1. Volume 87, Issue 2, Pages 396-408 (February 2015)
IL-37 inhibits IL-18-induced tubular epithelial cell expression of pro-inflammatory cytokines and renal ischemia-reperfusion injury 
Yunbo Yang, Zhu-Xu Zhang, Dameng Lian, Aaron Haig, Rabindra N. Bhattacharjee, Anthony M. Jevnikar 
Kidney International 
Volume 87, Issue 2, Pages (February 2015)
DOI: /ki
Copyright © 2015 International Society of Nephrology Terms and Conditions

2. Figure 1
Interleukin (IL)-37 inhibits hypoxia-induced inflammatory cytokine expression in renal tubular epithelial cells (TECs). Murine NG 1.1 cells and human PT-2 cells were incubated in hypoxia chambers for 45min at 37°C and then under normal conditions for 24h for reverse transcription-PCR and for 48h for enzyme-linked immunosorbent assay (ELISA). IL-37 dose-dependent effect on IL-1β mRNA expression (a; n=3, *P<0.05) and protein production (b; n=3, *P<0.05) in NG 1.1 cells. (c) Real-time PCR expression of TNFα, IL-6, and IL-1β mRNA in control NG 1.1, hypoxic NG 1.1, and in NG 1.1 TECs treated with IL-37 (300ng/ml) for 2h before hypoxia (n=4, *P<0.05, **P<0.01). (d) NG 1.1 TECs were treated with hypoxia or hypoxia plus IL-37 (300ng/ml). Levels of TNFα, IL-6, and IL-1β in cell cultured supernatant were detected by ELISA (n=4, *P<0.05). (e) TNFα, IL-6, and IL-1β mRNA in hypoxia PT-2 cells transfected with IL-37-expressing plasmid vector or empty vector (n=4, *P<0.05). β-Actin amplification was used as the endogenous control. (f) Human PT-2 cells were transfected with IL-37-expressing plasmid vector or empty vector before hypoxia treatment, and levels of TNFα, IL-6, and IL-1β in cell cultured supernatant were detected by ELISA (n=3, *P<0.05, **P<0.01). (g, h) IL-37 (300ng/ml) was added to PT-2 TECs after hypoxia treatment. IL-6 mRNA expression (f) and protein production (h) were tested (n=3, *P<0.05, **P<0.01). TNF, tumor necrosis factor.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

3. Figure 2
Effect of IL-37 on hypoxia-induced cell death. NG 1.1 TECs (a) and human PT-2 TECs (b) were treated with hypoxia or hypoxia plus IL-37 (300ng/ml) for 45min. IL-37 was added 2h before hypoxia. Annexin V-propidium iodide (PI) staining was performed to detect total cell death (Annexin V+ plus PI+ cells) 24h after hypoxia treatment (n=3–4). Data are representative of one experiment. IL, interleukin; TEC, tubular epithelial cell.
Kidney International  , DOI: ( /ki )
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4. Figure 3
Expression of interleukin (IL)-18Rα, IL-18Rβ, and IL-18BP in renal tubular epithelial cells (TECs). (a) IL-18Rα cell surface staining in TECs. NG 1.1 TECs and PT-2 TECs were incubated with anti-IL-18Rα antibody (2.5μg/105 cells) for 30min at 4°C, followed by a secondary staining of anti-rat IgG conjugated with fluorescein isothiocyanate for 30min at 4°C and then tested by fluorescence activated cell sorting analysis. (b) mRNA expression of IL-18Rα, IL-18Rβ, and IL-18BP in NG 1.1 TECs. Cells were treated with hypoxia for 45min, and mRNA was collected for real-time PCR assay at 24h (n=3, *P<0.05, **P<0.01). (c) NG 1.1 cells were treated with hypoxia. Whole cell lysate proteins were collected at 48h, and expression of IL-18Rα, IL-18Rβ, and IL-18BP was analyzed by western blot. Data are representative of three separate experiments. Densitometry for IL-18Rα, IL-18Rβ, and IL-18BP using β-actin controls is shown (n=3, *P<0.05). Ig, immunoglobulin; NS, nonsignificant.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

5. Figure 4
Exogenous interleukin (IL)-37 reduces IL-18-induced cytokine expression in murine NG 1.1 tubular epithelial cells (TECs). (a, b) IL-18 mRNA expression (a; n=3, ***P<0.001) and protein production (b; n=3, *P<0.05) after hypoxia or IFNγ treatment in murine NG 1.1 TECs. Cells were treated with IFNγ (20ng/ml) for 24 and 48h or incubated in a hypoxia chamber for 45min at 37°C, followed by continued culture at 37°C for 24 and 48h. RNA was collected for real-time PCR at 24h, and cell cultured supernatant was collected at 48h for enzyme-linked immunosorbent assay (ELISA). (c) IL-18 increased IL-6 protein production dose dependently in NG 1.1 TECs. NG 1.1 TECs were treated with IL-18 (1–100ng/ml) for 48h, and then cell culture supernatant was collected for ELISA (n=3, *P<0.05). (d, e) Effect of IL-37 on the IL-18-induced cytokine expression in NG 1.1 TECs. NG 1.1 TECs were pretreated with IL-37 (300ng/ml) for 2h before being treated with 50ng/ml of IL-18 for 24 or 48h. Data show TNFα, IL-6, and IL-1β mRNA expression (d; n=3, *P<0.05, **P<0.01) and protein levels (e; n=3, *P<0.05). β-Actin was used as the endogenous control. IFN, interferon; TNF, tumor necrosis factor.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

6. Figure 5
Exogenous interleukin (IL)-37 reduces IL-18-induced cytokine expression in human PT-2 tubular epithelial cells (TECs). (a) IL-18 increased IL-6 protein production dose dependently in human PT-2 TECs. Human PT-2 TECs were treated with IL-18 (1–100ng/ml) for 48h, and then the cell culture supernatant was collected for enzyme-linked immunosorbent assay (ELISA) (n=3, *P<0.05). (b, c) PT-2 TECs were treated with IL-18 (20ng/ml) or IL-18 plus IL-37 (300ng/ml) for 24h to detect mRNA expression of TNFα, IL-6, and IL-1β by real-time PCR (b; n=3, *P<0.05, **P<0.01) and for 48h to detect protein levels of these molecules in cell culture supernatant by ELISA (c; n=3, *P<0.05, **P<0.01). TNF, tumor necrosis factor.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

7. Figure 6
Interleukin (IL)-37 siRNA augments cytokine expression in renal tubular epithelial cells (TECs). (a, b) Inflammatory stimuli induced IL-37 expression in human PT-2 TECs. Cells were treated with IL-18 (20ng/ml), IFNγ (20ng/ml), or LPS (1μg/ml) for 24h for real-time PCR assay and for 48h for western blot assay. β-Actin was used as the endogenous control (n=3, *P<0.05, **P<0.01). (c) mRNA silencing of IL-37 mRNA. Human PT-2 TECs were transfected with 1μg of IL-37 siRNA or control scrambled siRNA. After 24h recovery, cells were treated with IL-18, IFNγ, or LPS for 24h for the real-time PCR assay (n=3, *P<0.05). (d) IL-37 protein expression after silencing. Human PT-2 TECs were treated for 48h as above and used for western blot assay. Data are representative of three separate experiments. Scrambled siRNA was used as control. Densitometry analysis of IL-37 using β-actin as control is shown (n=3, *P<0.05). (e, f, g) Effects of IL-37 silencing on the expression of pro-inflammatory cytokines. PT-2 cells were treated with IL-18, IFNγ, or LPS for 24h after IL-37 siRNA and scrambled siRNA transfection. mRNA expression of TNFα, IL-6, and IL-1β was analyzed by real-time PCR (n=4, *P<0.05, **P<0.01). (h) PT-2 TECs were treated with IL-18 (20ng/ml) for 48h after IL-37 siRNA and scrambled siRNA transfection, and levels of TNFα, IL-6, and IL-1β in cell culture supernatant were detected by enzyme-linked immunosorbent assay (n=3, *P<0.05, **P<0.01). IFN, interferon; LPS, lipopolysaccharides; siRNA, small interfering RNA; TNF, tumor necrosis factor.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

8. Figure 7
Interleukin (IL)-37 overexpression inhibits cytokine expression in renal tubular epithelial cells (TECs). Human PT-2 TECs were transfected with IL-37-expressing plasmid (pCMV6-XL5-IL-37) or empty vector (pCMV6-XL5). After 24h, cells were treated with normal medium containing IL-18 (20ng/ml) for 24h for real-time PCR (a; n=3, **P<0.01) and for 48h for western blot assay (b) to detect IL-37 mRNA and protein level, respectively. β-Actin was used as control. Data are representative of three separate experiments. (c) Real-time PCR detected TNFα, IL-6, and IL-1β mRNA upon IL-37 overexpression as above in PT-2 TECs (n=4, *P<0.05). (d) TNFα, IL-6, and IL-1β protein levels in cell culture supernatant after IL-37 overexpression and IL-18 treatment for 48h (n=3, *P<0.05, **P<0.01). TNF, tumor necrosis factor.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

9. Figure 8
Transgenic interleukin (IL)-37 expression in mouse kidney. (a) IL-37 mRNA expression in kidney after IL-37 plasmid injection. C57BL/6 WT mice were injected with pCMV6-XL5-IL-37 plasmid (50μg/mouse) by hydrodynamic injection through the tail vein to induce IL-37 expression. Mouse kidney samples were collected at 24 and 48h for real-time PCR (n=4, *P<0.05). (b) IL-37 immunohistochemical staining in mouse kidney. Mouse kidney was collected for immunohistochemistry staining 24 and 48h after IL-37 plasmid injection. Paraffin-embedded kidney sections were probed with anti-IL-37 antibody. Data are representative of three separate experiments. Magnification × 200. WT, wild type.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

10. Figure 9
Transgenic interleukin (IL)-37 expression inhibits cytokine expression in kidney following renal ischemia–reperfusion injury. (a, b) Expression of IL-18 Rα, IL-18 Rβ, and IL-18BP in mouse kidney after renal ischemia–reperfusion injury (IRI). Kidney samples were collected at 48h after renal IRI for real-time PCR assay (a) and western blot assay, and densitometry for IL-18 Rα, IL-18 Rβ, and IL-18BP using β-actin controls is shown (b). Sham mice (n=6) were used as control. IRI mice, n=8, **P<0.01. β-Actin amplification was used as the endogenous control. (c, d) Cytokine expression in mouse kidney after renal IRI. C57BL/6 WT mice were subjected to renal IRI 24h after hydrodynamic tail vein injection of pCMV6-XL5-IL-37 plasmid-DNA (50μg/mouse) or empty vector DNA (50μg/mouse). Following 48h of reperfusion, kidney samples were collected for real-time PCR to detect TNFα, IL-6, and IL-1β mRNA expression (c) and protein levels (d). IRI-empty vector: n=8; IRI-IL-37 plasmid: n=8; Sham: n=6, *P<0.05, **P<0.01. (e) IL-6 immunohistochemical staining in mouse kidney. C57BL/6 WT mice were subjected to renal IRI as above, and kidney samples were collected for immunochemical staining. The intensity of IL-6 immunostaining by automated image analysis is shown in f. Magnification × 200, n=4, *P<0.05, **P<0.01. NS, nonsignificant; TNF, tumor necrosis factor; WT, wild type.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

11. Figure 10
Interleukin (IL)-37 protects kidney dysfunction following renal ischemia–reperfusion injury. Mouse kidney samples were collected from naive, sham mice, and the samples from empty vector and IL-37 plasmid–treated mice were collected at 48h after renal ischemia–reperfusion injury (IRI). Serum creatinine levels were measured (a). Formalin-fixed and paraffin-embedded sections (3μm) were stained with hematoxylin and eosin. Tubule necrosis (b) was scored using a semiquantitative score (0: no change; 0.5: 10–25%; 1: 25–50%; 1.5: 50–75%; and 2: 75–100% area change). (c) Mouse kidney periodic acid–Schiff staining (magnification × 400). (d) CD3 immunohistochemistry (magnification × 200). (e) Infiltration of CD3+ T cells by immunohistochemistry was scored by a pathologist blinded to groups. CD3+ cells were counted with a × 400 power microscope and recorded as the average of at least 10 non-overlapping fields for each kidney. (f) Infiltration of mononuclear cells was scored by a pathologist blinded to groups by morphometry. The cells were counted with a × 400 power microscope and recorded as the average of at least 10 non-overlapping fields. IRI-empty vector: n=8; IRI-IL-37 plasmid: n=8; naive mice and sham: n=6; *P<0.05, **P<0.01, ***P< NS, nonsignificant.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions