ABSTRACT
In a variety of renal diseases, males progress at a more rapid rate and have a more fulminant course than females. This gender difference may be related to the direct effects of sex hormones on the cells of the kidney. To evaluate this hypothesis, we studied the effects of estrogens and testosterone on mesangial cell proliferation and collagen synthesis. At 48 hours, estradiol at 10 nM and 100 nM had a modest proliferative effect on cultured mesangial cells, as measured by 3H thymidine incorporation into DNA and direct cell counting. This estradiol effect was fully reversed by Tamoxifen (1 microM). Estradiol had no effect on cellular proliferation at 1 microM concentrations, but suppressed proliferation at 10 microM doses. Testosterone had a modest but statistically insignificant effect on proliferation at 10 nM and 100 nM concentrations but no effect at 1 microM or 10 microM. Neither estradiol nor testosterone at 10 microM affected total cellular protein accumulation. Estradiol at 1 microM and 10 microM, markedly suppressed total collagen synthesis as measured by 3H proline incorporation, and specifically suppressed the synthesis of collagen types I and IV, as measured by immunoprecipitation and gel electrophoresis. Testosterone did not affect collagen synthesis. Estradiol also reduced the steady state message for the alpha 2 chain of type I collagen, while testosterone had no effect. Neither estradiol nor testosterone affected the steady state message for TGF beta or EGF. The direct effects of estradiol on mesangial cell collagen generation may help explain the slower development of glomerulosclerosis in women and therefore the "protective" effect of female gender on the progression of renal disease.
Subject(s)
Collagen/biosynthesis , Estradiol/pharmacology , Kidney Glomerulus/physiology , Testosterone/pharmacology , Animals , Cell Division/drug effects , Cells, Cultured , Collagen/genetics , Dose-Response Relationship, Drug , Epidermal Growth Factor/analysis , Immunoblotting , Kidney Glomerulus/drug effects , Male , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Time Factors , Transforming Growth Factor beta/analysisABSTRACT
In this communication we describe the sequential use of standard and low-melting agarose in a single gel slab for the electrophoresis of DNA. This method has the advantages of high resolution and reproducibility characteristic of standard agarose and the ease of manipulation of DNA for direct cloning, sequential digestion and isolation, characteristic of low-melting agarose.
