Abstract
In renal proximal tubule, multidrug resistance protein 2 (Mrp2) actively transports many organic anions into urine, including drugs and metabolic wastes. Upon exposure to nephrotoxicants or during endotoxemia, both Mrp2 activity and expression are up-regulated. This may result from induced de novo synthesis of Mrp2 or post-transcriptional events involving specific signaling pathways. Here, we investigated glucocorticoid signaling to Mrp2 in killifish renal proximal tubules, a model system in which transport activity can be measured using a fluorescent substrate and confocal imaging. Exposure of tubules to dexamethasone rapidly increased Mrp2-mediated fluorescein methotrexate transport. Other glucocorticoid receptor (GR) ligands, cortisol and triamcinolone acetonide, also stimulated Mrp2-mediated transport. The GR antagonist, mifepristone 17β-hydroxy-11β-[4-dimethylamino phenyl]-17α-[1-propynyl]estra-4,9-dien-3-one (RU486), abolished stimulation by all three ligands, whereas the mineralocorticoid receptor antagonist, spironolactone, was ineffective. Consistent with action through a nongenomic mechanism, dexamethasone stimulation of Mrp2-mediated transport was insensitive to cycloheximide and actinomycin D, and immunohistochemistry revealed no alterations in Mrp2 expression at the luminal membrane. (9S-(9α,10β,12α))-2,3,9,10,11,12-hexahydro-10-hydroxy-10-(methoxycarbonyl)-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one (K252a), an inhibitor of the tyrosine receptor kinase subfamily, reduced the dexamethasone effect, as did the specific hepatocyte growth factor receptor (c-Met) tyrosine kinase inhibitor, (2R)-1-[[5-[(Z)-[5-[[(2,6-dichlorophenyl)methyl]sulfonyl]-1,2-dihydro-2-oxo-3H-indol-3-ylidene]methyl]-2,4-dimethyl-1H-pyrrol-3-yl]carbonyl]-2-(1-pyrrolidinylmethyl)pyrrolidine (PHA-665752). Hepatocyte growth factor (HGF), an endogenous ligand for c-Met, stimulated Mrp2-mediated transport. This effect was reversed by PHA-665752 but not by RU486. Inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK 1/2) also abolished the effects of dexamethasone and HGF. Our results disclose a novel mechanism by which glucocorticoids acting through GR, c-Met, and MEK1/2 cause rapid, nongenomic stimulation of Mrp2-mediated transport in renal proximal tubules. This up-regulation may be nephroprotective, enhancing efflux of metabolic wastes and toxicants during cell and tissue stress.
Footnotes
This work was supported by the Blum Halsey Fund for the visiting scientific team of Mount Desert Island Biological Laboratory (to R.M.); the Intramural Research Program National Institute of the Environmental Health Sciences/National Institute of Health (to D.M.); and the Radboud University Study Fund, Nijmegen, The Netherlands [Grant B090148] (to B.P.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.111.179689.
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ABBREVIATIONS:
- Mrp2
- multidrug resistance protein 2
- FXR
- farnesoid xenobiotic receptor
- ERK
- extracellular signal-regulated kinase
- ET
- endothelin
- FL-MTX
- fluorescein methotrexate
- c-Met
- hepatocyte growth factor receptor
- GR
- glucocorticoid receptor
- HGF
- hepatocyte growth factor
- MTS
- marine teleost saline
- MR
- mineralocorticoid receptor
- NHE
- Na+/H+ exchanger
- iNOS
- inducible nitric-oxide synthase
- PXR
- pregnane xenobiotic receptor
- MEK 1/2
- mitogen-activated protein kinase/extracellular signal-regulated kinase kinase
- BIM
- bisindolylmaleimide
- PKC
- protein kinase C
- RU486
- mifepristone [17β-hydroxy-11β-[4-dimethylaminophenyl]-17α-[1-propynyl]estra-4,9-dien-3-one]
- K252a
- (9S-(9α,10β,12α))-2,3,9,10,11,12-hexahydro-10-hydroxy-10-(methoxycarbonyl)-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one
- PHA-665752
- (2R)-1-[[5-[(Z)-[5-[[(2,6-dichlorophenyl)methyl]sulfonyl]-1,2-dihydro-2-oxo-3H-indol-3-ylidene]methyl]-2,4-dimethyl-1H-pyrrol-3-yl]carbonyl]-2-(1-pyrrolidinylmethyl)pyrrolidine
- LY-294002
- 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
- U0126
- 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene
- SB203580
- 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole.
- Received January 20, 2011.
- Accepted April 21, 2011.
- U.S. Government work not protected by U.S. copyright
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