In vivo Modeling Implicates APOL1 in Nephropathy: Evidence for Dominant Negative Effects and Epistasis under Anemic Stress

Abstract

African Americans have a disproportionate risk for developing nephropathy. This disparity has been attributed to coding variants (G1 and G2) in apolipoprotein L1 (APOL1); however, there is little functional evidence supporting the role of this protein in renal function. Here, we combined genetics and in vivo modeling to examine the role of apol1 in glomerular development and pronephric filtration and to test the pathogenic potential of APOL1 G1 and G2. Translational suppression or CRISPR/Cas9 genome editing of apol1 in zebrafish embryos results in podocyte loss and glomerular filtration defects. Complementation of apol1 morphants with wild-type human APOL1 mRNA rescues these defects. However, the APOL1 G1 risk allele does not ameliorate defects caused by apol1 suppression and the pathogenicity is conferred by the cis effect of both individual variants of the G1 risk haplotype (I384M/S342G). In vivo complementation studies of the G2 risk allele also indicate that the variant is deleterious to protein function. Moreover, APOL1 G2, but not G1, expression alone promotes developmental kidney defects, suggesting a possible dominant-negative effect of the altered protein. In sickle cell disease (SCD) patients, we reported previously a genetic interaction between APOL1 and MYH9. Testing this interaction in vivo by co-suppressing both transcripts yielded no additive effects. However, upon genetic or chemical induction of anemia, we observed a significantly exacerbated nephropathy phenotype. Furthermore, concordant with the genetic interaction observed in SCD patients, APOL1 G2 reduces myh9 expression in vivo, suggesting a possible interaction between the altered APOL1 and myh9. Our data indicate a critical role for APOL1 in renal function that is compromised by nephropathy-risk encoding variants. Moreover, our interaction studies indicate that the MYH9 locus is also relevant to the phenotype in a stressed microenvironment and suggest that consideration of the context-dependent functions of both proteins will be required to develop therapeutic paradigms.

A tripartite complex of suPAR, APOL1 risk variants and αvβ3 integrin on podocytes mediates chronic kidney disease

“…6). Although APOL1 risk-variant proteins were linked to the induction of nonfunctional autophagosomes 21,22 , transgenic animal models expressing high-risk APOL1 variants did not develop the expected kidney phenotypes 9,10 . Indeed, whereas the addition of both suPAR and APOL1 G1 and G2 proteins resulted in an increase in the number of cells that contained measurable levels of autophagosomes, the most dramatic effect observed when both proteins were present was substantial cell detachment.…”

“…Moreover, the APOL1 high-risk genotype is strongly associated with nephropathy in people with HIV, a state of immune dysregulation 7,8 . However, a puzzling observation is that not all individuals carrying the high-risk APOL1 genotype develop renal disease, and equally, not all transgenic animal models, including models of mice 9 and zebrafish 10 , expressing high-risk APOL1 variants display the expected kidney phenotypes. Taken together, these findings suggest the possibility of a ‘second hit’ in the pathogenesis of APOL1 -associated nephropathy 10 .…”

The Cell Biology of APOL1

“…Morpholino or CRISPR/Cas9 knockdown of zebrafish (zf) apol1 caused pericardial edema, depressed glomerular filtration and podocyte foot process effacement 29, 41 , which was complemented by human APOL1 G0 but not G1 or G2 41 , suggesting variant APOL1 have lost an activity necessary for normal function of the pronephric glomerular filtration barrier. zfApol1 is expressed in podocytes and endothelial cells in the zebrafish pronephros and has 38% identity to human APOL1 41, 42 . The rapid gene duplication of the human APOL1 gene cluster makes it difficult to determine homologues outside primate species.…”

Kidney Disease Genetics and the Importance of Diversity in Precision Medicine

“…In the present study, the three MYH9 SNPs targeted for genotyping are in strong linkage disequilibrium with one another in all three racial/ethnic groups (r 2 ranging from 0.86 -1.0). Reports have implicated APOL1 as the driving cause of racial/ethnic disparity in kidney disease [21], though other functional studies suggest MYH9 remains relevant to kidney disease risk [22]. The lack of association may be attributable to the combination of heterogeneous kidney diseases among individuals in the Mexican American and non-Hispanic black samples.…”