Urinalysis is more specific and urinary neutrophil gelatinase-associated lipocalin is more sensitive for early detection of acute kidney injury.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) protein is a promising biomarker to detect acute kidney injury (AKI). Earlier detection of AKI could facilitate evaluation of novel therapeutic strategies. METHODS: Random and 24-h urine samples were prospectively obtained from 125 normal volunteers for analytic validation of a urinary enzyme-linked immunosorbent assay for NGAL. For clinical validation of the test, urine from 363 emergency department patients admitted to the hospital was obtained for NGAL enzyme-linked immunosorbent assay and urinalysis and AKI was determined by the use of Acute Kidney Injury Network (AKIN) criteria. RESULTS: NGAL was stable in urine for 7 days when ambient, 4 °C or frozen (-20 or -70 °C). The assay was linear between 0.24 and 10,000 ng/mL with a limit of quantitation of 0.24 ng/mL. Intra- and inter-assay precision were excellent (coefficient of variation <5%); however, urinary white blood cells were associated with increased NGAL levels. The 95th percentile reference value for NGAL in females is ≤ 65.0 and ≤ 23.4 ng/mL in males. Urinary NGAL levels increased with AKI stage but had only fair sensitivity (65%) and specificity (65%) to differentiate no AKI versus Stages 1, 2 or 3 (area under the curve 0.70). Urinalysis with microscopy was very specific (91%) but not very sensitive (22%) with an area under the curve of 0.57. CONCLUSIONS: NGAL can be reliably measured in clinical urine samples, although pyuria is an important potential confounder. In our cohort, increased urinary NGAL was associated with AKI by the AKIN criteria; however, the sensitivity and specificity were only fair, in part because patients with pre-renal causes are not excluded by AKIN criteria. Conversely, findings on microscopic urinalysis are very specific for AKI.

Low- and high-molecular-weight urinary proteins as predictors of response to rituximab in patients with membranous nephropathy: a prospective study.

BACKGROUND: Selective urinary biomarkers have been considered superior to total proteinuria in predicting response to treatment and outcome in patients with membranous nephropathy (MN). METHODS: We prospectively tested whether urinary (U) excretion of retinol-binding protein (RBP), α1-microglobulin (α1M), albumin, immunoglobulin IgG and IgM and/or anti-phospholipase 2 receptor (PLA(2)R) levels could predict response to rituximab (RTX) therapy better than standard measures in MN. We also correlated changes in antibodies to PLA(2)R with these urinary biomarkers. RESULTS: Twenty patients with MN and proteinuria (P) >5 g/24 h received RTX (375 mg/m(2) × 4) and at 12 months, 1 patient was in complete remission (CR), 9 were in partial remission (PR), 5 had a limited response (LR) and 4 were non-responders (NR). At 24 months, CR occurred in 4, PR in 12, LR in 1, NR in 2 and 1 patient relapsed. By simple linear regression analysis, UIgG at baseline (mg/24 h) was a significant predictor of change in proteinuria at 12 months (Δ urinary protein) (P = 0.04). In addition, fractional excretion (FE) of IgG, urinary alpha 1 microglobulin (Uα1M) (mg/24 h) and URBP (µg/24 h) were also predictors of response (P = 0.05, 0.04, and 0.03, respectively). On the other hand, UIgM, FEIgM, albumin and FE albumin did not predict response (P = 0.10, 0.27, 0.22 and 0.20, respectively). However, when results were analyzed in relation to proteinuria at 24 months, none of the U markers that predicted response at 12 m could predict response at 24 m (P = 0.55, 0.42, 0.29 and 0.20). Decline in anti-PLA(2)R levels was associated with and often preceded urinary biomarker response but positivity at baseline was not a predictor of proteinuria response. CONCLUSIONS: The results suggest that in patients with MN, quantification of low-, medium- and high-molecular-weight urinary proteins may be associated with rate of response to RTX, but do not correlate with longer term outcomes.

Comparison of urinary albumin quantification by immunoturbidimetry, competitive immunoassay, and protein-cleavage liquid chromatography-tandem mass spectrometry.

BACKGROUND: Increased urinary albumin excretion is a well-documented diagnostic and prognostic biomarker for renal disease. Urinary albumin is typically measured in clinical settings by immunoassay methods. However, neither a reference method nor a urine albumin calibration reference material is currently available. METHODS: We quantified urinary albumin in patient samples by using 3 commercially available reagent systems: DiaSorin SPQ and Beckman Coulter LX 20 (immunoturbidimetric), and Siemens Immulite (competitive immunoassay). Results were compared to values obtained by protein-cleavage liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: In general, results from the 3 immunoassays agreed with results from LC-MS/MS. However, the SPQ results showed a negative bias across all ranges of albuminuria [(0-200 mg/L, y = 0.91x - 3.74 (CI 0.86-0.96); > 200 mg/L, y = 0.88x - 40.30 (CI 0.76-1.00)], whereas the LX 20 showed minimal bias in the 0-200 mg/L range [y = 0.97x - 88 (CI 0.92-1.02)] and the Immulite assay showed positive bias in the 0-200 mg/L range [y = 1.15x - 4.38 (CI 1.09-1.20)]. CONCLUSIONS: These results showed a reasonable quantification of urinary albumin by representative polyclonal and monoclonal immunoassays compared to an LC-MS/MS assay. In addition, the results do not suggest the presence of nonimmunoreactive albumin in urine. However, differences in analytic performance between assays support the need for a reference calibration material and reference method to standardize clinical laboratory measurements of urinary albumin.

Comparison between immunoturbidimetry, size-exclusion chromatography, and LC-MS to quantify urinary albumin.

BACKGROUND: The accurate and precise measurement of urinary albumin is critical, since even minor increases are diagnostically sensitive indicators of renal disease, cardiovascular events, and risk for death. To gain insights into potential measurement biases, we systematically compared urine albumin measurements performed by LC-MS, a clinically available immunoturbidimetric assay, and size-exclusion HPLC. METHODS: We obtained unused clinical urine samples from 150 patients who were stratified by degrees of albuminuria (<20 mg/L, 20-250 mg/L, >250 mg/L) as determined by the immunoturbidimetric assay used in our clinical laboratory (Roche Hitachi 912). Urine albumin was then remeasured via LC-MS and HPLC (Accumin) assays. RESULTS: The immunoturbidimetric assay, calibrated using manufacturer-supplied serum-derived calibrators (Diasorin), underestimated albumin compared with LC-MS. After calibration with purified HSA, this immunoturbidimetric assay correlated well with LC-MS. HPLC overestimated albumin compared with both LC-MS and immunoturbidimetry. The current LC-MS and HPLC assays both performed poorly at concentrations <20 mg/L. CONCLUSIONS: Efforts are needed to establish gold-standard traceable calibrators for clinical assays. LC-MS is a specific method to quantify albumin in native urine when concentrations exceed 20 mg/L, and therefore could be employed for standardization among assays.

The effects of aerobic exercise on glucose and counterregulatory hormone concentrations in children with type 1 diabetes.

OBJECTIVE: To examine the acute glucose-lowering effects of aerobic exercise in children and adolescents with type 1 diabetes. RESEARCH DESIGN AND METHODS: Fifty children and adolescents with type 1 diabetes (ages 10 to <18 years) were studied during exercise. The 75-min exercise session consisted of four 15-min periods of walking on a treadmill to a target heart rate of 140 bpm and three 5-min rest periods. Blood glucose and plasma glucagon, cortisol, growth hormone, and norepinephrine concentrations were measured before, during, and after exercise. RESULTS: In most subjects (83%), plasma glucose concentration dropped at least 25% from baseline, and 15 (30%) subjects became hypoglycemic (< or = 60 mg/dl) or were treated for low glucose either during or immediately following the exercise session. The incidence of hypoglycemia and/or treatment for low glucose varied significantly by baseline glucose, occurring in 86 vs. 13 vs. 6% of subjects with baseline values <120, 120-180, and >180 mg/dl, respectively (P < 0.001). Exercise-induced increases in growth hormone and norepinephrine concentrations were marginally higher in subjects whose glucose dropped < or = 70 mg/dl. Treatment of hypoglycemia with 15 g of oral glucose resulted in only about a 20-mg/dl rise in glucose concentrations. CONCLUSIONS: In youth with type 1 diabetes, prolonged moderate aerobic exercise results in a consistent reduction in plasma glucose and the frequent occurrence of hypoglycemia when preexercise glucose concentrations are <120 mg/dl. Moreover, treatment with 15 g of oral glucose is often insufficient to reliably treat hypoglycemia during exercise in these youngsters.

Measurement of urinary TGF-ß1 in patients with diabetes mellitus and normal controls.

OBJECTIVE: Increasing evidence links TGF-ß1 to progression of renal fibrosis including its association with diabetic nephropathy (DN). Current ELISA assays are not sensitive enough to measure TGF-ß1 in the urine of many clinically healthy individuals, even those with established renal disease. The objective of this study was to validate a sensitive urinary assay for TGF-ß1 and compare levels between healthy controls and patients with established DN. DESIGN AND METHODS: An ELISA method (R&D Systems) was utilized together with an amplification step to assay TGF-ß1 in urine samples from 190 patients with DN and 80 healthy controls. RESULTS: Using an ELAST (Perkin Elmer, Inc) amplification step, the ELISA for urinary TGF-ß1 had a limit of quantification of 15.6 pg/mL and limit of detection of 7 pg/mL. Preliminary studies demonstrated that TGF-ß1 was stable if urine was frozen promptly at -70°C without preservatives. Using this assay, 22/80 controls (27%) had detectable levels of urinary TGF-ß1 (range <7 to 40.9 pg/mL; mean±SD 6.4±11.1 pg/mL). This was significantly lower (p<0.0001) than in the DN group in whom 114/190 (60%) had detectable levels of urinary TGF-ß1 (range <7 to 526.4 pg/mL; mean±SD 20.4±45.8 pg/mL). Urinary protein and TGF-ß1 concentrations demonstrated modest correlation in patients with DN (r=0.47, P<0.001). TGF-ß1 measurement in patients with DN did not demonstrate significant association with progression of proteinuria or increase in serum creatinine during the next 12 months of follow-up. CONCLUSION: We have validated a sensitive ELISA assay for urinary TGF-ß1, and demonstrated correlations with the degree of proteinuria and higher levels in patients with DN compared to controls. Additional study will be necessary in order to determine if serial testing can predict renal prognosis independent of known prognostic factors for patients with DN.