Deletion of the pH sensor GPR4 decreases renal acid excretion

Abstract

Proton receptors are G protein-coupled receptors that accept protons as ligands and function as pH sensors. One of the proton receptors, GPR4, is relatively abundant in the kidney, but its potential role in acid-base homeostasis is unknown. In this study, we examined the distribution of GPR4 in the kidney, its function in kidney epithelial cells, and the effects of its deletion on acid-base homeostasis. We observed GPR4 expression in the kidney cortex, in the outer and inner medulla, in isolated kidney collecting ducts, and in cultured outer and inner medullary collecting duct cells (mOMCD1 and mIMCD3). Cultured mOMCD1 cells exhibited pH-dependent accumulation of intracellular cAMP, characteristic of GPR4 activation; GPR4 knockdown attenuated this accumulation. In vivo, deletion of GPR4 decreased net acid secretion by the kidney and resulted in a nongap metabolic acidosis, indicating that GPR4 is required to maintain acid-base homeostasis. Collectively, these findings suggest that GPR4 is a pH sensor with an important role in regulating acid secretion in the kidney collecting duct.

Figure 1.

GPR4 mRNA is expressed in all three kidney zones with higher levels of expression in the medulla than in the cortex. (A) Representative gel image, demonstrating GPR4 real-time RT-PCR end products (218 bp) in the mouse kidney cortex, outer medulla, and inner medulla. (B) Corresponding real-time RT-PCR growth curves for GPR4 and β-actin mRNA in the kidney zones. (C) Ratios of GPR4 mRNA/β-actin mRNA in the three kidney zones. GPR4 mRNA expression in the mouse kidney is 1.8 times higher in the outer medulla and 2.9 times higher in the inner medulla than in the cortex.

Figure 2.

GPR4 mRNA is expressed in isolated collecting ducts. (A) Representative gel image of GPR4 RT-PCR products, which were abundant in CCD (cortical collecting duct), could be detected in TAL (thick ascending limb), and were virtually absent from PT (proximal tubule). The specificity of the bands was confirmed by sequencing. The lower panel shows β-actin expression. Unlike GPR4 mRNA, we could efficiently amplify β-actin from the proximal tubule. (B) GPR4 could not be amplified from the nephron segments isolated from GPR4 knockout mice (upper panel), whereas it was amplified from the wild-type kidney tissue (the far right line), which served as a positive control. In addition, we were able to amplify β-actin from all nephron segments (middle panel). The lower panel shows that we could also positively identify the collecting duct of GPR4 knockouts by amplifying a gene specific to the collecting duct (aquaporin 2-AQP2), from the collecting duct, but not from the thick ascending limb.

Figure 3.

Cultured collecting duct cells derived from the mouse medulla exhibit abundant endogenous expression of GPR4. (A) GPR4 is detected as a 218-bp band in the mouse outer medullary (mOMCD1) and inner medullary (mIMCD3) collecting duct cell lines. (B) Corresponding real-time RT-PCR growth curves for GPR4 mRNA, illustrating the higher expression of GPR4 mRNA in mIMCD3 compared with mOMCD1 cells, similar to the mouse kidney tissue.

Figure 4.

The magnitude of pH-dependent cAMP response in mOMCD1 cells decreases with a decreased level of GPR4 expression. Intracellular cAMP was measured by column chromatography in the presence of unspecific phosphodiesterase inhibitor IBMX in mOMCD1 cells exposed to buffers of different pH. To ensure that GPR4 mediated pH-dependent changes in intracellular cAMP concentration, we measured cAMP in nontransfected cells and cells transfected with control or anti-GPR4 siRNA. (A) Real-time RT-PCR analysis of nontransfected mOMCD1 cells and mOMCD1 cells transiently transfected with synthetic anti-GPR4 siRNA or with the control siRNA that does not match any known sequence. The analysis demonstrated that GPR4 expression decreased approximately 50% compared with mOMCD1 cells transfected with control siRNA or nontransfected cells. (B) mOMCD1 cells incubated in buffers of different pH accumulated cAMP in a pH-dependent manner (full triangles) and so did the cells transiently transfected with the control siRNA. Correlating well with the decreased GPR4 mRNA, pH-dependent accumulation of cAMP decreased in the cells transiently transfected with synthetic anti-GPR4 siRNA (empty circles). These experiments were done in four different batches of mOMCD1 cells.

Figure 5.

Proton receptor knockout mice (GPR4^−/−) exhibit decreased net acid excretion and attenuated response to acid challenge. GPR4 knockouts (GPR4^−/−) and their wild types (GPR4^+/+) were placed in metabolic cages and acid-loaded with NH₄Cl for 4 days. Net acid excretion was measured in the urine collected over 24 hours under oil. Both GPR4^+/+ and GPR4^−/− increased acid secretion after acid challenge; however, this increase was blunted in GPR4^−/−. There were no differences in food, protein, water intake, and body weight between GPR4^−/− and GPR4^+/+ during the experiment (Supplement 4).

Figure 6.

Proton receptor knockout mice (GPR4^−/−) have slightly lower urine phosphate compared to wild-type mice (GPR4^+/+) at baseline and after the first day of acid loading. Because urine titratable acidity primarily represents urine phosphate, we also measured phosphate in 24-hour urine samples collected from GPR4^−/− and GPR4^+/+ in metabolic cages under oil before and during NH₄Cl loading.

Figure 7.

Increased urine calcium excretion in response to increased acid load is more pronounced in proton receptor knockout mice (GPR4^−/−) than the wild-type mice (GPR^+/+). Urine calcium was measured in 24-hour urine samples collected from GPR4^−/− and GPR4^+/+ in metabolic cages under oil before and during NH₄Cl loading. Hypercalciuria elicited by acid loading was significantly more pronounced in GPR4^−/− (n = 5) than in GPR^+/+ (n = 5) (*P < 0.05).