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J Am Soc Nephrol 14:303-310, 2003
© 2003 American Society of Nephrology

Pressure Natriuresis in AT2 Receptor–Deficient Mice with L-NAME Hypertension

Michael Obst*, Volkmar Gross*, Jürgen Janke{dagger}, Maren Wellner{dagger}, Wolfgang Schneider{dagger} and Friedrich C. Luft{dagger}

*Max-Delbrück-Center (MDC) for Molecular Medicine, HELIOS-Klinikum-Berlin, Franz Volhard Clinic, Medical Faculty of the Charité, {dagger}Humboldt University, Berlin, Germany.

Correspondence to Dr. Friedrich C. Luft, Franz Volhard Clinic, Wiltbergstrasse 50 13125 Berlin, Germany. Phone: 49-30-9417-2202; Fax: 49-30-9417-2206;


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. AT2 receptor-disrupted (AT2 -/-) mice provide a unique opportunity to investigate the cardiovascular and BP-related effects of NO depletion. This study compared the pressure-diuresis-natriuresis relationship in (AT2 -/-) and wild-type (AT2 +/+) mice after treating the animals with L-NAME (130 mg/kg body wt per day) for 1 wk. L-NAME increased mean arterial pressure (MAP) more in AT2 -/- than in AT2 +/+ mice (118 ± 2 versus 108 ± 4 mmHg). This difference occurred even though L-NAME–treated AT2 +/+ mice had a greater sodium excretion than AT2 -/- mice (10.9 ± 0.5 versus 8.0 ± 1.0 µmol/h). The pressure-natriuresis relationship in conscious AT2 -/- mice was shifted rightward compared with controls. RBF was decreased in AT2 -/- compared with AT2 +/+ mice. L-NAME decreased RBF in these mice further from 4.08 ± 0.43 to 2.79 ± 0.15 ml/min per g of kidney wt. GFR was not significantly different between AT2 +/+ and AT2 -/- mice (1.09 ± 0.08 versus 1.21 ± 0.09 ml/min per g of kidney wt). L-NAME reduced GFR in AT2 -/- to 0.87 ± 0.07 ml/min per g of kidney wt. Fractional sodium (FENa) and water (FEH2O) curves were shifted more strongly to the right by L-NAME in AT2 -/- mice than in AT2 +/+ mice. AT1 receptor blocker treatment lowered BP in both L-NAME–treated strains to basal values. It is concluded that the AT1 receptor plays a key role in the impaired renal sodium and water excretion induced by NO synthesis blockade. Changes in RBF, GFR, and tubular sodium and water reabsorption are involved and may be also responsible for the greater BP increase in L-NAME–treated AT2 -/- mice. E-mail: luft@fvk-berlin.de


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide (NO) and angiotensin II (AngII) are integrated in homeostatic mechanisms regulating renal function and BP. The effects of AngII are mediated by at least two receptor isoforms (AT1 and AT2). AngII stimulates proximal tubular sodium reabsorption and decreases sodium excretion by reducing renal medullary blood flow via the AT1 receptor. AngII also enhances sodium reabsorption indirectly by stimulating aldosterone release through the AT1 receptor. There is evidence that the AT2 receptor serves a counter-regulatory protective role (1). Tonically secreted intrarenal NO is also involved in the control of glomerular hemodynamics, tubuloglomerular feedback, renin release, and sodium and water excretion (2). NO-synthesis blockade by L-NAME lowers renal blood flow, reduces sodium and water excretion, shifts pressure-natriuresis curves toward the right (2–5), and increases BP (6–8). The renin-angiotensin system participates in the renal and systemic alterations induced by NO synthesis blockade (9). Chronic AT1 receptor or converting enzyme blockade prevents the development of L-NAME hypertension (6,10). However, pretreatment with losartan had no effect on the impaired pressure natriuresis produced by NO-synthesis blockade in another study (11), suggesting that the AT1 receptor may not or only partially be involved in L-NAME-induced changes. This suggestion is underscored by results suggesting that AT2 receptor blockade in L-NAME-pretreated rats shifts pressure natriuresis curves toward the left (12) and that AT2 receptor blockade with PD-123319 shifts pressure natriuresis toward lower pressure values (13). The AT1 receptor is upregulated in AT2 disrupted (-/-) mice compared with AT2 +/+ controls (14–16). Thus, AT2 -/- mice may provide an opportunity to study the relationship between the AT1 receptor and L-NAME effects without concomitant AT2 receptor-related effects. We tested that notion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All animals used in this study were obtained from breeder pairs supplied by the Vanderbilt University Medical Center (Nashville, TN) through the courtesy of Prof. T. Inagami, as described elsewhere (17). The mice were bred in our animal facility. Genotypes were regularly verified by PCR. The mice were allowed free access to standard chow (0.25% sodium, SNIFF Spezialitäten GmbH, Soest, Germany) and drinking water ad libitum. The experimental protocol was approved by the local council on animal care, whose standards correspond to those of the American Physiological Society. Renal function studies were conducted in mice aged 12 to 15 wk. Telemetry was performed in eight AT2 -/- mice and in seven AT2 +/+ mice. The body weights averaged 31 ± 1 g (AT2 -/-) and 33 ± 1 g (AT2 +/+) before surgery. The telemetric techniques we employed are described in detail elsewhere (18). The mice were synchronized to a light-dark schedule of 12:12 h with lights on at 06:00 h. All mice were allowed at least 9-d recovery before any measurements were made. Baseline values were thereafter continuously recorded for 7 d; the last 3 d of this period were used for statistical analyses. Thereafter, the mice were given L-NAME for 1 wk (5 mg L-NAME/10 ml tap water), and measurements were obtained. We quantified L-NAME uptake by weighing the drinking bottles and calculating daily water intake. We found no difference in water and L-NAME uptake in AT2 -/- and AT2 +/+ mice. The daily L-NAME uptake averaged 130 mg/kg of body wt per 24 h in both groups. In the literature, rats are commonly given no more than 100 mg/kg L-NAME per 24 h (19). Doubling the L-NAME amount in the drinking water (10 mg L-NAME per 10 ml tap water) disturbed the drinking behavior and led to mortality in AT2 -/- mice. The L-NAME uptake of these mice was approximately 180 mg/kg of body wt per 24 h.

In all instances, the last 3 d of the L-NAME period were used for statistical analyses. All values were sampled every 5 min for 10 s continuously day and night with a sampling rate of 1000 Hz. Values are shown as 24 h means. In another group of nine AT2 -/- mice (body weight 31 ± 1 g) and nine AT2 +/+ controls (body weight 29 ± 1 g), 24-h urine collections over 2 d were obtained after adaptation to the metabolic cages (UNO Roestvaststaal, Zevenaar, The Netherlands) under baseline conditions and after L-NAME treatment. Mean arterial BP values (MAP), urine volume (V), and sodium excretion (UNaV) values were used to construct pressure volume and pressure natriuresis curves for conscious AT2 -/- and AT2 +/+ mice before and after L-NAME treatment.

The effect of acutely increased renal perfusion pressure (RPP) on pressure-diuresis-natriuresis relationships and on total renal blood flow (RBF) was examined in seven AT2 -/- mice weighing 29 ± 1 g and ten AT2 +/+ mice weighing 30 ± 1 g under control conditions and in nine AT2-/- mice weighing 31 ± 0.5 g and eight AT2 +/+ mice weighing 29 ± 1 g after 1-wk L-NAME in the drinking water. All mice received the standard chow as outlined above. We relied on techniques described earlier (14), but without performing the unilateral nephrectomy. After surgery and a 30 to 45 min equilibration period, MAP and RBF were recorded continuously and urine was sampled in two 10 to 30 min collecting periods. Tying off the mesenteric and celiac arteries and by occluding the aorta below the kidney then increased RPP. Mean arterial BP (MAP) and renal blood flow (RBF) were calculated for each period by averaging all recorded values during that time period. Renal vascular resistance (RVR) was calculated as the MAP-to-RBF ratio. Urine flow (UV) was sampled and determined gravimetrically. Urinary sodium concentrations were determined by ion selective electrode (Konelab Microlyte 3+2, Frankfurt, Germany). Urine flow, sodium excretion (UNaV), and RBF were normalized per gram of kidney wet weight (kwt).

The effect of changes in RPP on GFR and fractional excretion of sodium (FENa) and fractional excretion of water (FEH2O) were examined in 14 AT2 -/- mice weighing 29 ± 1 g and 10 AT2 +/+ controls weighing 32 ± 1 g. Similarly, 16 AT2 -/- mice weighing 29 ± 1 g and 14 AT2 +/+ controls weighing 30 ± 1 g that received L-NAME for 1 wk were subjected to the same protocol. The mice were surgically prepared as described elsewhere (14). GFR was measured by inulin clearance. This determination required an additional catheter (PE 10) that was placed also into the jugular vein for infusion of a 1% FITC-inulin in 0.9% NaCl-solution (Sigma, St. Louis, MO).

We tested the effect of AT1 receptor blockade on L-NAME-induced BP increases in AT2 -/- and AT2 +/+ mice. Six AT2 +/+ and four AT2 -/- mice were outfitted with telemetry and given L-NAME at the maximally tolerable dose. Under these conditions, valsartan (50 mg/kg per d per gavage, Novartis) was administered. This dose that inhibits 80% of the vasopressor effect of exogenous AngII (20).

Gene Expression Analyses
Gene expression analyses were performed in ten kidneys from ten separate mice in each group (AT2 +/+, AT2 -/-, AT2 +/+ with L-NAME, AT2 -/- with L-NAME) to characterize the AT1 receptor. RNA was isolated from homogenized kidneys by the Qiagen RNeasy mini Kit, and DNA contamination was eliminated with the Qiagen RNase-free DNase kit, followed by determination of quality and quantity with the Agilent 2100 bioanalyser (Walbronn, Germany). Reverse transcription was performed with 2 µg of total RNA in a final volume of 20 µl using 100 U Superscript reverse transcriptase, 5.4 µg random primer, 0.5 mM dNTPs, 10 mM DTT and 1 x RT buffer. RNA and random primer were first denatured for 10 min at 65°C and then placed on ice and subsequently reverse transcribed for 1 h at 37°C. Relative quantitation of gene expression was performed with the ABI 5700 sequence detection system for real-time PCR (TaqMan) using the standard curve method. Gene expression analysis was performed for the AT1 receptor (AT1) with 1 ng/µl cDNA equal to reverse transcribed RNA. The sequence of the mouse AngII receptor isoforms 1a and 1b were derived from GenBank accession numbers S37484 and S37491. Primers and probe were designed to amplify both isoforms. The sequence of primers and probe were: 5'-TGG CCC TTC GGC AAT C-3' (forward primer, 300 nM final concentration), 5'-TGG CGT AGA GGT TGA AAC TGA-3' (reverse primer, 900 nM final concentration), 5' FAM-CCT ATG TAA GAT CGC TTC GGC CAG C-TAMRA 3' (probe, 175 nM final concentration). Rodent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen as the endogenous control (housekeeping gene). PCR were performed with the TaqMan Universal Master Mix and the TaqMan assay reagent for rodent GAPDH in a total volume of 25 µl. The two-step PCR conditions were 2 min at 50°C, 10 min at 95°C, followed by 45 cycles of 15 s at 95°C and 1 min at 62°C. Each sample was measured in quadruplicate. Materials from the following companies were used: RNeasy mini Kit, DNase Set (Qiagen, Hilden, Germany), Superscript reverse transcriptase, 1 x RT buffer, DTT, dNTPs, random primer (Life Technologies, Karlsruhe, Germany), AT1 Primer and probes (BioTez, Berlin, Germany), Universal Master Mix, TaqMan assay reagent for rodent GAPDH (PE Biosystems, Weiterstadt, Germany).

AT1 receptor expression was determined on the protein level by Western blotting in each group. Three kidneys of each group were homogenized in 500 µl of homogenization buffer (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 3 mM MgCl2, 300 mM sucrose, 1 mM EDTA, 0.2 mM PMSF, 1 µg/ml leupeptin, 0.5% Triton X100, 0.02% Na azide) for 10 min on ice. The protein concentration was determined using Bradfort reagent. For proteins with a mass of about 50 kD, 6% acrylamide gels were prepared and 30 µg of protein were loaded. The electrophoresis was carried out at 120 V. Protein transfer onto PVDF membranes was performed using a semidry blotting apparatus (Millipore) with 2.5 mA/cm2 for 1 h. Blocking was carried out in 5% nonfat skimmed milk (BioRad, Munich, Germany) for 1 h at RT. The antibody was diluted 1:1000 in 5% nonfat skimmed milk TBS-T and incubated overnight at 4°C. An anti-rabbit IgG horseradish peroxidase (1:1000) was used as the second antibody. The signal detection was performed using ECL-system (Amersham, Braunschweig, Germany).

For statistical analyses, we relied on the unpaired t test, the Mann-Whitney test, and the Kruskal-Wallis test. Significance was accepted at P < 0.05. Data are given as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The upper panel of Figure 1 shows 24-h MAP in AT2 -/- and AT2 +/+ mice before and after L-NAME treatment. MAP leveled at 105 ± 2 mmHg in AT2 -/- mice and at 101 ± 3 mmHg in AT2 +/+ mice. With L-NAME treatment, MAP increased to 118 ± 2 and 108 ± 4 mmHg in AT2 -/- and AT2 +/+ mice, respectively. The middle panel of Figure 1 shows the 24-h sodium excretion rates obtained before and after L-NAME treatment. Sodium excretion increased in AT2 +/+ from 8.5 ± 0.8 to 10.9 ± 0.5 µmol/h (P < 0.05) as L-NAME was applied. Contrary, in AT2 -/- mice, 24-h sodium excretion after L-NAME averaged 8.0 ± 1.0 µmol/h and was not significantly different from values measured under baseline conditions. To evaluate the effect of L-NAME on pressure natriuresis in conscious AT2 -/- and AT2 +/+ mice, the urinary sodium excretion rate (y-axis) was plotted against MAP (x-axis). The lower panel of Figure 1 shows that the pressure natriuresis curve was shifted rightward in AT2 -/- compared with AT2 +/+ mice.



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  		Figure 1. Mean arterial BP (MAP, upper panel), sodium excretion (UNaV, middle panel), and pressure-natriuresis relationships (lower panel) in AT2 -/- and wild-type (AT2 +/+) mice before (baseline) and after 1 wk of L-NAME (5 mg/10 ml drinking water). MAP values were measured continuously (24-h) by telemetry. Sodium excretion values represent the average of two 24-h urine collections. * P < 0.05, L-NAME increased BP more in AT2 -/- mice and had smaller effects on sodium excretion in AT2 -/- than in AT2 +/+ mice, shifting pressure natriuresis more towards the right.

 
To gain insight into the mechanisms that shifted the chronic pressure-natriuresis relationships in AT2 -/- mice toward the right, we repeated these experiments in anesthetized mice and measured RBF, GFR, and fractional sodium and water excretion. Figure 2 shows pressure-diuresis and pressure-natriuresis responses of AT2 -/- and AT2 +/+ mice with and without L-NAME treatment. In untreated AT2 +/+ mice, urine flow and sodium excretion averaged 25.7 ± 4.4 µl/min per g of kidney wt and 4.7 ± 0.8 µmol/min per g of kidney wt with the RPP level at 107 mmHg, respectively. Increasing the RPP to 143 mmHg in these mice increased urine flow and sodium excretion to 97.3 ± 12.3 µl/min per g of kidney wt and 18.2 ± 2.7 µmol/min per g of kidney wt, respectively. The pressure-diuresis and pressure-natriuresis curves of AT2 -/- mice were shifted rightward by about 10 mmHg. Thus, by increasing RPP to 147 mmHg, urine flow and sodium excretion leveled at 60.5 ± 11.6 µl/min per g of kidney wt and 11.1 ± 1.8 µmol/min per g of kidney wt, respectively. L-NAME shifted pressure-diuresis and pressure-natriuresis curves significantly rightward in AT2 -/- mice. At similar RPP levels of 140 mmHg, urine flow and sodium excretion were reduced by L-NAME treatment in AT2 +/+ mice about 1.5-fold, whereas a 2.5-fold reduction was observed in AT2 -/- mice.



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  		Figure 2. L-NAME treatment for 1 wk and the relationship between renal perfusion pressure and urine flow (left panel) and sodium excretion (right panel) in AT2 +/+ mice (upper panels) and AT2 -/- mice (lower panels). * P < 0.05, values compared at equivalent renal perfusion pressure levels. L-NAME shifted pressure-diuresis and pressure-natriuresis curves towards the right in AT2 -/- mice.

 
Figure 3 shows the relationships between RPP and RBF. RBF averaged between 5.0 ± 0.5 and 5.5 ± 0.5 ml/min per g of kidney wt in AT2 +/+ mice. In AT2 -/- mice, RBF was reduced and leveled between 3.7 ± 0.6 and 4.4 ± 1.0 ml/min per g of kidney wt. L-NAME reduced RBF in AT2 -/- mice to levels between 2.5 ± 0.3 and 3.2 ± 0.3 ml/min per g of kidney wt. In AT2 -/- mice RBF was significantly affected by L-NAME. The decrease in RBF was associated with an increase in RVR. RVR at baseline was 31.51 ± 8.20 mmHg/ml per min per g of kwt and increased as RPP was increased to 45.43 ± 15.18 mmHg/ml per min per g of kwt in AT2 -/- mice. In L-NAME–treated AT2 -/- mice, RVR leveled between 45.86 ± 5.02 and 62.59 ± 9.23 mmHg/ml per min per g of kwt.



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  		Figure 3. L-NAME treatment for 1 wk and the relationship between renal perfusion pressure and renal blood flow in AT2 +/+ (upper panel) and AT2 -/- mice (middle panel). * P < 0.05, values compared at equivalent renal perfusion pressure levels. Renal blood flow at all pressure levels in AT2 +/+ and AT2 -/- mice with and without L-NAME are shown in the lower panel. Renal blood flow was decreased in AT2 -/- compared with AT2 +/+ mice and reduced in L-NAME–treated AT2 -/- mice.

 
Figure 4 shows the relationships between RPP and GFR. No significant GFR differences were observed in AT2 +/+ and AT2 -/- mice. GFR at all pressure levels averaged 1.09 ± 0.08 in AT2 +/+ and 1.21 ± 0.09 ml/min per g of kidney wt in AT2 -/- mice, respectively. L-NAME decreased GFR in AT2 -/- mice to a level of 0.87 ± 0.07 ml/min per g of kidney wt (P < 0.05). The fractional water excretion and fractional sodium excretion curves for AT2 +/+ and AT2 -/- mice, with and without L-NAME treatment, are shown in Figure 5. The fractional excretion of sodium and water at the basal RPP level were not different between the groups. Both relationships were shifted rightward by L-NAME. This shift was more pronounced in AT2 -/- mice, compared with AT2 +/+ controls. At similar RPP levels of 150 mmHg, L-NAME–treated AT2 -/- mice displayed fractional sodium and water values of 3.24 ± 0.99 and 2.41 ± 0.58%, compared with 12.30 ± 3.08 and 10.58 ± 2.23% in AT2 +/+ mice, respectively.



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  		Figure 4. L-NAME treatment for 1 wk and the relationship between renal perfusion pressure and GFR in AT2 +/+ (upper panel) and AT2 -/- mice (middle panel). * P < 0.05, values compared at equivalent renal perfusion pressure levels. GFR at all pressure levels in AT2 +/+ and AT2 -/- mice with and without L-NAME are shown in the lower panel. GFR was not different between AT2 +/+ and AT2 -/- mice. L-NAME reduced GFR in AT2 -/- mice.

 


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  		Figure 5. L-NAME treatment for 1 wk and the relationship between renal perfusion pressure and fractional water excretion (left panel) and fractional sodium excretion (right panel) in AT2 +/+ mice (upper panels) and AT2 -/- mice (lower panels). * P < 0.05, values compared at equivalent renal perfusion pressure levels. L-NAME shifted the fractional water and sodium excretion curves rightward in AT2 +/+ and AT2 -/- mice. The rightward shift was more pronounced in AT2 -/- mice.

 
Finally, we tested the effect of AT1 receptor blockade in AT2 -/- and AT2 +/+ treated with L-NAME. Valsartan decreased mean BP in L-NAME–treated AT2 -/- mice from 127 ± 2 to 116 ± 3 mmHg and from 122 ± 2 to 103 ± 2 mmHg in AT2 +/+ mice. In AT2 +/+ mice, these values were lower than the BP without any manipulation.

AT1 Receptor Expression and Histology
AT1 receptor mRNA results are shown in Figure 6. AT1 receptor expression was significantly higher in AT2 -/- than in AT2 +/+ mice and leveled 2.11 ± 0.6 and 1.41 ± 0.36 arbitrary units, respectively. This finding suggests that the AT1 receptor expression was increased 150% in AT2 -/- compared with AT2 +/+ mice. L-NAME decreased the expression of the renal AT1 receptor only in AT2 +/+ mice (1.05 ± 0.29 arbitrary units). The decrease of the AT1 receptor by L-NAME to 1.56 ± 0.08 arbitrary units in AT2 -/- was NS. AT1 receptor protein levels confirmed the data of AT1 receptor mRNA levels. The protein expression of the AT1 receptor leveled in AT2 -/- mice 134.13 ± 5.22 compared with 120.10 ± 0.94 arbitrary units in AT2 +/+ mice. L-NAME did not affect these values (AT2 -/- mice with L-NAME 126.30 ± 7.76 compared with 128.67 ± 2.12 arbitrary units in AT2 +/+ mice with L-NAME). Kidneys in both groups, with or without L-NAME, showed normal renal morphology (data not shown).



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  		Figure 6. AT1 receptor gene expression in the kidney of AT2 +/+ and AT2 -/- mice with and without L-NAME treatment. * P < 0.05, the AT1 receptor was upregulated in AT2 -/- mice. L-NAME decreased AT1 receptor gene expression in AT2 +/+ mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The kidney is responsible for long-term BP regulation by the pressure-diuresis-, pressure-natriuresis mechanism that links RPP to the excretion of sodium and water (21,22). Therefore, any changes in kidney function induced by NO-synthase inhibition may be of major importance for the BP increase after L-NAME. Indeed, systemic or local renal inhibition of NOS activity by L-NAME induced sodium retention, a resetting of pressure-natriuresis relationships, and the development of hypertension in numerous earlier studies (4,5,8,12,23–25). With telemetric BP measurements, we observed similar or slightly increased basal BP values in AT2 -/- mice compared with AT2 +/+ mice and similar levels of sodium excretion. With NO inhibition, BP increased to a greater degree in AT2 -/- mice than in AT2 +/+ mice; the difference was about 15 mmHg. This increase was not associated with a greater salt consumption in AT2 -/- mice reflected by 24-h urine sodium excretion. The resulting pressure-natriuresis relationship in AT2 -/- mice compared with AT2 +/+ mice showed a flattening and rightward shift, consistent with a salt-sensitive form of hypertension in AT2 -/- mice. Our acute pressure-diuresis and pressure-natriuresis experiments in anesthetized mice confirmed that L-NAME shifts pressure-natriuresis and pressure-diuresis curves rightward in AT2 -/- mice. Renal blood flow was reduced by L-NAME in AT2 -/- mice. The same was the case for GFR. As a result, fractional water and sodium curves were shifted rightward; the shift in AT2 -/- mice was particularly pronounced after L-NAME treatment. Under the view of the key role of the pressure-natriuresis mechanism for long-term BP regulation, the observed kidney function changes in AT2 -/- mice under L-NAME may be responsible for the stronger BP increase in these mice.

The mechanisms for the BP increase and changes in renal sodium and water excretion and the relationships between these changes after administration of L-NAME are not fully understood. However, there is evidence that the renin-angiotensin system is largely responsible for renal and systemic alterations when NO synthesis is reduced. Chronic AT1 receptor blockade or converting enzyme inhibition prevents the development of hypertension after L-NAME (6). Furthermore, the L-NAME–induced BP increase was completely reversed by AT1 receptor blockade in both AT2 -/- and AT2 +/+ mice, underscoring the importance of the AT1 receptor for BP changes after L-NAME. Strikingly, AT1 receptor blockade or converting enzyme inhibition that prevented L-NAME hypertension and increased GFR and RBF were not able to normalize the pressure-natriuresis mechanism (4). On the other hand, a compensatory increase in NO activity counteracts the vasoconstrictor AT1 receptor–mediated influence of AngII on renal cortical blood flow (26). Also, the production of NO in the renal medulla is thought to be important for counteracting AngII-induced reduction of blood flow in the medulla (27). The pharmacologic blockade of the AT2 receptor subpopulation in L-NAME–treated rats shifted the slopes of pressure-diuresis, pressure-natriuresis curves toward control values (12), showing that renal effects of NO-blockade may depend also on the AT2 receptors.

In accordance with earlier studies (14–16), we showed in the present study that the AT1 receptor is upregulated in mice lacking the AT2 receptor. Furthermore, L-NAME treatment reduced in AT2 +/+ mice renal AT1 receptor expression, whereas the AT1 receptor expression in AT2 -/- mice was not significantly changed. L-NAME application produces an AngII-dependent form of hypertension (6). The physiologic results on sodium and water reabsorption and renal hemodynamic may have been aggravated in AT2 -/- mice by the deletion of the AT2 receptor and the upregulation of the AT1 receptor. In rats, AT1 receptor blockade reduced or abolished the renal vasoconstriction after L-NAME treatment (28,29). The AT2 receptor mediates vasodilation and sodium excretion and opposes the effects of AT1 receptor stimulation. The absence of the AT2 receptor, together with the upregulation of the AT1 receptor, may have led to increased AngII-related pressure-natriuresis rightward shifts in L-NAME treated AT2 -/- mice.

The AT1 receptor is responsible for AngII-induced sodium and water reabsorption in the renal tubules. Infusion of AngII induces sodium and water reabsorption by a marked increase in proximal tubular fractional sodium and water reabsorption (30,31). AngII also influences renal hemodynamics (32,33). The AT2 receptor appears to counterbalance the AT1 receptor-mediated vasoconstriction and sodium and water reabsorption (1). Chronic AngII infusion leads to impairment of sodium excretion, reduced RBF, and diminished GFR autoregulatory efficiency, as well as suppression of the pressure-natriuresis relationship and hypertension. AT1 receptor blockade prevented the suppression of pressure natriuresis and hypertension (31,34). Furthermore, in hypertensive (mRen2)27 rats, the pressure-natriuresis relationships were shifted leftward when the AT1 receptors were blocked (35).

RBF was reduced by L-NAME treatment in AT2 -/- mice compared with AT2 +/+ mice. Concomitantly, RVR increased more after L-NAME in AT2 -/- than in AT2 +/+ mice, indicating that NO and the AT2 receptor are important in counter-regulating AT1 receptor effects on vascular tone. We found that L-NAME reduced GFR in AT2 -/- mice and shifted fractional water and sodium excretion curves further toward the right. L-NAME treatment caused a decreased (4,12) or unchanged (36) GFR in rats. As with renal blood flow, GFR reduction induced by L-NAME were also prevented by AT1 receptor blockade (4), underscoring the importance of the AT1 receptor for L-NAME–induced renal hemodynamic changes. The AT1 receptor is responsible for AngII-induced tubular sodium and water reabsorption (37). Blockade of this receptor shifts curves of fractional sodium and water reabsorption in hypertensive rats toward normal (38,39). In addition to any direct tubular effects of NO inhibition, the increase in renal vascular resistance and decrease in RBF in L-NAME AT2 -/- mice would support a reduction in medullary blood flow and renal interstitial hydrostatic pressure in these mice (40,41). These reductions at any given renal perfusion pressure would provide an additional hemodynamic explanation for the increased tubular sodium and water reabsorption in L-NAME–treated AT2 -/- mice. NO is also an important modulator of the vasoconstrictor influence of AngII in the renal cortical circulation (26,42). Furthermore, changes in renal cortical blood flow appear to play an important role in the chronic regulation of sodium excretion and BP (43). This result could be important considering that in AT2 -/- mice the AT1 receptor is upregulated in cortical structures (16).

An alternative explanation may involve the recently described NO dependency of arterial pressure-induced changes in renal interstitial hydrostatic pressure. Renal arterial pressure and renal interstitial hydrostatic pressure are closely interrelated. Renal interstitial hydrostatic pressure has a bearing on natriuretic responses (44). During NO inhibition, the renal interstitial hydrostatic pressure responses were markedly attenuated and were not restored even during constant-rate infusion of NO donors in dogs (45). We did not measure renal interstitial hydrostatic pressure in AT2 -/- mice; however, a role for the disturbed AT1-AT2 receptor relationship in maintaining the relationship between renal perfusion pressure and renal interstitial hydrostatic pressure is conceivable.

Our study shows that the AT1 receptor is important for L-NAME–induced changes in renal function, sodium and water excretion, and hypertension. Absence of the AT2 receptor, which counter-regulates AT1 receptor-dependent AngII effects, aggravates the L-NAME–induced renal and BP effects. The AT1 receptor seems to be crucial to renal sodium excretion, pressure diuresis and natriuresis, and BP increase when NO production is impaired.


   Acknowledgments
 
This study was supported by a grant-in-aid from the Deutsche Forschungsgemeinschaft to Volkmar Gross. We are grateful to Sabine Grüger, Ilona Kamer, and Jana Czychi for technical assistance.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication May 31, 2002. Accepted for publication October 4, 2002.




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