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Differential expression and induction of mRNAs encoding two inducible nitric oxide synthases in rat kidney
M G Mohaupt1, J L Elzie, K Y Ahn
1DCI Laboratory of Molecular Biology in Nephrology, University of Florida College of Medicine, Gainesville.
Abstract:
We used quantitative PCR methods and renal microdissection to characterize the expression of inducible nitric oxide synthase (iNOS) mRNAs in rat kidney and cultured glomerular mesangial cells. A partial cDNA homologous to murine macrophage iNOS (macNOS), but distinct from rat vascular smooth muscle iNOS (vsmNOS), was cloned from normal rat kidney. macNOS was the principal iNOS isoform tonically expressed in microdissected glomeruli, proximal tubules, medullary thick ascending limbs (mTAL), cortical and inner medullary collecting ducts (IMCD), and cultured mesangial cells, whereas vsmNOS was the major isoform expressed in arcuate and interlobular arteries. Basal macNOS expression was greatest in mTALs and IMCDs. Restriction mapping of RT-PCR products indicated that basal expression of macNOS mRNA was comparable to that of vsmNOS in cortex, but greater than vsmNOS in outer and inner medulla. However, compared to controls, lipopolysaccharide (LPS)-treated rats exhibited a much greater proportion of vsmNOS mRNA and higher levels of total iNOS mRNA in each zone. Similarly, TNF alpha and IF-gamma preferentially induced expression of vsmNOS mRNA in cultured mesangial cells. We conclude that two iNOS isoforms are constitutively and heterogeneously expressed in the normal rat kidney, and that endotoxemia and cytokines differentially induce their expression.
Insights
Two inducible nitric oxide synthase (iNOS) isoforms are expressed in rat kidneys. Lipopolysaccharide and cytokines differentially regulate these iNOS isoforms, impacting kidney function.
Area of Science:
- Nephrology
- Molecular Biology
- Biochemistry
Background:
- Inducible nitric oxide synthase (iNOS) plays a role in kidney physiology and pathology.
- Understanding iNOS isoform expression is crucial for comprehending kidney responses to inflammation.
Purpose of the Study:
- To characterize the expression patterns of different inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) isoforms in the rat kidney.
- To investigate the differential regulation of iNOS isoforms by inflammatory stimuli like lipopolysaccharide (LPS) and cytokines.
Main Methods:
- Quantitative PCR (qPCR) and renal microdissection were employed to analyze iNOS mRNA expression.
- A partial cDNA for a murine macrophage-inducible nitric oxide synthase (macNOS) isoform was cloned and characterized.
- Restriction mapping of reverse transcription-PCR (RT-PCR) products differentiated between macNOS and rat vascular smooth muscle iNOS (vsmNOS) isoforms.
Main Results:
- The macNOS isoform was the primary iNOS isoform constitutively expressed in various kidney segments, including glomeruli, proximal tubules, and collecting ducts.
- The vsmNOS isoform was predominantly found in renal arteries.
- Lipopolysaccharide (LPS) treatment significantly increased the proportion of vsmNOS mRNA and total iNOS mRNA levels across all kidney zones.
- Tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) preferentially induced vsmNOS mRNA expression in cultured mesangial cells.
Conclusions:
- The rat kidney exhibits constitutive and heterogeneous expression of two distinct iNOS isoforms (macNOS and vsmNOS).
- Endotoxemia and inflammatory cytokines differentially regulate the expression of these iNOS isoforms in the kidney.