Abstract
Purpose :
The Lens Epithelia Glutathione (GSH) Synthesis KnockOut (LEGSKO) mouse lacks the catalytic subunit of gamma-glutamylcysteine ligase (GCLC) in its lens, resulting in a lack of GSH synthesis in this tissue. This models the natural GSH deficiency which occurs in the lens with age and is highly associated with age-related cataract. Our prior RNA-Seq study indicated that the urea transporter UT-B is one of the most upregulated transcripts in the LEGSKO lens compared to wildtype (approximately 6-fold greater in epithelia and 10-fold greater in cortical fibers).
Methods :
To further our understanding of the role of urea metabolism and transport in the lens, the concentration of urea in the lens and its rate of efflux was assessed using a colorimetric assay (Sigma). Western blotting was utilized to determine the presence and abundance of urea transporters and urea cycle enzymes, including UT-A and UT-B isoforms, OTC, ASS1, ARG1, ARG2, GluD1, ASL, and CPS1. We are further investigating the potential connection between GSH and urea by measuring homocitrulline in wildtype and LEGSKO lens protein digests via LC-MS/MS.
Results :
LEGSKO mouse lenses had ~50% lower steady state urea levels (P<0.01) and ~2-fold faster rate of urea efflux (P<0.05) in comparison to wildtype lenses. This efflux displayed saturation kinetics and the rate of efflux from the LEGSKO lenses could be decreased by incubating the lens with dimethylthiourea, a competitive inhibitor of urea transporters. The 35 kDa isoform of UT-B was found to have a 1.6-2.8-fold higher concentration in LEGSKO lenses across the tissues tested (P<0.05), while there were no significant differences in other isoforms of UT-B or UT-A. OTC was ~2-fold more abundant in LEGSKO lens capsule, cortex, and nucleus compared to wildtype (P<0.05), while all other urea cycle enzymes were unchanged.
Conclusions :
Urea is a common waste product produced in the lens and its export is an important and regulated process that is affected by GSH deficiency. These are not major changes in the production of urea accounting for its lower concentration in LEGSKO lenses. Although OTC levels were higher in LEGSKO, it is not involved in a rate-limiting step of urea production and would be expected to increase the concentration in LEGSKO lenses if it had an effect. The data indicate that noted differences between wildtype and LEGSKO are due primarily to changes in urea efflux, most likely as a result of increased UT-B activity.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.