Volume 78, Issue 1, Pages 60-68 (July 2010) Adipose tissue explants and MDCK cells reciprocally regulate their morphogenesis in coculture  Kazuma Udo, Shigehisa Aoki, Kazuyoshi Uchihashi, Maki Kawasaki, Aki Matsunobu, Yuji Tokuda, Akifumi Ootani, Shuji Toda, Jiro Uozumi  Kidney International  Volume 78, Issue 1, Pages 60-68 (July 2010) DOI: 10.1038/ki.2010.68 Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 1 Experimental system. (a) Madin–Darby canine kidney (MDCK) cells are seeded on an adipose tissue fragment (ATF)-embedded collagen gel layer in an inner dish with a nitrocellulose membrane in its bottom. The inner dish is placed in a larger outer dish, and then complete medium is added to both dishes. Cells are fed by sufficient medium from both the inner and outer dishes, owing to the permeability of the nitrocellulose membrane. (b and c) As a control, MDCK cells (b) or ATFs (c) alone are cultured. (d) To estimate the specificity of ATF-induced effects on MDCK cell behaviors, MDCK cells on a 3T3 fibroblast-embedded collagen gel layer are cultured. Kidney International 2010 78, 60-68DOI: (10.1038/ki.2010.68) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 2 Histology of Madin–Darby canine kidney (MDCK) cells cultured with or without either adipose tissue fragments (ATFs) or 3T3 fibroblasts at 7 days. (a) MDCK cells cultured alone have a flat-shape, with cytoplasm and flattened nuclei. (b and c) MDCK cells with ATFs, which were originated from subcutaneous tissues (b) and perirenal adipose tissues (c), show a large tall columnar shape with clear cytoplasm and vertically and basally situated nuclei. (d) The morphology of MDCK cells with 3T3 fibroblasts is similar to that of MDCK cells cultured alone, although the fibroblasts induce mild hypertrophy of MDCK cells. Kidney International 2010 78, 60-68DOI: (10.1038/ki.2010.68) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 3 Electron microscopy. Fine structures of Madin–Darby canine kidney (MDCK) cells with (b–d) or without (a) adipose tissue fragments (ATFs). MDCK cells with ATFs (b–d) organized microvilli (arrows) and basal lamina (arrowheads) at the apical and basal sides, respectively, more effectively than the cells without ATFs (a). MDCK cells with ATFs (b and c) showed the accumulation of glycogen granules in supranuclear sites more prominently than MDCK cells cultured alone (a). (c) A higher magnification of the smaller open rectangle in b. (d) A higher magnification of the larger open rectangle in b. N, nuclei; G, accumulation of glycogen granules. Kidney International 2010 78, 60-68DOI: (10.1038/ki.2010.68) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 4 Proliferation and apoptosis of Madin–Darby canine kidney (MDCK) cells cultured with or without adipose tissue fragments (ATFs) at 7 days. Growth and apoptosis of MDCK cells are analyzed by immunohistochemistry with bromodeoxyuridine (BrdU) (a, arrow) and single-stranded DNA (ssDNA) (b, arrow), respectively. BrdU and ssDNA expression of MDCK cells with ATFs is significantly lower than that of MDCK cells without ATFs (BrdU, P<0.05; and ssDNA, P<0.001). This indicates that ATFs inhibit the proliferation and apoptosis of MDCK cells. Kidney International 2010 78, 60-68DOI: (10.1038/ki.2010.68) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 5 Zonula occludens-1 (ZO-1), atypical protein kinase C (aPKC), protease-activated receptor 3 (Par3), phosphatase and tensin homolog deleted from chromosome 10 (PTEN), cell division cycle 42 (Cdc42), and pendrin expression of Madin–Darby canine kidney (MDCK) cells with or without adipose tissue fragments (ATFs) at 7 days by immunofluorescence. MDCK cells with ATFs express the tight junction protein, ZO-1, at the apical end of their cell–cell contact regions (arrowheads) more effectively than MDCK cells cultured alone. MDCK cells with ATFs express the polarity-related molecules, aPKC (arrowheads) and Cdc42, more effectively than MDCK cells cultured alone, whereas Par3 and PTEN expressions of MDCK cells with ATFs are similar to those of MDCK cells cultured alone. The aPKC and Par3 are detected at the apical tight junction regions of the cells. Cdc42 is detected at the apical end to basolateral regions of the cells. MDCK cells with ATFs express the chloride/iodide transporter, pendrin, at the apical surface more prominently than MDCK cells cultured alone. Kidney International 2010 78, 60-68DOI: (10.1038/ki.2010.68) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 6 Western blot and corresponding densitometry analyses. Western blotting (a) of zonula occludens-1 (ZO-1), atypical protein kinase C (aPKC), protease-activated receptor 3 (Par3), phosphatase and tensin homolog deleted from chromosome 10 (PTEN), cell division cycle 42 (Cdc42), and pendrin, and corresponding densitometry analyses (b) in Madin–Darby canine kidney (MDCK) cells cultured with or without adipose tissue fragments (ATFs) at 7 days. MDCK cells with ATFs express ZO-1, aPKC, Cdc42, and pendrin more prominently than MDCK cells cultured alone, whereas Par3 and PTEN expressions of MDCK cells with ATFs are similar to those of MDCK cells cultured alone. This supports the immunofluoresence results of Figure 5. Kidney International 2010 78, 60-68DOI: (10.1038/ki.2010.68) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 7 Expression of zonula occludens-1 (ZO-1), protease-activated receptor 3 (Par3), phosphatase and tensin homolog deleted from chromosome 10 (PTEN), cell division cycle 42 (Cdc42), and pendrin mRNAs in Madin–Darby canine kidney (MDCK) cells with or without adipose tissue fragments (ATFs) at 7 days in culture by real-time reverse transcriptase PCR. ZO-1, Cdc42, and PTEN mRNA expressions of MDCK cells with ATFs are significantly lower than those of MDCK cells cultured alone (*P<0.001). There is no significant difference in Par3 and pendrin mRNA expressions between MDCK cells with and without ATFs. Kidney International 2010 78, 60-68DOI: (10.1038/ki.2010.68) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 8 Adipokine treatment. Effects of leptin (b–e), its antibody (insets in b–e), and combination of leptin and adiponection (f) on morphology of Madin–Darby canine kidney (MDCK) cells at 7 days in culture. (a) (Control), MDCK cells without leptin and adiponectin treatment show a flat shape with cytoplasm and flattened nuclei. (b and c) 20 (b) or 50ng/ml (c) leptin does not affect the morphology of MDCK cells. (d and e) 100 (d) or 1000ng/ml (e) leptin allows MDCK cells to develop a tall and large columnar shape, with clear cytoplasm and vertically and basally situated nuclei. The leptin-induced morphology is similar to the adipose tissue fragment (ATF)-induced morphology of MDCK cells. Leptin antibody (L-ab, 25μg/ml) abolishes the MDCK cell morphology induced by 100 (inset in d) or 1000ng/ml leptin (inset in e), although the antibody does not affect the morphology of cells at the concentration of 20 (inset in b) or 50ng/ml (inset in c) leptin. (f) Adiponectin (10μg/ml) abolishes the MDCK cell morphology induced by 1000ng/ml leptin. Kidney International 2010 78, 60-68DOI: (10.1038/ki.2010.68) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 9 Adiponectin and leptin production of adipose tissue fragments (ATFs) in the absence or presence of Madin–Darby canine kidney (MDCK) cells in the supernatants of outer and inner dishes at 7 days in culture. In the cultures of ATFs alone, adiponectin and leptin are detected. Their concentration is not significantly different between the outer and inner dishes (P>0.05). In the supernatants of outer dishes, adiponectin concentrations in the cocultures of ATFs and MDCK cells are significantly higher than those in the cultures of ATFs alone (P<0.05), whereas leptin concentrations are not significantly different between cultures of ATFs with and without MDCK cells (P>0.05). In cocultures of ATFs and MDCK cells, adiponectin (P<0.01) and leptin (P<0.05) concentrations in the inner dishes were significantly lower than those in the outer dishes. Kidney International 2010 78, 60-68DOI: (10.1038/ki.2010.68) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 10 Effects of Madin–Darby canine kidney (MDCK) cells on development of CD44+/CD105+ mesenchymal stem cell (MSC)-like cells and preadipocytes from adipose tissue fragments (ATFs) at 7 days in culture. In cultures of ATFs alone (a), two spindle-shaped cell types that consist of CD44+/CD105+ MSC-like cells (c) and lipid droplet-laden preadipocytes (d) develop at the peripheral zone of ATFs. However, the development of these cell types from ATFs is clearly inhibited in cocultures of ATFs and MDCK cells (b). (c) Spindle-shaped cells express CD44 in red (upper panel). The same cells also express CD105 in green (middle panel). On the same spindle-shaped cells, CD44 and CD105 are merged, suggesting that these CD44+/CD105+ spindle-shaped cells (yellow) are MSC-like cells. (d) Lipid droplets (red) of preadipocytes are confirmed by oil red O staining. (e) The numbers of CD44+/CD105+ MSC-like cells (left graph) and preadipocytes (right graph) in the coculture of MDCK cells and ATFs are significantly lower than those that of MSC-like cells and preadipocytes in the cultures of ATFs alone. (a and b) Hematoxylin–eosin staining. (c) Immunofluorescence. (d) Oil red O stain. Kidney International 2010 78, 60-68DOI: (10.1038/ki.2010.68) Copyright © 2010 International Society of Nephrology Terms and Conditions