Proteomic analysis of urinary exosomes from patients of early IgA nephropathy and thin basement membrane nephropathy

To identify biomarker candidates associated with early IgA nephropathy (IgAN) and thin basement membrane nephropathy (TBMN), the most common causes presenting isolated hematuria in childhood, a proteomic approach of urinary exosomes from early IgAN and TBMN patients was introduced. The proteomic results from the patients were compared with a normal group to understand the pathophysiological processes associated with these diseases at the protein level. The urinary exosomes, which reflect pathophysiological processes, collected from three groups of young adults (early IgAN, TBMN, and normal) were trypsin-digested using a gel-assisted protocol, and quantified by label-free LC-MS/MS, using an MS(E) mode. A total of 1877 urinary exosome proteins, including cytoplasmic, membrane, and vesicle trafficking proteins, were identified. Among the differentially expressed proteins, four proteins (aminopeptidase N, vasorin precursor, α-1-antitrypsin, and ceruloplasmin) were selected as biomarker candidates to differentiate early IgAN from TBMN. We confirmed the protein levels of the four biomarker candidates by semi-quantitative immunoblot analysis in urinary exosomes independently prepared from other patients, including older adult groups. Further clinical studies are needed to investigate the diagnostic and prognostic value of these urinary markers for early IgAN and TBMN. Taken together, this study showed the possibility of identifying biomarker candidates for human urinary diseases using urinary exosomes and might help to understand the pathophysiology of early IgAN and TBMN at the protein level.     

Thioredoxin interacting protein expression in the urinary sediment associates with renal function decline in type 1 diabetes

Aims: Thioredoxin interacting protein (TXNIP), an inhibitor of antioxidant thioredoxin (Trx), is upregulated by hyperglycemia and implicated in pathogenesis of diabetes complications. We evaluated mRNA expressions of genes encoding TXNIP and Trx (TXN) in urinary sediment and peripheral blood mononuclear cells (PBMC) of type 1 diabetes (T1D) patients with different degrees of chronic complications. Methods: qPCR was employed to quantify target genes in urinary sediment (n?=?55) and PBMC (n?=?161) from patients sorted by presence or absence of diabetic nephropathy (DN), retinopathy, peripheral and cardiovascular neuropathy; 26 healthy controls and 13 patients presenting non-diabetic nephropathy (focal and segmental glomerulosclerosis, FSGS) were also included. Results: Regarding the urinary sediment, TXNIP (but not TXN) expression was higher in T1D (p?=?0.0023) and FSGS (p?=?0.0027) patients versus controls. Expressions of TXNIP and TXN were higher, respectively, in T1D patients with versus without DN (p?=?0.032) and in those with estimated glomerular filtration rate (eGFR)?<?60 versus?≥60 mL/min/1.73 m² (p?=?0.008). eGFR negatively correlated with TXNIP (p?=?0.04, r?=??0.28) and TXN (p?=?0.04, r?=??0.30) expressions. T1D patients who lost?≥5 mL/min/1.73 m² yearly of eGFR presented higher basal TXNIP expression than those who lost?<5 mL/min/1.73 m² yearly after median follow-up of 24 months. TXNIP (p?<?0.0001) and TXN (p?=?0.002) expressions in PBMC of T1D patients were significantly higher than in controls but no differences were observed between patients with or without chronic complications. Conclusions: TXNIP and TXN are upregulated in urinary sediment of T1D patients with diabetic kidney disease (DKD), but only TXNIP expression is associated with magnitude of eGFR decline.     

2型糖尿病患者尿视黄醇结合蛋白与视网膜病变的相关性

目的 探讨2型糖尿病患者尿视黄醇结合（RBP）与视网膜病变相关性。方法 采用酶联免疫法检测158例2型糖尿病患者24h尿视黄醇结合蛋白排泄（24hURBP）及24h尿白蛋白排泄（24hUAE）,并同时用眼底镜仔细检查其眼氏。结果 糖尿病视网膜病变（DR）发生率随着24hURBP、24hUAE增加而显著增高,24hURBP、24hUAE也随DR的程度加重而显著增加。结论 2型糖尿病患者DR与尿RBP、尿白蛋白呈正相关。     

高表达C3aR的肥大细胞在糖尿病肾病患者肾组织中的分布及病理意义分析

选取糖尿病肾病(diabetic nephropathy,DN)早期、中期、晚期以及正常移植供肾各15例,分别作为DN早期组、中期组、晚期组和正常对照组,利用各个病例的肾穿刺组织标本进行C3aR免疫组化染色,分析各组中高表达C3aR浸润细胞的数量和分布特点,及其与DN发生、发展的关系,利用连续切片、免疫荧光双套色染色、免疫电镜、及甲苯胺蓝染色等方法对DN患者肾组织中高表达C3aR的浸润细胞进行细胞类型鉴定.结果表明:a.光镜和免疫电镜的形态学分析显示DN患者肾组织中高表达C3aR的浸润细胞在形态上具有肥大细胞的特点,免疫荧光双套色染色的分析显示,C3aR浸润细胞CD45阴性、CD68和类胰蛋白酶阳性,与肥大细胞的情况一致,甲苯胺蓝特殊染色进一步证实这是一种肥大细胞.b.正常移植供肾组织中虽有肥大细胞分布,但数量很少;从DN早期组到DN中期组,再到DN晚期组,肾组织中肥大细胞的数量有一种不断增加的趋势;DN患者肾组织肥大细胞的数量与24 h尿蛋白和血肌酐水平均具有很好的线性相关性.上述工作不仅揭示了在DN发生发展过程中肥大细胞在DN患者肾组织中的数量及分布情况,提示肥大细胞很可能参与了DN的发生发展过程,同时,DN患者肾组织肥大细胞高表达过敏毒素C3a受体C3aR的发现,提示C3a/C3aR通路很可能在DN患者肥大细胞的招募和激活过程中有重要作用.     

Low plasma albumin levels are associated with increased plasma protein glycation and HbA1c in diabetes

Albumin is one of the most abundant plasma proteins and is heavily glycated in diabetes. In this study, we have addressed whether variation in the albumin levels influence glycation of plasma proteins and HbA1c. The study was performed in three systems: (1) streptozotocin (STZ)-induced diabetic mice plasma, (2) diabetic clinical plasma, and (3) in vitro glycated plasma. Diabetic mice and clinical plasma samples were categorized as diabetic high albumin plasma (DHAP) and diabetic low albumin plasma (DLAP) on the basis of their albumin levels. For the in vitro experiment, two albumin levels, high albumin plasma (HAP) and low albumin plasma (LAP), were created by differential depletion of plasma albumin. Protein glycation was studied by using a combination of two-dimensional electrophoresis (2DE), Western blotting, and LC-MS(E). In both mice and clinical experiments, an increased plasma protein glycation was observed in DLAP than in DHAP. Additionally, plasma albumin levels were negatively correlated with HbA1c. The in vitro experiment with differential depletion of albumin mechanistically showed that the low albumin levels are associated with increased plasma protein glycation and that albumin competes for glycation with other plasma proteins.     

Urinary chromium loss associated with diabetes is offset by increases in absorption

The results of the current study indicate that diabetic rats have increased urinary Cr loss as a result of their diabetes; however, this increased urinary Cr loss is offset by increased absorption of Cr. Insulin resistant, obese rats have alterations in the rates of Cr transport and distribution compared to lean rats but have similar levels of urinary Cr loss and Cr absorption. Thus, any increases in urinary Cr loss associated with insulin resistance or diabetes are offset by increased absorption. Given that dietary chromium is normally absorbed with only ∼ 1% efficiency, suitable Cr exists in the diet so that a standard diet possesses sufficient chromium to allow for the increases in absorption associated with diabetes. Consequently, supplementing the diet with nutritionally relevant quantities of chromium is not anticipated to have any beneficial effects. Similarly, beneficial effects on plasma variables, such as cholesterol, triglycerides, and insulin concentrations, from supra-nutritional doses of Cr(III) complexes should not arise from alleviation of chromium deficiency. These beneficial effects must arise from pharmacological effects of high dose Cr(III) administration.     

Increased serum urea and creatinine levels correlate with decreased retinal nerve fibre layer thickness in diabetic retinopathy

Abstract

Correlation of increased levels of serum urea and creatinine with retinal nerve fibre layer (RNFL) thinning on spectral domain optical coherence tomography (SD-OCT) was studied in diabetic retinopathy (DR). Sixty consecutive cases and 20 healthy controls were included. Cases were divided into three groups: without DR, non-proliferative DR with macular oedema and proliferative DR with oedema. Serum urea and creatinine were measured using a standard protocol. Average (RNFL) was measured using SD-OCT. Increased severity of DR was associated with decrease in levels of serum urea and serum creatinine levels. RNFL thinning correlated positively with increase in serum urea and creatinine levels.     

Cardiomyopathy associated with noninsulin-dependent diabetes

Summary Cardiovascular disease represents the major cause of morbidity and mortality in noninsulin-dependent diabetic patients. While it was once thought that atherosclerotic vascular disease was responsible for all of these adverse effects, recent studies support the notion that one of the major adverse complications of diabetes is the development of a diabetic cardiomyopathy characterized by defects in both diastolic and systolic function. Contributing to the development of the cardiomyopathy is a shift in myosin isozyme content in favor of the least active V₃ form. Also defective in the noninsulin-dependent diabetic heart is regulation of calcium homeostasis. While transport of calcium by the sarcolemmal and sarcoplasmic reticular calcium pumps are minimally affected by noninsulin-dependent diabetes, significant impairment occurs in sarcolemmal Na⁺-Ca²⁺ exchanger activity. This defect limits the ability of the diabetic heart to extrude calcium, contributing to an elevation in [Ca²⁺]_i. Also promoting the accumulation of calcium by the diabetic cell is a decrease in Na⁺, K⁺ ATPase activity, which is known to increase [Ca²⁺]_i secondary to a rise in [Na⁺]_i. In addition, calcium influx via the calcium channel is stimulated. Although the molecular mechanisms underlying these defects are presently unknown, the possibility that they may be related to aberrations in glucose or lipid metabolism are considered. The evidence suggests that classical theories of glucose toxicity, such as excessive polyol production or glycosylation, appear to be insignificant factors in heart. Also insignificant are defects in lipid metabolism leading to accumulation of toxic lipid amphiphiles or triacylglycerol. Rather, the major defects involve membrane changes, such as phosphatidylethanolamine N-methylation and protein phosphorylation, which can be attributed to the state of insulin resistance.     

Urinary nucleic acid oxidation product levels show differential associations with pharmacological treatment in patients with type 2 diabetes

The relationship between RNA and DNA oxidation and pharmacological treatment has not been systematically investigated in patients with type 2 diabetes (T2D). We aimed to investigate the association between pharmacological treatments and levels of urinary markers of nucleic acid oxidation in T2D patients. Vejle Diabetes Biobank cohort data was nested into nationwide registry data. Multiple logistic regression was used to associate drug usage with risk of high (above median) RNA and DNA oxidation. Data from 2664 T2D patients (64% male, age range: 25–75) were included. Questionnaire-validated lipid lowering drug use was associated with low RNA oxidation (Odds ratio, OR 0.71, 95% CI: [0.59–0.87]). Insulin and non-specific antidiabetic drugs were associated with low DNA oxidation (insulin: OR 0.60, 95% CI [0.49–0.73]). Oral antidiabetics were associated with high DNA oxidation and RNA oxidation (OR 1.30, 95% CI [1.10–1.53] and OR 1.26, 95% CI [1.07–1.29]). Our findings indicate that diabetes-related drugs are associated with RNA and DNA oxidation and further studies are required to determine causality in T2D patients.     

Plasma levels and diagnostic value of catestatin in patients with heart failure

Catestatin (CST) is an endogenous neuropeptide with multiple cardiovascular activities. The study is to investigate circulating CST levels in heart failure (HF) patients and to evaluate the role of CST as a biomarker for HF. Plasma CST concentrations were measured by enzyme-linked immunosorbent assay in 228 HF patients and 172 controls. Plasma CST gradually increased in patients from NYHA class I to class IV. No significant differences in CST were found among NYHA I, NYHA II patients and controls. Plasma CST in NYHA III and IV patients was higher compared to other groups. Plasma CST levels in HF patients after treatment were similar to admission, but still higher than controls. In a subgroup analysis among the patients with NYHA class III or IV, patients with ischemic etiology had significantly higher CST. Plasma CST levels were similar between patients with preserved and reduced ejection fraction. Multivariable analysis showed that NYHA classes, the etiology of HF (ischemic or not) and estimated glomerular filtration rate independently predicted plasma LogCST levels (P < 0.05). The area under ROC for CST and BNP in moderate to severe HF diagnosis was 0.626 and 0.831, respectively, combining CST and BNP did not improve the accuracy.     

Elevated plasma visfatin levels correlate with poor prognosis of gastric cancer patients

Visfatin is a proinflammmatory cytokine with accumulating evidence for its rise in circulation of cancer patients. This study aimed to evaluate the relationship between preoperative plasma visfatin level and prognosis of gastric cancers. Preoperative plasma visfatin levels of 262 patients with gastric cancers and plasma visfatin levels of 262 healthy individuals were determined using enzyme-linked immunosorbent assay. Preoperative plasma visfatin level was substantially higher in patients than in healthy subjects. Plasma visfatin levels were associated with invasion depth, lymph node metastasis, distant metastasis, peritoneal dissemination, tumor size and tumor node metastasis stage. Multivariate analysis revealed that high plasma visfatin level was an independent factor for death. Receiver operating characteristic curve analysis showed that plasma visfatin level predicted death with high area under curve. Multivariate Cox regression analysis identified plasma visfatin level as an independent predictor of overall survival. Thus, our results suggest that high preoperative plasma visfatin level is associated with prognostic factors for gastric cancer as well as may play a role as prognostic biomarker in gastric cancer survival.     

Bradykinin B2 receptor gene polymorphism is associated with altered urinary albumin/creatinine values in diabetic patients

Diabetic nephropathy (DN) is an important microvascular complication of both insulin-dependent and non-insulin-dependent diabetes mellitus. Considerable evidence exists that genetic predisposition is a major determinant in the development of DN. Progress in the understanding of the kinin receptor gene expression indicates their relevance in nephrology and renal pathology. In order to investigate whether clinically relevant polymorphisms of the kinin receptor genes contribute to the genetic predetermination of the renal complication of diabetes, we have initiated a retrospective study with a mixed population of 49 type 1 and 112 type 2 diabetic patients who have been followed for several years by an endocrinologist and (or) nephrologist with periodical functional tests relevant to DN (microalbuminuria, serum and urinary creatinine). The allelic frequencies of four kinin receptor polymorphisms, including three B2R polymorphisms (the C/T-58 promoter polymorphism, the exon 2 and exon 1 polymorphisms, all of them with assumed clinical significance) and the putative nephroprotective (G/C-699) B1R promoter polymorphism, were analyzed in all recruited diabetic patients. Our results indicate a significant association of the B2R exon 1 (+/-) genotype with increased urinary albumin/creatinine values (P = 0.026) and serum creatinine levels (P = 0.028). More importantly, the (+) allele of B2R exon 1 polymorphism was associated very significantly with lower albumin/creatinine values in these patients (P = 0.0087). Thus, the B2R exon 1 polymorphism may represent a susceptibility marker for nephropathy progression in diabetic patients.     

Cohort study of predictive value of urinary albumin excretion for atherosclerotic vascular disease in patients with insulin dependent diabetes.

OBJECTIVE: To examine whether slightly elevated urinary albumin excretion precedes development of atherosclerotic vascular disease in patients with insulin dependent diabetes independently of conventional atherogenic risk factors and of diabetic nephropathy. DESIGN: Cohort study with 11 year follow up. SETTING: Diabetes centre in Denmark. SUBJECTS: 259 patients aged 19-51 with insulin dependent diabetes of 6-34 years'' duration and without atherosclerotic vascular disease or diabetic nephropathy at baseline. MAIN OUTCOME MEASURES: Baseline variables: urinary albumin excretion, blood pressure, smoking habits, and serum concentrations of total cholesterol, high density lipoprotein cholesterol, sialic acid, and von Willebrand factor. End point: atherosclerotic vascular disease assessed by death certificates, mailed questionnaires, and hospital records. RESULTS: Thirty patients developed atherosclerotic vascular disease during follow up of 2457 person year. Elevated urinary albumin excretion was significantly predictive of atherosclerotic vascular disease (hazard ratio 1.06 (95% confidence interval 1.02 to 1.18) per 5 mg increase in 24 hour urinary albumin excretion, P = 0.002). Predictive effect was independent of age; sex; blood pressure; smoking; serum concentrations of total cholesterol, high density lipoprotein cholesterol, sialic acid, and von Willebrand factor; level of haemoglobin A(lc); insulin dose, duration of diabetes, and diabetic nephropathy (hazard ratio 1.04 (1.01 to 1.08) per 5 mg increase     

前列地尔联合益肾化湿颗粒对糖尿病肾病患者血糖、血脂、肾功能以及尿足细胞相关蛋白的影响

目的:分析前列地尔联合益肾化湿颗粒对糖尿病肾病患者血糖、血脂、肾功能以及尿足细胞相关蛋白的影响。方法:98例糖尿病肾病患者按抽签法分为对照组与实验组,各49例,对照组予以前列地尔治疗,实验组基于对照组加用益肾化湿颗粒治疗,比较两组疗效,糖化血红蛋白(Hb A1c)、血糖(FPG)、餐后2 h血糖低(2h PG),甘油三脂(TG)、总胆固醇(TC)、高密度脂蛋白(HDL-C)、低密度脂蛋白(LDL-C),血尿素氮(BUN)、肌酐(Cr)、β2微球蛋白(β2-MG)、胱抑素(Cys-C),尿足细胞标志蛋白(PCX)nephrin,安全性。结果:实验组总有效率高于对照组(P0.05)。治疗后,两组Hb Alc、FPG、2h PG比较无差异(P0.05)。实验组TG、TC、LDL-C、BUN、Cr、β2-MG、Cys-c、PCX、尿nephrin/尿Cr低于对照组(P0.05)。实验组HDL-C高于对照组(P0.05)。结论:前列地尔联合益肾化湿颗粒治疗对糖尿病肾病的疗效确切,可利于血糖、血脂、肾功能的改善,降低尿足细胞相关蛋白的浓度。     

周脂素在糖尿病肾病大鼠肾组织中的表达

目的观察大鼠糖尿病肾病(diabetic nephropathy,DN)模型的特点和尿LN对早期DN的诊断价值,探讨周脂素(perilipin,Plin)在DN大鼠肾脏中的表达情况。方法将14只SD雄性大鼠随机分为对照组(普通饲料)和糖尿病肾病模型组(高糖高脂饲料),对照组6只,模型组8只,饲养4周后模型组按照30mg/kg剂量注射1%链脲佐菌素(STZ),检测血糖≥16.7mmol/L,糖尿病模型制作成功,继续喂养6周,检测24h尿蛋白≥30mg/kg,糖尿病肾病模型制作成功。考马斯亮蓝检测24h尿蛋白、ELISA测尿层粘连蛋白,HE染色观察肾组织的病理变化,Real-time PCR及Western blot检测肾脏组织中perilipin表达情况。结果模型鼠24h尿蛋白≥30mg/kg,糖尿病肾病大鼠模型制作成功。和对照组大鼠相比,模型组的肾重/体重比明显增高(P<0.05),尿量、尿层粘连蛋白于5周出现升高、24h尿蛋白于6周时出现升高,且三项指标均随着时间不断增高。肾组织病理检查显示:肾小球肥大,基膜增生,微小血管瘤形成,肾小管管腔变形,上皮脱落、空泡样变,大量单核、淋巴等炎性细胞浸润,间质内胶原纤维增生。模型组大鼠肾组织Plin的mRNA及蛋白表达均明显的升高(P<0.05)。结论尿层粘连蛋白比24h尿蛋白升高得早,可作为早期糖尿病肾病的警示指标。Plin表达增高可能参与了糖尿病肾病肾病变过程,为进一步探讨糖尿病肾病的发病机制提供新的思路。     

Small ubiquitin-like modifier 4 M55V polymorphism is not associated with diabetic nephropathy in Iranian type 2 diabetes patients

INTRODUCTION:

We studied the impact of small ubiquitin-like modifier 4 (SUMO4) M55V polymorphism on susceptibility to diabetic nephropathy in Iranian type 2 diabetes patients.

MATERIALS AND METHODS:

The patient group consisted of 50 Iranian type 2 diabetes patients with nephropathy, and the control group consisted of 50 Iranian type 2 diabetes patients without nephropathy. Genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism method for the M55V.

RESULTS:

The frequency of SUMO4 AA, AG, and GG genotypes were 23%, 18%, and 9% in the patient group and 10%, 22%, and 18% in the control group. There was no significant difference in frequency of SUMO4 genotypes in patients compared to controls.

CONCLUSION:

These findings indicate that SUMO4 M55V polymorphism is not associated with diabetic nephropathy in Iranian type 2 diabetes patients.     

Upregulated microRNAs in membranous glomerulonephropathy are associated with significant downregulation of IL6 and MYC mRNAs

Membranous glomerulonephropathy (MGN) is a glomerulopathy characterized by subepithelial deposits of immune complexes on the extracapillary side of the glomerular basement membrane. Insertion of C5b-9 (complement membrane-attack complex) into the membrane leads to functional impairment of the glomerular capillary wall. Knowledge of the molecular pathogenesis of MGN is actually scanty. MicroRNA (miRNA) profiling in MGN and unaffected tissues was performed by TaqMan Low-Density Arrays. Expression of miRNAs and miRNA targets was evaluated in Real-Time polymerase chain reaction (PCR). In vitro transient silencing of miRNAs was achieved through transfection with miRNA inhibitors. Ten miRNAs (let-7a-5p, let-7b-5p, let-7c-5p, let-7d-5p, miR-107, miR-129-3p, miR-423-5p, miR-516-3p, miR-532-3p, and miR-1275) were differentially expressed (DE) in MGN biopsies compared to unaffected controls. Interleukin 6 (IL6) and MYC messenger RNAs (mRNAs; targets of DE miRNAs) were significantly downregulated in biopsies from MGN patients, and upregulated in A498 cells following let-7a-5p or let-7c-5p transient silencing. Gene ontology analysis showed that DE miRNAs regulate pathways associated with MGN pathogenesis, including cell cycle, proliferation, and apoptosis. A significant correlation between DE miRNAs and mRNAs and clinical parameters (i.e., antiphospholipid antibodies, serum creatinine, estimated glomerular filtration, proteinuria, and serum cholesterol) has been detected. Based on our data, we propose that DE miRNAs and their downstream network may be involved in MGN pathogenesis and could be considered as potential diagnostic biomarkers of MGN.