We have reported that the production of 20-HETE is reduced in the renal vasculature of Dahl S rats and that myogenic and TGF responses of afferent arteries (Af-Art) are impaired in Dahl S rats. In this study we generated CYP4A1 transgenic rats in the Dahl S inbred strain background utilizing the enhanced
Sleeping Beauty
(SB100X) transposon system to determine if upregulation of 20-HETE production can restore vascular reactivity and oppose the development of renal injury. Fertilized eggs collected from female Dahl S rats were microinjected with a transposon vector harboring the rat CYP4A1 cDNA under the control of the ubiquitous CAG promoter along with SB100X transposase mRNA to produce transgenic founders. Heterozygous founders were backcrossed to Dahl S rats, transgene insertion sites were identified by Ligation Mediated PCR and sequencing, and the progeny were brother-sister mated to derive homozygous transgenic lines. The expression of CYP4A protein was significantly elevated and the production of 20-HETE was 3-fold higher in the renal outer medullary tissue of CYP4A1 transgenic (n=17) compared to Dahl S rats (n=17). 20-HETE production was 10-fold higher in renal microvessels of CYP4A1 transgenic animals than Dahl S rats. (0.2±0.3, n=22 versus 1.9±0.1 pmol/mg/min, n=14). The luminal diameter of the Af-Art decreased significantly from 15.9 ± 0.6 to 14.1 ± 0.5 μm in CYP4A1 transgenic rats (n=5) when the perfusion pressure was increased from 60 to 120 mmHg, whereas it remained unaltered in Dahl S rats (from 19.4 ± 2.3 to 20.6 ± 5.6 μm, n=22). These studies further support the view that a deficiency in the formation of 20-HETE in the renal microcirculation contributes to the marked susceptibility of Dahl S rats to develop of hypertension and diabetic induced renal injury, and the new CYP4A1 transposon transgenic rat model may be useful for determining the mechanisms involved.
Generation of CYP4A1 transgenic rats using the Sleeping Beauty transposon system