title: Knockdown of NBCe1 in vivo compromises the corneal endothelial pump.
abstract: OBJECTIVE
  To evaluate the role of the sodium bicarbonate cotransporter (NBCe1) as a component of the corneal endothelial pump in the in vivo rabbit eye.
  
  
  METHODS
  Lentiviruses with NBCe1 shRNA and GFP expression cassettes were injected intracamerally. Knockdown efficacy was determined 1 week to 4 weeks later by immunofluorescence, Western blot analysis, and PCR. Functional effects were monitored by corneal thickness (CT) and brinzolamide sensitivity.
  
  
  RESULTS
  Within 24 hours there was a modest anterior chamber inflammation that resolved within 48 hours. At 4 × 10(6) IFU, more than 95% of the corneal endothelial surface showed GFP fluorescence above background within 7 days. At 14 to 21 days, signs of anterior chamber inflammation reemerged, and endothelial cell GFP fluorescence disappeared within 40 days after injection. The second phase of inflammation could be avoided by using GFP-less viruses. There was no significant difference in CT between scrambled sequence and NBCe1 shRNA-injected eyes over 3 weeks. Two drops of 1% brinzolamide produced 7.85% ± 3.3% corneal swelling within 5 hours of topical instillation. However, in corneas showing more than 25% NBCe1 knockdown (30 of 42 rabbits; 59% ± 15% knockdown), corneal swelling was significantly higher (10.1% ± 2.9%) relative to control eyes.
  
  
  CONCLUSIONS
  FIV-based lentiviral vectors can transfect CE with shRNA in rabbits. The response to GFP is consistent, with previous studies showing the production of anti-GFP antibodies. Partial knockdown of NBCe1 did not affect baseline CT, which is consistent with the corneal endothelium having a substantial functional reserve. Provocative testing using, brinzolamide, however, revealed an underlying deficiency, confirming the importance of NBCe1 bicarbonate transport and demonstrating the concerted action between NBCe1 and carbonic anhydrases.
authors.affiliation: School of Optometry, Indiana University, Bloomington, Indiana 47405, USA.
authors.forename: Cailing
authors.initials: C
authors.lastname: Liu
authors.forename: Qiang
authors.initials: Q
authors.lastname: Cheng
authors.forename: Tracy
authors.initials: T
authors.lastname: Nguyen
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Bonanno
mesh_terms: D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D001693:Biological Transport, Active / Q000187:drug effects; D015153:Blotting, Western; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000502:physiology; D004728:Endothelium, Corneal / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D020868:Gene Silencing / Q000502:physiology*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016089:Immunodeficiency Virus, Feline / Q000235:genetics; D007429:Intraocular Pressure; D026901:Membrane Transport Proteins / Q000502:physiology*; D008856:Microscopy, Fluorescence; D034622:RNA Interference; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D027982:Sodium-Bicarbonate Symporters / Q000502:physiology*; D013449:Sulfonamides / Q000494:pharmacology; D013843:Thiazines / Q000494:pharmacology; D014162:Transfection
chemical_list: D001639:Bicarbonates; D002257:Carbonic Anhydrase Inhibitors; D026901:Membrane Transport Proteins; D027982:Sodium-Bicarbonate Symporters; D013449:Sulfonamides; D013843:Thiazines; D049452:Green Fluorescent Proteins; C111827:brinzolamide; D002256:Carbonic Anhydrases
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