1. Volume 138, Issue 4, Pages 1384-1394.e2 (April 2010)
Tumor Necrosis Factor and Interferon-γ Down-regulate Klotho in Mice With Colitis 
Robert D. Thurston, Claire B. Larmonier, Pawel M. Majewski, Rajalakshmy Ramalingam, Monica Midura-Kiela, Daniel Laubitz, Alain Vandewalle, David G. Besselsen, Marcus Mühlbauer, Christian Jobin, Pawel R. Kiela, Fayez K. Ghishan 
Gastroenterology 
Volume 138, Issue 4, Pages e2 (April 2010)
DOI: /j.gastro
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2. Figure 1
The effects of TNBS colitis on renal expression of Klotho. (A) Klotho mRNA expression in control mice, TNBS-treated mice, and mice treated with TNBS and anti-TNF antibody. Anti-TNF antibody was given to mice (250 μg/mouse) 4 hours prior to TNBS enema and again on day 3 after TNBS treatment (n = 6–8). (B) ELISA analysis of renal Klotho protein in tissue lysates from control and TNBS-treated mice (n = 5). (C) Exogenous recombinant murine TNF (15 μg/100 g body weight) is not sufficient to down-regulate Klotho mRNA in vivo (n = 3). Decreased expression of Klotho protein correlates with the severity of colitis in individual TNBS-treated mice as exemplified in mice selected (animals numbered 4–6) by varying weight loss (D) and distal colonic histology (E).
Gastroenterology  , e2DOI: ( /j.gastro )
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3. Figure 2
Progressing colitis in SPF-colonized germ-free IL-10−/− mice results in decreased renal Klotho expression. (A) Representative microendoscopy picture of the distal colonic mucosa of WT mice or IL-10−/− mice 8 weeks after colonization with SPF microflora. (B) Real-time RT-PCR analysis of renal expression of Klotho mRNA in WT and colitic IL-10−/− mice. Values referred to age-matched WT mice maintained in SPF conditions (n = 5–9; *P < .05 t test WT vs IL-10−/−). (C) Representative Western blot analysis of Klotho protein in the renal lysates of WT and IL-10−/− mice 8 weeks postcolonization.
Gastroenterology  , e2DOI: ( /j.gastro )
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4. Figure 3
Renal Klotho expression is down-regulated in the adoptive T-cell transfer model of Crohn's disease. 0.5 × 106 Naïve CD4+CD45RBhigh lymphocytes were transferred into Rag-2−/− host. Control (PBS injected) and colitic mice were killed 8 weeks after transfer. (A) Real-time RT-PCR analysis of renal Klotho mRNA expression in control and adoptively transferred mice (n = 4). (B) Representative H&E-stained sections of the proximal and distal colon in control (PBS) mice and mice transferred with CD45RBhigh lymphocytes. (C) Secretion of IFN-γ, TNF, IL-1β, and IL-17 by the mesenteric lymph node (MLN) cells cultured in the presence of CD3/CD28 antibodies (bars, left axis) and by the colonic explant cultures (dashed line, right axis). Asterisks indicate statistical differences between groups (P < .05, Student t test; n = 4).
Gastroenterology  , e2DOI: ( /j.gastro )
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5. Figure 4
The effect of selected cytokines alone and in combination on Klotho mRNA expression mpkDCT4 cells. Immortalized mouse distal convoluted tubule epithelial cells were treated with TNF (20 ng/mL), IL-1β (10 ng/mL), IL-6 (10 ng/mL), IFN-γ (100 U/mL), combination of TNF and IFN-γ, or all 4 cytokines (Cytomix) for 24 hours. Klotho mRNA was analyzed by real-time RT-PCR. (*P < .05 control vs cytokine treatment; #P < .05 TNF vs TNF/IFN-γ; analysis of variation followed by Fisher protected least significant difference test; n = 4).
Gastroenterology  , e2DOI: ( /j.gastro )
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6. Figure 5
Time course analysis of the effects of TNF and TNF/IFN-γ combination on the expression of Klotho, iNOS, and NO production in mpkDCT4 cells. Cells were treated for 2–24 hours with TNF (20 ng/mL; open squares) or TNF/IFN-γ (20 ng/mL and 100 U/mL, respectively; open triangles), and expression of Klotho (A) and iNOS (B) was measured by real-time RT-PCR. (C) NO in the media was measured after conversion of nitrate to nitrite using nitrite reductase and colorimetric Griess reaction (Active Motif Nitric Oxide Quantitation Kit). (*P < .05 control vs cytokine treatment; #P < .05 TNF vs TNF/IFN-γ; analysis of variation followed by Fisher protected least significant difference test; n = 4).
Gastroenterology  , e2DOI: ( /j.gastro )
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7. Figure 6
The effects of nitric oxide on Klotho expression in mpkDCT4 cells. Cells were treated with SNAP for 20 hours with medium changed every 5 hours. Klotho expression was measured with real-time PCR (A), and the corresponding NO production was measured in medium using Nitric Oxide Quantitation Kit (Active Motif) (B). (*P < .05 control vs SNAP treatment; values above bars in B indicate the mean NOx concentration in medium).
Gastroenterology  , e2DOI: ( /j.gastro )
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8. Figure 7
iNOS inhibitor L-NIL restores Klotho expression in IFN-γ-treated cells, whereas JNK inhibition abrogates the effects of TNF, IFN-γ, or combination of the 2 cytokines. (A) Cells were pretreated with 1 mmol/L L-NIL prior to addition of IFN-γ (100 U/mL) or TNF (20 ng/mL) to the medium. Cells were harvested 24 hours later, and Klotho expression was analyzed by real-time RT-PCR. Curcumin (20 μmol/L) used as a nonspecific inhibitor of NF-κB and activator protein 1 effectively reversed TNF-induced down-regulation of Klotho expression (*P < .05 control vs cytokine treatment; n = 4). (B) Cells were pretreated with a selective inhibitor of JNK, 1,9-pyrazoloanthrone (SP600125; 20 μmol/L), prior to 24-hour cytokine treatment (TNF at 20 ng/mL, IFN-γ at 100 U/mL), and Klotho mRNA expression was analyzed by real-time RT-PCR (*P < .05 control vs cytokine treatment; n = 4).
Gastroenterology  , e2DOI: ( /j.gastro )
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9. Figure 8
Klotho is regulated by TNF and/or IFN-γ via a transcriptional mechanism. (A) De novo transcription was inhibited in mpkDCT4 cells with Actinomycin D (ActD), and Klotho transcript decay rate was followed over time by real-time RT-PCR analysis. No significant differences were observed in the rate of mRNA degradation in control (open circles), TNF-treated (open squares), or TNF/IFN-γ cotreated cells (open triangles). (B) The effects of the 2 cytokines on Klotho (bars) and iNOS (line) expression in mIMCD-3 cells mirror the effects observed in the mpkDCT4 cells. (C) The effects of TNF, IFN-γ, and their combination on murine Klotho gene promoter activity in transiently transfected mIMCD-3 cells. 5′-Deletion constructs spanning −1085 nt to −273 nt upstream of the transcription start site are depicted with predicted binding sites for the 3 prototypical JNK-regulated transcription factors, activator protein 1 (AP-1), Elk-1, and activating transcription factors (ATF)/CREB (*P < .05 control vs cytokine treatment, n = 4). Transcription start site, mapped by Shiraki-Iida et al,32 is indicated by a right-pointing arrow.
Gastroenterology  , e2DOI: ( /j.gastro )
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