Novel Therapies in APOL1-Mediated Kidney Disease: From Molecular Pathways to Therapeutic Options

Abstract

Apolipoprotein L1 (APOL1) high-risk variants confer an increased risk for the development and progression of kidney disease among individuals of recent African ancestry. Over the past several years, significant progress has been made in understanding the pathogenesis of APOL1-mediated kidney diseases (AMKD), including genetic regulation, environmental interactions, immunomodulatory, proinflammatory and apoptotic signaling processes, as well as the complex role of APOL1 as an ion channel. Collectively, these findings have paved the way for novel therapeutic strategies to mitigate APOL1-mediated kidney injury. Precision medicine approaches are being developed to identify subgroups of AMKD patients who may benefit from these targeted interventions, fueling hope for improved clinical outcomes. This review summarizes key mechanistic insights in the pathogenesis of AMKD, emergent therapies, and discusses future challenges.

The APOL1 gene resides on chromosome 22 and is part of the APOL gene family, present in humans and certain primates. Although the lack of endogenous APOL1 expression in commonly used mouse and rat models has been a barrier to disease modeling and drug discovery efforts, significant progress has been made since the original seminal work by Pollak et al. in 2010.¹ G1 and G2 risk alleles define the APOL1 risk genotypes (G1/G1, G2/G2, G1/G2), whereas the “non-risk” APOL1 allele is referred to as G0. The APOL1 G1 allele consists of 2 missense variants in high linkage disequilibrium (APOL1 p.S342G, rs73885319; APOL1 p.I384M, rs6091014), whereas the G2 allele is a 2–amino acid deletion (APOL1 p.delN388/Y389, rs60910145). G1 and G2 alleles exhibit a notably high prevalence in populations of recent African ancestry, likely reflecting the influence of natural selection in West and Central Africa to protect against African trypanosomiasis known as “sleeping sickness.” As a result of the slave trade and other migration patterns, APOL1 high-risk variants are widely disseminated, especially in the Americas.² High-risk variants of APOL1 are found in approximately 10% to 15% of African Americans.³ In African Americans, carrying 2 high-risk alleles confers a 1.49-fold increased risk of chronic kidney disease and a 1.88-fold risk of end-stage kidney disease compared to those with 0 or 1 risk allele.⁴

AMKD encompasses diverse clinical manifestations characterized by kidney function decline, variable proteinuria levels, and hypertension. A recent large Phenome-Wide Association Study confirmed the association of APOL1 with primarily kidney and kidney-associated pathologies.⁵ Some of the most frequent conditions studied within the spectrum of AMKD include focal segmental glomerulosclerosis (FSGS), to virus-related forms such as HIV-associated nephropathy and COVID-19-associated nephropathy, and the syndrome of solidified or diffuse glomerulosclerosis with low level proteinuria (often mislabeled arterionephrosclerosis or hypertensive nephropathy).6, 7, 8, 9 The role of APOL1 in conditions such as preeclampsia, sickle cell disease and some autoimmune diseases is not fully understood. Improved knowledge of APOL1 biology and its therapeutic targeting offers a unique opportunity to treat clinical entities of important public health relevance. In this review, we summarize mechanistic pathways implicated in AMKD and provide an overview of promising emerging therapeutic options.

APOL1 Expression and Upstream Regulation

The mode of inheritance in AMKD pathogenesis has been a topic of controversy.¹⁰ Although most autosomal recessive diseases are characterized by loss-of-function mutations, APOL1 is atypical, having a recessive model of inheritance for chronic kidney disease and apparent gain-of-function characteristics.¹¹ Recent data investigating the use of small molecule inhibitors and antisense oligonucleotides to reduce APOL1 expression and its downstream effects further support the gain-of-function model.^12,13 The fact that a functional APOL1 gene is found only in some African primate species and that individuals lacking APOL1 can still maintain normal kidney function suggests that this gene is not essential for normal kidney development or functioning.^14,15 In theory, inhibiting APOL1 toxicity should ameliorate kidney disease without major adverse effects. However, caution is warranted until the effects of APOL1 expression in other systems such as the endothelium, immune cells, and hepatocytes are fully understood. Furthermore, reducing APOL1 expression may result in deleterious implications in areas endemic for trypanosomiasis.

The expression of APOL1 is tightly controlled by a complex interplay between genetic and epigenetic factors. Numerous immune and inflammatory pathways have been identified that upregulate APOL1 expression, including the interferon family, the most extensively studied, interleukin-1β and proteins from the toll-like receptor family.16, 17, 18 The APOL1 promoter contains regulatory elements that interact with several transcription factors, including STAT2, STAT3, and interferon regulating factors 1, 2, and 3. Interferons and toll-like receptor agonists have been shown to increase APOL1 expression by up to 200-fold.¹⁶ Comparative promoter analyses have revealed differential effects on APOL1 expression. Toll-like receptor 3 activation exerts a more pronounced effect than toll-like receptor 4, whereas interferon γ has a greater impact on APOL1 expression compared to interferon β and α. These findings support the hypothesis that APOL1 is a cellular immune response gene.

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, a highly conserved pathway that regulates immunological and adaptative responses, has been well studied in the context of APOL1 regulation. This pathway consists of 4 JAK members (JAK1–JAK3 and receptor tyrosine kinase 2) and 7 STAT members (STAT1–STAT4, STAT5a, STAT5b, and STAT6), and it is essential for cell maintenance, immune fitness, and tissue repair.19, 20, 21 Extensive activation of the JAK/STAT pathway has been recognized as a critical step in kidney diseases such as diabetic nephropathy and autosomal dominant polycystic kidney disease.22, 23, 24, 25, 26 Most recently, COVID-19-associated nephropathy has been identified as a condition where COVID-19-induced cytokines (predominantly interleukin-6, interleukin-1β, and interleukin-18) are sufficient to activate the JAK/STAT signaling pathway.²⁶ Interestingly, the amplification of APOL1 expression in human podocytes and glomerular endothelial cells was seen even when interferon levels were negligible (indicating the presence of interferon-independent mechanisms), but still resulting in podocyte injury and loss. This was rescued by baricitinib (JAK inhibitor), providing a rationale for its use as a therapeutic agent in APOL1-induced cellular injury.

Host genetic factors may also affect the expression of APOL1. For example, gain-of-function mutations in TMEM173, the gene encoding stimulator of interferon genes (STING) can exacerbate the production of interferon, a condition that has been recently described as STING-associated vasculopathy with onset in infancy. Type I IFN triggers a positive feedback loop leading to activation of JAK1 and STAT1/STAT 2, and transcription of proinflammatory IFN-stimulated genes. The recently published case of a patient with APOL1 G1 and G2 risk alleles with a high interferon state due to STING-associated vasculopathy with onset in infancy, illustrated a human model where a high interferon state can lead to collapsing glomerulopathy.²⁷

Several studies continue to explore the role of epigenetic factors, copy number variants and SNPs in APOL1 nephropathy.²⁸ Genetic modifiers such as SMOC2, DEF1B, UBD, NUDT7, and GSTB1 have also been identified.29, 30, 31, 32, 33 In addition, environmental factors such as air pollution have been implicated in the development of AMKD in patients carrying APOL1 risk variants, probably due to cellular stress mechanisms.34, 35, 36, 37 In sum, AMKD has diverse clinical manifestations and it is likely that complex epistatic and environmental interactions result in differential pathological cellular programming.

APOL1 Mechanisms of Podocyte Injury

APOL1 as an Ion Channel

One of the most widely accepted proposed mechanisms of APOL1-mediated injury centers on its role as an ion channel.^38,39 Conflicting findings have been reported regarding the nature of these channels, especially their anionic or cationic activity. This is related to variability in localization (cellular vs. organelle), model studied (trypanosomal activity vs. podocyte-specific toxicity), and structural or biochemical factors, derived from pH environment, among others.40, 41, 42 Subcellular localization of APOL1 and/or affinity for binding partners have been described but there is a lack of consensus across models.43, 44, 45

Several studies support the hypothesis that APOL1 forms distinct anion-selective pores in unilamellar vesicular membranes, promoting chloride influx facilitated by the initial influx of extracellular sodium following its concentration gradient.^46,47 This osmotic imbalance leads to the passive entry of water into the cell, resulting in cell swelling and trypanosome lysis. However, other studies support the cationic nature of the channel, suggesting that trypanosome lysis requires an acidic pH for the initial steps to allow APOL-1 insertion into vacuolar lipid bilayers, to be subsequently transported to the plasma membrane, where it is exposed to a nonacidic pH allowing APOL1 to open pH-sensitive cationic channels, depolarizing the membrane and killing the trypanosome.⁴⁸ Another publication supports this view demonstrating that mammalian cells expressing APOL1 risk variants exhibit increased nonselective cation permeability, resulting in a net efflux of intracellular potassium through the plasma membrane, thereby inducing cell damage through the activation of stress-activated protein kinases, p38, mitogen-activated mitogen kinase, and JNK.⁴⁹ These seemingly discordant findings may be reconciled by the proposal that APOL1 ion-channel selectivity is pH-switchable. At pH of 5, APOL1 may promote chloride permeability through anionic channels, whereas at neutral pH, it facilitates potassium permeability.⁵⁰ Collectively, the body of evidence suggests that APOL1 channel activity depends on 3 factors that allow APOL1 to associate with pore-forming vesicles: delicate pH fine tuning, presence of negatively charged phospholipids in vesicle membranes, and low ionic strength.

APOL1-Associated Mitochondrial Stress

APOL1-induced mitochondrial dysfunction is believed to contribute to AMKD through various mechanisms.⁵¹ The induction of APOL1 G1 and G2 expression results in a significant reduction in the maximum oxygen consumption rate and respiratory reserve capacity compared to cells expressing the APOL1 G0 variant.⁵² After being transported to the mitochondria by incompletely understood mechanisms, APOL1 risk variants form higher-order oligomers within the mitochondria and activate pore opening, resulting in cell toxicity by increasing fatty acid oxidation, and decreased redox homeostasis, disruption of the mitochondrial membrane potential, and cell toxicity.⁴⁵ Another possible mechanism underlying APOL1-induced mitochondrial dysfunction is the increase in mitochondrial fragmentation (fission), as opposed to fusion. Physiologically, fission helps segregate the most severely damaged mitochondria to preserve the overall health of the mitochondrial network. However, when G1 and G2 variant-induced mitochondrial fission cannot be adequately compensated through mitophagy (possibly due to defective intracellular trafficking), cell death mechanisms are activated.53, 54, 55 Overexpression of APOL1 G1 and G2 in HEK293 cells promotes mitochondrial fragmentation thought active DRP1. The inhibition of mitochondrial fission using the DRP1 inhibitor, Mdivi-129, appears to preserve mitochondrial morphology, resulting in fewer fragmented mitochondria, and treatment restores cell viability in a dose-dependent manner.^56,57

APOL1-Associated Endoplasmic Reticulum and Lysosomal Stress

Other organelles that have been implicated in APOL1-induced cell damage are lysosomes and the endoplasmic reticulum (ER). APOL1 risk variants are associated with increased lysosomal permeability and compromised endolysosomal trafficking, suggesting their involvement in cellular perturbations.⁵⁸ Insertion of APOL1 into the lysosomal membrane triggers ion flux into the organelle, leading to osmotic damage and death.⁴⁶ Notably, APOL1 causes lysosomal dysfunction in cultured human renal cells as well as in parasites. The G1 and G2 variants of APOL1 decrease the number of lysosomes in podocytes, leading to leakage of lysosomal enzymes into the cytoplasm.^59,60

Recent studies have shown that APOL1 expression is localized to the ER. In their study using cultured HEK293 cells, Chun et al.⁶¹ uncovered a distinct pattern of localization for APOL1 risk variants, primarily within the ER, whereas wild type APOL1 localization predominantly to lipid droplets. Notably, when cells were subjected to treatments promoting lipid droplet formation, a notable shift in the localization of G1 and G2 variants occurred, moving from the ER to the lipid droplets, reducing autophagic flux and cytotoxicity. In addition, factors such as tissue hypoxia,⁶² oxidative stress, and chronic inflammation can further amplify ER stress, exacerbating kidney disease progression among those with high-risk APOL1 variants. Further research efforts are warranted to unravel the complex mechanisms of ER stress, its modulation by APOL1 variants, and its impact on disease pathogenesis. Consequently, therapeutic approaches targeting ER stress that hold promise for other clinical applications (e.g., cancer and metabolic diseases), could be an important opportunity for drug-repurposing.⁶³

Inflammation and APOL1

As previously described, inflammatory mechanisms induce the expression of APOL1. However, these mechanisms also appear to be involved in the induction and maintenance of APOL1-mediated damage. STING is an adjuvant protein on the ER that recognizes the cyclic dinucleotides generated by cyclic GMP-AMP synthase, that recognizes foreign and host DNA (incremented in states of cellular stress).⁶⁴ Once cyclic GMP-AMP synthase senses cytoplasmic dsDNA, it converts GTP and ATP into 2′,3′-cGAMP, which binds and activates STING, promoting the secretion of type I interferons and various proinflammatory cytokines.⁶⁵ However, when cyclic GMP-AMP synthase-STING pathway activation exceeds a certain threshold, STING induces necroptosis, apoptosis, and lysosome fragmentation via NOD-like receptor pyrin domain-containing 3 inflammasome-dependent pyroptosis.^66,67 The NOD-like receptor pyrin domain-containing 3 inflammasome is a multiprotein complex that forms part of the innate immune system and activates multiple inflammatory proteins such as interleukin 1β, interleukin-18, and the pore-forming gasdermin D, leading to cellular osmotic imbalance and the release of proinflammatory intracellular contents.⁶⁶ Recent studies have identified the STING-NOD-like receptor pyrin domain-containing 3 pathway as a pivotal mechanism underlying APOL1-induced cytotoxicity.⁶⁸ Emerging small-molecule-based strategies and biologics to therapeutically target cyclic GMP-AMP synthase-STING and NOD-like receptor pyrin domain-containing 3 signaling are gaining interest in several models of chronic inflammation (e.g., cancer and autoimmune disorders), and these findings support its potential value in the treatment of AMKD.

Other Mechanisms

APOL1-mediated cellular injury depends on complex molecular mechanisms that are intricately linked. In addition to those mentioned previously, other mechanisms implicated in AMKD that have been suggested include activation of protein kinase R,⁶⁹ which appears during viral infections and inhibits protein synthesis, increased autophagic cell death (mediated by the BCL2- homology 3 domain within the pore-forming domain of APOL1),⁷⁰ or the alteration of the ubiquitin-proteasome system, which reduces ubiquitin levels prolonging the intracellular retention of proteins (including APOL1 itself).³¹ In addition, APOL1 G1 and G2 variants have been shown to have high affinity for suPAR activated αvβ3 integrin on podocytes in the progression of chronic kidney disease.⁷¹ These and the aforementioned mechanisms are summarized in Figure 1.

Figure 1.

Purported mechanisms of APOL1-induced kidney injury. PKR, protein kinase R.

Emerging Therapies in AMKD

Drug discovery has traditionally relied on well-characterized disease targets, high-throughput screening methods to test large compound libraries, and prior experimental data.^72,73 Nonetheless, the emergence of novel technologies such as genomics, proteomics, and bioinformatics to generate and analyze large-scale data sets, enabled the identification of new mechanistic pathways and therefore more precise options such as peptide-based inhibitors, oligonucleotides, or gene therapies.74, 75, 76 These modalities have been extensively described in other fields such as oncology and rheumatology. The rapid development of drug repurposing methods, systems biology, and artificial intelligence in nephrology is supporting the emergence of novel approaches to tackle AMKD.77, 78, 79 Given the number of potential mechanisms that contribute to APOL1-mediated renal injury, it is not surprising that different approaches are currently being explored. A summary of phase 1 to 3 clinical trials for AMKD since its discovery is presented in Table 1. A brief discussion of these emerging agents (Figure 2) will be discussed below.

Table 1.

Recent and ongoing APOL1 therapeutic trials

NCT number	Drug	Mechanism of action	Status	Phase	Completion
NCT04340362	VX-147	APOL1 channel blocker (small molecule inhibitor)	Completed	Phase 2	December 2021
NCT05312879			Recruiting	Phase 2/3	June 2026
NCT05324410	VX-840	APOL1 channel blocker (small molecule inhibitor)	Completed	Phase 1	November 2022
NCT04269031	AZD2373	APOL1 antisense oligonucleotide	Completed	Phase 1	August 2021
NCT05351047			Active, not recruiting	Phase 1	July 2023
NCT05237388	Baricitinib	Janus Kinase-STAT Inhibition	Recruiting	Phase 2	March 2026

APOL1, apolipoprotein L1; STAT, signal transducer and activator of transcription.

Search of clinicaltrials.gov was performed on July 19, 2023.

Figure 2.

Mechanism of action of current APOL1 therapeutic agents in development.

APOL1 Small Molecule Inhibitors

Small molecules are a class of pharmacological agents that exhibit high specificity and potency in targeting enzymes or protein-protein interactions of disease-relevant pathways.⁷⁸ With their relatively low molecular weight and favorable physicochemical properties, small molecule inhibitors possess the ability to penetrate cellular membranes and enable access to intracellular components. These inherent characteristics offer significant advantages for therapeutic intervention by mitigating off-target effects and minimizing toxicity to normal cells. In addition, harnessing the modulatory potential of small molecule inhibitors holds great promise in advancing personalized medicine, particularly in the context of AMKD, because individual patients may exhibit distinct molecular drivers of disease within the spectrum of AMKD.^26,49,53,68 Moreover, the oral bioavailability of many small molecule inhibitors enables convenient administration.

A novel small molecule inhibitor on APOL1 channel called VX-147 or inaxaplin demonstrated promising results in reducing proteinuria in patients with APOL1-associated FSGS in a recently published phase 2a study.¹² After demonstration of efficacy in preclinical models (a reduction of cationic influx in vitro and a reduction of proteinuria in a transgenic APOL1 mouse model), inaxaplin was administered during 13 weeks to 16 participants who had 3 APOL1 high-risk variants, biopsy-proven FSGS, and estimated glomerular filtration rate ≥27 ml/min per 1.73 m². Among the 13 participants who were adherent to the treatment threshold, the mean change from the baseline urinary protein-to-creatinine ratio at week 13 was −47.6% (95% confidence interval, −60.0 to −31.3). Furthermore, in an analysis that included all 16 participants regardless of adherence to therapy, reductions were similar to those in the primary analysis in all but 1 participant. The drug showed a substantial decrease in proteinuria, even in patients receiving standard-of-care treatment. However, there are some limitations to note. In addition to statistical limitations and biases inherent to the single arm, small sample study design, most patients had FSGS with subnephrotic range proteinuria, and secondary FSGS could not be ruled out. Furthermore, baseline and concomitant therapy for FSGS were variable during the treatment phase and it remains unclear whether longer duration of inaxaplin would persistently suppress proteinuria. A larger phase 2/3 inaxaplin AMKD study (NCT05312879), not limited to FSGS, is currently actively enrolling. In addition, another small molecule inhibitor (VX-840) is currently being studied by the same sponsor (phase 1 completed in November 2022).

Recently, small molecule inhibitor, MZ-301 has been developed to potentially block APOL1 electrophysiological currents in response to a voltage ramp in HEK293 cells expressing APOL1 G2.⁸⁰ APOL1-mediated ion currents measured in HEK293 cells were noted to decrease in the presence of incremental doses of MZ-301, eventually rescuing them from APOL1-mediated cytotoxicity. Furthermore, MZ-301 was described to inhibit APOL1-dependent cytotoxicity in vitro in human immortalized podocytes, which translated into urine albumin-to-creatinine ratio reductions in APOL1 G2 mutant mice.

Antisense Oligonucleotides

Oligonucleotide therapeutics, such as those based on antisense oligonucleotides (ASOs), small interfering RNA, microRNA, aptamers, and decoys, are promising agents that have gained importance during the past decades in nephrology and other fields.81, 82, 83 APOL1 ASOs are oligonucleotide analogs that modify expression of specific RNAs and can alter protein synthesis. ASOs bind to select mRNA sequences and can cause RNase H1-mediated degradation (ceasing synthesis of the protein), splicing defects, or interfere with gene expression. In a recent study, a generation 2.5 APOL1 ASO (IONIS-APOL1_Rx) was selected as the APOL1 clinical candidate based on its consistent and potent activity in vitro as well as in vivo in genomic APOL1-transgenic mice. Subcutaneous administration of IONIS-APOL1_Rx to APOL1 G1-transgenic mice resulted in dose-dependent reductions in kidney and liver APOL1 mRNA, preventing dose-dependent interferon-induced proteinuria.⁸⁴ The agent was used in a first-in-human, single ascending dose, phase I study (NCT04269031), to evaluate the safety and assess the pharmacokinetics of escalating single doses of a subcutaneously administered ASO (ION532, also known as AZD2373), with results pending. Another phase I study (NCT05351047) is ongoing.

JAK/STAT Pathway Blockade

As described above, JAK/STAT plays a critical role in activating proinflammatory cell programs and its inhibition could efficiently decrease APOL1-associated cellular toxicity, which has been tested in preclinical models with promising results. A phase 2 trial (JUSTICE, NCT05237388) is currently recruiting to evaluate the efficacy of baricitinib, a JAK inhibitor approved for the treatment of rheumatoid arthritis and alopecia areata, in patients with AMKD. STAT3 inhibition using next-generation ASOs, including 2.5 ASO (e.g., AZD9150) has been explored for other clinical indications. STAT3 binds mRNA and silences gene expression through blocking translation or recruiting RNase H enzymes, which degrade the DNA-RNA heteroduplex.^19,20 AZD9150 is currently being studied in leukemia and lymphoma due to its proapoptotic and cell regulatory effects.^85,86

Future Directions and Considerations

Extensive studies have shed light on cellular pathways activated by APOL1 risk variants in cellular, animal, and human studies; however, several gaps remain. Evidence that less than 30% of individuals with 2 high-risk APOL1 variants develop AMKD warrants careful exploration to further define “second hits” and clarify patient populations that should undergo genetic screening. This will influence ongoing initiatives to potentially reduce adverse kidney and transplant outcomes, support the development of noninvasive biomarkers that could potentially anticipate the onset of kidney disease, provide risk-stratification algorithms and advance precision-based therapeutic approaches.

There are still other unresolved questions in developing APOL1 therapeutics. There is uncertainty regarding the impact of circulating APOL1 on the kidneys and consequently a lack of clarity regarding the relative efficacy of reducing systemic APOL1 levels versus inhibiting the function of mutant APOL1 protein. The multiple upstream regulatory and downstream pathogenic signaling cascades may also require potentially divergent therapeutic approaches in certain AMKD subpopulations. Safety will be a concern, especially in regions endemic for trypanosomiasis and where prolonged therapy may be required. Small molecule inhibitors can lead to off-target effects and prolonged genetic alterations. Advances are also required in improving kidney-specific drug delivery to reduce the potential for off-target effects of APOL1 inhibitors.

In summary, significant progress has been made in understanding the cellular injury mechanisms of APOL1 risk variants and the development of new therapies for AMKD. More work will be needed in molecular subphenotyping, precision-based targeted approaches, and careful investigation of the efficacy and safety profile of emerging therapies.

Disclosure

KNC reports consulting fees from Travere, Goldfinch, Chinook, ANI, and Aurinia and funds to his department for being a site principal investigator for studies sponsored by Vertex and Travere outside the submitted work. GVR and MC declare no competing interests.