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Location of an inducible nitric oxide synthase mRNA in the normal kidney
J J Morrissey1, R McCracken, H Kaneto
1Department of Medicine, Washington University School of Medicine, St. Louis, Missouri.
Abstract:
An inducible nitric oxide synthase (iNOS) mRNA was found primarily in the outer medulla of normal rat kidney. Identification of the mRNA was based upon the specificity of the oligonucleotide primers used for PCR amplification, PCR-Southern blot analysis and the nucleic acid sequence of the cloned PCR product. In addition to the outer medulla, glomeruli prepared from normal rat kidney contained significant amounts of an iNOS mRNA. These results suggest that there may be tonic influences in the outer medulla of the normal rat kidney resulting in the "steady-state" presence of an iNOS mRNA. Cortical tubules and the inner medulla were found to contain detectable but lesser amounts of the iNOS mRNA. The outer medulla was microdissected into proximal straight tubule (PST), medullary thick ascending limb (MTAL), medullary collecting duct (MCD) and vasa recta bundle (VRB). The iNOS mRNA was found primarily in the MTAL with minor amounts in the MCD and VRB of normal rat kidney. Animals were injected with lipopolysaccharide (LPS) and sacrificed 24 hours later. Treatment with LPS caused at least a 20-fold increase in the amount of iNOS mRNA in the liver or in macrophages isolated from the peritoneum. Endotoxin treatment led to over a 10-fold increase in iNOS mRNA content in glomeruli and the inner medulla. The iNOS mRNA level of the outer medulla was increased two- to threefold due to LPS treatment.
Insights
Inducible nitric oxide synthase (iNOS) mRNA is present in normal rat kidney, mainly in the outer medulla, particularly the medullary thick ascending limb. Lipopolysaccharide (LPS) treatment significantly increases iNOS mRNA levels in various kidney regions and other organs.
Area of Science:
- Nephrology
- Molecular Biology
- Immunology
Background:
- Inducible nitric oxide synthase (iNOS) plays a role in inflammation and immune responses.
- The localization and regulation of iNOS mRNA in the kidney are not fully understood.
Purpose of the Study:
- To investigate the distribution and abundance of iNOS mRNA in different regions of the normal rat kidney.
- To examine the effect of lipopolysaccharide (LPS) administration on iNOS mRNA levels in the rat kidney.
Main Methods:
- Polymerase Chain Reaction (PCR) amplification with specific oligonucleotide primers.
- PCR-Southern blot analysis for mRNA identification.
- Nucleic acid sequencing of the cloned PCR product.
- Microdissection of kidney outer medulla into specific tubular segments and vasa recta.
- Analysis of iNOS mRNA levels after LPS injection.
Main Results:
- iNOS mRNA was predominantly found in the outer medulla of normal rat kidneys, with significant amounts also detected in glomeruli.
- Within the outer medulla, iNOS mRNA was primarily localized to the medullary thick ascending limb (MTAL), with lesser amounts in the medullary collecting duct (MCD) and vasa recta bundle (VRB).
- LPS treatment led to a substantial increase (2- to 3-fold) in iNOS mRNA in the outer medulla, a >10-fold increase in glomeruli and inner medulla, and a >20-fold increase in the liver and peritoneal macrophages.
Conclusions:
- The outer medulla, particularly the MTAL, exhibits a steady-state level of iNOS mRNA in normal rats, suggesting tonic influences.
- Kidney iNOS expression is significantly upregulated by endotoxin (LPS), indicating its involvement in the inflammatory response within the kidney.