title: Nephron specific regulation of chloride channel CLC-K2 mRNA in the rat.
abstract: BACKGROUND
  This study investigated the influence of salt intake on the nephron specific gene expression of the kidney chloride channel CLC-K2. To this end, male Sprague-Dawley rats were fed a low (0.02% wt/wt), normal (0.6% wt/wt), or high salt (8% wt/wt) diet for ten days, or they received the loop diuretic furosemide (12 mg/kg/day) for six days.
  
  
  METHODS
  Expression and regulation of messenger RNA for CLC-K2 was demonstrated by RNase protection assay and in situ hybridization in kidney cortex, outer medulla and inner medulla. Tubular localization and regulation were determined precisely by reverse transcription-polymerase chain reaction (RT-PCR) and real time PCR of microdissected nephron segments.
  
  
  RESULTS
  In situ hybridization analysis and RNase protection assay of the total kidney revealed a down-regulation of CLC-K2 mRNA in the high salt diet rats and an up-regulation of CLC-K2 mRNA in furosemide treated rats, which were restricted to the outer medulla. Microdissection of collagenase treated kidney revealed CLC-K2 mRNA expression in the outer medullary thick ascending limb (mTAL), cortical thick ascending limb (cTAL), distal convoluted tubule (DCT), connecting tubule and cortical collecting duct (CNT/CCD), and outer medullary collecting duct (OMCD), whereas no signals were detected in proximal convoluted and straight tubules (PCT and PST), descending thin limb from the outer medulla (dTL), descending and ascending thin limb from the inner medulla (TL), inner medullary collecting duct (IMCD) and glomeruli (glom). Using RT-PCR and real time PCR, the changing levels of CLC-K2 mRNA after furosemide treatment or high salt diet were restricted to the mTAL, whereas CLC-K2 mRNA levels in cTAL and OMCD were not changed in furosemide or high salt rats compared to time paired controls.
  
  
  CONCLUSIONS
  Given that CLC-K2 expressed in the thick ascending limb of Henle's loop is responsible for net chloride reabsorption in this part of the nephron, our findings suggest that in states of surplus salt and in states of severe salt deprivation, selective regulation of CLC-K2 mRNA plays a role in the adaptation of the kidney to different salt loads.
authors.affiliation: Institut für Physiologie, Universität Regensburg, and Klinik und Poliklinik für Innere Medizin II, Klinikum der Universität Regensburg, Regensburg, Germany.
authors.forename: Helga
authors.initials: H
authors.lastname: Vitzthum
authors.forename: Hayo
authors.initials: H
authors.lastname: Castrop
authors.forename: Martina
authors.initials: M
authors.lastname: Meier-Meitinger
authors.forename: Günter A J
authors.initials: GA
authors.lastname: Riegger
authors.forename: Armin
authors.initials: A
authors.lastname: Kurtz
authors.forename: Bernhard K
authors.initials: BK
authors.lastname: Krämer
authors.forename: Konrad
authors.initials: K
authors.lastname: Wolf
mesh_terms: D000818:Animals; D027321:Anion Transport Proteins; D001477:Bartter Syndrome / Q000503:physiopathology; D018118:Chloride Channels / Q000235:genetics* / Q000378:metabolism*; D004210:Dissection; D004232:Diuretics / Q000494:pharmacology; D005665:Furosemide / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D017403:In Situ Hybridization; D008297:Male; D008565:Membrane Proteins; D009399:Nephrons / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D012260:Ribonucleases; D017673:Sodium Chloride, Dietary / Q000493:pharmacokinetics; D015854:Up-Regulation / Q000502:physiology
chemical_list: D027321:Anion Transport Proteins; D018118:Chloride Channels; C486572:Clcnkb protein, rat; D004232:Diuretics; D008565:Membrane Proteins; D012333:RNA, Messenger; D017673:Sodium Chloride, Dietary; D005665:Furosemide; D012260:Ribonucleases