Kidney fibrosis is independent of the amount of ascorbic acid in mice with unilateral ureteral obstruction.

In response to sustained damage to a kidney, fibrosis that can be characterized as the deposition of a collagenous matrix occurs and consequently causes chronic kidney failure. Because most animals used in experiments synthesize ascorbic acid (AsA) from glucose, the roles of AsA in fibrotic kidney diseases are largely unknown. Unilateral ureteric obstruction (UUO) mimics the complex pathophysiology of chronic obstructive nephropathy and is an ideal model for the investigation of the roles of AsA in kidney failure. We examined the impact of a deficiency of Akr1a, a gene that encodes aldehyde reductase and is responsible for the production of AsA, on fibrotic damage caused by UUO in mice. Oxidatively modified DNA was elevated in wild-type and Akr1a-deficient kidneys as a result of UUO to a similar extent, and was only slightly suppressed by the administration of AsA. Even though Akrla-deficient mice could produce only about 10% of the AsA produced by wild-type mice, no difference was observed in collagen I synthesis under pathological conditions. The data implied either a low demand for AsA or the presence of another electron donor for collagen I production in the mouse kidney. Next, we attempted to elucidate the potential causes for oxidative damage in kidney cells during the fibrotic change. We found decreases in mitochondrial proteins, particularly in electron transport complexes, at the initial stage of the kidney fibrosis. The data imply that a dysfunction of the mitochondria leads to an elevation of ROS, which results in kidney fibrosis by stimulating cellular transformation to myofibroblasts.

Feasibility and assessment of non-invasive in vivo redox status using electron paramagnetic resonance imaging.

PURPOSE: To test the feasibility of electron paramagnetic resonance imaging (EPRI) to provide non-invasive images of tissue redox status using redox-sensitive paramagnetic contrast agents. MATERIAL AND METHODS: Nitroxide free radicals were used as paramagnetic agents and a custom-built 300 MHz EPR spectrometer/imager was used for all studies. A phantom was constructed consisting of four tubes containing equal concentrations of a nitroxide. Varying concentrations of hypoxanthine/xanthine oxidase were added to each tube and reduction of the nitroxide was monitored by EPR as a function of time. Tumor-bearing mice were intravenously infused with a nitroxide and the corresponding reduction rate was monitored on a pixel-by-pixel basis using 2D EPR of the tumor-bearing leg and normal leg serving as control. For animal studies, nitroxides were injected intravenously (1.25 mmol/kg) and EPR projections were collected every 3 min after injection using a magnetic field gradient of 2.5 G/cm. The reduction rates of signal intensity on a pixel-by-pixel basis were calculated and plotted as a redox map. Redox maps were also collected from the mice treated with diethylmaleate (DEM), which depletes tissue thiols and alters the global redox status. RESULTS: Redox maps obtained from the phantoms were in agreement with the intensity change in each of the tubes where the signals were decreasing as a function of the enzymatic activity, validating the ability of EPRI to accurately access changes in nitroxide reduction. Redox imaging capability of EPR was next evaluated in vivo. EPR images of the nitroxide distribution and reduction rates in tumor-bearing leg of mice exhibited more heterogeneity than in the normal tissue. Reduction rates were found to be significantly decreased in tumors of mice treated with DEM, consistent with the depletion of thiols and the consequent alteration of the redox status. CONCLUSION: Using redox-sensitive paramagnetic contrast agents, EPRI can non-invasively discriminate redox status differences between normal tissue and tumors.

Development of dengue IgM-capture enzyme-linked immunosorbent assay with higher sensitivity using monoclonal detection antibody.

An IgM-capture enzyme-linked immunosorbent assay (IgM-ELISA) is used widely for serodiagnosis of dengue. A dengue IgM-ELISA with higher sensitivity has been developed. In the new ELISA, anti-dengue IgM antibody, which had been captured on the solid phase, was reacted with tetravalent dengue viral antigens, and detected by a flavivirus group specific monoclonal antibody, D1-4G2-4-15 (4G2). Reaction of 4G2 to viral antigens was similar to that of dengue patients' IgG. Non-specific reaction of 4G2 to the control antigen, which was prepared from uninfected cell culture fluid of mosquito C6/36 cells, was much lower than that of patients' IgG. Thus, specificity of the ELISA with 4G2 was much higher than that with patients' IgG, and lower levels of specific IgM was detected in the serum samples. These results suggest that the modified dengue IgM-ELISA with monoclonal antibody 4G2 has many advantages over the original "in-house" ELISA.

Serotype-cross-reactive immunoglobulin M responses in dengue virus infections determined by enzyme-linked immunosorbent assay.

We developed immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assays (ELISAs) with four monovalent dengue virus antigens. We attempted to determine whether IgM responses in dengue virus infections are serotype specific or serotype cross-reactive. Serum samples from 14 confirmed dengue cases were examined. In these 14 cases, which consisted of 12 Japanese and 2 non-Japanese patients, infecting dengue virus serotypes were defined by reverse transcription-PCR. Thirteen of the 14 cases were IgM positive in ELISA. IgM responses were serotype cross-reactive in these 13 cases but were highest against infecting dengue virus serotype in 9 of the 13 cases. These results indicate that IgM responses are generally dengue serotype cross-reactive but that IgM levels are highest against the infecting serotype in most dengue cases.