ApoL1 Overexpression Drives Variant-Independent Cytotoxicity
| Autor: | Martha Konieczkowski, Yaping Gu, Leslie A. Bruggeman, William P. Schilling, John F. O’Toole, Sethu M. Madhavan, John R. Sedor, Diana L. Kunze, Zhenzhen Wu, Liping Luo |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Programmed cell death Patch-Clamp Techniques Genotype p38 mitogen-activated protein kinases Gene Expression p38 Mitogen-Activated Protein Kinases Ion Channels 03 medical and health sciences Autophagy Humans Patch clamp Phosphorylation Protein kinase A Cytotoxicity Chemistry HEK 293 cells Cell Membrane Sodium Genetic Variation General Medicine Tetracycline Apolipoprotein L1 Cell biology 030104 developmental biology Basic Research HEK293 Cells Nephrology Potassium Calcium Kidney Diseases Intracellular |
| Popis: | Coding variants in the APOL1 gene are associated with kidney diseases in African ancestral populations; yet, the underlying biologic mechanisms remain uncertain. Variant-dependent autophagic and cytotoxic cell death have been proposed as pathogenic pathways mediating kidney injury. To examine this possibility, we conditionally expressed APOL1-G0 (reference), -G1, and -G2 (variants) using a tetracycline-regulated system in HEK293 cells. Autophagy was monitored biochemically and cell death was measured using multiple assays. We measured intracellular Na(+) and K(+) content with atomic absorption spectroscopy and APOL1-dependent currents with whole-cell patch clamping. Neither reference nor variant APOL1s induced autophagy. At high expression levels, APOL1-G0, -G1, and -G2 inserted into the plasma membrane and formed pH-sensitive cation channels, causing collapse of cellular Na(+) and K(+) gradients, phosphorylation of p38 mitogen-activated protein kinase, and cell death, without variant-dependent differences. APOL1-G0 and -G2 exhibited similar channel properties in whole-cell patch clamp experiments. At low expression levels, neither reference nor variant APOL1s localized on the plasma membrane, Na(+) and K(+) gradients were maintained, and cells remained viable. Our results indicate that APOL1-mediated pore formation is critical for the trypanolytic activity of APOL1 and drives APOL1-mediated cytotoxicity in overexpression systems. The absence of cytotoxicity at physiologic expression levels suggests variant-dependent intracellular K(+) loss and cytotoxicity does not drive kidney disease progression. |
| Databáze: | OpenAIRE |
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