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

Background and Purpose Therapeutic options to treat 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 suited for compound screening. Experimental Approach We isolated and cultured glomeruli from transgenic mice, expressing cyan fluorescent protein (CFP) under 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 of MFG was paralleled by a decrease in CFP and nephrin expression as determined by RT-PCR, Western blot and proteomics. Puromycin aminonucleoside (PAN), which damages podocytes, concentration-dependently induced a complete loss of MFG (IC50 = 0.3 µg/ml). Dexamethasone (25 μM) and pioglitazone (10 μM) markedly attenuated the effect of 0.6 µg/ml 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 suited for compound screening to identify drugs for the treatment of glomerulopathies. This article is protected by copyright. All rights reserved.