Abstract: FR-PO951
Protective Effect of Hydroxychloroquine on Cultured Mouse Podocytes Expressing the HIV Accessory Protein Vpr
Session Information
- Glomerular Diseases: Podocyte Biology - II
November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
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.