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NDT Advance Access originally published online on May 13, 2008
Nephrology Dialysis Transplantation 2008 23(10):3302-3306; doi:10.1093/ndt/gfn272
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


RAGE expression in the human peritoneal membrane

Lars P. Kihm1, Dennis Wibisono1, Sandra Müller-Krebs1, Friederike Pfisterer1, Christian Morath1, Marie L. Gross2, Michael Morcos1, Yuri Seregin1, Angelika Bierhaus1, Peter P. Nawroth1, Martin Zeier1 and Vedat Schwenger1

1 Department of Nephrology and Endocrinology 2 Department of Pathology, University of Heidelberg, Germany

Vedat Schwenger, Department of Nephrology, University of Heidelberg, Im Neuenheimer Feld 162, 69120 Heidelberg, Germany. Tel: +49-6221-91120; Fax: +49-6221-9112-229; E-mail: vedat.schwenger{at}med.uni-heidelberg.de



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Experimental animal models have demonstrated that the interaction of advanced glycation end-products (AGE) with their receptor RAGE is, at least in part, responsible for peritoneal damage. This study investigates the in vivo expression of RAGE in the peritoneal membrane of uraemic human patients.

Methods. Peritoneal biopsies of 89 subjects (48 uraemic and 41 healthy age-matched patients) were examined. The expression of CD3, IL-6, activated NF{kappa}Bp65, VEGF, transforming growth factor (TGF)-β1, smooth-muscle actin (SMA), methylglyoxal (MGO) and RAGE was analysed immunohistochemically. Additionally, in 4 of the 48 uraemic patients, peritoneal biopsies were repeated after 15 months at the time of catheter removal to analyse the above parameters and the extent of NF{kappa}B-binding activity determined by electrophoretic mobility shift assay (EMSA) in the long-term follow-up.

Results. In comparison to the healthy controls, uraemic patients showed a significant increase in fibrosis, angiogenesis, submesothelial thickness, MGO-derived protein adducts, RAGE, IL-6, VEGF, TGF-β1, SMA and NF{kappa}Bp65 in their peritonea. Four patients, followed up longitudinally from peritoneal dialysis (PD) catheter insertion to removal, demonstrated further significant increase in the above parameters, particularly in RAGE expression and NF{kappa}B activation.

Conclusions. Along with a higher expression of several indicators for inflammation, angiogenesis, fibrosis and AGE accumulation, the peritoneal membrane of the uraemic patients showed an increased submesothelial thickness and a marked induction of RAGE expression and NF{kappa}B-binding activity, which both further increased after PD treatment. These findings in human peritoneum support the concept of the AGE–RAGE interaction being crucial in peritoneal damage due to uraemia and PD.

Keywords: AGE; peritoneum; peritoneal damage; RAGE



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Carbonyl stress and resulting advanced glycation end-products (AGE) accumulation contribute to the damage of peritoneal membranes in peritoneal dialysis (PD) [1]. In addition to the altering effect of AGE on peritoneal integrity due to the cross-reaction with matrix molecules, such as collagen, multiple receptor-dependent damaging pathways have been identified. The interaction of AGE with RAGE, a well-characterized receptor of AGE [2], has been identified as a major cause of peritoneal damage in diabetic [3] and uraemic rats [4], and particularly in non-diabetic mice [5]. AGE–RAGE interaction activates key signal transduction pathways and subsequently induces the transcription of mediators for inflammation [5–7], angiogenesis [8] and fibrosis [3,5]. In vitro RAGE expression has recently been described in human mesothelial cells [9]. However, until now, no data are available on RAGE expression in human peritoneal tissue. Therefore this study was designed to investigate RAGE expression in vivo in the peritoneal membrane of uraemic patients.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Patients
The study protocol was approved by the local ethics committee, and a written informed consent was given by all participating patients. Peritoneal tissue was obtained from 41 patients with normal renal function without peritoneal alterations during unrelated abdominal surgery. Reasons of surgery were as follows: 17 pancreatic carcinoma, 10 colorectal carcinoma, 4 stomach-oesophageal carcinoma, 2 gall-bladder carcinoma, 2 liposarcoma and 6 hernias. Peritoneal tissue was taken from the left-sided omentum near to the mesenteric root, and from uraemic patients who had never previously undergone dialysis at the time of peritoneal catheter insertion. After a mean duration of 15 months of PD treatment, a peritoneal biopsy was collected at the time of catheter removal from the left-sided omentum near to the mesenteric root in four PD patients who received a kidney allograft and who had not previously exhibited any signs of peritonitis.

Material
The following primary antibodies were used: Anti-CD3 (DakoCytomation GmbH, Hamburg), Fast Red (DakoCytomation GmbH, Hamburg), Goat-Anti-Rabbit Link (Biogenex, San Ramon, CA, USA), Anti-IL-6 (Biotrend Chemikalien GmbH, Cologne), Anti-Methylglyoxal-AGE (Biologo, Kronshagen), Multilink (Biogenex, San Ramon, CA, USA), Anti-NF{kappa}Bp65 (Chemicon International Inc., Temecula, Canada), Rabbit-Anti-Sheep Link (Jackson ImmunoResearch Europe Ltd, Soham, Cambridgeshire, UK), Anti-{alpha}SMA (Sigma Aldrich, St Louis, MO, USA), Anti-RAGE (R&D Systems, Minneapolis, MN, USA), Anti- TGF-β1 (Santa Cruz Biotechnology Inc., USA) and Anti-VEGF (Genzyme Diagnostics, Cambridge, MA, USA).

Histological and immunohistochemical staining
Visceral peritoneal tissue samples were fixed in 6% phosphate-buffered formaline (pH 7.4) and embedded in paraffin. Four-µm-thick tissue sections were stained with haematoxylin & eosin, periodic acid Schiff reagent and Picro-Sirius red staining for detection of fibrous tissue and investigated by light microscopy. For immunohistochemical staining, tissue sections were deparaffinized, rehydrated and incubated in Tris-buffered saline (TBS). The following antibodies were used: methylglyoxal-AGE (MGO-AGE), CD3, IL-6, VEGF, NF{kappa}Bp65, and transforming growth factor (TGF)-β1. RAGE was pretreated in rabbit serum. The sections were incubated with the primary antibody overnight at 4°C and thereafter stained with the secondary antibody, AP-conjugated streptavidin, and fast red. Replacement of the primary antibodies with TBS served as control.

Quantification of histological and immunohistochemical findings
For each visceral peritoneal specimen, >30 cross-sections were evaluated in a blinded manner. Interstitial fibrosis was analysed using Sirius red-stained sections and a semi-quantitative score system: 0—no interstitial fibrosis; 1—mild interstitial fibrosis; 2—moderate interstitial fibrosis with thickening of the peritoneum and 3—severe interstitial fibrosis with marked thickening of the peritoneum. The maximal thickness of the submesothelial compact zone (in micrometers) was measured in sections oriented perpendicular to the serosal surface. For determination of inflammatory cell number per area, CD3+ T cells per area and vessel number per area, all cells/vessels on a 121-point grid (Leitz, Wetzlar, Germany) were counted (area 0.028 µm2) using the CD3/VEGF staining. For the analysis of VEGF, TGFβ-1, MGO-AGE, RAGE, IL-6, NF{kappa}Bp65 and {alpha}SMA, each peritoneal specimen was screened by light microscopy in a blinded fashion and scored using a semi-quantitative system, with 0—no reaction; 1—mild reaction; 2—moderate reaction and 3—severe reaction, according to a defined area (0.028 µm2). All samples were evaluated three times by one blinded observer. The results were re-evaluated as a random inspection by a second observer. The intra- and inter-observer variability was <10%.

Electrophoretic mobility shift assay
Peritoneal specimens were quick-frozen in liquid nitrogen, homogenized, transferred into cold buffer A (10 mM Hepes-KOH, pH 7.9, at 4°C, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 1 mM EDTA, 0.2 mM PMSF, 0.6% Nonidet® P-40), incubated on ice for 10 min and centrifuged for 5 min at 5400 g at 4°C. The supernatant was discarded, and the nuclear pellet was resuspended in 100 µl of buffer B (25% glycerol, 20 mM Hepes-KOH, pH 7.9, at 4°C, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, 2 mM benzamidine, 5 mg/ml leupeptin) and incubated on ice for 20 min. Cellular debris was removed after 2 min of centrifugation at 4°C, and the supernatant was quick-frozen at –80°C. Protein concentrations were determined by the Bradford assay. Nuclear extract (10 µg) was included in the binding reaction. Binding to an NF{kappa}B consensus oligonucleotide (5'-AGTTGAGGGGACTTTCCAGGC-3') was performed in 10 mM Hepes, pH 7.5, containing 0.5 mM EDTA, 100 mM KCl, 2 mM dithiothreitol, 2% glycerol, 4% Ficoll 400, 0.25% Nonidet P-40, 1 mg/ml BSA (DNAse-free) and 0.3 µg/µl poly(dI/dC) in a total volume of 20 µl. Specificity of binding was ascertained by comparison with a 160-fold molar excess of unlabelled consensus oligonucleotides and by characterisation with specific polyclonal antibodies (Santa Cruz Biotechnology).

Statistical analysis
All values are expressed as mean ± standard error of the mean (SEM). ANOVA and unpaired t-tests were used as appropriate to test the statistical significance. The significance level was set at P < 0.05. All statistical evaluations and representations were performed using PRISM (version 4.0, GraphPad Software Inc.).



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Patients
Peritoneal biopsies were collected from 89 individuals, including 41 healthy controls and 48 uraemic patients. Age, gender, incidence of diabetes and the MDRD-GFR of the patients are given in detail in Table 1.


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  		Table 1 Patient characteristics

 
AGE and RAGE expression
Immunohistochemistry for MGO-protein adducts (MGO-AGE) and RAGE was significantly increased in uraemic patients compared to control patients (Table 2, Figure 1). During the course of PD, the expression of MGO-AGE and RAGE was further enhanced (Table 2).


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  		Table 2 Histological and immunohistochemical analyses of the peritoneum

 

Figure 1
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  		Fig. 1 Representative light micrographs of staining for MGO-AGE of the peritoneal tissue from healthy controls (a), uraemic patients (b), RAGE of peritoneal tissue from healthy controls (c) and uraemic patients (d).

 
Inflammation and electrophoretic mobility shift assay
The peritoneum of uraemic patients demonstrated a higher expression of inflammatory mediator substances, such as NF{kappa}B and the NF{kappa}B-controlled cytokine IL-6 in comparison to patients with normal kidney function. There was no significant change in CD3+ cell number (Table 2). PD treatment was associated with a significant increase in CD3+ leukocytes and an enhanced upregulation of NF{kappa}Bp65 and IL-6 (Table 2). The increase in NF{kappa}B activation and nuclear translocation was confirmed by the electrophoretic mobility shift assay (EMSA), which detected elevated peritoneal DNA binding activity for NF{kappa}B during the course of PD (Table 3, Figure 2).


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  		Table 3 Electrophoretic mobility shift assay of NF{kappa}B activation—optical density

 

Figure 2
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  		Fig. 2 Electrophoretic mobility shift assay (EMSA) for activated NF{kappa}B in peritoneal tissue of four PD patients (#1–4) at baseline (t1) and treated (t2).

 
Angiogenesis
The peritoneal tissue of the uraemic patients revealed an increased number of vessels per area and a higher expression of VEGF compared to the healthy controls. The number of peritoneal vessels as well as VEGF expression was further enhanced after PD treatment (Table 2).

Fibrosis, submesothelial thickness and expression of {alpha}SMA
The fibrosis score obtained from the Picro-Sirius red staining and submesothelial thickness of the peritoneum were significantly elevated in uraemic patients and further increased after 15 months of PD therapy. The expression of TGF-β1 and {alpha}SMA was strongly upregulated in the peritoneum of uraemic patients and significantly increased after the course of PD therapy (Table 2).



   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Peritoneal damage in uraemic patients before the initiation of PD is supposed to be a setting in which RAGE ligands accumulate in the presence of increased RAGE expression. This study shows for the first time accumulation of MGO-AGE, paralleled by an enhanced expression of RAGE and in parallel an augmentation of inflammation (indicated by IL-6), angiogenesis (with enhanced expression of VEGF) and fibrosis (indicated by increased levels of TGF-β1 and {alpha}SMA) in peritoneal tissue of patients undergoing PD.

Highest RAGE expression was evident in patients undergoing PD treatment for several months when compared to uraemic patients not yet on PD; healthy age-matched controls showed the lowest RAGE expression in peritoneal tissue.

Since the AGE–RAGE interaction activates numerous signalling cascades and therefore results in an ascending spiral of tissue damage in animal experimental models [2], it is regarded as a major cause of peritoneal damage. However, only little is known about the relevance of this concept in the clinical situation. RAGE has been localized in human peritoneal mesothelial cells (HPMC) in vitro [9], confirming earlier observations of RAGE mRNA synthesis in HPMC in vitro [10]; however, biopsy studies from human peritoneum have not yet been performed. To the best of our knowledge, our results are the first to demonstrate that increased peritoneal MGO-AGE accumulation is paralleled by an enhanced expression of RAGE in the peritoneal membrane of uraemic patients in vivo. Over the course of PD with a mean duration of 15 months, peritoneal MGO-AGE and RAGE expression further increased.

However, there is no correlation between clinically estimated peritoneal equilibration tests performed 6 months after the initiation of PD (2 h D/P creatinine as well as 2 h D/P urea P > 0.05, data not shown) and histologically estimated RAGE expression. Whether this was due to the small number of samples or due to the relatively short follow-up cannot be dissolved. A further explanation might be that RAGE certainly plays a pivotal role in the pathogenesis of peritoneal damage in PD, but other factors contribute to the peritoneal damage, too.

Activation of NF{kappa}B is considered to be crucial in inflammation. Since RAGE has the unique ability to sustain NF{kappa}B activation through de novo synthesis of NF{kappa}Bp65-mRNA [6], engagement of RAGE results in perpetuated cell activation and functions as a master switch able to convert normally short-lasting proinflammatory responses into long-lasting cellular dysfunction [6,11]. In the peritoneum of uraemic patients, we found significantly increased levels of NF{kappa}Bp65 when compared to healthy age-matched subjects. These findings were accompanied by an elevation of NF{kappa}B-controlled cytokine expression.

In addition to this inflammatory state, the peritoneal membrane of uraemic patients revealed an increased number of blood vessels per area accompanied by an enhanced expression of VEGF. Although a correlation between the accumulation of AGE and VEGF expression has been demonstrated by de Vriese et al., the inhibition of the AGE–RAGE interaction with anti-RAGE antibodies did not prevent peritoneal neovascularization [3]. However, this study supports the hypothesis of upregulation of peritoneal angiogenesis by VEGF via the AGE–RAGE interaction related to uraemia [5,8,12–15]. The hypothesis is further strengthened by earlier observations of a PD-related peritoneal angiogenesis [12] and confirmed by our data, which revealed an elevated VEGF expression and an increased blood vessel count at the time of catheter removal compared to the level at catheter insertion. Besides this, there is evidence for other crucial pathways inducing peritoneal angiogenesis due to uraemia and PD treatment such as mast cell-derived activation of angiogenic factors [16].

The interaction of AGEs with RAGE has been shown to induce expression of TGF-β1 and thus to contribute to peritoneal fibrosis [3–5]. TGF-β1 is suspected to be pivotal in the process of epithelial-to-mesenchymal transition (EMT), through which cells of epithelial origin acquire myofibroblastic characteristics [17]. Myofibroblasts are involved in all conditions of pathological fibrosis and are typically identified by their expression of {alpha}SMA [18]. Along with accentuated interstitial fibrosis, our data showed a significant increase in TGF-β1 and {alpha}SMA in the peritoneum of uraemic patients. Fibrosis and expression of TGF-β1 and {alpha}SMA were significantly increased after PD treatment. To what extent an increase in {alpha}SMA contributes to EMT cannot be clarified. Nevertheless, our findings might support the hypothesis that RAGE-mediated activation of transcription factors, such as NF{kappa}B or TGF-β1 [3] might result in an ascending spiral of tissue damage and ultimately PD failure [19].

In conclusion, this study confirms an increase in AGE and RAGE in the peritoneal tissue of patients undergoing PD and thus continues to support the concept of AGE–RAGE interactions playing a pivotal role in peritoneal damage due to uraemia and particularly PD in vivo.



   Acknowledgments
 
The authors wish to thank Professor Dr Jan Schmidt from the Department of Surgery and Ms Heike Ziebart from the Department of Pathology, both at the University of Heidelberg, Germany. This work was supported in part by a grant from the Else-Kroener-Fresenius Foundation (Bad Homburg, Germany) and a grant from the Juvenile Diabetes Research Foundation (JDRF to A.B. and P.P.N.).

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Nakayama M, Kawaguchi Y, Yamada K, et al. Immunohistochemical detection of advanced glycosylation end-products in the peritoneum and its possible pathophysiological role in CAPD. Kidney Int (1997) 51:182–186.[Web of Science][Medline]
  2. Yan SF, Ramasamy R, Naka Y, et al. Glycation, inflammation, and RAGE: a scaffold for the macrovascular complications of diabetes and beyond. Circ Res (2003) 93:1159–1169.[Abstract/Free Full Text]
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  5. Schwenger V, Morath C, Salava A, et al. Damage to the peritoneal membrane by glucose degradation products is mediated by the receptor for advanced glycation end-products. J Am Soc Nephrol (2006) 17:199–207.[Abstract/Free Full Text]
  6. Bierhaus A, Schiekofer S, Schwaninger M, et al. Diabetes-associated sustained activation of the transcription factor nuclear factor-kappaB. Diabetes (2001) 50:2792–2808.[Abstract/Free Full Text]
  7. Schmidt AM, Hori O, Cao R, et al. RAGE: a novel cellular receptor for advanced glycation end products. Diabetes (1996) 45(Suppl_3):S77–S80.[Web of Science][Medline]
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Received for publication: 21. 6.07
Accepted in revised form: 18. 4.08


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