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First published online 16 March 2004
doi: 10.1242/jcs.01020

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Both mitogen activated protein kinase and the mammalian target of rapamycin modulate the development of functional renal proximal tubules in matrigel

¹ Department of Veterinary Physiology, College of Veterinary Medicine, Hormone Research Center, Chonnam National University, Gwangju 500-757, Korea
² Confocal Microscope and 2D Imaging Facility, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214, USA
³ Department of Pathology and Anatomical Sciences, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214, USA
⁴ Biochemistry Department, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214, USA

View larger version (146K): [in a new window]   Fig. 1. Tubule formation in response to EGF. Purified rabbit kidney proximal tubules were plated into matrigel in serum free medium supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 5x10–8 M hydrocortisone and 10 ng/ml EGF. Subsequently, the cultures were photographed under an inverted microscope. (a) Unbranched tubule, 1 week; (b) branched tubules, 1 week. Bars, 50 µm.

View larger version (100K): [in a new window]   Fig. 2. Tubule formation as a function of time. Matrigel cultures were initiated in serum free medium supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 5x10–8 M hydrocortisone and 10 ng/ml EGF, and then examined under an inverted microscope. (a) A microscope field after 1 day in culture. The same microscope field after (b) 2 days and (c) 4 days in culture. Bar in a, 50 µm (same scale for b and c).

View larger version (131K): [in a new window]   Fig. 3. Lumen formation by tubules. Lucifer Yellow accumulation (observed as green) in the lumen of a tubule in matrigel cultures, while DiI (observed as Yellow) acted as a membrane stain. Matrigel cultures were initiated as described in the legend to Fig. 2. Subsequently, the cultures were incubated for 20 hours in Phenol Red-free medium supplemented with 80 µM Lucifer Yellow, 1 µM DiI, 5 µg/ml bovine insulin, 5 µg/ml human transferrin, 5x10–8 M hydrocortisone and 10 ng/ml EGF. (A) Transverse section of a 2-week-old culture. (B) Longitudinal section of another tubule. (C) Section of a 1-week-old matrigel culture. Red arrow, DiI stained plasma membrane; Green arrow, Lucifer Yellow in lumen. (D) Cross-section of a tubule formed by a 1-week-old matrigel culture was examined by TEM, showing a lumen (L) and nucleus (N). Bar in A, 50 µm (same scale for B and C); Bar in D, 10 µm.

View larger version (153K): [in a new window]   Fig. 4. Specificity of substrate accumulation. To determine whether tubules in matrigel cultures transport a variety of fluorescent substrates, matrigel cultures were initiated as described in the legend to Fig. 2. Confocal micrographs of matrigel cultures incubated for 20 hours in Phenol Red-free medium supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 5x10–8 M hydrocortisone, 10 ng/ml EGF and either 5,6-carboxyfluorescein (5 µM), Rhodamine 123 (10 µM) or BODIPY FL verapamil (5 µM), also as described in Fig. 3. (A) Cultures with 5,6-carboxyfluorescein (5,6 FAM) shown as a fluorescent image (white present in the lumen represents 5,6 FAM), and the same section shown in (B) as a bright-field image. (C) Fluorescent image of a culture incubated with BODIPY FL verapamil (represented by white, which is present intracellularly). (D) Fluorescent image of a culture incubated as described above with Rhodamine 123 (represented by white in cells, and diffuse gray in the lumen). Bar in A, 50 µm (same scales for B, C and D).

View larger version (157K): [in a new window]   Fig. 5. Lectin binding by matrigel cultures. Matrigel cultures, initiated as described in the legend to Fig. 2 above, were incubated for 20 hours in Phenol Red-free medium supplemented with a lectin (as specified in A, B, C or D below), in addition to insulin, transferrin, hydrocortisone and Lucifer Yellow (as described in Fig. 3). Confocal images of matrigel cultures maintained 20 hours with particular lectins. (A) TRITC T. vulgare (30 µg/ml) (shown as red) + Lucifer Yellow (green) (insert shows a culture maintained for 20 hours as above with additional brefeldin A, 30 µg/ml), (B) FITC-Lotus Tetragonolobus purpurea (30 µg/ml) (green), (C) FITC-Dolichos biflorous (50 µg/ml) (green), and (D) TRITC-glycine max (30 µg/ml) (red) + Lucifer Yellow (green) are illustrated, as overlayed on bright-field images. The lumenal space shown in A is apparently still developing, not being contiguous, as indicated by the TRITC T. vulgare staining of the apical membrane, which sections off a portion of the lumen from the rest of the lumenal space. Bar in A, 50 µm (same scale for the insert in A).

View larger version (85K): [in a new window]   Fig. 6. Effect of HGF and other factors on tubule formation. (A) Comparison of the frequency of tubule formation in response to HGF and EGF. Matrigel cultures were initiated in Phenol Red-free medium supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 5x10–8 M hydrocortisone, and either 40 ng/ml HGF, 10 ng/ml EGF, 1 ng/ml TGF-ß, EGF + TGF-ß, or no further supplement. The frequency of tubule formation was determined as described in Materials and Methods. The tubules that formed in response to HGF were observed (B) under the inverted microscope, as well as (C) under the confocal microscope after incubation with Lucifer Yellow and DiI as described in the legend to Fig. 3, with the exception that HGF, rather than EGF, was utilized. Bars, 50 µm (B,C). The effect of 5x10–5 M PD98059 (PD) and 5x10–8 M wortmannin (Wort) on the frequency of tubule formation was examined either (D) in the presence or in the absence of 10 ng/ml EGF, or (E) in the presence or in the absence of 40 ng/ml HGF. Results were significantly (P<0.05) different from the control (*), EGF (**) or HGF (**).

View larger version (52K): [in a new window]   Fig. 7. Effect of PD98059, wortmannin and rapamycin on tubule formation. Matrigel cultures were maintained either with 5x10–5 M PD98059, 5x10–8 M wortmannin or 2.5x10–8 M rapamycin in addition to insulin, transferrin, hydrocortisone and EGF for 2 weeks. Before confocal microscopy, the cultures were maintained for 20 hours under the same conditions, with the addition of 80 µM Lucifer Yellow and 4 µg/ml Texas Red-labeled T. vulgare, and then examined under the confocal microscope. (A) PD98059; (B) wortmannin; (C) rapamycin. Bar in A, 50 µm (for B and C).

View larger version (111K): [in a new window]   Fig. 8. Cyst formation by matrigel cultures. Matrigel cultures incubated with 8-Br-cAMP were examined (a) under the inverted microscope, (b) after the preparation of paraffin sections, as well as (c) under the confocal microscope following incubation with Lucifer Yellow. Bars, 50 µm (a-c). The effects of 8-Br-cAMP and TPA on (d) cyst formation and (e) tubule formation were quantitated as described in Materials and Methods. An effect was found to be significant (*) when P<0.05. (f) Effect of 8-Br-cAMP on the level of the mRNA for the Na,K-ATPase subunit, the Na,K-ATPase ß subunit, mitochondrial PEPCK, and ß-actin was determined by northern analysis.

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