1. Volume 81, Issue 11, Pages 1098-1107 (June 2012)
TWEAK (tumor necrosis factor–like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation 
María Concepción Izquierdo, Ana B. Sanz, Sergio Mezzano, Julia Blanco, Susana Carrasco, María Dolores Sanchez-Niño, Alberto Benito-Martín, Marta Ruiz-Ortega, Jesús Egido, Alberto Ortiz 
Kidney International 
Volume 81, Issue 11, Pages (June 2012)
DOI: /ki
Copyright © 2012 International Society of Nephrology Terms and Conditions

2. Figure 1
TWEAK (tumor necrosis factor–like weak inducer of apoptosis) induces renal CXCL16 expression in vivo. (a) Quantification of CXCL16 mRNA by real-time quantitative reverse transcription-PCR in kidneys from mice 4h after the administration of TWEAK and/or parthenolide. *P<0.003 vs. control. #P<0.05 vs. TWEAK. Data were normalized with murine glyceraldehyde-3-phosphate dehydrogenase mRNA. (b) Quantification of CD3-positive cells 4h after TWEAK or/and parthenolide injection. *P<0.009 vs. control. #P<0.01 vs. TWEAK. Mean±s.e.m. of six animals per group. h.p.f, high power field.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

3. Figure 2
Localization of TWEAK (tumor necrosis factor–like weak inducer of apoptosis)-induced renal CXCL16 expression in vivo. In control kidneys CXCL16 located mainly to tubules. In TWEAK-injected animals, an increased number of CXCL16-expressing tubules is noted. CXCL16 immunohistochemistry. Original magnification × 400. Pictures are representative of six animals per group.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

4. Figure 3
TWEAK (tumor necrosis factor–like weak inducer of apoptosis) neutralization decreases CXCL16 and CXCR6 mRNA expression in experimental tubulointerstitial inflammation (TII). (a) During experimental TII, CXCL16 mRNA is increased. TWEAK neutralization decreased kidney CXCL16 mRNA (real-time quantitative reverse transcription-PCR (qRT-PCR)). *P<0.001 vs. control, **P<0.01 vs. TII, #P<0.05 vs. TII+IgG. Data were normalized with 18s eukaryotic ribosomal RNA. (b) During experimental TII, CXCR6 mRNA is increased. TWEAK neutralization decreased kidney CXCR6 mRNA (real-time qRT-PCR). *P<0.01 vs. control, **P<0.03 vs. TII. Data were normalized with 18s eukaryotic ribosomal RNA. (c) Histological assessment confirmed that cellular tubulointerstitial injury was decreased by anti-TWEAK antibodies. *P< vs. control, **P<0.003 vs. TII, #P<0.003 vs. TII+IgG. Mean±s.e.m. of eight animals per group.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

5. Figure 4
Localization of CXCL16 expression in experimental tubulointerstitial inflammation (TII). Note tubular cell localization of increased CXCL16 expression in mice with TII, when compared with healthy control or mice with TII treated with anti-TWEAK (tumor necrosis factor–like weak inducer of apoptosis). Immunohistochemistry. Original magnification × 400. Pictures representative of eight animals per group.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

6. Figure 5
TWEAK (tumor necrosis factor–like weak inducer of apoptosis) neutralization decreases interstitial CD3+ lymphocytes in experimental tubulointerstitial inflammation (TII). The increased number of interstitial lymphocytes stained with anti-CD3 (arrows) in kidneys with TII was reduced by anti-TWEAK antibody treatment. Original magnification × 200. Quantification as mean±SEM of eight animals per group. *P<0.01 vs. control, **P<0.01 vs. TII.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

7. Figure 6
Increased tubular fibroblast growth factor–inducible 14 (Fn14) and CXCL16 expression is associated with interstitial inflammatory infiltrates in human tubulointerstitial inflammation (TII). (a) CXCL16 immunohistochemistry. CXCL16 is observed in renal tubules (arrows) and in surrounding inflammatory infiltrates (arrowheads) in a case of TII secondary to glomerular injury (rapidly progressive glomerulonephritis), but not in minimal change disease (MCD). (b) Fn14 immunohistochemistry. Fn14 is observed in the same renal tubules (arrows) that were stained for CXCL16 in serial sections of the same case of TII, but not in MCD. Original magnifications × 200 and × 400. Control for the technique is shown in Supplementary Figure S3 online. Representative images of two patients with MCD, and two with rapidly progressive glomerulonephritis.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

8. Figure 7
Increased tubular fibroblast growth factor–inducible 14 (Fn14) and CXCL16 expression is associated with interstitial inflammatory infiltrates in human acute tubulointerstitial nephritis. (a) CXCL16 immunohistochemistry. CXCL16 is observed in renal tubules (arrows) and in surrounding inflammatory infiltrates (arrowheads). (b) Fn14 immunohistochemistry. Fn14 is observed in renal tubules (arrows) of the same case. Original magnification × 400.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

9. Figure 8
TWEAK (tumor necrosis factor–like weak inducer of apoptosis) modulates CXCL16 expression in cultured renal tubular cells. (a) MCT cells were stimulated with 100ng/ml TWEAK. Quantification of CXCL16 mRNA expression (real-time quantitative reverse transcription-PCR). Expression level at 6h control was considered to be 100%. Mean±s.e.m. of three independent experiments. *P<0.04 vs. control 6h; #P<0.05 vs. control 24h. (b) MCT cells were pretreated for 1h with 10μmol/l parthenolide and stimulated with TWEAK for 3h. The nuclear factor-κB inhibitor parthenolide prevented TWEAK-induced CXCL16 upregulation. Mean±s.e.m. of three independent experiments. *P<0.05 vs. vehicle; #P<0.05 vs. vehicle+TWEAK. Data were normalized with murine glyceraldehyde-3-phosphate dehydrogenase mRNA. (c) MCT cells stimulated with TWEAK for 6h or 24h were analyzed for CXCL16 surface expression by flow cytometry. Mean±s.e.m. of three independent experiments. *P<0.03 vs. control. (d) NP-1 cells were treated with 100ng/ml TWEAK for 24h. Soluble and cellular CXCL16 were quantified by ELISA. Mean±s.e.m. of three independent experiments. *P<0.05 vs. control.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

10. Figure 9
Confocal microscopy detection of CXCL16 and CXCR6 expression in cultured tubular cells. Cells were stimulated with 100ng/ml TWEAK (tumor necrosis factor–like weak inducer of apoptosis) or vehicle (control) for 24h. (a) Constitutive CXCR6 (green) and inducible CXCL16 (red) expression in proximal tubular MCT cells. Original magnification × 40. (b) Constitutive CXCR6 and inducible CXCL16 expression in proximal tubular MCT cells. Note the increased peripheral location of CXCL16 following TWEAK with areas of overlap (yellow). Original magnification × 40, zoom 7. (c) Constitutive CXCR6 and inducible CXCL16 expression in distal tubular NP1 cells. Original magnification × 40, zoom 7. Indirect immunofluorescence using anti-CXCR6 with secondary Alexa Fluor 488–conjugated antibody (green) and anti-CXCL16 antibodies with secondary Alexa Fluor 633–conjugated antibody (red). Nuclei were stained with 4′-6-diamidino-2-phenylindole (blue). Images are representative of three independent experiments.
Kidney International  , DOI: ( /ki )
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11. Figure 10
CXCL16 does not modulate tubular cell survival or proliferation. (a) Tubular MCT cell death and (b) proliferation were assessed by flow cytometry of DNA content after culture for 24h in the presence of 100ng/ml TWEAK and different concentrations of CXCL16. Hypodiploid cells were considered apoptotic and cells in S/G2/M phase were considered proliferating. Mean±s.e.m. of four independent experiments. *P<0.02 vs. control.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

12. Figure 11
CXCL16 modulates TWEAK (tumor necrosis factor–like weak inducer of apoptosis)-induced tubular inflammation. MCT cells were pretreated with 50ng/ml CXCL16 for 1h, and then stimulated with TWEAK for 6h or 24h. (a) ICAM-1 mRNA, *P<0.02 vs. control 6h, **P<0.03 vs. control 24h, #P<0.05 vs. TWEAK 6h, ##P<0.05 vs. TWEAK 24h. (b) MCP-1 mRNA, *P<0.01 vs. control 6h, **P<0.03 vs. control 24h, #P<0.05 vs. TWEAK 6h. (c) RANTES mRNA, *P<0.01 vs. control 6h, **P<0.03 vs. control 24h, ##P<0.05 vs. TWEAK 24h. Real-time quantitative reverse transcription-PCR. Values for mRNA were normalized to glyceraldehyde-3-phosphate dehydrogenase expression. Expression level at 6h control was considered to be 100%. Mean±s.e.m. of four independent experiments.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions