Abstract: TH-PO084

VDR Agonist (VDA) Facilitates Transition of Parietal Epithelial Cells (PECs) to Podocytes (PDS) Molecular Phenotype through Induction of APOL1

Session Information

Category: Glomerular

  • 1002 Glomerular: Basic/Experimental Pathology

Authors

  • Kumar, Vinod, Fienstine Institute for Medical Research, New York, New York, United States
  • Lan, Xiqian, Feinstein Institute for Medical Research, Great Neck, New York, United States
  • Marashi Shoshtari, Seyedeh Shadafarin, The Feinstein Institute for Medical Research, Manhasset, New York, United States
  • Aslam, Rukhsana, Feinstein Institute for medical research, Glenoaks, New York, United States
  • Ayasolla, Kamesh R., Feinstein Institute for Medical Research, Great Neck, New York, United States
  • Meyer-Schwesinger, Catherine, University of Hamburg, Hamburg, Germany
  • Malhotra, Ashwani, Feinstein Inst.Med research and NSLIJ, Manhasset, New York, United States
  • Skorecki, Karl, Rambam Health Care Campus, Haifa, Israel
  • Singhal, Pravin C., North Shore LIJ Health System, Great Neck, New York, United States
Background

APOL1 is expressed intracellularly and also a minor component of circulating lipid-rich trypanolytic multiprotein complexes in certain primate species including humans. However, the role of kidney cell , both in vitro and in vivo studies. We hypothesize that VDA is an inducer of APOL1 in PECs and plays an important role in the maintenance of PD homeostasis.

Methods

Immortalized human PECs proliferate at 33°C but enter into a transition mode (differentiated to PDs) after incubation in special media and collagen/fibronectin substrate at 37°C. PECs and differentiated PECs molecular phenotype (WT1 and podocalyxin vs. PAX2 and Claudin 1) was characterized. To determine the effect of VDA on the entry of PECs transition at 33°C, PECs were incubated in media containing either buffer or VDA (EB1089, 10 nM) for 48 hours at 33°C (n=4). TO evaluate the dose response effect, PECs were incubated in media containing different concentratios of VDA (0, 1, 10, 25, 50 and 100 nM) for 48 hours at 33°C (n=4).To determine the effect of other APOL1 stimulants, PECs were incubated in media containing variable concentrations of IFN-Y (0, 5, 10, and 20 nM) for 48 hours at 33°C. To examine a causal relationship, PECs were transfected with either control of miR193a plasmids/siRNA APOL1, followed by treatment with/without VDA (10 nM) or IFN-y (10 nm). Proteins and RNAs were extracted. Protein blots were probed for APOL1 and reprobed for WT1, podocalyxin, podocin, PAX2, Claudin 1, and GAPDH. cDNAs were probed for APOL1, WT1, and podocalyxin. RNAs were assayed for microRNA193a.

Results

VDA induced APOL1 protein expression in PECs in a dose dependent manner. IFN-y also induced APOL1 expression in PECs. Both VDA and IFN-γ also enhanced (2 to 3 fold) transcription of APOL1 in PECs. VDA-induced APOL1 expression was associated with down regulation of miR193a (2.3 fold), PAX2 (1.8 fold) and enhanced expression of WT1 (2.2 fold), podocalyxin (2.5 fold), and podocin (1.8 fold) (both mRNA and protein levels). However, the effects of VDA were partially reversed both by increasing miR193a or silencing APOL1 expressions.

Conclusion

VDA stimulates PECs transition through induction of APOL1 and down regulation of miR193a.

Funding

  • NIDDK Support