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Abstract: PO1659

Characterization of the Direct Effect of Mycophenolic Acid on Murine Podocytes

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Abo Zed, Seif El Din, Children’s and Adolescents’ Hospital, Faculty of Medicine and University Hospital of Cologne, Cologne, Germany
  • Hackl, Agnes, Children’s and Adolescents’ Hospital, Faculty of Medicine and University Hospital of Cologne, Cologne, Germany
  • Bohl, Katrin, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany
  • Fink, Gregor, Children’s and Adolescents’ Hospital, Faculty of Medicine and University Hospital of Cologne, Cologne, Germany
  • Nüsken, Eva, Children’s and Adolescents’ Hospital, Faculty of Medicine and University Hospital of Cologne, Cologne, Germany
  • Nüsken, Kai Dietrich, Children’s and Adolescents’ Hospital, Faculty of Medicine and University Hospital of Cologne, Cologne, Germany
  • Schermer, Bernhard, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany
  • Weber, Lutz Thorsten, Children’s and Adolescents’ Hospital, Faculty of Medicine and University Hospital of Cologne, Cologne, Germany

Group or Team Name

  • Nephrologisches Forschungslabor
Background

Mycophenolic Acid (MPA) is the active component of Mycophenolate Mofetil, a selective, noncompetitive inhibitor of the inosine monophosphate dehydrogenase. Blockade of the de novo purine synthesis depletes the pool of deoxyguanosine triphosphate, leading to a specific suppression of proliferation of B- and T-Lymphocytes. MMF has an established role as a therapeutic agent in childhood nephrotic syndrome, where it currently serves as a second line option for frequent relapsing and steroid dependent cases. Although its immunological functions are well studied, direct effects of MPA on podocytes remain largely unknown. With first preliminary results showing a protective effect in vivo, the present study aims to examine the direct effect of MPA on murine podocytes and its abilities to alter albumin-induced podocyte injury.

Methods

Cultured murine podocytes were exposed to albumin for 48 hours, with one group receiving treatment with MPA for the second 24 hours. Cells were stained with a Synaptopodin antibody and additional markers to visualize components of the cytoskeleton. Currently, we are analyzing apoptosis through a TUNEL assay as well as alterations in intracellular Calcium content with Fluo-4 and fura red. In addition, we study podocyte mobility under injury and with MPA intervention by migration assays. We will also study small GTPases content and activity through a pull-down assay for RhoA and Rac1. In an unbiased approach, podocytes were exposed to either 2 hours of 10 mg/l MPA or an additional 22 hours of 4 mg/l MPA. Total RNA was isolated and subjected to RNASeq anaylses.

Results

Synaptopodin immunofluorescence shows significant alterations of the actin cytoskeleton through albumin exposure. MPA treated cells reveal a restorative ability of the drug, with a recovery of stress fiber formation and a reduction of albumin-induced vacuoles. mRNA expression analyses are in progress.

Conclusion

First results show a promising effect of MPA on stress fiber formation. The additional functional assays will be finished by late summer this year and will give an important insight to MPAs ability to influence pathways, known to be affected during the development of proteinuric diseases. The RNAseq results will provide an objective and detailed picture of the direct effects of MPA on podocytes.