INTRODUCTION

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SUBSTANTIAL EXPERIMENTAL evidence supports the existence of a specific and synergistic interaction between adenosine and angiotensin II in regulating renal vascular resistance. Early studies have shown that the renal vasoconstrictor effect of bolus injections of adenosine was less pronounced or absent in NaCl-loaded animals (17, 31). Furthermore, the vasoconstrictor response to an infusion of adenosine was markedly attenuated in dogs during converting enzyme inhibition, suggesting that an intact renin-angiotensin system is necessary for the expression of the vasoconstrictor effect of adenosine (7). Evidence that the interaction between adenosine and angiotensin II occurs at the receptor level is suggested by the observations that the renal vasoconstriction caused by single injections of adenosine or of the adenosine type 1 receptor (A₁R)-specific agonist N⁶-cyclohexyladenosine (CHA; Ref. 3) was attenuated by saralasin, an AT receptor antagonist (4, 29, 34). Nevertheless, the renal vasoconstriction caused by adenosine or CHA does not appear to be always modified by inhibition of AT receptors (10, 21).

The availability of mice with a targeted deletion in the angiotensin II type 1A (AT_1A) receptor gene offers a new opportunity to investigate the interaction between adenosine and angiotensin II in the complete absence of the AT_1A receptor (9). Previous studies from our laboratory have shown that the constrictor effect of CHA at the glomerular vascular pole can be studied in vivo by measuring the response of stop-flow pressure (P_SF) to an addition of CHA to the fluid perfusing the loop of Henle of the index nephron or of an adjacent nephron (28). In the current experiments we have examined the effect of local tubular CHA application on P_SF in AT_1A receptor-deficient mice. The results of these studies indicate that the decrease in P_SF following local administration of CHA was markedly blunted in these genetically altered animals. This observation is consistent with the notion that functional AT_1A receptors are required for the expression of the full constrictor potential of A₁R activation.

	METHODS

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Experiments were performed in a strain of AT_1A germ line null mutant mice that has been generated as described by Ito et al. (9). All animals used in this study (wild-type AT_1A +/+, heterozygous AT_1A +/, and homozygous AT_1A /) were derived from a heterozygous breeder pair from the Duke University colony. At weaning, animals were ear-tagged, and a short piece of the tail was clipped off. Genomic DNA was extracted from the tail samples with a standard procedure involving digestion with proteinase K and purification of DNA with ethanol. The genotype of each DNA sample was determined by testing for the presence of wild-type or modified AT_1A sequence using PCR. AT_1A gene-specific primers were selected in the region of the first exon of the AT_1A gene that is deleted in the knockout mice, resulting in a 433-bp PCR product in AT_1A +/+ and AT_1A +/, but not AT_1A /. The mutant AT_1A gene was detected with Neo^r-specific primers amplifying a 457-bp product of the neomycin resistance gene (Neo^r) in AT_1A +/ and AT_1A /, but not in AT_1A +/+. A third PCR for -globin served as control to validate similar amounts of DNA in each sample. The sequence of the oligonucleotide primers and their location in the published sequence are as follows: sense AT_1A, 5'-GTCAAGTGGATTTCGAATAGTGTCTG-3' (bp 220-245); antisense AT_1A, 5'-TCTCAGCATCGACCGCTAC-3' (bp 634-652) (13); sense Neo^r, 5'-ACAACAGACAATCGGCTGCTCTGATG-3' (bp 225-250); and antisense Neo^r, 5'-GTTCGCCAGGCTCAAGGCGCGCA-3' (bp 660-682) (1). Amplification was carried out for 30 cycles (denaturation at 94°C for 40 s, annealing at 58°C for 40 s, and extension at 72°C for 40 s, followed by a final extension at 72°C for 8 min). After genotype analysis was completed, animals were separated according to genotype and gender.

Animals were maintained on a standard rodent diet and tap water. Male mice in a weight range between 23 and 30 g were anesthetized with 100 mg/kg ip Inactin and 100 mg/kg im ketamine. Body temperature was maintained at 38°C by placing the animals on an operating table with a servo-controlled heating plate. The trachea was cannulated, and a stream of 100% oxygen was blown toward the tracheal tube throughout the experiment. The femoral artery was cannulated with hand-drawn polyethylene tubing for measurement of arterial blood pressure. The femoral vein was cannulated for an intravenous maintenance infusion of 2.25 g bovine serum albumin/100 ml saline at a rate of 0.5 ml/h. In addition to the five AT_1A +/+, three AT_1A +/, and five AT_1A / mice studied at ambient blood pressures, three wild-type mice were prepared in which arterial blood pressure was lowered to the mean level observed in AT_1A knockouts by injections of small amounts of Inactin.

The left kidney was approached from a flank incision, freed of adherent fat and connective tissue, and placed in a Lucite cup adapted for the size of the mouse kidney. The kidney was covered with mineral oil. Measurements of P_SF during loop of Henle perfusion were done by identification of a late proximal tubule segment from the staining pattern following microperfusion of a randomly selected proximal segment with the FD&C-stained perfusate. The tubule was blocked with wax, the pump was inserted in the last superficial proximal segment, and the pressure pipette was inserted into an early proximal segment recognizable from the widening of the tubular lumen. After P_SF had stabilized, loop of Henle perfusion rate was increased to 40 nl/min to assess the maximal tubuloglomerular feedback (TGF) response. The perfusion pipette was then withdrawn and replaced with a pipette containing CHA, and TGF responses to an increase in flow to 40 nl/min were tested again. Subsequently, the CHA-containing pipette was inserted into a randomly chosen proximal segment adjacent, but not belonging to the index nephron. CHA solution was infused into the freely flowing tubule at a rate of 40 nl/min, and P_SF in the index nephron was recorded with its loop of Henle not being perfused. Control perfusate contained (in mM/l) 136 NaCl, 4 NaHCO₃, 4 KCl, 2 CaCl₂, 7.5 urea, 100 mg/100 ml FD&C green (Keystone), and 1 g/100 ml ethanol as a solvent control. CHA was prepared as a 10⁴ M solution in artificial tubular fluid (ATF) containing 10 g/100 ml ethanol and diluted 10-fold with ATF to yield a concentration of 10⁵ M. CHA-containing solutions were made fresh for each experiment. At the end of each experiment, the blood pressure response to intravenous angiotensin II (1, 5, and 10 ng in AT_1A +/+ and AT_1A +/; and 10, 50, and 100 ng in AT_1A / mice) was tested to functionally verify the animal's genotype.

Adenosine type 1 receptor mRNA. To determine whether AT_1A knockout mice express A₁R mRNA, we extracted total RNA from the cortex of kidneys harvested from AT_1A +/+ and AT_1A / mice. Methods for RNA extraction, cDNA synthesis, and RT-PCR have been described in detail in several earlier publications from this laboratory (35). The sequence of the oligonucleotide primers used for the amplification of the A₁R product was as follows: sense A₁R, 5'-GCA TGG AGT ACA TGG TCT AC-3' (bp 832-851), and antisense A₁R, 5'-AGT CCT CAG CTT TCT CCT CT-3' (bp 1266-1285) (11). PCR products of the expected size of 453 bp have been amplified with these primers in cDNA from both rats and mice, and they have been identified as part of the A₁R cDNA by restriction enzyme analysis.

Statistical comparisons were made with ANOVA in association with the Scheffé test, or the Student's t-test, using paired or unpaired comparisons, as appropriate.

	RESULTS

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Mean body weights and mean kidney weights were 25.4 ± 0.6 and 0.18 ± 0.01 g in five wild-type mice, 29.0 ± 1.6 and 0.17 ± 0.02 g in four heterozygous mice, and 27.6 ± 2.06 and 0.2 ± 0.02 g in five homozygous mice of the AT_1A receptor knockout strain generated by Ito et al. (9). Mean arterial blood pressure was 96.9 ± 2.8 mmHg in wild-type (range 77-108 mmHg), 97.7 ± 3.3 mmHg in heterozygous (range 77-110 mmHg), and 80 ± 1.9 mmHg in homozygous mice (range 64-97 mmHg). Similar to earlier observations in both conscious and anesthetized animals (9, 15, 28), mean arterial pressures in wild-type and heterozygote mice were significantly higher than in homozygous AT_1A knockout mice (P < 0.001). Blood pressure responses to exogenous angiotensin II, assessed at the end of the micropuncture experiments, were greatly attenuated in homozygous knockout animals. Bolus injections of 1, 5, and 10 ng angiotensin II increased mean arterial blood pressure by 11.3 ± 6, 31.7 ± 6.2, and 43.2 ± 9.9 mmHg in wild-type mice and by 8 ± 1.05, 15.3 ± 4.7, and 22.5 ± 6.6 mmHg in heterozygous mice. In AT_1A / mice, injections of 10, 50, and 100 ng angiotensin II were associated with mean pressure changes of 0.5 ± 0.6, 3.4 ± 1.4, and 0.3 ± 3.8 mmHg, respectively.

Table 1 summarizes measurements of P_SF during perfusion of the loops of Henle of both index and adjacent nephrons in the absence and presence of 10⁵ M CHA. In previous studies in rats, this dose of CHA had been found to produce the largest reductions in P_SF (22). Mean P_SF fell significantly in both AT_1A +/+ and AT_1A +/ mice when loop perfusion of the index nephron was increased from 0 to 40 nl/min using control Ringer solution. In agreement with our earlier observations, P_SF at zero loop flow was lower in AT_1A / mice than in wild-type mice, and there was no change in P_SF in response to loop perfusion at 40 nl/min (28). Perfusion of nephrons adjacent to the index nephrons with control solution did not cause significant changes in P_SF in any of the mice regardless of genotype. Adding CHA to the perfusate markedly augmented the fall of P_SF during perfusion of both index and adjacent nephrons in both AT_1A +/+ and AT_1A +/ mice. In AT_1A / mice, a small and significant change of P_SF was noted when CHA was present in the perfusate of index or adjacent nephrons, but these changes were significantly less than in AT_1A +/+ mice (P < 0.0001 for index or adjacent nephron comparison). Original recordings of the P_SF response to control Ringer perfusion without and with CHA in a wild-type and a homozygous knockout mouse are shown in Fig. 1. Figure 2 graphically depicts P_SF changes caused by increasing loop flow from 0 to 40 nl/min. It can be seen that the effect of CHA to augment TGF responses of P_SF was significantly diminished in both heterozygous and homozygous AT_1A transgenic compared with wild-type mice. Attenuation of the CHA response was found both in the presence (index nephron) and absence (adjacent nephron) of a superimposed TGF response.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Original recordings of stop-flow pressure (PSF; top tracing in each set) during changes of loop of Henle perfusion rate in an AT1A +/+ (top two tracings) and an AT1A / (bottom two tracings) mouse. During the time periods marked by the black bars, tubules were perfused at a rate of 40 nl/min with control Ringer solution or with control Ringer containing 105 M N6-cyclohexyladenosine (CHA). Perfusion was either into the index nephron or into a nephron adjacent to the index nephron. Bottom tracing in each pair represents arterial blood pressure (AP).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Mean changes in PSF in response to loop perfusion with control Ringer solution or with control Ringer + CHA (105 M) in AT1A +/+, AT1A +/, and AT1A / mice. A: infusion into the loop of Henle of the index nephron. B: infusion into the loop of Henle of an adjacent nephron. Error lines indicate SE (where line is missing, SE is smaller than the line). * P < 0.05 and ** P < 0.01 by ANOVA (comparison given is with PSF change in AT1A +/+ mice).

To examine the effect of arterial blood pressure per se on the vascular response to CHA, studies were performed in which blood pressure in three wild-type mice was reduced by supplemental doses of Inactin. Mean arterial blood pressure in these animals averaged 82.8 ± 1.3 mmHg, similar to mean blood pressures found in the AT_1A knockout mice (80 ± 1.9 mmHg). When perfusion rate was increased to 40 nl/min P_SF fell by 4.5 ± 0.3 mmHg, from 38.6 ± 2.5 to 34.1 ± 2.2 mmHg (n = 9; P = 0.06 compared with wild-type mice at ambient blood pressure). In the presence of CHA (10⁵ M) in the perfusate of the index nephron, P_SF fell by 9.4 ± 0.4 mmHg, from 35.7 ± 2 to 26.2 ± 1.8 mmHg (P = 0.0023 compared with mice at ambient blood pressures; P < 0.0001 compared with knockout mice).

Expression of A₁R mRNA in renal cortical tissue as determined by RT-PCR was not found to be different between AT_1A +/+ and AT_1A / animals. An example showing PCR products amplified from four different cDNA samples from AT_1A null transgenic and control mice is given in Fig. 3. Relative band intensities corrected for -actin were 0.98 ± 0.21 in AT_1A +/+ and 1.01 ± 0.23 in AT_1A / mice, not significantly different between the two strains of mice.

View larger version (55K): [in this window] [in a new window]   Fig. 3.   Phosphoimages of adenosine type 1 (A1) receptor and -actin RT-PCR products from cortical tissue of AT1A +/+ and AT1A / mice. Results are from 4 cDNA samples, with each sample being amplified at two cDNA dilutions (1× and 10× for A1 receptor, and 100× and 1,000× for -actin).

	DISCUSSION

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The current experiments show that the effects of CHA on P_SF in wild-type mice are qualitatively and quantitatively similar to those observed earlier in rats (5, 22, 27). When CHA was included in the perfusate of the index nephron, the fall in P_SF in response to a saturating flow increase was significantly larger than that caused by TGF alone. Furthermore, addition of CHA to the proximal fluid of an adjacent nephron caused a marked reduction of P_SF in the neighboring index nephron. Since the loop of Henle of the index nephron was not perfused at this time, the enhanced P_SF response occurred in the absence of TGF activation. These results are difficult to reconcile with the assumption that the CHA effect on glomerular arterioles is mediated through luminal A₁Rs, but suggests rather that CHA at least during adjacent nephron perfusion interacts with extratubular, presumably vascular receptors. A high density of A₁Rs in the terminal afferent arteriole is suggested by in situ hybridization of A₁R mRNA and by functional studies in isolated perfused afferent arterioles (32, 33).

The main finding in the present study is that the P_SF response magnitude to local administration of the A₁ agonist CHA was significantly reduced in both AT_1A +/ and AT_1A / mice compared with wild-type controls. The reason for the attenuation of A₁R-mediated vasoconstriction in AT_1A knockout mice is not clear, but a number of possibilities may be considered. Chronic absence of AT_1A receptor activity may cause downregulation of A₁R expression, but our assessment of A₁R mRNA abundance does not support this assumption. Alternate splicing can lead to variant transcripts of adenosine A₁R mRNA (14). However, it would seem highly speculative to assume that AT_1A mutant mice express an A₁R variant that is more angiotensin II dependent than that found in wild-type mice. Previous studies have shown that the renal vasoconstriction caused by adenosine was greatly attenuated in rats in which renal perfusion pressure had been reduced to 70 mmHg (6). Since AT_1A receptor knockout mice had a significantly lower blood pressure than wild-type mice, we examined whether lowering of arterial pressure per se could be accountable for the reduced response of P_SF to CHA in these animals. Both native TGF responses and CHA-induced constrictor responses were in fact found to be smaller in magnitude than those observed in wild-type mice at ambient blood pressures. However, CHA-induced responses were significantly larger than in knockout mice, suggesting that the diminished CHA constriction in the mutant animals for the most part was not a consequence of the lower blood pressure. Finally, in view of the abnormalities in renal vascular morphology described in angiotensin converting enzyme knockout mice it seems possible that structural alterations in the glomerular microvasculature of AT_1A receptor knockout mice may prevent vasoconstrictor responses in general (8). However, such structural changes have not been described in the heterozygous mice, suggesting that the significantly reduced constrictor responses to CHA in AT_1A +/ animals have some functional cause. Overall, data are compatible with the view that the blunting of constrictor responses to CHA is another expression of the specific and synergistic interaction between angiotensin II and adenosine that has been observed in earlier experiments using different approaches.

A regulatory mechanism in which adenosine and angiotensin II appear to interact to cause afferent arteriolar vasoconstriction is TGF, the response of afferent arteriolar resistance to changes in NaCl concentration at the macula densa (2). Nonspecific as well as adenosine A₁R-specific inhibitors were noted to markedly blunt the efficiency of signal transmission (5, 16, 23, 27). Inhibition of TGF responses was also caused by interference with the generation or action of angiotensin II, whereas peritubular or intravenous infusion of angiotensin II caused an augmentation of TGF responses (12, 18-20, 24-26, 30). Furthermore, results in a recent report as well as our current findings show that TGF responses are essentially obliterated in AT_1A receptor knockout mice (28). The coincidence of absent TGF responses and blunted responses to CHA in the AT_1A receptor-deficient mice is consistent with the view that an interaction between adenosine and angiotensin II is a major cause for macula densa-controlled changes in afferent arteriolar tone.

In summary, the present experiments show a marked reduction in the response of the glomerular vasculature to the constrictor effect of the adenosine A₁R agonist CHA in mice both heterozygous or homozygous for an AT_1A receptor knockout mutation. These data support the notion that adenosine-induced vasoconstriction of afferent arterioles is synergistically enhanced by angiotensin II. Furthermore, the coincident absence of both TGF responses and of normal constrictor responses to adenosine A₁R activation in AT_1A-deficient mice is consistent with the notion that adenosine A₁ and AT_1A receptors interact to induce macula densa-dependent regulation of afferent arteriolar tone.