EXPERIMENTAL CELL RESEARCH 236, 248–258 (1997) ARTICLE NO. EX973739 Rearrangements of the Cytoskeleton and Cell Contacts Induce Process Formation during Differentiation of Conditionally Immortalized Mouse Podocyte Cell Lines Peter Mundel, 1 Jochen Reiser, Aime ´e Zu ´n ˜ iga Mejı B a Borja,* Hermann Pavensta ¨ dt,† Gary R. Davidson,* Wilhelm Kriz, and Rolf Zeller* ,1 Department of Anatomy and Cell Biology, University of Heidelberg, D-69120 Heidelberg, *European Molecular Biological Laboratory, D- 69117 Heidelberg, Germany; and †Division of Nephrology, Department of Medicine, University of Freiburg, D-79106 Freiburg, Germany Key Words: epithelial-to-mesenchymal transdiffer- entiation; podocytes; process formation; synapto- Mature podocytes are among the most complex dif- podin; WT-1. ferentiated cells and possess a highly branched array of foot processes that are essential to glomerular fil- tration in the kidney. Such differentiated podocytes are unable to replicate and culturing of primary podo- INTRODUCTION cytes results in rapid growth arrest. Therefore, condi- tionally immortalized mouse podocyte clones (MPC) Podocytes are highly specialized cells with a complex were established, which are highly proliferative when cellular architecture contributing to many functions of cultured under permissive conditions. Nonpermissive the normal kidney glomerulus. In particular, podocyte conditions render the majority of MPC cells growth differentiation results in cells extending numerous pri- arrested within 6 days and induce many characteris- mary and secondary foot processes that function in ul- tics of differentiated podocytes. Both proliferating and trafiltration of the renal glomerulus ([1]; reviewed in differentiating MPC cells express the WT-1 protein and [2]). Furthermore, evidence has been obtained that de- an ordered array of actin fibers and microtubules ex- generation of podocytes is central to the development tends into the forming cellular processes during differ- of chronic renal failure. Differentiated podocytes of the entiation, reminiscent of podocyte processes in vivo. adult are unable to replicate (reviewed in [3]). How- These cytoskeletal rearrangements and process for- ever, isolation and culturing of glomeruli results in a mation are accompanied by the onset of synaptopodin fraction of podocytes reentering the cell cycle and the synthesis, an actin-associated protein marking spe- outgrowth of these cells from glomeruli. Such cells re- cifically differentiated podocytes. In addition, focal gain a limited proliferation potential and can be tran- contacts are rearranged into an ordered pattern in dif- siently maintained in culture. However, such cultiva- ferentiating MPC cells. Most importantly, electrophys- tion of podocytes induces dedifferentiation, as reflected iological studies demonstrate that differentiated MPC by loss of processes [4, 5] and differentiation-specific cells respond to the vasoactive peptide bradykinin by markers such as synaptopodin (formerly called pp44; changes in intracellular calcium concentration. These [6 – 8]). Synaptopodin is specifically expressed by pro- results suggest a regulatory role of podocytes in glo- cess-bearing, differentiated podocytes in vivo, but not merular filtration. Taken together, these studies estab- their precursors nor other glomerular cells [6]. Re- lish that conditionally immortalized MPC cells retain cently, cell culture conditions were modified to permit a differentiation potential similar to podocytes in vivo. Therefore, the determinative steps of podocyte differ- partial in vitro differentiation of cultured podocytes de- entiation and process formation are studied for the rived from rat and human kidneys. Such differentiation first time using an inducible in vitro model.  1997 results in process formation and synaptopodin expres- Academic Press sion, but is always associated with growth arrest [7]. Therefore, it would be advantageous to create cell lines retaining the potential to both proliferate and differen- tiate along the podocyte lineage. Such cell lines would 1 To whom correspondence and reprint requests should be ad- dressed. Peter Mundel: Department of Anatomy and Cell Biology, enable the study of molecular regulation of podocyte University of Heidelberg, Im Neuenheimer Feld 307, D-69120 Hei- differentiation and function, as little is currently delberg, Germany. Fax: /49-6221-544951. E-mail: peter.mundel@ known about these processes. urz.uni-heidelberg.de. Rolf Zeller: EMBL Meyerhofstrasse 1, D- Jat et al. [9] established transgenic mice expressing 69117 Heidelberg, Germany. Fax: /49-6221-387516. Zeller@ EMBL-Heidelberg.de. a temperature-sensitive SV40 large T antigen (tsA58 248 0014-4827/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.