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Abstract: FR-PO951

Protective Effect of Hydroxychloroquine on Cultured Mouse Podocytes Expressing the HIV Accessory Protein Vpr

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Kajiyama, Hiroshi, Saitama Medical University, Iruma, Saitama, Japan
  • Kopp, Jeffrey B., NIDDK, NIH, Bethesda, Maryland, United States
  • Mimura, Toshihide, Saitama Medical University, Iruma, Saitama, Japan
Background

Studies in HIV-transgenic mice have implicated the HIV accessory protein R (Vpr) in podocyte injury, culminating in HIV-associated nephropathy. Clinical studies indicate that hydroxychloroquine reduces kidney damage in lupus. In this study, we tested protective effects of hydroxychloroquine on cultured mouse podocytes expressing Vpr.

Methods

Stably-transfected mouse podocytes bearing three transgenes, expressing Vpr (thermosensitive SV40 T antigen, podocin-promoter-rtTA and tet-responsive element-Vpr) or control AI podocytes expressing two transgenes (thermosensitive SV40 T and podocin-promoter-rtTA) were grown at 33°C and differentiated at 37°C. Differentiated podocytes were plated on day 1, hydroxychloroquine at concentrations ranging from 0.63 to 80 μg/mL was added to podocyte cultures on day 3, and 1 μg/mL doxycycline (DOX) was added day 4). Cell death was observed by phase-contrast microscopy on day 6 and 9, and total cell number and dead cell number were counted in each condition to obtain the percentage of dead cells.

Results

AI control and Vpr-expressing podocytes tolerated hydroxychloroquine concentrations of 10 μg/mL of hydroxychloroquine or less but died at higher concentrations. Vpr podocytes died 48 h after 1 μg/mL DOX was added, likely due to induced expression of Vpr. DOX-treated Vpr podocytes were protected from cell death with 24 h pretreatment of 0.63, 1.25, 2.5, 5 and 10 μg/mL hydroxychloroquine (H), in a dose-dependent manner (H0: 30.2%, H0.63: 25.0%, H1.25: 20.8%, H2.5: 4.3%, H5: 0%, H10: 0%). DOX-untreated Vpr podocytes without hydroxychloroquine also underwent cell death on day 9 of 37°C culture due to leaky expression of Vpr, which was partly inhibited by 5 and 10 μg/mL of hydroxychloroquine pretreatment (H0: 56.3%, H5: 32.0%, H10: 2.6%).

Conclusion

In cultured mouse podocytes expressing Vpr, hydroxychloroquine showed protective effects at up to 10 μg/mL. Hydroxychloroquine has diverse effects on mammalian cells, including increasing lysosomal pH, which in turn alters protein processing such as glycosylation. Hydroxychloroquine also alters Toll-like receptor signaling. The effects of hydroxychloroquine on Vpr-induced podocyte injury deserve further study.