title: An in vivo method for adenovirus-mediated transduction of thick ascending limbs.
abstract: BACKGROUND
  The thick ascending limb of the loop of Henle (THAL) plays an important role in the maintenance of salt, water, and acid-base balance. While techniques for gene transfer of renal vascular cells and some tubular segments have been described, in vivo transduction of THALs has not been successful. We hypothesized that in vivo injection of adenoviral vectors into the renal medulla would result in efficient transduction of THALs.
  
  
  METHODS
  We injected recombinant adenoviruses containing the reporter gene, green fluorescent protein (GFP), driven by either the cytomegalovirus promoter (Ad-CMVGFP) or the promoter for the Na/K/2 Cl cotransporter (Ad-NKCC2GFP), which is THAL-specific, into the outer medullary interstitium of Sprague-Dawley rat kidneys. Kidneys were removed at various times after viral injection and analyzed for GFP expression.
  
  
  RESULTS
  Western blots revealed strong GFP expression in the outer medulla (which is composed primarily of THALs) 5 days after Ad-CMVGFP injection. We quantified THAL transduction efficiency by scoring the number of fluorescent tubules in THALs suspensions, which showed that at least 77 +/- 3% of THAL expressed GFP. To specifically transduce THALs, we injected Ad-NKCC2GFP into the medullary interstitium. As determined by Western blot, GFP expression was only detected in the outer medulla. Immunohistochemistry and confocal microscopy showed that GFP was localized to tubular cells positive for Tamm-Horsfall protein. Thus, GFP fluorescence was only detected in THALs, not in cortical, inner medulla or vascular cells. Time-course studies showed that GFP expression in THALs was measurable from 4 to 14 days, peaked at 7 days, and had returned to background levels by 21 days.
  
  
  CONCLUSIONS
  This method facilitates highly efficient, THAL-specific transduction. While application of this technique for gene therapy in humans is unlikely due to the transient gene expression observed and the impossibility for repeated injections of adenoviral vectors, this method provides a valuable tool for investigators studying regulation and mechanisms of THAL ion transport and its relationship to whole-kidney physiology and pathophysiology.
authors.affiliation: Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan 48202, USA.
authors.forename: Pablo A
authors.initials: PA
authors.lastname: Ortiz
authors.forename: Nancy J
authors.initials: NJ
authors.lastname: Hong
authors.forename: Craig F
authors.initials: CF
authors.lastname: Plato
authors.forename: Marisela
authors.initials: M
authors.lastname: Varela
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Garvin
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D003587:Cytomegalovirus / Q000235:genetics; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008138:Loop of Henle / Q000502:physiology*; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics; D064466:Solute Carrier Family 12, Member 1; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D007202:Indicators and Reagents; D008164:Luminescent Proteins; C578714:SLC12A1 protein, human; C578716:Slc12a1 protein, rat; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D049452:Green Fluorescent Proteins