Tumor necrosis factor-alpha mRNA expression in lipopolysaccharide-stimulated rat kidney. Chronological analysis of localization.
To study the time course of lipopolysaccharide-induced production of tumor necrosis factor-alpha (TNF-alpha) in the kidney, we utilized a highly sensitive non-radioisotopic in situ hybridization with 1-nm gold-conjugated anti-digoxigenin for localization of TNF-alpha mRNA expression after lipopolysaccharide administration. TNF-alpha mRNA expression localized by in situ hybridization showed a peak increment in proximal tubular epithelial cells and glomeruli at 2 hours and returned to almost normal levels at 6 hours. The intensity of the signal was much stronger in proximal tubules than in glomeruli. These findings were confirmed by the demonstration of similar kinetics in the increase of TNF-alpha message, measured by using amplification of the third and fourth exons of TNF-alpha gene by reverse transcription-polymerase chain reaction of microdissected proximal tubular segments and isolated glomeruli. Reverse transcription-polymerase chain reaction of cultured rat mesangial and glomerular epithelial cells demonstrated that mesangial cells, not glomerular epithelial cells, were responsible for the observed glomerular signals.
Noiri
E
E
First Department of Internal Medicine, University of Tokyo, Japan.
Kuwata
S
S
Nosaka
K
K
Tokunaga
K
K
Juji
T
T
Shibata
Y
Y
Kurokawa
K
K
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis / Q000235:genetics; D004848:Epithelium / Q000737:chemistry; D005260:Female; D005920:Glomerular Mesangium / Q000737:chemistry; D017403:In Situ Hybridization; D007668:Kidney / Q000737:chemistry* / Q000166:cytology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009399:Nephrons / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000235:genetics*
D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D004247:DNA