Early urinary markers of diabetic kidney disease: a nested case-control study from the Diabetes Control and Complications Trial (DCCT).

BACKGROUND: Urinary markers were tested as predictors of macroalbuminuria or microalbuminuria in patients with type 1 diabetes. STUDY DESIGN: Nested case-control of participants in the Diabetes Control and Complications Trial (DCCT). SETTING & PARTICIPANTS: 87 cases of microalbuminuria were matched to 174 controls in a 1:2 ratio, while 4 cases were matched to 4 controls in a 1:1 ratio, resulting in 91 cases and 178 controls for microalbuminuria. 55 cases of macroalbuminuria were matched to 110 controls in a 1:2 ratio. Controls were free of micro-/macroalbuminuria when their matching case first developed micro-/macroalbuminuria. PREDICTORS: Urinary N-acetyl-beta-d-glucosaminidase (NAG), pentosidine, advanced glycation end product (AGE) fluorescence, and albumin excretion rate (AER). OUTCOMES: Incident microalbuminuria (2 consecutive annual AERs > 40 but < or = 300 mg/d) or macroalbuminuria (AER > 300 mg/d). MEASUREMENTS: Stored urine samples from DCCT entry and 1-9 years later when macro- or microalbuminuria occurred were measured for the lysosomal enzyme NAG and the AGE pentosidine and AGE fluorescence. AER and adjustor variables were obtained from the DCCT. RESULTS: Submicroalbuminuric AER levels at baseline independently predicted microalbuminuria (adjusted OR, 1.83; P < 0.001) and macroalbuminuria (adjusted OR, 1.82; P < 0.001). Baseline NAG excretion independently predicted macroalbuminuria (adjusted OR, 2.26; P < 0.001) and microalbuminuria (adjusted OR, 1.86; P < 0.001). Baseline pentosidine excretion predicted macroalbuminuria (adjusted OR, 6.89; P = 0.002). Baseline AGE fluorescence predicted microalbuminuria (adjusted OR, 1.68; P = 0.02). However, adjusted for NAG excretion, pentosidine excretion and AGE fluorescence lost the predictive association with macroalbuminuria and microalbuminuria, respectively. LIMITATIONS: Use of angiotensin-converting enzyme inhibitors was not directly ascertained, although their use was proscribed during the DCCT. CONCLUSIONS: Early in type 1 diabetes, repeated measurements of AER and urinary NAG excretion may identify individuals susceptible to future diabetic nephropathy. Combining the 2 markers may yield a better predictive model than either one alone. Renal tubule stress may be more severe, reflecting abnormal renal tubule processing of AGE-modified proteins, in individuals susceptible to diabetic nephropathy.

Effects of advanced glycation end product modification on proximal tubule epithelial cell processing of albumin.

AIM: The goal of this work is to understand the cellular effects of advanced glycation end product (AGE)-modified protein on renal proximal tubule cells. BACKGROUND: A major function of the proximal tubule is to reabsorb and process filtered proteins. Diabetes is characterized by increased quantities of tissue and circulating proteins modified by AGEs. Therefore in diabetes, plasma proteins filtered at the glomerulus and presented to the renal proximal tubule are likely to be highly modified by AGEs. METHODS: The model system was electrically resistant polarized renal proximal tubular epithelial cells in monolayer culture. The model proteins comprise a well-characterized AGE, methylglyoxal-modified bovine serum albumin (MGO-BSA), and unmodified BSA. RESULTS: Renal proximal tubular cells handle MGO-BSA and native BSA in markedly disparate ways, including differences in: (1) kinetics of binding, uptake, and intracellular accumulation, (2) processing and fragmentation, and (3) patterns of electrical conductance paralleling temporal changes in binding, uptake and processing. CONCLUSION: These differences support the idea that abnormal protein processing by the renal tubule can be caused by abnormal proteins, thereby forging a conceptual link between the pathogenic role of AGEs and early changes in tubular function that can lead to hypertrophy and nephropathy in diabetes.

Serum vitamin E and oxidative protein modification in hemodialysis: a randomized clinical trial.

BACKGROUND: Patients with end-stage renal disease have increased circulating concentrations of oxidatively modified circulating proteins. Therefore, we examined the ability of vitamin E alpha (alpha-tocopherol) to alter levels of these modified proteins. STUDY DESIGN: Randomized clinical trial. SETTING & PARTICIPANTS: 27 clinically stable patients treated by means of hemodialysis in 4 freestanding outpatient dialysis units. INTERVENTION: Oral administration of 800 IU of vitamin E alpha or placebo daily. OUTCOMES & MEASUREMENTS: Plasma levels of alpha- and gamma-tocopherol and oxidative protein modifications reflecting 2 pathways for protein-oxidant damage. The advanced glycation end product pentosidine reflects glycoxidation. The lipid peroxidation products iso[4]-levuglandin E(2), (E)-4-hydroxy-2-nonenal, and (E)-4-oxo-2-nonenal are formed through covalent adduction. RESULTS: Circulating levels of all oxidative protein modifications were increased in patients with end-stage renal disease. Supplementation with alpha-tocopherol caused alpha-tocopherol levels to rise (13.2 +/- 3.7 to 27.3 +/- 14 mug/mL), but gamma-tocopherol levels to decrease (4.1 +/- 1.6 to 3.5 +/- 1.1 mug/mL). Control values were unchanged. There was no effect on oxidative protein modifications (placebo versus treatment; mean for pentosidine, 15.6 +/- 11.4 (SD): 95% confidence interval (CI), 8.2 to 23.1 versus 21.3 +/- 9.0 pg/mg protein; 95% CI, 16.1 to 26.6; iso[4]-levuglandin E(2), 8.31 +/- 2.55; 95% CI, 6.77 to 9.85 versus 8.46 +/- 2.37 nmol/mL; 95% CI, 7.09 to 9.84; (E)-4-hydroxy-2-nonenal, 0.51 +/- 0.11; 95% CI, 0.45 to 0.57 versus 0.51 +/- 0.08 nmol/mL; 95% CI, 0.46 to 0.56; (E)-4-oxo-2-nonenal, 189 +/- 44; 95% CI, 162 to 215 vs 227 +/- 72 pmol/mL; 95% CI, 183 to 271). LIMITATIONS: Sample size was adequate to show changes in alpha- and gamma-tocopherol levels in response to treatment. However, power was insufficient to show an effect on oxidative protein modifications. CONCLUSIONS: Intervention of oral supplementation with alpha-tocopherol did not result in changes in circulating oxidative protein modifications. A larger study may be required to show an effect in this clinical setting.

Processing advanced glycation end product-modified albumin by the renal proximal tubule and the early pathogenesis of diabetic nephropathy.

Diabetes is characterized by increased quantities of circulating proteins modified by advanced glycation end products (AGEs). Proteins filtered at the glomerulus and presented to the renal proximal tubule are likely to be highly modified by AGEs. The proximal tubule binds, takes up, and catabolizes AGE-modified albumin by pathways different from those of unmodified albumin. These differences were examined in polarized, electrically resistant proximal tubular cells grown in monolayer culture. In patients with type 1 diabetes, urinary excretion of a lysosomal enzyme predicted the development of nephropathy.

Glycation of mitochondrial proteins from diabetic rat kidney is associated with excess superoxide formation.

Chronic hyperglycemia causes structural alterations of proteins through the Maillard reaction. In diabetes, methylglyoxal (MGO)-induced hydroimidazolones are the predominant modification. In contrast to acute hyperglycemia, mitochondrial respiration is depressed in chronic diabetes. To determine whether MGO-derived protein modifications result in abnormalities in mitochondrial bioenergetics and superoxide formation, proteomics and functional studies were performed in renal cortical mitochondria isolated from rats with 2, 6, and 12 mo of streptozotocin-induced diabetes. MGO-modified proteins belonged to the following two pathways: 1) oxidative phosphorylation and 2) fatty acid beta-oxidation. Two of these proteins were identified as components of respiratory complex III, the major site of superoxide production in health and disease. Mitochondria from rats with diabetes exhibited a diminution of oxidative phosphorylation. A decrease in the respiratory complex III activity was significantly correlated with the quantity of MGO-derived hydroimidazolone present on mitochondrial proteins in both diabetic and control animals. In diabetes, isolated renal mitochondria produced significantly increased quantities of superoxide and showed evidence of oxidative damage. Administration of aminoguanidine improved mitochondrial respiration and complex III activity and decreased oxidative damage to mitochondrial proteins. Therefore, posttranslational modifications of mitochondrial proteins by MGO may represent pathogenic events leading to mitochondria-induced oxidative stress in the kidney in chronic diabetes.

Paradoxical effects of green tea (Camellia sinensis) and antioxidant vitamins in diabetic rats: improved retinopathy and renal mitochondrial defects but deterioration of collagen matrix glycoxidation and cross-linking.

We tested the hypothesis that green tea prevents diabetes-related tissue dysfunctions attributable to oxidation. Diabetic rats were treated daily with tap water, vitamins C and E, or fresh Japanese green tea extract. After 12 months, body weights were decreased, whereas glycated lysine in aorta, tendon, and plasma were increased by diabetes (P < 0.001) but unaffected by treatment. Erythrocyte glutathione and plasma hydroperoxides were improved by the vitamins (P < 0.05) and green tea (P < 0.001). Retinal superoxide production, acellular capillaries, and pericyte ghosts were increased by diabetes (P < 0.001) and improved by green tea and the vitamins (P variable). Lens crystallin fluorescence at 370/440 nm was ameliorated by green tea (P < 0.05) but not the vitamins. Marginal effects on nephropathy parameters were noted. However, suppressed renal mitochondrial NADH-linked ADP-dependent and dinitrophenol-dependent respiration and complex III activity were improved by green tea (P variable). Green tea also suppressed the methylglyoxal hydroimidazolone immunostaining of a 28-kDa mitochondrial protein. Surprising, glycoxidation in tendon, aorta, and plasma was either worsened or not significantly improved by the vitamins and green tea. Glucosepane cross-links were increased by diabetes (P < 0.001), and green tea worsened total cross-linking. In conclusion, green tea and antioxidant vitamins improved several diabetes-related cellular dysfunctions but worsened matrix glycoxidation in selected tissues, suggesting that antioxidant treatment tilts the balance from oxidative to carbonyl stress in the extracellular compartment.

Assessing 24-hour blood glucose patterns in diabetic paitents treated by peritoneal dialysis.

The minute-to-minute effect on blood glucose levels of high-dextrose peritoneal dialysate is not known. We arranged for 7 patients with diabetes, treated by peritoneal dialysis (PD), to wear a continuous glucose monitoring system (CGMS: Medtronic MiniMed, Northridge, CA, U.S.A.). A sensor was inserted subcutaneously into the skin of the patient's abdomen or back to measure glucose in the interstitial fluid. Readings were recorded every 5 minutes for up to 72 hours. The portion of the day during which the patient's blood glucose levels were greater than 180 mg/dL (calculated as a percentage of time) was recorded. Most of the patients participating in the study had elevated levels of glycohemoglobin and hemoglobin A1c, and, for a large percentage of the day, showed blood glucose tracings well above the recommended standards of control. Representative CGMS tracings from patients with type 1 and type 2 diabetes are shown.

Thrombotic events and markers of oxidation and inflammation in hemodialysis.

The goal of this study was to determine whether antioxidant therapy with vitamin E would alter the rate of vascular access complications or other macrovascular complications in hemodialysis (HD) patients. A secondary goal of the study was to explore the relationship between baseline pretreatment markers of oxidative stress (the advanced glycation end product pentosidine and basal levels of vitamin Ealpha and gamma) and the subsequent development of access failure. Thirty-five stable patients treated by HD were recruited for the study. Patients were provided with vitamin E (800 IU) or placebo capsules to be taken daily. Clinical variables, vascular access function (flow meter access flow measurements), and circulating blood markers were obtained initially and every 3 months throughout the study. Vitamin Ealpha levels rose in treated patients from 12.7 +/- 4.4 to 25.1 +/- 15.1 microg/mL at 3 months and 28.6 +/- 14.8 microg/mL at 6 months. Vitamin Egamma levels fell in treated patients from 3.9 +/- 1.7 to 2.3 +/- 1.5 microg/mL at 3 months and 1.7 microg/mL at 6 months. Patients who subsequently developed repeated thrombotic vascular access events were characterized by higher baseline pentosidine content of circulating proteins. Patients who developed a myocardial infarction had higher pentosidine, lower vitamin Ealpha, and much lower vitamin Egamma than patients who did not develop thrombotic events. These findings lead to the speculation that the anti-inflammatory effects of vitamin Egamma may play a more important role in thrombotic vascular events than the antioxidant effects of vitamin Ealpha. Additional studies of these interactions are in progress.

Effect of the peritoneal dialysis prescription on pentosidine in children.

Enhanced formation of advanced glycation end products (AGEs) by peritoneal dialysate containing high dextrose concentrations has been implicated as a source of peritoneal membrane toxicity and loss of viability in patients treated with peritoneal dialysis (PD). The goal of this project was to elucidate the relationship between the structurally defined AGE pentosidine accumulation on peritoneal and plasma proteins and peritoneal membrane function, and to identify clinical factors leading to alterations in these parameters. The study comprised 27 pediatric patients (14 continuous ambulatory PD, 13 chronic cycling PD) on PD for a mean duration of 37.0+/-22.8 months (range 1-120 months) and with a mean age of 13.3+/-4.4 years (range 2.4-20 years). The pentosidine contents of plasma and peritoneal proteins were significantly lower in patients with residual renal function than in patients who were anuric (plasma pentosidine 11.2+/-8.8 vs. 24.1+/-16.6, P=0.02, respectively, peritoneal pentosidine 14.9+/-11.9 vs. 31.1+/-3.7, P=0.01, respectively). There was no effect of treatment modality on plasma pentosidine (18.1+/-11.2, 18.8+/-19.3, CAPD vs. CCPD, P>0.05) or peritoneal pentosidine content (24.1+/-14.1, 24.9+/-19.6, CAPD vs. CCPD, P>0.05). There was no evidence that increased levels of pentosidine on peritoneal proteins reflect or affect peritoneal membrane function in these patients. Furthermore, there was no effect of peritonitis on the pentosidine content of peritoneal proteins or peritoneal function as measured by peritoneal equilibration test. In conclusion, PD represents a well-tolerated therapy in children with no evidence that current practice causes changes in peritoneal membrane function, or in the peritoneal clearance of plasma or peritoneal proteins rich in pentosidine.

Abdominal catastrophe revisited: the risk and outcome of enteric peritoneal contamination.

OBJECTIVE: Peritonitis from a visceral source is associated with striking morbidity and mortality in patients treated with peritoneal dialysis (PD). Surgical intervention for both diagnosis and repair is definitive. However, because the antecedents of enteric injury leading to peritonitis are unpredictable, no preventive strategy has been proposed or adopted. The goal of this study was to examine risk factors influencing the occurrence and outcome of anatomically documented peritonitis of enteric origin. DESIGN: Retrospective chart and database review. SETTING: Peritoneal dialysis unit in tertiary-care referral hospital. PATIENTS: 330 patients treated with PD for end-stage renal disease between 1988 and 2000. MAIN OUTCOME MEASURES: Prevalence of peritonitis of anatomically documented enteric origin over two consecutive time periods within the study interval: period 1, from 1 January 1988 through 30 June 1996; period 2, from 1 July 1996 through 30 June 2000. RESULTS: At least 1 episode of peritonitis occurred in 202 of 330 patients during the entire study period of 12.5 years (600.74 patient-years of care). There were 543 episodes of peritonitis. Anatomically documented visceral Injury caused bacterial peritonitis in 41 patients with a total of 63 discrete episodes, an incidence rate of 0.1048 per patient-year. Peritonitis-free survival was compared between the two periods using Kaplan-Meier analysis. The curve representing risk distribution for anatomically documented visceral peritonitis remained constant over the two periods, in contrast to improvements found in all other types of peritonitis, taken as a group (p= 0.044). Logistic regression modeling showed that the only risk factor associated with development of anatomically documented visceral peritonitis was older age. There was no influence of race, sex, time on PD, and underlying disease etiology. 31 deaths were attributed to peritonitis during the study period. The mortality rate from enteric peritonitis due to visceral injury was 46.3% (19/41 cases), compared to 7.5% for all other peritonitis taken as a group (12/161 cases, p < 0.0001). CONCLUSIONS: The experience at University Hospitals of Cleveland suggests that abdominal catastrophe occurs in approximately 10% of all patients treated with PD, and is associated with high mortality, which has not changed over time. Therefore, peritonitis due to spontaneous visceral injury presents a great diagnostic and therapeutic challenge. It is important to develop a research strategy to understand this devastating complication.

Alterations in renal mitochondrial respiration in response to the reactive oxoaldehyde methylglyoxal.

Chronic hyperglycemia has been linked to alterations in mitochondrial function, suggesting an important role in the pathophysiology of the complications of diabetes mellitus. In the diabetic kidney, ultrastructural changes in mitochondria are associated with impaired tubular function. The goal of this study was to determine if methylglyoxal (MGO), a dicarbonyl compound reaching high levels in hyperglycemic conditions, has direct toxicity for renal mitochondria. Intact mitochondria isolated from the renal cortex of rats were incubated with MGO to determine 1) its effect on mitochondrial respiration, 2) the conditions under which MGO exerts these effects, and 3) the potential mitochondrial targets of MGO influence. This study demonstrates that MGO has an inhibitory effect on both the tricarboxylic acid cycle and the electron respiratory chain. The modifications appear to be specific to certain mitochondrial proteins. Alterations of these proteins lead to disturbances in mitochondria that may play an important role in renal cellular toxicity and in the development of diabetic nephropathy.

Cellular oxidant stress and advanced glycation endproducts of albumin: caveats of the dichlorofluorescein assay.

In order to understand the mechanism by which advanced glycation endproducts (AGEs) elicit oxidative stress, macrophage-like RAW264.7 cells were exposed to various AGE-albumins, and oxidant stress was estimated from the fluorescence of oxidized dichlorofluorescein using the microtiter plate assay. Strongest fluorescence was observed with methylglyoxal modified albumin (MGO-BSA) compared with native albumin. Similar effects that were prevented by arginine coincubation were seen with phenylglyoxal-BSA. MGO-BSA had increased affinity for Cu(2+) and Ca(2+), but was conformationally similar to native albumin. Surprisingly, the mere addition of unmodified albumin to cells suppressed the fluorescence of oxidized DCF. While, several site-directed mutants of human serum albumin (HSA), including C34S and recombinant domains II and III retained fluorescence suppressing activity, proteolytic digests, recombinant domain I, and several nonalbumin proteins failed to suppress. Kinetic and ANS binding studies suggested albumin quenches DCF fluorescence by binding to hydrophobic pockets in domains II and III and that MGO-BSA is less hydrophobic than BSA. Finally, BSA also prevented H(2)O(2) catalyzed DCF fluorescence more potently than MGO-BSA. These studies reveal important caveats of the widely used dichlorofluorescein assay and suggest methods other than the microtiter plate assay are needed to accurately assess cellular oxidant stress in presence of native or modified albumin.