SUMMARY African Americans develop kidney failure at rates 4-5 fold higher than European
Americans. Coding variants in the APOL1 gene, found only in people of recent African ancestry, drive a
large fraction of this risk disparity. We have named these APOL1 variants G1 (S342G and I384M) and
G2 (del388N389Y). Risk inheritance is recessive: only individuals carrying two variant APOL1 alleles
have a markedly increased risk of kidney disease. In the U.S. approximately 13% of African Americans,
or about 4,000,000 people, have a high-risk genotype. Most recessive diseases are caused by loss-of-
function mutation. Surprisingly, there is data from model systems in kidney cells, mice, drosophila, and
yeast that support the idea that the APOL1 risk variants (RV) are actually gain-of-function mutations
that cause toxicity when expressed at high levels. The overarching goal of this proposal is to
understand the mechanism of APOL1 kidney disease such that two RVs are required, providing insight
into this fundamental question of recessive, gain-of-function toxicity. We propose that the G0, or wild-
type, APOL1 allele can alter the behavior of the risk variants and thus prevent their toxicity. In AIM 1,
we will use isogenic, Crispr-engineered APOL1 BAC transgenic mice to determine the differing
properties of G0 and risk-variant APOL1 in the full complexity of the glomerulus. We will test the effects
of different APOL1 variants singly and in combination in order to answer questions about gene dosage,
WT rescue of risk-variant toxicity, and possible differences in mechanism of action between the two
major APOL1 risk alleles (G1 and G2). In AIM 2, our goal is to understand APOL1 targeting to lipid
droplets. We have observed that lipid droplets (LDs) show prominent G0 but little or no G1 or G2
localization in cell systems and that co-expression of G0 facilitates the movement of G1 or G2 onto lipid
droplets. We will use imaging and biochemical analyses to examine determinates of APOL1 localization
to LDs, determine if there are differences in the binding of APOL1 to other LD-associated proteins, and
define the cellular consequence(s) of altered APOL1 risk variant targeting to the LD. We will test the
relevance of these experiments with in vivo studies. In AIM 3, we will determine the role of aggregation
in APOL1-mediated cytotoxicity. We have observed that the APOL1 RVs have much stronger
propensity than G0 to aggregate. Our data indicate that RV aggregation occurs in mitochondria. We
hypothesize that aggregation is a key element in RV toxicity, and that G0 can interfere with RV
aggregation. In cell-based systems we will characterize the APOL1 aggregates, determine whether
aggregation is essential for APOL1-mediated cytotoxicity, understand the effect of aggregates on
mitochondrial function, and test whether G0 can reverse RV aggregation and cytotoxicity. In vitro
results will be validated in kidney organoids, a mouse model, and in human tissues.