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Corresponding authors at: Department of Gastroenterology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, Zhejiang, China.
Corresponding authors at: Department of Gastroenterology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, Zhejiang, China.
Aquaporins (AQPs) maintain fluid homeostasis in the colon. The role of colonic AQPs in the pathophysiology of functional constipation (FC) remains largely unknown.
Aim
To explore variations in aquaporins and investigate their underlying mechanisms.
Methods
Colonic biopsies were collected from patients with FC and healthy controls. The expression and localization of AQPs were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR), western blot analysis, and immunofluorescence assays. Furthermore, osmotic pressure-induced cell model was used in vitro to investigate the potential relationship between AQP8 and osmotic pressure, and to reveal the underlying mechanisms.
Results
Upregulation of AQP3 and AQP8, and downregulation of AQP1, AQP7, AQP9, AQP10, and AQP11 were observed in the patients with functional constipation. Furthermore, cellular translocation of AQP8 from the cytoplasm to the plasma membrane was observed in patients with FC. Mechanistically, the increase in osmotic pressure could activate the Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways, and subsequently promote the upregulation and translocation of AQP8.
Conclusion
Upregulation of AQP8 and AQP3, and translocation of AQP8 were observed in colon biopsies from patients with FC. The p38 and JNK MAPK signaling pathways are involved in the regulation of osmotic pressure-induced AQP8 variation.
Functional constipation (FC) is a common gastrointestinal disorder of the gut-brain interaction, affecting the tenth of the general population according to the Rome IV criteria [
]. The chronic and fluctuating course of troublesome symptoms leads to an impaired quality of life. Patients with FC are more likely to seek physician visits, require prescription medications, and receive non-cancer-related abdominal surgeries compared to the general population, resulting in considerable economic impact on healthcare systems and society [
]. Commonly used targeted pharmacotherapies for FC include secretagogues, serotonin modulators and bile acid modulators. However, given the complex pathophysiology and chronic course of FC, broadening the understanding on the mechanisms involved in FC is important for the development of novel therapies.
Water recirculation in the digestive tract is important for maintaining body water homeostasis and controlling the fluid and ion balance [
]. To date, water absorption in the intestinal tract occurs by two mechanisms: paracellular route through the gap between cell junctions, and transcellular transport through apical and basolateral cell membranes [
. However, paracellular route as the dominant mechanism for water movement in the intestinal epithelia of the digestive tract is unlikely given the rather tight barriers formed by cell junction electrical resistance [
]. In the transcellular route, three mechanisms are involved: diffusion through aquaporins (AQPs), passive diffusion through phospholipids, and cotransport with ions and nutrients [
]. Moreover, the discovery of aquaporins has dramatically changed our understanding of how water is transported through epithelial barriers.
AQPs are a family of water channel proteins that play vital roles in gut homeostasis. The distribution and functions of AQPs are modulated under both physiological and pathophysiological conditions [
]. Furthermore, research showed that patients with irritable bowel syndrome-diarrhea had greater expression of AQP7 and lower levels of AQP3 in rectosigmoid mucosal biopsies [
]. In addition, the transport of water and solutes through the gut epithelia is essential for osmoregulation, digestion, and absorption. This passage is regulated by different AQP isoforms, and is characterized by distinctive distribution patterns in the gastrointestinal tract [
]. To date, 13 AQPs have been identified in mammals, among which AQP1, AQP3, AQP4, AQP7, AQP8, AQP10, and AQP11 are expressed at the mRNA level by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) [
]. However, the expression, localization, and molecular mechanisms of AQPs in patients with FC remain unknown. The study aimed to explore the variations in aquaporins in patients with FC, and investigate the underlying mechanisms.
2. Materials and methods
2.1 Ethics and colon biopsy collection
This research was approved by the Ethics Committee of Sir Run Shaw Hospital, Zhejiang University School of Medicine (Ethics number: 2022–379–01) prior to enrollment. Colon biopsies from healthy controls and patients with FC were obtained from donors who provided written informed consent. A total of 12 adult patients (10 women and 2 men; aged 21–60 years) meeting the Rome IV criteria for FC and 10 healthy controls (5 women and 5 men; aged 30–60 years) were recruited. Exclusion criteria included patients who received previous abdominal surgery other than appendectomy or hysterectomy, pregnancy, and use of medications including antibiotics, probiotics, or laxatives within 4 weeks prior to enrollment. The patient information is summarized in Supplementary Table 1.
2.2 RNA extraction and qRT-PCR
To evaluate AQPs activity at the RNA level in the colonic mucosa of patients with FC and healthy controls, total RNAs from colon biopsy tissues were extracted using the RNAisoPlus reagent (Kangwei Biotechnology, China) according to the manufacturer's instructions. Each RNA sample was reverse transcribed into cDNAs using TIANScript RT reagent kit (Kangwei Biotechnology, China). qRT-PCR analysis was performed in triplicate in ROCHE LightCycler480 System (Rotor gene 6000 Software, Sydney, Australia). Each reaction was tested in triplicate in a 10 µl reaction system containing SYBR Premix Ex Taq (Takara, Japan), primers and template cDNAs. Relative abundance was calculated using the -ΔCt method. GAPDH served as an internal reference gene. Following primer sets were used:
Human AQP1:
Forward: 5′-GATTAACCCTGCTCGGTCCT-3′,
Reverse: 5′-AGACCCCTTCTATTTGGGCTTC-3′;
Human AQP3:
Forward: 5′-ACCCCTCTGGACACTTGGATA-3′,
Reverse: 5′-AGGTGCCAATGACCAGGAC-3′;
Human AQP4:
Forward: 5′-GGAAGGCATGAGTGACAGAC-3′,
Reverse: 5′-TTGCAATGCTGAGTCCAAAGC-3′;
Human AQP7:
Forward: 5′-TACAGTTTGAGGGGATGGGC-3′,
Reverse: 5′-ACGGTGCTGACCCATTGTTA-3′;
Human AQP8:
Forward: 5′-GATAGCCATGTGTGAGCCTGA-3′,
Reverse: 5′-GTTGAAGTGTCCACCACTGATATT-3′;
Human AQP9:
Forward: 5′-ACGATTAAGCCACAGCCTCT-3′,
Reverse: 5′-CCACATCCAAGGACAATCAAGA-3′;
Human AQP10:
Forward: 5′-ATGCTGATTGTGGGGCTCTT-3′,
Reverse: 5′-GCCATTACCAGCACTGAAGAC-3′;
Human AQP11:
Forward: 5′-ATACTGGCTGGCTCCTTCTTT-3′,
Reverse: 5′-GTCCCAGTGATGAAAACTATAAGGC-3′;
Human GAPDH:
Forward: 5′-CGGATTTGGTCGTATTGGG-3′,
Reverse: 5′-CTGGAAGATGGTGATGGGATT-3′.
2.3 Western blot analysis
To evaluate AQPs activity at the protein level in the colonic mucosa of patients with FC and healthy controls, colonic mucosa tissues were collected using RIPA lysis buffer (Beyotime, China) containing protease inhibitors and phosphatase inhibitors (FDbio Science, China) on ice and quantified using the BCA Protein Assay Kit (Kangwei Biotechnology, China). Total proteins were loaded on to 10% polyacrylamide gels, and electrophoresed proteins were transferred onto PVDF membranes. The membranes were blocked in 5% bovine serum albumin (BSA) (FDbio Science, China) for 1.5 h at room temperature, and then incubated with primary and secondary antibodies. Subsequently, the signals were detected using an ECL Kit (FDbio Science, China) and the ChemiDoc Touch Imaging System (Bio-Rad, USA). Following primary antibodies were used: AQP1 (Santa Cruz, sc-25,287), AQP3 (Abcam, ab125219), AQP7 (Santa Cruz, sc-376,407), AQP8 (Bioss, bs-6786R), AQP9 (Santa Cruz, sc-74,409), AQP10 (Fabgenni, AQP10–1001AP), AQP11 (Novus, NBP1–86,208), Erk1/2 (Cell Signaling Technology, #4695), p-Erk1/2 (Cell Signaling Technology, #4370), p38 (Cell Signaling Technology, #8690), p-p38 (Cell Signaling Technology, #8690), JNK (Santa Cruz, sc-7345), p-JNK (Santa Cruz, sc-6254), Na, K-ATPase (Abcam, ab76020), β-actin (CWBIO, CW0096) and GAPDH (Proteintech, 10,494–1-AP).
2.4 Immunofluorescence assay
Colonic epithelial specimens fixed in 10% neutral buffered formalin were embedded in paraffin blocks and subjected to immunofluorescence staining. After routine deparaffinization, dewaxing, and antigen retrieval, the paraffin sections of colonic tissues were blocked in 5% BSA (FDbio Science, China) for 1 h at room temperature. Then the slides were stained with primary antibodies AQP1, AQP3, AQP7, AQP8, AQP9, AQP10 and AQP11 overnight at 4 °C in a wet chamber, and incubated with a goat anti-Rabbit IgG (H + L) secondary antibody (Alexa FluorⓇ 488 conjugate, green, 1:500; Beyotime) or a goat anti-Mouse IgG (H + L) secondary antibody (Alexa FluorⓇ 555 conjugate, red, 1:500; Beyotime) for 2 h at room temperature. The cell nuclei were stained with DAPI (blue, 1:1000; Beyotime). As for immunofluorescence assay of cell samples, the cell membrane was also stained with Alexa Fluor 555 WGA (1:200; Thermo Fisher Scientific). Images were collected using a confocal microscope (LSM 880, Zeiss) and processed using ZEN image software.
2.5 Cell culture
Different concentrations of magnesium sulfate (MgSO4) or mannitol were applied to generate an environment of osmotic pressure changes. Human normal colonic epithelial cell line NCM460 and colorectal cancer cell line HT29 were purchased from American Type Culture Collection (ATCC, USA) and cultured in DMEM medium (GIBCO, China) supplemented with 10% fetal bovine serum (Sijiqing, China) at 37 °C in a humidified 5% CO2 atmosphere.
2.6 Membrane and cytosol protein extraction
Membrane and cytosolic proteins were isolated using a Membrane and Cytosol Protein Extraction Kit (P0033; Beyotime, China) according to the manufacturer's instructions.
2.7 siRNAs synthesis and transfection
To downregulate the expression of AQP3, small interfering RNAs (siRNAs) targeting AQP3 were synthesized and designed by GenePharma (Shanghai, China). According to the manufacturer's instructions, the siRNAs were transfected into cells at 30% confluence using Lipofectamine RNAiMAX (Thermo Fisher Scientific, USA) in opti-MEM (Genom, China). Following sequences of siRNAs were used:
si-NC: 5′-UUCUCCGAACGUGUCACGUTT-3′;
si-AQP3 #1: 5′-CCUCUGGACACUUGGAUAUTT-3′;
si-AQP3 #2: 5′-GUGGUUUCCUCACCAUCAATT-3′;
si-AQP3 #3: 5′-CCAACGAGGAAGAGAAUGUTT-3′.
2.8 Plasmid construction and transfection
To upregulate the expression of AQP8, the pCDNA3.1 containing the full length of AQP8 was synthesized and designed by Tsingke Biological Technology (Beijing, China). Transient plasmid transfection was carried out using FuGENE HD transfection reagent (Promega, USA) according to the manufacturer's instructions.
2.9 Statistical analysis
Statistical analysis was performed using the GraphPad Prism software (version 7.0). Data were analyzed with paired or unpaired Student's t-test and Mann-Whitney test, as shown in the figure legends. P < 0.05 was considered statistically significant.
3. Results
3.1 Evaluation of colonic aquaporins expressions
On the whole, the overall study design and flowchart for each passage are presented in Fig. 1. Compared with healthy controls, the RNA levels of AQP3 and AQP8 were remarkably upregulated in ascending colons with statistically significant differences, accompanied by downregulation of AQP7, AQP9, AQP10, and AQP11 (Fig. 2A). Parallel findings of AQPs expression were observed at the protein level (Fig. 2B). Accordingly, increased expression of AQP3 and decreased levels of AQP1, AQP9, AQP10 and AQP11 were also observed in the descending colon mucosa, whereas no difference in AQP8 was observed (Fig. 2C-D). No difference in AQP4 expression was observed in patients in the FC and control groups, regardless of the colon segment.
Fig. 1Study overview. Flowchart conceptualizing the process or methods followed in this study.
Fig. 2Evaluation of colonic aquaporins expressions. (A-B) Quantitative RT-PCR analysis (A) and western blotting validation (B) of AQP1, AQP3, AQP4, AQP7, AQP8, AQP9, AQP10 and AQP11 in ascending colons of patients with FC and healthy controls. (C-D) Quantitative RT-PCR analysis (C) and western blotting validation (D) of AQP1, AQP3, AQP4, AQP7, AQP8, AQP9, AQP10 and AQP11 in descending colons of patients with FC and healthy controls. (E-F) Quantitative RT-PCR analysis of AQP3 in paired ascending and descending colons of healthy controls (E) and patients with FC (F). (G-H) Quantitative RT-PCR analysis of AQP8 in paired ascending and descending colons of healthy controls (G) and patients with FC (H). (I) Representative images of H&E staining colon mucosa of healthy controls and patients with FC were shown. Data are shown as mean ± SD. n.s, no significance, * p < 0.05, by Student's t-test.
]. Therefore, we further assessed the expression of AQPs using qRT-PCR analysis stratified by colon segments. No difference in AQP3 expression was observed between the paired ascending and descending colons (Fig. 2E-F). Overall, similar results were observed for other AQP isoforms in both patients with FC and healthy controls (Supplementary Fig. 1A-B). Interestingly, a higher expression of AQP8 were observed in the ascending colon of patients with FC compared to the control group (Fig. 2G-H), suggesting that AQP8 may be involved in increased fluid absorption capacity in ascending colons. Furthermore, hematoxylin and eosin (H&E) staining of colonic segments showed a lack of immune cell filtration or tissue injury in patients with FC, which is consistent with functional changes (Fig. 2I).
3.2 Location of aquaporin water channels in colon biopsies
Localization is closely related to protein function and may play an important mechanistic role. Data from several animal models showed that both superficial and deep crypt colonocytes can absorb water [
]. Here, we examined the location of AQPs using immunofluorescence assay (Fig. 3A), and summarized them in the form a table (Fig. 3B).
Fig. 3Location of aquaporin water channels in colon biopsies. (A) Representative images of immunofluorescence assay of AQP1 (red), AQP3 (green), AQP7 (orange), AQP9 (yellow), AQP10 (purple), AQP11 (turquoise) in colon biopsies from healthy controls, and the nuclei were counterstained with DAPI (blue). Scale bars, 5 µm. (B) The summary table of the location of AQPs. (C) Representative images of immunofluorescence staining of AQP8 (green) in colon tissues from patients with FC and healthy controls. Scale bars, 20 µm.
AQP trafficking is an important mechanism that controls cellular water/solute homeostasis of cells. As reported, vasopressin-dependent water channel AQP2 can translocate between the plasma membrane and intracellular vesicles, and subsequently mediate mineralocorticoid-regulated sodium- and vasopressin-regulated water reabsorption in the kidneys of mice [
]. In light of these findings, immunofluoresence assay of AQP subtypes was performed on colonic specimens in patients with FC and the controls. Intriguingly, our results revealed the translocation of AQP8 from the cytoplasm to the plasma membrane in the colonic epithelial cells in patients with FC (Fig. 3C).
3.3 Change of osmotic pressure could upregulate aquaporin-8 and promote its relocalization to cytomembrane
On average, 1.5–2 L of fluid daily could be absorbed against the osmotic gradient by the colon epithelium to generate dehydrated feces [
]. To further investigate the potential relationship between AQP8 and osmotic pressure, we designed an osmotic pressure-induced cell model in vitro. Different concentrations of magnesium sulfate (MgSO4) or mannitol were applied to generate an environment of osmotic pressure changes, as previously reported [
. NCM460 cells cultured in DMEM supplemented with various concentrations of MgSO4 (0, 7.5, 15, 30, and 60 mM) were subjected to western blot analysis of AQP8. As expected, with an increase in concentration, the protein level of AQP8 gradually increased from baseline (Fig. 4A). In addition, we incubated the NCM460 cells with 60 mM MgSO4 for 1 h, 2 h, 4 h, 6 h, and 24 h. The results showed that the expression of AQP8 started to increase 1 h after stimulation and reached peak levels at 24 h (Fig. 4B). Furthermore, mannitol was used to assess the effect of osmotic pressure change on the expression of AQP8. Similarly, there was a progressive increase in the level of AQP8 at higher concentrations (Fig. 4C), and the expression of AQP8 peaked at 24 h (Fig. 4D). Collectively, these data suggest that the expression of AQP8 was positively associated with osmotic pressure changes in a concentration- and time-dependent manner.
Fig. 4Change of osmotic pressure could upregulate aquaporin-8 and promote its relocalization to cytomembrane. (A) NCM460 cells treated with different concentrations of magnesium sulfate (MgSO4) were subjected to western blot analysis of the protein level of AQP8. (B) Western blot analysis of AQP8 in NCM460 cells stimulated with 60 mM MgSO4 at 1 h, 2 h, 4 h, 6 h and 24 h. (C) NCM460 cells treated with different concentrations of mannitol were subjected to western blot analysis of AQP8. (D) Western blot analysis of AQP8 in NCM460 cells stimulated with 1% mannitol at 1 h, 2 h, 4 h, 6 h and 24 h. (E) The distribution of AQP8 (green) in the indicated cells was measured by immunofluorescence assay, in which cell membranes were stained with a marker of cytomembrane (WGA, red), and the nuclei were counterstained with DAPI (blue). Scale bar, 10 µm. (F) The distribution of AQP8 was quantified by Pearson correlation analysis. (G) NCM460 cells stimulated with 60 mM MgSO4 were applied for Membrane and Cytosol Protein Extraction, and western blot analysis of the levels of AQP8 in the cytoplasm and membrane was performed. Na, K-ATPase, a membrane marker. (H) Western blot analysis of MAPK signaling pathway in colon tissues from patients with FC and healthy controls. Data are shown as mean ± SD. ** p < 0.01, by Mann- Whitney test (F).
To further elucidate the relocalization of AQP8, we performed an immunofluorescence assay of AQP8 (green), in which the cytomembrane was labeled with WGA (red), and the nuclei were counterstained with DAPI (blue). MgSO4 treatment promoted the cellular localization of AQP8 from the cytoplasm to the plasma membrane (Fig. 4E), and the results were quantified using Pearson's correlation analysis (Fig. 4F). In addition, we isolated the membrane and cytoplasmic components of NCM460 cells cocultured with MgSO4. Western blot analysis showed that the protein level of AQP8 was significantly decreased in the cytosol, whereas it was notably increased in the membrane (Fig. 4G), indicating the relocalization of AQP8 following osmotic pressure changes.
3.4 Osmotic pressure influenced aquaporin-8 through the activation of p38 and JNK MAPK signaling pathway
We next investigated the underlying mechanisms by which osmotic pressure upregulates the expression and facilitates its relocalization of AQP8. Several studies have reported that the mitogen-activated protein kinase (MAPK) pathways may play vital roles in osmotic stress [
]. Given that extracellular signal-regulated kinase 1/2 (Erk1/2), p38 MAPK, and c-Jun N-terminal kinase (JNK) are well-known members of the MAPK pathway, we performed a western blot analysis to examine the phosphorylation of Erk1/2, p38 and JNK in the colons of patients with FC and healthy controls. Our data showed that the MAPK signaling pathway was activated in FC colon tissues to a certain extent (Fig. 4H). Therefore, we further examined the activation of the MAPK pathway in NCM 460 cells under high osmotic pressure. As shown in (Fig. 5A-B), the phosphorylation levels of p38 and JNK were upregulated upon MgSO4 and mannitol treatment, while the protein levels of phospho- Erk1/2 were not significantly different. Collectively, these results revealed that p38 and JNK MAPK signaling pathways are activated under conditions of high osmotic pressure.
Fig. 5Osmotic pressure influenced aquaporin-8 through the activation of p38 and JNK MAPK signaling pathway. (A-B) NCM460 cells were stimulated with different concentrations of MgSO4 (A) and mannitol (B). Western blot analysis of total Erk 1/2, phosphor-Erk 1/2, total p38, phosphor-p38, total JNK and phosphor-JNK were performed. (C-D) NCM460 cells were stimulated with MgSO4 (C) and mannitol (D), and then treated with p38 inhibitor, HY-10,256. Western blot analysis was performed to detect the expression of AQP8. (E-F) NCM460 cells were stimulated with MgSO4 (E) and mannitol (F), and then treated with JNK inhibitor, HY-12,041. Western blot analysis was performed to detect the expression of AQP8. (G-H) NCM460 cells stimulated with MgSO4 were treated with p38 inhibitor (G) and JNK inhibitor (H), and subjected to Membrane and Cytosol Protein Extraction. Western blot analysis of AQP8 in the cytoplasm and membrane was performed. Na, K-ATPase, a membrane marker.
To evaluate the roles of p38 and JNK pathways in the regulation of AQP8, HY-10256, an inhibitor of p38 MAPK pathway, was employed to treat NCM460 cells stimulated with MgSO4 or mannitol. As expected, western blot analysis showed that the upregulation of AQP8 induced by high osmotic pressure was attenuated when the p38 MAPK pathway was blocked (Fig. 5C-D). Similar results were also observed for HY-12041, which is an inhibitor of the JNK MAPK pathway (Fig. 5E-F). Furthermore, the upregulation of AQP8 in membranes was attenuated when the p38 or JNK MAPK pathways were inhibited compared to the group treated with MgSO4 (Fig. 5G-H), highlighting the important roles of the p38 and JNK MAPK pathways in sustaining the functions of AQP8 in response to high osmotic pressure.
Taken together, these results indicate that high osmotic pressure treatment regulates AQP8 and facilitates its relocalization by activating the MAPK signaling pathway.
3.5 Dynamic balance of expressions among aquaporin-3, −8, and others
Recently, the role of AQPs in diverse disease processes including kidney diseases, loss of skin barrier function, and edema following cerebral or spinal stroke has been demonstrated [
]. To clarify the relationship among the different subtypes of AQPs, specific siRNAs targeting AQP3 were designed and transfected into NCM460 cells, and the siRNA with the best silencing efficiency was applied in the following studies (Fig. 6A). The immunofluorescence assay results also corroborated the decreased AQP3 levels in AQP3-knockdown NCM460 cells (Fig. 6B). Moreover, both qRT-PCR and western blot analysis showed that silencing of AQP3 resulted in upregulated expression of AQP1, AQP7, AQP8, AQP9, AQP10, and AQP11 (Fig. 6C-D). Similar results were observed in HT29 cells (Supplementary Fig. 2A).
Fig. 6Dynamic balance of expressions among aquaporin-3, −8, and others. (A) Quantitative RT-PCR of AQP3 was performed in NCM460 cells transfected with three siRNAs targeting AQP3 or control siRNAs. (B) Representative images of immunofluorescence assay of AQP3 in AQP3-knockdown NCM460 cells. Cells were stained with specific antibody against AQP3 (green), and the nuclei were counterstained with DAPI (blue). Scale bar, 50 µm. (C-D) NCM460 cells were transfected with siRNAs targeting AQP3 in triplicate. Quantitative RT-PCR analysis (C) and western blotting validation (D) of AQP3 and other AQPs were performed. (E-F) NCM460 cells were transfected with the indicated plasmids targeting AQP8 in triplicate. Quantitative RT-PCR analysis (E) and western blotting validation (F) of AQP8 and other AQPs were performed. Data are shown as mean ± SD. n.s, no significance, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, by Student's t-test.
To further confirm the dynamic balance among AQPs, we constructed plasmids targeting AQP8 to overexpress AQP8. Similarly, NCM460 cells transfected with the indicated plasmids were analyzed by qRT-PCR and western blotting. As expected, ectopic expression of AQP8 significantly inhibited the expression levels of AQP1, AQP3, AQP7, AQP9, AQP10, and AQP11 (Fig. 6E-F). Similar results were observed in HT29 cells (Supplementary Fig. 2B).
4. Discussion
In our study, we demonstrated the differential expression of AQP subtypes in the ascending and descending colon mucosa of patients with FC and healthy controls. Increased expression of AQP3 and AQP8, and downregulation of other AQP subtypes were observed in patients with FC compared to the control group. Furthermore, upregulation and relocalization of AQP8 were observed with osmotic pressure manipulation. Mechanistically, osmotic pressure could activate the p38 and JNK MAPK pathways, thereby upregulating AQP8 and facilitating its relocalization to the cytomembrane, which is critical for excessive fluid absorption in the colon of patients with FC.
Rapid water exchange enables intestinal tract to maintain physiological functions. The possible pathways for trans-epithelial water transport in the digestive system include the paracellular, transcellular, diffusion, and osmolality-dependent AQP pathway [
], and we summarized the main differences in each aquaporin type in Supplementary Table 2. Various AQPs have been identified in the human colonic mucosa, including AQP1, 3, 4, 7, 8, 9, 10 and 11 at the RNA level [
]. AQPs may play a vital role in functional constipation, with symptoms of lumpy or hard stools induced by disruption of the water transport system. In our study, we revealed the upregulation of AQP3 and AQP8, and downregulation of AQP1, AQP7, AQP9, AQP10, and AQP11 by qRT-PCR at the mRNA and western blot analysis at the protein levels, respectively. Consistently, several other prior studies have demonstrated the upregulation of AQP3 and AQP8 in loperamide-induced constipation in rats [
. This prompted us to explore AQPs expression in the ascending and descending colon. Although likely, due to limited sample size, there was no obvious difference in the expression levels of AQPs between them in our clinical cohort, except for AQP8. Future studies analyzing a more robust sample size in patients with FC will be of great interest to further elucidate the potential role of other AQP subtypes.
As for cellular localization, we were first to demonstrate the localization of AQP1, AQP3, AQP7, AQP8, AQP9, AQP10, and AQP11 by utilizing immunofluorescence assay in colonic tissue. Interestingly, our results also showed the translocation of AQP8 from the cytoplasm to the plasma membrane in the colonic epithelial cells in patients with FC. Similarly, the relationship between the unique subcellular distribution of AQP isoforms in organ sites and their functions have been documented [
and found that AQP8 was upregulated in normal human colonic epithelial cells (NCM460 cells) under different osmolality. Furthermore, a similar distribution of AQP8 to the cell membrane was observed in NCM460 cells upon increased osmotic pressure stimulation. Here, we confirmed the increasing levels of AQP3 and AQP8 expression and the effect of translocation AQP8, thus, uncovering a new pathological role in functional constipation.
Another key finding from this study was that the increase in osmotic pressure could activate the p38 and JNK signaling pathways, and subsequently promote the expression and translocation of AQP8. It is widely accepted that AQP expression is influenced by environmental, genetic, and disease-related factors. It is reported that single nucleotide polymorphism (rs9951307) at the 3′ end of AQP4 was associated with severe brain edema [
]. Except for difference in chromosomal localization, and number of exons of AQPs determining their physiological expression and location, altered protein abundance of AQPs is affected by environmental factors such as systemic hormones, local molecules, pH, divalent cation concentrations, and osmolality [
]. For example, Choi et al. found that extracellular pH affects phosphorylation and intracellular trafficking of AQP2 in inner medullary collecting duct cells [
]. In our study, activation of p38 and JNK signaling pathways was observed when cells were stimulated with mannitol or magnesium sulfate, which coincided with recent studies evaluating the role of MAPK [
]. When we blocked the MAPK pathway using small molecule inhibitors, the upregulation of AQP8 induced by high osmotic pressure was attenuated. These findings indicate that altering the activation of the p38 and JNK pathways may be sufficient to attenuate excessive water absorption in the colon. However, more work is needed in the future to elucidate the therapeutic effect of blocking the pathway in vivo.
Alterations in the AQP subtype provide new insights into the understanding and potential therapeutic strategies in FC. In our study, by transfecting small interfering RNAs or plasmids to alter the levels of AQP3 and AQP8, a dynamic balance among AQP subtypes was observed. As one of the emerging research areas, AQPs have attracted great interest as therapeutic targets for a number of disease processes associated with disorders in water homeostasis [
]. In a rat spinal cord injury model, interventions targeting AQP4 localization using trifluoperazine improved central nervous system (CNS) edema and accelerated functional recovery [
]. However, the pharmacological interventions targeting aquaporins are still in infancy, rapid progress is being made. Owning the limitations of laboratory techniques, we were unable to measure the water permeability of cells and tissues after altering the expression of AQPs. To date, no specific antibodies have been engineered to establish the important role of AQP8 in a constipation mouse model. Future studies examining unique permeability characteristics in colon-specific AQP-knockout mice or drug-like small molecules and bona fide AQP inhibitors in clinical intervention may yield useful information.
In summary, we are first to document the subtypes of AQPs in colonic tissue and their potential mechanisms involved in FC. The abundance and distribution of AQPs in the human colon indicate their important roles in the maintenance of gut water homeostasis and in the pathophysiology of FC. Finally, upregulation and translocation of AQP8 activated by the MAPK pathway provides opportunity for promising therapeutic targets for the treatment of a large number of patients with FC.
Conflict of interest
None declared.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (81800475, 81670487), Zhejiang Provincial Medical and Health Science and Technology Project (2019RC186, 2020KY583) and Natural Scientific Foundation of Zhejiang Province, China (LQ19H030006). The authors would like to thank Dr. Ying Zhang for her efforts for this paper.
Author contributions
C.L, Z.H and N.D designed the experiments and interpreted the results. C.L and H.H performed the experiments. F.C, X.Z, Y.Z and Z.F analyzed the data. X.Z, Y.Z and J.J.K improved the project design and interpreted the results. J.J.K and C.L wrote the manuscript, which was further refined by all authors. N.D supervised the overall study.
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