Lieske, John C.

Mammalian urine contains a range of macromolecule proteins that play critical roles in renal stone formation, among which Tamm-Horsfall protein (THP) is by far the most abundant. While THP is a potent inhibitor of crystal aggregation in vitro and its ablation in vivo predisposes one of the two existing mouse models to spontaneous intrarenal calcium crystallization, key controversies remain regarding the role of THP in nephrolithiasis. By carrying out a long-range follow-up of more than 250 THP-null mice and their wild-type controls, we demonstrate here that renal calcification is a highly consistent phenotype of the THP-null mice that is age and partially gene dosage dependent, but is gender and genetic background independent. Renal calcification in THP-null mice is progressive, and by 15 mo over 85% of all the THP-null mice develop spontaneous intrarenal crystals. The crystals consist primarily of calcium phosphate in the form of hydroxyapatite, are located more frequently in the interstitial space of the renal papillae than intratubularly, particularly in older animals, and lack accompanying inflammatory cell infiltration. The interstitial deposits of hydroxyapatite observed in THP-null mice bear strong resemblances to the renal crystals found in human kidneys bearing idiopathic calcium oxalate stones. Compared with 24-h urine from the wild-type mice, that of THP-null mice is supersaturated with brushite (calcium phosphate), a stone precursor, and has reduced urinary excretion of citrate, a stone inhibitor. While less frequent than renal calcinosis, renal pelvic and ureteral stones and hydronephrosis occur in the aged THP-null mice. These results provide direct in vivo evidence indicating that normal THP plays an important role in defending the urinary system against calcification and suggest that reduced expression and/or decreased function of THP could contribute to nephrolithiasis.

Abstract Background: Glomerular filtration rate (GFR) can be determined by measuring renal clearance of the radiocontrast agent iothalamate. Current analytic methods for quantifying iothalamate concentrations in plasma and urine using liquid chromatography or capillary electrophoresis have limitations such as long analysis times and susceptibility to interferences. We developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method to overcome these limitations. Methods: Urine and plasma samples were deproteinized using acetonitrile and centrifugation. The supernatant was diluted in water and analyzed by LC-MS/MS using a water:methanol gradient. We monitored 4 multiple reaction monitoring transitions: m/z 614.8–487.0, 614.8–456.0, 614.8–361.1, and 614.8–177.1. We compared the results to those obtained via our standard capillary electrophoresis (CE-UV) on samples from 53 patients undergoing clinical GFR testing. Results: Mean recovery was 90%–110% in both urine and plasma matrices. Imprecision was ≤15% for the m/z 614.8–487.0 and 614.8–456.0 transitions over a 10-day period at 1 mg/L. Method comparison for 159 patient samples (53 clearances) provided the following Passing–Bablok regressions: plasma iothalamate LC-MS/MS (y) vs CE-UV (x), y = 0.99x + 0.36; urine iothalamate LC-MS/MS vs CE-UV, y = 1.01x + 0.31; corrected GFR LC-MS/MS vs CE-UV, y = 1.00x + 0.00. Interfering substances prevented accurate iothalamate quantification by CE-UV in 2 patients, whereas these samples could be analyzed by LC-MS/MS. Conclusions: Iothalamate can be quantified by LC-MS/MS for GFR measurement. This method circumvents potential problems with interfering substances that occasionally confound accurate GFR determinations.

Abstract 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.

Abstract Background: Urinary albumin excretion is a sensitive diagnostic and prognostic marker for renal disease. Therefore, measurement of urinary albumin must be accurate and precise. We have developed a method to quantify intact urinary albumin with a low limit of quantification (LOQ). Methods: We constructed an external calibration curve using purified human serum albumin (HSA) added to a charcoal-stripped urine matrix. We then added an internal standard, 15N-labeled recombinant HSA (15NrHSA), to the calibrators, controls, and patient urine samples. The samples were reduced, alkylated, and digested with trypsin. The concentration of albumin in each sample was determined by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and linear regression analysis, in which the relative abundance area ratio of the tryptic peptides 42LVNEVTEFAK51 and 526QTALVELVK534 from albumin and 15NrHSA were referenced to the calibration curve. Results: The lower limit of quantification was 3.13 mg/L, and the linear dynamic range was 3.13–200 mg/L. Replicate digests from low, medium, and high controls (n = 5) gave intraassay imprecision CVs of 2.8%–11.0% for the peptide 42LVNEVTEFAK51, and 1.9%–12.3% for the 526QTALVELVK534 peptide. Interassay imprecision of the controls for a period of 10 consecutive days (n = 10) yielded CVs of 1.5%–14.8% for the 42LVNEVTEFAK51 peptide, and 6.4%–14.1% for the 526QTALVELVK534 peptide. For the 42LVNEVTEFAK51 peptide, a method comparison between LC-MS/MS and an immunoturbidometric method for 138 patient samples gave an R2 value of 0.97 and a regression line of y = 0.99x + 23.16. Conclusions: Urinary albumin can be quantified by a protein cleavage LC-MS/MS method using a 15NrHSA internal standard. This method provides improved analytical performance in the clinically relevant range compared to a commercially available immunoturbidometric assay.

Abstract Background: Urinary excretion of albumin indicates kidney damage and is recognized as a risk factor for progression of kidney disease and cardiovascular disease. The role of urinary albumin measurements has focused attention on the clinical need for accurate and clearly reported results. The National Kidney Disease Education Program and the IFCC convened a conference to assess the current state of preanalytical, analytical, and postanalytical issues affecting urine albumin measurements and to identify areas needing improvement. Content: The chemistry of albumin in urine is incompletely understood. Current guidelines recommend the use of the albumin/creatinine ratio (ACR) as a surrogate for the error-prone collection of timed urine samples. Although ACR results are affected by patient preparation and time of day of sample collection, neither is standardized. Considerable intermethod differences have been reported for both albumin and creatinine measurement, but trueness is unknown because there are no reference measurement procedures for albumin and no reference materials for either analyte in urine. The recommended reference intervals for the ACR do not take into account the large intergroup differences in creatinine excretion (e.g., related to differences in age, sex, and ethnicity) nor the continuous increase in risk related to albumin excretion. Discussion: Clinical needs have been identified for standardization of (a) urine collection methods, (b) urine albumin and creatinine measurements based on a complete reference system, (c) reporting of test results, and (d) reference intervals for the ACR.

Abstract 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.