Intracellular APOL1 Risk Variants Cause Cytotoxicity Accompanied by Energy Depletion

Abstract

Population genetic approaches have uncovered a strong association between kidney diseases and two sequence variants of the gene, called risk variant G1 and variant G2, compared with the nonrisk G0 allele. However, the mechanism whereby these variants lead to disease manifestation and, in particular, whether this involves an intracellular or extracellular pool of APOL1 remains unclear. Herein, we show a predominantly intracellular localization of APOL1 G0 and the renal risk variants, which localized to membranes of the endoplasmic reticulum in podocyte cell lines. This localization did not depend on the N-terminal signal peptide that mediates APOL1 secretion into the circulation. Additionally, a fraction of these proteins localized to structures surrounding mitochondria. overexpression of G1 or G2 lacking the signal peptide inhibited cell viability, triggered phosphorylation of stress-induced kinases, increased the phosphorylation of AMP-activated protein kinase, reduced intracellular potassium levels, and reduced mitochondrial respiration rates. These findings indicate that functions at intracellular membranes, specifically those of the endoplasmic reticulum and mitochondria, are crucial factors in APOL1 renal risk variant-mediated cell injury.

ApoL1 Overexpression Drives Variant-Independent Cytotoxicity

“…Building on APOL1's known trypanolytic activity, published in vitro evidence has suggested that variantdependent, autophagic, or cytotoxic cell death is responsible for progressive kidney disease. We did not detect variantdependent, differential cytotoxicity, findings which differ from several prior reports using HEK293 cells, 25,30,36,37 HEK293T cells, 38 or human podocytes. 26 The reasons that our data differ from earlier publications are unclear.…”

“…16 When associated with plasma membranes in cells, some report variant APOL1s permit greater intracellular potassium depletion than reference APOL1. 30,38 However, we found no variant-dependent differences in the intracellular potassium loss and found comparable channel activity in cells expressing APOL1-G0 compared with those expressing APOL1-G2. We characterized the mechanism of K + efflux for the first time in mammalian cells and found APOL1-G0 and APOL1-G2 both had pH-sensitive, nonselective, cation channel activity, consistent with previous reports.…”

APOL1 polymorphisms and kidney disease: loss-of-function or gain-of-function?

“…Despite strong evidence establishing CKD risk as a recessive trait (a mode of inheritance more commonly associated with loss-of-function mutations), most studies examining the disease mechanism have concluded the APOL1 variants produce a new protein function and represent gain-of-function mutations (18,26,28,36,44,48,62). The proposed new function endowed by the variants results in cytotoxicity; however, details of the biochemical mechanism are not fully established.…”

Recruitment of APOL1 kidney disease risk variants to lipid droplets attenuates cell toxicity

“…Several groups have investigated the intracellular localization and function of APOL1 in kidney cells, with differing conclusions. APOL1 has previously been reported to localize to the ER, partially to mitochondria, as well as to endosomes, lysosomes, and autophagosomes (10,19,20). Interestingly, although APOL1 has been well-characterized as an extracellular HDL-associated molecule (21), a hypothesized role as an intracellular lipid-binding protein has not been demonstrated (10,19).…”