Frances Kindt1,2, Elke Hammer3, Stefan Kemnitz4, Antje Blumenthal1, Paul Klemm1, Rabea Schlüter5, Susan E Quaggin6, Jens van den Brandt7, Georg Fuellen2, Uwe Völker3, Karlhans Endlich1, Nicole Endlich1. 1. Institute of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany. 2. Institute for Biostatistics and Informatics in Medicine and Ageing Research, Rostock University Medical Centre, Rostock, Germany. 3. Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany. 4. Computational Science Group, Institute of Physics, Ernst Moritz Arndt University, Greifswald, Germany. 5. Imaging Centre of the Faculty of Mathematics and Natural Sciences, Ernst Moritz Arndt University, Greifswald, Germany. 6. Feinberg Cardiovascular Research Institute and Division of Nephrology and Hypertension, Northwestern University, Chicago, IL, USA. 7. Central Core and Research Facility of Laboratory Animals, University Medicine Greifswald, Greifswald, Germany.
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-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.
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.
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