Time-resolved fluorescence immunoassay for urine retinol-binding protein is more sensitive than polyclonal and monoclonal assays.

BACKGROUND: Retinol-binding protein4 (RBP) assays using polyclonal antibodies (pRBP) have major problems of non-linearity of dilution and a very small useable dynamic range. Our objective was to develop a specific assay with a wider dynamic range to detect tubular proteinuria. METHODS: mRBP (monoclonal capture and second antibody with colorimetric detection) and fluoroimmunoassays for RBP (fRBP) (polyclonal capture and monoclonal second antibody with fluorescence detection) were developed and compared with pRBP. Four hundred and eighty-eight patient samples were collected; 290 samples were analysed by mRBP and 198 samples with fRBP and compared with pRBP. RESULTS: mRBP assay has the advantages of better linearity on dilution and wider analytical range over pRBP. It is limited by poor signal in the patients with albuminuria and glomerular proteinuria and inferior discrimination between patient groups. fRBP had an intra-assay and inter-assay CV of <6% and <8%, respectively, and analytical range was 2.3-599 µg/L. fRBP was linear on dilution within the analytical range. Correlation (r) was 0.8722 (95% CI 0.7621 to 0.9333, P< 0.0001); Mann-Whitney test revealed no significant difference (U = 18,877, n = 198, P = 0.5244) asserting that the medians of the two samples were identical. Bland-Altman test between pRBP and fRBP showed a mean negative bias of 16.43 (CI -994 to 1027) µg/mmol. CONCLUSIONS: The combination assay with fluorescence detection (fRBP) proved more discriminatory than a purely monoclonal system especially in patients with significant proteinuria and has advantages of better linearity on dilution and wider analytical range than the existing pRBP assay and compared extremely well with pRBP.

Artefactual 25-OH vitamin D concentration in multiple myeloma.

The most commonly used techniques to measure vitamin D are automated immunoassays which are known to be affected by interferences, especially from immunoglobulins present in the patient's serum. We present a case of a patient with myeloma in whom interference with the vitamin D assay was identified. An 83-year-old female, known to have IgG myeloma, was found to have a high concentration of 25-OH vitamin D on a routine test without any signs of vitamin D toxicity. She was not taking vitamin D supplements or any other multivitamin preparation and had minimal sun exposure. The initial and subsequent samples run by the ARCHITECT 25-OH vitamin D assay (chemiluminescent microparticle immunoassay technology, Abbott Laboratories, Abbott Park, IL) showed a high concentration of 25-OH vitamin D of 281 nmol/L and 327 nmol/L, respectively. Further fresh samples taken for 25-OH vitamin D and analysed by liquid chromatography-mass spectrometry (LC-MS/MS) and ARCHITECT analysis showed results of 49 nmol/L and 289 nmol/L, respectively. Our patient had high concentrations of circulating IgG paraproteins and had a long history of rheumatoid arthritis; paraproteins and rheumatoid factor may interfere in the assay. In conclusion, we report a case of a patient with IgG myeloma and rheumatoid arthritis with high concentrations of 25-OH vitamin D detected by the Abbott ARCHITECT, but not by a reference method (LC-MS/MS). The most likely cause of the discordant results is interference in the immunoassay by the paraprotein but interference from rheumatoid factor remains a possibility.

A novel COL4A1 frameshift mutation in familial kidney disease: the importance of the C-terminal NC1 domain of type IV collagen.

BACKGROUND: Hereditary microscopic haematuria often segregates with mutations of COL4A3, COL4A4 or COL4A5 but in half of families a gene is not identified. We investigated a Cypriot family with autosomal dominant microscopic haematuria with renal failure and kidney cysts. METHODS: We used genome-wide linkage analysis, whole exome sequencing and cosegregation analyses. RESULTS: We identified a novel frameshift mutation, c.4611_4612insG:p.T1537fs, in exon 49 of COL4A1. This mutation predicts truncation of the protein with disruption of the C-terminal part of the NC1 domain. We confirmed its presence in 20 family members, 17 with confirmed haematuria, 5 of whom also had stage 4 or 5 chronic kidney disease. Eleven family members exhibited kidney cysts (55% of those with the mutation), but muscle cramps or cerebral aneurysms were not observed and serum creatine kinase was normal in all individuals tested. CONCLUSIONS: Missense mutations of COL4A1 that encode the CB3 [IV] segment of the triple helical domain (exons 24 and 25) are associated with HANAC syndrome (hereditary angiopathy, nephropathy, aneurysms and cramps). Missense mutations of COL4A1 that disrupt the NC1 domain are associated with antenatal cerebral haemorrhage and porencephaly, but not kidney disease. Our findings extend the spectrum of COL4A1 mutations linked with renal disease and demonstrate that the highly conserved C-terminal part of the NC1 domain of the α1 chain of type IV collagen is important in the integrity of glomerular basement membrane in humans.

The urinary proteome and metabonome differ from normal in adults with mitochondrial disease.

We studied the extent and nature of renal involvement in a cohort of 117 adult patients with mitochondrial disease, by measuring urinary retinol-binding protein (RBP) and albumin; established markers of tubular and glomerular dysfunction, respectively. Seventy-five patients had the m.3243A>G mutation and the most frequent phenotypes within the entire cohort were 14 with MELAS, 33 with MIDD, and 17 with MERRF. Urinary RBP was increased in 29 of 75 of m.3243A>G patients, whereas albumin was increased in 23 of the 75. The corresponding numbers were 16 and 14, respectively, in the 42 non-m.3243A>G patients. RBP and albumin were higher in diabetic m.3243A>G patients than in nondiabetics, but there were no significant differences across the three major clinical phenotypes. The urine proteome (mass spectrometry) and metabonome (nuclear magnetic resonance) in a subset of the m.3243A>G patients were markedly different from controls, with the most significant alterations occurring in lysosomal proteins, calcium-binding proteins, and antioxidant defenses. Differences were also found between asymptomatic m.3243A>G carriers and controls. No patients had an elevated serum creatinine level, but 14% had hyponatremia, 10% had hypophosphatemia, and 14% had hypomagnesemia. Thus, abnormalities in kidney function are common in adults with mitochondrial disease, exist in the absence of elevated serum creatinine, and are not solely explained by diabetes.

Urine retinol-binding protein 4: a functional biomarker of the proximal renal tubule.

Measurement of retinol-binding protein 4 in urine (uRBP4) is arguably the most sensitive biomarker for loss of function of the human proximal renal tubule. Megalin- and cubilin-receptor-mediated endocytosis normally absorbs > 99% of the approximately 1.5 g/24 h of protein filtered by the renal glomerulus. When this fails there is "tubular proteinuria," comprising uRBP4, albumin, and many other proteins and peptides. This tubular proteinuria is a consistent feature of the renal Fanconi syndrome (FS) and measurement of uRBP4 appears to be an excellent screening test for FS. FS occurs in rare inherited renal diseases including cystinosis, Dent disease, Lowe syndrome, and autosomal dominant FS. Acquired FS occurs in paraproteinemias, tubulointerstitial renal disease, oncogenic osteomalacia, Chinese herbs nephropathy, and Balkan endemic nephropathy. Though poorly understood, FS may be associated with HIV disease and antiretroviral treatment; cadmium poisoning may cause FS. In addition to FS, uRBP4 measurement has a different role: the early detection of acute kidney injury. Urine RBP4 comprises several isoforms, including intact plasma RBP4, MW 21.07 kDa, and C-terminal truncated forms, des-L- and des-LL-RBP4, also probably plasma derived. In FS, uRBP4 levels are about 104-fold above the upper limit of normal and small increments are frequently seen in carriers of some inherited forms of FS and in acquired disease. The very high levels in disease, frequent assay nonlinearity, lack of defined calibrants, and multiple uRBP4 isoforms make accurate assay challenging; top-down mass spectrometry has brought advances. Assays for uRBP4 with defined molecular targets allowing good interlaboratory comparisons are needed.

Analysis of molecular forms of urine Retinol-Binding Protein in Fanconi Syndrome and design of an accurate immunoassay.

BACKGROUND: Retinol-Binding Protein in urine (uRBP), a biomarker for the proximal renal tubular disease of congenital and acquired Fanconi Syndrome (FS) occurs in multiple forms. However these have not had quantitative mass spectrometric (MS) analysis, nor is there a validated assay for defined molecular species of uRBP with linearity on sample dilution. METHODS: A 'Top-down' MS approach identified distinct forms of uRBP differing by only one amino acid. Based on this, we designed a dual-monoclonal antibody-based fluorescence immunoassay calibrated with intact plasma RBP4. RESULTS: LC-MS showed that uRBP in FS (one Dent disease urine) comprised intact plasma RBP4 and C-terminal-truncated RBP4, desL-RBP4 and desLL-RBP4 in molar ratio 2:2:1. DELFIA® assay calibrated with plasma RBP4, formulated with two monoclonal antibodies (HyTest, Finland), mAb48 for capture and biotinylated-mAb42 for detection, provided good sensitivity (1 µg/L), working range>500 µg/L and good linearity on sample dilution. The three predominant forms of uRBP were equipotent over the assay working range. uRBP reference range was <3 µg/mmol creatinine and FS patients had concentrations of 1000-5000 µg/mmol creatinine. CONCLUSIONS: Using 'Top-down' MS analysis of uRBP we devised an accurate, linear, fluorescence immunoassay with defined RBP molecular targets optimal for uRBP measurement. Discrimination of elevated uRBP from the upper limit of normal was some 10-fold greater than previous assays.

Remote ischemic preconditioning for renal protection during elective open infrarenal abdominal aortic aneurysm repair: randomized controlled trial.

We aimed to determine whether remote ischemic preconditioning (IP) reduces renal damage following elective open infrarenal abdominal aortic aneurysm (AAA) repair. Sequential common iliac clamping was used to induce remote IP in randomized patients. Urinary retinol binding protein (RBP) and albumin-creatinine ratio (ACR) were measured following induction and 3, 24, and 48 hours postoperatively. In controls (n = 22), median urinary RBP increased from 112 microg/mL (interquartile range [IQR] 96-173 microg/mL) preoperatively to 5919 microg/mL (IQR 283-17 788 microg/mL) at 3 hours. Preoperative urinary RBP in preconditioned patients was 96 microg/mL (IQR 50 to 229 microg/mL) preoperatively, rising to 1243 microg/mL (IQR 540 to 15400 microg/mL) at 3 hours. Although control patients' median urinary RBP level was 5 times greater at 3 hours, there were no statistically significant differences in renal outcome indices. This trial could not confirm that remote IP reduces renal injury following elective open aneurysm surgery.

Genetic loci influencing kidney function and chronic kidney disease.

Using genome-wide association, we identify common variants at 2p12-p13, 6q26, 17q23 and 19q13 associated with serum creatinine, a marker of kidney function (P = 10(-10) to 10(-15)). Of these, rs10206899 (near NAT8, 2p12-p13) and rs4805834 (near SLC7A9, 19q13) were also associated with chronic kidney disease (P = 5.0 x 10(-5) and P = 3.6 x 10(-4), respectively). Our findings provide insight into metabolic, solute and drug-transport pathways underlying susceptibility to chronic kidney disease.

Remote ischemic preconditioning for renal and cardiac protection during endovascular aneurysm repair: a randomized controlled trial.

PURPOSE: To report a randomized clinical trial designed to determine if remote ischemic preconditioning (IP) has the ability to reduce renal and cardiac damage following endovascular aneurysm repair (EVAR). METHODS: Forty patients (all men; mean age 76+/-7 years) with abdominal aortic aneurysms averaging 6.3+/-0.8 cm in diameter were enrolled in the trial from November 2006 to January 2008. Eighteen patients (mean age 74 years, range 72-81) were randomized to preconditioning and completed the full remote IP protocol; there were no withdrawals. Twenty-two patients (mean age 76 years, range 66-80) were assigned to the control group. Remote IP was induced using sequential lower limb ischemia. Serum and urinary markers of renal and cardiac injury were compared between the groups. RESULTS: Urinary retinol binding protein (RBP) levels increased 10-fold from a median of 235 micromol/L to 2356 micromol/L at 24 hours (p = 0.0001). There was a lower increase in the preconditioned group, from 167 micromol/L to 413 micromol/L at 24 hours (p = 0.04). The median urinary albumin:creatinine ratio was significantly lower in the preconditioned group at 24 hours (5 versus 8.8, p = 0.06). There were no differences in the rates of renal impairment or major adverse cardiac events. CONCLUSION: Remote preconditioning reduces urinary biomarkers of renal injury in patients undergoing elective EVAR. This small pilot trial was unable to detect an effect on clinical endpoints; further trials are warranted.

Subclinical tubular injury in HIV-infected individuals on antiretroviral therapy: a cross-sectional analysis.

BACKGROUND: Randomized control studies have not shown an association between treatment with tenofovir (TDF) and clinically significant kidney toxicity. However, multiple cases of renal tubular toxicity have been described in patients with HIV treated with TDF. It is unclear whether spot urine protein- or albumin-creatinine ratio is a sufficiently sensitive screening test to detect subclinical renal tubular toxicity in patients with HIV. STUDY DESIGN: Cross-sectional. SETTING & PARTICIPANTS: 99 patients with HIV with serum creatinine levels < 1.70 mg/dL and dipstick-negative proteinuria; 19 were antiretroviral treatment (ART) naive, 47 were on a TDF regimen, and 33 were on ART, but with no history of TDF exposure. PREDICTOR OR FACTOR: Exposure to TDF. OUTCOMES: Spot urine concentrations of retinol-binding protein (RBP; a low-molecular-weight protein normally reabsorbed by the proximal tubule), N-acetyl-beta-D-glucosaminidase (NAG; a proximal tubule lysosomal enzyme), albumin (A; a marker of glomerular disease), and protein (P; a standard clinical screening test for kidney pathological states) expressed as a ratio to creatinine (C; U(RBP/C), U(NAG/C), U(A/C), and U(P/C), respectively). RESULTS: There were no significant differences in median U(A/C) (ART-naive, 7.3 mg/g [range, 0-245.8 mg/g]; TDF, 9.0 mg/g [range, 0.1-184.1 mg/g]; and non-TDF, 10.5 mg/g [range, 2.6-261.6 mg/g]; P = 0.8). U(RBP/C) excretion was significantly higher in the TDF group (median, 214.2 microg/g [range, 26.8-17,454.5 microg/g]) than in the ART-naive group (92.5 microg/g [range, 21.3-3,969.0 microg/g]; P = 0.03); there was also a trend toward higher values than in the non-TDF group (111.6 microg/g [range, 31.0-6,136.3 microg/g]; P = 0.08). U(NAG/C) excretion was significantly higher in both the TDF (median, 394.7 micromol/h/g [range, 140.5-10,851.3 micromol/h/g]; P = 0.01) and non-TDF (406.8 micromol/h/g [range, 12.4-8,485.8 micromol/h/g]; P = 0.03) groups compared with the ART-naive group (218.6 micromol/h/g [range, 56.5-2,876.1 micromol/h/g]). U(P/C) was significantly higher in the TDF (median, 123.9 mg/g [range, 53.1-566.4 mg/g]) than the non-TDF group (97.3 mg/g [range, 0-451.3 mg/g]; P = 0.03). The proportion of patients with evidence of tubular dysfunction (increased U(RBP/C) and/or U(NAG/C)) was considerably higher than the proportion with an increase in U(A/C) or U(P/C) in all groups: for ART-naive, 52.6% vs 31.6% vs 25.0%; for TDF, 80.9% vs 29.8% vs 52.2%; and for non-TDF, 81.8% vs 39.4% vs 30.0%. The level of agreement among the different urinary test results was low. LIMITATIONS: Causality cannot be established from single measurements of urinary markers in a cross-sectional study. CONCLUSIONS: Patients with HIV had high rates of subclinical proteinuria, but neither U(P/C) nor U(A/C) is sufficiently sensitive alone to detect many of these cases. Patients using TDF have increased U(RBP/C) and U(P/C); the significance of this will need to be determined from longer-term outcome studies.

Does N-acetylcysteine prevent contrast-induced nephropathy during endovascular AAA repair? A randomized controlled pilot study.

PURPOSE: To examine if N-acetylcysteine (NAC) reduces the incidence of contrast nephropathy during endovascular abdominal aortic aneurysm repair (EVAR) as evidenced by changes in markers of renal function. METHODS: Twenty consecutive men (mean age 72 years, range 65-79) undergoing EVAR were randomized to receive standard intravenous fluid hydration or standard fluid hydration and NAC (600 mg BID orally, 4 doses). Venous blood and urine were collected prior to the procedure and for 5 postoperative days and analyzed blindly for serum creatinine, urinary retinol-binding protein (RBP), and albumin/creatinine ratio (ACR). RESULTS: There were no significant differences in baseline demographics between the groups. No patient developed acute renal failure. In both groups, urinary RBP rose significantly from baseline (median 15 microg/mmol to peak 699 microg/mmol in controls versus 17 to 648 microg/mmol in the treatment group, p<0.003). There were similar significant rises in ACR (p<0.02). There was, however, no significant difference in the postoperative RBP or ACR between the groups at any time point. CONCLUSION: EVAR causes significant acute renal injury in most patients. This was not attenuated by N-acetylcysteine. The causes of renal injury are probably multifactorial, the long-term clinical significance of which is unclear.

Urinary megalin deficiency implicates abnormal tubular endocytic function in Fanconi syndrome.

Normal reabsorption of glomerular filtrate proteins probably requires recycling of the endocytic receptors megalin (gp330) and cubilin. Both receptors are located on the luminal surface of the renal proximal tubule epithelium. Whether abnormal amounts of receptor are present in the urine of patients with Dent's disease, Lowe's syndrome, or autosomal dominant idiopathic Fanconi syndrome was explored. They are all forms of the renal Fanconi syndrome and are associated with tubular proteinuria. Urine samples of equal creatinine contents were dialyzed, lyophilized, and subjected to electrophoresis on nonreducing sodium dodecyl sulfate-5% polyacrylamide gels. Proteins were blotted and probed with anti-megalin IgG, anti-cubilin IgG, or receptor-associated protein. Megalin and cubilin levels detected by immunochemiluminescence were measured as integrated pixels and expressed as percentages of the normal mean values. A striking deficiency of urinary megalin, compared with normal individuals (n = 42), was observed for eight of nine families with Dent's disease (n = 10) and for the two families with Lowe's syndrome (n = 3). The family with autosomal dominant idiopathic Fanconi syndrome (n = 2) exhibited megalin levels within the normal range. The measured levels of cubilin were normal for all patients. These results are consistent with defective recycling of megalin to the apical cell surface of the proximal tubules and thus decreased loss into urine in Dent's disease and Lowe's syndrome. This defect would interfere with the normal endocytic function of megalin, result in losses of potential ligands into the urine, and produce tubular proteinuria.

Characterization of renal chloride channel (CLCN5) mutations in Dent's disease.

Dent's disease is an X-linked renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, and renal failure. The disease is caused by mutations in a renal chloride channel gene, CLCN5, which encodes a 746 amino acid protein (CLC-5), with 12 to 13 transmembrane domains. In this study, an additional six unrelated patients with Dent's disease were identified and investigated for CLCN5 mutations by DNA sequence analysis of the 11 coding exons of CLCN5. This revealed six mutations: four frameshift deletions involving codons 392, 394, 658, and 728, one nonsense mutation (Tyr617Stop), and an A to T transversion at codon 601 that would result in either a missense mutation (Asp601Val) or creation of a novel donor splice site. These mutations were confirmed by restriction endonuclease or sequence-specific oligonucleotide hybridization analysis and were not common polymorphisms. The frameshift deletions and nonsense mutation predict truncated and inactivated CLC-5. The effects of the putative missense Asp601Val mutant CLC-5 were assessed by heterologous expression in Xenopus oocytes, and this revealed a chloride conductance that was similar to that observed for wild-type CLC-5. However, an analysis of the mutant CLCN5 transcripts revealed utilization of the novel donor splice site, resulting in a truncated CLC-5. Thus, all of the six mutations are likely to result in truncated CLC-5 and a loss of function, and these findings expand the spectrum of CLCN5 mutations associated with Dent's disease.