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Volume 70, Issue 5, Pages (September 2006)

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Presentation on theme: "Volume 70, Issue 5, Pages (September 2006)"— Presentation transcript:

1 Volume 70, Issue 5, Pages 892-900 (September 2006)
Screening and identification of substances that regulate nephrin gene expression using engineered reporter podocytes  K. Yamauchi, Y. Takano, A. Kasai, K. Hayakawa, N. Hiramatsu, N. Enomoto, J. Yao, M. Kitamura  Kidney International  Volume 70, Issue 5, Pages (September 2006) DOI: /sj.ki Copyright © 2006 International Society of Nephrology Terms and Conditions

2 Figure 1 Establishment of conditionally immortalized REPON. (a) Structure of pN5.4-SEAP and pN8.5-SEAP. The 5.4 and 8.3kb promoter fragments of the murine nephrin gene were subcloned into pSEAP2-Basic upstream of the SEAP gene. MCS, multicloning sites. (b) Typical morphologic features of established REPON under subconfluent and confluent culture conditions. Phase-contrast microscopy. (c, d) Responses of REPON to ATRA. REPON5.4(C5) precultured under a non-permissive condition were treated with (+) or without (−) 10μM ATRA for 24h, and culture medium and cells were subjected to SEAP assay (c) and RT-PCR (d), respectively. Data were presented as means±s.d. An asterisk indicates a statistically significant difference (P<0.05). In (d), molecular markers are shown on the left, and expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as a loading control. RT(−), reaction without reverse transcriptase. Kidney International  , DOI: ( /sj.ki ) Copyright © 2006 International Society of Nephrology Terms and Conditions

3 Figure 2 Screening and identification of bioactive substances that regulate expression of the nephrin gene. REPON5.4(C5) cells precultured under a non-permissive condition were treated for 24h with various substances including ATRA (10μM), 1,25(OH)2D3 (10−7M), dexamethasone (1μM), IL-1β (20ng/ml), TNF-α (250U/ml), TPA (50nM), and PDGF (20ng/ml). After the treatment, 5μl of culture medium was collected from each well and subjected to SEAP assay to evaluate the activity of the nephrin gene promoter. Assays were performed in quadruplicate, and data were expressed as means±s.d. Asterisks indicate statistically significant differences (P<0.05). Kidney International  , DOI: ( /sj.ki ) Copyright © 2006 International Society of Nephrology Terms and Conditions

4 Figure 3 Activation of the nephrin gene promoter by ATRA. (a) REPON5.4(C5) cells precultured under a non-permissive condition were treated for 24h with ATRA at different concentrations (0–10μM), and culture media were subjected to SEAP assay. (b) REPON5.4(C5) cells were treated with or without 10μM of ATRA for the indicated time periods and subjected to SEAP assay. (c) REPON5.4(C5), REPON5.4(C17), and REPON8.3(C2) cells were treated with 10μM of ATRA for 24h and subjected to SEAP assay. Data were expressed as means±s.d., and asterisks indicate statistically significant differences (P<0.05). In (c), fold induction of SEAP vs unstimulated condition is shown. Kidney International  , DOI: ( /sj.ki ) Copyright © 2006 International Society of Nephrology Terms and Conditions

5 Figure 4 Activation of the nephrin gene promoter by 1,25(OH)2D3. (a) REPON5.4(C5) cells precultured under a non-permissive condition were treated for 24h with 1,25(OH)2D3 at different concentrations (0–10−6M), and culture media were subjected to SEAP assay. (b) REPON5.4(C5) cells were treated with (+) or without (−) 10−7M of 1,25(OH)2D3 for 24h, and expression of nephrin mRNA was evaluated by RT-PCR. The level of GAPDH mRNA is shown as a loading control. RT(−), reaction without reverse transcriptase. (c) REPON5.4(C5), REPON5.4(C17), and REPON8.3(C2) cells were treated with 10−7M of 1,25(OH)2D3 for 24h and subjected to SEAP assay. Data were expressed as means±s.d., and asterisks indicate statistically significant differences (P<0.05). Kidney International  , DOI: ( /sj.ki ) Copyright © 2006 International Society of Nephrology Terms and Conditions

6 Figure 5 Activation of the nephrin gene promoter by dexamethasone. (a) REPON5.4(C5) cells precultured under a non-permissive condition were treated for 24h with 1,25(OH)2D3 at different concentrations (0–100μM), and culture media were subjected to SEAP assay. (b) REPON5.4(C5) cells were treated with (+) or without (−) 1μM of dexamethasone for 24h, and expression of nephrin mRNA was evaluated by RT-PCR. (c) REPON5.4(C5), REPON5.4(C17), and REPON8.3(C2) cells were treated with 1μM of dexamethasone for 24h and subjected to SEAP assay. Data were expressed as means±s.d., and asterisks indicate statistically significant differences (P<0.05). Kidney International  , DOI: ( /sj.ki ) Copyright © 2006 International Society of Nephrology Terms and Conditions

7 Figure 6 Combinational effects of ATRA, 1,25(OH)2D3, and dexamethasone on the activity of the nephrin gene promoter. REPON5.4(C5) cells precultured under a non-permissive condition were treated for 24h with 10μM ATRA, 10−7M 1,25(OH)2D3 (VitD), or 1μM dexamethasone (Dex), alone or in various combinations, and subjected to SEAP assay. Assays were performed in quadruplicate, and data were expressed as means±s.d. *Significantly different vs untreated control (control) (P<0.05); †significantly different vs any single treatments (P<0.05); #significantly different vs any other combinational treatments, except for treatment with 1,25(OH)2D3 plus dexamethasone (P=0.186). Kidney International  , DOI: ( /sj.ki ) Copyright © 2006 International Society of Nephrology Terms and Conditions

8 Figure 7 Suppression of nephrin expression by inflammatory cytokines. (a) REPON5.4(C5) cells precultured under a non-permissive condition were treated for 24h with IL-1β (20ng/ml), TNF-α (250U/ml), PDGF (20ng/ml), or TPA (50ng/ml). After the treatment, the cells were subjected to formazan assay to evaluate the number of viable cells. (b) Conditionally immortalized reporter podocytes that constitutively secrete SEAP under the control of the SV40 promoter were treated for 24h with IL-1β, TNF-α, TPA, or PDGF and subjected to SEAP assay. Assays were performed in quadruplicate, and data were expressed as means±s.d. (c) REPON5.4(C5) cells were treated for 24h with IL-1β (20ng/ml), TNF-α (250U/ml), or TPA (50ng/ml) and subjected to RT-PCR. The level of GAPDH mRNA is shown as a loading control. RT(−), reaction without reverse transcriptase. Kidney International  , DOI: ( /sj.ki ) Copyright © 2006 International Society of Nephrology Terms and Conditions

9 Figure 8 Expression of nephrin in reporter podocytes with or without prolonged deprivation of IFN-γ. REPON5.4(C5) cells were preincubated without IFN-γ for 48h or 9 days and subjected to (a) chemiluminescence assay, (b) RT-PCR, and (c) Western blot analysis to compare the activity of the nephrin gene promoter and the levels of nephrin mRNA and protein. In (a), data were expressed as means±s.d. NS, not statistically significant. In (b) and (c), levels of GAPDH mRNA and β-actin protein are shown as loading controls, respectively. Kidney International  , DOI: ( /sj.ki ) Copyright © 2006 International Society of Nephrology Terms and Conditions

10 Figure 9 Responses of reporter podocytes to stimuli with or without prolonged deprivation of IFN-γ. REPON5.4(C5) cells were preincubated without IFN-γ for 48h or 9 days, treated with (a) ATRA (10μM), 1,25(OH)2D3 (VitD; 10−7M), or dexamethasone (Dex; 1μM), or (b) IL-1β (20ng/ml) or TNF-α (250U/ml) for 24h, and culture media and cells were subjected to SEAP assay and formazan assay, respectively. The levels of SEAP were normalized by the number of viable cells, and relative values vs untreated controls (100%) are shown. Data were expressed as means±s.d. NS, not statistically significant. Kidney International  , DOI: ( /sj.ki ) Copyright © 2006 International Society of Nephrology Terms and Conditions

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