原发性局灶节段硬化性肾小球肾炎患者循环microRNA表达谱研究

目的:本研究通过芯片分析原发性局灶节段硬化性肾小球肾炎(p FSGS)患者血浆中microRNA(miRNA)表达谱变化,寻找与p FSGS相关的miRNA及其与临床指标的相关性。方法:采用Exiqon miRNA表达谱芯片技术,检测人成熟miRNA在p FSGS患者和健康对照中表达水平差异,应用分层聚类分析获得p FSGS差异表达的miRNA谱。并采用实时荧光定量PCR在扩大样本的FSGS患者中验证芯片结果。结果:(1)MiRNA芯片检测结果表明存在p FSGS特异性的miRNA表达谱,共筛选出95个差异表达的miRNA,均为低表达(P0.05)。(2)选取的4个表达差异较为显著的miRNA(miR-3678-3p,miR-4670-5p,miR-583,miR-30c-2-3p)进行定量PCR检测,其表达均低于正常对照组,其中miR-4670-5p、miR-583及miR-30c-2-3p三个差异较明显,比值均在0.5以下(P0.05),结果与芯片相符性较好。(3)纳入的12例患者,顶端型4例,非特异型8例,不同病理亚型,上述验证的4个miRNAs表达差异均无统计学意义(P0.05);Pearson相关性分析显示:miR-4670-5p表达量与BMI显著正相关(r=0.638,P=0.035);miR-583表达量与Scr正相关(r=0.672,P=0.047)、与GFR负相关(r=-0.723,P=0.028);miR-3678-3p和miR-30c-2-3p与患者GFR、BMI、Scr、Alb、24 h尿蛋白定量等指标无明显相关性(P0.05)。结论:多种miRNA在p FSGS患者血浆中异常表达,提示它们可能在FSGS的发生发展中起调控作用。     

狼疮肾炎患者循环miRNA表达谱研究及循环miR-130b-3p的临床意义

目的分析处于不同进展阶段的狼疮肾炎(LN)患者循环miRNA的表达变化并探讨其可能存在的临床意义。 方法本研究共94份血清标本,包括58例LN患者和36名健康体检者(作为对照组)。挑选出其中性别、年龄匹配的12份血清标本用于循环miRNA表达谱筛选,包括早期LN组4份(CKD 1～3期),晚期LN组4份(CKD分期4～5期)及对照组4份作为芯片组,使用miRNA PCR芯片检测血清miRNA变化;剩余50例LN患者(40例早期LN患者、10例晚期LN患者)及32名对照者血清样本作为验证组,用于验证miRNA表达谱筛选结果;以Nanodrop 2000检测血清总RNA抽提浓度;选择miR-130b-3p和miR-1233-3p作为验证对象;进行Spearman相关性分析。 结果芯片组结果显示,与对照组相比,早期LN组中7个表达上升的miRNA差异具有统计学意义,分别是：miR-1233-3p (P＝0.019)、miR-130b-3p (P＝0.021)、miR-18a-3p (P＝0.021)、miR-628-3p (P＝0.023)、miR-1260b (P＝0.030)、miR-1539 (P＝0.041)和miR-378e (P＝0.047);晚期LN组分别与对照组及早期LN组比较,未发现表达上调大于2倍或者上调差异具有统计学意义的miRNA。验证组结果显示：晚期LN组患者分别与对照组、早期LN组比较,血清总RNA浓度下降(U＝4.5,P<0.001;U＝18.0,P<0.001);早期LN组与对照组比较,血清中miR-130b-3p表达升高[IQR 16.2(8.7,42.7)与9.6(4.8,17.4), U＝405.5,P＝0.008];晚期LN组分别与对照组及早期LN组比较,MiR-130b-3p和miR-1233-3p表达均下降(U＝69.0、P＝0.008,U＝46.0、P<0.001;U＝80.0、P＝0.019,U＝70.0、P＝0.002);相关性分析显示循环miR-130b-3p的相对表达量与24 h尿蛋白(r＝0.404,P＝0.010)、肾脏慢性活动指数(r＝0.389,P＝0.013)、甘油三酯(r＝0.376,P＝0.017)呈正相关。 结论严重肾功能衰竭患者可能存在循环miRNA广泛表达下调,循环miRNA-130b-3p在早期LN患者中表达升高并与肾脏损伤和血脂调节异常相关,在LN发生发展中可能起作用。     

2型糖尿病患者血清胆红素水平与糖尿病肾病的相关性

目的:探讨2型糖尿病患者血清胆红素水平与糖尿病肾病的相关性。方法:156例2型糖尿病患者,按照尿白蛋白排泄率水平分为正常蛋白尿组(n=54),微量蛋白尿组(n=50),大量蛋白尿组(n=52),比较患者一般资料及胆红素水平并进行分析。结果:三组间的总胆红素水平和直接胆红素水平,大量蛋白尿组微量蛋白尿组正常蛋白尿组,差异有统计学意义(P0.01)。Pearson相关分析表明,血清总胆红素与尿白蛋白排泄率呈负相关(r=-0.305,P0.01)。Logistic回归分析显示,总胆红素是糖尿病肾病的独立保护性因素(OR=0.832,95%CI:0.725~0.954,P0.01)。结论:总胆红素水平与尿白蛋白排泄率水平相关,胆红素的下降参与了糖尿病肾病的发生、发展。     

血清miR-135-5p及miR-337-5p在糖尿病肾病中的表达及其临床意义

目的探讨糖尿病肾病(DN)患者的血清miR-135-5p及miR-337-5p表达及其临床意义。方法选取2018年1月至2020年12月本院收治的105例2型DN患者和60例体检正常者(对照组)。根据尿白蛋白排泄率(UAER)将105例2型DN患者分为早期DN组57例(UAER为20～200 μg/min)和临床期DN组48例(UAER>200 μg/min)。比较各组血清miR-135-5p及miR-337-5p表达水平, 应用多因素logistic回归分析影响DN发生的危险因素。绘制受试者工作特征(ROC)曲线分析血清miR-135-5p及miR-337-5p表达水平在诊断DN中的价值。结果 DN组的血清miR-135-5p及miR-337-5p表达水平均明显高于对照组(均P<0.001)。临床期DN组的血清miR-135-5p及miR-337-5p表达水平均明显高于早期DN组(均P<0.001)。多因素logistic回归分析显示, 血清miR-135-5p及miR-337-5p表达水平升高是影响DN发生的独立危险因素(均P<0.001)。ROC曲线分析显...     

儿童IgA肾病血清miR-152-5p及miR-210-5p的表达及其临床价值

目的探讨miR-152-5p及miR-210-5p在儿童IgA肾病(IgAN)中的表达及其临床价值。方法选取2018年1月至2022年6月由本院收治的109例IgAN患儿(IgAN组)、100例非IgA肾病的肾炎患儿(非IgAN组)和60例体检正常者(对照组)作为研究对象。其中IgAN患儿根据牛津分型评分(MEST)分为MEST≥3分组(41例)和MEST<3分组(68例), 并根据尿蛋白量分为尿蛋白≥1.0 g/24 h组(56例)和尿蛋白<1.0 g/24 h组(53例)。比较各组的miR-152-5p、miR-210-5p、IgA/C3及半乳糖缺乏型IgA1分子(Gd-IgA1)水平。绘制受试者工作特征(ROC)曲线分析miR-152-5p、miR-210-5p、IgA/C3及Gd-IgA1诊断IgAN的价值。采用Pearson相关分析miR-152-5p、miR-210-5p表达水平与IgA/C3及Gd-IgA1的相关性。结果 IgAN组的miR-152-5p、miR-210-5p、IgA/C3及Gd-IgA1水平均明显高于非IgAN组和对照组(均P<0.00...     

IgA肾病外周血B淋巴细胞微核糖核酸差异表达及临床意义

目的通过检测人外周血B淋巴细胞微核糖核酸(microRNA)的差异表达,并分析其与IgA肾病临床病理特点、IgA1分子O-糖基化异常之间的关系,探索microRNA在IgA肾病发病中的可能机制。方法收集7例IgA肾病患者及4例正常人外周血5 ml,磁珠法分选出CD19~+B淋巴细胞,提取RNA,采用表达谱芯片筛选差异表达的microRNA。在另外29例IgA肾病患者和16例正常对照者中,对候选microRNAs用实时荧光定量聚合酶链反应(quantificational real-time polymerase chain reaction,QRT-PCR)进行验证,并分析它们与临床病理、IgA1分子半乳糖缺陷(galatose-defecient IgA1,Gd-IgA1)水平的关系。结果 IgA肾病患者外周血B淋巴细胞microRNA表达明显异常,其中85个明显上调,30个明显下调。对5个候选microRNA:miR-3189-5p、let-7g-5p、miR-4258-5p、miR-4695-3p及miR-99b-5p,进一步验证显示,其表达水平在IgA肾病患者与正常对照之间差异无统计学意义(P0.05)。且上述5个microRNA表达量与IgA肾病患者的临床病理指标没有明显的相关性。IgA肾病组血清IgA1、Gd-IgA1明显高于正常对照(P0.05或P0.01)。5个候选microRNA:miR-3189-5p、let-7g-5p、miR-4258-5p、miR-4695-3p及miR-99b-5p的水平与IgA肾病患者IgA1分子O-连接半乳糖缺陷之间没有明显相关性。结论 IgA肾病患者和正常人外周血CD19~+B淋巴细胞microRNA表达谱存在明显差异。初步验证显示miR-3189-5p、let-7g-5p、miR-4258-5p、miR-4695-3p及miR-99b-5p的表达水平在IgA肾病患者与正常对照之间差异无统计学意义,且与IgA肾病的临床病理特点和Gd-IgA1之间无明显相关性,提示这5个microRNA可能不是参与IgA肾病发病和进展的重要microRNA。     

血清miR-130b与糖尿病肾病患者肾脏损伤及远期预后的相关性研究

目的:探讨血清miR-130b与糖尿病肾病患者肾脏损伤及远期预后的相关性。方法:选择在杭州师范大学附属医院诊治的糖尿病肾病患者134例为观察组,同期选择杭州师范大学附属医院门诊正常体检者118例为对照组,测定两组miR-130b水平,分析miR-130b水平与BMI、蛋白尿定量、空腹血糖、糖尿病病程、血肌酐及e GFR水平的关系,分析肾脏损伤程度与miR-130b的关系。结果:观察组miR-130b水平低于对照组(P 0. 05); BMI、蛋白尿定量、空腹血糖、糖尿病病程、血肌酐水平越高,e GFR水平越低,血清miR-130b表达水平越低(P 0. 05);随着肾脏损伤程度的加重,肾小球分级、IFTA评分、间质炎症评分的增加,血清miR-130b水平逐渐降低(P 0. 01); miR-130b与肾小球分级、IFTA评分、间质炎症评分负相关明显(P 0. 05); IFTA评分、miR-130b两者的AUC相近,皆小于e GFR,miR-130b、IFTA评分诊断糖尿病肾病的灵敏度、特异度相近(P 0. 05),皆小于e GFR(P 0. 05); ESRD组血清miR-130b水平低于非ESRD组(P 0. 05),ESRD组血清miR-130b低表达率高于非ESRD组,miR-130b低水平组相对于高水平进入ESRD的风险升高13. 020倍(P 0. 05)。结论:miR-130b低表达能加速糖尿病患者肾脏损伤程度,降低糖尿病肾病患者预后; miR-130b可作为判定糖尿病肾病的生物学标志物,值得在临床上推广应用。     

香烟烟雾暴露后的雄性大鼠睾丸差异性microRNA的筛选及功能验证

目的:筛选香烟烟雾暴露后的雄性大鼠睾丸组织差异性表达的miRNA,寻找并鉴定吸烟所致睾丸凋亡损伤的早期分子标记物。方法:200只清洁级SD雄鼠,随机分为10支/d、20支/d、30支/d香烟烟雾暴露组和对照组(n=10),采用静式染毒法分别处理2、4、6、8和12周后,通过HE染色观察睾丸组织病理学变化,运用TUNEL法检测睾丸细胞凋亡情况,免疫组化法检测Caspase-3的表达,RT-PCR法和Western印迹检测Caspase-9的mRNA及蛋白表达。根据以上结果选择12周高剂量组与对照组各4个睾丸样本进行miRNA芯片筛选及生物信息学分析,运用RT-PCR法在所有受试动物中对差异性miRNA的表达进行验证。结果:与对照组相比,随香烟烟雾暴露剂量和时间的增加,暴露组大鼠睾丸组织逐渐出现萎缩等改变;染毒第6周起,睾丸凋亡细胞数量明显增多(P0.05);大鼠睾丸Caspase-3的表达从第6周中剂量组开始显著升高(P0.01);Caspase-9 mRNA和蛋白表达水平上调(P0.05);芯片筛选结果发现暴露组大鼠睾丸差异性表达的miRNA有5条,其中miR-138-5p、miR-181d-5p、miR-19a-3p及miR-3588表达下调,miR-155-5p表达上调,RT-PCR验证结果与芯片结果一致。且差异性miRNA的靶基因的主要功能为正向调节细胞凋亡。结论:miR-155-5p、 miR-138-5p、miR-181d-5p、miR-19a-3p及miR-3588在吸烟所致睾丸凋亡损伤中存在调控作用,并有望成为吸烟所致精子发生受阻的早期诊断和预测预后的生物标志物。     

miR-92a表达与药物联合治疗糖尿病肾病大量尿蛋白的疗效及预后关系探究

目的:探究miR-92a表达与前列地尔联合血管紧张素转化酶抑制剂(angiotensin-converting enzyme inhibitors, ACEI)或血管紧张素受体抗结剂(angiotensin receptor anticaking agent, ARB)治疗糖尿病大量蛋白尿疗效及预后的关系。方法:收集2014年5月—2016年10月我院收治的糖尿病肾病大量蛋白尿患者共106例,根据治疗方案不同分为研究组(n=66)与对照组(n=40),其中对照组患者给予ACEI/ARB治疗,研究组患者则在此基础上均联合前列地尔治疗。比较两组患者的疗效、预后及治疗前后的miR-92a水平。对患者进行24个月的随访,根据患者是否进入终末期肾病(ESRD),将患者分为ESRD组与非ESRD组,比较患者的miR-92a的表达情况,分析miR-92a水平对患者预后的预测作用。结果:与对照组患者相比,研究组患者的总有效率明显更高(P0.05);治疗后,研究组患者的miR-92a水平明显更低(P0.05);与对照组患者相比,研究组患者的发展为ESRD的数量明显更低(P0.05);与非ESRD组患者的miR-92a水平(1.8±0.8)相比,ESRD组患者的miR-92a水平(2.1±0.4)明显更高(P0.05)。miR-92a水平对糖尿病肾病大量尿蛋白患者预后预测的曲线下面积(AUC)为0.7041(95%CI:0.603 4～0.804 9,P0.000 5)。结论:前列地尔联合ACEI/ARB治疗糖尿病肾病大量尿蛋白患者的疗效显著,可明显降低患者的miR-92a水平,且miR-92a水平与患者的预后具有一定相关性,其水平的上升可预测患者不良预后的发生。     

自发性2型糖尿病KKAy小鼠肾小球microRNA表达谱和氯沙坦治疗效应

目的 分析糖尿病肾病(DN)发病过程中肾小球miRNA表达谱的变化,观察血管紧张素受体拮抗剂(ARB)氯沙坦对DN肾小球miRNA表达谱的影响,确认在DN发病过程中发挥关键作用的miRNA.方法 8周龄KKAy小鼠随机分为氧沙坦治疗组(10 mg·kg-1·d-1)和非治疗组,C57BL/6小鼠作为正常对照组.于20周龄检测体质量、随机血糖、尿微量白蛋白、尿肌酐,观察肾脏形态改变.应用磁珠灌注法分离肾小球,提取总RNA,应用Affymetrix GeneChip miRNA芯片,分析KKAv小鼠肾小球microRNA表达谱的变化,以及氯沙坦对microRNA表达谱的影响.结果 KKAy小鼠的体质量和血糖较正常对照C57BL/6组小鼠显著升高(均P＜ 0.05),氯沙坦治疗显著改善2型糖尿病KKAy小鼠的尿白蛋白/肌酐比值[( 539.71±100.23) mg/g比(728.00±177.19) mg/g,P＜0.05]和肾脏病理损害,而对血糖无影响.miRNA芯片分析结果发现,与正常对照C57BL/6小鼠相比,20周龄KKAy小鼠肾小球内10个miRNA的表达上调;12个miRNA的表达下调.与KKAy非治疗组小鼠相比,20周龄氯沙坦治疗组KKAy小鼠肾小球内共有4个miRNA表达下调,其中miR-503和miR-181d在KKAy非治疗组小鼠肾小球内的表达显著上调,氯沙坦治疗可抑制其过表达.结论 miR-503和miR-181d在糖尿病KKAy小鼠肾小球内的表达显著上调,氯沙坦治疗可抑制其在糖尿病状态下的异常表达,可能为糖尿病肾病新的治疗靶点.     

Increased serum high-molecular-weight complex of adiponectin in type 2 diabetic patients with impaired renal function

BACKGROUND/AIM: Adiponectin, an adipocyte-derived protein, has been shown to exert antidiabetic, anti-inflammatory, and antiatherosclerotic effects. Although recent reports show an increase in the total adiponectin levels in chronic kidney disease patients and in patients with end-stage renal disease, the nature of biodegradation and renal involvement of adiponectin is largely unknown. We aimed at determining whether the high-molecular-weight (HMW) complex of adiponectin is associated with renal insufficiency in type 2 diabetic patients. METHODS: A total of 179 type 2 diabetic patients were selected from among outpatients and divided into four groups according to their albumin-to-creatinine ratio: patients with normoalbuminuria (n = 86), patients with microalbuminuria (n = 44), patients with macroalbuminuria (n = 23), and patients on hemodialysis (n = 26). The serum HMW adiponectin was specifically assayed with a commercially available enzyme-linked immunosorbent assay kit. RESULTS: The HMW adiponectin levels were higher in patients on hemodialysis (17.1 +/- 8.2 microg/ml) and in those with macroalbuminuria (14.3 +/- 8.7 microg/ml) than in patients with normoalbuminuria (7.2 +/- 5.6 microg/ml) and microalbuminuria (10.8 +/- 7.0 microg/ml). Univariate linear regression analysis showed that the HMW adiponectin concentrations correlated negatively with the estimated glomerular filtration rate in patients with normoalbuminuria, microalbuminuria, and macroalbuminuria (r = -0.42, p < 0.001). Multiple stepwise regression analysis disclosed that estimated glomerular filtration rate, pioglitazone therapy, gender differences, and systolic blood pressure were independently associated with HMW adiponectin levels (r = 0.56). CONCLUSIONS: The serum HMW adiponectin concentrations are higher in type 2 diabetic patients with nephropathy, and these levels are also associated with renal insufficiency.     

Nephrinuria in diabetic nephropathy of type 1 diabetes

Diabetic nephropathy is the leading cause of end-stage renal disease. Because early diagnosis and treatment may prevent the complication, new tools for an early detection are needed. One of the key components of the glomerular filtration slit spanning between neighboring podocytes is nephrin. Its expression is altered in experimental models of diabetes and also in various human proteinuric diseases, including diabetes. We studied whether type 1 diabetic patients with or without nephropathy exhibit immunoreactive nephrin in the urine, reflecting early damage of the filtration barrier. Diabetic patients with normoalbuminuria (n = 40), with microalbuminuria (n = 41), and with macroalbuminuria (n = 39) and patients previously normoalbuminuric but now testing positive for microalbuminuria (newMicro, n = 39) were screened for nephrinuria with Western blotting using two affinity-purified anti-nephrin antibodies. Nondiabetic healthy subjects (n = 29) were also studied. Nephrinuria was present in 30% of normoalbuminuric, 17% of microalbuminuric, 28% of macroalbuminuric, and 28% of newMicro patients. Of female patients, 35% were nephrinuric compared with only 19% of male patients (P = 0.02). None of the control subjects was nephrinuric. In conclusion, glomerular filtration barrier may be affected in one-third of diabetic patients manifesting as early nephrinuria. Nephrinuria may have prognostic value and become a marker of susceptibility for kidney complications in diabetes.     

Increased levels of alpha-defensin (-1, -2 and -3) in type 1 diabetic patients with nephropathy.

OBJECTIVE: Diabetic nephropathy is associated with low-grade inflammation and activation of the complement system. Defensins, as part of the innate immune system, may play a regulatory role in the complement cascade and may also augment the production of proinflammatory cytokines. The aim of this study was therefore to elucidate whether alpha-defensin is associated with diabetic nephropathy, low-grade inflammation and lipid profiles. RESEARCH DESIGN AND METHODS: Data were obtained from 189 patients with type 1 diabetes selected from the FinnDiane Study. Patients were divided into three groups according to their albumin excretion rate (AER) in three consecutive overnight or 24-h urine collections: normoalbuminuria (AER <20 microg/min or <30 mg/24 h), microalbuminuria (20 200 microg/min or >300 mg/24 h). Alpha-defensin was determined by a novel, solid-phase radioimmunoassay (RIA) based on a monoclonal antibody, which recognizes alpha-defensin isoforms 1-3. RESULTS: Total serum alpha-defensin (-1, -2 and -3) concentrations were higher (P < 0.001) in patients with macroalbuminuria compared to micro- and normoalbuminuria, but no difference was observed between normoalbuminuria and microalbuminuria. In multiple linear regression analysis alpha-defensin was associated with systolic blood pressure (P = 0.032), HDL-cholesterol (P = 0.013), total cholesterol (P = 0.008), age (P = 0.001) and estimated glomerular filtration rate (P = 0.001), but not with low-grade inflammatory markers. CONCLUSIONS; Serum alpha-defensin (-1, -2 and -3) concentrations are increased in type 1 diabetic patients with diabetic nephropathy.     

Relationship of p22phox C242T polymorphism with nephropathy in type 2 diabetic patients

BACKGROUND: In this case-control study, we investigated the possible involvement of the p22phox C242T polymorphism in the development and progression of diabetic nephropathy (DN) in 535 Caucasian Brazilians with type 2 diabetes. We also evaluated the effects of the interaction of the C242T polymorphism with smoking and hypercholesterolemia on the susceptibility to nephropathy. METHODS: Genotype analysis was performed using polymerase chain reaction (PCR) followed by digestion with restriction enzyme. Logistic regression analysis was used to control for independent risk factors associated with nephropathy. RESULTS: The genotype frequencies in patients with overt DN (CC/CT/TT: 0.36/0.47/0.17) were not significantly different from those of diabetic individuals with normoalbuminuria (0.47/0.41/0.12) or microalbuminuria (0.42/0.48/0.10) (p=0.214). Likewise, there were no differences in the T allele frequency among patients with normoalbuminuria, microalbuminuria or overt DN (0.33, 0.34 and 0.40, respectively; p=0.111). However, the T allele was found to be more frequent among smokers with overt nephropathy (macroalbuminuria and/or in dialysis) than those who had normoalbuminuria (43 vs. 32%, p=0.045). The multiple logistic regression analysis confirmed that the CT+TT genotypes were independently associated with a higher risk of having overt nephropathy among smokers [odds ratio (OR)=6.76, 95% confidence interval (95% CI) 1.83-25.02]. CONCLUSIONS: Our study shows a gene-environment interaction associated with the increased risk of DN progression in Caucasian Brazilian smokers with type 2 diabetes. Further studies should be performed to clarify whether it exists, and to what extent there is a relationship between the p22phox C242T polymorphism and DN.     

Urinary excretion of podocytes in patients with diabetic nephropathy.

BACKGROUND: Detection of podocytes in the urinary sediments of children with glomerulonephritis has been shown to indicate severe injury to the podocytes. The aim of the present study was to determine whether podocytes are present in the urine sediments of adult patients with diabetes with and without nephropathy and whether trandolapril is effective for podocyte injury. METHODS: Fifty diabetic patients (10 with normoalbuminuria, 15 with microalbuminuria, 15 with macroalbuminuria and 10 with chronic renal failure) and 10 healthy controls were studied. Urinary podocytes were examined by immunofluorescence using monoclonal antibodies against podocalyxin, which is present on the surface of podocytes. In addition, we studied plasma metalloproteinase (MMP)-9 concentrations in all patients. RESULTS: Urinary podocytes were absent in healthy controls, diabetic patients with normoalbuminuria and diabetic patients with chronic renal failure. Podocytes were detected in the urine of eight diabetic patients with microalbuminuria (53%) and of 12 patients with macroalbuminuria (80%). The number of podocytes in the urine of patients with macroalbuminuria was significantly greater than in patients with microalbuminuria (P:<0.01). However, there was no relationship between urinary albumin excretion and urinary podocytes. In addition, plasma MMP-9 concentrations were significantly correlated with the number of urinary podocytes (P:<0.01). Twelve diabetic patients with macroalbuminuria and eight patients with microalbuminuria who had urinary podocytes were treated with the angiotensin-converting enzyme inhibitor trandolapril. Urinary albumin excretion, the number of podocytes and plasma MMP-9 concentrations were reduced by the trandolapril treatment. CONCLUSIONS: Podocytes in the urine may be a useful marker of disease activity in diabetic nephropathy. Trandolapril may be effective for podocyte injury.     

Influence of renal involvement on peripheral blood mononuclear cell expression behaviour of tumour necrosis factor-alpha and interleukin-6 in type 2 diabetic patients.

BACKGROUND: Type 2 diabetes is associated with a high cardiovascular risk, which is even increased if renal damage is superimposed. Peripheral blood mononuclear cells (PBMCs) and pro-inflammatory cytokines are key factors linking type 2 diabetes and atherosclerosis. We investigated the influence of renal damage on serum, urinary and PBMCs expression behavior of TNF-alpha and IL-6 in these patients. METHODS: PBMCs were isolated by density gradient centrifugation (Ficoll-Paque method) from fasting blood samples of 22 non-diabetic control subjects and 78 diabetic patients with normal renal function and different stages of diabetic nephropathy (18 with normoalbuminuria, 29 with microalbuminuria and 31 with macroalbuminuria). Expression levels of TNF-alpha and IL-6 were analyzed by real-time quantitative RT-PCR. Serum and urinary TNF-alpha and IL-6 concentrations were measured by a solid-phase, chemiluminescent immunometric assay. RESULTS: The mean percent increases in the serum and urinary levels of TNF-alpha and IL-6 in diabetic patients with respect to control subjects were 176% (P < 0.0001), 250% (P < 0.0001), 114% (P < 0.0001) and 39.6% (P = 0.01), respectively. The mRNA expression level of TNF-alpha was higher by 68.8% (P < 0.001) and IL-6 mRNA levels were higher by 64.1% (P < 0.001) with respect to non-diabetic controls. TNF-alpha mRNA expression in patients with macroalbuminuria was higher by 84.8% with respect to subjects with normalbuminuria (P < 0.001) and by 29% with respect to individuals with microalbuminuria (P < 0.05). Likewise, microalbuminuric patients showed a 44.5% increase in TNF-alpha mRNA expression compared to subjects with normoalbuminuria (P < 0.05). Concerning IL-6, the mRNA expression levels of this cytokine was higher by 63.1% with respect to normoalbuminuric subjects (P < 0.01), and by 23.1% with respect to patients with microalbuminuria (P < 0.05). However, with respect to controls, diabetic patients with normoalbuminuria had similar serum TNF-alpha and urinary excretion of IL-6, without any differences in the mRNA expression levels of these cytokines in PBMCs. Partial correlation and multiple regression analysis using TNF-alpha and IL-6 mRNA levels as the dependent variables showed that urinary albumin excretion (UAE) was direct and independently associated with the expression profile of these pro-inflammatory cytokines in PBMCs. CONCLUSIONS: These data show for the first time the relationship between inflammatory activation of PBMCs (reflected by enhanced mRNA expression of TNF-alpha and IL-6) and renal involvement (reflected by increased UAE) in type 2 diabetic patients. These results provide potential insights for the increased inflammation, accelerated atherosclerosis and cardiovascular risk associated with nephropathy in type 2 diabetes.     

Urinary Albumin Excretion and the Risk of Graft Loss and Death in Proteinuric and Non-proteinuric Renal Transplant Recipients

BACKGROUND: Microalbuminuria and macroalbuminuria constitute risk factors for ESRD and death in non-transplanted populations. Whether microalbuminuria (especially in non-proteinuric patients) and macroalbuminuria constitute risk factors for graft loss and death is presently unknown in renal transplantation. METHODS: We retrospectively assessed the association between urinary albumin excretion (UAE) and ESRD and death in renal transplantation. RESULTS: UAE was measured in 616 (397 proteinuric; 219 non-proteinuric patients) renal transplant recipients. They were grafted for 62 months (range: 6-192). During the 40 months (3.7-99) thereafter, 31 patients underwent dialysis and 32 died. Microalbuminuria (vs. normoalbuminuria) and macroalbuminuria (vs. microalbuminuria) were powerful risk factors for graft loss [OR: 14.25 (2.88-52.3) and 16.41 (7.46-36.0), respectively, both p < 0.0001], even after adjustments on renal function and diabetes. Among the 219 non-proteinuric patients, microalbuminuria (vs. normoalbuminuria) was a significant risk factor for graft loss [OR: 23.09 (1.93-276.4), p = 0.0132]. Both microalbuminuria (vs. normoalbuminuria) [OR: 5.55 (2.43-12.66), p < 0.0001] and macroalbuminuria (vs. microalbuminuria) [OR: 4.12 (1.65-10.29), p = 0.0024] were predictive of death. CONCLUSIONS: Microalbuminuria and macroalbuminuria are powerful independent predictors of ESRD and death. Microalbuminuria is a risk factor for graft loss even in non-proteinuric patients. UAE provides additional information on renal and patient prognosis as compared to proteinuria and renal function.     

Serum ionized magnesium levels in type 2 diabetic patients with microalbuminuria or clinical proteinuria

BACKGROUND/AIMS: The association between microalbuminuria and magnesium depletion is a controversial issue, and serum ionized magnesium levels have not been previously studied in patients with different grades of diabetic nephropathy. Therefore, the aim of this study was to evaluate circulating ionized magnesium concentrations in patients with non-insulin-dependent diabetes mellitus (NIDDM) and incipient or overt diabetic nephropathy. METHODS: We measured fasting plasma glucose, creatinine, creatinine clearance estimate, total cholesterol and triglycerides, and serum ionized magnesium (ion-selective electrodes, ISE) in 30 NIDDM patients with urinary albumin excretion rate (UAER) <20 microg/min (normoalbuminuria), 30 NIDDM patients with microalbuminuria (20 < UAER < 200 microg/min), 30 NIDDM patients with clinical proteinuria (UAER >200 microg/min), and 20 healthy subjects. RESULTS: Serum ionized magnesium levels were significantly reduced in diabetic patients when compared to control subjects (0.39 +/- 0.06 vs. 0.58 +/- 0.05 mmol/l, p < 0.001). Moreover, diabetic patients with microalbuminuria or clinical proteinuria showed a significant decrease in serum ionized magnesium with respect to normoalbuminuria group (normoalbuminuria: 0.45 +/- 0. 02 mmol/l; microalbuminuria: 0.36 +/- 0.05 mmol/l, p < 0.001; clinical proteinuria: 0.35 +/- 0.04 mmol/l, p < 0.001). Serum ionized magnesium showed a significant negative correlation with plasma HbA1c and triglycerides in both microalbuminuria and clinical proteinuria groups. Multiple linear regression analysis showed that circulating ionized magnesium levels decrease together with the increase of plasma HbA1c and triglycerides in NIDDM patients with incipient or overt nephropathy, also after adjusting for age, sex, BMI, diabetes duration, systolic and diastolic blood pressure, hypoglycemic therapy, plasma creatinine, creatinine clearance, plasma cholesterol and fasting glucose. CONCLUSIONS: Microalbuminuria and clinical proteinuria, as well as poor glycometabolic control and hypertriglyceridemia, are associated to relevant alterations in magnesium metabolism, and the measurement of serum ionized magnesium seems to represent a useful biochemical tool for the study of magnesium disturbances in patients with different grades of diabetic nephropathy.     

Chemokine receptor genotype is associated with diabetic nephropathy in Japanese with type 2 diabetes.

Glomerular infiltration of monocytes/macrophages occurs in diabetic patients with nephropathy, and chemokine receptor signals are thought to play a key role in the development of nephropathy. Recently, polymorphism of the chemokine receptor (CCR)2 coding region V64I and the CCR5 promoter region 59029 (G/A) have been identified. Accordingly, we evaluated the effects of these genotypes on diabetic nephropathy. CCR2 V64I and CCR5 59029 (G/A) were detected by polymerase chain reaction-restriction fragment-length polymorphism in 401 patients with type 2 diabetes who had a serum creatinine <2.0 mg/dl. Although the CCR2 V64I genotype showed no association with nephropathy, the frequency of the CCR5 59029 A-positive genotype (G/A or A/A) was significantly higher in patients with microalbuminuria (urinary albumin-to-creatinine ratio [ACR] > or = 30 and <300 mg/gCre, 86%) and patients with macroalbuminuria (ACR > or = 300 mg/gCre, 87%) than in patients with normoalbuminuria (ACR <30 mg/gCre, 75%; P = 0.0095). Polytomic logistic regression analysis showed that the CCR5 59029 A-positive genotype was associated with nephropathy (odds ratio 2.243, P = 0.0074). These results suggest that the CCR5 promoter 59029 A genotype may be an independent risk factor for diabetic nephropathy in patients with type 2 diabetes.     

Role of GLUT1 gene in susceptibility to diabetic nephropathy in type 2 diabetes

BACKGROUND: The XbaI polymorphism in the glucose transporter GLUT1 gene has been implicated in the development of diabetic nephropathy in Chinese type 2 diabetes patients. METHODS: To examine whether the XbaI polymorphism is involved in the development of diabetic nephropathy in Caucasian type 2 diabetes patients, a large case control study was performed. The study group of 444 patients with type 2 diabetes consisted of three subgroups: 162 patients with normoalbuminuria (only patients with duration of type 2 diabetes of at least 10 years after diagnosis); 150 with microalbuminuria; and 132 subjects with persistent proteinuria or chronic renal failure (CRF). The polymerase chain reaction (PCR)-based genotyping of the XbaI polymorphism was performed in each subject. RESULTS: The genotype distribution in the subgroups showed an increased frequency of the (+/+) genotype in patients with microalbuminuria (41%; OR 1.40, 95% CI, 0.89 to 2.24) and proteinuria/CRF (47%; OR 1.82, 95% CI, 1.13 to 2.93, P = 0.013) when compared with normoalbuminuria (33%). No difference in the genotype distribution was observed between type 2 diabetes patients and healthy controls. CONCLUSIONS: The results of this study in Caucasian patients with type 2 diabetes indicate that the XbaI(-) allele in the GLUT1 gene protects against the development of diabetic nephropathy. Our results are in contrast to the case control study in Chinese patients with type 2 diabetes in which the presence of the XbaI(-) allele appeared to have a strong association with the development of diabetic nephropathy.