SUMMARY: Two ancestry-associated coding variants in APOL1 drive much of the racial disparity in rates of
kidney disease. However, the presence of two risk alleles (a high-risk genotype) does not lead to kidney disease
in all or even most individuals, indicating that other factors must also be important. Several environmental 2nd
hits have been identified such as HIV, therapeutic interferon, and Covid19. APOL1 itself is an innate immune
factor induced by interferons. A unifying theme is that both the APOL1 high-risk genotype and factors that elevate
APOL1 expression are required for development of APOL1 kidney disease. In addition to APOL1 protein function
as a membrane pore, APOL1 mRNA 3’-UTR has inversely oriented alu repeats that form a hairpin loop of double-
stranded RNA (dsRNA). This loop acts as an inflammatory factor because dsRNA is recognized by pattern
recognition receptors (PRR). Activation of PRR induces an anti-viral state with production of additional
interferons, which can trigger more APOL1 expression in a vicious cycle. Several mechanisms act as brakes on
APOL1 activity to prevent excess toxicity including transcript repression by miRNA and prevention of the PRR
inflammatory response by the A-to-I editing enzyme ADAR. We believe that imbalance between APOL1
activation and suppression may trigger kidney disease. To continue these studies, we will now: (1) Examine how
miRNA activity at the APOL1 3-’UTR regulates APOL1 expression. miRNA regulate gene expression by binding
to mRNA (usually 3’-UTR), promoting its degradation. We identified ~80 miRNAs in a high-throughput screen
with the potential to reduce APOL1 mRNA levels. We will perform a counterscreen to identify the most potent
miRNA at preventing APOL1 upregulation by interferons. We will find the intersection between miRNA expressed
in the podocyte and those that repress APOL1. We will test whether human polymorphisms in the APOL1 3’-
UTR alter miRNA binding and gene expression. We will validate the most relevant miRNA in vivo in APOL1 BAC
transgenic mice. (2) Characterize environmental and endogenous factors that induce or modify APOL1
expression. We will test a wide range of exogenous and endogenous molecules for their ability to induce APOL1
expression and to modulate cytokine-driven APOL1 expression. We will test the most potent molecules in APOL1
BAC transgenic mice. (3) Examine the role of APOL1 mRNA as an innate immune signaling molecule. We will
determine the specific PRR that recognize APOL1 3’-UTR dsRNA and whether that recognition and the resulting
inflammation depends on APOL1 genotype. We will assess how A-to-I editing of the APOL1 3’-UTR by ADAR
or protection by mRNA binding proteins may prevent the inflammatory loop driven by PRR recognition of APOL1
mRNA. We will test the effect of ADAR editing on the stability of the APOL1 mRNA and how A-to-I editing alters
3’-UTR binding by miRNA. Together, these experiments will help us understand the positive and negative factors
that regulate the interferon-driven APOL1 feed-forward inflammatory loop, APOL1 expression levels, and
podocyte injury leading to kidney disease.