Solute-linked cotransporter, SLC4A11, a member of the bicarbonate transporter family, is an electrogenic H⁺ transporter activated by NH₃ and alkaline pH. Although SLC4A11 does not transport bicarbonate, it shares many properties with other members of the SLC4 family. SLC4A11 mutations can lead to corneal endothelial dystrophy and hearing deficits that are recapitulated in SLC4A11 knock-out mice. SLC4A11, at the inner mitochondrial membrane, facilitates glutamine catabolism and suppresses the production of mitochondrial superoxide by providing ammonia-sensitive H⁺ uncoupling that reduces glutamine-driven mitochondrial membrane potential hyperpolarization. Mitochondrial oxidative stress in SLC4A11 KO also triggers dysfunctional autophagy and lysosomes, as well as ER stress. SLC4A11 expression is induced by oxidative stress through the transcription factor NRF2, the master regulator of antioxidant genes. Outside of the corneal endothelium, SLC4A11’s function has been demonstrated in cochlear fibrocytes, salivary glands, and kidneys, but is largely unexplored overall. Increased SLC4A11 expression is a component of some “glutamine-addicted” cancers, and is possibly linked to cells and tissues that rely on glutamine catabolism.

Purpose: Inducible Slc4a11 KO leads to corneal edema by disruption of the pump and barrier functions of the corneal endothelium (CE). The loss of Slc4a11 NH₃-activated mitochondrial uncoupling leads to mitochondrial membrane potential hyperpolarization-induced oxidative stress. The goal of this study was to investigate the link between oxidative stress and the failure of pump and barrier functions and to test different approaches to revert the process. Methods: Mice which were homozygous for Slc4a11 Flox and Estrogen receptor –Cre Recombinase fusion protein alleles at 8 weeks of age were fed Tamoxifen (Tm)-enriched chow (0.4 g/Kg) for 2 weeks, and controls were fed normal chow. During the initial 14 days, Slc4a11 expression, corneal thickness (CT), stromal [lactate], Na⁺-K⁺ ATPase activity, mitochondrial superoxide levels, expression of lactate transporters, and activity of key kinases were assessed. In addition, barrier function was assessed by fluorescein permeability, ZO-1 tight junction integrity, and cortical cytoskeleton F-actin morphology. Results: Tm induced a rapid decay in Slc4a11 expression that was 84% complete at 7 days and 96% complete at 14 days of treatment. Superoxide levels increased significantly by day 7; CT and fluorescein permeability by day 14. Tight junction ZO-1 distribution and the cortical cytoskeleton were disrupted at day 14, concomitant with decreased expression of Cldn1, yet with increased tyrosine phosphorylation. Stromal lactate increased by 60%, Na⁺-K⁺ ATPase activity decreased by 40%, and expression of lactate transporters MCT2 and MCT4 significantly decreased, but MCT1 was unchanged at 14 days. Src kinase was activated, but not Rock, PKCα, JNK, or P38Mapk. Mitochondrial antioxidant Visomitin (SkQ1, mitochondrial targeted antioxidant) and Src kinase inhibitor eCF506 significantly slowed the increase in CT, with concomitant decreased stromal lactate retention, improved barrier function, reduced Src activation and Cldn1 phosphorylation, and rescued MCT2 and MCT4 expression. Conclusions: Slc4a11 KO-induced CE oxidative stress triggered increased Src kinase activity that resulted in perturbation of the pump components and barrier function of the CE.

Purpose: Inducible Slc4a11 KO leads to corneal edema by disruption of the pump and barrier functions of the corneal endothelium (CE). The loss of Slc4a11 NH3-activated mitochondrial uncoupling leads to mitochondrial membrane potential hyperpolarization-induced oxidative stress. The goal of this study is to investigate the link between oxidative stress and failure of pump and barrier functions and test different approaches to revert the process. Methods: Mice homozygous for Slc4a11 Flox and Estrogen receptor –Cre Recombinase fusion protein alleles at 8 weeks of age were fed Tamoxifen (Tm) enriched chow (0.4 g/Kg) for 2 weeks, and controls were fed normal chow. During the initial 14 days, Slc4a11 expression, corneal thickness (CT), stromal [lactate], Na+-K+ ATPase activity, mitochondrial superoxide levels, expression of lactate transporters, and activity of key kinases were assessed. In addition, barrier function was assessed by fluorescein permeability, ZO-1 tight junction integrity, and cortical cytoskeleton F-actin morphology. Results: Tm induced a rapid decay in Slc4a11 expression that was 84% complete at 7 days and 96% at 14 days of treatment. Superoxide levels increased significantly by day 7; CT and fluorescein permeability by day 14. Tight junction ZO-1 distribution and cortical cytoskeleton were disrupted at day 14 concomitant with decreased expression of Cldn1 yet an increase in tyrosine phosphorylation. Stromal lactate increased by 60%, Na+-K+ATPase activity decreased by 40%, and expression of lactate transporters MCT2 and MCT4 significantly decreased, but MCT1 was unchanged at 14 days. Src kinase was activated but not Rock, PKCα, JNK, or P38Mapk. Mitochondrial antioxidant Visomitin (SkQ1, mitochondrial targeted antioxidant) or Src kinase inhibitor eCF506 significantly slowed the increase in CT, with concomitant decreased stromal lactate retention, improved barrier function, reduced Src activation and Cldn1 phosphorylation, and rescued MCT2 and MCT4 expression. Conclusions: Slc4a11 KO-induced CE oxidative stress triggered increased Src kinase activity that results in perturbation of pump components and barrier function of the CE.

Recent studies from Slc4a11 KO mice have identified mitochondrial dysfunction as a major contributor toward oxidative stress and cell death in Congenital Hereditary Endothelial Dystrophy. Here we asked if this stress activated autophagy in the Slc4a11 KO cell line and in KO mouse endothelial tissue. Early indicators of autophagy, phospho-mTOR and LC3-II indicated activation, however P62 was elevated suggesting an impairment of autophagy flux. The activity and the number of lysosomes, the organelle responsible for the final degradation of autophagy substrates, were found to be reduced in the KO. In addition, the expression of the master regulator of lysosomal function and biogenesis, TFEB, was significantly reduced in the KO corneal endothelia. Also, we observed increased Unfolded Protein Response, as well as elevated expression of ER stress markers, BIP and CHOP. To test if lysosomal and ER stress stems from elevated mitochondrial ROS, we treated Slc4a11 KO corneal endothelial cells with the mitochondrial ROS quencher, MitoQ. MitoQ restored lysosomal enzymes as well as TFEB, reduced ER stress, and increased autophagy flux. MitoQ injections of Slc4a11 KO mice decreased corneal edema, the major phenotype associated with CHED. We conclude that mitochondrial ROS causes ER stress and lysosomal dysfunction with impairment of autophagy in Slc4a11 KO corneal endothelium. Our study is the first to identify the presence as well as cause of lysosomal dysfunction and ER stress in an animal model of CHED, and to characterize inter-organelle relationship in a corneal cell type.