1 Departments of Molecular Cell Biology and Immunology, 2 Department of Physiology, 3 Department of Nephrology and 4 Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands
Correspondence and offprint requests to: Mohammad Zareie, Department of Molecular Cell Biology and Immunology, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands. Email: m.zareie{at}vumc.nl
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Abstract |
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Methods. In a peritoneal dialysis (PD) rat model, we evaluated the effects of the addition of AG to the PDF on microcirculation and morphology of the peritoneum, by intravital microscopy and quantitative morphometric analysis.
Results. AG-bicarbonate effectively scavenged different GDPs from PDF. Daily exposure to PDF for 5 weeks resulted in a significant increase in leucocyte rolling in mesenteric venules, which could be reduced for 50% by addition of AG-bicarbonate (P<0.02). Vascular leakage was found in rats treated with PDF/AG-bicarbonate, but not with PDF alone. Evaluation of visceral and parietal peritoneum showed the induction of angiogenesis and fibrosis after PDF instillation. PDF/AG-bicarbonate significantly reduced vessel density in omentum and parietal peritoneum (P<0.04), but not in mesentery. PDF-induced fibrosis was significantly reduced by AG (P<0.02). PDF instillation led to AGE accumulation in mesentery, which was inhibited by supplementation of AG. Since addition of AG-bicarbonate to PDF raised pH from 5.2 to 8.5, a similar experiment was performed with AG-hydrochloride that did not change the fluid acidity. We could reproduce most of the results obtained with AG-bicarbonate; however, AG-hydrochloride induced no microvascular leakage and had a minor effect on angiogenesis.
Conclusion. The supplementation of either AG reduced a number of PDF-induced alterations in our model, emphasizing the involvement of GDPs and/or AGEs in the PDF-induced peritoneal injury.
Keywords: aminoguanidine; angiogenesis; fibrosis; peritoneal dialysis; rats
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Introduction |
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Several lines of evidence suggest aminoguanidine (AG) to be a candidate agent for preventing PDF-induced peritoneal alterations. AG scavenges GDPs [6], prevents the formation of AGEs [6,7] and inactivates inducible NO synthase [8], thereby possessing the ability to reduce the vascular proliferation by modulating the expression of growth factors [3]. In addition, intervention studies with AG in clinical and experimental diabetes have shown the reduction of diabetes-induced pathological changes [7,9]. However, a clinical trial of AG was terminated early for safety concerns. In the PD field, Lamb et al. [10] have previously found that the formation of AGEs could be inhibited by AG. Recently, the supplementation of AG to PDF showed inhibitory effects on peritoneal AGE accumulation, mesothelial denudation, submesothelial monocyte infiltration, peritoneal permeability and ultrafiltration [11], and preserved the functional capacity of peritoneal macrophages in the rat [12]. However, the formulation of AG used in most of these studies is not well documented and basic knowledge is lacking on the angiogenic and fibrotic responses as well as on leukocyteendothelium interactions of the peritoneal membrane after exposing to AG.
In the present study, we evaluated long-term effects of the addition of two different formulations of AG to conventional PDF on quantitative morphometric analysis of the peritoneal membrane, focusing on angiogenesis and fibrosis, as well as on the peritoneal microcirculation by intravital microscopy.
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Material and methods |
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Animals
Male Wistar rats (Harlan CPB, Horst, The Netherlands) weighing 180200 g at the beginning of the experiment were used throughout the study. They were allowed to acclimatize 1 week before the experiments started. Animals were maintained under conventional laboratory conditions and were allowed free access to food and water. The experiments were reviewed and approved by the local ethics committee on the use of laboratory animals.
Experimental peritoneal dialysis model
Peritoneal catheters connected to subcutaneous mini-vascular access ports were implanted, as previously described [4,5]. During the first week post-operation all animals, except control animals, received 2 ml of saline with 1 U/ml heparin to allow wound healing. Thereafter, 10 ml of PDF was given daily, for 5 weeks, between 0900 and 1200 hours without the addition of heparin or antibiotics.
Experimental design
Experiment A. Rats were divided into three groups. The first group (PDF, at the start: n = 10) received heat-sterilized, lactate-buffered PDF with a pH 5.2 and containing 3.86% glucose (Dianeal® PD4, Baxter Healthcare, Utrecht, The Netherlands), in the second group 1 g/l AG-bicarbonate (Sigma, St. Louis) was added to PDF (AG-bicarbonate, at the start: n = 11), and finally untreated rats (control, n = 7) served as controls. Thereafter, all animals were subjected to intravital microscopy in order to study the leukocyteendothelium interactions, such as leukocyte rolling and adhesion, as well as vascular permeability. In addition, the cellular/morphological changes of various peritoneal tissues were studied, including angiogenesis, fibrosis, omental milky spot reaction and mesothelial regeneration response.
Experiment B. Rats received the conventional PDF (Dianeal 3.86%, at the start: n = 10) or PDF supplemented with AG-hydrochloride (1 g/l, Sigma, St Louis; at the start: n = 15). Untreated rats served as controls (n = 7). In this experiment, vascular permeability of the mesenteric venules and cellular/morphological alterations of peritoneal tissues were studied.
Intravital microscopy
Experiment A. After 5 weeks of treatment, rats were anaesthetized and cannulated, as described previously [13]. Thereafter, blood samples were collected and cells were differentiated using a Helios cell counter (ABX Diagnostics) implemented with rat-specific software. Intravital microscopic observations of the mesenteric venules were performed as described previously [13]. Briefly, the distal ileum was exteriorized from the peritoneal cavity to allow microscopic observations. The area for analysis was selected in a standardized way, i.e. the most distally situated loop of the ileum. During a stabilization period of 30 min, the whole mesentery was video inspected. Thereafter, observation of the mesenteric microcirculation was performed during a 12 h period. In each individual rat, 1733 venules (101151 venules per group; total 360 venules in all animals) were analysed.
Venular diameter
The inner diameter was measured with a home-made video image shearing device [13].
Leukocyte rolling
Leucocytes were considered to be rolling if they could be seen moving along the vessel wall, by eye, at a significantly lower rate than the blood flow [13]. The number of rolling leukocytes in the venules was determined from the recorded video images by counting the rolling leukocytes per minute passing a reference point in the microvessel.
Leukocyte adhesion
The number of firmly adherent leukocytes was expressed as the number of leukocytes remaining stationary for 30 s or longer in a 100 µm segment of venule during 1 min [13].
Experiments A and B: Microvascular permeability. FITC-BSA, 200 mg/kg body weight, dissolved in 0.5 ml 0.9% NaCl, was infused i.v. during 23 min in all rats. The luminescent microvessels were observed and video-documented during a period of 4560 min. The experimental protocol was ended by superfusion with histamine (10 µg/ml) to determine its ability to induce hyperpermeability in the preparation.
Morphological analysis
Omentum and mesentery
Omental (two sections of approximately 4 cm2/rat) and mesenteric tissues (the most distally situated loop entirely) were dissected, spread on an object slide and stained with toluidine blue. Omental tissues were inspected for the presence of bacteria in order to exclude silent peritonitis and no bacteria were found. Since omental milky spots (local aggregates of immune cells) are the major route through which leucocytes migrate into the peritoneal cavity and because their number and size reflect the activated state of the omentum, we determined their number and size by light microscopy, using a scored eyepiece, as previously described [4,5]. The total milky spot surface area was calculated by multiplying both the parameters. Likewise, in 25 random areas, the number of blood vessels in omental and mesenteric tissues were counted and expressed as the number of vessels/cm2. Furthermore, in 10 randomly selected areas of the omentum and mesentery, mast cells were counted and expressed per mm2 [5]. Mast cells present in/on omental milky spots were excluded from this analysis. Stretch preparations of mesenteric tissues (the second most diatally situated loop of the ileum) were also stained with the monoclonal antibody 4B5 against glycated proteins [14].
Parietal peritoneum
Cryostat sections of large specimens (two portions of approximately 20 cm2) of the parietal peritoneum were cut (8 µm) and embedded in a standardized fashion. The thickness of the submesothelial extracellular matrix (ECM) was determined after Van Giesson staining (Merck KGaA, Darmstadt, Germany), as described previously [4,5]. Frozen sections were also used to quantify the number of submesothelial blood vessels, using anti-CD31 (PECAM) as the endothelial marker and expressed as the number of vessels per mm length of the mesothelial layer. Cryostat sections were also used to evaluate the formation of granulation tissue after staining with anti-CD31 and Van Giesson, which was characterized by thickening of the ECM (fibrosis), the formation of numerous blood vessels, and cellular infiltrates including mast cells, as previously described [5].
Liver
Mesothelial liver imprints were dried and stained by May Grünwald Giemsa, as described before [4,5]. The number of cells per 0.1 mm2 area was counted and the average of 16 areas was calculated for each slide and expressed as cells/mm2.
Electron microscopy of peritoneal tissues
In order to inspect the integrity of the mesothelial cell layer covering the peritoneal membrane, portions of the dissected omentum, mesentery, diaphragm, liver and parietal peritoneum of at least three animals/group were prepared for electron microscopy according to standard procedures [4,5]. Peritoneal fibrosis was measured based on overview electron micrographs of cross sections of mesenteric tissues. The distance between the two mesothelial cell layers was measured at various places on each micrograph and the mean was calculated for each photo. We analyzed 3 rats per group, 10 or more micrographs per rat [5].
Statistical analysis
Data are expressed as medians with the spread from 25th to 75th percentile (interquartile range) and the range from 10th to 90th percentile. Data were statistically analysed using either the non-parametric KruskalWallis, MannWhitney U-tests or Fisher's exact test. Because three groups were involved, P<0.05/n, thus P<0.03 was regarded as significant, according to the Bonferroni correction.
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Results |
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There was a drop-out of 30 and 27% of the animals in PDF and AG-hydrochloride group, respectively, due to omental wrapping around the tip of the catheter, which was not different between treated groups (Fisher's exact test; P = 0.26). After 5 weeks of fluid instillation, 7 and 11 rats in PDF and AG-hydrochloride had remained and all (seven) untreated animals were used for analysis.
Kinetic study on scavenging of GDPs by aminoguanidines
In a time-course study, four different concentrations of AG-bicarbonate and AG-hydrochloride (0.01, 0.1, 1 and 10 g/l) were added to the PDF and the amount of three different GDPs, namely GO, MGO and 3-DG, were measured. Addition of 0.01 g/l of both kinds of AG could not scavenge GDPs from PDF. Supplementation of 0.1 g/l of both AGs for 23 days scavenged GO and MG effectively, but had barely any effect on 3-DG concentrations. Moreover, 1 g/l of both AGs scavenged effectively GO, MGO and 3-DG from the PDF, but with different kinetics. AG-bicarbonate scavenged GDPs completely within 23 days, whereas AG-hydrochloride did not (Figure 1). A higher concentration (10 g/l) of AG-bicarbonate did not solve in PDF, while this concentration of AG-hydrochloride scavenged GDPs faster than lower concentrations (data not shown). Therefore, to ensure the quenching of GDPs, 1 g/l AG-bicarbonate was added to PDF 23 days before fluid injection and 1 g/l AG-hydrochloride was supplemented 5 days before fluid instillation. The supplementation of 1 g/l AG-bicarbonate resulted in an elevated pH value of the PDF (from 5.2 to 8.5), whereas AG-hydrochloride had no effect on pH as it remains at 5.2.
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AGE-accumulation
The vascular walls and mesothelial cells in mesenteric tissues from rats exposed to PDF were glycated, as shown by a positive staining with anti-AGE monoclonal antibody 4B5, whereas barely any staining was found in control rats and rats treated with AG-bicarbonate or AG-hydrochloride after staining with monoclonal antibody against AGEs (Figure 6).
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Mesothelial cells
The density of mesothelial cells on the liver was significantly increased in rats instilled with PDF, compared to control animals (median of 1656 vs 1406 cells/mm2; P<0.008). The addition of either AG to PDF did not influence the PDF-induced regenerative response of the mesothelial cells on the liver, as no significant differences were found between rats exposed to PDF and animals treated with PDF supplemented with AG-bicarbonate (P>0.99) or AG-hydrochloride (P = 0.52). Thus, compared to control rats, more mesothelial cells were found in the AG-bicarbonate group, although not quite statistically significant (P = 0.09), and in the AG-hydrochloride groups (P<0.0002).
The integrity of the mesothelial cell layers covering different peritoneal tissues (omentum, mesentery, liver, diaphragm and parietal peritoneum) were inspected by electron microscopy (Figure 7). The mesothelial cell layer on the peritoneal surface of both visceral and parietal peritoneum of control animals showed a normal (intact) appearance, thus mesothelial cells and their microvilli were present (Figure 7A). In contrast to control animals, chronic exposure to PDF resulted in a severe damage to the mesothelial cell layer covering the peritoneal membrane, which was characterized by focal loss of microvilli, vacuolization or complete loss of mesothelial cells (Figure 7B). Addition of both kinds of AG did not prevent the PDF-induced mesothelial cell damage (Figure 7C), with no difference between both AGs, and thus, severe damage was found in the AG-bicarbonate and AG-hydrochloride groups. In general, damage to the visceral mesothelial cell layer covering the omentum, mesentery, diaphragm and liver found in all treated groups was profound, whereas, this damage was clearly milder in the parietal peritoneum (data not shown).
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Discussion |
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Although the dysfunction of the vascular endothelium is a known risk factor for patients suffering from hyperglycaemia [3], little attention has been paid to the study of the leukocyteendothelium interactions of the peritoneum during chronic PD. Therefore, we focused on the study of the peritoneal microcirculation, including leukocyteendothelium interactions as well as vascular permeability, providing useful information regarding the functional status of the endothelium during peritoneal dialysis. We realized that the performance of intravital microscopy did not permit the study of the peritoneal transport parameters in the same animals; however, this has already been studied by others [11]. In the present study, the number of rolling leukocytes in mesenteric venules was approximately 4-fold increased in rats exposed to PDF, which could not be explained by changes in blood flow or systemic leukocyte count [13]. However, a recent study showed a decreased number of rolling leukocytes upon exposure to PDF in rats [15], which might be explained by a difference in experimental approach, i.e. acute vs chronic PDF exposure. Several lines of evidence suggest that chronic exposure to GDPs and/or AGE depositions are involved in the endothelial activation. We and others showed increased levels of soluble endothelial adhesion molecules in continous ambulatory peritoneal dialysis (CAPD) patients [16,17]. We recently showed that exposure of mesothelial cells to reactive aldehydes like MG and 3-DG resulted in cellular uptake of these compounds followed by an inflammatory response of the cells, including an upregulation of the vascular cell adhesion molecule-1 [17], which can potentially be involved in the leukocyte rolling process. The same holds true for AGEs [17]. Thus, we concluded that both neutralization of GDPs and prevention of AGE formation by AG might explain the reduced endothelial activation in this study. In line with this conclusion, our preliminary data revealed less profound leukocyteendothelium interactions in the mesenteric venules after chronic exposure to a GDP-poor, bicarbonate/lactate-buffered PDF, compared to the conventional PDF containing a high GDP content in our model (data not shown). Furthermore, in our study, the level of adherent leukocytes was not affected by PDF with or without AG, indicating that the elevated level of rolling found in the PDF group was not related to bacterial infection, which is always accompanied by enhanced leukocyte adhesion.
It has been shown that AG strongly reduced fibrosis in the lungs [8]. In agreement with this study, we now show that the addition of AG to PDF completely prevented the PDF-induced peritoneal fibrosis in the parietal peritoneum and largely reduced fibrosis in the mesentery in vivo.
Due to the bioincompatible effects of the conventional PDF, more physiological solutions have become available. We [4] and others [18] have already shown that the bicarbonate/lactate buffered, glucose-based PDF (Physioneal) better preserved the function and morphology of the peritoneum in the experimental PD model. The desirable effects of this solution can, partly, be explained by the formation of a lower amount of GDPs. However, the supplementation of AG appears to produce more beneficial effects on the peritoneal morphology and microvasculature than Physioneal. For instance, AG could almost completely inhibit the PDF-induced fibrosis, whereas animals treated with Physioneal developed substantially more fibrosis than control rats [4,18]. Furthermore, our preliminary data revealed more rolling leukocytes in the mesenteric venules after chronic treatment with low GDP content Physioneal compared to rats exposed to the conventional PDF supplemented with AG-bicarbonate (it will be communicated separately). Therefore, we suggest that the beneficial effects of AG on the peritoneal morphology and microcirculation are not only due to its action on GDPs, but also on AGEs [8] and/or NO production.
Besides common beneficial effects of both AG formulations, they also differ from each other in some aspects. First, exposure to PDF supplemented with AG-bicarbonate resulted in a clear albumin leakage in some of the mesenteric venules. No vascular leakage, however, was found in the AG-hydrochloride group. Second, the supplementation of AG-bicarbonate significantly reduced the PDF-induced angiogenesis in both the omentum and in the parietal peritoneum. In contrast, AG-hydrochloride had no effect on PDF-induced angiogenesis. Since major differences between both PDF-dissolved AG formulations are their acidity and the bicarbonate concentration, these data suggest that this differential response is related either to pH or the presence of bicarbonate. On one hand, it has been described that alkalosis is a driving force behind Vascular endothelial growth factor (VEGF) production [19]. This might also explain the increased microvascular permeability upon AG-bicarbonate supplementation to PDF. VEGF is also involved in angiogenesis, which we, however, showed to be repressed by AG-bicarbonate. Hence, at present the relation between high pH of PDF/AG-bicarbonate, albumin leakage and repressed angiogenesis remains unclear. On the other hand, the quick capacity of the peritoneum to correct the pH of the solutions has been well recognized. Therefore, we suggest that the differential response of both formulation of AG is likely due to the protective effect of bicarbonate.
It is worth noting that AG has an inhibitory effect on not only GDPs and AGEs, but also on inducible NO synthase [8], although we did not demonstrate that in this study. From the elegant studies of Devuyst et al. [3], it is known that NO is a critical determinant in the induction of functional and structural changes of the peritoneal membrane during PD and peritonitis. Beneficial effects of AG found in the present study could thus partly be explained by the ability of AG to inhibit NO synthesis. Furthermore, we cannot exclude the possibility that the reduction of PDF-induced leukocyte rolling by AG is (partly) due to a reduced NO activity. Since it is known that increased NO activity leads to reduced leukocyte rolling and adhesion [20], we believe that the AG-induced decreased rolling is most likely due to its inhibitory effects on GDPs and/or AGEs rather than on NO synthesis.
Despite the fact that AG was able to prevent various pathological changes, the oral application of high concentrations of AG was found to be toxic in diabetic patients [7]. Specifically, AG trapped pyridoxal and subsequently caused neurotoxic complications and vitamin B6 deficiency in experimental models for diabetic nephropathy [7]. In this respect, AG-pyridoxal adduct found to be superior to AG alone, as it not only prevents vitamin B6 deficiency, but is also better at controlling diabetic nephropathy [7]. Nevertheless, in PD patients, AG or related compounds, such as AG-pyridoxal adduct, could be supplemented with PDF and applied locally and in much lower concentrations than in the diabetes field. More importantly, our findings highlight the involvement of GDPs, AGEs and/or NO in the pathophysiology of PD-related complications.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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