diabetes mellitus is recognized as a leading cause of new cases of blindness among americans between the ages of 20 and 74 . these include excess formation of advanced glycation end - products , activation of pkc isoforms , activation of the polyol pathway , and excessive oxidative stress . considerable evidence points to excess conversion of glucose to sorbitol , mediated by aldose reductase ( akr1b1),as a key factor in diabetic cataract formation . akr1b1-mediated polyol accumulation causes osmotic imbalances that lead to fiber cell swelling , liquefaction , and eventually cataract . compelling evidence to support this hypothesis came from lee and coworkers , who created a transgenic mouse model that expressed high levels of akr1b1 in lens fiber cells . these mice developed cataracts following diabetes induction , demonstrating an essential role for akr1b1 in mediating high glucose - dependent cataract formation .   the role of akr1b1 during euglycemia is still unclear . the aldo - keto reductase ( akr ) gene superfamily includes several enzymes and proteins with similar structures and/or enzymatic activities . the akr1b subfamily contains two genes that are expressed at relatively high levels in human tissues . akr1b1 , which is equivalent to aldose reductase , is expressed in many tissues throughout the body . akr1b10 , which has been given the trivial names human small intestine reductase ( hsir ) and akr1b1-like protein 1 ( arl-1 ) , is also expressed in many tissues [ 8 , 9 ] . based on a blot analysis of multiple tissue rnas , gene transcript levels of akr1b10 closely parallel those of akr1b1 . the broad catalytic similarities between akr1b1 and akr1b10 make it difficult to map the distribution of these proteins in human tissues using enzyme activity assays . the enzymes utilize an overlapping array of substrates , and many so - called aldose reductase inhibitors effectively block both akr1b1 and akr1b10 . therefore , studies conducted over 2 decades ago to demonstrate expression of akr1b1 in tissues of the human eye may have lacked sufficient specificity to distinguish between these two closely related gene products [ 11 , 12 ] . in the current study , we have reexamined the expression pattern of these enzymes , taking into account the possibility that akr1b10 may contribute to the aldo - keto reductase profile of ocular tissues and thus may participate in the pathogenesis of diabetic eye disease . the current study also addressed the question of whether akr1b10 contributes to the onset and progression of cataracts in a mouse model of diabetes . human postmortem eyes were obtained from certified eye banks through the national disease research interchange . the time interval between death to enucleation ( < 8 hours ) and then to fixation ( usually 812 hours ) was rigorously controlled . once received in the laboratory , the cornea , iris , ciliary body , lens , and retinas were carefully dissected and used to prepare protein lysates . total rna was extracted from human ocular tissues using an rnase kit ( qiagen ) . after digesting genomic dna using dnase i ( roche ) , cdna was synthesized from 1  g total rna using retroscript kit ( ambion ) in 20  l volume . quantitative real - time pcr for akr1b1 and akr1b10 were done using an icycler iq detection system ( bio - rad , hercules , ca ) . reaction mixtures contained iq sybr green supermix ( bio - rad ) and primers 5for - cccaaagatgataaaggtaatgccatcggt-3 and 5rev - cgatctggaagtggctgaaattggaga-3 for akr1b10 ,   5for - tgagtgccacccatatctca-3 and 5rev - tgtcacagacttggggatca-3 for akr1b1 , or 5for - agaaggagatcactgccctggcacc and 5rev - cctgcttgctgatccacatctgctg for -actin . pcr condition was 1 cycle of 95c for 3 minutes followed by 40 cycles at 95c for 20 seconds , 65c for 30 seconds , and 72c for 30 seconds . all the samples were run in triplicate , and the results were averaged . specific amplification of akr1b1 , akr1b10 , and -actin ( 244  bp for akr1b1 , 133  bp for akr1b10 and 162  bp for -actin ) was confirmed by gel electrophoresis and melting curve analysis after pcr . in order to compare expression patterns among tissues , relative quantification of gene expression the quantification data of akr1b1 and akr1b10 was indicated as a relative ratio of its signal to that of -actin to normalize the starting amount of template cdna . proteins were isolated from human ocular tissues by treatment in lysis buffer ( 10  mm tris , ph 7.4 , 150  mm nacl , 1% triton x-100 , 1  mm edta , ph   8.0 , 1  mm egta , ph 7.0 , 0.2  mm sodium orthovanadate , 1  mm pmsf , 0.5% np-40 ) with freshly added aprotinin to a final concentration of 5  g / ml . protein concentration was determined with the bicinchoninic acid methods , using bsa as standard ( micro bca protein assay kit ; pierce , rockford , il ) . equivalent amounts of protein ( 40  g ) from total cell lysates or tissue lysates were boiled in nupage lds sample buffer ( invitrogen ) for 5 minutes and analyzed by 10% sds - page . separated proteins were transferred to hybond - p pvdf membrane and were blocked with tbs-0.1% tween-20 containing 5% nonfat milk for overnight . membranes were incubated with antibodies for akr1b1 ( 1  :  3000 ) or akr1b10 ( 1  :  3000 dilution ) , probed with horseradish peroxidase - conjugated antirabbit secondary antibody ( 1  :  8000 ) for 2 hours , and washed . immune complexes were visualized with the ecl plus system and scanned on a storm 860 phosphorimager . antibodies to akr1b1 and akr1b10 were prepared through a commercial service ( bethyl laboratories , montgomery , tx ) . antibodies to human akr1b1 were made by immunizing rabbits with recombinant human akr1b1 , purified as described previously . antibodies to akr1b10 were prepared using synthetic peptides derived from akr1b10 encompassing residues 120 to 134 ( cddlfpkddkgnaigg ) . in both cases , antibodies were purified by column chromatography using the immunogen bound to a solid phase support as the affinity ligand . antibody specificity was verified using purified recombinant akr1b1 and akr1b10 in a western blotting format ( data not shown ) . immunohistochemical analysis for akr1b1 and akr1b10 was done with the formalin - fixed , paraffin - embedded tissue . the sections were deparaffinized in xylene , incubated for 30 minutes in methanol containing 3% h2o2 to inhibit endogenous peroxidase activity , rehydrated through a series of graded alcohols , and stained for akr1b1 or akr1b10 via the immunoperoxidase technique . the tissue was covered with 20% inactivated normal donkey serum in tris - buffered saline , ph 7.6 , incubated for 30 minutes at room temperature , and blotted and incubated overnight with a 1  :  500 dilution of akr1b1 antiserum or a 1  :  500 dilution of rabbit anti - akr1b10 peptide antibody overnight at 4c . immunostaining was visualized using diaminobenzidine tetrahydrochloride ( dab ) , a horseradish peroxidase system ( vector laboratories , burlingame , ca ) . further details on the preparation and characterization of five independent founder lines of akr1b10 transgenic mice will be presented elsewhere . animals derived from founder line par30 used in this study were maintained by outbreeding to the c57bl6 strain obtained from jackson laboratories ( bar harbor , me ) . experimental diabetes was induced in transgenic and nontransgenic control mice by treatment with a low - dose regimen of streptozotocin as described . fasting blood sugars were measured starting 2 weeks after the final streptozotocin treatment and monthly after hyperglycemia was established . for blood sugar measurements , animals were fasted for 6 hours prior to collection of a drop of blood from the saphenous vein . glucose levels were measured immediately using a glucometer ( alphatrak , abbot laboratories , chicago , il ) . animals were included in the study if fasting glucose levels were 250350  mg / dl . we used a quantitative real - time pcr - based assay ( qrt - pcr ) for measuring the expression profiles of the akr1b1 and akr1b10 genes in human eye tissues . the rt - pcr method was chosen because it provided the specificity necessary to distinguish between akr1b1 and akr1b10 gene transcripts , unlike the case with standard nucleic acid hybridization methods such as northern blotting . the expression profiles for akr1b1 and akr1b10 mrna levels were measured in cornea , iris , ciliary body , lens , and retina . data on the apparent abundance of gene - specific transcripts were computed relative to -actin and are shown in figure 1 . transcripts derived from the akr1b1 gene are present in all tissues examined , and are the highest in lens followed by retina and cornea . in the case of akr1b10 gene transcripts , the highest transcript levels are found in cornea , with substantially lower levels found in iris , ciliary body , lens , and retina .   to examine the distribution of the akr1b1 and akr1b10 at the protein level , we carried out immunohistochemical staining of paraffin sections produced from human eyes using affinity purified antibodies prepared as described in section 2 . staining appeared to be stronger in the basal as compared to the superficial cell layer . intense staining for akr1b10 was observed in the corneal stroma whereas akr1b1 staining was relatively limited in this region . staining appeared to be stronger in the basal as compared to the superficial cell layer . intense staining for akr1b10 was observed in the corneal stroma whereas akr1b1 staining was relatively limited in this region . lensstaining for akr1b1 and akr1b10 was observed in the lens epithelium and fiber cells located in the superficial cortex . the staining was the greatest at the equator region and diminished in cells located deeper in the cortex and nucleus . 
  staining for akr1b1 and akr1b10 the staining was the greatest at the equator region and diminished in cells located deeper in the cortex and nucleus . retinaakr1b1 and akr1b10 stained heavily in cell nuclei in the inner nuclear layer as well as in some ganglion cells , especially near the perinuclear cytoplasm and inner limiting membrane . no significant immunohistochemical staining of akr1b10 could be observed around the retinal vessels . in all cases , akr1b1 and akr1b10 stained heavily in cell nuclei in the inner nuclear layer as well as in some ganglion cells , especially near the perinuclear cytoplasm and inner limiting membrane . no significant immunohistochemical staining of akr1b10 could be observed around the retinal vessels . in all cases , transgenic micebased on results from rt - pcr and immunostaining experiments , it appears that both akr1b1 and akr1b10 are expressed in the human lens . to assess whether high levels of akr1b10 can predispose the lens toward diabetic cataract , we produced transgenic mice designed for overexpression of the enzyme in the lens . as shown in figure 3 , akr1b10 was readily detected by western blotting of lens homogenates from transgenic animals but was absent in nontransgenic control lenses . immunohistochemical staining showed intense positivity in the outer cortical fiber cells of transgenic animals and no detectable staining of nontransgenic controls ( figure 3 ) . thus , expression of akr1b10 in the transgenic lens had a similar regional distribution as endogenous akr1b10 in the human lens . on a gross level , overexpression of akr1b10 did not have a measureable impact on lens development , as the wet weight and appearance of transgenic lenses was not significantly different from nontransgenic controls ( data not shown).we induced experimental diabetes in our transgenic mice to determine if over - expression of akr1b10 influences the susceptibility of the mouse lens to cataracts . both transgenic and nontransgenic animals with and without experimental diabetes were monitored for up to six months for the appearance of lens opacities . in all cases , the lenses remained essentially clear and developed only minor focal areas of light scattering in the lens nucleus , typical of the normal aging mouse lens . a refractive abnormality localized on the anterior epithelium was observed in 50% ( 3 of 6 ) of the akr1b10 mice with diabetes . this defect gave rise to light scattering when viewed through a slit lamp ophthalmoscope or after dissection and brightfield illumination ( figure 4 ) . histological examination showed that this abnormality was associated with a localized disorganization of epithelial cells , formation of large vacuoles , and disrupted contact between epithelial cells and the lens capsule . this defect was not observed in age - matched nontransgenic controls with equivalent duration of experimental diabetes ( n = 4 ) or in nondiabetic transgenic controls ( n > 6 ) . the epithelial defect we observed is fundamentally different from cortical opacities that characterize the majority of diabetic cataracts . based on results from rt - pcr and immunostaining experiments , it appears that both akr1b1 and akr1b10 are expressed in the human lens . to assess whether high levels of akr1b10 can predispose the lens toward diabetic cataract , we produced transgenic mice designed for overexpression of the enzyme in the lens . as shown in figure 3 , akr1b10 was readily detected by western blotting of lens homogenates from transgenic animals but was absent in nontransgenic control lenses . immunohistochemical staining showed intense positivity in the outer cortical fiber cells of transgenic animals and no detectable staining of nontransgenic controls ( figure 3 ) . thus , expression of akr1b10 in the transgenic lens had a similar regional distribution as endogenous akr1b10 in the human lens . on a gross level , overexpression of akr1b10 did not have a measureable impact on lens development , as the wet weight and appearance of transgenic lenses was not significantly different from nontransgenic controls ( data not shown ) . we induced experimental diabetes in our transgenic mice to determine if over - expression of akr1b10 influences the susceptibility of the mouse lens to cataracts . both transgenic and nontransgenic animals with and without experimental diabetes were monitored for up to six months for the appearance of lens opacities . in all cases , the lenses remained essentially clear and developed only minor focal areas of light scattering in the lens nucleus , typical of the normal aging mouse lens . a refractive abnormality localized on the anterior epithelium was observed in 50% ( 3 of 6 ) of the akr1b10 mice with diabetes . this defect gave rise to light scattering when viewed through a slit lamp ophthalmoscope or after dissection and brightfield illumination ( figure 4 ) . histological examination showed that this abnormality was associated with a localized disorganization of epithelial cells , formation of large vacuoles , and disrupted contact between epithelial cells and the lens capsule . this defect was not observed in age - matched nontransgenic controls with equivalent duration of experimental diabetes ( n = 4 ) or in nondiabetic transgenic controls ( n > 6 ) . the epithelial defect we observed is fundamentally different from cortical opacities that characterize the majority of diabetic cataracts . osmotic stress to lens fiber cells resulting from excessive production and/or accumulation of sorbitol has been proposed as a mechanism leading to diabetic cataracts in humans . varma and coworkers previously demonstrated a strong correlation between the abundance of polyol pathway metabolites sorbitol and fructose and blood glucose levels in cataracts extracted from diabetic patients . our gene expression profiling of akr1b1 and akr1b10 using gene - specific rt - pcr clearly demonstrated that both of these aldo - keto reductases are expressed not only in lens but also in cornea , retina , and ciliary body . this raised the possibility that diabetes - induced cataract and retinopathy , as well as increased risk for glaucoma and corneal abnormalities , may develop through multiple akr - linked mechanisms . we employed a genetic strategy to determine if akr1b10 contributes to the pathogenesis of diabetic cataract in a mouse model . other members of the akr1b subfamily , such as akr1b7 ( major vas deferens protein , mvdp ; 18 ) and akr1b8 ( fibroblast growth factor - induced protein 1 ; fr-1 ; 19 ) , are virtually undetectable in the mouse lens . previous studies by lee et al . demonstrated that transgenic mice that overexpress akr1b1 in the lens develop cataracts after induction of galactosemia or experimental diabetes . therefore , transgenic expression of the human akr1b10 in the mouse lens allowed us to assess the impact of this enzyme on diabetic cataract formation using a transgenic mouse model that had been validated for diabetic cataract in a previous study .   in our diabetic animal model studies , we intentionally sought to achieve modest ( 250350  mg / dl ) levels of hyperglycemia so as to closely mimic the situation experienced by human patients with poorly controlled diabetes . since the akr1b10 transgenic mice remained cataract - free throughout 6 months of experimentally induced diabetes , it seems reasonable to conclude that akr1b10 likely has a limited role in the pathogenesis of diabetic cataract . both akr1b1 and akr1b10 are produced in many tissues of the eye affected by diabetes , including cornea , iris , ciliary body , lens , and retina . because lens transparency was maintained in akr1b10 transgenic mice following 6 months of experimental diabetes , we conclude that akr1b10 has a limited role in the pathogenesis of cataract in human patients with diabetes .
aldose reductase ( akr1b1 ) is an nadph - dependent aldo - keto reductase best known as the rate - limiting enzyme of the polyol pathway . 
 accelerated glucose metabolism through this pathway has been implicated in diabetic cataract and retinopathy . 
 some human tissues contain akr1b1 as well as akr1b10 , a closely related member of the aldo - keto reductase gene superfamily . 
 this opens the possibility that akr1b10 may also contribute to diabetic complications . 
 the goal of the current study was to characterize the expression profiles of akr1b1 and akr1b10 in the human eye . using quantitative reverse transcriptase - pcr and immunohistochemical staining , we observed expression of both akr genes in cornea , iris , ciliary body , lens , and retina . 
 expression of akr1b1 was the highest in lens and retina , whereas akr1b10 was the highest in cornea . 
 lenses from transgenic mice designed for overexpression of akr1b10 were not significantly different from nontransgenic controls , although a significant number developed a focal defect in the anterior lens epithelium following 6 months of experimentally induced diabetes . 
 however , lenses from akr1b10 mice remained largely transparent following longterm diabetes . 
 these results indicate that akr1b1 and akr1b10 may have different functional properties in the lens and suggest that akr1b10 does not contribute to the pathogenesis of diabetic cataract in humans .