Abstract
Renal tubulo-interstitial inflammation is frequently associated with polyuria and urine concentration defects. This led us to investigate the effects of the major pro-inflammatory nuclear factor kappaB (NF-kappaB) pathway on aquaporin 2 (AQP2) expression by the collecting duct. Using immortalized collecting duct principal cells (mpkCCDcl4), we found that, acting independently of vasopressin, activation of NF-kappaB by lipopolysaccharide (LPS) decreased AQP2 mRNA and protein levels in a time- and dose-dependent manner but did not decrease AQP2 mRNA stability. Consistently, constitutively active IkappaB kinase beta decreased AQP2 expression. The LPS-induced decrease in AQP2 mRNA levels was confirmed in rat kidney slices and was reproduced both under conditions of elevated cAMP concentration and V(2) receptor antagonism. Computer analysis of the AQP2 promoter revealed two putative kappaB elements. Mutation of either kappaB element abolished the LPS-induced decrease of luciferase activity in cells expressing AQP2 promoter-luciferase plasmid constructs. Chromatin immunoprecipitation revealed that LPS challenge decreased p65, increased p50 and p52, and had no effect on RelB and c-Rel binding to kappaB elements of the AQP2 promoter. RNA-mediated interference silencing of p65, p50, and p52 confirmed controlled AQP2 transcription by these NF-kappaB subunits. We additionally found that hypertonicity activated NF-kappaB in mpkCCDcl4 cells, an effect that may counteract the Tonicity-responsive enhancer binding protein (TonEBP)-dependent increase in AQP2 gene transcription. Taken together, these findings indicate that NF-kappaB is an important physiological regulator of AQP2 transcription.
Highlights
The nuclear factor B (NF-B) family of transcription factors consists of five members (p65, p50/p105, p52/p100, RelB, and c-Rel) that contain a Rel-homology domain required for the formation of various combinations of homo- and heterodimers and for DNA binding
Combined silencing of NF-B subunits, site-directed mutagenesis of the aquaporin 2 (AQP2) promoter, and the chromatin immunoprecipitation assay revealed that reduced AQP2 transcription under conditions of elevated NF-B activity is largely mediated by decreased p65 and increased p50 and p52 binding to B elements of the AQP2 promoter
These observations highlight the importance of down-regulated transcriptional AQP2 activity in the physiological control of AQP2 expression and suggest a role for AQP2 in polyuria associated with renal tubulo-interstitial diseases
Summary
The NF-B family of transcription factors consists of five members (p65, p50/p105, p52/p100, RelB, and c-Rel) that contain a Rel-homology domain required for the formation of various combinations of homo- and heterodimers and for DNA binding. Computer-assisted analysis of the first 2 kilobases of the AQP2 promoter revealed two putative B binding elements Taken together these observations may suggest that tubulo-interstitial inflammation leads to activation of NF-B in CD principal cells and, down-regulation of AQP2 expression. Combined silencing of NF-B subunits, site-directed mutagenesis of the AQP2 promoter, and the chromatin immunoprecipitation assay revealed that reduced AQP2 transcription under conditions of elevated NF-B activity is largely mediated by decreased p65 and increased p50 and p52 binding to B elements of the AQP2 promoter These observations highlight the importance of down-regulated transcriptional AQP2 activity in the physiological control of AQP2 expression and suggest a role for AQP2 in polyuria associated with renal tubulo-interstitial diseases
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