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

Figures & data

Table 1 Characterization of the study subjects

	CD	UC	Non-IBD control
n	22	10	11
Male, n (%)	10 (45)	8 (80)	6 (55)
Age median (range)	30.7 (19.2–52.5)	30.2 (18.1–61.0)	32.1 (20.7–41.1)
Distribution
Ileitis, n (%)	4 (18)	0	0
Colitis, n (%)	8 (36)	10 (100)	0
Ileocolitis, n (%)	10 (45)	0	0
Symptoms
Abdominal pain, n (%)	12 (55)	5 (50)	7 (64)
Diarrhea, n (%)	15 (68)	9 (90)	5 (45)
Blood in stool, n (%)	5 (23)	9 (90)	3 (27)
Fever, n (%)	1 (5)	2 (20)	0
Smoking, n (%)	7 (32)	1 (11)a	3 (27)
CRP mg/L, median (range)	26 (5–108)	24 (7–94)	7 (7–7)b
Fecal calprotectin, mg/kg, median (range)	118 (1–6,591)	740 (190–10,825)	4.5 (1–28)
Clinical activity index median (range)	4.5 (1–29)c	5 (3–14)d

Notes:

a One missing value.

b Lower cutoff value 7 mg/L.

c Harvey–Bradshaw index.

d Simple Clinical Colitis Activity Index.

Abbreviations: CD, Crohn’s disease; UC, ulcerative colitis; IBD, inflammatory bowel disease; CRP, C-reactive protein.

Table 2 Disease location, clinical and endoscopic activity indices, CRP, and fecal calprotectin values for patients with Crohn’s disease and those with ucerative colitis

Patient ID	Location	Age, years	Sex	HBI	Modifieda SES-CD^Citation56	CRP, mg/L	Fecal calprotectin, mg/kg
Crohn’s disease
51	L1	22	F	17	2	38	285
31	L1	38	F	5	1	7	29
168	L1	39	F	1	2	25	721
204	L1	52	M	1	2	5	–
53	L2	27	M	29	2	43	–
73	L2	27	M	6	2	7	45
121	L2	29	F	3	1	12	52
26	L2	32	M	4	2	7	1
37	L2	35	F	2	2	7	30
81	L2	37	F	1	2	7	843
9	L2	44	M	8	1	7	32
4	L2	52	M	6	2	7	477
1,034	L3	19	F	6	2	91	–
165	L3	20	F	2	2	26	477
94	L3	21	F	13	2	47	956
107	L3	22	F	7	2	11	118
194	L3	26	M	3	1	7	–
146	L3	27	M	10	2	108	6,591
169	L3	32	F	4	1	11	1
137	L3	33	M	4	1	61	16
36	L3	39	F	4	1	7	–
49	L3	39	M	9	2	21	354
Patient ID	Extension	Age	Sex	SCCAI	Mayob score	CRP	Fecal calprotectin
Ulcerative colitis
86	E3	18	M	4	2	7	740
1,008	E3	19	M	3	1	7	–
59	E3	20	M	4	2	7	303
84	E3	20	M	4	2	7	707
19	E3	28	F	11	3	94	10,825
1,021	E3	31	M	14	3	–	–
1,009	E3	36	F	3	2	7	–
2,012	E3	41	M	6	2	13	7,221
44	E3	41	M	7	3	7	190
93	E3	61	M	11	2	68	3,878

Notes:

a Regarding the mucosal disease activity, in CD we differentiated patients with subtle mucosal injury (aphtous lesions only) from those with a more severe endoscopic grade of inflammation (ulcers or more) using a modification of the SES-CD. This simple two-category index assessment was similar to a previous report from the IBSEN cohort.

b Partial endoscopic Mayo score. – indicates missing value.

Abbreviations: CD, Crohn’s disease; HBI, Harvey–Bradshaw Index; ID, identification number; SCCAI, Simple Clinical Colitis Activity Index; F, female; M, male; SES-CD, Simple Endoscopic Score for Crohn’s Disease; CRP, C-reactive protein; L, location according to the Montreal classification; E, extension, according to the Montreal classification; IBSEN, Inflammatory Bowel Disease South-Eastern Norway.

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.

Table 3 Comparison of aquaporin mRNA levels in the small and large bowel biopsies from CD and UC patients and non-IBD patients controls

	Ileum vs right colon			Ileum vs left colon			Right vs left colon
	mRNA ratio	P-value	q-value	mRNA ratio	P-value	q-value	mRNA ratio	P-value	q-value
CD
AQP1	5.49	<0.001	<0.001	2.64	0.002	0.009	0.48	0.002	0.01
AQP3	4.92	<0.001	<0.001	4.84	<0.001	<0.001	0.98	0.45	0.71
AQP7	0.92	0.72	0.72	0.67	0.53	0.62	0.73	0.43	0.71
AQP8	0.01	<0.001	0.01	0.03	0.004	0.07	2.85	0.11	0.46
COX-2	1.36	0.45	0.51	1.48	0.96	0.96	1.08	0.63	0.84
TNF-α	3.22	<0.001	0.002	2.20	<0.001	0.01	0.68	0.28	0.71
IL-23A	3.30	0.38	0.51	2.69	0.04	0.07	0.82	1.0	1.0
UC
AQP1	12.85	<0.001	<0.001	6.64	<0.001	<0.001	0.52	0.11	0.51
AQP3	40.62	<0.001	<0.001	24.78	<0.001	<0.001	0.61	0.61	0.62
AQP7	2.18	0.04	0.07	1.68	0.3	0.38	0.77	0.64	0.68
AQP8	0.002	0.02	0.07	0.02	0.09	0.19	13.45	0.01	0.12
COX-2	0.78	0.12	0.15	0.43	0.12	0.19	0.54	0.41	0.68
TNF-α	1.54	0.41	0.41	1.10	0.86	0.86	0.72	0.41	0.68
IL-23A	0.47	0.32	0.37	0.26	0.08	0.19	0.55	0.54	0.68
Controls
AQP1	8.50	0.002	0.005	4.25	0.004	0.01	0.5	0.23	0.53
AQP3	28.25	<0.001	0.001	18.44	<0.001	<0.001	0.65	0.48	0.67
AQP7	1.46	0.31	0.52	0.59	0.24	0.48	0.40	0.04	0.28
AQP8	0.0003	<0.001	0.002	0.0003	<0.001	0.002	0.96	0.7	0.70
COX-2	0.76	0.54	0.66	0.93	0.65	0.75	1.23	0.43	0.67
TNF-α	0.77	0.61	0.66	1.45	0.89	0.89	1.88	0.22	0.53
IL-23A	1.61	0.1	0.22	1.36	0.58	0.75	0.85	0.7	0.70

Note: mRNA levels detected by qRT-PCR are given as mRNA ratios calculated from delta delta C_T values with P-values and multiple testing adjusted P-values (q-values).

Abbreviations: mRNA, messenger RNA; CD, Crohn’s disease; UC, ulcerative colitis; IBD, inflammatory bowel disease; AQP, aquaporin; COX-2, cyclooxygenase 2; TNF-α, tumor necrosis factor-α; IL, interleukin; qRT-PCR, quantitative real-time polymerase chain reaction; vs, versus.

Table 4 Summary of the general trends in AQP expression detected by mRNA levels

	AQP1	AQP3	AQP7	AQP8
CD
Ileum	↓	↓	–	–
Colon	–	–	–	–
UC
Ileum	–	–	↓	↓
Colon	–	–	↓	↓

Abbreviations: mRNA, messenger RNA; CD, Crohn’s disease; UC, ulcerative colitis; AQP, aquaporin; –, no trend.

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.

Table 5 Summary of key properties of AQPs

Aquaporins	Organism	Main location	Our findings in the GI tract	Comment	References
AQP0	Mammals	Lens	na	AQP	^Citation57
AQP1	Mammals	Red blood cells	Reduced amounts in ileum in CD	AQP	^Citation58
		Kidney			^Citation59
		Lung			^Citation60
		Vascular endothelium			This study
		Brain, eye: glial cells
		Neurons
AQP2	Mammals	Kidney	na	AQP	^Citation82
		Vas deferens			^Citation61
		Neurons			^Citation62
AQP3	Mammals	Kidney	Reduced amounts in ileum in CD	Aquaglyceroporin	^Citation63
		Skin		Water, glycerol, urea	This study
		Lung
		Eye
		Colon
AQP4	Mammals	Brain, muscle, kidney, lung, stomach, small intestine	–	AQP	^Citation64
				Main AQP in the brain	^Citation65
					^Citation66
		Neurons
		Vascular endothelium
AQP5	Mammals	Salivary/lacrimal gland	–	AQP	^Citation67
		Sweat gland			^Citation68
		Lung			^Citation27
		Cornea
AQP6	Mammals	Kidney	na	AQP	^Citation7
				Anion channel
AQP7	Mammals	Adipose tissue	Reduced amounts	Aquaglyceroporin	^Citation69
		Kidney	in ileum and	Water, glycerol, urea, arsenite	This study
		Testis	colon in UC
AQP8	Mammals	Testis, kidney, liver, pancreas, small intestine, colon	Reduced amounts in ileum and colon in UC	AQP	^Citation70
					This study
AQP9	Mammals	Liver, leukocytes, brain, testis	–	Aquaglyceroporin	^Citation71
				Water, glycerol, urea, arsenite
AQP10	Mammals	Small intestine	na	Aquaglyceroporin	^Citation72
		Kidney		Water, glycerol, urea	^Citation6
AQP11	Mammals	Kidney ER	na	Super-AQP	^Citation73
				Divergent NPA motif, sign cysteine
AQP12	Mammals	Pancreas	na	Super-AQP	^Citation74
				Divergent NPA motif, sign cysteine
AQPZ	E. coli, Mycobacterium abscessus*	na	na	AQP	^Citation75
					This study*
GlpF	E. coli, Haemophilus influenzae, Streptococcus pyogenes*	na	na	Aquaglyceroporin	^Citation75
					^Citation76
					This study*
AQY1/2	Yeast	na	na	AQP	^Citation77
					^Citation78
GLPY1/2	Yeast	na	na	Aquaglyceroporin	^Citation79
PIPs/TIPs/	Plants	na	na	AQP	^Citation80
NIPs/SIPs					^Citation81

Note: – indicates not detected above threshold in the gut.

Abbreviations: AQP, aquaporin; AQPZ, aquaporin-related pore; na, not applicable; E. coli, Escherichia coli; ER, endoplasmic reticulum; glpF, glycerol facilitator; GI, gastrointestinal; NPA, asparagine-proline-alanine; CD, Crohn’s disease; NIPs, nodulin 26-like intrinsic membrane proteins; PIPs, plasma membrane intrinsic proteins; SIPs, small basic intrinsic proteins; TIPs, tonoplast intrinsic proteins; UC, ulcerative colitis; AQY1/2, yeast aquaporin; GLPY1/2, yeast glycerol facilitation-like protein.

Figure S1 AQP expression in healthy human tissues.

Notes: The left panel (positive controls) shows micrographs with immunofluorescence labeling of human samples with AQP1, AQP3, and AQP7 antibodies. The tubular lumina are marked L. AQP1 is localized at the brush border and basolateral membranes of the renal proximal tubule (arrow, upper panel), AQP3 is localized at the basolateral membranes of the collecting ducts (arrowheads, middle panel) and AQP7 is localized at the plasma membrane of dipocytes (arrow, lower panel). The right panel (negative controls) shows sections from the same tissue as in the left panel, treated the same way, in absence of the primary antibody. No labeling is observed, excluding nonspecific labeling with the secondary antibodies. Scale bar: 50 μm.

Abbreviation: AQP, aquaporin.

Table S1 TaqMan gene expression assays employed to monitor aquaporin expression

Gene product	Substrate	ABI assay IDa	Interrogated sequence RefSeq	Translated protein RefSeq	NCBI location chromosome	Exon boundary
AQP1	H2O	Hs00166067_m1b	NM_198098.1	NP_932766.1	Chr. 7 – 30951415–30965131	1–2, assay loc: 492
AQP3	H2O Glycerol H2O2 NH4^+ Urea	Hs00185020_m1	NM_004925.3	NP_004916.1	Chr. 9 – 33441158–33447590	2–3 assay loc: 297
AQP4	H2O	Hs00242342_m1	NM_001650.4	NP_001641.1	Chr. 18 – 24432007–24445716	2–3 assay loc: 508
AQP5	H2O	Hs00387048_m1	NM_001651.2	NP_001642.1	Chr. 12 – 50355279–50359461	1–2 assay loc: 888
AQP7	H2O Glycerol Urea	Hs00357359_m1	NM_001170.1	NP_001161.1	Chr. 9 – 33384948–33402517	2–3 assay loc: 199
AQP8	H2O H2O2 NH4^+	Hs00154124_m1	NM_001169.2	NP_001160.2	Chr. 16 – 25228285–25240253	2–3 assay loc: 339
AQP9	H2O Glycerol Urea	Hs00175573_m1	NM_020980.3	NP_066190.2	Chr. 15 – 58430408–58478110	1–2 assay loc: 467
TNF-α		Hs99999043_m1	NM_000594.2	NP_000585.2	Chr. 6 – 31543350–31546112	1–2 assay loc: 360
IL-23A		Hs00372324_m1	NM_016584.2	NP_057668.1	Chr. 12 – 56732663–56734194	1–2 assay loc: 323
COX-2/PTGS2		Hs00153133_m1	NM_000963.2	NP_000954.1	Chr. 1 – 186640944–186649559	5–6 assay loc: 777

Notes:

a All assays used dye label FAM;

b An assay whose probe spans an exon junction and does not detect genomic DNA.

Abbreviations: HS, Homo sapiens; AQP, aquaporin; IL, interleukin; loc, location; COX-2, cyclooxygenase 2; TNF-α, tumor necrosis factor-α.

Table S2 Comparison of aquaporin mRNA levels in the small and large bowel biopsies from CD and UC patients and non-IBD patient controls

	Ileum vs right colon			Ileum vs left colon			Right vs left colon
	mRNA ratio	P-value	q-value	mRNA ratio	P-value	q-value	mRNA ratio	P-value	q-value
CD
AQP4	Nega	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg
AQP5	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg
AQP9	1.77	0.07	0.12	1.16	0.54	0.62	0.65	0.89	1.0
UC
AQP4	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg
AQP5	Neg	Neg	Neg	0.22	0.41	0.46	1.13	0.55	0.68
AQP9	0.33	0.04	0.07	0.31	0.12	0.19	0.95	0.68	0.7
Controls
AQP4	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg
AQP5	5.0	0.35	0.52	Neg	Neg	Neg	Neg	Neg	Neg
AQP9	0.55	0.66	0.66	0.21	0.51	0.75	Neg	Neg	Neg

Notes: mRNA levels detected by qRT-PCR are given as mRNA ratios calculated from delta delta C_T values with P-values and multiple testing adjusted P-values (q-values).

a Neg = negative/below detection level (very low levels or no mRNA detected/the concentration of corresponding mRNAs was below the threshold for detection by qRT-PCR).

Abbreviations: AQP, aquaporin; CD, Crohn’s disease; COX-2, cyclooxygenase 2; IBD, inflammatory bowel disease; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction; UC, ulcerative colitis; vs, versus.

Table S3 Comparison of aquaporin mRNA levels from CD and UC patients and non-IBD patient controls in the small and large bowel biopsies

	CD vs controls			CD vs UC			UC vs controls
	mRNA ratio	P-value	q-value	mRNA ratio	P-value	q-value	mRNA ratio	P-value	q-value
Ileum
AQP1	0.54	0.07	0.13	0.43	<0.001	0.002	1.28	0.37	0.61
AQP3	0.15	<0.001	0.003	0.16	<0.001	<0.001	0.94	0.87	0.87
AQP7	1.16	0.67	0.67	4.31	0.002	0.006	0.27	0.003	0.02
AQP8	7.33	0.29	0.36	260.96	0.005	0.01	0.03	0.07	0.17
COX-2	2.56	0.075	0.13	0.57	0.31	0.38	4.51	0.014	0.05
TNF-α	2.94	0.06	0.13	4.00	0.01	0.02	0.73	0.62	0.69
IL-23A	4.43	0.03	0.10	6.93	0.01	0.017	0.64	0.56	0.69
Right colon
AQP1	0.83	0.72	0.72	0.90	0.98	0.98	0.84	0.78	0.78
AQP3	0.82	0.63	0.72	1.26	0.53	0.77	0.65	0.40	0.45
AQP7	1.97	0.37	0.59	10.98	<0.001	0.001	0.18	0.06	0.17
AQP8	0.25	0.03	0.27	52.46	0.008	0.04	0.01	0.001	0.009
COX-2	1.41	0.21	0.51	0.32	0.02	0.051	4.35	0.003	0.02
TNF-α	0.76	0.68	0.72	2.06	0.07	0.12	0.37	0.17	0.31
IL-23A	2.57	0.21	0.51	1.17	0.77	0.87	2.19	0.27	0.35
Left colon
AQP1	0.87	0.77	0.86	1.06	0.84	0.84	0.82	0.67	0.77
AQP3	0.54	0.09	0.34	0.78	0.31	0.40	0.70	0.30	0.40
AQP7	1.09	0.86	0.86	11.65	<0.001	<0.001	0.09	<0.001	<0.001
AQP8	0.08	0.004	0.03	247.47	<0.001	0.002	0.0003	<0.001	<0.001
COX-2	1.60	0.37	0.49	0.16	0.003	0.007	9.81	<0.001	<0.001
TNF-α	2.08	0.26	0.42	2.17	0.03	0.06	0.96	0.95	0.95
IL-23A	2.66	0.13	0.43	0.79	0.58	0.65	3.36	0.06	0.12

Note: mRNA levels detected by qRT-PCR are given as mRNA ratios calculated from delta delta C_T values with P-values and multiple testing adjusted P-values (q-values).

Abbreviations: AQP, aquaporin; CD, Crohn’s disease; COX-2, cyclooxygenase 2, IBD, inflammatory bowel disease; IL, interleukin; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction; TNF-α, tumor necrosis factor-α; UC, ulcerative colitis; vs, versus.

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