A new enzyme-linked immunosorbent assay for urine and serum concentrations of the carboxyterminal domain (NCl) of collagen. IV. Application in type I (insulin-dependent) diabetes

The major collagenous component of glomerular basement membrane (GBM) is collagen IV. Serum concentrations of the carboxyterminal end (NCl) of collagen IV have been proposed to be related to GBM turnover, which has been suspected to increase in diabetes mellitus. For the quantification of serum and urinary concentrations of NCl, a specific, sensitive enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies was developed. The detection limit of the assay was 30 micrograms/l at the 50% intercept of the standard curve. The intra- and interassay coefficients of variation were 6.2% and 13.9% for serum, respectively, and 11.9% and 39.7% for urine, respectively. The levels of NCl in serum and urine in 67 insulin-dependent diabetics and in 90 sex- and age-matched controls were compared. There were no differences in the serum concentrations of NCl between the diabetics and healthy controls. As a group, the diabetics had a higher urinary excretion of NCl than the controls (20.1 vs 12.5 ng/min, 2p less than 0.05). Furthermore, the results showed that the excretion of NCl in the urine was normal when the urinary albumin excretion rate (AER) was normal (less than 6.5 micrograms/min). The excretion was increased during the early stage of incipient diabetic nephropathy (AER 6.5-30 micrograms/min) and decreased to normal values with progression to clinical diabetic nephropathy (AER above 500 micrograms/min). Thus, it is suggested that an increased urinary excretion of NCl may be an early marker for incipient diabetic nephropathy.     

Improved competitive enzyme-linked immunoassay (ELISA) for albuminuria

Microalbuminuria has been established as a marker strongly predictive of diabetic nephropathy. The appearance of microalbuminuria (30-150 micrograms/min) is considered to herald incipient nephropathy, and the progression to clinical proteinuria (greater than 200 mg/24 h) is believed to reflect a shift from reversible to irreversible renal damage in diabetic patients. Periodic monitoring of albumin excretion could thus detect diabetic renal disease at an early, potentially reversible stage. However, this is not routinely done, largely due to cumbersome collection and methodologic constraints. We therefore have developed a simplified competitive immunoassay (ELISA) that is sensitive to 10 ng and reproducibly quantifies urinary albumin levels between 0.1-10 micrograms/ml, a range appropriate to that demarcating normal from microalbuminuric patients. Examination of results obtained with aliquots of 24 h and fractional urine collections, without and with correction for creatinine (albumin: creatine ratio), in basal and post-exercise states indicates that (a) this assay can effectively measure urine albumin concentration in single void specimens, and albumin excretion rates in fractional collections, and (b) conversion to albumin:creatinine ratio is not necessary to discriminate normal from microalbuminuric states.     

Factors affecting the urinary excretion of albumin in insulin-dependent diabetes

To determine the specificity of the urine excretion of albumin as a measure of glomerular permeability in early insulin-dependent diabetic nephropathy, the effect of variable glomerular filtration and urine flow rates on albumin, beta 2-microglobulin excretion, and the fractional renal clearance of neutral dextran (Stokes Einstein Radius 24-46 A) was examined. Five insulin-dependent diabetic subjects with normal glomerular permeability (albumin excretion less than 30 micrograms/min) and one with elevated albumin excretion (195 micrograms/min) were studied pre and post strict glucose control with constant subcutaneous insulin infusion for 7 days. The albumin excretion in the 5 subjects never exceeded 30 micrograms/min during wide variations in glomerular filtration and urine flow rates. A positive correlation between beta 2-microglobulin excretion and urine flow (r = 0.81), and glomerular filtration (r = 0.77) rates was observed. In contrast, albumin excretion showed no correlation, indicating different factors affect the excretion rate of albumin and beta 2-microglobulin. Therefore, elevated albumin excretion (greater than 30 micrograms/min) in insulin-dependent diabetes is due to increased glomerular permeability and not changes in glomerular filtration and urine flow rates, and the albumin/ beta 2-microglobulin ratio may not be a valid indicator of changing glomerular permeability. The fractional neutral dextran clearances remained unchanged with variation in glomerular filtration and urine flow rates. The sieving curve was identical in all subjects for neutral dextran 40 A, the size of albumin, suggesting that reduced glomerular charge selectivity may contribute to increased albuminuria in progressive diabetic glomerulosclerosis.     

Kidney volume in type 1 (insulin-dependent) diabetic patients with normal or increased urinary albumin excretion: effect of long-term improved metabolic control

Forty-seven patients with type 1 (insulin-dependent) diabetes mellitus and 14 normal subjects had renal volume determined by an ultrasonic technique. Renal volume of 299 +/- 49 ml/1.73 m2 (mean +/- SD) in type 1 diabetic patients with normal urinary albumin excretion exceeded that in the normal subjects (245 +/- 53 ml/1.73 m2, p less than 0.05). Compared with diabetic patients with normal urinary albumin excretion, renal volume was significantly higher in patients with microalbuminuria (372 +/- 24 ml/1.73 m2, p less than 0.05) and patients with clinical nephropathy (352 +/- 48 ml/1.73 m2, p less than 0.05). In a multiple linear regression with HbA1c, urinary albumin excretion, age, diabetes duration and mean blood pressure as independent variables, variations in HbA1c could account for 33% of the variations in kidney volume (n = 47, r = 0.57, p less than 0.01). The other variables played no role. When only patients without clinical nephropathy were included, HbA1c and kidney volume remained significantly correlated (n = 34, r = 0.60, p less than 0.01). In those patients a strong correlation between kidney volume and function, i.e. the glomerular filtration rate was found (n = 34, r = 0.70, p less than 0.01); metabolic control and function were also correlated (n = 34, r = 0.62, p less than 0.01). The urinary albumin excretion accounted for only 6% of the variation of the kidney volume (NS). In nine patients with microalbuminuria the kidney volume could be reduced during 2 years of improved metabolic control by means of insulin infusion pumps.(ABSTRACT TRUNCATED AT 250 WORDS)     

尿微量白蛋白/肌酐比值对糖尿病肾病早期诊断价值

目的 探讨尿微量白蛋白/肌酐比值在糖尿病肾病早期诊断中的价值.方法 选取确诊为2型糖尿病患者293例和体检健康体检者70例,对其尿微量白蛋白/肌酐比值（晨起空腹及随机）、24小时尿微量白蛋白定量、尿微量白蛋白排泄率、尿素氮、血肌酐、尿常规等临床资料进行回顾性分析,观察上述不同检测方法对糖尿病早期诊断灵敏度.结果 晨起、随机尿微量白蛋白/肌酐值与尿微量白蛋白排泄率（urine albumin excretion rateUAER）、24 h尿微量白蛋白定量成显著正相关,晨起空腹尿ACR与UAER、24小时尿微量白蛋白定量的相关系数分别为r＝0.936（P＜0 01）,r＝0.906,（P＜0.01）;随机尿ACR与UAER和24h尿微量白蛋白相关系数分别为r＝0.756（P＜0.01）,r＝0.738,（P＜0.01）.2型糖尿病组尿ACR阳性组和阴性组之间比较尿蛋白、血肌酐、尿素氮水平无统计学差异,P＞0.05.将血肌酐、尿素氮、尿ACR诊断糖尿病肾病敏感性比较,尿ACR阳性率显著高于前两者,P＜0.01.结论 晨起空腹及随机尿微量白蛋白/肌酐比值两者均可以作为糖尿病肾病早期诊断的敏感指标.     

不同时段尿微量清蛋白对糖尿病早期肾损害的诊断价值

目的探讨2型糖尿病肾病患者不同时段尿微量清蛋白(u-MA)变化及其对2型糖尿病患者早期肾损伤的诊断价值。方法收集糖尿病肾病患者组(n=27)和健康对照组(n=30)连续3 d的晨尿、餐后尿、随机尿和24 h尿,进行尿微量清蛋白测定。结果糖尿病患者尿微量清蛋白排泌高峰为餐后尿(61.7±26.5 mg/L),其次为晨尿(58.9±23.5 mg/L)和24 h尿(56.6±13.2 mg/L),3者显著高于随机尿(37.2±21.4 mg/L)(P<0.05),且各时段尿微量清蛋白都有较大的日间差异,以随机尿差异最大CV=57.5%,餐后尿、晨尿、和24 h尿分别为42.9%、39.9%和23.3%,糖尿病肾病患者各时段尿微量清蛋白与对照组比较差异有统计学意义(P<0.05)。结论餐后尿和晨尿微量清蛋白含量的多次检测有利于提高2型糖尿病患者早期肾损害的诊断。     

糖尿病肾病患者尿转化生长因子-β_1、α_1微球蛋白及视黄醇结合蛋白水平的变化

目的：探讨糖尿病肾病（DN）患者尿转化生长因子β1（TGF-β1）、α1微球蛋白（α1-MG）及视黄醇结合蛋白（RBP）水平的变化。方法：根据尿白蛋白排泄率（UAER）将65例2型糖尿病患者分成3组,测定尿TGF-β1、α1-MG、RBP含量,并与对照组23名健康体检者进行比较。结果：DN患者尿TGF-β1、α1-MG、RBP水平均高于健康对照组（P〈0.05或0.01）。尿TGF-β1、α1-MG、RBP与UAER呈正相关（r分别为0.73,0.62,0.85,P均〈0.01）。结论：早期DN患者尿TGF-β1、α1-MG水平升高,可能在评价DN病情进展及预后转归方面有一定临床价值。     

胱抑素C及同型半胱氨酸的血清含量与糖尿病肾病患者肾小球滤过率的相关性研究

目的:通过检测血胱抑素C(CystatinC,CysC)、同型半胱氨酸(Homocysteine,Hcy)浓度及尿白蛋白清除率(urinealbumin excretionrate,UAER)在糖尿病肾病(diabetic nephropathy,DN)进程中的变化以及与肾小球滤过率(glomerular filtration rate,GFR)的相关性,探讨其在DN诊断中的价值。方法:共47例患者,根据肾小球滤过率将其分为早期糖尿病肾病组(early-DN组,GFR≥60mL/min)及晚期糖尿病肾病组(end-DN组,GFR<60mL/min),比较两组间CysC、Hcy的变化以及与肾小球滤过率的相关性。结果:end-DN组CysC、UAER均高于early-DN组(P<0.01)。相关分析显示肾小球滤过率与CysC、Hcy、UAER负相关(r=-0.584,P=0.000;r=-0.547,P=0.000;r=-0.507,P=0.000),在慢性肾脏病(chronic kidney disease,CKD)Ⅱ、Ⅲ期,肾小球滤过率与CysC、Hcy负相关(r=-0.617,P=0.000;r=-0.431,P=0.018)。结论:糖尿病患者中,伴随慢性肾脏病进程,CysC、Hcy、UAER逐渐升高,尤其在CKDⅡ、Ⅲ期,CysC、Hcy联合检测与UAER比较,能更好的反应糖尿病肾病肾小球滤过功能异常。     

Tubular secretion of Tamm-Horsfall protein in type 1 (insulin-dependent) diabetes mellitus using a simplified enzyme linked immunoassay.

The relationship between glomerular and tubular dysfunction and metabolic control in type 1 diabetes was studied. To that end the urinary excretion rates of albumin and Tamm-Horsfall protein as well as HbA1c levels were measured in 58 patients with different degrees of diabetic nephropathy and in 76 apparently healthy subjects matched for sex and age. The urinary Tamm-Horsfall protein levels were measured by a simplified enzyme linked immunoassay. The intra- and interassay variations were 8.9% and 13.6%, respectively. The intraindividual variation was 41% and the sensitivity of the assay was 4 micrograms/l. The Tamm-Horsfall protein excretion rate was 42.1 x/2.0 micrograms/min (geometric mean x/tolerance factor) in the diabetic patients compared to 34 x/1.9 micrograms/min in the control subjects (NS). The diabetic patients had higher albumin excretion rate (38.5 x/7.3 micrograms/min) than the control subjects (4.7 x/2.3 micrograms/min; P less than 0.001). By using multivariate analysis of variance, HbA1c level was found to be the only independent variable associated with Tamm-Horsfall protein excretion rate in diabetic patients (r = -0.28; P = 0.04), while no relationship was found between Tamm-Horsfall protein excretion rate and age, age at onset and duration of diabetes, gender, serum creatinine, diuresis, urinary albumin excretion rate, systolic and diastolic blood pressure levels and antihypertensive treatment. The urinary albumin excretion rate was associated with diastolic blood pressure (r = 0.34; P = 0.02) but not with HbA1c levels when testing the above variables by multivariate analysis of variance. In conclusion, these results may indicate a lack of relationship between glomerular and tubular dysfunction. The former was influenced only by diastolic blood pressure levels and the latter only by the degree of metabolic control. However, the correlations were weak and do not provide any insight into what is actually responsible for glomerular and tubular dysfunction.     

Urinary excretion of angiotensin-converting enzyme in NIDDM patients with nephropathy

Twenty-four-hour urinary excretion of angiotensin-converting enzyme (ACE) was investigated in relation to that of albumin and beta 2-microglobulin (beta 2M) in 25 non-insulin-dependent diabetes mellitus (NIDDM) patients without nephropathy, 13 NIDDM patients with incipient nephropathy, 18 NIDDM patients with overt nephropathy, and 14 nondiabetic subjects. NIDDM patients without nephropathy and nondiabetic subjects were similar in albumin, beta 2M, and ACE excretion. NIDDM patients with incipient nephropathy had elevated albumin excretion (P less than .01) and similar beta 2M and ACE excretion compared with nondiabetic subjects. On the other hand, NIDDM patients with overt nephropathy had elevated albumin, beta 2M, and ACE excretion compared with nondiabetic subjects (P less than .01). In all NIDDM patients studied, a positive correlation was found between ACE excretion and albumin excretion (r = 0.76, P less than .001) or beta 2M excretion (r = 0.52, P less than .01). These data suggest that elevated ACE excretion in NIDDM patients with overt nephropathy may be reflective of renal tubular damage.     

Acute and long-term effect of antihypertensive treatment on exercise-induced albuminuria in incipient diabetic nephropathy

The aim of the study was to clarify whether antihypertensive treatment could affect the systolic blood pressure (SBP) and urinary albumin excretion (UAE) in diabetics during exercise (450 kpm/min, followed by 600 kpm/min, 20 min each). Young male insulin-dependent diabetics with normal UAE (n = 9) and diabetics with incipient nephropathy (n = 7) were examined in an acute study. Five patients with incipient diabetic nephropathy participated in a long-term study. Incipient diabetic nephropathy is defined as persistently elevated UAE (greater than 15 micrograms/min), but no clinical proteinuria. In the acute study, using placebo/metoprolol 10 mg i.v. in patients with normal UAE, the maximal SBP at 600 kpm/min was reduced by 17 mmHg +/- 10 (SD) (2p less than 1.0%) and the maximal SBP at 600 kpm/min in the patients with incipient nephropathy was reduced by 15 mmHg +/- 11 (SD) (2p less than 1.0%). However, no difference was observed in UAE, in patients with normal UAE or those with incipient nephropathy. Five of the patients with incipient nephropathy were followed with repeated exercise tests before and during 2.6 years of antihypertensive treatment, using metoprolol 200 mg/24 h and subsequently also hydroflumethiazid 25 mg/24 h. The maximal SBP at 600 kpm/min at the end of the study compared to the pretreatment level was reduced by 38 mmHg +/- 12 (SD) (2p less than 1.0%), and furthermore the exercise-induced elevated UAE was reduced by 61% +/- 29 (SD) (2p = 2.0%).(ABSTRACT TRUNCATED AT 250 WORDS)     

醛固酮受体拮抗剂治疗糖尿病肾病的临床研究

目的:探讨应用醛固酮受体拮抗剂能否改善糖尿病肾病患者的炎症病变、减少糖尿病肾病患者的蛋白尿排泄。方法:采用前瞻性随机对照方法,对糖尿病肾病(Ⅲ、Ⅳ期)患者60例,按照1:1分为治疗组(螺内酯)和对照组(安慰剂),予ACEI类或AT1RA类的药物治疗基础上加用螺内酯片(20mg/d)或安慰剂,共治疗12周。检测治疗前后24h尿微量白蛋白、尿单核细胞趋化蛋白-1(MCP-1)、肾功能等情况。结果:治疗后两组的肌酐清除率均较治疗前升高,但治疗组治疗前后比较差异有非常显著性(59.4±13.4,61.2±13.5,P=0.000)。两组治疗前后的24h尿微量白蛋白及尿MCP-1比较差异均有显著性(P<0.05),但治疗组治疗前后24h尿微量白蛋白及尿MCP-1比较差异有非常显著性(1450.13±1369.84,779.33±656.57,P=0.001;39.69±9.71,32.79±6.74,P=0.000)。治疗前后二者定量呈明显正相关。结论:醛固酮受体拮抗剂能改善糖尿病肾病患者的炎症病变,明显减少蛋白尿排泄,延缓了糖尿病肾病的进展。     

血管内皮生长因子与糖尿病肾病的相关性研究

目的探讨血管内皮生长因子(VEGF)与2型糖尿病肾病的关系。方法采用ELISA法测定2型糖尿病患者216例(其中单纯2型糖尿病患者112例，糖尿病肾病患者104．例)和正常对照组98例血清中的VEGF，并分析其与2型糖尿病并发肾病的关系。结果糖尿病肾病患者血清VEGF水平显著高于单纯2型糖尿病组和正常对照组。血清VEGF与收缩压、糖化血红蛋白、尿白蛋白排泄率呈显著正相关，与年龄、性别、病程、体重指数、血脂等无显著相关性。结论血清VEGF与2型糖尿病肾病密切相关，其浓度的检测对监测2型糖尿病肾病的发生、发展有重要的临床意义。     

血清同型半胱氨酸等指标联合检测对糖尿病肾病的临床价值

目的探讨血清同型半胱氨酸、血清胱抑素C和尿β2-微球蛋白联合检测对糖尿病肾病的临床价值。方法将102例糖尿病肾病患者按尿液清蛋白排泄率分为A、B、C 3组：正常清蛋白组（A组,n=42）、微量清蛋白组（B组,n=35）、大量清蛋白组（C组,n=25）,同时以20例健康人作为健康对照组。分别检测血清同型半胱氨酸、血清胱抑素C、尿液清蛋白和尿β2-微球蛋白水平,对检测结果采用SAS统计软件进行统计学处理作对比分析。结果糖尿病肾病患者各组间血清同型半胱氨酸、血清胱抑素C和尿β2-微球蛋白检测值比较差异均有统计学意义（P〈0.01）。102例糖尿病肾病患者血清同型半胱氨酸、血清胱抑素C和尿β2-微球蛋白检测值采用多元线性相关分析呈正相关（r为0.250,0.410,0.365;P〈0.01）。结论血清同型半胱氨酸、血清胱抑素C和尿β2-微球蛋白联合检测可以推断糖尿病肾病的损害程度,更全面地评价早期糖尿病肾损害,提示临床应早期干预血清同型半胱氨酸水平。     

Impaired left-ventricular function in insulin-dependent diabetic patients with increased urinary albumin excretion

Cardiac function was studied in 30 patients with insulin-dependent diabetes mellitus. Three groups, matched for age and diabetes duration, were defined as: group I (n = 10), normal urinary albumin excretion less than 30 mg 24 h-1; group II (n = 10), incipient diabetic nephropathy (urinary albumin excretion in the range of 30-300 mg 24 h-1); and group III (n = 10), clinical diabetic nephropathy (urinary albumin excretion greater than 300 mg 24 h-1). Ten non-diabetic subjects matched for sex and age served as controls. The left-ventricular end-diastolic volume measured by radionuclide cardiography was, at rest and during exercise, lower in group II and III compared with controls (p less than 0.05), while intermediate values were found in group I. The cardiac output was similar in the control group and group I; it was reduced, but not significantly so (p = 0.10), in group III and was significantly lower in group II (p less than 0.05). Stroke volume was also lower in group II and III than in controls (p less than 0.05), but not so in group I. These differences could not be explained by differences in metabolic control, blood pressure, blood volume status, degree of autonomic neuropathy or frequency of coronary heart disease. Our results might suggest that insulin-dependent diabetic patients with slightly but persistently elevated urinary albumin excretion have reduced diastolic compliance of the left-ventricle leading to impaired cardiac performance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased collagen IV excretion in diabetes. A marker of compromised filtration function

OBJECTIVE: Increased albumin excretion in diabetes is believed to be derived from hemodynamic and/or permeability abnormalities, whereas mesangial matrix expansion gives rise to the reduction in glomerular filtration surface and decline in renal function in diabetic nephropathy. We postulated that the overproduction of extracellular matrix proteins underlying glomerulosclerosis in diabetes might be associated with the excretion of increased amounts of type IV collagen in the urine. RESEARCH DESIGN AND METHODS: To explore this hypothesis, we measured the urinary excretion of (human) collagen IV by immunoassay in 65 patients with type 1 or type 2 diabetes and various degrees of albuminuria and examined its relationship to filtration function assessed by the reciprocal of the serum creatinine (RSC). RESULTS: Collagen IV excretion showed a significant (P < 0.001) inverse correlation (r = -0.62) with the RSC, and this correlation pertained regardless of whether albumin cxcretion was in the low (< or =< or = 100 microg/mg creatinine r = -0.73) or high (>100 microg/mg; r = -0.53) range. In contrast, albumin excretion showed insignificant correlation with either collagen IV excretion (r = 0.12) or with the RSC (r = -0.20). Urinary collagen IV was significantly higher (P < 0.05) in patients with an RSC value < or = 100 (28.3 +/- 2.4 ng/mg creatinine) than in patients with an RSC value > 100 (16.0 +/- 0.8 ng/mg creatinine). CONCLUSIONS: Because not all patients with microalbuminuria progress to declining renal function and some patients who develop nephropathy do not manifest albuminuria, the findings in this cross-sectional analysis suggest that measurement of urine collagen IV may be a useful noninvasive indicator to detect diabetic renal disease entering a phase of compromised renal function.     

Development and evaluation of a practical ELISA for human urinary lipocalin-type prostaglandin D synthase

BACKGROUND: Urinary excretion of lipocalin-type prostaglandin D synthase (L-PGDS) is significantly increased in patients with chronic renal failure, but its diagnostic potential in less advanced stages of renal diseases remains to be elucidated. METHODS: Six mouse monoclonal antibodies (MAbs) were raised against recombinant human L-PGDS. We constructed a sandwich ELISA with two MAbs that recognized different epitopes with high affinities and assessed its assay performance and clinical utility with urine samples from healthy controls, diabetic patients, and patients with various renal diseases. RESULTS: Western blot analyses with NH(2)-terminus-truncated L-PGDS mapped the epitopes to Ala(23)-Val(28) (MAb-7F5 and -10A3), Ser(52)-Ala(73) (MAb-9A6), Tyr(107)-Val(120) (MAb-1B7 and -6F5), and Gly(140)-Pro(155) (MAb-6B9). A sandwich ELISA was constructed with MAb-1B7 and -7F5, the K(d) values of which were 3.6 and 3.9 nmol/L, respectively, for native L-PGDS. Recoveries were 91-111%, and intra- and interassay CVs were <6% and <9%, respectively. The ELISA showed parallelism of standard and urine samples and no significant interference by a variety of urinary constituents. Urinary L-PGDS excretion was significantly increased in patients with diabetic nephropathy, IgA nephropathy, and chronic glomerulonephritis even when serum creatinine was not increased. In patients with renal diseases, urinary L-PGDS was correlated with urinary albumin (r = 0.64; P <0.0001), N-acetyl-beta-D-glucosaminidase (r = 0.43; P <0.001), and serum creatinine (r = 0.66; P <0.0001). At a cutoff value of 284 mg/mol creatinine, the assay had sensitivities of 74% for diabetic nephropathy and 83% for chronic glomerulonephritis and a specificity of 93%. CONCLUSIONS: This ELISA system is suitable for measurement of urinary L-PGDS in a routine clinical assay and may be useful to detect less advanced stages of renal diseases.     

Urinary connective tissue growth factor excretion correlates with clinical markers of renal disease in a large population of type 1 diabetic patients with diabetic nephropathy

OBJECTIVE: Levels of connective tissue growth factor (CTGF; CCN-2) in plasma are increased in various fibrotic disorders, including diabetic nephropathy. Recently, several articles have reported a strong increase of urinary CTGF excretion (U-CTGF) in patients with diabetic nephropathy. However, these studies addressed too small a number of patients to allow general conclusions to be drawn. Therefore, we evaluated U-CTGF in a large cross-sectional study of patients with type 1 diabetes. RESEARCH DESIGN AND METHODS: Subjects were 318 type 1 diabetic patients and 29 normoglycemic control subjects. U-CTGF was measured by sandwich enzyme-linked immunosorbent assay. Groups were compared by Kruskal-Wallis and Mann-Whitney analysis. The relation between U-CTGF and markers of diabetic nephropathy was determined by regression analysis. RESULTS: U-CTGF in patients with diabetic nephropathy (n = 89, median 155 pmol/24 h [interquartile range 96-258]) was significantly higher than in microalbuminuric (n = 79, 100 [65-78]) and normoalbuminuric (n = 150, 85 [48-127]) patients and control subjects (n = 29, 100 [78-114]). U-CTGF correlated with urinary albumin excretion (UAE) (R = 0.31) and glomerular filtration rate (R = -0.38) in patients with diabetic nephropathy. A standardized increase in U-CTGF was associated with diabetic nephropathy (odds ratio 2.3 [95% CI 1.7-3.1]), which was comparable with the odds ratios for diabetic nephropathy of increased HbA(1c) (2.0 [1.5-2.7]), and blood pressure (2.0 [1.5-2.6]). CONCLUSIONS: This is the first large cross-sectional study addressing U-CTGF in human type 1 diabetes. The observed association of U-CTGF with UAE and glomerular filtration rate might reflect a role of CTGF as progression promoter in diabetic nephropathy.     

Biological variability of albumin excretion rate and albumin-to-creatinine ratio in hypertensive type 2 diabetic patients.

The importance of measuring microalbuminuria is well established. However, only scanty data are available concerning the biological variability of albumin excretion in type 2 diabetic subjects. We report our experience from a large clinical trial of a new antihypertensive drug (Lercanidipine) designed to reduce albumin excretion and blood pressure in type 2 diabetic patients with hypertension and microalbuminuria. Eighty seven patients with persistent microalbuminuria were studied within 1 year of the clinical trial. The measurements were performed on blood and timed urine samples frozen at -80 degrees C and shipped to a central laboratory unit. Preliminary experiments were performed to assess albumin stability in urine under various conditions (4 degrees C, -20 degrees C and -80 degrees C), particularly with regard to the albumin/creatinine ratio. Urine samples can be stored up to 3 weeks at 4 degrees C or up to 2 months at -80 degrees C. The biological variability of the albumin excretion rate was 25.7%, while that of the albumin/creatinine ratio was 13.4%. These data are useful in defining the analytical goals of imprecision for microalbuminuria (CV = 13% for albumin, and CV = 6% for albumin/creatinine ratio). No correlation between albumin/creatinine ratio and HbA1c was found in the cohort of 61 microalbuminuric patients who completed the trial. The results of this study confirm that the albumin/ creatinine ratio is much more suitable for monitoring albumin excretion in longitudinal studies than the albumin excretion rate.     

Nonimmunological assay of urinary albumin based on laser-induced fluorescence.

We describe the first nonimmunological assay of albumin in urine with a detection limit of 1 mg/L. The method is simple, rapid, and accurate. It is based on the probe Albumin Blue 670, which becomes highly fluorescent on binding to albumin. An inexpensive diode laser was used as the light source for measurement of laser-induced fluorescence. The assay was coupled to a flow-injection analysis system capable of running 20 samples per hour. The working range was 1-100 mg/L, which covered albumin concentrations found in nonpathological urine and in urine with slightly increased albumin. This range makes prediction of nephropathy possible at an early stage. Other serum proteins and hemoglobin do not interfere. The coefficients of variation were < 4% and < 7% within one day and from day to day, respectively. A correlation coefficient of 0.990 (n = 100) was obtained for comparison with the Behring nephelometric assay.