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1.
FIGURE 2.

FIGURE 2. From: NF-?B Modulates Aquaporin-2 Transcription in Renal Collecting Duct Principal Cells.

LPS decreases both AVP-induced and hypertonicity-enhanced AQP2 expression in cultured collecting duct cells. Cultured mpkCCDcl4 (A) or mCCDcl1 (B) cells were preincubated 24 h or not with either 10–10 m AVP or hypertonic (NaCl, 500 mosmol/kg) medium and were then stimulated for an additional 24 h with 100 ng/ml LPS before RNA extraction. Real-time PCR was performed using primers specific for AQP2. Results are expressed relative to control values determined in the absence of stimuli. Bars are the mean ± S.E. from three independent experiments. *, p < 0.05.

Udo Hasler, et al. J Biol Chem. 2008 October 17;283(42):28095-28105.
2.
FIGURE 3.

FIGURE 3. From: NF-?B Modulates Aquaporin-2 Transcription in Renal Collecting Duct Principal Cells.

LPS decreases AQP2 expression independently of the V2 receptor and intracellular cAMP in rat kidney. Wistar rat kidney slices were incubated for 6 h in the absence or presence of 100 ng/ml LPS with or without 10–6 m SR121463B (SR) or 10–3 m 8-bromo-cAMP before mRNA extraction. Real-time PCR was performed using primers specific for AQP2 (A) and interleukin-1β (IL-1β)(B). Results are expressed relative to control values determined in the absence of stimuli. Bars are the mean ± S.E. from triplicate measurements performed in three different animals. *, p < 0.05. Ctl, control.

Udo Hasler, et al. J Biol Chem. 2008 October 17;283(42):28095-28105.
3.
FIGURE 6.

FIGURE 6. From: NF-?B Modulates Aquaporin-2 Transcription in Renal Collecting Duct Principal Cells.

NF-κB activity is increased by hypertonicity in mpkCCDcl4 cells. A, transfected cells expressing NF-κB-driven luciferase were challenged with either isotonic (Ctl, 300 mosmol/kg), hypertonic (NaCl, 500 mosmol/kg), or urea-supplemented hyperosmotic (urea, 500 mosmol/kg) medium for 12 h or were stimulated for 12 h with 100 μg/ml LPS in isotonic medium before measuring luciferase activity. Bars are the mean ± S.E. from four independent experiments. *, p < 0.05. B–D, cells were challenged with isotonic medium (Ctl, 300 mosmol/kg) or with either 100 ng/ml LPS (B), NaCl-supplemented hypertonic (400 or 500 mosmol/kg) (B) or urea-supplemented hyperosmotic (400 or 500 mosmol/kg) (C) medium for 2 or 24 h before RNA extraction. Real-time PCR was performed using primers specific for TNFα, MCP-1, or IκBα. Results are expressed relative to control values determined for each gene after 2 h of incubation in isotonic medium. Bars are the mean ± S.E. from five independent experiments. *, p < 0.05.

Udo Hasler, et al. J Biol Chem. 2008 October 17;283(42):28095-28105.
4.
FIGURE 7.

FIGURE 7. From: NF-?B Modulates Aquaporin-2 Transcription in Renal Collecting Duct Principal Cells.

Hypertonicity-induced NF-κB activation is dependent on IKKβ activity, IκBα degradation, and p65 activation. A and B, cells were transfected with an expression vector that contained either eGFP or constitutively active IKKβ or IκBα mutants together with (A) or without (B) NF-κB-driven luciferase plasmid. Cells were challenged or not with either 100 μg/ml LPS or hypertonic (NaCl, 500 mosmol/kg) medium for 12 (A) or 3 (B) h before measurement of luciferase activity (A) or to RNA extraction (B). Real-time PCR was performed using primers specific for TNFα (B). Results are expressed relative to control values determined in cells transfected with eGFP-containing cDNA and subjected to isotonic medium. Bars are the mean ± S.E. from four independent experiments. *, p < 0.05. C, cells were transfected with either scrambled RNAi or RNAi targeting p65 NF-κB and then challenged or not with either 100 ng/ml LPS or hypertonic (NaCl, 500 mosmol/kg) medium for 3 h before RNA extraction. Real-time PCR was performed using primers specific for MCP-1, IκBα, or TNFα. Results are expressed relative to control values determined in cells transfected with scrambled RNAi and subjected to isotonic medium. Bars are the mean ± S.E. from four independent experiments. *, p < 0.05. Ctl, control.

Udo Hasler, et al. J Biol Chem. 2008 October 17;283(42):28095-28105.
5.
FIGURE 8.

FIGURE 8. From: NF-?B Modulates Aquaporin-2 Transcription in Renal Collecting Duct Principal Cells.

Schematic illustration of controlled AQP2 transcription in collecting duct principal cells. By binding to cis elements of the AQP2 promoter cAMP CREB and AP-1 (c-Fos/c-Jun) enhance AQP2 transcriptional activity in response to protein kinase A-mediated vasopressin (VP) stimulation. By binding to the TonE element(s) of the AQP2 promoter, TonEBP positively regulates AQP2 transcription under both base-line conditions and in response to hypertonicity. Positive AQP2 transcriptional regulation mediated by both CREB/AP-1 and TonEBP is repressed after activation of the NF-κB pathway in response to either LPS stimulation, characteristic of inflammatory diseases, or hypertonicity. This event is mediated by p65 release from and increased p50 and p52 binding to κB elements of the AQP2 promoter. Although AQP2 transcriptional activity is maintained at low levels under iso-osmotic conditions in the absence or presence of VP, the repressive effect of NF-κB is superseded by increased TonEBP activity after longer periods of hypertonic stimulation. PKA, protein kinase A.

Udo Hasler, et al. J Biol Chem. 2008 October 17;283(42):28095-28105.
6.
FIGURE 5.

FIGURE 5. From: NF-?B Modulates Aquaporin-2 Transcription in Renal Collecting Duct Principal Cells.

The effect of LPS is dependent on altered p65, p52, and p50 binding to the AQP2 promoter. A, cells were challenged or not with 100 ng/ml LPS for 3 h before DNA fragmentation and immunoprecipitation using anti-p65, p50, p52, RelB, or c-Rel antibodies. Real-time PCR was performed using primers flanking either the NF-κB binding element of the AQP2 promoter (–2030, open box; –666, hatched box) or using primers flanking NF-κB binding element of the IκBα promoter (closed box). Results are expressed as the ratio of values obtained in the presence and in the absence of LPS. Shown are results of one of three similar experiments. ChIP, chromatin immunoprecipitation. B and C, cells were transfected with either scrambled RNAi or RNAi targeting various NF-κB isoforms and then challenged or not with 100 ng/ml LPS for 3 h before protein (B) or RNA (C) extraction. B, Western blot analysis was performed on various NF-κB isoforms and on α-tubulin, used as a loading control. Representative images are shown. C, real-time PCR from RNA extracts of cells transfected with scrambled RNAi or RNAi against p65 (left panel), p50 (middle panel), or p52 (right panel) was performed using primers specific for NF-κB isoforms or AQP2. Results are expressed relative to control values determined in cells transfected with scrambled RNAi and subjected to isotonic medium. Bars are the mean ± S.E. from six independent experiments. *, p < 0.05.

Udo Hasler, et al. J Biol Chem. 2008 October 17;283(42):28095-28105.
7.
FIGURE 4.

FIGURE 4. From: NF-?B Modulates Aquaporin-2 Transcription in Renal Collecting Duct Principal Cells.

Two κB binding elements located in the AQP2 promoter mediate the decrease of AQP2 transcriptional activity in response to NF-κB activation. A, cells transfected with an expression plasmid that contained either eGFP or constitutively active IKKβ and were stimulated or not for 3 h with 100 ng/ml LPS before RNA extraction. Real-time PCR was performed using primers specific for AQP2. Results are expressed relative to control values determined in cells transfected with eGFP-containing plasmid in the absence of LPS. Bars are the mean ± S.E. from four independent experiments. *, p < 0.05. NS, no significant difference. B, cells were transfected with a luciferase reporter gene 5′-flanked by either the first 517 or 2043 bp of mouse AQP2 promoter, the first 517 bp of mouse AQP2 promoter of which the TonE sequence was mutated, or the first 2043 bp of mouse AQP2 promoter of which either or both putative κB binding sites were mutated. Transfected cells were challenged with either hypertonic (NaCl, 500 mosmol/kg) medium or LPS (100 ng/ml) for 24 h before measurement of luciferase activity. Results are expressed relative to control values determined in cells transfected with luciferase reporter gene 5′-flanked by the first 517 bp of mouse AQP2 promoter and incubated for 24 h in isotonic medium. Bars are the mean ± S.E. from three independent experiments. *, p < 0.05; NS, no significant difference. The positions, relative to the start codon, and sequences of putative κB binding sites between different species are shown in the inset. Ctl, control.

Udo Hasler, et al. J Biol Chem. 2008 October 17;283(42):28095-28105.
8.
FIGURE 1.

FIGURE 1. From: NF-?B Modulates Aquaporin-2 Transcription in Renal Collecting Duct Principal Cells.

Increased NF-κB activity decreases AQP2 transcription in cultured collecting duct cells. A and B, cells were preincubated or not with 10–10 m AVP for 24 h and then challenged for an additional 24 h with 0–1000 ng/ml LPS (A) or for 0–48 h with 10 ng/ml LPS (B) before RNA extraction. Real-time PCR was performed using primers specific for AQP2. Results are expressed relative to control values determined in the absence of LPS and in the absence (filled bars) or presence (open bars) of AVP. Bars are the mean ± S.E. from four independent experiments. *, p < 0.05. C, cells were immediately lysed or were preincubated with 5 × 10–6 m actinomycin D for 30 min before 3 or 5 h of incubation in the absence or presence of 100 ng/ml LPS (in the continuous presence of actinomycin D) before RNA extraction. Real-time PCR was performed using primers specific for AQP2 (diamonds and squares) or IκBα (triangles and ×'s). Results are expressed as the percentage of control values for each gene determined in the absence of actinomycin D. Data are the mean ± S.E. from three independent experiments. D, cells were preincubated for 24 h with 10–10 m AVP and then for an additional 3 or 24 h with 10 ng/ml LPS before protein extraction. Western blot analysis was performed on AQP2, and Na+, K+-ATPaseα1 subunit (NaKα) was used as a loading control. A representative image is shown. AQP2 abundance is expressed as the ratio of optical density values measured in the presence of LPS and that measured in the absence of LPS. Values are the mean ± S.E. from three independent experiments. *, p < 0.05.

Udo Hasler, et al. J Biol Chem. 2008 October 17;283(42):28095-28105.

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