ENGINEERING AND CHARACTERIZATION OF HUMAN RENAL PROXIMAL TUBULAR CELLS FOR APPLICATIONS IN IN VITRO TOXICOLOGY AND BIOARTIFICIAL KIDNEYS FARAH TASNIM (B. Sc. (Hons.), NUS) A THESIS SUMBITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2012 Acknowledgements I would like to thank the National University of Singapore and Institute of Bioengineering and Nanotechnology (IBN, A-STAR) for giving me the opportunity to pursue my Ph.D. studies. In particular, I would like to thank my supervisors Dr. Daniele Zink and Assoc Prof. Wang Shu for their support and guidance throughout the project. They have been inspiring and encouraging, even through difficult times in the Ph.D. pursuit. I am grateful for the wonderful learning experience that they have helped me obtain. The members of the lab have contributed immensely to my personal and professional time during my Ph.D. as well. I would like to thank all of them for their support, cooperation and helpful discussions. I thank Joscha Muck for his efforts in helping me improve my image analysis and compilation skills, Dr. Karthikeyan Kandasamy for some of the qPCR experiments and Dr. Rensheng Deng and Mohammed Shahrudin Ibrahim for providing the membranes and bioreactors. I also greatly appreciate all our internal (different labs at IBN) and external collaborators: Prof. Carol Pollock, Prof. Anantharaman Vathsala, Dr. Tiong Ho Yee, Dr. Thomas Thamboo and all staff of National University Health System Tissue Repository (NUHS-TR) for their wonderful support. Finally, I would like to thank the directors of IBN for their constant support and IBN, Biomedical Research Council (BMRC) and A-STAR for funding. Table of Contents Acknowledgements . Summary List of Tables . List of Figures 1. Introduction . 10 1.1 Structure and function of renal proximal tubular cells 10 1.2 Development of BAKs and applications of HPTC in such devices . 18 1.3 Genetic Engineering of HPTC and development of a BMP-7-producing BAK 24 1.4 Co-culture systems 26 2. Hypotheses and Goals . 28 3. Materials and Methods . 30 3.1 Isolation of HPTC . 30 3.2 Static culture of commercial HPTC 32 3.3 Static culture of myoblast cell line, fibroblasts and endothelial cells 32 3.4 Experimental set up of static cell culture 33 3.5 Live/dead assay . 33 3.6 Bioreactor set up and perfusion culture . 34 3.7 Treatment with recombinant BMP-2 and recombinantBMP-7 . 34 3.8 Treatment with human recombinant TGF-β1 and human recombinant A2M 35 3.9 Immunostaining and quantification of fluorescence intensities 35 3.10 Immunoblotting . 37 3.11 ELISA 38 3.12 Quantitative real-time polymerase chain reaction (qPCR) 38 3.13 Determination of GGT activity . 43 3.14 Determination of leucine aminopeptidase (LAP) activity . 44 3.15 Determination of the response to parathyroid hormone 45 3.16 Determination of alkaline phosphatase (AP) activity . 45 3.17 Generation of BMP-7-producing HPTC using non-viral systems 46 3.18 Generation of BMP-7-producing HPTC using a lentiviral system . 48 3.19 Statistics 49 4. Results 50 4.1. Isolation of HPTC and characterization of isolated and commercial HPTC 50 4.1.1 Isolation of HPTC . 50 4.1.2 Characterization of HPTC by immunoblotting 51 4.1.3 Characterization of HPTC by qPCR 52 4.1.4 Characterization of HPTC by immunofluorescence 54 4.1.5 Characterization of HPTC by functional assays . 59 4.2 Analysis of factors impacting HPTC performance under in vitro conditions 63 4.3. Effects of BMP-7 and BMP-2 on HPTC 67 4.3.1 Effects of BMP-7 on the maintenance of epithelia formed by HPTC 67 4.3.2 Effects of BMP-2 treatment 70 4.3.3 Quantification of α-SMA expression . 71 4.3.4 BMP-7 enhances cell type-specific functions of HPTC in bioreactors 73 4.4 Generation of BMP-7-producing HPTC for applications in BAK . 77 4.4.1 Generation of BMP-7-expressing HPTC using a non-viral system . 77 4.4.2 Generation of BMP-7-expressing HPTC using a lentiviral system . 82 4.4.3 Bioactivity of BMP-7 secreted by HPTC . 83 4.4.4 Effects of secreted BMP-7 on HPTC . 89 4.5. Establishment and characterization of a co-culture system 93 4.5.1 Effect of endothelial cells on HPTC . 93 4.5.2 The cross-talk between HPTC and HUVEC and soluble factors secreted by HUVEC . 99 4.5.3 TGF-β1 and its antagonist A2M regulate the maintenance of renal epithelia 101 5. Discussion . 105 5.1 Characterization of isolated and commercial HPTC . 105 5.2 Characterization of effects of BMP-7 on HPTC and generation of a BMP-7producing BAK 107 5.3 Co-culture of HPTC with endothelial cells . 112 6. References 117 7. Appendix: Abbreviations…………………………………………………………. 127 Summary Renal proximal tubular epithelial cells perform a wide variety of kidney-specific functions. Due to their function in glomerular filtrate concentration and drug transport, they are a major target of drug-induced toxicity and hence important for in vitro nephrotoxicology. However, respective approved in vitro models based on renal cells have not been developed yet. One major obstacle is cellular de-differentiation of human primary renal proximal tubular cells (HPTC), which are most interesting for such applications, under in vitro conditions. HPTC are also important for the development of bioartificial kidneys (BAKs) and also in this application cell performance is of critical importance. In order to establish a reliable source and to characterize cell performance, I established in the laboratory a protocol for isolating HPTC from human kidney samples. The freshly isolated HPTC were characterized using qPCR, immunostaining, immunoblotting and functional assays. In addition, I characterized commercial HPTC. The results showed that both freshly isolated and commercial HPTC displayed many characteristics of HPTC, but showed some changes in gene expression patterns and expressed some markers specific for other parts of the nephron. I also established a co-culture system between HPTC and human primary endothelial cells. The results showed that HPTC stimulated endothelial cells to secrete a mixture of growth factors, which in turn improved HPTC performance. HPTC showed improved proliferation, marker gene expression and enzyme activity in co-cultures. Also, the long- term maintenance of epithelia formed by HPTC was improved. In order to determine which growth factors were responsible for these effects, qPCR analysis was performed. The results pointed to a central role of transforming growth factor-β1 (TGF-β1) and its antagonist alpha-2-macroglobulin (A2M). The impact of these factors on HPTC was further confirmed by additional experimental approaches involving supplementation with recombinant growth factors. Overall, the results showed that HPTC induced endothelial cells to secrete increased amounts of specific growth factors, which balanced each other functionally and improved cell performance. Together, the results revealed that co-culture systems are useful for analyzing the cross-talk between these cell types which plays an important role in renal disease and repair. Furthermore, the characterization of defined microenvironments, which positively affect HPTC, is helpful for improving the performance of this cell type in in vitro applications. The central role of TGF-β1 and its antagonists in regulating HPTC performance was further confirmed by our findings that treatment with bone morphogenetic protein-7 (BMP-7), which is a TGF-β1 antagonist, improved maintenance of epithelia formed by HPTC for extended time periods. In addition, the functional performance of the HPTC was improved. The effects of BMP-7 were strongly concentration-dependent. Following these findings, I generated BMP-7-expressing HPTC by genetic engineering for the development of BMP-7-producing bioartificial kidneys. The hypothesis underlying this work was that HPTC-produced BMP-7 would improve cell performance in the device by autocrine/paracrine signaling. Furthermore, pre-clinical studies revealed beneficial effects of BMP-7 on kidney recovery and hence there is a substantial interest in using BMP-7 for the treatment of kidney disease. Apart from the improvement of cellular functions, a BMP-7-producing BAK would allow the delivery of the growth factor to kidney patients. My results showed that HPTC-produced BMP-7 was bioactive and improved HPTC performance through autocrine signaling. In addition, our results suggested that the amount of BMP-7 produced by HPTC would be sufficient for therapeutic applications. List of Tables Table 1: Details of primer pairs for human marker genes and human GAPDH for analyzing gene expression in HPTC. 40 Table 2: Details of primer pairs for murine osteogenic markers and murine GAPDH. 41 Table 3: Details of primer pairs used for the qPCR analysis of HUVEC gene expression. . 43 Table 4: HPTC performance at different concentrations of BMP-2 and BMP-7. . 67 Table 5: Changes in amino acid residues of BMP-7 which could potentially improve properties of the secreted protein. . 80 List of Figures Figure 1: Phase contrast image of confluent freshly isolated HPTC. 50 Figure 2: Immunoblotting with antibodies against various marker proteins. . 52 Figure 3: Gene expression levels of freshly isolated and commercial HPTC determined by qPCR 53 Figure 4: Detection of various markers by immunostaining. . 58 Figure 5: Double-immunostaining of E-CAD and N-CAD 59 Figure 6: (A) GGT and LAP activity in isolated and commercial HPTC. (B) AP activity in isolated and commercial HPTC. (C) Hormone responsiveness of isolated and commercial HPTC. . 61 Figure 7: Formation and disruption of epithelia formed by HPTC. . 65 Figure 8: Effects of BMP-7 and BMP-2. 69 Figure 9: Treatment with 25 ng/ml of BMP-7 improved the long-term maintenance of epithelia. . 70 Figure 10: Quantification of α-SMA expression. . 72 Figure 11: HPTC performance in bioreactors. 75 Figure 12: HPTC transfection efficiency. . 77 Figure 13: Levels of BMP-7 produced after transfection of HPTC and cytotoxicity of the procedure…………………………………………………………………………….… .79 Figure 14: Level of HPTC-produced BMP-7. 81 Figure 15: Characterization of BMP-7 expressed by genetically engineered HPTC . 83 Figure 16: Alkaline phosphatase activity 85 Figure 17: Immunostaining of phosphorylated Smad1/5/8 in C2C12 cells 86 Figure 18: Immunostaining of phosphorylated Smad2/3 and phosphorylated Smad1/5/8 in C2C12 cells . 87 Figure 19: Expression levels of osteogenic genes determined by qPCR 88 Figure 20: GGT activity of BMP-7-expressing HPTC. 90 Figure 21: HPTC gene expression levels determined by qPCR. 92 Figure 22: HPTC performance in mono- and co-cultures 94 Figure 23: Gene expression levels of HPTC determined by qPCR 96 Figure 24: GGT activity of HPTC 97 Figure 25: Cell numbers 98 Figure 26: Gene expression levels of HUVEC determined by qPCR 101 Figure 27: Amounts of TGF-β1 and A2M determined by ELISA . 102 Figure 28: Long-term performance of HPTC in the presence of hr TGF-β1 and/or hr A2M 104 Figure 29: Schematic of a BMP-7-producing BAK. . 109 Figure 30: Summary of the interactions between HPTC and endothelial cells in cocultures 112 1. Introduction 1.1 Structure and function of renal proximal tubular cells The functional unit of the kidney is the nephron (1). The essential parts of the nephron include the renal corpuscle (glomerulus and Bowman’ capsule), the proximal tubule, the thin and thick ascending and descending limbs of the loop of Henle, the distal tubule and the connecting tubule (1). The remaining collecting duct system is an important segment for urine concentration but is not strictly considered part of the nephron structure. The glomerulus is a capillary extension consisting of a network of thin blood vessels, lined by a thin layer of endothelial cells. The glomerulus acts as the filtration apparatus in the kidney and consists of three filtration layers. The glomerular endothelium has many pores in the range of 80-100 nm and forms the first filtration layer (2). Immediately beneath the endothelium is the glomerular basement membrane (GBM), a 300- to 350 nm-thick basal lamina rich in heparin sulfate and charged proteoglycans with an average pore size of nm (2-4). Behind the GBM are the visceral epithelial cells of the Bowman’s capsule called the podocytes, which form the third layer of the filter (4). The glomerular filtration apparatus, taken in its entirety acts as a semi-permeable membrane, allowing the passage of molecules based on shape, charge and, most importantly, size. The molecular weight cut off of the filtration apparatus is about 70,000 Daltons (2). Hence, cells and large proteins such as albumin are mostly retained whereas smaller molecules such as amino acids, glucose and ions pass through the filter freely. The filtered fluid that is produced as a result of glomerular filtration is called the ultrafiltrate. The components of the ultrafiltrate are essentially the same as those of blood plasma except that it contains no cells and large proteins. The ultrafiltrate flows into the proximal tubules. 10 Generation of such a microenvironment would be important after kidney injury during repair and it has indeed been shown that HGF, VEGF and TGF-β1 antagonists have beneficial effects in animal models of kidney injury/disease (96, 146, 149, 172). It is interesting to note that primary renal proximal tubular cells cultivated in vitro display many features of renal tubular cells after kidney injury. These features include enhanced proliferation, partial de-differentiation and expression of embryonic markers like PAX-2 (58). This is not too surprising, given that the primary cells have been obtained by disruption of the organ and are kept in an artificial environment. Thus, it is tempting to speculate that the HPTC used here generated “injury” signals that stimulated in turn the endothelial cells to contribute to the generation of an environment that promoted survival, proliferation and regenerative processes of both cell types. It will be highly interesting to identify such presumable “injury” signals of renal cells, and co-culture systems will be a useful model system for corresponding experimental work. One interesting question is why endothelial cells up-regulated both, TGF-β1 as well as A2M. TGF-β1 plays in vivo important roles in coordinating the response to injury and inflammation and TGF-β1 expression is consistently increased in different cell types in such situations. However, overshooting TGF-β1 activity has deleterious effects. Probably, in the in vivo situation a delicate balance is achieved between TGF-βs secreted by various sources and their antagonists, which could be also generated by various sources. 114 It is also worth mentioning that A2M binds not only to TGF-β1, but to a large variety of growth factors and proteases, which could be important in vivo. Also, binding to other factors in the HPTC medium in the absence of increased levels of TGF-β1 might explain why the HPTC epithelium was disrupted when the cells were treated with A2M only. One disadvantage of the model system used here is the fact that peritubular capillary endothelial cells could not be used because they were not available. Thus, it cannot be excluded that the response of this cell type that is relevant in vivo will be different. However, I included at least HRGEC in some experiments and the HPTC response to HRGEC and HUVEC was always similar. Thus, the effects did not appear to be specific for particular types of endothelial cells. Irrespective of the in vivo situation, the results obtained here will be valuable for improving HPTC performance under in vitro conditions. In this respect it is important to note that the expression levels of OAT1, OAT3 and OCT1 and of various other transporters were substantially increased in co-cultures. As mentioned earlier, dedifferentiation and low expression levels of drug transporters renders the cells insensitive to drug exposure; this is a particular problem in in vitro nephrotoxicology (50). The analysis of the co-culture microenvironment performed here will help to establish improved and more defined in vitro culture systems. As discussed before, also in bioartificial kidneys it is important to maintain a differentiated HPTC epithelium during prolonged time periods (60, 61) and also in this regard the results obtained here will be helpful. 115 Overall, establishment and characterization of such a co-culture system allowed us to identify another way to improve HPTC performance which would be an alternative to treatment with BMP-7. The results will be useful for establishing more defined in vitro culture systems. The co-culture system investigated here will also help to study renal injury and regeneration. 116 6. References 1. Brenner, B.M. Brenner and Rector's The Kidney. Philadelphia: Saunders Elsevier. 2008. 2. Pinnock, C., Lin, T., Smith, T. Fundamentals of Anaesthesia. Cambridge University Press, 2003. 3. Kanwar, Y.S., and Farquhar, M.G. Presence of heparan sulfate in the glomerular basement membrane. Proc Natl Acad Sci U S A 76, 1303, 1979. 4. Ogawa, S., Ota, Z., Shikata, K., Hironaka, K., Hayashi, Y., Ota, K., Kushiro, M., Miyatake, N., Kishimoto, N., and Makino, H. High-resolution ultrastructural comparison of renal glomerular and tubular basement membranes. Am J Nephrol 19, 686, 1999. 5. Helbert, M.J., Dauwe, S.E., Van der Biest, I., Nouwen, E.J., and De Broe, M.E. Immunodissection of the human proximal nephron: flow sorting of S1S2S3, S1S2 and S3 proximal tubular cells. Kidney Int 52, 414, 1997. 6. Anzai, N., Jutabha, P., Kanai, Y., and Endou, H. Integrated physiology of proximal tubular organic anion transport. Curr Opin Nephrol Hypertens 14, 472, 2005. 7. Lee, Y.J., and Han, H.J. Regulatory mechanisms of Na(+)/glucose cotransporters in renal proximal tubule cells. Kidney Int Suppl, S27, 2007. 8. Wright, S.H. Role of organic cation transporters in the renal handling of therapeutic agents and xenobiotics. Toxicol Appl Pharmacol 204, 309, 2005. 9. Curthoys, N.P., and Godfrey, S.S. Properties of rat kidney glutaminase enzymes and their role in renal ammoniagenesis. Curr Probl Clin Biochem 6, 346, 1976. 10. Soleimani, M. Na+:HCO3- cotransporters (NBC): expression and regulation in the kidney. J Nephrol 15 Suppl 5, S32, 2002. 11. Fraser, D.R., and Kodicek, E. Unique biosynthesis by kidney of a biological active vitamin D metabolite. Nature 228, 764, 1970. 12. Boswell, R.N., Yard, B.A., Schrama, E., van Es, L.A., Daha, M.R., and van der Woude, F.J. Interleukin production by human proximal tubular epithelial cells in vitro: analysis of the effects of interleukin-1 alpha (IL-1 alpha) and other cytokines. Nephrol Dial Transplant 9, 599, 1994. 13. Wahl, P., Schoop, R., Bilic, G., Neuweiler, J., Le Hir, M., Yoshinaga, S.K., and Wuthrich, R.P. Renal tubular epithelial expression of the costimulatory molecule B7RP-1 (inducible costimulator ligand). J Am Soc Nephrol 13, 1517, 2002. 14. Prodjosudjadi, W., Gerritsma, J.S., Klar-Mohamad, N., Gerritsen, A.F., Bruijn, J.A., Daha, M.R., and van Es, L.A. Production and cytokine-mediated regulation of monocyte chemoattractant protein-1 by human proximal tubular epithelial cells. Kidney Int 48, 1477, 1995. 15. Wilson, C.O., Block, J.H., Gisvold, O., Beale, J.M. Wilson and Gisvold's textbook of organic medicinal and pharmaceutical chemistry. Philadelphia: Lippincott Williams and Wilkins, 2004. 16. Rector, F.C., Jr. Sodium, bicarbonate, and chloride absorption by the proximal tubule. Am J Physiol 244, F461, 1983. 17. Maddox, D.A., and Gennari, F.J. The early proximal tubule: a high-capacity delivery-responsive reabsorptive site. Am J Physiol 252, F573, 1987. 18. Baum, M., and Quigley, R. Proximal tubule water transport-lessons from aquaporin knockout mice. Am J Physiol Renal Physiol 289, F1193, 2005. 117 19. Nielsen, S., Frokiaer, J., Marples, D., Kwon, T.H., Agre, P., and Knepper, M.A. Aquaporins in the kidney: from molecules to medicine. Physiol Rev 82, 205, 2002. 20. Monnens, L., Starremans, P., and Bindels, R. Great strides in the understanding of renal magnesium and calcium reabsorption. Nephrol Dial Transplant 15, 568, 2000. 21. Biber, J., Hernando, N., Forster, I., and Murer, H. Regulation of phosphate transport in proximal tubules. Pflugers Arch 458, 39, 2009. 22. Kempson, S.A., and Dousa, T.P. Current concepts of regulation of phosphate transport in renal proximal tubules. Biochem Pharmacol 35, 721, 1986. 23. Berkhin, E.B., and Humphreys, M.H. Regulation of renal tubular secretion of organic compounds. Kidney Int 59, 17, 2001. 24. Pritchard, J.B., and Miller, D.S. Mechanisms mediating renal secretion of organic anions and cations. Physiol Rev 73, 765, 1993. 25. Nakanishi, T., Fukushi, A., Sato, M., Yoshifuji, M., Gose, T., Shirasaka, Y., Ohe, K., Kobayashi, M., Kawai, K., and Tamai, I. Functional characterization of apical transporters expressed in rat proximal tubular cells (PTCs) in primary culture. Mol Pharm 8, 2142, 2011. 26. Christensen, E.I., and Birn, H. Megalin and cubilin: synergistic endocytic receptors in renal proximal tubule. Am J Physiol Renal Physiol 280, F562, 2001. 27. Christensen, E.I., and Nielsen, R. Role of megalin and cubilin in renal physiology and pathophysiology. Rev Physiol Biochem Pharmacol 158, 1, 2007. 28. Cui, S., Verroust, P.J., Moestrup, S.K., and Christensen, E.I. Megalin/gp330 mediates uptake of albumin in renal proximal tubule. Am J Physiol 271, F900, 1996. 29. Fujita, Y., Terashima, M., Kakuta, T., Itoh, J., Tokimasa, T., Brown, D., and Saito, A. Transcellular water transport and stability of expression in aquaporin 1transfected LLC-PK1 cells in the development of a portable bioartificial renal tubule device. Tissue Eng 10, 711, 2004. 30. Mount, D.B., Kwon, C.Y., and Zandi-Nejad, K. Renal urate transport. Rheum Dis Clin North Am 32, 313, 2006. 31. Soriano, J.R., Boichis, H., and Edelmann, C.M., Jr. Bicarbonate reabsorption and hydrogen ion excretion in children with renal tubular acidosis. J Pediatr 71, 802, 1967. 32. Curthoys, N.P. Role of gamma-glutamyltranspeptidase in the renal metabolism of glutathione. Miner Electrolyte Metab 9, 236, 1983. 33. Yokoyama, H. [Gamma glutamyl transpeptidase (gammaGTP) in the era of metabolic syndrome]. Nihon Arukoru Yakubutsu Igakkai Zasshi 42, 110, 2007. 34. Wuthrich, R.P., Glimcher, L.H., Yui, M.A., Jevnikar, A.M., Dumas, S.E., and Kelley, V.E. MHC class II, antigen presentation and tumor necrosis factor in renal tubular epithelial cells. Kidney Int 37, 783, 1990. 35. Yard, B.A., Daha, M.R., Kooymans-Couthino, M., Bruijn, J.A., Paape, M.E., Schrama, E., van Es, L.A., and van der Woude, F.J. IL-1 alpha stimulated TNF alpha production by cultured human proximal tubular epithelial cells. Kidney Int 42, 383, 1992. 36. van Dorp, W.T., van Wieringen, P.A., Marselis-Jonges, E., Bruggeman, C.A., Daha, M.R., van Es, L.A., and van der Woude, F. Cytomegalovirus directly enhances MHC class I and intercellular adhesion molecule-1 expression on cultured proximal tubular epithelial cells. Transplantation 55, 1367, 1993. 37. Benson, E.M., Colvin, R.B., and Russell, P.S. Induction of IA antigens in murine renal transplants. J Immunol 134, 7, 1985. 118 38. Rubin-Kelley, V.E., and Jevnikar, A.M. Antigen presentation by renal tubular epithelial cells. J Am Soc Nephrol 2, 13, 1991. 39. Nakhoul, N., and Batuman, V. Role of proximal tubules in the pathogenesis of kidney disease. Contrib Nephrol 169, 37, 2011. 40. Guay-Woodford, L.M. Molecular insights into the pathogenesis of inherited renal tubular disorders. Curr Opin Nephrol Hypertens 4, 121, 1995. 41. Santer, R., Kinner, M., Lassen, C.L., Schneppenheim, R., Eggert, P., Bald, M., Brodehl, J., Daschner, M., Ehrich, J.H., Kemper, M., Li Volti, S., Neuhaus, T., Skovby, F., Swift, P.G., Schaub, J., and Klaerke, D. Molecular analysis of the SGLT2 gene in patients with renal glucosuria. J Am Soc Nephrol 14, 2873, 2003. 42. Turk, E., Zabel, B., Mundlos, S., Dyer, J., and Wright, E.M. Glucose/galactose malabsorption caused by a defect in the Na+/glucose cotransporter. Nature 350, 354, 1991. 43. Tenenhouse, H.S., and Murer, H. Disorders of renal tubular phosphate transport. J Am Soc Nephrol 14, 240, 2003. 44. Santer, R., Groth, S., Kinner, M., Dombrowski, A., Berry, G.T., Brodehl, J., Leonard, J.V., Moses, S., Norgren, S., Skovby, F., Schneppenheim, R., Steinmann, B., and Schaub, J. The mutation spectrum of the facilitative glucose transporter gene SLC2A2 (GLUT2) in patients with Fanconi-Bickel syndrome. Hum Genet 110, 21, 2002. 45. Vallon, V. The proximal tubule in the pathophysiology of the diabetic kidney. Am J Physiol Regul Integr Comp Physiol 300, R1009, 2011. 46. Rahmoune, H., Thompson, P.W., Ward, J.M., Smith, C.D., Hong, G., and Brown, J. Glucose transporters in human renal proximal tubular cells isolated from the urine of patients with non-insulin-dependent diabetes. Diabetes 54, 3427, 2005. 47. Bland, R., Walker, E.A., Hughes, S.V., Stewart, P.M., and Hewison, M. Constitutive expression of 25-hydroxyvitamin D3-1alpha-hydroxylase in a transformed human proximal tubule cell line: evidence for direct regulation of vitamin D metabolism by calcium. Endocrinology 140, 2027, 1999. 48. Yoshida, T., Yoshino, J., Hayashi, M., and Saruta, T. Identification of a renal proximal tubular cell-specific enhancer in the mouse 25-hydroxyvitamin d 1alphahydroxylase gene. J Am Soc Nephrol 13, 1455, 2002. 49. Eckardt, K.U., Kurtz, A., and Bauer, C. Regulation of erythropoietin production is related to proximal tubular function. Am J Physiol 256, F942, 1989. 50. Pfaller, W., and Gstraunthaler, G. Nephrotoxicity testing in vitro--what we know and what we need to know. Environ Health Perspect 106 Suppl 2, 559, 1998. 51. Prieto, P. Barriers, nephrotoxicology and chronic testing in vitro. Altern Lab Anim 30 Suppl 2, 101, 2002. 52. Wu, Y., Connors, D., Barber, L., Jayachandra, S., Hanumegowda, U.M., and Adams, S.P. Multiplexed assay panel of cytotoxicity in HK-2 cells for detection of renal proximal tubule injury potential of compounds. Toxicol In Vitro 23, 1170, 2009. 53. Li, Y., Zheng, Y., Zhang, K., Ying, J.Y., and Zink, D. Effects of quantum dots on different renal proximal tubule cell models and on gel-free renal tubules generated in vitro. Nanotoxicology, 2011. 54. Olsavsky, K.M., Page, J.L., Johnson, M.C., Zarbl, H., Strom, S.C., and Omiecinski, C.J. Gene expression profiling and differentiation assessment in primary 119 human hepatocyte cultures, established hepatoma cell lines, and human liver tissues. Toxicol Appl Pharmacol 222, 42, 2007. 55. Guillouzo, A., and Guguen-Guillouzo, C. Evolving concepts in liver tissue modeling and implications for in vitro toxicology. Expert Opin Drug Metab Toxicol 4, 1279, 2008. 56. Bregoli, L., Chiarini, F., Gambarelli, A., Sighinolfi, G., Gatti, A.M., Santi, P., Martelli, A.M., and Cocco, L. Toxicity of antimony trioxide nanoparticles on human hematopoietic progenitor cells and comparison to cell lines. Toxicology 262, 121, 2009. 57. Baer, P.C., and Geiger, H. Human renal cells from the thick ascending limb and early distal tubule: characterization of primary isolated and cultured cells by reverse transcription polymerase chain reaction. Nephrology (Carlton) 13, 316, 2008. 58. Elberg, G., Guruswamy, S., Logan, C.J., Chen, L., and Turman, M.A. Plasticity of epithelial cells derived from human normal and ADPKD kidneys in primary cultures. Cell Tissue Res 331, 495, 2008. 59. Zhang, H., Tasnim, F., Ying, J.Y., and Zink, D. The impact of extracellular matrix coatings on the performance of human renal cells applied in bioartificial kidneys. Biomaterials 30, 2899, 2009. 60. Saito, A., Sawada, K., and Fujimura, S. Present status and future perspectives on the development of bioartificial kidneys for the treatment of acute and chronic renal failure patients. Hemodial Int 15, 183, 2011. 61. Tasnim, F., Deng, R., Hu, M., Liour, S., Li, Y., Ni, M., Ying, J.Y., and Zink, D. Achievements and challenges in bioartificial kidney development. Fibrogenesis Tissue Repair 3, 14, 2010. 62. Tumlin, J., Wali, R., Williams, W., Murray, P., Tolwani, A.J., Vinnikova, A.K., Szerlip, H.M., Ye, J., Paganini, E.P., Dworkin, L., Finkel, K.W., Kraus, M.A., and Humes, H.D. Efficacy and safety of renal tubule cell therapy for acute renal failure. J Am Soc Nephrol 19, 1034, 2008. 63. Humes, H.D., Fissell, W.H., Weitzel, W.F., Buffington, D.A., Westover, A.J., MacKay, S.M., and Gutierrez, J.M. Metabolic replacement of kidney function in uremic animals with a bioartificial kidney containing human cells. Am J Kidney Dis 39, 1078, 2002. 64. Humes, H.D., Weitzel, W.F., Bartlett, R.H., Swaniker, F.C., Paganini, E.P., Luderer, J.R., and Sobota, J. Initial clinical results of the bioartificial kidney containing human cells in ICU patients with acute renal failure. Kidney Int 66, 1578, 2004. 65. Ympa, Y.P., Sakr, Y., Reinhart, K., and Vincent, J.L. Has mortality from acute renal failure decreased? A systematic review of the literature. Am J Med 118, 827, 2005. 66. U. S. Renal Data System: USRDS 2008 Annual Data Report: Atlas of Chronic Kidney Disease and End-Stage Renal Disease in the United States National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 2008. 67. Bayat, S., Kessler, M., Briancon, S., and Frimat, L. Survival of transplanted and dialysed patients in a French region with focus on outcomes in the elderly. Nephrol Dial Transplant 25, 292, 2010. 68. McDonald, S.P., and Russ, G.R. Survival of recipients of cadaveric kidney transplants compared with those receiving dialysis treatment in Australia and New Zealand, 1991-2001. Nephrol Dial Transplant 17, 2212, 2002. 120 69. Wolfe, R.A., Ashby, V.B., Milford, E.L., Ojo, A.O., Ettenger, R.E., Agodoa, L.Y., Held, P.J., and Port, F.K. Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a first cadaveric transplant. N Engl J Med 341, 1725, 1999. 70. Kohn, O.F., Coe, F.L., and Ing, T.S. Solute kinetics with short-daily home hemodialysis using slow dialysate flow rate. Hemodial Int 14, 39, 2010. 71. Kraus, M., Burkart, J., Hegeman, R., Solomon, R., Coplon, N., and Moran, J. A comparison of center-based vs. home-based daily hemodialysis for patients with endstage renal disease. Hemodial Int 11, 468, 2007. 72. Davenport, A., Gura, V., Ronco, C., Beizai, M., Ezon, C., and Rambod, E. A wearable haemodialysis device for patients with end-stage renal failure: a pilot study. Lancet 370, 2005, 2007. 73. Gura, V., Davenport, A., Beizai, M., Ezon, C., and Ronco, C. Beta2microglobulin and phosphate clearances using a wearable artificial kidney: a pilot study. Am J Kidney Dis 54, 104, 2009. 74. Gura, V., Ronco, C., Nalesso, F., Brendolan, A., Beizai, M., Ezon, C., Davenport, A., and Rambod, E. A wearable hemofilter for continuous ambulatory ultrafiltration. Kidney Int 73, 497, 2008. 75. Aebischer, P., Ip, T.K., Panol, G., and Galletti, P.M. The bioartificial kidney: progress towards an ultrafiltration device with renal epithelial cells processing. Life Support Syst 5, 159, 1987. 76. Ip, T.K., and Aebischer, P. Renal epithelial-cell-controlled solute transport across permeable membranes as the foundation for a bioartificial kidney. Artif Organs 13, 58, 1989. 77. Uludag, H., Ip, T.K., and Aebischer, P. Transport functions in a bioartificial kidney under uremic conditions. Int J Artif Organs 13, 93, 1990. 78. Uludag, H., Panol, G., and Aebischer, P. Control of water flux in a bioartificial kidney. ASAIO Trans 35, 523, 1989. 79. Fissell, W.H., Dyke, D.B., Weitzel, W.F., Buffington, D.A., Westover, A.J., MacKay, S.M., Gutierrez, J.M., and Humes, H.D. Bioartificial kidney alters cytokine response and hemodynamics in endotoxin-challenged uremic animals. Blood Purif 20, 55, 2002. 80. Fissell, W.H., Lou, L., Abrishami, S., Buffington, D.A., and Humes, H.D. Bioartificial kidney ameliorates gram-negative bacteria-induced septic shock in uremic animals. J Am Soc Nephrol 14, 454, 2003. 81. Huijuan, M., Xiaoyun, W., Xumin, Y., Hengjin, W., and Xia, S. Effect of continuous bioartificial kidney therapy on porcine multiple organ dysfunction syndrome with acute renal failure. Asaio J 53, 329, 2007. 82. Chertow, G.M., and Waikar, S.S. Toward the promise of renal replacement therapy. J Am Soc Nephrol 19, 839, 2008. 83. Humes, H.D., Sobota, J.T., Ding, F., and Song, J.H. A selective cytopheretic inhibitory device to treat the immunological dysregulation of acute and chronic renal failure. Blood Purif 29, 183, 2010. 84. Humes, H.D., Buffington, D.A., MacKay, S.M., Funke, A.J., and Weitzel, W.F. Replacement of renal function in uremic animals with a tissue-engineered kidney. Nat Biotechnol 17, 451, 1999. 121 85. Inagaki, M., Yokoyama, T.A., Sawada, K., Duc, V.M., Kanai, G., Lu, J., Kakuta, T., and Saito, A. Prevention of LLC-PK(1) cell overgrowth in a bioartificial renal tubule device using a MEK inhibitor, U0126. J Biotechnol 132, 57, 2007. 86. Ozgen, N., Terashima, M., Aung, T., Sato, Y., Isoe, C., Kakuta, T., and Saito, A. Evaluation of long-term transport ability of a bioartificial renal tubule device using LLCPK1 cells. Nephrol Dial Transplant 19, 2198, 2004. 87. Sato, Y., Terashima, M., Kagiwada, N., Tun, T., Inagaki, M., Kakuta, T., and Saito, A. Evaluation of proliferation and functional differentiation of LLC-PK1 cells on porous polymer membranes for the development of a bioartificial renal tubule device. Tissue Eng 11, 1506, 2005. 88. Ni, M., Teo, J.C., Ibrahim, M.S., Zhang, K., Tasnim, F., Chow, P.Y., Zink, D., and Ying, J.Y. Characterization of membrane materials and membrane coatings for bioreactor units of bioartificial kidneys. Biomaterials 32, 1465, 2011. 89. Ueda, H., Watanabe, J., Konno, T., Takai, M., Saito, A., and Ishihara, K. Asymmetrically functional surface properties on biocompatible phospholipid polymer membrane for bioartificial kidney. J Biomed Mater Res A 77, 19, 2006. 90. Ni M, Z.P., Kandasamy K, Lai W, Li Y, Leong MF, Wan AC, and Zink D. The use of a library of industrial materials to determine the nature of substrate-dependent performance of primary adherent human cells. Biomaterials 33, 353, 2012. 91. Oo, Z.Y., Deng, R., Hu, M., Ni, M., Kandasamy, K., bin Ibrahim, M.S., Ying, J.Y., and Zink, D. The performance of primary human renal cells in hollow fiber bioreactors for bioartificial kidneys. Biomaterials 32, 8806, 2011. 92. Zhang, H., Lau, S.F., Heng, B.F., Teo, P.Y., Alahakoon, P.K., Ni, M., Tasnim, F., Ying, J.Y., and Zink, D. Generation of easily accessible human kidney tubules on twodimensional surfaces in vitro. J Cell Mol Med 15, 1287, 2011. 93. Grisaru, S., Cano-Gauci, D., Tee, J., Filmus, J., and Rosenblum, N.D. Glypican-3 modulates BMP- and FGF-mediated effects during renal branching morphogenesis. Dev Biol 231, 31, 2001. 94. Piscione, T.D., Phan, T., and Rosenblum, N.D. BMP7 controls collecting tubule cell proliferation and apoptosis via Smad1-dependent and -independent pathways. Am J Physiol Renal Physiol 280, F19, 2001. 95. Piscione, T.D., Yager, T.D., Gupta, I.R., Grinfeld, B., Pei, Y., Attisano, L., Wrana, J.L., and Rosenblum, N.D. BMP-2 and OP-1 exert direct and opposite effects on renal branching morphogenesis. Am J Physiol 273, F961, 1997. 96. Zeisberg, M., Hanai, J., Sugimoto, H., Mammoto, T., Charytan, D., Strutz, F., and Kalluri, R. BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat Med 9, 964, 2003. 97. Xu, Y., Wan, J., Jiang, D., and Wu, X. BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition in human renal proximal tubular epithelial cells. J Nephrol 22, 403, 2009. 98. Fan, J.M., Ng, Y.Y., Hill, P.A., Nikolic-Paterson, D.J., Mu, W., Atkins, R.C., and Lan, H.Y. Transforming growth factor-beta regulates tubular epithelial-myofibroblast transdifferentiation in vitro. Kidney Int 56, 1455, 1999. 99. Dudas, P.L., Argentieri, R.L., and Farrell, F.X. BMP-7 fails to attenuate TGFbeta1-induced epithelial-to-mesenchymal transition in human proximal tubule epithelial cells. Nephrol Dial Transplant 24, 1406, 2009. 122 100. Patel, S.R., and Dressler, G.R. BMP7 signaling in renal development and disease. Trends Mol Med 11, 512, 2005. 101. Simic, P., and Vukicevic, S. Bone morphogenetic proteins in development and homeostasis of kidney. Cytokine Growth Factor Rev 16, 299, 2005. 102. Gould, S.E., Day, M., Jones, S.S., and Dorai, H. BMP-7 regulates chemokine, cytokine, and hemodynamic gene expression in proximal tubule cells. Kidney Int 61, 51, 2002. 103. Simon, M., Maresh, J.G., Harris, S.E., Hernandez, J.D., Arar, M., Olson, M.S., and Abboud, H.E. Expression of bone morphogenetic protein-7 mRNA in normal and ischemic adult rat kidney. Am J Physiol 276, F382, 1999. 104. Vukicevic, S., Basic, V., Rogic, D., Basic, N., Shih, M.S., Shepard, A., Jin, D., Dattatreyamurty, B., Jones, W., Dorai, H., Ryan, S., Griffiths, D., Maliakal, J., Jelic, M., Pastorcic, M., Stavljenic, A., and Sampath, T.K. Osteogenic protein-1 (bone morphogenetic protein-7) reduces severity of injury after ischemic acute renal failure in rat. J Clin Invest 102, 202, 1998. 105. Wang, S.N., Lapage, J., and Hirschberg, R. Loss of tubular bone morphogenetic protein-7 in diabetic nephropathy. J Am Soc Nephrol 12, 2392, 2001. 106. Zeisberg, M. Bone morphogenic protein-7 and the kidney: current concepts and open questions. Nephrol Dial Transplant 21, 568, 2006. 107. Wang, S., Chen, Q., Simon, T.C., Strebeck, F., Chaudhary, L., Morrissey, J., Liapis, H., Klahr, S., and Hruska, K.A. Bone morphogenic protein-7 (BMP-7), a novel therapy for diabetic nephropathy. Kidney Int 63, 2037, 2003. 108. Zeisberg, M., and Kalluri, R. Reversal of experimental renal fibrosis by BMP7 provides insights into novel therapeutic strategies for chronic kidney disease. Pediatr Nephrol 23, 1395, 2008. 109. Hruska, K.A., Guo, G., Wozniak, M., Martin, D., Miller, S., Liapis, H., Loveday, K., Klahr, S., Sampath, T.K., and Morrissey, J. Osteogenic protein-1 prevents renal fibrogenesis associated with ureteral obstruction. Am J Physiol Renal Physiol 279, F130, 2000. 110. Sugimoto, H., Grahovac, G., Zeisberg, M., and Kalluri, R. Renal fibrosis and glomerulosclerosis in a new mouse model of diabetic nephropathy and its regression by bone morphogenic protein-7 and advanced glycation end product inhibitors. Diabetes 56, 1825, 2007. 111. Zeisberg, M., Bottiglio, C., Kumar, N., Maeshima, Y., Strutz, F., Muller, G.A., and Kalluri, R. Bone morphogenic protein-7 inhibits progression of chronic renal fibrosis associated with two genetic mouse models. Am J Physiol Renal Physiol 285, F1060, 2003. 112. Morrissey, J., Hruska, K., Guo, G., Wang, S., Chen, Q., and Klahr, S. Bone morphogenetic protein-7 improves renal fibrosis and accelerates the return of renal function. J Am Soc Nephrol 13 Suppl 1, S14, 2002. 113. Davies, M.R., Lund, R.J., and Hruska, K.A. BMP-7 is an efficacious treatment of vascular calcification in a murine model of atherosclerosis and chronic renal failure. J Am Soc Nephrol 14, 1559, 2003. 114. Bhogal, N., Grindon, C., Combes, R., and Balls, M. Toxicity testing: creating a revolution based on new technologies. Trends Biotechnol 23, 299, 2005. 123 115. Tumarkin, E., Tzadu, L., Csaszar, E., Seo, M., Zhang, H., Lee, A., Peerani, R., Purpura, K., Zandstra, P.W., and Kumacheva, E. High-throughput combinatorial cell coculture using microfluidics. Integr Biol (Camb) 3, 653, 2011. 116. Aydin, S., Signorelli, S., Lechleitner, T., Joannidis, M., Pleban, C., Perco, P., Pfaller, W., and Jennings, P. Influence of microvascular endothelial cells on transcriptional regulation of proximal tubular epithelial cells. Am J Physiol Cell Physiol 294, C543, 2008. 117. Bijuklic, K., Jennings, P., Kountchev, J., Hasslacher, J., Aydin, S., Sturn, D., Pfaller, W., Patsch, J.R., and Joannidis, M. Migration of leukocytes across an endothelium-epithelium bilayer as a model of renal interstitial inflammation. Am J Physiol Cell Physiol 293, C486, 2007. 118. Kim, B.S., Chen, J., Weinstein, T., Noiri, E., and Goligorsky, M.S. VEGF expression in hypoxia and hyperglycemia: reciprocal effect on branching angiogenesis in epithelial-endothelial co-cultures. J Am Soc Nephrol 13, 2027, 2002. 119. Linas, S.L., and Repine, J.E. Endothelial cells regulate proximal tubule epithelial cell sodium transport. Kidney Int 55, 1251, 1999. 120. Stoos, B.A., Carretero, O.A., Farhy, R.D., Scicli, G., and Garvin, J.L. Endothelium-derived relaxing factor inhibits transport and increases cGMP content in cultured mouse cortical collecting duct cells. J Clin Invest 89, 761, 1992. 121. Salerno, S., Campana, C., Morelli, S., Drioli, E., and De Bartolo, L. Human hepatocytes and endothelial cells in organotypic membrane systems. Biomaterials 32, 8848, 2011. 122. Takayama, G., Taniguchi, A., and Okano, T. Identification of differentially expressed genes in hepatocyte/endothelial cell co-culture system. Tissue Eng 13, 159, 2007. 123. Harimoto, M., Yamato, M., Hirose, M., Takahashi, C., Isoi, Y., Kikuchi, A., and Okano, T. Novel approach for achieving double-layered cell sheets co-culture: overlaying endothelial cell sheets onto monolayer hepatocytes utilizing temperature-responsive culture dishes. J Biomed Mater Res 62, 464, 2002. 124. Guzzardi, M.A., Vozzi, F., and Ahluwalia, A.D. Study of the crosstalk between hepatocytes and endothelial cells using a novel multicompartmental bioreactor: a comparison between connected cultures and cocultures. Tissue Eng Part A 15, 3635, 2009. 125. Liu, Y. Cellular and molecular mechanisms of renal fibrosis. Nat Rev Nephrol 7, 684, 2011. 126. Nangaku, M. Chronic hypoxia and tubulointerstitial injury: a final common pathway to end-stage renal failure. J Am Soc Nephrol 17, 17, 2006. 127. Phillips, A.O., and Steadman, R. Diabetic nephropathy: the central role of renal proximal tubular cells in tubulointerstitial injury. Histol Histopathol 17, 247, 2002. 128. Tang, S.C., Leung, J.C., and Lai, K.N. Diabetic tubulopathy: an emerging entity. Contrib Nephrol 170, 124, 2011. 129. Wagner, Z., Degrell, P., Lukats, B., Niwa, T., Molnar, G.A., Marko, L., Karadi, Z., and Wittmann, I. Accumulation of renin and imidazolone in peritubular capillary endothelial cells in insulin-resistant hypertensive rats. J Nephrol 24, 656, 2011. 130. Vesey, D.A., Qi, W., Chen, X., Pollock, C.A., and Johnson, D.W. Isolation and primary culture of human proximal tubule cells. Methods Mol Biol 466, 19, 2009. 124 131. Chung, S.D., Alavi, N., Livingston, D., Hiller, S., and Taub, M. Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium. J Cell Biol 95, 118, 1982. 132. Swencki-Underwood, B., Mills, J.K., Vennarini, J., Boakye, K., Luo, J., Pomerantz, S., Cunningham, M.R., Farrell, F.X., Naso, M.F., and Amegadzie, B. Expression and characterization of a human BMP-7 variant with improved biochemical properties. Protein Expr Purif 57, 312, 2008. 133. Shui, C., and Scutt, A.M. Mouse embryo-derived NIH3T3 fibroblasts adopt an osteoblast-like phenotype when treated with 1alpha,25-dihydroxyvitamin D(3) and dexamethasone in vitro. J Cell Physiol 193, 164, 2002. 134. Blaehr, H. Human renal biopsies as source of cells for glomerular and tubular cell cultures. Scand J Urol Nephrol 25, 287, 1991. 135. Kanwar, Y.S., Wada, J., Lin, S., Danesh, F.R., Chugh, S.S., Yang, Q., Banerjee, T., and Lomasney, J.W. Update of extracellular matrix, its receptors, and cell adhesion molecules in mammalian nephrogenesis. Am J Physiol Renal Physiol 286, F202, 2004. 136. Miner, J.H. Renal basement membrane components. Kidney Int 56, 2016, 1999. 137. Huang, J.T., and Lee, V. Identification and characterization of a novel human FOXK1 gene in silico. Int J Oncol 25, 751, 2004. 138. Brandenberger, R., Schmidt, A., Linton, J., Wang, D., Backus, C., Denda, S., Muller, U., and Reichardt, L.F. Identification and characterization of a novel extracellular matrix protein nephronectin that is associated with integrin alpha8beta1 in the embryonic kidney. J Cell Biol 154, 447, 2001. 139. Yoshioka, K., Hino, S., Takemura, T., Miyasato, H., Honda, E., and Maki, S. Isolation and characterization of the tubular basement membrane antigen associated with human tubulo-interstitial nephritis. Clin Exp Immunol 90, 319, 1992. 140. Zeisberg, M., Shah, A.A., and Kalluri, R. Bone morphogenic protein-7 induces mesenchymal to epithelial transition in adult renal fibroblasts and facilitates regeneration of injured kidney. J Biol Chem 280, 8094, 2005. 141. Tasnim, F., Kandasamy, K., Muck, J.S., Bin Ibrahim, M.S., Ying, J.Y., and Zink, D. Effects of bone morphogenetic proteins on primary human renal cells and the generation of bone morphogenetic protein-7-expressing cells for application in bioartificial kidneys. Tissue Eng Part A 18, 262, 2011. 142. Gregory, K.E., Ono, R.N., Charbonneau, N.L., Kuo, C.L., Keene, D.R., Bachinger, H.P., and Sakai, L.Y. The prodomain of BMP-7 targets the BMP-7 complex to the extracellular matrix. J Biol Chem 280, 27970, 2005. 143. Yanagita, M., Oka, M., Watabe, T., Iguchi, H., Niida, A., Takahashi, S., Akiyama, T., Miyazono, K., Yanagisawa, M., and Sakurai, T. USAG-1: a bone morphogenetic protein antagonist abundantly expressed in the kidney. Biochem Biophys Res Commun 316, 490, 2004. 144. Feng, X.H., and Derynck, R. Specificity and versatility in tgf-beta signaling through Smads. Annu Rev Cell Dev Biol 21, 659, 2005. 145. Kanellis, J., Fraser, S., Katerelos, M., and Power, D.A. Vascular endothelial growth factor is a survival factor for renal tubular epithelial cells. Am J Physiol Renal Physiol 278, F905, 2000. 146. Matsumoto, K., and Nakamura, T. Hepatocyte growth factor: renotropic role and potential therapeutics for renal diseases. Kidney Int 59, 2023, 2001. 125 147. Villegas, G., Lange-Sperandio, B., and Tufro, A. Autocrine and paracrine functions of vascular endothelial growth factor (VEGF) in renal tubular epithelial cells. Kidney Int 67, 449, 2005. 148. Ferrara, N. Vascular endothelial growth factor. Arterioscler Thromb Vasc Biol 29, 789, 2009. 149. Nakamura, T., and Mizuno, S. The discovery of hepatocyte growth factor (HGF) and its significance for cell biology, life sciences and clinical medicine. Proc Jpn Acad Ser B Phys Biol Sci 86, 588, 2011. 150. Schrijvers, B.F., Flyvbjerg, A., and De Vriese, A.S. The role of vascular endothelial growth factor (VEGF) in renal pathophysiology. Kidney Int 65, 2003, 2004. 151. You, W.K., and McDonald, D.M. The hepatocyte growth factor/c-Met signaling pathway as a therapeutic target to inhibit angiogenesis. BMB Rep 41, 833, 2008. 152. Miya, M., Maeshima, A., Mishima, K., Sakurai, N., Ikeuchi, H., Kuroiwa, T., Hiromura, K., Yokoo, H., and Nojima, Y. Enhancement of in vitro human tubulogenesis by endothelial cell-derived factors: implications for in vivo tubular regeneration after injury. Am J Physiol Renal Physiol 301, F387, 2011. 153. Montesano, R., Matsumoto, K., Nakamura, T., and Orci, L. Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor. Cell 67, 901, 1991. 154. Tufro, A., Norwood, V.F., Carey, R.M., and Gomez, R.A. Vascular endothelial growth factor induces nephrogenesis and vasculogenesis. J Am Soc Nephrol 10, 2125, 1999. 155. Tufro-McReddie, A., Norwood, V.F., Aylor, K.W., Botkin, S.J., Carey, R.M., and Gomez, R.A. Oxygen regulates vascular endothelial growth factor-mediated vasculogenesis and tubulogenesis. Dev Biol 183, 139, 1997. 156. Schluesener, H.J., and Meyermann, R. TGF-beta 1, beta 2, beta 1.2 and the bone morphogenetic protein BMP2: members of the transforming growth factor type beta supergene family with different morphogenetic effects on rat astrocyte cultures. Autoimmunity 9, 77, 1991. 157. Huang, S.S., and Huang, J.S. TGF-beta control of cell proliferation. J Cell Biochem 96, 447, 2005. 158. Tasnim, F., and Zink, D. Cross talk between primary human renal tubular cells and endothelial cells in cocultures. Am J Physiol Renal Physiol 302, F1055, 2012. 159. Kroening, S., Neubauer, E., Wullich, B., Aten, J., and Goppelt-Struebe, M. Characterization of connective tissue growth factor expression in primary cultures of human tubular epithelial cells: modulation by hypoxia. Am J Physiol Renal Physiol 298, F796, 2009. 160. Terryn, S., Jouret, F., Vandenabeele, F., Smolders, I., Moreels, M., Devuyst, O., Steels, P., and Van Kerkhove, E. A primary culture of mouse proximal tubular cells, established on collagen-coated membranes. Am J Physiol Renal Physiol 293, F476, 2007. 161. Valente, M.J., Henrique, R., Costa, V.L., Jeronimo, C., Carvalho, F., Bastos, M.L., de Pinho, P.G., and Carvalho, M. A rapid and simple procedure for the establishment of human normal and cancer renal primary cell cultures from surgical specimens. PLoS One 6, e19337, 2011. 162. Baer, P.C., Nockher, W.A., Haase, W., and Scherberich, J.E. Isolation of proximal and distal tubule cells from human kidney by immunomagnetic separation. Technical note. Kidney Int 52, 1321, 1997. 126 163. DeLeve, L.D., Wang, X., McCuskey, M.K., and McCuskey, R.S. Rat liver endothelial cells isolated by anti-CD31 immunomagnetic separation lack fenestrae and sieve plates. Am J Physiol Gastrointest Liver Physiol 291, G1187, 2006. 164. Wieser, M., Stadler, G., Jennings, P., Streubel, B., Pfaller, W., Ambros, P., Riedl, C., Katinger, H., Grillari, J., and Grillari-Voglauer, R. hTERT alone immortalizes epithelial cells of renal proximal tubules without changing their functional characteristics. Am J Physiol Renal Physiol 295, F1365, 2008. 165. Prozialeck, W.C., and Niewenhuis, R.J. Cadmium (Cd2+) disrupts Ca(2+)dependent cell-cell junctions and alters the pattern of E-cadherin immunofluorescence in LLC-PK1 cells. Biochem Biophys Res Commun 181, 1118, 1991. 166. Baer, P.C., Tunn, U.W., Nunez, G., Scherberich, J.E., and Geiger, H. Transdifferentiation of distal but not proximal tubular epithelial cells from human kidney in culture. Exp Nephrol 7, 306, 1999. 167. Chuman, L., Fine, L.G., Cohen, A.H., and Saier, M.H., Jr. Continuous growth of proximal tubular kidney epithelial cells in hormone-supplemented serum-free medium. J Cell Biol 94, 506, 1982. 168. Wilson, P.D., Dillingham, M.A., Breckon, R., and Anderson, R.J. Defined human renal tubular epithelia in culture: growth, characterization, and hormonal response. Am J Physiol 248, F436, 1985. 169. Powell, D.W., Mifflin, R.C., Valentich, J.D., Crowe, S.E., Saada, J.I., and West, A.B. Myofibroblasts. I. Paracrine cells important in health and disease. Am J Physiol 277, C1, 1999. 170. Tacke, F., Gabele, E., Bataille, F., Schwabe, R.F., Hellerbrand, C., Klebl, F., Straub, R.H., Luedde, T., Manns, M.P., Trautwein, C., Brenner, D.A., Scholmerich, J., and Schnabl, B. Bone morphogenetic protein is elevated in patients with chronic liver disease and exerts fibrogenic effects on human hepatic stellate cells. Dig Dis Sci 52, 3404, 2007. 171. Vukicevic, S., Latin, V., Chen, P., Batorsky, R., Reddi, A.H., and Sampath, T.K. Localization of osteogenic protein-1 (bone morphogenetic protein-7) during human embryonic development: high affinity binding to basement membranes. Biochem Biophys Res Commun 198, 693, 1994. 172. Lian, Y.G., Zhou, Q.G., Zhang, Y.J., and Zheng, F.L. VEGF ameliorates tubulointerstitial fibrosis in unilateral ureteral obstruction mice via inhibition of epithelial-mesenchymal transition. Acta Pharmacol Sin 32, 1513, 2011. 173. Webb, D.J., Wen, J., Karns, L.R., Kurilla, M.G., and Gonias, S.L. Localization of the binding site for transforming growth factor-beta in human alpha2-macroglobulin to a 20-kDa peptide that also contains the bait region. J Biol Chem 273, 13339, 1998. 127 Appendix: Abbreviations A2M: Alpha-2-macroglobulin α-SMA: α-smooth muscle actin ALPL: Alkaline phosphatase, liver, bone, kidney AP: Alkaline phosphatase AQP1: Aquaporin ARF: Acute renal failure ATCC: American Type Culture Collection AVP: Arginine vasopressin BAK: Bioartificial kidney BGLAP: Bone gamma carboxyglutamate protein BMP-2: Bone morphogenetic factor-2 BMP-7: Bone morphogenetic factor-7 BSA: Bovine serum albumin cAMP : Cyclic adenosine monophosphate CVVH : Continuous venovenous hemofiltration DAPI: 4’,6’-diamidino-2’-phenylindole DCN: Decorin DMEM: Dulbecco's Modified Eagle's Medium DMP1 : Dentin matrix protein E-CAD: E-cadherin ECM : Extracellular matrix coating EMT: Epithelial-to-mesenchymal transition ESRD: End stage renal disease FBS: Fetal bovine serum FGF: Fibroblast growth factor FST: Follistatin FSTL3: Follistatin-like GAPDH: Glyceraldehyde-3-phosphate dehydrogenase GBM: Glomerular basement membrane GGT: Gamma glutamyl transpeptidase GLUT: Glucose transporter HGF: Hepatocyte growth factor HPTC: Human primary renal proximal tubular cells HUVEC: Human umbilical vein endothelial cells HRGEC: Human renal glomerular endothelial cells IBMX: 3-isobutyl-1-methylxanthine IGF: Insulin-like growth factor LAP: Leucine aminopeptidase LLC-PK1: Lewis lung cancer-porcine kidney NUH: National University Hospital MDCK: Madin-Darby canine kidney MDR1: Multidrug resistance gene NBC1: Na+HCO - cotransporter N-CAD: N-cadherin 128 NUH: National University Hospital OAT: Organic anion transporter OCT: Organic cation transporter PBS: Phosphate-buffered saline PDGF: Platelet-derived growth factor PEPT: Proton-coupled peptide transporter PES: Polyethersulfone PHEX: Phosphate-regulating gene with homologies to endopeptidases on the X chromosome PI: Propidium iodide PSF: Polysulfone PSF-FC: Polysulfone–Fullcure PTC: Proximal tubular cells PTH: Parathyroid hormone PVP: Polyvinylpyrrolidone RAD: Renal tubule assist device RUNX2: Runt-related transcription factor SDS: Sodium dodecyl sulfate SGLT: Na+-glucose cotransporter SP7: Transcription factor SSP1: Secreted phosphoprotein TBS: Tris-buffered saline TBS-T: Tris-buffered saline containing 1% Tween-20 TGF-β1: Transforming growth factor β-1 THG: Tamm Horsfall glycoprotein TRIS: Tris hydroxymethyl aminomethane URO10: Uromodulin 10 VEGF: Vascular endothelial growth factor VIM: Vimentin Vit D3 Hydr: 25-hydroxyvitamin D3 1-hydroxylase ZO-1: Zona occludens-1 129 [...]... BAKs in clinical trials The distinguishing factor between BAKs and hemofiltration devices is the bioreactor unit containing renal cells As explained above, renal proximal tubule-derived cells have been used in BAK-related research, and for the clinical trials of BAKs, primary HPTC have been used However, most preceding in vitro and animal studies with BAKs were done with porcine primary renal proximal. .. used for studying the communication between renal tubular epithelial and endothelial cells in renal disease and repair Furthermore, such systems could then also be used for other applications, for instance in in vitro toxicology 27 2 Hypotheses and Goals Goals of my thesis were to: 1) a) Establish procedures for the isolation of HPTC from human renal tissues and b) characterize the isolated cells and. .. under in vitro conditions and this is one of the major obstacles for in vitro applications The underlying reason behind such a great interest in tissue models that contain more than one cell type is that a multiple cell type-system enables the study of cells in an environment more similar to that in the human body In the kidney, for example, the endothelial cells of the peritubular capillaries and the renal. .. sensitivity of the cells, and thus, thorough characterization of the cells is essential for applications in in vitro toxicology and kidney tissue engineering 17 One of the most important applications of HPTC is bioartificial kidney (BAK) development (60-62) Only this cell type has been approved for clinical applications (61, 63) BAKs containing HPTC have already been developed (62, 64); however, the work and. .. immunostaining, immunoblotting, qPCR and functional assays A thorough analysis and characterization of this primary cell type obtained from different donors and sources is essential for standardized applications of this cell type in in vitro systems and for the interpretation of results 2) Characterize the effects of BMPs on HPTC performance The hypothesis was that BMP-2 and/ or BMP-7 might inhibit epithelial... class I and class II antigens and adhesion molecules (34, 36, 37) PTC might also be involved in antigen presentation (38) and might interact with other cells in the renal cortex in producing or responding to costimulatory cytokines, i.e tumour necrosis factor alpha(35) In addition, PTC produce interleukin-6 in response to inflammatory cytokines (12) However, most of these studies were performed in vitro. .. to interspecies variability, it would be important to use primal renal proximal tubular cells of human origin Therefore, human primary renal proximal tubular cells (HPTC) would be most suitable for such applications However, the application of primary human cells is also associated with a variety of issues, which must be carefully addressed The costs of the primary cells are substantially higher and. .. addition of growth factors to HPTC Bone morphogenetic proteins (BMPs), in particular BMP-7 and bone morphogenetic factor-2 (BMP-2), are interesting candidates, based on their known effects on renal cells and tubule formation (93-95) In the following section, these growth factors, their effects on renal cells and possible applications will be discussed in detail 23 1.3 Genetic Engineering of HPTC and development... the pathophysiology of several diseases, renal PTC are considered one of the most important cell types for kidney tissue engineering Also, due to the function of renal tubular epithelial cells in glomerular filtrate concentration and drug transport (1, 15), in particular the renal PTC are a major target of drug-induced toxicity Therefore, this cell type is very important for in vitro toxicology studies... these findings suggest that transdifferentiation of HPTC and tubulogenesis (59, 92), which should be inhibited in BAKs, could probably be inhibited by application of BMPs Apart from the interesting effects on renal cells and tubulogenesis, BMP-7 has been FDA-approved for the treatment of human bone disease (release from local implant) There is also an increasing interest in the use of BMP-7 for the . ENGINEERING AND CHARACTERIZATION OF HUMAN RENAL PROXIMAL TUBULAR CELLS FOR APPLICATIONS IN IN VITRO TOXICOLOGY AND BIOARTIFICIAL KIDNEYS FARAH TASNIM (B de-differentiation of human primary renal proximal tubular cells (HPTC), which are most interesting for such applications, under in vitro conditions. HPTC are also important for the development of bioartificial. for in vitro toxicology studies (50-52). However, approved in vitro models based on renal cells have not been developed yet and this remains a major challenge. For applications of PTC to in vitro