Identifying the Intracellular Function of APOL1
Authors: Leslie A. Bruggeman, John F. O’Toole, John R. Sedor
Journal: Journal of the American Society of Nephrology (2017)

"We have to remember that what we observe is not nature herself, but nature exposed to our method of questioning" – Werner Heisenberg In 2010, nonsynonymous coding variants in the gene for APOL1 were strongly associated with nondiabetic CKD in blacks. A recessive model of inheritance best describes the association, and these kidney disease–associated variants are not present in individuals of other ethnicities. Although APOL1 kidney disease–associated variants are common (13% of United States blacks carry the risk genotype), only a subset of these individuals develops advanced CKD, which suggests that a second hit or environmental stressor is required to initiate disease. HIV-associated nephropathy, the CKD most strongly associated with APOL1 risk variants, is the clearest example of this environmental second hit theory. However, the biologic mechanisms underlying the APOL1 risk genotype and kidney pathogenesis have remained unclear. APOL1 is an abundant secreted protein that constitutively circulates as part of a subclass of HDL particles. This extracellular APOL1 has innate immune functions to lyse infecting flagellated parasites, such as trypanosomes. Both wild-type and variant APOL1s are endocytosed by the parasite and undergo a conformational change in an acid environment to insert into lipid membranes.1,2 Although the specific membrane target remains controversial, APOL1 forms an ion channel, ultimately resulting in the loss of membrane integrity and parasite death. However, circulating APOL1 levels fail to associate with CKD phenotypes,3,4 and kidney allograft outcomes track with the donor's not the recipient's, APOL1 genotype,5–7 suggesting that extracellular APOL1 is not contributing to CKD pathogenesis. APOL1 also is expressed in many tissues, including the kidney.8,9 Thus, investigations into the mechanisms of CKD have focused on intracellular APOL1 expressed in renal parenchymal cells, especially the podocyte, the key cell type injured in proteinuric CKDs associated with the APOL1 risk alleles. In this issue of the Journal of the American Society of Nephrology, three reports have explored the intracellular function of APOL1.10–12 Because APOL1 is only found in humans and a few other primates, novel methods and experimental systems are needed to investigate APOL1 function in renal cells. Fu et al.10 and Kruzel-Davila et al.11 both examined the role of intracellular APOL1 using the Gal4-UAS system to control tissue-specific gene expression in Drosophila. Human APOL1-G0 and risk variants G1 and G2 were expressed in the Drosophila nephrocyte (a detoxifying podocyte-proximal tubule hybrid cell), and nephrocyte function, nephrocyte death, and fly survival were assessed. Both studies made similar observations. APOL1 expression dysregulated the nephrocyte's normal endocytic activity, with an early transient increase followed by a later decrease in endocytic uptake of hemolymph proteins. Cells expressing G1 or G2 risk variants had more severe phenotypes than G0. With age, nephrocytes hypertrophied and died, and fly lifespan was reduced. Fly nephrocyte loss recapitulated the decreased podocyte density noted in mice expressing an APOL1-G2 transgene controlled by the Nphs1 promoter.13 However, unlike the flies, this mouse model and a similarly designed zebrafish model14 had no evidence for cytotoxicity or kidney pathology. Fu et al.10 also examined fly vacuole acidification. Both G0 and G1 reduced organelle acidification, an effect more pronounced with age, but again, the phenotype was more severe in nephrocytes expressing G1. Both studies came to similar conclusions: APOL1 dysregulates nephrocyte endocytic activity, but APOL1-G1 or -G2 caused more severe dysfunction, which was manifest by nephrocyte attrition in flies expressing the variant APOL1s. Kruzel-Davila et al.11 also studied APOL1 function in the yeast Saccharomyces cerevisiae. In contrast to Drosophila, APOL1-G0 caused significant toxicity when expressed in yeast cells, and the G1 and G2 variants were marginally more cytotoxic. The authors also systematically expressed APOL1 in various mutant yeast strains to identify the metabolic pathways in epistasis with cytotoxicity. Similar to the fly studies by Fu et al.,10 they also identified defective organelle acidification and additionally showed that strains defective in endosomal trafficking but not autophagy were more sensitive to APOL1-induced cytotoxicity compared with wild-type yeast, an effect that was similar for APOL1-G0, -G1, and -G2. Interestingly, studies of trypanolytic mechanisms show that APOL1 traffics through endolysosomal pathways to reach a target organelle.1 Data in both papers suggest the APOL1 risk variants G1 and G2 are inherently toxic (or more toxic) forms of the APOL1 protein, suggesting that they are gain of function mutations. This central conclusion, however, is difficult to reconcile with two well replicated observations in humans. First, a recessive model best explains APOL1-associated CKD risk, and humans heterozygous for either G1 or G2 are not at substantially increased risk for CKD. If the G1 and G2 risk variants had an inherent toxicity, an additive model should best fit CKD risk. Alternatively, a model has been proposed15 that addresses this inconsistency and speculates the membrane-associated APOL1 functions as a multimer where the G0 form is dominant negative when coexpressed with G1 or G2, thereby suppressing the variant-dependent toxicity. This hypothesis was not tested in any of the current reports but could be evaluated in the fly or yeast model systems. Second, the APOL1 risk genotype is insufficient to cause CKD in humans. Genetic evidence shows that CKD is caused when the risk genotype is combined with an environmental stressor. None of the fly or yeast experiments included a second hit trigger. The third study by Ma et al.12 used a human embryonic kidney cell line engineered for inducible expression of APOL1-G0 or the -G1 or -G2 risk variants. Using comparative pattern analyses of genome-wide transcriptomes, G1- or G2-expressing cells versus G0 cells were enriched in pathways related to mitochondrial function. In follow-up studies, proximal tubule cells were cultured from nephrectomy specimens from blacks without kidney disease. Gene expression profiles from proximal tubule cells from individuals with APOL1 high-risk genotypes were compared with those from individuals with no risk alleles. Although the authors did not compare endogenous levels of APOL1 in these cell lines, the gene most significantly increased was fibrinogen β-chain gene, and the genes most significantly decreased were involved in biosynthesis of ATP and nicotinate energy carriers, representative of mitochondrial pathways in pattern analysis. Functionally, APOL1-G1 and -G2 both impaired mitochondrial respiration compared with APOL1-G0, a finding that is consistent with APOL1's function in trypanolysis where APOL1 targets mitochondria, causing membrane depolarization that results in endonuclease release and DNA fragmentation (hallmarks of an apoptosis-like death).2 Thus, similar to the studies from flies and yeast, the results of the study by Ma et al.12 also suggest that variant APOL1s have a gain of function that negatively affects organelle functions requiring membrane integrity. The consistency among all three reports in that variant APOL1s differentially disrupt intracellular organelle function is most compelling when considered with established information regarding APOL1's role in trypanolysis and its known protein structure. APOL1 has a membrane binding domain, and extracellular APOL1 is always bound to HDL particles; thus, it is logical that, when APOL1 is intracellular, it too will be associated with lipid-containing structures and organelles. The APOL1 trypanosome killing function involves events that disrupt organelle membrane integrity, and the pathways identified in these three reports are consistent with some mechanisms proposed for trypanolysis through pore formation (Figure 1A). The candidate pathways identified in these new reports similarly show that APOL1 disrupts organellar function, either mitochondrial membrane potential in human kidney cells or endocytic trafficking and acidification in nephrocytes and yeast (Figure 1B). Given that APOL1 evolved as an immune effector molecule, modeling CKD mechanisms on its known immune function (trypanolysis) is attractive, and models based on trypanolytic mechanisms may explain kidney diseases from immune-mediated events, such as IFN infusion16 or direct HIV-1 infection of kidney cells. However, experiments robustly expressing APOL1 may not mimic the pathways responsible for kidney diseases that slowly progress over years. Most blacks with APOL1 risk genotypes do not develop kidney diseases, despite the fact that variant APOL1 protein is expressed in healthy human kidney. Like other innate immune effector proteins, APOL1 is always circulating but has no apparent function, that is, until the immune system is challenged and APOL1 is now usable to kill the invading pathogen. Extrapolating this to the intracellular function of APOL1 (and remembering that most vertebrates do not have or need APOL1 for normal kidney function), APOL1 expressed in the podocyte or proximal tubule cell may also have no function until a disease-causing stressor is introduced.Figure 1.: Summary of APOL1 killing mechanisms associated with lysosomal and mitochondrial membrane permeabilization by intracellular, membrane-associated APOL1. (A) Trypanolysis begins with endocytosis of HDL particles containing APOL1 (red). With acidification of endosomes, the engulfed APOL1 undergoes a conformation change that results in membrane insertion, forming a pore. Acidified APOL1 can be shuttled via kinesins to mitochondria, causing mitochondrial membrane depolarization and subsequent activation of apoptotic-like death triggers, or endosomes containing APOL1 can fuse with lysosomes and cause lysosomal membrane permeabilization and osmotic swelling. (B) In yeast, fly nephrocytes, and human kidney cells, APOL1 is produced by normal biosynthetic pathways within the cell. APOL1 caused an increase in endocytic activity and reduced organelle acidification, ultimately resulting in cell hypertrophy and death. Mitochondrial membrane potential was also reduced and preceded cell death. Cytotoxicity of APOL1 was pH dependent. Although not directly examined in these reports, others have observed that APOL1 may insert into plasma membranes (dashed lines) of both trypanosomes and human kidney cells.17 , 18 ER, endoplasmic reticulum; Hpr, Haptoglobin-related protein.Developing model organisms to study APOL1 is necessary and has been and will continue to be useful, but all model systems have limitations. These three studies highlight the tractability and power of Drosophila and yeast model organisms. However, when studies using model systems fail to replicate human observations, their interpretation requires conservative assessment. The value of data from model organisms will be greatest when the experimental design accurately replicates the human disease condition. For APOL1, model system studies will be incomplete unless they address the recessive inheritance pattern and the requirement of a second hit stressor. Disclosures None.