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

作者：Kindt Frances; Hammer Elke; Kemnitz Stefan; Blumenthal Antje; Klemm Paul; Schlueter Rabea; Quaggin Susan E; van den Brandt Jens; Fuellen Georg; Voelker Uwe; Endlich Karlhans; Endlich Nicole^*

来源：British Journal of Pharmacology, 2017, 174(2): 163-176.

DOI：10.1111/bph.13667

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 ofMFG. Dexamethasone (25 mu M) and pioglitazone (10 mu M) markedly attenuated the effect of 0.6 mu g center dot 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.

• 出版日期2017-1
• 单位西北大学