BASIC RESEARCH www.jasn.org 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 non- canonical 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 Na+ reabsorption by activated ENaC also enhances K+ excretion through the luminal potassium chan- Aldosterone plays critical roles in the control of nel, leading to kaliuresis in the collecting duct.4 salt homeostasis and BP through stimulating Na+ Loss-of-function mutations in the MR or ENaC reabsorption and K+ secretion.1 Upon binding to subunitgenesaccountforpseudohypoaldosteronism mineralocorticoid receptor (MR), the hormone– type 1 (PHA1),5,6 which is the principal form of aldo- receptor complex translocates into the nucleus sterone resistance and shows an autosomal-dominant and interacts with glucocorticoid receptor response element in specific promoter regions of the target Received April 7, 2011. Accepted October 6, 2011. genes, thereby activating their transcription, such Published online ahead of print. Publication date available at as a-subunit of the amiloride-sensitive epithelial www.jasn.org. sodium channel (aEnaC) in the aldosterone- Correspondence: Dr. Guang Ning, Shanghai Clinical Center for 2 sensitive distal nephron. The functional relevance Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai of ENaC to aldosterone-dependent Na+ reabsorp- Jiaotong University School of Medicine, 197 Ruijin 2nd Road, tion, and thus to the regulation of extracellular fluid Shanghai 200025, China. Email: [email protected] volume and BP, is well established.3 Promotion of Copyright © 2012 by the American Society of Nephrology J Am Soc Nephrol 23: 281–293, 2012 ISSN : 1046-6673/2302-281 281 BASIC RESEARCH www.jasn.org (adPHA1; OMIM #177735) and a recessive (arPHA1; OMIM trap–mutated Gpr48 ES cells into C57BL/6 blastocysts.27 #264350) transmission.7 First described in 1958, this rare con- An insertion of the trap vector into intron 1 of the Gpr48 dition features renal resistance to aldosterone action.8 Early in gene resulted in approximately 90% knockdown efficiency infancy, patients with adPHA1 present with dehydration and in the kidney and adrenal gland of adult Gpr48m/m mice (Fig- failure to thrive associated with salt wasting, hypotension, hy- ure 1, A–C). We observed that approximately half of Gpr48m/m perkalemia, and metabolic acidosis, despite increased plasma newborns died within 28 hours after birth, but no further aldosterone levels.6 These patients can improve with age, and deaths occurred in the following 20 hours (Figure 1D). some adult patients are usually asymptomatic and have fewer abnormal biochemical findings (e.g., only lifelong increases Increases in Water Intake and Urine Volume with in aldosterone or hyperkalemia).9,10 To date, approximately Partially Impaired Urine-Concentrating Ability 50 distinct mutations in the human MR gene and approxi- in Gpr48m/m Mice mately 20 mutations in ENaC genes responsible for PHA1 We explored the potential changes of water balance in Gpr48m/m have been described.11,12 However, some patients, especially mice using metabolic cages and found that Gpr48m/m mice those with sporadic PHA1, do not have genetic abnormalities showed a dramatic increase in 24-hour water and food intake in MR or ENaC.6,10,13–15 Because PHA1 is life-threatening and at 16 and 24 weeks compared with wild-type mice (Figure 2, many probands may be missed as a result of early death, addi- A and B). Their urine volume was also significantly increased tional genetic mechanisms might participate in its pathogene- during this time (Figure 2C). Meanwhile, urine osmolality sis. According to recent studies, aldosterone resistance may also declined markedly (Figure 2D). Female mice showed overall be associated with a reduction in MR expression, probably similar phenotypes (Supplemental Figure S1). We next per- mediated by transcriptional mechanisms.16–19 formed a water deprivation test to assess the urine-concentrating Gprotein–coupled receptor 48 (GPR48/ LGR4) belongs to the G protein–coupled receptor superfamily. It has recently been re- ported 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 Gpr48- mutant mice display early neonatal lethality.23 Our group and others have demonstrated that Gpr48 deficiency results in impaired func- tion of the male reproductive tract through downregulation of estrogen receptor a ex- pression.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 hypo- morphic mutant mice display a significant aldosterone resistance, which mimics a mild state of adPHA1 disease. We also dem- onstrate that GPR48 regulates MR expres- sion through the cAMP/protein kinase A (PKA) pathway. This study elucidates the potential role of GPR48 in electrolyte ho- meostasis and aldosterone resistance. 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 RESULTS 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 corre- sponding cDNA length is 471 bp, and the PCR products were verified to be wild-type Gpr48 Homozygous Mutant Mice Gpr48 by sequencing. (C) Approximately 10% of Gpr48 transcripts remained in the adrenal Show Hypomorphic Features gland of Gpr48m/m mice at age 16 weeks according to qualitative PCR (n=12). 36B4 was We obtained Gpr48 hypomorphic mutant used as internal control. (D) Survival curve of neonatal wild-type (Gpr48+/+)(n=29) and (Gpr48m/m) mice by microinjecting gene Gpr48m/m (n=23) mice within 48 hours after birth. Error bars represent SEM. ***P,0.001. 282 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 281–293, 2012 www.jasn.org BASIC RESEARCH between the two genotypes, nor did pH, 2 HCO3 , or base excess (Table 1 and Sup- plemental Table S1). We next examined plasma mineralocor- ticoid levels, which play fundamental roles in regulating water and salt homeostasis. Surprisingly, plasma aldosterone was sig- nificantly 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 pre- serving Na+ and water, suggesting aldoste- rone 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 en- zymes in the adrenal gland and the plasma levels of adrenergic hormones (Supple- mental Figure S3, B–E), suggesting that the phenotypes observed in Gpr48m/m Figure 2. Adult Gpr48m/m mice show polydipsia, polyphagia, severe water loss, and re- mice are not secondary to defects in the duced urine osmolality. Male Gpr48m/m mice at age 16 and 24 weeks displayed increased adrenal gland. Taken together, these data (A) water intake, (B) food intake, and (C) urine volume but (D) decreased urine osmolality suggest that Gpr48m/m mice displayed so- compared with their age-matched wild-type littermates. At 8 weeks, n=4; at 16 weeks, +/+ dium and water loss as well as hyperkale- n=9; at 24 weeks, n=10. White bar, wild-type mice (Gpr48 ); black bar, Gpr48-mutant mia, despite elevated aldosterone, thus mice (Gpr48m/m). Error bars represent SEM. **P,0.01; ***P,0.001. BW, body weight. resembling the mild state of adPHA1.6,32 ability of Gpr48m/m mice.