Hb egf

All GFs and chemokines were purchased in their mature forms, highly pure (>95% pure), carrier-free, and lyophilized^{35 (link)}. VEGF-A121, VEGF-A165, PlGF-1, PlGF-2, PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, FGF-1, FGF-2, FGF-6, FGF-7, FGF-9, FGF-10, FGF-18, BMP-2, BMP-3, BMP-4, BMP-7, β-NGF, NT-3, BDNF, IGF-1, IGF-2, HB-EGF, CXCL-11, and CXCL-12α were purchased from PeproTech. CXCL-12γ was purchased from R&D systems. Except for PDGF-DD and BMP-7, which were produced in eukaryotic cells, all GFs were produced in Escherichia coli and thus were not glycosylated. All GFs were reconstituted and stored according to the provider’s instructions to regain full activity and prevent loss of protein.

A monocyte migration assay was performed to investigate the chemotactic function of HB-EGF. First, 200 μL of serum-free RPMI 1640 containing 5 × 10⁵ THP-1 cells were added to the upper chamber (24-transwell inserts, pore size of 8 μm, Corning, NY, USA). Subsequently, 600 μL of serum-free RPMI 1640 medium containing 100 ng/mL HB-EGF (Peprotech, Rocky Hill, NJ, USA) was loaded into the bottom chamber. The migrated cells were observed using an inverted microscope and counted in four fields (200× magnification) at 2, 4, and 8 h. This experiment was repeated three times.
After 10⁵ THP-1 cells were differentiated into macrophages in the upper chamber for 48 h, the medium was discarded, and the cells were washed twice with phosphate-buffered saline (PBS) before using them in the migration assay. The same dose of HB-EGF was used in the bottom chamber to attract macrophages for 8 h. The non-migrated cells were gently scraped and washed with PBS. The migrated cells were fixed in ethanol and stained with crystal violet. At least four pictures of the migrated cells were obtained at 200× magnification and quantified using FIJI software. The experiments were repeated three times.

10⁵ keratinocytes were cultured in a 24 well plate for 16-20 hours in KBM-2. They were rendered quiescent by serum starvation for 16 to 20 h and subsequently treated with fresh supplement-free KBM-2 medium containing 10 ng/ml FGF7, 50 ng/ml IGF-1, 80 ng/ml IGF-2, 100 ng/ml HBEGF or 1ng/ml TGF β1 respectively (Pepro Tech France, Neuilly sur Seine, France), for 5 hours. Cells were harvested and counted or lysed with PBS 1% Triton prior to K15 western blotting experiments or for RNA isolation.

Magnetic beads infused-mouse kidneys were extracted, minced, and digested in 2 mg/ml collagenase I solution (Gibco) in RPMI 1640 (Invitrogen) at 37 °C for 3 min, then filtered through a 70-µm cell strainer and once more through a 40-µm cell strainer. The homogenate was centrifuged at 720×g for 6 min and the cells were plated. Podocyte primary cultures consisted of freshly isolated glomeruli plated in 6-well dishes in RPMI 1640 (Invitrogen) supplemented with 10% fetal calf serum (Biowest) and 1% penicillin–streptomycin (Invitrogen). Purity of culture of differentiated primary podocytes was verified as previously described^{13 (link), 69 (link)} and shown in Supplementary Fig. 3b. Podocyte primary cultures used in this study was always P0. The outgrowth of podocytes started between days 2 and 3. Podocyte outgrowth area was quantified at day 4 using ImageJ software. Differentiated podocytes were exposed to HB-EGF (10 ng/ml, Preprotech), AG1478 (1 µM, Calbiochem), anti-mIL-6 monoclonal antibody MP5-20F3, monoclonal rat IgG1, κ isotype control immunoglobulin (both functional grade purified, 10 µg/ml, eBiosciences), Stattic (2 µM, Calbiochem), or recombinant IL6 (10 ng/ml, Preprotech) for 16 h. After stimulation, podocytes were scrapped in Phosphosafe buffer (Novagen) for protein extraction or in Trizol (Invitrogen) for total RNA extraction.

Astrocytes were purified by immunopanning from the forebrains of P5 Sprague Dawley rats (Charles River) forebrains and cultured as previously described.^{60 (link)} In brief, cortices were enzymatically disrupted (using papain) and then mechanically dissociated to produce a single-cell suspension that was incubated on several negative immunopanning plates to remove microglia, endothelial cells and oligodendrocyte lineage cells. Positive selection for astrocytes was with an ITGB5-coated panning plate. Isolated astrocytes were cultured in a defined, serum-free base medium containing 50% neurobasal, 50% DMEM, 100 U mL⁻¹ penicillin, 100 μg mL⁻¹ streptomycin, 1 mM sodium pyruvate, 292 μg mL⁻¹l-glutamine, 1× SATO and 5 μg mL⁻¹ of N-acetyl cysteine. This medium was supplemented with the astrocyte-required survival factor HBEGF (Peprotech, 100-47) at 5 ng mL⁻¹.^{60 (link)} Cells were plated at 5000 cells per well in 12-well plates coated with poly-d-lysine and maintained at 10% CO₂.

Astrocytes were purified by immune-panning from P2-P5 C57BL/6J mice and cultured as previously described^{72 (link)}. Briefly, cortices were digested by Trypsin at 37 °C and then mechanically dissociated to generate single-cell suspension, which was incubated in successive negative immune-panning plates to remove macrophage and microglia cells before positive selection for astrocytes by an ITGβ5-coated panning plate. Isolated astrocytes were cultured in a defined, serum-free base medium containing 50% neurobasal, 50% DMEM/F12, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, 292 μg/ml l-glutamine, 1 × G5 supplement. This medium was supplemented with the astrocyte-required survival factor HBEGF (Peprotech, 100-47) at 5 ng/ml as previously described^{73 (link)}.