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.

In a cross-sectional population study to assess whether environmental exposure to cadmium is associated with renal dysfunction, 1699 subjects aged 20-80 years were studied as a random sample of four areas of Belgium with varying degrees of cadmium pollution. After standardisation for several possible confounding factors, five variables (urinary excretion of retinol-binding protein, N-acetyl-beta-glucosaminidase, beta 2-microglobulin, aminoacids, and calcium) were significantly associated with the urinary excretion of cadmium (as a marker of cadmium body burden), suggesting the presence of tubular dysfunction. There was a 10% probability of values of these variables being abnormal when cadmium excretion exceeded 2-4 micrograms/24 h. Excretion reached this threshold in 10% of non-smokers. There was also evidence that diabetic patients may be more susceptible to the toxic effect of cadmium on the renal proximal tubule.

Transepithelial transport of retinol is linked to retinol-binding protein (RBP), which is taken up and also synthesized in a number of epithelia. By immunocytochemistry of human, rat, and mouse renal proximal tubules, a strong staining in apical endocytic vacuoles, lysosomes, endoplasmic reticulum, Golgi, and basal vesicles was observed, in accordance with luminal endocytic uptake as well as a constitutive synthesis and basal secretion of RBP. Analysis of mice with target disruption of the gene for the major endocytic receptor of proximal tubules, megalin, revealed no RBP in proximal tubules of these mice. Western blotting and HPLC of the urine of the megalin-deficient mice instead revealed a highly increased urinary excretion of RBP and retinol, demonstrating that glomerular filtered RBP-retinol of megalin-deficient mice escapes uptake by proximal tubules. A direct megalin-mediated uptake of purified RBP-retinol was indicated by surface plasmon resonance analysis and uptake in immortalized rat yolk sac cells. Uptake was partially inhibited by a polyclonal megalin antibody and the receptor-associated protein. The present data show that the absence of RBP-binding megalin causes a significantly increased loss of RBP and retinol in the urine, demonstrating a crucial role of megalin in vitamin A homeostasis.

Latex immunoassay is a nonisotopic method based on agglutination, by protein, of calibrated latex particles coated with a specific antibody. The assay has been automated in a simple continuous-flow system by incubating the reaction mixture in a heated mixing coil for 25 min and measuring the agglutination with a cell counter. No external shaking of the latex suspension and no additional reagent is required for the agglutination. The method can accurately and precisely quantify a wide variety of proteins in plasma and urine, including human ferritin, beta 2-microglobulin, retinol-binding protein, and albumin. Depending on the antigen-antibody system, the detection limit ranges from 10(-10) to about 10(-12) mol/L. Within- and between-assay CVs are less than 10%. In the assay of ferritin, sera are pretreated to eliminate interferences from chylomicrons, complement, and rheumatoid factor.

The urinary excretion of retinol-binding protein (RBP), beta 2-microglobulin (beta 2-m), and beta-N-acetyl-D-glucosaminidase was monitored in patients with renal tubular damage secondary to multiple injuries, rhabdomyolysis, antibiotic treatment, or poisoning by various chemicals such as solvents, heavy metals, or pesticides. In almost all cases, RBP proved to be a more sensitive index of renal tubular damage than was beta-N-acetyl-D-glucosaminidase and, being more stable in acid urine, a more practical analyte to measure than was beta 2-m. We corroborated this finding by studying the relationships between these three analytes in more than 150 patients. On the average, an increase in the urinary excretion of beta-N-acetyl-D-glucosaminidase becomes detectable when urinary RBP already exceeds the normal value by 50- to 100-fold. In urines with pH greater than 6, RBP and beta 2-m concentrations are well correlated (r = 0.93, n = 150), beta 2-m tending to be more frequently positive (i.e., greater than 311 micrograms/L). But in urines with pH less than 6 (about 30-40% of the samples), the RBP/beta 2-m concentration ratio increases as pH decreases, up to 500 in some patients with massive tubular injury. Because the renal uptake of proteins involves a saturable process, the urinary excretion of RBP, like that of beta 2-m, specifically reflects the reabsorption capacity of proximal tubules only when the glomerular filtration rate is normal or slightly impaired (i.e., serum creatinine less than 20 mg/L). Under these conditions the determination of RBP protein in urine appears the most appropriate test when early detection of tubular injury is desirable.

The specificity, sensitivity and stability of beta 2-microglobulin (beta 2-m) and retinol-binding protein (RBP) in urine as indices of tubular proteinuria were compared. In vitro experiments show that RBP in urine is stable down to pH 4.5, whereas beta 2-m degrades below pH 5.5. This was confirmed by the relationships between pH and the concentration of both proteins in the urines from 150 healthy subjects. Urinary RBP was independent of pH (r = 0.125) but in contrast at pH below 6, beta 2-m concentration was inversely correlated with pH (r = 0.54, p less than 0.05). The comparison of urinary excretion of RBP and beta 2-m in 68 patients with renal diseases shows that in absence of a pH effect, the sensitivity and specificity of both proteins as indices of tubular proteinuria are rather similar. Therefore, in view of its greater stability in urine, RBP appears to be a more practical and reliable index of proximal tubular function than beta 2-m.

An enzyme-linked immunosorbent assay (ELISA) for urinary retinol-binding protein (RBP) has been developed and compared with urinary beta 2-microglobulin for the detection of tubular proteinuria. The assay has a working range of 10 to 250 micrograms of RBP per liter of urine. The within-assay CV was 3.2-7.1%, the between-assay CV 12.5%. A control population of 118 male subjects gave a geometric mean urinary RBP concentration of 7.7 micrograms per millimole of creatinine and a 95th centile of 22 micrograms per millimole of creatinine. Comparison of urinary RBP and beta 2-microglobulin concentrations in 80 control subjects and 117 subjects exposed to cadmium fumes gave correlations of r = 0.59 and 0.91, respectively. Of the 117 subjects exposed to cadmium fumes, 103 gave both RBP and beta 2-microglobulin concentrations on the same side of the upper 95th centile values of 22 and 38 micrograms per millimole of creatinine for RBP and beta 2-microglobulin respectively (Chi-square analysis p less than 0.001), demonstrating that RBP and beta 2-microglobulin detect tubular proteinuria with equal sensitivity and specificity. ELISA and an established latex immunoassay gave well-correlated results.

It is well established that the detection of microalbuminuria in a patient with diabetes mellitus indicates the presence of glomerular involvement in early renal damage. Recent studies have demonstrated that there is also a tubular component to renal complications of diabetes, as shown by the detection of renal tubular proteins and enzymes in the urine. In fact, tubular involvement may precede glomerular involvement, as several of these tubular proteins and enzymes are detectable even before the appearance of microalbuminuria. This review looks at the studies reported so far on serum and urinary markers of diabetic nephropathy, both glomerular and tubular, and their roles in the early detection of renal damage. The advantages and disadvantages of some of these markers are also discussed. The markers reviewed include (1) glomerular--transferrin, fibronectin, and other components of glomerular extracellular matrix, and (2) tubular--low molecular weight proteins (beta 2 microglobulin, retinol binding protein, alpha 1 microglobulin, urine protein 1), other proteins such as Tamm-Horsfall protein, beta 2 glycoprotein-1, urinary enzymes (N-acetyl-beta-D-glucosaminidase, cholinesterase, gamma glutamyltranspeptidase, alanine aminopeptidase), and tubular brush-border antigen.

Background: It has been shown that apical sodium transporters of the renal tubule can be detected by immunoblotting of urine membrane fraction from rats. We raised the hypothesis that protein levels of the Na(+)/H(+) exchanger isoform 3 (NHE3), the most abundant apical sodium transporter in renal tubule, should be increased in urine of patients presenting with acute renal failure (ARF) with severe tubular cell damage and thus might be a noninvasive marker of acute tubular necrosis (ATN). Methods: Sixty-eight patients admitted to the intensive care unit were studied prospectively (54 patients with ARF, 14 controls without renal dysfunction). Patients with ARF were divided into 3 subgroups as follows: prerenal azotemia, ATN, and intrinsic ARF other than ATN. Urinary NHE3 protein abundance was estimated from semiquantitative immunoblots of urine membrane fraction samples collected from patients. The amount of urinary NHE3 was compared with the fractional excretion of sodium (FeNa) and urinary retinol-binding protein (RBP). Results: NHE3 was not detected in urine from controls. Levels of urinary NHE3 normalized to urinary creatinine level were increased in patients with prerenal azotemia and 6 times as much in patients with ATN, without overlap (ATN, 0.78 +/- 0.36; prerenal azotemia, 0.12 +/- 0.08; P < 0.001). Conversely, urinary NHE3 protein was not detected in patients with intrinsic ARF other than ATN. Normalized NHE3 level correlated positively with serum creatinine level in patients with tubular injury (R(2)= 0.305; P = 0.0003). Values for FeNa and normalized urinary RBP did not discriminate ATN from intrinsic ARF other than ATN and prerenal azotemia, respectively. Conclusion: In patients with ARF, urinary NHE3 abundance might be a novel noninvasive marker of renal tubule damage, helping to differentiate prerenal azotemia, ATN, and intrinsic ARF other than ATN.

OBJECTIVES Cardiopulmonary bypass (CPB) is widely regarded as an important contributor to renal failure, a well recognized complication following coronary artery surgery (coronary artery bypass grafting (CABG)). Anecdotally off-pump coronary surgery (OPCAB) is considered renoprotective. We examine the extent of renal glomerular and tubular injury in low-risk patients undergoing either OPCAB or on-pump coronary artery bypass (ONCAB). METHODS Forty low-risk patients with normal preoperative cardiac and renal functions awaiting elective CABG were prospectively randomized into those undergoing OPCAB (n=20) and ONCAB (n=20). Glomerular and tubular injury were measured respectively by urinary excretion of microalbumin and retinol binding protein (RBP) indexed to creatinine (Cr). Daily measurements were taken from admission to postoperative day 5. Fluid balance, serum Cr and blood urea were also monitored. RESULTS No mortality or renal complication were observed. Both groups had similar demographic makeup, Parsonnet score, functional status and extent of coronary revascularization (2.1+/-1.0 vs. 2.5+/-0.7 grafts; P=0.08). Serum Cr and blood urea remained normal in both groups throughout the study. A significant and similar rise in urinary RBP:Cr occurred in both groups peaking on day 1 (3183+/-2534 vs. 4035+/-4079; P=0.43) before reapproximating baseline levels. These trends were also observed with urinary microalbumin:Cr (5.05+/-2.66 vs. 6.77+/-5.76; P=0.22). Group B patients had a significantly more negative fluid balance on postoperative day 2 (-183+/-1118 vs. 637+/-847 ml; P=0.03). CONCLUSIONS Although renal complication or serum markers of kidney dysfunction were absent, sensitive indicators revealed significant and similar injury to renal tubules and glomeruli following either OPCAB or ONCAB. These results suggest that avoidance of CPB does not offer additional renoprotection to patients at low risk of perioperative renal insult during CABG.

OBJECTIVE A case-control study was conducted to evaluate the effects of diabetes mellitus on serum levels of vitamin A, alpha-carotene, beta-carotene, alpha-tocopherol, serum and urine RBP. SUBJECTS One hundred and seven patients with Type 2 diabetes mellitus (28-74 y) were recruited from those attending a primary health care clinic in King Khalid University Hospital in Riyadh City (Saudi Arabia). They were matched for age and sex with 143 healthy individuals. METHODS Fasting blood samples and 10h urine collections were obtained from all subjects. Levels of vitamins and carotenoids in serum measured by high performance liquid chromatography (HPLC), and of retinol binding protein (RBP) in serum and urine by an enzyme-linked immunosorbent assay (ELISA). RESULTS The mean serum concentrations of retinol, alpha-carotene, and alpha-tocopherol were similar in both groups after correction of lipid soluble vitamins for serum lipids levels. However, serum beta-carotene concentration was significantly higher in control subjects than diabetics (P = 0.002). Serum and urine RBP concentrations were significantly higher in diabetics than in controls (P = 0.0001). In normal subjects (but not diabetics) serum concentrations of retinol and RBP were higher in men than in women (P = 0.02, P = 0.0001 respectively). In both normal and diabetic subjects, serum levels of alpha-tocopherol (P = 0.007) and urine RBP (P = 0.005), were higher in men than women. Urinary excretion of RBP was significantly higher in diabetic patients with renal impairment than other diabetics or controls (P = 0.0001). There was a negative correlation between fasting blood glucose (FBG) concentration and serum beta-carotene (P = 0.008) in the total combined group and a positive correlation between FBG and urinary RBP/creatinine (P = 0.009) in diabetic patients. CONCLUSION Serum beta-carotene concentration was significantly lower in diabetic patients than controls. Serum retinol concentration in patients with diabetes was normal, yet serum and urine RBP concentrations were significantly higher in diabetics than in controls.