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A novel assay to assess the effect of pharmaceutical compounds on the differentiation of podocytes
Author(s) -
Kindt Frances,
Hammer Elke,
Kemnitz Stefan,
Blumenthal Antje,
Klemm Paul,
Schlüter Rabea,
Quaggin Susan E,
Brandt Jens,
Fuellen Georg,
Völker Uwe,
Endlich Karlhans,
Endlich Nicole
Publication year - 2017
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/bph.13667
Subject(s) - podocyte , nephrin , synaptopodin , podocalyxin , genetically modified mouse , puromycin , chemistry , biology , microbiology and biotechnology , medicine , transgene , proteinuria , endocrinology , pharmacology , kidney , biochemistry , protein biosynthesis , gene
Background and Purpose 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. Experimental Approach 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. Key Results 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 −1 PAN on MFG. Conclusion and Implications 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.