Figure 5. PGE₂-induced organoid swelling is sensitive to inhibitors targeted to CFTR, NKCC1, KCNQ1 and KCNN4.

(a) mRNA of membrane-bound ion channels and transporters are expressed in cultured human small intestinal organoids. Semi-quantitative RT-PCR analysis shows expression of CFTR, NKCC1, KCNQ1 and KCNN4 in small intestine-derived organoids. Results acquired from the whole small intestinal tissue sample are shown as a reference. dH₂O served as a negative control. The number of bp indicates the length of the targeted amplification region for each gene. (b) Immunostaining of NKCC1 and KCNQ1 shows their expression at the basolateral membrane of cultured small intestinal organoids. Organoids were fixed and subjected to immunostaining using primary antibodies specific for NKCC1 or KCNQ1. The expression was visualized by FITC-labeled Tyramide (green). The stainings using non-immunized mouse IgG and rabbit IgG served as negative controls. Scale bar represents 100 μm. (c,d) Inhibitory effects of various compounds on the PGE₂-induced swelling of jejunal organoids that were established from the uninflamed region of a CD patient (c) and from the healthy mucosa of a non-IBD patient (d). Inhibitors were added 1 hour prior to PGE₂-induction at the following concentration: GlyH-101 (50 μM), PPQ-102 (10 μM), CFTR Inhibitor-172 (CFTRinh-172) (50 μM), glyburide (500 μM), 4-AP (1 mM), XE 991 (10 μM), HMR 1556 (10 μM), TRAM 34 (10 μM) and bumetanide (100 μM). PGE₂-induced swelling was quantified 30 min after the PGE₂-induction (10⁻⁹ M). Data are shown as the mean ± SEM of three independent wells. *indicates P < 0.05, **indicates P < 0.005, ***indicates P < 0.0005, ****indicates P < 0.0001 as determined by two-sided Student’s t-test compared to the data of Inhibitor (−). All results are representative of at least three independent experiments.