Evaluation of Advanced Glycation End–Products in Diabetic and Inherited Canine Cataracts

Abstract

Abstract: : Purpose: The purpose of the study was to immunolocalize cell–associated receptors of advanced glycation end–products (RAGE), and to determine whether there is a correlation between RAGE, DNA damage (gadd45), and cell proliferation (PCNA, p21) in canine diabetic and inherited cataracts. Methods: Samples included 32 anterior capsulotomies from 22 dogs. Eleven were diabetic (17 eyes) and eleven had inherited cataracts (16 eyes). Standard ABC immunohistochemical staining was performed using antibodies against RAGE, gadd45, p21, and PCNA. Appropriate controls were included. Western blot analysis was performed on 6 anterior lens capsules (3 diabetic and 3 inherited) to validate the antibodies. Results: All capsules demonstrated increased expression of RAGE and gadd45. Stain intensity varied among different populations of lens epithelial cells (LEC). Gadd45 immunostaining was mainly perinuclear and intranuclear. No differences were observed between diabetic and inherited specimens, stage or duration of cataract, or presence or absence of lens–induced uveitis at the time of sample collection. PCNA expression was increased in diabetic capsules and all mature and hypermature inherited samples in the nucleus and perinuclear area of the proliferating LECs that surrounded fibrotic plaques. PCNA expression was decreased in 42% of the inherited cataracts, but only in specimens from immature cataracts. p21 expression was increased in diabetic cataracts and decreased in inherited cataracts. Western blot analysis was consistent with these findings. Conclusions: Increased RAGE expression indicates the presence of AGE products in canine inherited and diabetic cataracts. There was no correlation between expression of RAGE in inherited and diabetic cataracts in either the Western blot or immunohistochemistry. LEC exposed to AGEs up–regulate RAGE and transdifferentiate into the pseudofibroblastic phenotype seen in cataract and after–cataract. Staining characteristics for RAGE and gadd45 suggest that increased expression of RAGE in LECs exposed to AGEs may contribute to pseudofibroblastic changes and altered cellular growth, establishing a correlation between oxidative stress, RAGE and DNA damage. Proliferation of LEC in diabetic cataracts and advanced stages of inherited cataracts are supported by increased PCNA expression. Significant elevation of p21 was observed in diabetic cataracts, this may occur in an attempt to exert its inhibitory action against abnormally proliferating LEC.

Keywords: diabetes • immunohistochemistry