Biomarkers for acute kidney injury (AKI) have been used to predict the progression of AKI but a systematic comparison of the prognostic ability of each biomarkers alone or in combination has not been performed. In order to assess this, we measured the concentration of 32 candidate biomarkers in the urine of 95 patients with AKIN stage 1 after cardiac surgery. Urine markers were divided into eight groups based on the putative pathophysiologic mechanism they reflect. We then compared the ability of the markers alone or in combination to predict the primary outcome of worsening AKI or death (23 patients) and the secondary outcome of AKIN stage 3 or death (13 patients). IL-18 was the best predictor of both outcomes (AUC of 0.74 and 0.89). L-FABP (AUC of 0.67 and 0.85), NGAL (AUC of 0.72 and 0.83) and KIM-1 (AUC of 0.73 and 0.81) were also good predictors. Correlation between most of the markers was generally related to their predictive ability but KIM-1 had a relatively weak correlation with other markers. The combination of IL-18 and KIM-1 had a very good predictive value with an AUC of 0.93 to predict AKIN 3 or death. Thus, combination of IL-18 and KIM-1 would result in improved identification of high risk patients for enrollment in clinical trials.
Kidney; renal failure; Outcomes; Postoperative care; Risk assessment; predictive modeling; biomarker discovery; Surgery; complications; Interleukin 18; Interleukin 6; Vascular endothelial growth factor; Monocyte chemotactic protein-1; Interleukin 1 receptor antagonist; Interleukin 8; Growth related oncogene alpha; Leukemia inhibitory factor; Interleukin 10; Eotaxin; Vascular cell adhesion molecule-1; RANTES; Regulated on activation; normal T cell expressed and secreted; Tumor necrosis factor alpha; Macrophage inflammatory protein-1alpha; Neutrophil gelatinase associated lipocalin; Kidney injury molecule-1; Liver type fatty acid binding protein; Hepatocyte growth factor; Netrin-1; Clusterin; Fetuin-A; Cystatin C; Albumin; Beta-2-microglobulin; Retinol binding protein; Alpha-1 antitrypsin; 8-Isoprostane; Trefoil factor 3; N-acetyl-beta-D-glucosaminidase; TRAIL; TNF-related apoptosis-inducing ligand
Domoic acid toxicosis (DAT) in California sea lions (Zalophus californianus) is caused by exposure to the marine biotoxin domoic acid and has been linked to massive stranding events and mortality. Diagnosis is based on clinical signs in addition to the presence of domoic acid in body fluids. Chronic DAT further is characterized by reoccurring seizures progressing to status epilepticus. Diagnosis of chronic DAT is often slow and problematic, and minimally invasive tests for DAT have been the focus of numerous recent biomarker studies. The goal of this study was to retrospectively profile plasma proteins in a population of sea lions with chronic DAT and those without DAT using two dimensional gel electrophoresis to discover whether individual, multiple, or combinations of protein and clinical data could be utilized to identify sea lions with DAT. Using a training set of 32 sea lion sera, 20 proteins and their isoforms were identified that were significantly different between the two groups (p<0.05). Interestingly, 11 apolipoprotein E (ApoE) charge forms were decreased in DAT samples, indicating that ApoE charge form distributions may be important in the progression of DAT. In order to develop a classifier of chronic DAT, an independent blinded test set of 20 sea lions, seven with chronic DAT, was used to validate models utilizing ApoE charge forms and eosinophil counts. The resulting support vector machine had high sensitivity (85.7% with 92.3% negative predictive value) and high specificity (92.3% with 85.7% positive predictive value). These results suggest that ApoE and eosinophil counts along with machine learning can perform as a robust and accurate tool to diagnose chronic DAT. Although this analysis is specifically focused on blood biomarkers and routine clinical data, the results demonstrate promise for future studies combining additional variables in multidimensional space to create robust classifiers.
Diabetic nephropathy is the leading cause of end stage renal disease. The urinary albumin to creatinine ratio is used as a predictor for the development of nephropathy but it is neither sensitive nor specific. Here we used liquid chromatography/mass spectrometry on urine of eight normoalbuminuric patients with type 2 diabetes from the VA Diabetes Trial to identify candidate markers for loss of renal function. Initial verification of 7 markers (agrin, haptoglobin, mannan-binding lectin serine protease 2, LAMP-2, angiotensinogen, NGAL and uromodulin) in the urine of an additional 30 patients showed that haptoglobin was the best predictor of early renal functional decline. We then measured this in the urine of 204 patients with type 2 diabetes who did not yet have significant kidney disease (eGFR stage 2 or better and an albumin to creatinine ratio less than 300 mg/g). In comparing the highest to lowest tertile, the odds ratio for having early renal function decline was 2.70 (CI 1.15, 6.32) using the haptoglobin to creatinine ratio compared to 2.50 (CI 1.14, 5.48) using the albumin to creatinine ratio after adjusting for treatment group and use of ACE inhibitors. Addition of the haptoglobin to creatinine ratio to a model using the albumin to creatinine ratio to predict early renal function decline resulted in improved predictive performance. Thus, the haptoglobin to creatinine ratio may be useful to predict patients with type 2 diabetes at risk of nephropathy prior to the development of macroalbuminuria or reduced GFR.
Diabetes; diabetic nephropathy; type 2 diabetes; urine; biological markers; chronic kidney disease
Unopposed angiotensin (Ang) II-mediated cellular effects may lead to progressive glomerulosclerosis. While Ang-II can be locally generated in the kidneys, we previously showed that glomerular podocytes primarily convert Ang-I, the precursor of Ang-II, to Ang-(1-7) and Ang-(2-10), peptides that have been independently implicated in biological actions opposing those of Ang-II. Therefore, we hypothesized that Ang-(1-7) and Ang-(2-10) could be renoprotective in the fawn-hooded hypertensive rat, a model of focal segmental glomerulosclerosis. We evaluated the ability of 8–12 week-long intravenous administration of either Ang-(1-7) or Ang-(2-10) (100–400 ng/kg/min) to reduce glomerular injury in uni-nephrectomized fawn-hooded hypertensive rats, early or late in the disease. Vehicle-treated rats developed hypertension and lesions of focal segmental glomerulosclerosis. No reduction in glomerular damage was observed, as measured by either 24-hour urinary protein excretion or histological examination of glomerulosclerosis, upon Ang-(1-7) or Ang-(2-10) administration, regardless of peptide dose or disease stage. On the contrary, when given at 400 ng/kg/min, both peptides induced a further increase in systolic blood pressure. Content of Ang peptides was measured by parallel reaction monitoring in kidneys harvested at sacrifice. Exogenous administration of Ang-(1-7) and Ang-(2-10) did not lead to a significant increase in their corresponding intrarenal levels. However, the relative abundance of Ang-(1-7) with respect to Ang-II was increased in kidney homogenates of Ang-(1-7)-treated rats. We conclude that chronic intravenous administration of Ang-(1-7) or Ang-(2-10) does not ameliorate glomerular damage in a rat model of focal segmental glomerulosclerosis and may induce a further rise in blood pressure, potentially aggravating glomerular injury.
Acute kidney injury (AKI) is an important cause of death among hospitalized patients. The two most common causes of AKI are acute tubular necrosis (ATN) and prerenal azotemia (PRA). Appropriate diagnosis of the disease is important but often difficult. We analyzed urine proteins by 2-DE from 38 patients with AKI. Patients were randomly assigned to a training set, an internal test set or an external validation set. Spot abundances were analyzed by artificial neural networks (ANN) to identify biomarkers which differentiate between ATN and PRA. When the trained neural network algorithm was tested against the training data it identified the diagnosis for 16/18 patients in the training set and all 10 patients in the internal test set. The accuracy was validated in the novel external set of patients where 9/10 subjects were correctly diagnosed including 5/5 with ATN and 4/5 with PRA. Plasma retinol binding protein (PRBP) was identified in one spot and a fragment of albumin and PRBP in the other. These proteins are candidate markers for diagnostic assays of AKI.
Acute kidney injury; Biomarkers; Diagnosis; Kidney; Urine
In the setting of early acute kidney injury (AKI), no test has been shown to definitively predict the progression to more severe stages.
We investigated the ability of a furosemide stress test (FST) (one-time dose of 1.0 or 1.5 mg/kg depending on prior furosemide-exposure) to predict the development of AKIN Stage-III in 2 cohorts of critically ill subjects with early AKI. Cohort 1 was a retrospective cohort who received a FST in the setting of AKI in critically ill patients as part of Southern AKI Network. Cohort 2 was a prospective multicenter group of critically ill patients who received their FST in the setting of early AKI.
We studied 77 subjects; 23 from cohort 1 and 54 from cohort 2; 25 (32.4%) met the primary endpoint of progression to AKIN-III. Subjects with progressive AKI had significantly lower urine output following FST in each of the first 6 hours (p<0.001). The area under the receiver operator characteristic curves for the total urine output over the first 2 hours following FST to predict progression to AKIN-III was 0.87 (p = 0.001). The ideal-cutoff for predicting AKI progression during the first 2 hours following FST was a urine volume of less than 200mls(100ml/hr) with a sensitivity of 87.1% and specificity 84.1%.
The FST in subjects with early AKI serves as a novel assessment of tubular function with robust predictive capacity to identify those patients with severe and progressive AKI. Future studies to validate these findings are warranted.
Two-dimensional gel electrophoresis (2DE) offers high-resolution separation for intact proteins. However, variability in the appearance of spots can limit the ability to identify true differences between conditions. Variability can occur at a number of levels. Individual samples can differ because of biological variability. Technical variability can occur during protein extraction, processing, or storage. Another potential source of variability occurs during analysis of the gels and is not a result of any of the causes of variability named above. We performed a study designed to focus only on the variability caused by analysis. We separated three aliquots of rat left ventricle and analyzed differences in protein abundance on the replicate 2D gels. As the samples loaded on each gel were identical, differences in protein abundance are caused by variability in separation or interpretation of the gels. Protein spots were compared across gels by quantile values to determine differences. Fourteen percent of spots had a maximum difference in intensity of 0.4 quantile values or more between replicates. We then looked individually at the spots to determine the cause of differences between the measured intensities. Reasons for differences were: failure to identify a spot (59%), differences in spot boundaries (13%), difference in the peak height (6%), and a combination of these factors (21). This study demonstrates that spot identification and characterization make major contributions to variability seen with 2DE. Methods to highlight why measured protein spot abundance is different could reduce these errors.
heart; proteomics; reproducibility; protein
Diagnosis of the type of glomerular disease that causes the nephrotic syndrome is necessary for appropriate treatment and typically requires a renal biopsy. The goal of this study was to identify candidate protein biomarkers to diagnose glomerular diseases. Proteomic methods and informatic analysis were used to identify patterns of urine proteins that are characteristic of the diseases. Urine proteins were separated by two-dimensional electrophoresis in 32 patients with FSGS, lupus nephritis, membranous nephropathy, or diabetic nephropathy. Protein abundances from 16 patients were used to train an artificial neural network to create a prediction algorithm. The remaining 16 patients were used as an external validation set to test the accuracy of the prediction algorithm. In the validation set, the model predicted the presence of the diseases with sensitivities between 75 and 86% and specificities from 92 to 67%. The probability of obtaining these results in the novel set by chance is 5 × 10−8. Twenty-one gel spots were most important for the differentiation of the diseases. The spots were cut from the gel, and 20 were identified by mass spectrometry as charge forms of 11 plasma proteins: Orosomucoid, transferrin, α-1 microglobulin, zinc α-2 glycoprotein, α-1 antitrypsin, complement factor B, haptoglobin, transthyretin, plasma retinol binding protein, albumin, and hemopexin. These data show that diseases that cause nephrotic syndrome change glomerular protein permeability in characteristic patterns. The fingerprint of urine protein charge forms identifies the glomerular disease. The identified proteins are candidate biomarkers that can be tested in assays that are more amenable to clinical testing.
Acute kidney injury (AKI) is commonly observed in the intensive care unit (ICU), where it can be caused by a variety of factors. The objective of this study was to evaluate the prognostic value of urinary angiotensinogen, a candidate prognostic AKI biomarker identified in post-cardiac surgery patients, in this heterogeneous population.
Urinary angiotensinogen was measured by ELISA and corrected for urine creatinine in 45 patients who developed AKI in the ICU. Patients were grouped by AKI etiology, and the angiotensinogen-to-creatinine ratio (uAnCR) was compared among the groups using the Kruskal-Wallis test. The ability of uAnCR to predict the following endpoints was tested using the area under the ROC curve (AUC): the need for renal replacement therapy (RRT) or death, increased length of stay (defined as hospital discharge > 7 days or death ≤ 7 days from sample collection), and worsening AKI (defined as an increase in serum creatinine > 0.3 mg/dL after sample collection or RRT).
uAnCR was significantly elevated in patients who met the composite outcome RRT or death (89.4 vs 25.4 ng/mg; P = 0.01), and it was a strong predictor of this outcome (AUC = 0.73). Patients with uAnCR values above the median for the cohort (55.21 ng/mg) had increased length of stay compared to patients with uAnCR ≤ 55.21 ng/mg (22 days vs 7 days after sample collection; P = 0.01). uAnCR was predictive of the outcome increased length of stay (AUC = 0.77). uAnCR was also a strong predictor of worsening of AKI (AUC = 0.77). The uAnCR of patients with pre-renal AKI was lower compared to patients with AKI of other causes (median uAnCR 11.3 vs 80.2 ng/mg; P = 0.02).
Elevated urinary angiotensinogen is associated with adverse events in AKI patients in the ICU. It could be used to identify high risk patients who would benefit from timely intervention that could improve their outcomes.
High molecular weight (HMW) adiponectin levels are reduced in humans with type 2 diabetes and insulin resistance. Similar to humans with insulin resistance, managed bottlenose dolphins (Tursiops truncatus) diagnosed with hemochromatosis (iron overload) have higher levels of 2 h post-prandial plasma insulin than healthy controls. A parallel reaction monitoring assay for dolphin serum adiponectin was developed based on tryptic peptides identified by mass spectrometry. Using identified post-translational modifications, a differential measurement was constructed. Total and unmodified adiponectin levels were measured in sera from dolphins with (n = 4) and without (n = 5) iron overload. This measurement yielded total adiponectin levels as well as site specific percent unmodified adiponectin that may inversely correlate with HMW adiponectin. Differences in insulin levels between iron overload cases and controls were observed 2 h post-prandial, but not during the fasting state. Thus, post-prandial as well as fasting serum adiponectin levels were measured to determine whether adiponectin and insulin would follow similar patterns. There was no difference in total adiponectin or percent unmodified adiponectin from case or control fasting animals. There was no difference in post-prandial total adiponectin levels between case and control dolphins (mean ± SD) at 763 ± 298 and 727 ± 291 pmol/ml, respectively (p = 0.91); however, percent unmodified adiponectin was significantly higher in post-prandial cases compared to controls (30.0 ± 6.3 versus 17.0 ± 6.6%, respectively; p = 0.016). Interestingly, both total and percent unmodified adiponectin were correlated with glucagon levels in controls (r = 0.999, p < 0.001), but not in cases, which is possibly a reflection of insulin resistance. Although total adiponectin levels were not significantly different, the elevated percent unmodified adiponectin follows a trend similar to HMW adiponectin reported for humans with metabolic disorders.
parallel reaction monitoring; marine mammal; assay; hemochromatosis; liver; diabetes
Using multiplex bead assays to measure urine proteins has a great potential for biomarker discovery, but substances in urine (the matrix) can interfere with assay measurements. By comparing the recovery of urine spiked with known quantities of several common analytes, this study demonstrated that the urine matrix variably interfered with the accurate measurement of low abundance proteins. Dilution of the urine permitted a more accurate measure of these proteins, equivalent to the standard dilution technique when the diluted analytes were above the limits of detection of the assay. Therefore, dilution can be used as an effective technique for over-coming urine matrix effects in urine immunoassays. These results may be applicable to other biological fluids in which matrix components interfere with assay performance.
biomarkers; body fluids urine; analysis/urine; standard addition; assay validation
Lupus nephritis is divided into six classes and scored according to activity and chronicity indices based on histologic findings. Treatment differs based on the pathologic findings. Renal biopsy is currently the only way to accurately predict class and activity and chronicity indices. We propose to use patterns of abundance of urine proteins to identify class and disease indices.
Urine was collected from 20 consecutive patients immediately prior to biopsy for evaluation of lupus nephritis. The International Society of Nephrology/Renal Pathology Society (ISN/RPS) class of lupus nephritis, activity, and chronicity indices were determined by a renal pathologist. Proteins were separated by two-dimensional gel electrophoresis. Artificial neural networks were trained on normalized spot abundance values.
Biopsy specimens were classified in the database according to ISN/RPS class, activity, and chronicity. Nine samples had characteristics of more than one class present. Receiver operating characteristic (ROC) curves of the trained networks demonstrated areas under the curve ranging from 0.85 to 0.95. The sensitivity and specificity for the ISN/RPS classes were class II 100%, 100%; III 86%, 100%; IV 100%, 92%; and V 92%, 50%. Activity and chronicity indices had r values of 0.77 and 0.87, respectively. A list of spots was obtained that provided diagnostic sensitivity to the analysis.
We have identified a list of protein spots that can be used to develop a clinical assay to predict ISN/RPS class and chronicity for patients with lupus nephritis. An assay based on antibodies against these spots could eliminate the need for renal biopsy, allow frequent evaluation of disease status, and begin specific therapy for patients with lupus nephritis.
lupus nephritis; biomarkers; urine; electrophoresis; two-dimensional gel
In spite of two-dimensional gel electrophoresis (2-DE) being an effective and widely used method to screen the proteome, its data standardization has still not matured to the level of microarray genomics data or mass spectrometry approaches. The trend toward identifying encompassing data standards has been expanding from genomics to transcriptomics, and more recently to proteomics. The relative success of genomic and transcriptomic data standardization has enabled the development of central repositories such as GenBank and Gene Expression Omnibus. An equivalent 2-DE-centric data structure would similarly have to include a balance among raw data, basic feature detection results, sufficiency in the description of the experimental context and methods, and an overall structure that facilitates a diversity of usages, from central reposition to local data representation in LIMs systems.
Results & Conclusion
Achieving such a balance can only be accomplished through several iterations involving bioinformaticians, bench molecular biologists, and the manufacturers of the equipment and commercial software from which the data is primarily generated. Such an encompassing data structure is described here, developed as the mature successor to the well established and broadly used earlier version. A public repository, AGML Central, is configured with a suite of tools for the conversion from a variety of popular formats, web-based visualization, and interoperation with other tools and repositories, and is particularly mass-spectrometry oriented with I/O for annotation and data analysis.
Variability is a major complicating factor in analysis by two-dimensional gel electrophoresis. Improvements in methodologies have focused on improving individual gel quality rather than reproducibility. We homogenized rat cardiac tissue and rehydrated using a matrix of buffers to determine the optimal sample conditions. Six buffers were used to solubilize the proteins. Solubilized proteins were separated by isoelectric focusing using four buffers. Gels were run in triplicate to assess the method of preparation yielding the least variability. Number of spots and variability were different between conditions. Proteins solubilized in a buffer containing 5 M urea, 2M thiourea, 2% CHAPS, 2% SB 3–10, ampholytes, DTT, and protease inhibitors and focused in a buffer containing 9 M urea and 4% NP40 had the lowest coefficient of variation. Variability was compared across isoelectric point ranges and was different. Minimizing technical variability in two-dimensional polyacrylamide gel electrophoresis is critical to identify differences between conditions. Sample preparation should be optimized to minimize variability as well as to maximize the number of spots seen.
Reproducibility; variability: two-dimensional gel electrophoresis; heart