Reduced expression of aquaporins in human intestinal mucosa in early stage inflammatory bowel disease

Abstract

Objectives: The aim of this study was to investigate the relationship between aquaporin (AQP) water channel expression and the pathological features of early untreated inflammatory bowel disease (IBD) in humans.

Methods: Patients suspected to have IBD on the basis of predefined symptoms, including abdominal pain, diarrhea, and/or blood in stool for more than 10 days, were examined at the local hospital. Colonoscopy with biopsies was performed and blood samples were taken. Patients who did not meet the diagnostic criteria for IBD and who displayed no evidence of infection or other pathology in the gut were included as symptomatic non-IBD controls. AQP1, 3, 4, 5, 7, 8, and 9 messenger RNA (mRNA) levels were quantified in biopsies from the distal ileum and colon by quantitative real-time polymerase chain reaction. Protein expression of selected AQPs was assessed by confocal microscopy. Through multiple alignments of the deduced amino acid sequences, the putative three-dimensional structures of AQP1, 3, 7, and 8 were modeled.

Results: AQP1, 3, 7, and 8 mRNAs were detected in all parts of the intestinal mucosa. Notably, AQP1 and AQP3 mRNA levels were reduced in the ileum of patients with Crohn's disease, and AQP7 and AQP8 mRNA levels were reduced in the ileum and the colon of patients with ulcerative colitis. Immunofluorescence confocal microscopy showed localization of AQP3, 7, and 8 at the mucosal epithelium, whereas the expression of AQP1 was mainly confined to the endothelial cells and erythrocytes. The reduction in the level of AQP3, 7, and 8 mRNA was confirmed by immunofluorescence, which also indicated a reduction of apical immunolabeling for AQP8 in the colonic surface epithelium and crypts of the IBD samples. This could indicate loss of epithelial polarity in IBD, leading to disrupted barrier function.

Conclusion: AQPs 1 and 8 and the aquaglyceroporins AQPs 3 and 7 are the AQPs predominantly expressed in the lower intestinal tract of humans. Their expression is significantly reduced in patients with IBD, and they are differentially expressed in specific bowel segments in patients with Crohn's disease and ulcerative colitis. The data present a link between gut inflammation and water/solute homeostasis, suggesting that AQPs may play a significant role in IBD pathophysiology.

Keywords: Crohn’s disease; aquaglyceroporins; aquaporins; inflammatory bowel disease; ulcerative colitis.

Figure 1

AQP1 expression is reduced in the ileum of CD patients. Notes: (A) 3D structural model of human AQP1. The model is drawn as a cartoon representation; only amino acids in the entrance and water selection region are shown as sticks. The numbering is consistent with the human AQP sequence. Complete sequence identities between bovine AQP1 (PDB ID 1J4N) and human AQP1, 3, and 7 are colored red, and nonidentical sequences are colored light blue. The water in the water channels is marked as W. (B) AQP1 mRNA levels relative to GAPDH in the ileum, right and left colon. ***P<0.001. (C) Immunofluorescense labeling of samples from control (upper panel), CD (middle panel), and UC (lower panel) with anti-AQP1 antibody (red) and DAPI staining of the cell nuclei (blue). For AQP1 in general, the labeling was not concentrated in the luminal epithelium, but mainly in the capillary endothelia and erythrocytes (arrowhead). Little or no staining was present in the mucosa epithelium. Scale bar: 50 μm. Abbreviations: 3D, three-dimensional; AQP, aquaporin; CD, Crohn’s disease; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ID, identification number; mRNA, messenger RNA; PDB, Protein Data Bank; UC, ulcerative colitis.

Figure 2

AQP3 expression is reduced in the ileum of CD patients. Notes: (A) 3D structural model of human AQP3. The model is drawn as a cartoon representation; only amino acids in the entrance and water selection region are shown as sticks. The numbering is consistent with the individual human AQP sequence. Complete sequence identities between bovine AQP1 (PDB ID 1J4N) and human AQP1, 3, and 7 are colored red, and nonidentical sequences are colored light blue. The water in the water channels is marked as W. (B) AQP3 mRNA levels relative to GAPDH in the ileum, right and left colon. **P<0.01. (C) Immunofluorescense staining with anti-AQP3 antibody (red) and DAPI staining of the cell nuclei (blue). Immunoreactivity of AQP3 was high in general. In the ileal biopsy of the control (upper panel) and the UC patient (lower panel), as well as in the sample from the colon of the control (upper panel), there was a distinct staining along the apical parts of the surface epithelium (arrows). The apical staining was reduced in the surface epithelium of the ileum biopsy from the CD patient (middle panel; arrowhead). Scale bar: 50 μm. Abbreviations: 3D, three-dimensional; AQP, aquaporin; CD, Crohn’s disease; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ID, identification number; mRNA, messenger RNA; PDB, Protein Data Bank; UC, ulcerative colitis.

Figure 3

AQP7 expression is reduced in the ileum and colon of UC patients. Notes: (A) 3D structural model of human AQP7. The model is drawn as a cartoon representation; only amino acids in the entrance and water selection region are shown as sticks. The numbering is consistent with the individual human AQP sequence. Complete sequence identities between bovine AQP1 (PDB ID 1J4N) and human AQP1, 3, and 7 are colored red, and nonidentical sequences are colored light blue. The water in the water channels is marked as W. (B) AQP7 mRNA levels relative to GAPDH in the ileum, right and left colon. **P<0.01, ***P<0.001. (C) Immunofluorescense staining with anti-AQP7 antibody (red) and DAPI staining of the cell nuclei (blue): AQP7 staining was mainly present in the basolateral parts of surface epithelia in the ileum and colon (arrows), but also in the basolateral parts of colonic crypts (arrow). Especially in CD, and also in UC, the staining was weaker in both crypts and surface epithelia of the colon (arrowheads). Scale bar: 50 μm. Abbreviations: 3D, three-dimensional; AQP, aquaporin; CD, Crohn’s disease; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ID, identification number; mRNA, messenger RNA; PDB, Protein Data Bank; UC, ulcerative colitis.

Figure 4

AQP8 expression is reduced in the ileum and colon of UC patients. Notes: (A) 3D structural model of human AQP8. The model is drawn as a cartoon representation; only amino acids in the entrance and water selection region are shown as sticks. The numbering is consistent with the individual human AQP sequence. Complete sequence identities between human AQP8 and the template model human AQP5 (PDB ID 3D9S) are colored red, and nonidentical sequences are colored light blue. The water in the water channels is marked as W. (B) AQP8 mRNA levels relative to GAPDH in the ileum, right and left colon. *P<0.05, **P<0.01, ***P<0.001. (C) Immunofluorescense staining with anti-AQP8 antibody (red) and DAPI staining of the cell nuclei (blue). A distinct AQP8 staining is present at the apical parts of the ileal and colonic surface epithelium (arrows), as well as the apical parts of colonic crypts (arrow). In ileal samples, there was no difference in the degree of staining between the control and CD and UC, whereas the signal was significantly decreased in intensity in the colonic samples of CD and UC (arrowheads). The distinct apical AQP8 staining in the colonic crypts and surface epithelium was lost and a faint basolateral labeling appeared in the CD and UC samples. Scale bar: 50 μm. Abbreviations: 3D, three-dimensional; AQP, aquaporin; CD, Crohn’s disease; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ID, identification number; mRNA, messenger RNA; PDB, Protein Data Bank; UC, ulcerative colitis.

Figure 5

Elevated COX-2 expression in UC and TNF-α and IL-23 expression in CD. Notes: (A) COX-2 mRNA levels relative to GAPDH in the ileum, right and left colon. *P<0.05, **P<0.01, ***P<0.001. (B) TNF-α mRNA levels relative to GAPDH in the ileum, right and left colon. *P<0.05. (C) IL-23A mRNA levels relative to GAPDH in the ileum, right and left colon. *P<0.05. Abbreviations: CD, Crohn’s disease; UC, ulcerative colitis; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL, interleukin; mRNA, messenger RNA; COX-2, cyclooxygenase 2; TNF-α, tumor necrosis factor-α.