GPR48 Increases Mineralocorticoid Receptor Gene Expression

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Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China; †Laboratory for Endocrine and. Metabolic ... Published online ahead of print. ..... (Kangchen Bio-tech). ... followed by sonication in an ultrasound bath on ice.
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GPR48 Increases Mineralocorticoid Receptor Gene Expression Jiqiu Wang,* Xiaoying Li,*† Yingying Ke,* Yan Lu,* Feng Wang,† Nengguang Fan,* Haiyan Sun,* Huijie Zhang,* Ruixin Liu,* Jun Yang,* Lei Ye,* Mingyao Liu,‡ and Guang Ning*† *Shanghai Clinical Center for Endocrine and Metabolic Diseases, Shanghai Institute of Endocrinology and Metabolism, Shanghai Key Laboratory for Endocrine Tumors and E-Institute of Shanghai Universities, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China; †Laboratory for Endocrine and Metabolic Diseases, Institute of Health Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences, Shanghai, China; and ‡Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas

ABSTRACT Aldosterone and the mineralocorticoid receptor (MR) are critical to the maintenance of electrolyte and BP homeostasis. Mutations in the MR cause aldosterone resistance known as pseudohypoaldosteronism type 1 (PHA1); however, some cases consistent with PHA1 do not exhibit known gene mutations, suggesting the possibility of alternative genetic variants. We observed that G protein–coupled receptor 48 (Gpr48/ Lgr4) hypomorphic mutant (Gpr48m/m) mice had hyperkalemia and increased water loss and salt excretion despite elevated plasma aldosterone levels, suggesting aldosterone resistance. When we challenged the mice with a low-sodium diet, these features became more obvious; the mice also developed hyponatremia and increased renin expression and activity, resembling a mild state of PHA1. There was marked renal downregulation of MR and its downstream targets (e.g., the a-subunit of the amiloride-sensitive epithelial sodium channel), which could provide a mechanism for the aldosterone resistance. We identified a noncanonical cAMP-responsive element located in the MR promoter and demonstrated that GPR48 upregulates MR expression via the cAMP/protein kinase A pathway in vitro. Taken together, our data demonstrate that GPR48 enhances aldosterone responsiveness by activating MR expression, suggesting that GPR48 contributes to homeostasis of electrolytes and BP and may be a candidate gene for PHA1. J Am Soc Nephrol 23: 281–293, 2012. doi: 10.1681/ASN.2011040351

INTRODUCTION

Aldosterone plays critical roles in the control of salt homeostasis and BP through stimulating Na+ reabsorption and K+ secretion.1 Upon binding to mineralocorticoid receptor (MR), the hormone– receptor complex translocates into the nucleus and interacts with glucocorticoid receptor response element in specific promoter regions of the target genes, thereby activating their transcription, such as a-subunit of the amiloride-sensitive epithelial sodium channel (aEnaC) in the aldosteronesensitive distal nephron.2 The functional relevance of ENaC to aldosterone-dependent Na+ reabsorption, and thus to the regulation of extracellular fluid volume and BP, is well established.3 Promotion of J Am Soc Nephrol 23: 281–293, 2012

Na+ reabsorption by activated ENaC also enhances K+ excretion through the luminal potassium channel, leading to kaliuresis in the collecting duct.4 Loss-of-function mutations in the MR or ENaC subunit genes account for pseudohypoaldosteronism type 1 (PHA1),5,6 which is the principal form of aldosterone resistance and shows an autosomal-dominant Received April 7, 2011. Accepted October 6, 2011. Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Guang Ning, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, 197 Ruijin 2nd Road, Shanghai 200025, China. Email: [email protected] Copyright © 2012 by the American Society of Nephrology

ISSN : 1046-6673/2302-281

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(adPHA1; OMIM #177735) and a recessive (arPHA1; OMIM #264350) transmission.7 First described in 1958, this rare condition features renal resistance to aldosterone action.8 Early in infancy, patients with adPHA1 present with dehydration and failure to thrive associated with salt wasting, hypotension, hyperkalemia, and metabolic acidosis, despite increased plasma aldosterone levels.6 These patients can improve with age, and some adult patients are usually asymptomatic and have fewer abnormal biochemical findings (e.g., only lifelong increases in aldosterone or hyperkalemia).9,10 To date, approximately 50 distinct mutations in the human MR gene and approximately 20 mutations in ENaC genes responsible for PHA1 have been described.11,12 However, some patients, especially those with sporadic PHA1, do not have genetic abnormalities in MR or ENaC.6,10,13–15 Because PHA1 is life-threatening and many probands may be missed as a result of early death, additional genetic mechanisms might participate in its pathogenesis. According to recent studies, aldosterone resistance may also be associated with a reduction in MR expression, probably mediated by transcriptional mechanisms.16–19 G protein–coupled receptor 48 (GPR48/ LGR4) belongs to the G protein–coupled receptor superfamily. It has recently been reported to bind R-spondin and mediate its signaling in intestinal crypt cells,20,21 yet its function has not been well investigated.22 GPR48 is critical in development, and Gpr48mutant mice display early neonatal lethality.23 Our group and others have demonstrated that Gpr48 deficiency results in impaired function of the male reproductive tract through downregulation of estrogen receptor a expression.24,25 GPR48 is also involved in colon carcinoma metastasis, development of ocular anterior segment, and bone formation through different downstream targets.26–28 However, the involvement of this protein in electrolyte balance has not been described. In this study, we find that Gpr48 hypomorphic mutant mice display a significant aldosterone resistance, which mimics a mild state of adPHA1 disease. We also demonstrate that GPR48 regulates MR expression through the cAMP/protein kinase A (PKA) pathway. This study elucidates the potential role of GPR48 in electrolyte homeostasis and aldosterone resistance.

RESULTS Gpr48 Homozygous Mutant Mice Show Hypomorphic Features

We obtained Gpr48 hypomorphic mutant (Gpr48m/m) mice by microinjecting gene 282

trap–mutated Gpr48 ES cells into C57BL/6 blastocysts. 27 An insertion of the trap vector into intron 1 of the Gpr48 gene resulted in approximately 90% knockdown efficiency in the kidney and adrenal gland of adult Gpr48m/m mice (Figure 1, A–C). We observed that approximately half of Gpr48m/m newborns died within 28 hours after birth, but no further deaths occurred in the following 20 hours (Figure 1D). Increases in Water Intake and Urine Volume with Partially Impaired Urine-Concentrating Ability in Gpr48m/m Mice

We explored the potential changes of water balance in Gpr48m/m mice using metabolic cages and found that Gpr48m/m mice showed a dramatic increase in 24-hour water and food intake at 16 and 24 weeks compared with wild-type mice (Figure 2, A and B). Their urine volume was also significantly increased during this time (Figure 2C). Meanwhile, urine osmolality declined markedly (Figure 2D). Female mice showed overall similar phenotypes (Supplemental Figure S1). We next performed a water deprivation test to assess the urine-concentrating

Figure 1. Residual levels of Gpr48 transcripts and neonatal survival rate of Gpr48m/m mice. (A) Approximately 10% of Gpr48 transcripts remained in the kidney of Gpr48m/m mice at age 16 weeks according to quantitative PCR (n=12). (B) Reverse transcription PCR for Gpr48 expression in the kidney of Gpr48m/m mice at age 16 weeks (n=6). The corresponding cDNA length is 471 bp, and the PCR products were verified to be wild-type Gpr48 by sequencing. (C) Approximately 10% of Gpr48 transcripts remained in the adrenal gland of Gpr48m/m mice at age 16 weeks according to qualitative PCR (n=12). 36B4 was used as internal control. (D) Survival curve of neonatal wild-type (Gpr48+/+) (n=29) and Gpr48m/m (n=23) mice within 48 hours after birth. Error bars represent SEM. ***P,0.001.

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Figure 2. Adult Gpr48m/m mice show polydipsia, polyphagia, severe water loss, and reduced urine osmolality. Male Gpr48m/m mice at age 16 and 24 weeks displayed increased (A) water intake, (B) food intake, and (C) urine volume but (D) decreased urine osmolality compared with their age-matched wild-type littermates. At 8 weeks, n=4; at 16 weeks, n=9; at 24 weeks, n=10. White bar, wild-type mice (Gpr48+/+); black bar, Gpr48-mutant mice (Gpr48m/m). Error bars represent SEM. **P,0.01; ***P,0.001. BW, body weight.

ability of Gpr48 m/m mice. The urine osmolalities of both genotypes were markedly increased by a similar extent after water deprivation; however, that of Gpr48m/m mice still did not reach the level of wild-type mice (Figure 3A), suggesting a partial urine-concentrating defect. However, neither genotype showed any difference in hypothalamic vasopressin expression or renal vasopressin receptor 2 and aquaporin 2 expression, which are all associated with diabetes insipidus (Figure 3, B–E).29–31 Aldosterone Resistance Exhibited by Gpr48m/m Mice

Water and electrolyte homeostasis are tightly controlled in the kidney. As shown in Table 1, the daily Na+ excretion was increased by 1.5 times in Gpr48m/m mice compared with excretion in wild-type mice. Although urinary K+ and creatinine excretion showed no significant change, plasma K+ was strikingly elevated in Gpr48m/m mice (6.760.3 versus 5.560.2 mM; P,0.001). The urinary Na+/K+ ratio was also higher. Plasma Na+, Cl2, creatinine, and BUN concentrations did not differ J Am Soc Nephrol 23: 281–293, 2012

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between the two genotypes, nor did pH, HCO32, or base excess (Table 1 and Supplemental Table S1). We next examined plasma mineralocorticoid levels, which play fundamental roles in regulating water and salt homeostasis. Surprisingly, plasma aldosterone was significantly higher in Gpr48m/m mice than in wild-type mice (302.6630.0 versus 155.3611.9 pg/ml; P,0.001) (Figure 4A). Their daily urinary aldosterone excretion was also increased (Figure 4B). However, this aldosterone excess did not have the expected effects of excreting K+ while preserving Na+ and water, suggesting aldosterone resistance in Gpr48m/m mice. Plasma renin activity (PRA) showed no difference but did have a tendency to increase in Gpr48m/m mice (Figure 4C). In addition, plasma corticosterone concentration did not show any difference (Figure 4, D–F). Morphologic analysis of the adrenal gland revealed no obvious changes in Gpr48m/m mice (Supplemental Figures S2 and S3A), nor did the gene expression of the key enzymes in the adrenal gland and the plasma levels of adrenergic hormones (Supplemental Figure S3, B–E), suggesting that the phenotypes observed in Gpr48 m/m mice are not secondary to defects in the adrenal gland. Taken together, these data suggest that Gpr48m/m mice displayed sodium and water loss as well as hyperkalemia, despite elevated aldosterone, thus resembling the mild state of adPHA1.6,32

Enhanced adPHA1 Phenotype in Gpr48m/m Mice on a Low-Sodium Diet

To exclude the effects of compensatory salt intake on Na+ balance, we challenged Gpr48m/m and wild-type mice with a low-sodium diet proportionate to their body weight. Urinary Na+ excretion was reduced in both groups (Figure 5A). However, except for a marked reduction on the first day after salt deprivation, Gpr48m/m mice overall displayed limited changes in Na+ excretion compared with wild-type mice and exhibited corresponding hyponatremia (Figure 5, A and B), suggesting impaired Na+ conserving ability. Hyperkalemia persisted in Gpr48m/m mice as under normal chow (Figure 5C). Meanwhile, obvious depletion in extracellular fluid volume was seen in Gpr48m/m mice, indicated by increased hematocrit and blood hemoglobin content (Figure 5, D and E). Consequently, the systolic, mean arterial, and diastolic BPs in Gpr48m/m mice were markedly decreased (Figure 5F). Gpr48m/m mice also showed higher PRA and plasma aldosterone levels (Figure 5, G and H). Consistent with PRA changes, the renal renin GPR48 in Aldosterone Resistance

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morphologic abnormalities or apparent pathologic defects were observed in the kidney of Gpr48m/m mice, except for reduced kidney size (Figure 6, B–D). After an examination of the expression of genes that regulate water and salt balance, we found that both the mRNA and protein levels of MR were dramatically reduced in the kidney of Gpr48m/m mice compared with wild type, whereas glucocorticoid receptor (GR) expression did not differ (Figure 6, E–G). Accordingly, the expression of two MR downstream targets, aENaC and Na/K-adenosine triphosphatase (ATPase) 1a (Na/K-ATPase 1a), also significantly decreased, but the expression of inwardly rectifying K+ channel showed no difference between the genotypes (Figure 6, E–G). Furthermore, both the distal tubules and collecting ducts displayed significant decreases in MR and Na/K-ATPase 1a immunostaining in Gpr48m/m mice (Figure 6H). These results suggest that the reduced expression of MR and MR-driven genes could be the main reason for the electrolyte disturbance observed in Gpr48m/m mice. GPR48 Regulates MR Gene Expression through the cAMP/PKA Pathway

We isolated and established mouse embryonic fibroblasts (MEFs) of both genotypes (Figure 7A), and MR expression was consistently decreased in Gpr48 m/m MEFs (MEFGpr48m/m) compared with wild type (Figure 7B and Supplemental Figure S4A). We further investigated whether m/m expresFigure 3. Partially impaired urine-concentrating ability in Gpr48 mice. (A) Urine GPR48 could directly regulate MRGpr48m/m osmolality of wild-type and Gpr48m/m mice aged 16 weeks was measured under 8-hour sion. As shown in Figure 7C, MEF water deprivation (n=11). (B–D) Relative mRNA expression levels of vasopressin (AVP) showed much lower MR promoter activity Gpr48+/+ ). Morein the hypothalamus (B) and vasopressin receptor 2 (AVPR2) (C) and aquaporin 2 (AQP2) than wild-type MEFs (MEF (D) in the kidney (n=11). (E) Representative immunohistochemical staining of aqua- over, GPR48 overexpression increased MR porin 2 in renal collecting ducts (scale bars, 50 mm). n.s., not significant. Error bars transcriptional activity in a dose-dependent represent SEM. manner (Figure 7D). The intracellular signaling and downstream targets of GPR48 expression was also increased in Gpr48m/m mice (Figure 5, are mediated by the cAMP/PKA pathway.27,28 As shown in I–K). These data demonstrate that Gpr48m/m mice displayed Figure 7, E and F, the adenylate cyclase agonist forskolin and more severe electrolyte and hormone abnormalities when dethe phosphodiester inhibitor 3-isobutyl-1-methyl-xanthine inprived of salt than did wild-type mice. creased MR promoter activity in MEFGpr48m/m or HEK293 cells. Consistently, the PKA inhibitor H89 (Figure 7G and SupMorphologic and Gene Expression Changes plemental Figure S4B) and the adenylate cyclase antagonist in the Kidney of Gpr48m/m Mice M182 (Supplemental Figure S4C) inhibited MR promoter acGpr48 was ubiquitously expressed in different tissues of mice, tivity in a dose-dependent manner. Moreover, H89 abolished with the highest expression in the kidney (Figure 6A), sug- GPR48-induced MR transcriptional activity (Figure 7H). The gesting its critical role in this organ. However, no gross classic cAMP-responsive element (CRE) reporter was used as a 284

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Table 1. Blood and urine measures of male wild-type and Gpr48m/m mice on the normal chow diet (n=10) Variable Blood Na+ (mM) K+ (mM) Cl2 (mM) creatinine (mM) BUN (mM) pH HCO32 (mM) BE (mM) hemoglobin (g/dl) hematocrit (% packed cell volume) Urine Na+ (mmol/g body wt per day) K+ (mmol/g body wt per day) Cl2 (mmol/g body wt per day) creatinine (pmol/g body wt per day) Na+/K+ ratio

Gpr48+/+

Gpr48m/m

144.260.7 5.560.2 117.261.1 16.961.6 9.260.4 7.2960.01 19.860.6 26.760.6 15.360.2 44.960.7

145.461.2 6.760.3a 120.062.3 19.562.6 10.861.0 7.2760.02 23.562.9 23.563.0 15.260.2 44.860.7

8.761.0 13.961.7 13.261.7 0.2560.02 0.6460.01

Values are expressed as the mean 6 SEM. BE, base excess. a P,0.001. b P,0.01.

13.061.1b 18.361.5 18.861.7b 0.2960.02 0.7160.02b

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positive control (Figure 7I). These results show that GPR48 regulates MR transcription through the cAMP/PKA pathway. By using two software programs (TFSEARCH and TESS), we found a noncanonical CRE, TAGCGTCA, located between 2472 and 2465 bp in the MR promoter. Subsequently, one CRE mutant and three truncated MR promoter luciferase constructs were generated and transfected into HEK293 cells. The CRE mutant and 2321 truncated MR promoter constructs displayed only half of the transcriptional activity of the full-length MR promoter, and H89 inhibited the transcriptional activity of the full-length MR promoter, but not the CRE-mutant or truncated promoters (Figure 8A). Moreover, the MR promoter with the mutant CRE failed to be activated by GPR48 overexpression (Figure 8B). These results suggest that CRE is essential for GPR48-activated MR transcription. We next examined the interaction of phospho-CRE binding protein (pCREB) with the CRE sequence. As shown in Figure 8C, abundant DNA fragments contained the CRE sequence from the MR promoter bound to pCREB in MEFGpr48+/+, whereas the binding was dramatically reduced in MEFGpr48m/m (Figure 8C). This CRE was confirmed in vitro using an electrophoresis mobility-shift assay (EMSA) with a wild-type or mutant CRE probe designed according to the mouse MR promoter (Figure 8D). The exact band was confirmed by gel shift assay with CREB antibody and a CRE-containing fragment from the mouse somatostatin promoter (positive control).33 These findings identify a functional CRE in the MR promoter that can directly bind to pCREB, thereby mediating GPR48-induced MR expression (Figure 8E).

DISCUSSION

In this study, we demonstrated that Gpr48m/m mice displayed salt loss, hyponatremia, and hyperkalemia despite aldosterone excess, which was associated with reduced renal expression of MR and its targets. These phenotypes, especially under a lowsodium diet challenge, resembled the characteristics exhibited by adPHA1 patients.6 Mild adPHA1 Phenotype Caused by a Gpr48 Hypomorphic Mutant-Induced MR Downregulation

Figure 4. Elevated plasma aldosterone levels and urine aldosterone excretion in Gpr48m/m mice. (A–C) Elevated aldosterone concentrations (n=9) (A), increased urinary aldosterone excretion (n=14) (B), and marginally unchanged plasma renin activity (n=9) (C) in Gpr48m/m mice. (D–F) Unaltered plasma corticosterone was observed in Gpr48m/m mice under normal conditions (n=8) (D), 8-hour fasting (n=8) (E), and stimulation (n=5) (F). Error bars represent SEM. ***P,0.001.

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adPHA1 caused by MR mutations is characterized by salt wasting, hypotension, and hyperkalemia despite elevated plasma aldosterone levels.6 The appearance of salt loss as well as hormone abnormalities in Gpr48m/m mice resembled a mild state of adPHA1. These phenotypes were similar to MR heterozygous mutant mice but less GPR48 in Aldosterone Resistance

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Figure 5. Gpr48m/m mice exhibit adPHA1 features on a low-sodium diet. (A) Impaired salt-conserving ability was observed in Gpr48m/m mice fed the low-sodium diet. Mice were placed into metabolic cages initially, with normal chow for 3 days (from D23 to D0), followed by the low-sodium diet for 5 days (from D0 to D5). Salt deprivation started on D0. (B and C) Reduced plasma Na+ and increased K+ in Gpr48m/m mice (n=12 for Gpr48+/+; n=8 for Gpr48m/m). (D and E) Dehydration indicated by increased hematocrit and hemoglobin content was observed in Gpr48m/m mice (n=12 for Gpr48+/+; n=8 for Gpr48m/m). (F) Systolic (SBP), mean arterial (MBP), and diastolic (DBP) BP were markedly decreased in Gpr48m/m mice on the low-sodium diet (n=14 for Gpr48+/+; n=8 for Gpr48m/m). (G and H) Elevated PRA and aldosterone in Gpr48m/m mice (n=8). (I–K) Renin mRNA (n=8) (I) and protein (two mice mixed as a pool, n=6–8) (J and K) were markedly elevated in the kidney of Gpr48m/m mice on the low-sodium diet. White bar, wild-type mice (Gpr48+/+); black bar, Gpr48-mutant mice (Gpr48m/m). Error bars represent SEM. *P,0.05; **P,0.01; ***P,0.001.

severe than MR homozygous mutant mice,34,35 which are most likely attributable to some functional MR proteins retained in the kidney of Gpr48m/m mice. Our results are also supported by the phenotypes of rats with MR knockdown, which exhibit Na+/K+ disturbance and a significant inverse correlation between MR downregulation and plasma aldosterone level.19 There is also clinical evidence for the association of low renal MR expression with aldosterone resistance in neonates.36 These findings suggest that in addition to MR mutations, the factors that affect MR expression could be another cause of adPHA1. The neural MR expression is tightly controlled by various hormone stimuli,37,38 but the factors regulating renal MR expression are unknown. Herein, we identified a direct role for 286

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GPR48 in the regulation of renal MR transcription via the cAMP/PKA pathway and identified a functional CRE site in the MR promoter. This is consistent with previous reports on the mechanism underlying GPR48-mediated target gene expression.24,27 Salt Deprivation Exacerbates Electrolyte and Hormone Abnormalities in Gpr48m/m Mice

Although Gpr48m/m mice fed a normal chow diet exhibited aldosterone resistance, we did not find any abnormalities in plasma Na+ level or PRA. This can also be observed in some adPHA1 carriers. The first index case of adPHA1 showed markedly elevated serum aldosterone levels and urinary Na+ excretion but normal serum Na+ and PRA at 28 years of age.9 J Am Soc Nephrol 23: 281–293, 2012

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Figure 6. Morphology and gene expression in the kidney of Gpr48m/m mice. (A) Relative expression pattern of Gpr48 in different tissues of C57BL/6 mice. (B and C) Kidney and body sizes of wild-type and Gpr48m/m mice (scale bar, 1 cm). (D) Representative hematoxylin and eosin staining of renal cortex and medulla in Gpr48m/m mice showed no defects at age 16 weeks (scale bar, 20 mm). (E–G) Relative mRNA and protein expression levels of genes involved in water and electrolyte reabsorption or excretion in both genotypes (n=8). (H) Representative immunohistochemical staining for MR and Na/K-ATPase 1a in cortical and medullary tubules of wild-type and Gpr48m/m mice (scale bar, 50 mm). White bar, wild-type mice (Gpr48+/+); black bar, Gpr48-mutant mice (Gpr48m/m). Error bars represent SEM. *P,0.05. AQP, aquaporin; ATPase, adenosine triphosphatase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GR, glucocorticoid receptor; ROMK1, inwardly rectifying K+ channel; NHE3, Na(+)/H(+) exchanger 3; NCC, thiazide-sensitive Na-Cl cotransporter; NKCC2, Na-K-2Cl cotransporter.

In addition, Geller and colleagues have prospectively screened individuals in two large Spanish pedigrees for various clinical indices, and only elevated aldosterone was seen in adPHA1 carriers with the R537X mutation in the MR gene.32 On the basis of our results and those of previous studies, we postulate that hyperkalemia, rather than hyponatremia, might be a J Am Soc Nephrol 23: 281–293, 2012

more sensitive serum electrolyte marker for mild adPHA1 animal models39–43 and adult patients with adPHA1.9,10,32 The normal plasma Na+ level in Gpr48m/m mice on normal chow may be attributable to increased food intake, which has been observed in rescued MR-null mice with NaCl substitutions;35 the normal Na+ is also consistent with the observation GPR48 in Aldosterone Resistance

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Figure 7. GPR48 regulates MR expression via the cAMP/PKA pathway. (A) Morphology of MEFs derived from wild-type and Gpr48m/m embryos (scale bars, 100 mm). (B) Reduced MR protein in MEF Gpr48m/m . (C) Decreased MR promoter transcriptional activity in MEFGpr48m/m. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (D) MR promoter transcriptional activity induced by hGPR48 overexpression (0.2, 0.5, 1.0, or 1.5 mg) in a dose-dependent pattern in HEK293 cells. (E) Forskolin (FSK, 10 mM) increased MR transcriptional activity in MEFGpr48m/m. (F and G) MR promoter transcriptional activity was induced by FSK plus 3-isobutyl-1-methyl-xanthine (IBMX) (5/125, 10/250, 15/375, 20/500, or 25/625 mM) treatment for 8 hours (F) and inhibited by H89 (5, 10, 20, 30, or 50 mM) treatment for 24 hours (G) in HEK293 cells in a dose-dependent pattern. (H) MR promoter transcriptional activity induced by hGPR48 overexpression was abrogated by H89. HEK293 cells were co-transfected with the MR promoter luciferase construct and pRL-TK Renilla luciferase construct, followed by treatment with FSK plus IBMX (20/500 mM) for 8 hours, or co-transfected with the hGPR48-expressing construct with or without 20 mM H89 treatment for 24 hours. In I, the classic CRE reporter was used as a positive control, which was subjected to the same treatments described for H. Each experiment was performed at least three times. Error bars represent SEM.

that the symptoms of patients with adPHA1 are alleviated with age because of higher dietary salt intake from food.6 Expectedly, when challenged with the low-sodium diet, Gpr48m/m mice displayed markedly decreased plasma Na+ concentration compared with wild-type mice. Consequently, renal renin expression and PRA were also augmented. These changes are consistent with the phenotypes of MRAQP2-cre mice deprived of salt.39 These findings confirm the role of GPR48 in the pathogenesis of adPHA1 and the regulation of electrolyte and hormone homeostasis. Unchanged Renal Morphology with Partial Urine-Concentrating Defects in Gpr48m/m Mice

Gpr48m/m mice displayed no apparent pathologic defects in the kidney except for the smaller size. These observations are consistent with those in the previous study by Mazerbourg and colleagues.23 However, the phenotypes reported by us and those authors are different from the findings of Kato and associates,44 who found that Gpr48-knockout mice can barely 288

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survive after birth, with higher lethality than our strains, and whose only surviving mouse showed dramatic morphologic defects in the kidney. In our study and Mazerbourg and colleagues’ study,23,27 Gpr48 gene expression was abolished by a secretory trap approach, whereas in Kato and coworkers’ study, Gpr48 was deleted by intercrossing Gpr48floxed/floxed mice with CAG-Cre “transgenic general deleter” mice.44 The gene-trap method can lead to residual Gpr48 transcripts,22,45 which was also seen in Gpr48m/m mice in our study, with approximately 10% residual Gpr48 transcripts in the kidney. This low residual expression may prevent Gpr48m/m mice from dying and displaying severe morphologic defects in the kidney. We thus speculate that different gene-targeting strategies and the residual Gpr48 transcripts may have caused the discrepancies observed in different strains of Gpr48m/m mice. Gpr48m/m mice displayed a partial urine-concentrating defect, which can also be observed in aldosterone deficiency conditions (e.g., aldosterone synthase knockout mice,46 adrenalectomized rats, 47 and patients with chronic adrenal J Am Soc Nephrol 23: 281–293, 2012

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Figure 8. A functional CRE-like element is located in the mouse MR promoter. (A) HEK293 cells transfected with the pGL4.15-control, +176 trunMR, 2174 trunMR, 2321 trunMR, 21165 mutMR (CRE mutant), or 21165 wtMR promoter luciferase reporter construct were treated with H89 (20 mM) or an equivalent volume of H2O for 24 hours before measurement. The mouse MR transcription start site is indicated by +1. (B) mutMR promoter transcription failed to be activated by hGPR48. ***P,0.001. (C) Chromatin immunoprecipitation assay. MEFGpr48+/+ and MEFGpr48m/m were subjected to fixation, lysis, sonication, and incubation with pCREB antibody. The immunoprecipitated DNA was amplified with specific primers. Primer 1 included the CRE region, whereas primer 2 was located in exon 9 as a negative control. IgG was used as a negative control for pCREB antibody. The relative binding level is indicated as the percentage of input DNA. (D) EMSA to analyze the binding activity of CREB to the mMR promoter in vitro. Lanes 1–4, nuclear extracts from mouse Leydig cells showed binding to the mMR-CRE probe; lanes 5 and 6, anti-CREB antibody (Ab) shifted the binding complex of the mMR-CRE probe; lanes 7–10, positive control with an mSom-CRE probe. Each experiment was performed at least three times. (E) Model illustrating how initial GPR48 activation promotes MR transcription in the kidney, which induces the expression of downstream targets (e.g., aEnaC) and leads to more salt reabsorption and thus hypertension, theoretically. Conversely, inactivation or loss of GPR48 protein could lead to reduced expression of MR and its targets, and, thus, aldosterone resistance occurs. FSK, forskolin; mMR, mouse MR; mSom, mouse somatostatin. Error bars represent SEM. J Am Soc Nephrol 23: 281–293, 2012

GPR48 in Aldosterone Resistance

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insufficiency 48). Moreover, administration of the MR antagonist spironolactone in patients can also increase diluted urine production.49 These findings demonstrate that aldosterone signaling defects could be one origin of urine-concentrating defect. As a result of aldosterone resistance, Gpr48m/m mice showed reduced expression of aENaC and Na/K-ATP 1a and thus impaired Na+ reabsorption in aldosterone-sensitive distal nephron, which led to less water reabsorption and hence more urine production. We further excluded as causative factors any abnormalities in the expression or cellular location of hypothalamic vasopressin or renal vasopressin receptor 2 and aquaporin 2, which are important regulators in water reabsorption.29–31 On the basis of the preceding findings, aldosterone resistance due to MR deficiency can probably be considered one major cause, if not the sole cause, of this urine-concentrating defect in Gpr48m/m mice. In summary, Gpr48m/m mice showed aldosterone resistance and resembled the manifestations of mild adPHA1, which could be explained by the reduced renal MR expression level. This study extends our understanding of GPR48 function in electrolyte homeostasis as well as BP control, and it provides another potential pathogenic mechanism of adPHA1, especially in cases without known MR or ENaC mutations. Further studies are needed to screen Gpr48 gene mutations in these patients to confirm the link between this gene and adPHA1.

CONCISE METHODS Mice

The generation of Gpr48m/m mice has been described in our previous study.27 Three PCR primers were used for genotyping: the common upstream primer A: 59- CCA GTC ACC ACT CTT ACA CAA TGG CTA AC-39; downstream primer B: 59-ATT CCC GTA GGA GATAGC GTC CTA G-39; and downstream primer C: 59-GGT CTT TGA GCA CCA GAG GAC-39. Gpr48m/m mice and their wild-type littermates were age-matched and produced by intercrosses of male and female heterozygous mutant mice throughout the experiments. All procedures were approved by the Animal Care Committee of Shanghai Jiaotong University School of Medicine. In metabolic balance studies, wild-type and Gpr48m/m mice were fed normal chow (0.25% sodium; Shanghai Laboratory Animal Center) or a low-sodium diet (global sodium content , 0.05%; Shanghai Laboratory Animal Center) in metabolic cages (Tecniplast) to assess their water and salt balance. The water deprivation test was conducted for 8 hours, and spot urine samples were collected by bladder massage. Urine osmolality was determined using the freezing point depression method according to standard procedures (Fiske Associates).

Hormone and Biochemical Measurements in Plasma and Urine Blood samples of mice fed normal chow or the low-sodium diet were drawn through retro-orbital bleeding, and urine samples were collected with the metabolic cage for biochemical measurements. Plasma and urine electrolytes were measured using a clinical biochemical 290

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autoanalyzer (Beckman). Blood gases and plasma K+ were measured within 2 minutes of blood collection with a portable blood gas analyzer (Abbott) using the EG7+ cartridge (i-STAT Corp.). RIAs for plasma renin activity (Immunotech) and plasma and urine aldosterone (Diagnostic Systems Laboratories) were performed using appropriate kits according to the manufacturers’ protocols. For plasma corticosterone measurement, mice were anesthetized with 1% pentobarbital sodium (Sigma-Aldrich) before blood collection between 1600 and 1800 hours (lights on 0700 hours). For the fasting condition, mice were deprived of food for 8 hours before blood collection. For the restrained condition, mice were placed in a clear acrylic tube for 30 minutes before blood collection. The RIA for plasma corticosterone (ICN Biomedicals) was performed by following standard procedures.

BP measurement We measured BP as described elsewhere.50 The operating room was kept quiet, and the room temperature was 28°C throughout the experiment. We put the conscious mouse into a properly sized holder with a darkened nose cone to reduce its stress, and then the holder was inserted into a warming chamber of 38°C. When the mouse was quiet for a while and its tail was fully extended, BP was measured with a tail cuff using a BP analyzer (Softron). Data were collected after mice acclimated to the instrument for 3 days. All measurements were completed at a fixed time by a skilled technician.

Tissue Collection, Quantitative PCR Analysis, and Immunoblot Assay Mouse kidneys were collected and stored in liquid nitrogen until use. One microgram of RNA was reverse-transcribed into cDNA using the Reverse Transcription System (Promega), and quantitative PCR was performed to verify gene expression in the kidney with a Roche LightCycler 480 Real-Time PCR System. Primers used in this study are shown in Supplemental Table S2. Immunoblot assay was performed using rabbit anti-MR (Santa Cruz) or monoclonal mouse anti-MR (6G1, a gift from Dr. Celso E. Gomez-Sanchez), rabbit antiGR (Santa Cruz), rabbit anti-aENaC (Santa Cruz), rabbit anti– inwardly rectifying K+ channel (Alomone Labs), mouse monoclonal anti-Na/K-ATPase a1 (Novus Biologicals), mouse monoclonal antirenin (Santa Cruz), rabbit anti-CYP11B1 (Santa Cruz), goat antiHSD3B1 (Santa Cruz), rabbit anti-steroidogenic acute regulatory protein (Santa Cruz), rabbit anti-tubulin (Cell Signaling Technology), and anti–glyceraldehyde 3-phosphate dehydrogenase antibodies (Kangchen Bio-tech). For histologic examination, tissue sections were stained with hematoxylin and eosin by routine methods. For immunohistochemistry, sections were stained with the indicated antibodies: rabbit anti-aquaporin 2 (Santa Cruz), mouse monoclonal anti-MR, and Na/K-ATPase a1. The Vectastain ABC system (Vector Laboratories) was used to detect the primary antibodies.

Cell Culture

MEFs from both Gpr48m/m mice and their wild-type littermates were isolated and established in our laboratory.24 These cells were grown in DMEM supplemented with 15% FBS at 37°C with 5% CO2 . HEK293 and Leydig cells (MA-10), which were used for luciferase

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assays and EMSA, respectively, were cultured in DMEM supplemented with 10% FBS. Chemicals used in this study were all obtained from Sigma and dissolved in appropriate solvents.

Plasmids and Luciferase Reporter Assay The pcDNA3.1-hGPR48 construct containing cDNA for human GPR48 (hGPR48) has been described elsewhere.27 The CRE reporter construct containing four tandem CREs was used to assess cAMPdependent gene transcription (Stratagene). The various lengths of mouse MR promoter constructs from –1165 to +235 were amplified from mouse genomic cDNA and inserted into the pGL4Basic vector (Promega). Mutations were introduced into the MR promoter using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene). All constructs were verified by DNA sequencing. For the luciferase reporter assay, HEK293 or MEFs seeded in 24-well plates were co-transfected with the indicated MR promoter constructs, pRL-TK (expressing Renilla luciferase) (Promega) and pcDNA3.1 or pcDNA3.1-hGPR48, followed by lysis and luciferase activity measurement using the Dual-Luciferase Reporter Assay System (Promega).

Chromatin Immunoprecipitation Assay To investigate the interaction of pCREB with the MR promoter, chromatin immunoprecipitation assay was performed using a commercial kit (Upstate Biotechnology) according to the manufacturer’s instructions. In brief, wild-type and Gpr48-mutant MEFs were treated with 1% formaldehyde to cross-link the proteins and DNA, followed by sonication in an ultrasound bath on ice. The chromatin was incubated with rabbit anti-pCREB antibody (Cell Signaling Technology) or rabbit IgG overnight at 4°C. The immunoprecipitated DNA fragments containing or lacking a CRE sequence were detected using quantitative PCR with primers shown in Supplemental Table S2.

EMSA Nuclear proteins were extracted from mouse Leydig cells using nuclear and cytoplasmic extraction reagents (Pierce Biotechnology). The DNA probe was derived from a 29-bp DNA fragment covering the CRE region in the mouse MR promoter, and a mutant probe contained eight nucleotide substitutions within the CRE sequence (Supplemental Table S2). Oligonucleotides were labeled with biotin (Invitrogen), and unlabeled probes were used to compete for the specific binding. EMSA was performed using the Lightshift Chemiluminescent EMSA kit (Pierce Biotechnology). For the DNA supershift assay, rabbit anti-CREB antibody (Cell Signaling Technology) was incubated on ice before being mixed with the labeled probes. As a positive control, the consensus CRE of the mouse somatostatin promoter was prepared in parallel.33 Electrophoresis binding reactions, transfer, cross-linking, and detection were performed according to standard procedures.

Statistical Analyses

All data are presented as mean 6 SEM. The data were analyzed using a two-tailed t test for Gpr48m/m mice versus wild-type mice. P,0.05 was considered to indicate a statistically significant difference. J Am Soc Nephrol 23: 281–293, 2012

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ACKNOWLEDGMENTS We are grateful to Imelda Lee for critical revision for the manuscript, Professor Celso E. Gomez-Sanchez (University of Mississippi Medical Center) for kindly providing MR antibodies, and Yan Ge and Jia Xu for immunohistochemical staining. This study is supported by the National Natural Science Foundation of China (Grants 81030011 and 30725037).

DISCLOSURE None.

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