Exploring the relationship between heavy metals and diabetic retinopathy: a machine learning modeling approach.

Diabetic retinopathy (DR) is one of the leading causes of adult blindness in the United States. Although studies applying traditional statistical methods have revealed that heavy metals may be essential environmental risk factors for diabetic retinopathy, there is a lack of analyses based on machine learning (ML) methods to adequately explain the complex relationship between heavy metals and DR and the interactions between variables. Based on characteristic variables of participants with and without DR and heavy metal exposure data obtained from the NHANES database (2003-2010), a ML model was developed for effective prediction of DR. The best predictive model for DR was selected from 11 models by receiver operating characteristic curve (ROC) analysis. Further permutation feature importance (PFI) analysis, partial dependence plots (PDP) analysis, and SHapley Additive exPlanations (SHAP) analysis were used to assess the model capability and key influencing factors. A total of 1042 eligible individuals were randomly assigned to two groups for training and testing set of the prediction model. ROC analysis showed that the k-nearest neighbour (KNN) model had the highest prediction performance, achieving close to 100% accuracy in the testing set. Urinary Sb level was identified as the critical heavy metal affecting the predicted risk of DR, with a contribution weight of 1.730632 ± 1.791722, which was much higher than that of other heavy metals and baseline variables. The results of the PDP analysis and the SHAP analysis also indicated that antimony (Sb) had a more significant effect on DR. The interaction between age and Sb was more significant compared to other variables and metal pairs. We found that Sb could serve as a potential predictor of DR and that Sb may influence the development of DR by mediating cellular and systemic senescence. The study revealed that monitoring urinary Sb levels can be useful for early non-invasive screening and intervention in DR development, and also highlighted the important role of constructed ML models in explaining the effects of heavy metal exposure on DR.

Urinary peptidome and diabetic retinopathy in the DIRECT-Protect 1 and 2 trials.

BACKGROUND: Given the association of diabetic retinopathy (DR) and kidney disease, we investigated the urinary peptidome to presence and deterioration of DR in a post hoc analysis of trials investigating the effect of candesartan on progression of DR in type 1 and type 2 diabetes, respectively. METHODS: Baseline urinary peptidomic analysis was performed on a random selection of 783 and 792 subjects in two randomized controlled trials, DIRECT-Protect 1 and 2, respectively. End points were two-step (RET2) and three-step (RET3) change in Early Treatment of Diabetic Retinopathy Study protocol (ETDRS) defined level. Peptide levels were correlated to baseline EDTRS level in a discovery set of 2/3 of the participants from DIRECT-Protect 1. The identified peptides were then validated cross-sectionally in the remaining 1/3 from DIRECT-Protect 1. Thereafter, peptides identified in the discovery set were assessed in the entire DIRECT-Protect 1 and 2 cohorts and significant peptides were tested longitudinally. RESULTS: Follow-up ranged 4.0-4.7 years. 24 peptides were associated with baseline DR in the discovery set. COL3A1 (seq: NTG~) and COL4A1 (seq: DGA~) were associated with baseline DR in the validation set (Rho: -.223, p < 0.001 and Rho: -.141, p = 0.024). Neither was significantly associated with end points. Assessing the 24 identified peptides in the entire cohorts, several collagen peptides were associated with baseline DR and end points; however, there was no overlap across diabetes types. CONCLUSIONS: We identified several urinary peptides (mainly collagen) associated with the presence of DR, however they could not be conclusively associated with worsening of DR.

Relationship between serum bilirubin levels, urinary biopyrrin levels, and retinopathy in patients with diabetes.

OBJECTIVE: Previous reports have indicated that serum bilirubin levels may be associated with diabetic retinopathy. However, the detailed mechanism is not fully understood. In this study, we evaluated the relationship between the severity of diabetic retinopathy and various factors including bilirubin levels and factors influencing bilirubin metabolism. METHODS: The study participants consisted of 94 consecutive patients with diabetes mellitus admitted to Kyushu University Hospital from April 2011 to July 2012. The patients were classified into three groups: no retinopathy (NDR), simple retinopathy (SDR), and pre-proliferative or proliferative retinopathy (PDR). The relationship between the severity of retinopathy and various factors was evaluated using univariate and logistic regression analyses. In addition, multivariate regression analysis was performed to evaluate the significant determinants for bilirubin levels. RESULTS: In univariate analysis, a significant difference was found among NDR, SDR and PDR in bilirubin levels, duration of diabetes, systolic blood pressure, and macroalbuminuria. Logistic regression analysis showed that PDR was significantly associated with bilirubin levels, duration of diabetes, and systolic blood pressure (OR 0.737, 95% CI 0.570-0.952, P = 0.012; OR 1.085, 95% CI 1.024-1.149, P = 0.006; OR 1.036, 95% CI 1.011-1.062, P = 0.005, respectively). In turn, multivariate regression analysis showed that bilirubin levels were negatively associated with high-sensitivity C-reactive protein levels and PDR, but positively correlated with urinary biopyrrin levels, oxidized metabolites of bilirubin. CONCLUSION: PDR was negatively associated with bilirubin levels. This negative association may be due to a decreased production of bilirubin rather than its increased consumption considering the positive association between bilirubin and biopyrrin levels.

Diabetic Retinopathy Screening Using a Gold Nanoparticle-Based Paper Strip Assay for the At-Home Detection of the Urinary Biomarker 8-Hydroxy-2'-Deoxyguanosine.

PURPOSE: We sought to assess a smartphone-based, gold nanoparticle-based colorimetric lateral flow immunoassay paper sensor for quantifying urine 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a biomarker for diabetic retinopathy (DR) screening. METHODS: Paper strips incorporate gold nanoparticle-8-OHdG antibody conjugates that produce color changes that are proportional to urine 8-OHdG and that are discernible on a smartphone camera photograph. Paper strip accuracy, precision, and stability studies were performed with 8-OHdG solutions of varying concentrations. Urine was collected from 97 patients with diabetes who were receiving DR screening examinations, including 7-field fundus photographs. DR was graded by standard methods as either low risk (no or mild DR) or high risk (moderate or severe DR). Paper sensor assays were performed on urine samples from patients and 8-OHdG values were correlated with DR grades. The differences in 8-OHdG values between the low- and high-risk groups were analyzed for outliers to identify the threshold 8-OHdG value that would minimize false-negative results. RESULTS: Lateral flow immunoassay paper strips quantitatively measure 8-OHdG and were found to be accurate, precise, and stable. Average urine 8-OHdG concentrations in study patients were 22 ± 10 ng/mg of creatinine in the low-risk group and 55 ± 11 ng/mg of creatinine in the high-risk group. Screening cutoff values of 8-OHdG >50 ng/mg of creatinine or urine creatinine >1.5 mg minimized screen failures, with 91% sensitivity and 81% specificity. CONCLUSIONS: Urinary 8-OHdG is a useful biomarker to screen DR. Quantitative 8-OHdG detection with the lateral flow immunoassay paper sensor and smartphone camera demonstrates its potential in DR screening. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Effect of microalbuminuria on macular thickness in patients with type-2 diabetes mellitus.

PURPOSE: To investigate the effect of microalbuminuria on macular thickness in patients with type-2 diabetes mellitus with no or mild diabetic retinopathy and to investigate the relationship between macular thickness and metabolic parameters. MATERIALS AND METHODS: Fifty eight eyes of 58 patients without diabetic retinopathy (group 1) in microalbuminuria stage, 42 eyes of 42 patients with mild diabetic retinopathy (group 2) in microalbuminuria stage, and 50 eyes of 50 patients without diabetic retinopathy and microalbuminuria (group 3) were included in this study. After detailed ophthalmologic examination, all patients underwent spectral domain-optical coherence tomography measurements. Macular thickness was noted from nine different areas (fovea, four parafoveal, and four perifoveal areas) and compared between groups. The correlations between macular thickness and age, duration of diabetes mellitus, microalbuminuria, serum urea, creatinine, glycosylated hemoglobin (HbAIc), albumin, sodium (Na), and urinary Na were evaluated. RESULTS: The mean age was 53.29 ± 6.49 in group 1, 55.86 ± 6.97 in group 2, and 52.98 ± 5.66 years in group 3 (p = 0.06). The macular thickness values of superior, inferior, and nasal parafoveal areas were significantly different between groups (p = 0.001, p = 0.006, and p = 0.03, respectively). Bonferroni post test revealed that this difference originated from the difference between group 2 and 3 (p < 0.05 for all values). There were significant negative correlations between the macular thickness values of parafoveal areas and serum urea, HbA1c, albumin, microalbuminuria levels (p < 0.05 for all values). CONCLUSION: In this study, a significantly decreased parafoveal macular thickness was measured in patients with mild diabetic retinopathy and microalbuminuria compared to patients without diabetic retinopathy and microalbuminuria.

Urinary neutrophil gelatinase-associated lipocalin in type 2 diabetes: Relation to nephropathy and retinopathy.

BACKGROUND: Diabetes mellitus is the leading cause of end stage renal disease worldwide. Early identification of diabetic nephropathy even before appearance of microalbuminuria is a challenge for early prevention of occurrence and progression of this complication. Neutrophil gelatinase-associated lipocalin is a small protein that belongs to the lipocalin protein. Urinary neutrophil gelatinase-associated lipocalin is a promising early marker in different renal problems. AIM OF THE WORK: To measure urinary neutrophil gelatinase-associated lipocalin in type 2 diabetic patients and to assess its role as an early marker for diagnosis of diabetic nephropathy and diabetic retinopathy. PATIENT AND METHODS: The current study included 60 subjects with type 2 diabetes and 20 healthy control subjects. Diabetic subjects were divided into 3 groups according to urinary albumin creatinine ratio; 20 normoalbuminuric patients, 20 micro-albuminuric patients and 20 macroalbuminuric patients. They were subjected to history taking, full clinical examination, fundus examination, anthropometric measurement, urinary neutrophil gelatinase-associated lipocalin and urinary albumin creatinine ratio. RESULTS: Urinary neutrophil gelatinase-associated lipocalin was higher in all diabetic groups than in the control group, with no difference in between diabetic groups. The difference was of great value when comparing normoalbuminuric group with control as albumin creatinine ratio was not different while the urinary neutrophil gelatinase-associated lipocalin was statistically significant (5.94 ±â€¯1.85 ng/dl vs 1.96 ±â€¯0.65, p < 0.001). No correlation was found with retinopathy. CONCLUSION: Urinary neutrophil gelatinase-associated lipocalin is a sensitive marker for early detection of diabetic nephropathy even in normoalbuminuric patients denoting early tubular damage before microalbuminuria. It is not correlated with retinopathy.

Glomerular Filtration Rate and/or Ratio of Urine Albumin to Creatinine as Markers for Diabetic Retinopathy: A Ten-Year Follow-Up Study.

AIMS: To determine the relationship between diabetic nephropathy and diabetic retinopathy on a population of type 2 diabetes mellitus patients. METHODS: A prospective ten-year follow-up population-based study. We determined differences between estimated glomerular filtration rate (eGFR) using the chronic kidney disease epidemiology collaboration equation and urine albumin to creatinine ratio. RESULTS: Annual incidence of any-DR was 8.21 ± 0.60% (7.06%-8.92%), sight-threatening diabetic retinopathy (STDR) was 2.65 ± 0.14% (2.48%-2.88%), and diabetic macular edema (DME) was 2.21 ± 0.18% (2%-2.49%). Renal study results were as follows: UACR > 30 mg/g had an annual incidence of 7.02 ± 0.05% (6.97%-7.09%), eGFR < 60 ml/min/1.73 m2 incidence was 5.89 ± 0.12% (5.70%-6.13%). Cox's proportional regression analysis of DR incidence shows that renal function studied by eGFR < 60 ml/min/1.73 m2 was less significant (p = 0.04, HR 1.223, 1.098-1.201) than UACR ≥ 300 mg/g (p < 0.001, HR 1.485, 1.103-1.548). The study of STDR shows that eGFR < 60 ml/min/1.73 m2 was significant (p = 0.02, HR 1.890, 1.267-2.820), UACR ≥ 300 mg/g (p < 0.001, HR 2.448, 1.595-3.757), and DME shows that eGFR < 60 ml/min/1.73 m2 was significant (p = 0.02, HR 1.920, 1.287-2.864) and UACR ≥ 300 mg/g (p < 0.001, HR 2.432, 1.584-3.732). CONCLUSIONS: The UACR has a better association with diabetic retinopathy than the eGFR, although both are important risk factors for diabetic retinopathy.

Synergistic interaction between prolonged increased glycemic exposure and mildly increased urinary albumin excretion on diabetic retinopathy.

The association of mild increase in urinary albumin excretion with diabetic retinopathy (DR) in clinical studies is controversial. The aim of this study is to clarify the interaction between increased glycemic exposure and mild increase in urinary albumin excretion on risk of DR.Data were collected from the National Health and Nutrition Examination Survey (NHANES) from 2005 to 2012. Overall, data from 953 participants without microalbuminuria (477 men and 476 women) were assessed. Logistic regression analysis was constructed to evaluate the association between DR and related clinical parameters, including urinary albumin-creatinine ratio (UACR, mg/g creatinine). The biological interaction of glycemic status and UACR on DR was evaluated by 3 indices: RERI, the relative excess risk due to the interaction; AP, the attributable proportion due to the interaction; and S, the additive interaction index of synergy.We found that UACR, glycated hemoglobin (HbA1c), and diabetic duration were deeply associated with increased risk of DR (UACR, odds ratio [OR] = 1.04, 95% confidence interval [CI] = 1.02-1.07; HbA1c, OR = 1.16, 95% CI = 1.04-1.30; diabetic duration, OR = 1.06, 95% CI = 1.04-1.07). Furthermore, our interaction analysis demonstrated that synergistic interaction between HbA1c and UACR on development of DR was prominent in participants with diabetic duration of ≥10 years (adjusted RERI = 0.92, 95% CI = 0.10-1.74; adjusted AP = 0.29, 95% CI = -0.82-1.41; adjusted S = 1.76, 95% CI = 1.27-2.25), but not subjects with shorter diabetic duration.These findings imply that there is the interaction between prolonged hyperglycemic exposure and increased urinary albumin excretion may exert additive synergistic effect on vascular endothelial dysfunction in the eye, even before the appearance of overt diabetic nephropathy.

Normal-to-mildly increased albuminuria predicts the risk for diabetic retinopathy in patients with type 2 diabetes.

Albuminuria is closely associated with diabetic retinopathy (DR), but the precise role of the albumin-to-creatinine ratio (ACR) in screening for DR remains to be determined. This study aimed to investigate an ACR threshold for predicting DR in patients with type 2 diabetes. A cross-sectional study was conducted on 1,102 type 2 diabetes patients, aged ≥30 years and recruited from the Korea National Health and Nutrition Examination Survey, 2010-2011. Participants were grouped by stage of DR: mild-to-moderate nonproliferative DR (NPDR), severe NPDR, and proliferative diabetic retinopathy (PDR). An early morning spot urine sample was obtained for ACR measurement. ROC curve analysis revealed that the optimal cut-off value of ACR for predicting DR was 2.26 mg/mmol (20 µg/mg). The prevalence of ACR ≥ 2.26 mg/mmol tended to increase with severity of DR. The risk for DR in patients with ACR ≥ 2.26 mg/mmol was higher than in those with ACR < 2.26 mg/mmol. The risk for severe NPDR and PDR also increased at ACR ≥ 2.26 mg/mmol. Normal-to-mildly increased albuminuria (an ACR of 2.26 mg/mmol) may predict the risk for DR development and progression in patients with type 2 diabetes.

Model for Risk-Based Screening of Diabetic Retinopathy in People With Newly-Diagnosed Type 2 Diabetes Mellitus.

Purpose: The purpose of this study was to evaluate the role of inflammatory/lipid markers and potential risk factors for diabetic retinopathy (DR) development in newly diagnosed patients with type 2 diabetes mellitus (T2DM). Methods: Participants in this study were 1062 patients with newly diagnosed T2DM. Demographic and clinical data of patients were collected. Assessment of DR status was performed using digital two-field photography. In addition, HbA1c (%), lipid profile, and urinary albumin were measured at recruitment. The following inflammatory markers were also measured: serum C-reactive protein, white blood cells, platelet, adiponectin, IL-4, IL-6, IL-10, vascular endothelial growth factor, tumor necrosis factor-α (TNF-α), IL-1b, IL-1 receptor antagonist (IL-1RA), and monocyte chemotactic protein-1. Univariate and multivariate analyses of the association of various potential risk factors and DR were conducted. Results: Univariate analysis showed that male sex, any cardiovascular event, and HbA1c were positively associated with DR, while IL-1RA, IL-1b, IL-6, and TNF-α were significantly negatively associated with presence of DR in the cohort. Risk factors that remained significantly associated with DR presence at the multivariate analysis were male sex, any cardiovascular event, HbA1c, and IL-1RA. Conclusions: Our study demonstrated that HbA1c levels, male sex, and previous cardiovascular events were risk factors for presence of DR in people with newly diagnosed T2DM, while IL-1RA seemed to have a protective role. The prevalence of DR in our population was 20.2%, reflecting current practice. Our findings may contribute to future risk-based modelling of screening for DR.

Urine Proteome Specific for Eye Damage Can Predict Kidney Damage in Patients With Type 2 Diabetes: A Case-Control and a 5.3-Year Prospective Cohort Study.

OBJECTIVE: The predictive value of microalbuminuria (MAU) for kidney damage is limited in type 2 diabetes (T2D). We studied whether a urine proteome specific for sight-threatening proliferative diabetic retinopathy (PDR) is an indicator to predict chronic renal insufficiency (CRI) in patients with T2D. RESEARCH DESIGN AND METHODS: A shotgun urine proteomic analysis was performed in patients with MAU and PDR (case subjects) and in patients with MAU and a duration of T2D for >10 years but without any degree of retinopathy (control subjects). In the cohort study, 210 patients with T2D with an estimated glomerular filtration rate (eGFR) ≥80 mL/min/1.73 m2 were followed for a median of 5.3 years. Urine proteins specific for PDR were used for predicting CRI (eGFR <60 mL/min/1.73 m2). RESULTS: The top two urine proteins with the highest difference in ratio of case subjects to control subjects were haptoglobin (8.7 times; P < 0.0001) and α-2-macroglobulin (5.7 times; P < 0.0001). In the cohort study, patients with baseline urinary haptoglobin ≥20 ng/min (haptoglobinuria) had a higher incidence of CRI than those without (hazard ratio [95% CI] 3.27 [1.41-7.58]; P = 0.006). The overall CRI rate was 3.2% for patients without haptoglobinuria or MAU, 9.5% for those with MAU, and 13.3% for those with haptoglobinuria. The highest rate for CRI (22.4%) was in patients with both MAU and haptoglobinuria (P < 0.001). CONCLUSIONS: Urine haptoglobin, which is specific for PDR, is a novel biomarker and complement to urine albumin for predicting kidney damage in patients with T2D.

Chronic Kidney Disease and Diabetic Retinopathy in Patients with Type 2 Diabetes.

PURPOSE: To explore the relationship between chronic kidney disease (CKD) and diabetic retinopathy (DR) in a representative population of type 2 diabetes mellitus (DM2) patients in Catalonia (Spain). METHODS: This was a population-based, cross-sectional study. A total of 28,344 patients diagnosed with DM2 who had recorded ophthalmologic and renal functional examinations were evaluated. Data were obtained from a primary healthcare electronic database of medical records. CKD was defined as an estimated glomerular filtration ratio (eGFR) of <60 ml/min/1.73 m2 and/or urine albumin to creatinine ratio (UACR) ≥30 mg/g. DR was categorized as non-vision threatening diabetic retinopathy and vision threatening diabetic retinopathy. RESULTS: CKD was associated with a higher rate of DR [OR], 95% confidence interval [CI], 1.5 (1.4-1.7). When we analyzed the association between different levels of UACR and DR prevalence observed that DR prevalence rose with the increase of UACR levels, and this association was significant from UACR values ≥10 mg/g, and increased considerably with UACR values ≥300 mg/g (Odds ratio [OR], 95% confidence interval [CI], 2.0 (1.6-2.5). This association was lower in patients with eGFR levels 44 to 30 mL/min/1.73 m2 [OR], 95% confidence interval [CI], 1.3 (1.1-1.6). CONCLUSIONS: These results show that CKD, high UACR and/or low eGFR, appear to be associated with DR in this DM2 population.

High Prolactin Excretion in Patients with Diabetes Mellitus and Impaired Renal Function.

BACKGROUND: The metabolic clearance of prolactin (PRL) is partially executed by the kidney. Here, we investigate the urine excretion of PRL in patients with Diabetes Mellitus and renal impairment. METHODS: Serum and urine samples were collected from male, mestizo patients in central Mexico employing a cross-sectional study design. Ninety-eight individuals had either no diabetes and normal renal function (control), diabetes and normal renal function, or diabetes with impaired renal function. PRL was determined by a chemiluminescent immunometric assay; protein, albumin, and creatinine were evaluated using quantitative colorimetric assays. The results were analyzed using ANOVA-testing. RESULTS: Patients with Diabetes Mellitus and renal impairment had significantly higher urine PRL levels than patients with Diabetes Mellitus and normal renal function and control patients. Higher urine PRL levels were associated with lower glomerular filtration rates, higher serum creatinine, and higher urinary albumin-to-creatinine ratios (UACR). Urine PRL levels correlated positively with UACR. Serum PRL levels were similar among groups. CONCLUSIONS: Patients with Diabetes Mellitus and impaired renal function demonstrate a high urinary PRL excretion. Urinary PRL excretion in the context of proteinuria could contribute to PRL dysregulation in renal impairment.

Decreased urine uric acid excretion is associated with diabetic retinopathy but not with lower limb atherosclerosis in hospitalized patients with type 2 diabetes.

OBJECTIVE: To explore the associations between urine uric acid excretion (UUAE) and diabetic retinopathy (DR)/lower limb atherosclerotic lesions in hospitalized Chinese patients with type 2 diabetes. METHODS: This cross-sectional study was conducted in 2529 hospitalized Chinese patients with type 2 diabetes. UUAE was determined enzymatically using a single 24-h urine collection. The subjects were stratified into quartile based on UUAE levels. DR was determined by digital fundus photography. Lower limb atherosclerotic lesions were assessed by Doppler ultrasound. Both DR and lower limb atherosclerosis were compared among the UUAE quartile groups, respectively. RESULTS: There was a significant decrease in the prevalence of DR in patients across the UUAE quartiles after adjustment for sex, age and diabetic duration (35.0%, 30.7%, 26.1%, and 21.5%, respectively, p = 0.000001 for trend). A fully adjusted multiple logistic regression analyses revealed that UUAE quartiles were markedly inversely associated with the presence of DR (p = 0.030). The prevalence of lower limb plaque (73.9% vs. 62.6%, p = 0.000044) and stenosis (16.3% vs. 9.7%, p = 0.000015) was markedly higher in the diabetics with DR than in those without DR. However, there was no statistical association between the UUAE and lower limb atherosclerotic lesions in type 2 diabetes. CONCLUSIONS: Decreased UUAE was an independent risk factor for DR but not for lower limb atherosclerosis in hospitalized Chinese patients with type 2 diabetes. In selected populations, such as those with type 2 diabetes, the role of uric acid in atherosclerosis may be result from other concomitantly atherosclerotic risk factors, such as DR.

Retinal nerve fiber layer loss is associated with urinary albumin excretion in patients with type 2 diabetes.

OBJECTIVES: To identify the factors associated with retinal nerve fiber layer (RNFL) loss in patients with type 2 diabetes. DESIGN: Cross-sectional study. PARTICIPANTS: Ninety-six nonglaucomatous patients with type 2 diabetes without renal impairment (estimated glomerular filtration rate, ≥60 ml/minute per 1.73 m(2)). METHODS: Eyes were divided into 2 groups based on the presence or absence of RNFL defects detected by red-free retinal fundus photography. All participants underwent an eye fundus examination, and the urinary albumin-to-creatinine ratio (ACR) was determined. A cardiovascular autonomic function test was performed using the following heart rate variability parameters: expiration-to-inspiration ratio, response to the Valsalva maneuver, and standing. Multiple logistic regression analyses were performed to determine potential risk factors related to the presence of RNFL defects in these patients. MAIN OUTCOMES AND MEASURES: The association between RNFL defects and diabetic complications. RESULTS: Among the patients, 43 (44.8%) had localized RNFL defects (group 1), whereas the others (55.2%) did not (group 2). The RNFL defects occurred more frequently on the superior side (75.6% and 71.0% in right and left eyes, respectively) compared with the inferior side (13.8% and 0.0% in right and left eyes, respectively). Patients with RNFL defects (group 1) had significantly higher rates of diabetic retinopathy (60.5%) compared with those without RNFL defects (group 2; 32.1%; P = 0.007). The urinary ACR was significantly higher in patients with RNFL defects than in those without defects (45.3±72.1 µg/mg vs. 15.4±17.3 µg/mg creatinine, respectively; P = 0.015), whereas autonomic function test grading was similar between the groups. The urinary ACR was the only factor related to visual field defect location in both univariate (P = 0.021) and multivariate (P = 0.036) logistic regression analyses after adjusting for age; gender; presence of diabetic retinopathy; diabetes duration; smoking; statin use; and antiplatelet, angiotensin-converting enzyme inhibitor or angiotensin receptor blocker treatment. CONCLUSIONS: Urinary albumin excretion was associated with nerve fiber layer loss in patients with type 2 diabetes. Careful examination of the optic nerve head may be necessary, particularly in patients with type 2 diabetes exhibiting albuminuria.

Changes in urine proteome accompanying diabetic nephropathy progression.

INTRODUCTION: Owing to the prevalence of type 2 diabetes, diabetic kidney disease (DKD) becomes the major cause of end-stage renal disease. The current markers of diabetic nephropathy are based on albuminuria and clinical signs of retinopathy. Sensitive and specific noninvasive diagnostic tools, unbiased by the presence of comorbidities, are needed, especially to detect the early stages of diabetic complications. OBJECTIVES: The aim of the study was to analyze changes in urinary protein excretion based on the stage of DKD using quantitative proteomics. PATIENTS AND METHODS: A total of 27 healthy controls were age- and sex-matched to 72 diabetes patients classified into 3 groups: no signs of retinopathy or nephropathy (n = 33), retinopathy but no microalbuminuria (n = 15), and diabetic nephropathy (DN) based on overt albuminuria or microalbuminuria with retinopathy (n = 24). To assess the intergroup differences, samples were partially pooled, tagged using 8-plex iTRAQ reagents, and the resulting peptide mixture was resolved by isoelectrofocusing. The obtained fractions were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Data were analyzed using the MASCOT software and dedicated in-house proteomic data analysis programs. RESULTS: The changes in the urine proteome following DKD progression involved some known protein markers of DN and several other proteins. Decreased levels of some proteins are presumably related to impaired secretory function of other organs affected by diabetes. In particular, a diminished excretion of pancreatic amylase and deoxyribonuclease I suggested exocrine pancreatic insufficiency (EPI), coexisting with type 2 diabetes. CONCLUSIONS: A decrease in the urinary excretion of some pancreatic enzymes suggests EPI associated with diabetes. This hypothesis is yet to be verified; nevertheless, renal and extrarenal confounders must be considered when interpreting the results of quantitative urinary proteomics.  

Urinary 6-sulfatoxymelatonin level in diabetic retinopathy patients with type 2 diabetes.

Melatonin is a powerful antioxidant. Decreased melatonin excretion has been reported to be associated with several oxidative stress-related diseases. The urinary metabolite of melatonin, 6-sulfatoxymelatonin (aMT6s), has proved to be a very reliable index of melatonin production. The present study aims to evaluate the level of urinary aMT6s in patients with type 2 diabetes mellitus and diabetic retinopathy. Urine samples were collected from 10 patients with diabetes and no diabetic retinopathy (NDR), 19 patients with nonproliferative diabetic retinopathy (NPDR), 38 patients with proliferative diabetic retinopathy (PDR), and 16 subjects without diabetes mellitus, who served as controls. The level of aMT6s in specimens was assayed by a commercial aMT6s ELISA kit, creatinine levels were also measured for each sample to get urinary aMT6s/creatinine ratio. Creatinine-adjusted urinary aMT6s values were compared among four groups. The urinary aMT6s (mean ± SD) levels were 9.95 ± 2.42, 9.90 ± 2.28, 8.40 ± 1.84 and 5.58 ± 1.33 ng/mg creatinine in the controls and in patients with NDR, NPDR, or PDR, respectively. The urinary aMT6s level of the PDR group was significantly lower than that of the control, NDR and DR groups. No significant difference was found among the control, NDR and DR groups. After adjustment for various factors (age, smoking, cancer, and coronary heart disease) that may influence the aMT6s level, the odds-ratio of urinary aMT6s comparing PDR patients to controls was 0.246 (95% confidence interval = 0.108-0.558, P = 0.001). Therefore, the urinary aMT6s level is significantly decreased in diabetic patients with PDR but not in diabetic patients without PDR, which indicates that decreased urinary aMT6s level may be associated with the pathogenesis of PDR.

Higher dietary salt intake is associated with microalbuminuria, but not with retinopathy in individuals with type 1 diabetes: the EURODIAB Prospective Complications Study.

AIMS/HYPOTHESIS: High dietary salt intake has been associated with elevated BP and may also have a deleterious effect on microvascular complications. We studied the cross-sectional associations between dietary salt intake (estimated from 24 h urinary sodium excretion) and urinary potassium excretion on the one hand, and the prevalence of microvascular complications on the other, in individuals with type 1 diabetes. METHODS: We measured sodium and potassium concentrations in two 24 h urine samples in 1,212 individuals with type 1 diabetes (40 ± 10 years old, 51% men) who participated in the EURODIAB Prospective Complications Study. We used multiple logistic regression analyses to investigate associations between dietary salt intake and microvascular complications adjusted for age and sex, and additionally for BMI, smoking, urinary potassium excretion, antihypertensive medication and physical activity, and total energy, protein, alcohol, saturated fat and fibre intake. RESULTS: After full adjustment, 1 g/day higher dietary salt intake was positively associated with the presence of microalbuminuria (OR 1.06 [95% CI 1.01, 1.10]), but not macroalbuminuria (OR 0.99 [95% CI 0.94, 1.05]), non-proliferative retinopathy (OR 1.00 (95% CI 0.96, 1.04]) or proliferative retinopathy (OR 1.02 (95% CI 0.95, 1.08]). After excluding individuals with cardiovascular disease and/or antihypertensive medication (n = 418), we found a non-significant association with microalbuminuria (OR 1.04 [95% CI 0.99, 1.10]) and macroalbuminuria (OR 1.05 [95% CI 0.96, 1.16]). The association between dietary salt intake and microalbuminuria was stronger in individuals with a BMI above 25 kg/m(2) (OR 1.11 [95% CI 1.04, 1.18]) than in those with BMI below 25 kg/m(2) (OR 1.03 [95% CI 0.97, 1.09]). No significant associations were found between urinary potassium excretion and microvascular complications. CONCLUSIONS/INTERPRETATION: In individuals with type 1 diabetes, higher dietary salt intake, as determined by 24 h urinary sodium excretion, may be positively associated with microalbuminuria, particularly in overweight individuals.

Atherogenic lipids and vascular complications in a selected diabetic population with normal urinary albumin/creatinine ratios.

OBJECTIVE: To test the hypothesis that at different urinary albumin/creatinine ratios within the normal ranges, diabetics have low but similar prevalence of metabolic and micro vascular disease. METHODS: The study sample consisted of normotensive diabetics not taking any medications known to effect blood pressure and lipids. The data were collected from the Diabetes Register. The diabetics were subgrouped according to the urinary albumin/creatinine ratios. MA is defined as present if the albumin/creatinine ratio (ACR) is more than 2 mg/mmol. RESULTS: MA was present in 16% of the 152 diabetics. Total cholesterol, systolic BP, and triglycerides were significantly high in diabetics with ACR≥1<2 compared with <1. The prevalence rates for retinopathy and neuropathy in the MA group were also significantly high. However, a large number of diabetics without MA had had established complications (37% retinopathy, 40% neuropathy, and 16% peripheral vascular disease). Because these results were based on single early morning urine samples, we looked at their MA in the past year. After exclusion of regressed and progressed groups, the complications rate remained the same. CONCLUSION: The high prevalence of metabolic and vascular complications seen even in absence of MA indicates an early intervention and those diabetics should not wait unitl CVD risk scores raise to receive preventive treatment.

Pursuing type 1 diabetes mellitus and related complications through urinary proteomics.

Diabetes mellitus is a chronic metabolic disease with multiple complications, and its successful management requires early diagnosis, to allow timely interventions. Here, we have comprehensively analyzed the proteome changes in urine of type 1 diabetic subjects with and without complications such as retinopathy and nephropathy. gel electrophoresis combined to liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) analysis of midstream urine highlighted the mechanisms involved in disease pathogenesis as, for instance wound healing and blood coagulation in all diabetics or altered ganglioside metabolism in retinopathy, and also some urinary proteins with potential diagnosis value. From these, gelsolin and antithrombin-III appear as promising diagnosis markers for type 1 diabetes mellitus (T1DM), whereas ephrin type-B receptor 4 and vitamin K-dependent protein Z seem to be promising markers for advanced T1DM disease state presenting retinopathy and nephropathy (T1DM-R + N). Data also suggest urinary ganglioside GM2 activator and beta-hexosaminidase subunit beta as potential urinary markers of retinopathy in diabetics. Taken together, the present exploratory urinary proteomic analysis might be seen as an important starting point for studies targeting specific urinary proteins aimed at the implementation of new biomarkers for the early detection of T1DM-related microvascular complications.