Differences in peritoneal response after exposure to low-GDP bicarbonate/lactate-buffered dialysis solution compared to conventional dialysis solution in a uremic mouse model

Differences in peritoneal response after exposure to low-GDP bicarbonate/lactate-buffered... Background Long-term exposure of conventional peritoneal dialysis (PD) fluid is associated with structural membrane alterations and technique failure. Previously, it has been shown that infiltrating IL-17-secreting CD4+T cells and pro-fibrotic M2 macrophages play a critical role in the PD-induced pathogenesis. Although more biocompatible PD solutions are rec- ognized to better preserve the peritoneal membrane integrity, the impact of these fluids on the composition of the peritoneal cell infiltrate is unknown. Materials and methods In a uremic PD mouse model, we compared the effects of daily instillation of standard lactate (LS) or bicarbonate/lactate-buffered solutions (BLS) and respective controls on peritoneal fibrosis, vascularisation, and inflammation. Results Daily exposure of LS fluid during a period of 8 weeks resulted in a peritoneal increase of αSMA and collagen accom- panied with new vessel formation compared to the BLS group. Effluent from LS-treated mouse showed a higher percentage + + of CD4 IL-17 cell population while BLS exposure resulted in an increased macrophage population. Significantly enhanced inflammatory cytokines such as TGFβ1, TNFα, INFγ, and MIP-1β were detected in the effluent of BLS-exposed mice when compared to other groups. Further, immunohistochemistry of macrophage subset infiltrates in the BLS group confirmed a higher ratio of pro-inflammatory M1 macrophages over the pro-fibrotic M2 subset compared to LS. Conclusion Development of the peritoneal fibrosis and angiogenesis was prevented in the BLS-exposed mice, which may + + underlie its improved biocompatibility. Peritoneal recruitment of M1 macrophages and lower number of CD4 IL-17 cells might explain the peritoneal integrity preservation observed in BLS-exposed mouse. Keywords Peritoneal dialysis · Macrophage · IL17 · Fibrosis · Angiogenesis Introduction accumulation of extracellular matrix, angiogenesis, and other structural alterations of the peritoneum resulting in Continuous and long-term treatment with peritoneal dialy- technique failure and serious clinical complications includ- sis (PD) promotes an inflammatory response which even- ing encapsulation peritoneal sclerosis [1]. To a large extent, tually leads to a progressive remodeling of the peritoneal these events seem to be driven by high glucose degrada- membrane [1]. These changes are characterized by the tion products (GDPs) content, the low pH, and the pres- ence of lactate, typically present in conventional PD fluids [2]. Therefore, the conversion of PD fluids towards more * M. G. Vervloet biocompatible solutions is recognized as an urgent unmet m.vervloet@vumc.nl clinical need to better preserve the peritoneal integrity. As a 1 consequence, lactate–bicarbonate buffered fluids with more Department of Nephrology, VU University Medical Center, physiological pH, a lower amount of GDPs, and alternative Amsterdam, The Netherlands 2 solutions in which glucose is replaced by alternative osmotic Amsterdam Cardiovascular Sciences, Amsterdam, agents such as icodextrin and amino-acids have been devel- The Netherlands 3 oped [3]. Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands Vol.:(0123456789) 1 3 1152 International Urology and Nephrology (2018) 50:1151–1161 The introduction of neutral pH and particularly bicarbo- MMP-4S-061108A, Access Technologies, Ridgeway, nate/lactate-buffered solutions seemed to offer advantages USA). 5/6 nephrectomy consisted in the complete removal in terms of peritoneal membrane preservation and perito- of the right kidney and the removal of the anterior and neal homeostasis control [4, 5]. In the Euro balance trial, it posterior 1/3 part of the left kidney by using a monopolar demonstrated a significant improvement in effluent markers electric blade as previously described [14]. All the animals of peritoneal membrane integrity, a decrease in systemic were housed under standard conditions and were given advanced glycation end products (AGEs) levels, less decline food and water ad libitum. Health conditions were checked in residual renal function and a decrease in peritoneal ultra- daily. The experimental protocols were approved by the filtration [6 ]. Moreover, such biocompatible fluids increase Animal Welfare Committee at the VU University Medical mesothelial cell markers, induce less systemic inflammation, Center, Amsterdam. and reduced the incidence of peritonitis [7–10]. Recent studies revealed that immunological responses underlie PD-induced peritoneal injury upon conventional Cell counting high GDPs lactate PD fluid exposure although the spe- cific mechanism remains unclear. These data pointed to At the end point, following the injection of 2-ml stand- the importance of IL-17-mediated inflammation as a novel ard PD fluid via a catheter, peritoneal effluents were col- player in the PD-induced injury in both PD patients and lected after 30 min; cells were isolated by centrifugation experimental models [11]. Furthermore, M2 macrophages and counted and stained with fluorochrome-conjugated are suggested to play a key role in the development of peri- mouse-specific antibodies against CD3, CD4, CD8α, toneal inflammation and fibrosis [12, 13]. However, while B220, CD11b, Ly6C, F480, and IL-17 purchased from experimental and clinical data suggest a better preservation eBiosciences. Before intracellular staining, cells were re- of peritoneal morphologic and functional features upon stimulated for 4 h with 50 ng/ml Phorbol 12-Myristate bicarbonate/lactate solution compared to conventional PD 13-Acetate (PMA) and 500 ng/ml ionomycin in the pres- solutions, the implication on the inflammatory cell popu- ence of 1 µg/ml BD Golgi Plug (eBiosciences). Samples lation in this novel solution has been poorly defined. To were analyzed in a BD FACS Fortessa (BD Biosciences) overcome this knowledge gap, our recently developed ure- flow cytometer and further analyses were performed with mic mouse PD exposure model [14] was used in the pre- FloJo software. sent study to compare a pH neutral low-GDP bicarbonate/ lactate-buffered solution (BLS) with a standard high GDPs lactate (LS) PD fluid in respect of inflammation, fibrosis, Histology and immunohistochemistry and vascularisation. Here, we demonstrate that the enhanced fibrotic and angiogenic response observed in LS-exposed Parietal peritoneal biopsies were collected from the opposite mouse was prevented upon BLS exposure. This preserva- side from the catheter installation. The biopsies were fixed tion of the peritoneal integrity by BLS was accompanied in Bouin’s solution, embedded in paraffin, cut into 5-µm sec- + + with a lower number of CD4 IL-17 cells, higher levels of tions and stained with Masson’s Trichrome. Peritoneal mem- macrophage-related pro-inflammatory cytokines and with a brane thickness was determined using a Carl Zeiss Micro- higher ratio of M1 macrophages over M2 subset. scope (GmbH, 37081, Göttingem, Germany). Microscope photographs were obtained by using an AxioCam ICc5. The peritoneal thickness of each animal was calculated by the Methods median of measurement taken every 50 µm from one side to the other of the tissue sample. Mouse PD model Biopsies were frozen in Tissue-Teck® (O.C.T.® Sakura) and cut into 5-µm sections. To identify myofibroblasts and C57BL/6J female mouse (Charles River, Maastricht, The vessels, samples were stained for anti-rat Alpha Smooth Netherlands) aged 12–14 weeks and weighing approxi- Muscle Actin (αSMA 1A4, DAKO, 1:500) combined with mately 20  g at the start of the study were used. Ani- anti-mouse-IgG (H+L) (Invitrogen) and Cluster of Differen- mals were organized as follows: 1 healthy control group tiation 31 (αCD31, PECAM, Serotec, Oxford, UK, 1:1000) (n = 10), 3 PD groups (n = 10 per group) daily exposed coupled to anti-mouse-IgG-555 according to the manufac- to 2  ml saline or standard lactate-buffered solution turer’s instructions. Nuclei were stained with DAPI. Fluores- (Dianeal®, Baxter) or bicarbonate/lactate-buffered solu- cence microscopy was performed with a Carl Zeiss Micro- tion (Physioneal®, Baxter) during a period of 8 weeks. scope and photographs were taken with an AxioCam HR R3. Mouse in all the PD groups underwent 5/6 nephrectomy The areas positive for CD31 were calculated by CellProfiler and catheter implantation (Customized mouse catheter software (2.1.1, Broad Institute, UK). 1 3 International Urology and Nephrology (2018) 50:1151–1161 1153 However, thickening of peritoneum did not differ between Immunoblotting the two PDF compositions (LS or BLS). In contrast, immu- nohistological analysis of peritoneal biopsies revealed that Lysates of the peritoneal membrane were prepared by homogenizing of preserved tissue in lysis buffer containing exposure to BLS significantly prevented (P = 0.01) the accumulation of α αSMA positive cells (myofibroblast) in Protease Inhibitor Cocktail (Roche Applied Science, Indi- anapolis, IN). Protein concentrations were determined using the parietal membrane when compared to conventional LS PDF (C: 0.020 ± 0.016, S: 0.026 ± 0.026, LS: 0.075 ± 0.072, the Pierce Micro BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). The following antibodies were used: CD31 BLS: 0.016 ± 0.014), (Fig. 1b, c). Similarly, statistically sig- nificant enhanced collagen I protein levels were detected in (Abbiotec; 1: 200), Collagen I (Abcam; 1:250), GAPDH (14C10) 1:1000 cell signaling, followed by donkey anti-rat/ peritoneal samples after exposure to LS fluid when com - pared to control, whereas upon BLS no significant effect was rabbit conjugated with HRP (Dako; 1:5000). Signal was vis- ualized using enhanced chemiluminescence (Life Sciences) shown (C: 0.33 ± 0.19, S: 0.40 ± 0.39, LS: 1.24 ± 0.94, BLS: 0.66 ± 0.13), (Fig. 1d, e). Analysis of the peritoneal effluents on LAS3000 (Fujifilm, Japan). Image J (NIH, Bethesda, Maryland) was used for analysis. collected after 8 weeks of daily exposure to the different treatments performed by standard peritoneal equilibrium test Quantification of cytokines (PET) revealed no differences between the groups regarding the volume of ultrafiltration (data not shown). Peritoneal effluents collected after 8 weeks of treatment Chronic treatment with LS resulted in a significant incre- ment (P = 0.02) of new vessel formation in the omentum, were made cell free by centrifugation (300G, 5 min, RT) and stored at − 20 °C. Protein levels of mouse Transforming represented by the increased abundance of CD31 protein- positive cells (indicative of endothelial cells) when com- Growth Factor β 1 (TGFβ1), Interleukin-1β (IL-1β), Tumor Necrosis Factor α (TNFα), Vascular Endothelial Growth pared with the BLS group (Fig.  2a, b). These data were confirmed by the detection in the peritoneal tissue of high Factor (VEGF), IL-17A, IL-6, Interferon γ (INFγ), IL-5, IL-4, Macrophages Inflammatory Proteins 1α and β (MIP-1α levels of CD31 protein upon LS fluid which was prevented (P = 0.02) in BLS-exposed animals (Fig.  2c, d). Taken and MIP-1β) were quantified by ProcartaPlex™ Multiplex Immunoassays (Affymetrix eBioscence). together, these results indicate that LS but not BLS fluid pro- moted a fibrotic and angiogenic response in the peritoneum Statistical analysis although the development of increased peritoneal thickness was not preserved in BLS-exposed mouse. Data were analyzed using GraphPad Prism software (La Analysis of inflammatory cell recruitment Jolla, CA). Statistical analysis was performed using One-way ANOVA test to compare the groups. A P value < 0.05 was in peritoneal cavity upon PD fluid exposure considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001). Data were shown as means ± SD. Exposure to PD fluid caused a numerical increase of the total number of cells in the peritoneal fluid compared to non- exposed animals, which however was not statistically sig- nificant (Table  1). No statistically significant variation was Results found in both CD4- and CD8-positive populations among groups while there was a ninefold statistically significant BLS exposure in mouse prevented the development of both peritoneal fibrosis and angiogenesis increase in CD4/IL-17 double positive lymphocytes (CD4 IL-17 ) found in the LS group compared to the BLS group, with no changes in thickness in which these double positive cells remained at the same level as controls. No significant differences were observed In order to mimic the situation in chronic kidney disease patients undergoing PD, a uremic mouse model, performed between the groups in the percentage of B cells and mono- + + cytes (Ly6G CD11b ), although the number of the latest by 5/6 nephrectomy, was exposed to daily PD fluid during a period of 8 weeks. Nephrectomized groups showed a twofold increased especially after BLS. As in our previous study in mouse [15], a slight increase in macrophages (F480 increase in both serum urea and creatinine concentrations 15 days after the surgery and remained stable for the duration of CD11b ) was shown after exposure to conventional LS, but this rise was more pronounced in the BLS group (*P = 0.04 the experiment (data not shown). As shown in Fig. 1a, after 8 weeks of daily exposure to the PDFs, there was a statisti- compared to control). Therefore, exposure to BLS clearly caused profound changes compared to LS primarily in terms cally significant increase (P = 0.04) in peritoneal thickness compared to the non-PDF-exposed control (C: 30.63 ± 3.66, of peritoneal inflammatory macrophages recruitment, and IL-17 expressing CD4 cells. S: 41.84 ± 12.05, LS: 78.78 ± 39.62, BLS: 78.11 ± 32.87). 1 3 1154 International Urology and Nephrology (2018) 50:1151–1161 Fig. 1 Exposure to low GDPs bicarbonate/lactate-buffered PD fluid (blue). Representative immunoblotting (d) and analysis (e) of Colla- prevents myofibroblast recruitment but does not protect from parietal gen I (Col I) levels in total protein lysates of peritoneal membrane. peritoneum thickness. Graph a represents peritoneal thickness (µm) GAPDH was used as loading control (n = 6). C control, S saline, measurements for each group (n = 10). Representative immunofluo- LS lactate PD fluid, BLS bicarbonate/lactate PD fluid. Data show rescence microscopy (b) and analysis (c) of parietal peritoneal sec- means ± SD. Differences were considered statistically significant for tions stained with αSMA marker (magnification ×10; n = 10). αSMA P < 0.05 using one-way ANOVA. *P < 0.05 positive cells are indicated in red. Nuclei were stained with DAPI significant for BLS only (P = 0.04). A similar pattern was Cytokine production changes in BLS‑treated mouse observed for IL-1β that was slightly enhanced in the LS group and further increased upon BLS exposure (Fig. 3b), We further explored the differences between the PD flu- albeit non-significant. Alternatively, IL-6 and VEGF release ids in terms of inflammatory cytokine responses. As increased as a consequence of PD fluid exposure but it did shown in Fig.  3a, an increment in TGFβ1 levels was not differ statistically between the PD regimens and con- detected upon both BLS (435.9 ± 266.3  pg/ml) and LS trol (Fig. 3c, e). On the other hand, a statistically significant (204.1 ± 211.8 pg/ml) treatment when compared to control increase of TNFα was shown only after BLS exposure but group (C:28.57 ± 22.85 pg/ml) although this was statistically 1 3 International Urology and Nephrology (2018) 50:1151–1161 1155 Fig. 2 Peritoneal angiogenesis is attenuated in bicarbonate/lactate- lotting (c) and analysis (d) of CD31 levels in total protein lysates of buffered-exposed mouse. Immunofluorescence microscopy (a) and peritoneal membrane (n = 6). GAPDH was used as loading control. analysis (b) of omentum sections stained for vasculature with CD31; C control, S saline, LS lactate PD fluid, BLS bicarbonate/lactate PD (green) marker (n = 10). Each value corresponds to an average (% sur- fluid. Data show means ± SD. Differences were considered statisti- face staining CD31) of 10 independent values of each mouse omen- cally significant for P < 0.05 using one-way ANOVA. *P < 0.05 tum taken each time (magnification ×20). Representative immunob- not after LS treatment (C: 10.38 ± 1.25, LS: 33.57 ± 24.82, BLS: 239 ± 186.8 pg/ml; P = 0.02) (Fig. 3d). Emerging evidence points to IL-17 as an important factor 6 in mediating peritoneal inflammation [11]. In this study, the Table 1 Quantification of total cell number (×10 ) composition of + + cells (%) in mouse peritoneal effluents after 8 weeks of exposure increment found in the CD4 IL-17 cell population upon LS exposure was accompanied by a nominal, but statistically Cell populations Groups (Mean ± SD) insignificant, increase of the levels of IL-17 measured in the C LS BLS peritoneal effluents (Fig.  3g). We found upregulation of IL-5 in the LS group which was not shown upon treatment with Total cells (×10 ) 2.93 ± 1.31 4.36 ± 2.06 5.38 ± 2.56 BLS (C: 18.44 ± 2.40, LS: 53.4 ± 33.09, BLS: 23.44 ± 9.13) CD4 (%) 29 ± 1.14 22.13 ± 13.73 17 ± 13.87 (Fig. 3i). A similar trend was found for IL-4 (C: 13 ± 0.70, CD8 (%) 14.35 ± 3.23 3.45 ± 3.97 6.51 ± 7.37 + + LS: 59.07 ± 51.03, BLS: 18 ± 6.06) (Fig. 3h), indicating that CD4 IL-17 (%) 1.98 ± 2.46 13.19 ± 8.23* 1.47 ± 2.49 involvement of T helper 2 (Th2) cells was more pronounced Monocytes (%) 7.45 ± 8.7 10.88 ± 10.09 14.8 ± 11.38 upon LS treatment over BLS. On the other hand, the sta- Macrophages (%) 2.03 ± 0.69 8.16 ± 6.84 25.07 ± 16.78* tistically significant high levels of INFγ (Fig.  3f) detected B cells (%) 37.85 ± 19.45 37.04 ± 23.18 35.03 ± 13.31 only after BLS exposure suggested that in BLS regimens *P < 0.05 an important role may be played by T helper 1 (Th1) cells Data show Means ± SD (n = 4). Differences were considered statisti- (C: 23.69 ± 5.65, LS: 42.18 ± 30.2, BLS: 180.3 ± 123.5; cally significant for P < 0.05 using one-way ANOVA. *P < 0.05. (Sta- P = 0.01). tistically significant differences were found in LS vs. C and LS vs. + + The presence of enhanced macrophage population in BLS for CD4 IL-17 , and BLS vs. C in Macrophages) BLS-treated mouse was further substantiated by the rise C control, LS lactate PD fluid, BLS bicarbonate/lactate PD fluid 1 3 1156 International Urology and Nephrology (2018) 50:1151–1161 Fig. 3 Exposure to a bicarbonate/lactate-buffered solution is associ- b IL-1β, c IL-6, d TNFα, e VEGF, f INFγ, g IL-17, h IL-4, i IL-5, j ated with increase of pro-inflammatory cytokines. Protein levels of MIP-1α, k MIP-1β. C control, LS lactate PD fluid, BLS bicarbonate/ the main pro-inflammatory cytokines detected in effluents collected lactate PD fluid. Differences were considered statistically significant from mouse after 8 weeks of PD fluid exposure. Cytokines levels for P < 0.05 using one-way ANOVA. *P < 0.05 (ρg/ml) are represented as means ± SD (n ≥ 7) as follows: a TGFβ1, of MIP-1α and MIP-1β (also known as CCL3 and CCL4) played a crucial role over other cell types in modulat- following BLS treatment although significant changes ing the response of BLS on peritoneal fibrosis and were only found in the MIP-1β measurements (MIP-1β angiogenesis. C: 12.81 ± 1.43, LS: 53.43 ± 49.73, BLS: 278.8 ± 246.8; P = 0.04) (Fig.  3j, k). Taken together, between the two PDFs, there are outspoken differences in levels of TGFβ1, TNFα, INFγ, and MIP-1β suggesting that macrophages 1 3 International Urology and Nephrology (2018) 50:1151–1161 1157 [16], in the peritoneum of BLS-treated mouse (Fig. 4b). In BLS exposure induced the recruitment of pro‑inflammatory macrophages in parietal addition, macrophages with classical morphology display high levels of both F480 and CD11b [17], which were peritoneum significantly increased in the BLS group compared to both the control and the LS groups (Fig. 4c). On the other hand, Given the parallels between the high percentage of mac- rophages in effluent and the elevated levels of chemokine staining for Dectin-1, a marker for anti-inflammatory (M2) macrophages, did not reveal any significant differences MIP-1α and 1β, we further explored macrophage subset population in the peritoneum by immunohistochemistry among groups (Fig. 4d). Overall, our results suggest that pro-inflammatory M1 phenotype modulated the response (Fig.  4a). Quantification shows high F480 expression, characteristic of classically activated macrophages (M1) in the peritoneum induced by BLS exposure. Fig. 4 Bicarbonate/lactate-buffered solutions mediate pro-inflamma- per mouse for each group. Significant increase of CD11b and F480 tory macrophages recruitment. Panel a shows representative perito- double positive and single F480-positive cells in the BLS group are neal membrane macrophages recruitment for each group in the ure- represented, respectively, in graphs (b) and (c). Graph d shows Dec- mic PD mouse model. Peritoneal staining for CD11b (violet), F480 tin-1-positive cells in each group (C control, LS lactate PD fluid, BLS (red), CD11b plus F480 double positive and Dectin-1 (green) are rep- bicarbonate/lactate PD fluid). Magnification ×20. Data show means ± resented in the rows from the top to the bottom panel, respectively. SD (n = 10). Differences were considered statistically significant for Nuclei were stained with DAPI (blue). Column bars represent number P < 0.05 using one-way ANOVA. *P < 0.05 of macrophages measured per field in three different pictures taken 1 3 1158 International Urology and Nephrology (2018) 50:1151–1161 structural changes and the increment of fibrosis [21– 23]. Discussion However, other factors such as the amount of cell infil- trate may contribute to the increment of peritoneal thick- The current study is the first one using a mouse PD expo- ness [24]. In addition, daily instillation with normal saline sure model comparing lactate and bicarbonate/lactate- also induces a slight increment of the thickness, indicating buffered solutions in a uremic setting, which more closely that peritoneal remodeling is not exclusive of PD fluid mimics the clinic status of PD patients when compared to effects, and these additional effects may have masked or the non-uremic animal models. Our findings support the overwhelmed any difference between types of PD solution notion that bicarbonate/lactate-buffered solution better pre- used. Furthermore, we previously reported that uremia per serves the peritoneal integrity status when compared with se also contributes to this event [14]. Overall, all those the conventional PD fluid. Specifically, we demonstrated + + factors may influence the lack of differences in thickness that the enhanced CD4 IL-17 cell population in effluent between PD groups. detected in LS-exposed mouse was prevented in the BLS In vitro experimental research has demonstrated the group although with no significant changes in IL-17 efflu - capacity of neutral pH, bicarbonate/lactate-buffered solu- ent concentrations. Importantly, increased macrophage tions to maintain mesothelial cellular integrity and function population together with high levels of chemokines that when compared to conventional fluid [18, 25]. Although regulate migration and infiltration of monocytes/mac- PD fluid may exert a direct impact on the mesothelial cell rophages suggested that macrophages played a key role stability, evidence points to the inflammatory response as during BLS treatment. While pro-fibrotic M2 macrophages key factor inducing PD-associated pathology. In this regard, are thought to promote fibrosis and angiogenesis upon con- in our previous studies, the chronic exposure to standard ventional PDF fluid treatment, in this study it was found dialysis fluids resulted in peritoneal Th17 response includ- that in BLS-exposed mouse the pro-inflammatory (M1) ing elevated IL-17 protein production [11]. Importantly, the phenotype was dominant in the peritoneal tissue over M2 modulation of IL-17 during treatment with PD fluid was subset suggesting a difference in inflammatory response shown to be an effective therapy for PD-mediated perito- between different PD fluid exposures. neal fibrosis and angiogenesis [11, 15, 26]. In our study, the The accumulation of extracellular matrix and fibrosis is + + increase in CD4 IL-17 cell population observed upon LS a characteristic peritoneal alteration induced by PD fluid treatment was prevented in the BLS groups. Although no exposure which leads to a progressive remodeling of the significant changes in IL-17 levels were observed between peritoneal membrane [1]. As previously demonstrated PD fluid exposed animals, it is possible that in this study [15], and confirmed by the present study, daily exposure of statistical significance for IL-17 was missed due to too low conventional PD fluid contributes to the fibrotic response power. by an accumulation of αSMA-positive cells in the parietal Alternatively, a striking enhanced INFγ concentration in peritoneum. Consistently, an increment of the extracel- the effluents of BLS group compared to the LS was found. lular matrix protein collagen I in peritoneal tissues of LS- In parallel, BLS slightly prevented the increment of IL-5 treated mouse was observed. In contrast, daily exposure to and IL-4 levels detected in the LS group. These findings BLS fluid prevented the accumulation of both αSMA and suggested that inflammatory mechanisms occurring during collagen I. This is in accordance with previous studies sug- exposure to bicarbonate/lactate buffer involve Th1 rather gesting that the use of low GDPs solutions was associated than Th2 cell subset while the opposite happened upon expo- with less αSMA expression in vitro and less development sure to high GDPs lactate solutions. Possibly, this particular of fibrotic response in the peritoneal membrane in rats inflammatory milieu can explain the differences observed in [4, 18, 19]. Importantly, other factors such as the neu- peritoneal remodeling between PD fluids. In this regard, T tral pH present in the more biocompatible fluid may also helper-related cells interact with many immune cells includ- contribute to the differences observed. In addition, as we ing macrophages, which also play a crucial role in chronic reported previously in a rat model with PD [4, 20], daily inflammation-induced fibrosis [27]. Enhanced levels of Th2 exposure to BLS prevented new vessel formation, which cell subsets together with pro-fibrotic cytokine microenvi- did occur when LS was used, indicating that BLS regimen ronment contribute to the polarization of peritoneal mac- is more biocompatible in terms of peritoneal angiogenesis. rophages towards the anti-inflammatory or pro-fibrotic M2 Our findings suggest reduced angiogenesis and peritoneal macrophages subset [27]. Particularly, M2 macrophages fibrosis as markers of better preservation of the structure subset demonstrated to be dominant in the cellular infiltrate of the peritoneum after exposure of BLS fluid. Despite the in PD patients and are suggested to drive peritoneal fibrosis differences reported, no changes in peritoneal thickness [12, 28]. In this study, BLS exposure mediated the increment were detected among the PD-treated groups. Numerous of the proteins of the chemotactic factors for macrophages studies have suggested a relationship between peritoneal MIP-1α and MIP-1β in the peritoneal effluents. It has been 1 3 International Urology and Nephrology (2018) 50:1151–1161 1159 shown that in the inflammatory phase, newly attracted mac- exposure to PD fluid [14]. As an additional limitation, no rophages present a more pro-inflammatory (M1) phenotype differences in the ultrafiltration capacity were detected but only after the switch to M2 they become pro-fibrotic between groups. This weakness, however, was previously [16, 27, 28]. Interestingly in our experiment, the increase reported in our previous study in uremic mice and rats with in the percentage of macrophages cell population in the PD [15]. Furthermore, an assumed reduction of the harmful peritoneal effluents after BLS exposure was associated with effects of the new generation of PD fluids may explain lim- enhanced recruitment of pro-inflammatory M1 macrophages ited peritoneal thickening after exposure to any PD treatment in the peritoneal membrane. Our results showed indeed an when compared to relevant effect showed in our previous accumulation of macrophages in the parietal peritoneum studies [33, 34]. This event suggests that longer exposure and prevalence of the pro-inflammatory (F480 ) over the with PD fluid is necessary to fully explore the different anti-inflammatory subset (Dectin-1 ) in the BLS group. In effects in thickness and ultrafiltration. Finally, our model line with these findings, inflammatory M1 macrophages would be closer to the clinical situation by the addition of secrete TNFα which were significantly enhanced in the the drainage of the PD fluid. This feature, however, was not effluent of the BLS-exposed mice when compared to the included since rodent models of PD absorb the fluid before group undergoing standard PD treatment. Furthermore, 24-h post-instillation. M1-polarization, or classical activation, is induced by INFγ In conclusion, a large difference exists in inflammatory which also was significantly incremented upon BLS fluid. response between conventional and low-GDP bicarbonate/ In contrast, BLS-treated mouse did have high levels of the lactate-based PD fluids. The use of the latter solution in pro-fibrotic cytokine TGFβ1, also known to be a stimulus our uremic mouse model leads to better preservation of the of M2 polarization [27], which, however, as outlined, did peritoneal membrane in terms of fibrosis and vasculariza- not occur. Nevertheless, in some PD studies, increased lev- tion. Moreover, peritoneal recruitment of M1 macrophages, els of TNFα, TGFβ1, and INFγ have been interpreted as a higher levels of macrophage-related pro-inflammatory + + consequence of improved mesothelial and macrophages cell cytokines and lower number of CD4 IL-17 cells might function as part of a pro-inflammatory process [5 , 29, 30]. explain the response observed in the peritoneal membrane However, further studies are needed to confirm the effects of BLS-exposed mice. Finally, we provide a better under- of these cytokines during longer periods of PD treatment standing of the inflammatory mediators during the exposure with a more biocompatible fluid. Overall, these findings of bicarbonate/lactate low GDPs buffered solution which suggest that the differences observed between the two PD might help to design new therapeutic approaches favoring fluids might rather indicate that an inflammatory process led the PD treatment. by an influx of M1-macrophages mainly occurs in the BLS Acknowledgements This work was supported by European Union, subgroup, while a more fibrotic response takes place in the Seventh Framework Program “EuTRiPD” under Grant agreement LS group. Importantly, we previously showed in vitro that Marie Curie ITN-GA-2011-287813. only M2 macrophages, and not M1, secrete factors induc- ing αSMA expression and fibrosis [31]. So, based on these Compliance with ethical standards findings, our present results showing a higher prevalence of peritoneal M1 over M2 macrophages in the BLS-treated ani- Conflict of interest The authors declare that they have no conflict of mals can be interpreted as preventive from the development interest. of fibrosis by BLS exposure as compared to conventional Ethical approval The animal study protocol was in compliance with PD fluid. animal welfare regulations according to the Dutch law and approved by Our study bears the limitation that the exact mechanism the Animal Welfare Committee at the VU University Medical Center, involved in the distinct effects of PD fluid exposure is not Amsterdam. defined. Moreover, we did not explore other inflammatory mediators such as T regulatory cells which are important to Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creat iveco regulate the activated T-cell expansion [32]. However, we mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- provide a characterization of the key immunological cells tion, and reproduction in any medium, provided you give appropriate dominating the peritoneal response upon standard high credit to the original author(s) and the source, provide a link to the GDPs lactate solution and we compare it with a low-GDP Creative Commons license, and indicate if changes were made. bicarbonate/lactate-buffered fluid. Although previous experi - mental research already suggested the capacity of bicarbo- nate/lactate-buffered solution in preserving morphologic parameters when compared with the conventional fluid, here we support previous findings and extend the data by using our well-established uremic model combined with long-term 1 3 1160 International Urology and Nephrology (2018) 50:1151–1161 14. Ferrantelli E, Liappas G, Keuning ED, Vila Cuenca M, González- References Mateo G, Verkaik M, López-Cabrera M, Beelen RH (2015) A novel mouse model of peritoneal dialysis: combination of uraemia 1. Devuyst O, Margetts PJ, Topley N (2010) The pathophysiology of and long-term exposure to PD fluid. Biomed Res Int 2015:106902. the peritoneal membrane. J Am Soc Nephrol 21(7):1077–1085. https ://doi.org/10.1155/2015/10690 2 https ://doi.org/10.1681/ASN.20090 70694 15. Ferrantelli E, Liappas G, Vila Cuenca M, Keuning ED, Foster TL, 2. 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Biomed Res Int 2015:468574. https://doi. rophages induces alpha-smooth muscle actin expression by dermal org/10.1155/2015/46857 4 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Urology and Nephrology Springer Journals

Differences in peritoneal response after exposure to low-GDP bicarbonate/lactate-buffered dialysis solution compared to conventional dialysis solution in a uremic mouse model

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Copyright © 2018 by The Author(s)
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Medicine & Public Health; Nephrology; Urology
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0301-1623
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Abstract

Background Long-term exposure of conventional peritoneal dialysis (PD) fluid is associated with structural membrane alterations and technique failure. Previously, it has been shown that infiltrating IL-17-secreting CD4+T cells and pro-fibrotic M2 macrophages play a critical role in the PD-induced pathogenesis. Although more biocompatible PD solutions are rec- ognized to better preserve the peritoneal membrane integrity, the impact of these fluids on the composition of the peritoneal cell infiltrate is unknown. Materials and methods In a uremic PD mouse model, we compared the effects of daily instillation of standard lactate (LS) or bicarbonate/lactate-buffered solutions (BLS) and respective controls on peritoneal fibrosis, vascularisation, and inflammation. Results Daily exposure of LS fluid during a period of 8 weeks resulted in a peritoneal increase of αSMA and collagen accom- panied with new vessel formation compared to the BLS group. Effluent from LS-treated mouse showed a higher percentage + + of CD4 IL-17 cell population while BLS exposure resulted in an increased macrophage population. Significantly enhanced inflammatory cytokines such as TGFβ1, TNFα, INFγ, and MIP-1β were detected in the effluent of BLS-exposed mice when compared to other groups. Further, immunohistochemistry of macrophage subset infiltrates in the BLS group confirmed a higher ratio of pro-inflammatory M1 macrophages over the pro-fibrotic M2 subset compared to LS. Conclusion Development of the peritoneal fibrosis and angiogenesis was prevented in the BLS-exposed mice, which may + + underlie its improved biocompatibility. Peritoneal recruitment of M1 macrophages and lower number of CD4 IL-17 cells might explain the peritoneal integrity preservation observed in BLS-exposed mouse. Keywords Peritoneal dialysis · Macrophage · IL17 · Fibrosis · Angiogenesis Introduction accumulation of extracellular matrix, angiogenesis, and other structural alterations of the peritoneum resulting in Continuous and long-term treatment with peritoneal dialy- technique failure and serious clinical complications includ- sis (PD) promotes an inflammatory response which even- ing encapsulation peritoneal sclerosis [1]. To a large extent, tually leads to a progressive remodeling of the peritoneal these events seem to be driven by high glucose degrada- membrane [1]. These changes are characterized by the tion products (GDPs) content, the low pH, and the pres- ence of lactate, typically present in conventional PD fluids [2]. Therefore, the conversion of PD fluids towards more * M. G. Vervloet biocompatible solutions is recognized as an urgent unmet m.vervloet@vumc.nl clinical need to better preserve the peritoneal integrity. As a 1 consequence, lactate–bicarbonate buffered fluids with more Department of Nephrology, VU University Medical Center, physiological pH, a lower amount of GDPs, and alternative Amsterdam, The Netherlands 2 solutions in which glucose is replaced by alternative osmotic Amsterdam Cardiovascular Sciences, Amsterdam, agents such as icodextrin and amino-acids have been devel- The Netherlands 3 oped [3]. Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands Vol.:(0123456789) 1 3 1152 International Urology and Nephrology (2018) 50:1151–1161 The introduction of neutral pH and particularly bicarbo- MMP-4S-061108A, Access Technologies, Ridgeway, nate/lactate-buffered solutions seemed to offer advantages USA). 5/6 nephrectomy consisted in the complete removal in terms of peritoneal membrane preservation and perito- of the right kidney and the removal of the anterior and neal homeostasis control [4, 5]. In the Euro balance trial, it posterior 1/3 part of the left kidney by using a monopolar demonstrated a significant improvement in effluent markers electric blade as previously described [14]. All the animals of peritoneal membrane integrity, a decrease in systemic were housed under standard conditions and were given advanced glycation end products (AGEs) levels, less decline food and water ad libitum. Health conditions were checked in residual renal function and a decrease in peritoneal ultra- daily. The experimental protocols were approved by the filtration [6 ]. Moreover, such biocompatible fluids increase Animal Welfare Committee at the VU University Medical mesothelial cell markers, induce less systemic inflammation, Center, Amsterdam. and reduced the incidence of peritonitis [7–10]. Recent studies revealed that immunological responses underlie PD-induced peritoneal injury upon conventional Cell counting high GDPs lactate PD fluid exposure although the spe- cific mechanism remains unclear. These data pointed to At the end point, following the injection of 2-ml stand- the importance of IL-17-mediated inflammation as a novel ard PD fluid via a catheter, peritoneal effluents were col- player in the PD-induced injury in both PD patients and lected after 30 min; cells were isolated by centrifugation experimental models [11]. Furthermore, M2 macrophages and counted and stained with fluorochrome-conjugated are suggested to play a key role in the development of peri- mouse-specific antibodies against CD3, CD4, CD8α, toneal inflammation and fibrosis [12, 13]. However, while B220, CD11b, Ly6C, F480, and IL-17 purchased from experimental and clinical data suggest a better preservation eBiosciences. Before intracellular staining, cells were re- of peritoneal morphologic and functional features upon stimulated for 4 h with 50 ng/ml Phorbol 12-Myristate bicarbonate/lactate solution compared to conventional PD 13-Acetate (PMA) and 500 ng/ml ionomycin in the pres- solutions, the implication on the inflammatory cell popu- ence of 1 µg/ml BD Golgi Plug (eBiosciences). Samples lation in this novel solution has been poorly defined. To were analyzed in a BD FACS Fortessa (BD Biosciences) overcome this knowledge gap, our recently developed ure- flow cytometer and further analyses were performed with mic mouse PD exposure model [14] was used in the pre- FloJo software. sent study to compare a pH neutral low-GDP bicarbonate/ lactate-buffered solution (BLS) with a standard high GDPs lactate (LS) PD fluid in respect of inflammation, fibrosis, Histology and immunohistochemistry and vascularisation. Here, we demonstrate that the enhanced fibrotic and angiogenic response observed in LS-exposed Parietal peritoneal biopsies were collected from the opposite mouse was prevented upon BLS exposure. This preserva- side from the catheter installation. The biopsies were fixed tion of the peritoneal integrity by BLS was accompanied in Bouin’s solution, embedded in paraffin, cut into 5-µm sec- + + with a lower number of CD4 IL-17 cells, higher levels of tions and stained with Masson’s Trichrome. Peritoneal mem- macrophage-related pro-inflammatory cytokines and with a brane thickness was determined using a Carl Zeiss Micro- higher ratio of M1 macrophages over M2 subset. scope (GmbH, 37081, Göttingem, Germany). Microscope photographs were obtained by using an AxioCam ICc5. The peritoneal thickness of each animal was calculated by the Methods median of measurement taken every 50 µm from one side to the other of the tissue sample. Mouse PD model Biopsies were frozen in Tissue-Teck® (O.C.T.® Sakura) and cut into 5-µm sections. To identify myofibroblasts and C57BL/6J female mouse (Charles River, Maastricht, The vessels, samples were stained for anti-rat Alpha Smooth Netherlands) aged 12–14 weeks and weighing approxi- Muscle Actin (αSMA 1A4, DAKO, 1:500) combined with mately 20  g at the start of the study were used. Ani- anti-mouse-IgG (H+L) (Invitrogen) and Cluster of Differen- mals were organized as follows: 1 healthy control group tiation 31 (αCD31, PECAM, Serotec, Oxford, UK, 1:1000) (n = 10), 3 PD groups (n = 10 per group) daily exposed coupled to anti-mouse-IgG-555 according to the manufac- to 2  ml saline or standard lactate-buffered solution turer’s instructions. Nuclei were stained with DAPI. Fluores- (Dianeal®, Baxter) or bicarbonate/lactate-buffered solu- cence microscopy was performed with a Carl Zeiss Micro- tion (Physioneal®, Baxter) during a period of 8 weeks. scope and photographs were taken with an AxioCam HR R3. Mouse in all the PD groups underwent 5/6 nephrectomy The areas positive for CD31 were calculated by CellProfiler and catheter implantation (Customized mouse catheter software (2.1.1, Broad Institute, UK). 1 3 International Urology and Nephrology (2018) 50:1151–1161 1153 However, thickening of peritoneum did not differ between Immunoblotting the two PDF compositions (LS or BLS). In contrast, immu- nohistological analysis of peritoneal biopsies revealed that Lysates of the peritoneal membrane were prepared by homogenizing of preserved tissue in lysis buffer containing exposure to BLS significantly prevented (P = 0.01) the accumulation of α αSMA positive cells (myofibroblast) in Protease Inhibitor Cocktail (Roche Applied Science, Indi- anapolis, IN). Protein concentrations were determined using the parietal membrane when compared to conventional LS PDF (C: 0.020 ± 0.016, S: 0.026 ± 0.026, LS: 0.075 ± 0.072, the Pierce Micro BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). The following antibodies were used: CD31 BLS: 0.016 ± 0.014), (Fig. 1b, c). Similarly, statistically sig- nificant enhanced collagen I protein levels were detected in (Abbiotec; 1: 200), Collagen I (Abcam; 1:250), GAPDH (14C10) 1:1000 cell signaling, followed by donkey anti-rat/ peritoneal samples after exposure to LS fluid when com - pared to control, whereas upon BLS no significant effect was rabbit conjugated with HRP (Dako; 1:5000). Signal was vis- ualized using enhanced chemiluminescence (Life Sciences) shown (C: 0.33 ± 0.19, S: 0.40 ± 0.39, LS: 1.24 ± 0.94, BLS: 0.66 ± 0.13), (Fig. 1d, e). Analysis of the peritoneal effluents on LAS3000 (Fujifilm, Japan). Image J (NIH, Bethesda, Maryland) was used for analysis. collected after 8 weeks of daily exposure to the different treatments performed by standard peritoneal equilibrium test Quantification of cytokines (PET) revealed no differences between the groups regarding the volume of ultrafiltration (data not shown). Peritoneal effluents collected after 8 weeks of treatment Chronic treatment with LS resulted in a significant incre- ment (P = 0.02) of new vessel formation in the omentum, were made cell free by centrifugation (300G, 5 min, RT) and stored at − 20 °C. Protein levels of mouse Transforming represented by the increased abundance of CD31 protein- positive cells (indicative of endothelial cells) when com- Growth Factor β 1 (TGFβ1), Interleukin-1β (IL-1β), Tumor Necrosis Factor α (TNFα), Vascular Endothelial Growth pared with the BLS group (Fig.  2a, b). These data were confirmed by the detection in the peritoneal tissue of high Factor (VEGF), IL-17A, IL-6, Interferon γ (INFγ), IL-5, IL-4, Macrophages Inflammatory Proteins 1α and β (MIP-1α levels of CD31 protein upon LS fluid which was prevented (P = 0.02) in BLS-exposed animals (Fig.  2c, d). Taken and MIP-1β) were quantified by ProcartaPlex™ Multiplex Immunoassays (Affymetrix eBioscence). together, these results indicate that LS but not BLS fluid pro- moted a fibrotic and angiogenic response in the peritoneum Statistical analysis although the development of increased peritoneal thickness was not preserved in BLS-exposed mouse. Data were analyzed using GraphPad Prism software (La Analysis of inflammatory cell recruitment Jolla, CA). Statistical analysis was performed using One-way ANOVA test to compare the groups. A P value < 0.05 was in peritoneal cavity upon PD fluid exposure considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001). Data were shown as means ± SD. Exposure to PD fluid caused a numerical increase of the total number of cells in the peritoneal fluid compared to non- exposed animals, which however was not statistically sig- nificant (Table  1). No statistically significant variation was Results found in both CD4- and CD8-positive populations among groups while there was a ninefold statistically significant BLS exposure in mouse prevented the development of both peritoneal fibrosis and angiogenesis increase in CD4/IL-17 double positive lymphocytes (CD4 IL-17 ) found in the LS group compared to the BLS group, with no changes in thickness in which these double positive cells remained at the same level as controls. No significant differences were observed In order to mimic the situation in chronic kidney disease patients undergoing PD, a uremic mouse model, performed between the groups in the percentage of B cells and mono- + + cytes (Ly6G CD11b ), although the number of the latest by 5/6 nephrectomy, was exposed to daily PD fluid during a period of 8 weeks. Nephrectomized groups showed a twofold increased especially after BLS. As in our previous study in mouse [15], a slight increase in macrophages (F480 increase in both serum urea and creatinine concentrations 15 days after the surgery and remained stable for the duration of CD11b ) was shown after exposure to conventional LS, but this rise was more pronounced in the BLS group (*P = 0.04 the experiment (data not shown). As shown in Fig. 1a, after 8 weeks of daily exposure to the PDFs, there was a statisti- compared to control). Therefore, exposure to BLS clearly caused profound changes compared to LS primarily in terms cally significant increase (P = 0.04) in peritoneal thickness compared to the non-PDF-exposed control (C: 30.63 ± 3.66, of peritoneal inflammatory macrophages recruitment, and IL-17 expressing CD4 cells. S: 41.84 ± 12.05, LS: 78.78 ± 39.62, BLS: 78.11 ± 32.87). 1 3 1154 International Urology and Nephrology (2018) 50:1151–1161 Fig. 1 Exposure to low GDPs bicarbonate/lactate-buffered PD fluid (blue). Representative immunoblotting (d) and analysis (e) of Colla- prevents myofibroblast recruitment but does not protect from parietal gen I (Col I) levels in total protein lysates of peritoneal membrane. peritoneum thickness. Graph a represents peritoneal thickness (µm) GAPDH was used as loading control (n = 6). C control, S saline, measurements for each group (n = 10). Representative immunofluo- LS lactate PD fluid, BLS bicarbonate/lactate PD fluid. Data show rescence microscopy (b) and analysis (c) of parietal peritoneal sec- means ± SD. Differences were considered statistically significant for tions stained with αSMA marker (magnification ×10; n = 10). αSMA P < 0.05 using one-way ANOVA. *P < 0.05 positive cells are indicated in red. Nuclei were stained with DAPI significant for BLS only (P = 0.04). A similar pattern was Cytokine production changes in BLS‑treated mouse observed for IL-1β that was slightly enhanced in the LS group and further increased upon BLS exposure (Fig. 3b), We further explored the differences between the PD flu- albeit non-significant. Alternatively, IL-6 and VEGF release ids in terms of inflammatory cytokine responses. As increased as a consequence of PD fluid exposure but it did shown in Fig.  3a, an increment in TGFβ1 levels was not differ statistically between the PD regimens and con- detected upon both BLS (435.9 ± 266.3  pg/ml) and LS trol (Fig. 3c, e). On the other hand, a statistically significant (204.1 ± 211.8 pg/ml) treatment when compared to control increase of TNFα was shown only after BLS exposure but group (C:28.57 ± 22.85 pg/ml) although this was statistically 1 3 International Urology and Nephrology (2018) 50:1151–1161 1155 Fig. 2 Peritoneal angiogenesis is attenuated in bicarbonate/lactate- lotting (c) and analysis (d) of CD31 levels in total protein lysates of buffered-exposed mouse. Immunofluorescence microscopy (a) and peritoneal membrane (n = 6). GAPDH was used as loading control. analysis (b) of omentum sections stained for vasculature with CD31; C control, S saline, LS lactate PD fluid, BLS bicarbonate/lactate PD (green) marker (n = 10). Each value corresponds to an average (% sur- fluid. Data show means ± SD. Differences were considered statisti- face staining CD31) of 10 independent values of each mouse omen- cally significant for P < 0.05 using one-way ANOVA. *P < 0.05 tum taken each time (magnification ×20). Representative immunob- not after LS treatment (C: 10.38 ± 1.25, LS: 33.57 ± 24.82, BLS: 239 ± 186.8 pg/ml; P = 0.02) (Fig. 3d). Emerging evidence points to IL-17 as an important factor 6 in mediating peritoneal inflammation [11]. In this study, the Table 1 Quantification of total cell number (×10 ) composition of + + cells (%) in mouse peritoneal effluents after 8 weeks of exposure increment found in the CD4 IL-17 cell population upon LS exposure was accompanied by a nominal, but statistically Cell populations Groups (Mean ± SD) insignificant, increase of the levels of IL-17 measured in the C LS BLS peritoneal effluents (Fig.  3g). We found upregulation of IL-5 in the LS group which was not shown upon treatment with Total cells (×10 ) 2.93 ± 1.31 4.36 ± 2.06 5.38 ± 2.56 BLS (C: 18.44 ± 2.40, LS: 53.4 ± 33.09, BLS: 23.44 ± 9.13) CD4 (%) 29 ± 1.14 22.13 ± 13.73 17 ± 13.87 (Fig. 3i). A similar trend was found for IL-4 (C: 13 ± 0.70, CD8 (%) 14.35 ± 3.23 3.45 ± 3.97 6.51 ± 7.37 + + LS: 59.07 ± 51.03, BLS: 18 ± 6.06) (Fig. 3h), indicating that CD4 IL-17 (%) 1.98 ± 2.46 13.19 ± 8.23* 1.47 ± 2.49 involvement of T helper 2 (Th2) cells was more pronounced Monocytes (%) 7.45 ± 8.7 10.88 ± 10.09 14.8 ± 11.38 upon LS treatment over BLS. On the other hand, the sta- Macrophages (%) 2.03 ± 0.69 8.16 ± 6.84 25.07 ± 16.78* tistically significant high levels of INFγ (Fig.  3f) detected B cells (%) 37.85 ± 19.45 37.04 ± 23.18 35.03 ± 13.31 only after BLS exposure suggested that in BLS regimens *P < 0.05 an important role may be played by T helper 1 (Th1) cells Data show Means ± SD (n = 4). Differences were considered statisti- (C: 23.69 ± 5.65, LS: 42.18 ± 30.2, BLS: 180.3 ± 123.5; cally significant for P < 0.05 using one-way ANOVA. *P < 0.05. (Sta- P = 0.01). tistically significant differences were found in LS vs. C and LS vs. + + The presence of enhanced macrophage population in BLS for CD4 IL-17 , and BLS vs. C in Macrophages) BLS-treated mouse was further substantiated by the rise C control, LS lactate PD fluid, BLS bicarbonate/lactate PD fluid 1 3 1156 International Urology and Nephrology (2018) 50:1151–1161 Fig. 3 Exposure to a bicarbonate/lactate-buffered solution is associ- b IL-1β, c IL-6, d TNFα, e VEGF, f INFγ, g IL-17, h IL-4, i IL-5, j ated with increase of pro-inflammatory cytokines. Protein levels of MIP-1α, k MIP-1β. C control, LS lactate PD fluid, BLS bicarbonate/ the main pro-inflammatory cytokines detected in effluents collected lactate PD fluid. Differences were considered statistically significant from mouse after 8 weeks of PD fluid exposure. Cytokines levels for P < 0.05 using one-way ANOVA. *P < 0.05 (ρg/ml) are represented as means ± SD (n ≥ 7) as follows: a TGFβ1, of MIP-1α and MIP-1β (also known as CCL3 and CCL4) played a crucial role over other cell types in modulat- following BLS treatment although significant changes ing the response of BLS on peritoneal fibrosis and were only found in the MIP-1β measurements (MIP-1β angiogenesis. C: 12.81 ± 1.43, LS: 53.43 ± 49.73, BLS: 278.8 ± 246.8; P = 0.04) (Fig.  3j, k). Taken together, between the two PDFs, there are outspoken differences in levels of TGFβ1, TNFα, INFγ, and MIP-1β suggesting that macrophages 1 3 International Urology and Nephrology (2018) 50:1151–1161 1157 [16], in the peritoneum of BLS-treated mouse (Fig. 4b). In BLS exposure induced the recruitment of pro‑inflammatory macrophages in parietal addition, macrophages with classical morphology display high levels of both F480 and CD11b [17], which were peritoneum significantly increased in the BLS group compared to both the control and the LS groups (Fig. 4c). On the other hand, Given the parallels between the high percentage of mac- rophages in effluent and the elevated levels of chemokine staining for Dectin-1, a marker for anti-inflammatory (M2) macrophages, did not reveal any significant differences MIP-1α and 1β, we further explored macrophage subset population in the peritoneum by immunohistochemistry among groups (Fig. 4d). Overall, our results suggest that pro-inflammatory M1 phenotype modulated the response (Fig.  4a). Quantification shows high F480 expression, characteristic of classically activated macrophages (M1) in the peritoneum induced by BLS exposure. Fig. 4 Bicarbonate/lactate-buffered solutions mediate pro-inflamma- per mouse for each group. Significant increase of CD11b and F480 tory macrophages recruitment. Panel a shows representative perito- double positive and single F480-positive cells in the BLS group are neal membrane macrophages recruitment for each group in the ure- represented, respectively, in graphs (b) and (c). Graph d shows Dec- mic PD mouse model. Peritoneal staining for CD11b (violet), F480 tin-1-positive cells in each group (C control, LS lactate PD fluid, BLS (red), CD11b plus F480 double positive and Dectin-1 (green) are rep- bicarbonate/lactate PD fluid). Magnification ×20. Data show means ± resented in the rows from the top to the bottom panel, respectively. SD (n = 10). Differences were considered statistically significant for Nuclei were stained with DAPI (blue). Column bars represent number P < 0.05 using one-way ANOVA. *P < 0.05 of macrophages measured per field in three different pictures taken 1 3 1158 International Urology and Nephrology (2018) 50:1151–1161 structural changes and the increment of fibrosis [21– 23]. Discussion However, other factors such as the amount of cell infil- trate may contribute to the increment of peritoneal thick- The current study is the first one using a mouse PD expo- ness [24]. In addition, daily instillation with normal saline sure model comparing lactate and bicarbonate/lactate- also induces a slight increment of the thickness, indicating buffered solutions in a uremic setting, which more closely that peritoneal remodeling is not exclusive of PD fluid mimics the clinic status of PD patients when compared to effects, and these additional effects may have masked or the non-uremic animal models. Our findings support the overwhelmed any difference between types of PD solution notion that bicarbonate/lactate-buffered solution better pre- used. Furthermore, we previously reported that uremia per serves the peritoneal integrity status when compared with se also contributes to this event [14]. Overall, all those the conventional PD fluid. Specifically, we demonstrated + + factors may influence the lack of differences in thickness that the enhanced CD4 IL-17 cell population in effluent between PD groups. detected in LS-exposed mouse was prevented in the BLS In vitro experimental research has demonstrated the group although with no significant changes in IL-17 efflu - capacity of neutral pH, bicarbonate/lactate-buffered solu- ent concentrations. Importantly, increased macrophage tions to maintain mesothelial cellular integrity and function population together with high levels of chemokines that when compared to conventional fluid [18, 25]. Although regulate migration and infiltration of monocytes/mac- PD fluid may exert a direct impact on the mesothelial cell rophages suggested that macrophages played a key role stability, evidence points to the inflammatory response as during BLS treatment. While pro-fibrotic M2 macrophages key factor inducing PD-associated pathology. In this regard, are thought to promote fibrosis and angiogenesis upon con- in our previous studies, the chronic exposure to standard ventional PDF fluid treatment, in this study it was found dialysis fluids resulted in peritoneal Th17 response includ- that in BLS-exposed mouse the pro-inflammatory (M1) ing elevated IL-17 protein production [11]. Importantly, the phenotype was dominant in the peritoneal tissue over M2 modulation of IL-17 during treatment with PD fluid was subset suggesting a difference in inflammatory response shown to be an effective therapy for PD-mediated perito- between different PD fluid exposures. neal fibrosis and angiogenesis [11, 15, 26]. In our study, the The accumulation of extracellular matrix and fibrosis is + + increase in CD4 IL-17 cell population observed upon LS a characteristic peritoneal alteration induced by PD fluid treatment was prevented in the BLS groups. Although no exposure which leads to a progressive remodeling of the significant changes in IL-17 levels were observed between peritoneal membrane [1]. As previously demonstrated PD fluid exposed animals, it is possible that in this study [15], and confirmed by the present study, daily exposure of statistical significance for IL-17 was missed due to too low conventional PD fluid contributes to the fibrotic response power. by an accumulation of αSMA-positive cells in the parietal Alternatively, a striking enhanced INFγ concentration in peritoneum. Consistently, an increment of the extracel- the effluents of BLS group compared to the LS was found. lular matrix protein collagen I in peritoneal tissues of LS- In parallel, BLS slightly prevented the increment of IL-5 treated mouse was observed. In contrast, daily exposure to and IL-4 levels detected in the LS group. These findings BLS fluid prevented the accumulation of both αSMA and suggested that inflammatory mechanisms occurring during collagen I. This is in accordance with previous studies sug- exposure to bicarbonate/lactate buffer involve Th1 rather gesting that the use of low GDPs solutions was associated than Th2 cell subset while the opposite happened upon expo- with less αSMA expression in vitro and less development sure to high GDPs lactate solutions. Possibly, this particular of fibrotic response in the peritoneal membrane in rats inflammatory milieu can explain the differences observed in [4, 18, 19]. Importantly, other factors such as the neu- peritoneal remodeling between PD fluids. In this regard, T tral pH present in the more biocompatible fluid may also helper-related cells interact with many immune cells includ- contribute to the differences observed. In addition, as we ing macrophages, which also play a crucial role in chronic reported previously in a rat model with PD [4, 20], daily inflammation-induced fibrosis [27]. Enhanced levels of Th2 exposure to BLS prevented new vessel formation, which cell subsets together with pro-fibrotic cytokine microenvi- did occur when LS was used, indicating that BLS regimen ronment contribute to the polarization of peritoneal mac- is more biocompatible in terms of peritoneal angiogenesis. rophages towards the anti-inflammatory or pro-fibrotic M2 Our findings suggest reduced angiogenesis and peritoneal macrophages subset [27]. Particularly, M2 macrophages fibrosis as markers of better preservation of the structure subset demonstrated to be dominant in the cellular infiltrate of the peritoneum after exposure of BLS fluid. Despite the in PD patients and are suggested to drive peritoneal fibrosis differences reported, no changes in peritoneal thickness [12, 28]. In this study, BLS exposure mediated the increment were detected among the PD-treated groups. Numerous of the proteins of the chemotactic factors for macrophages studies have suggested a relationship between peritoneal MIP-1α and MIP-1β in the peritoneal effluents. It has been 1 3 International Urology and Nephrology (2018) 50:1151–1161 1159 shown that in the inflammatory phase, newly attracted mac- exposure to PD fluid [14]. As an additional limitation, no rophages present a more pro-inflammatory (M1) phenotype differences in the ultrafiltration capacity were detected but only after the switch to M2 they become pro-fibrotic between groups. This weakness, however, was previously [16, 27, 28]. Interestingly in our experiment, the increase reported in our previous study in uremic mice and rats with in the percentage of macrophages cell population in the PD [15]. Furthermore, an assumed reduction of the harmful peritoneal effluents after BLS exposure was associated with effects of the new generation of PD fluids may explain lim- enhanced recruitment of pro-inflammatory M1 macrophages ited peritoneal thickening after exposure to any PD treatment in the peritoneal membrane. Our results showed indeed an when compared to relevant effect showed in our previous accumulation of macrophages in the parietal peritoneum studies [33, 34]. This event suggests that longer exposure and prevalence of the pro-inflammatory (F480 ) over the with PD fluid is necessary to fully explore the different anti-inflammatory subset (Dectin-1 ) in the BLS group. In effects in thickness and ultrafiltration. Finally, our model line with these findings, inflammatory M1 macrophages would be closer to the clinical situation by the addition of secrete TNFα which were significantly enhanced in the the drainage of the PD fluid. This feature, however, was not effluent of the BLS-exposed mice when compared to the included since rodent models of PD absorb the fluid before group undergoing standard PD treatment. Furthermore, 24-h post-instillation. M1-polarization, or classical activation, is induced by INFγ In conclusion, a large difference exists in inflammatory which also was significantly incremented upon BLS fluid. response between conventional and low-GDP bicarbonate/ In contrast, BLS-treated mouse did have high levels of the lactate-based PD fluids. The use of the latter solution in pro-fibrotic cytokine TGFβ1, also known to be a stimulus our uremic mouse model leads to better preservation of the of M2 polarization [27], which, however, as outlined, did peritoneal membrane in terms of fibrosis and vasculariza- not occur. Nevertheless, in some PD studies, increased lev- tion. Moreover, peritoneal recruitment of M1 macrophages, els of TNFα, TGFβ1, and INFγ have been interpreted as a higher levels of macrophage-related pro-inflammatory + + consequence of improved mesothelial and macrophages cell cytokines and lower number of CD4 IL-17 cells might function as part of a pro-inflammatory process [5 , 29, 30]. explain the response observed in the peritoneal membrane However, further studies are needed to confirm the effects of BLS-exposed mice. Finally, we provide a better under- of these cytokines during longer periods of PD treatment standing of the inflammatory mediators during the exposure with a more biocompatible fluid. Overall, these findings of bicarbonate/lactate low GDPs buffered solution which suggest that the differences observed between the two PD might help to design new therapeutic approaches favoring fluids might rather indicate that an inflammatory process led the PD treatment. by an influx of M1-macrophages mainly occurs in the BLS Acknowledgements This work was supported by European Union, subgroup, while a more fibrotic response takes place in the Seventh Framework Program “EuTRiPD” under Grant agreement LS group. Importantly, we previously showed in vitro that Marie Curie ITN-GA-2011-287813. only M2 macrophages, and not M1, secrete factors induc- ing αSMA expression and fibrosis [31]. So, based on these Compliance with ethical standards findings, our present results showing a higher prevalence of peritoneal M1 over M2 macrophages in the BLS-treated ani- Conflict of interest The authors declare that they have no conflict of mals can be interpreted as preventive from the development interest. of fibrosis by BLS exposure as compared to conventional Ethical approval The animal study protocol was in compliance with PD fluid. animal welfare regulations according to the Dutch law and approved by Our study bears the limitation that the exact mechanism the Animal Welfare Committee at the VU University Medical Center, involved in the distinct effects of PD fluid exposure is not Amsterdam. defined. Moreover, we did not explore other inflammatory mediators such as T regulatory cells which are important to Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creat iveco regulate the activated T-cell expansion [32]. However, we mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- provide a characterization of the key immunological cells tion, and reproduction in any medium, provided you give appropriate dominating the peritoneal response upon standard high credit to the original author(s) and the source, provide a link to the GDPs lactate solution and we compare it with a low-GDP Creative Commons license, and indicate if changes were made. bicarbonate/lactate-buffered fluid. 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International Urology and NephrologySpringer Journals

Published: May 4, 2018

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