ABSTRACT. Pharmacologic studies suggest that the release ofnitric oxide (NO) by endothelial NO synthase (eNOS) contributesto functional alterations of the peritoneal membrane (PM) inducedby acute peritonitis. In this study, peritoneal permeabilityparameters in a mouse model of peritoneal dialysis were characterized,and the effects of eNOS deletion on the PM structure and permeabilityat baseline and after catheter-induced bacterial peritonitiswere examined. Exposure of C57BL/6 mice to standard dialysateyielded a transport of urea and glucose, a sodium sieving, anda net ultrafiltration that were remarkably similar to the valuesobtained in rats. In comparison with controls, mice with catheter-inducedperitonitis were characterized by structural changes in thePM (mononuclear cells infiltrate, vascular proliferation), upregulationof endothelial and inducible NOS, increased permeability forurea and glucose, decreased ultrafiltration, and increased proteinloss in the dialysate. Comparison of eNOS wild-type and knockoutmice revealed that the permeability modifications and structuralchanges induced by acute peritonitis were significantly reversedin eNOS knockout mice, resulting in a net increase in ultrafiltration.In contrast, the deletion of eNOS in mouse peritoneum was notreflected by permeability modifications or structural changesat baseline. These results are the first to take advantage ofa knockout mouse model to demonstrate directly the crucial importanceof eNOS in the permeability and structural modifications causedby acute peritonitis. The characterization of this mouse modelsuggests that genetically modified mice represent useful toolsto investigate the molecular bases of the peritoneal changesduring peritoneal dialysis.
Despite technological advances and accumulating clinical experience,acute peritonitis remains the most frequent and serious complicationof peritoneal dialysis (PD) (1). Understanding the molecularmechanisms that operate in acute peritonitis thus is an essentialgoal to reduce the functional and structural changes associatedwith the condition. Studies in rat and rabbit models of PD havedemonstrated that transport across the peritoneal membrane (PM)depends on (1) the intrinsic permeability to each solute and(2) the effective peritoneal surface area (EPSA) reflectingthe number of perfused capillaries within the peritoneum (2).The capillary endothelium, which expresses both the endothelialnitric oxide (NO) synthase (eNOS) and the water channel aquaporin-1(AQP1), constitutes the major barrier for solutes and watertransport during PD (3,4). Acute peritonitis is characterizedby an increased EPSA, with increased permeability for smallsolutes and glucose, a faster-than-normal dissipation of theosmotic gradient, a decrease of free-water permeability, anda loss of ultrafiltration (UF) (5,6). These modifications areassociated with mononuclear cell infiltrate and vascular proliferationwithin the PM (6).
During the past decade, NO has emerged as a crucial mediatorinvolved in countless biologic processes, including regulationof vascular tone and permeability (7), interference with growthfactors to modulate angiogenesis (8), and posttranslationalcontrol of protein activity by S-nitrosylation (9). All of theseproperties are relevant for the PM, because a significant NOSactivity, mostly as a result of the Ca2+-dependent eNOS, isdetected in the peritoneum (6,10). The exact nature of the roleof NO and eNOS in the PM remains debated (11). In control conditions,NOS inhibitors showed no effect on peritoneal permeability parameters(12,13). In acute peritonitis, a condition characterized byincreased NOS activity as a result of upregulation of eNOS andinducible NOS (iNOS) (6,14), addition of the NOS inhibitor NG-nitro-L-argininemethyl ester (L-NAME) to the dialysate resulted in a significantreduction of peritoneal permeability with an improvement ofUF (14).
One limitation of the current rat and rabbit models of PD isthat investigations of the molecular mechanisms of peritonealpermeability in vivo are mostly based on intervention studiesusing pharmacologic agents, blocking antibodies, or variousexpression systems (15). These models are often limited by thelack of specificity or the side effects of most interventions,as well as the transient efficacy of expression systems, includingadenoviral vectors (15,16). The use of genetically modifiedmice to investigate the molecular counterparts of PD could providean attractive alternative to the above models. Although smallerin size, mice are economical and easy to breed and maintain,and an extensive number of knockout or transgenic mice havenow been used for investigations that may be relevant for thePM.
In this study, we have characterized the parameters of peritonealpermeability, the structure of the PM, and the expression ofNOS isoforms and endothelial markers (AQP1 and CD31) in C57BL/6Jmice, both in basal state and after catheter-induced bacterialperitonitis. We then show that deletion of eNOS in this geneticbackground had significant protective effects on the permeabilityand structure of the PM exposed to acute peritonitis. Thesedata substantiate the role of eNOS in the regulation of peritonealpermeability and demonstrate the usefulness of genetically modifiedmouse models in studies of peritoneal transport and physiology.
Animals, Experimental Groups, and Acute Peritonitis Model
Experiments were conducted using male C57 BL/6J mice (Iffa Credo,Brussels, Belgium). The C57 BL/6J eNOS+/+ (wild-type [WT]) andeNOS-/- (knockout [KO]) mice were generated as described previously(17) and obtained from the Jackson Laboratory (Bar Harbor, ME).Male Wistar rats, aged 8 to 10 wk, were used for comparativestudies (6,10,14). All animals had access to appropriate standarddiet and tap water ad libitum. The experiments were conductedin accordance with the National Institutes of Health Guide forthe Care and Use of Laboratory Animals and were approved bythe local Ethics Committee.
A group of 12 control mice was used to characterize the permeabilityparameters at the basal state, in comparison with those of controlrats (n = 12) and mice with acute peritonitis (n = 6). Anothergroup of mice with acute peritonitis (n = 6) was used to assessthe differential leukocyte counts in the dialysate at day 1,day 3, and day 7. In a second series of experiments, 11 pairsof eNOS WT and KO mice matched for age were investigated incontrol conditions (n = 5) or after acute peritonitis (n = 6).Acute peritonitis was generated by insertion of a peritonealcatheter as described previously (6,14). At day 0, mice wereanesthetized with ketamine (100 mg/kg subcutaneously; Merial,Brussels, Belgium) and xylazine (10 mg/kg subcutaneously; Bayer,Brussels, Belgium). A silicone catheter (Terumo, Leuven, Belgium)was implanted into the peritoneal cavity without aseptic precautionsand subcutaneously tunneled to the neck. A daily infusion of2 ml of dialysate (3.86% Dianeal; Baxter, Nivelles, Belgium)was performed for 6 d. In addition, animals were intraperitoneallyadministered Staphylococcus epidermidis (107/ml colony-formingunits, diluted in 3.86% dialysate) at day 1 and day 3. On day7, mice were submitted to a 2-h PD exchange to measure permeabilityparameters followed by tissue sampling.
Peritoneal Permeability Measurement and Tissue Sampling
A peritoneal equilibration test similar to that used in rat(6,14) was used to investigate peritoneal permeability parametersin mice. After anesthesia with ketamine and xylazine, mice wereplaced on a thermopad at 37°C. The right common carotidartery was cannulated for the measurement of mean arterial BPusing an isotonic BP transducer (Harvard Apparatus, Holliston,MA). The right jugular vein was catheterized for saline infusion(0.9% NaCl, 0.3 ml/h). After 30 min of stabilization, a siliconcatheter (Venflon 22 GA, Baxter) was inserted into the peritonealcavity and 2.0 ml of a standard dialysate (Dianeal; Baxter)containing either 3.86% or 7% glucose was instilled. Blood anddialysate samples (100 µl) were taken from the carotidartery and the PD catheter at time 0 and at 30 min, 60 min,and 120 min of dwell time. Before each sampling, 50 to 100 µlof the dialysate was flushed back and forth, and the abdomenwas agitated gently to facilitate fluid mixing. Hematocrit wasmeasured before PD exchange. At the end of the dwell, the dialysatewas recovered from the peritoneal cavity through the catheter,whereas the remaining portion was subsequently removed fromthe open cavity with gauze tissues that were weighted. As describedpreviously (6), net UF corresponds to the difference betweenthe total volume of dialysate collected (sampling through catheterand dialysate weight) and the volume instilled in the cavity.The intra-assay and interassay variability parameters of thelatter method in mice are 6% and 5%, respectively.
Dialysate white blood cells (WBCs) were counted using a hemocytometer(Marienfeld, Lauda, Germany), and differential leukocyte countswere obtained from peritoneal cells plated to a glass slideby cytospin (Thermoshandon, Pittsburgh, PA) and stained withPapanicolaou. Urea, glucose, sodium, and total protein wereassayed using a Kodak Ektachem DT60 II and DTE II analyser (EastmanKodak Company, Rochester, NY). PD parameters were also obtainedin rats, using 15 ml of standard dialysate containing either3.86% glucose (n = 6) or 7% glucose (n = 6) as described previously(6,14).
At the end of the dwell, animals were killed and peritoneumsamples were processed for fixation and protein extraction asdescribed previously (6,10,14). Samples from the visceral andparietal peritoneum were fixed for 3 h at 4°C in 4% paraformaldehydein 0.1 M phosphate buffer, rinsed, and embedded in paraffin.Samples from the visceral peritoneum were dissected, snap-frozenin liquid nitrogen, and stored at -80°C.
Antibodies
The NOS isoforms were detected with monoclonal antibodies againsteNOS, neuronal NOS (nNOS), and iNOS (Transduction Laboratories,Lexington, KY) (10). Other antibodies included a rabbit antibodyagainst AQP1 (Chemicom International, Temecula, CA), a goatantibody against CD31 (Santa Cruz Biotechnology, Santa Cruz,CA), a rabbit antibody against vascular endothelial growth factor(VEGF; Santa Cruz), a goat antibody against vascular cell adhesionmolecule-1 (VCAM-1; Santa Cruz), and a monoclonal antibody against-actin (Sigma, St. Louis, MO).
Immunoblot and ELISA Analyses
SDS-PAGE and immunoblotting were performed as described (6,10).Protein extracts from the visceral peritoneum were separatedon 7.5% or 12% acrylamide slabs and transferred to nitrocellulose.Efficiency of transfer to nitrocellulose was tested by Ponceaured (Sigma) staining and immunoreactivity for -actin. The membraneswere blocked for 30 min, incubated overnight with the primaryantibody at 4°C, washed, incubated for 1 h with peroxidase-labeledsecondary antibodies (Dako, Glostrup, Denmark), and visualizedwith enhanced chemiluminescence (Amersham, Little Chalfont,UK). The immunoblots were performed at least in duplicate, andspecificity of the signal was determined by comparison withpositive controls. Densitometry analysis was performed witha StudioStar Scanner (Agfa-Gevaert, Mortsel, Belgium) usingthe NIH-Image V1-57 software. The concentrations of VEGF inmouse plasma and dialysate were measured by quantitative sandwichELISA specific for mouse VEGF (R&D Systems, Minneapolis,MN).
Tissue Staining and Immunohistochemistry
Trichrome blue staining and immunostaining were performed asdescribed previously (6,14). After blocking in 0.3% H2O2 andincubation with 10% normal serum, sections were incubated successivelyfor 45 min each with the primary antibody, biotinylated IgG,and avidin-biotin peroxidase (Vector Laboratories, Burlingame,CA). The MOM immunodetection kit (Vector) was used for monoclonalantibodies. Antigen retrieval was performed by incubating sectionsfor 30 min in 0.01 M citrate buffer (pH 5.8), in a water bathheated at 97°C, before rinsing in water. Immunolabelingwas visualized using aminoethylcarbazole (Vector). Sectionswere viewed under a Leica DMR coupled to a Leica DC 300 camera(Leica, Heerbrugg, Switzerland). The specificity of immunolabelingwas confirmed by incubation without primary antibody and withnonimmune IgG (Dako).
Quantification of Vascular Immunoreactivity
The use of CD31 to quantify microvascular density has been previouslyreported in detail (18,19). Peritoneal sections stained forCD31 were viewed through a Zeiss microscope coupled to a DAGE-MTICCD 72 camera (Michigan City, IL) and analyzed through a KS400system (Kontron, Munich, Germany). The stained areas of thevisceral peritoneum were digitized, to outline the vessel walls.The density of vascular structures (in N vessels/mm2), the relativeendothelial area (i.e., the ratio between the cumulative endothelialarea and the peritoneum area, in %), and the mean vascular radius(in µm) were measured as described previously (20). Allslides were coded and analyzed on a simple-blind basis by thesame operator. The analysis included a mean of 10 fields persample and four mice in each group. The intensity of the mononuclearcells infiltrate was graded as 0, absent; 1, moderate; 2, mild;and 3, severe in WT versus KO eNOS mice (n = 5 in each group).
Statistical Analyses
Data are presented as mean ± SEM. Comparisons betweenresults from different groups were performed using two-tailedt test or Mann-Whitney U test, as appropriate.
Clinical and Biological Parameters
Mice were similar in terms of body weight, hematocrit, plasmaurea, and glycemia at baseline (Table 1), and none of them diedprematurely during the protocol. In comparison with controls,mice with acute peritonitis were characterized by positive dialysatecultures and cloudy dialysates with increased WBC counts (Table 1).None of the cultures obtained from controls, eNOS WT, andeNOS KO mice was positive. The differential leukocyte countsin the dialysate of mice with peritonitis showed that the majority(60 to 80%) of inflammatory cells were polymorphonuclear leukocytesat day 1. That proportion decreased to 34 to 45% at day 3 andfurther to 8 to 20% at day 7, in parallel with a progressiveincrease in mononuclear leukocytes (macrophages and lymphocytes).A comparison between eNOS WT and KO mice did not show any significantdifference in the differential leukocyte count at day 7 (datanot shown). As expected (17), eNOS KO mice were characterizedby a mean arterial BP higher than eNOS WT mice in control conditionsand, to a lower extent, during acute peritonitis (Table 1).
Table 1. Clinical and biological parameters in the different groups of C57BL/6J micea
Characterization of Peritoneal Permeability in Mouse: Effects of Acute Peritonitis and eNOS Deletion
Control mice were submitted to a 2-h exchange with 3.86% versus7% glucose dialysate (Figure 1, Table 2). Exposure to both dialysatesinduced a progressive increase in the dialysate-to-plasma ratiofor urea (Figure 1A), a progressive reabsorption of glucosefrom the dialysate (Figure 1B), and a fall in the dialysate-to-plasmaratio of sodium during the first 30 min of the dwell (sodiumsieving; Figure 1C). At drainage, the dialysate was clear inall animals. Exposure to 7% dialysate induced a significantincrease in the sodium sieving (Figure 1C, Table 2), whereasit had no effect on other parameters (Figure 1, A and B, Table 2).It must be noted that the PD parameters obtained in micewere remarkably similar to those obtained in rats, as shownby similar values for cumulative urea and glucose transport,sodium sieving, and net UF (normalized to body weight; Table 2).
Figure 1. Functional parameters of peritoneal dialysis (PD) in mice at baseline. The dialysate-to-plasma (D/P) ratio of urea (A) and sodium (C) and the progressive removal of glucose from the dialysate (D/D0 glucose; B) were determined during a 2-h exchange with 2 ml of 3.86% () versus 7% glucose () dialysate. Exposure to 7% glucose induces a significant increase in the sodium sieving, whereas the permeability to urea and glucose remains similar. *P < 0.05 versus 3.86% glucose dialysate.
Table 2. Peritoneal permeability parameters in control mice and ratsa
The effects of acute peritonitis were investigated in controland eNOS mice (Figure 2, Table 3). Acute peritonitis induceda major increase in peritoneal permeability for urea, a fasterglucose absorption from the dialysate, and a loss of the sodiumsieving in control and eNOS WT mice (Figure 2). In both groups,the changes were reflected by a similar increase in the cumulativetransport of urea and glucose, a fall in UF, and an increasedprotein loss in the dialysate (Table 3). Thus, the magnitudeof the changes induced by acute peritonitis was similar in controland eNOS WT mice. However, in comparison with the latter, eNOSKO mice with acute peritonitis were characterized by a significantreduction in the hyperpermeability to urea and glucose, a significantincrease in UF and sodium sieving, and a decreased protein lossin the dialysate (Figure 2, Table 3). At baseline, the deletionof eNOS was not reflected by changes in peritoneal permeabilityparameters (Table 3).
Figure 2. Functional parameters of PD in mice: Effect of acute peritonitis and endothelial nitric oxide synthase (eNOS) deletion. The D/P ratio of urea (A) and sodium (C), the progressive removal of glucose from the dialysate (D/D0 glucose; B), and net ultrafiltration (UF; volume out–volume in; D) were determined in eNOS wild-type (WT) mice (), eNOS WT mice with peritonitis (WT-p; ), and eNOS knockout (KO) mice with peritonitis (KO-p; ) during a 2-h exchange with 2 ml of 3.86% glucose dialysate. Acute peritonitis induces a major increase in the permeability for urea and glucose, with a loss of the sodium sieving and a loss of UF. All of these modifications are significantly reversed in eNOS KO mice. #P < 0.05 between WT and WT-p mice; *P < 0.05 between WT-p and KO-p mice; **P < 0.05 between KO-p and WT mice.
Table 3. Peritoneal permeability parameters in mice: Effects of acute peritonitis and eNOS deletion
Structural Changes in the Mouse Peritoneum: Morphology and Immunostaining
Morphologic examination of the visceral and parietal peritoneumshowed that acute peritonitis in mice was reflected by a massive,submesothelial infiltrate of mononuclear cells, together witha discrete edema (Figure 3). These structural modificationswere similar in eNOS WT and KO mice, although the mononuclearcell infiltrate was less marked in the latter (median intensityof the mononuclear cells infiltrate, 3.0 versus 2.0; n = 5;P = 0.033, Mann-Whitney U test).
Figure 3. Effects of acute peritonitis on the structure of the peritoneum in eNOS WT and KO mice. Trichrome blue staining in the visceral (A through D) and parietal (E through G) peritoneum of eNOS WT (A, B, E, and F) and eNOS KO (C, D, and G) mice in control conditions (A, C, and E) or acute peritonitis (B, D, F, and G). The deletion of eNOS is not reflected by structural changes of the peritoneal membrane at baseline (A versus C). Acute peritonitis is reflected by a massive infiltrate of mononuclear cells in the visceral and parietal peritoneum. The changes induced by acute peritonitis are similar in eNOS WT and KO mice, although the infiltrate is less important in the latter. Magnifications: x150 in A through D, x300 in E through G.
Immunostaining showed that acute peritonitis was associatedwith an increased signal for eNOS in capillary endothelium (Figure 4, A and B),whereas no specific staining was detected in eNOSKO mice (Figure 4C). Contrasting with the absence of signalin control mice, a strong staining for iNOS was detected inmononuclear cells infiltrating the peritoneum of mice with acuteperitonitis (Figure 4, D and E). Of note, the intensity of iNOSstaining was lower in eNOS KO versus eNOS WT mice (compare Figure 4, F and E).Acute peritonitis was reflected by a marked increasein the immunoreactivity for CD31, located in both endothelialcells and macrophages (Figure 4, G and H). The signal for CD31was lower (intensity and density) in eNOS KO versus eNOS WTmice (compare Figure 4, I and H). The morphologic and immunocytochemicalmodifications induced by acute peritonitis were similar in thevisceral and parietal peritoneum.
Figure 4. Effects of acute peritonitis and eNOS deletion on NOS isoforms and CD31 expression in the peritoneum. Immunostaining for eNOS (A through C), inducible NOS (iNOS; D through F), and CD31 (G through I) in the visceral peritoneum of eNOS WT mice (A, D, and G), eNOS WT mice with peritonitis (B, E, and H), and eNOS KO mice with peritonitis (C, F, and I). Immunoreactivity for eNOS (1:250) is located in the endothelium lining peritoneal blood vessels in eNOS WT mice (A). The staining intensity and the density of capillaries and blood vessels stained for eNOS are markedly increased in eNOS WT mice with peritonitis (B), whereas no signal is detected in eNOS KO mice (C). In contrast with the lack of signal in eNOS WT mice (D), an intense immunoreactivity for iNOS (1:100) is detected in mononuclear cells infiltrating the visceral peritoneum of eNOS WT mice with peritonitis (E, inset). The signal for iNOS, less intense than in WT mice, is also detected in eNOS KO mice with peritonitis (F, inset). Staining for CD31 (1:50) is detected in the endothelium lining peritoneal vessels and capillaries in eNOS WT mice (G). Acute peritonitis is associated with an increased density of CD31-positive blood vessels and capillaries that is significantly more marked in eNOS WT (H) than in eNOS KO (I) mice. Magnification, x300.
Quantification of vascular density and relative endothelialarea in the visceral peritoneum (Figure 5) confirmed that thedeletion of eNOS in mice had no effect on both parameters atbaseline. Peritonitis induced a significant increase in vasculardensity and relative endothelial area in both eNOS WT and KOmice, but the effect was significantly attenuated in eNOS KOmice. Peritonitis induced a significant increase in the meanradius of stained vessels in both eNOS WT-p (63 ± 8 versus198 ± 19 µm; P = 0.03) and eNOS KO-p (64 ±21 versus 115 ± 16 µm; P = 0.03) mice. The increase,which was predominantly observed in intermediate and large-sizedvessels, was significantly attenuated in eNOS KO-p versus eNOSWT-p mice (115 ± 16 µm versus 198 ± 19 µm,respectively; P = 0.03).
Figure 5. Effects of acute peritonitis and eNOS deletion on the vascular density and relative endothelial area in the visceral peritoneum. Quantification of vascular density and relative endothelial area was performed on sections stained for CD31 (see text for details). The morphometry was performed on four samples in each group, with a mean of 10 fields analyzed per sample. #P < 0.05 versus eNOS WT; *P < 0.05 versus eNOS KO; P < 0.05 versus eNOS WT-p.
Expression of NOS Isoforms and AQP1 in the Mouse Peritoneum: Immunoblotting
Acute peritonitis induced a significant upregulation of theendothelial (140 kD) and inducible (130 kD) NOS isoforms, anda discrete upregulation of AQP1 (relative optical density versuscontrol, 141 ± 17%; P = 0.20) in the mouse peritoneum(Figure 6A). As described previously (10), the anti-iNOS antibodycross-reacted with eNOS (see the upper band on the gel), butthe specific band corresponding to iNOS (lower band of the gel)was identified by its molecular mass (130 kD) and co-migrationwith macrophage extract (positive control). The induction ofiNOS was variable (6,10), ranging from weak to very strong.In control conditions, eNOS and iNOS isoforms were not detectedin the peritoneum of eNOS KO mice, whereas the expression ofAQP1 was similar in eNOS WT and KO mice (data not shown). Acuteperitonitis in eNOS WT and KO mice was reflected by a variableinduction of iNOS in the peritoneum (Figure 6B). Of note, theexpression of iNOS was lower in eNOS KO (relative optical densityversus eNOS WT, 64 ± 22%; P = 0.55), whereas that ofAQP1 was higher (relative optical density versus eNOS WT, 175± 54%; P = 0.42). A weak expression of nNOS was alsodetected within the peritoneum of control and eNOS mice andshowed no significant variation in case of acute peritonitis(data not shown). Preliminary experiments (limited to threepairs of eNOS mice with peritonitis) also indicated that therewas a 20% decrease in the expression of the adhesion moleculeVCAM-1 in the peritoneum of eNOS KO mice versus eNOS WT micewith peritonitis (data not shown).
Figure 6. Effects of acute peritonitis on the expression of eNOS and iNOS and aquaporin-1 (AQP1) in the peritoneum. (A) Representative immunoblots for eNOS (1:5,000), iNOS (1:5,000), and AQP1 (1:20,000) in peritoneum extracts prepared from control mice and mice with acute peritonitis (20 µg protein/lane). Extracts from bovine aortic endothelial cells (eNOS), mouse macrophages (iNOS), and rat kidney (AQP1) were used as positive controls (lane C). The blots were stripped and reprobed with an mAb against -actin (1:10,000) for loading accuracy. A consistent upregulation of eNOS (140 kD) is detected in mice with acute peritonitis. The signal for iNOS (130 kD, lower band of the blot), never detected in control samples, is upregulated with variable intensity in mice with acute peritonitis. Note that the antibody against iNOS cross-reacts with eNOS (upper band at 140 kD), as previously reported (10). The core (28 kD) and glycosylated (35 to 50 kD) AQP1 isoforms are identified in the mouse peritoneum, with a slight upregulation in mice with peritonitis. Densitometry analysis confirms that peritonitis induces a major upregulation of eNOS (relative optical density, 312 ± 41%; P = 0.001), whereas the increase in AQP1 does not reach statistical significance (relative optical density, 141 ± 17%; P = 0.20). *P < 0.05 versus control mice. (B) Representative immunoblots for eNOS, iNOS, and AQP1 in the visceral peritoneum of eNOS WT and KO mice with peritonitis. The blots were performed as in A. As expected, eNOS KO mice do not express eNOS in the peritoneum. Although iNOS is detected in all samples from eNOS WT and KO mice with peritonitis, the upregulation is less intense in eNOS KO mice (relative optical density, 64 ± 22%; P = 0.55). The latter are also characterized by the upregulation (175 ± 54%; P = 0.42) of AQP1. However, these differences do not reach statistical significance.
Determination of VEGF Concentrations in the Plasma and Dialysate
A specific ELISA was used to determine the concentrations ofVEGF in the plasma and dialysate of the eNOS mice in controlconditions and after catheter-induced acute peritonitis (Table 4).The deletion of eNOS in mice was not reflected by significantchanges in VEGF concentrations. Peritonitis induced a majorincrease in dialysate VEGF in both eNOS WT and eNOS KO mice;the increase in that parameter was slightly blunted in eNOS-KOmice, but the difference with eNOS WT mice was NS (36 ±11 versus 61 ± 24 pg/ml, respectively; P = 0.42). Immunoblottingstudies also failed to demonstrate a significant differencein the expression of VEGF in peritoneal samples from eNOS WTand KO mice (Figure 7).
Figure 7. Expression of vascular endothelial growth factor (VEGF) in the peritoneum of eNOS WT and KO mice with peritonitis. Representative immunoblot for monomeric VEGF (21 kD; 1, 1000) in the visceral peritoneum of eNOS WT and KO mice with peritonitis (20 µg protein/lane, reducing conditions). Recombinant VEGF was used as positive control (lane C). The blots were probed with Ponceau red (Sigma) for transfer accuracy. The band corresponding to VEGF is detected in all samples from eNOS WT and KO mice with peritonitis. The expression of VEGF in the peritoneum is similar in both groups (relative optical density, eNOS KO-p versus eNOS WT-p mice, 115 ± 16%; P = 0.63).
In this study, we describe the structure, permeability parameters,and expression of NOS isoforms and endothelial markers in thePM of a mouse model of PD at baseline and after catheter-inducedbacterial peritonitis. The latter condition is reflected bymajor structural and functional changes in the PM, similar tothose observed in PD patients. These alterations are significantlyreversed in eNOS KO mice. Importantly, the deletion of eNOSin mouse has no effect on the PM at baseline, demonstratingthe importance of a tight regulation of this enzyme.
The permeability of the PM was investigated in C57 BL/6J mice,which represent one of the most common genetic backgrounds forgene-targeted mutations. The permeability for small solutes,the reabsorption of glucose, the free-water permeability (asassessed by the sodium sieving), and the net UF normalized forbody weight were actually similar in mice and rats exposed tothe same dwell. Moreover, exposure to higher osmolality wasreflected by a similar increase in sodium sieving and net UFin both species (Table 2, Figure 1). We next used a model ofcatheter-induced bacterial peritonitis (6,14) to investigatestructural and permeability modifications in the mouse. To modelizefurther the situation encountered in PD-related peritonitis,the catheter contamination from skin flora was completed byan inoculum of coagulase-negative Staphylococci—the mostcommon organism responsible for peritonitis in PD patients (14,21).As compared with controls, mice with peritonitis showed cloudydialysates with positive cultures and high WBC counts in thedialysate with a majority of polymorphonuclear leukocytes atday 1 followed by a progressive increase in macrophages andlymphocytes; mononuclear cells infiltrates and vascular proliferationin the peritoneum at day 7 (Figures 3 through 5); upregulationof eNOS and iNOS (Figures 4 and 6); increased permeability forurea and glucose, with a loss of sodium sieving (Figure 2);and a combination of decreased UF and increased protein lossin the dialysate (Table 3). These changes, which confirm ourprevious observations in rat models (6,14), are characteristicof acute peritonitis in PD patients (5). These functional andstructural studies thus demonstrate the relevance of the mouseas a model for studying peritoneal transport at baseline andduring acute peritonitis.
The role of NO in the regulation of peritoneal permeabilityis suggested by its influence on vascular tone and permeability,particularly during infection or inflammation (7,22), as wellas its interaction with angiogenic growth factors such as VEGF(8). Addition of the NO donor nitroprusside to the dialysateincreases EPSA in rat models (23) and stable PD patients (24).An increased release of NO is also involved in the increasedEPSA and the loss of UF that are observed during acute peritonitis(25). An upregulation of eNOS and iNOS has been documented ina rat model of acute peritonitis (6,14), and addition of L-NAMEto the dialysate has been shown to restore UF and decrease proteinloss in this model (14). However, the acute nature and the lackof specificity of these pharmacologic interventions againstNOS limit the relevance of these earlier studies. Consideringthat the endothelium lining peritoneal vessels is the majorfunctional barrier during PD and because eNOS accounts for mostof the NOS activity in the peritoneum (6,10), it was temptingto investigate the permeability and structure of the PM in eNOSKO mice.
Mice that lack eNOS show equal survival to infection but areknown to develop high BP and end-organ damage with aging (17,26).To limit systemic consequences of high BP, our studies wereperformed in young mice (8 to 10 wk). Although eNOS KO micehad a mild but consistent increase in mean BP before the exchange,this had no consequences on the morphology of the peritonealblood vessels and the basal PD parameters (Table 2, Figure 3).These data confirm previous observations in rats (14) suggestingthat increased BP has no effect on peritoneal permeability—atleast in these experimental settings. The lack of effect ofeNOS deletion on peritoneal permeability at baseline is consistentwith pharmacologic studies in rabbits (13) and rats (12,14),suggesting that low levels of NO generated by eNOS do not criticallyaffect peritoneal permeability (10). In a striking contrast,the permeability modifications associated with acute peritonitiswere significantly reversed in eNOS KO mice, resulting in improvedUF (Table 3, Figure 2). Furthermore, mice that lack eNOS showedan attenuation of the intensity of the infiltrate together witha lower level of iNOS induction (Figures 3 and 4) and, mostimportant, a significant decrease in vascular proliferation(Figures 4 and 5). These results, which are the first to takeadvantage of a KO mouse model to demonstrate a molecular mechanismregulating peritoneal permeability, have several implications.
First, they indicate that a tight regulation of eNOS is criticalto regulate permeability parameters in PD. The positive effectsobserved in eNOS KO mice are very similar to those observedacutely with L-NAME addition to the dialysate (14), which supportsthe relative specificity of L-NAME toward constitutive NOS (27)and emphasizes the therapeutic potential of such NOS inhibitorsfor functional alterations encountered during acute peritonitis(14).
Second, our observation of a significant reduction in vascularproliferation in eNOS KO mice, mirrored by lower endothelialarea and permeability parameters, completes several lines ofevidence suggesting that eNOS plays a predominant role in growthfactor–induced angiogenesis and vascular permeability.For instance, eNOS-deficient mice show impaired wound healingand angiogenesis in response to VEGF or angiopoietin-1 (28,29).Deficiency in eNOS also impairs myocardial angiogenesis (30)or angiogenesis in the ischemic hindlimb (31). The exact mechanismof the impaired angiogenesis in these conditions remains tobe determined. There are no significant modifications of VEGFlevels in mice that lack eNOS (32) (Table 4, Figure 7), butinformation about the state of VEGF receptors in these miceis lacking. Nevertheless, the role of eNOS in mediating angiogenesisinduced by VEGF and other growth factors (33,34) is particularlyimportant for the PM, because neovascularization is a majorcause for functional changes and loss of UF in PD patients (35).
Third, the protective effect of eNOS deletion on the intensityof the infiltrate and the induction of iNOS suggests that eNOSmay regulate the vascular permeability of inflammatory mediatorssuch as leukotrienes or histamine (7). As suggested by our preliminaryobservations, eNOS activity could interfere with the expressionof adhesion molecules such as VCAM-1 (36). In turn, such aninterference could modify leukocyte-endothelial cell interactions(37) and inflammation at the microvascular level (38).
Finally, despite its spectacular effects, the deletion of eNOSdoes not totally reverse the permeability changes induced byacute peritonitis in mice. This incomplete reversibility isparticularly evident for the free-water permeability and netUF, as already observed in acute studies with L-NAME (14). Consideringthat the water channel AQP1 is upregulated in these conditions(Figure 6), the lack of total reversibility could be due tovasoactive substances and proinflammatory cytokines liberatedduring the course of infection/inflammation (39) or, alternatively,to posttranslational modifications of target proteins (9). Ofinterest, our studies also confirm the differential regulationof the two endothelial proteins eNOS and AQP1, as already observedin rat models (6,40) and PD patients (4).
In conclusion, we have characterized and validated a model ofPD in mouse and provided a direct demonstration of the importanceof eNOS for structural and functional changes associated withacute peritonitis. These results suggest that the use of geneticallymodified mice will be useful to characterize the molecular basesfor the alterations of the PM during PD.
Acknowledgments
These studies were supported in part by the Fonds National dela Recherche Scientifique, the Fonds de la Recherche ScientifiqueMédicale, an Action de Recherche Concertée (ARC00/05-260), and grants from Baxter.
We thank M. Delmée, J. Gigi, G. Gillerot, E. Goffin,N. Lameire, B. Lindholm, and T. Wang for providing invaluablesuggestions and critiques. The expert technical assistance ofY. Cnops, H. Debaix, A. Saliez, and L. Wenderickx is highlyappreciated.
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Received for publication June 5, 2003.
Accepted for publication September 13, 2003.
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Top
Abstract
Introduction
-actin (Sigma, St. Louis, MO). 
) versus 7% glucose (
) dialysate. Exposure to 7% glucose induces a significant increase in the sodium sieving, whereas the permeability to urea and glucose remains similar. *P < 0.05 versus 3.86% glucose dialysate.
), eNOS WT mice with peritonitis (WT-p; 
P < 0.05 versus eNOS WT-p.
