Regulation of sodium balance and blood pressure by the AT(1A) receptor for angiotensin II.

To examine the role of the angiotensin II (AT)(1A) receptor in the regulation of blood pressure and sodium balance, we measured systolic blood pressure responses in AT(1A) receptor-deficient (Agtr1a-/-) and wild-type (Agtr1a+/+) mice while dietary sodium content was systematically altered. On a 0.4% sodium diet, systolic blood pressures were significantly lower in Agtr1a-/- than in +/+ mice. In Agtr1a+/+ mice, changing dietary sodium content did not affect blood pressure. In contrast, when Agtr1a-/- mice were fed a high-salt diet (6% NaCl), their systolic blood pressures increased significantly from 79+/-4 to 94+/-4 mm Hg (P<0.006). The low blood pressures of Agtr1a-/- mice decreased further while on a low-salt diet from 82+/-3 to 69+/-3 mm Hg (P<0.03). On the high-salt diet, urinary sodium excretion increased to similar levels in Agtr1a+/+ and -/- mice. Although urinary sodium excretion was substantially reduced in both groups during the low-salt diet, cumulative sodium balances became negative in Agtr1a-/- mice despite a 6-fold increase in urinary aldosterone. We infer, therefore, that the reduced blood pressures in Agtr1a-/- mice on a normal diet are caused by depletion of sodium and extracellular volume. Their "sodium sensitivity" suggests a critical role for renal AT(1A) receptors to modulate sodium handling.

Abnormal water metabolism in mice lacking the type 1A receptor for ANG II.

Mice lacking AT(1A) receptors for ANG II have a defect in urinary concentration manifested by an inability to increase urinary osmolality to levels seen in controls after thirsting. This defect results in extreme serum hypertonicity during water deprivation. In the basal state, plasma vasopressin levels are similar in wild-type controls and Agtr1a -/- mice. Plasma vasopressin levels increase normally in the AT(1A) receptor-deficient mice after 24 h of water deprivation, suggesting that the defect in urine concentration is intrinsic to the kidney. Using magnetic resonance microscopy, we find that the absence of AT(1A) receptors is associated with a modest reduction in the distance from the kidney surface to the tip of the papilla. However, this structural abnormality seems to play little role in the urinary concentrating defect in Agtr1a -/- mice since the impairment is largely reproduced in wild-type mice by treatment with an AT(1)-receptor antagonist. These studies demonstrate a critical role for the AT(1A) receptor in maintaining inner medullary structures in the kidney and in regulating renal water excretion.

Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II.

The classically recognized functions of the renin-angiotensin system are mediated by type 1 (AT1) angiotensin receptors. Whereas man possesses a single AT1 receptor, there are two AT1 receptor isoforms in rodents (AT1A and AT1B) that are products of separate genes (Agtr1a and Agtr1b). We have generated mice lacking AT1B (Agtr1b -/-) and both AT1A and AT1B receptors (Agtr1a -/-Agtr1b -/-). Agtr1b -/- mice are healthy, without an abnormal phenotype. In contrast, Agtr1a -/-Agtr1b -/- mice have diminished growth, vascular thickening within the kidney, and atrophy of the inner renal medulla. This phenotype is virtually identical to that seen in angiotensinogen-deficient (Agt-/-) and angiotensin-converting enzyme-deficient (Ace -/-) mice that are unable to synthesize angiotensin II. Agtr1a -/-Agtr1b -/- mice have no systemic pressor response to infusions of angiotensin II, but they respond normally to another vasoconstrictor, epinephrine. Blood pressure is reduced substantially in the Agtr1a -/- Agtr1b -/- mice and following administration of an angiotensin converting enzyme inhibitor, their blood pressure increases paradoxically. We suggest that this is a result of interruption of AT2-receptor signaling. In summary, our studies suggest that both AT1 receptors promote somatic growth and maintenance of normal kidney structure. The absence of either of the AT1 receptor isoforms alone can be compensated in varying degrees by the other isoform. These studies reaffirm and extend the importance of AT1 receptors to mediate physiological functions of the renin-angiotensin system.

Renal growth and development in mice lacking AT1A receptors for angiotensin II.

To examine the role of the type 1A (AT1A) angiotensin receptor in renal growth and development, we analyzed F2 progeny from a series of crosses between F1 mice that were heterozygous for a targeted disruption of the AT1A receptor gene [Agtr1A-(+/-)]. Among 21-day-old weanling F2 mice, we found that 194 (32%) were homozygous for the wild-type allele Agtr1A-(+/+), 299 (49%) were Agtr1A-(+/-), and 119 (19%) were Agtr1A-(-/-). This differed significantly from the proportions predicted by Mendelian genetics (P = 0.01), suggesting that the complete absence of AT1A receptors is associated with a mild survival disadvantage. Agtr1A-(-/-) mice grew normally, and we found no significant differences in body weight or heart and kidney weights in Agtr1A-(+/+) and Agtr1A-(-/-) mice examined at 21, 60, and 100 days. Protein and DNA content of kidneys and hearts were also similar in weanling or adult Agtr1A-(+/+) and Agtr1A-(-/-) mice. By light microscopy with immunohistochemistry, kidneys from Agtr1A-(-/-) were essentially normal, with two exceptions: 1) there was marked hypertrophy of the juxtaglomerular apparatus (JGA) and proximal expansion of renin-producing cells along the afferent arterioles, and 2) some glomeruli showed evidence of mesangial expansion. We did not find the severe renal vascular lesions or papillary atrophy that have been observed in angiotensinogen- or angiotensin converting enzyme-deficient animals. We conclude that the AT1A receptor is not essential for the normal organogenesis of the kidney; however, its absence is associated with mild mesangial expansion and JGA hypertrophy.

Angiotensin II responses in AT1A receptor-deficient mice: a role for AT1B receptors in blood pressure regulation.

Most of the classic functions of the renin-angiotensin system are mediated by type 1 (AT1) angiotensin receptors, of which two subtypes, AT1A and AT1B, have been identified. However, distinct functions for these two AT1 receptors have been difficult to separate. We examined the pressor effects of angiotensin II in Agtr1A -/- mice, which lack AT1A receptors. In enalapril-pretreated Agtr1A -/- mice, angiotensin II caused significant and dose-proportional increases in mean arterial pressure. This pressor response was not blocked by pretreatment with sympatholytic agents but was completely inhibited by the AT1-receptor antagonists, losartan and candesartan, suggesting that it is directly mediated by AT1B receptors. Chronic treatment of Agtr1A -/- mice with losartan reduced systolic blood pressure from 80 +/- 5 to 72 +/- 4 mmHg (P < 0.04), suggesting a role for AT1B receptors in chronic blood pressure regulation. These studies provide the first demonstration of in vivo pressor effects mediated by AT1B receptors and demonstrate that, when AT1A receptors are absent, the AT1B receptor contributes to the regulation of resting blood pressure.

Regulation of blood pressure by the type 1A angiotensin II receptor gene.

The renin-angiotensin system plays a critical role in sodium and fluid homeostasis. Genetic or acquired alterations in the expression of components of this system are strongly implicated in the pathogenesis of hypertension. To specifically examine the physiological and genetic functions of the type 1A receptor for angiotensin II, we have disrupted the mouse gene encoding this receptor in embryonic stem cells by gene targeting. Agtr1A(-/-) mice were born in expected numbers, and the histomorphology of their kidneys, heart, and vasculature was normal. AT1 receptor-specific angiotensin II binding was not detected in the kidneys of homozygous Agtr1A(-/-) mutant animals, and Agtr1A(+/-) heterozygotes exhibited a reduction in renal AT1 receptor-specific binding to approximately 50% of wild-type [Agtr1A(+/+)] levels. Pressor responses to infused angiotensin II were virtually absent in Agtr1A(-/-) mice and were qualitatively altered in Agtr1A(+/-) heterozygotes. Compared with wild-type controls, systolic blood pressure measured by tail cuff sphygmomanometer was reduced by 12 mmHg (1 mmHg = 133 Pa) in Agtr1A(+/-) mice and by 24 mmHg in Agtr1A(-/-) mice. Similar differences in blood pressure between the groups were seen when intraarterial pressures were measured by carotid cannulation. These studies demonstrate that type 1A angiotensin II receptor function is required for vascular and hemodynamic responses to angiotensin II and that altered expression of the Agtr1A gene has marked effects on blood pressures.

Genetic control of blood pressure and the angiotensinogen locus.

Variants of the human angiotensinogen gene have been linked in some studies to increased circulating angiotensinogen levels and essential hypertension. To test for direct causality between genotypes at the angiotensinogen locus and blood pressures, we have studied mice carrying zero, one, two, three, or four functional copies of the murine wild-type angiotensinogen gene (Agt) at its normal chromosomal location. Plasma angiotensinogen levels increase progressively, although not linearly, from zero in the zero-copy animals to 145% of normal in the four-copy animals. Mice of all genotypes are normal at birth, but most zero-copy animals die before weaning. The kidneys of the zero-copy animals show pathological changes as adults, but the kidneys are normal in the other genotypes. One adult zero-copy male tested was fertile. The blood pressures of the one-copy through four-copy animals show significant and almost linear increases of approximately 8 mmHg per gene copy despite their normal compensatory mechanisms being intact. These results establish a direct causal relationship between Agt genotypes and blood pressures.

Renotrophic factors in urine.

Our previous observations of increased renal protein synthesis in rats subjected to the constant intravenous reinfusion of half their urine output has suggested that the circulatory retention of renotrophic factors in urine is capable of stimulating renal growth. In the present studies, using this same model of "half-urine-reinfusion," which is designed to produce a selective halving of renal excretory function, we have demonstrated significant increases in total DNA content and the incorporation of tritiated thymidine in renal DNA. In addition, a bioassay method was developed in which an assay rat, given an intravenous infusion of urine from another rat, exhibited increases in the incorporation of thymidine into renal DNA and the incorporation of radiolabelled choline into renal phospholipid. This renotrophic activity in the urine was only minimally decreased by heating to 100 degrees C for 30 min and was confined to ultrafiltration fractions retained on a membrane with a nominal 10,000-dalton solute rejection. Removal of one kidney from the rats from which the urine was obtained led to only a modest and transient reduction in the excretion of renotrophic activity, suggesting that the urinary renotrophic factors are of circulatory, not renal, origin. Isolated renal cortical fragments incubated with an ultrafiltration retentate of urine displayed a dose-dependent increase in choline incorporation into phospholipid, suggesting a direct action of the factors on kidney tissue. Finally, no evidence of stimulation of either DNA or phospholipid synthesis could be seen in hepatic tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Ureteral catheter system for renal function studies in conscious rabbits.

To overcome the problem of obstruction of ureteral catheters with blood clots, a ureteral catheter was constructed which had a double lumen. Irrigation of blood from the catheter was accomplished by perfusion of heparinized saline through a small inner catheter. In renal function studies performed in 12 rabbits using this system, no instances of catheter obstruction occurred.

Slowly dialyzable stimulators of renal protein synthesis in urine.

Previously, we demonstrated that continuous intravenous reinfusion of half the urine output (1/2UR) in rats for 1 wk led to increased renal mass. This suggested that reduced renal excretory function, or the retention of urinary factors, was capable of stimulating renal growth. The present study was designed to examine renal protein synthesis during the early phase of this growth and to better define the nature of the stimuli. Compared with matched sham-manipulated control rats, rats subjected to 24 h of 1/2UR displayed significant increases in both the incorporation of tritiated leucine into protein and in the cellular uptake of leucine by renal cortical tissue in vitro. In addition, total protein content of the kidneys, but not of the liver, was significantly increased after 24 h of 1/2UR. Dialysis of urine prior to its reinfusion did not diminish, but rather augmented, the incorporation of leucine into renal protein. These results suggest that renal protein synthesis can be stimulated by the retention of factors in the urine that are poorly dialyzable.