USA) and a refractive index detector; and total HMW fractions were estimated as the difference between total icodextrin and LMW G2-G7 fractions, as previously described11-14. The amount of carbohydrates (CHO) absorbed from the peritoneal cavity was calculated as the difference between the mass of CHO (glucose and icodextrin) infused into the peritoneal cavity and the mass removed.

Mouse three-pore model. The mouse three-pore model is essentially the same as that used in the initial version15 where LpS and other transport parameters have been adapted to the mouse based on separate experimental measurements of the osmotic conductance for glucose in +/+ -/- Aqp1 and Aqp1 mice. The αC parameter was set to 0.04 (i.e. slightly higher than in PD patients) to match the difference between Aqp1+/+ and Aqp1-/- mice (using 3.86% glucose as the osmotic agent) and the osmotic water transport for icodextrin (which is essentially independent of g and can therefore be used to estimate LpS). In their original version of the three-pore model, Rippe et al. had to inflate the mass transfer area coefficient for glucose to compensate for the fact that the rate of glucose disappearance from the peritoneal cavity was higher than that expected from the calculated rate of diffusion of glucose15. This correction was used unchanged in the present modelling studies. All relevant transport parameters were rescaled by BW0.75 from man to mouse16 and are shown in Table S1. The net flow of water to/from the mouse peritoneal cavity (equal to the change in the intraperitoneal volume VD per unit time t) is described by the ordinary differential equation

= 퐽 + 퐽 + 퐽 − 퐿 (S1) , , , where, Jv,C, Jv,S, and Jv,L are the flows of water (in µL/min) across the aquaporins, the highly selective pathways (the “small pores”) and the weakly selective pathways (the “large pores”),

4 respectively. L is the lymphatic flow (Table S1). The dialysate concentration for each solute i

(denoted dCD,i/dt in mmol/L/min) as a function of time was calculated from

, ,,,, ,,, = − 퐶, (S2) where Js,S,i and Js,L,i are the flows of solute i across the small pores and the large pores, respectively. The initial conditions are VD(0) = 2500 µL, CD,glucose(0) = see Table 1, CD,Na+(0)

= 133 mmol/L for Icodextrin and 132 mmol/L for glucose fluids (Table 1), CD,urea(0) = 0 mmol/L, and CD,albumin(0) = 0. The initial concentrations for the icodextrin fractions were set according to Table S3. The initial value problem consisting of the ordinary differential equations S1 and S2 (one for each solute) and the initial conditions were solved with the Dormand-Prince (DOPRI) method17. The solute flow (in mmol/min) over each pathway is calculated according to the non-linear flux equation

, ,, 퐽,, = 퐽,1− 휎, (S3) ,

, ,, 퐽,, = 퐽,(1 − 휎,) (S4) ,

Here CP,i is the plasma concentration of solute i (set according to experimentally measured values); S,i and L,i the small- and large pore reflection coefficients (calculated from pore 15 theory, see Eq. 4 in reference ); PeS,i=Jv,S(1-σS,i)/MTACS,i an PeL,i=Jv,L(1-σL,i)/MTACL,i are the Péclet numbers for the small and large pore pathway, respectively. The small- and large diffusion capacities, MTACS,i and MTACL,i (in µL/min), are either set according to Table S1 or calculated according to pore theory, see Eq. 11 in reference15. All reflection coefficients are calculated according to theory, see Eq. 4 in reference15. The flow of water across each pore population is calculated using

퐽 = 훼퐿푆Δ푃 − 19.3 ∑ 휑퐶, − 퐶, (S5)

퐽 = 훼퐿푆Δ푃 − 19.3 ∑ 휑휎,퐶, − 퐶, (S6)

퐽 = 훼퐿푆Δ푃 − 19.3 ∑ 휑휎,퐶, − 퐶, (S7)

Here αC, αS and αL are the contributions for each of the different pathways to the barrier hydraulic conductance (see Table 1), φi is the osmotic coefficient of solute i set to 0.93 for sodium and related anions, cf. reference15, and 1.0 for the other solutes. The hydraulic pressure gradient ΔP was set to 8 mmHg. Icodextrin fractions were the same as those used by

5 Rippe and Levin in their landmark publication, including trace amounts (~2 mmol/L) of glucose15. Additional simulations using the adapted version of the three-pore model showed that considering or not this negligible amount of glucose did not significantly impact net UF obtained with icodextrin (248 µl vs. 236 µl considering 8 icodextrin fractions or only the 7 fractions with a molecular weight >180 Da; <5% difference).

Estimation of icodextrin HMW distributions from DLS intensity data. The icodextrin molecules are relatively small compared to the wavelength of the incident light (λ=632.8 nm) and thus the intensity I of the scattered light can be estimated using the Rayleigh scattering equation

퐼 = 퐼 (1) where I0 is the incident light intensity; ae the hydrodynamic radius of the solute, d the distance between the point of observation and the particle, N the particle to medium refractive index ratio and θ the angle between the incident and scattered light. Knowing the molecular weight MW and molecular density ρ of the solute, the above equation can be written

퐼 = 퐼 (2)

As a good approximation the molecular density ρ and refractive index for polymers are

related via the well-known Lorenz–Lorentz relation 휌 = and thus, at a 90° angle of detection

퐼 = 퐼 푀푊 (3) where r denotes the specific refraction (a tentative measure of properties like polymer shape and dipole strength) often assumed to be constant for a specific polymer-solvent system18. Moreover, the intensity of the scattered light is proportional to the molar concentration n yielding

퐼 =Λ푀푊푛 (4)

where all the constants have been incorporated into Λ. Thus the parameter 휙 = is proportional to n and the fractional molarity n% of each fraction is π

푛%= (5) ∑

6 where summation is performed over all fractions K. If the net molar concentration nnet is known then the molar concentration of each fraction is 푛 = 푛%∙ 푛. The distribution of molecular weights of icodextrin was estimated using an empirical Mark-Houwink-Kuhn- Sakurada (MHKS) relationship

푎 = 퐾 푀푊 (6)

15 The MHKS-parameters K´ (0.486) and  (0.385) were applied and nnet was adjusted to give a mass concentration of 7.5%, 2.48% and 1.44% for un-fractionated and the two fractionated icodextrins, respectively. The scattered light intensity is proportional to the square of the solute molecular weight vide infra and DLS is therefore especially well suited to detect even minute amounts of high molecular weight solutes. For the same reason DLS data are inherently biased toward higher MWs, making LMW fractions undetectable in the current experiment. This effect was particularly evident for the unfractionated icodextrin solution but apparently less so for the fractionated solutions. LMW fractions were added in the TPM simulations (180 Da, 540 Da and 1 kDa fractions, identical to those used previously15) and, for unfractionated icodextrin, a 3 kDa fraction was added. Using the above procedure we obtained a number average Mn and mass average Mw for icodextrin of 6.3 kDa and 34.0 kDa, respectively, and a net molarity of 11.9 mmol/L (compared to ~12 mmol/L in reference15).

Statistical methods analysis. Data are presented as mean ± SEM. Comparisons between results from different groups were performed using t test, one- or two-way ANOVA, as appropriate. Significance level was indicated in the legend of each figure (*P<0.05, **P<0.01, ***P<0.001).

7 SUPPLEMENTARY FIGURES

Supplementary Figure 1. Dynamic changes in UF rate and osmotic gradient using glucose or icodextrin in the mouse model of PD. (A) Changes in UF rate and osmotic gradient over time using either 3.86% glucose (blue) or 7.5% icodextrin (orange) as osmotic agent in wild-type mice. (B) Changes in the osmotic gradient between the dialysate and plasma (D/P osmolality) over time on PD in wild-type mice, using hypertonic glucose (blue) or icodextrin (orange).

Supplementary Figure 2. Correlation and agreement between intraperitoneal (IP) volume measured in vivo and predicted by the three-pore model (TPM). (A) Correlation line between IP volume measured by direct volumetry in the mouse model of PD and that predicted by computer simulations based on the TPM. (B) Bland-Altman analysis plot comparing both methods to assess IPV. Every circle represents the mean of IPV measured at 30, 60, 90 and 120 min of a dwell with either 3.86% glucose or 7.5% icodextrin in Aqp1+/+or Aqp1-/- animals. Dotted lines in the Bland-Altman graph represent mean ± 95% limits of agreement. Bias (±SD) was -3.6 (±76.0), and 95% limits of agreement, -152.5 to 145.4.

Supplementary Figure 3. Effect of lipopolysaccharide-induced modulation of peritoneal solute transport on water removal achieved by hypertonic glucose vs. icodextrin in the mouse model of PD. (A) Relative dialysate-over-plasma (D/P) urea ratio 24-h after exposure to intraperitoneal (IP) lipopolysaccharide (LPS), during dialysis with either 3.86% glucose (blue circles) or 7.5% icodextrin (orange circles). (B) Relative net ultrafiltration (UF) at the end of 2-h dwells in the same animals. (C) Relationship between net UF and solute transport using either hypertonic glucose (blue) or icodextrin (orange), in basal conditions (closed circles) and after LPS exposure (open circles). Data are mean ± SEM; each circle represents a mouse.

Supplementary Figure 4. Role of AQP1 in osmotic water transport induced by combinations of icodextrin and glucose. (A) Predictions from the three-pore model (TPM) showing the changes in the intraperitoneal (IP) volume over time during P with a combination of icodextrin and glucose (CIG) 3.86%, using a fractional ultrafiltration coefficient for ultrasmall pores of 0.04 (solid line) or 0.00 (dotted line), respectively. (B) Changes in IP volume over time during PD with CIG 3.86% in Aqp1 wild-type (open circles, solid line) vs. knockout (close circles, dotted line) mice. (C) Net ultrafiltration (UF) at the end of 2-h dwells with CIG 1.36% or CIG 3.86% in Aqp1 mice. Data are mean ± SEM; n = 4-6/group.

Supplementary Figure 5. Relative concentration of low molecular weight icodextrin metabolites (G2-G7) to the total icodextrin concentration in the peritoneal effluent of Aqp1 mice. At the end of a 2-h dwell with 7.5% icodextrin, smaller low molecular weight icodextrin metabolites (G2-G4) predominate over the larger ones (G5-G7), suggesting intraperitoneal metabolism of the glucose polymers. No difference is observed between Aqp1+/+ and Aqp1-/- mice. Data are mean ± SEM; n=6 in each group.

Supplementary Figure 6. Molecular mechanisms of osmosis. (A) Brownian motion of water molecules through a membrane pore with water on both sides. The amount of water molecules that diffuses in the two directions is balanced so precisely that zero net movement of water occurs. (B) Osmotic flow through a water filled membrane pore from the water side towards a dilute solution containing large, near impermeable, solute molecules in a concentration CA. Osmotic water flow will continue until the solute concentration becomes equal on both sides of the membrane. The osmotic pressure exerted by particles in solution is determined by the number of particles per unit volume of fluid and their reflection coefficients, not by the mass of the particles. (C) Osmotic flow between solutions of equimolar concentration (isotonic flow). Although the concentrations are equal, osmotic flow proceeds from left to right because the reflection coefficient (σ) for solute A is larger than for solute B. From a conceptual point of view, situation B is like the osmotic water flow at the level of solute-impermeable water-only channels (transcellular route, ultrasmall pores). On the contrary, situation C may reflect water transport across interendothelial clefts (paracellular route, small pores). Hydrostatic pressure is considered equal on both sides of the membrane in all three situations. Jv, osmotic water flow; Lp, conductance; R, gas constant; T, absolute temperature. Adapted from reference19.

13 SUPPLEMENTARY TABLES

Supplementary Table 1. Parameters of the modified, mouse-adapted three-pore model.

Aqp1+/+ Aqp1-/- Transperitoneal hydrostatic pressure gradient (ΔP) - mmHg 8 8 Transperitoneal oncotic pressure gradient (Δπ) - mmHg 22 22

Unrestricted pore area over unit diffusion distance (A0/Δx) - cm 70 70 MTAC for glucose - µL/min 30 30 MTAC for sodium - µL/min 20 20 MTAC for urea - µL/min 50 50 UF coefficient (LpS) - µl/min/mmHg 0.15 0.12

Osmotic conductance to glucose (LpS σg) – nl/min/mmHg 10 3.5 Peritoneal lymph flow (L) - µl/min 0.8 0.8

Fractional LpS small pores (αs) 0.88 0.92

Fractional LpS ultrasmall pores (αc) 0.04 0.00

Fractional LpS large pores (αL) 0.08 0.08 MTAC, mass transfer area coefficient; UF, ultrafiltration

14 Supplementary Table 2. Clinical and biological characteristics of Aqp1 mice at baseline. Body weight Hematocrit Plasma sodium Solution Groups (g) (%) (mmol/L) 1.36% glucose Aqp1+/+ 27.20.8 48.80.3 143.80.5 Aqp1-/- 26.40.9 48.50.6 144.81.6 3.86% glucose Aqp1+/+ 26.80.5 48.00.5 144.50.6 Aqp1-/- 24.70.6 47.30.5 147.70.8 1.1% aminoacids Aqp1+/+ 26.10.7 50.20.8 144.01.5 Aqp1-/- 25.41.3 48.50.6 146.81.3 7.5% icodextrin Aqp1+/+ 24.80.7 49.80.9 144.21.1 Aqp1-/- 25.21.0 50.51.0 141.21.9

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17 SIGNIFICANCE STATEMENT

Osmosis sustains the efficiency of peritoneal dialysis to restore fluid balance in patients with ESRD. The water channel aquaporin-1 (AQP1) plays a critical role in glucose-driven (crystalloid) osmosis across the peritoneal membrane, but it is not known whether it contributes to ultrafiltration induced by colloid osmotic agents such as icodextrin. On the basis of studies in Aqp1 mice, biophysical experiments, and computer simulations, these data re- examine mechanisms of crystalloid versus colloid osmosis and demonstrate that colloidal fractions of icodextrin induce an osmotic flow that is independent of AQP1. They also show the role of large icodextrin fractions to generate colloid osmosis and provide a rationale for using combinations of osmotic agents to improve fluid volume control in patients treated with peritoneal dialysis.