Decreased renal Na-K-2Cl cotransporter abundance in mice with heterozygous disruption of the Gsalpha gene

Carolyn A. Ecelbarger1, Shuhua Yu2, Alanna J. Lee1, Lee S. Weinstein2, and Mark A. Knepper1

1 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute; and 2 Metabolic Diseases Branch, National Institute of Diabetes, Digestive, and Kidney Diseases, Bethesda, Maryland 20892


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transport processes along the nephron are regulated in part by hormone stimulation of adenylyl cyclases mediated by the heterotrimeric G protein Gs. To assess the role of this pathway in the regulation of Na-K-2Cl cotransporter abundance in the renal thick ascending limb (TAL), we studied mice with heterozygous disruption of the Gnas gene, which codes for the alpha -subunit of Gs. Outer medullary Gsalpha protein abundance (as assessed by semiquantitative immunoblotting) and glucagon-stimulated cAMP production were significantly reduced in the heterozygous Gsalpha knockout mice (GSKO) relative to their wild-type (WT) littermates. Furthermore, Na-K-2Cl cotransporter protein abundance in the outer medulla was significantly reduced (band density, 48% of WT). In addition, GSKO mice had a significantly reduced (72% of WT) urinary osmolality in response to a single injection of 1-deamino-[8-D-arginine]vasopressin (DDAVP), a vasopressin analog. In contrast, outer medullary protein expression of the type 3 Na/H exchanger (NHE-3) or Tamm-Horsfall protein did not differ between the GSKO mice and their WT littermates. However, abundance of type VI adenylyl cyclase was markedly decreased in the outer medullas of GSKO mice, suggesting a novel feed-forward regulatory mechanism. We conclude that expression of the Na-K-2Cl cotransporter of the TAL is dependent on Gsalpha -mediated hormone stimulation, most likely due to long-term changes in cellular cAMP levels.

vasopressin; urinary concentrating mechanism; adenosine 3',5'-cyclic monophosphate; sodium-potassium-adenosinetriphosphatase; aquaporins


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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TRANSPORT FUNCTIONS are regulated throughout the length of the nephron by hormone stimulation of adenylyl cyclases mediated by the heterotrimeric G protein Gs. For example, activation of this signaling pathway via vasopressin binding to V2 receptors in rat renal collecting duct and the thick ascending limb (TAL) (18, 19) is key in renal water conservation. In the TAL, acute exposure to vasopressin has been shown to increase NaCl transport in isolated perfused tubule experiments (12, 14, 27). Furthermore, chronic administration of vasopressin to Brattleboro rats has been reported to increase TAL chloride transport (3). An increase in NaCl transport by the TAL would enhance counter-current multiplication and thus the ability to form a concentrated urine. Increased TAL chloride transport could be due to increased expression of the apical Na-K-2Cl cotransporter, perhaps as a result of increased cAMP production. The finding of a cAMP regulatory element (CRE) in the 5'-flanking region of the NKCC-2 gene (17), which codes for the Na-K-2Cl cotransporter of the TAL, is consistent with the hypothesis that the expression level of this cotransporter is regulated by adenylyl cyclase activation. We recently showed an increase in outer medullary expression of the apically located Na-K-2Cl cotransporter of the TAL in rats by 1-deamino-[8-D-arginine]vasopressin (DDAVP) administration or water restriction (conditions which raise intracellular cAMP levels) (20). Furthermore, the expression of aquaporin-2, another vital protein in the urinary concentrating mechanism, is thought to be mediated by cAMP stimulation of cAMP regulatory element binding protein (CREB) phosphorylation (16, 26, 35). Phosphorylated CREB has been proposed to activate aquaporin-2 gene transcription by binding to a CRE and possibly other elements in the 5'-flanking region of the aquaporin-2 gene.

A new animal model, recently introduced, is useful in addressing the role of cAMP in the regulation of the expression of the Na-K-2Cl cotransporter, namely mice with heterozygous disruption of the Gnas gene, which codes for the heterotrimeric G protein subunit, Gsalpha . These Gsalpha knockout (GSKO) mice were created by targeted disruption of Gnas through insertion of a GK-Neo cassette into exon 2 (36). The homozygotes do not develop beyond the embryonic stage. However, the heterozygotes survive and manifest an interesting phenotype that is dependent upon whether the mutant gene is derived from the mother (m-/p+) or from the father (m+/p-). This phenotypic variability is due to genomic imprinting, i.e., epigenetic inactivation of one of the two alleles coding for Gnas (36). Thus proximal tubules of m+/p- mice express normal (wild-type, WT) levels of Gsalpha protein and manifest normal cAMP responses to parathyroid hormone, whereas proximal tubules of m-/p+ mice express low levels of Gsalpha protein and manifest depressed cAMP responses to parathyroid hormone.

The primary objective of the present study was to test the effect of reduced Gsalpha protein expression in the heterozygous GSKO mice on signaling and transporter expression in the TAL of Henle's loop. Specifically, the goals were to evaluate 1) whether Gsalpha protein expression differs between m-/p+ GSKO mice and m+/p- GSKO mice in a manner consistent with imprinting of Gnas in TAL cells, 2) whether the TALs of GSKO mice manifest a reduction in Na-K-2Cl cotransporter expression or a reduction in expression of other TAL proteins (NHE-3, Tamm-Horsfall protein, or Na-K-ATPase subunits), 3) whether a reduction in Gsalpha expression in the TALs of GSKO mice is associated with reduced cellular cAMP production in TAL cells, and 4) whether GSKO mice have a defect in urinary concentrating capacity.


    MATERIALS AND METHODS
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Knockout mouse model. The GSKO mice used in this study were originally described by Yu et al. (36). Homozygous GSKO mice did not develop beyond the embryonic stage. Consequently, heterozygous knockout mice were used in these studies. Heterozygous GSKO mice were obtained by mating either a heterozygous GSKO female mouse to a WT male (to generate m-/p+ mice) or by mating a heterozygous male to a WT female (to generate m+/p- mice). Genotyping of offspring was performed by Southern hybridization or duplex PCR (34) by using primers for the beta -globin gene promoter and "neo-specific" primers as described in the initial publication of Yu et al. (36). In some tissues and cell types, Gnas is subject to imprinting or inactivation of one parent's donated allele, presumably by methylation (2). Therefore, it is important to distinguish between the two types of heterozygotes. In this report, we present and statistically analyze the immunoblotting data from the two groups of heterozygotes both separately and in a pooled format.

Experimental design. Four studies were conducted using the GSKO mice. In the first, we examined expression of TAL proteins in the two groups of heterozygous mice by immunoblotting. Four m-/p+ mice and four m+/p- mice were compared with four each of their respective WT littermates. The mice were killed by cervical dislocation, the kidneys were removed, and samples were prepared for immunoblotting as described below. In the second study, m+/p- mice were injected intramuscularly with 1 ng DDAVP (Peninsula Laboratories, Belmont, CA), and urine was collected 1 h later to assess the ability to increase urinary osmolality (model 5100C; Wescor, Logan, UT). In a third study (for cAMP measurement), six additional untreated heterozygous GSKO mice were used (three m-/p+ mice and three m+/p- mice) along with their respective WT littermates. In these mice, after death, TAL and inner medullary collecting duct (IMCD) tubule suspensions were prepared from both right and left kidney outer or inner medullas, respectively, as described below. In the fourth study, six m-/p+ mice and six littermates were deprived of water for 48 h prior to urine collection to assess Na-K-2Cl cotransporter expression and final urine concentration in the context of a maximal long-term antidiuretic stimulus. After measurement of urinary osmolality, kidneys from these water-deprived mice were harvested, and whole homogenates were prepared from the outer medullas of the left kidneys for immunoblotting.

Antibodies. We used a rabbit polyclonal anti-Na-K-2Cl cotransporter antibody (L224) (9) raised against a synthetic peptide corresponding to amino acids 109-129 of the amino-terminal tail of the rat Na-K-2Cl cotransporter based on the cloned sequence reported by Gamba et al. (11). The antibody is highly specific for the TAL form of the Na-K-2Cl cotransporter and does not recognize either the secretory form of the Na-K-2Cl cotransporter or the thiazide-sensitive Na-Cl cotransporter. Similarly, rabbit polyclonal antibodies were raised against 1) amino acids 809-831 of the type 3 Na/H exchanger (NHE-3) (L546) (10), 2) against amino acids 267-292 of aquaporin-3 water channel (L178) (8), and 3) against amino acids 911-929 of UTA1, the vasopressin-regulated urea channel of the IMCD (L403) (30), by immunizing rabbits with synthetic peptides corresponding to these regions in the carboxy-terminal tail of each protein. A rabbit polyclonal anti-aquaporin-2 antibody (L414), likewise, was raised against a 23-amino acid synthetic peptide corresponding to the carboxy-terminal tail of aquaporin-2. The immunizing peptide was identical to that used for the previously described polyclonal antibody (L127) (6), and the two anti-aquaporin-2 antibodies have been found to give equivalent labeling on immunoblots (unpublished data). These polyclonal antibodies were affinity purified on columns prepared with the corresponding synthetic peptides. An affinity-purified polyclonal anti-Gsalpha antibody (33) was kindly provided by Dr. Paul Goldsmith (National Institute of Diabetes and Digestive and Kidney Diseases). Rabbit anti-Na-K-ATPase (alpha 1- and beta -subunit) polyclonal antibodies were obtained from Upstate Biotechnology (Lake Placid, NY). A polyclonal anti-Tamm-Horsfall antibody (15) was kindly provided by Dr. John Hoyer (University of Pennsylvania). A polyclonal adenylyl cyclase VI antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). This antibody recognizes adenylyl cyclase VI, which is expressed in the TAL of the collecting duct (4), as well as adenylyl cyclase V (not expressed in the TAL). For immunoblotting, antibodies were used at the following concentrations: Na-K-2Cl cotransporter, 0.21 µg/ml; aquaporin-2, 0.29 µg/ml; Na-K-ATPase (alpha 1-subunit), 0.10 µg/ml, Na-K-ATPase (beta -subunit), 0.10 µg/ml; Gsalpha , 0.73 µg/ml; adenylyl cyclase VI, 0.20 µg/ml, NHE-3, 0.40 µg/ml; UTA1, 0.21 µg/ml; and aquaporin-3, 0.12 µg/ml.

Immunoblotting sample preparation. Left kidneys were removed from the mice and were dissected with small, curved scissors into inner medulla, outer medulla, and cortex. Outer medulla as dissected consisted primarily of inner stripe and thus did not contain a significant proportion of proximal straight tubule. Each region was homogenized separately using a tissue homogenizer (Omni 2000 fitted with a 7 or 10 mm micro-sawtooth generator) in chilled membrane-isolation solution (1 ml for outer or inner medulla, 5 ml cortex) containing 250 mM sucrose, 10 mM triethanolamine (Calbiochem, La Jolla, CA), 1 µg/ml leupeptin (Bachem, Torrance, CA) and 0.1 µg/ml phenylmethylsulfonyl fluoride (US Biochemical, Toledo, OH) adjusted to pH 7.6. The homogenates were then centrifuged in a Beckman L8-M ultracentrifuge fitted with a type 80TI rotor at 200,000 g for 1 h to obtain a total membrane pellet. This pellet was resuspended in a small amount of isolation solution, and the protein concentration was measured (BCA protein assay reagent kit; Pierce, Rockford, IL). All samples were then diluted with isolation solution to a final protein concentration of 1 µg/µl and solubilized at 60°C for 15 min in Laemmli sample buffer. Samples were stored at -80°C until ready to run on gels.

Electrophoresis and blotting of membranes. SDS-PAGE was done on precast minigels of 7.5, 10, or 12% polyacrylamide (Bio-Rad, Hercules, CA). The proteins were transferred from the gels electrophoretically to nitrocellulose membranes. After a 30-min 5% milk block, membranes were probed overnight at 4°C with the appropriate primary antibody. For this, the antibodies were diluted into a solution containing 150 mM NaCl, 50 mM sodium phosphate, 10 mg/dl sodium azide, 50 mg/dl Tween 20, and 0.1 g/dl bovine serum albumin (pH 7.5). The secondary antibody was donkey anti-rabbit IgG conjugated to horseradish peroxidase (Pierce no. 31458) used at a concentration of 0.16 µg/ml. Sites of antibody-antigen reaction were visualized using luminol-based enhanced chemiluminescence (LumiGLO; Kirkegaard and Perry Laboratories, Gaithersburg, MD) before exposure to X-ray film (no. 165-1579 Scientific Imaging Film; Kodak, Rochester, NY). Immunoblot band densities were quantitated by laser densitometry (Molecular Dynamics, San Jose, CA).

cAMP measurements in tubule suspensions. Immediately after death, both kidneys were rapidly removed from the mouse. The outer medulla (inner stripe) and inner medulla were dissected out of both kidneys and separately minced into small cubes (<1 mm) with a razor blade, while the tissue was kept moist. Pieces of outer medulla (or inner medulla) were incubated in a 50-ml centrifuge tube at 37°C in 10 ml collagenase digestion solution which contained 50 mg collagenase B (Worthington type; Boehringer, Mannheim, Germany), 50 mg bovine serum albumin (fraction V; Sigma, St. Louis, MO), and 5 mg DNase I (DN-25, Sigma) dissolved in 50 ml of solution 1, which was composed of DMEM-F12 (Sigma) with added 4 mM glycine (Sigma, no. G6388) and 1 mM heptanoate (Sigma, no. H9278). A light stream of oxygen was blown across the surface of the solution during the incubation, which was done in a heated shaking water bath. After 8 min, the tube was removed, and the partially digested outer (or inner) medullary tissue was pipetted up and down (15 times) into a 10-ml disposable pipette to facilitate digestion. Chunks were allowed to settle out in the pipette by gravity. Large chunks were then redigested for 8 min more. The remaining digested material (primarily short segments of TALs and collecting ducts for the outer medullary suspensions and collecting ducts and thin limbs for inner medullary suspensions) was added to 25 ml of ice-cold solution 1 (which diluted out enzymes). After three total rounds of digestion, the digested tubules were centrifuged at 100 g for 5 min to softly pellet tubules and concentrate the TALs (for outer medullary suspensions) or collecting ducts (for inner medullary suspensions). In this preparation, for the outer medullary suspensions, thin limbs and free-floating collecting duct cells generally stayed in supernatant, as assayed by microscopic analysis of the fractions. After the gentle centrifugation, the outer medullary suspension consisted of ~80-90% TALs compared with other tubule components. This was based on microscopic analysis of several preparations and counting of the tubules present of each type under the microscope. For the inner medullary suspensions, this protocol has been previously shown to result in relative enrichment of IMCD vs. other tubule components, such as thin limbs (21).

The outer (or inner) medullary tubules were resuspended in 0.5 ml fresh solution 1 immediately before beginning the incubation. Incubations for cAMP production were performed as previously described (7). After an initial 10-min incubation with 0.5 mM IBMX to inhibit phosphodiesterases, 50-µl aliquots of the suspension were incubated for 5 min with one level of hormone or vehicle. For the TAL suspensions, treatments were as follows: vehicle, 0.1, 1, 10, or 100 nM glucagon. The IMCD reportedly expresses one receptor subtype coupled to Gsalpha , the vasopressin receptor (28). Therefore, the IMCD suspensions were exposed to vehicle or 0.1 or 10 nM DDAVP. The reactions were terminated by the addition of 10% trichloroacetic acid. After centrifugation at 2,300 g for 10 min, cAMP was measured in the supernatant using an ELISA-based approach (enzyme immunoassay kit no. 581001; Cayman Chemical, Ann Arbor, MI). Protein content of the pellet was measured by a Bradford analysis kit (Bio-Rad).


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Thick Ascending Limb

Gsalpha expression. The mice evaluated in these studies were heterozygous for the alpha -subunit of the heterotrimeric GTP-binding protein, Gs, and were previously characterized by Yu et al. (36). Heterozygotes were derived from the mating of a heterozygous female to a WT male (m-/p+, where "m" indicates the maternally derived allele and "p" indicates the paternally derived allele) or from the mating of a heterozygous male to a WT female (m+/p-). Imprinting (epigenetic inactivation of one allele) of the gene that codes for Gsalpha (Gnas) has been demonstrated in the kidney proximal tubule, but not in IMCD, in this mouse model (36). Because of the potential for imprinting in the TAL, we first evaluated outer medullary tissue (primarily inner stripe, see MATERIALS AND METHODS) from both types of GSKO mice separately. Figure 1 shows Gsalpha immunoblots prepared from gels loaded with outer medullary membrane samples. Each lane was loaded with 5 µg of total protein prepared from a different mouse. Figure 1A shows an immunoblot prepared with crude membrane samples prepared from outer medullas of m-/p+ mice with respective WT littermates. Figure 1B shows an immunoblot loaded with membrane samples prepared from m+/p- mice and their respective wild-type WT littermates. Two bands were present at ~50 and 45 kDa (33), secondary to alternative splicing of Gnas transcript. Gsalpha expression was approximately equally reduced in the outer medullas of both groups of heterozygotes (Table 1). Because there was an approximately equal decrease (but not elimination) of Gsalpha expression in both groups of GSKO mice (Table 1), we conclude that imprinting of Gnas does not occur in the TAL, whose cells make up most of the cellular volume of the outer medulla (22).


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Fig. 1.   Immunoblots of total outer medullary membranes (200,000 g pellet) of wild-type (WT) and Gsalpha knockout (GSKO) mice probed with anti-Gsalpha antibody. A: m-/p+ mice vs. WT littermates. B: m+/p- vs. WT littermates. For both blots, each lane was loaded with 5 µg protein from a different animal. Gsalpha -antibody was affinity purified and used at an IgG concentration of 0.73 µg/ml. Preliminary 12% polyacrylamide gels were run in each case and were stained with Coomassie blue to confirm equality of loading (not shown). Gsalpha abundance was significantly reduced in the GSKO mice in both groups (see Table 1).


                              
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Table 1.   Summary of densitometry of immunoblots from GSKO mice

Reduced expression of the Na-K-2Cl cotransporter. Figure 2A shows an immunoblot of outer medullary membranes (same samples as in Fig. 1A) from GSKO mice (m-/p+) and their WT littermates. This immunoblot demonstrates a decrease in Na-K-2Cl cotransporter expression in GSKO mice relative to WT mice from the same litter. [In membrane fractions, commonly there are two broad bands associated with the Na-K-2Cl cotransporter centered at ~161 and at 320 kDa. We have recently shown by cross-linking studies in rat (25) that the upper band (320 kDa) is most likely due to the natural existence of a homodimer of the Na-K-2Cl cotransporter in the plasma membrane. The dimer tends to be stabilized under low ionic strength conditions such as exists in the solution used for membrane isolation in the present studies.] Figure 2B shows normalized densitometry data comparing pooled band densities from m-/p+ and m+/p- GSKO mice (n = 8) and the pooled band densities of their respective WT controls (n = 8). This pooled analysis confirmed the decrease in band density for the Na-K-2Cl cotransporter in the GSKO mice (band density for GSKO mice: 49 ± 6% of WT controls, means ± SE). Before running immunoblots, Coomassie-stained "loading gels" were run on all samples and evaluated by laser densitometry to confirm equality of loading on the immunoblots (data not shown).


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Fig. 2.   A: Na-K-2Cl cotransporter immunoblot of total outer medullary membranes (200,000 g pellet; 1 µg/lane) from 4 WT and 4 m-/p+ GSKO heterozygotes. Each lane was loaded with a sample from a different animal. Na-K-2Cl antibody was affinity purified and used at an IgG concentration of 0.21 µg/ml. Preliminary 12% polyacrylamide gels were run in each case and were stained with Coomassie blue to confirm equality of loading (not shown). B: densitometry of the Na-K-2Cl cotransporter immunoblot bands from GSKO mice plotted as a percentage of their WT littermates. Data were pooled from both groups of mice. Na-K-2Cl abundance was significantly reduced in the GSKO mice in both m-/p+ and m+/p- groups (see also Table 1). * P < 0.05.

Na-K-2Cl cotransporter expression also was reduced in the cortex. When data were pooled from both groups of GSKO heterozygotes, Na-K-2Cl cotransporter band density was significantly reduced (73 ± 11% of WT littermates, means ± SE, P < 0.05).

Lack of effect on other TAL apical proteins. We also measured the abundance of other apically expressed proteins in the outer medullary TAL by semiquantitative immunoblotting. Figure 3A shows an immunoblot for NHE-3 (the type 3 Na/H exchanger) in the outer medulla using m-/p+ GSKO heterozygotes and their WT littermates. The single band we observe for NHE-3 in mouse runs at ~84 kDa, similar to the band seen in rat kidney (1). Densitometric analysis revealed that the expression level of NHE-3 did not differ significantly between the GSKO mice and their WT littermates. Table 1 shows that similar results were obtained in the m+/p- GSKO mice.


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Fig. 3.   Immunoblots of total outer medullary membranes (200,000 g pellet) from 4 WT and 4 m-/p+ heterozygous Gsalpha -knockout mice. A: blot probed with anti-NHE-3 antibody, 0.40 mg/ml, with 10 µg protein from a different rat loaded in each lane. B: blot probed with anti-Tamm-Horsfall antibody, 1.4 µg/ml IgG, with 5 µg protein from a different rat loaded in each lane. Preliminary 12% polyacrylamide gels were run and were stained with Coomassie blue to confirm equality of loading (not shown). Neither NHE-3 nor Tamm-Horsfall protein abundance was significantly affected by Gsalpha knockout (see Table 1).

Figure 3B shows an immunoblot for Tamm-Horsfall protein in the outer medullas of m-/p+ GSKO mice compared with WT littermates. Band density was not different between the GSKO mice and the WT controls. Likewise, no difference in expression of Tamm-Horsfall protein was seen in outer medullas of m+/p- GSKO mice vs. their WT littermates (Table 1).

Na-K-ATPase abundance in outer medullas of GSKO mice. Figure 4 shows immunoblots of outer medullary membranes from m-/p+ GSKO mice and their WT controls probed with antibodies to the alpha 1-subunit (Fig. 4A) and beta -subunit (Fig. 4B) of Na-K-ATPase. As shown in Fig. 4 and summarized in Table 1, the expression level of the beta -subunit was significantly reduced in the GSKO mice (equally so in both groups of heterozygotes). Although the band density for the alpha 1-subunit also appeared to be lower in the GSKO mice relative to the WT mice (especially in the m-/p+ group), a significant decrease was not detected in the pooled groups (Table 1).


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Fig. 4.   Immunoblots of total outer medullary membranes (200,000 g pellet) from 4 WT and 4 m-/p+ heterozygous Gsalpha -knockouts (GSKO). A: blot probed with anti-alpha 1-subunit Na-K-ATPase antibody, 0.10 µg/ml, with 10 µg protein from a different rat loaded in each lane. B: blot probed with anti-beta -subunit Na-K-ATPase antibody, 0.10 mg/ml, with 10 µg protein from a different rat loaded in each lane. Preliminary 12% polyacrylamide gels were run and were stained with Coomassie blue to confirm equality of loading (not shown). The alpha 1-subunit of Na-K-ATPase was not significantly changed in either group of GSKO heterozygotes, whereas beta -subunit of Na-K-ATPase was significantly reduced in both groups of GSKO heterozygotes (Table 1).

Decreased cAMP production in TAL suspensions. To assess a potential mechanism for the decrease in Na-K-2Cl cotransporter expression in these mice, we first tested whether the observed reduction in Gsalpha protein was associated with a corresponding reduction in cAMP production by the TAL cells. In the TAL, Gs is coupled to four different receptors: glucagon, vasopressin V2, calcitonin, and parathyroid hormone (28). In the present studies, glucagon was used. Figure 5 shows a dose response of outer medullary suspensions to one of four levels of glucagon (0.1, 1, 10 or 100 nM) or vehicle. The cAMP production rate in outer medullary TAL suspensions from the GSKO mice was reduced significantly (by 50% or more) relative to the WT mice, regardless of whether they were exposed to glucagon or vehicle alone.


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Fig. 5.   cAMP production in 50-µl aliquots of outer medullary suspensions prepared from 6 WT mice (black bars) and 6 GSKO heterozygotes (striped bars) (3 m-/p+ and 3 m+/p- GSKO mice were used with their respective littermates). Samples were incubated for 5 min with the agonist or vehicle in presence of the phosphodiesterase inhibitor, IBMX (0.25 mM). Each reaction was terminated by the addition of trichloroacetic acid. Aliquots were incubated with glucagon (0.1, 1, 10 or 100 nM) or vehicle. cAMP accumulation was significantly reduced in the GSKO mice throughout the dose profile. * P < 0.05.

Decreased adenylyl cyclase abundance in the TAL. Figure 6 shows immunoblots of the outer medullary membranes probed with an antibody to type VI adenylyl cyclase, the adenylyl cyclase isoform expressed in the TAL (4). The m-/p+ group is shown in Fig. 6A, and the m+/p- group is shown in Fig. 6B. The abundance of type VI adenylyl cyclase was significantly reduced in the outer medullas from both groups of GSKO mice relative to their WT littermates (Table 1).


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Fig. 6.   Immunoblots of total outer medullary membranes (200,000 g pellet) from WT and heterozygous (GSKO) mice probed with anti-adenylyl cyclase (V, VI) antibody, 0.20 mg/ml. A: m-/p+ mice. B: m+/p- mice. For both blots, individual lanes were loaded with 10 µg protein from a different animal. Preliminary 12% polyacrylamide gels were run in each case and were stained with Coomassie blue to confirm equality of loading (not shown). Expression of adenylyl cyclase (VI) was significantly reduced in both m-/p+ and m+/p- knockouts (Table 1).

Decreased urinary concentrating capacity. A decrease in Na-K-2Cl cotransporter abundance in the TAL would predict a reduction in countercurrent multiplication by the loop of Henle and decreased medullary solute concentrations. Therefore, one would predict a reduction in maximum urinary osmolality achieved in response to acute stimulation by vasopressin. To assess this possibility, six m+/p- GSKO mice and six WT littermates were injected intramuscularly with DDAVP, a V2 receptor-selective vasopressin agonist. After 1 h, urine was collected from all 12 mice for measurement of osmolality. Figure 7 is a plot of individual measurements obtained from the urine of each mouse. Average urinary osmolality was significantly lower in the GSKO mice relative to their WT littermates (2,449 ± 211 vs. 3,399 ± 60 mosmol/kgH2O, respectively; P <=  0.005).


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Fig. 7.   Final urine osmolality (mosmol/kgH2O) 1 h after injection of 1 ng of 1-deamino-[8-D-arginine]vasopressin (DDAVP, im) to 6 m+/p- Gsalpha knockout (GSKO) mice and 6 WT littermates. Each symbol represents a different litter. All mice were in the basal (untreated) state before injection of DDAVP. Urinary osmolality of the GSKO mice was significantly lower than their WT littermates, P < 0.01.

Obliteration of effect on Na-K-2Cl cotransporter expression by water restriction. Finally, to test whether the impairment of outer medullary Na-K-2Cl cotransporter expression in GSKO mice could be overcome by a chronic increase in circulating vasopressin, five m-/p+ GSKO mice and five WT littermates were thirsted for 48 h. Analysis of urine samples collected just prior to death revealed that urinary osmolality was not significantly different between the two groups of mice. Final urine osmolalities for the WT mice and their GSKO littermates were 3,518 ± 82 and 3,465 ± 96 mosmol/kgH2O, respectively, as previously reported (36). Immunoblots of outer medullary membrane samples from these mice (Fig. 8) revealed that after the water deprivation, Na-K-2Cl cotransporter abundance was no longer lower in outer medullas of the GSKO mice than in their WT littermates (band density for GSKO was 140 ± 58% of WT littermates, mean ± SE; not significant).


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Fig. 8.   Immunoblot (probed with anti-Na-K-2Cl cotransporter antibody, 0.21 µg/ml) of outer medullary homogenates from 6 WT and 6 heterozygous (m-/p+) Gsalpha -knockouts (GSKO) after a 48-h water deprivation period. Each lane was loaded with 1 µg total protein from a different mouse. Each letter (A-E) designates a different litter. (Note: absence of observable 320-dimer band is most likely due to the fact that these were whole homogenates rather than membrane fractions, as were used in Fig. 2.) Preliminary 12% polyacrylamide gels were run and were stained with Coomassie blue to confirm equality of loading (not shown). Expression of the Na-K-2Cl cotransporter was not significantly different between the two groups.

Collecting Duct

Aquaporin-2 expression is reduced in renal cortex, but not medulla. Because the urinary concentrating defect we observed in the GSKO mice could be partly due to a defect in collecting duct water and/or solute transport, we also assessed the expression level of several important collecting duct transporters. Figure 9A shows an immunoblot of cortical membranes from m-/p+ GSKO mice and their WT littermates probed with anti-aquaporin-2 antibody demonstrating a decrease in aquaporin-2 expression in the GSKO mice. As shown in Table 1, the expression level of aquaporin-2 protein was significantly lower in cortical membranes from the GSKO mice relative to their WT littermates in both groups (Table 1). When data were pooled from both groups, aquaporin-2 band density of GSKO mice was 55 ± 6% of WT littermates (means ± SE, P < 0.05). Figure 9B shows an immunoblot of the outer medullary membranes (m-/p+ vs. WT controls) probed with anti-aquaporin-2 antibody. In contrast to the cortex, in the outer medulla, no significant difference was observed between the GSKO mice and their WT littermates when data were analyzed separately or when data from both groups were pooled (Fig. 9B, Table 1). Similarly, there was no significant difference in aquaporin-2 expression in the inner medulla between the GSKO mice and their WT littermates (data not shown).


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Fig. 9.   A: aquaporin-2 immunoblot of renal cortical membranes (200,000 g pellet; 10 µg protein/lane) from 4 WT and 4 m-/p+ heterozygous Gsalpha -knockout mice (GSKO). B: aquaporin-2 immunoblot of renal outer medullary membranes (200,000 g pellet; 3 µg protein/lane) from 4 WT and 4 m-/p+ heterozygous Gsalpha -knockout (GSKO) mice. Both blots were probed with anti-aquaporin-2 antibody, 0.29 mg/ml. In each case, preliminary 12% polyacrylamide gels were run and were stained with Coomassie blue to confirm equality of loading (not shown).

Aquaporin-3 expression modestly decreased while urea transporter expression was unchanged. Aquaporin-3 is a major basolateral water transporter in kidney collecting duct. Interestingly, aquaporin-3 protein was significantly reduced in the m-/p+ mice (40 ± 14% of WT mean; mean ± SE) outer medulla, but not in the m+/p- mice, or when data were pooled. (see Table 1). Finally, immunoblotting of the inner medulla revealed no significant differences in basal expression levels of the vasopressin-regulated urea transporter (UTA1), the apical urea transporter of the IMCD (Table 1). Thus the decreased urinary concentrating capacity is not likely to be due to decreased urea transporter abundance in the collecting duct.

No differences in IMCD cAMP production. IMCD suspensions prepared from three m-/p+ and three m+/p- mice were exposed to vehicle or one of two doses of DDAVP (0.1 and 10 nM). Although cAMP production tended to be lower in the GSKO mice at both doses, there were no significant differences between the two groups (Fig. 10). Thus this is consistent with our finding of no differences in aquaporin-2 protein expression (see Fig. 9, Table 1).


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Fig. 10.   cAMP production in 50-µl aliquots of inner medullary suspensions prepared from 6 WT mice (solid bars) and 6 GSKO heterozygotes (hatched bars) (3 m-/p+ and 3 m+/p- GSKO mice were used with their respective littermates). Samples were incubated for 5 min with DDAVP (0.1 or 10 nM) or vehicle in presence of the phosphodiesterase inhibitor, IBMX (0.25 mM). Each reaction was terminated by the addition of trichloroacetic acid. cAMP production was not significantly different between the groups at any concentration.

No change in adenylyl cyclase abundance in the inner medulla. Figure 11 shows immunoblots of the outer medullary membranes probed with an antibody to type VI adenylyl cyclase, the adenylyl cyclase isoform expressed in the collecting duct (4). The m-/p+ group is shown in Fig. 11A, and the m+/p- group is shown in Fig. 11B. The abundance of type VI adenylyl cyclase in GSKO was not significantly different from the abundance in their WT littermates.


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Fig. 11.   Immunoblots of inner medullary homogenates from WT and heterozygous (GSKO) mice probed with anti-adenylyl cyclase (V, VI) antibody, 0.20 mg/ml. A: WT vs. GSKO (m-/p+) mice. B: WT vs. GSKO (m+/p-) mice. For both blots, individual lanes were loaded with 5 µg protein from a different animal. Preliminary 12% polyacrylamide gels were run in each case and were stained with Coomassie blue to confirm equality of loading (not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The knockout mice used in these studies had a heterozygous disruption of Gnas, the gene coding for the alpha -subunit of Gs. The model was previously characterized by Yu et al. (36). Homozygous knockout mice do not develop in utero and are not born. To obtain heterozygotes, heterozygous females were mated with WT males, yielding m-/p+ mice, and WT females were mated with heterozygous males yielding m+/p- mice. Because of imprinting in various tissues, the two groups of heterozygotes have distinct phenotypes. For example, m-/p+ mice are born larger than their WT littermates, and m+/p- mice are born smaller than their WT littermates. Furthermore, as discussed in the introduction, proximal tubules from the two groups of heterozygotes manifest different levels of Gsalpha expression and different rates of cAMP production in response to parathyroid hormone (36). Therefore, it was important initially to evaluate the two groups separately with regard to Gsalpha expression in the TAL. We found that expression of Gsalpha protein (as assessed by immunoblotting band density) was ~50% reduced in the outer medullas obtained from heterozygotes relative their WT littermates, regardless of whether m-/p+ or m+/p- heterozygotes were used (Table 1). Thus there is no imprinting of Gnas in the TAL of these mice, whose cells make up the greatest proportion of cell volume in the inner stripe of the outer medulla (22). Lack of Gnas imprinting has also been reported for inner medulla, where collecting ducts are the predominant structure (36).

The chief new finding in this study was that the abundance of the Na-K-2Cl cotransporter was reduced in the TALs of both groups of heterozygotes (band density for GSKO mice 48% of WT) in association with a significant reduction in acute urinary concentrating capacity and decreased cAMP production by TAL cells. The most direct explanation for the decreased expression of the Na-K-2Cl cotransporter in the GSKO mice is that cotransporter expression is regulated by cAMP and that cAMP levels in the TAL cells are reduced as a result of reduced Gsalpha expression. An unexpected finding from these studies was that adenylyl cyclase type VI abundance was dramatically reduced in the outer medulla of the GSKO mice (Fig. 6, Table 1). Thus it appears possible that the decline in cAMP production was a consequence of the decrease in adenylyl cyclase expression rather than the decrease in Gsalpha expression per se.

However, other explanations should be considered in this relatively complex animal model, i.e., the Gsalpha heterozygote, where many systems and cells are variably affected (36). First, a reduction in expression of the alpha -subunit of Gs protein would hypothetically result in an increase in the amount of free beta -gamma subunits. Free beta -gamma (like free GTP-bound alpha ) has well defined effects on several components of classic signaling pathways, e.g., phospholipase C (beta 2 and beta 3) and adenylyl cyclase (subtypes II, IV, VII, I) (13).

Second, it would be predicted that a fall in Gsalpha expression in the proximal tubule would increase salt and water absorption (by decreasing cAMP production) in this segment (23). This would reduce flow of both salt and water to downstream segments of the tubule, which conceivably could affect expression of various transporters. Increased NaCl load to the TAL has been shown to increase sodium reabsorption by the loop of Henle (as assessed by micropuncture) (24), and we have shown that increased NaCl in the drinking water will increase the expression of the Na-K-2Cl cotransporter in the outer medulla (9), possibly due to flow effects. With this model, we can get some estimate of the importance of altered proximal tubule fluid reabsorption in affecting expression of the Na-K-2Cl cotransporter, because of the existence of Gnas imprinting in the proximal tubule (36). The m-/p+ GSKO mice express very low levels of Gsalpha in the proximal tubule, whereas m+/p- GSKO mice have normal expression levels. Therefore, if the decrease in Na-K-2Cl cotransporter expression in the TAL were due entirely to reduced salt and water flow from the proximal tubule, then we would have expected a relatively greater decrease in Na-K-2Cl cotransporter expression in the m-/p+ mice. Instead, we see no difference in the relative decrease in Na-K-2Cl cotransporter expression between the m-/p+ mice (47% of WT) and the m+/p- mice (51% of WT) (Table 1). However, it is possible that factors other than Gsalpha expression in the proximal tubule might affect salt delivery to the TAL.

Third, it is possible that factors, such as aberrations in hormonal status, autacoids, or sympathetic nervous activity in the GSKO mice might directly or indirectly influence TAL protein expression. To date, we have no evidence to suspect that any of these factors as influence expression of the Na-K-2Cl cotransporter.

Thus it appears that the most plausible explanation for the decrease in the expression of the Na-K-2Cl cotransporter in the GSKO mice is decreased production of cAMP secondary to decreased Gsalpha expression and/or the associated decrease in adenylyl cyclase expression. Several G protein-coupled receptors, for example, vasopressin, glucagon, and parathyroid hormone, are linked to adenylyl cyclase activation in the kidney TAL (28). Because the TAL cells in this model had a markedly diminished ability to produce cAMP when stimulated in vitro with glucagon (Fig. 5), we suggest that chronically depressed cAMP levels in the TAL in these GSKO mice may be responsible for the reduction in outer medullary Na-K-2Cl cotransporter expression. Consistent with this mechanism, Igarashi et. al. (17) have reported the existence of a CRE in the 5'-flanking region of the Na-K-2Cl cotransporter gene. This motif potentially could have an important role in regulation of cotransporter abundance. Furthermore, we have recently reported studies in rats showing that DDAVP infusion and restriction of water intake to amounts matching respiratory losses (conditions which increase cellular cAMP production via the V2 receptor) both strongly upregulate Na-K-2Cl cotransporter abundance (20).

The effect of the Gsalpha knockout on Na-K-2Cl cotransporter abundance appeared to be selective and not due to general atrophy of the TAL because the expression levels of other apical TAL proteins, such as NHE-3 and Tamm-Horsfall protein, were not similarly reduced (Fig. 3). However, the expression level of the beta -subunit of Na-K-ATPase was markedly decreased in outer medullas of GSKO mice, whereas the alpha 1-subunit was much less profoundly affected. TAL segments are thought to account for at least 90% of the total Na-K-ATPase in the outer medulla. Therefore, measurements of Na-K-ATPase subunit abundance in the outer medulla is tantamount to measurement of the abundance in the TAL. Consequently, we conclude that GSKO mice had reduced amounts of Na-K-ATPase in their TALs. The activity of Na-K-ATPase has been reported to be increased acutely by vasopressin in the TAL (5). However, we are unaware of studies investigating the long-term effect of vasopressin on Na-K-ATPase activity in the TAL. The mechanism by which a reduction in Gsalpha expression leads to decreased Na-K-ATPase beta -subunit expression has not been defined by the present studies. Na-K-ATPase activity has been reported to be influenced by intracellular sodium concentration (31, 32). Conceivably, the reduction in the beta -subunit expression could be secondary to the reduced expression of the Na-K-2Cl cotransporter and the resulting fall in apical sodium entry.

An unexpected finding from these studies was that adenylyl cyclase type VI abundance was dramatically reduced in the outer medulla of the GSKO mice (Fig. 6 and Table 1). Adenylyl cyclase VI is expressed in both the TALs and the collecting ducts of the outer medulla. Little is known about long-term regulation of adenylyl cyclase abundance. Possibly, the half-life of the type VI adenylyl cyclase is influenced by its state of activation by Gsalpha or by concomitant changes in cellular signaling such as changes in cAMP level or protein kinase A activation. The reduction in expression of adenylyl cyclase in the GSKO mice might also be responsible, and certainly would be expected to contribute, to the decrease in cAMP production measured in the TAL suspensions (Fig. 5).

The expression of aquaporin-2 protein of the collecting duct would be predicted to be decreased in the GSKO mice, because, like the Na-K-2Cl cotransporter, the abundance of aquaporin-2 is thought to be regulated through a CRE in the 5'-flanking region of the gene. As we predicted, aquaporin-2 abundance was significantly reduced in the renal cortex regardless of whether data were pooled (band density of GSKO on average 55% of WT) or whether the two groups of heterozygotes were analyzed separately (Table 1). However, in the inner and outer medulla, aquaporin-2 protein expression was not reduced in either group of heterozygotes or when data were pooled (Table 1). Consistent with the lack of change in aquaporin-2 expression, we also observed no significant differences in cAMP production by the IMCD suspensions from the GSKO mice compared with their WT littermates. Therefore, it appears that decreased Gsalpha expression does not necessarily result in decreased cAMP production and thus Gsalpha is not rate-limiting for cAMP production in the IMCD. It is interesting that, unlike the TAL, the IMCD did not show any decrease in adenylyl cyclase VI expression. Thus it seems likely that the decrease in Na-K-2Cl cotransporter expression in the outer medulla was critically dependent on the observed fall in adenylyl cyclase VI abundance. These observations point to the regulation of the abundance of adenylyl cyclase as a possible control site for cAMP-mediated cell signaling and call for additional studies to investigate the mechanisms involved.

Finally, in the basal (untreated state), the ability of the GSKO mice to concentrate their urine after a short-term (1 h) DDAVP challenge was significantly compromised (Fig. 7). This may be attributable to the downregulation of the Na-K-2Cl cotransporter in the TAL, which would substantially impair countercurrent multiplication. However, it is also possible that decreased collecting duct water or solute permeability could have contributed to the concentrating defect. Indeed, the expression level of aquaporin-3 was significantly decreased in the m-/p+ group in the outer medulla, although significant changes in aquaporin-2 or UTA1 expression were not seen.

After a 48-h thirsting period, there were no differences in final urine osmolality between the heterozygotes and their WT littermates. Furthermore, after the 48-h thirst, the whole kidney expression of the Na-K-2Cl cotransporter (Fig. 8) or aquaporin-2 (data not shown) was not significantly different between the two groups. Presumably, prolonged stimulation with high levels of vasopressin can overcome the limiting effects of decreased Gsalpha expression on cotransporter expression. These findings therefore provide additional evidence for the importance of expression level of the Na-K-2Cl cotransporter in the TAL of the outer medulla of rodents as a determinant of urine concentrating capacity, although decreased expression of aquaporin-2 protein in the cortical collecting duct and aquaporin-3 in the outer medullary collecting duct might also play a role.

Human patients with heterozygous inactivating mutations of the Gsalpha gene (pseudohypoparathyroidism type Ia) did not manifest evidence for a decrease in concentrating ability after overnight fluid restriction or in response to exogenous vasopressin (29), suggesting that impairment of countercurrent multiplication may be less severe in these human patients than in mice with analogous mutations.


    ACKNOWLEDGEMENTS

We thank Dr. Paul Goldsmith for the anti-Gsalpha antibody and for assistance with the ELISA measurements, Dr. Brian Doctor for advice with regard to preparation of tubule suspensions, and Dr. K. Spring for careful reading of the manuscript.


    FOOTNOTES

These studies were supported by the intramural research budgets of the National Heart, Lung, and Blood Institute and the National Institute of Diabetes and Digestive and Kidney Diseases.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. A. Knepper, National Institutes of Health, 10 Center Dr., MSC 1603, Bldg. 10, Rm. 6N260, Bethesda, MD 20892-1603 (E-mail: knep{at}helix.nih.gov).

Received 4 September 1998; accepted in final form 26 April 1999.


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ABSTRACT
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RESULTS
DISCUSSION
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