Genome-wide analysis of DNA methylation in subjects with type 1 diabetes identifies epigenetic modifications associated with proliferative diabetic retinopathy.

BACKGROUND: Epigenetic variation has been linked to several human diseases. Proliferative diabetic retinopathy (PDR) is a major cause of vision loss in subjects with diabetes. However, studies examining the association between PDR and the genome-wide DNA methylation pattern are lacking. Our aim was to identify epigenetic modifications that associate with and predict PDR in subjects with type 1 diabetes (T1D). METHODS: DNA methylation was analyzed genome-wide in 485,577 sites in blood from cases with PDR (n = 28), controls (n = 30), and in a prospective cohort (n = 7). False discovery rate analysis was used to correct the data for multiple testing. Study participants with T1D diagnosed before 30 years of age and insulin treatment within 1 year from diagnosis were selected based on 1) subjects classified as having PDR (cases) and 2) subjects with T1D who had had diabetes for at least 10 years when blood DNA was sampled and classified as having no/mild diabetic retinopathy also after an 8.7-year follow-up (controls). DNA methylation was also analyzed in a prospective cohort including seven subjects with T1D who had no/mild diabetic retinopathy when blood samples were taken, but who developed PDR within 6.3 years (converters). The retinopathy level was classified by fundus photography. RESULTS: We identified differential DNA methylation of 349 CpG sites representing 233 unique genes including TNF, CHI3L1 (also known as YKL-40), CHN2, GIPR, GLRA1, GPX1, AHRR, and BCOR in cases with PDR compared with controls. The majority of these sites (79 %) showed decreased DNA methylation in cases with PDR. The Natural Killer cell-mediated cytotoxicity pathway was found to be significantly (P = 0.006) enriched among differentially methylated genes in cases with PDR. We also identified differential DNA methylation of 28 CpG sites representing 17 genes (e.g. AHRR, GIPR, GLRA1, and BCOR) with P <0.05 in the prospective cohort, which is more than expected by chance (P = 0.0096). CONCLUSIONS: Subjects with T1D and PDR exhibit altered DNA methylation patterns in blood. Some of these epigenetic changes may predict the development of PDR, suggesting that DNA methylation may be used as a prospective marker of PDR.

Nuclear factor of activated T cells regulates osteopontin expression in arterial smooth muscle in response to diabetes-induced hyperglycemia.

OBJECTIVE: Hyperglycemia is a recognized risk factor for cardiovascular disease in diabetes. Recently, we reported that high glucose activates the Ca(2+)/calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in arteries ex vivo. Here, we sought to determine whether hyperglycemia activates NFAT in vivo and whether this leads to vascular complications. METHODS AND RESULTS: An intraperitoneal glucose-tolerance test in mice increased NFATc3 nuclear accumulation in vascular smooth muscle. Streptozotocin-induced diabetes resulted in increased NFATc3 transcriptional activity in arteries of NFAT-luciferase transgenic mice. Two NFAT-responsive sequences in the osteopontin (OPN) promoter were identified. This proinflammatory cytokine has been shown to exacerbate atherosclerosis and restenosis. Activation of NFAT resulted in increased OPN mRNA and protein in native arteries. Glucose-induced OPN expression was prevented by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. The calcineurin inhibitor cyclosporin A or the novel NFAT blocker A-285222 prevented glucose-induced OPN expression. Furthermore, diabetes resulted in higher OPN expression, which was significantly decreased by in vivo treatment with A-285222 for 4 weeks or prevented in arteries from NFATc3(-/-) mice. CONCLUSIONS: These results identify a glucose-sensitive transcription pathway in vivo, revealing a novel molecular mechanism that may underlie vascular complications of diabetes.

Tumor necrosis factor-alpha does not mediate diabetes-induced vascular inflammation in mice.

OBJECTIVE: Vascular inflammation is a key feature of both micro- and macrovascular complications in diabetes. Several lines of evidence have implicated the cytokine tumor necrosis factor (TNF) alpha as an important mediator of inflammation in diabetes. In the present study we evaluated the role of TNF alpha in streptozotocin (STZ)-induced diabetes on vascular inflammation in C57BL/6 wild-type and apoE-/- mice. METHODS AND RESULTS: Diabetes increased the expression of vascular cell adhesion molecule (VCAM)-1 in cerebral arteries 150 m in diameter as well as the macrophage accumulation in aortic root atherosclerotic plaques in apoE-/- mice. A more pronounced vascular inflammatory response was observed in diabetic TNF alpha-deficient apoE-/- mice. These mice were also characterized by increased accumulation of IgG and IgM autoantibodies in atherosclerotic lesions. Diabetes also increased VCAM-1 expression and plaque formation in apoE-competent TNF alpha -/- mice, whereas no such effects were observed in C57BL/6 wild-type mice. CONCLUSIONS: The present findings suggest that TNF alpha does not mediate diabetic-induced vascular inflammation in mice and reveal an unexpected protective role for TNF alpha. These effects are partly attributable to a direct antiinflammatory role of TNF alpha, but may also reflect a defective development of the immune system in these mice.

The aldose reductase inhibitor fidarestat suppresses ischemia-reperfusion-induced inflammatory response in rat retina.

Recent studies suggest that increased aldose reductase (AR) activity plays an important role in ischemia-reperfusion injury in the retina. The mechanisms are not completely understood, but may be linked to inflammation. In the present study, we investigated whether the AR inhibitor fidarestat suppressed the retinal inflammatory response induced by ischemia-reperfusion in a rat model. The inflammatory response was manifested by increased gene expression of tumor necrosis factor-alpha and intercellular adhesion molecule-1 (ICAM-1) as well as elevated protein levels of soluble ICAM-1. This response was partially suppressed by the AR inhibitor fidarestat. The findings may reveal beneficial effects of AR inhibition on retinal inflammation associated with ischemia-reperfusion and are in agreement with recent developments in pharmacological research suggesting that pathological conditions other than diabetes may benefit from AR inhibitors.

Multiethnic Genome-Wide Association Study of Diabetic Retinopathy Using Liability Threshold Modeling of Duration of Diabetes and Glycemic Control.

To identify genetic variants associated with diabetic retinopathy (DR), we performed a large multiethnic genome-wide association study. Discovery included eight European cohorts (n = 3,246) and seven African American cohorts (n = 2,611). We meta-analyzed across cohorts using inverse-variance weighting, with and without liability threshold modeling of glycemic control and duration of diabetes. Variants with a P value <1 × 10-5 were investigated in replication cohorts that included 18,545 European, 16,453 Asian, and 2,710 Hispanic subjects. After correction for multiple testing, the C allele of rs142293996 in an intron of nuclear VCP-like (NVL) was associated with DR in European discovery cohorts (P = 2.1 × 10-9), but did not reach genome-wide significance after meta-analysis with replication cohorts. We applied the Disease Association Protein-Protein Link Evaluator (DAPPLE) to our discovery results to test for evidence of risk being spread across underlying molecular pathways. One protein-protein interaction network built from genes in regions associated with proliferative DR was found to have significant connectivity (P = 0.0009) and corroborated with gene set enrichment analyses. These findings suggest that genetic variation in NVL, as well as variation within a protein-protein interaction network that includes genes implicated in inflammation, may influence risk for DR.

A genome-wide association study suggests new evidence for an association of the NADPH Oxidase 4 (NOX4) gene with severe diabetic retinopathy in type 2 diabetes.

PURPOSE: Diabetic retinopathy is the most common eye complication in patients with diabetes. The purpose of this study is to identify genetic factors contributing to severe diabetic retinopathy. METHODS: A genome-wide association approach was applied. In the Genetics of Diabetes Audit and Research in Tayside Scotland (GoDARTS) datasets, cases of severe diabetic retinopathy were defined as type 2 diabetic patients who were ever graded as having severe background retinopathy (Level R3) or proliferative retinopathy (Level R4) in at least one eye according to the Scottish Diabetic Retinopathy Grading Scheme or who were once treated by laser photocoagulation. Controls were diabetic individuals whose longitudinal retinopathy screening records were either normal (Level R0) or only with mild background retinopathy (Level R1) in both eyes. Significant Single Nucleotide Polymorphisms (SNPs) were taken forward for meta-analysis using multiple Caucasian cohorts. RESULTS: Five hundred and sixty cases of type 2 diabetes with severe diabetic retinopathy and 4,106 controls were identified in the GoDARTS cohort. We revealed that rs3913535 in the NADPH Oxidase 4 (NOX4) gene reached a p value of 4.05 × 10-9 . Two nearby SNPs, rs10765219 and rs11018670 also showed promising p values (p values = 7.41 × 10-8 and 1.23 × 10-8 , respectively). In the meta-analysis using multiple Caucasian cohorts (excluding GoDARTS), rs10765219 and rs11018670 showed associations for diabetic retinopathy (p = 0.003 and 0.007, respectively), while the p value of rs3913535 was not significant (p = 0.429). CONCLUSION: This genome-wide association study of severe diabetic retinopathy suggests new evidence for the involvement of the NOX4 gene.

Modifying a standard method allows simultaneous extraction of RNA and protein, enabling detection of enzymes in the rat retina with low expressions and protein levels.

The aim of the study was to evaluate messenger RNA and protein expression in limited amounts of tissue with low protein content. The Chomczynski method was used for simultaneous extraction of RNA, and protein was modified in the protein isolation step. Template mass and cycling time for the complementary DNA synthesis step of real-time reverse transcription-polymerase chain reaction (RT-PCR) for analysis of catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, the catalytic subunit of glutamylcysteine ligase, glutathione peroxidase 1, and the endogenous control cyclophilin B (CypB) were optimized before PCR. Polymerase chain reaction accuracy and efficacy were demonstrated by calculating the regression (R2) values of the separate amplification curves. Appropriate antibodies, blocking buffers, and running conditions were established for Western blot, and protein detection and multiplex assays with CypB were performed for each target. During the extraction procedure, the protein phase was dissolved in a modified washing buffer containing 0.1% sodium dodecyl sulfate, followed by ultrafiltration. Enzyme expression on real-time RT-PCR was accomplished with high reliability and reproducibility (R2, 0.990-0.999), and all enzymes except for glutathione peroxidase 1 were detectable in individual retinas on Western blot. Western blot multiplexing with CypB was possible for all targets. In conclusion, connecting gene expression directly to protein levels in the individual rat retina was possible by simultaneous extraction of RNA and protein. Real-time RT-PCR and Western blot allowed accurate detection of retinal protein expressions and levels.

Expression of antioxidant enzymes in rat retinal ischemia followed by reperfusion.

To evaluate the expression and protein levels of antioxidant enzymes in the rat retina exposed to oxidative stress induced by ischemia-reperfusion injury. Retinal ischemia was induced in female Wistar rats by ligation of the optic nerve and vessels behind the left eye bulb, and was followed by reperfusion for 0, 3, 6, or 24 hours. The right eye served as control. RNA and protein were extracted simultaneously from each retina. Expressions of the endogenous antioxidant enzymes glutathione peroxidase (GPx1), catalase (CAT), copper/zinc superoxide dismutase, manganese superoxide dismutase, and the catalytic subunit of glutamylcysteine ligase (GCLc) were analyzed with real-time reverse transcription polymerase chain reaction and related to the endogenous control cyclophilin B. Protein levels were measured with Western blot analysis. During the early phase (0 or 3 hours) of reperfusion, no changes were seen in enzyme expression. After 6 hours, GCLc expression increased by a factor of 1.14 (P = .034), followed by a decline of 0.80 after 24 hours (P = .00004), according to the comparative Ct method. After 24 hours of reperfusion, GPx1 expression increased by a factor of 1.14 (P = .028), and CAT had decreased by 0.82 (P = .022). Expressions of copper/zinc superoxide dismutase and manganese superoxide dismutase showed a tendency toward a decrease by factors of 0.86 (P = .055) and 0.88 (P = .053), respectively, after 24 hours. Protein levels did not differ for any of the antioxidants, regardless of reperfusion time. The slightly increased messenger RNA expression of GPx1 after 24 hours of reperfusion with a concomitant very modest decrease in CAT and GCLc expression and no change in protein levels indicate a very modest, if any, response to oxidative stress generated by ischemia followed by reperfusion in rat retina.

Increased serum levels of the specific advanced glycation end product methylglyoxal-derived hydroimidazolone are associated with retinopathy in patients with type 2 diabetes mellitus.

Advanced glycation end products (AGEs) are thought to play a major pathogenic role in diabetic retinopathy. The most important AGE is unknown, but as increased serum methylglyoxal-derived hydroimidazolone has been demonstrated in patients with type 2 diabetes mellitus, the aim of the present study was to elucidate possible associations between serum levels of hydroimidazolone and retinopathy in patients with type 2 diabetes mellitus. We recruited 227 patients with type 2 diabetes mellitus and retinopathy ranging from none to proliferative. Level of retinopathy was determined from 7 standard field stereo photographs per eye according to the Early Treatment Diabetic Retinopathy Study. The patients were 66 +/- 11 years old, with a known diabetes duration of 14 +/- 9 years. Serum levels of hydroimidazolone were determined with a competitive immunoassay. Serum levels of hydroimidazolone were increased in nonproliferative (median, 4.50 U/mL; interquartile range, 3.69-5.77 U/mL) and proliferative retinopathy (median, 4.88 U/mL; interquartile range, 3.70-6.52 U/mL) compared with patients without retinopathy (median, 4.02 U/mL; interquartile range, 3.47-4.88 U/mL) (P = .008 and .002, respectively). There was no association between hydroimidazolone and hemoglobin A1c (r = 0.04, P = .57). In addition, patients with proliferative retinopathy and a relatively short known duration of diabetes, that is, less than the median of 14 years, had increased serum levels of hydroimidazolone (median, 6.91 U/mL; interquartile range, 4.70-8.91 U/mL) compared with those with nonproliferative retinopathy (median, 4.34; interquartile range, 3.86-5.53U/mL, P = .015). Serum levels of hydroimidazolone are increased in type 2 diabetic patients with retinopathy. This association is independent of hitherto known associated factors, such as hemoglobin A1c.

Effects of focal/grid laser treatment on the central visual field in diabetic macular oedema: a 2-year follow-up study.

PURPOSE: To assess the long-term effects of focal/grid laser treatment on the central visual field in subjects with clinically significant diabetic macular oedema (CSMO). METHODS: Twenty-six subjects with CSMO were included in the study. Fundus photography, assessment of retinal thickness by time-domain optical coherence tomography (OCT), measurement of visual acuity (VA) and standard automated perimetry of the central 10° of the visual field were performed at baseline before focal/grid laser treatment and thereafter at four follow-up visits 6 months apart, when the laser treatment was also repeated in subjects with persistent oedema. Changes in VA and retinal thickness between baseline and study end at 24-month follow-up were calculated. Trends in changes in the visual field were assessed by linear regression of number of significantly depressed test points over time. RESULTS: On average after the laser treatment, retinal thickness decreased by 17% and VA decreased by three letters. In most eyes, visual fields were stable over time: the mean of the trend slopes (0.05 points/month) did not differ significantly from 0 (p = 0.63). The number of depressed test points at follow-up was not correlated with either the total number of laser effects or the number of laser sessions. CONCLUSION: Focal/grid laser treatment for CSMO did not affect retinal sensitivity in the central visual field in the subjects included in this 2-year follow-up study.

Bovine retinal pericytes are resistant to glucose-induced oxidative stress in vitro.

Diabetic retinopathy is a sight-threatening complication of diabetes, and loss of pericytes represents early signs of its development. We tested the hypothesis that high glucose levels may induce signs of oxidative stress in cultured bovine retinal pericytes. Pericytes were exposed to either normal (5.5 mM) or high (22 mM) glucose levels for 1, 3, and 5 days. Signs of oxidative stress were measured by expression of copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, and glutathione peroxidase using real-time RTPCR. To elucidate the role of oxidative stress, we also measured glutathione (GSH) concentration in the cells and investigated the impact of thiol-reactive metal ions and hydrogen peroxide (H(2)O(2)) on intracellular GSH. Despite the stimulation with high glucose, thiol-reactive metal ions, or H(2)O(2), there was no clear increased expression of antioxidant enzymes or influence of GSH levels. Lipid peroxidation (malondialdehyde level) was increased in bovine aortic smooth muscle cells, but not in bovine retinal pericytes. The data indicate that pericytes do not develop oxidative stress in response to hyperglycemia. However, it is not definitively excluded that oxidative stress may occur after longer time periods of glucose stimulation.

Photocoagulation of human retinal pigment epithelium in vitro: unravelling the effects on ARPE-19 by transcriptomics and proteomics.

PURPOSE: Despite the extensive use of retinal photocoagulation for ischaemia and vascular leakage in retinal vascular disease, the molecular mechanisms behind its clinical beneficial effects are still poorly understood. One important target of laser irradiation is the retinal pigment epithelium (RPE). In this study, we aimed at identifying the isolated effects of photocoagulation of RPE at both the mRNA and protein expression levels. METHODS: Human ARPE-19 cells were exposed to photocoagulation. Gene expression and protein expression were compared to untreated cells using microarray and liquid chromatography-mass spectrometry analysis. Genes and proteins queried by microarray and mass spectrometry were subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database pathway analyses. RESULTS: Laser irradiation resulted in an induction of the cytoprotective heat-shock protein subfamily Hsp70 as well as in a suppression of the vascular permeability factor carbonic anhydrase 9 (CA9). These expression patterns were evident at both the mRNA and protein levels. KEGG pathway analyses revealed genes and proteins involved in cellular turnover, repair and inflammation. CONCLUSIONS: By characterizing the transcriptional and translational effects of laser coagulation on the RPE cells in culture, we have revealed responses, which might contribute to some of the beneficial effects obtained by photocoagulation for ischaemia and vascular leakage in retinal vascular disease.

Automated detection of fundus photographic red lesions in diabetic retinopathy.

PURPOSE: To compare a fundus image-analysis algorithm for automated detection of hemorrhages and microaneurysms with visual detection of retinopathy in patients with diabetes. METHODS: Four hundred fundus photographs (35-mm color transparencies) were obtained in 200 eyes of 100 patients with diabetes who were randomly selected from the Welsh Community Diabetic Retinopathy Study. A gold standard reference was defined by classifying each patient as having or not having diabetic retinopathy based on overall visual grading of the digitized transparencies. A single-lesion visual grading was made independently, comprising meticulous outlining of all single lesions in all photographs and used to develop the automated red lesion detection system. A comparison of visual and automated single-lesion detection in replicating the overall visual grading was then performed. RESULTS: Automated red lesion detection demonstrated a specificity of 71.4% and a resulting sensitivity of 96.7% in detecting diabetic retinopathy when applied at a tentative threshold setting for use in diabetic retinopathy screening. The accuracy of 79% could be raised to 85% by adjustment of a single user-supplied parameter determining the balance between the screening priorities, for which a considerable range of options was demonstrated by the receiver-operating characteristic (area under the curve 90.3%). The agreement of automated lesion detection with overall visual grading (0.659) was comparable to the mean agreement of six ophthalmologists (0.648). CONCLUSIONS: Detection of diabetic retinopathy by automated detection of single fundus lesions can be achieved with a performance comparable to that of experienced ophthalmologists. The results warrant further investigation of automated fundus image analysis as a tool for diabetic retinopathy screening.

Application of quantitative competitive polymerase chain reaction for measurements of mRNA from antioxidative enzymes in the diabetic rat retina and kidney.

This present study applied quantitative competitive polymerase chain reaction (QC-PCR) in the analyses of mRNA expression of the endogenous antioxidative enzymes CuZn superoxide dismutase (SOD), MnSOD, catalase, and glutathione peroxidase in tissue samples from the retina and kidney cortex of diabetic rats. RNA was extracted from snap-frozen retinas and pieces of the kidney cortex of male Wistar rats with streptozotocin (STZ)-induced diabetes and control rats. The mRNA levels were analyzed using QC-PCR. The animals were kept in the laboratory for 1 and 6 months, respectively, and fed a normal or probucol- (1% wt/wt) enriched diet. By using QC-PCR, relative mRNA levels of all antioxidative enzymes could be estimated in the retina and kidney cortex. In the retina, the relative catalase mRNA concentration was about 1/10 that of the other enzymes. After 6 months of diabetes, there was a 100% increase of the catalase (median, 0.012 [range, 0.008 to 0.017] v 0.006 [0.005 to 0.010]; P =.011) and a 50% increase of the glutathione peroxidase mRNA levels (0.88 [0.44 to 1.12] v 0.52 [0.31 to 0.79]; P =.044). In the kidney cortex, the relative glutathione peroxidase mRNA level was 10 to 15 times higher, and catalase mRNA level about half of those of CuZnSOD and MnSOD. After 1 month of diabetes, there was an increase only of the glutathione peroxidase mRNA levels, by 170% (17.59 [6.19 to 29.49] v 6.96 [2.34 to 9.04]; P =.047). We conclude that quantification of mRNA can provide difficulties when the amount of sample RNA is limited and/or the gene expression is low. The present study shows QC-PCR to be useful as a tool for measuring expression of mRNA not only in the kidney cortex but also in small tissue samples like the retina. Our results indicate modestly increased mRNA expression of catalase and glutathione peroxidase in the retina and likewise modestly increased mRNA expression of glutathione peroxidase in the kidney cortex of rats with STZ-induced diabetes. Extended studies, also including enzyme activities, are needed before any effect of hyperglycemia on the overall enzyme activity can be established.

Putative role of polymorphisms in UCP1-3 genes for diabetic nephropathy.

Increased production of reactive oxygen species (ROS) has been suggested as a cause of diabetic complications. Uncoupling proteins (UCPs) have been ascribed a role in reducing the formation of ROS, and genetic variation in genes encoding for UCPs could thus be putative candidate genes for diabetic nephropathy. To test this hypothesis we searched for association between the A-->G (-3862) variant in UCP1, the insertion/deletion (I/D) polymorphism in exon 8 in UCP2, and the C-->T (-55) polymorphism in UCP3 and diabetic nephropathy in 218 diabetic patients with normal urinary albumin excretion rate (AER), 216 with micro- or macroalbuminuria, and in 106 control subjects without a family history of diabetes. We did not find any association between the different polymorphisms and diabetic nephropathy, nor did we observe any difference in AER among carriers of different UCP1-3 genotypes. We could, however, confirm the reported association between BMI and the UCP3 -55 C-->T polymorphism; patients carrying the T allele had higher BMI than patients homozygous for the C allele (26.4+/-4.2 vs. 25.3+/-4.3 kg/m(2); P=.01). We conclude that studied polymorphisms in the UCP1-3 genes do not play a major role in the development of micro- or macroalbuminuria in Scandinavian diabetic patients.

HLA-DRB1, -DQA1, and -DQB1 subtypes or ACE gene polymorphisms do not seem to be risk markers for severe retinopathy in younger Type 1 diabetic patients.

The aim of this study was to test the hypothesis that HLA-DRB1, -DQA1, and -DQB1 subgroups or angiotensin-converting enzyme (ACE) gene polymorphisms are associated with severe retinopathy in younger Type 1 diabetic patients. Twenty-four Type 1 diabetic patients who had received panretinal photocoagulation for severe nonproliferative or proliferative diabetic retinopathy were compared with 24 Type 1 diabetic patients (participating in a photographic screening program with regular fundus examinations) with no or minimal retinopathy, matched for age at onset and duration of diabetes. The HLA-DRB1-DQA1-B1 haplotype 04-03-0302 represented 22/48 (46%) in the severe and 21/48 (44%) in the no/minimal retinopathy group, respectively (n.s.). The most common genotype, 03-0501-0201/04-03-0302, occurred in 8/24 (33%) with severe and 10/24 (42%) with no/minimal retinopathy, respectively (n.s.). There were no statistical differences between patients with severe and no/minimal retinopathy whether DRB1, DQA1, or DQB1 alleles, haplotypes, or genotypes were analysed. The ACE gene polymorphism was almost identical between patients with severe and no/minimal retinopathy, and serum ACE levels did not differ. Thus, in the present study on a small group with carefully characterised diabetic retinopathy phenotypes, there was no indication that HLA-DRB1, -DQA1, and -DQB1 subtypes or ACE gene polymorphisms were associated with severe retinopathy in younger Type 1 diabetic patients.

Effects of inhibition of glycation and oxidative stress on the development of diabetic nephropathy in rats.

We investigated whether aminoguanidine (AG), an inhibitor of advanced glycated end product formation, or probucol (PB), a free radical scavenger, could influence signs of glomerular and distal tubular function and morphological changes in kidneys of male Wistar rats after 6 months of streptozocin (STZ)-induced diabetes. Diabetic rats had a higher kidney weight/body weight ratio (P<.001), but neither AG nor PB influenced the increased ratio. Diabetes caused an increased urinary albumin excretion (P<.05), which was normalized by AG, but further exaggerated by PB (P<.001). Diabetes also caused an increase in the urinary excretion of Tamm-Horsfall protein (P<.001). Both AG and PB attenuated this increase (P<.05 for both). A few glomeruli displayed focal thickening of varying degrees. Silver staining disclosed the glomerulopathy to be intercapillary glomerulosclerosis. Rats on PB-enriched diet displayed less pronounced changes than untreated rats (P<.01), while AG had no effect. The results suggest that oxidative stress could be involved in the development of diabetic nephropathy.

Progression of early retinal dysfunction in diabetes over time: results of a long-term prospective clinical study.

We explored signs of retinal dysfunction over time in diabetic subjects before or early in the course of retinopathy. Patients with no, mild, or moderate retinopathy were consecutively recruited and underwent standard automated perimetry, visual acuity measurement, and fundus photography. These examinations and measurements of HbA1c and blood pressure were repeated for up to 5 years from baseline. Visual field improvement/deterioration in diabetic subjects was evaluated using significance limits for change. Progression or regression of retinopathy was defined as a two-step change on the Early Treatment Diabetic Retinopathy Study final severity scale. Seventy-four subjects completed at least 3 years of follow-up, and 22% showed visual field worsening, defined as repeated significant deterioration at ≥10% of the test points, whereas only 1% showed field improvement. Worsening occurred in subjects both with and without vascular lesions. The degree of retinopathy was stable throughout the observation period in 68 of 74 eyes, improved in 4, and worsened in 2. Visual field deterioration was not correlated with a change in retinopathy. By using perimetry with an analysis tailored for monitoring diabetic subjects, we were able to demonstrate progression of retinal dysfunction over time, which may represent early signs of retinal neurodegeneration.

Profile of intraocular tumour necrosis factor-α and interleukin-6 in diabetic subjects with different degrees of diabetic retinopathy.

PURPOSE: To assess and correlate the levels of inflammatory mediators in the eyes from non-diabetic and diabetic subjects without retinopathy (NDR), with non-proliferative diabetic retinopathy (NPDR) or with proliferative diabetic retinopathy (PDR) to corresponding erum levels. METHODS: The levels of interleukin 1ß, interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) were analysed by an ELISA-mimicking technique in the vitreous from 26 diabetic subjects with active PDR and 27 non-diabetic subjects, or by a multiplex bead assay in the aqueous humour from 35 diabetic subjects with NDR/NPDR and 40 non-diabetic subjects. Intraocular protein production was estimated in vitreous specimens by calculating a vitreous/serum ratio. RESULTS: In the vitreous, IL-6 was higher in diabetic [157.5 (25.0-1401.0) pg/ml; median (min-max)] than in non-diabetic subjects [44.0 (5.0-4425) pg/ml; p = 0.021]. The vitreous/serum ratio was high (55.5:1 and 16:1, respectively), suggesting intraocular production. TNF-α was lower in diabetic [18.0 (8.0-46.0) pg/ml] than in non-diabetic subjects [22.0 (13.0-47.0) pg/ml; p = 0.034], but the vitreous/serum ratio was elevated in both groups (2:1 and 3.4:1, respectively). TNF-α levels were higher in serum from diabetic subjects [9.0 (5.0-53.0) pg/ml versus 6.7 (3.0-11.0) pg/ml; p < 0.001]. Aqueous levels of inflammatory mediators did not differ between diabetic subjects with NDR/NPDR and non-diabetic subjects despite elevated TNF-α in serum [27.8 (6.8-153.7) pg/ml versus 16.4 (4.1-42.4) pg/ml; p = 0.021]. CONCLUSION: Intraocular inflammation seems to be involved in PDR but does not seem to be prominent in early retinopathy stages, i.e. NDR or NPDR. Diabetic subjects have an overall increased inflammatory activity compared to non-diabetic subjects, as demonstrated by increased serum levels of TNF-α.