Mutation of a caveolin binding motif in Na/K-ATPase activates the Nrf2 signaling pathway and attenuates experimental ischemic retinopathy

Abstract

Purpose : Increases in oxidative stress is well documented in ischemic retinopathy. The α1 Na/K-ATPase (NKA) has an enzyme independent receptor function through its interaction with signaling proteins such as Src and caveolin-1. Moreover, the α1 NKA/Src receptor complex serves as a reactive oxygen species (ROS) amplifier. In this study, we investigated how the disruption of α1 NKA signaling secondary to a loss of caveolin binding motif (CBM) affects ischemic retinopathy in the mouse model.

Methods : Mice expressing the mutated CBM (mCBM) α1 NKA were obtained by knocking-in of F97A and F100A substitution in α1 NKA. The homozygous mCBM genotype was embryonic lethal, but heterozygous (Het) mice were viable. Oxygen induced retinopathy (OIR) was triggered in mCBM Het neonatal mice and wild-type (Wt) littermates by exposure to 75% oxygen from postnatal day (P) 7 to 12. Eyes were collected and fixed at P12 and P17. Retinas were isolated, stained with lectin, flat-mounted and imaged using fluorescence microscopy. Retinal vascular obliteration and neovascularization (NV) were quantified using SWIFT_NV macro and Image J. Marker genes for angiogenesis, inflammation and the Nrf2 pathway were determined by quantitative RT-PCR. Results were expressed as mean ± SEM. Data were analyzed using unpaired t-test or one-way ANOVA followed by Tukey’s correction for multiple comparisons.

Results : At P12, there was no significant difference of avascular area (AV) between mCBM Het and Wt litter mates. However, at P17, mCBM Het mice had significantly reduced AV and NV compared to the Wt. Consistently, the expression of gene markers for angiogenesis (Vegf, Angpt2 and Epo), inflammation (Tnfa and Cxcl10) was significantly lower in mCBM Het mice. In room air environment, the basal levels of Nrf2-targeted gene markers were increased in mCBM Het mice at P17 compared to Wt. Under OIR conditions, this increase was augmented significantly in mCBM Het mice.

Conclusions : α1 NKA signaling is involved in the mouse model of OIR. The loss of CBM in α1 NKA protects mice from OIR and is associated with increased levels of Nrf2-targeted genes.

This is a 2021 ARVO Annual Meeting abstract.

 

Fig. 1. Retinal AV and NV at P12and P17. n=8 (from 4-6 litters) per group.

 

Fig. 2. Decreased expression of angiogenetic and inflammatory gene markers and increased Nrf2-targeted marker genes in mCBM retinas. n=4-9 (from 4-6 litters) per group.