Knockdown of NBCe1 in vivo compromises the corneal endothelial pump

Invest Ophthalmol Vis Sci. 2010 Oct;51(10):5190-7. doi: 10.1167/iovs.10-5257. Epub 2010 May 5.


Purpose: 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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Bicarbonates / metabolism
  • Biological Transport, Active / drug effects
  • Blotting, Western
  • Carbonic Anhydrase Inhibitors / pharmacology
  • Carbonic Anhydrases / physiology
  • Endothelium, Corneal / metabolism*
  • Gene Knockdown Techniques
  • Gene Silencing / physiology*
  • Genetic Vectors
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Immunodeficiency Virus, Feline / genetics
  • Intraocular Pressure
  • Membrane Transport Proteins / physiology*
  • Microscopy, Fluorescence
  • RNA Interference
  • Rabbits
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sodium-Bicarbonate Symporters / physiology*
  • Sulfonamides / pharmacology
  • Thiazines / pharmacology
  • Transfection


  • Bicarbonates
  • Carbonic Anhydrase Inhibitors
  • Membrane Transport Proteins
  • Sodium-Bicarbonate Symporters
  • Sulfonamides
  • Thiazines
  • Green Fluorescent Proteins
  • brinzolamide
  • Carbonic Anhydrases