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TNF production by the medullary thick ascending limb of Henle's loop
C M Macica1, B A Escalante, M S Conners
1Department of Pharmacology, New York Medical College, Valhalla.
Abstract:
Medullary thick ascending limb of Henle's loop (mTALH) tubules, isolated from kidneys of male Sprague-Dawley rats, expressed the gene for tumor necrosis factor (TNF) and released this cytokine when challenged with lipopolysaccharide (LPS). The TNF produced was biologically active, as determined by cytotoxic activity present in supernatants from LPS-stimulated mTALH, using the TNF-sensitive murine fibrosarcoma cell line, WEHI 164. The amount of TNF produced, approximately 75 nM, has previously been shown to affect ion transport in the mTALH. The TNF-mediated cytotoxicity (and ion transport effects) were completely neutralized with a polyclonal anti-TNF antisera. Further, immunoprecipitation experiments demonstrated that the 17 kDa TNF monomer was formed by de novo protein synthesis. In contrast, the mTALH did not produce the related cytokine, lymphotoxin (LT). Production of TNF was confirmed by demonstrating the accumulation of a 1.6 kb TNF mRNA by Northern blot analysis; mRNA for LT was not detected. Expression of the TNF gene in the mTALH was confirmed by the polymerase chain reaction (PCR). Southern blot analysis and ethidium bromide staining of the resultant PCR products revealed the expected 276 bp sequence of TNF DNA for the mTALH. We have demonstrated that mTALH tubules stimulated with LPS express the gene for TNF, but not LT, and release biologically active TNF. TNF is an important mediator of septic shock and may contribute to changes in renal function associated with endotoxemia. Production of TNF by the mTALH may be an important autocrine regulatory mechanism for this nephron segment.
Insights
Kidney tubules produce tumor necrosis factor (TNF) when exposed to lipopolysaccharide (LPS). This biologically active TNF may influence kidney function during endotoxemia.
Area of Science:
- Nephrology
- Immunology
- Molecular Biology
Background:
- The medullary thick ascending limb of Henle's loop (mTALH) is crucial for renal function.
- Tumor necrosis factor (TNF) is a key cytokine in inflammatory responses, including septic shock.
- The role of mTALH in producing inflammatory mediators like TNF during endotoxemia is not well understood.
Purpose of the Study:
- To investigate whether mTALH tubules express the gene for TNF and release this cytokine.
- To determine if the produced TNF is biologically active and affects mTALH function.
- To examine the expression of lymphotoxin (LT), a related cytokine, in mTALH.
Main Methods:
- Isolation of mTALH tubules from male Sprague-Dawley rats.
- Stimulation of mTALH with lipopolysaccharide (LPS).
- Assays for TNF biological activity (cytotoxicity using WEHI 164 cells).
- Neutralization of TNF effects with anti-TNF antisera.
- Immunoprecipitation to confirm TNF monomer synthesis.
- Northern blot analysis for TNF mRNA.
- Polymerase chain reaction (PCR) and Southern blot analysis for TNF gene expression.
Main Results:
- LPS-stimulated mTALH tubules expressed TNF gene and released biologically active TNF.
- TNF production was confirmed by de novo protein synthesis and TNF mRNA accumulation.
- TNF-mediated cytotoxicity and effects on ion transport were neutralized by anti-TNF antisera.
- mTALH tubules did not produce lymphotoxin (LT) or its mRNA.
- PCR and Southern blot confirmed the presence of TNF DNA sequence in mTALH.
Conclusions:
- mTALH tubules express the TNF gene and release biologically active TNF upon LPS stimulation.
- TNF production by mTALH may play a role in renal dysfunction during endotoxemia.
- TNF may act as an autocrine regulator within the mTALH segment.