Quantitation of α2u-Globulin in Rat Kidney Cytosol by Capillary Electrophoresis

Abstract

The renal accumulation of α2u-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 α2u-globulin. This binding impairs the renal degradation of α2u-globulin, resulting in lysosomal overload, cell death, increased cell proliferation, and, presumably, renal tumor formation. To support the role of α2u-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 α2u-globulin are available for quantifying α2u-globulin content; however, the procedure is time-consuming and complicated. We developed a method for the quantitation of α2u-globulin in renal cytosol using capillary electrophoresis. Renal cytosol fractions were analyzed by capillary electrophoresis as protein–SDS complexes. Using α2u-globulin purified from urine of male rats, the limit of detection was 10 μg/ml sample in routine analyses. Excellent run to run reproducibility in migration time (CV ≤ 4%) and peak areas corresponding to α2u-globulin (CV ≤ 3%) after normalization to the internal standard was observed. Significant increases in renal α2u-globulin content (up to 85% of total protein content) compared to controls (approx 15%) were observed in kidney cytosol of rats treated with α2u-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 α2u-globulin contents determined by capillary electrophoresis and immunoblotting with an α2u-globulin-specific antibody (r2= 0.997) was obtained. Capillary electrophoresis provides a simple, rapid, and highly reproducible quantitation of α2u-globulin accumulation for renal tumorigens and may assist in the risk assessment process for these chemicals.