Induction of Angiotensin II Infused Hypertension.
Male-Sprague Dawley rats were housed separately in cages and maintained in a temperature- and light controlled room. Throughout the experiments, animals had free access to standard rat chow. All experiments were approved by the Tulane University Animal Care and Use Committee. Surgical procedures were performed with rats under pentobarbital anesthesia and with sterile procedures. Rats weighing 180 to 200 g were divided into two experimental groups: one group received sham surgery and a second group was subjected to angiotensin II infusion. Angiotensin II was infused at a continuous rate (60 ng/min) via an osmotic minipump implanted subcutaneously at the dorsum of the neck. Systolic blood pressure was measured in conscious rats by tail-cuff plethysmography to monitor the progression of hypertension. Renal microvascular studies were performed at 5-7 or 11-13 days after the start of the angiotensin II infusion.

In Vitro Perfused Juxtamedullary Preparation.
Animals were anesthetized with sodium pentobarbital and the kidney was isolated and perfused in vitro for study of the juxtamedullary vasculature. The kidney was removed from the rat, placed in an organ chamber, and continuously superfused with a warmed (37 °C) physiological salt solution containing 1% albumin. The kidney was perfused with a reconstituted blood (Hct = 20%). Perfusion pressure was monitored using a pressure transducer and set at 100 mmHg. Renal microvascular responses to vasoactive agonist and changes in perfusion pressure were monitored by videomicroscopy (Figure 1).

Fig. 1:

Interlobular and afferent arteriolar diameter were measured using a digital image shearing monitor which was calibrated by a stage micrometer and measurements were reproducible within 0.5 µm. In the first series of experiments, renal perfusion pressure was lowered to 80 mmHg and the pressure-diameter relationship determined. Following the 15 min equilibration period, perfusion pressure was varied in steps form 80 to 120 to 160 mmHg. In the second series of experiments, measurements of resting afferent arteriolar diameters were made at a renal perfusion pressure of 100 mmHg. Angiotensin II (0.1-10 nM) or norepinephrine (10 nM-1µM) were administered by superfusion and diameter changes monitored for 5 minutes. Renal perfusion pressure was increased to 150 mmHg and the response to angiotensin II redetermined in animals that received angiotensin II infusion for 11-13 days.

Statistical Analysis.
All data are presented as mean ± SEM. Difference between and within groups were analyzed by two-way ANOVA with repeated measures on one factor and Fisher’s least significance post hoc test. A value of &It; 0.5 was considered significant.

Development of Hypertension.
Angiotensin II infusion results in a slowly developing hypertension. Systolic blood pressure increased significantly above control values by day 6 and reached 184 ± 6 mmHg by day 12 in animals receiving angiotensin II infusion (Figure 2).

Fig. 2:

Enhanced Renal Microvascular Tone in Angiotensin II Induced Hypertension. Renal microvascular responses to increasing renal perfusion pressure were observed utilizing the in vitro juxtamedullary nephron preparation. At a renal perfusion pressure of 80 mmHg interlobular and afferent arteriolar diameters of normotensive animals averaged 28 ± 1 and 18 ± 1 µm; respectively. Diameters of the interlobular arteriole at renal perfusion pressures of 80, 120, and 160 mmHg were 12-20% smaller in hypertensive animals; however, arterioles from both groups responded to increases in renal perfusion pressure with similar 21-25% decreases in diameter (Figure 3).

Fig. 3:

Enhanced Renal Microvascular Responsiveness to Angiotensin II in Hypertensive Animals. Angiotensin II infused hypertensive rats studied at 11-13 days had an enhanced afferent arteriolar responsiveness to angiotensin II at renal perfusion pressures of 100 and 150 mmHg (Figure 4).

Fig. 4:

Afferent arteriolar responses to angiotensin II and norepinephrine at a renal perfusion pressure of 100 mmHg were observed in angiotensin II infused hypertensive’s and normotensive controls 5-7 days following osmotic minipump implantation. Angiotensin II (10 nM) decreased afferent arteriolar diameter by 27 ± 3% in the 5-7 say hypertensive group compared to 20 ± 2% in normotensive controls. In contrast, the afferent arteriolar response to norepinephrine was not altered after 5-7 days of angiotensin II infusion (Figure 5).

Fig. 5:

SUMMARY

1.) Angiotensin II infused hypertensive rats have reduced interlobular diameters and maintained reactivity to changes in renal perfusion pressure.

2.) Afferent arterioles of hypertensive rats infused with angiotensin II for 11-13 days have an enhanced responsiveness to angiotensin II, "autopotentiation".

3.) The afferent arteriolar response to angiotensin II but not norepinephrine is enhanced following 5-7 days of angiotensin II-induced hypertension.

CONCLUSION

These data demonstrate an elevated renal microvascular tone and enhanced preglomerular reactivity which is selective for angiotensin II during the early stages of hypertension following infusion of angiotensin II. Thus, excessive renal vasoconstriction is a contributing factor to the initial defect in renal function leading to the development of hypertension.

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