Knockdown of NBCe1 in vivo compromises the corneal endothelial pump.
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.
Liu
Cailing
C
School of Optometry, Indiana University, Bloomington, Indiana 47405, USA.
Cheng
Qiang
Q
Nguyen
Tracy
T
Bonanno
Joseph A
JA
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
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
Gene Ther. 2010 Apr;17(4):486-93
20010626
Exp Eye Res. 1984 Sep;39(3):335-42
6499954
J Physiol. 1974 Sep;241(3):647-75
4215880
Arch Ophthalmol. 2003 Feb;121(2):252-8
12583793
Arch Soc Esp Oftalmol. 2002 Mar;77(3):139-44
11967735
Hum Mol Genet. 2008 Jul 15;17(14):2084-100
18385099
Am J Ophthalmol. 1999 Apr;127(4):403-6
10218692
Pflugers Arch. 1994 Oct;428(5-6):577-82
7838680
Hum Gene Ther. 2007 Oct;18(10):871-80
17892416
J Virol. 2004 Oct;78(20):11327-33
15452253
J Gene Med. 2001 Sep-Oct;3(5):437-49
11601757
Invest Ophthalmol Vis Sci. 2004 Sep;45(9):3091-8
15326125
Am J Hum Genet. 2007 Jul;81(1):127-35
17564969
Invest Ophthalmol Vis Sci. 1994 Feb;35(2):560-7
8113007
Vision Res. 2002 Feb;42(4):551-8
11853772
Vision Res. 2007 Apr;47(9):1202-8
17292939
Acta Ophthalmol (Copenh). 1991 Apr;69(2):149-54
1651641
J Physiol. 1972 Feb;221(1):43-54
5016991
Am J Physiol. 1992 Jan;262(1 Pt 1):C98-103
1733238
Invest Ophthalmol Vis Sci. 1995 Jan;36(1):103-12
7822137
Invest Ophthalmol Vis Sci. 2010 Jan;51(1):236-48
19684004
Acta Ophthalmol Scand. 1998 Feb;76(1):78-9
9541439
Invest Ophthalmol Vis Sci. 1992 Oct;33(11):3068-79
1399410
Invest Ophthalmol Vis Sci. 1998 Jan;39(1):23-9
9430541
Am J Ophthalmol. 2000 Feb;129(2):144-50
10682965
J Physiol. 1976 Dec;263(3):563-77
828203
Arch Ophthalmol. 2003 May;121(5):621-5
12742838
J Biol Chem. 2007 Mar 23;282(12):9042-52
17192275
J Physiol. 1978 Feb;275:377-89
633130
Exp Eye Res. 2003 Sep;77(3):287-95
12907161
Invest Ophthalmol Vis Sci. 1992 Oct;33(11):3058-67
1328110
Exp Eye Res. 1966 Jul;5(3):221-8
5914654
Prog Retin Eye Res. 2003 Jan;22(1):69-94
12597924
Hum Gene Ther. 2005 Jun;16(6):741-51
15960605
Invest Ophthalmol Vis Sci. 2007 Oct;48(10):4407-20
17898259
Jpn J Ophthalmol. 2005 Jul-Aug;49(4):332-3
16075340
J Enzyme Inhib Med Chem. 2004 Jun;19(3):231-6
15499994
J Gene Med. 2004 Apr;6(4):395-404
15079814
Chin Med J (Engl). 2002 Nov;115(11):1641-4
12609078
Am J Physiol Cell Physiol. 2005 Mar;288(3):C739-46
15548570
Curr Eye Res. 1982-1983;2(3):183-6
7151464
Gene Ther. 2001 Nov;8(21):1665-8
11895005
Am J Physiol Cell Physiol. 2000 Nov;279(5):C1648-55
11029313
Hum Gene Ther. 2009 Mar;20(3):191-200
19301472
Exp Eye Res. 1990 May;50(5):487-93
2373152
Arch Ophthalmol. 2004 Jul;122(7):1089; author reply 1090
15249392
Exp Eye Res. 1995 Apr;60(4):425-34
7789422
Mol Vis. 2005 Jun 21;11:425-30
15988411
Invest Ophthalmol Vis Sci. 2008 Mar;49(3):1048-55
18326729