Biomarker discovery in mass spectrometry-based urinary proteomics.

Urinary proteomics has become one of the most attractive topics in disease biomarker discovery. MS-based proteomic analysis has advanced continuously and emerged as a prominent tool in the field of clinical bioanalysis. However, only few protein biomarkers have made their way to validation and clinical practice. Biomarker discovery is challenged by many clinical and analytical factors including, but not limited to, the complexity of urine and the wide dynamic range of endogenous proteins in the sample. This article highlights promising technologies and strategies in the MS-based biomarker discovery process, including study design, sample preparation, protein quantification, instrumental platforms, and bioinformatics. Different proteomics approaches are discussed, and progresses in maximizing urinary proteome coverage and standardization are emphasized in this review. MS-based urinary proteomics has great potential in the development of noninvasive diagnostic assays in the future, which will require collaborative efforts between analytical scientists, systems biologists, and clinicians.

[Recent Advances in Urinalysis as a Diagnostic Indicator of Renal Diseases].

Urinary sediments may be associated with the pathogenesis of renal diseases. It is important to examine the presence of dysmorphic red blood cells, macrophages, tubular epithelial cells, fibrin casts, glomerular epithelial cells, and ammonium acid urate crystals in urine for evaluating the clinical status of glomerulonephritis. Collecting evidence regarding the relationship between urinary sediments and renal disease activity may provide cost-effective and prompt clinical information to improve clinical practice.

[Soluble fibrin is not excreted in urine and its plasma level is elevated in nephrotic syndrome].

Soluble fibrin (SF) is produced by activated blood coagulation reaction and is useful to diagnose thrombotic diseases. We measured plasma and urine SF levels in nephritic patients to assess the hypercoagulability state associated with the disease. Before they received anti-coagulation or anti-platelet therapies, 60 patients underwent measurement of plasma SF and D-dimer levels by Latex agglutination turbidimetric immnoassay (LA). Urinary SF levels were also measured by LA. Plasma and urinary thrombin antithrombin III complex (TAT) levels were measured by enzyme immunoassay (EIA). Plasma SF levels showed a good correlation with plasma TAT levels but only weak positive correlations were observed between plasma D-dimer and SF or TAT levels. Plasma SF and D-dimer levels were significantly higher in the Iatients with nephrotic-range hypoalbuminemia (< or =3 g/dL) than those without it. Contrarily there was no significant difference in plasma TAT levels between these two groups of patients. In almost all patients, urinary SF levels were under the detection limit. However, TAT was excreted into urine more frequently in patients showing the nephrotic range of hypoalbuminemia at 38.2% than in non-nephrotic patients at 8.0%. Thus, plasma SF levels more precisely indicate activated blood coagulation reaction than plasma TAT levels in nephrotic patients, probably because the plasma SF is not excreted into urine, while plasma TAT is.

Urinary fibrin and fibrinogen degradation products and the origin of hematuria.

We examined urinary fibrin and fibrinogen degradation product (U-FDP) concentrations in pediatric patients with hematuria using the rapid and highly-sensitive latex particle agglutination test (LPAT), and assessed the value of this test for the localization of the site of hematuria. Patients with hematuria were divided into two groups: 60 with glomerular hematuria and 46 with non-glomerular hematuria. If U-FDP concentrations less than 0.25 microgram/ml are accepted as an indicator of glomerular bleeding, the sensitivity and specificity of localization of glomerular hematuria in the present study were 78% (47/60) and 89% (41/46), respectively. The high U-FDP concentrations observed in patients with non-glomerular hematuria may reflect direct bleeding into the urinary tract. Since all 13 patients with glomerular hematuria and U-FDP concentrations of 0.25 microgram/ml or more had coexistent erythrocyte cylindruria, the U-FDP test seems to be compensated with combined urinalysis for the relatively lower sensitivity. We conclude that a knowledge of U-FDP concentrations by LPAT can be of help in localizing the site of bleeding in hematuria.

Importance of immunoenzyme histochemical reaction in diagnosis of disseminated intravascular coagulation in human and animal material.

Renal tissues from 208 human necropsies were observed histologically for disseminated intravascular coagulation (DIC). The tissues were stained with hematoxylin-eosin, Mallory's phosphotungstic acid hematoxylin (PTAH) and cationic ferric hydroxide colloid stabilized with cacodylate (Fe-Cac), and tested by immunoenzyme histochemical (IEH) reaction for fibrin-related materials (FRMs). The use of the IEH method increased FRM recognition, and FRMs were detected in a total of 80 cases (38.5%). In 26 cases diagnosed clinically as DIC, FRMs were shown in 23 of the cases (88.5%). Thus, 57 patients with FRMs were clinically asymptomatic. In rats with DIC induced by endotoxin injection, glomerulus FRM was effluxed into the tubulus through the Bowman's capsule and was excreted into urine. The electric charge was reduced on the endothelial surface of the glomerular capillaries in both human and rat DIC. Under the scanning electron microscopy, the endothelial surface appeared coarse in the glomerular capillary and fibrin degradation was present. Our conclusions are: (a) PTAH is non-specific for FRMs, (b) IEH aids the pathohistological diagnosis of DIC, especially in asymptomatic forms including the compensated DIC state, (c) FRMs in tubuli suggest DIC, and (d) DIC is possibly initiated by a reduction in the capillary electric surface charge.

[Effects of tissue-type plasminogen activator from a culture of calf kidney cells on hemostasis and fibrinolysis in experimental nephritis]. / Vliianie aktivatora plazminogena tkanevo tipa iz kul'tury kletok pochki telenka na gemostaz i fibrinoliz pri éksperimental'nom nefrite.

The plasminogen activator 960 IU/mg protein activity isolated from cultured fluid of the calf kidney cells was introduced to albino rats (180-200 g) with experimental Heynmann nephritis every day during 4 days. Nephritis caused activation of haemostasis and inhibition of fibrinolysis in the blood. There was increased excretion of the fibrin, fibrinogen degradation products in urine as a results of the local fibrin deposition in diseased kidneys. The fibrinolytic activity of the cortical zone of kidney was markedly decreased. The plasminogen activator, infused to experimental animals, resulted in normalization of the altered indexes.

A diagnostic-prognostic test for bladder cancer using a monoclonal antibody-based enzyme-linked immunoassay for detection of urinary fibrin(ogen) degradation products.

An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody was developed to determine the clinical value of urinary fibrinogen/fibrin degradation product levels for the identification and management of patients with bladder cancer. Assays were performed on 286 serial urine specimens from 56 bladder carcinoma patients. Specimens were grouped according to whether the patient had an evident tumor at the time of specimen collection (134 specimens, 41 patients) or was clinically disease-free following treatment (152 specimens, 38 patients). Many patients contributed specimens to both groups as determined by their clinical status at the time of collection. In addition, 45 specimens from 33 patients with inflammation of the urogenital tract and 81 specimens from 19 patients with renal or prostatic cancer were assayed for urinary fibrin degradation products. The ELISA, using a high-sensitivity procedure, identified 83% of the specimens from bladder cancer-positive patients with an overall accuracy with all specimens of 78% and a false-negative rate of 5% for all specimens tested. The high-sensitivity ELISA appeared most appropriate for monitoring bladder cancer patients for recurrence of tumor after surgery. The ELISA using a high-specificity procedure appeared most appropriate for screening. The high-specificity ELISA accurately identified 96% of urine specimens from non-bladder cancer patients with a false-positive rate of only 5%. These results demonstrate that the ELISA is an efficient, reliable, quantitative, and noninvasive immunoassay that can be useful both for the identification of bladder cancer patients and for monitoring the course of the disease.

Protein composition of urinary casts from healthy subjects and patients with glomerulonephritis.

Urinary casts from 46 healthy volunteers and 60 patients with glomerulonephritis were examined for the presence of Tamm-Horsfall glycoprotein and other proteins. All samples gave immunofluorescence evidence of Tamm-Horsfall protein in casts. Casts from 59 of the patients but only three of the controls contained other proteins in addition (p less than 0.001). Immunoglobulins (IgG, IgM, IgA) were detected in casts from 53 of the patients but none of the healthy volunteers. Examination of urinary casts for immunoglobulins, complement, and fibrin provides a non-invasive method for distinguishing patients with active glomerular disease.

Comparison of serum and urine fibrin split products and urinary beta-glucuronidase in the diagnosis of renal transplant rejection.

This study compares the usefulness of serum and urine fibrin split products and the urinary enzyme, beta-glucuronidase, in the diagnosis and management of renal transplant rejection. Fibrin split products, determined by a tanned human red cell agglutination inhibition immunoassay, were measured as a reflection of the secondary fibrinolysis from fibrin deposited in the renal microvasculature as a result of rejection. Urinary beta-glucuronidase, expressed as the ratio of enzyme activity to creatinine concentration, was determined by a colorimetric technique following dialysis of urine to remove endogenous activators and inhibitors. Activity of this lysosomal enzyme is thought to reflect tubular injury. Twenty-nine renal transplant recipients (15 from living donors and 14 from cadaver donors) were evaluated. Both serum and urinary fibrin split products and urinary beta-glucuronidase were markedly elevated in the immediate postoperative period, probably reflecting ischemic trauma. Acute rejection occurring within the first three months was associated with elevations of fibrin split products (particularly urine) and beta-glucuronidase. Elevated values returned to normal following successful treatment with steroids and/or heparin, but remained high in the presence of continued rejection. After the first 48 hours post-transplant, in the absence of rejection, values for fibrin split products were within the normal range. Urinary beta-glucuronidase remained elevated if the transplanted kidney was recovering from acute tubular necrosis. Fibrin split products and urinary beta-glucuronidase were usually normal in chronic rejection.

Study of the correlation between glomerular and urinary sediment deposits using immunofluorescent technique.

Correlations between immunofluorescent protein deposits in the glomeruli, urinary sediment and proteinuria selectivity pattern have been attempted in different glomerulonephritis. The overall study showed following results: a) Glomerular and urinary casts deposits of immunoglobulins, C3 and fibrin/fibrinogen are related to moderately selective proteinuria; b) IgG and IgA are most often found in urinary sediment and c) Glomerular deposits of IgG and IgA are well correlated with the presence of these immunoglobulins in urinary casts. Analysis according to different histological types of nephritis: a) In "minimal changes" nephropathy, deposits are infrequent and well correlated with urinary sediment, when they are present; b) In lupus nephritis, constant and intense glomerular deposits of immunoglobulins, fibrin/fibrinogen are correlated with the same proteins found in urinary casts; c) Inconstant correlations are found in membranous nephritis and in IgA-IgG nephropathy.

Failure of heparin therapy to affect the clinical course of severe pre-eclampsia.

In view of the association between pre-eclampsia and disseminated intravascular coagulation, three patients presenting with severe pre-eclampsia before the 28th week of pregnancy were treated with heparin. In all three patients, there was deterioration of hypertension and proteinuria that necessitated the withdrawal of treatment after five to six days. During treatment, serum and urinary fibrinolytic degradation products (FDPs) continued to rise or remained unaltered, plasminogen levels showed a steady fall, and the platelet count remained at a reduced level. These data suggest that heparin was an ineffective form of treatment and did not prevent the intravascular fibrin deposition associated with severe pre-eclampsia.

[Diagnostic significance of the demonstration of fibrin and fibrinogen degradation products in the urine]. / Diagnostische Bedeutung des Nachweises von Fibrin-/Fibrinogen-Spaltprodukten im Urin

The concentration of fibrin/fibrinogen degradation products in urine (FDPu) was measured in samples obtained from 114 patients and 63 clinically healthy volunteers, once or repeatedly. The FDP titers measured by the hemagglutination inhibition (HI) method corresponded to less than 2.6 mug/ml FDP in all samples from healthy controls. Slightly elevated FDP concentrations were found in urine obtained from a few patients with disorders not primarily involving the urinary tract. The clinical importance of these isolated findings remains unclear. Urinary tract infections were not frequently accompanied by elevated FDPu concentrations. In patients with glomerulonephritis FDP excretion correlated somewhat better with severity of the renal affection. A further group of patients showed an unequivocal correlation between FDP excretion in the urine and postoperative complications following renal transplantation. However, the clinical diagnosis of acute rejection crisis was usually established at the same time or even before an increase in FDPu was found. Our results suggest that among diagnostic procedures the measurement of FDPu contributes little specific information for the evaluation of urinary tract disease. FDPu measurements in the immediate postoperative phase following renal transplantation may however be important for prognostic evaluation and, in individual cases, predict transplant rejection. We also attempted to define the FDPu qualitatively by simultaneous measurements using HI and the staphylococcal clumping test (SCT). Immunoelectrophoresis confirmed the well-known fact that the SCT detects only high-molecular FDP; this limits its clinical usefulness, despite its high sensitivity.

Origin of urinary fibrin/fibrinogen degradation products in glomerulonephritis.

To elucidate the origin of the fibrin/fibrinogen degradation products (F.D.P.) occurring in the urine in glomerulonephritis 28 patients with glomerulonephritis were examined for renal fibrinolytic activity, F.D.P. in urine and serum, and blood fibrinolytic activators and blood fibrinolytic activators and inhibitors. Unlike the glomerful of healthy kidneys, which were fibrinolyticly inactive, those of kidneys with glomerulonephritis constantly showed fibrinolytic activity. The presence or absence of fibrin in the glomeruli was almost always accompanied by, respectively, the presence or absence of urinary F.D.P., which suggested a renal origin of urinary F.D.P. in glomerulonephritis. The low fibrinolytic activity of the blood and the absence of F.D.P. in the serum of these patients make it unlikely that the urinary F.D.P. in glomerulonephritis result from glomerular filtration.

Urinary fibrin-fibrinogen degradation products in nephrotic syndrome.

The urinary concentration of fibrin-fibrinogen degradation products (F.D.P.) was measured in 90 patients with proteinuria above 2 g/1 and correlated with proteinuria, differential protein clearances, serum urea and creatinine, and renal biopsy findings. There was a linear correlation (r equals 0-7; P less than 0-001) between the urinary F.D.P. excretion and the selectivity of the proteinuria such that patients with highly selective proteinuria excreted only small amounts of F.D.P. whereas those with non-selective proteinuria excreted much higher levels. There was a significant correlation between the urinary F.D.P. excretion and the urine:serum (U:S) ratio of IgG excretion but not with the U:S ratio or urinary excretion of albumin or transferrin. Sephadex G200 column chromatography of the concentrated urine in 26 cases showed that patients with highly selective proteinuria excreted predominantly F.D.P. of low molecular weight in the urine whereas those with non-selective proteinuria excreted mainly fibrinogen and products of high molecular weight. Hence the type and quantity of F.D.P. in the urine are determined primarily by the differential filtration of fibrinogen and the various degradation products from the plasma through the glomerular basement membrane, which in turn is determined by the "pore size" of the basement membrane. In clinical nephrology measurement of the urinary F.D.P. level provides a rapid and convenient means of estimating the differential protein clearance.