Advances in Peritoneal Dialysis, Vol. 18, 2002

Protein OxidativeStress in DialysisPatients

From: Nephrology Service, Fundaci Puigvert, Barcelona;1Endocrinology and Nutrition Service, Hospital Sant Pau,Autonomous University of Barcelona, Barcelona; and2Ci ncies M diques Service, University of Lleida, Lleida,Spain.

Teresa Do ate, Alba Herreros, Ester Martinez, JoaquinMartinez, Enric Andr s, Antonio Cabezas, Angeles Ortiz, 1

Ana de Prado,1 Jos M. Pou, 1 Reinald Pamplona,2 ManuelPortero Otin,2 Maria J. Bellmunt2

Inflammatory status is observed in patients withchronic renal failure (CRF). The relationship be-tween oxygen free radical production and dialysiscould play an important role in protein oxidation.Carbonyl protein plasma level is an important toolin the study of protein stress, and it is related tothe arterial intima thickness in the atherosclerosisprocess.

We studied protein oxidative stress in 21 peri-toneal dialysis (PD) patients and 42 hemodialysis(HD) patients as compared with 32 undialyzedpatients with CRF. Carbonyl protein plasma levelswere measured in nanomoles per milligram pro-tein by the ELISA method (Winterbourn et˚al).

Dialysis patients had a higher protein carbon-yl content than did CRF patients (0.1265˚±0.04˚nmol/mg vs. 0.1594 ±̊ 0.03˚nmol/mg, p˚<0.0002). Patients on PD had a lower level thanpatients on HD (0.1452˚± 0.03˚nmol/mg vs.0.1665˚± 0.04, p˚< 0.004).

Glucose administration in PD is known to beable to increase glucose degradation products(GDPs) and advanced glycosylation end-products(AGEs) with high carboxylic and oxidative stress.In our study, the carbonyl protein level was higherin HD patients than in PD patients, perhaps be-cause more protein oxidative stress is associatedwith hemodialysis technique or because the PDpatients had greater residual renal function.

Key wordsChronic renal failure, hemodialysis, oxidative stress,carbonyl protein

IntroductionInflammatory status is observed in dialyzed andundialyzed chronic renal failure (CRF) patients.The relationship between oxygen free radical pro-duction and dialysis could play an important rolein protein oxidation. Oxidative reactions most fre-quently involve free-radical intermediates that havedirect or indirect participation in the inflammatoryresponse (1). That response is amplified by thehemobioincompatibility of dialysis systems and so-lutions, which worsen the pro-oxidant status ofuremic patients or which, by activating signallingcascades, mediate proliferation, differentiation, andcell death (2).

The accumulation of oxidized proteins dependsupon the balance between pro-oxidant, antioxidant,and proteolytic activity. That oxidatively modifiedforms of proteins have been demonstrated to accu-mulate during oxidative stress and in some patho-logic conditions has focused attention onphysiologic and non physiologic mechanisms forthe generation of reactive oxygen species (3,4)(Figure˚1).

Hemodialysis is associated with increased oxi-dant stress. That observation appears to be due toan increased production of free radicals duringhemodialysis, a net reduction of many antioxidants,and factors relative to the uremic state. Severalstudies show the amplified inflammatory responseduring hemodialysis being produced by variousfactors and mechanisms (Figure˚2). The phenom-enon relates to the interaction of uremic toxins,endotoxins, dialysate, and membrane (5,6).

Glucose in peritoneal dialysis solutions in-creases the auto-oxidation and protein glycation,increasing oxidative factors and reducing the anti-oxidant response by the polyol pathway. Oxidativestress is associated with an accelerated atheroscle-rosis process and is accompanied by proteinchanges (7—9) (Figure˚3).

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The carbonyl content of plasma proteins is an im-portant tool in the study of protein oxidative stress,and it is related to the arterial intima thickness in theatherosclerosis process. Many ways exist to produce

Do ate et al

protein carbonyls; they result from the interactionbetween free radicals and proteins or carbohydrates,and from auto-oxidation of lipids (10).

The aim of our study was to evaluate protein oxi-dative stress in peritoneal dialysis (PD) patients, he-modialysis (HD) patients, and undialyzed CRF patients.

FIGURE 1 Protein oxidative stress (adapted from reference 10). UV˚=ultraviolet; NOS˚= nitric oxide synthase; MCO˚= metal-catalyzedoxidation systems; ROS˚= reactive oxygen species.

FIGURE 3 Oxidative stress in peritoneal dialysis. LDL˚= low den-sity lipoprotein.FIGURE 2 Oxidative stress in hemodialysis. IL-1β˚= interleukin-

1β; TNFα˚= tumor necrosis factor α; IL-6˚= interleukin-6; ROS˚=reactive oxygen species; LDL˚= low density lipoprotein; AOPP˚=advanced oxidative protein products.

OXIDATEDPROTEIN

17Protein Oxidative Stress in Dialysis Patients

Patients and methods

PatientsThe study involved

¥ 21 PD patients [10˚men, 11˚women; mean age:59.9˚years (range: 16˚— 84˚years); mean time on PD:38 ±̊ 8˚months], using the Fresenius Medical CareStayÆSafe system with glucose as the osmotic agent.

¥ 42 HD patients [22˚men, 20˚women; mean age:60.5˚years (range: 38˚— 86˚years); time on HD:51 ±̊ 12˚months], using Fresenius Medical Carepolysulfone low-flux (F8) membranes.

¥ 32 undialyzed patients with CRF (22˚men,10˚women; mean age: 57.6˚years (range: 20˚—86˚years).

No patients were diabetic.

Analysis of protein carbonyl contentIn all of the patients, the carbonyl content of plasmaproteins was measured in nanomoles per milligramby the ELISA method [Winterbourn et al (5)].

ResultsTable˚I shows the results of the ELISA measurements.

DiscussionGlucose administration in PD is know to be able toincrease glucose degradation products (GDPs) and ad-vanced glycation end-products (AGEs) with high car-bonyl and oxidative stress. In our study, the carbonylprotein level was higher in HD patients than in PDpatients, perhaps because more protein oxidative stressis associated with hemodialysis technique or becausethe PD patients had greater residual renal function.

Conclusions1. Dialysis patients had higher carbonyl protein

plasma levels than did undialyzed CRF patients.2. Patients on PD had lower carbonyl protein plasma

levels than did patients on HD.

References1 Morena M, Cristol JP, Canaud B. Why hemodialysis

patients are in a prooxidant state? What could bedone to correct the pro/antioxidant imbalance. BloodPurif 2000; 18:191—9.

2 Wratten ML, Tetta C, Ursini F, et al. Oxidant stress inhemodialysis: prevention and treatment strategies.Kidney Int 2000; 76(Suppl):S126—32.

3 Miyata T, Kurokawa K, Van Ypersele de Strihou C.Relevance of oxidative and carbonyl stress to long-term uremic complications. Kidney Int 2000;76(Suppl):389—99.

4 Descamps—Latscha B, Witko—Sarsat V. Importance ofoxidatively modified proteins in chronic renal failure.Kidney Int 2001; 78(Suppl):S108—13.

5 Winterbourn CC, Buss IH, Chan TP, et al. Proteincarbonyl measurements show evidence of earlyoxidative stress in critically ill patients. Crit CareMed; 28:143—9.

6 Miyata T, Inagi R, Asahi K, et al. Generation of pro-tein carbonyls by glycoxidation and lipoxidation re-actions with autoxidation products of ascorbic acidand polyunsaturated fatty acids. FEBS Lett 1998;437:24—8.

7 Weiss MF, Saxena AK, Monnier VM. Pharmacologi-cal modulation of AGEs: a unique role for redox-active metal ions. Perit Dial Int 1999; 19(Suppl 2):S62—7.

8 Dawnay A, Millar DJ. The pathogenesis and conse-quences of AGE formation in uraemia and its treat-ment. Cell Mol Biol 1998; 44:1081—94.

9 Honda K, Nitta K, Horita S, et al. Accumulation ofadvanced glycation end products in the peritonealvasculature of continuous ambulatory peritonealdialysis patients with low ultra-filtration. NephrolDial Transplant 1999; 14:1541—9.

10 Berlett BS, Stadtman ER. Protein oxidation in aging,disease, and oxidative stress. J˚Biol Chem 1997; 272:20313—16.

Corresponding author:Teresa Do ate, MD, Fundaci Puigvert, C/˚Cartagena 340,Barcelona 08025 Spain.

TABLE I Results of ELISA measurements for carbonyl proteinplasma level in the study patients

Patient group Results of ELISA (nmol/mg) p˚Valuea

Undialyzed CRF 0.1265±0.04All dialysis 0.1594±0.03 <0.0002Peritoneal dialysis 0.1452±0.03Hemodialysis 0.1665±0.04 <0.004

a By Student t-test.CRF˚= chronic renal failure.

Advances in Peritoneal Dialysis, Vol. 18, 2002

Endothelial Nitric OxideSynthase Gene Polymorphismin Dialysis Patients

From: Nephrology Service, Fundaci Puigvert, Barcelona,and 1Endocrinology and Nutrition Service, Hospital de SantPau, Autonomous University of Barcelona, Barcelona,Spain.

Ana de Prado,1 Teresa Do ate, Ester Martinez, AlbaHerreros, Antonio Cabezas, Enric Andr s, Angeles Ortiz, 1

Jos M. Cubero, 1 Jos M. Pou 1

Nitric oxide is an important factor in the regulationof vasodilator tone. In vascular cells, NO is synthe-sized by endothelial nitric oxide synthase, a key en-zyme of the endogenous vasodilator system. Somestudies have described the interaction between NOand the other factors that promote vasodilatation invascular smooth muscular cells. Some of those fac-tors are angiotensin-converting enzyme (ACE), trans-forming growth factor˚β (TGFβ), and endothelialoxide nitric synthase (eNOS).

Polymorphism that can alter the expression or thefunction of the eNOS protein has been identified inthe eNOS gene in the promoter and codification zones.

We studied the Glu298Asp variant of the eNOSgene in 52 hemodialysis (HD) patients, 22 peritonealdialysis (PD) patients, and 93 healthy controls. Iden-tification of the Glu298Asp variant in exon˚7 was per-formed by enzymatic amplification and restrictionfragment length polymorphism (RFLP) analysis.

The frequencies of eNOS genotypes in the controlgroup were GG, 39.8%; GT, 43%; and TT, 17.2%. InHD patients, the frequencies were GG, 40.3%; GT,38.7%; and TT, 21.7%. In PD patients, they were GG,41.6%; GT, 50%; and TT, 8.6%.

No significant differences were seen between thecontrol group and the dialysis patients, or betweenthe HD and the PD patients.

Key wordsNitric oxide, endothelial nitric oxide synthase gene,hemodialysis

IntroductionNitric oxide is an important factor for the regulationof vasodilator tone. In response to stimuli such ashypoxia and shear stress, vascular endothelial cells

synthesize NO by endothelial nitric oxide synthase(eNOS) (1). The NO produced enzymatically fromL-arginine by eNOS in the endothelium diffuses to thevascular smooth cells (VSMCs) and increases cGMPlevels, which mediate muscular relaxation.

Some studies have described the interaction be-tween NO and other factors such as angiotensin-con-verting enzyme (ACE), transforming growth factor β̊(TGFβ), and eNOS, which all promote vasodilatationin VSMCs (Figure˚1).

Polymorphism that can alter the expression or thefunction of the eNOS protein has been identified inthe eNOS gene in the promoter and codification zones.One of the recently identified variants of the eNOSgene is a G-to-T conversion at nucleotide position˚894within exon˚7 of the eNOS cDNA, resulting in a re-placement of glutamic acid by aspartic acid atcodon˚298 (Glu298Asp). That mutation could be re-lated to hypertension (2) and worse prognosis innephropathy (3) prevalence and evolution.

The mutation of the eNOS gene has been studiedto determine if it could be related to cardiovascularproblems (4). The aim of the present study was to in-vestigate whether the Glu298Asp variant in the eNOSgene varied among patients on dialysis.

Patients and methodsThe Glu298Asp genotype of eNOS gene was deter-mined in 62˚patients on hemodialysis (HD) and 29on continuous ambulatory peritoneal dialysis (PD).The dialysis groups included 40˚men and 34˚women(mean age: 55.9˚± 14.4; time on dialysis: 44.5˚±10˚months). The two dialysis groups were also com-pared to 109 healthy control subjects (mean age: 56 ±̊7˚years).

Identification of the Glu298Asp variant in exon˚7was performed by enzymatic amplification andrestriction fragment length polymorphism (RFLP)analysis (5). The genotyping of the polymorphism wasperformed by polymerase chain reaction (PCR)amplification with primers 5’-CATGAGGCTCAG-

19

CCCCAGAAC-3’ (sense) and 5’-AGTCAATCCCTT-TGGTGCTCAC-3’ (antisense), followed by MboIrestriction endonuclease. The 206˚bp PCR product wascleaved into 119˚bp and 87˚bp fragments in thepresence of a T at nucleotide˚894. The presence of a Tcorresponds to the amino acid aspartic acid (Asp).

ResultsIn the control group, the observed frequencies ofeNOS genotype were GG, 39.8%; GT, 43%; and TT,17.2%. In patients on dialysis, the frequencies were(HD subgroup) GG, 40.3%; GT, 38.7%; and TT,21.7%; and (PD subgroup) GG, 41.6%; GT, 50%;and TT, 8.6%.

We found no difference in the distribution of thestudied polymorphism between the 93 controls sub-jects and the 74 dialysis patients (Table˚I). No signifi-cant difference was seen between the two groups ofdialysis patients.

DiscussionVarious studies have reported that the Glu298Aspvariant can be associated with hypertension, acutemyocardial infarction, vascular problems, and progres-

de Prado et al

sion of diabetic nephropathy (4).The etiology for mostof the renal diseases is not clear, although vascularchanges can be a possible origin. For this reason, eNOSmay play a role in renal diseases.

The Glu298Asp variant is not correlated with dif-ferent levels of NO. Differences are seen between Japa-nese and Caucasian populations. Although eNOS maybe implicated in renal alterations, it may not play amain role; or perhaps the defective eNOS DNA vari-ants manifest in kidney disease only as local patho-physiologic disturbances. Further studies in groups ofpatients with varying degrees of renal and cardiovas-cular disease must be performed before conclusionscan be drawn.

C 1 2 3 4 5 6 7 8 9 10 M

206 bp ♦♦♦♦♦

FIGURE 1 (A)˚Amplification by polymerase chain reaction of exon˚7 endothelial nitric oxide synthase (eNOS) gene with specific prim-ers. (B)˚Digestion of the amplified product (206˚bp) with MboI enzyme, 16˚hours at 37°C.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 M

♦♦♦♦♦♦♦♦♦♦♦♦♦♦♦

206 bp

119 bp87 bp

TABLE I Distribution of the Glu298Asp variant of the eNOS genein hemodialysis (HD) and peritoneal dialysis (PD) patients, and inhealthy controls

Controls HD PD HD+PD

GG 42 (38.5%) 25 (40.3%) 12 (41.4%) 37 (40.6%)GT 46 (42.2%) 24 (38.7%) 9 (31.0%) 33 (36.3%)TT 21 (19.3%) 13 (21.0%) 8 (27.6%) 21 (23.1%)

20 eNOS Gene Polymorphism

References1 Neugebauer S, Baba T, Watanabe T. Association of

the nitric oxide synthase gene polymorphism with anincreased risk for progression to diabetic nephropa-thy in type˚2 diabetes. Diabetes 2000; 49:500—3.

2 Wang XL, Wang J. Endothelial nitric oxide synthasegene sequence variations and vascular disease. MolGenet Metab 2000; 70:241—51.

3 Miyamoto Y, Saito Y, Kajiyama N, et al. Endothelialnitric oxide synthase gene is positively associatedwith essential hypertension. Hypertension 1998;32:3—8.

4 Hibi K, Ishigami T, Tamura K, et al. Endothelialnitric oxide synthase gene polymorphism and acutemyocardial infarction. Hypertension 1998; 32:521—6.

5 Hingorani A, Liang CF, Fatibene J, et al. A commonvariant of the endothelial nitric oxide synthase(Glu298 >Asp) is a major risk factor for coronaryartery disease in the U.K. Circulation 1999;100:1515—20.

Corresponding author:Teresa Do ate, MD, Fundaci Puigvert, C/˚Cartagena340, Barcelona 08025 Spain.

Advances in Peritoneal Dialysis, Vol. 18, 2002

Glucose Suppresses PeritonealInflammatory Reactions andMesothelial Hyperplasia Causedby Intraperitoneal Saline Infusion

From: Department of Pathophysiology, University Medi-cal School of Poznan, Poland, and 1Division of Nephrol-ogy, University of Toronto, Canada.

Arkadiusz Styszynski, Renata Podkowka, KatarzynaWieczorowska—Tobis, Beata Kwiatkowska, KrzysztofKsiazek, Andrzej Breborowicz, Dimitrios G. Oreopoulos1

In the past, we had observed that infusion of normalsaline into the peritoneal cavity stimulates an inflam-matory response. In the present study, we examinedwhat effect the addition of glucose to normal salinewould have on the peritoneal inflammatory responseand change in peritoneal morphology.

After catheter implantation, rats were infused in-traperitoneally (IP) for 3˚days with Dianeal 1.36%(Baxter Healthcare Corporation, Deerfield, IL,U.S.A.). Dialysate samples were collected on day˚3after a 4-hour dwell. Next, rats were exposed to ei-ther NaCl (n˚=˚7) or NaCl with glucose 250˚mmol/L(Glu, n˚= 7) twice daily for 4˚weeks. After 2˚weeksand 4˚weeks of the study, dialysate samples were col-lected after a 4-hour dwell to analyze the activity ofinflammatory reaction. At the end of the experiment,imprints of peritoneal mesothelium were taken. Con-trol animals (C, n̊˚=˚6) did not undergo catheter im-plantation or the dialysis procedure.

The inflammatory reaction cell count, cell dif-ferentiation, nitric oxide production, protein loss, andmonocyte chemoattractant protein-1 (MCP-1) concen-tration in dialysate expressed as a percentage of theinitial value did not change during the study in ratsexposed to NaCl. On the other hand, in Glu-treatedanimals, the protein concentration was decreased after4˚weeks of the study (74% ±̊ 23%, p˚< 0.05), as wasMCP-1 (24%˚± 12%, p˚< 0.05). The nitrites concen-tration was decreased after 2˚weeks (72% ±̊ 19%; p˚<0.05). Intraperitoneal adhesions were found in 6˚ratsof the NaCl group (86%) and in only 4˚rats (57%) ofGlu group. In the NaCl rats, a higher density of me-sothelial cells was observed (2792 ±̊ 510˚cells/mm2)as compared with Glu rats (2028 ±̊ 561˚cells/mm2; p˚<0.05) and with control rats (1629 ±̊ 422˚cells/mm2, p˚<0.05). The NaCl group also showed a higher

nucleus:cytoplasm˚surface ratio (0.25 ±̊ 0.03) as com-pared with the Glu group (0.18 ±̊ 0.02, p˚< 0.01) andwith the control group (0.14 ±̊ 0.01, p˚< 0.01).

Addition of glucose to normal saline suppressesthe peritoneal inflammatory response and mesothe-lial hyperplasia occurring with intraperitoneal infu-sion of NaCl solution alone.

Key wordsGlucose, intraperitoneal inflammation, mesothelialhyperplasia

IntroductionLong-term exposure of the peritoneum to dialysis fluidin dialyzed patients results in progressive changes inperitoneal membrane permeability to solutes and wa-ter (1). Simultaneously, alterations in morphologyoccur, including both mesothelial cell changes anddisorganization of the peritoneal interstitium (2). Inthe absence of bacterial peritonitis, such histologic andphysiologic alterations are believed to result frombioincompatibility of peritoneal dialysis fluids.

Among the potential bioincompatible componentsof the dialysis solutions, glucose is one of the mostimportant. Glucose is used as an osmotic agent in mostcommercially available peritoneal dialysis solutions.Toxic effects of glucose have at least two possiblemechanisms: direct metabolic alterations owing to thehigh glucose concentration itself and alterations sec-ondary to the hyperosmolality of peritoneal dialysisfluids (3,4).

On the other hand, peritoneal changes also appearto be related to chronic intraperitoneal inflammationinduced by the process of peritoneal dialysis (5). In-traperitoneal infusion of any fluid was shown to re-sult in mechanical irritation of the peritoneum,followed by activation of intraperitoneal inflamma-tory cells (6). In our previous studies, we showed thatphosphate-buffered saline (PBS) which is an isotonicsolution with a physiologic pH (and therefore may be

22

considered a biocompatible dialysis fluid) caused asevere inflammatory response in rats after intraperi-toneal administration (7). An intriguing conclusionof our study was that glucose suppressed the perito-neal inflammation caused by PBS, as measured bycell count and neutrophil:macrophage ratio in dialy-sate, and by nitrites in dialysate (an index of nitrousoxide synthesis). However, the effects on the morphol-ogy of mesothelial cells of the inflammatory reactioncaused by PBS alone and PBS supplemented with glu-cose have not been studied.

In the present study, we tried to estimate the effectof the addition of glucose to normal saline on the peri-toneal inflammatory response and mesothelial cellmorphology in chronically dialyzed rats.

Materials and methodsThe study was performed on 20 male Wistar ratsweighting between 250˚g and 350˚g. At the beginningof the study, peritoneal catheters were implanted into14˚rats according to a previously described method(8). (The 6 rats that did not undergo catheter implan-tation and dialysis made up the control group.)

For the first 2˚days, all rats with implanted cath-eters were injected intraperitoneally with Dianeal1.36% (Baxter Healthcare Corporation, Deerfield, IL,U.S.A.) supplemented with antibiotics [gentamicin5˚mg/L (Polfa, Tarchomin, Poland) and cefazolin50˚mg/L (Eli Lilly, Florence, Italy)]. The fluid wasallowed to absorb. On the third day, after a 4-hourdwell, dialysate samples (5˚mL from each rat) werecollected to characterize the activity of the inflamma-tory reaction at baseline. The rats were then randomlydivided into two groups and were infused twice dailyfor a further 4˚weeks with 20˚mL of various solutionsalso supplemented with antibiotics:

¥ Group NaCl (n˚= 7): 0.9% NaCl (Polfa, Kutno,Poland)

¥ Group Glu (n˚= 7): 0.9% NaCl supplemented withglucose, 250˚mmol/L (Sigma, St.˚Louis, MO,U.S.A.)

To analyze the activity of the inflammatory reac-tion for the given dialysate after 2˚weeks and 4˚weeksof the study, samples of dialysate (5˚mL from eachrat) were collected 4˚hours after injection of the givensolution in the respective group.

At the end of the study, the rats were humanelykilled by bleeding, and the peritoneal cavity was

Styszynski et al

opened to estimate the presence of adhesions in theperitoneal cavity. Also, imprints of mesothelial cellsfrom the visceral peritoneum of the liver were takento characterize their morphology.

Analysis of the inflammatory reactionImmediately after dialysate drainage, a cell count (ina hemocytometer) and cell differentiation measure-ments were performed manually. The neutrophil:mac-rophage ratio (Ne:Ma, expressed as percentage) wasthen calculated for every rat.

In dialysate samples, total protein was measuredusing the colorimetric method described by Lowry(9). Additionally, the concentration of dialysate ni-trites (an index of nitrous oxide synthesis) was deter-mined after reduction of nitrates to nitrites with nitratereductase (Roche, Mannheim, Germany). The nitriteconcentration in each dialysate sample was measuredusing Griess reagent (10), and the monocytechemoattractant protein-1 (MCP-1) concentration wasmeasured using an ELISA kit (Biosource, Camarillo,CA, U.S.A.).

Mesothelial cell imprintsImprints of the mesothelial monolayer from the vis-ceral peritoneum were taken after the peritoneal cav-ity was opened. Glass slides coated with 1% agar(Sigma, St.˚Louis, MO, U.S.A.) were stuck to the liversurface for 30˚seconds, allowing mesothelial cells tobe peeled off that surface. Next, fixation was doneusing 96% ethanol. Slides were stained with hema-toxylin (Quimica Clinica Aplicada, Amposta, Spain).The density of mesothelial cells and thenucleus:cytoplasm˚surface ratio was determined bylight microscopy (observed at 200∞ magnification)supported with digital image analyzing equipment(Screen Measurement 4.0, Prague, Czech Republic).

Statistical analysisThe results obtained in the dialysate from each groupafter 2 and 4˚weeks of exposure to dialysis fluids werecompared with the initial values and between thegroups in both periods.

Results are expressed as mean ±˚standard devia-tion. The statistical analysis was performed using re-peated-measures ANOVA and post-hoc analysis withthe nonparametric Dunn test or the Mann—Whitneytest, as appropriate. A p˚value of less than 0.05 wasconsidered significant.

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Results

Inflammatory reactionIn the NaCl group, the activity of the intraperitonealinflammatory parameters did not change during the4-week experiment. It was the same at the end of4˚weeks as it was immediately after catheter insertion(Figure˚1). In contrast, the intensity of the intraperi-toneal inflammation decreased significantly in Glugroup during the study. The total protein concentra-tion was significantly reduced after 4˚weeks expo-sure as compared with the initial results [p˚< 0.05,Figure˚1(C)]. However, the nitrite concentration was

already diminished after 2˚weeks (p˚< 0.05), and thedecrease was even more strongly expressed after4˚weeks [p˚< 0.01; Figure˚1(D)].

Additionally, after 4˚weeks exposure, the dialy-sate cell count was significantly lower in the Glugroup than in NaCl group (1443˚± 516˚cells/mm3 vs.2771 ±̊ 1600˚cells/mm3, p˚< 0.05), as was the dialy-sate total protein concentration (328 ±̊ 101˚mg/dL vs.418˚± 81˚mg/dL, p˚< 0.05) and MCP-1 level (134 ±̊57˚pg/mL vs. 363 ±̊ 292˚pg/mL, p˚< 0.05). TheNe:Ma ratio was also lower (49% ±̊ 34% vs. 66% ±̊29%); however, the difference was not statisticallysignificant.

Glucose and Peritoneal Inflammation

FIGURE 1 Peritoneal inflammation markers after 2 and 4˚weeks of exposure to 0.9% NaCl alone (white bars) and to 0.9% NaClsupplemented with glucose 250˚mmol/L (striped bars). * p̊˚< 0.05, ** p̊˚< 0.01 (both with respect to initial value). Ne:Ma˚= neutrophil tomacrophage ratio.

*

**

**

24

Alterations in morphologyIntraperitoneal adhesions were found in 6˚rats of theNaCl group (86%) and in only 4˚rats (57%) of theGlu group.

Rats exposed to NaCl revealed higher mesothe-lial cell density than did rats exposed to Glu (2792 ±̊510˚cells/mm2 vs. 2028 ±̊ 561˚cells/mm2, p˚< 0.05) andthe non dialyzed control rats (1629˚± 422˚cells/mm2,p˚< 0.05). The nucleus:cytoplasm˚surface ratio wasalso higher in NaCl rats than in Glu rats (0.25 ±̊ 0.03vs. 0.18˚± 0.02, p˚< 0.01) and in non dialyzed controlrats (0.14 ±̊ 0.01, p˚< 0.01, Figure˚2).

DiscussionIn the present study, we showed that physiologic sa-line solution causes severe inflammatory reactionwhen administered intraperitoneally. In rats that wereinjected with physiologic saline, the dialysate cellcount, peritoneal permeability to proteins, and nitricoxide concentration remained high for 4˚weeks of theexperiment. On the other hand, supplementation ofthe same solution with glucose resulted in lower con-centrations of the mentioned factors (Figure˚1).

Because we did not drain the entire quantity ofintraperitoneal fluid after a 4-hour exposure, we donot know the cumulative values of inflammatory mark-ers. Their concentrations depend on the volume ofdialysate, which may be larger in the Glu group owingto the hypertonicity of the injected solution. There-fore, the effect of dilution of the inflammatory mark-ers must be considered in that group.

Still, the alterations in the morphology of the peri-toneal mesothelium and the prevalence of intraperito-neal adhesions confirm our hypothesis that theconcentration of inflammatory markers, rather thantheir cumulative value, is a predictive factor for peri-toneal injury (Figure˚2). Our observations lead us toconclude that glucose suppresses the intraperitonealinflammation and mesothelial hyperplasia that occurduring chronic intraperitoneal administration of 0.9%saline.

Previously, we observed that chronic exposure ofthe peritoneum to PBS resulted in a severe inflamma-tory reaction and that that reaction was milder whenPBS was supplemented with glucose or mannitol (7).Earlier Wang et al also reported that physiologic sa-line cannot be considered a biocompatible dialysissolution owing to its influence on lymphatic flow andprobable damaging effect on peritoneal tissues (11).

In contrast to our findings of mesothelial cell ab-normalities, Hekking et al observed, in a study in rats,a higher density of mesothelial cells after chronic ex-

FIGURE 2 Imprints of mesothelial monolayer obtained from a ratdialyzed with 0.9% saline alone (upper photograph), a rat dialyzedwith saline supplemented with glucose (middle photograph) and froma non dialyzed control rat (lower photograph). Slides were stainedwith hematoxylin and were observed at 200∞ magnification.

Styszynski et al

25

posure to dialysis fluid containing high glucose con-centrations as compared with saline fluid (12). Yet, inthat study, the volume of dialysis fluid was small (only10˚mL, once daily) and the duration of the experimentwas shorter than that in our experiment.

Is the observed suppressive effect of glucose ben-eficial or is it not?

Inhibition of mesothelial hyperplasia may be areflection of a lower degree of peritoneal injury, but itmay also indicate ineffective mechanisms of regen-eration. Shostak et al noticed a higher prevalence ofapoptotic mesothelial cells and a depletion of growthcapabilities after chronic in vivo exposure to glucose(13). Lower concentrations of nitric oxide and MCP-1are probably related to low activity of peritoneal mac-rophages and a lower degree of peritoneal damage.But the peritoneal cavity may be more susceptible toperitonitis when phagocyte function is depleted (14).

Further studies have to be performed (based onthe same experimental system) to evaluate the func-tion of peritoneal phagocytes and the microscopicchanges of the peritoneum in connection with inflam-matory reaction. The influence of other osmotic sol-utes, such as mannitol, on peritoneal inflammation andmesothelial hyperplasia caused by saline also need tobe examined to distinguish the hypertonic effect ofglucose from its metabolic action.

References1 Davies SJ, Bryan J, Phillips L, Russell GI. Longitudi-

nal changes in peritoneal kinetics: the effects ofperitoneal dialysis and peritonitis. Nephrol DialTransplant 1996; 11:498—506.

2 Di Paolo N, Sacchi G, De Mia M, et al. Morphologyof the peritoneal membrane during continuous ambu-latory peritoneal dialysis. Nephron 1986; 44:204—11.

3 Breborowicz A, Rodela H, Oreopoulos DG. Toxicityof osmotic solutes on human mesothelial cells invitro. Kidney Int 1992; 41:1280—5.

4 Cendoroglo M, Sundaram S, Groves C, Ucci AA,

Jaber BL, Pereira BJ. Necrosis and apoptosis ofpolymorphonuclear cells exposed to peritoneal dialy-sis fluids in vitro. Kidney Int 1997; 52:1626—34.

5 Dobbie JW. Pathogenesis of peritoneal fibrosingsyndromes (sclerosing peritonitis) in peritonealdialysis. Perit Dial Int 1992; 12:14—27.

6 Bos HJ, Meyer F, de Veld JC, Beelen RH. Peritonealdialysis fluid induces change of mononuclear phago-cyte proportions. Kidney Int 1989; 36:20—6.

7 Wieczorowska—Tobis K, Styszynski A, PolubinskaA, Radkowski M, Breborowicz A, Oreopoulos DG.Hypertonicity of dialysis fluid suppresses intraperito-neal inflammation. Adv Perit Dial 2000; 16:262—6.

8 Moore HL. Chronic catheter model for exchanges inthe ambulatory rat [Abstract]. Perit Dial Int 1992;12(Suppl 2):S141.

9 Lowry OH, Rosenbrough NJ, Rarr AL, Randall RJ.Protein measurement with the folin phenol reagent.J˚Biol Chem 1951; 193:265—75.

10 Gilliam MB, Sherman MP, Griscavage JM, IgnarroLJ. A spectrophotometric assay for nitrate usingNADPH oxidation by Aspergillus nitrate reductase.Anal Biochem 1993; 212:359—65.

11 Wang T, Heimb r ger O, Qureshi AR, Waniewski J,Bergstr m J, Lindholm B. Physiological saline is nota biocompatible peritoneal dialysis solution. Int JArtif Organs 1999; 22:88—93.

12 Hekking LHP, Aalders MC, Van Gelderop E, et al.Effect of peritoneal dialysis fluid measured in vivo ina rat model of continuous peritoneal dialysis. AdvPerit Dial 1998; 14:14—18.

13 Shostak A, Wajsbrot V, Gotloib L. High glucoseaccelerates the life cycle of the in vivo exposed me-sothelium. Kidney Int 2000; 58:2044—52.

14 Liberek T, Topley N, J rres A, Coles GA, Gahl GM,Williams JD. Peritoneal dialysis fluid inhibition ofphagocyte function: effects of osmolality and glucoseconcentration. J Am Soc Nephrol 1993; 3:1508—15.

Corresponding author:Arkadiusz Styszynski, Department of Pathophysiology,University Medical School of Poznan, ul.˚Swiecickiego 6,Poznan 60-781 Poland.

Glucose and Peritoneal Inflammation

Advances in Peritoneal Dialysis, Vol. 18, 2002

Small-Solute and Middle-Molecule ClearancesDuring˚Continuous FlowPeritoneal Dialysis

From: 1Research Service, Salt Lake City VA Health CareSystem and Departments of Internal Medicine and Bioengi-neering, University of Utah, Salt Lake City, Utah, and 2Sec-tion of Nephrology, Wake Forest University School ofMedicine, Winston—Salem, North Carolina, U.S.A.

John K. Leypoldt,1 John M. Burkart2

Previous theoretic and clinical studies have shown thatcontinuous flow peritoneal dialysis (CFPD) providesa high dose of small-solute removal; however, the doseof middle-molecule removal with CFPD therapy hasnot been evaluated. We used a variable-volume, two-compartment model to calculate theoretical steady-state solute kinetic profiles during CFPD, continuousambulatory peritoneal dialysis (CAPD), and hemodi-alysis using a high-flux dialyzer (HFHD), for an an-uric 70-kg patient and two measures of dose: equivalentrenal clearance (EKR) and standard Kt/V (stdKt/V).

Dose measures during each therapy were calcu-lated for five solutes: urea, creatinine, vitamin˚B12,inulin, and β2-microglobulin. Fluid (1˚L daily) wasassumed to accumulate in and to be removed fromthe extracellular space, and non renal clearance wasassumed to be zero for all solutes exceptβ2-microglobulin.

Calculated doses for CFPD were higher than forCAPD or HFHD when assessed by either EKR orstdKt/V. Dose enhancements for CFPD were highestfor small solutes, but were still considerable for middlemolecules. We conclude that CFPD achieves higherdoses than CAPD or HFHD for both small-solute andmiddle-molecule removal.

Key wordsβ2-Microglobulin, continuous flow peritoneal dialy-sis, dose, middle molecules, urea

IntroductionSmall-solute clearances during current forms of peri-toneal dialysis (continuous ambulatory peritoneal di-

alysis and automated peritoneal dialysis) are limitedpredominantly by low dialysate flow rates. Early ex-perience with peritoneal dialysis using a high and con-tinuous flow of dialysis solution [most frequentlycalled continuous flow peritoneal dialysis (CFPD)]showed impressive increases in clearances of smallsolutes, such as urea and creatinine (1—3). Those earlyclinical experiences have been confirmed in recentyears (4—7), identifying a potential strategy for increas-ing the dose of small-solute removal during perito-neal dialysis. The impact of a high and continuousflow of dialysate on peritoneal clearances of middlemolecules during CFPD has not previously beenstudied.

The present study describes theoretical predictionsfor peritoneal clearances of small solutes and middlemolecules during CFPD. The predictions were madeusing a variable-volume, two-compartment model ofsolute kinetics, similar to that described previously(8). Using two measures of dose [the equivalent renalclearance (EKR) described by Casino and Lopez (9)and the standard Kt/V (stdKt/V) described by Gotch(10,11)], solute clearances for CFPD were comparedwith those for continuous ambulatory peritoneal di-alysis (CAPD) and for hemodialysis using a high-fluxhemodialyzer (HFHD). Solute clearances and dosemeasures were computed for urea as well as for othermarker molecules of various molecular weights.

MethodsSolute kinetics were simulated theoretically using avariable-volume, two-compartment model (8) for threedifferent therapies: CFPD, CAPD, and HFHD. Steady-state solute concentration profiles were simulated forfive surrogate uremic solutes spanning a broad rangeof molecular weights (Table˚I). No binding to plasmaproteins was assumed to occur for any solute.

The total volume of distribution for each solutewas partitioned into a perfused compartment from

27

which solute was directly removed and a non per-fused compartment from which solute transport oc-curred only into the perfused compartment. Solutevolumes of distribution were allowed to vary withtime owing to interdialytic fluid intake and intra-dialytic fluid removal. The total volume of distribu-tion for urea, creatinine, and vitamin˚B12 was assumedto be the volume of total body water. Of that total,one third was assumed to be within the perfused (ex-tracellular) compartment, and two thirds, within thenon perfused (intracellular) compartment. The totalvolume of distribution for inulin and β2-microglobulinwas assumed to be the extracellular compartment,with intravascular (one quarter of extracellular vol-ume) and interstitial (three quarters of extracellularvolume) spaces as the perfused and non perfused com-partments respectively.

All computer simulations were performed for ananephric patient with a post-dialysis weight of 70˚kg;total body water was assumed to be 50% of the post-dialysis weight. That latter value was a compromisebetween the value estimated from anthropometricequations (12) and the lower value estimated from ureakinetics during chronic hemodialysis therapy (13,14).All fluid gained by and removed from the patient wasassumed to move into and out of the extracellular com-partment; the relative amounts removed from the intra-vascular and interstitial fluids were assumed to beproportional to the compartment volumes (one quar-ter and three quarters, respectively).

Solute generation was assumed to occur onlywithin the perfused compartment. Endogenous solutegeneration rates were assumed as constant for urea,creatinine, and β2-microglobulin [(8) Table˚I].Vitamin˚B12 and inulin are not naturally generatedwithin the body; nevertheless, to predict steady-statesolute concentration profiles for calculating dose mea-

Leypoldt and Burkart

sures, it was assumed that those solutes were gener-ated at constant rates.

Solute transfer between the compartments was as-sumed as proportional to the solute concentration dif-ference; the proportionality constant was defined asthe intercompartmental transfer constant, or clearanceKIC. The values of KIC for each solute were assumedto be identical to those reported previously [(8)Table˚I].

Excess fluid removal during HFHD was assumedas constant throughout the treatment. During a CFPDdwell, the rate of fluid removal was assumed as con-stant. During a CAPD dwell, the rate of fluid removaldepends on the glucose concentration in the dialysate;it was assumed as equal to the time-dependent ultra-filtration rate predicted by the model of Pyle [(15) seeAppendix]. Weekly fluid removal was matched toweekly fluid accumulation so that overall patient vol-ume was at steady state. The rate of fluid intake oraccumulation was assumed as constant and equal to1˚L daily.

Solute removal from the body was evaluated dif-ferently for each therapy; the mathematical details ofthe relationships are described in the Appendix. DailyCFPD therapy consisted of an 8-hour, 2-L CFPD treat-ment and two 8-hour, 2-L CAPD dwells using 1.5%glucose—containing dialysis solution. Daily CAPDtherapy consisted of four 4-hour, 2-L CAPD dwellsusing 1.5% glucose—containing dialysis solution andone 8-hour, 2-L CAPD dwell using 4.25% glucose—containing dialysis solution. Solute transport acrossthe peritoneal membrane was calculated accountingfor both diffusive and convective transport (see Ap-pendix for details). Solute transport rates by diffusionand convection were assumed to be related to the per-meability-area product or mass transfer-area coeffi-cient (PA), and 1 minus the solute reflection coefficient(σ), respectively, for the peritoneal membrane. Table˚IIlists the assumed values of those parameters for CAPD.Previous work has shown that PA values for urea andcreatinine during CFPD are substantially higher thanthose during CAPD (18). We therefore assumed thatPA values during CFPD are twice those during CAPDfor all solutes.

For HFHD, three 3-hour treatments per week weresimulated on a traditional Monday/Wednesday/Fridayschedule. Solute clearances during HFHD were cal-culated assuming the use of a high-flux dialyzer, ablood flow rate of 400˚mL/min and a dialysate flow

TABLE I Solute characteristics

Ga KICb

Solute Molecular weight (mg/min) (mL/min)

Urea 60 6.25 600Creatinine 113 1.0 275Vitamin B12 1,355 0.2 125Inulin 5,200 0.3 90β2-Microglobulin 11,800 0.17 40

a Solute generation rate.b Intercompartmental transfer constant or clearance.

28

rate of 800˚mL/min to achieve a single-pool urea Kt/Vof 1.4 per treatment. The dialyzer mass transfer-areacoefficients for each solute in the present study weresimilar to those assumed by Clark et al (8) and areshown in Table˚II. Dialyzer solute clearances duringHFHD were also adjusted for ultrafiltration (see Ap-pendix). Non renal clearance (KNR) was assumed tobe zero except for β2-microglobulin, where it was as-sumed to be 3˚mL/min (19).

For each solute, a variable time-step, fourth-fifth—order Runge—Kutta algorithm (20) was used to com-pute steady-state solute concentration profiles byiterative solution of the mass balance equations (seeAppendix) over a 10-week interval. From the steady-state concentration profiles, several indices were cal-culated. The time-averaged concentration (TAC) wasobtained for each solute and therapy combination as atime-weighted and distribution˚volume—weighted av-erage of the intradialytic and interdialytic concentra-tions, assuming that those concentrations were linearduring the intradialytic, post-dialysis rebound, andinterdialytic periods (8). The immediate post-dialysisrebound period was assumed to be 1˚hour for urea,creatinine, and vitamin˚B12, and 4˚hours for inulin andβ2-microglobulin. With the assumed solute generationrate (G) and calculated whole-body TAC valuesknown, the EKR was then calculated (9) as

EKR˚= G˚/ TAC [1]

The mean pretreatment concentration (MPC) wascalculated as the highest daily concentration in the

perfused compartment (during CFPD and CAPD) orby averaging the three pretreatment concentrations inthe perfused compartment (during HFHD). The stan-dard Kt/V (stdKt/V) for each solute was then calcu-lated by the equation

stdKt/V˚= G˚∞ t˚/ MPC˚/ V [2]

where t and V denote the total weekly time and vol-ume of distribution for the solute of interest.

ResultsSolute concentrations during CAPD were relativelytime-independent; the concentrations in the perfusedand non perfused compartments were very similar inmagnitude, but dependent on the solute. Concentra-tions in the perfused compartment decreased duringan 8-hour CFPD treatment by 24% for urea, 13% forcreatinine, 7% for vitamin˚B12, 5% for inulin, and —6%for β2-microglobulin. However, the differences in sol-ute concentration between the perfused and non per-fused compartments were uniformly small(<˚1˚mg/dL), indicating little solute disequilibrationduring CFPD. Post-dialysis concentration reboundsof urea and inulin in the perfused compartment afterHFHD were approximately 25% and 73% respectively.Those values are larger than the values reported pre-viously by Clark et al (8), reflecting the higher effi-ciency of hemodialysis treatments simulated in thepresent study.

Table˚III shows calculated values of EKR andstdKt/V. Values of EKR were higher during CFPD thanduring CAPD or HFHD for all solutes. Values ofstdKt/V were also higher during CFPD than duringCAPD or HFHD for all solutes, except for β2-micro-globulin. Figures˚1 and˚2 show the magnitude of theincreases in EKR and stdKt/V for each solute duringCFPD over CAPD and HFHD.

DiscussionBecause of rapid removal rates, two-compartmentmodels are required for simulations of solute concen-tration profiles during intermittent artificial kidneytherapies. The concentration profiles simulated in thepresent study for HFHD confirm previous work indi-cating that significant post-dialysis rebound occurs inthe perfused compartment for all solutes (8), but espe-cially for middle molecules (21). The present studyalso shows that the magnitude of solute disequilibrium

CFPD and Middle-Molecule Clearances

TABLE II Solute transport characteristics during continuous ambu-latory peritoneal dialysis (CAPD) and high-flux hemodialysis(HFHD)

HFHDCAPD In vivo KoAc

Solute PAa (mL/min) 1˚— σb (mL/min)

Urea 20 0.6 650Creatinine 10 0.55 450Vitamin B12 5 0.5 225Inulin 2 0.4 100β2-microglobulin 1 0.1 32

a Permeability area for the peritoneal membrane (15—17).b Solute reflection coefficient for the peritoneal membrane (15).c Mass transfer-area coefficient for high-flux dialyzers [modifiedslightly from reference (8) because of the higher assumed dialy-sate flow rate].

29

during CFPD and CAPD is quite small, suggesting thatfuture kinetics studies of those therapies may be per-formed using simpler single-compartment models.

The results of the present study show that dosesfor small-solute and middle-molecule removal are bothhigher during CFPD than during either CAPD orHFHD. The magnitude of the increase in dose dependson whether EKR or stdKt/V is used for evaluation;nevertheless, the primary conclusion of the presentstudy is relatively independent of the dose measureemployed.

The mechanisms for the higher dose during CFPDas compared with CAPD depend on the solute. Forexample, the increase in urea and creatinine removalduring CFPD is attributable both to the increase inthe concentration difference across the peritonealmembrane and to the increase in PA. In contrast, theincreased dose for middle-molecule removal wasattributable primarily to the increase in˚PA.

It should be emphasized that our clearance anddose estimates are likely conservative. We have as-sumed that PA values during CFPD are twice thoseobserved during CAPD; however, limited experimen-tal studies have shown even larger increases in clear-ances and PA values for small solutes such as ureaand creatinine (1,3,18). Because the magnitude of theincrease in PA for middle molecules has not beenevaluated experimentally, future studies regardingperitoneal transport during CFPD should include in-vestigations of middle-molecule removal in additionto evaluations of small-solute removal.

Two limitations of the present study should bementioned. First, we have assumed that no proteinbinding of solutes occurs. That assumption is likelygood for some small solutes, such as urea and creati-nine, but it may not be realistic for other molecules ofinterest, especially vitamin˚B12 (22). Second, our theo-retical predictions are limited by the accuracy of the

TABLE III Calculated dose measures during continuous flow peritoneal dialysis (CFPD), continuous ambulatory peritoneal dialysis(CAPD), and high-flux hemodialysis (HFHD)

EKRa (mL/min) stdKt/Vb

Solute CFPD CAPD HFHD CFPD CAPD HFHD

Urea 16.58 6.94 11.48 4.07 1.99 2.25Creatinine 10.63 5.55 9.22 2.58 1.60 1.93Vitamin B12 6.63 3.87 6.22 1.60 1.12 1.43Inulin 3.99 2.57 3.08 2.88 2.22 2.36β2-microglobulin 5.08 4.10 4.43 3.63 3.56 4.07

a Equivalent renal clearance (as defined in Methods ).b Standard Kt/V (as defined in Methods )

Leypoldt and Burkart

FIGURE 2 Calculated percentage increases of standard Kt/V(stdKt/V) for urea, creatinine, vitamin˚B12, inulin, andβ2-microglobulin (Beta-2-M) for continuous flow peritonealdialysis (CFPD) over those for continuous ambulatory peritonealdialysis (white bars) and high-flux hemodialysis (black bars).

Urea Creatinine VitaminB12

Inulin Beta-2-Mstd

Kt/

V In

crea

se (

%)

Fo

r C

FP

D

150

125

100

75

50

25

0

-25 Urea Creatinine Vitamin Inulin Beta-2-MB12

FIGURE 1 Calculated percentage increases of equivalent renal clear-ance (EKR) for urea, creatinine, vitamin˚B12, inulin, and β2-micro-globulin (Beta-2-M) for continuous flow peritoneal dialysis (CFPD)over those for continuous ambulatory peritoneal dialysis (whitebars) and high-flux hemodialysis (black bars).

30

assumed transport parameters. For example, differ-ences in peritoneal membrane transport characteris-tics among patients may yield clinical results differentfrom those predicted here. Future studies should evalu-ate differences in solute transport during CFPD andCAPD with respect to patient transport status.

ConclusionWe conclude that CFPD achieves higher doses for bothsmall-solute and middle-molecule removal than doesCAPD or HFHD.

AppendixThe equations governing solute concentrations in theperfused (p) and non perfused (np) compartments arebased on mass balances within each compartment asdescribed previously (8):

d (̊˚CpVp )̊˚/ dt˚

= G˚— KIC (̊˚Cp˚— Cnp )̊˚— Js˚— KNR˚∞ Cp [A1]

d (̊˚CnpVnp )̊˚/ dt˚= KIC (̊˚Cp˚— Cnp )̊ [A2]

dVp˚/ dt˚= —J̊v˚, and [A3]

dVnp˚/ dt˚= 0 ,̊ [A4]

where Js and Jv denote solute and volume removalrates, respectively; and Cp and Cnp denote solute con-centrations in plasma water. For HFHD, Js was setequal to 0 during inter-treatment intervals.

The two preceding equations describe changes inintercompartmental volumes only for urea, creatinine,and vitamin˚B12. When considering inulin and β2-micro-globulin, changes in volume for the perfused and nonperfused compartments were assumed as follows:

dVp˚/ dt˚= —Jv˚/ 4 [A5]

dVnp˚/ dt = —3∞̊ Jv˚/ 4˚. [A6]

Solute and volume removal rates depended on thetreatment modality. Solute removal rates during CFPDand CAPD were calculated from this equation (15):

Js˚= PA˚(̊Cp˚— Cd)˚+ (̊1˚— σ )̊˚∞ Qf˚∞ Cp˚, [A7]

where PA and 1˚— σ during CAPD dwells are as re-ported in Table˚II, and Qf denotes the transperitoneal

ultrafiltration rate. During CFPD treatment, values ofPA were assumed to be twice the values reported inTable˚II.

The change in the dialysate solute concentrationwith time during CAPD dwells is governed by thisequation:

d˚(̊CdVd )̊˚/ dt˚= Js˚— QL˚∞ Cd ,̊ [A8]

where Vd denotes the volume of dialysis solutionwithin the peritoneal cavity and QL denotes the rateof fluid absorption from the peritoneal cavity.

During CFPD, changes in the dialysate solute con-centration are governed alternatively by this equation:

d˚(̊CdVd )̊˚/ dt˚= Js˚— (˚QL˚+ Qpd˚+ Qf )̊˚∞ Cd˚, [A9]

where Qpd denotes the continuous flow of dialysissolution into the peritoneal cavity during that therapy(assumed as 200˚mL/min).

Dialysate solute concentration entering the peri-toneal cavity during CFPD was assumed to be 0, andthe initial concentration during CAPD was assumedto be 0. During CFPD, the transperitoneal ultrafiltra-tion rate was assumed as constant during the treat-ment. The constant value was assigned to maintainoverall weekly fluid balance.

During CAPD dwells, the transperitoneal ultrafil-tration rate was assumed to be time-dependent as de-scribed previously (15,23). The coefficients of theexponential equation describing transperitoneal ultra-filtration depended on the assumed glucose concen-tration of the freshly infused dialysis solution for eachexchange. The fluid absorption rate from the perito-neal cavity was assumed to be the rate defining thedecrease in peritoneal volume after osmotic equili-bration between plasma and the dialysis solution (15),and residual volume between exchanges was assumedto be 0.

During HFHD, the solute removal rate was de-scribed by this equation:

Js˚= (̊Kd˚+ 0.4 ∞̊ Qf )̊˚∞ Cp˚, [A10]

where Kd denotes solute clearance in the absence ofultrafiltration, which was calculated using standardformulas (24) from the blood water and dialysate flowrates and the in vivo KoA values reported in Table˚II.The second term in the parentheses of equation˚[A10]

CFPD and Middle-Molecule Clearances

31

denotes an approximate correction factor for increasedsolute removal owing to ultrafiltration. That correc-tion factor has been shown to be approximately validfor both urea and β2-microglobulin (25).

AcknowledgmentThis material is the result of work supported with theresources and use of the facilities at the Salt Lake CityVA Health Care System and by the Dialysis ResearchFoundation (Ogden, UT).

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Corresponding author:John K. Leypoldt, PHD, Dialysis Program, University ofUtah, 85˚N.˚Medical Drive, East Room˚201, Salt Lake City,Utah 84112-5350 U.S.A.

Leypoldt and Burkart