Analysis Of Urinary Proteins Research Articles

Urinary Sodium Dodecyl Sulfate Electrophoresis With Silver Staining: A Noninvasive Diagnostic Tool for Obstructive Uropathy in Children

Obstructive uropathy such as ureteropelvic junction obstruction in the newborn is a major diagnostic and therapeutic dilemma. We investigated whether urinary sodium dodecyl sulfate electrophoresis with polyacrylamide gel electrophoresis with silver staining could be used to discriminate between children requiring and those not requiring pyeloplasty. In a pilot study we analyzed the urine of 18 children (mean age 2.7 years) with grade III or IV hydronephrosis according to the Society for Fetal Urology classification. A total of 44 healthy children were studied as controls. Children with hydronephrosis were followed using ultrasound, (99m)technetium mercaptoacetyltriglycine diuretic renography and voiding cystourethrography. Urine was obtained by spontaneous voiding and studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis with silver staining using Melzer's modification. After the study period test results were compared to outcomes, ie whether patients required surgery, and to normalization of previously abnormal protein excretion patterns. All but 1 of the healthy controls had a normal electrophoresis assessment. Of 9 patients followed for hydronephrosis 7 had an abnormal electrophoresis result preoperatively. One child had to be operated on twice because of relapse of ureteropelvic junction obstruction. Six children returned to a normal electrophoresis result postoperatively, including the child who was operated on twice. All children with an initially normal electrophoresis assessment displayed persistent normal values, except 1. Children shifting from a normal to an abnormal electrophoresis result underwent surgery after exclusion of urinary tract infection. Sodium dodecyl sulfate polyacrylamide gel electrophoresis with silver staining seems to be a good predictive test for clinically relevant ureteropelvic junction obstruction. Further studies are being performed to see whether the test can stand against the gold standard, (99m)technetium mercaptoacetyltriglycine diuretic renography.

Urine proteomics: the present and future of measuring urinary protein components in disease

For centuries, physicians have attempted to use the urine for noninvasive assessment of disease. Today, urinalysis, in particular the measurement of proteinuria, underpins the routine assessment of patients with renal disease. More sophisticated methods for assessing specific urinary protein losses have emerged; however, albumin is still the principal urinary protein measured. Changes in the pattern of urinary protein excretion are not necessarily restricted to nephrourological disease; for instance, the appearance of beta-human chorionic gonadotropin in the urine of pregnant women is the basis for all commercially available pregnancy kits. Similarly, microalbuminuria is a clinically important marker not only of early diabetic nephropathy but also of concomitant cardiovascular disease. With the emergence of newer technologies, in particular mass spectrometry, it has become possible to study urinary protein excretion in even more detail. A variety of techniques have been used both to characterize the normal complement of urinary proteins and also to identify proteins and peptides that may facilitate earlier detection of disease, improve assessment of prognosis and allow closer monitoring of response to therapy. Such proteomics-based approaches hold great promise as the basis for new diagnostic tests and as the means to better understand disease pathogenesis. In this review, we summarize the currently available methods for urinary protein analysis and describe the newer approaches being taken to identify urinary biomarkers.

Detection of Subclinical Tubular Injury After Renal Transplantation: Comparison of Urine Protein Analysis With Allograft Histopathology

Tubulointerstitial injury due to rejection leads to tubular atrophy (TA)/interstitial fibrosis (IF) followed by deterioration of allograft function. This study investigated whether urinary tubular injury biomarkers can detect subclinical tubulitis found in protocol biopsies allowing for a noninvasive screening procedure. Four rigidly defined groups (stable transplants with normal tubular histology [n=24], stable transplants with subclinical tubulitis [n=38], patients with clinical tubulitis Ia/Ib [n=18], and patients with other clinical tubular pathologies [n=20]) were compared for differences in urinary intact/cleaved beta2-microglobulin (i/cbeta2m), retinol-binding protein (RBP), neutrophil-gelatinase-associated lipocalin (NGAL), and alpha1-microglobulin (alpha1m). Tubular proteinuria was present in 38% (RBP) to 79% (alpha1m) of patients in the stable transplant with normal tubular histology group. The stable transplant with subclinical tubulitis group had slightly higher levels of i/cbeta2m (P=0.11), RBP (P=0.17), alpha1m (P=0.09), and NGAL (P=0.06) than the stable transplant with normal tubular histology group with a substantial overlap. The clinical tubulitis Ia/Ib and the other clinical tubular pathology groups had significantly higher levels of RBP, NGAL, and alpha1m than stable transplants with normal tubular histology or stable transplants with subclinical tubulitis (P<0.002). None of the investigated biomarkers allow for clear differentiation between stable transplants with normal tubular histology and stable transplants with subclinical tubulitis. Therefore, the protocol allograft biopsy currently remains the preferred tool to screen for subclinical tubulitis. Further longitudinal studies should determine whether tubular proteinuria in stable transplants with normal tubular histology indicates a clear risk for early development of TA/IF.

Einsatz der Siliziumchiptechnologie zur Detektion von Tumormarkern auf Proteinbasis beim Harnblasenkarzinom

The protein structure of human tumor tissue has a significant influence on the molecular attributes. It was demonstrated that the individual prognosis of tumor patients is among other things dependent on molecular tumor tissue characteristics.A promising marker is E-cadherin, an adhesion glycoprotein which plays a central role in the mediation of cell-cell contacts. Aberrant E-cadherin expressions were described in several tumors such as in bladder cancer. This was also found to be correlated with tumor invasion and survival. There are hardly any fast, quantitative and easily automated protein assays in everyday practice which can analyze several markers at the same time. With silicon chip technology we have a new detection and measurement method which makes it possible to give a quantitative analysis of numerous different proteins in tissue, urine, or serum in a few minutes.

Pseudoparaproteinemia Detected by CE due to Omnipaque™ Infusion: A Case Report

Serum or plasma protein electrophoresis is often performed in clinical laboratories to detect and monitor M-components. During the last decade, capillary electrophoresis (CE) has emerged as an interesting alternative to traditional analysis of serum, plasma and urine proteins by agarose gel electrophoresis. We here report a case of pseudoparaproteinemia detected by capillary electrophoresis after Omnipaque™ (iohexol) infusion in a patient with normal kidney function. It is important for the laboratories to be aware of this source of erroneous results and not misclassify it as monoclonal gammopathies.

Diagnostic performance of combined specific urinary proteins and urinary flow cytometry in urinary tract pathology

Urinalysis comprises three techniques: urinary flow cytometry, test strip analysis and determination of specific urinary proteins. We investigated the diagnostic possibilities of combining these methods for a cohort of patients with a variety of well-documented urological and nephrological pathology. Urinary samples from 407 in- and out-patients with nephrological or urological pathology were retrospectively included in our study. Test strip analysis (URISYS 2400), urinary flow cytometry (UF-100) and urinary protein analysis [albumin, total protein, alpha1-microglobulin (A1M), alpha2-macroglobulin (A2M)] were performed. In discriminating upper and lower urinary tract infections, A1M and A1M/log(white blood cells) can be used, whereas pathological casts only give poor discrimination. The ratio A2M/log(red blood cells; RBC) allows differentiation between cystitis and pyelonephritis, while glomerular diseases can be recognised by the log(RBCxurinary total protein). Combining A2M and urinary albumin allows the determination of acute prostatitis. Using combined parameters provided by various urinalysis techniques, ratios can be proposed with diagnostic value for classifying renal and urological conditions. Possible integration in computer-based knowledge systems may offer valuable information for clinicians dealing with these pathologies.

Prediction of renal allograft rejection by urinary protein analysis using ProteinChip Arrays (surface-enhanced laser desorption/ionization time-of-flight mass spectrometry)

Objectives To develop a noninvasive method for the detection of renal transplant rejection using ProteinChip Arrays (surface-enhanced laser desorption/ionization time-of-flight mass spectrometry). Methods A total of 23 urine samples were collected from 13 patients showing biopsy-proven renal allograft rejection and from 10 patients without histologic signs of rejection. All 23 patients had clinical symptoms and signs of acute allograft rejection and underwent renal biopsy. Samples were centrifuged, and supernatants were directly spotted onto the ProteinChip arrays with different chromatographic surfaces. The obtained spectra in a range from 2 to 200 kDa were subjected to bioinformatic analysis using the method of Fuzzy c-means, followed by the establishment of rule bases and evaluation using the relevance index according to Kiendl. Results Several protein peaks were identified allowing differentiation between rejection and no rejection. Using two different ProteinChip surfaces, we found two biomarkers at 25.71 kDa and 28.13 kDa that gave a diagnostic sensitivity of 90% and 93% and a specificity of 80% (SAX2) and 85% (CM10), respectively. Conclusions Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry appears to be a promising new diagnostic tool for distinguishing renal transplant patients with no rejection from those with acute rejection.

Concanavalin A-captured Glycoproteins in Healthy Human Urine

Both the urinary proteome and its glycoproteome can reflect human health status, and more directly, functions of kidney and urinary tracts. Because the high abundance protein albumin is not N-glycosylated, the urine N-glycoprotein enrichment procedure could deplete it, and urine proteome could thus provide a more detailed protein profile in addition to glycosylation information especially when albuminuria occurs in some kidney diseases. In terms of describing the details of urinary proteins, the urine glycoproteome is even a better choice than the proteome itself. Pooled urine samples from healthy volunteers were collected and acetone-precipitated for proteins. N-Linked glycoproteins enriched with concanavalin A affinity purification were separated and analyzed by SDS-PAGE-reverse phase LC/MS/MS or two-dimensional LC/MS/MS. A total of 225 urinary proteins were identified based on two-hit criteria with reliability over 97% for each peptide. Among these proteins, 94 were identified in previous urine proteome works, 150 were annotated as glycoproteins in Swiss-Prot, and 43 were predicted as glycoproteins by NetNGlyc 1.0. A number of known biomarkers and disease-related glycoproteins were identified. Because changes in protein quantity or the glycosylation status can lead to changes in the concanavalin A-captured glycoprotein profile, specific urine glycoproteome patterns might be observed for specific pathological conditions as multiplex urinary biomarkers. Knowledge of the urine glycoproteome is important in understanding kidney and body function.

Proteomic Analysis of Protein Expression and Oxidative Modification in R6/2 Transgenic Mice

Huntington disease (HD) is a hereditary neurodegenerative disorder characterized by motor, psychiatric, and cognitive symptoms. The genetic defect responsible for the onset of the disease, expansion of CAG repeats in exon 1 of the gene that codes for huntingtin on chromosome 4, has been unambiguously identified. On the other hand, the mechanisms by which the mutation causes the disease are not completely understood yet. However, defects in energy metabolism of affected cells may cause oxidative damage, which has been proposed as one of the underlying molecular mechanisms that participate in the etiology of the disease. In our effort to investigate the extent of oxidative damage occurring at the protein level, we used a parallel proteomic approach to identify proteins potentially involved in processes upstream or downstream of the disease-causing huntingtin in a well established HD mouse model (R6/2 transgenic mice). We have demonstrated that the expression levels of dihydrolipoamide S-succinyltransferase and aspartate aminotransferase increase consistently over the course of disease (10-week-old mice). In contrast, pyruvate dehydrogenase expression levels were found to be decreased in 10-week-old HD transgenic mice compared with young (4-week-old) mice. Our experimental approach also led to the identification of oxidatively modified proteins. Six proteins were found to be significantly oxidized in old R6/2 transgenic mice compared with either young transgenic mice or non-transgenic mice. These proteins are alpha-enolase, gamma-enolase (neuron-specific enolase), aconitase, the voltage-dependent anion channel 1, heat shock protein 90, and creatine kinase. Because oxidative damage has proved to play an important role in the pathogenesis and the progression of Huntington disease, our results for the first time identify specific oxidatively modified proteins that potentially contribute to the pathogenesis of Huntington disease.

Proteome Analysis of Gelatin-Bound Urinary Proteins from Patients with Bladder Cancers

Objectives: Proteome analysis with protein separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and mass spectrometric measurement is utilized for comprehensive identification of proteins in various biological samples. However, this technique has not been widely applied for the analysis of body fluid for clinical assessments, because large amounts of proteins in fluid such as albumin and globulin hinder the detection of small amounts of proteins with high clinical values. The aim of the present study is to develop a method of proteomic analysis for urine of patients with bladder cancers. Materials and methods: Urine samples collected from 40 patients with bladder cancers, 32 healthy volunteers, and 7 old volunteers with benign prostate hypertrophy were treated with gelatin-beads as a group-specific affinity carrier. The gelatin-bound proteins were subjected to the proteome analysis. Results: Various amounts of matrix metalloproteinase-2 (MMP-2), -9 (MMP-9), fibronectin (FN), and their fragments were identified on 2-D PAGE gels from cancer-bearing patients, but not from normal individuals. Importantly, not only total amounts of these proteins, but also patterns of their distributions on the gels were well matched with the extent of invasion diagnosed with histopathological examinations. Thus, the score including such qualitative patterning of these proteins predicts pathological stages of the cancer. Conclusion: The proteomic analysis with gelatin-affinity purification of urine samples is useful not only for the diagnosis of bladder cancers, but also for estimating the extent of tumor invasion.

Complete Chemical and Enzymatic Treatment of Phosphorylated and Glycosylated Proteins on ProteinChip Arrays

A simple and quick protocol for chemical treatment, enzymatic digestion, and subsequent identification of proteins on ProteinChip arrays is presented. Chicken ovalbumin, bovine fetuin and a heavily posttranslationally modified protein, human epidermal growth factor receptor extracellular domain, were employed as model proteins to evaluate the novel protocol. The chemical treatment includes denaturation, reduction, and alkylation, while enzymatic digestion encompasses deglycosylation, dephosphorylation, and digestion by various proteases. All reactions were carried out on-chip in a sequential fashion. Peptide mass fingerprinting identified several peptides derived from the three proteins. This protocol was also applied to the analysis of urinary proteins from a male rat with puromycin-induced proteinuria. alpha-2u-Globulin, the major urinary protein in the normal male rat and albumin, the most abundant in the treated animal, were readily identified. This procedure demonstrates that complete on-chip treatment can be used for rapid protein identification and structural characterization.

Different techniques for urinary protein analysis of normal and lung cancer patients

Many components in urine are useful in clinical diagnosis and urinary proteins are known as important components to define many diseases such as proteinuria, kidney, bladder and urinary tract diseases. In this study, we focused on the comparison of different sample preparation methods for isolating urinary proteins prior to protein analysis of pooled healthy and lung cancer patient samples. Selective method was used for preliminary investigation of some putative urinary protein markers. Urine samples were passed first through a gel filtration column (PD-10 desalting column) to remove high salts and subsequently concentrated. Remaining interferences were removed by ultrafiltration or four precipitation methods. The analysis of urinary proteins by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed many similarities in profiles among preparation methods and a few profiles were different between normal and lung cancer patients. In contrast, the results of two-dimensional gel electrophoresis (2-DE) showed more distinctly different protein patterns. Our finding showed that the sequential preparation of urinary proteins by gel filtration and ultrafiltration could retain most urinary proteins which demonstrated the highest protein spots on 2-D gels and able to identify preliminary urinary protein markers related to cancer. Although sequential preparation of urine samples by gel filtration and protein precipitation resulted in low amounts of proteins on 2-D gels, high Mr proteins were easily detected. Therefore, there are alternative choices for urine sample preparation for studying the urinary proteome and identifying urinary protein markers important for further preclinical diagnostic and therapeutic applications.

Urinary Protein Analysis in Pre- and Postoperative Cancer Patients

Urinary proteins from six patients with esophageal cancer and two with stomach cancer were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Analyses were performed on days-1 to 3, 5, 7, 10, 14, and 21 (or 22) after surgery. The protein patterns were scanned by densitometry and divided into nine fractions. The main proteins in the fractions (Fr.) were identified as follows: immunoglobulin G in Fr. A, Tamm-Horsfall glycoprotein (THP) in Fr. B, transferrin in Fr. C, albumin in Fr. D, alpha(1)-acid glycoprotein in Fr. E, alpha(1)-microglobulin in Fr. F, retinol binding protein in Fr. G, and beta(2)-microglobulin in Fr. I. The protein in Fr. H was not identified. The percentage of each fraction was calculated from the densitometry pattern of each lane. The percentage values were averaged among all the patients, and pre- and postoperative data were compared. The percentage of Frs. E, F, and G increased on days 1-7, and the changes in these three proteins were similar to changes in serum C-reactive protein (CRP). In particular, the percentage of Fr. G peaked within 1 day of operation, which was faster than for CRP. Conversely, other fractions decreased. These results suggest that urinary protein analysis is useful for monitoring the response to surgical stress.

Anti-Vascular Endothelial Growth Factor Receptor-2 Antibody Accelerates Renal Disease in the NZB/W F1 Murine Systemic Lupus Erythematosus Model

Anti-Vascular Endothelial Growth Factor Receptor-2 Antibody Accelerates Renal Disease in the NZB/W F1 Murine Systemic Lupus Erythematosus Model

Size-selective extraction of peptides from urine for mass spectrometric analysis.

Protein excretion in urine has been suggested as an indicator of kidney disease since the time of Hippocrates. In the early 1800s, Bright further established approaches for studying proteinuria as a marker for kidney disease (1). As methods for quantitative and qualitative analysis have become more sophisticated, it has become possible to detect earlier stages of kidney disease and to differentiate different patterns of protein excretion (1)(2)(3). Quantitative immunoassays of selected urinary components such as α1-microglobulin, albumin, IgG, and α2-macroglobulin have been shown to be useful in characterizing the nature of proteinuria (4). Two-dimensional electrophoresis has provided a method for simultaneous analysis of numerous proteins in urine (5)(6). Recently, a new dimension has been added to analysis of urinary components by mass spectrometric techniques, which detect many small peptide components below the size resolution of electrophoresis (7)(8). The highly complex mixtures of small peptides in urine offer the potential for information-rich patterns for clinical diagnosis. Concentrations of urinary peptides serve not only as markers for kidney function but also as markers of other systemic physiologic processes. As examples, immunoassays for specific peptides provide measures of thrombosis and fibrinolysis (9)(10) and endocrine function (11). In the present study, we sought to identify a simple method to prepare urine specimens for the analysis of small peptide components. Sample preparation represents one of the major challenges for analysis of peptide components in urine specimens by mass spectrometry. Ideally, sample preparation needs to accomplish three tasks: (a) concentration of relatively dilute peptide components; (b) removal of salts that suppress peptide ionization in mass spectrometry; and (c) depletion of albumin and other high-molecular-weight components that comprise most of the total protein mass in urine. Standard methods that have been applied …

NHE3 Na+/H+ exchanger supports proximal tubular protein reabsorption in vivo.

Proximal tubular receptor-mediated endocytosis (RME) of filtered proteins prevents proteinuria. Pharmacological and genetic studies in cultured opossum kidney cells have shown that the apical Na(+)/H(+) exchanger isoform 3 (NHE3) supports RME by interference with endosomal pH homeostasis and endocytic fusion events. However, it is not known whether NHE3 also supports proximal tubular RME in vivo. We analyzed proximal tubular protein reabsorption by microinfusion experiments in rats and investigated renal protein excretion in NHE3 knockout (Nhe3 -/-) mice. Inhibition of NHE3 by EIPA or S-3226 reduced the fractional reabsorption of [(14)C]cytochrome c by approximately 50% during early proximal microinfusion. During early distal microinfusion, no protein reabsorption could be detected. Urinary protein excretion of Nhe3 -/- or heterozygous mutant mice was significantly higher compared with wild-type mice. SDS-PAGE analysis of urinary proteins revealed that Nhe3 -/- animals excreted proteins the size of albumin or smaller. Thus a reduction in NHE3 activity or abundance causes tubular proteinuria. These data show that NHE3 supports proximal tubular RME of filtered proteins in vivo.

1857: Prediction of Renal Allograft Rejection by Urinary Protein Analysis Using Seldi Mass Spectroscopy

1857: Prediction of Renal Allograft Rejection by Urinary Protein Analysis Using Seldi Mass Spectroscopy

7 Prediction of renal allograft rejection by urinary protein analysis using seldi mass spectroscopy

7 Prediction of renal allograft rejection by urinary protein analysis using seldi mass spectroscopy

Cardiac Amyloidosis: Heterogenous Pathogenic Backgrounds

Cardiac amyloidosis is a fatal disorder which develops on the basis of the different pathologic conditions in systemic amyloidosis: the most common underlying disease is immunoglobulin light chain-derived primary amyloidosis and the next is transthyretin-related hereditary amyloidosis; the latter disorder, typically represented by familial amyloid polyneuropathy, was long regarded as an endemic disease. However, this disorder has now been shown to involve a highly variable clinical picture due to a large number of transthyretin gene mutations, and many patients with diverse ancestors suffer from severe cardiac amyloidosis. Additionally, senile systemic amyloidosis is now noted as a cause of cardiac dysfunction in elderly individuals. Echocardiogram and myocardial technetium-99m-pyrophosphate scintigraphy can provide characteristic findings. Immunohistochemistry on tissue amyloid, biochemical analysis of serum and urine proteins, and DNA sequencing are usually employed to determine the disease-related amyloid fibril protein. Although systemic amyloidosis has become treatable, the prognosis of each patient who received up-to-date and effective, but nevertheless stressful, therapy depends on the severity of cardiac involvement by amyloid deposition.

Does Human Ochratoxin A Aggravate Proteinuria in Patients with Chronic Renal Disease?

Background. Ochratoxin A (OTA) is a nephrotoxic metabolite occurring in foodstuffs. In the last decade, OTA‐induced nephropathy in man and animals have been confirmed by previous literature. The correlation between OTA and the severity of CRI and nephrotic syndrome was also researched. Therefore, this study was designed to determine whether OTA also played an important role in renal insufficiency of patients with chronic renal diseases in Taiwan. Methods. The patients in this study were divided into nonnephrotic syndrome and nephrotic syndrome groups, first, to look for the relation between urine protein and OTA. And then these patients were also divided into six groups: (I) patients with chronic glomerulonephritis; (II) patients with chronic interstitial nephritis; (III) patients with diabetes mellitus; (IV) patients with hypertension; (V) patients with other diseases; (VI) patients with unknown reasons. For all groups, laboratory evaluation of kidney such as serum creatinine, urinary creatinine, creatinine clear rate, urinary protein, and urinary analysis were carried out coupled with determination of ochratoxin A level in urine. Results. Higher levels of OTA were found in patients with nephrotic syndrome. There was a significantly positive correlation (P < 0.001) between 24‐hr OTA and 24‐hr urine protein. On the other hand, the mean excretion of OTA in DM group (group III) was found significantly higher compared to the other groups (P < 0.05). Distinct differences (P < 0.01) were found especially when DM group was compared with patients with chronic glomerulonephritis (group I; P = 0.0019), patients with chronic interstitial nephritis (group II; P = 0.0032) and patients with hypertension (group IV; P = 0.0062). Conclusion. The results could lead to the conclusion that OTA could play an important role in proteinuria of patients with chronic renal diseases in Taiwan. And OTA may play a role in diabetes patients with nephropathy. Further longitudinal study is needed to clarify the role of OTA in diabetic nephropathy.