Dopamine regulates renal osmoregulation during hyposaline stress via DRD1 in the spotted scat (Scatophagus argus)

Dopamine is an important regulator of renal natriuresis and is critical for the adaptation of many animals to changing environmental salinity. However, the molecular mechanisms through which dopamine promotes this adaptation remain poorly understood. We studied the effects of dopamine on renal hypo-osmoregulation in the euryhaline fish Scatophagus argus (S. argus) during abrupt transfer from seawater (SW) to freshwater (FW). Following the transfer, serum dopamine concentration was decreased, and dopamine activated expression of the dopamine receptor 1 (designated SaDRD1) in the kidney, triggering the osmoregulatory signaling cascade. SaDRD1 protein is expressed in the renal proximal tubule cells in vivo, and is localized to the cell membrane of renal primary cells in vitro. Knockdown of SaDRD1 mRNA by siRNA significantly increased Na+/K+-ATPase (NKA) activity in cultured renal primary cells in vitro, suggesting that expression of SaDRD1 may oppose the activity of NKA. We demonstrate that exogenous dopamine enhances the response of NKA to hyposaline stress after transferring primary renal cells from isosmotic medium to hypoosmotic medium. Our results indicate that dopamine regulation via SaDRD1 ignited the renal dopaminergic system to balance the osmotic pressure through inhibiting NKA activity, providing a new perspective on the hyposaline adaptation of fish.

The lack of specific agonists and antagonists for each D 1 -like receptor subtype has made it challenging to distinguish the individual roles of DRD1 and DRD5 in the kidney, although some progress has been made through other methods 20,21 . In rats, blocking or down-regulating the DRD1 activity attenuates the natriuretic response to salt infusion in the kidney, suggesting a role distinct from that of DRD5 22 . DRD1 appears to play a greater role than DRD5 in the D 1 -like receptor-mediated regulation of the cAMP-dependent protein kinase signaling pathway in renal proximal tubule cells, as DRD1 activation increases cAMP production to a much greater extent than DRD5 activation 23 . Furthermore, it is likely that the natriuretic effect of dopamine is due mainly to the DRD1 in nephron segments of human 15 . However, other researches imply that DRD5 may be involved in the maintenance of normal sodium and water balance when dietary salt intake is increased 14 . Furthermore, DRD1 and DRD5 may be able to form a heteromeric receptor, cooperatively regulating sodium transport in renal cells 24 . Together, these studies demonstrate a need for further research to clarify the roles of each D 1 -like receptor subtype in renal osmotic regulation 15 .
Euryhaline fishes offer a valuable model for the study of osmoregulation in the kidney, as they frequently move between areas of high and low salinity, and must rapidly adapt to these conditions. Thus, these fish present a unique opportunity to study physiological responses in osmoregulatory organs as they occur. Most previous studies on euryhaline fish have focused on changes in Na + /K + -ATPase (NKA) activity during periods of osmotic and ionic stresses 3,4,25 . Here, we seek to gain a greater understanding of the poorly-understood hormonal control of NKA as this important enzyme responds to changing salinity 26 . In the present study, we studied role of dopamine and the D 1 -like receptors on the renal response of the euryhaline fish Scatophagus argus (the spotted scat) to hyposmotic stress. We employed a targeted molecular approach to specifically knock down SaDRD1 levels in cultured primary S. argus kidney cells, and observed the resulting response to osmotic stress in the presence and absence of exogenous dopamine. These results bring us closer to understanding the mechanisms of osmotic regulation in this fish.

Dopamine concentration detection in vivo and in vitro.
During acclimation of S. argus to FW, dopamine concentrations were significantly reduced in serum from the treatment group (**p < 0.01) at 1 and 3 hours post-transfer (hpt) compared to the control. Serum dopamine in the treated fish then rose, peaking at 12 hpt before declining. Compared with the control group, an increase of about 20% (from 346.5 pg/ml ± 18.2 pg/ml to 418.2 ± 26.7 pg/ml) of dopamine (DA) concentration was observed 12 hpt (p = 0.249). Thereafter, dopamine expressions in both groups were similar (Fig. 1).
Dopamine concentration in the culture medium of primary renal cells exposed to hyposmotic shock (100 mOsmol/L) was decreased significantly (*p < 0.05) at 0.5 h and 3 h (Fig. 1).
Cloning and sequence analysis of SaDRD1. A full-length cDNA encoding S. argus DRD1, designated SaDRD1, was cloned. The full-length of SaDRD1 cDNA was 2001 bp, containing a 1392-bp ORF, and encoded a precursor protein of 463 amino acids (aa) with 7 transmembrane domains ( Fig. 2A). Compared with DRD1 paralog amino acid sequences in other species, SaDRD1 showed 94% identity with Haplochromis burtoni, 93% identity with Oreochromis niloticus, 92% identity with Oryzias latipes, 90% identity with Fugu rubripes, and 71% identity with Homo sapiens (Fig. 2B). Phylogenetic analysis showed that SaDRD1 was clustered with teleost DRD1 paralogs in a cluster distinct from that of Homo sapiens (Fig. 2C).
Tissue distribution of DRD1 in S. argus. The expression patterns of SaDRD1 mRNA were examined in SW-reared and FW-shocked S. argus using semi-quantitative real-time RT-PCR (sqRT-PCR). In SW-reared fish, SaDRD1 mRNA was broadly expressed in the eye, brain and heart, and its expression was also observed argus. Dopamine contration in the serum of SW-reared S. argus was set as the control group, and that of the FW-shock fish was regarded as the treatment group. (B) Dopamine concentration in the culture medium of S. argus renal primary cells exposed to hypoosmotic conditions (100 mOsmol/L) in *p < 0.05; **p < 0.01.
Scientific RepoRts | 6:37535 | DOI: 10.1038/srep37535 in osmoregulatory organs, including the kidney and gill (Fig. 2D). Expression patterns of SaDRD1 mRNA in FW-shocked fish at different time points were shown in Supplementary Fig. S1. In the tissues of central nervous system (brain and notochord), SaDRD1 expression was detected throughout the period of FW-shock. In osmoregulatory organs (e.g. kidney, gill and skin), expression varied greatly within this period, suggesting that SaDRD1 was involved in acclimation to FW (Fig. S1). mRNA expression of D 1 -like receptors in vivo under hyposaline conditions. To determine effects of the hyposaline shock on the expression of D 1 -like receptors in the kidney of S. argus, SaDRD1 and SaDRD5 mRNA levels were measured by real-time quantitative RT-PCR (RT-qPCR) at different time points after hyposaline shock. SaDRD1 mRNA expression in the kidney fluctuated highly, with significant upregulation at 3 hpt and 12 hpt (> 2-fold) (*p < 0.05) (Fig. 3A). In contrast, SaDRD5 mRNA in the kidneys of fish transferred from SW to FW exhibited no significant changes, and was undetectable at 6 hpt and 1 dpt (Fig. 3B). The original data (Ct values of SaDRD1 and SaDRD5 mRNA expression) was presented in Supplementary Tables S1 and S2. Immunohistochemical experiments were conducted to determine the localization of SaDRD1 protein in the kidney of SW-acclimated S. argus and the subcellular localization in renal primary cells under isosmotic culture (approximately 300 mOsmol/L). Renal sections were double-stained with polyclonal rabbit antibody against SaDRD1 (green) and DAPI (blue), while the red channel was set as the background. The arrows and letters in The change of NKA activity in the kidney of spotted scat under hyposaline conditions. Kidneys were isolated from fish that had either been maintained in SW or transferred to FW for varying periods of time. For the first 3 hours after transfer to FW, there was no significant change of NKA activity in the kidney of spotted scat. At 6 hpt NKA activity increased sharply (7.14 ± 0.03 μ molPi/mgprot/h) but transiently, subsequently dropping to 3.62 ± 0.02 μ molPi/mgprot/h at 12 hpt. After 1 day (24hpt), NKA activity returned to levels similar to controls (*p < 0.05) (Fig. 6A).

Effects of dopamine on expression of SaDRD1 mRNA and NKA activity in vitro.
The effects of exogenous dopamine on cultured primary kidney cells were tested under isosomotic and hyposmotic conditions. The addition of exogenous dopamine to cells maintained under the isosmotic condition (300 mOsm/L) caused a significant change of SaDRD1 mRNA at 180 min after treatment with either 1 μ M dopamine (18.9 fold) or 10 μ M dopamine (6.7-fold) (***p < 0.001), although no increase was detected at 30 minutes (Fig. 6B).
Exposure of cultured kidney cells to hypoosmotic medium (100 mOsm/L) for 30 minutes did not alter the expression of SaDRD1 mRNA in the absence of exogenous dopamine. In contrast, when the hypoosmotic shock was accompanied by exogenous dopamine (1 μ M), SaDRD1 mRNA levels increased significantly at this time point (*p < 0.05). After a longer exposure to hypoosmotic medium, for 180 min, SaDRD1 mRNA expression increased significantly (***p < 0.001) in both the dopamine-treated cells and the control cells. At 180 minutes post-transfer, SaDRD1 mRNA expression levels were elevated in cells in both the presence and absence of exogenous dopamine. Notably, the increase in expression was greater in the absence of added dopamine at 180 min, showing a 26.3-fold increase, compared to a 4.4-fold increase in the medium containing exogenous dopamine (Fig. 6C). The result indicates that exogenous dopamine initially elevates SaDRD1 mRNA expression compared to no-dopamine cells when kidney cells are transferred to hyposmotic medium, but that this elevated expression is not sustained. The expression levels of SaDRD1 and SaDRD5 relative to β-actin in kidney during hyposaline shock were evaluated by RT-qPCR, and all the data was normalized to control group (SW → SW). The results are presented as the mean ± S.E.M. of the data with 3 replicates. *p < 0.05; ND means "no detection".
NKA activity was observed at 30 min and 180 min after exposure to the hyposaline medium in the presence and absence of 1 μ M exogenous dopamine. While the NKA activity was not significantly altered in no-dopamine cells at 30 min, treatment with 1 μ M exogenous dopamine significantly inhibited NKA activity (from 5.07 ± 0.84 μ molPi/mgprot/h to 3.45 ± 0.71 μ molPi/mgprot/h). The significantly inhibition of NKA activity was observed at 180 min post-transfer in both the dopamine-treated and no-treated cells (Fig. 6D).

Discussion
Salinity adaptation in euryhaline teleosts is a complex process involving physiological responses in several osmoregulatory organs. The endocrine system mediates many of these processes in order to maintain salt and water balance when fish are challenged with changing environmental salinity 27,28 . Dopamine, a catecholamine hormone, directly inhibits Na + transporters, thereby favoring natriuresis through paracrine and autocrine pathways 29,30 . Dopamine accumulates in the hypothalamus of tilapia (Sarotherodon mossambicus) during SW to FW    31 . Dopamine is known to inhibit the expression of prolactin, which has been identified as a major osmoregulatory hormone during SW acclimation 32 . However, little is understood about the direct effects of dopamine on the regulation of osmotic balance in the peripheral non-neuronal tissues of teleost fishes 32 .
In mammals, dopamine is synthesized in dopaminergic neurons of the brain, where it serves as a neurotransmitter. A substantial amount of dopamine circulates in the bloodstream, but its origins and functions outside of the brain are not entirely clear 11,33 . Due to the presence of blood-brain barrier, dopamine synthesis and function in peripheral areas is expected to be independent of its role in the brain. We have chosen to study the role of dopamine in the transition of fish from high to low salinity conditions, in order to increase our understanding of the role of this neurotransmitter in osmotic regulation in vertebrates. Unlike mammalian models such as rats and mice, for which many cell lines are available, few such resources exist for fish 34,35 . Thus, we have developed a protocol for the culture of primary renal cells in our laboratory over the past 3 years 36 , which we have used here to reveal molecular and physiological insights into dopamine function during hypoosmotic acclimation.
In the present study, the serum dopamine level of S. argus decreased significantly during the first 24-hours after transfer into FW, with the exception of the 12 hpt collection (Fig. 1). This 12 hpt result parallels findings from one of our previous studies, in which we observed that serum osmotic pressure in S. argus returned to normal levels at 12 h after SW to FW shock, following an initial decrease 7 . This phenomenon indicates that during FW acclimation, the serum dopamine in S. argus is associated with the short-term regulation of osmotic balance.
Several tissues can secrete dopamine into the circulatory system, and the pancreas is regarded as an important source of non-neuronal dopamine in the rat 37 . Some studies support the view that in humans, free (unconjugated) dopamine in the serum is derived largely from sympathetic noradrenergic nerves 38 . The fish kidney is regarded to play an important role in ionic regulation during environmental adaptation, and during freshwater acclimation, the primary function of the kidney is to drain excess water and retain sodium for the blood to maintain homeostasis 39 . Thus, we were interested in the ability of the fish kidney, and cultured renal cells, to produce and respond to dopamine.
Renal primary cells of S. argus were employed here to explore the role of dopamine in osmoregulation in vitro. After cells were exposed to hypotonic culture medium (100 mOsmol/L), dopamine contents in the culture medium were reduced substantially at 0.5 h and 3 h in comparison with those exposed to isosmotic culture medium (Fig. 1). This demonstrates the synthesis and release of dopamine can be achieved in the renal cells of S. argus. Our results indicate that the renal contribution to serum dopamine may play a prominant role. In the kidney, dopamine can be synthesized and secreted by renal tubular cells and transported to target organs or tissues by hemodynamic mechanisms 22,40 . In the kidney, the natriuretic effect of dopamine is mediated by inhibition of sodium transporters (e.g. NKA) via activation of D 1 -like receptors 12 . It is accepted that D 1 -like receptors (DRD1 and DRD5) are the principal mediators of the natriuretic effects of dopamine 19,41 . However, roles of DRD1/DRD5 in renal osmotic homeostasis are equivocal 14 . In present study, the mRNA expression of DRD5 in the kidney remained constant during 24 hours after the transfer from SW to FW, and was undetectable at later time points (Fig. 3B). In contrast, FW shock significantly increased renal SaDRD1 expression (Fig. 3A). Thus, DRD1 is the primary subtype of D 1 -like receptors regulating dopamine-mediated FW adaption in S. argus.
The SaDRD1 gene is predicted to encode a protein of 463 amino acids with 7 transmembrane domains ( Fig. 2A). Sequence alignment indicates that SaDRD1 is highly homologous with the DRD1 protein of other vertebrate species (Fig. 2B). The phylogenetic tree in Fig. 2C reveals that SaDRD1 is clearly grouped with DRD1 homologs from other teleosts.
A 127 aa extracellular region of SaDRD1 was chosen for antigen preparation, and the specificity of SaDRD1 antibody was tested by western blot analysis. The anti-SaDRD1 recognized a single band of about 51 kDa in the kidney of S. argus (Fig. 4A), the predicted molecular size of DRD1 in the opossum rat 42 . There was no cross-reactivity between anti-SaDRD1 antibody and rat DRD1 (Fig. 4A), which means the high specificity of SaDRD1 antibody. Immunohistochemistry experiment with this antibody showed that SaDRD1 expression is predominantly localized to the renal proximal tubule of S. argus (Fig. 4B a-h). Staining of renal primary cells of S. argus showed that SaDRD1 is localized to the cell membrane (Fig. 4B i-n), a finding similar to earlier reports in rats 43 .
We assessed changes of SaDRD1 expression in S. argus during hypo-osmostic acclimation in vitro and in vivo, respectively. In vivo, renal SaDRD1 mRNA expression was significantly up-regulated at 3 hpt and 12 hpt, and was relatively stable after 12 hpt (Fig. 3A). In renal primary cells, SaDRD1 mRNA expression was increased more than 25-fold at 3 h following transfer from isotonic medium to hypoosmotic medium (Fig. 6C). The expression pattern of SaDRD1 in response to hypoosmotic stress was opposite to renal NKA activity both in vivo and in vitro ( Fig. 6A and D). In the renal proximal tubule of the rat, it is known that dopamine can inhibit NKA activity by activating DRD1 to regulate the renal sodium excretion 44,45 . Thus, we sought to explore the functions of SaDRD1 in the regulation of NKA activity in renal primary cells of the spotted scat by using RNAi to reduce SaDRD1 expression. si-SaDRD1 reduced the expression and protein levels of SaDRD1. This treatment also affected expression of the protein subunit NKA α 1 and the activity of NKA, which was significantly higher in treated cells than in control group ( Fig. 5A and B). NKA α 1 is one of catalytic subtypes that cleaves high-energy phosphate bonds and exchanges intracellular Na + for extracellular K + ions, and is predominantly expressed in the kidney 46 . Our results showed that the inhibition of SaDRD1 expression can significantly increase NKA activity through upregulating the protein expression of NKA α 1. Under isosmotic condition, exogenous dopamine (both 1 μ M and 10 μ M) stimulates the expression of SaDRD1 in renal primary cells at 3 h after exposure. Higher expression of SaDRD1 was observed in the group induced with 1 μ M dopamine (18.9-fold) than with 10 μ M dopamine (6.7-fold) (Fig. 6B). There are some evidences suggesting that dopamine can increase dopamine receptor expression in levodopa-treated mice 47 . In addition, recruitment of DRD1 protein to the plasma membrane is also regulated by dopamine 48 . Thus, our results imply that dopamine can promote the high expression of SaDRD1 in the short-term, and the SaDRD1 density is altered in plasma membrane through the recruitment from cytoplasm to the plasma membrane in a dose-and time-dependent manner.
The effects of exogenous dopamine (1 μ M) on NKA activity in renal primary cells were explored. The results show that dopamine enhances the NKA response early when renal cells are faced with hyposaline stress by activating dopaminergic system through SaDRD1 (Fig. 6C and D). The upregulation of SaDRD1 mRNA occurred in a dose-dependent manner, with the higher dose of dopamine (10 μ M) resulting in the blunted dopaminergic system activity, in accordance with a previous study in the rat 49 . Thus, we conclude that the activation of SaDRD1 by dopamine participates in FW acclimation by igniting dopaminergic system to inhibit the activity of NKA, leading to maintenance of osmotic homeostasis in the kidney of S. argus.
During the acclimation to FW shock in S. argus, intrarenal dopamine plays an important role in the regulation of osmotic balance by activating renal SaDRD1, while SaDRD5 does not appear to be involved. SaDRD1 is expressed in the proximal tubule cells, and subcellularly localized to the cell membrane of cultured renal cells. During hypoosmotic shock of cultured renal cells, exogenous dopamine enhanced the early response of NKA to hyposaline stress through SaDRD1. Based on our results, we hypothesize that the dopaminergic system responds to hypoosmotic shock via DRD1 activation, which leads to inhibition of NKA activity and maintenance of osmotic balance in the fish exposed to acute hypoosmotic shock (Fig. 7). Further mechanistic studies are necessary to explore these intriguing hypotheses in detail.
Scientific RepoRts | 6:37535 | DOI: 10.1038/srep37535 Methods Collection, maintenance, and treatment of fish and collection of tissue samples. Scatophagus argus (32.6 ± 7.4 g) were collected from Zhuhai (N22°09′ 16.82″ , E113°21′ 41.50″ ), Guangdong Province, China and reared for 3 weeks in 25‰ seawater (SW, approximately 620 mOsmol/L) at 27 ± 2 °C. Fish for hyposaline challenge were divided into a treatment group (n = 105) and a control group (n = 105). Treated individuals were transferred from 25‰ SW to fresh water (FW, 0 mOsmol/L). Control individuals were transferred to another SW tank (identical salinity). Fifteen individuals were removed from each group at each experimental time point and anaesthetized 7 . Each set of 15 individuals was divided into 3 groups of 5 individuals, each group comprising 1 biological replicate. Samples were collected at 1 hour post-transfer (hpt), 3 hpt, 6 hpt, 12 hpt, 24 hpt, 2 days post-transfer (dpt), and 7 dpt, and inlcuded brain, kidney, gill, eye, liver, spleen, notochord, skin, intestines, heart, muscle, pituitary, ovary, spermary and blood. Tissue was frozen in liquid nitrogen and stored at − 80 °C. Serum was separated from blood cells by centrifugation (4200 × g for 10 min) and assayed for dopamine concentration. Animal welfare and experimental procedures were in accordance with the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006) and were approved by the animal ethics committee of Shanghai Ocean University.
Preparation and culture of primary kidney cells. Kidney tissue was collected from healthy SW-acclimated fish weighing ~40 g and used for primary cell culture as described in Zhou et al. 36 . The cultures of primary cells were maintained at 28 °C. Approximately 80% of the primary cells adhered to the culture plate.
For hypoosmotic stress experiments, primary cells were subjected to hypoosmotic medium (100 mOsmol/L), while control cultures were exposed to fresh isosmotic medium (300 mOsmol/L) (survival rate > 95%). Culture medium and cells were collected at 0.5 h and 3 h post-challenge, respectively, and used for hormone assays, RNA extraction and NKA activity measurement.
Additional cultures of primary cells in isosmotic medium were maintained at a density of approximately 2 × 10 4 /cm 2 at 28 °C for transfection and immunohistochemistry staining. Total RNA isolation and cDNA synthesis. Total RNA was isolated using Trizol Reagent (Invitrogen, USA) following the manufacturer's protocol. RNA was treated with DNase I (Invitrogen, USA) and quantified via Nanodrop-2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was verified by gel electrophoresis. Only samples with A260/A280 ratios 1.8 to 2.0 were used. cDNA was synthesized by reverse transcription of 1 μ g total RNA with oligo(dT) primers using the PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan) and stored at − 20 °C.
Cloning of full-length SaDRD1. To obtain the partial sequences of SaDRD1, primers (Table 1) were designed using Primer Premier v5.0 (Premier, Canada) based on homologous sequences obtained from the National Center for Biotechnical Information (NCBI, http://www.ncbi.nlm.nih.gov/). SaDRD1 was amplified from S. argus brain cDNA. PCR products were purified by gel extraction (Axygen, USA) and cloned into the pMD18-T vector (Takara, Japan) for sequencing. To obtain the full-length cDNA sequence of SaDRD1, rapid amplification of cDNA ends (RACE) was performed (SMART RACE amplification Kit, Clontech, USA). Primers are listed in Table 1. The amplified PCR product was sequenced using an ABI PRISM 377 DNA Sequencer (Applied Biosystems, USA). Sequence analysis. The nucleotide sequence of SaDRD1 was aligned with Clustal X 1.81 and analyzed using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) to predict the amino acid sequence and open reading frames (ORFs). Transmembrane domains were predicted using TMHMM v. 2.0 (http://www.cbs.dtu.dk/services/ TMHMM-2.0/). A phylogenetic tree was constructed using MEGA 6.0 software with neighbor-joining method and 1000 boot-strap replicates 51 .
Tissue distribution of SaDRD1 mRNA. Semi-quantitative reverse transcriptase PCR (SqRT-PCR) was conducted to assess tissue expression patterns of SaDRD1 in S. argus. Primers are listed in Table 2. The annealing temperature and PCR conditions were: 60 °C, 28 cycles for β-actin; 55 °C, 28 cycles for SaDRD1. The PCR products were visualized by 2% gel electrophoresis and expression was quantified using densitometric analysis (Image Lab, Bio-Rad, USA). SaDRD1 expression was normalized against β-actin.
Kidneys of S. argus and rat were homogenized on ice in normal saline (0.86% NaCl) and lysates were centrifuged at 12000 rpm 4 °C for 10 min. Supernatant was collected and protein concentration determined using a BCA Protein Assay Kit (Merck, Germany). About 75 μ g total protein of S. argus or rat kidney was separated on 12% SDS-polyacrylamide gels and transferred to PVDF membranes. Following blocking in 5% skim milk in PBS (Bio-Rad, USA), membranes were incubated with rabbit anti-SaDRD1 (1:2000 dilution) in PBS overnight at 4 °C. Membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (Abcam, UK) (1:5000 dilution) for 2 h at room temperature. The signals were detected by chemiluminscence using Universal HoodII (Bio-Rad, USA).