DNA Microarray Analysis of the Epithelial Mesenchymal Transition of Mesothelial Cells in a Rat Model of Peritoneal Dialysis

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1 Advances in Peritoneal Dialysis, Vol. 27, 2011 Toshimi Imai, Ichiro Hirahara, Yoshiyuki Morishita, Akira Onishi, Makoto Inoue, Shigeaki Muto, Eiji Kusano DNA Microarray Analysis of the Epithelial Mesenchymal Transition of Mesothelial Cells in a Rat Model of Peritoneal Dialysis Long-term peritoneal dialysis induces peritoneal hyperpermeability, and the subsequent loss of ultrafiltration causes patients to discontinue peritoneal dialysis. Glucose degradation products (GDPs) in peritoneal dialysis fluids (PDFs) are probably one of the primary causes for peritoneal injury. In the present study, we used a transcriptome analysis to determine the mechanism of peritoneal injury by GDPs. Rats were administered 20 mmol/l methylglyoxal (MGO) in PDF or 20 mmol/l formaldehyde in PDF (100 ml/kg) intraperitoneally for 21 days. The peritoneal membrane in rats that received MGO showed increased thickness and fibrosis. Mesenchymal-like cells over-proliferated on the surface of the peritoneum. A DNA microarray analysis revealed that the expression of 168 genes had increased by more than a factor of 4. The upregulated genes included those that code for extracellular matrix components (such as types III and IV collagen, among others), cell division cycle 42 (Cdc42), an enabled/vasodilator stimulated phosphoprotein-like protein [Ena/VASP (Evl)], and actin-related protein 2/3 complex subunits (Arp2/3). In conclusion, a rat model of peritoneal injury by GDPs induced mesothelial cells to redifferentiate and proliferate, with upregulation of Cdc42, the Evl Ena/VASP, and Arp2/3, suggesting that GDPs induce fibrous thickening of the peritoneal membrane by redifferentiation of mesothelial cells, resulting in hyperpermeability of the peritoneum. Key words Peritoneal injury, glucose degradation products, methylglyoxal, epithelial mesenchymal transition, Cdc42 From: Division of Nephrology, Department of Medicine, Jichi Medical University, Tochigi, Japan. Introduction Peritoneal dialysis (PD) is an established therapy for patients with end-stage renal disease, but successful long-term PD is difficult because of peritonitis and peritoneal dysfunction after exposure to PD fluids (PDFs). Many PDFs contain glucose degradation products (GDPs) as a result of heat sterilization. These GDPs for example, methylglyoxal (MGO) and formaldehyde (FA) are thought to damage the peritoneum, resulting in peritoneal fibrosis and loss of ultrafiltration (1,2), but the pathogenesis remains unclear. We therefore administered PDFs with GDPs intraperitoneally to rats every day and used a transcriptome analysis to determine the mechanism of peritoneal injury by GDPs. Methods Intraperitoneal injection of PDFs in animals The experiments were performed using male Sprague Dawley Crj:(CD)SD-IGS rats, 5 weeks of age, weighing about 215 g (Charles River Japan, Kanagawa, Japan), according to the US National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animals were housed in an air-conditioned room kept at a constant temperature of 23 ± 2 C, with a relative humidity of 50% ± 10%, a 12-hour light/dark cycle, and free access to sufficient pellet food and water. The PDFs were prepared by adding 20 mmol/l MGO or 20 mmol/l FA to a PDF (2.5% glucose, 100 mmol/l NaCl, 35 mmol/l sodium lactate, 2 mmol/l CaCl 2, and 0.7 mmol/l MgCl 2 at ph 5.0), which was then sterilized by filtration. The rats were divided into three groups (n = 6 per group) and given the following solutions intraperitoneally for 21 days: group 1, 100 ml/kg of PDF without

2 12 Imai et al. GDPs; group2, 100 ml/kg of PDF containing MGO; group 3, 100 ml/kg of PDF containing FA. On the 22nd day of the experiment, the parietal peritoneum was sampled to perform either a histologic analysis or gene expression analysis. Analysis of gene expression by DNA microarray Gene expression in the parietal peritoneum was assessed using a DNA microarray. Total RNA was extracted from the peritoneum using the RNeasy Kit with the DNase Set (Qiagen, Hilden, Germany). Total RNA from the peritoneum was reverse transcribed using Superscript reverse transcriptase (Gibco, Rockville, MD, U.S.A.). The resulting complementary DNA was analyzed using microarrays representing approximately 24,000 rat genes (NimbleGen, Reykjavík, Iceland). Statistics Data are presented as mean ± standard deviation. Statistical comparisons use an analysis of variance. A p value less than 0.05 was considered significant. Results Cellular proliferation was prominent in the surface of the peritoneum in MGO-treated rats, with deposition of collagen fibers and neoangiogenesis [Figure 1(B,E)]. By contrast, FA-treated rats showed only mild fibrotic changes, with interstitial edema and no cellular proliferation [Figure 1(C,F)]. In control rats, the surface of the peritoneum was lined with a single layer of mesothelial cells [Figure 1(A,D)]. In MGO-treated rats, the expression of 168 genes in parietal peritoneum increased by a factor of more than 4. The upregulated genes included those coding for extracellular matrix components such as types I, III, IV, V, and VI collagen and fibronectin (Figure 2, Table I). In addition, expression of some proteins related to remodeling of the actin skeleton was upregulated. In MGO-treated rats, compared with control rats, gene expression of Cdc42, an enabled/ vasodilator stimulated phosphoprotein-like protein [Ena/VASP (Evl)], and actin-related protein 2/3 complex (Arp2/3) subunit 1B were increased by factors of 2.7, 2.2, and 6.8 respectively. The expression of many interleukins and cytokines tended to increase, but the difference was not significant, except in the case of transforming growth factor β. Discussion Long-term PD is limited by peritoneal dysfunction after exposure to dialysis solutions. Peritoneal dysfunction is associated with structural changes: mesothelial loss and thickening of the submesothelial compact zone. Glucose degradation products, one of the most harmful substances in PDFs, are thought to induce peritoneal dysfunction and structural change (1,2). The GDPs MGO and FA, which can both be found in commercially available PDFs, are characterized by severe toxicity to the peritoneum (1). Therefore, they are useful for elucidating the mechanism of peritoneal injury by GDPs. The GDPs induced increases in peritoneal thickness and decreases in peritoneal function. The peritoneal thickening was characterized by cellular and fibrous proliferation, which was consistent with the results of the DNA microarray analysis. The expression of collagen fibers and cytoskeletal proteins was enhanced in MGO-treated rats, but not in FA-treated rats. Tissue inhibitor of matrix metalloproteinase 1 inhibits the degradation of extracellular matrix by matrix metalloproteinases. Gene expression of that inhibitor was found to have significantly increased, which may account for the fibrosis and proliferation in MGO-treated rats. Mesenchymal-like cells were recognized on the surface of the peritoneum in MGO-treated rats but not in FA-treated rats. These cells are derived by the epithelial mesenchymal transition (EMT) of mesothelial cells (2). In mesothelial cells, MGO may induce EMT. Transforming growth factor β, enhanced in this study, is a master regulator of EMT (3). Protrusive structures formed by migrating and invading cells are called filopodia, lamellipodia, and invadopodia or podosomes (4). These morphology changes occur during EMT in carcinoma cells and are associated with actin cytoskeleton remodeling via Cdc42 (5), which plays an essential role in invadopodia formation. Lamellipodia contain a large actin assembly controlled mainly by Arp2/3, which is able to nucleate the polymerization of new actin filaments (6), and filopodia formation is associated with Cdc42 (5). The EMT of mesothelial cells may be accompanied by actin cytoskeleton remodeling, as occurs in carcinoma cells, because EMT gives mesothelial cells a migratory capacity (3,7). In MGO-treated rats, compared with control rats, expression of Cdc42, Evl, and Arp2/3 increased. Membrane type 1 matrix metalloproteinase

3 DNA Microarray Analysis of EMT of Mesothelium 13 1 Histopathology findings in parietal peritoneum of the study rats. (A) Control rat, hematoxylin and eosin (HE) stain. (B) Methylglyoxal (MGO) treated rat, HE stain. (C) Formaldehyde (FA) treated rat, HE stain. (D) Control rat, Azan stain. (E) MGO-treated rat, Azan stain. (F) FA-treated rat, Azan stain. All 100 magnification. figure 2 Scatter plot of gene expression in parietal peritoneum of the study rats. Gene expression levels in parietal peritoneum were analyzed using a DNA microarray. (A) Control rats compared with methylglyoxal (MGO) treated rats. (B) Control rats compared with formaldehyde (FA) treated rats. figure

4 14 Imai et al. ta b l e i Changes in gene expression of proteins in peritoneum exposed to glucose degradation products Parameter Change factor MGO vs. C FA vs. C MGO vs. FA Extracellular matrix Collagen type I α /7 8.3 Collagen type I α Collagen type III α Collagen type IV α Collagen type V α Collagen type V α Collagen type V α Collagen type VI α Collagen type VI α Collagen type VI α Fibronectin Matrix metalloproteinase (MMP) MMP Membrane type 1 MMP Tissue inhibitor of MMP Cytokine (growth factor) Epidermal growth factor Fibroblast growth factor Fibroblast growth factor Transforming growth factor β Vascular endothelial growth factor Interleukin Interleukin Interleukin Interleukin Interleukin Interleukin Interleukin Interleukin Cytoskeleton-related proteins Cdc Evl Actin-related protein 2/3 complex (Arp2/3) subunit 1B Dedicator of cytokinesis Tubulin α subunit Tubulin α Actin β Myosin, heavy polypeptide Calponin Tropomyosin Filamin A Fibulin Housekeeping protein GAPDH C = control group; MGO = methylglyoxal-treated group; FA = formaldehyde-treated group; Cdc42 = cell division cycle 42, Evl = protein of the enabled/vasodilator-stimulated phosphoprotein-like family; GAPDH = glyceraldehyde 3-phosphate dehydrogenase. (MT1-MMP) is a key enzyme in tumor-cell invasion (8), which is reported to be expressed in invadopodia after EMT. The Ena/Vasp family protein Evl, which is associated with remodeling of the actin cytoskeleton, is localized to the leading edges of lamellipodia (9). The foregoing results suggest that mesothelial

5 DNA Microarray Analysis of EMT of Mesothelium 15 cells undergo EMT and acquire motility by a mechanism similar to that in carcinoma cells, and that they move from the surface of the peritoneum to the submesothelial compact zone. Conclusions Glucose degradation products induced mesothelial cells to redifferentiate and proliferate, with upregulation of Cdc42, Arp2/3, MT1-MMP, and Evl. These results suggest that GDPs in PDFs induce peritoneal fibrous thickening through redifferentiation of mesothelial cells, resulting in hyperpermeability of the peritoneum. A Western blot analysis of these proteins is necessary to reveal the mechanisms of morphology change in mesothelial cells. Acknowledgment This study was funded by Terumo, Tokyo, Japan. Disclosures There was no financial involvement that might raise any question of bias in regard to this work or the conclusions, implications, and opinions stated therein. References 1 Hirahara I, Kusano E, Yanagiba S, et al. Peritoneal injury by methylglyoxal in peritoneal dialysis. Perit Dial Int 2006;26: Hirahara I, Ishibashi Y, Kaname S, Kusano E, Fujita T. Methylglyoxal induces peritoneal thickening by mesenchymal-like mesothelial cells in rats. Nephrol Dial Transplant 2009;4: Yáñez Mó M, Lara Pezzi E, Selgas R, et al. Peritoneal dialysis and epithelial-to-mesenchymal transition of mesothelial cells. N Engl J Med 2003;348: Yamaguchi H, Condeelis J. Regulation of the actin cytoskeleton in cancer cell migration and invasion. Biochim Biophys Acta 2007;1773: Yilmaz M, Christofori G. EMT, the cytoskeleton, and cancer cell invasion. Cancer Metastasis Rev 2009;28: Pollard TD. Regulation of actin filament assembly by Arp2/3 complex and formins. Annu Rev Biophys Biomol Struct 2007;36: Margetts PJ, Bonniaud P, Liu L, et al. Transient overexpression of TGF-β1 induces epithelial mesenchymal transition in the rodent peritoneum. J Am Soc Nephrol 2005;16: Poincloux R, Lizárraga F, Chavrier P. Matrix invasion by tumour cells: a focus on MT1 MMP trafficking to invadopodia. J Cell Sci 2009;122: Kwiatkowski AV, Gertler FB, Loureiro JJ. Function and regulation of Ena/VASP. Trends Cell Biol 2003;13: Corresponding author: Ichiro Hirahara, Division of Nephrology, Department of Medicine, Jichi Medical University, Yakushiji, Shimotsuke-shi, Tochigi Japan. hirahara@rpf.jp