Chronic Uremia Induces Permeability Changes, Increased Nitric Oxide Synthase Expression, and Structural Modifications in the Peritoneum
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J Am Soc Nephrol 12: 2146–2157, 2001 Chronic Uremia Induces Permeability Changes, Increased Nitric Oxide Synthase Expression, and Structural Modifications in the Peritoneum SOPHIE COMBET,*‡ MARIE-LAURE FERRIER,* MIEKE VAN LANDSCHOOT,§ MARIA STOENOIU,* PIERRE MOULIN,† TOSHIO MIYATA,¶ NORBERT LAMEIRE,§ and OLIVIER DEVUYST* Departments of *Nephrology and †Pathology, Universite´Catholique de Louvain Medical School, Brussels, Belgium; ‡Department of Cell Biology, CEA, Saclay, France; §Department of Nephrology, Rijksuniversiteit Gent, Gent, Belgium; and ¶Institute of Medical Science and Department of Internal Medicine, Tokai University School of Medicine, Kanagawa, Japan. Abstract. Advanced glycation end products (AGE), growth failure. Focal areas of vascular proliferation and fibrosis were factors, and nitric oxide contribute to alterations of the perito- detected in uremic rats, in relation with a transient up-regula- neum during peritoneal dialysis (PD). These mediators are also tion of vascular endothelial growth factor and basic fibroblast involved in chronic uremia, a condition associated with in- growth factor, as well as vascular deposits of the AGE car- creased permeability of serosal membranes. It is unknown boxymethyllysine and pentosidine. Correction of anemia with whether chronic uremia per se modifies the peritoneum before erythropoietin did not prevent the permeability or structural PD initiation. A rat model of subtotal nephrectomy was used to changes in uremic rats. Thus, in this rat model, uremia induces measure peritoneal permeability after 3, 6, and 9 wk, in parallel permeability and structural changes in the peritoneum, in par- with peritoneal nitric oxide synthase (NOS) isoform expression allel with AGE deposits and up-regulation of specific NOS and activity and structural changes. Uremic rats were charac- isoforms and growth factors. These data suggest an indepen- terized by a higher peritoneal permeability for small solutes dent contribution of uremia in the peritoneal changes during and an increased NOS activity due to the up-regulation of PD and offer a paradigm to better understand the modifications endothelial and neuronal NOS. The permeability changes and of serosal membranes in uremia. increased NOS activities correlated with the degree of renal The unique structure of the peritoneal membrane (PM) allowed glucose concentrations, with eventual accumulation of ad- the development of peritoneal dialysis (PD), a therapeutic vanced glycation end products (AGE), release of growth fac- modality that now accounts for 15% of the total number of tors, and increased production of nitric oxide (NO) (4–6). The patients worldwide who are undergoing dialysis (1). The long- latter is mediated by regulation of specific NO synthase (NOS) term use of PD is limited by progressive alterations in the PM. isoforms within the PM (6). These alterations include fibrosis and vascular proliferation, Thus far, the potential contribution of uremia per se to the the latter being responsible for an increased effective peritoneal changes of PM structure and function has not been considered. surface area (EPSA), an increased permeability for small sol- Among a constellation of symptoms known for decades, ure- utes and glucose, and, thereby, an impaired ability to sustain mia is associated with an increase in the permeability of serosal ultrafiltration (2). The relevance of the problem has been membranes with eventual pleural or pericardial effusion (7). confirmed by recent studies demonstrating that high PM per- Several molecular mechanisms associated with uremia might meability is associated with increased mortality and morbidity have potential effects on the PM, irrespective of exposure to in patients undergoing PD (3). Studies in both animal models glucose-based dialysate. NO is an obvious candidate, because and patients undergoing PD have shown that these structural it regulates vascular tone and reactivity and is involved in changes are mainly related to the exposure of the PM to high vascular proliferation (8). However, the issue of the L-argi- nine–NO pathway in uremia appears to be complex. Evidence for decreased excretion of NO metabolites (9) and reduced Received October 19, 2000. Accepted March 14, 2001. whole-body NO production (10) have suggested low basal NO Correspondence to Dr. Olivier Devuyst, Division of Nephrology, Universite´ production in uremia. In contrast, other studies have shown Catholique de Louvain Medical School, Avenue Hippocrate 10, B-1200 Brus- increased NO production in the systemic circulation of uremic sels, Belgium. Phone: ϩ32-2-764-1855; Fax: ϩ32-2-764-5455; E-mail: [email protected] rats (11) and patients (12). The availability of L-arginine, the 1046-6673/1210-2146 NOS substrate, in uremia has also been debated (9,12), al- Journal of the American Society of Nephrology though renal synthesis of endogenous arginine appears to be Copyright © 2001 by the American Society of Nephrology maintained in uremic rats (13) and patients with end-stage J Am Soc Nephrol 12: 2146–2157, 2001 Peritoneal Modifications in Chronic Uremia 2147 renal disease (12). Interestingly, an up-regulation of NOS erlands) implanted in the carotid artery and connected to a Linear- isoforms in extrarenal tissues has been documented in uremic corder polygraph (Ankersmit, Groot-Bijgaarden, Belgium). The BP rats (14). Uremic patients are also characterized by high levels values were obtained after 30-min equilibration, i.e., before starting of circulating AGE (15) and vascular endothelial growth factor the dwell. Blood and dialysate samples were obtained before (t0) and at 30 min (t ), 60 min (t ), and 120 min (t ) of dwell time. At the (VEGF) (16). Several growth factors, including the basic fi- 30 60 120 end of the dwell, animals were killed by exsanguination, and the broblast growth factor (FGF2) and VEGF, have been impli- dialysate was collected. Urea, creatinine, hematocrit, glucose, sodium, cated in structural changes induced by experimental uremia in potassium, total protein, and osmolality were assayed by standard the remnant kidney (17) or in the heart (18). methods (22). Plasma levels of prealbumin were measured in 6-wk In this study, we tested the hypothesis that chronic uremia control and uremic rats (four randomly selected samples in each induces functional and structural changes in the PM of rats that group) by nephelometry (Behring Nephelometer Analyzer II, Dade have undergone subtotal nephrectomy. This procedure pro- Behring, Marburg, Germany) and amino acids by ion-exchange chro- vides a classical model of chronic uremia that has been exten- matography (Biochrom 20 Analyzer, Pharmacia Biotech, Rosendaal, sively used for descriptive and interventional studies focused The Netherlands). White blood cells were counted in a Bu¨rker cham- thus far on the remnant kidney or other target organs such as ber, and dialysate cultures were routinely obtained (22). Identical the heart (18) or the bone (19). Our investigations of peritoneal samples from the visceral and parietal peritoneum were processed for permeability parameters were correlated with determinations fixation in 4% paraformaldehyde or protein extraction as previously detailed (22,23). All experiments were conducted in accord with local of NOS expression and enzymatic activities, as well as with prescriptions and the NIH Guide for the Care and Use of Laboratory expression of AGE and growth factors in the peritoneum. The Animals. role of anemia in the genesis of the modifications was further investigated in uremic rats treated with erythropoietin (EPO). Antibodies The NOS isoforms were detected with monoclonal antibodies Materials and Methods raised against human endothelial NOS (eNOS) and neuronal NOS Rat Model of Chronic Uremia (nNOS) and mouse inducible NOS (Transduction Laboratories, Lex- Adult (8 wk) male Sprague-Dawley rats (Iffa Credo, Brussels, ington, Kentucky) (23). Other antibodies included affinity-purified Belgium) weighing 300 Ϯ 3 g were randomly assigned to uremic and rabbit antibodies against FGF2 and a monoclonal antibody against sham-operated groups. Uremia was induced by a standard procedure VEGF (both from Santa Cruz Biotechnology, Santa Cruz, California); of tissue removal (5/6 nephrectomy followed 1 wk later by contralat- a goat anti–type III collagen (Southern Biotechnology Associates, eral nephrectomy), as described previously (20). The amount of renal Birmingham, Alabama); a monoclonal antibody against human tissue removed (approximately 85%) was quantified by weighting the ␣–smooth muscle actin (Dako, Glostrup, Denmark); affinity-purified tissue, and less than 10% variation was observed between series (20). rabbit antibodies against carboxymethyllysine (CML) and pentosidine Sham-operated (control) rats were subjected to flank incisions sepa- (24), and a monoclonal antibody against CML (25); and a monoclonal rated by 1 wk. All rats had access to a standard rat laboratory diet antibody against -actin (Sigma, St. Louis, Missouri). Positive im- (Pavan, Oud-Turnhout, Belgium) containing 21% protein (1.3% L- munoblot controls included lysates from bovine aortic endothelial arginine) and tap water ad libitum until the evening before they were cells (eNOS), mouse macrophages (iNOS), rat pituitary gland killed. A first series of uremic and control rats were studied 3, 6, and (nNOS), and recombinant FGF2 and VEGF (Santa Cruz 9 wk after the last surgery/incision. Subsequently, a second series of Biotechnology). 8-wk-old male Sprague-Dawley