Effects of AT1A receptor deletion on blood pressure and sodium excretion during altered dietary salt intake

Amy J. Mangrum1, R. Ariel Gomez2, and Victoria F. Norwood2

Departments of 1 Internal Medicine and 2 Pediatrics, University of Virginia School of Medicine, Charlottesville, Virginia 22908


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study was performed to investigate the role of type 1A ANG II (AT1A) receptors in regulating sodium balance and blood pressure maintenance during chronic dietary sodium variations in AT1A receptor-deficient (-/-) mice. Groups of AT1A (-/-) and wild-type mice were placed on a low (LS)-, normal (NS)-, or high-salt (HS) diet for 3 wk. AT1A (-/-) mice on an LS diet had high urinary volume and low blood pressure despite increased renin and aldosterone levels. On an HS diet, (-/-) mice demonstrated significant diuresis, yet blood pressure increased to levels greater than control littermates. There was no effect of dietary sodium intake on systolic blood pressures in wild-type animals. The pressure-natriuresis relationship in AT1A (-/-) mice demonstrated a shift to the left and a decreased slope compared with wild-type littermates. These studies demonstrate that mice lacking the AT1A receptor have blood pressures sensitive to changes in dietary sodium, marked alterations of the pressure-natriuresis relationship, and compensatory mechanisms capable of maintaining normal sodium balance across a wide range of sodium intakes.

kidney; pressure-natriuresis relationship; angiotensin II; plasma renin concentration; aldosterone


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EXTRACELLULAR FLUID VOLUME is normally maintained within narrow limits despite considerable variations in daily salt and water intake. Therefore, renal sodium excretion is generally considered a crucial variable in the control of extracellular fluid volume and blood pressure. The kidneys determine the long-term blood pressure response by altering sodium excretion and maintaining extracellular volume. In normal conditions, small elevations in blood pressure result in increases in urinary excretion of sodium and water, reducing the blood pressure to baseline levels. The relationship between sodium excretion and blood pressure is called pressure-natriuresis (5, 8-11). The renin-angiotensin system (RAS) and pressure-natriuresis relationship are closely coordinated mechanisms that are crucial for maintaining sodium balance and systemic blood pressure (8).

The RAS is the major hormone system regulating sodium balance. At physiological concentrations, ANG II stimulates proximal tubular sodium reabsorption. ANG II may also act to decrease sodium excretion and increase urinary concentrating ability by reducing renal medullary blood flow. Through indirect effects, ANG II enhances sodium reabsorption through stimulation of aldosterone release from the adrenal gland.

On the basis of pharmacological criteria, ANG II exerts its actions via two subtypes of receptors, AT1 and AT2. Most of the classic functions of ANG II are mediated through the AT1 receptor. In rodents, the AT1 receptor is divided into two subtypes, AT1A and AT1B. With the use of gene-targeting techniques, the AT1A gene has been inactivated in mice, resulting in the functional deletion of the AT1A receptor (4). Previous studies using these mice demonstrated that the AT1A receptor has a critical role in regulating blood pressure (2, 4, 13, 15, 19, 20). In these mice, loss of AT1A resulted in lower blood pressure, decreased ability to conserve sodium, and an inability to appropriately concentrate the urine. However, the interaction between the AT1A receptor deletion and the pressure-natriuresis relationship has not been defined. The present study was designed to assess the importance of the AT1A receptor in chronically regulating blood pressure and sodium balance across a spectrum of varying dietary sodium intakes.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Mice heterozygous at the AT1A locus, originally derived from the line described by Coffman et al. (13) and backcrossed more than five generations into the C57/B6 strain, were bred to generate wild-type (+/+), heterozygous (+/-), and homozygous null (-/-) littermates. The genotype at the AT1A locus was determined by PCR analysis of genomic DNA isolated from tail biopsies. The chosen primers [5'-ACCAACTCAACCCAGAAAAGC-3' (upstream) and 5'-CCAGGATGTTCTTGGTTAGG-3' (downstream)] amplify both the wild-type (620-bp) and mutant (1.2-kb) sequences, using an annealing temperature of 55°C for 1 min and an extension temperature of 72°C for 1.2 min. Genotyping was accomplished by size differential of amplified fragments after gel electrophoresis.

Dietary sodium manipulation. At 3 wk of age, (+/+) and (-/-) littermates were weaned to one of three commercially prepared isocaloric diets (Harlan Tekland, Madison, WI): low [LS; 0.03% (g NaCl/100 g diet); (+/+) n = 10; (-/-) n = 9]; normal [NS; 0.43%; (+/+) n = 8; (-/-) n = 10]; or high salt [HS; 3.14%; (+/+) n = 11; (-/-) n = 8]. The animals were allowed free access to distilled water. After 3 wk of ad libitum access to these diets, the animals were weighed and placed in metabolic cages (described below) for 2 days before euthanasia. Body weights were documented at 3 and 6 wk of age.

Systolic blood pressure measurements in conscious mice. At 6 wk of age, after 3 wk of dietary sodium manipulation, blood pressure in conscious animals was measured using a computerized noninvasive tail-cuff system that determines tail blood flow using a photoelectric sensor (Visitech Systems, Apec, NC). With the use of protocols previously validated for this system (13, 15, 16), systolic blood pressure was measured 10 times/animal and averaged. Two measurements/day were recorded for 3 consecutive days.

Metabolic studies. At 6 wk of age, daily water and food intake were quantitated and 24-h urine collections were obtained for 2 consecutive days using Rodent Metabolic Cages (Nalgene, VWR Scientific, Suwanee, GA). Identical cages were used in the same temperature-controlled vivarium room. Urine was collected at the same time each day, and the cage was in an enclosed system to decrease evaporative losses. Urine collections were centrifuged to remove any particles and stored at -80°C.

Blood and tissue harvesting. After blood pressure recordings and urine collections, the animals were euthanized under anesthesia with ketamine/xylazine (0.1 mg/100 kg ip). Blood samples were obtained by cardiac puncture. The plasma was rapidly separated by centrifugation and frozen at -80°C until assayed. Kidney, heart, and liver weights were documented.

Blood and urine chemistries. Plasma and urine sodium and potassium concentrations were measured using a flame photometer (Instrumentation Laboratories, Lexington, MA). All urine and serum osmolalities were measured using a vapor pressure osmometer (Wescor 5100C, Logan, UT). Creatinine was measured by the Jaffe reaction (Sigma Diagnostics, St. Louis, MO).

Plasma renin concentration. Plasma renin concentration (PRC) was determined by the method of Sealy and Laragh (22).

Aldosterone concentrations. Plasma aldosterone concentrations were measured by radioimmunoassay (Coat-A-Count RIA, Diagnostic Products, Los Angeles, CA).

Tissue preparation. Kidneys were fixed in 10% buffered formaldehyde or Bouin's solution and embedded in paraffin or immediately frozen in liquid nitrogen and stored at -80°C.

Histochemistry. Kidney sections were stained with hematoxylin and eosin, periodic acid-Schiff, and Masson-Trichrome.

Statistical analysis. Data are expressed as means ± SE. Comparisons between the AT1A (-/-) and wild-type littermates were made by t-test. Comparisons of animals on LS, NS, and HS diets within a genotype were made by one-way ANOVA. Statistical significance was defined as P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Dietary intake. To ensure that daily caloric intake was unaffected by the salt content of the diet, daily food intake was quantitated for 2 days. As shown in Table 1, AT1A null mice consumed equivalent quantities of food as their wild-type controls. Both genotypes consumed higher quantities of the high-salt chow than the normal or salt-depleted diets. Table 1 also shows that AT1A null mice consumed equivalent quantities of sodium as their wild-type littermates.

                              
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Table 1.   Effect of dietary sodium content on food intake

Body, kidney, heart and liver weights. At the initiation of the study (3 wk of age), there were no significant differences in body weight between (+/+) and (-/-) mice. However, as shown in Table 2, at 6 wk of age, sodium-restricted AT1A null mice had lower body weights than their wild-type littermates [15.9 ± 0.9 (+/+) vs. 13.8 ± 0.5 g (-/-), P = 0.02] despite equal caloric intake.

                              
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Table 2.   Effect of chronic dietary sodium manipulation on body weight and organ weight

Kidney weight-to-body weight and liver weight-to-body weight ratios were unaffected by sodium intake or genotype. AT1A-deficient mice on the HS diet showed significantly higher heart weights compared with the wild-type littermates, NS and HS diet null animals.

Water balance. Figure 1A shows that daily water intake in AT1A null mice was approximately two times greater than wild-type controls regardless of sodium intake. Likewise, 24-h urine output in (-/-) mice was approximately twice that of wild-type controls (Fig. 1B).


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Fig. 1.   Water intake (A) and urinary volume (B) during manipulation of sodium intake in control mice [(+/+); filled bars] or in mice lacking the ANG II type 1A (AT1A) receptor [(-/-); open bars] on low (LS)-, normal (NS)-, and high-salt (HS) diets. Values are means ± SE and represent the average of 2 days in metabolic cages. *P < 0.005.

Sodium excretion. Table 3 illustrates 24-h urinary sodium excretion rates obtained after 3 wk of dietary sodium manipulation. To ensure adequacy of urine collections, daily urine creatinine excretion was checked in all mice placed in the metabolic cage. No significant differences between urine collection accuracy were found among the groups.

                              
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Table 3.   Effect of chronic dietary sodium manipulation on urinary sodium excretion

No significant differences in daily sodium excretion were found between (+/+) and (-/-) littermates on any diet. As expected, animals on an HS diet excreted significantly more sodium than animals on the NS or LS diet (Table 3).

Serum sodium and potassium concentrations. Serum sodium and potassium concentrations were unaffected by genotype or dietary sodium intake (Table 4, A and B).

                              
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Table 4.   Effect of chronic dietary sodium manipulation on serum sodium, net sodium balance, and serum potassium

Sodium balance. Sodium balance was calculated as sodium intake minus sodium excretion and expressed as a function of body weight. All animals remained in positive sodium balance regardless of genotype (Table 4). Positive sodium balance increased as dietary intake of sodium increased. There were no differences in net sodium balance between (+/+) and (-/-) mice.

Blood pressure. Figure 2 shows that (-/-) mice on the LS diet had significantly lower systolic blood pressures compared with their (+/+) littermates (P = 0.019). On the HS diet, AT1A (-/-) mice had systolic pressures similar to (+/+) littermates. Interestingly, the systolic blood pressures of the null mice on the HS diet were higher than the null mice on both LS and NS diets. In contrast, there was no effect of dietary sodium intake on systolic blood pressure in wild-type animals. Similarly, there was no significant difference in systolic blood pressure between (+/+) and (-/-) mice on the NS diet.


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Fig. 2.   Systolic blood pressure (SBP) in AT1A (+/+) and AT1A (-/-) mice (filled and open bars, respectively) on LS [(+/+), n = 10; (-/-), n = 9]; NS [(+/+), n = 8; (-/-), n = 10]; and HS [(+/+), n = 11; (-/-), n = 8] diets. Values are means ± SE and represent the average of measurements on 3 consecutive days. *P < 0.05.

Pressure natriuresis. To evaluate the effect of the AT1A receptor on the pressure-natriuresis relationship, the urinary sodium excretion rate (y-axis) was plotted against the average systolic blood pressure (x-axis). Figure 3 shows that AT1A-deficient mice had a marked decrease in the slope of the pressure-natriuresis curve compared with (+/+) littermates. Furthermore, the pressure-natriuresis curve was significantly shifted to the left in the AT1A-deficient mice compared with wild-type littermates.


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Fig. 3.   Steady-state relationship between SBP and urinary sodium excretion (UNaV; mmol Na · mg creatinine-1 · g BW-1 · day-1) in control mice (+/+) and in mice with the AT1A receptor deleted (-/-). Values are means ± SE and represent data obtained at the end of the study period at each level of sodium intake and blood pressure. Dashed line, pressure-natriuresis relationship between mice of the same genotype on different sodium intakes (LS, NS, and HS); Ucr, urinary creatinine.

PRC. AT1A (-/-) mice on the LS diet had PRCs that were significantly higher than (+/+) controls. This difference between genotypes was not apparent in mice on the NS or HS diet. As expected, the PRC was inversely related to sodium intake in all animals (Fig. 4).


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Fig. 4.   Plasma renin concentration (PRC) in AT1A (+/+) and AT1A (-/-) mice (filled and open bars, respectively) on LS, NS, and HS diets. Values are means ± SE; n = >= 6 mice/group. *P < 0.001.

Plasma aldosterone concentrations. Aldosterone concentrations were not significantly different between the (+/+) and (-/-) mice on any diet (Fig. 5). However, plasma aldosterone concentration in the (-/-) mice decreased significantly as the sodium content in the diet was increased. Serum and urine potassium levels were not significantly different between the genotypes.


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Fig. 5.   Aldosterone in AT1A (+/+) and AT1A (-/-) mice (filled and open bars, respectively) on LS, NS, and HS diets. Values are means ± SE; n = >= 4 mice/group. *P < 0.05.

Urine osmolality. On normal sodium intake, urine osmolality was not different between the null and wild-type littermates. However, in animals on either the LS or HS diet, urine osmolalities were lower in AT1A null mice compared with wild-type littermates (Fig. 6).


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Fig. 6.   Urine osmolality in AT1A (+/+) and AT1A (-/-) mice (filled and open bars, respectively) on LS, NS, and HS diets. Values are means ± SE; n = >= 8 mice/group. *P < 0.01.

Histology. Renal histopathology was significantly altered by the absence of AT1A but was not affected by diet. Macroscopically, the AT1A receptor-deficient kidneys showed an irregular lobulated surface compared with the smooth surface of control kidneys. Figure 7, A and B, shows periodic acid-Schiff-stained kidney sections from (+/+) and (-/-) kidneys, respectively. Atrophic tubules with flattened tubular epithelia were present in all (-/-) mice. Thickened basement glomerular basement membranes were also apparent. Trichrome staining also revealed abnormal renal architecture [Fig. 7, C (+/+) and D (-/-)]. Focal areas of increased hypercellular interstitial tissue (Fig. 7D, arrow) in the (-/-) kidneys were not present in the wild-type controls. Cortical glomeruli in (-/-) mice were variable in size and appeared immature (arrowhead). Juxtamedullary glomeruli appeared normal. In stark contrast to (+/+) kidneys in which glomeruli were buried within a layer of proximal and distal tubules located just below the capsule (Fig. 7C), the glomeruli of the (-/-) kidneys were found adjacent to the renal capsule, possibly suggesting loss of tubular mass (Fig. 7D, arrowhead).


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Fig. 7.   A: wild-type control kidney. B: AT1A receptor-deficient kidney showing atrophic tubules with flattened tubular epithelia, luminal dilatation, decreased number of tubules and thickened tubular basement membrane (arrows). C: wild-type control. D: AT1A receptor-deficient kidney showing focal areas of increased interstitial tissue (arrow). Cortical glomeruli are variable in size, and some appear immature (arrowhead). Juxtamedullary glomeruli appear to be normal. A and B: periodic acid-Schiff staining, magnification ×200. C and D: Trichrome staining, magnification ×400.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The RAS is one of the most powerful hormone systems for regulating blood pressure and body fluid volumes. Most RAS functions are exerted through ANG II actions on AT1 receptors. The direct vasoconstrictor effects of ANG II on blood pressure are closely intertwined with indirect control of volume homeostasis through effects on renal excretion of salt and water (7). The major objectives of this study were to examine the role of the AT1A receptor on sodium excretion, blood pressure, and the pressure-natriuresis relationship during chronically high and low sodium intakes in a mouse line that lacks the AT1A receptor. The results provide evidence supporting the notions that 1) blood pressure is regulated in a salt-sensitive manner in the absence of the AT1A receptor; 2) this regulation is dependent on changes mediated through the pressure-natriuresis relationship; and 3) the AT1A receptor is not required to maintain normal sodium homeostasis.

Our data show that (-/-) mice on a LS diet had a mean decrease in blood pressure of 11 mmHg compared with (-/-) mice on a NS diet whereas there were no differences in blood pressure in (+/+) mice on a LS diet. To determine whether the drop in blood pressure in AT1A (-/-) mice was due to differences in sodium balance, we compared the sodium excretion rates for these animals on a LS diet. When challenged, the (-/-) mice were able to reduce urinary sodium excretion about ninefold, from 3.42 µmol Na/mg creatinine on an NS diet to 0.40 µmol Na/mg creatinine on a LS diet. Sodium balance studies show that AT1A (-/-) mice can maintain a minimally positive sodium balance and maintain normal serum sodium when faced with sodium restriction. However, the sodium-restricted (-/-) mice had lower body weights at the end of the study, suggesting that the minimally positive sodium accretion rate may not have been sufficient to allow for normal growth. Alternatively, extracellular fluid volume in (-/-) mice could be decreased in the presence of sodium restriction. Lower body weights cannot be attributed to low caloric intake because there were no differences in the daily food consumption in (-/-) mice compared with their wild-type controls.

On a HS diet, the AT1A receptor-deficient mice excreted the sodium load similarly to their (+/+) littermates. Despite similar sodium excretions, blood pressure increased in the (-/-) mice from 87 mmHg (NS) to 103 mmHg (HS). It is possible that even in the face of elevated water excretion, the concomitant elevation of water intake in salt-loaded (-/-) mice resulted in net volume expansion and increased blood pressure. In our study, the body weights of (-/-) mice did not differ from either the (+/+) controls on the HS diet or the (-/-) mice on NS intake, suggesting that body water was not significantly altered. However, Cervenka et al. (2) showed that 7-8% body weight volume expansion elicited marked increases in sodium excretion and increases in blood pressure without changing body weight. Our animals could also have experienced clinically significant volume expansion without statistically evident changes in body weight. This possible explanation is additionally supported by Oliverio et al. (19), who also showed that a HS diet resulted in an increase in blood pressure in AT1A (-/-) mice to similar levels as the wild-type controls. The authors believed that these changes are due to alterations in effective blood volume, although further supporting evidence was not given. A further potential explanation is found in work by Inokuchi et al. (12) that showed that AT1A receptor-deficient mice have abnormal vasculature. While not thoroughly investigated in our experiments, our animals also exhibit intrarenal arterial wall thickening that may also alter resistance and blood pressure. Therefore, in this study, it is possible that the AT1A-deficient mice experienced volume expansion in a setting of abnormal vascular resistance, resulting in increased blood pressure.

While a pressure-natriuresis relationship is maintained in AT1A-deficient mice, the tight coupling between changes in blood pressure and changes in sodium excretion is significantly altered. The leftward shift in the pressure-natriuresis relationship demonstrated by AT1A (-/-) mice shows that 1) they are more natriuretic at lower blood pressures than (+/+) mice and 2) they require a larger change in blood pressure to excrete additional sodium compared with wild-type controls.

There are three predominant mechanisms involved in pressure-natriuresis: 1) proximal tubule sodium reabsorption, 2) medullary blood flow and 3) interstitial perfusion pressure, which is closely linked to the medullary circulation (7, 8, 10, 11). It is probable that a significant portion of the increased baseline natriuresis seen in AT1A-deficient mice is due to the loss of sodium reabsorptive capacity by the proximal tubule in the absence of the AT1 receptor. However, this should be balanced to some degree by increased sodium reabsorption in the distal tubule and collecting duct driven by aldosterone- and nonaldosterone-mediated effects on the collecting duct. In rodents, aldosterone secretion is mediated through both AT1A and AT1B receptors (3, 18). Therefore, the preservation of aldosterone regulation in the AT1A receptor-deficient mice should be maintained by the remaining AT1B receptors.

Another possible mechanism for the shift in the pressure-natriuresis relationship and increased natriuresis in AT1A-deficient mice is abnormal regulation of medullary blood flow. While medullary blood flow was not directly measured in our study, there are reasons to suspect it may be altered. Loss of the efferent arteriolar AT1A receptor is expected to result in an increase in medullary blood flow by dilation of the postglomerular circulation. In a pharmacological approach to this question, Cowley et al. (17) demonstrated an increase in medullary blood flow and sodium excretion and a drop in blood pressure after treatment with captopril in normal animals. Additionally, micropuncture studies have shown that AT1A receptor-deficient mice have a complete absence of tubuloglomerular feedback response, an important mediator of salt balance and autoregulation (21). Although not measured, our mice behave as if they have lost their autoregulatory response, resulting in a glomerular filtration rate and renal blood flow rates that are more pressure dependent, as demonstrated by the shifted pressure-natriuresis curve. Whether these AT1A-deficient mice have an inability to regulate medullary blood flow or alterations of interstitial pressure remains to be investigated.

As ANG II activation of the AT1 receptor at the juxtaglomerular apparatus suppresses renin release, we evaluated the expression of renin in mice lacking the AT1A receptor. Basal renin levels were elevated in the LS AT1A (-/-) mice, confirming AT1A receptor involvement in the negative feedback of renin release (1, 14, 24). Under LS conditions, the differences between these two groups are merely the deletion of the AT1A receptor and lower blood pressure in the (-/-) animals. Hence, the AT1A receptor is involved with the basal regulation of renin release, but regulation of renin is also mediated by baroreceptor mechanisms, chiefly, low blood pressure.

In contrast to previous studies, the blood pressures of our AT1A receptor-deficient mice on NS intakes were not lower than in the wild-type controls. The earlier reports utilized F2 generations of AT1A receptor-deficient mice that demonstrated blood pressures significantly lower than wild-type controls. The mice utilized in the present study have been backcrossed more than six generations into a C57BL/6 background, thereby reducing the genetic heterogeneity in the originally described mice. In the first descriptions of the AT1A knockouts, only minimal renal abnormalities were found. These changes included hypertrophy of the juxtaglomerular apparatus and proximal expansion of renin-producing cells along the afferent arterioles (20). In an attempt to ascertain the reasons behind the differences between our baseline blood pressure data and the previous reports, we evaluated the renal histology in the inbred AT1A line. Our inbred AT1A receptor-deficient mice exhibit more significant renal pathology, consisting of delayed glomerular maturity and tubular atrophy. These findings are similar in many aspects to the studies in which angiotensin production was inhibited by the angiotensin-converting enzyme or the deletion of the angiotensinogen gene (6, 15, 23, 25, 26). Hence, background genetic variability clearly affects the renal phenotype of these animals and may manifest physiologically as differences in blood pressure.

In contrast to previous studies that suggest AT1A deficiency results in renal salt wasting (19), our experiments revealed no differences between (+/+) and (-/-) mice with respect to sodium excretion. These results may differ due to the chronic nature of the dietary sodium manipulation in our study compared with the shorter time period in other studies. Careful correction of urinary sodium excretion for creatinine excretion, and/or the previously mentioned differences in renal histopathology, may also account for these differences. Clearly, our results indicate an ability to maintain normal sodium excretion in the absence of the AT1A receptor.

In summary, our results indicate that AT1A receptor-deficient mice are able to reduce urinary sodium excretion under conditions of sodium restriction and excrete a sodium load on an HS diet. However, AT1A receptor-deficient mice require a larger change in blood pressure to achieve the same degree of sodium excretion than their wild-type littermates. Maintenance of equivalent sodium excretion between (+/+) and (-/-) animals on LS, NS, and HS diets suggests that the AT1A receptor is not the primary mechanism by which chronic renal sodium balance is controlled. Compensatory mechanisms such as aldosterone and AT1B receptors are likely to be involved. Importantly, however, blood pressure directly correlates with salt intake in AT1A receptor-deficient mice, while blood pressure is unaffected by dietary sodium manipulation in wild-type mice. This is illustrated by a leftward shift in the pressure-natriuresis curve compared with that for the (+/+) control mice. Although, these hemodynamic effects may be the direct result of deletion of the AT1A receptor, the architectural abnormalities seen in these mice cannot be ruled out as contributory processes. This study confirms the significant role of the AT1A receptor in the pressure-natriuresis relationship.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Thomas Coffman (Dept. of Medicine, Duke University, Durham, NC) for the gift of AT1A receptor-deficient mice.


    FOOTNOTES

A. Mangrum was supported by National Institutes of Health Grant DK-22360. V. F. Norwood was supported by an American Heart Association Clinician Scientist Award (96004380). A. R. Gomez was supported by National Institutes of Health Grants DK-52613 and GM-20069. Portions of this work have been published previously in abstract form (J Am Soc Nephrol 9: 312A, 1998).

Address for reprint requests and other correspondence: A. Mangrum, Dept. of Internal Medicine, Div. of Nephrology, Univ. of Virginia Health Sciences Ctr., Box 133, Charlottesville, VA 22908 (E-mail: ajm7p{at}virginia.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

April 10, 2002;10.1152/ajprenal.00259.2001

Received 17 August 2001; accepted in final form 29 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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