Preserved macula densa-dependent renin secretion in A1 adenosine receptor knockout mice

Frank Schweda1, Charlotte Wagner1, Bernhard K. Krämer2, Jürgen Schnermann3, and Armin Kurtz1

1 Institut für Physiologie and 2 Klinik und Poliklinik für Innere Medizin, Universität Regensburg, 93040 Regensburg, Germany; and 3 National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recent studies demonstrated that the influence of the macula densa on glomerular filtration is abolished in adenosine A1 receptor (A1AR) knockout mice. Inasmuch as the macula densa not only regulates glomerular filtration but also controls the activity of the renin system, the present study aimed to determine the role of the A1AR in macula densa control of renin synthesis and secretion. Although a high-salt diet over 1 wk suppressed renin mRNA expression and renal renin content to similar degrees in A1AR+/+, A1AR+/-, and A1AR-/- mice, stimulation of Ren-1 mRNA expression and renal renin content by salt restriction was markedly enhanced in A1AR-/- compared with wild-type mice. Pharmacological blockade of macula densa salt transport with loop diuretics stimulated renin expression in vivo (treatment with furosemide at 1.2 mg/day for 6 days) and renin secretion in isolated perfused mouse kidneys (treatment with 100 µM bumetanide) in all three genotypes to the same extent. Taken together, our data are consistent with the concept of a tonic inhibitory role of the A1AR in the renin system, whereas they indicate that the A1AR is not indispensable in macula densa control of the renin system.

loop diuretics; low salt; high salt


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE KIDNEYS PLAY A KEY ROLE in maintenance of fluid and electrolyte balance of the body as well as in blood pressure regulation. Multiple extra- and intrarenal factors cooperate in the adjustments of renal function that underlie body fluid homeostasis. A specific intrarenal control element for NaCl excretion is the juxtaglomerular apparatus, the anatomic substrate of a mechanism in which changes in tubular salt delivery are sensed and translated to changes in afferent arteriolar tone [tubuloglomerular feedback (TGF)] and renin synthesis and secretion (macula densa-mediated renin release). An increase in NaCl concentration at the macula densa results in vasoconstriction of the afferent arteriole, reducing glomerular filtration rate and tubular salt load, and inhibition of the renin-angiotensin system; a decrease in macula densa NaCl concentration has the opposite effect. The nature of the extracellular signaling events between macula densa cells and vascular smooth muscle or renin-producing effector cells is still a matter of debate. Besides cyclooxygenase-2-derived prostanoids (3, 9, 10, 12, 16, 31), the nucleoside adenosine has been proposed to be centrally involved in macula densa control of the renin system and glomerular filtration, inasmuch as adenosine is an inhibitor of the renin system as well as a vasoconstrictor of the afferent arteriole (13, 14), both effects being mediated by the A1 adenosine receptor (A1AR). The inhibitory effect of the A1AR on the renin system has been demonstrated in vitro and in vivo, inasmuch as selective A1AR agonists suppress renin secretion and pharmacological blockade of the A1AR results in stimulation of renin secretion (1, 5, 6, 17, 24). The putative role of adenosine in the salt-dependent regulation of the renin system is underlined by several studies suggesting a relationship between tubular salt load and adenosine concentration in the kidney. Infusion of hypertonic saline or a high dietary sodium intake, both of which are associated with an inhibition of the renin system, led to elevated adenosine concentrations in the kidney (23, 27, 37). In contrast, dietary sodium restriction, known to stimulate the renin system, resulted in reduced renal interstitial concentrations of adenosine (27). Therefore, an increase in adenosine concentrations due to a high tubular salt load could mediate vasoconstriction and inhibition of the renin system, whereas a decrease in renal adenosine concentration resulting from a reduced salt load could cause vasodilatation and stimulation of the renin system. The A1AR agonist cyclohexyladenosine (CHA) suppressed stimulation of renin secretion in response to a perfusion medium containing a low NaCl concentration in the isolated juxtamedullary apparatus, and blockade of the A1AR diminished the reduction in renin secretion caused by high luminal NaCl concentrations (35), supporting the involvement of adenosine in macula densa control of the renin system. However, in a similar experimental setup, application of exogenous adenosine did not fully mimic the inhibitory effects of increasing tubular NaCl concentration on renin secretion (19), which would be expected from a mediator of macula densa control of the renin system.

Recent investigations have provided direct evidence that adenosine is required for the vasoconstriction caused by TGF. Consistent with earlier studies showing that inhibition of adenosine production (29) or selective blockade of the A1AR (26, 36) blunts TGF, mice with a genetic deletion of the A1AR lack the TGF response to an increase in tubular NaCl concentration (2, 28). Inasmuch as the effector cells of the TGF, namely, the vascular smooth muscle cells of the afferent arteriole, are located in the immediate vicinity of the renin-producing juxtaglomerular cells, it is reasonable to assume that adenosine mediating the TGF also influences the renin system. Therefore, the present experiments were performed to determine whether adenosine and A1AR may be centrally involved in macula densa control of the renin system. Utilizing A1AR knockout mice, we determined whether the absence of the A1AR is associated with altered expression or secretion of renin consistent with tonic inhibition of the renin-angiotensin system by adenosine. Furthermore, the macula densa mechanism is believed to be critically involved in adjustment of the renin system to different salt loads of the body, with a high sodium intake inhibiting and a low sodium intake stimulating the renin system (8, 18, 25, 33). Pharmacological blockade of macula densa NaCl transport with loop diuretics is an intervention that, similar to a low-salt diet, stimulates the renin-angiotensin system by diminishing the NaCl transport-dependent, renin-inhibitory signal to the granular cells (11). Therefore, we investigated the influence of a high- and a low-salt diet and furosemide on the renin system in mice with a genetic deletion of the A1AR. Finally, to assess the more acute modulation of renin secretion by the macula densa, we investigated the effects of the loop diuretic bumetanide on the rate of renin secretion in isolated perfused kidneys of A1AR knockout mice and their wild-type controls. The isolated perfused kidney model is ideally suited to investigate renin secretion in the absence of interindividual differences in systemic factors that may influence the renin system, such as variations in blood pressure or renal sympathetic nerve activity, for example.


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

A1AR knockout mice. A1AR knockout mice were generated as described by Sun et al. (28). The mice were derived from two heterozygous breeder pairs. For genotyping, tail biopsies were performed, and DNA was extracted and tested for the presence of wild-type and mutant genes using A1AR- and Neo-R-specific PCR primers (28).

Experimental procedures in vivo. In the first set of experiments, 10 mice of each genotype (A1AR+/+, A1AR+/-, and A1AR-/-, 20-24 g body wt) were fed a low-salt (0.02% NaCl) or a high-salt (8% NaCl) diet for 7 days. As controls, 10 mice of each genotype were fed normal mouse chow (0.6% NaCl).

In the second set of experiments, five mice of each genotype were treated with furosemide (1.2 mg/day; Dimazon, Intervet) administered via osmotic minipumps (Alzet, Durect) for 6 days. As controls, five mice of each genotype were infused with physiological saline. Surgical insertion of the pumps was performed under inhalation anesthesia (Sevofluran, Abbot). The mice had free access to standard mouse chow (0.6% NaCl), tap water, and an electrolyte solution containing 0.9% NaCl and 0.1% KCl.

After the experimental periods, the animals were killed by decapitation, and blood was collected for determination of serum electrolyte concentration by flame photometry (model PFP7, Jenway, Dunmow). Kidneys were removed rapidly, frozen in liquid nitrogen, and stored at -80°C until further processing.

Determination of preprorenin mRNA and cytosolic beta -actin by RNase protection assay. After isolation of total RNA from the frozen kidney using the method of Chomczynski and Sacchi (4), renin was measured by an RNase protection assay using an antisense RNA probe suitable for detecting mRNA levels from the Ren-1 and Ren-2 genes as described previously (32). Cytosolic beta -actin was measured by an RNase protection assay as described elsewhere (32). For semiquantification of Ren-1 and Ren-2 mRNA abundance, the hybridization signals were related to those obtained for beta -actin mRNA. beta -Actin mRNA levels were not different between the different genotypes and the different experimental maneuvers (not shown).

Determination of renal renin content. The renal renin content was determined by measuring the capacity of homogenized kidneys to generate angiotensin I according to a modification of the method described by Norling et al. (22). Frozen kidneys were halved, homogenized in 1 ml of homogenization buffer [5% (vol/vol) glycerol, 0.1 mM phenylmethylsulfonyl fluoride, 10 mM EDTA, and 0.1 mM 4-(2-aminomethyl)benzenesulfonyl fluoride] for 30 s (Ultra-Turrax, IKA Labortechnik), and centrifuged at 4°C at 14,000 g for 5 min. The supernatants were frozen at -20°C and then thawed three times by alternating the temperature between -20°C and 4°C. Supernatants were incubated with saturating concentrations of rat renin substrate, and the generated angiotensin I was assayed with a commercial radioimmunoassay kit (Byk and DiaSorin).

Isolated perfused mouse kidney. Male A1AR-/-, A1AR+/-, and A1AR+/+ mice (20-23 g body wt) with free access to commercial pellet chow and tap water were used as kidney donors. The animals were anesthetized with an intraperitoneal injection of 5-ethyl-5-(1-methylbutyl)-2-thiobarbituric acid (100 mg/kg; Trapanal, Byk Gulden) and ketamine HCl (80 mg/kg; Curamed, Germany) and placed on a heating table. The abdominal cavity was opened by a midline incision, and the aorta was clamped distal to the right renal artery so that the perfusion of the right kidney was not disturbed during the subsequent insertion of the perfusion cannula into the abdominal aorta distal to the clamp. The mesenteric artery was ligated, and a metal perfusion cannula (0.8 mm OD) was inserted into the abdominal aorta. After removal of the aortic clamp, the cannula was advanced to the origin of the right renal artery and fixed in this position. The aorta was ligated proximal to the right renal artery, and perfusion was started in situ with an initial flow rate of 1 ml/min. With the use of this technique, a significant ischemic period of the right kidney was avoided. Finally, the right kidney was excised, placed in a thermostated moistening chamber, and perfused at constant pressure (100 mmHg). Perfusion pressure was monitored within the perfusion cannula (Isotec pressure transducer, Hugo Sachs Elektronik), and the pressure signal was used for feedback control (model SCP 704, Hugo Sachs Elektronik) of a peristaltic pump. Finally, the renal vein was cannulated (1.5-mm-OD polypropylene catheter). The venous effluent was drained outside the moistening chamber and collected for determination of renin activity and venous blood flow.

The basic perfusion medium, supplied from a thermostated (37°C) 200-ml reservoir, consisted of a modified Krebs-Henseleit solution containing all physiological amino acids at 0.2-2.0 mM, 8.7 mM glucose, 0.3 mM pyruvate, 2.0 mM L-lactate, 1.0 mM alpha -ketoglutarate, 1.0 mM L-malate, and 6.0 mM urea. The perfusate was supplemented with 6 g/100 ml bovine serum albumin, 1 mU/100 ml vasopressin 8-lysine, and freshly washed human red blood cells (10% hematocrit). Ampicillin (3 mg/100 ml) and flucloxacillin (3 mg/100 ml) were added to inhibit possible bacterial growth in the medium. To improve the functional preservation of the preparation, the perfusate was continuously dialyzed against a 10-fold volume of the same composition but without erythrocytes and albumin. For oxygenation of the perfusion medium, the dialysate was gassed with 94% O2-6% CO2. Perfusate flow was calculated by collection and gravimetric determination of the venous effluent. Perfusion pressure was continuously monitored by a potentiometric recorder.

After constant perfusion pressure was established, perfusate flow rates usually stabilized within 15 min. Stock solutions of the drugs to be tested were added to the dialysate.

For determination of perfusate renin activity, venous effluent was collected over a period of 1 min at intervals of 3 min. The samples were centrifuged at 1,500 g for 10 min, and the supernatants were stored at -20°C until assayed for renin activity. For determination of renin activity, the perfusate samples were incubated for 1.5 h at 37°C with plasma from bilaterally nephrectomized male rats as renin substrate. The generated angiotensin I (ng · ml-1 · h-1) was determined by radioimmunoassay (Byk and DiaSorin). Renin secretion rates were calculated as the product of the renin activity and the venous flow rate (ml · min-1 · g kidney wt-1).

Statistical analysis. Values are means ± SE. Differences between groups were analyzed by ANOVA and Bonferroni's adjustment for multiple comparisons. In the isolated perfused kidney experiments, all values obtained within an experimental period (n = 4) were averaged and compared with the average values of an adjoining experimental period. Student's paired t-test was used to calculate levels of significance within individual kidneys. P < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Serum concentrations of sodium, chloride, or potassium were not different between any of the genotypes or the treatment groups (not shown).

Basal renin expression. Basal renal renin content was 1.5-fold higher in A1AR-/- than in wild-type mice (Fig. 1A).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1.   Renal renin expression of A1 adenosine receptor (A1AR)-/- (n = 8), A1AR+/- (n = 18), and A1AR+/+ (n = 7) mice under basal conditions. A: renal renin content of A1AR-/-, A1AR+/-, and A1AR+/+ mice under basal conditions. B: autoradiography of a renin RNase protection assay using an antisense probe that is able to discriminate between Ren-1 and Ren-2 mRNA. C: semiquantification of Ren-1 and Ren-2 mRNA hybridization signals. NS, not significant.

For determination of the renal renin mRNA abundance, we used an RNase protection assay with an antisense probe that was able to discriminate between Ren-1 and Ren-2 mRNA. Autoradiographic band intensity of the RNase protection assays (Fig. 1B) as well as semiquantification of renin mRNA expression by beta -actin correction (Fig. 2C) revealed a similar abundance of Ren-1 mRNA in each of the genotypes. In contrast, Ren-2 mRNA expression showed distinct differences between the genotypes: whereas the expression levels of Ren-1 and Ren-2 were similar in A1AR-/- mice, Ren-2 mRNA was not found in A1AR+/+ mice. A1AR+/- mice showed an intermediate abundance of Ren-2 gene expression: Ren-2 mRNA levels were ~50% of Ren-1 mRNA levels.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.   Renin mRNA expression in A1AR-/-, A1AR+/-, and A1AR+/+ mice fed a high- or a low-salt diet. Controls were fed a normal-salt diet. Values are means ± SE (n = 10). *P < 0.05; #P < 0.001 vs. normal salt.

Effect of a high-salt diet on renin expression. A high-salt diet for 1 wk resulted in inhibition of Ren-1 mRNA expression irrespective of the genotype (0.6-, 0.54-, and 0.66-fold of control for A1AR-/-, A1AR+/-, and A1AR+/+, respectively, all P < 0.05; Fig. 2A). Moreover, Ren-2 mRNA levels were suppressed by the high-salt diet in the kidneys of A1AR-/- and A1AR+/- mice, whereas no Ren-2 mRNA signal was detectable in the kidneys of A1AR+/+ mice (Fig. 2B). According to the changes in Ren-1 and Ren-2 gene expression, total renin mRNA expression was significantly suppressed by a high-salt diet in all three genotypes (Fig. 2C). In parallel with the changes in renin gene expression, renal renin content was lowered to ~0.6-fold of control by a low-salt diet in A1AR-/-, A1AR+/-, and A1AR+/+ mice (P < 0.05; Fig. 3A).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Renal renin content in A1AR-/-, A1AR+/-, and A1AR+/+ mice. A: effects of high-, normal-, or low-salt diet. B: effects of treatment with vehicle or furosemide via osmotic minipumps. *P < 0.05; #P < 0.001 vs. control (normal-salt diet or vehicle treatment).

Effect of a low-salt diet on renin expression. Dietary salt restriction stimulated the renin system in all three groups of animals. However, in contrast to the changes due to a high-salt diet, there were marked differences between the genotypes: stimulation was most pronounced in A1AR-/- mice, in which Ren-1 mRNA levels increased 2.5-fold compared with control animals fed a normal-salt diet, whereas in A1AR+/+ mice only a 1.2-fold increase was detected. A1AR+/- mice showed an intermediate stimulation of renin mRNA, with Ren-1 mRNA levels showing a twofold upregulation (Fig. 2A).

Similar to the expression of Ren-1, Ren-2 mRNA expression levels were augmented by a low-sodium intake in A1AR-/- and A1AR+/- mice. In the kidneys of wild-type mice, no Ren-2 signal was detectable, even in animals fed the low-salt diet (Fig. 2B). As a result of the differences in Ren-2 expression, total renin mRNA was significantly higher in A1AR-/- than in A1AR+/- mice and wild-type controls (Fig. 2B).

In parallel with renal renin gene expression, salt restriction caused a twofold increase in renal renin content in A1AR-/- and A1AR+/- mice compared with control values. In contrast, in A1AR+/+ mice, in which total renin gene expression was only slightly stimulated by the low-salt diet, no significant stimulation of renal renin content was detected (Fig. 3A).

Effect of furosemide on renin expression. Blockade of thick ascending limb and macula densa salt transport with furosemide administration for 6 days stimulated Ren-1 expression in A1AR-/-, A1AR+/-, and A1AR+/+ mice to similar degrees, so that no differences in Ren-1 expression exist between genotypes (Fig. 4). Again, Ren-2 mRNA was not detectable in A1AR+/+ mice, whereas it was significantly stimulated in A1AR-/- and A1AR+/- mice (Fig. 4). Total renin mRNA expression was stimulated by furosemide in all groups, with the highest abundance detectable in the A1AR-/- mice. Furosemide also augmented renal renin content in all three groups of mice to a similar extent, so that no significant differences were detected between the different genotypes (Fig. 3C).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   Renin mRNA expression in A1AR-/-, A1AR+/-, and A1AR+/+ mice treated with vehicle or furosemide. Values are means ± SE (n = 5). *P < 0.05; #P < 0.001 vs. vehicle.

Effect of bumetanide administration on renin secretion in isolated perfused mouse kidneys. To investigate the acute effects of loop diuretics on renin secretion, we adapted the model of the isolated perfused rat kidney to the anatomic conditions of mice. This model allows us to study the acute regulation of renin secretion without interference by confounding systemic side effects of the experimental drug or systemic counterregulations. Basal renin secretion rates of isolated perfused kidneys were similar in A1AR-/-, A1AR+/-, and A1AR+/+ mice (Fig. 5). Blockade of thick ascending limb and macula densa salt transport by bumetanide resulted in significant and comparable increases in renin secretion in A1AR+/+, A1AR+/-, and A1AR-/- mice: 3.1-, 3.0-, and 2.8-fold of control, respectively. During subsequent administration of the A1AR agonist CHA, renin secretion rates returned to basal levels in kidneys of A1AR+/+ and A1AR+/- mice, whereas CHA was without effect in A1AR-/- mice (Fig. 5).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of blockade of macula densa salt transport and subsequent administration of cyclohexyladenosine (CHA) on renin secretion rates of isolated perfused kidneys in A1AR+/+, A1AR+/-, and A1AR-/- mice. Values are means ± SE (n = 5).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present experiments in A1AR knockout mice aimed to assess the chronic role of A1AR in the renal expression of renin under basal conditions as well as in the macula densa control of the renin system. Previous pharmacological studies provided evidence for a direct inhibitory role of adenosine on renin expression and renin secretion, an effect that appeared to be mediated by A1AR (1, 5, 15, 17, 24, 35). The present observation that renal renin content under basal conditions is elevated in A1AR knockout mice supports the concept of a tonic inhibition of the renin-angiotensin system through A1AR mediation. However, besides the direct disinhibition of the renin system by A1AR deletion, an enhanced sodium excretion reported previously in A1AR-/- mice (2) as well as after acute pharmacological blockade of A1AR (36) might also account, in part, for the higher renin content in A1AR-/- mice. The finding that A1AR-/- mice possess two renin genes (Ren-1d and Ren-2), whereas wild-type mice harbor only one renin gene (Ren-1c), and that this discrepancy is related, as discussed in detail below, to the different mouse strains used in the generation of the knockout mice somewhat complicates the straightforward interpretation of our data. Inasmuch as, in general, plasma renin activities and concentrations appear to be markedly higher in two-renin than in one-renin gene strains (20, 34), it is conceivable that the higher renin content in the A1AR-/- animals is the consequence of their expression of Ren-1d and Ren-2. However, the differences in Ren-2 expression do not explain the marked enhancement of Ren-1 mRNA stimulation in A1AR-/- animals by a low-salt diet, so this result further supports the concept of a tonic inhibitory role of A1AR in the renin system.

The main intention of our study was to clarify the specific role of A1AR in the macula densa control of the renin system. The rationale for the assumption of a central role of the A1AR in this process was as follows: 1) adenosine inhibits the renin system via the A1AR, 2) adenosine concentration in the kidney changes in parallel with the sodium load of the kidney, and 3) A1AR is essentially required for control of glomerular filtration by the macula densa. According to the hypothesis that the macula densa-controlled changes in renin expression and secretion are related to salt-dependent changes in the intrarenal adenosine concentration, the amplitude of the inhibition of the renin system due to a high-salt diet or the stimulation due to a low-salt diet should be attenuated or even blunted in mice lacking the A1AR. However, our results demonstrate that a high-salt diet suppressed renal renin mRNA expression and renal renin content to the same extent in A1AR-/- and A1AR+/+ mice, clearly arguing against a role of the A1AR in mediation of this process. Moreover, stimulation of the renin system by a low-salt diet was not diminished, but was even enhanced, by the genetic deletion of the A1AR, a further result that is not compatible with a role of A1AR in mediation of this stimulation. If stimulation of renal renin content and mRNA expression by the low-salt diet were related to the known decrease in renal adenosine concentration and the subsequent disinhibition of the A1AR, this should not be possible in mice lacking this receptor. However, as stated above, the pronounced stimulation of Ren-1 mRNA expression due to salt restriction is highly consistent with a tonically inhibitory role of the A1AR on the renin system, which is absent in A1AR-/- mice. The conclusion that the A1AR is not causally involved in regulation of the renin system by the macula densa is further supported by the intact stimulation of the renin gene expression and renin content by blockade of the macula densa salt transport with furosemide in A1AR-/- animals. Interpretation of the in vivo data is limited by the fact that salt restriction or furosemide treatment might affect renin expression through pathways independent from or in addition to the macula densa mechanism, for example, by alterations in blood pressure or in sympathetic nervous system activity. We therefore investigated the effects of loop diuretics on renin secretion in the isolated perfused kidney model. In this preparation, administration of loop diuretics would appear to act solely through the macula densa, because perfusion pressure is experimentally controlled and changes in sympathetic nerve activity are unlikely. Even under these experimental conditions, bumetanide stimulated renin secretion to the same extent in kidneys of A1AR-/- mice and their wild-type controls, arguing against a role of this receptor in mediation of this process. However, the complete reversal of the stimulated renin secretion by the selective A1AR agonist CHA in A1AR+/+ and A1AR+/- mice underlines the direct suppressive effects of the A1AR on renin secretion, as has been demonstrated in previous studies (1, 5, 6, 15, 19). Besides the advantages of constant experimental conditions, the isolated perfused mouse kidney model is suitable for use in investigating the acute effects of an inhibition of macula densa salt transport on renin secretion and, therefore, in examining the renin system in a time frame similar to that used in the studies demonstrating the absence of a TGF response in A1AR-/- mice (2, 28). Because the TGF response has been found to be abolished by pharmacological blockade or genetic deletion of the A1AR (2, 26, 28, 29, 36), regulation of glomerular filtration rate and control of the renin system by the macula densa appear to follow different pathways.

A further interesting result of our study is the discovery of a linkage between the A1AR mutation and the renin gene locus that causes homozygosity in the A1AR knockout genotype to be invariably associated with the two-renin gene constellation. In contrast, the wild-type phenotype, homozygous for the absence of the A1AR mutation, always contains a single renin gene. The foundation for this linkage is the fact that the genes encoding the A1AR and renin are localized on chromosome 1 in close vicinity, as first shown in humans (7, 30). Analysis of available mouse genomic sequences has confirmed that the renin and A1AR genes in the mouse are also located on chromosome 1 in relative close juxtaposition, separated by ~850 kb of DNA containing several putative gene loci. As is commonly done, the embryonic stem cells used for targeted disruption of the A1AR gene were derived from the 129J mouse strain, one of several mouse strains with two renin genes, designated Ren-1d and Ren-2 (21). By propagating the A1AR mutation in the one-renin gene C57BL/6 background, A1AR-/- mice will carry the 129J background in the area of the mutated A1AR gene and will therefore possess two renin genes. On the other hand, A1AR+/+ mice will have to carry the C57BL/6 background in the area of the native A1AR gene and will therefore have only one renin gene, designated Ren-1c. Breeding strategies will be used to segregate the A1AR knockout mutation from the two-renin gene constellation by backcrossing into the C57BL/6 background or to maintain the A1AR mutation in a two-renin gene background by backcrossing into the 129J or Swiss strain. Although this genetic artifact does not limit the conclusion that the A1AR is not required for mediation of macula densa control of the renin system, it has to be carefully considered when absolute values of renal renin content are compared between the genotypes. Inasmuch as a linkage of a targeted gene and the neighboring gene loci supposedly unaffected by the knockout procedure potentially occurs in every knockout model derived from different mouse strains, our results emphasize the necessity of careful interpretation of data comparing knockout with wild-type mice.

Taken together, our results support the concept of a tonic inhibitory role of the A1AR on the renin system, but they argue against a role of the A1AR in mediation of the macula densa control of the renin system, as has been demonstrated previously for macula densa control of glomerular filtration.


    ACKNOWLEDGEMENTS

We thank Maggy Schweiger and Susanne Lukas for expert technical assistance.


    FOOTNOTES

This study was financially supported by Deutsche Forschungsgemeinschaft Grant Schw 778/2-1.

Address for reprint requests and other correspondence: F. Schweda, Institut für Physiologie, Universität Regensburg, 93040 Regensburg, Germany (E-mail: frank.schweda{at}klinik.uni-regensburg.de).

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.

First published December 10, 2002;10.1152/ajprenal.00280.2002

Received 5 August 2002; accepted in final form 3 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Albinus, M, Finkbeiner E, Sosath B, and Osswald H. Isolated superfused juxtaglomerular cells from rat kidney: a model for study of renin secretion. Am J Physiol Renal Physiol 275: F991-F997, 1998[Abstract/Free Full Text].

2.   Brown, R, Ollerstam A, Johansson B, Skott O, Gebre-Medhin S, Fredholm B, and Persson AEG Abolished tubuloglomerular feedback and increased plasma renin in adenosine A1 receptor-deficient mice. Am J Physiol Regul Integr Comp Physiol 281: R1362-R1367, 2001[Abstract/Free Full Text].

3.   Castrop, H, Schweda F, Schumacher K, Wolf K, and Kurtz A. Role of renocortical cyclooxygenase-2 for renal vascular resistance and macula densa control of renin secretion. J Am Soc Nephrol 12: 867-874, 2001[Abstract/Free Full Text].

4.   Chomczynski, P, and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 62: 156-159, 1997.

5.   Churchill, PC, and Bidani A. Renal effects of selective adenosine receptor agonists in anesthetized rats. Am J Physiol Renal Fluid Electrolyte Physiol 252: F299-F303, 1987[Abstract/Free Full Text].

6.   Churchill, PC, Rossi NF, and Churchill MC. Renin secretory effects of N-6-cyclohexyladenosine: effects of dietary sodium. Am J Physiol Renal Fluid Electrolyte Physiol 252: F872-F876, 1987[Abstract/Free Full Text].

7.   Deckert, J, Nothen MM, Bryant SP, Ren H, Wolf HK, Stiles GL, Spurr NK, and Propping P. Human adenosine A1 receptor gene: systematic screening for DNA sequence variation and linkage mapping on chromosome 1q31-321 using a silent polymorphism in the coding region. Biochem Biophys Res Commun 214: 614-621, 1995[ISI][Medline].

8.   Hackenthal, E, Paul M, Ganten D, and Taugner R. Morphology, physiology, and molecular biology of renin secretion. Physiol Rev 70: 1067-1116, 1990[Free Full Text].

9.   Harding, P, Sigmon DH, Alfie ME, Huang PL, Fishman MC, Beierwaltes WH, and Carretero OA. Cyclooxygenase-2 mediates increased renal renin content induced by low-sodium diet. Hypertension 29: 297-302, 1997[Abstract/Free Full Text].

10.   Harris, RC, McKanna JA, Akai Y, Jacobson HR, Dubois RN, and Breyer MD. Cyclooxygenase-2 is associated with the macula densa of rat kidney and increases with salt restriction. J Clin Invest 94: 2504-2510, 1994[ISI][Medline].

11.   He, XR, Greenberg SG, Briggs JP, and Schnermann J. Effects of furosemide and verapamil on the NaCl dependency of macula densa-mediated renin secretion. Hypertension 26: 137-142, 1995[Abstract/Free Full Text].

12.   Höcherl, K, Kammerl MC, Schumacher K, Endemann D, Grobecker HF, and Kurtz A. Role of prostanoids in regulation of the renin-angiotensin-aldosterone system by salt intake. Am J Physiol Renal Physiol 283: F294-F301, 2002[Abstract/Free Full Text].

13.   Holz, FG, and Steinhausen M. Renovascular effects of adenosine receptor agonists. Renal Physiol 10: 272-282, 1987[ISI][Medline].

14.   Inscho, EW, Carmines PK, and Navar LG. Juxtamedullary afferent arteriolar responses to P1 and P2 purinergic stimulation. Hypertension 17: 1033-1037, 1991[Abstract].

15.   Itoh, S, Carretero OA, and Murray RD. Possible role of adenosine in the macula densa mechanism of renin release in rabbits. J Clin Invest 76: 1412-1417, 1985[ISI][Medline].

16.   Kammerl, MC, Nüsing RM, Seyberth HW, Riegger GA, Kurtz A, and Krämer BK. Inhibition of cyclooxygenase-2 attenuates urinary prostanoid excretion without affecting renal renin expression. Pflügers Arch 442: 842-847, 2001[ISI][Medline].

17.   Kurtz, A, Della Bruna R, Pfeilschifter J, and Bauer C. Role of cGMP as second messenger of adenosine in the inhibition of renin release. Kidney Int 33: 798-803, 1988[ISI][Medline].

18.   Lorenz, JN, Weihprecht H, He XR, Skott O, Briggs JP, and Schnermann J. Effects of adenosine and angiotensin on macula densa-stimulated renin secretion. Am J Physiol Renal Fluid Electrolyte Physiol 265: F187-F194, 1993[Abstract/Free Full Text].

19.   Lorenz, JN, Weihprecht H, Schnermann J, Skott O, and Briggs JP. Renin release from isolated juxtaglomerular apparatus depends on macula densa chloride transport. Am J Physiol Renal Fluid Electrolyte Physiol 260: F486-F493, 1991[Abstract/Free Full Text].

20.   Meneton, P, Ichikawa I, Inagami T, and Schnermann J. Renal physiology of the mouse. Am J Physiol Renal Physiol 278: F339-F351, 2000[Abstract/Free Full Text].

21.   Morris, BJ. Molecular biology of renin. I. Gene and protein structure, synthesis and processing. J Hypertens 10: 209-214, 1992[ISI][Medline].

22.   Norling, LL, Smith TM, and Ingelfinger JR. Stored renin isoforms in the developing rat. Am J Hypertens 11: 213-218, 1998[ISI][Medline].

23.   Osswald, H, Nabakowski G, and Hermes H. Adenosine as a possible mediator of metabolic control of glomerular filtration rate. Int J Biochem 12: 263-267, 1980[ISI][Medline].

24.   Pfeifer, CA, Suzuki F, and Jackson EK. Selective A1 adenosine receptor antagonism augments beta -adrenergic-induced renin release in vivo. Am J Physiol Renal Fluid Electrolyte Physiol 269: F468-F479, 1995.

25.   Schnermann, J. Juxtaglomerular cell complex in the regulation of renal salt excretion. Am J Physiol Regul Integr Comp Physiol 274: R263-R279, 1998[Abstract/Free Full Text].

26.   Schnermann, J, Weihprecht H, and Briggs JP. Inhibition of tubuloglomerular feedback during adenosine 1 receptor blockade. Am J Physiol Renal Fluid Electrolyte Physiol 258: F553-F561, 1990[Abstract/Free Full Text].

27.   Siragy, HM, and Linden J. Sodium intake markedly alters renal interstitial fluid adenosine. Hypertension 27: 404-407, 1996[Abstract/Free Full Text].

28.   Sun, D, Samuelson LC, Yang T, Huang Y, Paliege A, Saunders T, Briggs J, and Schnermann J. Mediation of tubuloglomerular feedback by adenosine: evidence from mice lacking adenosine 1 receptors. Proc Natl Acad Sci USA 98: 9983-9988, 2001[Abstract/Free Full Text].

29.   Thomson, S, Bao D, Deng A, and Vallon V. Adenosine formed by 5'-nucleotidase mediates tubuloglomerular feedback. J Clin Invest 106: 289-298, 2000[Abstract/Free Full Text].

30.   Townsend-Nicholson, A, Baker E, Schofield PR, and Sutherland GR. Localization of the adenosine A1 receptor subtype gene (ADORA1) to chromosome 1q321. Genomics 26: 423-425, 1995[ISI][Medline].

31.   Traynor, TR, Smart A, Briggs JP, and Schnermann J. Inhibition of macula densa-stimulated renin secretion by pharmacological blockade of cyclooxygenase-2. Am J Physiol Renal Physiol 277: F706-F710, 1999[Abstract/Free Full Text].

32.   Wagner, C, Gödecke A, Ford M, Schnermann J, Schrader J, and Kurtz A. Regulation of renin gene expression in kidneys of eNOS- and nNOS-deficient mice. Pflügers Arch 439: 567-572, 2000[ISI][Medline].

33.   Wagner, C, and Kurtz A. Regulation of renal renin release. Curr Opin Nephrol Hypertens 7: 437-441, 1998[ISI][Medline].

34.   Wang, Q, Hummler E, Nussberger J, Clement S, Gabbiani G, Brunner HR, and Burnier M. Blood pressure, cardiac, and renal responses to salt and deoxycorticosterone acetate in mice: role of renin genes. J Am Soc Nephrol 13: 1509-1516, 2002[Abstract/Free Full Text].

35.   Weihprecht, H, Lorenz JN, Schnermann J, Skott O, and Briggs JP. Effect of adenosine 1-receptor blockade on renin release from rabbit isolated perfused juxtaglomerular apparatus. J Clin Invest 85: 1622-1628, 1990[ISI][Medline].

36.   Wilcox, CS, Welch WJ, Schreiner GF, and Belardinelli L. Natriuretic and diuretic actions of a highly selective adenosine A1 receptor antagonist. J Am Soc Nephrol 10: 714-720, 1999[Abstract/Free Full Text].

37.   Zou, AP, Feng W, Li PL, and Cowley AW. Effect of chronic salt loading on adenosine metabolism and receptor expression in renal cortex and medulla in rats. Hypertension 33: 511-516, 1999[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 284(4):F770-F777