Inducible Slc4a11 Knockout Triggers Corneal Edema Through Perturbation of Corneal Endothelial Pump

Abstract

The conventional Slc4a11 knockout (KO) shows significant corneal edema at eye opening, a fact that complicates the study of the initial events leading to edema. An inducible KO would provide opportunities to examine early events following loss of Slc4a11 activity. METHODS.Slc4a11 Flox (SF) mice were crossed with mice expressing the estrogen receptor Cre Recombinase fusion protein and fed tamoxifen (Tm) for two weeks. Corneal thickness (CT) was measured by OCT. At eight weeks endpoint, oxidative damage, tight junction integrity, stromal lactate concentration, endothelial permeability, differentially expressed transporters, and junction proteins were determined. Separately, a keratocyte only inducible Slc4a11 KO was also examined. RESULTS.At four weeks post-Tm induction Slc4a11 transcript levels were 2% of control. Corneal thickness increased gradually and was 50% greater than Wild Type (WT) after eight weeks with significantly altered endothelial morphology, increased nitrotyrosine staining, significantly higher stromal lactate, decreased expression of lactate transporters and Na-K ATPase activity, higher ATP, altered expression of tight and adherens junctions, and increased fluorescein permeability. No significant differences in CT were found between WT and keratocyte only Slc4a11 KO. CONCLUSIONS.The Slc4a11 inducible KO shows development of a similar phenotype as the conventional KO, thereby validating the model and providing a tool for further use in examining the sequence of cellular events by use of noninvasive in vivo physiological probes.

Rescue of the Congenital Hereditary Endothelial Dystrophy Mouse Model by Adeno-Associated Virus–Mediated Slc4a11 Replacement

“…Endothelial pump function relies on both lactate transport and an intact osmotic barrier 20,21 . Changes in cell shape in Slc4a11 KO corneal endothelium is previously reported 9 . In Slc4a11 KO corneal endothelial cells, aberrations in actin cytoskeleton assembly, tight junction organization, cell shape changes, and increased paracellular permeability were observed 9 .…”

“…The expression pattern of ZO-1 is incomplete, which accounts for a weak endothelial barrier that allows the passage of nutrients and other molecules into the stroma 2 . Increased cell size is observed in Slc4a11 KO corneal endothelium 5,9 . In order to assess cell size, corneal cups were stained with ZO-1.…”

“…For negative controls, 11 weeks old Slc4a11 -/animals were injected in one eye with 0.5 -0.8 l AAV9-CAG-Null virus (0.9*10 12 GC/L). AAV9 serotype J o u r n a l P r e -p r o o f transduction efficiency into the corneal endothelium was determined by the injection of 0.5 l AAV9-GFP (3.5×10 9 Viral Genome) into the anterior chamber of 11-week-old Slc4a11 -/animals. A total of 124 animals (62 female, and 62 male) were used in this study.…”

“…F-actin staining was conducted using Alexa Fluor 488 Phallodin (#A12379, Thermo Fisher Scientific) following manufacturer's instructions, and examined by Zeiss LSM 800 confocal microscope (Zeiss, Oberkochen, Germany). Fluorescence intensity for phalloidin staining and cell area measurements were performed as described previously 9 . Two female and one male animal per group per staining condition were used.…”

“…Mutations in Slc4a11, which codes for a plasma membrane H + transporter and an ammonia sensitive mitochondrial uncoupler 6 , lead to CHED 4 . Slc4a11 -/-(Knockout, KO) mice recapitulate the progressive corneal edema, endothelial cell loss, and corneal opacity, seen in the disease phenotype 4,5,8 along with a loss of lactate transporters and accumulation of stromal lactate 9,10 . The only treatment available for CHED is corneal transplantation 8,11 .…”