Development of a time-resolved immunochromatographic test strip for rapid and quantitative determination of retinol-binding protein 4 in urine.

Urine retinol-binding protein 4 (RBP4) has recently been reported as a novel earlier biomarker of chronic kidney disease (CKD) which is a global public health problem with high morbidity and mortality. Accurate and rapid detection of urine RBP4 is essential for early monitor of impaired kidney function and prevention of CKD progression. In the present study, we developed a time-resolved fluorescence immunochromatographic test strip (TRFIS) for the quantitative and rapid detection of urine RBP4. This TRFIS possessed excellent linearity ranging from 0.024 to 12.50 ng/mL for the detection of urine RBP4, and displayed a good linearity (Y = 239,581 × X + 617,238, R2 = 0.9902), with the lowest visual detection limit of 0.049 ng/mL. This TRFIS allows for quantitative detection of urine RBP4 within 15 min and shows high specificity. The intra-batch coefficient of variation (CV) and the inter-batch CV were both < 8%, respectively. Additionally, this TRFIS was applied to detect RBP4 in the urine samples from healthy donors and patients with CKD, and the results of TRFIS could efficiently discern the patients with CKD from the healthy donors. The developed TRFIS has the characteristics of high sensitivity, high accuracy, and a wide linear range, and is suitable for rapid and quantitative determination of urine RBP4.

A candidate panel of eight urinary proteins shows potential of early diagnosis and risk assessment for diabetic kidney disease in type 1 diabetes.

Diabetic kidney disease (DKD) poses a significant health challenge for individuals with diabetes. At its initial stages, DKD often presents asymptomatically, and the standard for non-invasive diagnosis, the albumin-creatinine ratio (ACR), employs discrete categorizations (normal, microalbuminuria, macroalbuminuria) with limitations in sensitivity and specificity across diverse population cohorts. Single biomarker reliance further restricts the predictive value in clinical settings. Given the escalating prevalence of diabetes, our study uses proteomic technologies to identify novel urinary proteins as supplementary DKD biomarkers. A total of 158 T1D subjects provided urine samples, with 28 (15 DKD; 13 non-DKD) used in the discovery stage and 131 (45 DKD; 40 pDKD; 46 non-DKD) used in the confirmation. We identified eight proteins (A1BG, AMBP, AZGP1, BTD, RBP4, ORM2, GM2A, and PGCP), all of which demonstrated excellent area-under-the-curve (AUC) values (0.959 to 0.995) in distinguishing DKD from non-DKD. Furthermore, this multi-marker panel successfully segregated the most ambiguous group (microalbuminuria) into three distinct clusters, with 80% of subjects aligning either as DKD or non-DKD. The remaining 20% exhibited continued uncertainty. Overall, the use of these candidate urinary proteins allowed for the better classification of DKD and offered potential for significant improvements in the early identification of DKD in T1D populations.

Urinary retinol binding protein predicts renal outcome in systemic immunoglobulin light-chain (AL) amyloidosis.

Renal risk stratification in systemic immunoglobulin light-chain (AL) amyloidosis is according to estimated glomerular filtration rate (eGFR) and urinary protein creatinine ratio (uPCR), the latter attributed to glomerular dysfunction, with proximal tubular dysfunction (PTD) little studied. Urinary retinol binding protein 4 (uRBP), a low molecular weight tubular protein and highly sensitive marker of PTD, was prospectively measured in 285 newly diagnosed, untreated patients with systemic AL amyloidosis between August 2017 to August 2018. At diagnosis, the uRBP/creatinine ratio (uRBPCR) correlated with serum creatinine (r = 0·618, P < 0·0001), uPCR (r = 0·422, P < 0·0001) as well as both fractional excretion of phosphate and urate (r = 0·563, P < 0·0001). Log uRBPCR at diagnosis was a strong independent predictor of end-stage renal disease {hazard ratio [HR] 2·65, [95% confidence interval (CI) 1·06-6·64]; P = 0·038}, particularly in patients with an eGFR >30 ml/min/1.73 m2 [HR 4·11, (95% CI 1·45-11·65); P = 0·008] and those who failed to achieve a deep haematological response to chemotherapy within 3 months of diagnosis [HR 6·72, (95% CI 1·83-24·74); P = 0·004], and also predicted renal progression [HR 1·91, (95% CI 1·18-3·07); P = 0·008]. Elevated uRBPCR indicates PTD and predicts renal outcomes independently of eGFR, uPCR and clonal response in systemic AL amyloidosis. The role of uRBPCR as a novel prognostic biomarker merits further study, particularly in monoclonal gammopathies of renal significance.

Development of a multiplex mass spectrometry method for simultaneous quantification of urinary proteins related to respiratory health.

Respiratory health of children is a health priority. Club cell protein (CC16) is an interesting biomarker of lung diseases and adverse effects towards the airway epithelium integrity. Osteopontin (OPN) and nuclear factor-kappa B (NF-κB) also play a role in respiratory health. The use of urine as biomarker source is useful in studies involving children but necessitates proper adjustment for physiological confounders influencing the urinary excretion, potentially characterized with beta-2-microglobulin (ß2M), retinol binding protein 4 (RBP4) or myoglobin (MYO), as well as adjustment for possible renal dysfunction, characterized by human serum albumin (HSA). The simultaneous quantification of all these proteins in urine could facilitate children's health monitoring. A multiple reaction monitoring method (MRM) was developed and validated for the relative quantification of the seven mentioned urinary proteins. A total of nine proteotypic peptides were selected and used for the relative quantification of the seven proteins. The MRM method was completely validated for all proteins and partially for OPN. LOQ's ranged from 0.3 to 42.8 ng/ml, a good reproducibility and a good linearity were obtained across the analytical measurement range (r2 > 0.98). The method yielded varying correlations (r2 of 0.78, 0.71, 0.34 and 0.15 for CC16, ß2M, RBP4 and HSA respectively) with available immunoassay data. It also allowed the identification and successful quantification of ß2M and RBP4 as a protein candidate for adjustment of renal handling and dysfunction. All proteins were detected in the urine samples except for MYO and NF-κB. Our validated MRM-method is able to simultaneously quantify in urine biomarkers of airway epithelium integrity and biomarkers of variation in renal function and urinary dilution. This will allow to investigate further in future studies if urine can be used as a good surrogate source for biomarkers of airway epithelium integrity, and to understand the complex relationship between cause and effect in children's respiratory health monitoring.

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.

Ginkgo Biloba Extract EGB761 Ameliorates the Extracellular Matrix Accumulation and Mesenchymal Transformation of Renal Tubules in Diabetic Kidney Disease by Inhibiting Endoplasmic Reticulum Stress.

The study is aimed at investigating the effects of Ginkgo biloba extract EGB761 on renal tubular damage and endoplasmic reticulum stress (ERS) in diabetic kidney disease (DKD). A total of 50 C57BL/6 N mice were randomly divided into the normal group, DKD group, DKD+EGB761 group (36 mg/kg), and DKD+4-phenylbutyrate (4-PBA) group (1 g/kg). The DKD model was replicated by high-fat diet combined with intraperitoneal injection of streptozotocin (STZ). Renal tubular epithelial cells (HK-2) were divided into the control group, high-glucose group (30 mmol/L), EGB761 group (40 mg/L, 20 mg/L, 10 mg/L), TM group, and TM+4-PBA group. After 8 weeks of administration, expressions of serum creatinine (Scr), blood urea nitrogen (BUN), 24 h urinary protein (24 h Pro), fasting blood glucose (FBG), ß 2-microglobulin (ß 2-MG), and retinol binding protein 4 (RBP4) of mice were tested. The pathological changes of renal tissue were observed. The expressions of extracellular matrix (ECM) accumulation and epithelial-mesenchymal transition (EMT) markers α-smooth muscle actin (α-SMA), E-cadherin, fibronectin, and collagen IV, as well as the ERS markers GRP78 and ATF6, were tested by Western blot, qPCR, immunohistochemistry, or immunofluorescence. EGB761 could decrease the Scr, BUN, 24 h Pro, and FBG levels in the DKD group, alleviate renal pathological injury, decrease urine ß 2-MG, RBP4 levels, and decrease the expression of α-SMA, collagen IV, fibronectin, and GRP78, as well as ATF6, while increase the expression of E-cadherin. These findings demonstrate that EGB761 can improve renal function, reduce tubular injury, and ameliorate ECM accumulation and EMT in DKD kidney tubules, and the mechanism may be related to the inhibition of ERS.

Urinary DPP4 correlates with renal dysfunction, and DPP4 inhibition protects against the reduction in megalin and podocin expression in experimental CKD.

This study investigated the molecular mechanisms underlying the antiproteinuric effect of DPP4 inhibition in 5/6 renal ablation rats and tested the hypothesis that the urinary activity of DPP4 correlates with chronic kidney disease (CKD) progression. Experiments were conducted in male Wistar rats who underwent 5/6 nephrectomy (Nx) or sham operation followed by 8 wk of treatment with the DPP4 inhibitor (DPP4i) sitagliptin or vehicle. Proteinuria increased progressively in Nx rats throughout the observation period. This increase was remarkably mitigated by sitagliptin. Higher levels of proteinuria in Nx rats compared to control rats were accompanied by higher urinary excretion of retinol-binding protein 4, a marker of tubular proteinuria, as well as higher urinary levels of podocin, a marker of glomerular proteinuria. Retinol-binding protein 4 and podocin were not detected in the urine of Nx + DPP4i rats. Tubular and glomerular proteinuria was associated with the reduced expression of megalin and podocin in the renal cortex of Nx rats. Sitagliptin treatment partially prevented this decrease. Besides, the angiotensin II renal content was significantly reduced in the Nx rats that received sitagliptin compared to vehicle-treated Nx rats. Interestingly, both urinary DPP4 activity and abundance increased progressively in Nx rats. Additionally, urinary DPP4 activity correlated positively with serum creatinine levels, proteinuria, and blood pressure. Collectively, these results suggest that DPP4 inhibition ameliorated both tubular and glomerular proteinuria and prevented the reduction of megalin and podocin expression in CKD rats. Furthermore, these findings suggest that urinary DPP4 activity may serve as a biomarker of renal disease and progression.

Fractional excretion as a new marker of tubular damage in children with chronic kidney disease.

OBJECTIVES: Vitamin D-binding protein (VDBP), retinol-binding protein (RBP)4, and heat shock proteins (hsp) are markers of tubular function and apoptosis, accompanying chronic kidney disease (CKD) from its earliest stages. Fractional excretion of proteins with urine is a marker of tubular damage. The aim of study was to assess the usefulness of fractional excretion (FE) of VDBP, RBP4, HSF1 and Hsp27 as markers of tubular damage in the course of CKD. METHODS: The study group consisted of 70 children with CKD stages 1-5, treated conservatively, and 12 age-matched controls with normal kidney function. The serum and urine concentrations of VDBP, RBP4, HSF1 and Hsp27 were assessed by ELISA. The fractional excretion of analyzed parameters was calculated according to the formula: ([parameter urine concentration] × [creatinine serum concentration]) / ([parameter serum concentration] × [creatinine urine concentration])×100%. RESULTS: The FE values of all parameters exceeded 1% in CKD stage 2. However, the values of FE have raised significantly versus control group no sooner than CKD stage 2 (RBP4 and HSF1), stage 3 (VDBP) or stage 4 (Hsp27). CONCLUSION: Fractional excretion of RBP4 and HSF1 with urine may become a valuable marker, assessing the damage of tubular cells in children with CKD.

Development of a mouse IgA monoclonal antibody-based enzyme-linked immunosorbent sandwich assay for the analyses of RBP4.

Elevated circulating Retinol-binding protein 4 (RBP4) has been associated with insulin resistance, dyslipidemia, and hypertension. However, many commonly used RBP4 ELISAs have limited dynamic range. We therefore developed an enzyme-linked immunosorbent sandwich assay (ELISA) employing a novel immunoglobulin A (IgA)-type capture mAb called AG102 instead of IgG subtypes, which was selected for its stability, capture efficiency, and specificity for human RBP 4. These features of RBP4 have hampered the development of quantitative immunological assays. Molecular analysis of AG102 revealed IgA heavy and light chains and a J chain, as expected. AG102 demonstrated notable detection of both bacterial- and HEK293-expressed RBP4 in Western blots. Serial and internal deletion experiments suggested that a putative epitope may be located in the first 35 amino acids of the mature RBP4. Compared with commercial ELISAs, the AG102-based system exhibited more significant recovery of RBP4 from serum or urine at any given dilution factor. To substantiate its quantitation capacity, comparison between RBP4 measurements from quantitative western blots and the AG102-based ELISA demonstrated a significant correlation (R2 = 0.859). After measurement for those analytes, our data suggested that IgA-based ELISA could be adapted for quantitative measurement of those analytes existing as major serum proteins or as multi-protein complexes like RBP4.

Human plasma retinol-binding protein (RBP4) is also a fatty acid-binding protein.

RBP4 (plasma retinol-binding protein) is the 21 kDa transporter of all-trans retinol that circulates in plasma as a moderately tight 1:1 molar complex of the vitamin with the protein. RBP4 is primarily synthesized in the liver but is also produced by adipose tissue and circulates bound to a larger protein, transthyretin, TTR, that serves to increase its molecular mass and thus avoid its elimination by glomerular filtration. This paper reports the high resolution three-dimensional structures of human RBP4 naturally lacking bound retinol purified from plasma, urine and amniotic fluid. In all these crystals we found a fatty acid molecule bound in the hydrophobic ligand-binding site, a result confirmed by mass spectrometry measurements. In addition we also report the 1.5 Šresolution structures of human holo-RBP4 and of the protein saturated with palmitic and lauric acid and discuss the interaction of the fatty acids and retinol with the protein.

Urinary Kininogen-1 and Retinol binding protein-4 respond to Acute Kidney Injury: predictors of patient prognosis?

Implementation of therapy for acute kidney injury (AKI) depends on successful prediction of individual patient prognosis. Clinical markers as serum creatinine (sCr) have limitations in sensitivity and early response. The aim of the study was to identify novel molecules in urine which show altered levels in response to AKI and investigate their value as predictors of recovery. Changes in the urinary proteome were here investigated in a cohort of 88 subjects (55 AKI patients and 33 healthy donors) grouped in discovery and validation independent cohorts. Patients' urine was collected at three time points: within the first 48 h after diagnosis(T1), at 7 days of follow-up(T2) and at discharge of Nephrology(T3). Differential gel electrophoresis was performed and data were confirmed by Western blot (WB), liquid chromatography/mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA). Retinol binding protein 4 (RBP4) and kininogen-1 (KNG1) were found significantly altered following AKI. RBP4 increased at T1, and progressively decreased towards normalization. Maintained decrease was observed for KNG1 from T1. Individual patient response along time revealed RBP4 responds to recovery earlier than sCr. In conclusion, KNG1 and RBP4 respond to AKI. By monitoring RBP4, patient's recovery can be anticipated pointing to a role of RBP4 in prognosis evaluation.

Association of urinary RBP4 with insulin resistance, inflammation, and microalbuminuria.

OBJECTIVE: Serum concentrations of retinol-binding protein 4 (RBP4) are elevated in type 2 diabetes and associated with the severity of insulin resistance; however, there are few data about the relationship between urinary RBP4 levels and metabolic parameters. We assessed urinary RBP4 as a new biomarker by establishing its relationship with clinical parameters associated with insulin resistance and urinary albumin excretion. DESIGN AND METHODS: We measured RBP4 in the serum and urine of 689 subjects with diverse glucose tolerance status. We also evaluated the relationship between urinary RBP4 and cardiometabolic risk factors, including insulin resistance, high-sensitivity C-reactive protein (hsCRP), arterial stiffness, and microalbuminuria. RESULTS: Urinary RBP4 levels were higher in insulin-resistant subjects with prediabetes or type 2 diabetes than in subjects with normal glucose tolerance (NGT) (type 2 diabetes>prediabetes>NGT; all P<0.001). Urinary RBP4 correlated strongly with homeostasis model assessments of insulin resistance (HOMA-IR), fasting glucose, triglycerides, blood pressure, hsCRP, arterial stiffness, estimated glomerular filtration rate, and urinary albumin-to-creatinine ratio (all P<0.01). HOMA-IR and arterial stiffness were found to be independent determinants of urinary RBP4 concentration. Furthermore, urinary RBP4 was highly predictive of microalbuminuria (odds ratio 2.6, 95% CI 1.6-4.2), even after adjustment for other metabolic parameters. The area under the ROC curve for urinary RBP4 to detect the presence of microalbuminuria was 0.80±0.02 (95% CI 0.76-0.84) and the cut-off value was 157.01 µg/gCr. CONCLUSIONS: Urinary RBP4 concentrations were elevated in patients with dysregulation of glucose metabolism and were related to various cardiometabolic risk factors including insulin resistance, inflammation, and microalbuminuria.

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.

Urine protein biomarkers for the diagnosis and prognosis of necrotizing enterocolitis in infants.

OBJECTIVES: To test the hypothesis that an exploratory proteomics analysis of urine proteins with subsequent development of validated urine biomarker panels would produce molecular classifiers for both the diagnosis and prognosis of infants with necrotizing enterocolitis (NEC). STUDY DESIGN: Urine samples were collected from 119 premature infants (85 NEC, 17 sepsis, 17 control) at the time of initial clinical concern for disease. The urine from 59 infants was used for candidate biomarker discovery by liquid chromatography/mass spectrometry. The remaining 60 samples were subject to enzyme-linked immunosorbent assay for quantitative biomarker validation. RESULTS: A panel of 7 biomarkers (alpha-2-macroglobulin-like protein 1, cluster of differentiation protein 14, cystatin 3, fibrinogen alpha chain, pigment epithelium-derived factor, retinol binding protein 4, and vasolin) was identified by liquid chromatography/mass spectrometry and subsequently validated by enzyme-linked immunosorbent assay. These proteins were consistently found to be either up- or down-regulated depending on the presence, absence, or severity of disease. Biomarker panel validation resulted in a receiver-operator characteristic area under the curve of 98.2% for NEC vs sepsis and an area under the curve of 98.4% for medical NEC vs surgical NEC. CONCLUSIONS: We identified 7 urine proteins capable of providing highly accurate diagnostic and prognostic information for infants with suspected NEC. This work represents a novel approach to improving the efficiency with which we diagnose early NEC and identify those at risk for developing severe, or surgical, disease.

Proteomic identification of human urinary biomarkers in diabetes mellitus type 2.

BACKGROUND: During the proteomic era, one of the most rapidly growing areas in biomedical research is biomarker discovery, particularly using proteomic technologies. The urinary proteome is known to be a valuable field of study and has become one of the most attractive subdisciplines in clinical proteomics for human diseases. We have described the levels of protein biomarkers specific to diabetes mellitus type 2 in the Pakistani population using proteomic technology. METHODS: One hundred type 2 diabetes patients with 50 age- and sex-matched normal healthy controls were recruited from Sheikh Zayed Hospital, Lahore, Pakistan. Urinary proteins were analyzed by two-dimensional liquid chromatography, using chromatofocusing in the first dimension and reverse-phase chromatography in the second, followed by mass spectrometric analysis. Levels of the proteins, which were found to vary in the diabetes type 2 patients compared to the controls, were then determined by enzyme-linked immunosorbent assay in all the samples. RESULTS: Levels of transthyretin, α-1-microglobulin/bikunin precursor, and haptoglobin precursor decreased by 30.8%, 55.2%, and 81.45%, whereas levels of albumin, zinc α2 glycoprotein, retinol binding protein 4, and E-cadherin increased by 486.5%, 29.23%, 100%, and 693%, respectively, in the diabetes patients compared to the controls. CONCLUSIONS: Variation in the levels of these identified protein biomarkers have been reported in other pathological states. Assessment of the levels of these biomarkers will be helpful not only in early diagnosis but also in prognosis of diabetes mellitus type 2.