title: Sepsis-induced urinary concentration defect is related to nitric oxide-dependent inactivation of TonEBP/NFAT5, which downregulates renal medullary solute transport proteins and aquaporin-2.
abstract: OBJECTIVE
  Acute kidney injury associated with reduced urinary concentration is a frequent and severe complication during sepsis. The present study addressed the effect of endotoxemia on the functional and molecular mechanisms that determine urinary concentrating ability. Efficient urinary concentration depends on, amongst other factors, the expression of the Cl channel kidney-specific chloride channel 1 and its subunit Barttin, the urea transporter-A1, and the water channel aquaporin 2, all of which are regulated by the transcription factor TonEBP/NFAT5.
  
  
  METHODS
  Experimental animal and cell culture model.
  
  
  METHODS
  University laboratory.
  
  
  METHODS
  Wistar rats and Madin-Darby canine kidney cells.
  
  
  METHODS
  Rats were injected with lipopolysaccharide (5 mg/kg bodyweight intraperitoneal) or vehicle (phosphate-buffered saline) as control. After 24 hrs, urine, blood, and tissue samples from various kidney zones were analyzed for parameters that determine urinary concentration ability. Madin-Darby canine kidney cells were treated under isotonic or hypertonic conditions with the nitric oxide donor S-nitroso-N-acetylpenicillamine.
  
  
  RESULTS
  In rats injected with lipopolysaccharide, urine osmolality was reduced by ~40%, along with medullary induction of inducible nitric oxide synthase and a dramatic increase in urinary nitric oxide degradation products nitrite/nitrate. Concomitantly, expressions of ClC-K1, Barttin, urea transporter-A1, and aquaporin 2 were significantly lower. This was associated with the appearance of S-nitrosylated TonEBP/NFAT5, as monitored by the biotin-switch assay and immunoprecipitation, and reduced TonEBP/NFAT5 DNA binding activity in the renal inner medulla. These results were confirmed in Madin-Darby canine kidney cells transfected with a reporter construct driven by the urea transporter-A promoter, in which the nitric oxide donor S-nitroso-N-acetylpenicillamine reduces urea transporter-A reporter activity under isotonic and hypertonic conditions.
  
  
  CONCLUSIONS
  The present data demonstrate that lipopolysaccharide increases medullary nitric oxide production by iNOS induction, resulting in impairment of the transcriptional activity of TonEBP/NFAT5 by S-nitrosylation. The consequence thereof is reduced expression of TonEBP/NFAT5 target genes ClC-K1, Barttin, urea transporter-A1, and aquaporin 2 that are required for urinary concentration. Our findings may provide further insight into the molecular mechanisms underlying the urinary concentration defect in sepsis.
authors.affiliation: Department of Physiology, University of Munich, Munich, Germany. Christoph.Kueper@lrz.uni-muenchen.de
authors.forename: Christoph
authors.initials: C
authors.lastname: Küper
authors.forename: Maria-Luisa
authors.initials: ML
authors.lastname: Fraek
authors.forename: Hans-Helge
authors.initials: HH
authors.lastname: Müller
authors.forename: Franz-Xaver
authors.initials: FX
authors.lastname: Beck
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Neuhofer
mesh_terms: D000818:Animals; D051399:Aquaporin 2 / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D004285:Dogs; D015536:Down-Regulation; D019446:Endotoxemia / Q000378:metabolism / Q000503:physiopathology; D007671:Kidney Concentrating Ability / Q000502:physiology*; D007679:Kidney Medulla / Q000378:metabolism*; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D018805:Sepsis / Q000503:physiopathology* / Q000652:urine; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D051399:Aquaporin 2; D002352:Carrier Proteins; C492199:Nfat5 protein, rat; D014157:Transcription Factors; D009569:Nitric Oxide