1. IL-17A is a novel player in dialysis-induced peritoneal damage
Raquel Rodrigues-Díez, Luiz S. Aroeira, Macarena Orejudo, M-Auxiliadora Bajo, José Jiménez Heffernan, Raúl R Rodrigues-Díez, Sandra Rayego-Mateos, Alberto Ortiz, Guadalupe Gonzalez-Mateo, Manuel López-Cabrera, Rafael Selgas, Jesús Egido, Marta Ruiz- Ortega 
Kidney International 
Volume 86, Issue 2, Pages (August 2014)
DOI: /ki
Copyright © 2014 International Society of Nephrology Terms and Conditions

2. Figure 1
Interleukin-17A (IL-17A)-expressing cells were found in peritoneal biopsies from peritoneal dialysis (PD) patients. (a) In control human peritoneal biopsies, there was almost no IL-17A immunostaining, whereas in samples from PD patients, a marked increase in the number of IL-17A-positive cells in the submesothelial layer was observed. Magnification × 200. (b) In long-treated PD patients (>3 years), peritoneal IL-17A immunostaining was detected, presenting IL-17A-positive cells (arrows) associated with the loss of the mesothelial layer integrity (star), whereas no signal was found in its corresponding predialysis biopsy. Panel b shows two representative cases of 4 performed with similar results. Magnification × 200 in case 1 and × 400 in case 2. (c) IL-17A immunostaining quantification expressed as % of positive-stained area vs. total analyzed area of eight control and nine PD patients. *P<0.05 vs. control. (d) A positive Spearman's correlation between IL-17A immunostaining and time in PD treatment was found (n=17 samples).
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3. Figure 2
Interleukin-17A (IL-17A) immunostaining was associated with inflammatory cell infiltration. (a) In a representative case of a peritoneal dialysis (PD) patient, elevated number of infiltrating cells (CD68+, CD3+, and CD4+ cells) in the same areas of IL-17A staining was observed. Magnification × 200. (b) IL-17A+ staining detected by conventional immunohistochemistry was observed in infiltrating cells, characterized by cell morphology (magnification × 400). (c) To characterize IL-17A-expressing cells, double immunofluorescence was performed. IL-17A was labeled with a secondary Alexa 633 (red) and the different cells types were determined using a specific antiCD4 antibody (for CD4+/Th17 cells), an antiTCR γδ antibody (γδ lymphocytes), myeloperoxidase (MPO; for neutrophils), and tryptase (mast cells) labeled with a secondary Alexa 488 (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). The figure shows a confocal microscopy analysis of one PD patient of nine performed with similar results. DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride.
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4. Figure 3
Elevated interleukin-17A (IL-17A) concentrations were detected in peritoneal effluents. IL-17A concentrations were measured in effluents of peritoneal dialysis patients. These patients were divided in two groups depending on the duration of the treatment: more (n=17) or less (n=24) than 3 years. Data are expressed as mean±s.e.m. *P<0.05 vs. >3 years.
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5. Figure 4
Interleukin-17A (IL-17A) in vivo induced the recruitment of inflammatory cells in the submesothelial zone and increased the gene expression of proinflammatory and profibrotic factors in mouse peritoneum. IL-17A recombinant protein (10ng/g body weight) was intraperitoneously injected in mice and the peritoneal membrane was studied 10 days later. Inflammatory cells, characterized as described in Materials and Methods, were found in IL-17A-injected mice but not in saline-injected mice used as controls. Panel a shows a representative picture of each group (magnification × 200) and in b the quantification of the immunostaining. (c–e) Gene expression was evaluated by real-time PCR. Data are expressed as mean±s.e.m. of seven animals per group. *P<0.05 vs. control. α-SMA, α-smooth muscle actin; TGF-β, transforming growth factor-beta.
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6. Figure 5
Interleukin-17A (IL-17A) in vivo induced peritoneal fibrosis in mice. IL-17A recombinant protein was injected weekly (10ng/g body weight, intraperitoneously) and the peritoneal membrane was studied after 35 days. Figure (a) shows a representative immunostaining of fibronectin (FN), FSP-1, and α-SMA of each group (magnification × 200), and (b) the quantification of the staining. (c) Protein levels were evaluated by western blotting. Data were obtained from densitometric analysis and expressed as protein/GAPDH ratio as n-fold over control. Figure shows a representative experiment and data expressed as mean±s.e.m. of seven animals per group. *P<0.05 vs. control. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
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7. Figure 6
Exposure of the peritoneal membrane to peritoneal dialysis fluid (PDF) in mice increased Th17, but not Th1 and Th2 hallmark-cytokine levels. A vascular access port with the end of the catheter into the peritoneal cavity was implanted in mice. Then, animals were instilled daily with PDF or saline for 7 and 30 days. (a) In protein extracts from peritoneum of mice daily exposed to PDF for 7 and 30 days, the levels of IL-17A (Th17 hallmark-cytokine), but not interferon-γ (Th1) and IL-4 (Th2), were elevated compared with control mice. (b) Interleukin-17A (IL-17A) gene expression was also upregulated in the peritoneum after daily exposure to PDF for 7 and 30 days. Enzyme-linked immunosorbent assay and real-time PCR data are shown as fold change vs. control mice. Mean±s.e.m. of six to nine animals per group. *P<0.05 vs. control. (c) Only in the peritoneum of PDF-treated mice IL-17A staining was observed, whereas no signal was found in control mice. Nuclei are in blue (4′,6-diamidino-2-phenylindole staining) and IL-17A was visualized by secondary Alexa 633 antibody (red), and evaluated by indirect confocal immunofluorescence. (d) IL-17A-producing CD4+ and γδ cells were present in the damaged peritoneum. Double immunofluorescence was performed with IL-17A and an antiCD4+ antibody or antiTCR γδ antibody, followed by an Alexa 488 antibody (green). The figure shows a representative confocal microsocopy analysis of five mice studied in each group.
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8. Figure 7
Interleukin-17A (IL-17A) levels were elevated in the peritoneal cavity of peritoneal dialysis fluid (PDF)-treated mice. (a) After killing, the peritoneal cavity was lavaged with 2ml of saline solution and IL-17A levels were measured in the recovered volume. Data are shown as mean±s.e.m. of six to nine animals per group analyzed by enzyme-linked immunosorbent assay. *P<0.05 vs. control. (b) Correlation between intraperitoneal IL-17A and peritoneal thickness by Spearman’s test.
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9. Figure 8
Exposure of the peritoneal membrane to peritoneal dialysis fluid (PDF) in mice increased Th17-related cytokines (a) and transcription factors (b). (a) Peritoneal IL-6 and transforming growth factor-beta (TGF-β) mRNA levels are increased in response to peritoneal damage in mice daily instilled with PDF for 7 days, compared with the untreated group (used as control). Real-time PCR data are expressed as mean±s.e.m. of six to nine animals per group. *P<0.05 vs. control. (b) Increased peritoneal expression of RORγt and phosphorylated STAT3 in PDF-treated mice after 7 days. The figure shows a representative experiment (bottom) of total RORγt and phosphorylated STAT3 protein determined in 30μg of peritoneal tissue protein by western blotting. Data (top) were obtained from densitometric analysis and expressed as protein/GAPDH ratio as n-fold over control. Mean±s.e.m. of six to nine animals per group. *P<0.05 vs. control.
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10. Figure 9
Blockade of the Th17 response, using a neutralizing antibody against interleukin-17A (IL-17A), diminished peritoneal dialysis fluid (PDF)-induced inflammation in mouse peritoneum. PDF-instilled mice were treated with a neutralizing antibody against IL-17A or its corresponding control, a mouse IgG1-K isotype antibody (100μg every 7 days, via the peritoneal catheter), starting 24h before daily PDF instillation. As control, a group of saline-instilled mice was used. Inflammatory cells, characterized as described in Materials and Methods, were found in the submesothelial zone of IgG-treated PDF-instilled mice after 30 days. In the groups of anti-IL-17A-treated PDF-instilled mice and in saline-instilled mice, the inflammatory cell infiltration was markedly diminished. Panel a shows a representative picture of each group (magnification × 200) and in b the quantification. (c) Gene expression of chemokines (CXCL2 and CCL2) evaluated by real-time PCR. Data are mean±s.e.m. of eight to nine animals per group. *P<0.05 vs. saline. #P<0.05 vs. mouse IgG PDF-treated mice.
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11. Figure 10
Neutralization of interleukin-17A (IL-17A) diminished PDF-induced peritoneal fibrosis. Peritoneal dialysis fluid (PDF)-instilled mice were treated with anti-IL-17A or mouse IgG1-K isotype antibodies; saline-instilled mice were used as control. Panel a shows a representative mouse of each group. (a) Masson staining shows peritoneal fibrosis. Activated fibroblasts were determined by α-SMA, and fibrosis by fibronectin expression, evaluated by immunohistochemistry (a, b), gene expression (c), and western blotting (d). Panel a shows a representative mouse of each group, in b the immunohistochemistry quantification, in c the gene expression by real-time PCR, and in d protein levels by western blotting. Panel d shows a representative western blot (top) and densitometric values. Data are mean±s.e.m. of eight to nine animals per group. *P<0.05 vs. saline. #P<0.05 vs. mouse IgG PDF-treated mice.
Kidney International  , DOI: ( /ki )
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