Introduction: G protein-coupled bile acid receptor (TGR5), the first G protein-coupled receptor for bile acids identified, is capable of activating a variety of intracellular signaling pathways after interacting with bile acids. TGR5 plays an important role in multiple physiological processes and is considered to be a potential target for the treatment of various metabolic diseases, including type 2 diabetes. Evidence has emerged that genetic deletion of TGR5 results in an increase in basal urine output, suggesting that it may play a critical role in renal water and salt reabsorption. The present study aims to elucidate the effect and mechanism of TGR5 activation on urine concentration. Methods: Mice were treated with TGR5 agonists (LCA and INT-777) for 3 days. The 24-h urine of mice was collected and analyzed for urine biochemical parameters. The mRNA expressions were detected by real-time PCR, and the protein expressions were detected by western blot. Immunohistochemistry and immunofluorescence were performed to examine the cellular location of proteins. The cultured primary medullary collecting duct cells were pretreated with H89 (a PKA inhibitor) for 1 h, followed by 12-h treatment of LCA and INT-777. Luciferase reporter assays were used to detect the effect of CREB on the gene transcription of AQPs. Gel electrophoretic mobility shift assays were used to analyze DNA–protein interactions. Results: Treatment of mice with the TGR5 agonist LCA and INT-777 markedly reduced urine output and increased urine osmolality, accompanied by a marked increase in AQP2 and AQP3 protein expression and membrane translocation. In cultured primary medullary collecting duct cells, LCA and INT-777 dose-dependently upregulated AQP2 and AQP3 expression in a cAMP/PKA-dependent manner. Mechanistically, both AQP2 and AQP3 gene promoter contains a putative CREB-binding site, which can be bound and activated by CREB as assessed by both gene promoter-driven luciferase and gel shift assays. Conclusion: Collectively, our findings demonstrate that activation of TGR5 can promote urine concentration by upregulation of AQP2 and AQP3 expression in renal collecting ducts. TGR5 may represent an attractive target for the treatment of patients with urine concentration defect.

The kidney is a key organ in maintaining water and electrolyte homeostasis. Each day approximately 180 L of fluid are filtered into the Bowman's capsule to form the primary urine. About 99% of the primary urine is reabsorbed along various renal tubules, with only 1–2 L of urine excreted. Aquaporins (AQPs) play a crucial role in water reabsorption in the kidneys. Thus far, 8 aquaporins have been found to be expressed in the kidney including AQP1, AQP2, AQP3, AQP4, AQP5, AQP6, AQP7, and AQP11 [1]. Among them, AQP1 is expressed on both the apical and basolateral membrane surfaces of the proximal tubule and the descending thin limbs of the loop of Henle and in the vasa recta [2], responsible for about 80% of the water reabsorption of the primary urine. AQP2 is localized on the apical membrane of principal cells of the collecting ducts, where its expression is mainly regulated by vasopressin (AVP) [3], while AQP3 is constitutively expressed on the basolateral membrane of principal cells of the cortical and outer medullary collecting ducts, where its expression is regulated by AVP and aldosterone [4]. Similar to AQP3, AQP4 is distributed on the basolateral membrane of the principal cells. However, AQP4 is reported to be mainly regulated by protein kinase C (PKC) and dopamine [5]. Approximately 15% of the primary urine is reabsorbed by apical AQP2 and basolateral AQP3 and AQP4 in the collecting ducts. Mice lacking AQP1, AQP2, AQP3, or AQP4 exhibit a robust polyuria phenotype [6].

As a metabolite of cholesterol, bile acid not only promotes transportation and reabsorption of lipids and vitamins in the small intestine [7‒9], but also plays an important role in the pathogenesis of various diseases via binding and activating two receptors, i.e., the nuclear receptor FXR (farnesoid X receptor) and the membrane G protein-coupled receptor TGR5 (Takeda-G-protein-receptor-5). Increasing evidence has shown that bile acid receptors are critical in regulating renal water and sodium reabsorption. We have previously reported that FXR plays an important role in the maintenance of water homeostasis, and the activation of FXR increases urinary concentrating capacity through upregulating AQP2 expression at the transcriptional level [10]. A recent study has also shown that TGR5 can improve LiCl-induced diabetes insipidus, presumably related to the upregulation of AQP2 expression [11]. However, the effect and underlying mechanism of TGR5 on AQP2, AQP3, and AQP4 expression in the renal collecting duct remain incompletely understood.

In the present study, we reported that treatment of mice with both endogenous TGR5 agonist LCA and synthetic TGR5 agonist INT-777 can promote urine concentration with a marked increase in the expression of AQP2 and AQP3, but not AQP4. TGR5 activation by LCA and INT-777 dose-dependently increases AQP2 and AQP3 expression in cultured primary collecting duct cells via the cAMP-PKA-CREB pathway.

Animal Treatment

Male wild-type C57BL/6 mice (8–12 wk) were purchased from Liaoning Changsheng Company. All animals were cage-housed and maintained in a temperature-controlled room on a 12-h light-dark cycle with free access to water. Mice were divided into 3 groups. (1) Control group; the mice were intraperitoneally injected with vehicle (DMSO). (2) LCA group; the mice were intraperitoneally injected with LCA at 0.125 mg/g body weight (BW) for 3 days. (3) INT-777 group; the mice were intraperitoneally injected with INT-777 at 30 mg/kg BW dissolved in DMSO for 3 days. All animal experiments in the current study were approved by the Ethical Committee of Dalian Medical University and conformed to international guidelines for animal usage in research.

Metabolic Cage Experiment

All animals were placed in metabolic cages to pre-acclimatize to the environment for 24 h. Throughout the study, each mouse had free access to food and water, and then the voided urine was collected continuously for 24 h. At the end of the experiment, total water intake, food intake, and urine output were measured. On the final day, mice were euthanized after 24-h urine collection, and the kidneys were harvested for further analysis.

Urine Collection and Osmolality Analysis

BW, urine output, and food and water intake were measured. 24-h urine samples were collected at the end of treatment. Urine samples were centrifuged at 3,000 g at 4°C for 10 min, and the supernatants were collected. Urine osmolality was determined by freezing-point depression. Urine ion concentration was detected by a urine ion analyzer.

Western Blot Analysis

Protein from mouse kidneys or renal cell lines was extracted using a radio immunoprecipitation assay buffer containing protease or phosphatase inhibitor cocktail (Sigma). Protein concentrations were measured by a BCA assay (Thermo Fisher Scientific). Total protein (30∼60 μg) mixed with 6× loading buffer was separated on a 10% SDS-PAGE gel and transferred to PVDF membrane. Membranes were then incubated for 1 h in 5% milk (1× TBST) to block unspecific binding. Immunostaining was performed overnight at 4°C using the following primary antibodies against AQP2 (ab199975), AQP3 (ab125219), AQP4 (sc-32739), p-CREB (CST#9198), CREB (CST#9197), and β-actin (Abclonal #AC004). After being washed for 5 min with TBST buffer for 5 times, the membrane was then incubated with 1:10,000 HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 h at room temperature. The membrane was washed and exposed to the SuperLumia ECL Plus HRP Substrate Kit (K22030, Abbkine), then the immune complexes were detected by Tanon-5200 (Tanon, Shanghai, China), and the densitometry was performed with ImageJ software.

Immunofluorescence

Primary cultured rat inner medullary renal collecting duct (IMCD) cells were grown to proper confluence and fixed in 4% paraformaldehyde for 15 min on a rocking platform. After three 5-min washes with PBS, the cells were permeabilized in 0.5% Triton X-100 in PBS for 10 min at room temperature. After being blocked by 5% BSA in PBS for 60 min, the cells were incubated with primary antibodies at 4°C overnight and then immunostained with the following antibodies: anti-AQP2, anti-AQP3, and anti-AQP4. After being washed, the cells were incubated with appropriate DyLight 488 (green) secondary antibodies at 37°C for 30 min. The nuclei were stained with DAPI. Images were obtained through a confocal microscope.

Immunohistochemistry

Paraffin-embedded kidney sections (3 μm thickness) were deparaffinized and hydrated. Heat-induced epitope retrieval was employed. The sections were then treated using 0.3% H2O2 at room temperature for 10 min to quench endogenous peroxidase. Tissue sections were blocked in 5% bovine serum albumin, followed by incubation with primary antibody overnight at 4°C. After rinsing with PBS, the sections were incubated with biotin-conjugated secondary antibody at 37°C for 30 min and then HRP-conjugated streptavidin at 37°C for 30 min. Finally, reactions were developed with the Peroxidase Stain DAB kit to visualize the positive staining. PBS was used to replace the primary antibody for the negative control.

Real-Time PCR

Total RNA from tissues and cells was extracted using TRIZOL reagent (Biotek) and then reverse-transcribed to cDNA by using the RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, USA) according to the manufacturer’s instructions. Real-time PCR was carried out by using cDNA as a template in the PCR reaction with SYBR Green Mix (Bio-Rad, Hercules, CA, USA). The reaction conditions included 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 59°C for 30 s, 72°C for 30 s, with a final extension at 72°C for 5 min β-actin was used as an internal control. Quantitative values were obtained as the threshold PCR cycle number (Ct). Each sample was measured in duplicate or triplicate in each experiment. The primer pairs used for amplifying mouse and rat genes interested were listed in online supplementary Table1 (for all online suppl. material, see https://doi.org/10.1159/000538107).

Preparation and Treatment of Primary IMCD Cells

The rats were anesthetized by CO2, and renal inner medulla samples were taken and cut into small pieces. Tissues were digested in a solution with 0.1% hyaluronidase and 0.2% collagenase for 40 min. The cells were then centrifuged, washed three times, and resuspended in a fully supplemented medium. The cells were then seeded into culture dishes. Since IMCD cells are resided in a high osmotic environment in renal medulla, Dulbecco’s modified Eagle’s medium (DMEM) was adjusted to 600 mOsmol/L by adding 100 mm NaCl and 100 mm urea, which was used to establish the growth conditions with preferential selectivity for the IMCD cells. The cells were finally cultured in DMEM/F12 medium supplemented with 10% bovine serum albumin, 100 μg/mL streptomycin, and 100 U/mL penicillin at 37°C in a humid atmosphere with 5% CO2. Cells were grown to a confluence of 80% and stimulated with LCA and INT-777. After the treatment, the cells were collected for RT-PCR analysis or western blot analysis.

siRNA Transfection

Rat primary inner medullary collecting duct cells were cultured until 50–70% confluence. The cells were then transfected with the TGR5 siRNA or negative control (NC) siRNA (100 nm) in serum-free Opti-MEM. After 6 h, the medium was replaced by a fresh complete growth medium containing serum and incubated for an additional 48 h. The siRNA oligonucleotides for TGR5 were designed and synthesized by GenePharma (Shanghai, China). The sequences were as follows: (1) siRNA-TGR5: sense 5′-GGG​CCU​GUA​ACU​CUG​UUA​UTT-3′, antisense 5′-AUA​ACA​GAG​UUA​CAG​GCC​CTT′; (2) siRNA-NC: sense 5′-UUC UCC GAA CGU GUC ACG UTT-3′, antisense 5′-ACG UGA CAC GUU CGG AGA ATT-3′.

Measurement of cAMP Levels

To measure the intracellular concentration of cAMP, primary rat IMCD cell suspensions were treated with LCA (5 μm), INT-777 (5 μm), or vehicle for 6 h. After treatment, rat IMCD cells were harvested with 0.1 M HCl and centrifuged at 600 g for 5 min. The supernatants were directly assayed for cAMP concentrations by an cAMP-ELISA assay (Bioyears), normalized to total protein, and expressed as pmol of cAMP/mg of protein.

Luciferase Assay

A 510-bp fragment of mouse AQP2 gene promoter (−395 to +115) and a 708-bp fragment of mouse AQP3 gene promoter (−625 to +83) were amplified by PCR using mouse tail DNA. The sequences of primers used are as follows: (1) AQP2: forward primer 5′-CGC​CCA​CAT​TTC​CTC​ACA​GTT-3′, reverse 5′-GAC​CGG​AGT​TCC​CAC​ATG​CT-3′; (2) AQP3: forward primer 5′-GGG​GTA​CCC​CGT​CGA​CCC​ATG​GCG​CGA​GGC-3′, reverse 5′-TCC​CCC​GGG​GGA​CAA​GGG​GGA​AGG​TCC​ACA​AG-3′. PCR products were then subcloned in the pGL3-control vector to construct AQP2 gene promoter- and AQP3 gene promoter-driven luciferase reporters. All plasmids were confirmed by DNA sequencing. 293T cells were grown to 60–70% confluence and transfected with an empty pGL3 vector and the AQP2 gene promoter- or AQP3 gene promoter-driven luciferase reporter. The luciferase assay was performed using the Promega Dual Firefly and Renilla Luciferase Assay Kit. After 48 h transfection, the lysis products were collected using the supplied lysis buffer and plated in duplicate on 96-well plates. Each well was injected with 100 μL of luciferase assay reagent or Renilla assay reagent, and fluorescence was recorded using a microplate reader. The relative luminescence units per well were then calculated by dividing the luciferase reagent reading by the Renilla assay reagent. The experiment was repeated three times.

Electrophoretic Mobility Shift Assay

The nuclear extracts were prepared as shown in the NE-PER Nuclear and Cytoplasmic Extraction Kit (#78833, Thermo Scientific) and stored at −80°C. The oligonucleotide probes used in the electrophoretic mobility shift assay (EMSA) were based on computer-assisted search using promoter scanning software and labeled with the Biotin 3 ′End DNA labeling Kit (#89818, Thermo Scientific). According to the manufacturer's protocol, EMSA is performed using the LightShift Chemiluminescent EMSA Kit (#20148, Thermo Scientific). Each binding mixture (20 μL) for EMSA contained nuclear extract (10 µg protein), 20 fmol of biotin end-labeled double-stranded probe, 1 μg of poly-dI/dC, and 2 μL of 10× reaction buffer. It was incubated at room temperature for 20 min. The protein-DNA complexes were then separated on 6% nondenaturing polyacrylamide gels and transferred to a nylon membrane. The nylon membrane was UV cross-linked, detected by streptavidin-HRP conjugate, and incubated with chemiluminescent substrate for chemiluminescence detection. For the competition experiment, unlabeled rival oligonucleotides were preincubated with labeled probes at a 100-fold excess.

Statistical Analyses

Statistical analyses were performed using GraphPad Prism v7 (GraphPad Software, San Diego, CA, USA). Two-tailed t test was used for comparison between two groups. One-way ANOVA, followed by a Tukey multiple comparisons test, was used to analyze the differences between means. p value of <0.05 was considered significant. Error bars show ±SEM.

TGR5 Activation Increases Urine Concentration

In order to clarify the effect of TGR5 activation on urine concentration, we first chose male 8-week-old wild-type (WT) mice on the C57Bl/6 background and divided them into three groups, i.e., Control, LCA and INT-777 groups. 24-h urine was recorded and used for analyzing urine biochemical parameters. Results showed that LCA or INT-777 treatment for 3 days significantly decreased urine output (Fig. 1a), with a significant increase in urine osmolality (Fig. 1b). Little change in daily food and water intake and 24-h urinary excretion of Na+, K+, and Ca+ was observed after LCA and INT-777 treatment (online suppl. Fig. S1a, b; S2a–c). These findings demonstrate that activation of TGR5 can increase urine concentration ability in mice.

Fig. 1.

TGR5 activation increases urine concentration ability. Male mice on a C57Bl/6 background were intraperitoneally administrated with LCA and INT-777 for 3 days. The daily urine samples were collected at the end of treatment. a The 24-h urine volume was significantly decreased after a 3-day LCA and INT-777 treatment. b The urine osmolality was significantly increased following LCA and INT-777 treatment for 3 days. Data represent the mean ± SEM. *p < 0.05, **p < 0.01 compared to the control group, n = 8.

Fig. 1.

TGR5 activation increases urine concentration ability. Male mice on a C57Bl/6 background were intraperitoneally administrated with LCA and INT-777 for 3 days. The daily urine samples were collected at the end of treatment. a The 24-h urine volume was significantly decreased after a 3-day LCA and INT-777 treatment. b The urine osmolality was significantly increased following LCA and INT-777 treatment for 3 days. Data represent the mean ± SEM. *p < 0.05, **p < 0.01 compared to the control group, n = 8.

Close modal

TGR5 Activation Induces AQP2 and AQP3 Expression in Renal Medulla

To determine the effects of TGR5 activation on the expression of renal aquaporins, we measured AQP2, AQP3, and AQP4 expression at both mRNA and protein levels as assessed by real-time PCR and western blot, respectively. The results showed that both LCA and INT-777 treatment significantly upregulated the mRNA and protein expression of AQP2 and AQP3, with little effect on AQP4 (Fig. 2a–c). These results suggest that LCA and INT-777 treatment promote urine concentration, possibly by upregulating the expression of AQP2 and AQP3 in the renal medulla.

Fig. 2.

TGR5 activation increases the expression of AQP2 and AQP3 in the renal inner medulla. Renal medullary RNA and protein were extracted after LCA and INT-777 treatment for 3 days. a Quantitative PCR analysis showing the mRNA levels of AQP2, AQP3, and AQP4 in the renal medulla. b Western blot analysis showing the protein levels of AQP2, AQP3, and AQP4 in Control, LCA, and INT-777 groups. c Semi-quantification of western blot assays. Data represent the mean ± SEM. *p < 0.05, **p < 0.01 compared to the Control group, n = 4. Note: Little effect of LCA and INT-777 on AQP4 mRNA and protein expression was observed.

Fig. 2.

TGR5 activation increases the expression of AQP2 and AQP3 in the renal inner medulla. Renal medullary RNA and protein were extracted after LCA and INT-777 treatment for 3 days. a Quantitative PCR analysis showing the mRNA levels of AQP2, AQP3, and AQP4 in the renal medulla. b Western blot analysis showing the protein levels of AQP2, AQP3, and AQP4 in Control, LCA, and INT-777 groups. c Semi-quantification of western blot assays. Data represent the mean ± SEM. *p < 0.05, **p < 0.01 compared to the Control group, n = 4. Note: Little effect of LCA and INT-777 on AQP4 mRNA and protein expression was observed.

Close modal

TGR5 Activation Promotes Membrane Translocation of AQP2 and AQP3 Protein in Renal Collecting Duct Cells

To explore the expression and localization of AQP2 and AQP3 proteins in the renal medulla following TGR5 activation, immunohistochemical staining and immunofluorescence analysis were performed. The results showed that under normal conditions, AQP2 was selectively expressed on the apical membrane, while AQP3 and AQP4 were mainly localized on the basolateral membrane of the medullary collecting ducts (Fig. 3a–f; online suppl. Fig. S3a, b). After LCA and INT-777 treatment, total expression levels of AQP2 and AQP3 proteins were markedly induced. In addition, activation of TGR5 by LCA and INT-777 significantly increased the apical expression of AQP2 and the basolateral expression of AQP3 (Fig. 3a, b), with no significant impact on AQP4 in renal medullary collecting ducts (online suppl. Fig. S3a, b). These findings demonstrate that TGR5 activation not only induces AQP2 and AQP3 expression but also increases their membrane translocation in renal medullary collecting ducts.

Fig. 3.

TGR5 activation by LCA and INT-777 induces AQP2 and AQP3 protein membrane translocation in mouse renal inner medullary collecting ducts. a Immunohistochemical assay showing that treatment of mice with LCA and INT-777 markedly increased AQP2 and AQP3 protein expression and their membrane translocation renal inner medullary. b Immunofluorescence study showing that treatment of mice with LCA and INT-777 markedly increased AQP2 and AQP3 protein expression and their membrane translocation in renal inner medullary. c, d Semi-quantification of mean integrated optical density of AQP2 and AQP3 in (a). e, f Semi-quantification of mean integrated optical density of AQP2 and AQP3 in (b). Note: Apical AQP2 protein expression and basolateral AQP3 protein expression were dramatically increased after LCA and INT-777 treatment.

Fig. 3.

TGR5 activation by LCA and INT-777 induces AQP2 and AQP3 protein membrane translocation in mouse renal inner medullary collecting ducts. a Immunohistochemical assay showing that treatment of mice with LCA and INT-777 markedly increased AQP2 and AQP3 protein expression and their membrane translocation renal inner medullary. b Immunofluorescence study showing that treatment of mice with LCA and INT-777 markedly increased AQP2 and AQP3 protein expression and their membrane translocation in renal inner medullary. c, d Semi-quantification of mean integrated optical density of AQP2 and AQP3 in (a). e, f Semi-quantification of mean integrated optical density of AQP2 and AQP3 in (b). Note: Apical AQP2 protein expression and basolateral AQP3 protein expression were dramatically increased after LCA and INT-777 treatment.

Close modal

LCA- and INT-777-Induced AQP2 and AQP3 Expression Is Dependent on TGR5

To test whether LCA- and INT-777-induced AQP2 and AQP3 expression is dependent on TGR5, we first utilized a siRNA-based approach to knockdown TGR5 expression in primary cultured rat collecting duct cells (online suppl. Fig. S4a, b) and then treated the cells with LCA (5 μm) and INT-777 (5 μm) for 12 h. By using quantitative PCR and western blot assays, we found that TGR5 knockdown almost completely abolished LCA- and INT-777-induced AQP2 and AQP3 expression at both mRNA and protein levels (Fig. 4). These results demonstrate that LCA- and INT-777-induced AQP2 and AQP3 expression is dependent on the activation of TGR5.

Fig. 4.

LCA and INT-777 promote the expression of AQP2 and AQP3 in a TGR5-dependent manner. Rat IMCDs were transfected with TGR5 siRNA or negative control for 36 h and then stimulated with 5 μm LCA and INT-777 for 12 h. The mRNA expression of AQP2, AQP3, and AQP4 was measured by quantitative PCR. a–c Knockdown of TGR5 abolished the effect of LCA on AQP2 and AQP3 expression. LCA treatment significantly upregulated AQP2 (a) and AQP3 (b) mRNA expression, with little effect on AQP4 (c). LCA-induced AQP2 and AQP3 mRNA expression were completely abolished by the knockdown of TGR5. d–f Knockdown of TGR5 abolished the effect of INT-777 on AQP2 and AQP3 expression. INT-777 treatment significantly upregulated AQP2 (d) and AQP3 (e) mRNA expression, with little effect on AQP4 (f). INT-777-induced AQP2 and AQP3 mRNA expression were completely abolished by the knockdown of TGR5. g, h The protein levels of AQP2, AQP3, and AQP4 were measured by Western blot assay. LCA (g) and INT-777 (h) treatment induced AQP2 and AQP3 protein expression, which was markedly attenuated by the knockdown of TGR5. Data represent the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the Control group; #p < 0.05, ##p < 0.01, and ###p < 0.001 compared to the LCA or INT-777 group, n = 3.

Fig. 4.

LCA and INT-777 promote the expression of AQP2 and AQP3 in a TGR5-dependent manner. Rat IMCDs were transfected with TGR5 siRNA or negative control for 36 h and then stimulated with 5 μm LCA and INT-777 for 12 h. The mRNA expression of AQP2, AQP3, and AQP4 was measured by quantitative PCR. a–c Knockdown of TGR5 abolished the effect of LCA on AQP2 and AQP3 expression. LCA treatment significantly upregulated AQP2 (a) and AQP3 (b) mRNA expression, with little effect on AQP4 (c). LCA-induced AQP2 and AQP3 mRNA expression were completely abolished by the knockdown of TGR5. d–f Knockdown of TGR5 abolished the effect of INT-777 on AQP2 and AQP3 expression. INT-777 treatment significantly upregulated AQP2 (d) and AQP3 (e) mRNA expression, with little effect on AQP4 (f). INT-777-induced AQP2 and AQP3 mRNA expression were completely abolished by the knockdown of TGR5. g, h The protein levels of AQP2, AQP3, and AQP4 were measured by Western blot assay. LCA (g) and INT-777 (h) treatment induced AQP2 and AQP3 protein expression, which was markedly attenuated by the knockdown of TGR5. Data represent the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the Control group; #p < 0.05, ##p < 0.01, and ###p < 0.001 compared to the LCA or INT-777 group, n = 3.

Close modal

TGR5 Activation Upregulates AQP2 and AQP3 mRNA Expression in an cAMP-PKA-Dependent Manner

It has been previously reported that TGR5 is constitutively expressed in renal medullar collecting duct cells and exerts its biological actions through the cAMP-PKA pathway [12, 13]. To clarify the underlying mechanism by which TGR5 upregulates AQP2 and AQP3 expression, we treated rat primary medullary collecting duct cells with LCA (5 μm) and INT-777 (5 μm) for 6 h and found both TGR5 agonists significantly increased intracellular cAMP levels (online suppl. Fig. S5a, b). To further examine the role of the cAMP-PKA pathway in the regulation of AQP2 and AQP3 expression by TGR5, primary IMCDs were pretreated with the PKA inhibitor H89 for 1 h followed by 12-h treatment of LCA (Fig. 5a–c) and INT-777 (Fig. 5d–f). The results showed that LCA- and INT-777-induced mRNA expression of AQP2 and AQP3 was almost completely abolished by the H89 treatment (Fig. 5a, b, d, e). However, LCA and INT-777 treatment had no effect on AQP4 mRNA expression (Fig. 5c, f). These results demonstrate that the activation of TGR5 upregulates AQP2 and AQP3 expression at the transcriptional level via the cAMP-PKA pathway.

Fig. 5.

TGR5 activation induces AQP2 and AQP3 mRNA expression via a PKA-dependent manner in primary cultured rat IMCDs. Rat IMCDs were pretreated with vehicle or H89 (10 μm) for 1 h and then stimulated with 5 μm LCA and INT-777 for 12 h. The mRNA expression of AQP2, AQP3, and AQP4 was measured by quantitative PCR. a–c H89 treatment abolished the effect of LCA on AQP2 and AQP3 expression. LCA treatment significantly upregulated AQP2 (a) and AQP3 (b) mRNA expression, with little effect on AQP4 (c). LCA-induced AQP2 and AQP3 mRNA expression were completely abolished by H89 treatment. d–f H89 treatment abolished the effect of INT-777 on AQP2 and AQP3 expression. INT-777 treatment significantly upregulated AQP2 (d) and AQP3 (e) mRNA expression, with little effect on AQP4 (f). INT-777-induced AQP2 and AQP3 mRNA expression were completely abolished by H89 treatment. Data represent the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the Control group; #p < 0.05, ##p < 0.01, and ###p < 0.001 compared to the LCA or INT-777 group, n = 3.

Fig. 5.

TGR5 activation induces AQP2 and AQP3 mRNA expression via a PKA-dependent manner in primary cultured rat IMCDs. Rat IMCDs were pretreated with vehicle or H89 (10 μm) for 1 h and then stimulated with 5 μm LCA and INT-777 for 12 h. The mRNA expression of AQP2, AQP3, and AQP4 was measured by quantitative PCR. a–c H89 treatment abolished the effect of LCA on AQP2 and AQP3 expression. LCA treatment significantly upregulated AQP2 (a) and AQP3 (b) mRNA expression, with little effect on AQP4 (c). LCA-induced AQP2 and AQP3 mRNA expression were completely abolished by H89 treatment. d–f H89 treatment abolished the effect of INT-777 on AQP2 and AQP3 expression. INT-777 treatment significantly upregulated AQP2 (d) and AQP3 (e) mRNA expression, with little effect on AQP4 (f). INT-777-induced AQP2 and AQP3 mRNA expression were completely abolished by H89 treatment. Data represent the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the Control group; #p < 0.05, ##p < 0.01, and ###p < 0.001 compared to the LCA or INT-777 group, n = 3.

Close modal

TGR5 Activation Promotes AQP2 and AQP3 Protein Expression and CREB Nuclear Translocation in Cultured Primary IMCDs

As expected, TGR5 activation by LCA and INT-777 significantly induced AQP2 and AQP3 protein expression in cultured IMCDs, which was completely abolished by the H89 treatment (Fig. 6a, b; online suppl. Fig. S6a–d). In addition, LCA and INT-777 treatment significantly increased the Ser133 phosphorylation of CREB protein, suggesting TGR5 activation can promote CREB activity (Fig. 6a–d). In support, LCA and INT-777 treatment resulted in a marked increase in the nuclear translocation of CREB protein (Fig. 6c, d). These findings demonstrate that TGR5 activation can upregulate AQP2 and AQP3 expression, possibly via PKA-induced CREB phosphorylation and nuclear translocation.

Fig. 6.

Effect of H89 on LCA- and INT-777-induced AQP2 and AQP3 protein expression and the phosphorylation and nuclear translocation of CREB protein in primary cultured IMCDs Rat IMCDs were pretreated with DMSO (vehicle) or H89 (10 μm) for 1 h and then stimulated with LCA (5 μm) or INT-777 (5 μm) for 12 h. a, b Western blot assay showing that LCA (a) and INT-777 (b) treatment markedly induced AQP2 and AQP3 protein expression and the phosphorylation and nuclear translocation of CREB protein levels. c, d Immunofluorescence analysis demonstrating that LCA and INT-777 promoted nuclear translocation of CREB, which was markedly attenuated by the pretreatment with H89.

Fig. 6.

Effect of H89 on LCA- and INT-777-induced AQP2 and AQP3 protein expression and the phosphorylation and nuclear translocation of CREB protein in primary cultured IMCDs Rat IMCDs were pretreated with DMSO (vehicle) or H89 (10 μm) for 1 h and then stimulated with LCA (5 μm) or INT-777 (5 μm) for 12 h. a, b Western blot assay showing that LCA (a) and INT-777 (b) treatment markedly induced AQP2 and AQP3 protein expression and the phosphorylation and nuclear translocation of CREB protein levels. c, d Immunofluorescence analysis demonstrating that LCA and INT-777 promoted nuclear translocation of CREB, which was markedly attenuated by the pretreatment with H89.

Close modal

AQP2 and AQP3 Are Transcriptionally Induced by CREB

To determine the role of CREB in AQP2 and AQP3 gene transcription, we analyzed the sequences of mouse AQP2 and AQP3 promoter regions using commercially available software and found both AQP2 and AQP3 gene promoters contain a putative CREB-binding element (Fig. 7a, b). Luciferase reporter activity assay showed that CREB can significantly increase the promoter activity of AQP2 and AQP3 genes (Fig. 7c, d), possibly due to the binding of CREB protein to its binding element located in the promoter regions of AQP2 and AQP3 genes. This speculation was further confirmed by the gel EMSA, in which CREB protein was shown to directly bind to the predicted CREB-binding element in the promoter region of AQP2 and AQP3 genes, respectively (Fig. 7e, f). These results demonstrate that CREB can directly increase the transcription of both the AQP2 and AQP3 genes.

Fig. 7.

CREB transcriptionally induces AQP2 and AQP3 gene expression. a, b Schematic demonstration of the location of a putative CREB-binding element located in the mouse AQP2 (a) and AQP3 (b) gene promoter. c, d Luciferase reporter assay showing that CREB overexpression significantly increased the promoter activity of AQP2 gene (c) and AQP3 gene (d). ****p < 0.0001, n = 8. e, f Gel EMSA demonstrating that CREB can directly bind to the predicted CREB-binding site located in the promoter region of AQP2 (e) and AQP3 (f) gene. Note: Excessive unlabeled probe markedly attenuated the binding of CREB protein to the labeled AQP2 and AQP3 promoter probe.

Fig. 7.

CREB transcriptionally induces AQP2 and AQP3 gene expression. a, b Schematic demonstration of the location of a putative CREB-binding element located in the mouse AQP2 (a) and AQP3 (b) gene promoter. c, d Luciferase reporter assay showing that CREB overexpression significantly increased the promoter activity of AQP2 gene (c) and AQP3 gene (d). ****p < 0.0001, n = 8. e, f Gel EMSA demonstrating that CREB can directly bind to the predicted CREB-binding site located in the promoter region of AQP2 (e) and AQP3 (f) gene. Note: Excessive unlabeled probe markedly attenuated the binding of CREB protein to the labeled AQP2 and AQP3 promoter probe.

Close modal

TGR5, as a membrane receptor for bile acids, can be bound and activated by various endogenous bile acids, especially LCA. A large body of evidence demonstrates that it plays an important role in multiple physiological processes and the pathogenesis of many metabolic diseases [14‒16]. TGR5 is constitutively expressed in renal medullary collecting ducts (MCDs), where its activation can prevent the reduction of AQP2 expression induced by LiCl treatment and renal ischemia/reperfusion (I/R) [11, 17]. However, whether TGR5 affects AQP3 and AQP4 expression in the MCDs and what underlying mechanism might be involved remain largely unknown. The present study showed that treatment with the TGR5 agonist LCA and INT-777 markedly reduced mouse urine output and increased urine osmolality. TGR5 activation-induced urine concentration was accompanied by a marked increase in AQP2 and AQP3 protein expression and membrane translocation, with little effect on AQP4. In primary cultured MCDs, LCA and INT-777 upregulated AQP2 and AQP3 expression through the cAMP-PKA-CREB pathway in a TGR5-dependent manner. Collectively, our findings demonstrate that TGR5 activation can promote urine concentration by increasing AQP2 and AQP3 expression and function in renal medullary collecting ducts.

The principal cells of renal medullary collecting ducts play a critical role in maintaining body water homeostasis by three water channels, i.e., AQP2, AQP3, and AQP4 [5, 18, 19]. AQP2 is localized at the apical membrane, allowing the influx of water from the lumen into the MCD cells, while AQP3 and AQP4 are located at the basolateral membrane, mediating the efflux of water from MCD cells into the interstitium [20]. A large body of evidence demonstrates that AQP2 expression and its membrane translocation are under the tight control of the AVP-V2R system [21, 22] and PGE2-EP4 axis [23] in a cAMP/PKA/CREB-dependent manner. Consistent with previous reports, the present study found that AQP2 is also regulated by TGR5, suggesting a novel mechanism involved in local modulation of AQP2 expression and function. Unlike AQP2, little is known about the mechanism responsible for the regulation of AQP3 and AQP4 in the MCDs. In the present study, we provided evidence that AQP3, but not AQP4, is modulated by TGR5 in the MCDs, where TGR5 activation by both the endogenous agonist LCA and the synthetic agonist INT-777 induces AQP3 expression and membrane translocation. We further provide evidence that LCA- and INT-777-induced AQP2 and AQP3 expression is dependent on TGR5, since siRNA-mediated TGR5 knockdown abolished the effect of LCA and INT-777 on AQP2 and AQP3 mRNA and protein expression. Together, these findings reveal a novel mechanism by which TGR5 activation promotes water reabsorption in the MCDs by upregulating both AQP2 and AQP3 expression and function.

TGR5 is a Gs protein-coupled receptor for bile acids. Upon activation, TGR5 increases intracellular cAMP to stimulate cAMP-dependent PKA activity, which in turn activates the transcription factor CREB, resulting in target gene transcription [12]. The present study found that treatment with two structurally distinct TGR5 agonist, LCA and INT-777, markedly increased AQP2 and AQP3 expression at both mRNA and protein levels in renal medullary collecting duct cells in a cAMP-PKA-dependent manner. Further study revealed that LCA and INT-777 treatment promoted the Ser133 phosphorylation of CREB protein and its nuclear translocation, suggesting AQP2 and AQP3 may be regulated by CREB. This speculation was further supported by the finding that both AQP2 and AQP3 gene promoters contain a putative CREB-binding site which can be bound and activated by CREB. We also noticed that LCA treatment led to higher protein levels of AQP2 and AQP3 compared to INT-777. The underlying mechanism is not clear. Since LCA, a secondary bile acid naturally present in the body, has the ability to activate both TGR5 and FXR and the synthetic INT-777 is designed to selectively activate TGR5, the distinct activation patterns may be attributed to the specific properties of the drug. Together, these data demonstrate that AQP2 and AQP3 represent novel target genes of TGR5. Activation of TGR5 can induce AQP2 and AQP3 expression via the cAMP-PKA-CREB signaling pathway.

Bile acids are produced in the liver and are important in the digestion and absorption of lipophilic nutrients and vitamins in the small intestine. Increasing evidence demonstrates that bile acids may act as systemic hormone-like regulators and play an important role in many other tissues [24]. Previous study reported that AQP2 is a direct target of the bile acid nuclear receptor FXR in the renal medullary collecting duct, where chenodeoxycholic acid (CDCA) can induce AQP2 expression via directly increasing AQP2 gene transcription in an FXR-dependent manner [10, 11]. The present study further reported that the bile acid lithocholic acid (LCA) can also increase AQP2 expression via activating TGR5 to initiate the cAMP-PKA-CREB signaling pathway. In addition, bile acid-mediated TGR5 activation also increases AQP3 expression via the same mechanism. Taken together, endogenous bile acids can activate both FXR and TGR5 to increase AQP2 and AQP3 expression and membrane translocation, promoting water reabsorption in renal medullary collecting duct cells.

In conclusion, our study reports that the activation of TGR5 enhances urine concentration ability in vivo via increasing the expression and membrane translocation of AQP2 and AQP3 in the renal medullary collecting ducts. The TGR5 agonist LCA and INT-777 upregulate AQP2 and AQP3 gene expression in primary cultured medullary collecting duct cells through the cAMP-PKA-CREB pathway. The present study demonstrates that TGR5 plays an important role in the regulation of renal water reabsorption. Dysregulation or dysfunction of TGR5 might be involved in the pathogenesis of the hepatorenal syndrome, a severe renal complication of end-stage liver disease. Our findings suggest that TGR5 may serve as a novel target for the treatment of clinical diseases related to urinary concentration defect.

This animal study protocol was reviewed and approved by the Ethics Committee of East China Normal University (m20211205).

The authors have no conflicts of interest to declare.

This research was funded by the National Natural Science Foundation of China Grants 82270703 (to X.Y.Z.), 81970606 (to X.Y.Z.), and 81970595 (to Y.F.G.); and the East China Normal University Medicine and Health Joint Fund (2022JKXYD03001).

Y.L.G. and R.F.Q. performed research, luciferase, and electrophoretic mobility shift assay; analyzed data; and wrote the manuscript. Y.L.G. contributed to animal experiments. G.X.X., Y.Y., Y.X.S., and C.X.D. designed and performed in vitro experiments. Y.F.G. and X.Y.Z. designed and supervised this work. X.Y.Z. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Additional Information

Yanlin Guo and Rongfang Qiao contributed equally to this work.

All data generated or analyzed during this study are included in this article and its online supplementary material files. Further inquiries can be directed to the corresponding author.

1.
Noda
Y
,
Sohara
E
,
Ohta
E
,
Sasaki
S
.
Aquaporins in kidney pathophysiology
.
Nat Rev Nephrol
.
2010
;
6
(
3
):
168
78
. doi: .
2.
Chou
CL
,
Knepper
MA
,
Hoek
AN
,
Brown
D
,
Yang
B
,
Ma
T
, et al
.
Reduced water permeability and altered ultrastructure in thin descending limb of Henle in aquaporin-1 null mice
.
J Clin Invest
.
1999
;
103
(
4
):
491
6
. doi: .
3.
Ren
H
,
Yang
B
,
Ruiz
JA
,
Efe
O
,
Ilori
TO
,
Sands
JM
, et al
.
Phosphatase inhibition increases AQP2 accumulation in the rat IMCD apical plasma membrane
.
Am J Physiol Renal Physiol
.
2016
;
311
(
6
):
F1189
97
. doi: .
4.
Hara-Chikuma
M
,
Satooka
H
,
Watanabe
S
,
Honda
T
,
Miyachi
Y
,
Watanabe
T
, et al
.
Aquaporin-3-mediated hydrogen peroxide transport is required for NF-κB signalling in keratinocytes and development of psoriasis
.
Nat Commun
.
2015
;
6
(
1
):
7454
. doi: .
5.
Ma
T
,
Song
Y
,
Yang
B
,
Gillespie
A
,
Carlson
EJ
,
Epstein
CJ
, et al
.
Nephrogenic diabetes insipidus in mice lacking aquaporin-3 water channels
.
Proc Natl Acad Sci U S A
.
2000
;
97
(
8
):
4386
91
. doi: .
6.
Yang
B
,
Ma
T
,
Verkman
AS
.
Erythrocyte water permeability and renal function in double knockout mice lacking aquaporin-1 and aquaporin-3
.
J Biol Chem
.
2001
;
276
(
1
):
624
8
. doi: .
7.
Hofmann
AF
,
Borgstroem
B
.
The intraluminal phase of fat digestion in man: the lipid content of the micellar and oil phases of intestinal content obtained during fat digestion and absorption
.
J Clin Invest
.
1964
;
43
(
2
):
247
57
. doi: .
8.
Coleman
R
.
Bile salts and biliary lipids
.
Biochem Soc Trans
.
1987
;
15
(
Suppl
):
68s
80s
.
9.
Sanyal
AJ
,
Hirsch
JI
,
Moore
EW
.
Premicellar taurocholate enhances calcium uptake from all regions of rat small intestine
.
Gastroenterology
.
1994
;
106
(
4
):
866
74
. doi: .
10.
Zhang
X
,
Huang
S
,
Gao
M
,
Liu
J
,
Jia
X
,
Han
Q
, et al
.
Farnesoid X receptor (FXR) gene deficiency impairs urine concentration in mice
.
Proc Natl Acad Sci U S A
.
2014
;
111
(
6
):
2277
82
. doi: .
11.
Li
S
,
Qiu
M
,
Kong
Y
,
Zhao
X
,
Choi
HJ
,
Reich
M
, et al
.
Bile acid G protein-coupled membrane receptor TGR5 modulates aquaporin 2-mediated water homeostasis
.
J Am Soc Nephrol
.
2018
;
29
(
11
):
2658
70
. doi: .
12.
Qi
Y-C
,
Duan
GZ
,
Mao
W
,
Liu
Q
,
Zhang
YL
,
Li
PF
.
Taurochenodeoxycholic acid mediates cAMP-PKA-CREB signaling pathway
.
Chin J Nat Med
.
2020
;
18
(
12
):
898
906
. doi: .
13.
Yang
W-J.
,
Han
FH
,
Gu
YP
,
Qu
H
,
Liu
J
,
Shen
JH
, et al
.
TGR5 agonist inhibits intestinal epithelial cell apoptosis via cAMP/PKA/c-FLIP/JNK signaling pathway and ameliorates dextran sulfate sodium-induced ulcerative colitis
.
Acta Pharmacol Sin
.
2023
;
44
(
8
):
1649
64
. doi: .
14.
Chen
B
,
Bai
Y
,
Tong
F
,
Yan
J
,
Zhang
R
,
Zhong
Y
, et al
.
Glycoursodeoxycholic acid regulates bile acids level and alters gut microbiota and glycolipid metabolism to attenuate diabetes
.
Gut Microbes
.
2023
;
15
(
1
):
2192155
. doi: .
15.
Tian
F
,
Xu
W
,
Chen
L
,
Chen
T
,
Feng
X
,
Chen
J
, et al
.
Ginsenoside compound K increases glucagon-like peptide-1 release and L-cell abundance in db/db mice through TGR5/YAP signaling
.
Int Immunopharmacol
.
2022
;
113
(
Pt A
):
109405
. doi: .
16.
Reich
M
,
Spomer
L
,
Klindt
C
,
Fuchs
K
,
Stindt
J
,
Deutschmann
K
, et al
.
Downregulation of TGR5 (GPBAR1) in biliary epithelial cells contributes to the pathogenesis of sclerosing cholangitis
.
J Hepatol
.
2021
;
75
(
3
):
634
46
. doi: .
17.
Han
M
,
Li
S
,
Xie
H
,
Liu
Q
,
Wang
A
,
Hu
S
, et al
.
Activation of TGR5 restores AQP2 expression via the HIF pathway in renal ischemia-reperfusion injury
.
Am J Physiol Renal Physiol
.
2021
;
320
(
3
):
F308
21
. doi: .
18.
Kwon
TH
,
Frøkiær
J
,
Nielsen
S
.
Regulation of aquaporin-2 in the kidney: a molecular mechanism of body-water homeostasis
.
Kidney Res Clin Pract
.
2013
;
32
(
3
):
96
102
. doi: .
19.
van Hoek
AN
,
Ma
T
,
Yang
B
,
Verkman
AS
,
Brown
D
.
Aquaporin-4 is expressed in basolateral membranes of proximal tubule S3 segments in mouse kidney
.
Am J Physiol Renal Physiol
.
2000
;
278
(
2
):
F310
6
. doi: .
20.
Su
W
,
Cao
R
,
Zhang
XY
,
Guan
Y
.
Aquaporins in the kidney: physiology and pathophysiology
.
Am J Physiol Renal Physiol
.
2020
;
318
(
1
):
F193
203
. doi: .
21.
Boone
M
,
Deen
PMT
.
Physiology and pathophysiology of the vasopressin-regulated renal water reabsorption
.
Pflugers Arch
.
2008
;
456
(
6
):
1005
24
. doi: .
22.
Schrier
RW
.
Vasopressin and aquaporin 2 in clinical disorders of water homeostasis
.
Semin Nephrol
.
2008
;
28
(
3
):
289
96
. doi: .
23.
Gao
M
,
Cao
R
,
Du
S
,
Jia
X
,
Zheng
S
,
Huang
S
, et al
.
Disruption of prostaglandin E2 receptor EP4 impairs urinary concentration via decreasing aquaporin 2 in renal collecting ducts
.
Proc Natl Acad Sci U S A
.
2015
;
112
(
27
):
8397
402
. doi: .
24.
Kliewer
SA
,
Mangelsdorf
DJ
.
Bile acids as hormones: the FXR-FGF15/19 pathway
.
Dig Dis
.
2015
;
33
(
3
):
327
31
. doi: .