3846 The Journal of Experimental Biology 212, 3846-3856 Published by The Company of Biologists 2009 doi:10.1242/jeb.034157 The responses of zebrafish (Danio rerio) to high external ammonia and urea transporter inhibition: nitrogen excretion and expression of rhesus glycoproteins and urea transporter proteins Marvin H. Braun1,*, Shelby L. Steele2 and Steve F. Perry2 1Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1 and 2University of Ottawa, 30 Marie Curie, Ottawa, ON, Canada K1N 6N5 *Author for correspondence ([email protected]) Accepted 18 August 2009 SUMMARY While adult zebrafish, Danio rerio, possess ammonia and urea transporters (Rh and UT proteins, respectively) in a number of tissues, they are most heavily concentrated within the gills. UT has a diffuse expression pattern within Na+–K+-ATPase (NKA)-type mitochondrion-rich cells and Rh proteins form a network similar to the arrangement seen in pufferfish gills (Nakada et al., 2007b). Rhag expression appeared to be limited to the pillar cells lining the blood spaces of the lamellae while Rhbg was localized to the outer layer of both the lamellae and the filament, upon the pavement cells. Exposure to high external ammonia (HEA) or phloretin increased tissue levels of ammonia and urea, respectively, in adult and juvenile zebrafish; however, the responses to these stressors were age dependent. HEA increased mRNA levels for a number of Rh proteins in embryos and larvae but did not elicit similar effects in adult gills, which appear to compensate for the unfavourable ammonia excretory gradient by increasing expression of V-type H+-ATPase. Phloretin exposure increased UT mRNA levels in embryos and larvae but was without effect in adult gill tissue. Surprisingly, in both adults and juveniles, HEA increased the mRNA expression of UT and phloretin increased the mRNA expression of Rh proteins. These results imply that, in zebrafish, there may be a tighter link between ammonia and urea excretion than is thought to occur in most teleosts. Key words: Rh glycoproteins, urea transporter, ammonia transport, gills, gene expression. INTRODUCTION supports the work on seawater-adapted Japanese eels demonstrating How animals excrete the toxic products of amino acid catabolism UT protein expression on the basolateral surface of chloride cells (specifically ammonia and urea) has been a topic of study for nearly (Mistry et al., 2001). a century (Smith, 1929). While it was originally thought that Information regarding the location and function of Rh proteins ammonia and urea moved passively through tissues along partial in teleosts began with the discovery that pufferfish (Takifugu pressure or concentration gradients, it is now known that they require rugripes) possess a number of ammonia-transporting Rh proteins transporters [Rh proteins for ammonia (Marini et al., 1997), UT (Nakada et al., 2007b). Within the gills, specific members of the proteins for urea (Levine et al., 1973; You et al., 1993)] to efficiently Rh family are expressed in discrete cell layers, as ammonia moving cross plasma membranes. While much of the original work was from the blood to the water passes from Rhag to Rhbg to done on mammalian models and cell lines, in recent years there has Rhcg1/Rhcg2, a pattern similar to that within the mammalian kidney also been a great deal of interest regarding the functional (Eladari et al., 2002; Quentin et al., 2003; Verlander et al., 2003). arrangement of these transporters in teleost fish (Braun et al., 2009; However, while recent publications have revealed the existence of Hung et al., 2008; Hung et al., 2007; Nakada et al., 2007a; Nakada one or more Rh proteins in rainbow trout (Hung et al., 2008; Nawata et al., 2007b; Nawata et al., 2007; Shih et al., 2008; Tsui et al., et al., 2007; Tsui et al., 2009), mangrove killifish Krytobelias 2009). marmoratus (Hung et al., 2007) and zebrafish (Braun et al., 2009; While most adult teleosts are ammonotelic (producing ammonia Nakada et al., 2007a; Shih et al., 2008), our knowledge of the specific as the dominant nitrogenous excretory product), urea excretion plays expression pattern of Rh proteins in adult gills remains limited to a vital role during development, as the young of several fish species the initial work on pufferfish. exhibit ureotely (Barimo et al., 2004; Chadwick and Wright, 1999; Excretory stress in fish [high external ammonia (HEA) to limit Essex-Fraser et al., 2005; Steele et al., 2001; Wright et al., 1995). ammonia efflux or phloretin to limit urea efflux] often results in However, urea excretion also appears to be important in adults short-term inhibition, followed by the resumption of normal because UT proteins are present not only in the ureotelic toadfish nitrogenous excretion (Cameron, 1986; Claiborne and Evans, 1988; (Opsanus beta) (Walsh et al., 2000) and Lake Magadi tilapia Steele et al., 2001; Wilson et al., 1994). This pattern may reflect, (Alcolapia grahami) (Walsh et al., 2001) but also in ammonotelic in part, a re-establishment or an increase of the outwardly directed teleosts such as the rainbow trout (Oncorhynchus mykiss) (Pilley gradients for ammonia and urea, but it may also be due to the ability and Wright, 2000), Japanese eel (Anguilla japonica) (Mistry et al., of fish to increase transcription of Rh proteins in response to 2001) and zebrafish (Braun et al., 2009). Recent data from toadfish excretory stressors (Hung et al., 2007; Nawata et al., 2007; Nawata suggest that within the gill, UT mRNA is only expressed by and Wood, 2008), potentially increasing excretory flux. Among the mitochondrion-rich cells (McDonald et al., 2009), a finding which reasons why a complete understanding of this response is THE JOURNAL OF EXPERIMENTAL BIOLOGY Excretory stress and transport proteins 3847 confounded is the fact that different species regulate different Rh USA) equipped with an argon laser. Images were collected using proteins in different tissues (Hung et al., 2007; Nawata et al., 2007; Fluoview 2.1.39 graphics software (Fluoview, Melville, NY, USA). Nawata et al., 2008) and because there is little known regarding the effect of excretory stress on UT. Western blots In the present study, we tested the hypothesis that part of the Proteins were prepared from fresh tissues by homogenization on zebrafish response to the excretory stressors HEA and phloretin is ice in 1:5 w/v of extraction buffer containing 50mmoll–1 TrisHCl, an increased expression of Rh proteins and UT. However, 150mmoll–1 NaCl, 1% NP-40, 0.5% sodium deoxycholate, embryonic and larval zebrafish excrete ammonia and urea in a 2mmoll–1 sodium fluoride, 2mmoll–1 EDTA, 0.1% SDS and fundamentally different manner from adults, due in part to a variable protease inhibitor cocktail (Roche, Laval, Quebec, Canada). The expression of transporter proteins with age (Braun et al., 2009). samples were incubated on ice for 10min and briefly sonicated to Therefore, while we hypothesized that they both possess the ability break up any DNA that might have been extracted. The samples to regulate the expression of Rh and UT proteins when exposed to were centrifuged at 16,000g for 20min at 4°C, and the supernatants high levels of ammonia and urea, we predicted that the responses were stored at –20°C until use. Protein concentrations were to both HEA and phloretin would be different between adults and determined using a bicinchoninic acid protein assay (Pierce, juvenile fish. Rockford, IL, USA) with BSA as standard. Samples (100g) were size fractionated by reducing SDS-PAGE using 10% separating and MATERIALS AND METHODS 4% stacking polyacrylamide gels and transferred to nitrocellulose Animals membranes (Bio-Rad, Mississanga, ON, Canada). After protein Adult zebrafish (Danio rerio Hamilton-Buchanan 1822) were kept transfer, each membrane was blocked for 2h in 5% milk in the University of Ottawa Aquatic Care Facility where they were powder/TBS-T (1ϫTBS, 0.1% Tween 20) before probing. maintained in plastic tanks (10l) supplied with aerated, dechlorinated To test the efficacy of the new antibodies (Rhbg and UT), three City of Ottawa tap water at 28°C. Fish were subjected to a constant lanes of a gel were loaded with extracted gill proteins and each lane 10h L:14h D photoperiod and were fed daily with No.1 crumble- was incubated for 3h at room temperature with pre-immune serum ZeiglerTM (Aquatic Habitats, Apopka, FL, USA). (1:2000), one of the two primary antibodies (Rhbg, 1:2000; UT, Embryos were obtained using standard techniques for zebrafish 1:2000), or 1:2000 of preabsorbed antibody (antibodies were breeding (Westerfield, 1995) and newly spawned eggs were incubated with 10ϫ the respective antigenic peptide overnight at collected from random groups of adult breeders and kept in rearing 4°C). The membranes were washed (4ϫ5min) in PBS and incubated tanks at 28°C until needed. All procedures for animal use were for 1h at room temperature with peroxidase-conjugated secondary carried out according to institutional guidelines and in accordance anti-rabbit Ig (Rhbg, 1:50,000; UT, 1:20,000). The specific bands with those of the Canadian Council on Animal Care (CCAC). were detected by enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Pierce). The protein size marker Antibody production used was obtained from Fermentas Life Sciences (Burlington, Affinity purified polyclonal rabbit antibodies against zebrafish Rhbg Ontario, Canada). (accession no. NM_200071.2) and UT (accession no. AY788989.1) For semi-quantitative assessments of protein changes, gill proteins were generated by 21st Century Biochemicals (Marlboro, MA, were extracted from adult zebrafish exposed to one of three USA). The peptide sequences used as antigens were: treatment groups for 5days (for treatment procedures see below). UT: Ac-TDEKKQQGLEKINSGQRFKANLC-amide (amino Western blots were performed on the proteins using antibodies for acid nos 48–69) Rhag, Rhbg, Rhcg1, UT and -tubulin (H-300, Santa Cruz Rhbg: CLNEVSTQNEVEKLNS-OH (amino acid nos 445–459).