Novel Role for Mast Cells in Omental Tissue Remodeling and Cell Recruitment in Experimental Peritoneal Dialysis
Mohammad Zareie*,
Paolo Fabbrini,
Liesbeth H.P. Hekking*,
Eelco D. Keuning*,
Piet M. ter Wee,
Robert H.J. Beelen* and
Jacob van den Born*
Departments of * Molecular Cell Biology & Immunology and Nephrology, VU University Medical Centre, Amsterdam, The Netherlands; and Department of Clinical Nephrology, Ospedale San Gerardo, Monza, Italy
Address correspondence to: Dr. Jacob van den Born, Department of Molecular Cell Biology & Immunology, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands. Phone: +31-20-444-8078/8080; Fax: +31-20-444-8081; E-mail: j.vandenborn{at}vumc.nl
Received for publication November 9, 2005.
Accepted for publication August 31, 2006.
Because of its dynamic structure, the omentum plays a key rolein the immunity of the peritoneal cavity by orchestrating peritonealcell recruitment. Because mast cells accumulate in the omentumupon experimental peritoneal dialysis (PD) and may produce angiogenic/profibroticfactors, it was hypothesized that mast cells mediate omentaltissue remodeling during PD. Daily treatment with conventionalPD fluid (PDF) for 5 wk resulted in a strong omental remodelingresponse, characterized by an approximately 10-fold increasein mast cell density (P < 0.01), an approximately 20-foldincrease in vessel density (P < 0.02), an approximately 20-foldincrease in the number of milky spots (P < 0.01), and a four-foldincrease in submesothelial matrix thickness (P < 0.0003)in wild-type rats. In contrast, all PDF-induced omental changeswere significantly reduced in mast cell–deficient Ws/Wsrats or in wild-type rats that were treated orally with a mastcell stabilizer cromoglycate. A time-course experiment showedmast cell accumulation immediately before the formation of bloodvessels and milky spots. Functionally, PDF evoked a peritonealcell influx, which was significantly reduced in Ws/Ws rats (P< 0.04) and in wild-type rats that were treated with cromoglycate(P < 0.03). Cromoglycate treatment also completely preventedPDF-induced omental adhesions to the catheter tip (P = 0.0002).Mesothelial damage, angiogenesis, and fibrosis of mesenteryand parietal peritoneum as well as glucose absorption rate andultrafiltration capacity proved to be mast cell independent.Data strongly support the hypothesis that mast cells mediatePDF-induced omental tissue remodeling and, subsequently, peritonealcell influx and adhesion formation, providing therapeutic possibilitiesof modulating omental function.
Over several decades, omental transposition has been appliedto many human disorders, resulting in an accelerated wound-healingprocess (1–6). In the tissue engineering field, new organswere developed successfully by implantation of embryonic tissues,for example kidney and pancreas, into the omentum of the recipient(7,8). Previously, we showed that the omentum exerts an importantrole in the peritoneal defense mechanism against pathogens byproviding not only the primary site for neutrophil exudationbut also the local site for peritoneal leukocyte proliferationand macrophage differentiation (9–11). Despite that thisbroad range of omental applications and functions is due mainlyto its unique angiogenic properties, the underlying (cellular)mechanism of omental angiogenesis has not yet been identifiedfully.
The clinical application of peritoneal dialysis (PD) has introducedthe peritoneum, including omentum, into the nephrology field.A number of research groups now are using omentum as a modelfor investigating various aspects of PD-related pathophysiology(12,13). We previously reported that long-term exposure to PDfluid (PDF) evoked a chronic inflammatory response in an animalmodel, which typically is associated with a rapid influx ofinflammatory cells toward the peritoneal cavity, followed byangiogenesis in various peritoneal tissues, including omentum,and serosal injury to mesothelial cells that cover the peritoneumas well as fibrogenesis (14,15). Clinically, these pathologicalterations ultimately result in impaired ultrafiltration capacity,leading to treatment failure in a significant number of PD patients.It is interesting that we found that angiogenesis in the omentumwas accompanied y the accumulation of omental mast cells, locatedin close contact with vessels, and their number correlated stronglyto the number of omental vessels in our PD model (15,16). Thelatter also was found in other inflammatory conditions (17,18).To test the hypothesis that mast cells are the key cells thatcontrol PDF-induced omental tissue remodeling and, subsequently,peritoneal cell influx, we treated "white spotting" (Ws/Ws)mast cell–deficient rats and their normal +/+ littermateswith PDF daily. The Ws/Ws rats are affected by a 12-base deletionat the tyrosine kinase domain of c-kit gene (19), which is essentialnot only for mast cell development but also for hematopoiesis,erythropoieses, and gametogenesis (20,21). The Ws/Ws rats provedto be a powerful tool to determine the definite role of mastcells in many pathologic conditions as well as in normal woundhealing (22–25). In addition, we orally administered amast cell stabilizer, disodium cromoglycate, to wild-type ratsthat were treated with PDF. Our data strongly indicate an essentialrole for mast cells in PDF-induced omental tissue remodelingand, subsequently, peritoneal cell recruitment.
Laboratory Animals
In the first experiment, we used male mast cell–deficientWs/Ws rats and their normal wild-type littermates (KarolinskaInstitute, Stockholm, Sweden) that weighed 180 to 240 g at thestart of the experiment. For other experiments, we used malewild-type Wistar rats (Harlan CPB, Horst, The Netherlands) thatweighted 280 to 300 g at the start of the experiment. Rats weremaintained under conventional laboratory conditions and weregiven free access to water and food. Body weights of all animalswere monitored weekly, and no significant differences were foundamong the groups in each experiment (data not shown). Experimentswere reviewed and approved by the local ethical committee onthe use of laboratory animals and are in accordance with theNational Institutes of Health Guide for the Care and Use ofLaboratory Animals.
Experimental Design Experiment 1.
Mast cell–deficient (Ws/Ws; n = 10) and wild-type rats(+/+; n = 10) were exposed daily to 10 ml of conventional PDF(Dianeal PD4, 3.86% glucose, pH 5.2; Baxter R&D, Utrecht,The Netherlands), via a permanent peritoneal catheter connectedto a subcutaneous mini vascular access port (14,15) for 5 wk.Gender- and age-matched untreated mast cell–deficient(n = 8) and wild-type (n = 12) rats served as controls. To determineperitoneal performance and total cell influx upon PDF instillation,we performed a peritoneal equilibration test (PET) after 5 wkof PDF treatment in half of the PDF-treated animals (five ratsper group) as well as in all controls. A 1.5-h, PET was performed,by using 30 ml of the conventional PDF. Glucose levels weredetermined in PET effluents by the hexokinase method on a Roche/Hitachi(Basel, Switzerland) Modular P800, and percentage of glucoseabsorption was calculated as [1 – (final dialysate glucoseconcentration) x (drained volume)/200 mM x 30 ml] x 100. Cell-freesupernatants of PET effluents were stored at –20°Cfor measurement of vascular endothelial growth factor (VEGF),monocyte chemoattractant protein-1 (MCP-1), IL-1, and TNF-.Furthermore, a peritoneal wash was performed in the remaininganimals in both PDF-treated groups (five rats per group), asdescribed previously (14). Different peritoneal tissues werecollected for morphometric analysis.
Experiment 2.
Wild-type Wistar rats were exposed to the conventional PDF with(n = 7) or without (n = 8) oral disodium cromoglycate, a mastcell stabilizer, for 5 wk. The cromoglycate was dissolved in1 ml of 95% polyethyleneglycol and administered via gavage (50mg/kg per d). The first control group (n = 8) received onlythe vehicle (polyethyleneglycol), whereas the second controlgroup (n = 6) received only cromoglycate orally. Thereafter,PET was performed, followed by morphologic and cellular analysis.Effectiveness of cromoglycate treatment was controlled by directinjection of 10 nmol of degranulating compound 48/80 into theright hind paw in a volume of 50 µl. The same volume ofvehicle was injected into the left hind paw. Degranulation ofmast cells in the right hind paw resulted in paw swelling, whichcan be measured. Right/left paw thickness ratio was measuredafter 60 min and was increased from 1.01 (before injection)to 1.08 in control rats without cromoglycan. In rats that weretreated with PDF + cromoglycan, right/left ratio remained stablefrom 1.01 at baseline to 1.00 at 60 min, showing a completeprevention of mast cell–mediated right hind paw swellingby cromoglycate treatment.
Experiment 3.
Omental mast cell density was quantified in several groups ofWistar rats that were exposed to conventional lactate-bufferedPDF, bicarbonate/lactate-buffered PDF (Physioneal, 3.86% glucose,low glucose degradation products content, pH 7.4; Baxter R&D;n = 8), or bicarbonate/lactate buffer without glucose (pH 7.4;n = 7). Eight gender- and age-matched rats served as controls.
To exclude peritonitis, we checked peritoneal cell-free supernatants(obtained form peritoneal wash and PET effluents) for the presenceof bacteria by agar plating, and no bacteria were found. Omentaltissues also were inspected for the presence of bacteria and/oradherent neutrophils, and no bacteria or abnormal numbers ofneutrophils were found. Furthermore, the well-being of all animalswas monitored daily, but no apparent abnormalities were observed.Development of body weight of all animals also was normal.
Quantification of Mast cells Omentum and Mesentery.
Large whole-mount preparations of omental tissue (approximately4 cm2/rat) and the mesentery (the most distally situated loopentirely) were spread on a glass slide for light microscopicexamination. The sections were stained with 1% toluidine blue,and the number of mast cells was counted and expressed as thenumber of cells per square millimeter, using a scored eyepiece,as described previously (15,16).
Parietal Peritoneum.
The whole parietal peritoneum (two portions of approximately20 cm2) was dissected and frozen in liquid nitrogen. Cryostatsections were cut (8 µm) and stained with toluidine blue.The number of mast cells within the submesothelial extracellularmatrix (ECM) was quantified and expressed as the number of mastcells per millimeter length of the mesothelial cell layer, asdescribed previously (15).
Quantification of Blood Vessels Omentum.
Two stretched preparations of the omentum (approximately 4 cm2/rat)were obtained for quantification of blood vessels in each ratin a standardized manner. One sample was stained with toluidineblue (14,15). The blood vessels per area of 4 mm2 were counted,and 25 random areas were selected from each preparation (total1 cm2). The second sample was stained with a specific endothelialmarker, anti-CD31 (platelet-endothelial cell adhesion molecule;Serotec, Oxford, UK), as described previously for parietal peritoneum(15).
Mesentery.
The number of blood vessels was quantified in the mesentericwhole-mount preparations after staining with toluidine blue,as described for omentum (14,15).
Parietal Peritoneum.
The number of blood vessels within submesothelial ECM was quantified,using anti-CD31, and expressed as the number of vessels permillimeter length of the mesothelial cell layer (15).
Quantification of Milky Spots in Omentum
The number and the size of milky spots was determined by lightmicroscopy using a scored eyepiece, as described previously(14,15). Total milky spot surface area was calculated by multiplyingboth parameters.
Electron Microscopy of Peritoneal Tissues
Portions of dissected omentum, mesentery, and parietal peritoneumfrom at least three rats per group were prepared for electronmicroscopy (14).
Fibrosis Omentum and Mesentery.
Overview electron micrographs that were made from omental andmesenteric tissues were used to determine the thickness of ECM,as fibrotic marker (15). We thus measured the distance betweenboth mesothelial cell layers at various places on each electronmicrograph, and the mean value was calculated for each micrograph.We analyzed at least three rats per group and at least 10 photomicrographsper rat and therefore at least 30 measurements per group.
Parietal Peritoneum.
The thickness of the submesothelial ECM layer was determinedafter Van Gieson staining (Merck KGaA, Darmstadt, Germany),and the average of 10 independent measurements was calculatedfor each rat and expressed in microns (14,15).
Peritoneal Lavage Fluid
PET fluids and peritoneal washes were centrifuged, and cellswere counted and differentiated according standard procedures.PET cell-free peritoneal fluids were 20 times concentrated (AmiconUltra-4), and VEGF concentration was measured by ELISA accordingto the manufacturer’s instruction (Quantikine M, VEGFkit; R&D Systems, Minneapolis, MN). The detection levelwas 15 pg/ml. MCP-1 (Pharmingen, San Diego, CA), IL-1, and TNF-(NIBSC, South Mimms, UK) were determined by ELISA, detectionlevel 60, 30, and 30 pg/ml, respectively.
Statistical Analyses
Data are expressed as medians and 25th to 75th interquartileranges and the spread from 10th to 90th percentile. In caseof multiple comparisons, independent groups first were analyzedstatistically by nonparametric ANOVA (Kruskal-Wallis test),followed by the Mann Whitney U test, corrected according toBonferroni (P = 0.05/ number of comparisons). In experiment1, four comparisons are made: (1) +/+ rats with and withoutPDF, (2) Ws/Ws rats with and without PDF, (3) both untreatedgroups, and (4) both PDF-treated groups (P < 0.025 levelof significance). In experiment 2, three comparisons are made:(1) PDF-treated rats versus control rats, (2) PDF + cromoglycate–treatedrats versus control rats, and (3) PDF + cromoglycate–treatedrats versus control rats (P < 0.03 level of significance).In experiment 3, three comparisons are made: Dianeal treatedrats versus (1) physioneal-treated rats, (2) buffer-treatedrats, and (3) untreated control rats (P < 0.03 level of significance).Spearman rank correlation test was used for correlation analysis,and Fisher exact test was used for analysis of contingency table.
Mast Cells Mediate Omental Tissue Remodeling Mast Cell Density.
Light microscopic observation of the omentum, mesentery, andparietal peritoneum confirmed the absence of mast cells in theWs/Ws rats before and after PDF treatment. Data from peritonealwash also confirmed the lack of mast cells and eosinophils inthe peritoneal fluid of Ws/Ws rats before and after PDF treatment.Furthermore, analysis of the peritoneal wash revealed that mastcells almost disappeared from the peritoneal cavity of +/+ animalsthat were treated with PDF, whereas 5 to 7% of total peritonealcells were mast cells in control +/+ rats (P < 0.0005). Mastcells accumulated preferably within omental milky spots, asthe number of mast cells within milky spots was approximately10-fold higher in the +/+ rats that were treated with PDF comparedwith control +/+ rats (P < 0.005). In addition, the mastcell density was significantly increased in omental tissuesbetween milky spots of +/+ rats upon PDF treatment (P < 0.004;Figures 1 and 2). Compared with controls, rats that were exposedto PDF with or without cromoglycate also showed an increasednumber of mast cells within and between omental milky spots,with no significant differences between both treated groups(Figures 1 and 2).
Figure 1. Overview light micrographs of omental tissues. Representative low-magnification light micrographs of whole-mount preparations of omental tissues after toluidine blue staining of untreated wild-type (A) and untreated mast cell–deficient Ws/Ws rats (B), as well as wild-type and mast cell–deficient Ws/Ws rats exposed to peritoneal dialysis fluid (PDF; C and D, respectively). (E) PDF-treated wild-type rats that were administered cromoglycate orally. Instillation of PDF resulted in the accumulation of mast cells, along with the induction of new blood vessels and milky spots (MS) in the omental tissues of wild-type rats (A versus C) but not of mast cell–deficient Ws/Ws rats (B versus D). In the PDF + cromoglycate group (E), mast cells did accumulate; however, without the formation of new vessels and milky spots (E versus C). MS, mast cells (arrowheads), and blood vessels (arrows) are visualized clearly.
Figure 2. Accumulation of omental mast cells upon exposure to PDF. Mast cell density (number of cells/mm2) counted in omental tissues between (A and C) or within (B and D) the MS. In A and B, wild-type rats with or without PDF treatment were compared. In C and D, control Wistar rats were compared with Wistar rats treated with PDF in the presence or absence of oral cromoglycate intervention.
Angiogenesis.
Exposure to PDF induced a severe angiogenesis in the omentumof +/+ rats (P < 0.0005; Figures 1, 3, and 4), whereas approximatelysix-fold fewer blood vessels were formed in the omental tissuesof Ws/Ws rats (P < 0.0001). Oral administration of cromoglycatelargely prevented the PDF-induced omental angiogenesis (P <0.03; Figures 1, 3, and 4). When all wild-type rats from bothexperiments were combined, a strong correlation was found betweenomental blood vessels and mast cells (r = 0.79; P = 0.0004).
Figure 3. Visualization of whole-mount omental vasculature by immunofluorescence. Representative photomicrographs of omental tissues after CD31 (platelet-endothelial cell adhesion molecule) staining of untreated wild-type (A) and untreated mast cell–deficient Ws/Ws rats (B), as well as wild-type and mast cell–deficient Ws/Ws rats exposed to PDF (C and D, respectively). (E) PDF-treated wild-type rats administered cromoglycate orally. (F) A typical MS with its unique vascular network. Note the induction of severe angiogenesis upon PDF treatment in wild-type rats (C versus A), which was significantly less induced in mast cell–deficient rats (D versus B) and in wild-type rats treated orally with cromoglycate (E versus C).
Figure 4. Quantification of omental vasculature. (A) The number of blood vessels in the omental tissues of untreated wild-type (control+/+) and untreated mast cell–deficient (Control Ws/Ws) rats, as well as wild-type (PDF +/+) and mast cell–deficient Ws/Ws rats exposed to PDF (PDF Ws/Ws). (B) We compared PDF-induced new vessel formation in wild-type Wistar rats with and without cromoglycate therapy. Note the increased number of blood vessels in wild-type rats treated with PDF, which was significantly prevented in mast cell–deficient Ws/Ws and wild-type rats treated orally with cromoglycate.
In addition to histochemical staining, CD31 staining was usedto identify blood vessels more specifically (Figure 3). Theresults of these two methods were consistent and showed a lessprofound angiogenesis in Ws/Ws rats treated with PDF as wellas in wild-type rats treated with PDF and cromoglycate, comparedwith +/+ rats exposed to PDF.
Milky Spots.
Instillation of PDF increased the number (P < 0.0005) andthe size (P < 0.0005) of milky spots in +/+ rats (Figure 1,Table 1). Importantly, PDF treatment induced a lower numberof milky spots in the Ws/Ws rats (P < 0.0005), although thesize of milky spots was equal in both PDF-treated groups (P= 0.72). Compared with the PDF group, oral administration ofcromoglycate significantly reduced the number of milky spots(P < 0.005), but it had no effect on the size of milky spots(P > 0.99). Correlation analysis in all wild-type rats revealeda strong positive relationship between the number of milky spotsand mast cells in the omentum (r = 0.78; P = 0.001).
Fibrosis.
Daily exposure to PDF resulted in an increased omental thicknessin +/+ rats (P < 0.003), whereas PDF did not lead to omentalfibrosis in Ws/Ws rats (Figures 5 and 6). Oral administrationof cromoglycate completely prevented the PDF-induced fibrosisin the omentum (P < 0.0001; Figures 5 and 6). In controlrats, cromoglycate alone (thus without PDF instillation) hadno effect on omental mast cell density, angiogenesis, milkyspot response, and fibrosis (data not shown).
Figure 5. Transmission electron microscopy of omental tissues. Representative images are shown from an untreated wild-type rat (A) as well as a PDF-treated wild-type rat (B), a PDF-treated mast cell–deficient Ws/Ws rat (C), and a PDF-treated wild-type rat administrated with cromoglycate orally (D). Note a significant increase in the thickness of the omentum from wild-type rats that were treated with PDF (B), which was completely prevented in PDF-treated mast cell–deficient Ws/Ws rats (C) or PDF-treated wild-type rats + cromoglycate (D). Original magnification: x3000.
Figure 6. Measurement of omental fibrosis. Thickness of the submesothelial extracellular matrix layer measured in electron microscopy photomicrographs of untreated wild-type (Control +/+) and untreated mast cell–deficient (Control Ws/Ws) rats compared with PDF treatment in these rats (A). (B) We compared PDF-induced fibrosis in wild-type rats with and without cromoglycate therapy. Note the increased matrix layer in wild-type rats that were treated with PDF, which was completely prevented in mast cell–deficient Ws/Ws and wild-type rats that were treated orally with cromoglycate.
Time-Course Study
The kinetics of the PDF-mediated omental mast cell accumulationand angiogenesis was determined by exposing several groups ofrats to PDF (n = 2 to 4/group) for 1 to 5 wk (Figure 7). Mastcells started to accumulate markedly from the third week. Itis interesting that the formation of new blood vessels and milkyspots started from the fourth week, whereas this was not observedin the first 3 wk. These data indicate that omental mast cellssignificantly increased immediately before the formation ofblood vessels and milky spots.
Figure 7. Kinetics of PDF-induced omental changes. Time-course study of mast cell accumulation and the induction of blood vessels in the omentum. Note the accumulation of mast cells immediately before the formation of blood vessels.
Mast Cells Regulate Leukocyte Recruitment and Omental Adhesion Formation
Because mast cells mediated the PDF-induced omental angiogenesisand milky spot response and because milky spots reflect a functionalaspect of the omentum in terms of peritoneal cell influx, wecounted the number of cells in the peritoneal cavity upon PDFinstillation. PDF exposure induced a significant influx of cells(Figure 8), mostly macrophages, into the peritoneal cavity duringthe PET procedure in the +/+ (P < 0.002) and to a far lesserextent in the Ws/Ws rats (P = 0.082), compared with their controls.It is interesting that more cells were recruited toward theperitoneal cavity in the +/+ rats compared with Ws/Ws rats afterpretreatment with PDF (P < 0.03). We found no significantdifferences in the percentage of macrophages (P = 0.90), lymphocytes(P = 0.73), or neutrophils (P = 0.55) between both treated groups.Absolute numbers of macrophages, neutrophils, and lymphocytesall were reduced in PDF-treated Ws/Ws rats, suggesting hamperedperitoneal cell recruitment in general. Similar to the Ws/Wsrats, administration of cromoglycate significantly reduced PDF-inducedcell influx (P < 0.03). MCP-1, a major chemoattractant formonocytes/macrophages, was significantly increased in cell-freePET fluids from all rats that were treated by PDF; however,no differences were found between PDF-treated +/+ and Ws/Wsrats or between PDF-treated rats with or without cromoglycate(Tables 2 and 3). The proinflammatory cytokines IL-1 and TNF-were below detection limits in cell-free PET fluids of all rats.
Figure 8. Functional role of mast cells in peritoneal leukocyte recruitment. The number of recruited leukocytes in the peritoneal cavity during PET-test of untreated wild-type (Control +/+) and untreated mast cell–deficient (Control Ws/Ws) rats compared with PDF treatment in these rats (A). (B) We compared PDF-induced cell influx in wild-type rats with and without cromoglycate therapy. Note a strong cell influx to ward the peritoneal cavity in wild-type rats treated with PDF, which was significantly prevented in mast cell–deficient Ws/Ws and wild-type rats treated orally with cromoglycate.
Table 3. Morphometric/functional parameters of peritoneal tissues and PET: Experiment 2
In experiment 2 (intervention with cromoglycate), when the ratswere killed, we scored the presence or absence of omental adhesionformation around the catheter tip. All PDF-treated rats (n =7) showed variable omental adhesion to the catheter. In strikingcontrast, in none (n = 8) of the PDF + cromoglycate rats thiswas observed (Fisher exact test: P = 0.0002).
Mast Cell–Dependent Omental Tissue Remodeling Is Not Related to Effluent VEGF
Daily exposure to PDF significantly increased the VEGF levelsin the peritoneal effluents of both wild-type (median 46 pg/ml)and Ws/Ws rats (median 51 pg/ml), compared with control wild-type(median 16 pg/ml) and control Ws/Ws rats (median 12 pg/ml).No significant differences were found between treated +/+ andWs/Ws rats (P = 0.69). Oral administration of cromoglycate hadno effect on increased VEGF levels after PDF treatment (P =0.63).
Mast Cells Are Not Involved in Tissue Remodeling of Other Peritoneal Tissues
In contrast to the omentum, mast cells did not accumulate inthe mesentery (P = 0.38) or parietal peritoneum (P = 0.90) of+/+ rats upon PDF exposure (Tables 2 and 3). A similar numberof blood vessels were formed in the mesentery (P > 0.99)and parietal peritoneum (P = 0.19) of PDF-exposed Ws/Ws and+/+ rats as well as cromoglycate-treated rats (Tables 2 and3). Furthermore, the number of blood vessels did not correlateto the number of mast cells in the mesentery (r = 0.02; P =0.96) or in the parietal peritoneum (r = 0.10; P = 0.86). Nodifferences were found between +/+ and Ws/Ws rats after treatmentwith PDF with respect to fibrosis (Tables 2 and 3) in the mesentery(P = 0.25) or parietal peritoneum (P = 0.54). In addition, cromoglycatedid not reduce PDF-induced fibrosis in the mesenteric tissues(P = 0.11) or in parietal peritoneum of Wistar rats (P = 0.13).
Mast Cells Are Not Involved in PDF-Induced Mesothelial Cell Injury
Compared with controls, exposure to PDF resulted in a strongregenerative response of mesothelial cells on liver in both+/+ (P < 0.0003; Tables 2 and 3) and Ws/Ws rats (P < 0.009),without significant difference between both treated groups (P= 0.63). Cromoglycate had no impact on PDF-induced mesothelialregenerative response (data not shown). Using electron microscopy,we observed focal damage to the mesothelial cell layer coveringthe omentum (Figure 5), mesentery, and parietal peritoneum inboth +/+ and Ws/Ws rats after exposure to PDF, as well as inthe cromoglycate-treated rats, along with adhesion of macrophages,with no difference among treated groups.
Mast Cells Are Not Involved in Microvascular Peritoneal Transport
Chronic exposure to PDF was associated with loss of ultrafiltrationcapacity (Tables 2 and 3), accompanied by increased glucoseabsorption from peritoneal cavity, in both +/+ and Ws/Ws ratscompared with control rats, without significant differencesbetween both treated groups with respect to ultrafiltrationcapacity (P = 0.41) or glucose absorption rate (P = 0.19), althoughultrafiltration capacity and glucose absorption seem to be somewhatlower in the Ws/Ws strain. Oral administration of cromoglycatehad no significant impact on PDF-induced ultrafiltration dysfunction(P = 0.10) or on the increased glucose reabsorption (P = 0.12).
Less Profound Mast Cell Accumulation upon Treatment with Bicarbonate/Lactate-Buffered PDF
Increased mast cell accumulation upon treatment with lactate-bufferedPDF was significantly reduced after exposure to bicarbonate/lactate-bufferedPDF or the buffer alone (Figure 9). The data presented hereare in good agreement with our earlier data showing a milderomental tissue remodeling after treatment with the bicarbonate/lactate-bufferedPDF and the buffer alone (14,26).
Figure 9. Less profound omental mast cell accumulation upon exposure to bicarbonate/lactate-buffered PD solution. Mast cell density (number of cells/mm2) counted in omental tissues between the MS from wild-type rats exposed to conventional lactate-buffered PDF (ConPDF), bicarbonate/lactate-buffered PDF (Bic/Lac), or bicarbonate/lactate buffer without glucose, as well as untreated control rats (C). The number of mast cells in the ConPDF group was significantly increased compared with all other groups (P < 0.002). The number of mast cells also was increased in the Bic/lac (P < 0.002) and the buffer group (P < 0.003), compared with control rats.
In this report, we provide evidence for the critical involvementof mast cells in PDF-induced omental tissue remodeling and,subsequently, peritoneal cell influx and omental catheter adhesionduring experimental PD. We previously showed that omental milkyspots are active sites for leukocyte migration and peritonealleukocyte supply during inflammation (9–11). In addition,milky spots are the sites for local proliferation, maturation,and differentiation of macrophages (10). It has been reportedthat the inherited mast cell deficiency in experimental peritonitismodels significantly impaired the cell influx (27,28); however,no explanation was provided. Jippo et al. (29) elegantly showedthat tissue mast cells, not those in the peritoneal fluid, areessential for a proper innate immunity against bacterial invasion.In agreement with these studies, we observed that the PDF-inducedcell influx was significantly reduced in Ws/Ws rats or in wild-typerats that were treated with cromoglycate. Along with a poorcell influx, Ws/Ws rats and rats that were treated with cromoglycatedeveloped a weak milky spot response, which might explain thepoor influx response that was found in mast cell–deficientrats in this study and during peritonitis (27,28). We thereforesuggest a novel role for mast cells in the induction of a properomental milky spot response, providing new opportunities toregulate peritoneal cell recruitment. Formally, we cannot excludethe possibility that a reduced peritoneal cell recruitment inmast cell–deficient rats results in a hampered omentaltissue remodeling. However, the absence of the proinflammatorymediators IL-1 and TNF- do not support the idea that these exudatecells actively influence tissue remodeling.
A better understanding of how mast cells participate in omentalangiogenesis is critical to increase our knowledge about vasculardysfunction and tissue remodeling. Several lines of evidencesuggest a key role for mast cells in the induction of angiogenesis.Mast cells often are found near blood vessels and accumulatein various pathologic conditions (30–33). There is a significantcorrelation between mast cell numbers and blood vessel densityin different kinds of cancer (34) and in this study. Most important,mast cells are able to produce and release many potent angiogenicfactors, including tryptase, chymase, VEGF, basic fibroblastgrowth factor, and IL-8 (35). Similar to the chick chorioallantoicmembrane model for angiogenesis (36), our time-course studyrevealed an increased number of mast cells immediately beforethe formation of new blood vessels. It is interesting that theinhibition of PDF-induced omental angiogenesis did not improvethe ultrafiltration capacity and glucose absorption, confirmingthe idea that the transport function of the peritoneum is dominatedby blood vessels of the parietal peritoneum.
At this time, we do not know which mast cell mediator(s) is(are)involved in omental remodeling during PD. That therapeutic interventionusing cromoglycate yielded exactly the same results as usingmast cell–deficient rats clearly indicates mast cell degranulationto be essential for the observed response and excludes a delayedmast cell response via de novo synthesis of inflammatory mediators.In contrast to an earlier report (37), we found no role forVEGF in the omental tissue remodeling, because an equal amountof VEGF was released into the peritoneal cavity of wild-typeand mast cell–deficient rats after PDF treatment, whichcould be explained by the fact that VEGF is not stored in mastcell granules (38). Nevertheless, we cannot exclude the possibilitythat VEGF concentration at the tissue level might be differentbetween both treated groups.
Mast cells also participate in fibrotic processes (39). Mastcells are found in fibrogenic lesions in various diseases andcontain potent profibrotic mediators. Using mast cell–deficientanimals, some studies showed no difference in the degree offibrosis between mast cell–deficient and +/+ animals (23,40),whereas other studies suggested a key role for mast cells inthis process (41). These seemingly opposing results are reflectednicely in our study, in which we show omental fibrosis to bemast cell dependent, whereas the fibrotic response in the mesenteryand parietal peritoneum is mast cell unrelated. The completeabsence of omental adhesion formation to the catheter tip inthe PDF + cromoglycan group is in striking contrast to the 100%presence of these adhesions in the PDF group. This might bea clinically important observation that warrants further investigation.The role of mast cells in adhesion formation had been documented(42).
To our knowledge, this study is the first to unravel the underlyingcellular regulatory mechanism of omental tissue remodeling,which is responsible for the recruitment of inflammatory cellsduring PD. We therefore propose that PDF activates omental endotheliumto promote the adhesion and migration of leukocytes, includingmast cells. We indeed have shown by electron microscopy an activatedhigh endothelial venule-like phenotype of endothelial cellswithin milky spot vessels upon PDF instillation (14). In addition,we previously reported an increased number of rolling leukocytesin peritoneal venules of PDF-treated rats, pointing toward endothelialactivation (43). Once migrated, these recruited perivascular(mast) cells stay around vessels and do not migrate toward theperitoneal cavity but rather form a new milky spot. This explainsthe accumulation of mast cells in the omentum (especially inthe milky spots) and the absence of mast cells in the peritonealfluid. These perivascular mast cells locally degranulate andthereby promote new vessel formation and fibrosis. As long asperitoneal exposure to PDF is present, this omental remodelingwill continue. As soon as the trigger stops (peritoneal rest),the whole omental tissue remodeling response reverses to normal,as we recently reported (44). Importantly, the use of bicarbonate/lactate-bufferedPDF resulted in a less profound mast cell accumulation, alongwith less omental remodeling, which might be clinically relevant.We believe that increasing our knowledge of omental cell biology,especially the critical role of mast cells therein, will providenew opportunities to regulate omental function not only duringPD treatment but also during chirurgical omental transposition,tissue engineering, peritoneal defense, adhesion formation,and tumor growth.
Acknowledgments
This study was financially supported by the Dutch Kidney Foundation(grant C00.1888).
We thank Ann Hagstrom (Department of Physiology, KarolinskaInstitute, Stockholm, Sweden) for providing mast cell–deficientrats.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
, and TNF-
. Furthermore, a peritoneal wash was performed in the remaining animals in both PDF-treated groups (five rats per group), as described previously (14). Different peritoneal tissues were collected for morphometric analysis. 
number of comparisons). In experiment 1, four comparisons are made: (1) +/+ rats with and without PDF, (2) Ws/Ws rats with and without PDF, (3) both untreated groups, and (4) both PDF-treated groups (P < 0.025 level of significance). In experiment 2, three comparisons are made: (1) PDF-treated rats versus control rats, (2) PDF + cromoglycate–treated rats versus control rats, and (3) PDF + cromoglycate–treated rats versus control rats (P < 0.03 level of significance). In experiment 3, three comparisons are made: Dianeal treated rats versus (1) physioneal-treated rats, (2) buffer-treated rats, and (3) untreated control rats (P < 0.03 level of significance). Spearman rank correlation test was used for correlation analysis, and Fisher exact test was used for analysis of contingency table. 
