Podocyte protection by darbepoetin: preservation of the cytoskeleton and nephrin expression.

Podocyte injury is a significant contributor to proteinuria and glomerulosclerosis. Recent studies have shown a renoprotective effect of erythropoietin (EPO) during ischemic kidney disease. In this study, we examine mechanisms by which a long acting recombinant EPO analog, darbepoetin, may confer renoprotection in the puromycin aminonucleoside-induced model of nephrotic syndrome. Darbepoetin decreased the proteinuria of rats treated with puromycin. This protective effect was correlated with the immunohistochemical disappearance of the podocyte injury markers desmin and the immune costimulator molecule B7.1 with the reappearance of nephrin expression in the slit diaphragm. Podocyte foot process retraction and effacement along with actin filament rearrangement, determined by electron microscopy, were all reversed by darbepoetin treatment. The protective effects were confirmed in puromycin-induced nephrotic rats that had been hemodiluted to normal hematocrit levels. Furthermore, puromycin treatment of rat podocytes in culture caused actin cytoskeletal reorganization along with deranged nephrin distribution. All these effects in vitro were reversed by darbepoetin. Our study demonstrates that darbepoetin treatment ameliorates podocyte injury and decreases proteinuria by a direct effect on podocytes. This may be accomplished by maintenance of the actin cytoskeleton and nephrin expression.

Renal mineral handling in normal rats treated with sevelamer hydrochloride (Renagel), a noncalcemic phosphate binder.

The effects of sevelamer hydrochloride (Renagel); hereafter referred to as sevelamer), a noncalcemic phosphate binder, on renal mineral handling were examined in rats. Normal rats were fed a diet containing 0.3, 1, 3, and 5% sevelamer for 8 days, and serum, urine, and the immunohistochemical localization of the type II Na/Pi cotransporter protein in the kidney were analyzed. Rats treated with 3 or 5% sevelamer showed significant decreases in serum phosphorus (P) and parathyroid hormone (PTH) levels, with no changes in serum calcium (Ca), magnesium (Mg), or 1,25(OH)2D3 levels. Increases were observed in urinary excretions of Ca and Mg associated with a reduction in the PTH level in rats treated with 3 or 5% sevelamer. Rats treated with 1% or higher concentrations of sevelamer showed significant dose-dependent and marked reductions of the urinary P excretion, and the tubular reabsorption of P was maximized to almost 100% in the 5% sevelamer group. The hypophosphaturia in rats treated with 3 or 5% sevelamer was accounted for by the reductions in serum PTH and P per se, and immunohistochemical analysis showed that the expression of type II Na/Pi cotransporter protein was markedly increased at the brush border membranes of the deep and superficial nephrons in rats treated with 5% sevelamer as compared with rats given a normal diet. In conclusion, sevelamer rapidly lowered serum P and PTH levels in normal rats. Sevelamer treatment also produced a marked hypophosphaturia associated with translocation of type II Na/Pi cotransporter protein and increased urinary Ca and Mg excretions by the reduction of PTH.

Antigen binding of an ovomucoid-specific antibody is affected by a carbohydrate chain located on the light chain variable region.

We cloned the variable regions of heavy and light chain genes of an anti-ovomucoid monoclonal antibody (MAb-OM21) produced by the mouse hybridoma cell line OM21. DNA sequence analysis showed that the light chain of the MAb-OM21 has only one potential N-glycosylation consensus sequence in the complementarity determining region 2 of the light chain. To find whether carbohydrate chains are located on the light chain, we assayed for the size of the light chain, after treatment with N-glycosidase, by western blotting, and also detection of the carbohydrate chains on the light chain was done using the lectin blot assay. A N-linked carbohydrate chain has been shown to bind to the light chain. To clarify the role of this carbohydrate chain in the light chain, we produced carbohydrate variant antibodies by N-deglycosylation using glycosidase or by expressing the antibody from different host cells. The N-deglycosylated variant antibody has greater antigen binding, and the antibody produced from the different host cells showed a reduced antigen binding activity and acquired the ability to react to ovalbumin. These results suggest that antigen binding of the ovomucoid specific antibody MAb-OM21 can be affected by the carbohydrate chain on the light chain variable region.

Morphologic and functional characterization of perforin-deficient lymphokine-activated killer cells.

Mice deficient in perforin, a key mediator of lymphocyte-mediated cytolysis, have recently been generated using the gene knockout technique. CTL and NK cells derived from these mice have been shown to be defective in the granule-dependent cytolytic pathway. To investigate whether the granule-formation process has been altered in these perforin-deficient cytotoxic cells, rendering them defective in using the other granule mediators, we have examined in the present study the morphologic and functional characteristics of perforin-deficient LAK cells. Perforin-deficient LAK cells, similar to wild-type LAK cells, were shown to contain a large number of granules in their cytoplasm. By electron microscopy, the morphology of the granules present in these two cell populations appeared indistinguishable. The complete depletion of perforin in LAK cells derived from perforin gene-knockout mice was further confirmed by immunoelectron microscopy using anti-perforin antiserum. The expression of other cytolytic mediators, present either within the granules (granzymes A and B) or elsewhere (Fas ligand), appeared to be unperturbed, as investigated using the reverse transcription-PCR technique. Like the CTL and NK cells isolated from perforin-deficient mice, perforin-deficient LAK cells could lyse only target cells that express high levels of Fas molecule. Furthermore, these perforin-deficient LAK cells, similar to wild-type LAK cells and a CTL clone, were resistant to perforin-mediated cytolysis.

Establishment and characterization of monoclonal antibodies against Chuzan virus K-47.

We established sixteen mouse monoclonal antibodies reactive to Chuzan virus K-47 strain using P3-X63-Ag8-U1 cells as fusion partner cells. Among them, CG53/2/4 recognized a 100K structural protein of the virus. The 100K antigen lost it's antigenicity for CG53/2/4 after mild periodate oxidation treatment, suggesting that the 100K viral antigen is a glycoprotein. In addition, CG53/2/4 neutralized the viral infectivity. This indicates that the 100K glycoprotein is essential for the infection of the virus. The other monoclonal antibodies reacted with a 41K antigen of the virus. Especially CG1/1 showed the highest reactivity to the virus. Forward step sandwich assay using CG1/1 and biotinylated CG53/2/4 could detect the virus at 10TCID50/ml. Therefore, these monoclonal antibodies can eventually predict the virus infection to the animals before their sideration.

Establishment and characterization of monoclonal antibodies against bovine ephemeral fever virus.

We established fourteen monoclonal antibodies (MAbs) reactive to bovine ephemeral fever virus YHL strain, and characterized six representatives including three IgG1s (YG3/4, YG5/8, and YG6/7) and three IgMs (YM4/9, YM2/6, and YM6/8). Among them, YG3/4 and YM4/9 gave especially strong reactivities to the virus. YM4/9 reacted specifically with a 43K antigen of the virus, corresponding to the matrix protein 1. The other MAbs reacted most strongly with the 43K antigen, but also reacted with unknown 23K and 21K antigens. By a simultaneous two-site method using YM4/9 and YG3/4, it was possible to detect 10(4.10)TCID50/ml of the virus, in the presence of serum.

Bacillus thermoglucosidasius sp. nov., a New Species of Obligately Thermophilic Bacilli.

A group of 6 strains of obligately thermophilic spore-formers is described as a novel species named Bacillus thermoglucosidasius. The strains are strictly aerobic, neutrophilic, amylolytic, Gram-positive, azide sensitive, produce terminally swollen sporangia and exo-oligo-1,6-glucosidase in large amounts. Strain KP 1006 (DSM 2542) is designated as the type strain. Growth occurs at temperatures from 42°C to 69°C with an optimum at 61-63°C, and at an initial pH of 6.5-8.5. The guanine + cytosine content of DNA, estimated by thermal DNA denaturation, is 45-46 mol%.