Electronic supplementary material (ESM) J Mol Med Glucose promotes secretion-dependent renal cyst growth

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1 Electronic supplementary material (ESM) J Mol Med 15 Glucose promotes secretion-dependent renal cyst growth Andre Kraus 1, Gunnar Schley 1, Karl Kunzelmann, Rainer Schreiber, Dorien Peters 3, Ruth Stadler, Kai-Uwe Eckardt 1, Bjoern Buchholz 1 Corresponding author: Bjoern Buchholz, MD Department of Nephrology and Hypertension Friedrich-Alexander-University Erlangen-Nürnberg Ulmenweg 18 D 915 Erlangen, Germany Phone: Fax: Bjoern.Buchholz@uk-erlangen.de

2 Supplemental Table 1: Real-time PCR Primer Gene Species Primer sequence 5'-3' ANO1 Canine fw CTGCACGACGGAGACTACGA rev GTAGCTCGCCCACTCTTGGT ANO6 Canine fw AAGCAGCCCTTGGACCTTATC rev AGTGTAGTAGCCCAGCCAAGC PYR Canine fw ACAGGTGGGACAGAACTGACG rev TGTCCGCCTAGAATCCTCACT NKCC1 Canine fw GATGATTTGAGAGAAGGTGCAGCAT rev GACAAGTGTGTTTGGCTTCATACG CFTR Canine fw CGGAGACAACGAAGACAGTCTGT rev CTTCGGTGAATGCTCTGACCTT HPRT Canine fw CGGCTTGCTCGAGATGTGAT rev GAGCACACAGAGGGCTACGAT ANO1 Mouse fw TGAGGGTGACAACGTTGAGTTC rev CGTAACTTGCCCATTCCTCATAC ANO6 Mouse fw TGGCAACCTCAACTGGTTCA rev ACTCCTGCTCTGGCTTGATGA PYR Mouse fw GGGACGAACTGGGATACAAGTG rev ACACGGGCAACAGCACGTA NKCC1 Mouse fw GGTGGTGCAATTGGCTTGAT rev CGAATCCGACAACATACATAGCA CFTR Mouse fw CAGCTCAAACAACTGGAATCTGA rev GCTCGAAGTGTCCAGAGTCCTT PKD1 reference PKD1 deletion Mouse Mouse fw TTACGGGCTGCAGGAATTCGAT rev ATGCCCAAGATGAGGACAATGC fw TGCTCTTGGTAGCTGTAGCTGT rev AGCTGCTTGAGAGAAGCCACAT 18S Mouse fw TGATTAAGTCCCTGCCCTTTGTA rev CGATCCGAGGGCCTCACTA

3 Supplemental Figure 1 a b c I SC initial [µa/cm²] 1 I SC camp [µa/cm²] I SC UTP [µa/cm²] peak UTP 1µM plateau UTP 1µM Supplemental figure 1. Changes in transepithelial conductances by elevated glucose concentration is not referable to changes in medium osmolality. plmdck cells were grown as polarized monolayers on permeable supports and pre-incubated with either low (LG, 1 mg/dl), high glucose (, 5 mg/dl) or low glucose medium supplemented with mm mannitol (Ma) in order to increase osmolality to that of high glucose medium for 5 days. Then, cells were mounted into a perfused micro Ussing chamber and the luminal and basolateral surfaces of the epithelium were perfused continuously with ringer solution supplemented with either low glucose, high glucose, or mannitol. a Summary of the obtained initial short circuit currents (Isc) at the beginning of the experiment which was significantly elevated in cells but not in LG or Ma cells. b Summary of Isc upon perfusion of the cells with 1 µm 3-Isobutyl-1-methylxanthin (IBMX) together with 1 µm forskolin (Isc camp). c Summary of Isc upon application of 1 µm UTP (Isc UTP). P<.5, ANOVA. N=5-18.

4 Supplemental Figure a I SC [µa/cm²].. Flow stop Flow on b ΔI SC Flow on [µa/cm²].. Supplemental figure. The initial short circuit currents obtained in high glucose cells are not referable to changes in bath flow. plmdck cells were grown as polarized monolayers on permeable supports and pre-incubated with either low (LG, 1 mg/dl), high glucose (, 5 mg/dl) or low glucose medium supplemented with mm mannitol (Ma). Then, cells were mounted into a micro Ussing chamber and perfused continuously with ringer solution supplemented with either low glucose, high glucose, or mannitol. a Summary of the short circuit currents (Isc) obtained by stopping bath flow (Flow stop) and by switching on bath flow again (Flow on). b Summary of the change in Isc (ΔIsc) between switching bath flow off/on. N=6-8.

5 Supplemental Figure 3 a LG b c I SC UTP peak [%] min I SC UTP peak [%] min I SC UTP [µa/cm²] min peak UTP 1µM plateau UTP 1µM d 1µM UTP V te [mv] min Supplemental figure 3. The reduction of Isc UTP in high glucose treated cells may be refered to emptied calcium stores. plmdck cells were grown as polarized monolayers on permeable supports and pre-incubated with either low (LG, 1 mg/dl) or high glucose (, 5 mg/dl) medium. Then, cells were mounted into a micro Ussing chamber and perfused continuously with ringer solution supplemented with low glucose or high glucose. a Repetitive application of 1 µm UTP starting at time point, and repeated after 5 and 1 minutes led to a significant reduction of Isc UTP compared to the first application in (a) low glucose and (b) high glucose treated cells. Further application of 1 µm UTP in a 1 minute interval resulted in partial recovery of Isc UTP in (a) low glucose and (b) high glucose cells. c Application of UTP 3 minutes after the initial Isc obtained in high glucose cells resulted in much stronger Isc UTP compared to Isc UTP obtained after 5 minutes. d Original trace of the transepithelial potential (Vte) of high glucose () cells showing recovery of the negative deflection of Vte upon administration of UTP if applied 3 minutes after the initial deflection of Vte. *p<.5, paired t-test. N=5-1.