Nature Medicine: doi: /nm.2171

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4 Table 1. Anti-cystic effect of Genz in jck mouse model of PKD No No of animals Gender Dose (% in feed) Body weight (g) K/BW ratio (%) Cystic volume (%BW) BUN (mg dl 1 ) 1 Vehicle 10 F ± ± ± ± Genz F ± ± 0.16* 0.5 ± 0.05* 26.4 ± 1.3* 3 Genz F ± 0.5* 3.35 ± 0.08* 0.5 ± 0.03* 32.3 ± 2.3* 4 Vehicle 9 M ± ± ± ± Genz M ± ± 0.24* 1.5 ± 0.1* 48.3 ± 3.1* 6 Genz M ± 0.6* 4.36 ± 0.14* 0.8 ± 0.1* 35.0 ± 4.5* * indicates P<0.05 for reduction in Genz treated vs. vehicle treated animals

5 Table 2. Anti-cystic effect of Genz in pcy model of PKD No No of Body weight K/BW ratio Cystic Fibrosis (%) animals (g) (%) volume (%BW) 1 Vehicle ± ± ± ± Genz ± 0.66* 2.64 ± 0.13* 0.40 ± 0.05* 17.0 ± 1.3* * indicates P<0.05 for reduction in Genz treated vs. vehicle treated animals

6 Table 3. Anti-cystic effect of Genz in Pkd1 cko mice No No of animals Body weight (g) K/BW ratio (%) Cystic volume (%BW) BUN (mg dl 1 ) 1 Vehicle ± ± ± ± Genz ± ± 0.29* 0.80 ± 0.12* ± 9.73* * indicates P<0.05 for reduction in Genz treated vs. vehicle treated animals

7 SUPPLEMENTARY METHODS Generation of Pkd1 conditional knockout animals: To generate the Pkd1 conditional knockout mice (Pkd1 tm1gztn ), a positive/negative gene replacement vector containing LoxP sites flanking exons 21, 22 and 23 of the Pkd1 gene, as well as a selectable cassette, was electroporated into 129S7- strain AMK ES cells. Correctly targeted ES cells were expanded, and a Cre-expression plasmid was transiently introduced to delete the drug selection cassette, yet retain the floxed exons Correctly recombined ES cells were microinjected into C57BL/6 mouse blastocysts to generate chimeric mice. All animals were backcrossed to C57BL/6 mice for >4 generations before use. Cell culture: The rat kidney epithelial cell line NRK-52E (NRK; ATCC) was maintained in high glucose Dulbecco s modified Eagle s medium (DMEM) containing 10% fetal bovine serum, 1 mm Glutamine, 100 U ml 1 penicillin, and 100 g ml 1 streptomycin (Invitrogen) at 37 C under 5% CO 2 in a humidified incubator. We dispersed cells using 0.25% Trypsin-EDTA (Invitrogen), and plated them at 5x10 5 cells per 10 cm petri dish for routine culture. We established WT and jck kidney epithelial cells from wild-type or jck mouse kidneys as follows. Briefly, we disaggregated 64 day old jck kidneys with collagenase Class IV (Worthington Biochemicals) in DMEM containing DNase I (Sigma- Aldrich) as described 1. We plated the resulting single cell suspension onto collagen I- coated plates (Becton-Dickinson) in DMEM containing 10% fetal calf serum for 24 hours, then maintained them in REGM (Lonza) until confluent. We transfected the cells with an SV40 T-ag (T-Ag P427L ) expression vector using a Nucleofector apparatus (Lonza) 2, then isolated clonal lines by serial dilution. We selected epithelial cells based

8 on phenotypic characterization of marker expression. Both WT and jck cells express SV40 T-Ag, cytokeratins, Na + K + -ATPase and AQP2 and are negative for expression of AQP1 and Tamm-Horsfall Glycoprotein. We maintained cells on collagen-i coated plates at 33 C. For IGF-I induction experiments, cells were serum starved in DMEM media containing 0.1% BSA with or without 2 M Genz for 16 hours prior to the addition of IGF-1 (RnD Systems) for the indicated times. All cells used for glycosphingolipid analysis were washed twice with TBS before homogenization in distilled water. sirna transfection: We obtained sirna reagents from Applied Biosystems. We transiently transfected GCS specific (Ugcg, catalog #s136139) and negative control (Negative Control #1, catalog # ) small interfering RNAs (sirnas) into NRK cells for 8 hours using 150 pmole sirna and 20 l Lipofectamine RNAiMAX (Invitrogen) per 100 mm petri dish according to the manufacturer s guidelines. We harvested cells for glycosphingolipid analysis and quantitative RT-PCR analysis 48 hours after transfection. Quantitative RT-PCR analysis: We extracted RNA and processed it for RT-PCR as previously described 3. We performed quantitative RT-PCR for glucosylceramide synthase (Ugcg) using Applied Biosystems predesigned TaqMan Gene Expression Assays Rn _m1 (rat) or Mm _m1 (mouse), and normalized to rodent 18S RNA.

9 Cell cycle analysis: To synchronize cells in the G0/G1 phase of the cell cycle, we treated them for 16 hours with 1.5 µg ml 1 aphidicolin (Sigma-Aldrich), then washed them twice with PBS before incubating them for 6 hours in growth media or media containing Genz where indicated. For GlcCer synthase sirna knock down studies, we performed sirna-mediated knockdown as described above. 24 hours after sirna transfection, we synchronized the cells as described above, then released them back into the cell cycle for 6 hours in growth media. We then disassociated the cells using trypsin/edta, fixed them with 70% ethanol, washed them twice with PBS and stained them in PBS containing 50 g ml 1 propidium iodide and 10 g ml 1 RNase A (Sigma-Aldrich). We analyzed samples on a FACSCalibur flow cytometer (Becton-Dickinson) and analyzed the data using FlowJo software (Tree Star Inc). 1. Humes, H.D., et al. Metabolic replacement of kidney function in uremic animals with a bioartificial kidney containing human cells. Am J Kidney Dis 39, (2002). 2. Loeber, G., Tevethia, M.J., Schwedes, J.F. & Tegtmeyer, P. Temperaturesensitive mutants identify crucial structural regions of simian virus 40 large T antigen. J Virol 63, (1989). 3. Natoli, T.A., et al. Pkd1 and Nek8 mutations affect cell-cell adhesion and cilia in cysts formed in kidney organ cultures. Am J Physiol Renal Physiol 294, F73-83 (2008).