The dipeptide alanyl-glutamine ameliorates peritoneal fibrosis and attenuates IL-17 dependent pathways during peritoneal dialysis
2016; Elsevier BV; Volume: 89; Issue: 3 Linguagem: Inglês
10.1016/j.kint.2015.12.005
ISSN1523-1755
AutoresEvelina Ferrantelli, Georgios Liappas, Marc Vila Cuenca, Eelco D. Keuning, Thomas L. Foster, Marc G. Vervloet, Manuel López–Cabrera, Robert H.J. Beelen,
Tópico(s)Eosinophilic Esophagitis
ResumoPeritoneal dialysis (PD) can result in chronic inflammation and progressive peritoneal membrane damage. Alanyl-Glutamine (Ala-Gln), a dipeptide with immunomodulatory effects, improved resistance of mesothelial cells to PD fluids. Recently, interleukin-17 (IL-17) was found to be associated with PD-induced peritoneal damage. Here we studied the capacity of intraperitoneal Ala-Gln administration to protect against peritoneal damage by modulating IL-17 expression in uremic rat and mouse PD exposure models. Supplementation of PD fluid with Ala-Gln resulted in reduced peritoneal thickness, αSMA expression and angiogenesis. Addition of Ala-Gln also attenuated the IL-17 pathway expression induced by PD, reflected by substantial reduction or normalization of peritoneal levels of IL-17, transforming growth factor β, IL-6, and the transcription factor retinoic acid receptor–related orphan receptor gamma T. Moreover, increased levels of IL-17 were associated with PD-induced peritoneal thickening. Conversely, Ala-Gln treatment prevented peritoneal extracellular matrix deposition, an effect seen with IL-17 blockade. Thus, intraperitoneal administration of Ala-Gln, a stable dipeptide commonly used in parenteral nutrition, ameliorates PD-induced peritoneal damage in animal models, in part by modulating IL-17 expression. Hence, Ala-Gln supplementation of dialysate may be a potential strategy to ameliorate peritoneal deterioration during PD. Peritoneal dialysis (PD) can result in chronic inflammation and progressive peritoneal membrane damage. Alanyl-Glutamine (Ala-Gln), a dipeptide with immunomodulatory effects, improved resistance of mesothelial cells to PD fluids. Recently, interleukin-17 (IL-17) was found to be associated with PD-induced peritoneal damage. Here we studied the capacity of intraperitoneal Ala-Gln administration to protect against peritoneal damage by modulating IL-17 expression in uremic rat and mouse PD exposure models. Supplementation of PD fluid with Ala-Gln resulted in reduced peritoneal thickness, αSMA expression and angiogenesis. Addition of Ala-Gln also attenuated the IL-17 pathway expression induced by PD, reflected by substantial reduction or normalization of peritoneal levels of IL-17, transforming growth factor β, IL-6, and the transcription factor retinoic acid receptor–related orphan receptor gamma T. Moreover, increased levels of IL-17 were associated with PD-induced peritoneal thickening. Conversely, Ala-Gln treatment prevented peritoneal extracellular matrix deposition, an effect seen with IL-17 blockade. Thus, intraperitoneal administration of Ala-Gln, a stable dipeptide commonly used in parenteral nutrition, ameliorates PD-induced peritoneal damage in animal models, in part by modulating IL-17 expression. Hence, Ala-Gln supplementation of dialysate may be a potential strategy to ameliorate peritoneal deterioration during PD. Dialysis is a life-saving renal replacement therapy for about 2 million patients with end-stage renal disease worldwide; >200,000 patients are treated with peritoneal dialysis (PD).1Jain A.K. Blake P. Cordy P. et al.Global trends in rates of peritoneal dialysis.Am Soc Nephrol. 2012; 23: 533-544Crossref PubMed Scopus (377) Google Scholar PD is based on the ability of the peritoneum to function as a dialyzing membrane, allowing the exchange of solutes and waste products between the blood stream and the peritoneal dialysis fluid (PDF) instilled through a permanent catheter.2Krediet R.T. The peritoneal membrane in chronic peritoneal dialysis.Kidney Int. 1999; 55: 341-356Abstract Full Text PDF PubMed Scopus (170) Google Scholar, 3Baroni G. Schuinski A. de Moraes T.P. et al.Inflammation and the peritoneal membrane: causes and impact on structure and function during peritoneal dialysis.Mediators Inflamm. 2012; 2012: 912595Crossref PubMed Scopus (42) Google Scholar, 4Chaimovitz C. Peritoneal dialysis.Kidney Int. 1994; 45: 1226-1240Abstract Full Text PDF PubMed Scopus (45) Google Scholar, 5Krishnan M. Tam P. Wu G. et al.Glucose degradation products (GDP's) and peritoneal changes in patients on chronic peritoneal dialysis: will new dialysis solutions prevent these changes?.Int Urol Nephrol. 2005; 37: 409-418Crossref PubMed Scopus (16) Google Scholar, 6Krediet R.T. Lindholm B. Rippe B. Pathophysiology of peritoneal membrane failure.Perit Dial Int. 2000; 20: S22-S42PubMed Google Scholar This home-based treatment preserves the patient's residual renal function and provides better quality of life while ensuring equivalent patient survival compared with hemodialysis. Unfortunately, long-term PD is associated with the development of functional and structural alterations to the peritoneal membrane.7Di Paolo N. Sacchi G. De Mia M. et al.Morphology of the peritoneal membrane during continuous ambulatory peritoneal dialysis.Nephron. 1986; 44: 204-211Crossref PubMed Scopus (146) Google Scholar Peritoneal fibrosis, thickening of the submesothelial extracellular matrix and vascular changes,8Mateijsen M.A. van der Wal A.C. Hendriks P.M. et al.Vascular and interstitial changes in the peritoneum of CAPD patients with peritoneal sclerosis.Perit Dial Int. 1999; 19: 517-525PubMed Google Scholar, 9Williams J.D. Craig K.J. Topley N. et al.Morphologic changes in the peritoneal membrane of patients with renal disease.J Am Soc Nephrol. 2002; 13: 470-479PubMed Google Scholar together with epithelial-to-mesenchymal transition,10Aguilera A. Yanez-Mo M. Selgas R. et al.Epithelial to mesenchymal transition as a triggering factor of peritoneal membrane fibrosis and angiogenesis in peritoneal dialysis patients.Curr Opin Investig Drugs. 2005; 6: 262-268PubMed Google Scholar, 11Yanez-Mo M. Lara-Pezzi E. Selgas R. et al.Peritoneal dialysis and epithelial-to-mesenchymal transition of mesothelial cells.N Engl J Med. 2003; 348: 403-413Crossref PubMed Scopus (619) Google Scholar, 12Aroeira L.S. Aguilera A. Selgas R. et al.Mesenchymal conversion of mesothelial cells as a mechanism responsible for high solute transport rate in peritoneal dialysis: role of vascular endothelial growth factor.Am J Kidney Dis. 2005; 46: 938-948Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar are major causative factors in the development of ultrafiltration failure, necessitating withdrawal from PD. These pathologic changes are the consequences of inflammatory processes generated in part by the continuous exposure to conventional glucose-based dialysis solutions. Glucose is used as an osmotic agent to allow the exchange of solutes and is itself implicated in the alterations that occur in peritoneal cells, together with glucose degradation products formed during heat sterilization of the solutions.13Bajo M.A. Perez-Lozano M.L. Albar-Vizcaino P. et al.Low-GDP peritoneal dialysis fluid ('balance') has less impact in vitro and ex vivo on epithelial-to-mesenchymal transition (EMT) of mesothelial cells than a standard fluid.Nephrol Dial Transplant. 2011; 26: 282-291Crossref PubMed Scopus (63) Google Scholar, 14Schilte M.N. Celie J.W. Wee P.M. et al.Factors contributing to peritoneal tissue remodeling in peritoneal dialysis.Perit Dial Int. 2009; 29: 605-617PubMed Google Scholar Several other factors can contribute to peritoneal deterioration in patients undergoing PD, including peritonitis and mechanical injury during instillation of PDF.13Bajo M.A. Perez-Lozano M.L. Albar-Vizcaino P. et al.Low-GDP peritoneal dialysis fluid ('balance') has less impact in vitro and ex vivo on epithelial-to-mesenchymal transition (EMT) of mesothelial cells than a standard fluid.Nephrol Dial Transplant. 2011; 26: 282-291Crossref PubMed Scopus (63) Google Scholar In the past decade, "biocompatible" PD solutions have entered the market, many of which have been tested in various experimental PD models and clinical trials and are currently the first choice in the most developed countries. However, many of these solutions still feature high osmolarity, high glucose concentration, and low pH, features that are potentially harmful to the peritoneal membrane and its integrity. Glutamine is the most abundant nonessential free amino acid in the body. It is involved in nitrogen transport and hence represents an important fuel source during stress conditions. It is commonly used as a nutritional supplement in critically ill patients in whom its use is associated with a reduction in hospital mortality,15Wischmeyer P.E. Dhaliwal R. McCall M. et al.Parenteral glutamine supplementation in critical illness: a systematic review.Crit Care. 2014; 18: R76Crossref PubMed Scopus (137) Google Scholar and it is thought to have a protective immunomodulatory role by reducing tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 expression.16Raspe C. Czeslick E. Weimann A. et al.Glutamine and alanine-induced differential expression of intracellular IL-6, IL-8, and TNF-alpha in LPS-stimulated monocytes in human whole-blood.Cytokine. 2013; 62: 52-57Crossref Scopus (27) Google Scholar Alanyl-glutamine (Ala-Gln) is a dipeptide featuring a glutamine amino group joined to an alanyl residue. This more stable and soluble Ala-Gln dipeptide form is able to restore the stress proteome of mesothelial cells when exposed to PDF.17Kratochwill K. Boehm M. Herzog R. et al.Alanyl-glutamine dipeptide restores the cytoprotective stress proteome of mesothelial cells exposed to peritoneal dialysis fluids.Nephrol Dial Transplant. 2012; 27: 937-946Crossref PubMed Scopus (33) Google Scholar In lung injury, the dipeptide is able to reduce inflammation by modulating the T helper 17 (Th17)/regulatory T-cell balance.18Hou Y.C. Pai M.H. Liu J.J. et al.Alanyl-glutamine resolves lipopolysaccharide-induced lung injury in mice by modulating the polarization of regulatory T cells and T helper 17 cells.J Nutr Biochem. 2013; 24: 1555-1563Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar Th17 cells are a subset of T helper cells related to many autoimmune and inflammatory diseases.19Waite J.C. Skokos D. Th17 response and inflammatory autoimmune diseases.Int J Inflam. 2012; 2012: 819467Crossref PubMed Scopus (167) Google Scholar Naive T cells differentiate to Th17 cells under the combined effects of transforming growth factor-β (TGF-β) and IL-6. The main effector cytokine secreted by Th17 cells is IL-17, which is expressed in Th17 cells under the control of the transcription factor retinoic acid receptor–related orphan receptor gamma T.20Ivanov II, McKenzie B.S. Zhou L. et al.The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells.Cell. 2006; 126: 1121-1133Abstract Full Text Full Text PDF PubMed Scopus (3962) Google Scholar Elevated IL-17 levels have been found in experimental animal models of multiple sclerosis,21Matusevicius D. Kivisakk P. He B. et al.Interleukin-17 mRNA expression in blood and CSF mononuclear cells is augmented in multiple sclerosis.Mult Scler. 1999; 5: 101-104Crossref PubMed Scopus (629) Google Scholar psoriasis,22van der Fits L. Mourits S. Voerman J.S. et al.Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.J Immunol. 2009; 182: 5836-5845Crossref PubMed Scopus (1348) Google Scholar collagen-induced arthritis,23Nakae S. Nambu A. Sudo K. et al.Suppression of immune induction of collagen-induced arthritis in IL-17-deficient mice.J Immunol. 2003; 171: 6173-6177Crossref PubMed Scopus (1078) Google Scholar and other autoimmune diseases. A key factor in the development of fibrosis is the interplay between IL-17 and TGF-β.24Mi S. Li Z. Yang H.Z. et al.Blocking IL-17A promotes the resolution of pulmonary inflammation and fibrosis via TGF-beta1-dependent and -independent mechanisms.J Immunol. 2011; 187: 3003-3014Crossref PubMed Scopus (277) Google Scholar Notably, a role for IL-17 in inducing peritoneal fibrosis has recently been shown in an experimental PD model.25Rodrigues-Diez R. Aroeira L.S. Orejudo M. et al.IL-17A is a novel player in dialysis-induced peritoneal damage.Kidney Int. 2014; 86: 303-315Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar In this study, we used uremic rat and mice PD exposure models to examine the effects of supplementing PDF with pharmacologic doses of Ala-Gln dipeptide on IL-17 expression, Th17 balance, and peritoneal fibrosis. Histologic analysis of peritoneal sections showed that fibrosis took place in our uremic models. Masson trichrome staining of peritoneal sections, obtained in rats after 5 weeks of daily exposure to the PD fluid, clearly showed a significant increase of peritoneal thickness compared with the control group, as well as an induced expression of other fibrosis markers, including fibronectin (FBN) and hyaluronic acid (HA) (Figure 1). Ala-Gln dipeptide plays a role in immunomodulation and is associated with improved resistance of mesothelial cells to PDF exposure.16Raspe C. Czeslick E. Weimann A. et al.Glutamine and alanine-induced differential expression of intracellular IL-6, IL-8, and TNF-alpha in LPS-stimulated monocytes in human whole-blood.Cytokine. 2013; 62: 52-57Crossref Scopus (27) Google Scholar Thus, we examined its effect in our uremic rat PD model. Parietal peritoneal sections from the Ala-Gln PDF group showed reduced peritoneal thickness compared with the standard PDF group. Masson trichrome staining revealed that the peritoneal thickening in the Ala-Gln PDF group and the control group was comparable, indicating a protective effect of the dipeptide (Figure 1a and b). Decreased gene expression of FBN and protein expression of HA were found in the Ala-Gln group when compared with the PDF group (Figure 1c and d). Immunofluorescence analysis of peritoneal biopsy samples showed that exposure to Ala-Gln PDF resulted in a significant reduction of α–smooth muscle actin (α-SMA)-positive cells accumulating in the parietal membrane on daily instillation with conventional PDF (Figure 2a and b ). A positive linear correlation was also observed between the number of α-SMA–positive cells and the peritoneal thickness (Figure 2c). Additionally, the number of CD31+ vessels was lowered in the Ala-Gln–enriched group (Figure 2d and e). Regarding ultrafiltration, there was a nonsignificant minor decline with PDF exposure that was reversed with Ala-Gln (Supplementary Figure S1A online). The total number of peritoneal cells increased in the PDF groups with and without the addition of Ala-Gln is seen in Supplementary Table S1 online. A white cell differential demonstrated that these changes were mainly caused by an increase in lymphocytes and macrophages and a decrease in eosinophils, in accordance with previous observations after PDF exposure.14Schilte M.N. Celie J.W. Wee P.M. et al.Factors contributing to peritoneal tissue remodeling in peritoneal dialysis.Perit Dial Int. 2009; 29: 605-617PubMed Google Scholar, 26Loureiro J.S. Schilte M. Aguilera A. et al.BMP-7 blocks mesenchymal conversion of mesothelial cells and prevents peritoneal damage induced by dialysis fluid exposure.Nephrol Dial Transplant. 2010; 4: 1098-1108Crossref Scopus (77) Google Scholar Recent findings have shown activation of the Th17 immune response in patients undergoing PD and preservation of peritoneal membrane integrity by blockade of IL-17 in an experimental mouse PD model.25Rodrigues-Diez R. Aroeira L.S. Orejudo M. et al.IL-17A is a novel player in dialysis-induced peritoneal damage.Kidney Int. 2014; 86: 303-315Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Therefore, we quantified IL-17 in the current study. IL-17 was elevated by PDF exposure in peritoneal membrane and effluent and was reduced by addition of Ala-Gln (Figure 3a and b ). Examination at the mRNA level of IL-17 regulatory transcription factor retinoic acid receptor–related orphan receptor gamma T revealed expression in PDF Ala-Gln comparable to a control group, in contrast with the elevated levels of the PDF group (Figure 3c). Moreover, a positive Spearman correlation between IL-17 protein and peritoneal thickness was shown in rats (Figure 3d). To elucidate molecular mechanisms underlying the protective role of Ala-Gln on the peritoneal membrane, we examined the effect of IL-17 antagonism in a mouse model of peritoneal fibrosis. As previously shown in the rat model, Ala-Gln in mice was also associated with reduced peritoneal thickness (drastically increased after 8 weeks of daily exposure to PDF). Moreover, increased levels of IL-17 were related to higher PD-induced peritoneal thickening, because the addition of recombinant IL-17 significantly increased extracellular matrix thickness in PDF-treated mice. Conversely, IL-17 blockade prevented formation of peritoneal extracellular matrix in this group (Figure 3e and f). Thus, IL-17–dependent thickening of the peritoneal membrane was directly demonstrated in the mouse model by i.p. injections of recombinant IL-17 or IL-17–blocking antibody. The same interventional model was used to demonstrate effects of Ala-Gln on IL-17–dependent thickening of the peritoneal membrane. Addition of Ala-Gln reduced peritoneal thickness (Figure 3e and f) and both protein and mRNA IL-17 levels (Figure 3g and h) with combined exposure to recombinant IL-17 and PDF, whereas blockade of IL-17 reduced peritoneal extracellular matrix in the PDF group and further decreased it in the Ala-Gln PDF-exposed group (Figure 3e and f). Ala-Gln thus appeared to act in a way comparable with blockade of IL-17 expression. To further investigate whether the protective role played by the addition of Ala-Gln to PDF is caused by the modulation of IL-17 expression, key factors involved in peritoneal Th17 cell differentiation were studied. In the rat model, a significant upregulation of TGF-β (Figure 4a and b ) and IL-6 (Figure 4c and d) in the PDF-only group and downregulation in the PDF–Ala-Gln group when compared with the control group was noted. In the mouse model, no significant differences were found between groups regarding both mRNA and protein levels of IL-6 (Figure 4e and f), interferon gamma (Figure 4g and h), and IL-4 (Figure 4i and j) measured respectively in peritoneal effluents and parietal peritoneal biopsies. Nevertheless, IL-6 protein expression was reduced in the Ala-Gln group, as previously observed in rats. Similar to the rat model, in mice not significant differences regarding ultrafiltration were found, and the resultant total number of peritoneal cells increased in the PDF-exposed groups (without reaching significance) (Supplementary Table S2 online). No significant variation was found in the number of macrophages and lymphocytes, but further subtyping suggested that differences in lymphocytes were restricted to CD4+/IL-17+ lymphocytes. Analysis of infiltrating cells by confocal microscopy indeed showed a clear reduction in CD4+/IL-17+ parietal peritoneum–infiltrating T cells when the Ala-Gln–enriched group was compared with the PD-treated group (Figure 5a). Moreover analysis of the effluent showed that Ala-Gln was able to quantitatively reduce PDF-enhanced recruitment of CD4+/ IL-17+ T lymphocytes (Figure 5b). In this study it is shown that the addition of Ala-Gln to PD fluid is protective against peritoneal fibrosis in both uremic rat and mouse models. This is substantiated by decreases in peritoneal thickening, expression of epithelial-to-mesenchymal transition markers, pathologic neovascularization, and a reduction in inflammatory/immune cell numbers in both the effluent and the parietal peritoneal tissue. Finally, these data provide compelling evidence that Ala-Gln plays its protective role, at least partly, by modulating the expression of IL-17, a key factor involved in peritoneal membrane integrity. Although PD represents a life-saving treatment for many patients with chronic kidney disease, long-term exposure to conventional PDF causes a progressive increase of the peritoneal compact zone thickness, vascular changes, fibrosis and inflammatory events that ultimately result in treatment failure.6Krediet R.T. Lindholm B. Rippe B. Pathophysiology of peritoneal membrane failure.Perit Dial Int. 2000; 20: S22-S42PubMed Google Scholar, 7Di Paolo N. Sacchi G. De Mia M. et al.Morphology of the peritoneal membrane during continuous ambulatory peritoneal dialysis.Nephron. 1986; 44: 204-211Crossref PubMed Scopus (146) Google Scholar, 8Mateijsen M.A. van der Wal A.C. Hendriks P.M. et al.Vascular and interstitial changes in the peritoneum of CAPD patients with peritoneal sclerosis.Perit Dial Int. 1999; 19: 517-525PubMed Google Scholar, 9Williams J.D. Craig K.J. Topley N. et al.Morphologic changes in the peritoneal membrane of patients with renal disease.J Am Soc Nephrol. 2002; 13: 470-479PubMed Google Scholar, 10Aguilera A. Yanez-Mo M. Selgas R. et al.Epithelial to mesenchymal transition as a triggering factor of peritoneal membrane fibrosis and angiogenesis in peritoneal dialysis patients.Curr Opin Investig Drugs. 2005; 6: 262-268PubMed Google Scholar, 11Yanez-Mo M. Lara-Pezzi E. Selgas R. et al.Peritoneal dialysis and epithelial-to-mesenchymal transition of mesothelial cells.N Engl J Med. 2003; 348: 403-413Crossref PubMed Scopus (619) Google Scholar The PD procedure is a well-recognized stimulus for inflammation, but uremia itself also activates inflammatory pathways, resulting in a state of microinflammation.9Williams J.D. Craig K.J. Topley N. et al.Morphologic changes in the peritoneal membrane of patients with renal disease.J Am Soc Nephrol. 2002; 13: 470-479PubMed Google Scholar, 27Combet S. Ferrier M.L. Van Landschoot M. et al.Chronic uremia induces permeability changes, increased nitric oxide synthase expression, and structural modifications in the peritoneum.J Am Soc Nephrol. 2001; 12: 2146-2157PubMed Google Scholar In this study, we wanted to mimic as closely as possible the patient situation by combining uremia with long-term exposure to PDF in both rat and mouse models. In congruence with patient studies,8Mateijsen M.A. van der Wal A.C. Hendriks P.M. et al.Vascular and interstitial changes in the peritoneum of CAPD patients with peritoneal sclerosis.Perit Dial Int. 1999; 19: 517-525PubMed Google Scholar this investigation showed peritoneal membrane thickening and upregulated expression of fibrotic markers after extended PDF exposure. Interestingly, thickness of the peritoneal membrane and fibrosis (indicated by HA, FBN, and TGF-β upregulation) have been successfully restored by the addition of Ala-Gln dipeptide to the PDF. Apart from this, a possible involvement of Ala-Gln in preventing alterations of the peritoneal membrane regarding myofibroblast development and new blood vessel formation was found. Ala-Gln–enriched PDF did not cause any significant alteration regarding fibrosis and new vessel formation when compared with the control group, whereas an increased number of α-SMA and CD31+ cells appeared in the PDF-exposed group not treated with Ala-Gln. Because Ala-Gln is known to play a role in immunomodulation,14Schilte M.N. Celie J.W. Wee P.M. et al.Factors contributing to peritoneal tissue remodeling in peritoneal dialysis.Perit Dial Int. 2009; 29: 605-617PubMed Google Scholar we investigated whether this dipeptide could protect against peritoneal fibrosis by modulating the Th17 response. IL-17, which is the main cytokine produced by Th17 cells, is involved in many inflammatory diseases and is responsible for pulmonary,28Gasse P. Riteau N. Vacher R. et al.IL-1 and IL-23 mediate early IL-17A production in pulmonary inflammation leading to late fibrosis.PLoS One. 2011; 6: e23185Crossref PubMed Scopus (158) Google Scholar cardiac,29Cortez D.M. Feldman M.D. Mummidi S. et al.IL-17 stimulates MMP-1 expression in primary human cardiac fibroblasts via p38 MAPK- and ERK1/2-dependent C/EBP-beta, NF-kappaB, and AP-1 activation.Am J Physiol Heart Circ Physiol. 2007; 293: H3356-H3365Crossref PubMed Scopus (195) Google Scholar and liver fibrosis30Tan Z. Qian X. Jiang R. et al.IL-17A plays a critical role in the pathogenesis of liver fibrosis through hepatic stellate cell activation.J Immunol. 2013; 191: 1835-1844Crossref PubMed Scopus (225) Google Scholar and for peritoneal damage in experimental PD models. IL-17–injected mice had increased peritoneal thickness and upregulation of inflammatory cytokines, whereas IL-17 blockade diminished fibrotic responses in the peritoneum.25Rodrigues-Diez R. Aroeira L.S. Orejudo M. et al.IL-17A is a novel player in dialysis-induced peritoneal damage.Kidney Int. 2014; 86: 303-315Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar These data point to IL-17 as a good candidate for therapeutic strategies. In our study, the increase of the submesothelial zone thickness correlated with IL-17 expression in the peritoneal cavity. Our results showed that Ala-Gln not only was able to reduce peritoneal fibrosis but also produced a drastic decrease in IL-17 expression in both peritoneal membrane biopsy samples and peritoneal effluents. Retinoic acid receptor–related orphan receptor gamma T is the transcription factor responsible for IL-17 expression and drives Th17 cell differentiation.20Ivanov II, McKenzie B.S. Zhou L. et al.The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells.Cell. 2006; 126: 1121-1133Abstract Full Text Full Text PDF PubMed Scopus (3962) Google Scholar Our results demonstrated a downregulation of this transcription factor in rats treated with Ala-Gln, and this evidence suggests that Ala-Gln plays a protective role by regulating T-cell differentiation and subsequent IL-17 production. Our uremic PD model showed elevated expression of the Th17-related cytokines IL-6 and TGF-β and their drastic drop as a consequence of the Ala-Gln treatment. In addition to being a strong profibrotic cytokine,31Border W. Transforming growth factor beta in tissue fibrosis.N Engl J Med. 1994; 10: 1286-1292Google Scholar TGF-β also works in a coordinated manner with IL-6 to drive T-cell differentiation through the Th17 lineage. Consistent with this evidence, our data showed an analogue pattern for IL-17 levels and the expression of its positive regulators TGF-β and IL-6. Although previous studies observed a synergic function of IL-6 with interferon gamma (INF gamma) in driving peritoneal fibrosis,32Fielding C.A. Jones G.W. McLoughlin R.M. et al.Interleukin-6 signaling drives fibrosis in unresolved inflammation.Immunity. 2014; 40: 40-50Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar our results did not showed any significant differences between groups regarding INF gamma expression levels. Treated animals appear to have massive induction of INF gamma at the protein level, but because of the large interanimal variation, it is not possible to conclude any presence or absence of Th1 cell involvement. Because of contrasting results about IL-4, mRNA expression, and protein levels and lack of significant differences between groups, the role of Th2 cells remain still unclear. Taken together, our data suggest that IL-17, partly in synergy with TGF-β and IL-6, is the central player of the Ala-Gln–dependent protective effect against peritoneal fibrosis. In addition to that, in our mouse PD model, the number of CD4+/IL-17+ T cells detected in peritoneal effluents significantly increased after PDF exposure and decreased in the presence of Ala-Gln dipeptide. This result was also reflected by the presence/absence of CD4+/IL-17+ T cells infiltrated into the peritoneal membrane after PDF/PD–Ala-Gln treatment. Finally, a positive correlation between the increment of IL-17 levels and peritoneal thickening and the comparable effect of IL-17 blockade and Ala-Gln treatment after injections of recombinant IL-17 or IL-17 antibody provided direct evidence that Ala-Gln modulates the regulation of IL-17 levels and that it plays its protective role at least in part by means of the IL-17 pathway. In conclusion, exposure to conventional PDF caused peritoneal fibrosis and increased expression of IL-17. Supplementation of PDF with pharmacologic doses of Ala-Gln prevented the peritoneal thickness and preserved original peritoneal vasculature. In our uremic PD models, Ala-Gln had a protective role by modulating the IL-17 pathway. The results obtained in the mouse PD model confirmed and strengthened this role and indicated the use of Ala-Gln as a possible therapeutic strategy to preserve the integrity of the peritoneal membrane in patients undergoing PD. Further work is needed to study the clinical significance of Ala-Gln–mediated protection against the nonphysiologic, morphologic, and structural changes occurring in the peritoneal cavity during PD. Male Wistar rats (Charles River, Maastricht, The Netherlands) weighing 250 to 275 g were used. Fifteen rats were made uremic by 5/6 nephrectomy. These animals were then split into 3 groups. A control group (n = 5) that did not have catheters implanted, a PD-exposed group that was injected daily for 5 weeks with standard PDF (Dianeal 4; 3.86% glucose; pH, 5.2; Baxter R&D, Utrecht, The Netherlands) as previously described,33Hekking L.H. Aalders M.C. Van Gelderop E. et al.Effect of peritoneal dialysis fluid measured in vivo in a rat-model of continuous peritoneal dialysis.Adv Perit Dial. 1998; 14: 14-18PubMed Google Scholar, 34Hekking L.H. Zareie M. Driesprong B.A. et al.Better preservation of peritoneal morphologic features and defense in rats after long-term exposure to a bicarbonate/lactate-buffered solution.J Am Soc Nephrol. 2001; 12: 2775-2786PubMed Google Scholar and a PD group that was also injected daily for 5 weeks but with Dianeal enriched with 8 mM Ala-Gln dipeptide (Dipeptiven; Fresenius Kabi Austria, Graz, Austria). After 1 week of acclimatization and handling, a 5/6 nephrectomy was performed to induce uremia. The nephrectomy involved the complete removal of the right kidney and ligation of 1 to 3 branches of the arteries supplying the left kidney to obtain residual kidney function of about one sixth of the total capacity. To verify uremia induction, urea and creatinine serum levels were measured at day 0 (before 5/6 nephrectomy) and day 21 (data shown in Supplementary Figure 2A and B online). After 2 weeks of recovery, a peritoneal catheter connected to a subcutaneous access port (Venflon Pro, BD Medical, Franklin Lakes, NJ) was implanted as previously described.34Hekking L.H. Zareie M. Driesprong B.A. et al.Better preservation of peritoneal morphologic features and defense in rats after long-term exposure to a bicarbonate/lactate-buffered solution.J Am Soc Nephrol. 2001; 12: 2775-2786PubMed Google Scholar For 1 week after catheter implantation, 2 ml of saline with 1 IU/ml heparin was administered each day (n = 10). Animals were randomized to 2 treatment groups. For the following 5 weeks, rats were instilled daily with 10 ml of standard PDF (n = 5) or PDF enriched with Ala-Gln (n = 5). C57BL/6J female mice (Charles River, Maastricht, The Netherlands) aged 12 to 14 weeks and weighing approximately 20 g at the start of the study were used. Animals were organized as follows: 1 healthy control group (n = 5), 3 PD groups (n = 5 per group) exposed daily to 2 ml standard Dianeal during a period of 8 weeks, and 3 PD groups (n = 5 per group) exposed daily for 8 weeks to Dianeal enriched with Dipeptiven (Ala-Gln). PD groups were injected i.p. weekly with IL-17 recombinant protein (human recombinant protein; R&D Systems, Minneapolis, MN) at a dose of 10 ng/g of body weight, anti–IL-17 antibody (eBioMM17F3; eBioscience, San Diego, CA) 100 μl/mouse, or no additional injection during the whole experiment. Mice in all the PD groups underwent 5/6 nephrectomy and catheter implantation (customized mouse catheter MMP-4S-061108A, Access Technologies, Ridgeway, Skokie, IL). The 5/6 nephrectomy consisted of the complete removal of the right kidney and the removal of the anterior and posterior thirds of the left kidney by using a monopolar electric blade, as described previously.35Ferrantelli E. Liappas G. Keuning E.D. et al.A novel mouse model of peritoneal dialysis: combination of uraemia and long-term exposure to PD fluid.Biomed Res Int. 2015; 2015: 106902Crossref Scopus (9) Google Scholar To verify uremia induction, urea and creatinine serum levels were measured at day 0 (before 5/6 nephrectomy), at day 15, and at day 70 (end point) (Supplementary Figure 2C and D online). All the animals were housed under standard conditions and were given food and water ad libitum. Health conditions were checked daily. The weight of the animals was checked daily after surgery during a period of 10 days and weekly for the remainder of the experiment. Animals that lost more than 20% of their initial body weight or showed abnormal activity were excluded from the experiment. The experimental protocols were approved by the Animal Welfare Committee at the VU University Medical Center, Amsterdam. Five hundred and 200 μl of blood were drawn through tail and facial vein puncture in rats and mice at the time points previously indicated. At all the time points, serum samples were analyzed for urea and creatinine levels. For determination of urea levels, a kinetic test with urease and glutamate dehydrogenase was used. Creatinine levels were detected by indirect immunofluorescence assay. Measurements were performed by using spectrophotometer Cobas8000 C702 (Roche Diagnostics, Risch-Rotkreuz, Switzerland). At the end point after the injection of 2 ml standard PDF by catheter in mice or 30 ml through an extra catheter (Venflon Pro) in rats, peritoneal effluents were collected (after 30 and 90 minutes, respectively) and cells were isolated by centrifugation and counted. Cytocentrifuge preparations were made and cells number determined by May Grünwald staining for rat cells. Cell suspensions obtained from peritoneal lavage in mice were stained with fluorochrome-conjugated mouse-specific antibodies against CD3, CD4, CD8α, B220, CD11b, Ly6C, F480, and IL-17 purchased from eBiosciences (San Diego, CA). Before intracellular staining, cells were restimulated for 4 hours with 50 ng/ml phorbol myristate acetate (PMA) and 500 ng/ml ionomycin in the presence of 1 μg/ml BD Golgi Plug (eBiosciences). Samples were analyzed in a BD FACS Fortessa (BD Biosciences, San Jose, CA) flow cytometer, and further analyses were performed with FlowJo software. Parietal peritoneal biopsy samples were collected from the side opposite the catheter installation. The biopsy samples were fixed in Bouin solution, embedded in paraffin, cut into 5-μm sections, and stained with Masson trichrome. Peritoneal membrane thickness was determined using light microscopy (Leica CTR6000 with a Leica Microsystems LAS-AF6000, Leica Biosystems, Inc., Buffalo Grove, IL). Microscope photographs were obtained using an Olympus BX41 clinical microscope and an Olympus DP20 digital camera (Olympus, Center Valley, PA) using cell acquisition software. Peritoneal thickness of each animal was calculated by the median of measurements taken every 50 μm from 1 side to the other of the tissue sample. Biopsy samples were frozen in Tissue-Tek O.C.T. (Sakura, Finetek, Europe B.V., The Netherlands) and cut into 5-μm sections. To identify myofibroblasts and vessels, samples were stained for anti-rat Alp α-SMA (α-SMA 1A4, Dako North America, Inc., Carpinteria, CA; 1:500) combined with anti-mouse IgG (H+L) AF (Invitrogen/Life Technologies Corp., Grand Island, NY) and CD31 (αCD31; PECAM, Serotec, Oxford, UK; 1:1000) coupled to anti-mouse IgG-555 (Invitrogen/Life Technologies Corp., Grand Island, NY) according to the manufacturer's instructions. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Florescence microscopy was performed with a Zeiss microscope (Zeiss LM; Zeiss, Oberkochen, Germany). The areas positive for CD31 were calculated by CellProfiler software (2.1.1, Broad Institute, Cambridge, MA). To identify peritoneal infiltrated CD4+/IL-17+ T cells, double immunofluorescence was performed by using an anti-mouse/rat IL-17 (APC) (eBio17B7, ref: 17-7177-81, eBioscience) and an anti-mouse/rat CD4 (PE) (Lot: 3130616, ref: 553730, BD eBiosciences). Nuclei were stained with 4′,6-diamidino-2-phenylindole. Microscopy was performed with a confocal microscope (Leica TCS SPE with LAS0AF software, version 2.0.1, build 2043). Parietal peritoneum biopsy samples were mechanically homogenized, and total RNA was extracted using TRIzol Reagent (Invitrogen). Reverse transcription into cDNA was performed using a Reverse Transcription System Kit (Promega, Madison, WI). The synthesized cDNA was amplified with a standard quantitative polymerase chain reaction protocol including the use of SYBR Green (Applied Biosystems/Life Technologies) and rat or mouse specific primers were used (Supplementary Table S3 online). The relative amount of mRNA was calculated using comparative Ct (δδ Ct) methods. All samples were analyzed in triplicate and averages compared. Amplification products were normalized against glyceraldehyde 3-phosphate dehydrogenase mRNA, which was amplified in the same reaction as the internal control for each analyzed gene. Supernatants were made cell free by centrifugation (300 g, 5 minutes, room temperature) and stored at –20°C. Protein levels of rat IL-17A, TGF-β, and HA were quantified by enzyme-linked immunoassay (ELISA)-based assay (Rat Platinum ELISA kit; eBioscience). Protein levels of mouse IL-17A, IL-6, INF gamma, and IL-4 were quantified by ProcartaPlex Multiplex Imuunoassays (Affymetrix, Santa Clara, CA). Data were analyzed using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA). Statistical analysis was performed using 1-way analysis of variance to compare the groups. Bonferroni correction was used to correct for multiple analyses. Data were shown as means ± SEM. Confidence intervals used were 95%. Correlations were assessed using the Spearman correlation tests (GraphPad Prism, version 5.03). P < 0.05 was considered statistically significant (in figures, * = P < 0.05, ** = P < 0.01, *** = P < 0.001). All the authors declared no competing interests. This work was supported by the European Union, Seventh Framework Program "EuTRiPD" under grant agreement Marie Curie ITN-GA-2011-287813 (EF, GL, TF, MVC). We wish to thank Prof D. Fraser (Cardiff University) for providing English-language editing of the final manuscript and NJ Paauw for his technical help. Tables S1–S3View Large Image Figure ViewerDownload (PPT)
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