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. 2011 May 30;191(1-3):346-50.
doi: 10.1016/j.cbi.2011.02.028. Epub 2011 Mar 3.

Aldose reductase deficiency protects sugar-induced lens opacification in rats

Affiliations

Aldose reductase deficiency protects sugar-induced lens opacification in rats

Aramati B M Reddy et al. Chem Biol Interact. .

Abstract

Aldose reductase (AKR1B1), which catalyzes the reduction of glucose to sorbitol and lipid aldehydes to lipid alcohols, has been shown to be involved in secondary diabetic complications including cataractogenesis. Rats have high levels of AKR1B1 in lenses and readily develop diabetic cataracts, whereas mice have very low levels of AKR1B1 in their lenses and are not susceptible to hyperglycemic cataracts. Studies with transgenic mice that over-express AKR1B1 indicate that it is the key protein for the development of diabetic complications including diabetic cataract. However, no such studies were performed in genetically altered AKR1B1 rats. Hence, we developed siRNA-based AKR1B1 knockdown rats (ARKO) using the AKR1B1-siRNA-pSuper vector construct. Genotyping analysis suggested that more than 90% of AKR1B1 was knocked down in the littermates. Interestingly, all the male animals were born dead and only 3 female rats survived. Furthermore, all 3 female animals were not able to give birth to F1 generation. Hence, we could not establish an AKR1B1 rat knockdown colony. However, we examined the effect of AKR1B1 knockdown on sugar-induced lens opacification in ex vivo. Our results indicate that rat lenses obtained from AKR1B1 knockdown rats were resistant to high glucose-induced lens opacification as compared to wild-type (WT) rat lenses. Biochemical analysis of lens homogenates showed that the AKR1B1 activity and sorbitol levels were significantly lower in sugar-treated AKR1B1 knockdown rat lenses as compared to WT rat lenses treated with 50mM glucose. Our results thus confirmed the significance of AKR1B1 in the mediation of sugar-induced lens opacification and indicate the use of AKR1B1 inhibitors in the prevention of cataractogenesis.

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Figures

Figure 1
Figure 1. pSuper.gfp/neo vector map showing the multiple cloning site, AKR1B1-siRNA insertion site and the AKR1B1-siRNA sequence
Rat specific ARSiRNA was custom designed and synthesized as described in methods. The oligo nucleotide sequence was subcloned between BglII and HindIII restriction sites downstream of the H1 promoter in the pSuper.gfp/neo vector. The figure describes the complete map of the pSuper vector from manufacturer's technical literature along with the AKR1B1-siRNA sequence.
Figure 2
Figure 2. RT-PCR and Western blot confirming the knock-down of AKR1B1 by siRNA
A) Equal amount of total RNA isolated from the thoracic muscle tissue of AKR1B1 knockdown rats was subjected to RT-PCR. B) Equal amount of protein obtained from muscle tissue homogenates was subjected to Western blot analysis using AKR1B1 antibodies. Beta–actin and GAPDH were used as internal standards for RT-PCR and Western blot analysis, respectively. #1, 2, 3 are ARKO rats, Control: Wild-type rats.
Figure 3
Figure 3. Hyperglycemia-induced lens opacification was not observed in ARKO rat lenses
ARKO and age-matched control female rats (10-12 weeks; Fischer-344) were killed and the lenses were immediately dissected out and cultured in medium 199 containing 1% penicillin and streptomycin with or without high glucose (50 mM) as described in methods. On different days the lenses were periodically photographed and observed under the transmission light using a Nikon inverted microscope. The photographs show the lens opacification on days 1, 4 and 9. The opacification is represented by transmittance of light in distinct colors in decreasing order at the nuclear region of the lens. Day 1 corresponds to 24 h after the lens was incubated in the culture media. WT: Wild-type, WT+HG: Wild- type with high glucose treated, ARKO+HG, AKR1B1 knockout with high glucose treated.

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