title: A novel assay to assess the effect of pharmaceutical compounds on the differentiation of podocytes.
abstract: Therapeutic options for treating glomerulopathies, the main cause of chronic kidney disease, are limited. Podocyte dedifferentiation is a major event in the pathogenesis of glomerulopathies. The goal of the present study was, therefore, to develop an assay to monitor podocyte differentiation suitable for compound screening.
  
  
  
  We isolated and cultured glomeruli from transgenic mice, expressing cyan fluorescent protein (CFP) under the control of the promoter of nephrin, a marker of podocyte differentiation. Mean CFP fluorescence intensity per glomerulus (MFG) was determined by summation of all glomerular voxels from confocal z-stacks in the absence and presence of pharmaceutical compounds.
  
  
  
  In untreated cultured glomeruli, MFG remained fairly stable during the first 5 days, when foot processes were already effaced, and the level of many podocyte-specific proteins was only mildly affected, as revealed by proteomics. Between day 6 and 9, MFG decreased to almost zero. The decrease in MFG was paralleled by a decrease in CFP and nephrin expression, as determined by RT-PCR, western blots and proteomics. Puromycin aminonucleoside (PAN), which damages podocytes, concentration-dependently induced a complete loss of MFG. Dexamethasone (25 μM) and pioglitazone (10 μM) markedly attenuated the effect of 0.6 μg·mL-1PAN on MFG.
  
  
  
  In summary, we established a novel assay to assess the effect of pharmaceutical compounds on the differentiation of podocytes in situ. Our assay is suitable for compound screening to identify drugs for the treatment of glomerulopathies.
authors.affiliation: Institute of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany.|Institute for Biostatistics and Informatics in Medicine and Ageing Research, Rostock University Medical Centre, Rostock, Germany.
authors.forename: Frances
authors.initials: F
authors.lastname: Kindt
authors.affiliation: Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Elke
authors.identifier: 0000-0002-1507-0402
authors.initials: E
authors.lastname: Hammer
authors.affiliation: Computational Science Group, Institute of Physics, Ernst Moritz Arndt University, Greifswald, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Kemnitz
authors.affiliation: Institute of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Antje
authors.initials: A
authors.lastname: Blumenthal
authors.affiliation: Institute of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Paul
authors.initials: P
authors.lastname: Klemm
authors.affiliation: Imaging Centre of the Faculty of Mathematics and Natural Sciences, Ernst Moritz Arndt University, Greifswald, Germany.
authors.forename: Rabea
authors.initials: R
authors.lastname: Schlüter
authors.affiliation: Feinberg Cardiovascular Research Institute and Division of Nephrology and Hypertension, Northwestern University, Chicago, IL, USA.
authors.forename: Susan E
authors.initials: SE
authors.lastname: Quaggin
authors.affiliation: Central Core and Research Facility of Laboratory Animals, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: van den Brandt
authors.affiliation: Institute for Biostatistics and Informatics in Medicine and Ageing Research, Rostock University Medical Centre, Rostock, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Fuellen
authors.affiliation: Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Völker
authors.affiliation: Institute of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Karlhans
authors.identifier: 0000-0001-6052-6061
authors.initials: K
authors.lastname: Endlich
authors.affiliation: Institute of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Endlich
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects*; D003907:Dexamethasone / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D051379:Mice; D008822:Mice, Transgenic; D008855:Microscopy, Electron, Scanning; D000077205:Pioglitazone; D050199:Podocytes / Q000166:cytology / Q000187:drug effects*; D011692:Puromycin Aminonucleoside / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D045162:Thiazolidinediones / Q000494:pharmacology*
chemical_list: D045162:Thiazolidinediones; D011692:Puromycin Aminonucleoside; D003907:Dexamethasone; D000077205:Pioglitazone