APOL1-mediated kidney disease: a narrative review of the lessons learnt from the past 15 years

The APOL1 genetic risk variants, G1 and G2, are among the most powerful common risk variants for kidney disease that have been found in African ancestry. These two variants never occur on the same allele due to complete negative linkage disequilibrium [ 13 ]. Since these variants confer an adaptive advantage against certain trypanosome infections endemic to sub-Saharan Africa, this explains their high frequencies among populations of African descent. However, the allele frequencies of G1 and G2 vary greatly both globally and within Africa, averaging approximately 23% and 15%, respectively in African Americans [ 12 , 36 , 37 ]. Within in sub-Saharan Africa, an area with high burden of sleeping sickness, the frequencies of these variants tend to be higher, reaching their peak in West Africa, where G1 frequencies exceed 40% and G2 range from 6 to 24%, with the highest levels reported in Ghana and Nigeria [ 29 , 38 ]. Therefore, just like the haemoglobin mutation in individuals with sickle cell disease (SCD), APOL1 risk variants were inherited through positive selection, even though this has led to increased risk of kidney disease [ 33 ]. Unlike most common complex disease variants that follow additive or multiplicative patterns, APOL1-mediated kidney disease follows essentially autosomal-recessive inheritance. The presence of two alleles in homozygous (G1/G1 or G2/G2) or compound heterozygous (G1/G2) form, termed “ APOL1 high-risk genotype (HRG)”, is necessary to result in a drastically increased risk of developing kidney disease [ 1 , 13 ]. Over the last 15 years, numerous studies have confirmed the significant effect of these genetic variants on kidney disease. Individuals carrying the APOL1 HRG have an approximately 4-fold increased risk of developing lupus nephritis, a 7-fold increased risk of developing hypertensive nephrosclerosis, a 17-fold increased risk of primary FSGS, and an astounding 29 to 86-fold increased risk of HIVAN, when compared to individuals carrying the APOL1 low-risk genotype (LRG: G0/G0, G1/G0, and G2/G0) [ 1 , 13 , 29 , 39 – 43 ]. Additionally, as shown in Fig. 2 , data indicate a link between APOL1 HRG and increased risk of both sickle cell nephropathy (SCN) [ 1 , 44 – 49 ] and coronavirus-associated nephropathy [ 50 – 53 ]. Fig. 2 Spectrum of APOL1-mediated kidney disease. APOL1 risk variants are associated with a range of kidney diseases, including lupus nephritis, focal segmental glomerulosclerosis, human immunodeficiency virus (HIV)-associated nephropathy, and sickle cell nephropathy Spectrum of APOL1-mediated kidney disease. APOL1 risk variants are associated with a range of kidney diseases, including lupus nephritis, focal segmental glomerulosclerosis, human immunodeficiency virus (HIV)-associated nephropathy, and sickle cell nephropathy Overall, approximately 50% of individuals of African ancestry carry at least one APOL1 risk variant, and 6–30% possess an HRG [ 37 , 38 , 44 , 54 ]. This high allele frequency, large effect size, and the recessive inheritance pattern have led researchers to question whether APOL1-mediated kidney disease might be more accurately characterized as a Mendelian disease with significant modifiers rather than a complex disease [ 33 ]. Importantly, most people with APOL1 HRG do not develop APOL1-mediated kidney disease, and multiple studies have found variations in odds ratios across various aetiologies linked to the APOL1 risk variants. These findings suggest that carrying an APOL1 HRG does not guarantee the development of kidney disease [ 12 , 38 , 55 ], implying the need for a “second hit” factor in conjunction with a genetic predisposition. However, some evidence challenges the strictly recessive model of APOL1-mediated kidney disease [ 38 , 56 , 57 ]. Although it was initially proposed that carrying a single risk variant does not confer an increased risk of kidney disease [ 58 ], emerging data—including a recent study by the H3Africa Kidney Disease Research Network (KDRN)—have revealed that a single copy of an APOL1 risk variant may still increase the risk of kidney disease [ 38 ], as well as kidney allograft loss and rejection [ 59 ]. These findings highlight the need to refine our understanding of APOL1-associated risk, considering both gene dosage and modifiers in disease pathogenesis. APOL1 is only expressed in higher primates, which presents considerable challenges in understanding its impact on human health through the available experimental models. Although podocytes are considered the main target of APOL1-induced cytotoxicity in human kidneys [ 18 ], APOL1 protein is also present in other kidney cell types, including extraglomerular vascular endothelial cells [ 60 , 61 ]. Moreover, a growing body of evidence suggests extra-podocyte functions of APOL1 risk variants [ 62 ]. For example, Wu et al. proposed that the presence of APOL1 risk variants in endothelial cells could explain the observed association between these risk variants and hypertension, preeclampsia, and hypertensive kidney disease—conditions that typically affect the endothelium [ 63 ]. Thus, the wide range of proposed mechanisms underlying APOL1-mediated kidney disease may be partly attributed to the diverse cellular and animal models used across different studies, as outlined below. HEK293 cells are the most commonly used cellular model for studying APOL1 biology, despite not being native to the kidney. They are widely favoured due to their rapid proliferation, ease of maintenance, and high efficiency of transfection in a variety of methods [ 64 ]. As HEK293 cells do not endogenously express APOL1, these cells have been utilized in overexpression systems, through either stable or transient expression of the APOL1 protein [ 65 – 67 ]. This model has been pivotal in demonstrating that APOL1 risk variants can induce significantly greater cytotoxicity compared to the G0 allele [ 65 , 67 , 68 ], although conflicting results have been reported [ 69 , 70 ]. Moreover, HEK293 cells have been instrumental in elucidating the dysregulated intracellular pathways associated with APOL1 risk variants [ 66 , 67 , 71 – 73 ]. For instance, research has shown that APOL1 risk variants cause an ion homeostasis imbalance, resulting in cellular stress and cell death [ 66 , 67 ]. While HEK293 cells provide a valuable platform for understanding the fundamental molecular and cellular mechanisms underlying APOL1-mediated kidney disease, their major limitations lie in their lack of specialized functions inherent to podocytes or any other kidney cells [ 74 ]. Moreover, the 2-dimensional (2D) culture system used for HEK293 cells lacks the complex physiological environment of kidney tissues, making it difficult to fully recapitulate the in vivo conditions necessary to accurately model APOL1-induced cytotoxicity. Studies exploring the link between APOL1 risk variants and kidney disease are largely conducted in podocytes. Podocytes are specialized glomerular epithelial cells that form the slit diaphragms, a crucial component of the glomerular filtration barrier [ 75 , 76 ]. Human podocytes used in APOL1 research are typically derived from tissue or urine of human subjects and can be utilized as either primary cells or conditionally immortalized cells [ 61 , 77 , 78 ]. The immortalization process is usually achieved using the temperature-sensitive (tsA58) T large antigen variant of simian virus 40 and human telomerase reverse transcriptase [ 79 ]. Many functional studies of APOL1 in podocytes employ either an overexpression system [ 66 , 74 ] or induce endogenous APOL1 variants through inflammatory cytokines such as IFN-γ and polyinosinic-polycytidylic acid (poly I: C) [ 78 , 80 , 81 ] or gene editing [ 80 ]. Although these approaches have greatly advanced our understanding of APOL1-mediated kidney disease pathophysiology, the use of 2D podocyte cultures still presents inherent limitations in fully capturing in vivo cellular complexity [ 79 , 82 ]. While APOL1 has been predominantly studied in the context of podocytes, emerging evidence suggests it may have significant non-podocyte functions [ 62 ]. Recently, APOL1 risk variants have been implicated in conditions characterized by heightened inflammation and endothelial cell activation, such as sepsis [ 63 , 83 ] and acute kidney injury [ 51 ]. Using human umbilical vein endothelial cells (HUVECs), Blazer et al. [ 57 ] investigated the effects of APOL1 risk variants on endothelial cells. Their study demonstrated that IFN-γ stimulation significantly upregulated APOL1 expression across all genotypes. However, HUVECs carrying two copies of APOL1 risk variants exhibited both reduced baseline and maximum mitochondrial oxygen consumption and impaired mitochondrial networking compared to cells with the non-risk G0 allele. To further corroborate the cytotoxic effects of APOL1 risk variants in endothelial cells, the authors reported that HUVECs with two copies of the risk variants had defective autophagic flux, evidenced by a contracted lysosomal compartment and an accumulation of autophagosomes. These cytotoxic effects occurred in both the presence and absence of IFN-γ stimulation and followed an additive model of disease, as cells with one risk variant showed intermediate mitochondrial and autophagic flux defects [ 57 ]. Additional studies have reinforced the role of APOL1 in endothelial cell dysfunction [ 60 , 63 ]. Carracedo et al. [ 60 ] suggested that APOL1 may serve as an inducer of endothelial cell activation in humans. Wu et al. [ 63 ] found that circulating serum APOL1 levels correlated with sepsis, and single-cell RNA sequencing of human kidneys revealed high APOL1 expression in endothelial cells. Their study further demonstrated that APOL1 G2 transgenic mice with endothelial-specific expression of APOL1 developed endotheliopathy, characterized by increased endothelial inflammation, vascular permeability, elevated adhesion molecule expression, and defects in the endothelial glycocalyx. Notably, the APOL1 G2 variant caused mitochondrial dysfunction and mitophagy defects, which activated the NLRP3 inflammasome and intracellular cytosolic nucleotide-sensing pathways. Interestingly, similar effects were observed in mice with podocyte-specific APOL1 risk variant expression, highlighting a shared mechanism [ 84 ], thus lending support for the potential use of endothelial cells as a model for studying the mechanism of APOL1-mediated kidney disease. APOL1 is widely expressed across cell types, making it challenging to study APOL1-mediated kidney disease in a single cell type. However, cellular microenvironments, critical for regulating cell function in vivo, are poorly replicated in 2D cultures [ 85 ]. In contrast, three-dimensional (3D) culture systems and kidney organoids derived from induced pluripotent stem cells (iPSCs) better mimic kidney-specific functions, native genomic contexts, and cell-type heterogeneity, offering valuable models for studying APOL1-mediated kidney disease [ 50 , 82 , 86 , 87 ]. In 2020, Liu et al. generated isogenic APOL1 G0 and G1 organoids by applying footprint-free CRISPR-Cas9 genome editing to introduce APOL1 G1 variant into iPSCs from a non-African ancestry, APOL1 G0/G0 donor. Single-cell RNA sequencing revealed cell-type-specific responses to APOL1 induction in G1 organoids [ 86 ]. Similarly, Chun et al. used single-stranded donor oligonucleotide CRISPR-Cas9 genome editing to create isogenic APOL1 G0/G0 and G2/G2 organoids. The G2 organoids exhibited significantly higher APOL1 expression after IFN-γ treatment, leading to the downregulation of genes associated with fatty acid metabolism and lipid droplet biogenesis [ 87 ]. Additionally, Nystrom et al. [ 50 ] and Juliar et al. [ 82 ] independently identified the Janus kinase / signal transducers and activators of transcription (JAK/STAT) signalling pathway as key to APOL1 induction in organoids. Despite their advantages, kidney organoids have important limitations. They resemble early-stage kidneys and lack full maturation, vascularization, and structural complexity, limiting their ability to model adult-onset or chronic diseases. Cellular composition varies across batches and may include off-target cell types, and organoids generally lack immune components and physiological cues such as fluid flow or systemic signalling [ 88 ]. These limitations necessitate caution when extrapolating findings to human kidney pathology. It is worth noting that kidney 3D cell cultures can be generated from immortalized cells, as is the case in bioartificial kidneys and kidney-on-chip technologies [ 89 ]. These systems provide dynamic platforms to study kidney disease mechanisms and test therapeutic interventions. Bioartificial kidneys simulate filtration and metabolic functions, while organ-on-chip systems replicate microenvironments, fluid dynamics, and cell-cell interactions. Integrating these approaches may enhance disease model and advance our understanding of kidney disease. Saccharomyces cerevisiae is a well-established model organism, widely used for genetic manipulations and biochemical analysis [ 90 ]. Although yeast lacks endogenous APOL1 , researchers have employed this model to elucidate the mechanism of APOL1-mediated kidney disease [ 70 , 91 , 92 ]. Kruzel-Davila et al. demonstrated that APOL1 expression in yeast leads to cell lethality across all variants, with a significantly higher degree of lethality in the risk variants. Furthermore, they identified that yeast strains defective in endosomal trafficking or organelle acidification—but not those defective in autophagy—were hypersensitive to APOL1-induced cytotoxicity [ 91 ]. In a recent study, the same authors showed that yeast strains expressing APOL1 variants with the deletion of the first six amino acids of exon 4 (ΔMSALFL) exhibited reduced sensitivity to cell injury, an effect attributed to impaired APOL1 translocation to the endoplasmic reticulum (ER). Supporting the role of ER translocation, the authors found that APOL1 G2-induced lethality was partially abrogated in yeast strains with deletions of genes encoding ER translocon proteins, such as SBH2, SEC72, and HUT1 [ 70 ]. Similarly, Chidiac et al. demonstrated that the ectopic expression of APOL1 in yeast leads to its mitochondrial localization, which is associated with alterations in mitochondrial morphology, disruption of mitochondrial function, and dissipation of the mitochondrial membrane potential, ultimately affecting yeast respiratory function [ 92 ]. Drosophila melanogaster , also known as the fruit fly, is a model frequently used for studying protein function and protein-protein interaction because they are easy to genetically manipulate [ 93 , 94 ]. Although Drosophila lacks endogenous APOL1 and a mammalian kidney-like excretory organ, it has nephrocytes (kidney cells) around the heart and oesophagus, which share structural and functional similarity with human podocytes and proximal tubule cells [ 91 , 93 ]. An APOL1 transgenic Drosophila model has been generated by 3 independent groups [ 91 , 93 , 95 ]. Fu et al. demonstrated that the ubiquitous expression of human APOL1 in Drosophila resulted in lethal phenotypes, with the effect being more pronounced in the APOL1 G1 transgenic flies compared to G0. Additionally, the expression of the transgene in nephrocytes impaired nephrocyte function, leading to hypertrophy and cell death, an effect that was more severe in APOL1 G2 transgenic flies. Moreover, APOL1 variants also disrupt organelle acidification in nephrocytes [ 93 ]. Supporting these findings, Kruzel-Davila et al. [ 91 ] reported that the ubiquitous expression of APOL1 risk variants G1 and G2 in Drosophila caused a near-complete lethality, while G0 had no effect. They further noted that the expression of APOL1 G1 and G2, but not G0, in nephrocytes led to endolysosomal defects, resulting in nephrocyte loss in adult flies [ 91 ]. In a recent study, Gerstner et al. demonstrated that the expression of human APOL1 risk variants in the podocyte-like garland cells of Drosophila nephrocytes triggered an ER stress response, which preceded cytotoxicity. They further stated that this cytotoxic effect could be rescued by inhibiting ER stress signalling [ 95 ]. Zebrafish ( Danio rerio ) express proteins that are highly homologous to human APOL1, thus making the zebrafish the second most employed animal model to study APOL1 functions [ 94 , 96 ]. Knocking out APOL1 in zebrafish led to pericardial oedema, compromised glomerular filtration, and disruption of the glomerular ultrastructure. The complementation of APOL1 morphants with human APOL1 G0 mRNA rescued these defects, while the complementation with human APOL1 G1 or G2 did not [ 96 ]. This suggests that APOL1 plays an essential function in the glomerular function of the fish. Kotb et al. further confirmed the role of APOL1 in zebrafish by injecting morpholino against APOL1 into zebrafish eggs and larvae. The authors reported that the injection of morpholino induced severe oedema and morphological changes in the glomerulus, which were accompanied by reduced expression of nephrin in zebrafish podocytes [ 97 ]. To understand the effect of the APOL1 risk variants in the zebrafish, Olabisi et al. used a Gal4-UAS system to express human APOL1 variants in podocytes and endothelial cells under podocin/Flk promoters, respectively. The authors reported that the human APOL1 risk variants were associated with histologic abnormalities in zebrafish glomeruli, although the kidney function remained normal, implying that a second hit is necessary for the development and progression of APOL1-mediated kidney disease [ 98 ]. Nevertheless, pathway analysis of differentially expressed transcripts in APOL1 G0 and G2 of zebrafish podocytes indicated that the autophagy pathways are essential for APOL1 G2-associated kidney dysfunction [ 99 ]. The mouse model is the most frequently used animal model to investigate APOL1-mediated kidney disease, even though no orthologs for human APOL1 are found in mice [ 94 ]. Bruggeman et al. reported the first transgenic APOL1 mouse model in 2016 [ 100 ]. These authors used the mouse nephrin ( Nphs1) promoter to target the expression of APOL1 G0 and G2 transgene to the podocytes. They demonstrated that the APOL1 expression did not cause overt kidney disease. However, these mice showed decreased podocyte density and more preeclampsia, an effect that was more pronounced in the APOL1 G2 mice [ 100 ]. Beckerman et al. used an alternative approach and generated APOL1 transgenic mice with podocyte-specific, conditionally inducible APOL1 expression under a doxycycline-controlled Nphs1 promoter [ 101 ]. They showed that mice with Nphs1 promoter-driven overexpression of the APOL1 risk variant developed proteinuria and global and segmental glomerulosclerosis, in contrast to APOL1 G0 mice, which had no kidney phenotype. This model recapitulates APOL1-mediated kidney disease in humans [ 101 ]. Kumar et al. have used this same strategy in developing TetOn3G-APOL1 transgenic mice [ 102 ] to further investigate APOL1 functions. The main limitation of the above transgenic mouse models lies in the difficulty of relating transgene expression levels to those observed in humans. To circumvent this issue, Okamoto et al. [ 103 ]. and Aghajan et al. [ 104 ] have developed a physiologically relevant mouse model of APOL1-mediated kidney disease that can be used to study APOL1 systemically and across cell types. Their strategies enable studies of APOL1 gene regulation by administering physiologic inflammatory stimuli, serving as a second hit [ 94 , 103 , 104 ]. Okamoto et al. cloned an approximately 47-kb human DNA, encompassing only the APOL1 gene with 5’ and 3’ flanking regions, and including the exons 1 and 2 of APOL2 and exons 39–41 of MYH9 into a bacterial artificial chromosome (BAC). Using this model, the authors showed that the BAC-APOL1-G2 variant mice exhibited significantly worse proteinuria following podocyte injury after the initiation of IFN-γ (to induce APOL1 expression), puromycin aminonucleoside and basic fibroblast growth factor (to induce podocyte injury) compared to the BAC-APOL1-G0 allele mice [ 103 ]. This same BAC approach has been used by McCarthy et al. to develop another APOL1 mouse model [ 105 ]. On the other hand, Aghajan et al. generated a human APOL1-transgenic mouse model by using approximately 32-kb fosmid fragment containing the entire human APOL1 gene as well as >5-kb upstream and downstream regions [ 104 ]. The authors reported that administration of IFN-γ to this mouse model induces proteinuria only in the APOL1 G1 mice, despite IFN-γ inducing kidney APOL1 expression in both the G0 and G1 transgenic mice. Additionally, other groups have developed dual-transgenic or triple-transgenic mouse models. Bruggeman et al. studied the effect of APOL1 variants in HIVAN by creating a dual-transgenic mouse model between transgenic 26 (Tg 26), HIV transgenic mouse line [ 106 ], and Nphs1.APOL1 transgenic mice. Their study showed that among the APOL1ⅹTg26 dual-transgenic mice, APOL1 G0ⅹTg26 showed less podocyte loss when compared to the APOL1 G2ⅹTg26 mice or the Tg26 mice [ 107 ]. Likewise, Ge et al. used a triple transgenic BAC/APOL1 ⅹ podocin-rtTA ⅹTRE/NFATc1nuc mouse model to investigate the role of APOL1 risk variants in lipid-mediated podocyte injury [ 108 ]. These authors indicated that the expression of APOL1 risk variants increases the susceptibility to lipid-mediated podocyte injury, leading to mitochondrial dysfunction [ 108 ]. Despite these advances, a major limitation remains that no mouse model truly replicates the chronic and progressive nature of human APOL1-mediated kidney disease, as disease manifestations in mice typically occur within days or weeks rather than over years. For a more comprehensive overview of mouse and other animal models used to study APOL1-mediated kidney disease, please refer to Yoshida et al. [ 94 ]. As highlighted above, inheriting APOL1 HRG does not guarantee the development of kidney disease. Furthermore, odds ratios differ across the various aetiologies associated with APOL1-mediated kidney disease. These findings suggest that APOL1 risk variants require an additional “second hit”—either environmental, genetic, or a combination of both—for the onset and progression of kidney disease [ 109 ]. Several environmental factors, such as viral infections, including HIV, John Cunningham virus, coronavirus, polyomavirus, and factors of immune response, have been stated as the most important modifying factors of APOL1-mediated kidney disease. The association between viruria (virus infection in the urine) and APOL1-mediated kidney disease suggests that viruses can activate an APOL1 response, which initiates kidney damage [ 18 , 40 , 110 ]. HIV appears to provide the most powerful evidence for the primacy of environmental influences on APOL1-mediated kidney disease [ 39 , 41 ]. The overwhelming interaction between HIV infection and the APOL1 risk variants increases the chances of developing HIVAN by 29 to 87-fold [ 1 , 39 , 41 , 54 ]. Similarly, recent studies have shown that patients with the APOL1 HRG infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can present with collapsing glomerulopathy, comparable to that observed in HIVAN [ 111 ]. Markers of inflammatory immune response, such as several types of interferons (IFN-γ) or tumour necrosis factor (TNF-α), have also been reported as mediators of APOL1-mediated kidney disease via the toll-like receptor (TLR) 3 pathway [ 81 , 112 ]. Interferons have been shown to increase APOL1 mRNA expression by tens to hundreds of fold in different models and in a study involving interferon treatments in patients, all individuals with APOL1 HRG developed collapsing glomerulopathy after the therapeutic administration of interferon [ 81 ]. Although the TLR3 signalling pathway has long been recognized for its role in APOL1 expression [ 113 ], the pathway activation is infrequent in other diseases, such as SCN, even though these are also associated with APOL1 risk variants [ 1 , 45 , 49 , 114 ]. This implies a nuanced interplay of pathways and modifiers in APOL1-mediated kidney disease, varying across different conditions. Indeed, the TLR4 pathway has also emerged as a possible pathway for inducing APOL1-mediated kidney disease. Nichols et al. have demonstrated its ability to activate APOL1 expression in podocytes and endothelial cells upon stimulation with lipopolysaccharide, a TLR4 agonist [ 81 ]. More recently, other environmental factors have been proposed. Grampp et al. suggested that hypoxia and drugs that stabilize hypoxia-inducible transcription factors (HIFs) can induce APOL1 expression in kidney cells through an active regulatory DNA-element upstream of APOL1 , that interacts with HIFs [ 115 ]. This pathway might be of crucial importance in APOL1-mediated SCN [ 45 ], a disease that is well characterized by hypoxia [ 49 ]. Likewise, Paranjpe et al. utilized a Bio Me Biobank cohort of 4800 self-identifying black participants to demonstrate that air pollution, measured by fine particulate matter < 2.5 μm (PM 2.5 ), can be an additional environmental trigger of APOL1-mediated kidney disease in individuals with African ancestry living in New York [ 116 ]. In their study, the authors found a multiplicative interaction between PM 2.5 and APOL1: individuals with APOL1 HRG had an increased adjusted odds ratio of 1.54 of worse kidney outcome for every 10 µg/m 3 increase in PM 2.5 , while those with APOL1 LRG had a better kidney outcome (increased adjusted odds ratio of 1.11) for every 10 µg/m 3 increase in PM 2.5 [ 116 ]. Besides, another study by Hong et al. demonstrated that pregnant women of African ancestry with APOL1 HRG living in the Boston area, who were born within the United States, were at greater risk for preeclampsia when compared with those who migrated to the United States [ 117 ]. This finding lends further credence to the importance of environmental modifiers in modulating APOL1 function. Due to the absence of a clear association between APOL1 HRG and any specific gene in the genome-wide association study (GWAS) by Langefeld et al., the authors suggested that environmental factors, rather than gene-gene interactions, may play a more significant role in triggering kidney disease in individuals with APOL1 HRG [ 118 ]. However, as outlined below, recent clinical studies, experimental research, and GWAS findings (Table 1 ) did identify several genetic modifiers that can influence the impact of APOL1 on kidney disease development and progression. Table 1 Genome-wide association study-identified genetic modifiers interacting with APOL1 risk variants Gene Protein Reference SNP identifier Reference NPHS2 Podocin rs16854341 Divers et al. 2014 [ 119 ] SDCCAG8 Serologically defined colon cancer antigen 8 rs2802723 Divers et al. 2014 [ 119 ] BMP4 Bone morphogenetic protein 4 rs8014363 Divers et al. 2014 [ 119 ] DEFB1 β-defensin-1 rs73188225 Vy et al. 2022 [ 120 ] SMOC2 SPARC-related modular calcium-binding protein 2 rs2181251, rs62423451, rs11751195, rs4286744, rs62422403 Chaudhary et al. 2022 [ 121 ] Genome-wide association study-identified genetic modifiers interacting with APOL1 risk variants APOL1 exhibits a diverse range of genetic variations that are organized into distinct haplotypes, including at least 8 relatively common non-risk haplotypes with unique coding sequences. Interestingly, this specific combination of genetic variations within an individual’s APOL1 haplotype can influence their susceptibility to kidney disease [ 65 ]. The reference haplotype (E150, M288, R255: EMR) represents a non-risk sequence that is widely documented in databases and encoded by commercially available APOL1 cDNA plasmids [ 36 ]. Lannon et al. reported that the APOL1 cytotoxicity is dependent on the haplotype background, with G1 and G2 being more toxic in HEK cells when expressed in their native, African haplotype background (E150, I228 and K255: EIK) compared to their expression in either the reference or the wild-type (K150, I228 and K255: KIK) haplotype background [ 65 ]. Besides, the authors observed that this increased cytotoxicity of APOL1 G1 and G2 variants in the African haplotype was substantially reduced when expressed in African haplotype plasmid constructs containing the p.N264K missense variant [ 65 ]. The p.N264K is present in a small fraction of both the G0 allele and the G2 variant (African haplotype) but is absent in the G1 variant [ 122 ]. Two different studies have highlighted the protective effect against APOL1-mediated kidney disease when individuals with APOL1 G2/G2 genotypes co-inherit the p.N264K variant. Gupta et al. reported that individuals with APOL1 G2/G2 are at least 8.3 times less likely to develop FSGS if they carry one copy of the p.N264K missense variant compared to those lacking this variant [ 122 ]. These findings are in alignment with the data from the Million Veteran Program, showing a reduced risk for CKD and kidney failure in individuals with APOL1 G2/G2 who also inherit the p.N264K variant [ 123 ]. The mechanisms by which these haplotypes modulate cytotoxicity remain unclear but are thought to involve regulation of the cation-selective pore-forming property of the risk variants, ultimately affecting the activation of cytotoxic pathways [ 124 ]. Supporting this, Gupta et al. reported that the efflux of intracellular [K + ] from APOL1-containing liposomes correlates with the haplotype-dependent cytotoxicity data. Specifically, the African haplotype conducts more intracellular [K + ] than the APOL1 wild-type haplotype, which in turn conducts more than the reference haplotype [ 74 ]. This hypothesis also applies to the p.N264K missense variant. Overexpression of APOL1 G2 with the p.N264K missense variant in HEK cells resulted in a complete lack of intracellular [Ca 2+ ] influx compared to the [Ca 2+ ] influx observed with the comparable expression of APOL1 G2 without p.N264K variant [ 123 ]. Therefore, these findings highlight the potential value of implementing genotyping of APOL1 haplotypes and the p.N264K variant into patient management and clinical research. They also underscore the need for further structural studies of full-length APOL1 to identify additional modifying loci. Variation at the UBD (FAT10) locus has been reported as a genetic modifier of the risk of FSGS in individuals with APOL1 HRG [ 125 ]. Like APOL1, UBD is upregulated in response to proinflammatory stimuli, including IFN-γ [ 126 ]. This protein encodes a ubiquitin-like protein modifier that targets proteins to the 26S proteasome for degradation [ 127 , 128 ]. Using a cellular model, Zhang et al. showed that expression of APOL1 risk variants, G1 and G2 and not the G0, led to upregulation of the UBD transcript. Despite this increased mRNA expression, there was a reduction in detectable UBD protein, indicating rapid UBD protein turnover in the presence of the APOL1 risk variants. Additionally, overexpression of UBD led to a decrease in APOL1 protein in cells overexpressing the G1 and G2 variants compared to the G0 allele [ 125 ]. These data suggest that UBD may mitigate the toxic effects of G1 and G2 by targeting the risk variants for destruction [ 125 ]. Hence, variants in UBD that genetically encode differences in UBD expression can modify kidney phenotypes associated with APOL1-mediated kidney disease. Therefore, human genetic studies are warranted to identify genetic variants that influence UBD expression and, in turn, modulate the course of APOL1-mediated kidney disease. This encodes the enzyme GSTM1, a member of the glutathione-S-transferases family, which plays a crucial role in metabolizing reactive oxygen species and reactive aldehydes, both of which are end products of lipid peroxidation [ 129 , 130 ]. A complete deletion of this gene, known as the GSTM1 null variant, results in the absence of the GSTM1 enzyme. This null variant is highly prevalent, with a homozygous null variant frequency of about 50% in Caucasians and 27% in Africans [ 130 , 131 ]. Similar to the risk variants of APOL1 , the GSTM1 null variant has been linked to an increased susceptibility to kidney disease [ 132 ]. In the context of APOL1-mediated kidney disease, a study by Bodonyi-Kovacs et al. within the African American Study of Kidney Disease and Hypertension cohort revealed that individuals carrying at least one copy of the GSTM1 null variant along with the APOL1 HRG exhibited the lowest baseline kidney function despite having the lowest mean arterial blood pressure. Additionally, these individuals had the lowest survival rate from incident kidney failure [ 130 ]. The precise mechanism by which GSTM1 modulates APOL1 remains unclear. However, it is known that the GSTM1 enzyme plays a role in regulating oxidative stress [ 130 , 133 ]. In patients with epilepsy, the presence of the GSTM1 null variant has been associated with higher circulating levels of malondialdehyde, a marker of oxidative stress [ 133 ]. This suggests that the null variant could contribute to increased oxidative stress, potentially exacerbating the overexpression of APOL1 in patients with the HRG. Although some studies have not observed a significant association between the GSTM1 null variant and kidney disease [ 134 ], further longitudinal and mechanistic studies are needed to validate and elucidate any potential interplay between APOL1 and GSTM1 in kidney disease. TMEM173 encodes the protein, stimulator of interferon genes (STING), an important protein in type 1 interferon response to viral double-stranded DNA [ 135 ]. However, gain-of-function mutations in TMEM173 lead to a life-threatening auto-inflammatory disease called STING-associated vasculopathy with onset in infancy (SAVI), which was first described in 2014 [ 136 , 137 ]. Such mutations in TMEM173 result in aberrant activation of STING, which enhances the production of type 1 interferons. These interferons initiate a positive feedback loop leading to the activation of the JAK1, STAT1, and STAT2, ultimately driving the transcription of pro-inflammatory interferon-stimulated genes [ 135 , 137 ]. An association between TMEM173 and APOL1-mediated kidney disease has been observed. Abid et al. reported a case involving an infant with the APOL1 G1/G2 genotype, who developed collapsing glomerulopathy due to elevated circulating interferon levels associated with his underlying SAVI condition [ 135 ]. It is worth mentioning that most of these proposed genetic modifiers lack reproducibility across studies, and as such many of them remain unvalidated. Future studies are therefore warranted to systematically validate these modifiers and define their mechanistic relevance. Furthermore, beyond the aforementioned environmental and genetic factors, other variables such as APOL1 isoform [ 70 , 138 , 139 ], intracellular localization of APOL1 [ 73 , 87 ], and APOL1 copy number [ 105 , 140 ] may impact APOL1-related cellular toxicity. Thus, understanding the role of these factors is also crucial for unravelling the mechanisms underlying APOL1-mediated kidney disease and their impact on cellular processes. Overexpression of APOL1 kidney risk variants in various experimental systems has been demonstrated to lead to a significant increase in cell death when compared to the non-risk, G0 allele [ 101 ]. However, despite the demonstrated deleterious effect of APOL1 risk variants and the localization of APOL1 in several cell types including the vascular endothelial cells and podocytes in both normal biopsies and biopsies of people with APOL1-mediated kidney disease [ 61 , 141 , 142 ], the mechanisms underlying the predisposition to the spectrum of nephropathies related to APOL1 remain incompletely understood. Evidence suggests that the risk variants exhibit a toxic gain-of-function effect rather than a loss-of-function [ 15 , 67 , 71 , 72 , 91 , 143 , 144 ]. This is supported by several lines of evidence: APOL1 is present only in humans and a few primates, and its absence in other mammals indicates that it is not essential for kidney development or function [ 15 ]. A human completely lacking APOL1 due to a null mutation exhibited normal kidney function, suggesting that APOL1 is not required for normal kidney physiology [ 144 ]. Experimental studies further demonstrate that APOL1, particularly its risk variants, exerts enhanced cytotoxic effects [ 67 , 71 , 72 , 91 , 143 ]. Moreover, the pathogenic effects of APOL1 risk variants on kidney disease are thought to be largely confined to podocytes, and, to some extent, endothelial cells [ 60 , 63 , 101 , 145 ]. Furthermore, evidence from kidney transplantation studies indicates that APOL1 expressed within the kidney, rather than circulating APOL1, is responsible for disease pathogenesis. Specifically, the risk of kidney allograft failure correlates with the donor kidney APOL1 genotype [ 146 – 148 ], whereas the recipient genotype has no impact on allograft survival [ 149 ]. As illustrated in Fig. 3 and highlighted below, multiple hypotheses have been proposed to elucidate how APOL1 risk variants induce kidney damage. These mechanisms include endolysosomal function disruption and autophagy [ 101 , 143 , 150 , 151 ], pyroptosis [ 101 ], mitochondrial dysfunction [ 152 ], impaired vacuolar acidification [ 91 ], enhanced pore-forming activity [ 66 – 68 ], activation of stress-activated kinases [ 67 ], ER stress [ 95 ], and cholesterol efflux [ 153 ]. In addition, APOL1 risk variants have been shown to have a high affinity for soluble urokinase plasminogen activator receptor (suPAR) and α v β 3 integrin on the podocyte cell membrane [ 154 ]. However, little or no consensus on the pathways involved has been reached. Fig. 3 Potential pathophysiological mechanisms of APOL1-mediated kidney disease. Overexpression of APOL1 can drive podocyte injury through multiple mechanisms. APOL1 can form cation pores or channels at the plasma membrane, allowing abnormal flux of monovalent and divalent cations. This ionic imbalance may depolarize the plasma membrane and further trigger calcium (Ca²⁺) release from the endoplasmic reticulum (ER), leading to ER stress. Elevated APOL1 expression and increased cytosolic Ca²⁺ can also impair mitochondrial function, promoting mitochondrial fragmentation, increased reactive oxygen species (ROS) production, and the release of mitochondrial DNA (mtDNA). These mitochondrial events can amplify inflammation by inducing pro-inflammatory cytokine release. Furthermore, APOL1 expression has been associated with lysosomal depolarization, increased autophagy, and reduced global protein synthesis, contributing to cellular dysfunction and kidney injury Potential pathophysiological mechanisms of APOL1-mediated kidney disease. Overexpression of APOL1 can drive podocyte injury through multiple mechanisms. APOL1 can form cation pores or channels at the plasma membrane, allowing abnormal flux of monovalent and divalent cations. This ionic imbalance may depolarize the plasma membrane and further trigger calcium (Ca²⁺) release from the endoplasmic reticulum (ER), leading to ER stress. Elevated APOL1 expression and increased cytosolic Ca²⁺ can also impair mitochondrial function, promoting mitochondrial fragmentation, increased reactive oxygen species (ROS) production, and the release of mitochondrial DNA (mtDNA). These mitochondrial events can amplify inflammation by inducing pro-inflammatory cytokine release. Furthermore, APOL1 expression has been associated with lysosomal depolarization, increased autophagy, and reduced global protein synthesis, contributing to cellular dysfunction and kidney injury APOL1 has been observed to colocalize with mitochondria, where it contributes to mitochondrial dysfunction through multiple mechanisms, as illustrated in Fig. 3 [ 72 , 143 , 152 ]. The overexpression of APOL1 risk variants disrupts mitochondrial function and membrane potential, as evidenced by a significant decrease in maximum respiration rate and reserve respiration capacity, without a concomitant reduction in mitochondrial mass [ 152 ]. Granado et al. similarly demonstrated that in APOL1-inducible HEK cells, the induction of the risk variants leads to a decrease in ATP concentration and activation of AMP-activated protein kinase (AMPK) signalling, thereby impairing mitochondrial function [ 143 ]. Additionally, oxidative stress may worsen APOL1-induced mitochondrial dysfunction. Indeed, cells expressing APOL1 G1 and G2 variants show reduced levels of superoxide dismutase 2 and catalase transcripts, which can further exacerbate mitochondrial dysfunction [ 152 ]. Unlike the non-risk G0 allele, APOL1 risk variants can form oligomers within mitochondria, translocating to the mitochondrial matrix via the TOMM20-dependent pathway. This process results in the opening of the mitochondrial permeability transition pore, leading to rapid mitochondrial depolarization, thus driving risk variant-dependent cytotoxicity [ 72 ]. Another way in which APOL1 risk variants contribute to mitochondrial dysfunction is by increasing mitochondrial fragmentation through enhanced mitochondrial fission [ 155 ]. Overexpression of APOL1 risk variants upregulates dynamic-1-like protein (DRP1), a key gene in the mitochondrial fission pathway. However, inhibition of DRP1 using Mdivi-1 has been shown to prevent mitochondrial fragmentation and restore mitochondrial membrane potential and cell viability in cells overexpressing the APOL1 risk variants [ 155 ]. It is important to note that the study by Scales et al. contested the localization of APOL1 to the mitochondria. The authors argued that the presence of APOL1 in the mitochondria may result from transfection-related artifacts, as mitochondrial import sequences are typically recognized in the cytoplasm rather than the secretory pathway [ 142 ]. Therefore, further research is needed to clarify the mechanisms of APOL1’s mitochondrial localization and its effects on cellular function. The ER is considered the primary intracellular localization for APOL1 [ 73 , 142 , 156 ]. Wen et al. were the first to propose an association between APOL1 and ER stress, observing a significant elevation of ER stress biomarkers—glucose-regulated protein 78 (GRP78) and the phosphorylated eukaryotic initiation factor 2 alpha subunit (eIF-2α)–in human podocytes stably expressing APOL1 risk variants compared to those expressing the G0 allele or the empty vector [ 157 ]. Their finding demonstrated that the elevated expression of these biomarkers was associated with APOL1-mediated podocyte injury, as inhibitors targeting these biomarkers were able to significantly reduce APOL1 risk variant-induced necrosis [ 157 ]. This association between APOL1 risk variants and ER stress has been corroborated by other researchers. Gerstner et al., using Drosophila nephrocytes, reported that the expression of APOL1 risk variants induced inositol-requiring enzyme 1-dependent ER stress, as indicated by ER swelling and the induction of the ER chaperone protein disulfide-isomerase, ultimately leading to cytotoxicity [ 95 ]. The translocation of APOL1 to the ER appears to be critical for its cytotoxicity [ 70 , 73 ]. Although the mechanism by which APOL1 induces ER stress remains unclear, the risk variants have been proposed to act as misfolded proteins [ 157 ]. However, Gerstner et al. challenged this assumption, as their control transgene lacking 9 amino acids in the PFD of the APOL1 showed no impact on ER stress [ 95 ], indicating that other mechanisms may be responsible for APOL1-induced ER stress. Further research is warranted to fully elucidate the relationship between APOL1 and ER stress, as well as the broader effects of this interaction. Pérez-Morga et al. demonstrated that APOL1 promotes pore formation in lysosomal membranes leading to lysosomal membrane depolarization [ 19 ]. Similarly, lysosomal dysfunction is observed in human kidney cells expressing APOL1 risk variants G1 and G2. These variants are linked to a reduced number of lysosomes and increased lysosomal membrane permeability in podocytes, leading to leakage of lysosomal enzymes into the cytoplasm. This disruption compromises the actin cytoskeleton, causing podocyte injury and necrotic cell death [ 150 ]. As highlighted in Fig. 3 , lysosomal dysfunction also interferes with autophagy, a crucial cellular process for maintaining homeostasis by degrading and recycling damaged organelles and proteins. Autophagosome maturation and degradation (flux) depend on functional lysosomes to complete the autophagic cycle [ 158 ]. Studies have shown that podocytes [ 78 , 159 ] and endothelial cells [ 57 ] expressing APOL1 risk variants exhibit defective autophagic flux. This impairment has been linked to the downregulation of the mechanistic target of rapamycin (mTOR), a central regulator of cellular metabolism and autophagy, attenuation of phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) transcription, and enhanced expression of Rubicon, a negative regulator of autophagy [ 159 ]. Chronic inflammation is a contributor to glomerular disease [ 160 , 161 ]. Proinflammatory stimuli have been shown to increase APOL1 expression [ 81 ]. Notably, systemic lupus erythematosus, an autoimmune disease that involves type 1 interferon-mediated inflammation, can exacerbate APOL1-mediated kidney disease [ 21 , 42 ]. Data from various studies [ 81 , 84 , 101 , 139 , 162 , 163 ], underscore the role of APOL1 expression in modulating inflammatory signalling pathways. In patients with glomerular disease, data from the NEPTUNE study have demonstrated that APOL1 risk variants enhance the glomerular expression of chemokines CXCL9 and CXCL11—induced in response to proinflammatory cytokines such as TNF, IL-1 and TLR signalling [ 139 , 163 ]. The upregulation of these chemokines is particularly significant, as CXCL9 and CXCL11 mediate leukocyte recruitment by attracting CXCR3-expressing immune cells, such as activated T-lymphocytes. This promotes local infiltration of immune cells and amplifies local inflammatory signalling within the glomerulus, thereby accelerating podocyte injury and glomerular damage [ 163 – 165 ]. Several key inflammatory pathways have been implicated in APOL1-mediated kidney disease. Activation of the retinoic acid inducible gene 1/nuclear factor kappa B (RIG-1/NF- Κ B) signalling pathway has been reported to mediate inflammation in the presence of APOL1 expression [ 162 ]. APOL1 has been shown to interact with and regulate the nucleotide-binding oligomerization domain-like receptor (NLR) pyrin domain (PYD)-containing protein 12 (NLRP12), contributing to inflammatory response [ 139 ]. Furthermore, APOL1 risk variants activate both the STING pathway and the NLPR3 inflammasome signalling, further amplifying pro-inflammatory signalling [ 84 ]. In addition to the aforementioned, APOL1 risk variants mRNA can form a stable double-stranded RNA structure that promotes protein kinase R (PKR) autophosphorylation, leading to podocyte damage in transgenic mice and in cultured podocytes [ 103 ]. Overexpression of the G1 and G2 variants in cultured human podocytes activates PKR, which triggers a type 1 interferon response. This response results in the phosphorylation of eIF-2α, inhibiting protein synthesis and ultimately leading to podocyte injury and death [ 103 ]. Conformational changes in the C-terminal domain of APOL1 risk variants have been shown to disrupt its interactions with other proteins such as APOL3 and vesicle-associated membrane protein 8 (VAMP8), thereby resulting in podocyte dysfunction [ 80 , 166 ]. Just like APOL1, APOL3 belongs to the APOL gene family and exhibits increased expression under inflammatory conditions associated with resistance against infection [ 167 ]. Uzureau et al. demonstrated that APOL1 can directly bind to APOL3, an interaction enhanced by the alteration in leucine zipper 1 or 2, as seen in the presence of the risk variants [ 80 ]. This binding, however, prevents APOL3 binding to neuronal calcium sensor-1 (NCS-1), thereby inhibiting the interaction of NCS-1 with phosphatidylinositol-4-kinase IIIB (PI4KB). This protein-protein interaction blockage results in inhibition of phosphatidylinositol-4-phosphate (PI(4)P) synthesis by Golgi PI4KB, thereby inducing actomyosin reorganization in the podocytes with a truncated APOL1 C-terminal helix or APOL3 knock out. Ultimately, this reorganization causes alterations in organellar trafficking, including mitochondrial fission, further implicating APOL1 in mitochondrial dysfunction, subsequently resulting in podocyte damage [ 80 ]. Interestingly, the authors observed a significantly reduced level of PI(4)P in urine-derived podocyte cell lines as well as in the glomeruli of patients with APOL1 HRG [ 80 ]. To bolster this point, a null variant in APOL3 (p. Q58*) has been reported to be associated with kidney disease in humans, independent of the APOL1 genotype [ 168 , 169 ]. Beyond APOL3, APOL1 risk variants have been proposed to interfere with VAMP8, a SNARE protein critical for intracellular vesicular trafficking. Using computational modelling and biochemical assays, Madhavan et al. demonstrated that the risk variants adopt a more rigid, “autoinhibited” C-terminal conformation compared to the flexible G0 protein [ 166 ]. This structural alteration significantly reduces the risk variants’ binding affinity for VAMP8, disrupting normal vesicular transport in podocytes. This impaired APOL1–VAMP8 interaction may lead to vesicle accumulation and cellular dysfunction, which may eventually result in CKD in individuals with APOL1 HRG [ 166 ]. However, Scales et al. challenged this model, arguing that a direct APOL1–VAMP8 interaction is unlikely due to differences in subcellular localization and isoform specificity. They noted that VAMP8 is cytoplasmically oriented, whereas APOL1 predominantly localizes to the luminal face of intracellular membranes. Although certain cytoplasmic APOL1 isoforms (vB3 and vC) could theoretically interact with VAMP8, these forms are not associated with cytotoxicity. Instead, toxic effects are mainly linked to secretory isoforms (vA and vB1), which lack cytoplasmic exposure [ 142 ]. Therefore, direct VAMP8 binding is unlikely to explain APOL1-mediated cytotoxicity. Nevertheless, VAMP8 may act indirectly via APOL3, which binds VAMP8 and promotes vesicular membrane fusion [ 170 ]. A parallelism has been suggested between APOL1’s trypanolytic activity and its cytotoxicity in mammalian kidney cells [ 68 , 171 ]. The trypanolytic function of APOL1 is believed to involve endocytosis, allowing APOL1 to enter the endosome membranes. The low pH inside the trypanosome causes an APOL1 conformation change that induces pore formation in the membranes of lysosomes and mitochondria, leading to lysosomal swelling, mitochondrial membrane depolarization and fenestration, and ultimately, parasite death [ 171 , 172 ]. These mechanisms, though distinct from mammalian cellular processes, hint at a conserved ability of APOL1 to form an ion pore or channel. This pore-forming ability is essential for trypanolysis and has been proposed to underlie APOL1-induced cytotoxicity in human kidney cells [ 66 , 74 , 173 ]. As illustrated in Fig. 3 , it is widely accepted that APOL1 can form pores in cellular membranes, permitting ion flux that ultimately leads to cell death [ 23 ]. However, details on this pore-forming activity, such as the pore’s ion selectivity, remain unclear, partly due to the experimental models and the influence of pH. As summarized in Table 2 , even the most fundamental aspect of the pore, its charge, has been controversial. APOL1 has been reported to confer pH-switchable ion permeability, with pH-dependent conformational changes altering ion selectivity: promoting anion permeability under acidic conditions and cation permeability when shifted to neutral pH [ 174 ]. Some studies suggested that APOL1 facilitates the conductance of positive ions, such as K + and Na + [ 67 , 68 , 175 ], while others proposed a flux of negatively charged ions, such as Cl − [ 19 , 174 , 176 , 177 ]. However, research using mammalian cellular models has largely reached a consensus that APOL1’s cationic pore-forming activity is the proximal event preceding cytotoxicity in APOL1-mediated kidney disease [ 66 – 68 , 173 ]. Olabisi et al. demonstrated that APOL1 forms a K + and Na + permissive channel at the plasma membrane in HEK cells, leading to intracellular K + depletion and Na + influx. This imbalance triggers the activation of stress-activated protein kinases (SAPKs), p38-mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinases (JNKs), ultimately causing cytotoxicity in a risk variant-dependent manner [ 67 ]. While most studies [ 66 , 74 , 173 , 178 ] support the hypothesis of intracellular K + depletion, the precise nature of the cations conducted by APOL1 remains contested [ 68 , 69 , 84 ]. For example, O’Toole et al. reported no variant-dependent K + loss or Na + gain among the APOL1 variants [ 69 ]. To further complicate this issue, Giovinazzo et al. used planar lipid bilayers to show that APOL1 forms an active channel at the plasma membrane, which allows Na + and Ca 2+ influx from the extracellular medium, an upstream event leading to risk variant-dependent cytotoxicity [ 68 ]. More recently, Datta et al. added to the ongoing debate by suggesting that APOL1 functions as a monovalent cation channel, facilitating Na + /K + transport. Their study proposed that APOL1 risk variant G1 causes the plasma membrane to depolarize through Na + and K + flux, leading to the activation of the inositol triphosphate receptor via a series of cascades involving a G-protein-coupled receptor and phospholipase C. These events ultimately result in intracellular Ca 2+ release from the ER into the cytoplasm, and the resulting ER/Ca 2+ signalling cascade has been implicated as the key driver for the multiple downstream cytotoxic phenotypes—such as mitochondrial dysfunction, increased autophagy, and reduced global protein synthesis—associated with APOL1 [ 66 ]. Given the contradictory evidence surrounding APOL1’s pore-forming activity, further research is needed to elucidate its exact role in kidney disease, particularly which ions are being conducted and whether APOL1 functions inherently as an ion channel, forms a pore, or modulates the activity of existing ion channels such as transient receptor potential canonical 6 (TRPC6) or the Piezo1 channel in human podocytes. Table 2 Studies describing contradictory pore-forming activity of APOL1 Models Experimental approach Ion Selectivity Comments about conductance Authors Truncated recombinant APOL1 with pore-forming domain Planar lipid bilayer and liposome assay Anion APOL1 causes an influx of Cl − , which was higher at acidic pH. Pérez-Morga et al. 2005 [ 19 ] TLF1 construct Planar lipid bilayer and unilamellar vesicles Cation An increase in TLF1-mediated influx of Na + is sufficient to cause a net Cl − influx via pre-existing Cl − channels. Molina-Portela et al. 2005 [ 171 ] Delipidated APOL1 protein Unilamellar liposomes Anion Permeabilizes lipid bilayer at low pH. Harrington et al. 2009 [ 176 ] Full-length recombinant APOL1 G0 Planar lipid bilayer Cation Conductance is first activated at pH 5.3 and increased by a magnitude of 3000-fold when the environment is alkalinized. Thomson et al. 2015 [ 23 ] APOL1 G0 cRNA injection into Xenopus laevis oocytes Two-electrode voltage clamp Cation and anion APOL1 causes an influx of Cl − and Ca 2+ , which was inhibited by acidic extracellular solution. Heneghan et al. 2015 [ 179 ] T-Rex-293 stable cell lines expressing APOL1 variants X-ray fluorescence Cation (K + and Na + ) Increased efflux of K + and influx of Na + in cells expressing the risk variants. Olabisi et al. 2016 [ 67 ] Recombinant APOL1 G0 Unilamellar vesicles and membrane association assay Cation and anion APOL1 has a pH-switchable ion-selective permeability. Cl − selectivity is observed at acidic pH during the membrane insertion stage. However, upon neutralization, this induces a structural transition resulting in the activation of K + selectivity. Bruno et al. 2017 [ 174 ] T-Rex-293 stable cell lines expressing APOL1 variants Atomic absorption spectroscopy and whole-cell patch clamping Cation APOL1 expression leads in a variant-independent manner to the loss of K + and a gain of Na + without a change in the cell’s Ca 2+ homeostasis. O’Toole et al. 2018 [ 69 ] Recombinant APOL1 and FT293-APOL1 cells Planar lipid bilayer and live-cell microscopy with calcium indicator, GcaMP6f Cation (Ca 2+ and Na + ) Requires a pH < 6 for irreversible membrane insertion, followed by neutralization to open and enhance conductivity by hundredfold. Increased Ca 2+ and Na + in cells expressing the risk variants. Giovinazzo et al. 2020 [ 68 ] Recombinant APOL1 G0 Planar lipid bilayer Cation APOL1 forms a cation-selective conductance after the pH is shifted from acidic (pH 5.5–6.0) to neutral pH. The APOL1 pH gating and cation selectivity are governed by the aspartate 348 residue. Schaub et al. 2020 [ 24 ] Recombinant APOL1 variants Unilamellar vesicles and membrane association assay Cation and anion Cation permeability was greater in the risk variants, while the anion permeability was comparable across all variants. Bruno et al. 2021 [ 180 ] Recombinant APOL1 G0 Planar lipid bilayer Cation (K + ) Requires an acidic environment for membrane insertion to form an ion conductance channel. Pant et al. 2021 [ 175 ] Recombinant APOL1 G0 Planar lipid bilayer Cation APOL1 cation-selectivity is pH sensitive and requires a C-terminal leucine zipper for its cation channel-forming activity. Schaub et al. 2021 [ 181 ] T-Rex-293 stable cell lines expressing APOL1 variants and primary human podocytes Whole-cell patch clamping Cation APOL1 shows a non-specific cation selectivity. Vandorpe et al. 2023 [ 182 ] T-Rex-293 stable cell lines expressing APOL1 variants X-ray fluorescence Monovalent cation (K + and Na + ) Increased efflux of K + and influx of Na + in cells expressing the risk variants. Datta et al. 2024 [ 66 ] T-Rex-293 stable cell lines expressing APOL1 variants Whole-cell patch clamping Monovalent cation (Na + , K + and Cs + ) Outward current amplitude was approximately twofold higher in APOL1 G1- and G2-mediated currents compared to those mediated by APOL1 G0. Zimmerman et al. 2025 [ 173 ] Abbreviations: TLF1: Trypanosome lytic factor 1, APOL1: Apolipoprotein L1, FT293: Flp-In ™ T-REx ™ 293 cells, HEK: Human embryonic kidney, T-Rex-293: Tetracycline-regulated expression-293, Ca 2+ : Calcium ion, K + : Potassium ion, Na + : Sodium ion, Cl - : Chloride ion Studies describing contradictory pore-forming activity of APOL1 Abbreviations: TLF1: Trypanosome lytic factor 1, APOL1: Apolipoprotein L1, FT293: Flp-In ™ T-REx ™ 293 cells, HEK: Human embryonic kidney, T-Rex-293: Tetracycline-regulated expression-293, Ca 2+ : Calcium ion, K + : Potassium ion, Na + : Sodium ion, Cl - : Chloride ion Currently, there are no specific drugs approved for the treatment of APOL1-mediated kidney disease. As illustrated in Fig.  4 , the complex pathophysiological mechanisms underlying this condition have identified several targetable options for intervention. Multiple therapeutic strategies are under investigation, with some advancing to phase 2 and 3 clinical trials. Below, we highlight promising key approaches. Fig. 4 Proposed therapeutic targets for APOL1-mediated kidney disease. Small-molecule and oligonucleotide-based therapies targeting APOL1 and its downstream signalling pathways represent promising strategies to mitigate APOL1-mediated kidney disease. APOL1 expression in human podocytes is transcriptionally upregulated by pro-inflammatory cytokines such as interferon-gamma (IFN-γ) and interleukin-1 (IL-1) via the JAK/STAT signalling pathway. This pathway can be inhibited using JAK inhibitors (e.g., Baricitinib) or cytokine antagonists (e.g., IFN-γ and IL-1 antagonists). Also, antisense oligonucleotides (e.g., ION532) can reduce APOL1 mRNA levels and subsequent protein expression. At the plasma membrane, APOL1 forms cation pores or channels that disrupt sodium, potassium, and calcium ion homeostasis, leading to risk variant-dependent cytotoxicity. Small-molecule inhibitors developed by Vertex Pharmaceuticals (VX-147, VX-840) and Maze Therapeutics (MZ-301, MZE829) are designed to block APOL1 pore-forming activity. Downstream of APOL1 expression, mitochondrial damage can activate the STING and NLRP3 inflammasome pathways, promoting inflammation and Gasdermin D (GSDMD)-mediated pyroptotic cell death. These inflammatory cascades can be therapeutically targeted using C176 (STING inhibitor), MCC950 (NLRP3 inflammasome inhibitor), or Disulfiram (GSDMD inhibitor), thereby reducing pro-inflammatory cytokine release and preventing cell death Proposed therapeutic targets for APOL1-mediated kidney disease. Small-molecule and oligonucleotide-based therapies targeting APOL1 and its downstream signalling pathways represent promising strategies to mitigate APOL1-mediated kidney disease. APOL1 expression in human podocytes is transcriptionally upregulated by pro-inflammatory cytokines such as interferon-gamma (IFN-γ) and interleukin-1 (IL-1) via the JAK/STAT signalling pathway. This pathway can be inhibited using JAK inhibitors (e.g., Baricitinib) or cytokine antagonists (e.g., IFN-γ and IL-1 antagonists). Also, antisense oligonucleotides (e.g., ION532) can reduce APOL1 mRNA levels and subsequent protein expression. At the plasma membrane, APOL1 forms cation pores or channels that disrupt sodium, potassium, and calcium ion homeostasis, leading to risk variant-dependent cytotoxicity. Small-molecule inhibitors developed by Vertex Pharmaceuticals (VX-147, VX-840) and Maze Therapeutics (MZ-301, MZE829) are designed to block APOL1 pore-forming activity. Downstream of APOL1 expression, mitochondrial damage can activate the STING and NLRP3 inflammasome pathways, promoting inflammation and Gasdermin D (GSDMD)-mediated pyroptotic cell death. These inflammatory cascades can be therapeutically targeted using C176 (STING inhibitor), MCC950 (NLRP3 inflammasome inhibitor), or Disulfiram (GSDMD inhibitor), thereby reducing pro-inflammatory cytokine release and preventing cell death The absence of the APOL1 gene in chimpanzees, our closest relatives, and the discovery of an individual lacking the APOL1 gene who was otherwise healthy but susceptible to Trypanosoma evansi infection [ 144 ], suggest that APOL1 may not be essential for normal growth, development, or kidney function. Instead, its primary role appears to be protective, offering resistance to African trypanosomiasis. These findings indicate that directly inhibiting APOL1 synthesis, expression, or function in the kidney could represent a safe and effective therapeutic strategy for managing APOL1-mediated kidney disease. Based on this, as summarised in Table 3 , several clinical trials have been initiated to evaluate drugs targeting APOL1 synthesis and expression. As mentioned earlier, the JAK/STAT signalling pathway plays a crucial role in driving the pro-inflammatory interferon-stimulated gene expression. Thus, it is a key inducer of APOL1 expression [ 50 ]. Therefore, targeting this pathway represents a promising therapeutic approach for APOL1-mediated kidney disease. The JAK1/2 inhibitor, Baricitinib has been shown to effectively block IFN-γ-induced APOL1 expression and mitigate cytotoxicity in iPSCs-derived organoids and podocytes from individuals carrying the APOL1 G1/G2 genotype [ 50 ]. Recent findings from this group further highlight that the JAK/STAT pathway is significantly upregulated in podocytes derived from patients with FSGS who carry APOL1 HRG, compared to podocytes from individuals with HRG who do not exhibit kidney disease [ 183 ]. These results have paved the way to the randomized, double-blind, placebo-controlled, phase 2 JUSTICE clinical trial ( NCT05237388 ), designed to evaluate the safety and efficacy of Baricitinib in reducing proteinuria in individuals with APOL1-associated FSGS and hypertension-attributed CKD [ 183 ]. Another promising approach involves antisense oligonucleotides (ASOs), as investigated in a study by Ionis Pharmaceuticals [ 104 ]. ASOs are oligonucleotide analogues designed to bind specific RNA sequences, modulating gene expression by inducing RNA degradation through RNAse H1, altering splicing, or preventing translation. In a preclinical study, treatment of APOL1 G1-transgenic mice with an APOL1-specific ASO (IONIS-APOL1RX) prior to IFN-γ challenge inhibited APOL1 mRNA expression in the liver and the kidneys and protected against IFN-γ-induced proteinuria in a dose-dependent manner [ 104 ]. This promising therapeutic agent, licensed to AstraZeneca (ION532 or AZD2373), has progressed to clinical trials. A phase 1 first-in-human, single ascending dose study ( NCT04269031 ) was conducted to evaluate the safety and pharmacokinetics of escalating single doses of the ASO in 48 healthy men of African descent. A subsequent phase 1 trial ( NCT05351047 ) was conducted in healthy male participants of sub-Saharan African ancestry to further evaluate the safety and tolerability of AZD2373. Results from these trials are still pending [ 184 , 185 ]. More recently, in March 2025, AstraZeneca initiated a phase 2 randomized, double-blind trial ( NCT06824987 ) to assess the efficacy and safety of AZD2373 in individuals with APOL1-mediated kidney disease carrying APOL1 HRG. Moreover, a STAT3 ASO (AZD9150) is currently being investigated for leukaemia and lymphoma [ 186 , 187 ]. Future studies should explore its potential protective effects in APOL1-mediated kidney disease. Targeting APOL1-mediated kidney disease through small-molecule inhibitors offers another promising therapeutic strategy. These inhibitors, developed by Vertex Pharmaceuticals (VX-147 and VX-840) [ 184 , 188 ] and Maze Therapeutics (MZ-301 and MZE829) [ 189 , 190 ], aim to block the pathological function of APOL1. Although their precise mechanisms of action remain unclear, these small-molecules are believed to inhibit pore-forming function of APOL1, thereby reducing proteinuria as demonstrated in transgenic APOL1 mouse models [ 66 , 188 , 189 ]. Inhibition of APOL1 may lower cytosolic calcium levels, preventing downstream mitochondrial dysfunction, impaired ATP production, and disrupted protein synthesis, mechanisms that have been reported to exacerbate APOL1-mediated kidney injury [ 66 ]. A phase 2a study ( NCT04340362 ) by Vertex Pharmaceuticals investigating the safety and efficacy of VX-147 in patients with APOL1-associated FSGS revealed that VX-147 significantly reduced proteinuria [ 188 ]. Currently, a larger phase 2/3 AMPLITUDE trial ( NCT05312879 ) is actively enrolling participants with APOL1 HRG and kidney disease to further evaluate its efficacy. Additionally, Vertex has conducted a phase 1 clinical trial ( NCT05324410 ) on VX-840, another generation of APOL1 small-molecule inhibitors, with results pending [ 184 ]. Parallel preclinical investigation further demonstrated that a close analogue of VX-147 (Compound 3) effectively inhibits APOL1 pore-forming activity, thereby preventing and reducing APOL1-dependent proteinuria as well as preserving glomerular integrity [ 173 ]. Similarly, Phase 1 trial by Maze Therapeutics showed MZE829 to be safe, well-tolerated, and exhibiting promising pharmacokinetic properties in healthy individuals [ 190 ]. Based on this result, Maze Therapeutics has recently launched a phase 2 clinical trial ( NCT06830629 ). These small-molecule inhibitors represent a significant step forward in addressing APOL1-mediated kidney disease, potentially transforming patient outcomes with targeted and effective therapies. Nevertheless, concerns remain regarding their long-term safety, particularly in regions endemic for trypanosomiasis, where APOL1 plays a protective role against infection. In addition, small-molecule inhibitors carry a risk of off-target effects and prolonged genetic alterations [ 184 ]. Table 3 APOL1-targeting therapeutics in clinical development Mechanism of action Name ClinicalTrials.gov identifier Trial population Phase Status Completion JAK/STAT pathway inhibitor Baricitinib NCT05237388 Adults with FSGS or hypertension-attributed CKD Phase 2 Recruiting March 2026 Antisense oligonucleotide AZD2373 NCT04269031 Healthy adult males of African ancestry Phase 1 Completed August 2021 NCT05351047 Healthy adult males of sub-Saharan West African ancestry Phase 1 Completed July 2023 NCT06824987 Adults with APOL1-mediated kidney disease Phase 2 Recruiting August 2027 APOL1 small-molecule inhibitors Inaxaplin (VX-147) NCT04340362 Adults with APOL1-mediated FSGS Phase 2a Completed December 2021 NCT06794996 Adults with proteinuric APOL1-mediated kidney disease Phase 2b Recruiting December 2026 NCT05312879 Adults and children with APOL1-mediated proteinuric kidney disease Phase 2/3 Recruiting June 2026 VX-840 NCT05324410 Healthy adults Phase 1 Completed November 2022 MZE829 NCT06830629 Adults with proteinuric APOL1-mediated kidney disease Phase 2 Recruiting September 2026 APOL1-targeting therapeutics in clinical development Several potential therapeutic targets have been identified in the downstream signalling pathways of APOL1. Wu et al. highlighted that the NLRP3 inflammasome and STING pathways are key downstream mediators of APOL1-induced toxicity in podocytes. Treatment of APOL1 G2 transgenic mice with selective inhibitors of NLRP3 and STING resulted in reduced albuminuria and improved kidney function, even when treatment was initiated after the onset of albuminuria [ 84 ]. Additionally, APOL1 overexpression has been associated with mitochondrial dysfunction and ER stress, both of which are critical contributors to podocyte injury and have also been implicated in other kidney diseases. Therapies targeting the NLRP3 inflammasome, STING pathway, mitochondrial dysfunction, and ER stress are already available and should be systematically evaluated in the context of APOL1-mediated kidney disease [ 185 , 191 ]. This approach may enable the rapid repurposing of these treatments to address APOL1-mediated kidney disease. At present, the cost-effectiveness of the aforementioned emerging therapeutic strategies remains uncertain. For instance, ASOs such as Spinraza, used for spinal muscular atrophy cost approximately $125,000 per injection, or $750,000 in the first year of treatment [ 192 ]. If similar pricing were applied to therapies for APOL1-mediated kidney disease, such intervention would be unaffordable in sub-Saharan Africa, the region bearing the highest disease burden. This underscores the urgent need for more accessible and cost-effective therapeutic options. ACEIs, widely used for decades in the management of proteinuric kidney diseases, reduce proteinuria by inhibiting angiotensin II, thereby promoting efferent arteriole vasodilation and lowering intraglomerular pressure [ 45 , 193 ]. In a recent study by Karreci et al., the effects of lisinopril, an ACEI, were compared with dapagliflozin, a sodium-glucose cotransporter-2 (SGLT2) inhibitor, in isogenic APOL1 BAC-transgenic mice carrying either the APOL1 G1/G1 or G2/G2 genotype [ 193 ]. In G1/G1 mice, a standard lisinopril dose (75 mg/L) reduced proteinuria by 90-fold and mitigated glomerulosclerosis. These effects were independent of hemodynamic changes, as neither hydralazine nor dapagliflozin conferred similar benefits. However, in G2/G2 mice, the same dose of lisinopril failed to significantly protect against kidney dysfunction despite comparable serum angiotensin converting enzyme (ACE) levels in both genotypes. Doubling the lisinopril dose to 150 mg/L provided modest benefits in G2/G2 mice, including a sevenfold reduction in proteinuria and slight improvement in glomerulosclerosis, although these beneficial effects were significantly less pronounced than those observed in G1/G1 mice. The authors suggested that these dose-dependent genotype-specific differences may reflect distinct disease mechanisms between APOL1 variants or differences in disease severity, as G2/G2 mice exhibited more severe kidney dysfunction. They proposed that lisinopril might not directly counteract APOL1-induced cytotoxicity but rather help preserve the survival of remaining podocytes by preventing further injury and detachment following the initial damage [ 193 ]. The translatability of these findings to humans remains uncertain. While randomized controlled trials are unlikely due to ethical concerns, existing cohort studies suggest potential benefits of ACEIs in reducing kidney disease progression in individuals with APOL1 HRG [ 194 , 195 ]. For example, data from the African American Study of Kidney Disease and Hypertension (AASK) [ 194 ] and a University of Illinois cohort of individuals with SCD [ 195 ] suggest a potential protective role of ACEIs. Nevertheless, the small sample sizes of these studies underscore the need for larger, long-term investigations to confirm the efficacy of ACEIs, ideally through randomized trials comparing ACEIs with angiotensin receptor blockers. Such research is particularly urgent for sub-Saharan Africa, where the prevalence and burden of APOL1 risk variants are highest. If proven effective, ACEIs could provide a cost-effective solution for reducing APOL1-mediated kidney disease progression in this resource-limited region.

Despite significant progress over the past 15 years and with the promise of new therapeutic interventions, several key questions regarding APOL1-mediated kidney disease remain. The observation that less than 30% of individuals with the APOL1 HRG develop kidney disease, coupled with the variability in odds ratios across different kidney disease aetiologies in HRG carriers, highlights the need to identify second hit factors that increase the risk. In addition, protective alleles in other genes or haplotypes within the APOL1 locus itself may also play a role. For example, the missense p.N264K variant, present in a small fraction of the G2 risk variant, has been shown to confer a protective effect to individuals with APOL1 HRG, thereby reducing penetrance of the risk variant [ 122 , 123 ]. A deeper understanding of these factors and their interplay with the APOL1 genotypes will be crucial for accurate risk stratification and personalized interventions. To achieve this, a multi-modal approach, combining clinical studies, in vitro, in vivo, and in silico modelling, will be essential. This integrated approach will facilitate the translation of basic science discoveries into clinically relevant therapies. Furthermore, the precise mechanisms by which APOL1 triggers kidney disease remain unclear. Importantly, it is likely that the molecular pathways involved vary depending on the specific aetiology of kidney disease. For example, the mechanisms underlying APOL1-mediated kidney disease in HIVAN may differ significantly from those in hypertension-attributed kidney disease or SCN. In support of this, Yoshida et al. compared two different APOL1 G1 mouse models: HIVAN and IFN-γ, and found both shared common pathways, yet also displayed divergent pathways. Specifically, the eIF-2 pathway was downregulated in the HIVAN model expressing the APOL1 G1 variant, whereas it was upregulated in the APOL1 G1 IFN-γ mice model [ 196 ]. These findings highlight that APOL1-mediated kidney disease should not be considered a one-size-fit-all condition. Therefore, future research should prioritize investigating these aetiology-specific pathways to develop more targeted therapeutic interventions. Looking ahead, the future of APOL1-mediated kidney disease research lies in a multi-faceted approach that includes integrating genetic screening into clinical practice. At present, however, there is insufficient evidence to support routine APOL1 genetic testing, and no established treatments for managing APOL1-mediated kidney disease are currently available [ 197 ]. In the United States of America, where structural inequities and systematic injustices have historically contributed to cycles of health disparities among racial and ethnic minorities, the implementation of APOL1 testing also raises concerns. Specifically, there is a risk that genetic testing could exacerbate existing inequities faced by individuals of African ancestry carrying APOL1 HRG, potentially exposing them to forms of racialized medicine, such as higher insurance premiums or reduced access to care. Nevertheless, genetic testing remains pivotal for identifying individuals at-risk and enabling early preventive strategies, particularly for individuals living with SCD, and for those living in resource-limited settings where kidney transplantation is often not a viable option. Importantly, the implementation of APOL1 genetic testing in Africa remains challenging. Although APOL1 genotyping is typically performed using quantitative polymerase chain reaction or sequencing, these techniques require specialized technical expertise and costly equipment, resources that are often limited in sub-Saharan Africa. Encouragingly, recent advances have led to the development of rapid, low-cost, point-of-care diagnostic assays for APOL1 genotyping, which hold promise for deployment in such settings [ 198 , 199 ].

Non-communicable diseases, such as chronic kidney disease (CKD), are becoming more prevalent globally [ 1 ]. In the United States of America, African Americans are about four times more at risk of kidney failure than European Americans [ 2 , 3 ]. This racial disparity has been linked to various factors including socioeconomic status, lifestyle, and clinical factors [ 1 , 2 , 4 , 5 ]. However, much of the increase remains unexplained. In African populations, studies looking for evidence of natural selection suggested that there had been a powerful selection sweep at the locus on chromosome 22 that is associated with kidney disease. In 2008, two studies leveraged the disparities in kidney disease prevalence to identify this locus that confers part of the increased risk for nondiabetic kidney disease in African Americans. The large excess of African ancestry at this single locus on chromosome 22 among patients with focal segmental glomerulosclerosis (FSGS), hypertension-associated kidney failure, or human immunodeficiency virus (HIV)-associated nephropathy (HIVAN) confirmed a genetic basis for the observed ancestry-related disparity in kidney disease prevalence [ 6 , 7 ]. Given the large differences in disease prevalence between ancestral groups, it is likely that relatively common genetic variants account for these disparities. By applying admixture linkage disequilibrium mapping, the two independent groups identified a locus on chromosome 22q12 containing African ancestry risk variants associated with certain forms of kidney failure [ 6 , 7 ]. This region encompassed 35 genes, including non-muscle myosin heavy chain 9 ( MYH9 ) [ 7 ]. MYH9 encodes non-muscle myosin heavy chain IIa, a key cytoskeletal motor protein expressed in various cell types, including the podocytes, where it plays a crucial role in maintaining podocyte cytoskeleton integrity [ 7 ]. Mutations in MYH9 are known to cause Giant Platelet Syndromes, which can be accompanied by glomerular abnormalities and CKD [ 8 – 11 ]. Consequently, MYH9 was initially considered a strong candidate gene responsible for the observed genetic association with CKD at this locus. However, no MYH9 variants with definite causal or functional effect were identified [ 6 , 7 ]. In 2010, two independent research groups re-examined the chromosome 22 locus using data from the International HapMap and 1000 Genomes Projects to search for alternative causal variants outside MYH9 [ 12 , 13 ]. These studies revealed that the apolipoprotein L1 ( APOL1 ) gene has two different coding risk variants (G1 and G2) that partly explain the racial discrepancy of kidney disease in the African population [ 12 , 13 ].