Abstract 497: H2O2 in Fumarase Insufficiencies in Dahl Salt-Sensitive Rats.
We have shown previously that renal insufficiencies of fumarase, an enzyme in the tricarboxylic acid cycle, appear to contribute to salt-induced hypertension in Dahl salt-sensitive (SS) rats. The present study examined the molecular mechanism underlying fumarase insufficiencies in SS rats. Protein abundance and enzymatic activity of fumarase was significantly lower in the renal medulla of SS rats compared to salt-insensitive Sprague-Dawley rats. We screened several factors including TGFβ1, TNFα, and H2O2 for their effects on fumarase expression. These factors were selected for screening because they had been reported to be elevated in the kidneys of the SS rat and they represent a pro-fibrotic, pro-inflammatory, and oxidative milieu present in the kidneys of the SS rat. Cultured human renal epithelial cells were treated with TGFβ1 (3 ng/ml), TNFα (25 ng/ml), or H2O2 (500 μM) for 24 hours. H2O2 substantially reduced fumarase mRNA levels (normalized by 18S rRNA) to less than 10% of control. TNFα tended to suppress fumarase expression, but the effect did not reach statistical significance. TGFβ1 did not have significant effects on fumarase mRNA levels. The greater than 90% loss of fumarase following the H2O2 treatment was accompanied by significant cell death. To determine if the down-regulation of fumarase was simply a consequence of the cytotoxicity of H2O2, we treated HRE cells with 100 μM of H2O2 for 48 hours. The lower dose of H2O2 did not cause significant cell death. Yet, fumarase mRNA was still down-regulated to 79.6% ± 4.7% of control (n=4, P<0.05 vs. control). Moreover, we repeated the treatment in cells cultured in the Dulbecco’s Modified Eagle Medium (DMEM) instead of the regular epithelial growth medium. HRE cells cultured in DMEM were highly resistant to the cytotoxicity of H2O2. H2O2 (500 μM) did not cause any significant cell death in HRE cultured in DMEM, yet it again significantly suppressed fumarase expression to 77.1% ± 6.6% of control (n=7-8, P<0.05 vs. control). The result suggests that elevated levels of H2O2 in the renal medulla might contribute to fumarase insufficiencies in SS rats.
- © 2012 by American Heart Association, Inc.