Quantitative reverse transcription polymerase chain reaction shows that ethanol enhances interleukin-6 mRNA expression in cultured mesangial cells.
IgA nephropathy, a form of mesangial glomerulonephritis, is associated with chronic ethanol ingestion in humans and in a rat model. We investigated the hypothesis that ethanol has a direct effect on a mesangial cell cytokine, interleukin-6 (IL-6). We measured IL-6 mRNA in cultured mesangial cells using a novel method, quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR). Primers were used to amplify a 346-bp segment. To quantify the results, we generated an internal standard using site-directed mutagenesis which resulted in a restriction site for EcoRI, absent in target IL-6 cDNA. In the presence of the same primers, the mutated internal standard (exogenous template) amplifies with equal efficiency as the target IL-6 cDNA. Q-RT-PCR, using 250 ng of total RNA, showed that after ethanol incubation, IL-6 mRNA was 65 (+/-30) attomol, a 1.5-fold increase from 44 (+/-28) attomol in control cells. This study shows that in vitro, ethanol enhances IL-6 mRNA expression in rat mesangial cells.
Meleg-Smith
S
S
Department of Pathology and Laboratory Medicine, Tulane University, School of Medicine, New Orleans, LA 70112, USA.
Zheng
Q
Q
Wu
J
J
Leung
W C
WC
Baricos
W
W
D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D000431:Ethanol / Q000494:pharmacology*; D005920:Glomerular Mesangium / Q000187:drug effects* / Q000378:metabolism*; D015850:Interleukin-6 / Q000096:biosynthesis / Q000235:genetics*; D008297:Male; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis*; D012194:RNA-Directed DNA Polymerase; D051381:Rats; D017207:Rats, Sprague-Dawley
D000276:Adjuvants, Immunologic; D015850:Interleukin-6; D012333:RNA, Messenger; D000431:Ethanol; D012194:RNA-Directed DNA Polymerase