Am J Physiol Renal Physiol 306: F588–F596, 2014. First published February 5, 2014; doi:10.1152/ajprenal.00196.2013.
Regulation of renalase expression by D5 dopamine receptors in rat renal proximal tubule cells Shaoxiong Wang,1,2* Xi Lu,1,2* Jian Yang,1,2* Hongyong Wang,1,2 CaiYu Chen,1,2 Yu Han,1,2 Hongmei Ren,1,2 Shuo Zheng,1,2 Duofen He,1,2 Lin Zhou,1,2 Laureano D. Asico,3 Wei Eric Wang,1,2# Pedro A. Jose,3 and Chunyu Zeng1,2# 1
Department of Cardiology, Daping Hospital, Third Military Medical University, Chongqing, People’s Republic of China; Chongqing Institute of Cardiology, Chongqing, People’s Republic of China; and 3Division of Nephrology, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 2
Submitted 8 April 2013; accepted in final form 15 January 2014
Wang S, Lu X, Yang J, Wang H, Chen C, Han Y, Ren H, Zheng S, He D, Zhou L, Asico LD, Wang WE, Jose PA, Zeng C. Regulation of renalase expression by D5 dopamine receptors in rat renal proximal tubule cells. Am J Physiol Renal Physiol 306: F588–F596, 2014. First published February 5, 2014; doi:10.1152/ajprenal.00196.2013.—The dopaminergic and sympathetic systems interact to regulate blood pressure. Our previous studies showed regulation of ␣1-adrenergic receptor function by D1-like dopamine receptors in vascular smooth muscle cells. Because renalase could regulate circulating epinephrine levels and dopamine production in renal proximal tubules (RPTs), we tested the hypothesis that D1-like receptors regulate renalase expression in kidney. The effect of D1-like receptor stimulation on renalase expression and function was measured in immortalized RPT cells from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs). We found that the D1-like receptor agonist fenoldopam (10⫺7–10⫺5 mol/l) increased renalase protein expression and function in WKY RPT cells but decreased them in SHR cells. Fenoldopam also increased renalase mRNA levels in WKY but not in SHR cells. In contrast, fenoldopam increased the degradation of renalase protein in SHR cells but not in WKY cells. The regulation of renalase by the D1-like receptor was mainly via the D5 receptor because silencing of the D5 but not D1 receptor by antisense oligonucleotides blocked the stimulatory effect of the D1-like receptor on renalase expression in WKY cells. Moreover, inhibition of PKC, by the PKC inhibitor 19 –31, blocked the stimulatory effect of fenoldopam on renalase expression while stimulation of PKC, by a PKC agonist (PMA), increased renalase expression, indicating that PKC is involved in the process. Our studies suggest that the D5 receptor positively regulates renalase expression in WKY but not SHR RPT cells; aberrant regulation of renalase by the D5 receptor may be involved in the pathogenesis of hypertension. dopamine receptor; renalase; renal proximal tubule cells; renal proximal tubule cells; hypertension
a neurotransmitter in the central nervous system, has been reported to be an important regulator of renal and adrenal function, sodium balance, and blood pressure and to be relevant to the pathogenesis and/or maintenance of hypertension (11, 24, 33, 36, 45, 47). Dopamine receptors are classified into two families: the D1-like receptor subfamily includes the D1 and the D5 receptor, while the D2-like subfamily includes the D2, D3, and D4 receptors. Whereas the D1-like receptors couple to the stimulatory G-pro-
tein G␣S and thus activate adenylyl cyclase, all of the D2-like receptors couple to the inhibitory G-protein G␣i/G␣o and inhibit adenylyl cyclase and calcium channels and modulate potassium channels (11, 24, 36, 45, 47). There are interactions between dopamine receptors and the sympathetic nervous system (13, 30, 34, 35, 43). Our previous studies found that, as an antihypertensive factor, activation of dopamine receptors, especially D1-like and D3 receptors, inhibits the ␣1-adrenergic receptor-mediated proliferation of vascular smooth muscle cells and relaxes the resistant artery preconstricted by norepinephrine (27). Moreover, stimulation of D1-like receptors inhibits the catecholamine production, partly via inhibition of tyrosine hydroxylase expression (18). Renalase, a flavin adenine dinucleotide-dependent amine oxidase, plays an important role in the control of blood pressure by regulating sympathetic tone (5, 14, 15, 26, 40, 48). It is known that renalase increases the degradation of epinephrine in the blood circulation (14, 15, 26, 40). There is evidence showing renalase expression in renal proximal tubules (26, 40). Renalase metabolizes circulating epinephrine and dihydroxyphenylanine, the latter is converted to dopamine by renal proximal tubules, the major source of renal dopamine (24, 45). Because renalase could regulate circulating epinephrine levels and dopamine production in renal proximal tubules (RPTs), we tested the hypothesis that D1-like receptors regulate renalase expression and function in kidney. The effect of D1-like receptor on renalase expression was studied in immortalized RPT cells from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs). Because the specific D1-like receptor involved in the regulation of dopamine release or metabolism is unknown, we also determined which D1-like receptor is involved in the process.
DOPAMINE, WELL KNOWN AS
MATERIALS AND METHODS
* S. Wang, X. Lu, and J. Yang contributed equally to this work. # W. E. Wang and C. Zeng contributed equally to this work. Address for reprint requests and other correspondence: C. Zeng, Dept. of Cardiology, Daping Hospital, The Third Military Medical Univ., Chongqing, P.R. China (e-mail: [email protected]).
WKY and SHRs. Male WKY and SHRs (SLRC Laboratory Animals, Shanghai, China), ranging in age from 9 to 16 wk, fed regular rat chow with normal sodium (0.4% NaCl) content, were used. Food but not water was withheld 24 h before the study. The rats were anesthetized with pentobarbital (50 mg/kg body wt ip) (21, 27). Following a laparotomy, the kidneys were harvested, snap frozen in liquid nitrogen, and stored at ⫺70°C. All studies were approved by the Daping Hospital Animal Care and Use Committee. Cell culture. Immortalized RPT cells from WKY and SHRs (22, 42, 46) were cultured at 37°C in 95% air-5% CO2 atmosphere in FBS% DMEM/F-12 culture media, as previously described (41). The cells (80% confluence) were extracted in ice-cold lysis buffer (PBS with 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 10⫺3 mol/l EDTA, 10⫺3 mol/l EGTA, 10⫺3 mol/l PMSF, 10 g/ml aprotinin, and 10 g/ml leupeptin),
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1931-857X/14 Copyright © 2014 the American Physiological Society
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REGULATION OF D5 RECEPTORS ON RENALASE IN RPT CELLS
sonicated, kept on ice for 1 h, and centrifuged at 16,000 g for 30 min. The supernatants were stored at ⫺70°C until use for immunoblotting. Immunoblotting. Uniform amounts of proteins, separated by SDSpolyacrylamide gel electrophoresis, were electrophoretically transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in Dulbecco’s PBS with 0.05% Tween-20 and then probed with diluted affinity-purified goat polyclonal antibody to renalase (1:350; Sigma-Aldrich) (26, 40), rabbit polyclonal antibody to D1 receptor (1:200; Santa Cruz Biotechnology), D5 receptor (1:500; Santa Cruz Biotechnology), or -actin antibody (1:600; Santa Cruz Biotechnology) overnight (19). After being washed for five times (10 min each time), the membranes were incubated with diluted peroxidase-labeled goat anti-rabbit IgG or rabbit anti-goat IgG (Santa Cruz Biotechnology) in 5% milk for 1.5 h at room temperature. The signal was detected using chemiluminescence and developed on X-ray film. The density of the bands was quantified by densitometry using Quantiscan (Ferguson, MO), as previously reported (22, 42, 46). The amount of protein transferred onto the membranes was verified by immunoblotting for -actin. Quantitative RT-PCR. Total RNA was isolated and quantified as described previously (22, 46). For real-time quantitative RT-PCR (qRT-PCR) analysis, cDNA was synthesized from 0.5 mg of total RNA with a cDNA synthesis kit (High Capacity RNA-to-cDNA Kit; Takara, Tokyo, Japan). In the thermal cycle, 2 l cDNA were used per 25-l final reaction volume. PCRs were carried out with the Brilliant SYBR Green QPCR Master Mix kit (High Capacity RNA-to-cDNA Kit; Takara) in a total volume of 25 l. For renalase, the forward primer was 5=-TGCCAACAGTCCTCATAATCC-3= and the reverse primer was 5=-TCCTTCCTTCACTTCCATTCC-3= (GenBank Accession No. BC 005100). -Actin served as a housekeeping/reference gene for normalization. For -actin, the forward primer was 5=AGTGTGACGTTGACATCCGT-3= and the reverse primer was 5=GACTCATCGTACTCCTGCTT-3= (GenBank Accession No. BC
ed l bl ro m a nt r o c C S
NA siR
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ed bl m a cr
063166). The amplification profile for renalase and -actin used on the BIO-RAD CFX96 (Bio-Rad Laboratories) was 95°C for 3 min followed by 40 cycles of 95°C per 10 s and 72°C per 30 s. qRT-PCR experiments were repeated for three times. Antisense oligonucleotides. The effects of 50 nM of propyne/ phosphorothioate-modified antisense oligonucleotides for rat D1 receptor, 5=-GGTAGAAGTGTTAGGAGCCAT-3= (GenBank Accession No. NC-005116) and D5 receptor, 5=-CAGCATGTCCGCTGAGT-3= (GenBank Accession No. NC-005113) were compared with sense sequence controls D1 receptor, 5=-ATGGCTCCTAACACTTCTACC-3= and D5 receptor, 5=-ACTCAGCGCGACATGCTG-3= (42, 44). Briefly, cells were grown in six-well plates until 50% confluence, and 50 nM antisense or scrambled oligonucleotides were mixed with 6 l of oligofectamine in Optimem medium (Invitrogen Life Technologies) and incubated for 24 h and then switched to growth medium and incubated for an another 24 h. Fenoldopam (10⫺6 mol/l) was then added to the medium without growth factors for 24 h, lysed, and processed for immunoblotting for renalase and D1 and D5 receptors (19, 49). To determine the specificity of renalase expression and activity measurements, we used renalase small interfering (si)RNA to inhibit the renalase expression. The antisense oligonucleotides for renalase was 3=-TdTCGGUCAUGUAGUGGUCAUU-5=, and the scramble oligonucleotides was 5=-GCCAGUACAUCACCAGUAAdTdT-3=. Renalase activity. The kidneys and cells were harvested as above. The supernatants were used for assay of renalase activity, as reported previously (38). Renalase, as a FAD-dependent oxidoreductase, uses NADH as a cofactor to reduce FAD (14, 15, 26, 38, 40). Therefore, the amount of NADH oxidized to NAD⫹ (NADH oxidation) is considered to indicate renalase activity (14, 15, 26, 38, 40). The reaction was initiated by adding 40 g of kidney lysate or cell protein to 200 l of assay buffer in 96-well plates (0.6-cm path length). Forty micrograms of BSA served as a negative control. Absorbance at 340 nM was measured in a plate reader at 37°C every 2 min for up to 30
Renalase Dimer 70 kDa Monomer 37 kDa
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Fig. 1. Specificity of renalase antibody. Wistar-Kyoto (WKY) renal proximal tubule (RPT) cells were incubated with renalase small interfering (si)RNA (5 ⫻ 10⫺8 mol/l) for 48 h; renalase expression was determined by immunoblotting. The 70-kDa band represents renalase protein (n ⫽ 4; *P ⬍ 0.05 vs. others).
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min. The amount of NADH oxidized to NAD⫹ was calculated from the decrease in absorbance at 340 nm using a molar extinction coefficient of 0.00622 l·M⫺1·cm⫺1. The background, i.e., changes in absorbance obtained with BSA, was subtracted from all the readings (16, 38). Statistical analysis. In the our study, the data are expressed as means ⫾ SE. Statistical analyses were performed by one-way ANOVA followed by Holm-Sidak’s post hoc multiple comparison test (for comparison of more than 2 groups) or Student’s t-test (for
A
comparison of 2 groups). A value of P ⬍ 0.05 was considered significant. RESULTS
D1-like receptors increase renalase expression in WKY RPT cells but decrease it in SHR RPT cells. The specificity of the renalase antibody was verified in WKY RPT cells with renalase siRNA; after incubation with renalase siRNA (5 ⫻ 10⫺8
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Fig. 2. Effect of fenoldopam on renalase protein expression in RPT cells from WKY and spontaneously hypertensive rats (SHRs). A–D: effect of fenoldopam on renalase expression in RPT cells from WKY (A and B) and SHRs (C and D), treated with fenoldopam with different concentrations (10⫺7–10⫺5 mol/l) and different durations (0 – 48 h). Results are expressed as the ratio of renalase to -actin densities (n ⫽ 5– 6; *P ⬍ 0.05 vs. control). E: basal renalase expression in WKY and SHR RPT cells. Renalase expression was determined by immunoblotting (n ⫽ 5; *P ⬍ 0.05 vs. WKY). F and G: effect of the D1-like receptor agonist fenoldopam (Fen; 10⫺6 mol/l) and the D1-like receptor antagonist SCH23390 (SCH; 10⫺6 mol/l) on renalase expression in WKY (F) and SHR cells (G). Cells were incubated with the indicated reagents for 24 h. Results are expressed as the ratio of renalase to -actin densities (n ⫽ 5; *P ⬍ 0.05 vs. others). AJP-Renal Physiol • doi:10.1152/ajprenal.00196.2013 • www.ajprenal.org
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expression in WKY cells and inhibitory effect in SHR cells (Fig. 2, F and G). Renalase has intrinsic NADH oxidase activity (14 –16, 38). Treatment of RPT cells with varying concentrations of fenoldopam (10⫺7–10⫺5 mol/l) for 24 h caused a concentration-dependent increase in NADH oxidation in WKY cells and WKY cell culture medium and a concentration-dependent decrease in NADH oxidation in SHR cells and SHR cell culture medium (Fig. 3, A and B). To determine the mechanisms involved in the differential regulation of D1-like receptor on renalase expression in RPT cells from WKY and SHRs, qRT-PCR was used to study the effect of fenoldopam on renalase transcription. We found that fenoldopam increased renalase transcription in WKY RPT cells in a concentration-dependent manner (Fig. 4A) but had no significant effect in SHR cells (Fig. 4B). To study potential posttranscriptional mechanisms of renalase regulated by D1-like receptors, we examined protein expression in the presence of 10 g/ml cycloheximide (CHX) to inhibit de novo protein synthesis (10); steady-state levels of renalase were determined by immunoblotting (Fig. 5, A and B). Fenoldopam (Fen, 10⫺6 mol/l) accelerated the degradation of
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Fenoldopam -Log [M] Fig. 3. Effect of fenoldopam on renalase activity in RPT cells from WKY and SHRs. RPT cells from WKY and SHRs were treated with different concentrations of fenoldopam (10⫺7–10⫺5 mol/l) for 24 h, and the renalase activities in cell lysates (A) and culture medium (B) were measured by the NADH oxidation method. Renalase activity is expressed as NADH oxidation (nmol·min⫺1·mg protein⫺1 or ml medium; n ⫽ 5– 6; *P ⬍ 0.05 vs. individual control).
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mol/l) for 48 h, renalase expression in WKY RPT cells was suppressed ⬃72.39% (Fig. 1). Stimulation of D1-like receptors with fenoldopam increased renalase expression in a concentration- and time-dependent manner in WKY RPT cells (Fig. 2, A and B). The stimulatory effect was evident at 10⫺6 mol/l with a 49.5% increase in renalase expression. In contrast, in RPT cells from SHR, fenoldopam treatment decreased renalase expression (Figs. 2, C and D). The basal level of renalase expression was also higher in WKY than SHR cells (Fig. 2E). The specificity of fenoldopam as a D1-like receptors agonist was determined by studying the effect of the D1-like receptor antagonist SCH23390 (24, 27, 44). Consistent with the results shown in Fig. 2, A and C, fenoldopam (10⫺6 mol/l for 24 h) increased renalase expression in RPT cells from WKY cells and decreased renalase expression in SHR cells. The D1-like receptor antagonist SCH23390 (10⫺6 mol/l), by itself, had no effect on renalase expression in both WKY and SHR cells but reversed the stimulatory effect of fenoldopam on renalase
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Fenoldopam -Log[M] Fig. 4. Effects of fenoldopam on renalase mRNA expressions in RPT cells from WKY and SHRs. RPT cells from WKY (A) and SHRs (B) were treated with fenoldopam (10⫺7–10⫺5 mol/l) for 24 h; the expression of renalase mRNA was determined by quantitative real time RT-PCR (n ⫽ 5; *P ⬍ 0.05 vs. control).
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Fig. 5. Effect of fenoldopam on renalase degradation in RPT cells from WKY and SHRs. RPT cells from WKY (A) and SHRs (B) were incubated with cycloheximide (CHX; 10 g/ ml) with or without fenoldopam (10⫺6 mol/l) for the indicated times (h). RPT cells from WKY (C) and SHRs (D) were incubated with or without fenoldopam (10⫺6 mol/l) for the indicated times. Results are expressed as the ratio of renalase to -actin densities (n ⫽ 5– 6; *P ⬍ 0.05 vs. CHX alone).
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renalase protein in SHR cells (Fig. 5B) but not in WKY rat cells (Fig. 5A). Consistent with Fig. 2, B and D, there was no effect of vehicle or fenoldopam on renalase expression for up to 4 –5 h without CHX treatment (Fig. 5, C and D). To determine whether or not the regulation of D1-like receptor on renalase has physiological relevance, we measured renalase expression in the kidneys from WKY and SHRs. We found that renalase expression was lower in kidneys from SHRs than those from WKY rats [ratio of renalase to -actin: WKY ⫽ 1.21 ⫾ 0.13 (relative units); SHR ⫽ 0.78 ⫾ 0.09 (relative units); n ⫽ 7; P ⬍ 0.05]. Moreover, NADH oxidation in kidneys was also lower in SHRs than in WKY rats (WKY ⫽ 2.43 ⫾ 0.25 nmol·min⫺1·mg protein⫺1; SHR ⫽ 1.34 ⫾ 0.32 nmol·min⫺1·mg protein⫺1; n ⫽ 8; P ⬍ 0.05). Role of PKC in the stimulatory effect of fenoldopam on renalase expression in RPT cells from WKY rats. To determine the second messengers involved in the regulation of D1-like receptor on renalase expression, different signaling agonists and antagonists were used. The PKC inhibitor peptide 19 –31 (10⫺6 mol/l) (9, 44), by itself, had no effect on renalase expression but blocked the stimulatory effect of fenoldopam on renalase expression in RPT cells (Fig. 6A), indicating that PKC was involved in the stimulatory action of fenoldopam. To confirm this finding, we also evaluated the effect of PKC activator (PMA) (25, 28) on renalase expression in RPT cells.
control
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PMA increased renalase expression in a concentration-dependent way in WKY RPY cells (Fig. 6B). A PKA inhibitor (PKA inhibitor 14 –22, 10⫺6 mol/l) (4) and a calcium channel blocker (nicardipine, 10⫺6 mol/l) (23) had no effect on the stimulatory effect of fenoldopam on renalase expression in WKY RPT cells (data not shown). Role of D5 receptor on the stimulatory effect of the D1-like receptor on renalase expression in WKY cells. To determine whether the D1- or the D5-receptor was responsible for the fenoldopam-induced increase in renalase expression, we used antisense oligonucleotides to reduce the expression of either the D1 or D5 receptor (19). D1- and D5-receptor proteins were quantified by immunoblotting. D1 and D5 receptor expressions were inhibited by their corresponding antisense oligonucleotides but not by scrambled oligonucleotides (Fig. 7, A and B). Treatment with fenoldopam increased renalase expression in WKY cells incubated with D1-receptor antisense but not with D5-receptor antisense oligonucleotides, indicating that the D5, not D1 receptor, was involved in the stimulatory effect of the D1-like receptor on renalase expression in WKY cells (Fig. 7, C and D). DISCUSSION
There are several novel observations in our study. We showed that the D1-like receptor agonist fenoldopam increased
AJP-Renal Physiol • doi:10.1152/ajprenal.00196.2013 • www.ajprenal.org
REGULATION OF D5 RECEPTORS ON RENALASE IN RPT CELLS
A Renalase
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PMA -Log [M] Fig. 6. Role of PKC in the stimulatory effect of fenoldopam on renalase expression in WKY cells. A: effects of fenoldopam and the PKC inhibitor 19 –31 on renalase protein in RPT cells from WKY rats. The cells were incubated with the indicated reagents [fenoldopam (Fen), 10⫺6 mol/l; PKC inhibitor 19 –31 (PKCI), 10⫺6 mol/l] for 24 h. Results are expressed as the ratio of renalase to -actin densities (n ⫽ 6; *P ⬍ 0.05 vs. others). B: concentrationdependent effect of PKC activator on renalase expression in WKY RPT cells. The cells were incubated with the PKC activator (PMA; 10⫺8–10⫺6 mol/l) for 24 h. Results are expressed as the ratio of renalase to -actin densities (n ⫽ 6; *P ⬍ 0.05 vs. control).
renalase expression in RPT cells from WKY rats but, in contrast, decreased it in SHR cells. The effect was clearly exerted at D1-like receptors because a D1-like receptor antagonist SCH23390 (24, 27, 44) blocked the effect of fenoldopam in WKY and SHR cells. The stimulatory effect of D1-like receptors on renalase expression was mainly mediated by the D5 receptor through the PKC pathway in WKY cells. D1-like receptor stimulation by fenoldopam had no effect on renalase transcription in SHR cells but accelerated renalase
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degradation, indicating that, in contrast to that in WKY cells, D1-like receptors, by increasing renalase degradation, downregulated renalase expression and activity in SHR cells. As indicated in the Introduction, there are interactions between dopamine and adrenergic receptors outside neural cells (13, 27, 30, 33–35, 43). For example, activation of D1-like receptors induces relaxation of resistance arteries preconstricted by norepinephrine (27, 33). Moreover, D1-like receptors inhibit the ␣1-adrenergic receptor-mediated proliferation of vascular smooth muscle cells (27). Norepinephrine increases oxidative stress, which is inhibited in the presence of a dopamine-receptor agonist (36, 45, 47). In addition to dopamineand adrenergic-receptor interaction, activation of dopamine receptors, including D1-like receptors, inhibits the catecholamine production (12, 13, 18, 30, 35). Damase-Michel and colleagues (13, 35) showed that infusion of the D1-like receptor agonist fenoldopam reduces plasma norepinephrine levels in conscious sinoaotic denervated dogs. Ferrari et al. (18) found that the D1-like receptor-mediated decrease of catecholamine production is mainly via inhibition of tyrosine hydroxylase expression in human lymphocytes. Ten to fifteen percent of catecholamines in the renal peritubular blood are metabolized (1). As an important amino oxidase, activation of renalase increases the degradation of epinephrine and dihydroxyphenylanine in the circulation (14, 15, 26, 40). Dihydroxyphenylanine taken up by renal proximal tubules is the major source or renal dopamine production (7). Renalase is abundantly expressed in RPTs (26, 40), but whether or not dopamine receptor has an effect on renalase expression and therefore on epinephrine levels is not clear. Dopamine may negatively interact with the sympathetic nervous system, including epinephrine (45). Our present study shows that activation of D1-like receptor increases renalase expression in RPT cells from WKY rats, indicating that in addition to an inhibitory effect of fenoldopam on tyrosine hydroxylase activity, the regulation of fenoldopam on renalase expression and activity could be involved in the regulation of epinephrine catabolism in kidney. The D1-like receptor family is comprised of D1 and D5 receptors. Activation of the D1 or D5 receptor increases adenylyl cyclase activity (11, 24, 33, 36, 45, 47). However, the effect of D1 or D5 receptor is not always the same, for example, the D5 receptor, not the D1 receptor, is responsible for the degradation of AT1 receptors in RPT cells (19). In the kidney the D5 receptor, but not the D1 receptor, is linked to G␣12 and G␣13. Stimulation of the D5 receptor increases the association between the D5 receptor and G␣12 or G␣13 and may impair the stimulatory effect of G␣12/G␣13 on sodium hydrogen exchanger 3 activity (49). Previous studies have shown that the D5 receptor may be important in the regulation of catecholamine release and plasma concentration. Dahmer and Senogles (12) demonstrated that the D5 receptor inhibits norepinephrine and epinephrine release from adrenal chromaffin cells. D5receptor-deficient mice develop sympathetic-dependent hypertension (43, 49). Our present study shows that inhibition of D5-receptor but not D1-receptor expression could block the stimulatory effect of fenoldopam on renalase expression in WKY cells. Renalase may be involved in the pathogenesis of hypertension. Renalase-deficient mice develop hypertension, and renalase polymorphisms are associated with essential hyperten-
AJP-Renal Physiol • doi:10.1152/ajprenal.00196.2013 • www.ajprenal.org
F594
REGULATION OF D5 RECEPTORS ON RENALASE IN RPT CELLS
A
B
D1 receptor
70kDa
β-actin
43kDa
50kDa
D5 receptor β−actin
43kDa
1.4 1.4 1.2
1.0
D5R/β-actin
D1R/β-actin
1.2
0.8
*
0.6 0.4 0.2
1.0 0.8
*
0.6 0.4 0.2
0.0
0.0 Control
AS
Scrambled
Control
C
AS Scrambled
D
Renalase
70kDa
Renalase
70kDa
β-actin
43kDa
β-actin
43kDa
1.2
*
1.6
*
1.0 0.8 0.6 0.4
1.2 1.0 0.8 0.6 0.4
0.2
0.2
0.0
0.0 AS AS+Fen
sion (5, 14, 15, 26, 40, 48). We found that activation of the D1-like receptor increases renalase expression in WKY cells but decreases it in SHRs. The stimulatory effect of the D1-like receptor on renalase expression could be explained by an increase renalase transcription because fenoldopam increases renalase mRNA expression in WKY rats. The inhibitory effect of fenoldopam on renalase expression in SHR cells is not due to a decrease in renalase transcription but could be ascribed to the augmented renalase degradation. These in vitro findings have relevance in vivo because renalase expression and activity are lower in kidneys from SHRs than WKY rats. Because renalase increases the degradation of epinephrine, the decreased expression and activity of renalase would lead to increased epinephrine concentration in kidney in SHRs, which would increase sodium transport and vascular smooth muscle contractility (6, 45). However, the increased degradation of dihydroxyphenylanine by renalase could also decrease the production of dopamine (24). Therefore, studying the regulation of renalase expression and activity in kidney is important. Our present study found that stimulation of D1-like receptor with fenoldopam increases renalase expression in WKY cells but not in SHR cells. We have reported that the renal D1-like receptor function is impaired in SHRs due to the hyperphosphorylation of the D1 receptor (17, 31), which has been ascribed to increased constitutive activity of G protein-coupled receptor kinase 4 (GRK4) (17, 31). Whether or not this strategy
*
1.4
Renalase/β-actin
1.4
Renalase/β-actin
Fig. 7. Role of the D5 receptor in the effect of fenoldopam on renalase expression in RPT cells from WKY rats. A and B: effects of D1or D5-receptor antisense oligonucleotides on D1- or D5-receptor expression in WKY cells. The cells were incubated with vehicle, D1- or D5-receptor antisense (AS) or scrambled oligonucleotides (50 nmol/l) for 48 h. D1- or D5-receptor protein expressions determined by immunoblotting were expressed as the ratio of D1- or D5-receptor to -actin densities (n ⫽ 5; *P ⬍ 0.05 vs. others). C and D: effects of D1- or D5-receptor antisense and scrambled oligonucleotides (SC) on the stimulatory of fenoldopam on renalase expression in RPT cells. The RPT cells were incubated with D1 (C)- or D5 (D)-receptor antisense and scrambled oligonucleotides for 48 h and then incubated with fenoldopam (10⫺6 mol/l) or vehicle for 24 h. The D1- or D5-receptor expression was determined by immunoblotting. Results were expressed at the ratio of renalase to -actin (n ⫽ 5; *P ⬍ 0.05 vs. corresponding controls).
SC SC+Fen
AS AS+Fen
SC SC+Fen
is helpful in increasing renalase expression and activity in kidney of SHRs needs to be confirmed in the future. In summary, we have demonstrated that the D1-like receptor, via the D5 receptor, increases renalase expression in RPT cells from WKY rats but, in contrast, decreases it in SHR cells. The stimulatory effect of the D5 receptor on renalase expression is ascribed to increased renalase transcription in WKY cells, while the inhibitory effect of the D1-like receptor on renalase expression in SHR cells may be due to augmented degradation. GRANTS These studies were supported in part by National Natural Science Foundation of China Grants 31130029, 81100500, 30925018, and 81100111; National Basic Research Program of China (973 Program) Grants 2012CB517801 and 2008CB517308, Natural Science Foundation Project of Chongqing Grants CSTC2009BA5044 and CSTC2011BB5033; and National Heart, Lung, and Blood Institute Grant R01-HL-092196. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: S.W., X.L., J.Y., C.C., Y.H., H.R., S.Z., and D.H. performed experiments; S.W., X.L., J.Y., H.W., C.C., Y.H., H.R., S.Z., and D.H. analyzed data; S.W. and X.L. prepared figures; S.W., J.Y., and H.W. drafted manuscript; S.W., J.Y., H.W., C.C., Y.H., H.R., S.Z., D.H., L.Z., L.D.A., W.E.W., P.A.J., and C.Z. approved final version of manuscript; X.L., H.W., L.Z., W.E.W., P.A.J., and C.Z. interpreted results of experiments; J.Y.,
AJP-Renal Physiol • doi:10.1152/ajprenal.00196.2013 • www.ajprenal.org
REGULATION OF D5 RECEPTORS ON RENALASE IN RPT CELLS H.W., W.E.W., and C.Z. conception and design of research; L.Z., L.D.A., W.E.W., P.A.J., and C.Z. edited and revised manuscript. 22.
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46. Zeng C, Liu Y, Wang Z, He D, Huang L, Yu P, Zheng S, Jones JE, Asico LD, Hopfer U, Eisner GM, Felder RA, Jose PA. Activation of D3 dopamine receptor decreases angiotensin II type 1 receptor expression in rat renal proximal tubule cells. Circ Res 99: 494 –500, 2006. 47. Zeng C, Villar VA, Yu P, Zhou L, Jose PA. Reactive oxygen species and dopamine receptor function in essential hypertension. Clin Exp Hypertens 31: 156 –178, 2009. 48. Zhao Q, Fan Z, He J, Chen S, Li H, Zhang P, Wang L, Hu D, Huang J, Qiang B, Gu D. Renalase gene is a novel susceptibility gene for essential hypertension: a two-stage association study in northern Han Chinese population. J Mol Med (Berl) 85: 877–885, 2007. 49. Zheng S, Yu P, Zeng C, Wang Z, Yang Z, Andrews PM, Felder RA, Jose PA. G␣12-and G␣13-protein subunit linkage of D5 dopamine receptors in the nephron. Hypertension 41: 604 –610, 2003.
AJP-Renal Physiol • doi:10.1152/ajprenal.00196.2013 • www.ajprenal.org
Regulation of renalase expression by D5 dopamine receptors in rat renal proximal tubule cells Shaoxiong Wang,1,2* Xi Lu,1,2* Jian Yang,1,2* Hongyong Wang,1,2 CaiYu Chen,1,2 Yu Han,1,2 Hongmei Ren,1,2 Shuo Zheng,1,2 Duofen He,1,2 Lin Zhou,1,2 Laureano D. Asico,3 Wei Eric Wang,1,2# Pedro A. Jose,3 and Chunyu Zeng1,2# 1
Department of Cardiology, Daping Hospital, Third Military Medical University, Chongqing, People’s Republic of China; Chongqing Institute of Cardiology, Chongqing, People’s Republic of China; and 3Division of Nephrology, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 2
Submitted 8 April 2013; accepted in final form 15 January 2014
Wang S, Lu X, Yang J, Wang H, Chen C, Han Y, Ren H, Zheng S, He D, Zhou L, Asico LD, Wang WE, Jose PA, Zeng C. Regulation of renalase expression by D5 dopamine receptors in rat renal proximal tubule cells. Am J Physiol Renal Physiol 306: F588–F596, 2014. First published February 5, 2014; doi:10.1152/ajprenal.00196.2013.—The dopaminergic and sympathetic systems interact to regulate blood pressure. Our previous studies showed regulation of ␣1-adrenergic receptor function by D1-like dopamine receptors in vascular smooth muscle cells. Because renalase could regulate circulating epinephrine levels and dopamine production in renal proximal tubules (RPTs), we tested the hypothesis that D1-like receptors regulate renalase expression in kidney. The effect of D1-like receptor stimulation on renalase expression and function was measured in immortalized RPT cells from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs). We found that the D1-like receptor agonist fenoldopam (10⫺7–10⫺5 mol/l) increased renalase protein expression and function in WKY RPT cells but decreased them in SHR cells. Fenoldopam also increased renalase mRNA levels in WKY but not in SHR cells. In contrast, fenoldopam increased the degradation of renalase protein in SHR cells but not in WKY cells. The regulation of renalase by the D1-like receptor was mainly via the D5 receptor because silencing of the D5 but not D1 receptor by antisense oligonucleotides blocked the stimulatory effect of the D1-like receptor on renalase expression in WKY cells. Moreover, inhibition of PKC, by the PKC inhibitor 19 –31, blocked the stimulatory effect of fenoldopam on renalase expression while stimulation of PKC, by a PKC agonist (PMA), increased renalase expression, indicating that PKC is involved in the process. Our studies suggest that the D5 receptor positively regulates renalase expression in WKY but not SHR RPT cells; aberrant regulation of renalase by the D5 receptor may be involved in the pathogenesis of hypertension. dopamine receptor; renalase; renal proximal tubule cells; renal proximal tubule cells; hypertension
a neurotransmitter in the central nervous system, has been reported to be an important regulator of renal and adrenal function, sodium balance, and blood pressure and to be relevant to the pathogenesis and/or maintenance of hypertension (11, 24, 33, 36, 45, 47). Dopamine receptors are classified into two families: the D1-like receptor subfamily includes the D1 and the D5 receptor, while the D2-like subfamily includes the D2, D3, and D4 receptors. Whereas the D1-like receptors couple to the stimulatory G-pro-
tein G␣S and thus activate adenylyl cyclase, all of the D2-like receptors couple to the inhibitory G-protein G␣i/G␣o and inhibit adenylyl cyclase and calcium channels and modulate potassium channels (11, 24, 36, 45, 47). There are interactions between dopamine receptors and the sympathetic nervous system (13, 30, 34, 35, 43). Our previous studies found that, as an antihypertensive factor, activation of dopamine receptors, especially D1-like and D3 receptors, inhibits the ␣1-adrenergic receptor-mediated proliferation of vascular smooth muscle cells and relaxes the resistant artery preconstricted by norepinephrine (27). Moreover, stimulation of D1-like receptors inhibits the catecholamine production, partly via inhibition of tyrosine hydroxylase expression (18). Renalase, a flavin adenine dinucleotide-dependent amine oxidase, plays an important role in the control of blood pressure by regulating sympathetic tone (5, 14, 15, 26, 40, 48). It is known that renalase increases the degradation of epinephrine in the blood circulation (14, 15, 26, 40). There is evidence showing renalase expression in renal proximal tubules (26, 40). Renalase metabolizes circulating epinephrine and dihydroxyphenylanine, the latter is converted to dopamine by renal proximal tubules, the major source of renal dopamine (24, 45). Because renalase could regulate circulating epinephrine levels and dopamine production in renal proximal tubules (RPTs), we tested the hypothesis that D1-like receptors regulate renalase expression and function in kidney. The effect of D1-like receptor on renalase expression was studied in immortalized RPT cells from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs). Because the specific D1-like receptor involved in the regulation of dopamine release or metabolism is unknown, we also determined which D1-like receptor is involved in the process.
DOPAMINE, WELL KNOWN AS
MATERIALS AND METHODS
* S. Wang, X. Lu, and J. Yang contributed equally to this work. # W. E. Wang and C. Zeng contributed equally to this work. Address for reprint requests and other correspondence: C. Zeng, Dept. of Cardiology, Daping Hospital, The Third Military Medical Univ., Chongqing, P.R. China (e-mail: [email protected]).
WKY and SHRs. Male WKY and SHRs (SLRC Laboratory Animals, Shanghai, China), ranging in age from 9 to 16 wk, fed regular rat chow with normal sodium (0.4% NaCl) content, were used. Food but not water was withheld 24 h before the study. The rats were anesthetized with pentobarbital (50 mg/kg body wt ip) (21, 27). Following a laparotomy, the kidneys were harvested, snap frozen in liquid nitrogen, and stored at ⫺70°C. All studies were approved by the Daping Hospital Animal Care and Use Committee. Cell culture. Immortalized RPT cells from WKY and SHRs (22, 42, 46) were cultured at 37°C in 95% air-5% CO2 atmosphere in FBS% DMEM/F-12 culture media, as previously described (41). The cells (80% confluence) were extracted in ice-cold lysis buffer (PBS with 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 10⫺3 mol/l EDTA, 10⫺3 mol/l EGTA, 10⫺3 mol/l PMSF, 10 g/ml aprotinin, and 10 g/ml leupeptin),
F588
1931-857X/14 Copyright © 2014 the American Physiological Society
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REGULATION OF D5 RECEPTORS ON RENALASE IN RPT CELLS
sonicated, kept on ice for 1 h, and centrifuged at 16,000 g for 30 min. The supernatants were stored at ⫺70°C until use for immunoblotting. Immunoblotting. Uniform amounts of proteins, separated by SDSpolyacrylamide gel electrophoresis, were electrophoretically transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in Dulbecco’s PBS with 0.05% Tween-20 and then probed with diluted affinity-purified goat polyclonal antibody to renalase (1:350; Sigma-Aldrich) (26, 40), rabbit polyclonal antibody to D1 receptor (1:200; Santa Cruz Biotechnology), D5 receptor (1:500; Santa Cruz Biotechnology), or -actin antibody (1:600; Santa Cruz Biotechnology) overnight (19). After being washed for five times (10 min each time), the membranes were incubated with diluted peroxidase-labeled goat anti-rabbit IgG or rabbit anti-goat IgG (Santa Cruz Biotechnology) in 5% milk for 1.5 h at room temperature. The signal was detected using chemiluminescence and developed on X-ray film. The density of the bands was quantified by densitometry using Quantiscan (Ferguson, MO), as previously reported (22, 42, 46). The amount of protein transferred onto the membranes was verified by immunoblotting for -actin. Quantitative RT-PCR. Total RNA was isolated and quantified as described previously (22, 46). For real-time quantitative RT-PCR (qRT-PCR) analysis, cDNA was synthesized from 0.5 mg of total RNA with a cDNA synthesis kit (High Capacity RNA-to-cDNA Kit; Takara, Tokyo, Japan). In the thermal cycle, 2 l cDNA were used per 25-l final reaction volume. PCRs were carried out with the Brilliant SYBR Green QPCR Master Mix kit (High Capacity RNA-to-cDNA Kit; Takara) in a total volume of 25 l. For renalase, the forward primer was 5=-TGCCAACAGTCCTCATAATCC-3= and the reverse primer was 5=-TCCTTCCTTCACTTCCATTCC-3= (GenBank Accession No. BC 005100). -Actin served as a housekeeping/reference gene for normalization. For -actin, the forward primer was 5=AGTGTGACGTTGACATCCGT-3= and the reverse primer was 5=GACTCATCGTACTCCTGCTT-3= (GenBank Accession No. BC
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A S
ed bl m a cr
063166). The amplification profile for renalase and -actin used on the BIO-RAD CFX96 (Bio-Rad Laboratories) was 95°C for 3 min followed by 40 cycles of 95°C per 10 s and 72°C per 30 s. qRT-PCR experiments were repeated for three times. Antisense oligonucleotides. The effects of 50 nM of propyne/ phosphorothioate-modified antisense oligonucleotides for rat D1 receptor, 5=-GGTAGAAGTGTTAGGAGCCAT-3= (GenBank Accession No. NC-005116) and D5 receptor, 5=-CAGCATGTCCGCTGAGT-3= (GenBank Accession No. NC-005113) were compared with sense sequence controls D1 receptor, 5=-ATGGCTCCTAACACTTCTACC-3= and D5 receptor, 5=-ACTCAGCGCGACATGCTG-3= (42, 44). Briefly, cells were grown in six-well plates until 50% confluence, and 50 nM antisense or scrambled oligonucleotides were mixed with 6 l of oligofectamine in Optimem medium (Invitrogen Life Technologies) and incubated for 24 h and then switched to growth medium and incubated for an another 24 h. Fenoldopam (10⫺6 mol/l) was then added to the medium without growth factors for 24 h, lysed, and processed for immunoblotting for renalase and D1 and D5 receptors (19, 49). To determine the specificity of renalase expression and activity measurements, we used renalase small interfering (si)RNA to inhibit the renalase expression. The antisense oligonucleotides for renalase was 3=-TdTCGGUCAUGUAGUGGUCAUU-5=, and the scramble oligonucleotides was 5=-GCCAGUACAUCACCAGUAAdTdT-3=. Renalase activity. The kidneys and cells were harvested as above. The supernatants were used for assay of renalase activity, as reported previously (38). Renalase, as a FAD-dependent oxidoreductase, uses NADH as a cofactor to reduce FAD (14, 15, 26, 38, 40). Therefore, the amount of NADH oxidized to NAD⫹ (NADH oxidation) is considered to indicate renalase activity (14, 15, 26, 38, 40). The reaction was initiated by adding 40 g of kidney lysate or cell protein to 200 l of assay buffer in 96-well plates (0.6-cm path length). Forty micrograms of BSA served as a negative control. Absorbance at 340 nM was measured in a plate reader at 37°C every 2 min for up to 30
Renalase Dimer 70 kDa Monomer 37 kDa
C
t on
l ro
N siR
A
ed l bl ro am nt r Co Sc
N siR
A
b m ra Sc
led C
ol tr on
N siR
A
ed bl am r Sc
β−actin 43 kDa
Fig. 1. Specificity of renalase antibody. Wistar-Kyoto (WKY) renal proximal tubule (RPT) cells were incubated with renalase small interfering (si)RNA (5 ⫻ 10⫺8 mol/l) for 48 h; renalase expression was determined by immunoblotting. The 70-kDa band represents renalase protein (n ⫽ 4; *P ⬍ 0.05 vs. others).
1.4
Renalase/β-actin
Co
l ro nt
F589
1.2 1.0 0.8 0.6
*
0.4 0.2 0.0 Control
siRNA
Scrambled
AJP-Renal Physiol • doi:10.1152/ajprenal.00196.2013 • www.ajprenal.org
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REGULATION OF D5 RECEPTORS ON RENALASE IN RPT CELLS
min. The amount of NADH oxidized to NAD⫹ was calculated from the decrease in absorbance at 340 nm using a molar extinction coefficient of 0.00622 l·M⫺1·cm⫺1. The background, i.e., changes in absorbance obtained with BSA, was subtracted from all the readings (16, 38). Statistical analysis. In the our study, the data are expressed as means ⫾ SE. Statistical analyses were performed by one-way ANOVA followed by Holm-Sidak’s post hoc multiple comparison test (for comparison of more than 2 groups) or Student’s t-test (for
A
comparison of 2 groups). A value of P ⬍ 0.05 was considered significant. RESULTS
D1-like receptors increase renalase expression in WKY RPT cells but decrease it in SHR RPT cells. The specificity of the renalase antibody was verified in WKY RPT cells with renalase siRNA; after incubation with renalase siRNA (5 ⫻ 10⫺8
B
C
Renalase
70 kDa
Renalase
70kDa
Renalase
70 kDa
β−actin
43 kDa
β−actin
43kDa
β-actin
43 kDa
1.4
*
1.2 1.0 0.8 0.6 0.4
*
0.30 0.25
*
0.20 0.15 0.10
1.6
7
6
5
Control 6
Fenoldopam -Log [M]
D
12
24
WKY
β−actin
43kDa
1.0
0.6
*
0.20 0.18
*
0.16 0.14 0.12 control 6
12 24 Time (hrs)
43 kDa
1.6 1.4 1.2 1.0
*
0.8 0.6 0.4 0.2 0.0
Renalase
70kDa
β-actin
43kDa
1.4
*
1.2 1.0 0.8 0.6 0.4 0.2 0.0
WKY
48
5
F
Renalase/β-actin
*
0.22
6
dimer 70 kDa
β-actin
Renalase/β-actin
0.24
7
Control
1.6
0.26
*
Fenoldopam -Log [M]
monomer 37 kDa
0.28
*
0.8
48
SHR
Renalase 70kDa
*
1.2
Time (hrs)
E
Renalase
1.4
0.4
0.05 Control
Renalase/β-actin
*
Renalase/β-actin
Renalase/β-actin
Renalase/β-actin
0.35
*
1.6
SHR
Control Fen
SCH Fen+SCH
G 70kDa
β-actin
43kDa
Renalase/β-actin
Renalase
1.2 1.0
*
0.8 0.6 0.4 0.2 0.0 Control
Fen SCH Fen+SCH
Fig. 2. Effect of fenoldopam on renalase protein expression in RPT cells from WKY and spontaneously hypertensive rats (SHRs). A–D: effect of fenoldopam on renalase expression in RPT cells from WKY (A and B) and SHRs (C and D), treated with fenoldopam with different concentrations (10⫺7–10⫺5 mol/l) and different durations (0 – 48 h). Results are expressed as the ratio of renalase to -actin densities (n ⫽ 5– 6; *P ⬍ 0.05 vs. control). E: basal renalase expression in WKY and SHR RPT cells. Renalase expression was determined by immunoblotting (n ⫽ 5; *P ⬍ 0.05 vs. WKY). F and G: effect of the D1-like receptor agonist fenoldopam (Fen; 10⫺6 mol/l) and the D1-like receptor antagonist SCH23390 (SCH; 10⫺6 mol/l) on renalase expression in WKY (F) and SHR cells (G). Cells were incubated with the indicated reagents for 24 h. Results are expressed as the ratio of renalase to -actin densities (n ⫽ 5; *P ⬍ 0.05 vs. others). AJP-Renal Physiol • doi:10.1152/ajprenal.00196.2013 • www.ajprenal.org
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REGULATION OF D5 RECEPTORS ON RENALASE IN RPT CELLS
9
* *
8 7
*
6 5
* WKY
4
SHR
3 Control
7
6
5
Fenoldopam -Log [M]
B 3.8
* NADH oxidation (nmol/min/ml medium)
3.6
WKY
*
SHR 3.4 3.2
expression in WKY cells and inhibitory effect in SHR cells (Fig. 2, F and G). Renalase has intrinsic NADH oxidase activity (14 –16, 38). Treatment of RPT cells with varying concentrations of fenoldopam (10⫺7–10⫺5 mol/l) for 24 h caused a concentration-dependent increase in NADH oxidation in WKY cells and WKY cell culture medium and a concentration-dependent decrease in NADH oxidation in SHR cells and SHR cell culture medium (Fig. 3, A and B). To determine the mechanisms involved in the differential regulation of D1-like receptor on renalase expression in RPT cells from WKY and SHRs, qRT-PCR was used to study the effect of fenoldopam on renalase transcription. We found that fenoldopam increased renalase transcription in WKY RPT cells in a concentration-dependent manner (Fig. 4A) but had no significant effect in SHR cells (Fig. 4B). To study potential posttranscriptional mechanisms of renalase regulated by D1-like receptors, we examined protein expression in the presence of 10 g/ml cycloheximide (CHX) to inhibit de novo protein synthesis (10); steady-state levels of renalase were determined by immunoblotting (Fig. 5, A and B). Fenoldopam (Fen, 10⫺6 mol/l) accelerated the degradation of
A
3.0
1.6
2.8
*
2.6 2.4 Control
7
6
*
1.4
*
5
Fenoldopam -Log [M] Fig. 3. Effect of fenoldopam on renalase activity in RPT cells from WKY and SHRs. RPT cells from WKY and SHRs were treated with different concentrations of fenoldopam (10⫺7–10⫺5 mol/l) for 24 h, and the renalase activities in cell lysates (A) and culture medium (B) were measured by the NADH oxidation method. Renalase activity is expressed as NADH oxidation (nmol·min⫺1·mg protein⫺1 or ml medium; n ⫽ 5– 6; *P ⬍ 0.05 vs. individual control).
Renalase/β-actin
NADH oxidation (nmol/min/mg protein)
A
*
1.2 1.0 0.8 0.6 0.4 Control
7
6
5
Fenoldopam -Log[M]
B 5
4
Renalase/β-actin
mol/l) for 48 h, renalase expression in WKY RPT cells was suppressed ⬃72.39% (Fig. 1). Stimulation of D1-like receptors with fenoldopam increased renalase expression in a concentration- and time-dependent manner in WKY RPT cells (Fig. 2, A and B). The stimulatory effect was evident at 10⫺6 mol/l with a 49.5% increase in renalase expression. In contrast, in RPT cells from SHR, fenoldopam treatment decreased renalase expression (Figs. 2, C and D). The basal level of renalase expression was also higher in WKY than SHR cells (Fig. 2E). The specificity of fenoldopam as a D1-like receptors agonist was determined by studying the effect of the D1-like receptor antagonist SCH23390 (24, 27, 44). Consistent with the results shown in Fig. 2, A and C, fenoldopam (10⫺6 mol/l for 24 h) increased renalase expression in RPT cells from WKY cells and decreased renalase expression in SHR cells. The D1-like receptor antagonist SCH23390 (10⫺6 mol/l), by itself, had no effect on renalase expression in both WKY and SHR cells but reversed the stimulatory effect of fenoldopam on renalase
3
2
1
0 Control
7
6
5
Fenoldopam -Log[M] Fig. 4. Effects of fenoldopam on renalase mRNA expressions in RPT cells from WKY and SHRs. RPT cells from WKY (A) and SHRs (B) were treated with fenoldopam (10⫺7–10⫺5 mol/l) for 24 h; the expression of renalase mRNA was determined by quantitative real time RT-PCR (n ⫽ 5; *P ⬍ 0.05 vs. control).
AJP-Renal Physiol • doi:10.1152/ajprenal.00196.2013 • www.ajprenal.org
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REGULATION OF D5 RECEPTORS ON RENALASE IN RPT CELLS
A
B
Renalase
70kDa
Renalase
β-actin
43kDa
β-actin
4h 3h 2h 1h 0h 1h 2h 3h 4h 1.4
1.2 1.1
CHX CHX+Fen
1.0 0.9 0.8 0.7
Renalase/β-actin
Renalase/β-actin
1h 2h 3h 4h CHX+Fen
1.8
1.3
Fig. 5. Effect of fenoldopam on renalase degradation in RPT cells from WKY and SHRs. RPT cells from WKY (A) and SHRs (B) were incubated with cycloheximide (CHX; 10 g/ ml) with or without fenoldopam (10⫺6 mol/l) for the indicated times (h). RPT cells from WKY (C) and SHRs (D) were incubated with or without fenoldopam (10⫺6 mol/l) for the indicated times. Results are expressed as the ratio of renalase to -actin densities (n ⫽ 5– 6; *P ⬍ 0.05 vs. CHX alone).
43kDa 4h 3h 2h 1h 0h CHX
CHX+Fen
CHX
70kDa
0.6
1.6 1.4
*
*
*
2
3
4
1.2
CHX CHX+Fen
1.0 0.8 0.6 0.4 0.2
Control
1
2
3
Control
4
1
Time (hours)
Time (hours)
C
D
Renalase
70kDa
Renalase
β-actin
43kDa
β-actin
70kDa 43kDa 4h 3h 2h 1h 0h 1h 2h 3h 4h Vehicle Fen
4h 3h 2h 1h 0h 1h 2h 3h 4h Fen Vehicle 1.4 1.2
1.0 0.8 0.6 0.4
Vehicle Fen
0.2
Renalase/β-actin
Renalase/β-actin
1.2
1.0 0.8 0.6 0.4
Vehicle Fen
0.2 0.0
0.0 control
1
2
3
4
Time (hours)
renalase protein in SHR cells (Fig. 5B) but not in WKY rat cells (Fig. 5A). Consistent with Fig. 2, B and D, there was no effect of vehicle or fenoldopam on renalase expression for up to 4 –5 h without CHX treatment (Fig. 5, C and D). To determine whether or not the regulation of D1-like receptor on renalase has physiological relevance, we measured renalase expression in the kidneys from WKY and SHRs. We found that renalase expression was lower in kidneys from SHRs than those from WKY rats [ratio of renalase to -actin: WKY ⫽ 1.21 ⫾ 0.13 (relative units); SHR ⫽ 0.78 ⫾ 0.09 (relative units); n ⫽ 7; P ⬍ 0.05]. Moreover, NADH oxidation in kidneys was also lower in SHRs than in WKY rats (WKY ⫽ 2.43 ⫾ 0.25 nmol·min⫺1·mg protein⫺1; SHR ⫽ 1.34 ⫾ 0.32 nmol·min⫺1·mg protein⫺1; n ⫽ 8; P ⬍ 0.05). Role of PKC in the stimulatory effect of fenoldopam on renalase expression in RPT cells from WKY rats. To determine the second messengers involved in the regulation of D1-like receptor on renalase expression, different signaling agonists and antagonists were used. The PKC inhibitor peptide 19 –31 (10⫺6 mol/l) (9, 44), by itself, had no effect on renalase expression but blocked the stimulatory effect of fenoldopam on renalase expression in RPT cells (Fig. 6A), indicating that PKC was involved in the stimulatory action of fenoldopam. To confirm this finding, we also evaluated the effect of PKC activator (PMA) (25, 28) on renalase expression in RPT cells.
control
1
2
3
4
Time (hours)
PMA increased renalase expression in a concentration-dependent way in WKY RPY cells (Fig. 6B). A PKA inhibitor (PKA inhibitor 14 –22, 10⫺6 mol/l) (4) and a calcium channel blocker (nicardipine, 10⫺6 mol/l) (23) had no effect on the stimulatory effect of fenoldopam on renalase expression in WKY RPT cells (data not shown). Role of D5 receptor on the stimulatory effect of the D1-like receptor on renalase expression in WKY cells. To determine whether the D1- or the D5-receptor was responsible for the fenoldopam-induced increase in renalase expression, we used antisense oligonucleotides to reduce the expression of either the D1 or D5 receptor (19). D1- and D5-receptor proteins were quantified by immunoblotting. D1 and D5 receptor expressions were inhibited by their corresponding antisense oligonucleotides but not by scrambled oligonucleotides (Fig. 7, A and B). Treatment with fenoldopam increased renalase expression in WKY cells incubated with D1-receptor antisense but not with D5-receptor antisense oligonucleotides, indicating that the D5, not D1 receptor, was involved in the stimulatory effect of the D1-like receptor on renalase expression in WKY cells (Fig. 7, C and D). DISCUSSION
There are several novel observations in our study. We showed that the D1-like receptor agonist fenoldopam increased
AJP-Renal Physiol • doi:10.1152/ajprenal.00196.2013 • www.ajprenal.org
REGULATION OF D5 RECEPTORS ON RENALASE IN RPT CELLS
A Renalase
70kDa
β-actin
43kDa
1.6
*
Renalase/β-actin
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Control
PKCI
Fen
Fen+PKCI
B Renalase
70kDa
β-actin
43kDa
1.6
*
Renalase/β-actin
1.4
*
1.2
* 1.0 0.8 0.6 0.4 Control
8
7
6
PMA -Log [M] Fig. 6. Role of PKC in the stimulatory effect of fenoldopam on renalase expression in WKY cells. A: effects of fenoldopam and the PKC inhibitor 19 –31 on renalase protein in RPT cells from WKY rats. The cells were incubated with the indicated reagents [fenoldopam (Fen), 10⫺6 mol/l; PKC inhibitor 19 –31 (PKCI), 10⫺6 mol/l] for 24 h. Results are expressed as the ratio of renalase to -actin densities (n ⫽ 6; *P ⬍ 0.05 vs. others). B: concentrationdependent effect of PKC activator on renalase expression in WKY RPT cells. The cells were incubated with the PKC activator (PMA; 10⫺8–10⫺6 mol/l) for 24 h. Results are expressed as the ratio of renalase to -actin densities (n ⫽ 6; *P ⬍ 0.05 vs. control).
renalase expression in RPT cells from WKY rats but, in contrast, decreased it in SHR cells. The effect was clearly exerted at D1-like receptors because a D1-like receptor antagonist SCH23390 (24, 27, 44) blocked the effect of fenoldopam in WKY and SHR cells. The stimulatory effect of D1-like receptors on renalase expression was mainly mediated by the D5 receptor through the PKC pathway in WKY cells. D1-like receptor stimulation by fenoldopam had no effect on renalase transcription in SHR cells but accelerated renalase
F593
degradation, indicating that, in contrast to that in WKY cells, D1-like receptors, by increasing renalase degradation, downregulated renalase expression and activity in SHR cells. As indicated in the Introduction, there are interactions between dopamine and adrenergic receptors outside neural cells (13, 27, 30, 33–35, 43). For example, activation of D1-like receptors induces relaxation of resistance arteries preconstricted by norepinephrine (27, 33). Moreover, D1-like receptors inhibit the ␣1-adrenergic receptor-mediated proliferation of vascular smooth muscle cells (27). Norepinephrine increases oxidative stress, which is inhibited in the presence of a dopamine-receptor agonist (36, 45, 47). In addition to dopamineand adrenergic-receptor interaction, activation of dopamine receptors, including D1-like receptors, inhibits the catecholamine production (12, 13, 18, 30, 35). Damase-Michel and colleagues (13, 35) showed that infusion of the D1-like receptor agonist fenoldopam reduces plasma norepinephrine levels in conscious sinoaotic denervated dogs. Ferrari et al. (18) found that the D1-like receptor-mediated decrease of catecholamine production is mainly via inhibition of tyrosine hydroxylase expression in human lymphocytes. Ten to fifteen percent of catecholamines in the renal peritubular blood are metabolized (1). As an important amino oxidase, activation of renalase increases the degradation of epinephrine and dihydroxyphenylanine in the circulation (14, 15, 26, 40). Dihydroxyphenylanine taken up by renal proximal tubules is the major source or renal dopamine production (7). Renalase is abundantly expressed in RPTs (26, 40), but whether or not dopamine receptor has an effect on renalase expression and therefore on epinephrine levels is not clear. Dopamine may negatively interact with the sympathetic nervous system, including epinephrine (45). Our present study shows that activation of D1-like receptor increases renalase expression in RPT cells from WKY rats, indicating that in addition to an inhibitory effect of fenoldopam on tyrosine hydroxylase activity, the regulation of fenoldopam on renalase expression and activity could be involved in the regulation of epinephrine catabolism in kidney. The D1-like receptor family is comprised of D1 and D5 receptors. Activation of the D1 or D5 receptor increases adenylyl cyclase activity (11, 24, 33, 36, 45, 47). However, the effect of D1 or D5 receptor is not always the same, for example, the D5 receptor, not the D1 receptor, is responsible for the degradation of AT1 receptors in RPT cells (19). In the kidney the D5 receptor, but not the D1 receptor, is linked to G␣12 and G␣13. Stimulation of the D5 receptor increases the association between the D5 receptor and G␣12 or G␣13 and may impair the stimulatory effect of G␣12/G␣13 on sodium hydrogen exchanger 3 activity (49). Previous studies have shown that the D5 receptor may be important in the regulation of catecholamine release and plasma concentration. Dahmer and Senogles (12) demonstrated that the D5 receptor inhibits norepinephrine and epinephrine release from adrenal chromaffin cells. D5receptor-deficient mice develop sympathetic-dependent hypertension (43, 49). Our present study shows that inhibition of D5-receptor but not D1-receptor expression could block the stimulatory effect of fenoldopam on renalase expression in WKY cells. Renalase may be involved in the pathogenesis of hypertension. Renalase-deficient mice develop hypertension, and renalase polymorphisms are associated with essential hyperten-
AJP-Renal Physiol • doi:10.1152/ajprenal.00196.2013 • www.ajprenal.org
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REGULATION OF D5 RECEPTORS ON RENALASE IN RPT CELLS
A
B
D1 receptor
70kDa
β-actin
43kDa
50kDa
D5 receptor β−actin
43kDa
1.4 1.4 1.2
1.0
D5R/β-actin
D1R/β-actin
1.2
0.8
*
0.6 0.4 0.2
1.0 0.8
*
0.6 0.4 0.2
0.0
0.0 Control
AS
Scrambled
Control
C
AS Scrambled
D
Renalase
70kDa
Renalase
70kDa
β-actin
43kDa
β-actin
43kDa
1.2
*
1.6
*
1.0 0.8 0.6 0.4
1.2 1.0 0.8 0.6 0.4
0.2
0.2
0.0
0.0 AS AS+Fen
sion (5, 14, 15, 26, 40, 48). We found that activation of the D1-like receptor increases renalase expression in WKY cells but decreases it in SHRs. The stimulatory effect of the D1-like receptor on renalase expression could be explained by an increase renalase transcription because fenoldopam increases renalase mRNA expression in WKY rats. The inhibitory effect of fenoldopam on renalase expression in SHR cells is not due to a decrease in renalase transcription but could be ascribed to the augmented renalase degradation. These in vitro findings have relevance in vivo because renalase expression and activity are lower in kidneys from SHRs than WKY rats. Because renalase increases the degradation of epinephrine, the decreased expression and activity of renalase would lead to increased epinephrine concentration in kidney in SHRs, which would increase sodium transport and vascular smooth muscle contractility (6, 45). However, the increased degradation of dihydroxyphenylanine by renalase could also decrease the production of dopamine (24). Therefore, studying the regulation of renalase expression and activity in kidney is important. Our present study found that stimulation of D1-like receptor with fenoldopam increases renalase expression in WKY cells but not in SHR cells. We have reported that the renal D1-like receptor function is impaired in SHRs due to the hyperphosphorylation of the D1 receptor (17, 31), which has been ascribed to increased constitutive activity of G protein-coupled receptor kinase 4 (GRK4) (17, 31). Whether or not this strategy
*
1.4
Renalase/β-actin
1.4
Renalase/β-actin
Fig. 7. Role of the D5 receptor in the effect of fenoldopam on renalase expression in RPT cells from WKY rats. A and B: effects of D1or D5-receptor antisense oligonucleotides on D1- or D5-receptor expression in WKY cells. The cells were incubated with vehicle, D1- or D5-receptor antisense (AS) or scrambled oligonucleotides (50 nmol/l) for 48 h. D1- or D5-receptor protein expressions determined by immunoblotting were expressed as the ratio of D1- or D5-receptor to -actin densities (n ⫽ 5; *P ⬍ 0.05 vs. others). C and D: effects of D1- or D5-receptor antisense and scrambled oligonucleotides (SC) on the stimulatory of fenoldopam on renalase expression in RPT cells. The RPT cells were incubated with D1 (C)- or D5 (D)-receptor antisense and scrambled oligonucleotides for 48 h and then incubated with fenoldopam (10⫺6 mol/l) or vehicle for 24 h. The D1- or D5-receptor expression was determined by immunoblotting. Results were expressed at the ratio of renalase to -actin (n ⫽ 5; *P ⬍ 0.05 vs. corresponding controls).
SC SC+Fen
AS AS+Fen
SC SC+Fen
is helpful in increasing renalase expression and activity in kidney of SHRs needs to be confirmed in the future. In summary, we have demonstrated that the D1-like receptor, via the D5 receptor, increases renalase expression in RPT cells from WKY rats but, in contrast, decreases it in SHR cells. The stimulatory effect of the D5 receptor on renalase expression is ascribed to increased renalase transcription in WKY cells, while the inhibitory effect of the D1-like receptor on renalase expression in SHR cells may be due to augmented degradation. GRANTS These studies were supported in part by National Natural Science Foundation of China Grants 31130029, 81100500, 30925018, and 81100111; National Basic Research Program of China (973 Program) Grants 2012CB517801 and 2008CB517308, Natural Science Foundation Project of Chongqing Grants CSTC2009BA5044 and CSTC2011BB5033; and National Heart, Lung, and Blood Institute Grant R01-HL-092196. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: S.W., X.L., J.Y., C.C., Y.H., H.R., S.Z., and D.H. performed experiments; S.W., X.L., J.Y., H.W., C.C., Y.H., H.R., S.Z., and D.H. analyzed data; S.W. and X.L. prepared figures; S.W., J.Y., and H.W. drafted manuscript; S.W., J.Y., H.W., C.C., Y.H., H.R., S.Z., D.H., L.Z., L.D.A., W.E.W., P.A.J., and C.Z. approved final version of manuscript; X.L., H.W., L.Z., W.E.W., P.A.J., and C.Z. interpreted results of experiments; J.Y.,
AJP-Renal Physiol • doi:10.1152/ajprenal.00196.2013 • www.ajprenal.org
REGULATION OF D5 RECEPTORS ON RENALASE IN RPT CELLS H.W., W.E.W., and C.Z. conception and design of research; L.Z., L.D.A., W.E.W., P.A.J., and C.Z. edited and revised manuscript. 22.
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