A Tumor Necrosis Factor-Alpha-Mediated Pathway Promoting Autosomal Dominant Polycystic Kidney Disease.

Transcription

1 A Tumor Necrosis Factor-Alpha-Mediated Pathway Promoting Autosomal Dominant Polycystic Kidney Disease. Xiaogang Li, Brenda S. Magenheimer, Sheng Xia, Teri Johnson, Darren P. Wallace, James P. Calvet and Rong Li a. TNF-α (16 hours) Golgi merge TNF-α (0 hour) tub merge c. TNF-α (16 hours) tub merge Supplemental Figure 1. TNF-α signaling disrupts cilia localization in IMCD cells. a, After TNF-α treatment was strongly enriched in perinuclear regions that partially overlapped the Golgi marker, GM-130. b, c, Double immunofluorescence staining of endogenous (green) using the YCC2 aniti- antibody and acetylated tubulin (tub, red) before (b) or after (c) treatment with TNF-α for 16 hr, observed with confocal microscopy. TNF-α stimulation results in the loss of localization along the primary cilia, however, with this antibody can still be detected in the basal body regions in some of the treated cells.

2 Li et al., Supplemental Figure 2 TUNEL DIC TNF-α induction (16 hours) TNF-α induction (0 hour) Positive control Supplemental Figure 2. TUNEL staining of the TNF-α treated IMCD cells. The loss of proper localization of was not due to cell death, as the TNF-α-treated cells showed normal morphology and were negative for TUNEL staining. Fixed cells treated with TACS-nuclease were used as the positive control.

3 Li et al., Supplemental Figure 3 a. sirna UN sirna UN 0h 16h sirna UN 0h 16h FIP2 05 FIP2 08 FIP2 Actin Actin Actin c. TNF-α (0 hour) tub merge d. TNF-α (16 hours) tub merge Supplemental Figure 3. Transfection of sirna against FIP2 into IMCD cells inhibited FIP2 expression and rescued the TNF-α effect on localization. a, IMCD cells transfected with 4 different sirna against FIP2 showing diminished FIP2 expression. UN, untransfected control. b, Western blot to determine the FIP2 levels in IMCD cells that were transfected with sirna#05 and #08 and then treated with TNF-α for 16 hours. c, d, Immunostaining with YCC2 anti- antibody of the IMCD cells transfected with sirna#08 before and after TNF-α induction shows that the sirna rescued the cilia localization in cells treated with TNF-α.

4 Li et al., Supplemental Figure 4 a. TNF-α 0 h 4 h 16 h TNF-α 0 h 4 h 16 h PC1 PC1 185 kda 185 kda 121 kda 121 kda IP: IB: PC1 IP: PC1 IB: PC1 IP: IB: IP: PC1 IB: c e TNF-α (0 h) TNF-α (16 h) d f Supplemental Figure 4. TNF-α disrupts PC1 and complex formation but not PC1 cilia localization. a and b, Reciprocal co-immunoprecipitation of PC1 and. After TNF-α induction, PC1 failed to be co-immunoprecipitated by a antibody (a), or by a PC1 antibody (b), even though the same amounts of PC1 and were immunoprecipitated with their respective antibodies at all time points. c and d, Merged double immunofluorescence staining of endogenous PC1 (green) and acetylated tubulin (red) before (c) or after (d) treatment with TNF-α, observed with confocal microscopy. The images shown are projections of all confocal sections. e and f show enlarged cilia images in the boxed regions in c and d, respectively.

5 Li et al., Supplemental Figure 5 a. -TNF-α +TNF-α 10X DBA a. 100X 10X LTA b. 100X Supplemental Figure 5. TNF-α initiates cyst formation from collecting ducts and proximal tubules. TNF-α treated (right panels) or untreated (left panels) Pkd2 +/- embryonic kidneys were stained with either DBA (a), a collecting duct marker, or LTA (b), a proximal tubule marker. a and b show enlarged tubule or cyst images in the boxed regions in a and b, respectively.

6 Li et al., Supplemental Figure 6 + TNF-α Supplemental Figure 6. TNF-α caused formation of multiple small cysts in Pkd2 +/ mouse kidneys. Two Pkd2 +/ mice treated with TNF-α from week 8.5 to week 18.5 showed the presence of multiple kidney cysts. The inset in each image shows cyst in the boxed region at a higher magnification.

7 Supplemental Methods Primary cultures of human kidney cells Primary cultures of ADPKD and normal human kidney (NHK) cells were generated with the assistance of the PKD Biomaterials Research Core laboratory at the University of Kansas Medical Center (KUMC). Normal regions of human kidneys, confirmed by histological examination, were collected from nephrectomy specimens removed for the treatment of renal carcinomas. ADPKD kidneys were obtained from UMKC or hospitals participating in the Polycystic Kidney Research Retrieval Program with the assistance of the PKD Foundation (Kansas City, MO). The kidneys were packaged within ice and delivered to the laboratory overnight. A protocol for the use of discarded human tissues complies with federal regulations and was approved by the Institutional Review Board at KUMC. Primary cell cultures were prepared as described 1. Cells are propagated in DMEM/F12 supplemented with 5% FBS, 5 µg/ml insulin, 5 µg/ml transferrin and 5 ng/ml sodium selenite (ITS) and 100 IU/ml penicillin G and 0.1 mg/ml streptomycin. Primary cultures of ADPKD and NHK cells appear epithelial 1-3 and stain with Arachis hypogaea and Dolichos biflorus, lectins that bind the collecting ducts and distal tubules 4. Reference: 1. Wallace, D.P., Grantham, J.J. & Sullivan, L.P. Chloride and fluid secretion by cultured human polycystic kidney cells. Kidney Int 50, (1996). 2. Yamaguchi, T. et al. camp stimulates the in vitro proliferation of renal cyst epithelial cells by activating the extracellular signal-regulated kinase pathway. Kidney Int 57, (2000). 3. Neufeld, T.K. et al. In vitro formation and expansion of cysts derived from human renal cortex epithelial cells. Kidney Int 41, (1992). 4. Yamaguchi, T. et al. Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys. Kidney Int 63, (2003).