Abstract
Purpose :
The conventional Slc4a11 knock out (KO) shows significant corneal edema at eye opening; a fact that complicates the study of the initial events leading to edema. We have reported that Slc4a11 is a NH3-activated mitochondrial uncoupler that facilitates glutamine catabolism and suppresses mitochondrial superoxide production. Here we tested the hypothesis that inducible Slc4a11 KO in the adult mouse leads to progressive corneal edema characterized by oxidative stress and alterations of pump and barrier functions of the corneal endothelium (CE).
Methods :
Slc4a11 Flox (SF) mice were crossed with mice expressing the estrogen receptor–Cre Recombinase fusion protein and at 8 weeks of age were fed Tamoxifen (Tx) enriched chow (0.4 g/Kg) for 2 weeks, followed by normal chow; and controls were fed normal chow. Corneal thickness (CT) was measured by OCT. Oxidative damage was detected by nitrotyrosine staining, tight junctions by ZO-1 and adherens junctions by F-actin staining. Pump function was tested by stromal lactate concentration and barrier function by paracellular permeability to fluorescein. To generate inducible Slc4a11 KO mice strictly in keratocytes, SF mice were crossed with Kera-rtTA;tetO-Cre driver mice (IOVS 2017, 58: 4800–4808) and were fed DOX since embryonic day 0 until test at 3 months of age.
Results :
Tx induced a 98% decrease in Slc4a11 expression in corneal tissue. Tx produced gradual corneal edema with an increase in CT of 34% at 2 weeks and 49% at 8 weeks of treatment. Cell density was not changed but morphology was significantly altered. Oxidative stress was evident as nitrotyrosine abundance increased. Tx induced a decrease in the CE pump function evidenced by an increased stromal lactate accumulation (Ctrl: 4.24±1.19 nmol/mg tissue vs Tx: 9.71±1.36, p=0.02, n=5), and down regulation of MCT2 and Atp1b2. However, Tx increased CE ATP levels by 12%. Tx increased paracellular permeability (decreased barrier function) by 16% and altered tight and adherens junctions. No significant differences in CT were found between WT mice and Slc4a11 KO in keratocytes only.
Conclusions :
These data confirm that oxidative stress and perturbation of pump and barrier functions of the CE are early events following diminution of Slc4a11 expression. Slc4a11 expression is also high in keratocytes, however specific KO did not lead to corneal edema.
This is a 2021 ARVO Annual Meeting abstract.