Technical Reference #1661

1661.

AT1 Receptor Activation Regulates the mRNA Expression of CAT1; CAT2; Arginase-1; and DDAH2 in Preglomerular Vessels From Angiotensin II Hypertensive Rats Michael Hultstro¨m; Frank Helle; and Bjarne M. Iversen, Haukeland University Hospital, American Journal of Physiology: Renal Physiology, 297(1661), (2009)
Link To Paper

Abstract:
Previously we found increased expression of l-arginine metabolizing enzymes in both kidneys from two-kidney one-clip (2K1C) hypertensive rats (Helle F Hultstrom M Skogstrand T Palm F Iversen BM. Am J Physiol Renal Physiol 296: F78-F86 2009). In the present study we investigate whether AT(1) receptor activation can induce the changes observed in 2K1C. Four groups of rats were infused with 80 ng/min ANG II or saline for 14 days and/or given 60 mg x kg(-1) x day(-1) losartan. Gene expression was studied in isolated preglomerular vessels by RT-PCR. Dose-responses to ANG II were studied in isolated preglomerular vessels with and without acute NOS inhibition [10(-4) mol/l N(G)-nitro-l-arginine methyl ester (l-NAME)]. Expressions of endothelial nitric oxide synthase (eNOS) caveolin-1 and arginase-2 were not changed by ANG II infusion. CAT1 (0.3 8 +/- 0.07 to 0.73 +/- 0.12 P < 0.05) CAT2 (1.14 +/- 0.29 to 2.74 +/- 0.48) DDAH2 (1.09 +/- 0.27 to 2.3 +/- 0.46) and arginase-1 (1.08 +/- 0.17 to 1.82 +/- 0.22) were increased in ANG II-infused rats. This was prevented by losartan treatment which reduced the expression of eNOS (0.97 +/- 0.26 to 0.37 +/- 0.11 in controls; 0.8 +/- 0.16 to 0.36 +/- 0.1 in ANG II-infused rats) and caveolin-1 (2.49 +/- 0.59 to 0.82 +/- 0.24 in controls and 2.59 +/- 0.61 to 1.1 +/- 0.25 in ANG II-infused rats). ANG II (10(-10) mol/l) caused vessels from ANG II-infused animals to contract to 53 +/- 15% of baseline diameter and 90 +/- 5% of baseline diameter in controls (P < 0.05) and was further enhanced by l-NAME to 4 +/- 4% of baseline diameter (P < 0.05). In vivo losartan treatment reduced the reactivity of isolated vessels to 91 +/- 2% of baseline in response to 10(-7) mol/l ANG II compared with 82 +/- 3% in controls (P < 0.05) and prevented the increased responsiveness caused by ANG II infusion. In conclusion CAT1 CAT2 DDAH2 and arginase-1 expression in renal resistance vessels is regulated through the AT(1) receptor. This finding may be of direct importance for NOS and the regulation of preglomerular vascular function.

Keywords:
vaceolin-1; afferent arteriole; contraction; losartan

Materials & Methods:
Isolation and preparation of renal preglomerular vessels for diameter measurements. Renal resistance vessels were isolated using an agarose infusion enzyme digestion technique as described earlier (7). In short rats were anesthetized with pentobarbital sodium (50–70 mg/kg). The abdominal aorta was freed cannulated and the kidneys were infused with 3–4 ml Seaprep agarose solution (2%) in Ca2 -free Roswell Park Memorial Institute solution (RPMI; 37°C) to create an elastic core of agarose inside the renal microvessels. About 100- mthick slices were cut from the cortex of the kidney with a Thomas slicer (Thomas Scientific) and incubated at 37°C for 1 h in Ca2 -free RPMI containing 0.5 mg/ml protease (Sigma P3417 0.5 U/ml) 0.3 mg/ml collagenase (Sigma C5138 700 U/ml) and 0.05 mg/ml trypsin inhibitor. Afferent fragments were picked with a 150- m-diameter glass pipette and fastened by self-adhesion to a glass coverslip in a petri dish (MatTek P35G-0–14-C) containing 3 ml RPMI without Ca2 . Before the experiments Ca2 concentration in the medium was increased to 2 mmol/l in three steps (20 mol/l 200 mol/l and 2 mmol/l) with a 5-min incubation between each step. A typical preparation of agar-infused preglomerular vessels is seen in Fig. 1.

Microscopic Technique
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