Introduction

The kidney plays a major role in the long-term regulation of blood pressure.1 Natriuresis and diuresis are regulated by numerous hormones and humoral factors, including angiotensin II (Ang II) and dopamine. Dopamine and Ang II are two important regulators of sodium and water transport in the kidney, but they serve counteracting functions.1,2 The activation of AT1 receptors by low concentrations of Ang II causes an increase in renal sodium reabsorption.3 By contrast, the activation of D1-like (D1 and D5 subtypes) and D2-like dopaminergic receptors (D2, D3, and D4 subtypes) causes a decrease in renal sodium reabsorption.4, 5, 6

Na+–K+ ATPase, which provides the driving force for sodium across the apical membrane in the proximal tubule of the kidney, plays an important role in sodium reabsorption. Na+–K+ ATPase is a heterodimer consisting primarily of α and β subunits. The α-subunit is the catalytic unit that binds sodium, potassium and ATP. The β-subunit is essential for targeting the α-subunit to the cell membrane and stabilizing the Na+–K+ ATPase.7, 8, 9 The dopaminergic D1 and D2-like receptors can inhibit Na+–K+ ATPase activity, thereby blocking tubule sodium reabsorption and inducing natriuresis.10, 11, 12, 13 Thus, D1 and D2-like receptors regulate water–sodium retention and balance the blood pressure through a Na+–K+ ATPase-dependent mechanism.

Dopaminergic and AT1 receptors are both expressed in the brush border and basolateral membranes of the renal proximal tubule (RPT).14, 15, 16 The interaction between dopaminergic receptors and Ang II receptors has been found in previously published papers.17, 18, 19 The activation of D3 dopaminergic receptors decreases AT1 receptor expression in RPT cells from Wistar-Kyoto (WKY) rats.18 Conversely, the stimulation of AT1 receptors decreases D3 receptor expression in WKY RPT cells.19 The D4 receptor is an important dopaminergic receptor for the function of natriuresis and diuresis in human and rodent renal proximal tubules. It antagonizes vasopressin- and aldosterone-dependent sodium reabsorption in the cortical collecting duct, and D4 dopaminergic receptor-deficient mice develop hypertension.20 However, whether stimulation of AT1 receptors affects the expression and function of D4 receptors is unknown. Our previous study proved that D4 and AT1 receptors colocalized in RPT cells, and stimulation of D4 receptors decreases AT1 receptor expression in RPT cells from WKY rats21 as well as in vascular smooth muscle cells.22

In this study, we aimed to identify the effects of AT1 receptors on D4 receptor expression, Na+–K+ ATPase activity and natriuresis and to investigate the underlying mechanism regulating this process. The AT1 and D4 receptor interaction in immortalized RPT cells from WKY rats and spontaneously hypertensive rats (SHRs) was detected. These immortalized RPT cells were used because they behave similarly to freshly isolated RPT cells in response to the stimulators and inhibitors of dopaminergic and AT1 receptors and of other G protein-coupled receptors (GPCRs).23

Methods

Cell culture

Immortalized RPT cells (Taconic, Germantown, NY, USA) from WKY rats and SHRs were cultured at 37 °C in 95% air and 5% CO2 in DMEM/F-12 (HyClone, Logan, UT, USA) with transferrin (5 μg ml−1), insulin (5 μg ml−1), epidermal growth factor (10 ng ml−1), dexamethasone (4 μg ml−1) and fetal bovine serum 10% in a 100-mm Petri dish.18 Cells were incubated in media without fetal bovine serum for 2 h before the addition of reagents.

Preparation of kidney and RPT cells

The WKY rats and SHRs (SLRC Laboratory Animals, Shanghai, China) were anesthetized with pentobarbital (50 mg kg−1), after which the kidneys were removed and the rats were killed (pentobarbital, 100 mg kg−1). The renal cortices or cultured RPT cells were homogenized in an ice-cold lysis buffer (phosphate-buffered saline with 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mmol l−1 EDTA, 1 mmol l−1 EGTA, 1 mmol l−1 PMSF, 10 μg ml−1 aprotinin and 10 μg ml−1 leupeptin), sonicated, kept on ice for 1 h and centrifuged at 16 000g for 30 min. The supernatants were stored at −80 °C until use.24 All experiments were approved by the Third Military Medical University Animal Use and Care Committee.

Western blot analysis

Rats and RPT cells were treated with vehicle (dH2O), an AT1 receptor agonist (Ang II; Sigma-Aldrich, St Louis, MO, USA), or an AT1 receptor antagonist (losartan; Merck, Darmstadt, Germany). Cells were serum-starved 2 h before cell lysis for immunoblotting. Immunoblotting was performed as previously reported.25 The blots were probed with polyclonal goat anti-D4 receptor antibodies (1:300; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and α-actin (Santa Cruz Biotechnology) was used for the normalization.18

Determination of the signaling pathways involved in the regulation of D4 receptor expression by AT1 receptors in WKY cells

To determine the regulatory pathways involved in the AT1 receptor-mediated regulation of D4 receptor expression in RPT cells, several inhibitors or agonists, including a protein kinase C inhibitor (19–31, 10−6 mol l−1; Sigma-Aldrich), a protein kinase A inhibitor (14–22, 10−6 mol l−1; Calbiochem, Darmstadt, Germany) and a calcium channel blocker (nicardipine, 10−7 mol l−1; Sigma-Aldrich) were used. These reagents were added into the incubation medium 15 min before the addition of Ang II.

Na+–K+ ATPase activity assay

Na+–K+ ATPase activity was determined as the rate of inorganic phosphate release in the presence or absence of ouabain.26 Rat RPT cells were pre-treated with Ang II or vehicle (dH2O) for 24 h. After washing for 15 min, the cells were treated with a D4 receptor agonist (PD168077; Tocris Cookson, Bristol, UK) or vehicle (dH2O) for 15 min. The cellular lysates of RPT cells were collected and centrifuged at 48 000g for 25 min. The pellet (membrane fraction) was washed two times and suspended in 10 mmol l−1 Tris containing 1 mmol l−1 EDTA (pH 7.4). Protein concentrations were determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA) and adjusted to 1 mg ml−1. Na+–K+ ATPase activity was measured by adding 100 μl of membrane fraction to an 800 μl reaction mixture, which was pre-incubated for 5 min in a water bath at 37 °C. The amount of phosphate produced was quantified by the addition of 1 ml of coloring reagent to the reaction mixture. The mixture was then mixed thoroughly and centrifuged at 3000 g for 10 min. The resulting phosphomolybdate was quantified spectrophotometrically at 740 nm using a standard curve prepared from K2HPO4. The difference between total and ouabain-insensitive ATPase activity was taken as Na+–K+ ATPase activity and expressed as μmol phosphate released per mg protein per minute. Protease inhibitors (1 mmol l−1 phenylmethylsulfonyl fluoride, 10 μg ml−1 each leupeptin and aprotinin) and a phosphatase inhibitor (50 μmol l−1 sodium orthovanadate) were used to eliminate the effect of proteases and phosphatases.27

Reverse transcriptase-polymerase chain reaction of D4 receptors

The total RNA was extracted from RPT cells. The cDNA was synthesized by using the extracted RNA that served as a template to amplify D4 receptors and β-actin. For D4 receptors, the forward primer was 5′-GAT GTG TTG GAC GCC TTT CT-3′ and the reverse primer was 5′-TCG GCA TTC AAG ATG GTG TA-3′. The amplification conditions of D4 receptors were as follows: 35 cycles of denaturation at 94 °C for 2 min, annealing for 30 s at 52.5 °C and extension for 45 s at 72 °C. For β-actin, the forward primer was 5′-GTG GGT ATG GGT CAG AAG GA-3′ and the reverse primer was 5′-AGC GCG TAA CCC TCA TAG AT-3′. The amplification conditions of β-actin were the same as the D4 receptor.28 The D4 receptor was corrected based on the density of β-actin.

Immunoprecipitation

Equal amounts of cell lysates (1000 μg protein ml−1 supernatant) were incubated with polyclonal anti-phosphoserine antibody (Abcam, Cambridge, MA, USA; D4 receptor phosphorylation; 2 μg ml−1) overnight at 4 °C and then with protein-G agarose for 2 h at room temperature. The immunoprecipitate was subjected to immunoblotting with the anti-D4 receptor antibody.

Determination of the effect of PD168077 on urine volume and urine sodium of WKY rats and SHRs

The rats were pre-treated with Ang II (10 ng kg−1 per day, for 1 week) or vehicle (dH2O). Urine and sodium excretions were measured in WKY rats and SHRs kept in metabolic cages for 24 h after the injection of PD168077 (0.3 mg kg−1) via tail vein. Urine sodium was measured by an electrolyte analyzer.

Statistical analysis

The data are expressed as the means±s.e.m. Comparison within groups was made by repeated measures ANOVA (or paired t-test when only two groups were compared), and comparison among groups (or t-test when only two groups were compared) was made by factorial ANOVA using the Holm–Sidak test. A value of P<0.05 was considered significant.

Results

AT1 receptors increase D4 receptor expression in RPT cells

Immortalized RPT cells from WKY rats were treated with varying concentrations and durations of the AT1 receptor agonist Ang II for 24 h. We found that Ang II increased D4 receptor expression in a concentration- (10−10–10−6 mol l−1) and time- (2–30 h) dependent manner. The stimulatory effect was evident at 10−9 mol l−1 (Figure 1a) and the stimulatory effect of Ang II (10−7 mol l−1) was noted as early as 2 h and was maintained for at least 30 h (Figure 1b). To determine whether the increase in D4 receptor expression was induced by AT1 receptor activation in RPT cells, we pre-treated WKY RPT cells with the AT1 receptor antagonist losartan (10−7 mol l−1) for 30 min. The results showed that the Ang II-induced increase of D4 receptor protein expression was blocked by the pre-treatment of losartan. Losartan alone did not affect the D4 receptor expression in RPT cells (Figure 1c). Meanwhile, we treated immortalized RPT cells from SHRs with varying concentrations of Ang II for 24 h. The results showed that Ang II also increased D4 receptor expression in a dosage-dependent manner in SHR RPT cells. Like that in WKY RPT cells, the stimulatory effect was significant at 10−9 mol l−1 in SHR RPT cells (Figure 1d).

Figure 1
figure 1

Effect of Ang II on D4 receptor protein expression in RPT cells. (a) Concentration response of D4 receptor protein expression in WKY RPT cells treated with varying concentrations of the AT1 receptor agonist (Ang II) for 24 h. The results are expressed as the ratio of D4 receptor and α-actin densities (n=7; *P<0.05 vs. control). (b) Time course of D4 receptor protein expression in WKY RPT cells treated with Ang II (10−7 mol l−1) for varying durations of incubation. The results are expressed as the ratio of D4 receptor and α-actin densities (n=7; *P<0.05 vs. control). (c) Effect of Ang II and an AT1 receptor antagonist (losartan) on D4 receptor expression in RPT cells from WKY rats. The cells were incubated with the indicated reagents (Ang II, 10−7 mol l−1; losartan, 10−7 mol l−1) for 24 h. The results are expressed as the ratio of the D4 receptor and α-actin densities (n=4; *P<0.05 vs. others). (d) Concentration response of D4 receptor protein expression in SHR RPT cells treated with varying concentrations of Ang II for 24 h. The results are expressed as the ratio of D4 receptor and α-actin densities (n=5; *P<0.05 vs. control).

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Calcium mediates the stimulatory effect of AT1 receptors on D4 receptor expression in RPT cells

To investigate the mechanism of AT1 receptor downregulating D4 receptor expression, RPT cells from WKY rats were treated with several agonists or antagonists. As protein kinase A, protein kinase C and Ca2+ were key cell signaling pathways, the protein kinase A inhibitor (14–22, 10−6 mol l−1), the protein kinase C inhibitor (19–31, 10−6 mol l−1) and the L-type Ca2+ channel blocker (nicardipine, 10−7 mol l−1) were added into the incubation medium 15 min before the addition of Ang II. D4 receptor expressions were detected by immunoblotting. The results showed that nicardipine blocked the stimulatory effect of Ang II on D4 receptor expression (Figure 2) while the protein kinase A inhibitor and the protein kinase C inhibitor did not (data not shown). Nicardipine alone had no effect on D4 receptor expression (Figure 2).

Figure 2
figure 2

Role of calcium in the stimulatory effect of AT1 receptors on D4 receptor expression in WKY RPT cells. Ang II (10−7 mol l−1) and the calcium channel blocker, nicardipine (10−7 mol l−1), were used to treat WKY RPT cells for 24 h. The results are expressed as the ratio of the D4 receptor and α-actin densities (n=6; *P<0.05 vs. others).

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The comparison of the regulation of AT1 receptors on D4 receptor expression in WKY rats and SHRs

We next studied whether there was different regulation of AT1 receptors on D4 receptor expression in RPT cells from WKY rats and SHRs. We compared D4 receptor expression in WKY RPT cells and SHR RPT cells after Ang II (10−7 mol l−1, 24 h) treatment by using a reverse transcriptase-PCR (RT-PCR) analysis and immunoblotting. It was noticed that basal D4 receptor mRNA and protein expression were higher in RPT cells from SHRs than that from WKY rats (Figures 3a and b). The results also showed that Ang II increased mRNA and protein expressions of D4 receptors in RPT cells from both WKY rats and SHRs (Figures 3a and b). This result was also confirmed in vivo. After treatment with Ang II (10 ng kg−1 per day) for 1 week, the renal cortices were collected and processed for immunoblotting of D4 receptors. The results showed that Ang II increased the protein expression of D4 receptors in WKY rats and SHRs (Figure 3c).

Figure 3
figure 3

Effect of Ang II on D4 receptor expression in RPT cells from both WKY rats and SHRs. (a, b) Effect of Ang II on D4 receptor expression in RPT cells from both WKY rats and SHRs. After treatment with the AT1 agonist, Ang II (10−7 mol l−1), for 24 h, the cells were collected and processed for reverse transcriptase-PCR (a) or immunoblotting (b) for the D4 receptor. The results are expressed as the ratio of D4 receptor and α-actin densities (n=5–6; *P<0.05 vs. WKY control; #P<0.05 vs. SHR control). (c) Effect of Ang II on D4 receptor expression in the kidneys of both WKY rats and SHRs. After treatment with the AT1 agonist (Ang II), the renal cortices were collected for immunoblotting for D4 receptor. The results are expressed as the ratio of the D4 receptor and α-actin densities (n=4, *P<0.05 vs. control).

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Ang II augmented D4 receptor-medicated inhibition of Na+–K+ ATPase in WKY RPT cells but not in SHR RPT cells

To investigate the influence of stimulation of the AT1 receptor on D4 receptor function, we evaluated the activity of the D4 receptor downstream molecule Na+–K+ ATPase. In RPT cells from both WKY rats and SHRs, the cells were pre-treated with Ang II (10−7 mol l−1) for 24 h. After washing for 15 min, the cells were treated with a D4 receptor agonist (PD168077, 10−11 mol l−1) for 15 min. The results showed that the activity of Na+–K+ ATPase was higher in SHR than WKY RPT cells at the basal level. PD168077 decreased Na+–K+ ATPase activities in WKY RPT cells, but not in SHR RPT cells. Pre-treatment with Ang II (10−7 mol l−1) for 24 h increased the inhibitory effect of PD168077 on Na+–K+ ATPase activity in WKY RPT cells. However, in SHR RPT cells, pre-treatment with Ang II had no effect on the D4-mediated inhibitory effect of Na+–K+ ATPase activity in SHR RPT cells (Figure 4a). Further, PD168077 significantly increased urine volume and sodium excretion in Ang II-pre-treated WKY rats, but not in SHRs (Figures 4b and c). Additionally, we used immunoprecipitation to investigate the phosphorylation of D4 receptors in WKY and SHR RPT cells. The results showed that the phosphorylation of D4 receptors was higher in SHR RPT cells than WKY RPT cells (Figure 4d).

Figure 4
figure 4

Effect of AT1 receptor agonists on the function of D4 receptors. (a) Effect of pre-treatment with Ang II on the inhibitory effect of D4 receptors on Na+–K+ ATPase activity in WKY and SHR RPT cells. The cells were pre-treated with Ang II (10−7 mol l−1) or vehicle (dH2O) for 24 h. After washing for 15 min, the cells were treated with PD168077 (10−11 mol l−1) for 15 min. The results are expressed as μmol phosphate released per mg protein per minute (n=5; *P<0.05 vs. control; #P<0.05 vs. PD168077 alone). (b) Effect of pre-treatment with Ang II on the inhibitory effect of D4 receptors on urine volume in WKY rats and SHRs. The rats were pre-treated with Ang II, or vehicle (dH2O) for 1 week. Urine volume (normalized by body weight) was measured for 24 h in rats after PD168077 or vehicle injection (n=5; *P<0.05 vs. control; #P<0.05 vs. PD168077 alone). (c) Effect of pre-treatment with Ang II on the inhibitory effect of D4 receptors on urine sodium in WKY rats and SHRs. Urine sodium excretion was measured in PD168077-treated or vehicle-treated rats (n=5; *P<0.05 vs. control; #P<0.05 vs. PD168077 alone). (d) D4 receptor phosphorylation in WKY and SHR RPT cells. The RPT cell lysate protein was immunoprecipitated with an anti-phosphoserine antibody and immunoblotted using an anti-D4 antibody (n=3; *P<0.05 vs. WKY).

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Discussion

In a physiological state, the activation of renal D4 receptors decreases Na+–K+ ATPase activity, an important sodium transporter, in basolateral membranes in RPT cells, which thereby increases sodium and water excretion and lowers blood pressure.29 In this study, we found that stimulation of AT1 receptors can increase D4 receptor expression in rat RPT cells, which may be of physiological significance since it enhances the effect of D4 receptors on sodium excretion to balance the blood pressure. The regulation of AT1 stimulation on D4 receptor expression is associated with an AT1-mediated calcium influx. The D4 receptor protein expression is higher in SHR than in WKY RPT cells. However, the D4 receptor agonist could not decrease Na+–K+ ATPase activity in SHR RPT cells, indicating that there were D4 receptor dysfunctions in a hypertensive state. Thus, the increasing D4 receptor expression by Ang II would augment the inhibitory effect of the D4 receptor on Na+–K+ ATPase activity in WKY RPT cells, but this effect is absent in SHR RPT cells.

The dopaminergic and renin–angiotensin systems play an important role in regulating the blood pressure. High NaCl intake increases the renal synthesis of dopamine and dopaminergic receptor activity, which decreases epithelial sodium transport, whereas a sodium deficit activates the renin–angiotensin-aldosterone system, which increases epithelial sodium transport.30 Several studies have shown that the dopaminergic and renin–angiotensin systems interact to regulate renal function.18, 19, 21, 31, 32 For example, D1-like receptor agonists antagonize the stimulatory effects of Ang II on NaCl uptake in rat RPT cells and brush border membrane vesicles.32 Additionally, an AT1 receptor blockade enhances the natriuretic effect of D1-like agonists.31 Moreover, activation of D3 dopaminergic receptors decreases AT1 receptor expression in WKY RPT cells but increases AT1 receptor expression in SHR RPT cells.18 In return, AT1 receptors negatively regulate the expression of D3 receptors in rat RPT cells.19 The interaction between the renin–angiotensin and dopaminergic systems has also been determined in our studies. Our previous study proved that stimulation of D4 receptors influences AT1 receptor expression in RPT cells.21 In this study, we demonstrated that stimulation of AT1 receptors, in return, increases D4 receptor expression in both WKY and SHR RPT cells. These findings indicate that the interaction between renin–angiotensin and dopaminergic systems is also involved in maintaining sodium balance.

The mechanism for the increase in D4 receptor expression caused by the AT1 receptor in WKY rats was also investigated in this study. We found that stimulation of AT1 receptors increases D4 receptor mRNA expression in both WKY and SHR RPT cells. This result is consistent with changes at the protein level and indicates that the regulation occurs at the transcriptional level. We also found that calcium mediates the stimulatory effect of the AT1 receptor on D4 receptor expression in RPT cells. It is known that the response of intracellular calcium concentrations to Ang II is mediated by AT1 receptors in the podocytes of the intact glomerulus and is partly due to Ca2+ entry from the extracellular space.33 L-type Ca2+ channels are major Ca2+ channels that mediate Ca2+ influx in RPT cells.34 Ca2+ influx through L-type Ca2+ channels triggers Ca2+ dynamics and various Ca2+-dependent signaling pathways. For example, Ca2+ is needed for the opening of the inositol 1,4,5-trisphosphate receptor (InsP3R), which activates a cascade of Ca2+ release from the endoplasmic reticulum store.35 Thus, we speculate that the AT1 receptor-mediated regulation of D4 receptor expression is dependent on the L-type Ca2+ channel-mediated Ca2+ influx in RPT cells and affects D4 receptor expression at the transcriptional level. Further studies are needed to investigate how calcium signals stimulate D4 receptor expression.

Our data suggest that D4 receptor function is aberrant in SHRs. It is known that phosphorylation plays an important role in regulating the function of the dopaminergic receptors, which are G protein-coupled receptors.36 Phosphorylation makes dopaminergic receptors high-affinity binding partners for arrestin proteins, which stop further G protein activation and promote receptor internalization, recycling or degradation.37 Therefore, our results indicate that the difference in the phosphorylation level of D4 receptor proteins might be associated with the aberrant function of D4 receptors in SHRs.

In conclusion, we have demonstrated that AT1 receptors upregulate D4 receptor expression in RPT cells via the activation of calcium channels. The inhibitory effect of D4 receptors on Na+–K+ ATPase activity was enhanced by AT1 receptor stimulation in WKY RPT cells but not in SHR RPT cells. These results were also confirmed in vivo. This effect might be involved in the pathogenesis of essential hypertension.