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Identification of pleiotrophin as a mesenchymal factor involved in ureteric bud branching morphogenesis

Departments of Pediatrics and Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0693, USA

View larger version (60K): [in a new window]   Fig. 1. BSN-CM is necessary for isolated ureteric bud branching morphogenesis. Phase contrast photomicrographs of isolated ureteric buds cultured for 14 days in the presence (B) or absence (C) of BSN-CM in the presence of 10% FCS, 125 ng/ml GDNF, and 250 ng/ml FGF1. In the presence of BSN-CM, the T-shaped ureteric bud (A) underwent extensive branching morphogenesis (B). In the absence of BSN-CM, no significant growth/morphogenesis was observed (C). Scale bar, 500 µm.

View larger version (58K): [in a new window]   Fig. 2. Purification of the morphogenetic factor. (A) Silver-stained SDS-PAGE gel of active fractions from column chromatography of BSN-CM. Lane 1, whole BSN-CM; lane 2, active fraction from heparin sepharose column; lane 3, active fraction from the Resource phenyl sepharose hydrophobic interaction column. (B) Elution profile from the Resource S cation exchange column of the active fraction from Resource phenyl sepharose column. A single, sharp protein peak was eluted at 0.4-0.6 M NaCl. Each of the individual 1 ml fractions eluted from the column are indicated by the numbers (1-8) above the x axis. (C) Phase-contrast photomicrographs of isolated ureteric buds cultured for 7 days in the presence of each 1 ml fraction from the Resource S cation exchange column (1-8 in B) supplemented with 10% FCS, 125 ng/ml GDNF and 250 ng/ml FGF1. Fraction 4, which corresponded to the protein peak on the elution profile (B) exhibited potent morphogenetic activity. Scale Bar, 500 µm. (D) Silver-stained SDS-PAGE gel of each fraction (1-8) eluted from the Resource S cation exchange column (B). Fraction 4, which possessed potent morphogenetic activity (C) contained a single low molecular mass band, which was identified as pleiotrophin by mass spectrometry. (E) Immunoblot analysis of the individual fractions eluted from the Resource S cation exchange column (1-8 in B). The blot was probed with anti-pleiotrophin antibodies. Rh-PTN; 250 ng of human recombinant pleiotrophin as a positive control.

View larger version (41K): [in a new window]   Fig. 3. Gel filtration chromatography of the eluate from the Resource S cation exchange column. (A) Elution profile from a Superdex 200 gel filtration column of the peak fraction from the Resource S cation exchange column (Fig. 2B, fraction 4). A single protein peak was eluted at 15.93 ml, which corresponds to a relative molecular mass of 18 kDa. Each of the individual 1 ml fractions are indicated by the numbers (1-5) along the x axis. (B) Immunoblot analysis of fraction 3 (A) from the gel filtration column demonstrated the presence of pleiotrophin. rh-PTN, human recombinant pleiotrophin used as a positive control. (C) Phase contrast photomicrograph of isolated ureteric bud grown for 7 days in the presence of fraction 3 supplemented with 10% FCS, 125 ng/ml GDNF and 250 ng/ml of FGF1. Scale bar, 500 µm.

View larger version (75K): [in a new window]   Fig. 4. Adsorption of pleiotrophin abolishes morphogenetic activity. (A) Silver-stained SDS-PAGE gel of morphogenetically active fraction from Resource S cation exchange column. Lane 1, whole fraction; Lane 2, fraction incubated with polyA-sepharose beads. The protein band at 18 kDa was not detected following treatment with polyA-sepharose beads. (B) Immunoblot analysis of the morphogenetically active fraction from Resource S cation exchange column. Lane 1, recombinant human pleiotrophin (positive control); Lane 2, active fraction; Lane 3, active fraction treated with polyA-sepharose beads; Lane 4, protein bound to beads. The blot was probed with anti-pleiotrophin antibodies. PolyA-sepharose beads adsorb pleiotrophin present in the fraction eluted from the Resource S cation exchange column. (C) Phase contrast photomicrographs of isolated ureteric buds grown for 7 days in the morphogenetically active fraction eluted from the Resource S cation exchange column with or without exposure to polyA-sepharose beads. In either case, the fraction was supplemented with 10% FCS, 125 ng/ml GDNF and 250 ng/ml FGF1. Scale bar, 500 µm.

View larger version (70K): [in a new window]   Fig. 5. Pleiotrophin-mediated UB branching morphogenesis is concentration dependent. (A) Phase contrast photomicrographs of isolated ureteric buds grown for 7 days in DMEM/F12 supplemented with increasing concentration of purified pleiotrophin. In each case, the growth medium was also supplemented with 10% FCS, 125 ng/ml GDNF and 250 ng/ml FGF1. The numbers in the upper-left-hand corner of each picture indicate the concentration of pleiotrophin in µg/ml. Clear differences in the phenotype depending on the concentration of pleiotrophin are exhibited. (B) Phase contrast photomicrographs of isolated ureteric buds grown for 11 days in the absence or presence of 250 ng/ml FGF1 diluted in DMEM/F12 supplemented with 2.5-5 µg/ml pleiotrophin (PTN), 10% FCS, and 125 ng/ml GDNF. Scale bar, 500 µm.

View larger version (49K): [in a new window]   Fig. 6. Pleiotrophin induced UB cell tubulogenesis in vitro. (A) Bar graph demonstrating the morphogenetic effects of pleiotrophin on UB cells grown in three-dimensional extracellular matrix gels. UB cells were suspended in 20% Matrigel, 80% collagen gel mixture and grown for 4 days in the absence (control) or presence of purified pleiotrophin (PTN; 0.1-2.5 µg/ml). Whole BSN-CM served as a positive control. All conditions were supplemented with 1% FCS. The percentage of cells and/or colonies with processes was counted as an indicator for tubulogenic activity. 20 cells and/or colonies were counted in three randomly selected fields for each condition. Data is presented as mean ± s.e.m., *P<0.05 (by unpaired Student’s t-test). (B) Phase contrast photomicrographs of UB cells grown for 8 days in DMEM/F12 supplemented with 1% FCS (a, control) and either BSN-CM (b) or purified pleiotrophin (c). BSN-CM and pleiotrophin induced the formation of branching tubules with lumens (compare b and c). Scale bar, 50 µm.

View larger version (55K): [in a new window]   Fig. 7. Pleiotrophin expression in the embryonic kidney. (A) Immunoblot detection of pleiotrophin. Lane 1, extract of whole E13 rat kidney; lane 2, conditioned medium collected from UB cells; lane 3, conditioned medium from BSN cells. Whole kidney and BSN-CM were positive for pleiotrophin (arrowheads). (B) Frozen sections of E13 mouse kidney stained with anti-pleiotrophin antibody. Pleiotrophin is present at the basement membrane of developing UB (a). Treatment with normal goat IgG did not result in significant staining (b). Scale bar, 100 µm.

View larger version (41K): [in a new window]   Fig. 8 Effect of exogenous pleiotrophin on UB morphology in whole kidney organ culture. Fluorescence photomicrographs of E13 rat kidneys cultured for 7 days in DMEM/F12 supplemented with 10% FCS in the absence (A, control) or presence of pleiotrophin (B, 2.5 µg/ml; C, 5 µg/ml). The UB was visualized E13 mouse with FITC-conjugated lectin from Dolichos biflorus. Scale bar, 500 µm.