Abstract
Purpose :
We recently described SLC4A11 as a novel NH3:2H+ transporter, suggesting it may play a crucial role in removing toxic ammonia generated by ATP producing mitochondrial glutaminolysis in corneal endothelium. Thus, we examined the Slc4a11 knock-out mouse cornea in comparison to wild-type for signs of ammonia toxicity: 1) morphological changes; 2) protein nitrosylation, a widely accepted marker for ammonia intoxication; 3) changes in enzyme expression of glutamine metabolic pathways.
Methods :
Endothelial expression of mitochondrial glutaminase (GLS1 and GLS2) and membrane glutaminase gamma-glutamyl-transpeptidase (GGT) was compared by QPCR in 12 week old mice. Age matched (40 weeks) mouse cornea paraffin sections were prepared for H&E or immunofluorescence staining of protein nitrosylation, GLS1, GLS2 and GGT. Fluorescence images were acquired with AxioImager M1 microscope (Zeiss) and AxioCam MRm camera (Zeiss). Image post processing and mean fluorencence intensity (MFI, mean±SEM) quantification of corneal epithelium, keratocytes and corneal endothelium were conducted using ImageJ.
Results :
On H&E staining, we observed vacuolated corneal endothelium with thickened Descemet’s membrane, significantly thickened hypercellluar corneal stroma with disorganized collagen fibers and desquamated corneal epithelium with focal hyperplasia in Slc4a11 knock-out mouse. Immunostaining revealed SLC4A11 is highly expressed in cornea endothelium and basolateral surface of corneal epithelium basal cells in Slc4a11 wild-type, but no expression in knock-out mouse. We observed increased protein nitrosylation of corneal endothelium (MFI 5274.2±491.45 a.u. vs 3874.48±390.20 a.u., p=0.044) and stromal keratocytes (MFI 4619.08±522.90 a.u. vs 3437.14±470.81 a.u., p=0.031) in Slc4a11 knock-out compared to wild-type, implicating ammonia toxicity resulted from loss of NH3:H+ transporter SLC4A11. QPCR revealed endogenous wild-type expression of kidney-type glutaminase (GLS1) is 10 fold higher than liver-type glutaminase (GLS2). In Slc4a11 knock-out, GLS1 is upregulated by 3 folds whereas GLS2 is diminished. The changes of GLS1 and GLS2 were further confirmed at the protein level with immunofluorescence. Membrane GGT has an endogenously low expression, and was diminished in Slc4a11 knock-out by QPCR.
Conclusions :
The observed phenotype in Slc4a11 knock-out mouse cornea is consistent with ammonia intoxication.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.