老年2型糖尿病合并慢性肾脏病患者肠道菌群变化

目的 探讨合并慢性肾脏病的老年2型糖尿病患者肠道菌群变化情况,为后续研究提供参考。 方法 选择2018年11月至2019年7月在我院内科确诊的老年2型糖尿病及并发慢性肾脏病患者作为糖尿病组(20例)及糖尿病肾病组(20例),选择本院健康老年体检者(30例)为对照组。测定各组对象血清甲状旁腺激素(PTH)、超敏C反应蛋白(hs CRP)、血肌酐(Scr)、血磷(P)、血钙(Ca2+)、碱性磷酸酶(ALP)、糖化血红蛋白(HbA1c)水平,同时分析各组对象肠道大肠埃希菌、双歧杆菌、乳杆菌、酵母菌、真杆菌的差异。 结果 糖尿病肾病组患者肠道大肠埃希菌数量高于糖尿病组和对照组,糖尿病组患者肠道大肠埃希菌数量高于对照组(均P2+水平高于糖尿病组和对照组,糖尿病组患者血清Scr、P、Ca2+水平高于对照组(均P2+、HbA1c、ALP、hs CRP水平呈正相关,双歧杆菌、真杆菌数量与Scr、P、Ca2+、HbA1c、ALP、hs CRP水平呈负相关(均P结论 糖尿病肾病患者肠道菌群失调,其特征为大肠埃希菌数量增多,双歧杆菌、真杆菌数量减少。大肠埃希菌数量升高,双歧杆菌和真杆菌数量降低均为糖尿病并发慢性肾脏病的危险因素。     

Increased oxidative damage to all DNA bases in patients with type II diabetes mellitus

Gas chromatography-mass spectrometry was used to measure the oxidative DNA damage in diabetic subjects and controls. Levels of multiple DNA base oxidation products, but not DNA base de-amination or chlorination products, were found to be elevated in white blood cell DNA from patients with type II diabetes as compared with age-matched controls. The chemical pattern of base damage is characteristic of that caused by an attack on DNA by hydroxyl radical. An increased formation of the highly reactive hydroxyl radical could account for many of the reports of oxidative stress in diabetic subjects. There was no evidence of an increased DNA damage by reactive nitrogen or chlorine species.     

Basal damage and oxidative DNA damage in children with chronic kidney disease measured by use of the comet assay

One consequence of chronic kidney disease (CKD) is an elevated risk for cancer. There is sufficient evidence to conclude that there is an increased incidence of at least some cancers in kidney-dialysis patients. Cancer risk after kidney transplantation has mainly been attributed to immunosuppressive therapy. There are no data evaluating DNA damage in children with CKD, in dialysis patients, or following kidney transplantation. In this study, the comet assay and the enzyme-modified comet assay - with the use of endonuclease III (Endo III) and formamidopyrimidine glycosylase (FPG) enzymes - were conducted to investigate the basal damage and the oxidative DNA damage as a result of treatment in peripheral blood lymphocytes of children. Children at various stages of treatment for kidney disease, including pre-dialysis patients (PreD) (n=17), regular hemodialysis patients (HD) (n=15), and those that received kidney transplants (Tx) (n=17), comprised the study group. They were compared with age- and gender-matched healthy children (n=20) as a control group. Our results show that the %DNA intensity, a measure of basal damage, was significantly increased in children with CKD (mean ± SD) (5.22 ± 1.57) and also in each of the PreD, HD, and Tx groups [(4.92 ± 1.23), (4.91 ± 1.35), and (5.79 ± 1.94), respectively, vs the healthy children (2.74 ± 2.91) (p<0.001). Significant increases in oxidative DNA damage were only found in the FPG-sensitive sites for the PreD and Tx groups, compared with control and HD groups (p<0.05), suggesting that basal DNA damage was more evident for the PreD, HD, and Tx groups. The findings of the present study indicate a critical need for further research on genomic damage with different endpoints and also for preventive measures and improvements in treatment of pediatric patients, in order to improve their life expectancy.     

2型糖尿病与糖尿病肾病患者微炎症及肠道微生物多样性分析

目的 分析2型糖尿病(T2DM)及糖尿病肾病(DKD)患者微炎症情况和肠道微生物多样性。 方法 将2016年4月至2019年7月在我院进行治疗的68例T2DM患者(T2DM组)和57例DKD患者(DKD组)纳入研究,选择同期于我院进行健康体检的36例志愿者作为对照组。收集3组对象一般资料、血液标本和粪便标本,测定血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDLC)、高密度脂蛋白胆固醇(HDLC)、糖化血红蛋白(HbAlc)、空腹血糖(FBG)、超敏C反应蛋白(hsCRP)、白细胞介素6(IL6)水平,并对肠道细菌进行16S rDNA序列测序。比较3组对象一般资料,血液指标,肠道菌群门水平构成情况,肠道菌群多样性,肠道菌属差异性,并对患者炎性指标与菌群种类进行相关性分析。 结果 DKD组患者糖尿病病程长于T2DM组(P结论 2型糖尿病患者普遍存在微炎症和肠道菌群失衡,微炎症程度与肾脏病变和肠道菌群数量密切相关。T2DM与DKD患者在肠道菌群结构上具有一致性和差异性,肠道菌群检测有可能成为预测T2DM患者发生肾脏病变的风险指标。     

Genetic factors in the development of chronic kidney disease in patients with diabetes mellitus

Patients with diabetes mellitus (DM) represent a risk group for developing chronic kidney disease (CKD), the terminal stages of which require renal replacement therapy. The study of genetic predisposition to CKD is of special significance from the perspective of the prediction and identification of risk groups at the preclinical stage. The present study is a review of the world literature on the genetics of CKD in diabetes and also includes data of our own studies.     

Simvastatin treatment prevents oxidative damage to DNA in whole blood leukocytes of dyslipidemic type 2 diabetic patients

Type 2 diabetes (T2D) is associated with increased oxidative stress as indicated by elevated levels of lipid peroxidation and protein oxidation products. Since reactive oxygen species (ROS) can cause damage to biological macromolecules including DNA, this study investigated oxidative damage to DNA using the alkaline (pH > 13) comet assay in peripheral whole blood leukocytes sampled from 15 dyslipidemic T2D patients treated with simvastatin (20 mg/day), 15 dyslipidemic T2D patients not treated with simvastatin, 20 non‐dyslipidemic T2D patients, and 20 healthy individuals (controls). Our results showed a greater DNA migration in terms of damage index (DI) (p < 0.01) in the dyslipidemic T2D patients not treated with statin (DI = 67.70 ± 10.89) when compared to the dyslipidemic T2D patients under statin treatment (DI = 47.56 ± 7.02), non‐dyslipidemic T2D patients (DI = 52.25 ± 9.14), and controls (DI = 13.20 ± 6.40). Plasma malondialdehyde (MDA) and C‐reactive protein (CRP) levels were also increased and total antioxidant reactivity (TAR) and paraoxonase activity (PON1) decreased in non‐dyslipidemic T2D patients and dyslipidemic T2D non‐treated with simvastatin. We also found that DI was inversely correlated with TAR (r = ?0.61, p < 0.05) and PON1 (r = ?0.67, p < 0.01). In addition, there was a significant positive correlation between DI and CRP (r = 0.80, p < 0.01). Our results therefore indicate that simvastatin treatment plays a protective role on oxidative damage to DNA in dyslipidemic T2D patients probably reflecting a general decrease in oxidative stress in these patients. Copyright © 2010 John Wiley & Sons, Ltd.     

Avocado oil induces long-term alleviation of oxidative damage in kidney mitochondria from type 2 diabetic rats by improving glutathione status

Hyperglycemia and mitochondrial ROS overproduction have been identified as key factors involved in the development of diabetic nephropathy. This has encouraged the search for strategies decreasing glucose levels and long-term improvement of redox status of glutathione, the main antioxidant counteracting mitochondrial damage. Previously, we have shown that avocado oil improves redox status of glutathione in liver and brain mitochondria from streptozotocin-induced diabetic rats; however, the long-term effects of avocado oil and its hypoglycemic effect cannot be evaluated because this model displays low survival and insulin depletion. Therefore, we tested during 1 year the effects of avocado oil on glycemia, ROS levels, lipid peroxidation and glutathione status in kidney mitochondria from type 2 diabetic Goto-Kakizaki rats. Diabetic rats exhibited glycemia of 120–186 mg/dL the first 9 months with a further increase to 250–300 mg/dL. Avocado oil decreased hyperglycemia at intermediate levels between diabetic and control rats. Diabetic rats displayed augmented lipid peroxidation and depletion of reduced glutathione throughout the study, while increased ROS generation was observed at the 3rd and 12th months along with diminished content of total glutathione at the 6th and 12th months. Avocado oil ameliorated all these defects and augmented the mitochondrial content of oleic acid. The beneficial effects of avocado oil are discussed in terms of the hypoglycemic effect of oleic acid and the probable dependence of glutathione transport on lipid peroxidation and thiol oxidation of mitochondrial carriers.     

仙灵脾对2型糖尿病大鼠血糖及氧化应激的影响

目的:研究仙灵脾对2型糖尿病大鼠血糖及氧化应激的影响。方法:利用链脲佐菌素建立2型糖尿病大鼠动物模型,将wistar大鼠随机分为对照组、单纯造模组、仙灵脾组及二甲双胍组。治疗8周,观察治疗期间及治疗后大鼠的一般状况、血糖、胰岛素、MCP-1、8-iso-PGF2α及24h尿白蛋白排泄量,计算ISI。结果:仙灵脾能改善2型糖尿病大鼠基本状况,仙灵脾组大鼠的血糖、胰岛素、8-iso-PGF2α及24h尿白蛋白排泄量均较单纯造模组降低,ISI明显提高;降低尿白蛋白优于二甲双胍组(P0.05),改善ISI、降低血糖不及二甲双胍组明显,胰岛素、MCP-1、8-iso-PGF2α,两组无明显差异(P0.05)。结论:仙灵脾对2型糖尿病大鼠有降糖、改善氧化应激作用,其降糖作用可能与降低MCP-1、8-iso-PGF2α有关。     

Adiponectin gene polymorphisms in Egyptian type 2 diabetes mellitus patients with and without diabetic nephropathy

Recently, several reports addressed the associations of adiponectin (ADIPOQ) gene polymorphisms with abnormal adiponectin serum levels, type 2 diabetes mellitus (T2DM), and diabetic nephropathy (DN); however, results are inconsistent. This study aimed to investigate the possible association of ADIPOQ gene polymorphisms with T2DM and/or DN and whether they affect serum adiponectin levels in Egyptian population. Two hundred and ninety-six T2DM patients (100 normoalbuminuric patients, 103 microalbuminuric patients, and 93 macroalbuminuric patients) and 209 controls were enrolled in the present study. Polymorphisms of +45, ?11391, and +276 of the ADIPOQ gene were detected using polymerase chain reaction restriction fragment length polymorphism. Serum adiponectin was measured using ELISA. Our results revealed that ADIPOQ +45 TG and GG genotypes and G allele were significantly associated with T2DM, micro/macroalbuminuria, and decreased serum adiponectin level. ADIPOQ ?11391 AA genotype frequency was significantly increased in T2DM group. Moreover, GA and AA genotypes and A allele of ADIPOQ ?11391 were significantly associated with susceptibility to macroalbuminuria despite increased serum adiponectin concentrations. While, ADIPOQ +276 TT genotype and T allele were protective factors regarding the susceptibility to T2DM and micro/macroalbuminuria, and they were significantly associated with increased adiponectin levels. We observed also that the decrease of the serum Adiponectin level was accompanied by an insulin resistance, albuminuria, as well as an increase of serum creatinine. We concluded that ADIPOQ +45; ADIPOQ ?11391 gene polymorphisms are associated with T2DM and/or DN in Egyptian population. While, ADIPOQ +276 gene polymorphism is a protective factor regarding T2DM and/or DN susceptibility.     

Superoxide dismutase type 1 in monocytes of chronic kidney disease patients

We analyzed proteomic profiles in monocytes of chronic kidney disease (CKD) patients and healthy control subjects. Two-dimensional electrophoresis (2-DE) and silver staining indicated differences in protein pattern. Among the analyzed proteins, superoxide dismutase type 1 (SOD1), which was identified both by MS/MS mass-spectrometry and immunoblotting, was reduced in kidney disease. We characterized SOD1 protein amount, using quantitative in-cell Western assay and immunostaining of 2-DE gel blots, and SOD1 gene expression, using quantitative real-time polymerase chain reaction (PCR), in 98 chronic hemodialysis (HD) and 211 CKD patients, and 34 control subjects. Furthermore, we showed that different SOD1 protein species exist in human monocytes. SOD1 protein amount was significantly lower in HD (normalized SOD1 protein, 27.2 ± 2.8) compared to CKD patients (34.3 ± 2.8), or control subjects (48.0 ± 8.6; mean ± SEM; P < 0.05). Analysis of SOD1 immunostaining showed significantly more SOD1 protein in control subjects compared to patients with CKD or HD (P < 0.0001, analysis of main immunoreactive protein spot). SOD1 gene expression was significantly higher in HD (normalized SOD1 gene expression, 17.8 ± 2.3) compared to CKD patients (9.0 ± 0.7), or control subjects (5.5 ± 1.0; P < 0.0001). An increased SOD1 gene expression may indicate increased protein degradation in patients with CKD and compensatory increase of SOD1 gene expression. Taken together, we show reduced SOD1 protein amount in monocytes of CKD, most pronounced in HD patients, accompanied by increased SOD1 gene expression.     

Oxidative protein damage in plasma of type 2 diabetic patients.

In this study, we evaluated protein oxidation in 84 patients with Type 2 diabetes with no complications and in 61 healthy volunteers who formed the control group, whose ages matched those of the patients. We determined plasma carbonyl and plasma thiol levels as markers of oxidative protein damage and erythrocyte glutathione, plasma ceruloplasmin and transferrin as markers of free radical scavengers. The concentrations (mean +/- SD) of both of plasma carbonyl (1.24 +/- 0.46 vs. 0.72 +/- 0.17 nmole/mg protein; p < 0.0001) and lipid hydroperoxides (1.8 +/- 0.63 vs. 1.3 +/- 0.21 micromole/l; p < 0.0001) were increased, and the concentration of plasma transferrin (3.85 +/- 0.65 vs. 4.59 +/- 0.79 g/l; p < 0.05) was decreased, respectively, in Type 2 diabetic patients compared with those of the controls. There were no significant differences in the concentrations of plasma thiol (0.0064 +/- 0.001 vs. 0.0068 +/- 0.001 micromole/mg protein), erythrocyte glutathione (2.54 +/- 0.57 vs. 2.65 +/- 0.56 mg/g Hb), plasma ceruloplasmin (548 +/- 107.30 vs. 609 +/- 93.34 mg/l) between the patients and the controls. These changes observed in diabetic patients contribute to the imbalance in the redox status of the plasma. We attribute this imbalance to oxidative protein damage in Type 2 diabetic patients clinically free of complications.     

Altered plasma acylcarnitine and amino acid profiles in type 2 diabetic kidney disease

Introduction

Dysregulation of acylcarnitines (AcylCNs) and amino acids metabolism have implicated in abnormality of fatty acid oxidation in type 2 diabetes (T2D). However, it is not well known whether altered plasma AcylCN, and amino acid profiles are associated with albuminuria or diabetic nephropathy (DN) in T2D.

Objective

The aim of this study was to elucidate alterations in plasma levels of AcylCNs and amino acids with respect to the T2D patients with various stages of albuminuria.

Methods

We recruited 52 healthy subjects as control, and 156 T2D patients which were divided into 52 normoalbuminuria, 52 microalbuminuria, and 52 macroalbuminuria. Plasma 37 AcylCNs and 12 amino acids were analyzed by tandem mass spectrometry.

Results

We found that T2D with normoalbuminuria and microalbuminuria had lower shot-, medium-, and long-chain AcylCNs, whereas T2D with macroalbuminuria had higher short-and medium-chain AcylCNs and lower long-chain AcylCNs than healthy subjects. Moreover, estimated glomerular filtration rate (eGFR) was a negative, independent and significant predictor of albumin to creatinine ratio (ACR) levels (β = ?0.376, P < 0.001), whereas plasma Low-density lipoprotein cholesterol (LDL-C) was significantly and positively associated with ACR levels (β = 0.169, P = 0.049). Furthermore, multivariate ordinal logistic regression analysis revealed that isobutyrylcarnitine (C4) was a positive, independent, and significant predictor of ACR levels with higher odds of having T2D patients with progression normoalbuminuria to microalbuminuria [OR = 9.93, 95 % CI (3.51–28.05), P < 0.001].

Conclusions

The findings suggest that plasma C4 may serve as a potential biomarker for the early stages of DN.

     

Mitochondrial defects and oxidative damage in patients with peripheral arterial disease

Abnormal mitochondrial function is present in patients with peripheral arterial disease and may contribute to its clinical manifestations. However, the specific biochemical mitochondrial defects and their association with increased oxidative stress have not been fully characterized. Gastrocnemius muscle was obtained from peripheral arterial disease patients (n = 25) and age-matched controls (n = 16) and mitochondrial parameters were measured. Complexes I through IV of the electron transport chain were individually evaluated to assess for isolated defects. Muscle was also evaluated for protein and lipid oxidative changes by measuring the levels of protein carbonyls, lipid hydroperoxides, and total 4-hydroxy-2-nonenal binding and for the activities of the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase. Mitochondrial electron transport chain complexes I, III, and IV in arterial disease patients demonstrated significant reductions in enzymatic activities and mitochondrial respiration compared to controls. Oxidative stress biomarker analysis demonstrated significantly increased levels of protein carbonyls, lipid hydroperoxides, and 4-hydroxy-2-nonenal compared to control muscle. Antioxidant enzyme activities were altered, with a significant decrease in superoxide dismutase activity and significant increases in catalase and glutathione peroxidase. Peripheral arterial disease is associated with abnormal mitochondrial function and evidence of significant oxidative stress.     

Evaluation of method performance for oxidative stress biomarkers in urine and biological variations in urine of patients with type 2 diabetes mellitus and diabetic nephropathy

Background

Oxidative stress biomarkers such as superoxide dismutase (CuZnSOD), catalase (CAT) and malondialdehyde (MDA) play an important role in the pathogenesis or progression of numerous diseases. Data regarding the biological variation and analytical quality specifications (imprecision, bias and total error) for judging the acceptability of method performance for oxidative stress biomarkers in urine are conspicuously lacking in the literature. Such data are important in setting analytical quality specifications, assessing the utility of population reference intervals (index of individuality) and assessing the significance of changes in serial results from an individual (reference change value; RCV).

Materials and methods

20 patients with type 2 diabetes mellitus (T2DM), 20 patients with diabetic nephropathy (DN) and 14 healthy individuals as control were involved in this study. Timed first morning urine samples were taken from patients and healthy groups on the zero, 1st, 3rd, 5th, 7th, 15th and 30th days. Index of individuality and reference change value were calculated from within-subject and between-subject variations. Methods of oxidative stress biomarkers in human blood were adopted in human urine and markers were measured as spectrophotometrically. Also, analytical quality specifications for evaluation of the method performance were established for oxidative stress biomarkers in urine.

Results

Within-subject variations of oxidative stress biomarkers were significantly higher in patients with DN and T2DM compared to healthy subjects. MDA showed low individuality, and within-subject variances of MDA were larger than between-subject variances in all groups. However, CAT and CuZnSOD showed strong individuality, but within-subject variances of them were smaller than between-subject variances in all groups. RCVs of all analytes in diabetic patients were relatively higher, because of high within-subject variation, resulting in a higher RCV. Also, the described methodology achieves these goals, with analytical CVs of < 3.5% for all analytes. Goals for bias and total error were 6.0-7.9% and 12.5-23.3%, respectively.

Conclusions

RCVs concept for predicting the clinical status in diabetic patients represents an optimization of laboratory reporting and could be a valuable tool for clinical decision. Furthermore, for oxidative stress biomarkers’ measurements in urine, the desirable imprecision goals based on biological variation are obtainable by current methodologies.     

DNA damage and repair in type 2 diabetes mellitus

DNA damage may be associated with type 2 diabetes mellitus (T2DM) and its complications mainly through oxidative stress. Little is known about DNA repair disturbances potentially contributing to the overall extent of DNA damage in T2DM, which, in turn, may be linked with genomic instability resulting in cancer. To assess whether DNA repair may be perturbed in 2DM we determined: (1) the level of endogenous basal DNA damage, this means damage recognized in the alkaline comet assay (DNA strand breaks and alkali labile sites) as well as endogenous oxidative and alkylative DNA damage (2) the sensitivity to DNA-damaging agents hydrogen peroxide and doxorubicin and the efficacy of removing of DNA damage induced by these agents in peripheral blood lymphocytes of T2DM patients and healthy individuals. The level of DNA damage and the kinetics of DNA repair was evaluated by the alkaline single cell gel electrophoresis (comet assay). Oxidative and alkylative DNA damage were assayed with the use of DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), recognizing oxidized DNA bases and 3-methyladenine-DNA glycosylase II (AlkA) recognizing alkylated bases. The levels of basal endogenous and oxidative DNA damage in diabetes patients were higher than in control subjects. There was no difference between the level of alkylative DNA in the patients and the controls. Diabetes patients displayed higher susceptibility to hydrogen peroxide and doxorubicin and decreased efficacy of repairing DNA damage induced by these agents than healthy controls. Our results suggest that type 2 diabetes mellitus may be associated not only with the elevated level of oxidative DNA damage but also with the increased susceptibility to mutagens and the decreased efficacy of DNA repair. These features may contribute to a link between diabetes and cancer and metrics of DNA damage and repair, measured by the comet assay, may be markers of risk of cancer in diabetes.     

Association between M/L55-polymorphism of paraoxonase enzyme and oxidative DNA damage in patients with type 2 diabetes mellitus and in control subjects

The paraoxonase enzyme (PON) gene polymorphism causes a change of methionine (M-allele) to leucine (L-allele). PON may reduce low density lipoprotein oxidation and prevent atherosclerosis. Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a sensitive index of oxidative DNA damage. We have studied the association between the PON genotypes and the urinary excretion of 8-OHdG. The study population consisted of 93 Finnish type 2 diabetes patients and 106 non-diabetic control subjects. The 24-h excretion of 8-OHdG was significantly higher in diabetic patients than in control subjects (P<0.001). In control subjects, the ratio of the 8-OHdG/glomerular filtration rate increased in order of genotype from MM to ML to LL (P<0.0412). These results suggest that lipid peroxidation may have an effect on DNA oxidation.     

Lignin-derived lignophenols attenuate oxidative and inflammatory damage to the kidney in streptozotocin-induced diabetic rats

Abstract

This study investigated the effects of lignin-derived lignophenols (LPs) on the oxidative stress and infiltration of macrophages in the kidney of streptozotocin (STZ)-induced diabetic rats. The diabetic rats were divided into four groups with 0%, 0.11%, 0.33% and 1.0% LP diets. The vehicle-injected controls were given a commercial diet. At 5 weeks, superoxide (O₂^?) production, macrophage kinetics, the degree of fibrosis in glomeruli and mRNA expression for monocyte chemoattractant protein-1 (MCP-1) were examined. The NADPH-stimulated O₂^? levels in the kidney of the diabetic rats treated with 1.0% LP were significantly lower than those in untreated diabetic rats. The number of macrophages, levels of MCP-1 mRNA expression and degree of glomerular fibrosis increased in untreated LP and these levels were significantly lower in 1.0%LP-treated rats. The results suggested that LPs suppress the excess oxidative stress, the infiltration and activation of macrophages and the glomerular expansion in STZ-induced diabetic kidneys.     

Immunologic autoagression in patients with diabetic retinopathy under type I diabetes mellitus

Presence of IgG autoantibodies to broad spectrum of tissues' antigens in patients with diabetic retinopathy under type I diabetes mellitus was studied. Increased levels of antibodies to different antigens were observed in 56% of patients. Increased levels of antibodies to hypophysis, denaturated DNA, and myelin basic protein were detected most frequently. Correlation in presence of autoantibodies of different specificity with diabetes length as well as with frequency of respiratory infections was established. There was relation between level of antibodies to DNA and increased permeability of microvascular network of retina as well as between presence of autoantibodies to myelin basic protein and encephalopathy in patients with type I diabetes mellitus.