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  • Title: Quantitation of alpha2u-globulin in rat kidney cytosol by capillary electrophoresis.
    Author: Pähler A, Blumbach K, Herbst J, Dekant W.
    Journal: Anal Biochem; 1999 Feb 01; 267(1):203-11. PubMed ID: 9918672.
    Abstract:
    The renal accumulation of alpha2u-globulin has been implicated in the tumorigenicity of many nongenotoxic chemicals to the kidney of the male rat. Several chemicals inducing renal tumors in the male rat were shown to bind to alpha2u-globulin. This binding impairs the renal degradation of alpha2u-globulin, resulting in lysosomal overload, cell death, increased cell proliferation, and, presumably, renal tumor formation. To support the role of alpha2u-globulin accumulation in the renal toxicity of a chemical, a demonstration of the accumulation of this protein in the kidney of the male rat is one prerequisite. Monoclonal antibodies to alpha2u-globulin are available for quantifying alpha2u-globulin content; however, the procedure is time-consuming and complicated. We developed a method for the quantitation of alpha2u-globulin in renal cytosol using capillary electrophoresis. Renal cytosol fractions were analyzed by capillary electrophoresis as protein-SDS complexes. Using alpha2u-globulin purified from urine of male rats, the limit of detection was 10 microg/ml sample in routine analyses. Excellent run to run reproducibility in migration time (CV </= 4%) and peak areas corresponding to alpha2u-globulin (CV </= 3%) after normalization to the internal standard was observed. Significant increases in renal alpha2u-globulin content (up to 85% of total protein content) compared to controls (approx 15%) were observed in kidney cytosol of rats treated with alpha2u-globulin nephropathy-inducing agents such as trimethylpentane or the alkylphosphonates dimethyl methylphosphonate and diethyl ethylphosphonate, but not in kidney cytosol of male rats treated with tris-(2-chloroethyl)phosphate or the nephrotoxic agent hexachlorobutadiene. A good correlation of the alpha2u-globulin contents determined by capillary electrophoresis and immunoblotting with an alpha2u-globulin-specific antibody (r2 = 0.997) was obtained. Capillary electrophoresis provides a simple, rapid, and highly reproducible quantitation of alpha2u-globulin accumulation for renal tumorigens and may assist in the risk assessment process for these chemicals.
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