Abstract
Purpose :
Congenital Hereditary Endothelial Dystrophy (CHED) is a clinical condition marked by early onset corneal edema and opacity. CHED is caused by autosomal recessive inheritance of mutations in the proton transporter SLC4A11 and congenital corneal edema is modeled in Slc4a11-null mice. A similar phenotype is exhibited by mice lacking NBCe1-B, the corneal sodium bicarbonate transporter. We examined the ocular transcriptomes of both mouse models to identify common disturbances.
Methods :
We extracted RNA from freshly enucleated eyes and dissected corneas of wild-type, Slc4a11-null, and NBCe1-B-null mice using the RNeasy Plus Universal Mini Kit (QIAGEN). Whole-eye cDNAs, synthesized using the cDNA iScript kit, were sent for next generation sequencing (Azenta US Inc), followed by analysis of differentially expressed genes (DEGs) with DESeq2. We confirmed select results by real-time qPCR of whole-eye and corneal cDNAs from wild-type and NBCe1-B-KO mice using BioRad Prime PCR assays, with relative quantification assessed by the Livak equation using GAPDH as reference.
Results :
Among the hundreds of transcription disturbances unique to each mouse model, we identified 13 common transcript disturbances (n=3 of each mouse). Among them is the common downregulation of FXYD4 (Slc4a11-null, 4-fold, Padj = 5.56E-08; NBCe1-B-null, 7-fold, Padj = 1.63E-06), one of seven alternate regulatory subunits of the Na/K-pump. qPCR confirms that FXYD4 is 3-fold reduced in the corneas of NBCe1-B KO mice relative to wild-type (1-sample t-test P<0.01, n=4). qPCR of all seven FXYD isoforms in wild-type mice reveals that FXYD1 and FXYD6 are the most abundant in whole-eye cDNA, while FXYD3 and FXYD4 are >10-fold enriched in corneal versus whole-eye cDNAs (n=4).
Conclusions :
FXYD4 is enriched in the cornea. Studies by others in heterologous systems report that FXYD4 increases the Na+ affinity of the Na/K-pump (Beguin et al, EMBO J, 2001). Thus, downregulation of FXYD4 in the cornea could lower Na/K-pump activity. This represents a previously uncharted connection in the intricate pathway that ties mutations in acid-base transporters with the failure of Na/K-pump function that is a common feature of corneal endothelial dystrophies.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.