Collagen coated petri dishes

Mouse kidney TECs were isolated and cultured as described previously^{65 (link)}. In brief kidneys were perfused with saline then removed. Kidney cortices were dissected into approximately 1 mm³ pieces and digested in HBSS containing 3 mg/mL of collagenase at 37 °C for 25 minutes, followed by washing in DMEM/F12 medium (Invitrogen). The kidney digest was washed through a series of sieves (mesh diameters of 250, 150, 75 and 40 µm) then spun down at 300 g for 5 minutes. The cell pellet was re-suspended in defined K1 medium: DMEM/F12 supplemented with 25 ng/mL epidermal growth factor, 1 ng/mL PGE1, 5 × 10-11 M triiodothyronine, 5 × 10-8 M hydrocortisone (Sigma-Aldrich), ITSS media supplement, 1% penicillin/streptomycin, 25 mM HEPES, and 5% FCS (Invitrogen). The cell suspension was then seeded on cell culture Petri dishes and incubated at 37 °C for 2–3 hours to facilitate adherence of contaminating glomeruli. The non-adherent tubules were collected and cultured on collagen-coated Petri dishes (BD Biosciences) in K1 medium. Expression of the epithelial cell marker cytokeratin was verified by immunofluorescent staining with an anti-cytokeratin antibody (Sigma-Aldrich). Cells were >95% cytokeratin positive. Experiments were commenced after cells had reached 80% confluence.

We isolated PTECs from normal adjacent kidney tissue specimens from patients with renal cell carcinoma according to guidelines approved by the Institutional Review Board of Seoul National University Hospital (IRB no. 1506-097-681). After dissecting the cortex, the unaffected specimens were minced and digested with Hank’s balanced salt solution (HBSS) containing 3 mg/mL collagenase (Sigma-Aldrich, St. Louis, MO, USA). After centrifugation for 5 minutes at 500 g, cortical tubular cells were isolated. The cells were then incubated in DMEM/F12. After 4 hours of incubation, the tubules were collected and cultured on collagen-coated petri dishes (BD Biosciences, Franklin Lakes, NJ, USA) until colonies of epithelial cells were established, and 2 ± 3 passages were used in the current study. After 3 days of culture, the cells were detached from the dishes with a 3 mM EDTA solution and a minimal amount of trypsin. Cells (2 × 10⁵/well) were seeded on 8-well chamber slides in serum-free medium for 24 hours and then washed twice with PBS. Next, recombinant TGFβ (R&D Systems, Minneapolis, MN, USA) was added (final concentration, 10 ng/ml) except where indicated. Cells were incubated in the presence or absence of either recombinant HGF (10 and 20 mg/ml) or agonistic cMet antibody (250, 500 and 1000 ng/ml) for 48 hours. rHGF and cMet Ab were treated simultaneously with rTGFβ.

The protocol for obtaining and processing human kidney specimens was reviewed and approved by the Institutional Review Board of Seoul National University Hospital (IRB No. 2110-026-1260). We dissected the proximal renal tubular segments from kidneys of patients diagnosed with renal cell carcinoma [30 (link)]. After dissecting the cortex, the unaffected specimens were minced and digested with Hank’s balanced salt solution (HBSS) containing 1.5 mg/mL collagenase (Sigma-Aldrich) at 37 °C for 1 h. The digested kidney sample was then washed through a series of sieves (150, 120, 70, and 40 μm) with 1 × PBS and centrifuged at 500× g for 5 min. After recovering cells from the pellet, they were incubated in DMEM/F12 (Lonza, Basel, Switzerland) for 4 h. Tubules floating in the media were collected and cultured on collagen-coated Petri dishes (BD Biosciences, Franklin Lakes, NJ, USA) until colonies of epithelial cells formed; 2–3 passages of these epithelial cells were used in the current study. We added 2 ng/mL of recombinant transforming growth factor β (rTGFβ) and cultured for 48 h to induce cellular injury including fibrotic change and apoptosis. Various concentrations of cystamine were administrated simultaneously with rTGFβ in the treatment group.