Cellulose Acetate Membrane Electrophoresis Based Urinary Proteomics for the Identification of Characteristic Proteins.

BACKGROUND: Analysis of urinary proteins using cellulose acetate membrane electrophoresis (CAME) is a useful and challenging method for the recognition of damaged sites in the kidney. However, protein content of each CAME fraction is still not completely understood. METHODS: In this study, an effective method of protein extraction from each band fractionated by CAME was established, which enabled us to examine the extracted proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. RESULTS: Proteins were extracted from the gel and analyzed by mass spectrometry. In all, 31 proteins were identified, including 20 urinary proteins that were newly identified in the CAME-based analysis. CONCLUSION: This methodology was useful for identifying the proteins responsible for creating unique bands on CAME in a urine sample of a patient with drug-induced interstitial nephritis. These findings provide in-depth characterization of urinary protein contents in each CAME fraction.

[Proteomic analysis of urinary exosomes].

Exosomes are 40-100-nm membrane vesicles secreted into the extracellular space by various types of cell in many biological fluids, including serum, saliva, breast milk, amniotic fluid, and urine. Exosomes, which contain several key proteins, lipids, mRNAs, and microRNAs, were considered as an alternative secretion pathway. In addition, recent findings suggest that the exosome itself is a functional biomolecule involved in intracellular communication; thus, its components can be transferred to recipient cells by fusion, changing the function of the target cell. Recently, urinary exosomes have attracted much attention because some of their proteins have been identified as biomarkers related to certain physiological events and disease-related metabolism of the kidney. This review provides an overview of urinary exosomes, including methods of isolation and associated problems, and focuses on urinary exosomes as protein biomarker sources involved in numerous physiological and pathophysiological processes.

[Comprehensive analysis of urinary proteins for identification of renal disease markers].

Urine can be obtained noninvasively and relatively easily to provide a large sample and it is an important source of biomarker discovery in kidney diseases. Today, urinary albumin screening is not only useful for detecting early kidney diseases, but also recognized as a risk factor for cardiovascular diseases. We have shown different kinds of post-translational modification of urinary albumin in patients with glomerulonephritis, such as high- and low-molecular mass forms of albumin and highly carbonylated albumin. These characteristic molecular forms of albumin are not detected in serum samples obtained from the same patients or urine of healthy subjects, reflecting the kidney diseases processes. Furthermore, measuring the levels of albumin and other urinary proteins enables comprehensive kidney examination. The urinary protein profile using cellulose acetate membrane electrophoresis coupled with colloidal silver staining allows us to construct urinary protein profiles. Basically, urinary protein patterns in patients with glomerulonephritis show 5 fractions, the same as the serum pattern; however, characteristic bands of cathode ß and anode ß fractions, reflecting ß2-microglobulin and retinol binding protein, appear in patients with tubular dysfunction. This method is useful to diagnose damaged portions of the kidney, and it provides supportive data for renal biopsy. Most recently, exosome-derived proteins are considered as a new marker source which reflects in-situ alterations in the urology and renal diseases. Different forms of electrophoresis reveal slight alterations in the protein profile on the analysis of urinary proteins including exosomes. This report introduces the electrophoresis-based urinary proteome results generated by our research group.

Redox state of urinary albumin in patients with IgA nephropathy.

We examined the relationship between oxidative damage and molecular instability of urinary albumin in the urine of patients with IgA nephropathy (IgAN). As measure of oxidation, we detected the free thiol group at Cys34 in albumin (albumin-Cys34) using maleimide-PEG2-biotin reagent; because decreased levels of albumin-Cys34 are correlated with increased oxidation. The urine albumin-Cys34 level in all 30 patients with IgAN was decreased to varying extents. No correlations were found between urinary albumin/creatinine ratio and decreased urinary albumin-Cys34 level. Furthermore, decreases in urinary albumin-Cys34 were not accompanied by changes in serum albumin-Cys34. In diagonal-two-dimensional SDS PAGE analysis which reveals reduction-induced degradation of H2O2-treated human serum albumin, a similar pattern of albumin degradation was also detected in urine samples from patients with IgAN, indicating that structural alterations resulting from oxidative stress may be involved. Our findings suggest that redox state, in addition to urinary albumin concentration, may be an important indicator of post-translational modification and a potentially useful predictor of the kidney disease.

Benzene risk estimation using radiation equivalent coefficients.

We estimated benzene risk using a novel framework of risk assessment that employed the measurement of radiation dose equivalents to benzene metabolites and a PBPK model. The highest risks for 1 microg/m(3) and 3.2 mg/m(3) life time exposure of benzene estimated with a linear regression were 5.4 x 10(-7) and 1.3 x 10(-3), respectively. Even though these estimates were based on in vitro chromosome aberration test data, they were about one-sixth to one-fourteenth that from other studies and represent a fairly good estimate by using radiation equivalent coefficient as an "internal standard."

[Analysis of proteins in urinary tract stones and urine of urolithic patients].

In order to investigate the mechanism of urinary tract stone formation, we analyzed protein components in urine and the stone. Urinary proteins of healthy subjects and urolithic patients as well as protein components urinary tract stone of the urolithic patients were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoretic patterns of urinary proteins of the patients differed from those of healthy subjects after separating protein patterns into those larger than 66kDa or smaller than 30kDa. Protein constituents of urinary tract stone were mainly separated into 18 bands ranging from 26.8 to 143 kDa. Major bands among these 18 bands differed among stones from different patients. On western blotting, the developed intensities of Tamm-Horsfall protein (THP) were fainter than those of healthy subjects. Whereas intensities of albumin (ALB) were stronger than those of healthy subjects. Moreover, blotting patterns of THP of the patients on non-reducing SDS-PAGE were obviously broad. Thus, we suggest that analysis of fractionated urinary proteins or protein components of urinary tract stone may provide a tool for monitoring the prognosis or relapse in the patients.

Molecular heterogeneity of urinary albumin in glomerulonephritis: comparison of cardiovascular disease with albuminuria.

BACKGROUND: Despite the unstable structure of urinary albumin in kidney diseases, urinary albumin fragments have been identified by denaturing methods such as two-dimensional electrophoresis. This study examined the relationship between the structural heterogeneity of urinary albumin and protease effects. METHODS: Urine samples from patients with glomerulonephritis (GN), cardiovascular diseases (CVD), and healthy subjects were analyzed by non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), Western blot, diagonal 2-dimensional non-reducing/reducing (d2D) SDS PAGE, and albumin zymography. RESULTS: The major band was monomer albumin in CVD and healthy subjects; however, 13 urinary albumin bands ranging from 55 to 172 kDa were identified by non-reducing SDS PAGE in GN. The results from d2D SDS PAGE showed urinary albumin polymerization between disulfide bridges, interactions with other proteins, and reduction induced degradation in GN patients. The results from albumin zymography showed that low-molecular mass forms of albumin did not necessarily correspond to high protease activity. Furthermore, concentrated healthy urine showed similar protease digestion as in GN without low-molecular mass of albumin. CONCLUSIONS: The molecular alterations observed cannot be explained only by urinary proteases. The specific alteration of urinary albumin molecules in GN can be attributed to different mechanisms to CVD.

Development of a physiologically based pharmacokinetic model for bisphenol A in pregnant mice.

Bisphenol A (BPA) is a weakly estrogenic monomer used to produce polymers for food contact and other applications, so there is potential for oral exposure of humans to trace amounts via ingestion. To date, no physiologically based pharmacokinetic (PBPK) model has been located for BPA in pregnant mice with or without fetuses. An estimate by a mathematical model is essential since information on humans is difficult to obtain experimentally. The PBPK model was constructed based on the pharmacokinetic data of our experiment following single oral administration of BPA to pregnant mice. The risk assessment of bisphenol A (BPA) on the development of human offspring is an important issue. There have been limited data on the exposure level of human fetuses to BPA (e.g. BPA concentration in cord blood) and no information is available on the pharmacokinetics of BPA in humans with or without fetuses. In the present study, we developed a physiologically based pharmacokinetic (PBPK) model describing the pharmacokinetics of BPA in a pregnant mouse with the prospect of future extrapolation to humans. The PBPK model was constructed based on the pharmacokinetic data of an experiment we executed on pregnant mice following single oral administration of BPA. The model could describe the rapid transfer of BPA through the placenta to the fetus and the slow disappearance from fetuses. The simulated time courses after three-time repeated oral administrations of BPA by the constructed model fitted well with the experimental data, and the simulation for the 10 times lower dose was also consistent with the experiment. This suggested that the PBPK model for BPA in pregnant mice was successfully verified and is highly promising for extrapolation to humans who are expected to be exposed more chronically to lower doses.

A physiologically based pharmacokinetic model for endosulfan in the male Sprague-Dawley rats.

Endosulfan, an organochlorine (OC) insecticide belonging to the cyclodiene group, is one of the most commonly used pesticides to control pests in vegetables, cotton, and fruits. To date, no physiologically based pharmacokinetic (PBPK) model has been located for endosulfan in animal species and humans. The estimation by a mathematical model is essential since information on humans can scarcely be obtained experimentally. The PBPK model was constructed based on the pharmacokinetic data of our experiment following single oral administration of (14)C-Endosulfan to male Sprague-Dawley rats. The model was parameterized by using reference physiological parameter values and partition coefficients that were determined in the experiment and optimized by manual adjustment until the best visual fit of the simulations with the experimental data were observed. The model was verified by simulating the disposition of (14)C-Endosulfan in vivo after single and multiple oral dosages and comparing simulated results with experimental results. The model was further verified by using experimental data retrieved from the literature. The present model could reasonably predict target tissue dosimetries in rats. Simulation with three-time repeated administration of (14)C-Endosulfan and experimental data retrieved from the literature by the constructed model fitted fairly well with the experimental results; thus suggesting that the newly developed PBPK model was developed. Sensitivity analyses were used to determine those input parameters with the greatest influence on endosulfan tissue concentrations.

Development of an in vitro blood-brain barrier model to study the effects of endosulfan on the permeability of tight junctions and a comparative study of the cytotoxic effects of endosulfan on rat and human glial and neuronal cell cultures.

Endosulfan, an organochlorine (OC) insecticide that belongs to the cyclodiene group, is one of the most commonly used pesticides to control pests in vegetables, cotton, and fruits. Porcine brain microvascular endothelial cells were used to develop a model to study the effects of endosulfan on the permeability of tight junctions in the blood-brain barrier (BBB). BBB permeability, measured as transendothelial electrical resistance, decreased in a dose- and time-dependent manner when treated with alpha-endosulfan, beta-endosulfan, or endosulfan sulfate. Cytotoxicity testing revealed that the three endosulfans did not cause cell death at concentrations of 10 microM and below. The ratio of the average permeability of the filter-grown endothelial cell monolayer to 14C-endosulfan (Pe) going from the outer to the inner compartments with that going from the inner to the outer compartments was approximately 1:1.2-2.1 after exposure to concentrations of 0.01-10 microM. alpha-Endosulfan, beta-endosulfan, and endosulfan sulfate had cytotoxic effects on rat glial (C6) and neuronal (PC12) cell cultures as well as on human glial (CCF-STTG1) and neuronal (NT2) cell cultures. The effects of alpha-endosulfan were highly selective, with a wide range of LC50 values found in the different cultures, ranging from 11.2 microM for CCF-STTG1 cells to 48.0 microM for PC12 cells. In contrast, selective neurotoxicity was not so manifest in glial and neuronal cell cultures after exposure to endosulfan sulfate, as LC50 values were in the range of 10.4-21.6 microM. CCF-STTG1 cells were more sensitive to alpha-endosulfan and endosulfan sulfate, whereas NT2 cells were more sensitive to beta-endosulfan.

Presence of immunounreactive albumin in the urine of diabetic patients.

Recent studies have demonstrated that conventional immunochemical assays underestimate urinary albumin concentration because of the presence of immunounreactive albumin. It has been reported that intact urinary albumin in 24-hr diabetic urine samples could be detected as total concentration (immunoreactive+immunounreactive) by an HPLC method based on size exclusion chromatography. The aim of this study was to investigate urinary albumin concentration in diabetic spot urine samples by comparing the HPLC method with several other methods. The albumin concentrations on 80 diabetic spot urine specimens were measured by turbidimetric immunoassay (TIA), high performance liquid chromatography (HPLC), and a dipstick method. In addition, they were also analyzed by reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and native polyacrylamide gel electrophoresis (Native PAGE). The albumin concentrations derived from diabetic spot urine samples measured by the HPLC method were higher than those of the other methods except for five of 80 samples. Furthermore, the albumin concentrations analyzed by Native PAGE were higher than SDS PAGE in 61 (76.2%) of 80 samples. This study suggests the need for evaluating diabetes not only by HPLC, but also by combining it with another method.

Toxicokinetics of 14C-endosulfan in male Sprague-Dawley rats following oral administration of single or repeated doses.

Endosulfan (ES), an organochlorine (OC) insecticide that belongs to the cyclodiene group, is one of the most commonly used pesticides to control pests in vegetables, cotton, and fruits. The toxicokinetics of 14C-endosulfan following oral administration of a single dose of 5 mg/kg body weight was investigated in male Sprague-Dawley rats. Three rats were sacrificed 30 min, 1 h, 2 h, 4 h, and 8 h after dosing. 14C-endosulfan radioactivity was detected in all tissues at each time point. In a separate experiment urine and feces were collected for 96 h. The total radioactivity recovered in the excreta for 4 days was 106.8% +/- 26.2%, with fecal elimination the major route of elimination route (94.4% +/- 21.4%). The cumulative excretion in the urine for 4 days was 12.4% +/- 4.8%. Radioactivity 8 h after administration was highest in gastrointestinal (GI) tract tissue (20.28 +/- 16.35 mg ES eq./L) and lowest in muscle (0.18 +/- 0.06 mg ES eq./L). The toxicokinetic parameters obtained from 14C-endosulfan-derived radioactivity in blood were distribution half-life (T1/2 x) = 31 min and terminal elimination half-life (T1/2 y) = 193 h. Blood concentration reached its maximum (Cmax) of 0.36 +/- 0.08 mg ES eq./L 2 h after the oral dose. Endosulfan was rapidly absorbed into the GI tract in rats, with an absorption rate constant (ka) of 3.07 h(-1).

Comparison of mutagenic potentials and mutation spectra of benzene metabolites using supF shuttle vectors in human cells.

Benzene is a human leukemogen and the metabolites are thought to be deeply involved in benzene leukemogenesis. In a previous study we reported the molecular analysis of p-benzoquinone (p-BQ) mutagenesis by using a supF shuttle vector plasmid and here we report the mutagenesis of the other metabolites, hydroquinone (HQ) and trans, trans-muconaldehyde (MUC). HQ is a precursor of p-BQ and MUC is produced by a ring-opening metabolic pathway. We found that the HQ redox cycle produced an oxidative lesion in plasmid DNA and significant differences among the mutagenic potentials of MUC, HQ and p-BQ. HQ has stronger mutagenicity than the others. It is about 20 and 600 times stronger than p-BQ and MUC, respectively. Furthermore, we found notable differences in each mutational feature. The MUC mutational type was characterized by a high frequency of tandem base substitutions that could be due to crosslinks produced by its aldehyde moieties, while HQ was characterized by frequent deletion. This HQ feature is the same as in vivo benezene mutagenesis of Big Blue mice reported by Provost et al. in 1996 and is also quite similar to a hydrogen peroxide mutational feature. Therefore, we presume that HQ and reactive oxygen species may play an important role in benzene carcinogenesis.