The Evolving Story Of Apolipoprotein L1 Nephropathy: The End Of The Beginning Ⅱ

Geographic distribution of APOL1 alleles 

The APOL1 G1 and G2 variants likely arose in West Africa after the out-of-Africa migrations 60,000 years ago1,31 and are thus found only in individuals with sub-Saharan African ancestry. Regionally, the G1 and G2 alleles are most prevalent in West Africa, with a combined allele prevalence of over 40% among major ethnic groups in Ghana and Nigeria13,32–34. However, the distribution of allele frequencies is complex and varies widely among African ethnic populations, even within the same geographical region or country, likely because of the topical endogamy within ethnic groups and migratory patterns (Fig. 2).

Trypanosoma brucei (T.b.) gambiense and T.b. rhodesiense are intracellular parasites that cause African human trypanosomiasis (African sleeping sickness). T. b. rhodesiense is distributed in eastern and southern Africa and causes the acute form of the disease, accounting for 2% of all trypanosomiasis cases. The chronic form of sleeping sickness, which is caused by T. b. gambiense, is found in west and central Africa and accounts for 98% of cases35,36 (Fig. 2). Trypanosoma brucei brucei is a subspecies that is physically indistinguishable from the other two subspecies; it infects many vertebrates, including domestic cattle and horses37, but not humans. This host selectivity is likely the result of co-evolution, with primates and trypanosomes each developing measures and countermeasures to ensure survival. It has been known for almost a century that some trypanosomes cannot infect humans as they are lysed by a component of normal human serum, now understood to be associated with a subclass of human HDL38.

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Fig. 2 | Global distribution of APOL1 high-risk genotypes and endemic areas of Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. The highest prevalence of APOL1 high-risk genotypes, defined as the presence of two risk alleles (i.e. G1/G1, G1/G2 or G2/G2), is in West Africa, but carriage of high-risk genotypes is found throughout sub-Saharan Africa and among those with African ancestry in the Americas and elsewhere. Allele frequency data are derived from Nadkarni et al.4 and Limou et al.7 ) and unpublished data (C.A. Winkler, unpublished work).

In 2003, researchers made the seminal observation that APOL1 contributes to trypanosome lysis via the actions of trypanosome lytic factors39. They also identified a T. b. rhodesiense gene that encodes a lysosomal SRA, which interacts with the C-terminal domain of APOL1 (Fig. 1a) and confers resistance to T. b. rhodesiense lysis by APOL1 in vitro39. Subsequent studies demonstrated that maximal anti-trypanosomal activity requires the assembly of APOL1 with other components40 present within the HDL particle and that this assembly is required to protect humans from T.b. brucei and T.b. evansi infection40,41. The G1 missense variants and the G2 deletion occur within the SRA protein binding domain and promote APOL1 trypanolytic activity in vitro by reducing or preventing SRA binding, respectively, by T.b. rhodesiense1,31. However, neither variant restores trypanolytic activity against T.b.gambiense1.

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The extent of natural protection against human African trypanosomiasis (HAT) provided by the variant proteins is not fully understood. Although trypanosomemal lytic activity has been attributed to the APOL1 G2 variant protein and, to a lesser degree, the G1 variant protein, using in vitro assays1, and delayed parasitemia has been demonstrated in transgenic mice expressing the APOL1 G1 variant31, the effects of these variants on natural infection and development of acute and chronic HAT caused by T.b. rhodesiense and T.b. gambiense, respectively, is not fully understood. Findings from the case-control studies from HAT endemic regions of sub-Saharan Africa indicate that protection conveyed by the G1 and G2 variants is complex (Fig. 1c). In one case-control study, the G2 variant prevented infection by T.b. rhodesiense, endemic to East Africa, consistent with in vitro findings. However, whereas G2 was associated with the development of symptomatic HAT in those infected by T.b. gambiense, endemic to West and Central Africa42, the G1 variant was associated with asymptomatic T.b. gambiense parasitemia but notably did not affect the risk of infection by either T.b. Rhodesense or T.b. gambiense42. These differential effects were confirmed by a second study, which found that the G2 variant was associated with a higher risk of developing T.b. gambiense-associated HAT, whereas the G1 variant was associated with lower risk43 (Fig. 1c). By contrast, a 2018 study observed no significant associations between the presence of APOL1 risk alleles and HAT in two Ugandan populations, which were exposed to both trypanosomal species44. These studies suggest that the effects on trypanosome infection and HAT severity may differ regionally owing to local adaptations by the host and pathogen. The discordance between the geographic distribution of allele frequencies and trypanosomal sensitivity also raises the possibility of an as yet unidentified source of selective pressure acting on APOL1.

Thus, a complex interplay exists between APOL1 evolution and trypanosomal infections, and associated selective pressures across sub-Saharan Africa are likely the primary driver underlying the emergence of APOL1 risk alleles and the associated end-organ toxicities observed in clinical practice today

APOL1 and kidney disease 

Following the discovery of the APOL1 variants, several studies reported strong associations between the APOL1 risk variants and kidney disease, particularly FSGS and HIVAN (odds ratios of 17 and 29, respectively, in the United States (and 89 for HIVAN in South Africa)), but also with sickle cell nephropathy and particular immune-mediated kidney diseases, including lupus nephritis13,23,45–48. In patients with certain forms of CKD, the presence of two APOL1 risk alleles was associated with more rapid disease progression and an increased risk of kidney failure49. Other studies showed that although few otherwise healthy, young-to-middle-aged adults with two APOL1 risk alleles, normal estimated glomerular filtration rate (eGFR), and no or low-grade proteinuria develop kidney disease during their lifetimes, some of these individuals do develop incident kidney disease9,50,51. These findings suggested that a "second hit" is required to initiate kidney injury in individuals with APOL1 risk genotypes52. Factors considered as second hits included genetic variants beyond APOL1, systemic factors, and environmental factors45,53,54. The most established systemic factors are viral infections (for example, HIV) and, more generally, systemic elevations in interferon levels. It is now apparent that APOL1 is expressed in an interferon-dependent manner55.

In the past couple of years, APOL1 risk allele status has been observed to modify the risk of kidney injury associated with SARS-CoV-2 infection. Cases of collapsing glomerulopathy have been reported in individuals with APOL1 risk variants with COVID-19 and hyper-inflammatory features, potentially reflecting a high-interferon state56,57. Of note, however, one case report described a patient with COVID-19-related collapsing glomerulopathy in the setting of one APOL1 risk allele58, suggesting a potential dominant effect or the existence of another unknown second hit.

Studies in the past few years have demonstrated that kidney transplant recipients with APOL1 high-risk genotype donor kidneys have lower eGFR at follow-up and poorer allograft survival15,59–61. However, whether the presence of two APOL1 risk alleles in the transplant recipient affects the outcome of the transplanted kidney is unclear, with studies showing conflicting results 62,63. Furthermore, kidney donors with two APOL1 risk alleles have an increased risk of reduced kidney function following kidney donation, which has the potential to progress to kidney failure, suggesting that counseling should be considered to inform potential kidney donors with APOL1 risk variants about the associated risks61. The most compelling case for genetic testing of APOL1 in the clinical setting at present is therefore in kidney transplantation, particularly for potential living donors64,65. An ongoing NIH-funded study - the APOL1 Long-term Kidney Transplantation Outcomes study (APOLLO) - is aimed at prospectively determining outcomes in living kidney donors with African ancestry and in recipients of living and deceased donor kidneys from individuals with African ancestry, to better define the effect of APOL1 risk alleles on transplantation outcomes, and will provide data with which to guide patients and clinicians in judging the risk of transplantation for living donors and recipients66,67. APOL1 risk status has also been associated with kidney disease in children, most strongly in the setting of HIV infection 68,69. Reports from two large pediatric cohorts - the Chronic Kidney Disease in Children (CKiD) and the Nephrotic Syndrome Study Network (NEPTUNE) study - show that children with a high-risk genotype have a higher prevalence of FSGS than those with a low-risk genotype and that the disease often follows an aggressive course70. To date, no significant correlation has been identified between APOL1 plasma levels and kidney function, with the possible exception of a kidney transplant, as discussed below 71. Moreover, studies in mouse models of APOL1-associated kidney disease have shown that expression of APOL1 risk variants in podocytes is associated with functional (albuminuria and azotemia), histomorphological (podocyte foot-process effacement, and glomerulosclerosis), and characteristic molecular (gene-expression) changes72. Together with the kidney transplant experience, these findings suggest that kidney-expressed APOL1 is the primary driver of kidney injury rather than circulating APOL1 in plasma15,61.

APOL1 localization in the kidneys. 

In 2011, immunohistochemistry studies demonstrated the localization of APOL1 to podocytes, arteriolar endothelium, and proximal tubular epithelium in kidney tissue from healthy individuals and patients with FSGS or HIVAN73. Interestingly, and despite the preservation of podocyte markers, fewer podocytes in glomeruli from patients with FSGS and HIVAN expressed APOL1 compared with podocytes in normal kidneys. On the other hand, increased APOL1 staining was observed in the vasculature of diseased kidneys, with expression located in a subset of α-smooth muscle actin-positive cells, the media of medium-sized arteries, and arterioles. The expression patterns were similar in both HIVAN and FSGS cases, leading the researchers to propose that the robust expression of APOL1 in specific kidney cells is a marker of de novo APOL1 synthesis in kidney tissue and not a consequence of kidney deposition of plasma APOL1.

Subsequent evaluation of this hypothesis by other researchers led to the conclusion that APOL1 in kidney cells is derived from both cellular synthesis and uptake from plasma or the glomerular filtrate. Using indirect immunofluorescence microscopy of kidney tissue from healthy individuals, these researchers noted more APOL1 protein in podocytes and less protein in tubular epithelial cells. Fluorescence in situ hybridization studies revealed expression of APOL1 mRNA in podocytes, endothelial cells, and proximal tubules, confirming the endogenous production of APOL1 in a restricted set of kidney cells. These results are consistent with studies of kidney-derived cell lines, in which both APOL1 mRNA and protein were detected in proximal tubular epithelial cells and glomerular endothelial cells, with lower expression in podocytes. Moreover, these studies demonstrated the uptake of APOL1 protein by cultured podocytes, but not by cultured glomerular endothelial cells and proximal tubular cells74. Studies of transgenic zebrafish models also showed co-localization of APOL1 with podocyte markers and to a lesser extent, with endothelial cells. In podocytes, APOL1 expression was more marked in the podocyte body than in the foot processes75. Together, these data demonstrate that APOL1 production occurs in the kidney and may drive local injury

APOL1 levels in plasma. 

Despite the importance of kidney-expressed APOL1 for kidney injury, the majority of APOL1 is produced in the liver and circulates in plasma. Several groups have assessed the implications of circulating APOL1 and showed that levels do not correlate with kidney disease risk, underscoring the essential role of kidney-expressed APOL1 in kidney disease71,76,77. A more recent study proposed that the circulating APOL1 variants have an immunomodulatory role, associated with T cell-mediated rejection and death-censored allograft loss63. Further studies are required to establish the role of circulatory APOL1 levels. Renal transplant studies provide a unique setting to distinguish between the effects of systemic APOL1 expression and kidney-limited APOL1 expression.

Models of APOL1-associated kidney disease. 

The APOL1 gene is absent from all experimental animals used to model human disorders and thus the development of animal models of APOL1-associated disorders has required transgenic approaches. Several groups have developed transgenic mouse models of APOL1 nephropathies. Mice with podocyte-specific expression of the APOL1 G2 variants under a nephrin promoter did not demonstrate overt manifestations of kidney disease but had a lower podocyte density than APOL1 G0-transgenic mice at about 6 months of age. However, APOL1 G2 transgenic mice, and to a lesser extent, APOL1 G0 transgenic mice, showed a pregnancy-related preeclampsia-like phenotype78. Transgenic mouse models with a podocyte-specific and tubule-specific expression of APOL1 G0, G1, or G2 isoforms have also been generated using a tetracycline-inducible enhanced green fluorescent protein (EGFP)-expressing plasmid vector. Mice with a glomerular expression of either APOL1 risk allele, but not the common variant, developed proteinuria and glomerulosclerosis, with the extent of glomerulosclerosisis correlating with the level of APOL1 expression72. By contrast, mice with tubule-specific expression of APOL1 did not demonstrate kidney abnormalities, underscoring the importance of glomerular, and particularly podocyte, APOL1 expression72. Transgenic mice with doxycycline-induced expression of APOL1 in podocytes also exhibited proteinuria and FSGS lesions79.

Other approaches to studying APOL1 kidney disease have involved the hydrodynamic delivery of human APOL1 mRNA to mice. Using this technique, mice receiving APOL1 risk variants showed more liver and kidney injury than those with the APOL1 G0 variant31,41,80.

Other model organisms have also been used. The zAPOL1 gene in zebrafish is homologous to the human APOL1 gene75,81,82. This gene is essential for normal glomerular function in zebrafish, as loss of function is associated with derangement of glomerular architecture. In zebrafish with deletion of zAPOL1, expression of the human APOL1 G0 variant leads to improvement in glomerular function, whereas expression of the G1 and G2 variants does not82,83. Moreover, transgenic expression of the G1 and G2 variants results in subtle alterations in renal structure that are apparent only on electron microscopy; these alterations are not present in APOL1-G0 transgenic zebrafish. Similar findings have been reported in studies of Drosophila nephrocytes, which show functional and structural similarities to podocytes. In these cells, the expression of two APOL1 G1 and G2 variants causes defects in intra-organelle acidification, leading to cellular hypertrophy and subsequent death 84,85.

APOL1 disease associations beyond the kidney 

APOL1 risk variants may be associated with diseases other than kidney diseases86, with some evidence supporting an association with cardiovascular diseases87, including atherosclerosis88,89, hypertension90,91, acute coronary syndrome, and myocardial infarction88, stroke92, and heart failure93,94. However, data regarding these associations are inconsistent. Although some studies have reported higher cardiovascular mortality among patients with APOL1 risk variants95, other studies, including a meta-analysis of 21,305 Black individuals from eight cohorts, do not support a strong correlation between APOL1 risk variants and cardiovascular diseases51,93,96–98.

Associations may also exist between APOL1 risk variants and preeclampsia - a pregnancy complication that is more common in populations of sub-Saharan African descent than in other populations99. A 2018 study reported that although maternal APOL1 risk genotype was not associated with preeclampsia, mothers of fetuses with two APOL1 risk alleles had a higher risk of preeclampsia than mothers of fetuses with low-risk alleles14, with an odds ratio of 1.8. This finding was confirmed and extended in a second study, which also found that APOL1 allelic mismatch between fetus and mother was associated with nearly three-fold increased odds of preeclampsia100. However, as described earlier, a study of APOL1 transgenic mice showed an eclampsia-like phenotype to be associated with the G2 risk allele and also unexpectedly with the G0 genotype; the effect of the G1 risk allele was not studied78. The occurrence of a preeclampsia phenotype in dams lacking an orthologous APOL1 gene may be due to the expression of the fetal APOL1 transgene in the placenta, resulting in a fetal-maternal incompatibility, or to the general toxicity of high levels of APOL1 protein, irrespective of genotype. Other studies have also demonstrated an association of APOL1 genotype with sepsis in Black adults101,102. The mechanism for this effect remains obscure and warrants further attention.

Mechanisms of cell injury 

Various mechanisms of APOL1-mediated cell toxicity have been proposed that might underlie the association between the APOL1 risk variants and kidney disease. Several of these are outlined below (Fig. 3; Table 1).

APOL1 and lysosomal dysfunction.

In 2007, researchers showed that APOL1 kills trypanosomes by forming pores in the trypanosome lysosomal membrane, to which APOL1 traffics following uptake into the parasite103. Using endocytosis, the parasite takes up APOL1 in complex with HDL, from which the parasite obtains lipids and iron20. The structure of the pH-sensitive membrane-addressing domain of APOL1 contains a hairpin region that connects two α-helices. This structural element undergoes a conformational change in the acidic environment of the lysosome, which allows the insertion of APOL1 into the lysosome membrane and activation of the APOL1 pore-forming domain. APOL1-mediated pore formation disrupts the lysosomal membrane, triggering an uncontrolled influx of ions from the cytoplasm into the lysosome. This process leads to osmotic swelling of the lysosome and eventual trypanosomal death25,104,105.

In 2003, a carboxy-terminal α-helical domain was identified in APOL1, which interacts with the amino-terminal of SRA in T. b. rhodesiense(Fig. 1a). As described earlier, this SRA domain confers resistance to APOL1, eluding host defenses and enabling the trypanosome to cause human disease39. A subsequent study showed that interaction with SRA altered the intracellular trafficking of APOL1, resulting in its localization to SRA-containing cytoplasmic vesicles instead of to the lysosomes, thereby inhibiting membrane toxicity in the parasite106. Additional studies of the C-terminal helix of APOL1 confirmed that interaction of the SRA domain with this region of APOL1 inhibits APOL1 toxicity by preventing pore-forming activity, thus protecting the trypanosome107.

Of note, APOL1 causes lysosomal dysfunction in cultured human kidney cells as well as in parasites. The APOL1 G1 and G2 variants decrease the number of lysosomes in podocytes, which results in leakage of lysosomal enzymes into the cytoplasm108. Over-expression of the APOL1 risk variants in cultured podocytes is associated with podocyte lysosomal swelling; tlsosomal dysfunction might occur through the functional downregulation of the mechanistic target of rapamycin (mTOR)109.

Table 1 | Possible mechanisms of APOL1-associated glomerular injury

passage of calcein, a 623Da negatively charged molecule at neutral pH. APOL1 channel activity was found to have three requirements that allow APOL1 to associate with pore-forming vesicles: low pH, presence of negatively charged phospholipids in vesicle membranes, and low ionic strength108.

In agreement with these findings, a 2005 study reported that APOL1 forms distinct anion-selective pores in unilamellar vesicular membranes, thereby promoting chloride influx. The researchers speculated that the passive entry of chloride through anion-selective channels is facilitated by the initial influx of extracellular sodium down its concentration gradient. The resulting osmotic imbalance leads to the passive entry of water to the cell, which promotes cell swelling and trypanosome lysis111.

However, a 2015 study reported quite different results, showing that APOL1-mediated trypanosome lysis requires an acidic pH for the first steps, in which APOL1 interacts with and inserts into vacuolar lipid bilayers. The researchers reported that APOL1 is subsequently trafficked to the plasma membrane, where it is exposed to a non-acidic pH, allowing APOL1 to open pH-sensitive non-selective cationic channels, dipolarsizing the trypanosome membrane and killing the tripanosome112. A subsequent publication supported the cation-selective nature of these ion channels by showing that mammalian cells expressing APOL1 risk variants have increased non-selective cation permeability and promote a net efflux of intracellular potassium through plasma membranes113. More recently, a population of plasma membrane cation channels for sodium and calcium was noted to be selectively expressed in HEK293 cells expressing APOL1 risk variants114.

These contradictory findings may be explained by the suggestion that APOL1 ion-channel selectivity is pH-switchable. At pH 5, APOL1 may promote chloride permeability through anionic channels; at neutral pH, it facilitates cationic (potassium) permeability, whereas it has almost no channel activity in a basic environment. Permeability to both ions requires negatively charged phospholipids for maximal activity. Moreover, potassium channels require calcium ions for proper function. This pH-switchable ion-selective permeability may explain both the anionic and cationic channel activity previously reported for APOL1 in intracellular and plasma membrane environments, respectively, as the differences could be due to pH differences in the various intracellular compartments under particular experimental conditions115.