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Peritoneal Dialysis International, Vol. 28, pp. 487–496Printed in Canada. All rights reserved.

0896-8608/08 $3.00 + .00Copyright © 2008 International Society for Peritoneal Dialysis

487

A PYRUVATE-BUFFERED DIALYSIS FLUID INDUCES LESS PERITONEALANGIOGENESIS AND FIBROSIS THAN A CONVENTIONAL SOLUTION

Roos van Westrhenen,1 Machteld M. Zweers,1 Cindy Kunne,1 Dirk R. de Waart,2

Allard C. van der Wal,3 and Raymond T. Krediet1

Division of Nephrology,1 Department of Medicine; Department of Experimental Hepatology 2 andDepartment of Cardiovascular Pathology,3 Academic Medical Center, University of

Amsterdam, Amsterdam, The Netherlands

Correspondence to: R.T. Krediet, Division of Nephrology,F4-215, Academic Medical Center, Meibergdreef 9, 1105 AZ,Amsterdam, The Netherlands.

c.n.deboer@amc.uva.nlReceived 17 August 2007; accepted 24 March 2008.

♦♦♦♦♦ Background: Conventional lactate-buffered peritonealdialysis (PD) fluids containing glucose and glucose degra-dation products are believed to contribute to the develop-ment of f ibrosis and angiogenesis in the dialyzedperitoneum. To reduce potential negative effects of lactate,pyruvate was substituted as a buffer and its effects on peri-toneal pathological alterations were studied in a chronicperitoneal exposure model in the rat.♦♦♦♦♦ Methods: 20 Wistar rats were infused intraperitoneallywith pyruvate-buffered (n = 9) or lactate-buffered PD fluid.After 20 weeks of daily infusion, peritoneal function wasassessed. In omental peritoneal tissue, the number of bloodvessels was analyzed following alpha-smooth muscle actinstaining. The degree of fibrosis was quantitated in PicroSirius Red-stained sections and by assessment of the hy-droxyproline content. Plasma lactate/pyruvate and beta-hydroxybutyrate/acetoacetate (BBA/AA) ratios weredetermined. Plasma and dialysate vascular endothelialgrowth factor (VEGF) levels were quantitated by ELISA.♦♦♦♦♦ Results: The mass transfer area coefficient of creatininewas higher and the dialysate-to-plasma ratio of sodium waslower in pyruvate-treated animals compared to the lactate-treated group (0.11 vs 0.05 mL/min, p < 0.05, and 78% vs89%, p < 0.05). The BBA/AA ratio tended to be lower in thepyruvate animals (p = 0.07). The number of blood vesselswas lower in pyruvate-treated animals (16 vs 37 per field,p < 0.001). Total surface area, luminal area, and wall/total

area of the vessels were larger in the pyruvate group. Thedegree of fibrosis was lower in intersegmental and perivas-cular areas of pyruvate-exposed animals. Effluent VEGF washigher in the pyruvate group.♦♦♦♦♦ Conclusions: Replacement of lactate by pyruvate resultedin changes in peritoneal solute transport, accompanied bya reduction in both peritoneal membrane angiogenesis andfibrosis, suggesting potentially novel mechanisms to reduceglucose-driven alterations to the peritoneal membrane inPD patients.

Perit Dial Int 2008; 28:487–496 www.PDIConnect.com

KEY WORDS: Pyruvate; angiogenesis; pseudohypoxia;polyol pathway; peritoneal fibrosis.

Ultrafiltration failure is an important peritonealtransport abnormality in long-term peritoneal di-

alysis (PD) patients (1). The most frequent cause of ul-trafiltration failure in these patients is associated withhigh transport rates of low molecular weight solutes,leading to a rapid dissipation of the osmotic gradient(2). This suggests that the development of an enlarge-ment of the vascular peritoneal surface area is a featureof long-term PD in some individuals (3). Increased vas-cularity has been observed in peritoneal biopsies fromPD patients. It was related to the duration of treatment(4) and was most prevalent in those individuals withmembrane failure (5,6).

In animal models of dialysis solution exposure, in-creased peritoneal vessel formation is a major feature

ORIGINAL ARTICLES

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glucose-driven alterations to the peritoneal membranein PD patients.

SUBJECTS AND METHODS

ANIMALS

All studies were performed in accordance with theregulations of the local ethics committee for animal ex-per imentation at the Academic Medical Center,Amsterdam. Male Wistar rats (n = 20; HSD; Harlan, Zeist,The Netherlands) weighing 299 ± 17 g were housed in in-dividual cages under controlled conditions (temperature19°C, relative humidity 50% ± 5%, 12/12-hour light/darkcycle) and fed standard chow (Hope Farms, Woerden, TheNetherlands) and water ad libitum. All rats acclimatizedfor 1 week before insertion of a peritoneal catheter underanesthesia [0.1 mL/100 g body weight (BW) of a mixtureof ketamine:xylazine:atropine , 8 mg, 4 mg, 5 µg/100 gBW]. One day before the operation, a bolus of antibioticswas given (enrofloxacin 0.02 mL/100 g BW subcutane-ously). A Fr. 7 catheter (lumen 1.1 mm, with a Dacron feltcuff glued at 2 cm) was introduced proximal of the umbi-licus and tunneled subcutaneously to the neck via the leftflank. The length of the catheter was adjusted for eachrat and a titanium/silicone device (Rat-o-Port, MTINC,7IS; Access Technologies, Norfolk Medical, Skokie, Illi-nois, USA) was attached to the catheter and implantedsubcutaneously. Adequate analgesia was given(buprenorphine 0.3 mg/mL, 0.2 mL/rat subcutaneously).From the next day on, daily intraperitoneal infusion wasperformed by puncture of the subcutaneous device im-planted in the neck of the rat, using a 0.6 mm wing-endinfusion set (microperfuser; Vygon, Ecouen, France). Priorto infusion, the solution was warmed to 37°C. Peritonealhealing was allowed for 1 week after catheter insertionby daily infusion of 1 mL of heparinized saline (5 IU/mLNaCl 0.9%), after which the experimental period startedand 20 mL per day was infused per rat.

The rats were randomly allocated into two groups: Theexperimental group (n = 11) was daily infused with heatsterilized, 3.86% glucose containing, pyruvate-buffereddialysis fluid prepared by the hospital pharmacy. The con-trol group (n = 9) received standard heat sterilized, 3.86%glucose containing, lactate-buffered dialysate (Dianeal;Baxter S.A., Castlebar, Ireland). The concentration of bothbuffers was 35 mmol/L. Also, the concentrations of Na+,Ca2+, Mg2+, Cl–, and glucose were identical. At least onceduring the experimental period, blood was drawn by tailvein puncture from each rat 60 minutes after infusion withdialysate for determination of plasma lactate/pyruvateratios and beta-hydroxybutyrate/acetoacetate ratios, as

following daily infusion of 3.86% glucose-containinglactate-buffered dialysis solution (7).

The above findings suggest that long-term exposureto components of dialysis solution is a major factor driv-ing the development of peritoneal angiogenesis. Thepresence of high concentrations of glucose and/or glu-cose degradation products (GDPs) derived from the heatsterilization process are thought to play a pivotal role inthis process, partly as a result of their ability to stimu-late the production of proangiogenic factors such as vas-cular endothelial growth factor (VEGF) from mesothelialand endothelial cells (8,9). Local production of VEGF isbelieved to be important in the development of newblood vessels (10) and its production is increased dur-ing ischemic conditions (11). At the cellular level, is-chemia is characterized by an increased NADH/NAD+

ratio. This ratio is also increased in hyperglycemia dueto the generation of NADH during glucose metabolism,both by glycolysis and by activation of the polyol path-way (12–15). In diabetes mellitus, elevation of theNADH/NAD+ ratio has therefore also been described aspseudohypoxia (15). In the context of PD, it has beenshown that locally produced VEGF in peritoneal effluentis related to the mass transfer area coefficient (MTAC) ofcreatinine and thus possibly reflects the peritoneal vas-cular surface area (16). These authors also showed in alongitudinal analysis that locally produced VEGF in-creases with the duration of PD and is decreased in pa-tients using a non-glucose dialysis regimen. Thissuggests that glucose and/or GDP exposure is importantin driving local production of VEGF (17).

The enzyme lactate dehydrogenase (LDH) stimulatesthe production of NAD+ from NADH by enhancing theconversion of pyruvate into lactate and is therefore anescape route to reduce the potential toxicity associatedwith high concentrations of glucose. The use of high con-centrations of lactate as a buffer in PD solutions is, how-ever, likely to inhibit LDH activity and may thereforedirectly augment glucose toxicity (18). In addition, thepresence of high lactate concentrations increases fluxthrough the polyol pathway, which can result in cellularactivation with the production of both proinflammatoryand profibrotic mediators (13,14).

The aim of the present study was to investigatewhether the replacement of lactate by pyruvate as abuffer in a heat-sterilized glucose-based dialysis solu-tion would result in reduced glucose-mediated angiogen-esis and f ibrosis within the chronically exposedperitoneal cavity. Our results show that substitution oflactate with pyruvate results in a reduction in both peri-toneal membrane angiogenesis and fibrosis, and sug-gests potentially novel mechanisms to reduce

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a reflection of the mitochondrial NADH/NAD+ ratio (19).After the experimental period of 20 weeks, a standard-ized peritoneal permeability analysis adapted for the rat(SPARa) was performed. The animals were sacrificed im-mediately thereafter. Two animals in the experimentalgroup were left out of the analysis because of peritonitis.This was diagnosed based on the presence of extensiveinflammatory infiltrates in the histological sections ofperitoneal tissue.

STANDARD PERMEABILITY ANALYSIS IN THE RAT (SPARa)

The SPARa is based on the human SPA (20) and adaptedfor the rat (21). In short, an intravenous infusion needlewith a PVC sheath was inserted intraperitoneally in theleft lower lateral quadrant of the abdomen. Thereafter,to avoid possible effects of a residual volume present be-fore the onset of the analysis, a rinsing step of the peri-toneal cavity was performed with 20 mL 3.86% glucose/lactate dialysate solution prior to the test. During a4-hour dwell with 30 mL 3.86% glucose/lactate-based di-alysate solution (Dianeal), dialysate samples were takenat 0 minutes (before instillation of the test solution) andat 10, 30, 60, 120, 180, and 240 minutes after the start.Outflow was accomplished by gravity. Prior to each sam-pling, 1 mL of dialysate was flushed back and forth 5 timesthrough the catheter with a syringe. Dextran 70, 5 g/L(Hyskon; Medisan Pharmaceuticals AB, Uppsala, Sweden),was added to the test solution as a volume marker for cal-culation of fluid kinetics. Directly after drainage of thedialysate used for the test, another rinsing step was per-formed using 20 mL 1.36% glucose/lactate-based dialy-sis solution to calculate the loss of dextran 70 and theresidual volume after the experiment. All solutions werepreheated to 37°C prior to instillation. During this wholeprocedure, intramuscular administration of anesthetics(a mixture of ketamine, xylazine, and atropine, 8 mg,4 mg, 5 µg per 100 g BW) was applied. Blood was obtainedby tail vein puncture before the experiment and by car-diac puncture at the end of the experiment. Body tem-perature was kept constant throughout the experimentsby placing the animal on a heating pad at 37°C.

ASSAYS

Total dextran 70 was measured in all the dialysatesamples by high performance liquid chromatography aspreviously described (22). In both plasma and dialysate,urea (Hitachi H747; Boehringer Mannheim, Mannheim,Germany) and creatinine (Hitachi H911, BoehringerMannheim) were measured using enzymatic methods. Glu-cose concentration was assessed by the glucose oxidase-

peroxidase assay (SMA II; Technicon, Terrytown, New Jer-sey, USA). Albumin was measured with ELISA (goat anti-rat albumin, GARa/Alb/7S; Nordic Immunology, Tilburg,The Netherlands). In short, 100 µL was pipetted in coat-ing buffer (Na2CO3 and NaHCO3 dissolved in aqueduct),after which pH was adjusted to 9.6. After overnight incu-bation, washing blocking buffer was added. Finally, con-jugate was added and absorbance was read at 490 nm.

Plasma concentrations of lactate, pyruvate, beta-hydroxybutyrate, and acetoacetate were determined inblood samples after protein precipitation. Briefly, per-chloric acid was added to the blood samples in a 1:1 ratiodirectly after the tail puncture. The mixture was put onice for 10 minutes and then centrifuged (3000 rpm for10 minutes). Quantitative enzymatic determination ofthese solutes in plasma was performed (Cobas-Fara;Hoffman–La Roche, Basel Switzerland). Lactate andpyruvate standards were run in parallel with the plasmasamples (19). VEGF was measured in ×10 concentrated(Centripep centrifugal filter device of 15 mL with a YM-10membrane; Amicon, Danvers, Massachusetts, USA) di-alysate and neat in blood samples taken after the SPARaby ELISA (MMVOO, mouse VEGF Quantikine kit; R&D Sys-tems, Minneapolis, Minnesota, USA). The antibody in thiskit cross reacts with rat VEGF and recognizes both the164 and 120 amino acid forms of VEGF. The lower detec-tion limit of the assay is 15.0 ng/L, as determined byrecovery experiments.

PERITONEAL TRANSPORT

A peritoneal transport line was computed for each ratbased on least squares regression analysis of the dialy-sate-to-plasma (D/P) ratio and free diffusion coefficientsin water (D20,w) when plotted on a double logarithmicscale. This method has been extensively described pre-viously (23). The following values were used for D20,w:albumin = 6.1, fibrinogen = 2.19, IgG = 4.0 cm2s–110–7.By interpolation of the D20,w of VEGF (=5.98) in the re-gression equation, the expected D/P ratios were calcu-lated assuming the dialysate concentrations would onlybe determined by transport from the circulation. Theconcentration of this growth factor attributed to localproduction was defined as the difference between themeasured and the expected dialysate concentrations.

Peritoneal fluid and solute kinetics were calculatedas previously described (24). Transcapillary ultrafiltra-tion was calculated from the dilution of the volumemarker dextran 70. The change in intraperitoneal vol-ume during the dwell can be calculated from the dilu-tion of the volume marker after correcting for incompleterecovery. Net ultrafiltration rate is defined as the change

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in intraperitoneal volume divided by dwell time. The MTACsof urea and creatinine were calculated according to themodel of Waniewski et al. (25), in which solute concentra-tion is expressed per volume of plasma water. Glucose ab-sorption was estimated as the difference between theinstilled and the recovered amounts of glucose, relative tothe instilled quantity of glucose. Free water transport wasestimated by the sieving of sodium, expressed as the mini-mum D/P ratio of sodium. A diffusion correction was madebecause a concentration difference between initial dialy-sate and plasma concentrations causes Na+ diffusion fromthe circulation to the dialysate, which leads to an under-estimation of the actual sodium sieving. This was doneusing the MTAC of creatinine as described previously (26).

HISTOLOGICAL ASSESSMENT

Omental and parietal peritoneal tissue samples(lower abdominal quadrant) were obtained from eachrat and fixed in freshly prepared 4% paraformaldehydeand embedded in paraffin. Serial sections (5 µm) werestained with hematoxylin and eosin and Picro Sirius RedF3B, the latter providing a brick red staining of all fibril-lary collagen.

For visualization of blood vessels, adjacent sectionswere stained with monoclonal antibodies reactive withendothelial cells (anti-von Willebrand factor, dilution1: 100; Dako, Glostrup, Denmark) and anti-α1-smoothmuscle actin reactive with vascular smooth muscle cellsand pericytes [smooth muscle actin-1 (SMA-1), dilution1:800; Dako]. A streptavidin–biotin–peroxidase detectionmethod was used for visualization of antibody reactivity.The sections were deparaffinized in xylene and rehydratedin ethanol, followed by incubation with hydrogen perox-ide 0.3% in methanol to block endogenous peroxidaseactivity. Sections were initially blocked with 10% normalgoat serum followed by incubation with one of the pri-mary antibodies. Before this, incubation with PBS-NHScontaining 5% normal rat serum and horseradish peroxi-dase-conjugated streptAB complex (Dako) was per-formed. Peroxidase activity was detected with 1 mg/mL3,3-diaminobenzidine tetrahydrochloride (Sigma, St.Louis, Missouri, USA) and 0.015% H2O2 in 50 mmol/L Tris-HCl buffer, pH 7.6. All slides were counterstained withMayer’s hematoxylin, dehydrated through a series ofethanol concentrations, and mounted with Pertex mount-ing medium (Histolab, Göteborg, Sweden).

VESSEL AND FIBROSIS SCORING

Two blinded observers (interobserver variability was<10%) assessed scoring of the number of vessels per field

and the amount of fibrosis. The number of vessels perfield of peritoneal tissue section was counted in SMA-1-stained omental tissue, using a light microscope (LeitzDialux 20; Leica, Rijswijk, The Netherlands) with a ×25flat field objective (×10 ocular). The SMA-1 antibodystains not only smooth muscle cells in the media of allvessels up to the level of venules, but also vessels with apericyte lining only (capillaries). Five non-overlappingfields from the upper left to the lower right were inves-tigated throughout the specimen, in which all vesselswere counted and measured (only distinct lumen-form-ing structures were counted). The thickening of the vas-cular wall was also measured using the SMA-1-stainedsection.

Measurements of surface areas and diameters ofblood vessels were performed with image analysis soft-ware (Image Pro 4.5; Media Cybernetics, Silver Spring,Maryland, USA). Digital images (0.172 mm2 per section,5 non-overlapping fields for each section) were acquiredwith a Roper Coolsnap CP digital camera (Roper Scien-tific, Vianen, The Netherlands) mounted on an OlympusBX60 microscope (Olympus, Zoeterwoude, The Nether-lands). Values in square micrometers of total surfacearea and luminal area were generated by the above-men-tioned measuring system. Utilizing these values, thewall/total ratio could be calculated. Wall thickness wascalculated as the difference between the total surfacearea and the luminal area. Vessels were divided into cap-illaries (diameter <8 µm), medium sized (8 – 20 µm),and larger vessels (>20 µm).

Fibrosis was assessed by a semiquantitative score andquantitatively. The semiquantitative score was per-formed as previously described (4) using both omentaland parietal sections stained with Picro Sirius Red. Scor-ing was as follows: 0 = normal presence of fibrous tissue(compared to tissues from untreated rats), 1 = mild ex-cess, 2 = moderate, 3 = severe. Scoring was assessed inthree areas of omental tissue: submesothelial, perivas-cular, and intersegmental. Also, an overall fibrotic scorewas assessed by adding the different scores in differenttissues to a total fibrotic index per animal. Quantitativedetermination of fibrosis was performed in parietal peri-toneum by measurement of the hydroxyproline content.Therefore, a portion of parietal peritoneum was frozenin liquid nitrogen directly after sacrifice and its hydroxy-proline content was determined with HPLC (27).

STATISTICAL ANALYSIS

Medians and interquartile ranges are given, or mean ±SD when appropriate. Mann–Whitney tests were used forall analyses.

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RESULTS

ANIMALS

All rats remained healthy, as assessed by weight gainand general condition, during the whole 20-week experi-mental period. After 20 weeks of infusion, the averageweight was somewhat higher in the pyruvate group(544 ± 31 g) than in the lactate group (504 ± 30 g), p =0.015. Plasma creatinine, glucose, urea, sodium, andalbumin measured at the end of the experiment were notdifferent between the lactate- and the pyruvate-exposedgroups (Table 1).

PERITONEAL TRANSPORT

The peritoneal transport results are summarized inTable 2. The MTAC creatinine was higher in the pyruvategroup compared to the lactate group (p = 0.04). Sodiumsieving was significantly less in the lactate group com-pared to the pyruvate animals (p = 0.01). No differencewas found in fluid kinetics.

VEGF

Based on the individual peritoneal transport line de-termined for each animal, dialysate VEGF concentrationswere all higher than expected if its generation was re-lated only to diffusion, suggesting that local productionwas occurring. The concentration of VEGF attributed tolocal production was significantly higher in the pyruvate-

infused group (23.4 ± 20.6 ng/L) than in the lactate-infused group (5.3 ± 1.5 ng/L, p = 0.015).

LACTATE/PYRUVATE RATIO AND BETA-HYDROXYBUTYRATE/ACETOACETATE RATIO

The plasma lactate/pyruvate ratio was within the nor-mal range and was not significantly different betweenthe two groups: 20.7 ± 7.4 (pyruvate-infused group) ver-sus 23.5 ± 3.5 (lactate-infused group), p = 0.2. There was,however, a trend toward a lower beta-hydroxybutyrate/acetoacetate ratio in the pyruvate-exposed group (3.1 ±1.8) compared to the lactate-exposed animals (6.8 ± 6.4)although this did not reach statistical significance (p =0.07).

BLOOD VESSEL ANALYSIS

Immunostaining with anti-von Willebrand factor an-tibody and αSMA revealed staining of both epitopes inall vessels, including capillaries. There were no differ-ences noted in the number of vessels stained with bothantibodies. Quantitation of the number of vessels re-vealed a significantly lower number of vessels in the pyru-vate-infused group [16 (11 – 22) per high power field]compared to the lactate group [37 (34 – 47)/hfp, p <0.001]. This is illustrated in Figure 1. On examining otherparameters of vessel morphology by computer analysis(Table 3), the pyruvate-exposed animals had a largertotal surface area and luminal area, and a lower wall/total ratio than those in the lactate-exposed group. Thiswas especially evident for vessels with a diameter

TABLE 1Plasma Concentrations of Various Solutes During the

Standardized Peritoneal Permeability Analysis Adaptedfor the Rat in the Two Groups (Medians and

Interquartile Ranges)

L (n=9) P (n=9)

Creatinine (µmol/L) 120 (85–138) 116 (95–150)Urea (mmol/L) 16 (15–17) 18 (17–21)Glucose (mmol/L) 35 (32–38) 34 (32–38)Sodium (mmol/L) 136 (134–138) 138 (133–139)Albumin (g/L) 39 (38–41) 39 (37–41)Lactate (mmol/L) 1.6 (1.4–1.8) 1.6 (1.2–2.1)Pyruvate (µmol/L) 65 (63–75) 79 (66–86)β-hydroxybutyrate (µmol/L) 40 (30–50) 30 (30–40)Acetoacetate (µmol/L) 113 (109–472) 85 (77–128)a

VEGF (ng/L) 84 (60–87) 70 (50–114)

VEGF = vascular endothelial growth factor; L = lactate-buff-ered solution; P = pyruvate-buffered solution.a p < 0.05.

TABLE 2Transport Parameters Measured During the Standardized

Peritoneal Permeability Analysis Adapted for the Rat(Medians and Interquartile Ranges)

L (n=9) P (n=9)

TCUFR (µL/min) 77 (70–80) 74 (62–100)nUFR (µL/min) 56 (44–60) 55 (45–70)Glucose absorption (%) 56 (52–58) 55 (40–64)MTAC creat (mL/min) 0.05 (0.04–0.1) 0.11 (0.06–0.30)a

MTAC urea (mL/min) 0.14 (0.11–0.14) 0.15 (0.11–0.19)D/P sodium (%) 89 (83–90) 78 (77–83)b

TCUFR = transcapillary ultrafiltration rate; nUFR = net ultra-filtration rate; MTAC = mass transfer area coefficient; creat =creatinine; D/P = dialysate-to-plasma ratio; L = lactate-buff-ered solution; P = pyruvate-buffered solution.a p < 0.05.b p < 0.01.

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>20 µm, but the differences were also present in thesmall (<8 µm) and medium-sized vessels (8 – 20 µm).

FIBROSIS SCORING

The semiquantitative scoring of fibrosis is shown inTable 4. Pyruvate-exposed animals had a lower fibrosisscore in intersegmental areas and in perivascular areas,although the significance of the latter was borderline.

No difference was found in the submesothelial areas. Theoverall fibrotic index tended to be lower in the pyruvate-exposed animals (p = 0.06). Parietal per itoneumhydroxyproline levels also showed a nonsignificant ten-dency to be lower in the pyruvate-exposed animals.

DISCUSSION

Animal infusion models using heat-sterilized lactate-buffered dialysis solutions containing 3.86% glucosehave shown clear changes in both new blood vessel for-mation and fibrotic alterations in the parietal and vis-ceral peritoneal membrane (7,28–30). This wasconfirmed in the present series where exposure to con-ventional lactate-buffered dialysis fluid resulted in sig-nificant changes in both angiogenesis and fibrosis. Incontrast, 20 weeks’ exposure to a dialysis solution ofidentical formulation but buffered with 35 mmol/Lsodium pyruvate resulted in significantly less bloodvessel formation, as assessed in visceral peritoneal

TABLE 3Vessel Measurements as Assessed with Image Analysis Pro Plus, in alpha-Smooth Muscle Actin-Stained Omental Tissue of

Rats Infused with Lactate (L; n = 9) or Pyruvate (P; n = 9) -Buffered Dialysate. Medians and interquartileranges of all vessels in five overlapping fields are given.

Diameter <8 µm Diameter 8–20 µm Diameter >20 µmL P L P L P

Wall area (µm2) 49 (39–63) 43 (31–58) 138 (91–225) 104 (78–166)a 705 (465–984) 1499 (708–5258)a

Luminal area (µm2) 16 (11–25) 20 (11–29) 44 (27–85) 51 (29–95)a 253 (113–544) 557 (240–2378)a

Wall/total ratio 0.74 0.69 0.73 0.67 0.76 0.70(0.66–0.80) (0.62–0.76)a (0.66–0.81) (0.57–0.77)a (0.66–0.83) (0.54–0.78)a

a p < 0.05 (L vs P).Image Analysis Pro Plus manufactured by Media Cybernetics, Silver Spring, Maryland, USA.

Figure 1 — Immunohistology of rat omental tissue usingsmooth muscle actin-1 antibody shows an example from thelactate-infused group (upper panel) and an example from thepyruvate-infused group (lower panel). The graph shows thenumber of vessels in all animals in the two groups, as assessedin omental tissue. (Original magnification ×10 for both).

TABLE 4Semiquantitative (Histological) and Quantitative (Biochemi-cal) Assessment of Fibrosis in Omentum of Rats Infused with

Lactate (L) or Pyruvate (P)-Buffered Dialysate (Mediansand interquartile ranges are given.)

L (n=9) P (n=9)

Fibrosis (0–3)Submesothelial area 1 (1–1.5) 1 (0.75–1.5)Intersegmental area 2 (1–2) 1 (1–1.25)a

Perivascular area 2 (1–2) 1 (0.5–1.75)b

Overall fibrotic index 4 (2–6) 4 (1–4.5)c

OH-proline content (mg/g) 1.7 (1.2–1.7) 1.3 (1.2–1.4)

a p < 0.001.b p < 0.05.c p = 0.06.

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samples, and some reduction in the peritoneal fibroticindex. It could be questioned whether once-daily ad-ministration gives sufficient exposure of peritoneal tis-sue. Taking into account that the peritoneal fluidabsorption rate averaged 20 µL/minute during theSPARa (data not shown), it can be calculated that theduration of exposure was 17 hours. Based on the al-most identical molecular weights of pyruvate and lac-tate, their absorption rates are likely to have an orderof magnitude of 70% – 80%. The pyruvate solution waswell tolerated, as illustrated by the higher BW of therats after 20 weeks.

Most studies using pyruvate as a buffer in dialysis so-lution have been done in vitro using various cell types.Exposure of cultured mesothelial cells to pyruvate-buff-ered dialysis solutions was associated with better pres-ervation of these cells, as judged by lower release of LDHcompared to lactate-buffered solutions, higher thymi-dine incorporation rates, and a higher production ofinterleukin-1 receptor antagonist (31). Pyruvate alsoprevented the negative effects of H2O2 exposure to thesecells (32). Exposure of peripheral blood neutrophils toan acidic pyruvate solution caused less decrease in intra-cellular pH than a similar lactate-buffered fluid (33).Also, a better preservation of neutrophilic superoxideproduction and nitric oxide generation (34,35) has beendescribed. A study in peritoneal macrophages showedsimilar results (36).

A single intraperitoneal administration of a nonacidicsterilized pyruvate-buffered dialysis solution in rats wasassociated with a lower MTAC glucose than a conventionalacidic heat sterilization solution, but this was not foundfor the MTAC or EDTA (37). Twice-daily intraperitonealadministration of a non glucose-containing pyruvate-buffered dialysis solution for 5 weeks in rats showed thatthe amount of angiogenesis induced was in between thatinduced by lactate (greatest) and bicarbonate (lowest),but was not significantly different from these (38). Itcan be concluded from these studies that a pyruvate-buffered glucose-containing dialysis solution has bet-ter in vitro biocompatibility than conventionallactate-buffered solutions, but has no significant pro-tective effect on angiogenesis in the absence of glucoseafter an intermediate exposure time in rats.

The control group of our study consisted of a 3.86%glucose, lactate-buffered conventional PD solution. Nocontrol group exposed to a solution without glucose wasincluded. However, in a previous study using the sametechnique we found that the number of omental vesselsin nonexposed animals averaged 7.9/field, and 4.1/fieldin animals exposed to Ringers lactate for 16 – 20 weeks(39). In that study, the number of vessels after expo-

sure to the same conventional dialysis solution was 32.6/field, which is very similar to the value found in the con-trol group of the present study.

Our finding that a smaller number of vessels was foundin the pyruvate-exposed animals is in accordance withthe contention that pyruvate induces less pseudohypoxiathan lactate at the cellular level, a situation that mightoccur during exposure to glucose-containing dialysissolutions. This is supported by the tendency to a lowerbeta-hydroxybutyrate/acetoacetate ratio in the pyru-vate group. Failure to reach statistical significance couldhave been caused by the relatively small number of ani-mals or by the fact that plasma ratios are only a weakrepresentation of processes at the mitochondrial level.In addition to the lower number of vessels in the pyru-vate group, the animals also had a larger luminal areaand, with the exception of vessels >20 µm, a smaller wallarea compared to the lactate group. This suggests thepresence of a larger endothelial surface area per vesseland thereby a larger number of interendothelial smallpores and transcellular water channels.

The vascular peritoneal surface area is the determi-nant of low molecular weight solute transport, asreflected in the MTAC of creatinine (40). Indeed, rela-tionships have been described between the number ofperitoneal vessels and peritoneal transport (41). There-fore, lower solute transport parameters would have beenexpected, which was not the case. However, vascularsurface area is not determined only by the number ofperfused microvessels, but also by their state of vasodi-lation or vasoconstriction. For example, animal experi-ments have suggested that the MTAC of low molecularweight solutes is not determined by splanchnic bloodflow, but by splanchnic blood volume (42). The largerluminal surface area of the peritoneal vessels in the pyru-vate group suggests some degree of vasodilation. It maytherefore be that the effect of a smaller number of ves-sels is counteracted by increased vasodilation. The MTACcreatinine was even higher in the pyruvate group, whilesodium sieving was more pronounced, suggesting morefree water transport. The interpretation of MTACs is dif-ficult in nonuremic animals due to the (near) equilib-rium and the low plasma and effluent concentrations,leading to inaccuracies in the calculation of the MTAC.The values were, however, in the range we reported pre-viously (21).

Vascular endothelial growth factor is an importantgrowth factor in diabetic retinopathy (9). In previousstudies in humans, we found that peritoneal effluentconcentrations of VEGF were higher than could be ex-plained by diffusion from the circulation (16), suggest-ing local production in peritoneal tissues or cells. In vitro

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studies have shown that human peritoneal mesothelialcells produce VEGF (8). Also in the present study, wefound evidence for local VEGF production. The highestvalues were found in the pyruvate-exposed animals,which would argue against a direct role of this growthfactor in driving new blood vessel formation in ourmodel. VEGF was, however, only measured at the end ofthe experiments, without full time course studies. Wecannot exclude that changes in its concentration mayhave been different between the groups at some timepoints earlier in the experiments. Another possibility isthat the mesothelium was preserved better in the pyru-vate group, but this was not studied.

The impact of replacing lactate with pyruvate on in-terstitial fibrosis was less pronounced than the impacton blood vessel formation. Significant differences in fi-brosis were present in perivascular and intersegmentalareas. The overall fibrotic index showed only a tendencyto be lower in the pyruvate-exposed group, as was alsothe case for the hydroxyproline content in the parietalperitoneum. It may be that the changes indicated by di-rect assessment were too subtle for detection in theassay, and/or contamination of the specimens with an-terior abdominal wall muscle occurred. This also containshigh levels of hydroxyproline and may therefore haveinfluenced the results obtained (27).

The limited effect of the pyruvate solution on the de-velopment of interstitial fibrosis was somewhat unex-pected given its potential to directly antagonizelactate-induced glucose-driven polyol pathway activa-tion (31). Previous in vitro studies with mesothelial cellshave shown that lactate acts to enhance glucose-drivenpolyol pathway activation, as evidenced by specific in-hibition with an aldose reductase inhibitor, and drivesintracellular sorbitol accumulation (43). In these samestudies, pyruvate was shown to have a potential antago-nistic effect on glucose/lactate-induced transforminggrowth factor-beta1 and monocyte chemoattractant pro-tein-1 synthesis. In these studies, however, glucose wasused at a much lower concentration (60 mmol/L, equiva-lent to 1.36% glucose PD fluids) to activate the polyolpathway. It may well be that, at 214 mmol/L/3.86% glu-cose, maximal activation of this pathway is facilitatedand that the potential antagonistic effect of pyruvate isthereby reduced. Further studies using lower glucoseconcentrations will be needed to address this issuespecifically.

Another potential explanation for the lack of dramaticeffects of pyruvate on the fibrotic process is that both ofthe PD fluids used were heat sterilized and thereforeprobably contained similarly high concentrations ofGDPs. Both GDPs themselves (44) and more directly GDP-

driven advanced glycation end product (AGE) formationhave been strongly implicated in the fibrotic alterationsin both animal models (37) and humans (45). Signifi-cant deposition of AGE has been observed in human peri-toneal biopsies from long-term PD patients (46). We werenot able to examine the deposition of AGE in our stud-ies. Previous data, however, have shown significantdeposition within the vessel walls, the interstitium, andin the mesothelium (30,47,48).

Intravenously administered pyruvate in high doseshas been used for years in the so-called pyruvate-load-ing test to detect mitochondrial defects in oxidativemetabolism in children, for instance in those suspectedto have Leigh syndrome (49). In this test, a high dose of910 mmol is administered in 10 minutes. This is usuallywell tolerated although tachycardia and trembling de-velops in some patients. Lately, pyruvate has been re-ported to have cardioprotective effects (50–53), whichmight be a positive systemic side effect in PD patients.

In conclusion, we have demonstrated that replace-ment of the lactate buffer by pyruvate in a glucose-basedheat-sterilized PD solution resulted in changes in peri-toneal transport, accompanied by reduced formation ofblood vessels, but with a larger luminal area and a moremarginal effect on the fibrotic process. Although thesedata do not provide definitive proof of the potential ofpyruvate as an alternative buffer in PD patients, takentogether with previously published data, they do sug-gest that further investigations of its use and mecha-nism of action are warranted. It would appear to havepotentially significant advantages as a buffer for glu-cose-based PD solutions that might reduce some of thenegative consequences associated with both glucose andlactate exposure.

ACKNOWLEDGMENTS

We thank K. Hiralall and A.B. van der Wardt for their excellenttechnical assistance. We also thank Dr. M. Duran for his helpwith the lactate/pyruvate and beta-hydroxybutyrate/aceto-acetate determinations and Dr. O. de Boer for assistance withthe vessel measurements. We are grateful to Professor Dr. B.Lindholm and Professor Dr. N. Topley for reading and carefuldiscussion of the manuscript.

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