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Intracellular Enhanced Cyan Fluorescent Protein/Angiotensin II Does Not Modify Angiotensinogen Accumulation in
Akannsha Singh1, Jason Vitko, Richard N Re
1Molecular Genetics Laboratory, Institute for Translational Research, and.
Background:
Several studies suggest that extracellular angiotensin can upregulate renin and angiotensinogen (AGT). We have shown that enhanced cyan fluorescent protein/angiotensin II (ECFP/AngII) transgenic mice, in which AngII is fused downstream of ECFP and regulated by the mouse metallothionein housekeeping gene, possess elevated blood pressure and kidney thrombotic microangiopathy. The present study evaluated the effect of intracellular AngII on AGT messenger RNA (mRNA) and protein levels in ECFP/AngII transgenic mice.
Methods:
The traditional guanidinium thiocyanate method was used to extract total mRNA. Proteins were extracted by homogenization in a tissue extraction reagent buffer. Northern blots for AGT mRNA and an 18S ribosomal RNA control were performed. Immunoblots for AGT protein levels with actin and tubulin controls were evaluated.
Results:
Northern blot densitometry showed liver mRNA levels an average of 12-fold greater than levels in the brain or kidney in both Lines A and D (different copies of the transgene) with no quantifiable differences between wild-type (WT) and homozygous (HO) transgenic mice. Immunoblots showed liver AGT protein levels 3.2-fold greater than levels in the brain or kidney, with no differences observed between WT and HO transgenic mice.
Conclusion:
ECFP/AngII transgene expression does not alter AGT mRNA or protein levels in major organs (kidney, liver, and brain) of transgenic mice. The altered blood pressure and kidney thrombosis observed in these transgenic mouse lines are not the result of increased intracellular AGT synthesis and resultant increases in free extracellular AngII. This finding is consistent with our published studies that indicate no increase in circulating AngII by radioimmunoassay.
