Oxidative stress during peritoneal dialysis: Implications in functional and structural changes in the membrane
2006; Elsevier BV; Volume: 69; Issue: 11 Linguagem: Inglês
10.1038/sj.ki.5001506
ISSN1523-1755
AutoresHyunjin Noh, J.S. Kim, Ki‐Hwan Han, G.T. Lee, Jisun Song, Sung Hee Chung, J.S. Jeon, Hunjoo Ha, H.B. Lee,
Tópico(s)Coenzyme Q10 studies and effects
ResumoProgressive peritoneal fibrosis, membrane hyperpermeability, and ultrafiltration failure have been observed in patients on long-term peritoneal dialysis (PD). The present study tested the hypothesis that reactive oxygen species (ROS) generated by conventional PD solution (PDS) mediate functional and structural alterations of peritoneal membrane in vivo. Sprague–Dawley rats were randomized to control, PDS, PDS with an antioxidant, and PDS with an angiotensin II (Ang II) receptor blocker. Commercial PDS containing 3.86% glucose (20–30 ml) with or without N-acetylcystein (NAC) 10 mM or losartan 5 mg/kg was administered intraperitoneally twice a day for 12 weeks. Control rats received sham injection. Rats treated with PDS had significantly lower drain volume and D4/D0 glucose, but higher D4/P4 creatinine and increased membrane thickness and endothelial NOS (eNOS) expression compared to control rats. Omental transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF), collagen I, and heat-shock protein (hsp) 47 expression and lipid peroxide levels and dialysate VEGF and Ang II concentrations were significantly increased in rats treated with PDS compared to control. All of these changes were prevented by both NAC and losartan. In conclusion, the present study demonstrates that ROS generated by conventional PDS are, in large part, responsible for peritoneal fibrosis and membrane hyperpermeability. We suggest that antioxidants or Ang II receptor blockers may allow better preservation of the structural and functional integrity of the peritoneal membrane during long-term PD. Progressive peritoneal fibrosis, membrane hyperpermeability, and ultrafiltration failure have been observed in patients on long-term peritoneal dialysis (PD). The present study tested the hypothesis that reactive oxygen species (ROS) generated by conventional PD solution (PDS) mediate functional and structural alterations of peritoneal membrane in vivo. Sprague–Dawley rats were randomized to control, PDS, PDS with an antioxidant, and PDS with an angiotensin II (Ang II) receptor blocker. Commercial PDS containing 3.86% glucose (20–30 ml) with or without N-acetylcystein (NAC) 10 mM or losartan 5 mg/kg was administered intraperitoneally twice a day for 12 weeks. Control rats received sham injection. Rats treated with PDS had significantly lower drain volume and D4/D0 glucose, but higher D4/P4 creatinine and increased membrane thickness and endothelial NOS (eNOS) expression compared to control rats. Omental transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF), collagen I, and heat-shock protein (hsp) 47 expression and lipid peroxide levels and dialysate VEGF and Ang II concentrations were significantly increased in rats treated with PDS compared to control. All of these changes were prevented by both NAC and losartan. In conclusion, the present study demonstrates that ROS generated by conventional PDS are, in large part, responsible for peritoneal fibrosis and membrane hyperpermeability. We suggest that antioxidants or Ang II receptor blockers may allow better preservation of the structural and functional integrity of the peritoneal membrane during long-term PD. Long-term peritoneal dialysis (PD) is associated with progressive increase in the thickness of peritoneal membrane, predominantly in the submesothelial compact collagenous zone,1.Williams J.D. Craig K.J. Topley N. Peritoneal Biopsy Study Group et al.Morphologic changes in the peritoneal membrane of patients with renal disease.J Am Soc Nephrol. 2002; 13: 470-479PubMed Google Scholar and membrane hyperpermeability.2.Davies S.J. Phillips L. Naish P.F. Russell G.I. Peritoneal glucose exposure and changes in membrane solute transport with time on peritoneal dialysis.J Am Soc Nephrol. 2001; 12: 1046-1051PubMed Google Scholar The mechanisms involved in these structural and functional changes remain unclear, but prolonged exposure of the membrane to conventional PD solution (PDS) containing high concentrations of glucose and glucose degradation products (GDP) may play an important role. We have previously demonstrated that conventional high glucose-containing PDS increases transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), and procollagen III N-terminal peptide secretion by human peritoneal mesothelial cells (HPMC),3.Ha H. Cha M.K. Choi H.N. Lee H.B. Effects of peritoneal dialysis solutions on the secretion of growth factors and extracellular matrix proteins by human peritoneal mesothelial cells.Periton Dial Int. 2002; 22: 171-177PubMed Google Scholar and that high glucose-induced activation of protein kinase C mediates TGF-β1 and fibronectin upregulation in HPMC.4.Ha H. Yu M.R. Lee H.B. High glucose-induced PKC activation mediates TGF-beta 1 and fibronectin synthesis by peritoneal mesothelial cells.Kidney Int. 2001; 59: 463-470Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar We also showed that conventional PDS5.Ha H. Song J.S. Yu M.R. Lee H.B. Oxidative stress in peritoneal dialysis.Lect Notes ICB Semin. 2004; 65: 32-39Google Scholar and high glucose6.Lee H.B. Yu M.R. Song J.S. Ha H. Reactive oxygen species amplify protein kinase C signaling in high glucose-induced fibronectin expression by peritoneal mesothelial cells.Kidney Int. 2004; 65: 1170-1179Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar increase cellular reactive oxygen species (ROS) in HPMC, that high glucose-induced ROS generation is dependent on activation of protein kinase C, nicotinamide adenine dinucleotide phosphate oxidase, and mitochondrial metabolism, and that ROS provide signal amplification in high glucose-induced fibronectin expression by HPMC.6.Lee H.B. Yu M.R. Song J.S. Ha H. Reactive oxygen species amplify protein kinase C signaling in high glucose-induced fibronectin expression by peritoneal mesothelial cells.Kidney Int. 2004; 65: 1170-1179Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar Recently, we demonstrated that high glucose increases cellular and secreted angiotensin II (Ang II) in HPMC, that Ang II induces TGF-β1 and fibronectin upregulation through generation of cellular ROS, and that losartan and captopril significantly reduce high glucose-induced TGF-β1 and fibronectin secretion by HPMC.7.Noh H. Ha H. Yu M.R. et al.Angiotensin II mediates high glucose-induced TGF-β1 and fibronectin upregulation in HPMC through reactive oxygen species.Periton Dial Int. 2005; 25: 38-47PubMed Google Scholar Mortier et al.8.Mortier S. Faict D. Schalkwijk C.G. et al.Long-term exposure to new peritoneal solutions: effects on the peritoneal membrane.Kidney Int. 2004; 66: 1257-1265Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 9.Mortier S. Faict D. Lameire N.H. De Vriese A.S. Benefits of switching from a conventional to a low-GDP bicarbonate/lactate-buffered dialysis solution in a rat model.Kidney Int. 2005; 67: 1559-1565Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar demonstrated that chronic exposure of rat peritoneum to conventional PDS containing high concentrations of glucose and GDP resulted in loss of ultrafiltration capacity, increased vascular density, development of fibrosis, and higher accumulation of advanced glycation end-products but not when exposed to new solution containing low concentration of GDP, suggesting that GDP may play a role in the PDS-induced peritoneal changes. GDP can generate intracellular ROS and signal through ROS. Methylglyoxal-induced heparin-binding epidermal growth factor-like growth factor mRNA expression in rat aortic smooth muscle cells10.Che W. Asahi M. Takahashi M. et al.Selective induction of heparin-binding epidermal growth factor-like growth factor by methyllglyoxal and 3-deoxyglucosone in rat aortic smooth muscle cells.J Biol Chem. 1997; 272: 18453-18459Crossref PubMed Scopus (104) Google Scholar and methylglyoxal-induced VEGF secretion by HPMC (HB Lee et al., unpublished data) were shown to be suppressed by antioxidant N-acetylcystein (NAC). Acetaldehyde-induced α1(I) procollagen gene expression in mouse hepatic stellate cells was also blocked by catalase.11.Greenwel P. Dominguez-Rosales J.A. Mavi G. et al.Hydrogen peroxide: a link between acetaldehyde-elicited alpha1(I) collagen gene up-regulation and oxidative stress in mouse hepatic stellate cells.Hepatology. 2000; 31: 109-116Crossref PubMed Scopus (166) Google Scholar GDP are precursors of advanced glycation end-products, which also induce cellular ROS by direct action12.Yim M.B. Yim H.S. Lee C. et al.Protein glycation: creation of catalytic sites for free radical generation.Ann NY Acad Sci. 2001; 928: 48-53Crossref PubMed Scopus (144) Google Scholar and through binding their receptor for advanced glycation end-products13.Wautier M.P. Chappey O. Corda S. et al.Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE.Am J Physiol. 2001; 280: E685-E694PubMed Google Scholar and signal through ROS.14.Lal M.A. Brismar H. Eklof A.C. Aperia A. Role of oxidative stress in advanced glycation end product-induced mesangial cell activation.Kidney Int. 2002; 61: 2006-2014Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar ROS, in turn, promote advanced glycation end-products formation15.Nishigawa T. Edelstein D. Du X.L. et al.Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage.Nature. 2000; 404: 787-790Crossref PubMed Scopus (3441) Google Scholar and can thereby amplify advanced glycation end-products signal. In this study, we tested our hypothesis that ROS generated by conventional PDS, regardless of whether they are from high glucose, GDP, or Ang II, may play a role in peritoneal membrane alterations in vivo and that antioxidant may prevent such changes. We specifically examined the effect of Ang II type 1 receptor blocker in PDS-induced membrane alterations in view of the fact that Ang II generates and signals through ROS in HPMC.7.Noh H. Ha H. Yu M.R. et al.Angiotensin II mediates high glucose-induced TGF-β1 and fibronectin upregulation in HPMC through reactive oxygen species.Periton Dial Int. 2005; 25: 38-47PubMed Google Scholar At the end of 12 weeks, rats treated with PDS had significantly lower drain volume and D4/D0 glucose, but higher D4/P4 creatinine compared to control rats. Both NAC and losartan prevented the decrease in drain volume and D4/D0 glucose and the increase in D4/P4 creatinine (Figure 1). Subhepatic peritoneal membrane showed intact mesothelial cell monolayer in control rats (Figure 2a). In rats treated with PDS, a significant increase in peritoneal thickness with collagen deposition in submesothelial layer was observed (Figure 2a). Both NAC and losartan prevented the increase in thickness of both visceral and parietal peritoneum (Figure 2a and b). In control rats, a faint signal for endothelial NOS (eNOS) was located in the endothelial lining. In rats treated with PDS, there was an intense eNOS signal along the vessel wall. Both NAC and losartan attenuated the increase in the signal (Figure 3). Omental collagen I and heat-shock protein (hsp) 47 expression was significantly increased in rats treated with PDS compared to control rats. Both NAC and losartan prevented the increase (Figure 4). Omental TGF-β1 (Figure 5a) and VEGF (Figure 5b) expression was significantly increased in rats treated with PDS compared to control. Both NAC and losartan significantly inhibited PDS-induced increase in TGF-β1 and VEGF. Omental lipid peroxide (LPO) (Figure 5c) also increased in rats treated with PDS, although the difference did not reach statistical significance. Losartan significantly reduced LPO in omentum when compared to rats treated with PDS without NAC or losartan. Dialysate TGF-β1 also increased in rats treated with PDS and the treatment with both NAC and losartan attenuated the increase, although the changes did not reach statistical significance (Figure 6a). Dialysate VEGF (Figure 6b) and Ang II (Figure 6c) concentrations significantly increased in rats treated with PDS compared to control and both NAC and losartan significantly inhibited PDS-induced increase in VEGF and Ang II. In this study, we hypothesized that cellular ROS generated by conventional PDS may play a role in structural and functional peritoneal changes observed during long-term PD and that antioxidant may prevent such changes. In our animal model of PD where the peritoneum was exposed to PDS over 12 weeks, we observed changes similar to those observed in long-term PD patients with increased transport of creatinine and glucose, decreased ultrafiltration capacity, and increased peritoneal thickening and new vessel formation. In rats treated with PDS, thickening of peritoneal membrane was observed in both parietal and visceral peritoneum and this was associated with increased omental collagen I and hsp 47 expression. Hsp 47 is a collagen-specific molecular chaperon during the synthesis and/or secretion of procollagen.16.Nakai A. Satoh M. Hirayoshi K. Nagata K. Involvement of the stress protein HSP 47 in procollagen processing in the endoplasmic reticulum.J Cell Biol. 1992; 117: 903-914Crossref PubMed Scopus (218) Google Scholar In various renal diseases including renal interstitial fibrosis induced by unilateral ureteral obstruction,17.Moriyama T. Kawada N. Ando A. et al.Up-regulation of HSP 47 in the mouse kidneys with unilateral ureteral obstruction.Kidney Int. 1998; 54: 110-119Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar experimental mesangioproliferative glomerulonephritis,18.Razzaque M.S. Taguchi T. Collagen-binding heat shock protein (HSP) 47 expression in anti-thymocyte serum (ATS)-induced glomerulonephritis.J Pathol. 1997; 183: 24-29Crossref PubMed Scopus (78) Google Scholar and age-related nephropathy,19.Razzaque M.S. Shimokawa I. Nazneen A. et al.Age-related nephropathy in the Fischer 344 rat is associated with overexpression of collagens and collagen-binding heat shock protein 47.Cell Tissue Res. 1998; 294: 471-578Crossref Scopus (57) Google Scholar the expression of hsp 47 was shown to be correlated with the degree of collagen expression. Recently, Mishima et al.20.Mishima Y. Miyazaki M. Abe K. et al.Enhanced expression of heat shock protein 47 in rat model of peritoneal fibrosis.Periton Dial Int. 2003; 23: 14-22PubMed Google Scholar showed marked increase in hsp 47 expression in mesothelial cells and in submesothelial connective tissue in chlorhexidine gluconate-induced peritoneal fibrosis. These observations together with our finding in this study suggest that hsp 47 is a good marker of collagen accumulation in the kidney and peritoneum. Our finding that antioxidant NAC prevented PDS-induced collagen I and hsp 47 accumulation in the omentum strongly suggests that ROS are the major mediator of peritoneal fibrosis. TGF-β1 is a multifunctional cytokine that is central in the process of fibrogenesis.21.Border W.A. Noble N.A. Transforming growth factor β in tissue fibrosis.N Engl J Med. 1994; 331: 1286-1292Crossref PubMed Scopus (2899) Google Scholar It is generally accepted that TGF-β1 is locally produced in the peritoneum3.Ha H. Cha M.K. Choi H.N. Lee H.B. Effects of peritoneal dialysis solutions on the secretion of growth factors and extracellular matrix proteins by human peritoneal mesothelial cells.Periton Dial Int. 2002; 22: 171-177PubMed Google Scholar and is likely to be the main growth factor for functional and structural changes of peritoneal membrane.22.Margetts P.J. Kolb M. Galt T. et al.Gene transfer of transforming growth factor-β1 to the rat peritoneum: effects on membrane function.J Am Soc Nephrol. 2001; 12: 2029-2039PubMed Google Scholar, 23.Margetts P.J. 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: 425-436Crossref PubMed Scopus (234) Google Scholar TGF-β1 gene transfer to rat peritoneum was shown to induce peritoneal fibrosis, neoangiogenesis, increase of peritoneal solute transport,22.Margetts P.J. Kolb M. Galt T. et al.Gene transfer of transforming growth factor-β1 to the rat peritoneum: effects on membrane function.J Am Soc Nephrol. 2001; 12: 2029-2039PubMed Google Scholar and epithelial–mesenchymal transition.23.Margetts P.J. 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: 425-436Crossref PubMed Scopus (234) Google Scholar Our observations that omental and dialysate TGF-β1 increased in rats treated with PDS, that the expression of TGF-β1 coincided with functional and structural changes in peritoneal membrane, and that the increase in TGF-β1 was effectively prevented by NAC strongly suggest the involvement of ROS in peritoneal alterations. VEGF is a mitogen for endothelial cells and a regulator of normal and abnormal angiogenesis24.Ferrara N. Role of vascular endothelial growth factor in the regulation of angiogenesis.Kidney Int. 1999; 56: 794-814Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar and induces vascular hyperpermeability by a direct action on endothelial cells.25.Hippenstiel S. Krüll M. Ikemann A. et al.VEGF induces hyperpermeability by a direct action on endothelial cells.Am J Physiol. 1998; 274: L678-L684PubMed Google Scholar VEGF is locally produced in the peritoneum,3.Ha H. Cha M.K. Choi H.N. Lee H.B. Effects of peritoneal dialysis solutions on the secretion of growth factors and extracellular matrix proteins by human peritoneal mesothelial cells.Periton Dial Int. 2002; 22: 171-177PubMed Google Scholar, 26.Combet S. Miyata T. Moulin P. et al.Vascular proliferation and enhanced expression of endothelial nitric oxide synthase in human peritoneum exposed to long term peritoneal dialysis.J Am Soc Nephrol. 2000; 11: 717-728PubMed Google Scholar, 27.Zweers M.M. Struijk D.G. Smit W. Krediet R.T. Vascular endothelial growth factor in peritoneal dialysis: a longitudinal follow-up.J Lab Clin Med. 2001; 137: 125-132Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar has been identified in biopsy samples from long-term PD patients,26.Combet S. Miyata T. Moulin P. et al.Vascular proliferation and enhanced expression of endothelial nitric oxide synthase in human peritoneum exposed to long term peritoneal dialysis.J Am Soc Nephrol. 2000; 11: 717-728PubMed Google Scholar and dialysate VEGF was shown to correlate with solute transport.27.Zweers M.M. Struijk D.G. Smit W. Krediet R.T. Vascular endothelial growth factor in peritoneal dialysis: a longitudinal follow-up.J Lab Clin Med. 2001; 137: 125-132Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar eNOS also plays a role in the control of vascular tone and permeability28.Kubes P. Nitric oxide affects microvascular permeability in the intact and inflamed vasculature.Microcirculation. 1995; 2: 235-244Crossref PubMed Scopus (99) Google Scholar, 29.Kone B.C. Nitric oxide in renal health and disease.Am J Kidney Dis. 1997; 30: 311-333Abstract Full Text PDF PubMed Scopus (187) Google Scholar as well as angiogenesis via an interaction with VEGF.30.Papapetropoulos A. Garcia-Cardena G. Madri J.A. Sessa W.C. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells.J Clin Invest. 1997; 100: 3131-3139Crossref PubMed Scopus (983) Google Scholar, 31.Murohara T. Asahara T. Silver M. et al.Nitric oxide synthase modulates angiogenesis in response to tissue ischemia.J Clin Invest. 1998; 101: 2567-2578Crossref PubMed Scopus (1057) Google Scholar Functional significance of eNOS in the peritoneal membrane permeability has been reported in acute peritonitis mouse model.32.Ni J. Moulin P. Gianello P. et al.Mice that lack endothelial nitric oxide synthase are protected against functional and structural modifications induced by acute peritonitis.J Am Soc Nephrol. 2003; 14: 3205-3216Crossref PubMed Scopus (48) Google Scholar In this study, PDS-induced increase in omental and dialysate VEGF and peritoneal eNOS expression was significantly prevented by NAC, again suggesting the involvement of ROS in functional as well as structural changes in the peritoneum. Ang II is a growth factor that regulates cell proliferation, apoptosis, and fibrosis.33.Ruiz-Ortega M. Lorenzo O. Suzuki Y. et al.Proinflammatory actions of angiotensins.Curr Opin Nephrol Hypertens. 2001; 10: 321-329Crossref PubMed Scopus (326) Google Scholar, 34.Wolf G. Neilson E.G. Angiotensin II as a renal growth factor.J Am Soc Nephrol. 1993; 3: 1531-1540PubMed Google Scholar, 35.Mezzano S.A. Ruiz-Ortega M. Egido J. Angiotensin II and renal fibrosis.Hypertension. 2001; 38: 635-638Crossref PubMed Scopus (399) Google Scholar Recently, we demonstrated that high glucose increases cellular and secreted Ang II in cultured HPMC, that Ang II generates cellular ROS, and that Ang II-induced upregulation of TGF-β1 and fibronectin was inhibited by catalase and diphenyleneiodinium, an nicotinamide adenine dinucleotide phosphate oxidase inhibitor, in HPMC.7.Noh H. Ha H. Yu M.R. et al.Angiotensin II mediates high glucose-induced TGF-β1 and fibronectin upregulation in HPMC through reactive oxygen species.Periton Dial Int. 2005; 25: 38-47PubMed Google Scholar These observations suggested that the effect of Ang II on peritoneal changes is mediated by ROS. Our current observation that losartan effectively lowers omental LPO level and, at the same time, prevents peritoneal hyperpermeability, eNOS expression, and membrane thickening confirms that the effect of Ang II on peritoneal changes is mediated by ROS. The involvement of nicotinamide adenine dinucleotide phosphate oxidase in Ang II-induced ROS generation has also been identified in vascular smooth muscle cells36.Griendling K.K. Sorescu D. Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease.Circ Res. 2000; 86: 494-501Crossref PubMed Scopus (2526) Google Scholar and endothelial cells.37.Li J.M. Shah A.M. Mechanism of endothelial cell NADPH oxidase activation by angiotensin II.J Biol Chem. 2003; 278: 12094-12100Crossref PubMed Scopus (261) Google Scholar A study by Hsieh et al.38.Hsieh T.J. Zhang S.L. Filep J.G. et al.High glucose stimulates angiotensinogen gene expression via reactive oxygen species in rat kidney proximal tubular cells.Endocrinology. 2002; 143: 2975-2985Crossref PubMed Scopus (147) Google Scholar showed that high glucose enhances angiotensinogen gene expression via ROS generation in rat kidney proximal tubular cells. Interestingly, we found that NAC decreases dialysate Ang II. This suggests that ROS are not only downstream but also upstream signaling molecules to Ang II in PDS-induced peritoneal injury. We also observed that the increase in dialysate Ang II induced by PDS was prevented by losartan, suggesting a positive feedback system by which the internalized Ang II is responsible for enhanced generation of Ang II. Such a link between Ang II receptor activation and stimulation of local Ang II formation is in agreement with previous studies,39.Zou L.X. Imig Z.D. Von Thun A.M. et al.Receptor-mediated intrarenal angiotensin II augmentation in angiotensin II-infused rats.Hypertension. 1996; 28: 669-677Crossref PubMed Scopus (175) Google Scholar, 40.Nishiyama A. Seth D.M. Naver L.G. Angiotensin II type 1 receptor-mediated augmentation of renal interstitial fluid angiotensin II in angiotensin II-induced hypertension.J Hypertens. 2003; 21: 1897-1903Crossref PubMed Scopus (50) Google Scholar, 41.Kobori H. Prieto-Carrasquero M.C. Ozawa Y. Naver L.G. AT1 receptor mediated augmentation of intrarenal angiotensinogen in angiotensin II-dependent hypertension.Hypertension. 2004; 43: 1126-1132Crossref PubMed Scopus (135) Google Scholar which showed that Ang II type 1 receptor stimulation augments intrarenal Ang II in Ang II-induced hypertension. The beneficial effect of losartan in the present study is consistent with an earlier study showing that intraperitoneal enalapril improves ultrafiltration capacity and decreases peritoneal thickening.42.Duman S. Wieczorowska-Tobis K. Styszynski A. et al.Intraperitoneal enalapril ameliorates morphologic changes induced by hypertonic peritoneal dialysis solutions in rat peritoneum.Adv Periton Dial. 2004; 20: 31-36PubMed Google Scholar GDP can induce intracellular ROS and signal through ROS10.Che W. Asahi M. Takahashi M. et al.Selective induction of heparin-binding epidermal growth factor-like growth factor by methyllglyoxal and 3-deoxyglucosone in rat aortic smooth muscle cells.J Biol Chem. 1997; 272: 18453-18459Crossref PubMed Scopus (104) Google Scholar, 11.Greenwel P. Dominguez-Rosales J.A. Mavi G. et al.Hydrogen peroxide: a link between acetaldehyde-elicited alpha1(I) collagen gene up-regulation and oxidative stress in mouse hepatic stellate cells.Hepatology. 2000; 31: 109-116Crossref PubMed Scopus (166) Google Scholar and are likely to contribute to conventional PDS-induced peritoneal alterations. We, however, did not use a control GDP-depleted PDS in this study because we found that GDP-depleted PDS generated almost as much ROS in HPMC as PDS containing high concentration of GDP (data not shown) probably from high glucose. In conclusion, the present study strongly suggests that ROS generated by conventional PDS, regardless of whether produced by high glucose, Ang II, or GDP, may be responsible for progressive membrane hyperpermeability, neoangiogenesis, accumulation of extracellular matrix, and eventual peritoneal fibrosis. Thus, antioxidants or Ang II receptor blockers may allow better preservation of the structural and functional integrity of the peritoneal membrane during long-term PD. All chemicals and materials, unless otherwise stated, were obtained from Sigma Chemical Company (St Louis, MO, USA) and Becton Dickinson Labware (Lincoln Park, NJ, USA). Male Sprague–Dawley rats weighing 250 g were purchased from Bio Genomics Inc. (Seoul, Korea) and housed in a room under controlled temperature (23±1°C) and humidity (45–65%), 12-h light/dark cycle, and free access to water and chow (Samyang rat food, Seoul, Korea). All animal experiments were conducted in accordance with the National Institute of Health guide for the care and use of laboratory animals. Rats were randomized to four groups (n=8 for each group): control, PDS, PDS with an antioxidant, and PDS with an Ang II receptor blocker. Commercial PDS containing 3.86% glucose, 20–30 ml depending upon the body weight change over time, with or without NAC 10 mM or losartan 5 mg/kg was administered intraperitoneally twice a day for 12 weeks. Dose of NAC was chosen based on the previous study43.Pat B. Yang T. Kong C. et al.Activation of ERK in renal fibrosis after unilateral obstruction: modulation by antioxidants.Kidney Int. 2005; 67: 931-943Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar and our own preliminary data using 5, 10, and 50 mM and that of losartan based on the previous study.44.Ferrario C.M. Jessup J. Chappell M.C. et al.Effect of angiotensin converting enzyme inhibition and angiotensin II receptor blockers on cardiac angiotensin converting enzyme 2.Circulation. 2005; 111: 2605-2610Crossref PubMed Scopus (962) Google Scholar pH of PDS was neutralized by NaOH and the final pH was 7.4. Control rats received sham injection (puncture of the peritoneal cavity without PDS) twice a day over 12 weeks. Peritoneal equilibration test was performed over 4 h before being killed. Rats were killed using pentobarbital sodium, blood and dialysate samples were collected, and omental tissue, liver, and anterior abdominal wall apart from injection site were removed and frozen until analysis. There was no evidence of infection at the puncture site or in the peritoneal cavity from repeated punctures. One animal each from control and losartan groups died during the study period. Peritoneal equilibration test was performed using 30 ml of 3.86% glucose PDS. Four hours later, a tail blood sample and a dialysate sample were obtained. The dialysate to plasma creatinine ratio at 4 h (D4/P4 creatinine) was used as the solute transport parameter. The drained volume was calculated as the sum of peritoneal fluid aspirated and the weight of (wet-dry) gauze at 4 h. The creatinine concentration in the PD effluent was measured by modified Jaffe method and corrected for glucose interference by a correction factor derived by our laboratory. Total TGF-β1 (after acid activation) and VEGF were measured in dialysate and omental tissue using a commercial sandwich enzyme-linked immunosorbent assay (ELISA) kit for TGF-β1 (R&D Systems, Minneapolis, MN, USA) and VEGF (R&D systems) according to the manufacturer's descriptions. Omental tissue was homogenized in a standard lysis buffer containing protease inhibitors (20 mM Tris-HCl, 1% Triton X-100, 137 mM sodium chloride, 5 mM ethylenediaminetetraaceticacid, 1 mM ethyleneglycol tetraacetate, 0.2 mM phenylmethylsulfonylfluoride, 10 μM leupeptin, 1 μg/ml aprotinin). Ang II was measured in dialysate using a commercial ELISA kit (Peninsula Laboratories, Belmont, CA, USA). Before the detection of Ang II, 1 ml of dialysate samples were lyophilized and reconstituted in 60 μl of H2O. Protein was extracted from frozen omental tissue by homogenization in a lysis buffer with the same composition as was used for ELISA. After electrophoresis, the proteins were transferred onto a nitrocellulose membrane using a transblot chamber with Tris buffer (0.025 M Tris-HCl, 0.192 M glycine, and 20% MeOH). Equal amount of samples were verified by Ponceau S staining. The membrane was blocked for 1 h at room temperature with 5% non-fat milk in Tris-buffered saline-Tween 20. Membranes were incubated at 4°C overnight with rat monoclonal antibody to collagen I (Southern Biotech, Birmingham, AL) or hsp 47 (Stressgen, Victoria, BC, Canada). After extensive washing in Tris-buffered saline-Tween 20, the membranes then were incubated with horseradish peroxidase-conjugated anti-mouse immunoglobulin G or anti-goat immunoglobulin G for 1 h at room temperature. After washing, the membranes were incubated with enhanced chemiluminescence system detection kit (Amersham Life Science, Little Chalfont, UK). Positive immunoreactive bands were quantified densitometrically. The omental tissue samples were homogenized in a buffer solution containing 50 mM Tris-HCl (pH 7.4) and 1.15% KCl, and then centrifuged. The supernatant was used for the assay. The levels of lipid peroxidation were measured by thiobarbituric acid method with a modification as described previously.45.Ha H. Yu M.R. Kim K.H. Melatonin and taurine reduce early glomerulopathy in diabetic rats.Free Radic Biol Med. 1999; 26: 944-950Crossref PubMed Scopus (130) Google Scholar Tissue samples obtained from the liver and anterior abdominal wall were fixed in a sufficient amount of 4% phosphate-buffered formaldehyde for 24 h, paraffin-processed and embedded, and 3 μm sections were cut. Cut sections were stained with Masson's trichrome stain. In histological assessment, attention was paid to the morphologic features of the mesothelial surface and submesothelial compact zone. The integrity of the mesothelial cell monolayer was assessed and the maximal thickness of the submesothelial compact zone was measured in subhepatic and parietal peritoneal membrane using Image Pro Plus (Media Cybernetics, Silver Spring, MD, USA). Four-micrometer paraffin sections were processed for immunohistochemistry using avidin–biotin–horseradish peroxidase technique, as described previously.46.Han K.H. Jung J.Y. Cha J.H. et al.1,25-Dihydroxyvitamin D3 stimulates osteopontin expression in rat kidney.Nephron Physiol. 2003; 93: 76-86Crossref Scopus (8) Google Scholar, 47.Han K.H. Lim J.M. Kim W.Y. et al.Expression of endothelial nitric oxide synthase in developing rat kidney.Am J Physiol Renal Physiol. 2005; 288: F694-F702Crossref PubMed Scopus (42) Google Scholar The sections were deparaffined with xylene and ethanol, rinsed in tap water, and incubated with 3% H2O2 for 30 min to eliminate endogenous peroxidase activity. Before incubation with primary antibody, the sections were permeabilized by incubation for 15 min in 0.5% Triton X-100 in phosphate-buffered saline, blocked subsequently with blocking serum for 30 min, and incubated overnight at 4°C with the antibody against eNOS (1:1000; Transduction Lab., Lexington, KY, USA) diluted in phosphate-buffered saline. After washing in phosphate-buffered saline, the sections were incubated for 2 h with the biotinylated secondary antibody against mouse immunoglobulin G, and subsequently with the Vectastain ABC reagent (Vectastain ABC kit; Vector Laboratories, Burlington, CA, USA) for 2 h. After rinsing with phosphate-buffered saline, the sections were incubated with the peroxidase substrate solution, a mixture of 0.05% 3,3′-diaminobenzidine, and 0.01% H2O2 for 2 min at room temperature. After rinsing with Tris-HCl buffer, the sections were dehydrated with graded ethanol and xylene, mounted in Canada balsam, and examined by light microscope. The mean values obtained from each group were compared by analysis of variance with subsequent Fisher's significant difference method. Non-parametric analyses were also used where appropriate. A P-value <0.05 was used as the criterion for a statistically significant difference. This work was supported by a grant from Department of Health and Welfare, Republic of Korea (Grant No. 02-PJ1-PG3-21499-0005).
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