Increased Retinal Expression of Carbonic Anhydrase, Anion and Proton Exchanger in Response to Systemic Acidosis

Abstract

Purpose: Changes in retinal pH may occur in response to altered metabolism and may contribute to disease processes in a variety of eye diseases including diabetic retinopathy. To study the effects of acidosis alone, we produced systemic metabolic acidosis and hypothesized that the retina may respond to changes in acidosis with altered expression of genes involved in acid/base regulation.

Methods: Adult Long-Evans rats were treated with NH₄Cl in the drinking water to promote systemic metabolic acidosis. Tail blood samples were taken every other day and at the end of two weeks, venous pH was 7.20 ± 0.08 (SD) in acidotic animals and 7.42 ± 0.02 in control animals not subjected to acidosis. [HCO₃^-] was 18.7 ± 4.8 mM in acidotic animals and 30.7 ± 1.0 mM in controls confirming the presence of metabolic acidosis. Rats were euthanized and retinas were harvested rapidly for RNA or protein expression or alternatively were prepared for sectioning and immunofluorescence (IF) analysis. Specific mRNAs were quantified by total RNA isolation followed by RT qPCR using Taqman specific primer sets and ddCt analysis. Protein content was measured by Western blot, and confocal microscopy identified retinal location. Two-tailed Student’s t-tests were used for statistical analysis.

Results: mRNA for carbonic Anhydrase (CA)-II increased 75% (p<0.005) and CA-XIV increased 40% (p<0.005), 14 days after induction of acidosis. Acid sensing ion channel (ASIC)-1 increased 50% (p<0.005) and ASIC-4 increased 2-fold (p<0.005). Anion exchanger protein (AEP)-3 showed a 65% (p<0.0001) increase in expression and Na+/H+ exchanger (NHE)-1 increased 50% (p<0.0001). Western blotting revealed a similar trend towards an increase in protein content for CA-II, CA-XIV, and NHE-1 with an increase in AEP3 (p<0.001). Confocal microscopy demonstrates that CAII and NHE-1 co-localize with vascular markers and CA-XIV is located in the neural parenchyma.

Conclusions: These studies show that the retina responds to systemic acidosis with increased expression of proton and bicarbonate exchangers and carbonic anhydrase. While responses to acidosis are usually associated with renal regulation, these studies suggest that the neural retina can locally adapt to help control its acid/base environment.