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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
-Aminobutyric Acid Receptor Subunits in Renal Proximal Tubular CellsHarvard Center for Neurologic Diseases, Brigham and Women's Hospital, Cambridge, Massachusetts (S.S.S., S.R.G.); Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina (S.M.L., B.S.C., R.G.S.); and National Institutes of Environmental Health Sciences, Research Triangle Park, North Carolina (D.D.B.)
Received for publication
August 17, 2007
Accepted
October 22, 2007.
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Abstract |
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1, 2, 4, 5, β1, 
1, 2, and 
subunits in the kidney and to determine whether these subunits coassemble to form an active renal epithelial cell GABAA receptor. Using reverse transcriptase products from RNA isolated from rat and rabbit kidney cortex and brain or cerebellum through polymerase chain reaction (PCR) and sequencing of the PCR products, we revealed that rat kidney cortex contained the 1, 5, β1, 1, and 2 subunits and that they were similar to the neuronal subunits. Sequencing of the PCR products revealed that the rabbit kidney cortex contained the 1 and 2 subunits and that they were similar to their neuronal counterparts. Immunoprecipitation and immunoblot studies using GABAA receptor subunit-specific antibodies and detergent-solubilized rat kidney cortex membranes identified a GABAA receptor complex containing 5, β1, and 1. Isolated rat renal proximal tubular cells exhibited GABA-mediated, picrotoxin-sensitive 36Cl- uptake. These studies demonstrate the presence of numerous GABAA receptor subunits in the kidneys of two species, the assembly of the subunits into at least one novel receptor complex, and an active GABAA receptor in renal proximal tubular cells.
). GABAA receptor subunits have been also identified outside the CNS, including in the liver, heart, retina, uterus, spermatozoa, adrenal medulla, kidney, anterior and intermediate pituitary lobes, and pancreatic -cells (Erdó, 1984; Berman et al., 1994; Grigorenko and Yeh, 1994; Parramón et al., 1994; Hedblom and Kirkness, 1997; Whiting et al., 1997; Sarang et al., 2001; Hu et al., 2002). Molecular biological approaches have revealed that GABAA receptors exist as a family of polypeptides composed of at least seven different GABAA receptor subunits, namely 1–6, β1–3, 1–3, , 
122, 
, 
, 
, and 1–3 (Hedblom and Kirkness, 1997; Whiting et al., 1997; Barnard et al., 1998; Rudolph and Mohler, 2006). The neuronal GABAA receptor is composed of five hetero-oligomer subunits that form a pentameric structure with a Cl- channel. Binding sites for benzodiazepines, barbiturates, convulsant compounds (e.g., picrotoxin), and neurosteroids have been identified on specific GABAA receptor subunits (Sieghart et al., 1999).
Heterologous expression systems showed that various combinations of the GABAA receptor , β, and subunits form functional Cl- channels (Korpi et al., 2002). Similarly, double- and triple-label immunofluorescence studies with subunit-specific antibodies have determined that 1β2/32, 3β2/32, and 13β2/32 complexes colocalize in neurons in the CNS (Fritschy and Mohler, 1995). Fritschy et al. (1992), using detergent-solubilized brain membranes, GABAA receptor subunit-specific antibodies, immunoprecipitation, and radioligand binding techniques determined that 3β2/32 and 13β2/32 coassemble in the rat brain., 1992). Finally Sur et al. (1998) identified an 5β33 receptor in the rat and human brain.
Evidence exists for GABAA receptor subunits in renal tubular epithelial cells. For example, ligand binding studies and autoradiography using the GABAA receptor agonist [3H]muscimol specifically bound to proximal convoluted tubules of the rat renal cortex with the highest density in the outer stripe of the medulla (Sur et al., 1998). In a preliminary report, Molony et al. (1993) identified the mRNA for the 1 subunit of the GABAA receptor in the thick ascending limb of the loop of Henle but not in the proximal tubule or in glomeruli., 1993). Sarang et al. (2001) used reverse transcriptase (RT)-polymerase chain reaction (PCR) to demonstrate expression of the GABAA receptor β2 and β3 subunits in the rat, rabbit, and human kidney. Furthermore, the sequences of the GABAA receptor β2 and β3 subunits were similar to those of their neuronal counterparts. Recently, Sasak's laboratory reported that GABAA receptor β-chain and GABAB receptor 1 staining were observed in proximal tubules, but its subcellular localization was not elucidated (Sasaki et al., 2007).
There is limited information available concerning the expression of GABAA receptor subunits in the kidney, whether they coassemble to form a GABAA receptor or what their physiological role might be. In this study, we focused on identifying the GABAA receptor 1, 2, 4, 5, β1, 1, 2, and subunits in the kidneys from three species for the purpose of determining whether these subunits coassemble in the rat kidney to form a GABAA receptor and to ascertain the activity of renal epithelial GABAA receptors.
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Materials and Methods |
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). Total cellular RNA was isolated from renal cortex, cerebellum, and whole brain using TRIzol reagent (Invitrogen, Grand Island, NY) following the manufacturer's instructions. RNA concentration was measured and the RNA stored in RNase-free water at -80°C.
RT-PCR Amplification. Rat, mouse, and human neuronal GABAA receptor 1, 2, 4, 5, β1, 1, 2, and subunit nucleotide and amino acid sequences were identified in the GenBank database (see Table 1 for accession numbers). PCR primers were designed for the receptor 1, 2, 4, 5, β1, 1, 2, and subunits (Table 1).
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First-strand cDNA was synthesized from total cellular RNA using Moloney murine leukemia virus RT (PerkinElmer, Foster City, CA), and a downstream antisense primer (Table 1) according to the manufacturer's instructions. Two controls were used, and neither RNA template nor reverse transcriptase was added to the RT reaction tubes. RT experiments consisted of one cycle of 60 min at 42°C, 5 min at 99°C, and 5 min at 5°C. The products from the RT reactions were amplified by PCR.
The samples for the PCR experiments were brought to a final concentration of 1x Vent ThermolPol Reaction Buffer, including 0.4 mM concentrations of nucleotide triphosphates, 2 mM MgCl2, 0.5 M sense and antisense primers, and 5 ng of cDNA template. After heating at 94°C for 2 min,1Uof VentR DNA polymerase (New England Biolabs, Beverly, MA) was added to the sample and mixed. The first round of PCR was then performed for 1 cycle of 2 min at 94°C and 30 s at 72°C. This was followed by 30 cycles of 30 s at 94°C, 30 s at 45 to 59°C (Table 1, annealing temperatures for each subunit), and 30 s at 72°C. Finally, 1 cycle of 5 min at 72°C was performed. PCR reamplification using nested primers was performed for 30 cycles of 30 s at 94°C; 30 s at 50 to 59°C (Table 1), and 30 s at 72°C. The PCR DNA products were analyzed by electrophoresis in 1.5% agarose gels containing 0.5 g/ml ethidium bromide and visualized with UV light.
A DNA fragment of the predicted size for each of the GABAA receptor subunits (Table 1) was isolated from the agarose gel using the QIAquick Gel Extraction Kit (QIAGEN, Chatsworth, CA). These fragments were directly sequenced using an automated DNA sequencer (PerkinElmer). In some experiments, PCR products were subjected to restriction endonuclease digestion according to the manufacturer's instructions (Invitrogen).
Preparation of Rat Kidney Cortex and Brain Membranes. A polyethylene catheter (PE 100) was inserted into the lower abdominal aorta of the euthanized rat, and the aorta was clamped above the renal arteries. The veins were cut, and the kidneys were perfused with ice-cold 0.3 M sucrose containing, 25 mM HEPES (pH 7.0), 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM leupeptin, and 0.2 mM pepstatin (buffer A). The plasma membranes were isolated as described by Sheikh and Moller (1987) with the following modifications. The kidney cortex was homogenized in ice-cold buffer A using a Potter-Elvehjem glass homogenizer and a Teflon pestle (1500 rpm for 20 strokes). The kidney homogenates were then centrifuged at 900g for 10 min (all centrifugation steps were preformed at 4°C), and the supernatant was collected and stored on ice; this process was repeated, and the supernatants were combined. The combined supernatants were homogenized as described above and centrifuged at 9200g for 10 min. The white and the fluffy upper parts of the pellet were resuspended using a Pasteur pipette and combined with the supernatant. The suspension was homogenized by hand and then centrifuged at 9200g for 10 min. The pellet was resuspended with the supernatant, homogenized by hand again, and then centrifuged at 48,000g for 30 min. The pellet representing the plasma membrane fraction was stored at -80°C.
Neuronal plasma membranes were prepared according to the method of Mernoff et al. (1983), with some modifications. The whole rat brain was homogenized as described above and centrifuged at 1000g for 8 min. The supernatant was removed and centrifuged at 78,000g for 30 min, the pellet was resuspended in 50 mM Tris-HCl, pH 7.4 (buffer B) containing the protease inhibitor cocktail as above (4 ml of buffer B/brain). The mixture was centrifuged at 30,000g for 10 min, and the pellet was resuspended in buffer B (2 ml/brain) and frozen for 2 hat -80°C. After thawing the mixture, buffer B was added to the tissue to a final volume of 12 ml/brain and centrifuged at 78,000g for 30 min. The membranes were resuspended in buffer B and stored at -80°C.
Immunoprecipitation and Immunoblot Studies. Rat kidney cortex and brain membranes (4 and 1 mg of protein, respectively) were solubilized in 1 to 2 ml of RIPA buffer for 45 to 90 min at 4°C with gentle rocking [RIPA buffer: 10 mM Tris-HCl (pH 7.4), 0.137 M NaCl, 1% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 2 mM EDTA, 2 mM EGTA, 0.3 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 0.5 mM leupeptin, and 0.2 mM pepstatin]. The solubilized membranes were centrifuged at 100,000g for 1 h. The supernatants were collected, RIPA buffer was exchanged with binding buffer [0.14 M NaCl, 0.008 M Na2HPO4, 0.002 M K3PO4, and 0.01 M KCl (pH 7.4)] and concentrated to a final volume of 200 µl using Ultrafree-4 centrifugal filter units, 30,000 molecular weight cutoff (Millipore Corporation, Billerica, MA). Protein concentrations were determined before and after solubilization using the bicinchoninic acid assay (Montrose-Rafizadeh et al., 1989). For immunoprecipitation studies Seize X Protein A Immunoprecipitation Kit (Pierce Chemical, Rockford, IL) was used. GABAA receptor 5 subunit, β1 subunit, and 1 subunit polyclonal antibodies (150 µlof each antibody) were immobilized on the protein A support (0.4 ml of 50% slurry) using the cross-linking agent disuccinimidyl suberate (all antibodies were a generous gift of Dr. R. M. McKernan, Merck, Harlow, UK). Anti-rat IgG (Sigma-Aldrich, St. Louis, MO) was used as a control. Kidney and brain solubilized membranes (200 µl) were incubated with immobilized antibodies overnight at 4°C.
Immunoprecipitated subunits were eluted, neutralized, subjected to SDS-polyacrylamide gel electrophoresis, and blotted onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). The membranes were blocked with 2.5% casein and incubated with a 1:100 dilution of GABAA receptor 5 subunit, β1 subunit, or 1 subunit polyclonal antibodies overnight at 4°C with gentle rocking. The membranes were washed, incubated with 1:5000 goat anti-rabbit horseradish peroxidase-conjugated IgG (Invitrogen), and the proteins were detected using the ECL system (GE Healthcare, Buckinghamshire, UK).
RPT Cell Cl- Uptake Studies. RPT cells were isolated as previously described (Smith et al., 1985). Cell viability was confirmed by the exclusion of trypan blue and was greater than 90% (Lash and Tokarz, 1989). The uptake of Cl- into rat PT cells was determined using 36Cl-. Briefly, aliquots of proximal tubular cells were treated with either buffer control, 1 µM GABA, 1 µM muscimol, and/or 10 µM picrotoxin at 37°C, immediately before the addition of 36Cl-. Reactions were immediately stopped by addition of excess cold buffer (time 0) or allowed to proceed for 10 s. All data were normalized for the amount of protein in each reaction.
Statistical Analysis. PCR and immunoprecipitation/immunoblot experiments were repeated three to four times with different preparations of RNA and plasma membranes, respectively. For the 36Cl- uptake experiments, PT cells were isolated and combined from two rats to represent one experiment (n = 1). Experiments were repeated until n = 3 to 4 was reached. Data are presented as means ± S.E.M. Significant differences between treatment groups (p < 0.05) were determined using SigmaStat (SPSS, Inc., San Rafael, CA) one-way analysis of variance as necessary, and Student-Newman-Keuls tests were used to compare multiple means.
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1, 2, 4, 5, β1, 1, 2, and and subunit specific primers (Table 1). The first round of PCR amplification of rat and rabbit kidney cortex and cerebellum RNA using 1 subunit-specific primers identified a 600-bp fragment (data not shown), and upon further amplification using nested primers, a 434-bp fragment was obtained (Fig. 1A). Similar results were observed from RNA isolated from rabbit RPT S2 segments and human and rat kidney cDNA libraries (Fig. 1A). This region encodes three of the four transmembrane domains (M1, M2, and M3) and intracellular and extracellular loops of the 1 subunit (Lolait et al., 1989). Sequencing of the PCR products and sequence alignment revealed that the rat kidney cortex and rat neuronal 1 subunits were identical in nucleotide composition. The rabbit kidney cortex and rabbit neuronal 1 subunits were also identical in nucleotide composition.
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2 subunit-specific primers yielded a 395-bp product in the rat whole brain only (Fig. 1B). No product was detected in the rat kidney cortex. The whole-brain PCR product was subjected to restriction endonuclease digestion using EcoO 109 I endonuclease. Digestion of the PCR product resulted in the expected 247- and 148-bp fragments (Fig. 1C). PCR amplification of rat kidney cortex and whole-brain RNA using 4 subunit-specific primers yielded 503-bp products (Fig. 1D). Restriction endonuclease digestion of the rat whole-brain 503-bp product using the RcaI endonuclease yielded the expected 308- and 195-bp fragments, whereas the rat kidney cortex band did not digest (Fig. 1E). PCR amplification of rat kidney cortex and whole-brain RNA using 5 primers identified 294-bp products (Fig. 1F). Sequencing of the PCR products and sequence alignment revealed that the rat kidney cortex and whole-brain GABAA 5 subunits were identical in nucleotide composition. PCR amplification of rat kidney cortex and whole-brain RNA using β1 subunit-specific primers yielded 426-bp products (Fig. 2A). Sequencing of the PCR products and sequence alignment revealed that the rat kidney cortex and whole-brain GABAA β1 subunits PCR were identical in nucleotide composition.
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1 subunit specific primers identified a 464-bp fragment (data not shown) and, upon further amplification using nested primers, a 429-bp fragment was obtained (Fig. 2B). Sequencing of the PCR products revealed that the rat kidney cortex and rat neuronal GABAA receptor 1 subunits were identical in nucleotide composition. However, the rabbit kidney cortex, rabbit RPT S2 segments (data not shown) and rabbit cerebellum did not result in the predicted size products (Fig. 2B). PCR amplification of rat kidney cortex and cerebellum RNA using 2 subunit-specific primers identified a 550-bp fragment (data not shown), and upon further amplification using nested primers, a 426-bp fragment was obtained (Fig. 2C). Sequencing of the PCR products revealed that the rat kidney cortex and rat neuronal GABAA receptor 2 subunits were identical in nucleotide composition. Similar results were obtained from rabbit kidney cortex, RPT S2 segments, and cerebellum (Fig. 2C). Sequencing of the rabbit kidney cortex and cerebellum revealed that they were 89% similar in nucleotide composition.
PCR amplification of rat kidney cortex and whole-brain RNA using GABAA subunit-specific primers yielded 404-bp products (Fig. 2D). The subunit PCR product was subjected to restriction endonuclease digestion using MboI endonuclease. Digestion of the rat whole-brain subunit bands yielded the expected 252- and 152-bp fragments; however, the rat kidney cortex PCR product band did not digest (Fig. 2E). Collectively, these results provide evidence for the expression of the GABAA receptor 1, 5, β1, 1, and 2 subunits in the rat kidney cortex, and 1 and 2 subunits in the rabbit kidney cortex.
Immunoprecipitation was used to enhance the detection and to assess coassembly of GABAA receptor subunits. GABAA receptor 5, β1, and 1 subunit-specific antibodies immunoprecipitated GABAA receptor subunit proteins from both the rat kidney cortex and brain detergent-solubilized membranes. Immunoprecipitation of kidney and brain solubilized membrane proteins using the 5 subunit specific antibody and subsequent probing of the immunoprecipitates with the 5 subunit antibody identified a 55-kDa protein corresponding to the 5 subunit (Fig. 3). Probing the same kidney and brain membrane protein immunoprecipitates with the β1 and 1 subunit-specific antibodies revealed immunoreactive proteins in the region of 51 to 54 and 54 kDa, corresponding to the β1 and 1 subunits, respectively (Fig. 3). Kidney membrane proteins were immunoprecipitated using an anti-IgG negative control and then probed using 5, β1, and 1 subunit antibodies. In all cases no immunoreactive proteins were identified (data not shown).
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5 and 1 subunit-specific antibodies identified immunoreactive proteins in the region of 55 and 54 kDa, corresponding to the 5 and 1 subunits, respectively (Fig. 3).
Immunoprecipitation of solubilized kidney and brain membrane proteins with the GABAA receptor 1 subunit-specific antibody and subsequent probing of the immunoprecipitates with the 1 subunit antibody identified an immunoreactive protein in the 54-kDa region corresponding to the GABAA receptor 1 subunit (Fig. 3). Probing the same kidney and brain membrane protein immunoprecipitates with the 5 and β1 subunit antibodies identified immunoreactive proteins in the region of 55 and 51 to 54 kDa, corresponding to the 5 and β1, subunits, respectively (Fig. 3). These results support the PCR data and show that the GABAA receptor 5, β1, and 1 subunits are present in the kidney and demonstrate that the receptor subunits coassemble in the kidney and form at least one novel receptor complex comprised of 5, β1, and 1 subunits.
To determine whether the identified assembled subunits form an active GABAA receptor, rat proximal tubular cells were isolated, and 36Cl- uptake was measured in the presence and absence of GABA, the GABAA receptor agonist muscimol, and the GABAA receptor antagonist picrotoxin. The addition of 1 µM GABA or 1 µM muscimol to the cells increased 36Cl- uptake 1.3- and 1.7-fold, respectively (Fig. 4). Whereas picrotoxin alone had no effect on basal 36Cl- uptake, picrotoxin in the presence of GABA or muscimol blocked 36Cl- uptake. These studies suggest that a GABAA receptor is active in the renal proximal tubular cells.
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). In a preliminary report, Molony et al. (1993) identified the mRNA for the 1 subunit of the GABAA receptor in the thick ascending limb of the loop Henle but not in the proximal tubule or in glomeruli. The first part of the present study provided evidence for the expression of GABAA receptor 1, 5, β1, 1, and 2 subunits in rat kidney, and 1 and 2 in rabbit kidney. Thus, rats and rabbits express two to five different GABAA receptor subunits in the kidney.
Sequencing of the PCR products revealed that the rat kidney and rat neuronal GABAA receptor 1, 5, β1, 1, and 2 subunits were identical in nucleotide composition. The human kidney and human neuronal GABAA receptor 1 subunit, and the rabbit kidney and rabbit neuronal 1 subunit were identical in nucleotide composition. However, the rabbit kidney and rabbit neuronal β2 subunit were 99% similar in nucleotide composition. Sequencing of the rabbit kidney cortex and rabbit neuronal 2 subunit revealed that the subunits were 89% similar in nucleotide composition; suggesting that the rabbit kidney expresses a novel isoform of the 2 subunit that is significantly different from that of the neuronal 2 subunit. With the exception of the rabbit renal 2 subunit, these results suggest that GABAA receptor subunits expressed in the kidney are identical to those expressed in the nervous system.
The method of immunoprecipitation followed by reprobing with GABAA receptor subunit specific antibodies to determine the coassembly of GABAA receptor subunits in the CNS has been used extensively (Fritschy et al., 1992; Mertens et al., 1993; Pollard et al., 1995; McKernan and Whiting, 1996). Immunoprecipitation studies using GABAA receptor 5, β1, and 1 subunit-specific antibodies and detergent-solubilized membranes from both the rat kidney cortex and rat brain successfully immunoprecipitated their respective GABAA subunits. Furthermore, reprobing of each of the immunoprecipitated subunits from the kidney and brain membranes with the 5, β1, and 1 subunit antibodies identified the fact that these subunits coassembled to form a novel GABAA receptor complex: 5β11. It is not clear at this time whether the renal GABAA receptor complex is composed of five subunits or what subunits comprise the missing two subunits.
To determine whether the renal GABAA receptor complexes are functional, a proof of concept study was conducted using 36Cl- uptake, freshly isolated rat renal proximal tubular cells and the agonist GABA, the GABAA agonist muscimol, and the antagonist picrotoxin. The presence of GABA and muscimol increased renal proximal tubular cell 36Cl- uptake and the concurrent addition of picrotoxin blocked 36Cl- uptake. These functional studies show that the GABAA receptor complexes in the rat kidney form active GABAA receptors.
To our knowledge, a neuronal GABAA receptor composed of 5, β1, and 1 subunits has not been reported and may represent a unique GABAA receptor complex in the kidney. Neuronal GABAA 5 and 1 subunits have been reported in very limited areas of the brain (Pirker et al., 2000). At this time, neuronal 5-containing receptors have been associated with β3 and 2 but not β1 and 1 subunits (McKernan and Whiting, 1996). Recently a GABAA receptor composed of 5, β1, and β3 subunits was found in the rat spermatozoa (Hu et al., 2002). Thus, a renal GABAA receptor composed of 5, β1, and 1 subunits is novel and may serve a specific function in proximal tubular cells.
The physiological function of the 5, β1, and 1 complex in the kidney is not known. Our evidence suggests that it is located on the plasma membrane, is activated by GABA and muscimol, and mediates Cl- influx. As such, Cl- uptake through this receptor may represent the first step in transepithelial Cl- movement or Cl- cycling across the plasma membrane. Interestingly, Sasaki et al. (2007) reported that treatment of rats with five-sixths kidney volume with GABA for 60 days inhibited progression of tubular fibrosis and atrophy. Although these results suggest a novel role for GABA receptors in the kidney, additional studies are needed to explore this observation.
In summary, PCR studies, immunoprecipitation coupled with immunoblotting and 36Cl- uptake studies provide compelling evidence for 1) expression of GABAA receptor 1, 5, β1,1, and 2 subunits in the kidney, 2) the fact that at least one GABAA receptor complex comprising 5, β1, and 1 subunits exists in the kidney, and 3) the kidney possesses a GABA agonist- and antagonist-sensitive receptor complex. Collectively, these results indicate that the kidney expresses the GABAA receptor complex and that this complex may form an active ligand-gated Cl- channel.
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Acknowledgements |
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5, β1, and 1 subunit antibodies, Dr. D. C. Zeldin for kindly providing the human kidney cDNA library, Drs. P. R. Mayeux and C. Zhang for assistance isolating the membranes, and Dr. J. F. Harriman for isolating the rabbit RPT S2 segments. |
Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: CNS, central nervous system; RT, reverse transcriptase; PCR, polymerase chain reaction; RPT, renal proximal tubule; bp, base pair.
1 Current affiliation: Alcon Laboratories, Fort Worth, Texas. 
2 Current affiliation: Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia.
Address correspondence to: Dr. Rick G. Schnellmann, Department of Pharmaceutical Sciences, Medical University of South Carolina, 280 Calhoun St., POB 250140, Charleston, SC 29425. E-mail: schnell{at}musc.edu
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