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. 2020 Mar 18;98(5):659–671. doi: 10.1007/s00109-020-01894-y

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — M1 renal organoid and cyst model. a RT-PCR analysis of mRNA expression of ion channels and receptors in mouse kidney and M1 collecting duct cells: TMEM16A, TMEM16F, αβγ-ENaC, NKCC1, PKD1, PKD2, and the receptors patched 1,2 (Ptch1,2). Similar expression patterns were found in mouse kidney and M1 cells. ± indicate presence/absence of reverse transcriptase. b Time-dependent development of renal organoids in Matrigel (n = 20). Bars = 20 μm. c Reconstructed 3D image from a renal M1 organoid with a view inside the organoid. Bar = 20 μm. d, e Differential interference contrast (DIC) image and immunocytochemistry of a cross-section of an organoid. Green, primary cilia; red, CFTR; blue, DAPI. Bars = 20 μm. f, g Increase of the volume of M1 organoids by shRNA-knockdown of PKD1 or PKD2. The presence of the NKCC1-inhibitor bumetanide (100 μM) did not change the size of control organoids (treated with scrambled RNA; scrbld), but inhibited further enlargement by shRNA-knockdown of PKD1 or PKD2, indicating fluid secretion upon knockdown of PKD1,2. Bars = 50 μm. h Increase of proliferative activity in shPKD1/shPKD2 organoids as indicated by Ki-67 staining. Bars = 20 μm. Mean ± SEM (number of organoids measured). ^#Significant difference when compared with scrambled (p < 0.05; unpaired t test). ^§Significant difference when compared to absence of bumetanide (p < 0.05; unpaired t test)