A novel assay to assess the effect of pharmaceutical compounds on the differentiation of podocytes

Frances Kindt, Elke Hammer, Stefan Kemnitz, Antje Blumenthal, Paul Klemm, Rabea Schlüter, Susan E. Quaggin, Jens van den Brandt, Georg Fuellen, Uwe Völker, Karlhans Endlich, Nicole Endlich*

*Corresponding author for this work

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

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−1PAN 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.

Original languageEnglish (US)
Pages (from-to)163-176
Number of pages14
JournalBritish journal of pharmacology
Volume174
Issue number2
DOIs
StatePublished - 2017

ASJC Scopus subject areas

  • Pharmacology

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    Kindt, F., Hammer, E., Kemnitz, S., Blumenthal, A., Klemm, P., Schlüter, R., Quaggin, S. E., van den Brandt, J., Fuellen, G., Völker, U., Endlich, K., & Endlich, N. (2017). A novel assay to assess the effect of pharmaceutical compounds on the differentiation of podocytes. British journal of pharmacology, 174(2), 163-176. https://doi.org/10.1111/bph.13667