APOL1 and kidney cell function

Abstract

The apolipoprotein L1 (APOL1) gene is unique to humans and gorillas and appeared ~33 million years ago. Since the majority of the mammals do not carry APOL1, it seems to be dispensable for kidney function. APOL1 renal risk variants (RRVs; G1 and G2) are associated with the development as well as progression of chronic kidney diseases (CKDs) at higher rates in populations with African ancestry. Cellular expression of two APOL1 RRVs has been demonstrated to induce cytotoxicity, including necrosis, apoptosis, and pyroptosis, in several cell types including podocytes; mechanistically, these toxicities were attributed to lysosomal swelling, K⁺ depletion, mitochondrial dysfunction, autophagy blockade, protein kinase receptor activation, ubiquitin D degradation, and endoplasmic reticulum stress; notably, these effects were found to be dose dependent and occurred only in overtly APOL1 RRV-expressing cells. However, cellular protein expressions as well as circulating blood levels of APOL1 RRVs were not elevated in patients suffering from APOL1 RRV-associated CKDs. Therefore, the question arises as to whether it is gain or loss of function on the part of APOL1 RRVs contributing to kidney cell injury. The question seems to be more pertinent after the recognition of the role of APOL1 nonrisk (G0) in the transition of parietal epithelial cells and preservation of the podocyte molecular phenotype through modulation of the APOL1-miR-193a axis. With this background, the present review analyzed the available literature in terms of the known function of APOL1 nonrisk and how the loss of these functions could have contributed to two APOL1 RRV-associated CKDs.

INTRODUCTION

The apolipoprotein L1 (APOL1) gene appeared ~33 million ago, after the divergence of Old World primates and New World primates, and is unique to humans and gorillas (96). APOL1 is secreted by liver cells and is a component of the circulating HDL complex (22). It is also expressed by several cell types, including the kidney, brain, pancreas, and macrophages. Initially, the role of circulating APOL1 as a trypanolytic factor was recognized (96). However, the role of the expressed protein in general, and in kidney cells in particular, was not appreciated (99). During the present decade, APOL1 mutants (G1 and G2) have been reported for their association with a higher incidence of chronic kidney disease (CKD) in patients with African ancestry and considered to be renal risk variants (RRVs). The G1 and G2 alleles are mutually exclusive and follow a recessive pattern of inheritance. The APOL1 RRVs are characterized as two G1 (homozygous G1/G1), two G2 (homozygous G2/G2), or one G1 and one G2 (compound heterozygous G1/G2) risk alleles.

In the Atherosclerosis Risk in Communities Study, a higher rate of end-stage kidney disease (ESKD) was observed among African American individuals carrying APOL1 RRVs compared with APOL1 nonrisk (NR) (34). Findings from the longitudinal studies including the African American Study of Kidney Disease and Hypertension, Chronic Renal Insufficiency Cohort study, and Coronary Artery Risk Development in Young Adults study also revealed that African Americans with APOL1 RRVs have higher rates of decline in kidney function, accelerated progression to ESKD, and higher occurrence of albuminuria compared with European American individuals (81, 82). Since concentrations of serum APOL1 did not correlate with the presence or severity of CKD (11, 50), it appeared that this effect of APOL1 is related to direct cellular expression rather than its endocrine or paracrine effects. This notion has been validated by other investigators. The toxic effect of APOL1 RRVs has been demonstrated in several cell types, including podocytes (35, 56–58). Both in vitro and in vivo studies have pointed toward gain of function by expression of APOL1 RRVs in podocytes as well as in other experimental cells (56–58, 91, 100). However, the results of other investigators differed with this concept: 1) recessive inheritance is often associated with loss of function rather than gain of function, 2) renal biopsy specimens of the affected individuals showed decreased expression of APOL1 in the involved cells (67, 69), and 3) several in vitro and in vivo experimental models with normal APOL1 expression do not develop expected phenotype (13, 78). Based on these findings, other investigators suggested that the disparity between in vitro and animal experimental model manifestation may be related to the higher expression levels of APOL1 RRVs (10, 78). Interestingly, the recent recognition of the APOL1-miR-193a axis (AMA) has provided some insight into the purpose of APOL1's evolution in humans as an effort to enhance the podocyte renewal reservoir to support the increased longevity of humans. As a part of the AMA, APOL1 facilitates the maintenance of the podocyte molecular phenotype and their renewal in adverse milieus (53, 55).

In the present review, we analyzed the prevalent theories about APOL1 nonrisk (NR)- and APOL1 RRV-associated CKDs and in vitro experimental data. However, if one believes in the loss of function theory, the function of cellular expression of APOL1 in kidney cells needs to be understood. At present, the majority of studies have been carried out in human embryonic kidney (HEK) cells, yeast, and endothelial cells in vitro and in zebrafish, fly, and mouse models in vivo. Only a limited number of studies have been carried out in human podocytes in vitro and in analyzed human kidney tissues of patients with focal segmental glomerular sclerosis (FSGS) and human immunodeficiency virus (HIV)-associated nephropathy (HIVAN) (10, 46, 55, 69). The present review focused on the function of APOL1 in podocytes and is an attempt to examine whether the podocyte phenotype in CKDs is a consequence of gain or loss of function by APOL1 RRVs.

APOL1 NR (G0) FUNCTION IN PODOCYTES

The function of cellular expression of APOL1 is still under investigation. The known role of cellular expression of APOL1 NR (G0) in podocytes is shown in Table 1. The loss of function occurring as a consequence of mutations in APOL1 RVVs (G1 and G2) contributing to cellular injury is also shown in Table 1. We highlighted the role of APOL1 NR in the maintenance of the podocyte molecular phenotype and their integrity in multiple ways and explained the compromised podocyte integrity in the APOL1 RRV milieu. Additionally, we have provided a list of reported cellular injuries in cells expressing APOL1 RRVs in Table 2; although these manifestations could be considered as a gain of function, it is not clear whether these were a consequence of overt expression of APOL1 RRVs in experimental conditions in vitro and also happen in vivo.

Table 1.

Presumed function of APOL1 G0 (NR)

Function	APOL1 G0 (NR)	APOL1 G1/G2 (RRV)	Reference(s)
APOL1-miR-193a axis	Functional	Disrupted	Kumar et al. (52, 55); Mishra et al. (72)
Human immunodeficiency virus survival	Compromised	Not Compromised	Mikulak et al. (71)
Adherens complex	Stabilized	De-stabilized	Kumar et al. (53)
Actin cytoskeleton	Maintained	Disorganized	Kumar et al. (53); Lan et al. (56)
Autophagy	Enhanced	Blocked	Beckerman et al. (10)
Parietal epithelial cells transition	Facilitated	Compromised	Kumar et al. (55)
Recruitment of APOL1 G1/G2 to lipid droplets	Enhanced	Compromised	Chun et al. (17)
K+ efflux	Maintained	Accelerated	Olabisi et al. (80)
Mitochondria	Maintained	Depolarized	Ma et al. (66);  Granado et al. (35);  Bruggeman et al. (13)
Endoplasmic reticulum	Normal	Stressed	Wen et al. (100); Cheng et al. (16); Chun et al. (17)
Cholesterol Efflux	Normal	Decreased	Ryu et al. (85)

APOL1, apolipoprotein L1; NR, nonrisk; RRV, renal risk variant.

Table 2.

Presumed gain of function by APOL1 RRVs

APOL1 G1/G2 (RRV)	Manifestations	Reference(s)
G1 and G2	Lysosomal dysfunction	Lan et al. (56)
G1 and G2	Mitochondrial dysfunction	Ma et al. (66); Bruggeman et al. (13)
G1 and G2	Channel/pore formation	Bruno et al. (14); Olabisi et al. (80)
G1 and G2	Autophagy blockade	Beckerman et al. (10)
G1 and G2	Pyroptosis	Beckerman et al. (10)
G1 and G2	Protein kinase R activation	Okamoto et al. (79)
G1 and G2	αvβ3-integrin activation	Hayek et al. (39)
G1 and G2	APOL1-miR-193a axis	Kumar et al. (52, 53); Mishra et al. (72)
G1 and G2	Actin cytoskeleton	Kumar et al. (53); Lan et al. (56)

APOL1, apolipoprotein L1; RVVs, renal risk variants

Experimental data suggest that cytokines carry the potential to enhance APOL1 NR expression in several cell types in vitro (71, 99, 106). In contrast, human specimens have not been reported to display enhanced expression of APOL1 RRV proteins (69); however, increased transcript levels of APOL1 RRVs have been reported in human renal biopsy specimens (10). Since the cellular toxicity of an APOL1 RRV is dependent on its protein expression, it is possible that studies carrying out enhanced expression of APOL1 RRVs in vitro may be determining its biological effects and may not be relevant in understanding the associated pathological conditions. On the contrary, in a recent report (79), an increase in mRNA transcript of APOL1 RRVs activated protein kinase receptor protein and associated downstream signaling. Thus, it appears that APOL1 RRVs execute a different modus operandi to inflict cellular injury. Alternatively, it may be that lack of APOL1 NR function by RRVs contribute to the development and progression of so-called APOL1 nephropathy (53, 79). This concept also forecasts a large role of APOL1 NR in the maintenance of glomerular health under normal physiology and pathological states (Fig. 1).

Fig. 1.

Podocyte phenotype in apolipoprotein L1 (APOL1) G0 [nonrisk (NR)] and APOL1 G1/G2 [renal risk variant (RRV)] milieus. Moderate expression of APOL1 G0 (NR) optimizes the podocyte molecular phenotype, intact actin cytoskeleton, adherens complexes, and cytosolic organelle homeostasis. In contrast, overt expression of APOL1 G1/G2 (RRVs) in podocytes is associated with a stressed endoplasmic reticulum, depolarized mitochondria, lysosomal swelling, autocrine cytokine-mediated activation, K⁺ depletion, autophagy blockade, pyroptosis, protein kinase receptor (PKR) activation, proteasomal degradation, ubiquitin D (UBD) activation, disorganization of the actin cytoskeleton, destabilization of adherens complexes, dedifferentiation, αβ-integrin signaling activation, unregulated endosomal trafficking, and a disrupted APOL1-miR-193a axis. All these changes culminate into podocyte loss and the development of kidney disease. CD2AP, CD2-associated protein; IFN-γ, interferon-γ; TGF-β, transforming growth factor-β.

In experimental design, the function of APOL1 has been evaluated by silencing of APOL1 in cells that normally express APOL1, such as podocytes (53, 72). Several of APOL1's functions are dependent on an intact AMA. APOL1 inversely regulates miR-193a (53, 55, 72). On that account, silencing of APOL1 would be associated with enhanced expression of mi-R193a (72). Interestingly, miR-193a negatively regulates Wilms’ tumor 1 (WT1), a master transcriptor of podocyte markers such as podocalyxin and nephrin (33). Therefore, APOL1 silencing in podocytes is associated with dedifferentiation of podocytes or loss of their molecular phenotype (72). The potential of APOL1 as an inducer of podocyte markers was evident by the induction of WT1, podocin, and podocalyxin in HEK cells transfected with APOL1 (55).

THE APOL1-miR-193A AXIS

MicroRNA mediate the posttranscriptional regulation of gene expression and targets cellular mRNAs (15). The altered expression of miRNAs under the expression of APOL1 NR and RRVs is unknown. In a recent study (55), we have shown that miR-193a is in a bifunctional relationship with APOL1; APOL1 NR can downregulate miR-193a expression, whereas miR-193a inversely regulates expression of APOL1 NR (Fig. 2). miR-193a targets the 3′-untranslated region of APOL1. In contrast, this axis is disrupted in APOL1 RRVs. APOL1 RRVs escalated rather than downregulated expression of miR-193a in podocytes; the latter mediated APOL1 RRV-induced toxicity. miR-193a is a known inducer of oxidative stress and stimulates cell death in podocytes (33). We have recently reported the role of miR-193a in adherens junction destabilization, disorganization of the actin cytoskeleton, and autophagy blockade in podocytes (52, 53). All these mechanisms carry the potential to dedifferentiate the molecular phenotype and can lead to eventual cell death in podocytes. Since APOL1 NR downregulates miR-193a, it would preserve the podocyte molecular phenotype and protect against cell death (Fig. 1).

Fig. 2.

Relationship between parietal epithelial cells (PECs) and podocytes (PDs). A: PEC-to-podocyte transition. Podocyturia as a consequence of detachment of PDs in the urinary space occurs under normal physiological conditions. PECs, which share their origin as well as reside adjacent to PDs, undergo transition. To sustain PD homeostasis, PECs or other progenitor cells are required to undergo an efficient transition. Patterns of PEC transition (efficient or inefficient) are shown in specified zones. Levels of concentration of miR-193a and apolipoprotein L1 (APOL1) are indicated by green and yellow colors, respectively. B: bifunctional APOL1-miR-193a axis (AMA). A magnified view of the specified zone is shown. PECs do not express APOL1 protein, but PDs overtly express APOL1 protein. Both APOL1 and miR-193a regulate each other inversely. The PEC transition is associated with the emergence of APOL1 and downregulation of miR-193a expression. Downregulation of miR-193a increases the expression of Wilms’ tumor 1 (WT1), which stimulates the transcription of podocyte markers in transiting PECs. C: disruption in the AMA. APOL1 renal risk variants (RRVs; G1/G2) are not capable of downregulating but escalate the expression of miR-193a, indicating a disruption in the AMA. On that account, the induction of APOL1 RRVs in PECs will keep a low expression of WT1, and that would prevent their transition. Similarly, the disrupted AMA in PDs would downregulate WT1 and would invoke their dedifferentiation (acquiring a PEC phenotype), resulting in their detachment and loss in urine. D: the AMA in adverse milieus. Adverse milieus have the potential to enhance as well as downregulate miR-193a expression. Patterns of the PEC transition are shown in specified zones. Adverse milieus such as human immunodeficiency virus (HIV) infection and interferon (IFN)-γ downregulate miR-193a expression in PECs and result in increased expression of WT1, stimulating their transition. On that account, downregulation of miR-193 in APOL1 nonrisk (NR; G0) PECs would induce APOL1 expression and upregulate WT1, stimulating their transition. Additionally, HIV also enhances the generation of IL-1β by podocytes, which induces proliferation in PECs. Therefore, proliferating G0 PECs in HIV milieu would be efficiently able to renew the lost PDs. In contrast, RRV (G1/G2) PECs would not be able to transit and partially transited proliferating PECs would accumulate in Bowman's space. E: magnified view of the specified zone. Proliferating G0 PECs are transiting to PDs. F: magnified view of the specified zone. Partially transited G1/G2 PECs are accumulating in Bowman's space. VDR, vitamin D receptor; PAX2, paired box gene 2; CD2AP, CD2-associated protein.

CONDUCTIVE PROPERTIES OF APOL1 ION CHANNELS

Initial reports have suggested that APOL1 acts as an anion channel in the vacuolar membrane resulting in the disruption of the vacuolar compartment (80, 83). These investigators used an internal segment of the APOL1 sequence to model it as a pore-forming region that generated modestly Cl⁻-selective channels in planar lipid bilayers. They also reconstituted full-length APOL1 into lipid membranes at neutral pH and demonstrated that Cl⁻ uptake by them was not inhibited by the Cl⁻ channel inhibitor 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid and that the uptake was higher at acidic pH. Other investigators have also shown an activity that generated pores in phospholipid vesicles with the use of full-length APOL1 (38). These pores allowed the passage of four negative charges at neutral pH. However, to allow a physical association and formation of the pores, the required conditions included low pH, the presence of negatively charged phospholipid, and low ionic strength. Another group of investigators used a lipid bilayer approach and showed insertion of APOL1 into the vacuolar membrane and its trafficking to the plasma membrane and activation of cation channel activity at neutral extracellular pH (96). Another report (80) also showed that expression of APOL1 increased plasma membrane nonselective cation channel activity in mammalian cells. Thus, it appears that insertion of APOL1 causes selective anion channel activity at lower pH but nonselective cation channel activity at neutral pH. However, in these models, comparative analysis of APOL1 NR and RRV proteins was not conducted. Nonetheless, cellular expression of APOL1 RRVs has been shown to enhance K⁺ efflux and activation of the MAPK pathway in HEK-293T cells (14, 80). Bioinformatics studies have suggested that APOL1 RRVs carried the potential to insert multiple APOL1 channels (K⁺ transporter) into the plasma membrane. Moreover, APOL1 RRVs alter the structures of channels that facilitate K⁺ transport (unpublished observations); interestingly, enhanced K⁺ efflux was associated with the formation of inflammasomes. However, glyburide (glibenclamide) prevented inflammasome formation through inhibition of K⁺ flux in podocytes expressing APOL1 RRVs (unpublished observations). Glyburide binds with the ATP-sensitive K⁺ channel inhibitory regulatory subunit and inhibits K⁺ efflux.

ACCELERATION OF AUTOPHAGY

Podocytes are terminally differentiated cells and require accelerated autophagy flux to maintain their integrity (24); on that account, even a partial blockade of autophagy results in a loss of their molecular phenotype (23). APOL1 NR has been reported to be an inducer of autophagy (105). Therefore, moderate expression of APOL1 may be maintaining adequate autophagy flux in podocytes under normal physiological conditions. In contrast, overexpression of APOL1 led to autophagic cell death in several cell types (40, 105). In a recent report (10), APOL1 RRVs invoked autophagy blockade in podocytes. Further studies revealed that APOL1 RRVs navigated autophagy blockade by multiple mechanisms (52). First, APOL1 RRVs stimulated the formation of rubicon-ultraviolet radiation resistance-associated gene protein (UVRAG) complexes, which inhibited the maturation of autophagosomes (52). Second, APOL1 RRVs downregulated the expression of mammalian target of rapamycin (52), resulting in the reduction of reformed lysosomes available to fuse with autophagosomes (18). Finally, high expression of miR-193a (in APOL1 RRV milieu) attenuated transcription of the regulatory unit of phosphatidylinositol 3-kinase catalytic subunit type 3 (PI3KC3), causing decreased assembly of the PI3KC3-autophagy-related protein 14L and PI3KC3-UVRAG complexes (52) needed for nucleation and maturation of autophagosomes (42). Interestingly, APOL1 RRV-induced autophagy blockade also promoted podocyte dedifferentiation by decreased WT1, nephrin, and CD2-associated protein (CD2AP) expression (52, 53). In brief, APOL1 NR facilitates autophagy through enhanced nucleation and maturation of autophagosomes, whereas APOL1 RRVs lack this property.

PRESERVATION OF THE PODOCYTE MOLECULAR PHENOTYPE IN ADVERSE MILIEUS

The original case-control studies and Dallas Heart Study showed an absence of association between the presence of APOL1 RRVs and CKD in patients with diabetes (29, 74). However, the Atherosclerosis Risk in Communities Study reported an association between two APOL1 RRVs and patients with diabetes with CKD (97). Parsa et al. (81) analyzed APOL1 loci in the Chronic Renal Insufficiency Cohort study and showed an association between the progression and diabetic kidney disease.

Podocyte dedifferentiation or reversal to an embryonic phenotype in the form of loss of podocyte markers and gain of paired box gene 2 [PAX2; a parietal epithelial cell (PEC) marker] is a common finding in FSGS (86), collapsing glomerulopathy (8), and diabetic glomerulosclerosis (65). Mishra et al. (72) highlighted the role of APOL1 NR in the induction of podocyte injury in the diabetic milieu. These investigators hypothesized that APOL1 NR preserves podocytes in the differentiated state in the nondiabetic milieu. High glucose inhibited expression of APOL1 NR and invoked dedifferentiation in podocyte (Fig. 3, A and B). This effect of high glucose in podocytes was mediated through enhanced generation of miR-193a (72); the latter inversely regulated transcription of WT1 in podocytes (33). Since WT1 forms a repressor complex on the PAX2 gene (a marker of dedifferentiation), its downregulation was associated with enhanced podocyte PAX2 expression in high glucose milieu.

Fig. 3.

Role of the apolipoprotein L1 (APOL1)-miR-193a axis in podocyte functioning. A: APOL1 NR (G0) podocyte. APOL1 G0 is a component of the adherens complex [CD2-associated protein (CD2AP)-nephrin-dendrin]. Additionally, APOL1 G0 enhances the expression of Wilms’ tumor 1 (WT1) by downregulating the expression of miR-193a. Increased expression of WT1 enhances the expression of podocyte proteins such as nephrin and podocalyxin and downregulates paired box gene 2 (PAX2; a marker of parietal epithelial cells). B: APOL1 nonrisk (NR; G0) podocyte in adverse milieus. High glucose (HG) and puromycin amino nucleoside (PAN) enhance the expression of miR-193a and downregulate the expression of APOL1 G0. Loss of any of the constituents destabilizes the adherens complex and results in the nuclear import of dendrin, escalating the transcription of cathepsin L (CTSL); the latter degrades dynamin and synaptopodin, which causes disorganization of the actin cytoskeleton in podocytes. Similarly, enhanced levels of miR-193a degrade WT1, decreasing the transcription of CD2AP, nephrin, and vitamin D receptor (VDR). All these changes contribute to dedifferentiation and loss of podocytes. C. APOL1 renal risk variant (RRV; G1/G2) podocytes. APOL RRV podocytes have elevated miR-193a levels de novo. On that account, these podocytes would have downregulation of WT1, destabilized adherens complex, disorganization of the actin cytoskeleton, and other features of RRVs. D: effect of VDR agonist (VDA) on APOL RRV podocytes and APOL1 NR podocytes in adverse milieus. VDR agonists carry the potential to inhibit expression of miR-193a. On that account, they would optimize the podocyte phenotype such as enhancement of WT1 expression both in RRV (G1/G2) podocytes and NR (G0) podocytes treated with HG/PAN. The increased levels of WT1 would transcribe podocyte proteins like CD2AP and nephrin, which would stabilize the adherens complex and prevent the nuclear import of dendrin.

Additionally, WT1 acts as a transcription factor for nephrin and podocalyxin; therefore, downregulation of WT1 also attenuated transcription of these podocyte proteins. Since APOL1 NR preserves the molecular phenotype of podocytes, decreased expression of APOL1 in diabetic milieu would accelerate dedifferentiation and loss of podocytes (Fig. 3). On that account, the majority of patients with diabetes are vulnerable to develop podocytopathy. In contrast, only 4% of patients carrying APOL1 develop FSGS (29, 46); thus, it appears that high glucose milieu is more damaging compared with APOL1 RRV milieu. Thus, the following question arises: when these two milieus are together, why does the occurrence of CKD not increase? However, this presumption is controversial, currently (81, 97). Nonetheless, if this is true, there could be two possibilities: 1) high glucose is more toxic and would be able to mask the kidney cell injury inflicted by a minor hit like APLOL1 RRVs and 2) since high glucose downregulates APOL1 NR, it may also be downregulating the expression of APOL1 RRVs in podocytes; therefore, the APOL1 RRV-induced injury to podocytes will be diminished relatively in high glucose milieu. However, the latter notion remains conjecture as there are no reported data that high glucose milieu downregulates expression of APOL1 RRVs in podocytes.

STABILIZATION OF THE ADHERENS COMPLEX AND MAINTENANCE OF THE PODOCYTE ACTIN CYTOSKELETON

Dynamic kinetics of repressor and activator protein complexes on podocyte genes such as WT1, nephrin, and podocin maintain their expression levels to sustain the podocyte molecular phenotype. Additionally, the rate of degradation of these proteins determines their expression levels. Podocytes provide support to the glomerular basement membrane through their complex organization of the actin cytoskeleton and prevent the collapse of the capillary lumen; moreover, loss of the actin cytoskeleton results in cell death (92). In a recent study (53), APOL1 NR preserved the actin cytoskeleton, whereas expression of APOL1 RRVs was associated with disorganization of the actin cytoskeleton (Fig. 3, A and C). Slit diaphragm proteins such as nephrin and CD2AP form a complex with dendrin known as the adherens complex (5, 53). Destabilization of the adherens complex results in the nuclear import of dendrin, which escalates the transcription of cathepsin L (CTSL); the latter degrades dynamin and synaptopodin, contributing to disorganization of the actin cytoskeleton (53, 104). In these studies, APOL1 was observed to be part of the podocyte adherens complexes. Podocytes expressing APOL1 NR exhibited stabilization of the adherens complexes, whereas podocytes expressing APOL1 RRVs showed destabilization of the adherens complex and nuclear import of dendrin both in vitro and in vivo (53). The authors attributed the disorganization of the actin cytoskeleton in podocytes expressing APOL1 RRVs as a consequence of the enhanced generation of CTSL in response to the nuclear import of dendrin.

In an initial in vivo study (13), podocyte-specific constitutive expression of APOL1 RRVs did not show any abnormality in adult mice. However, G2 transgenic mice demonstrated a reduction of podocyte density by 200 days compared with G0 transgenic mice (13). These findings suggest that transgenic expression of APOL1 G2 in podocytes may promote podocyte loss, but it remained clinically occult. Since G0 transgenic mice did not show any loss of podocytes, one could presume that APOL1 G0 was either not harmful or preserved podocyte integrity. In other studies, overt expression of APOL1 RRVs, both podocyte specific or globally, was associated with podocyte injury and the development of FSGS in transgenic mice (10, 53, 79).

ROLE OF APOL1 IN THE PEC TRANSITION

PECs have been reported to serve as progenitor cells for podocyte generation in juvenile mice (4, 90). However, the role of PECs in podocyte renewal in adult mice is controversial (61). It has been suggested that mice have an only limited pool of PECs that are predestined to serve as progenitor cells for podocytes; however, this limited pool is exhausted by the growing glomeruli in the postnatal period. In a recent report (55), it was proposed that to facilitate PECs to serve as progenitor cells for podocyte renewal, they require the expression of molecules such as APOL1. Human PECs do not express APOL1 because of their high constitutive expression of miR-193a (45, 67, 69). Nonetheless, downregulation of miR-193a in PECs not only induces APOL1 expression but also stimulates them to acquire a podocyte molecular phenotype (55). Interferon-γ, HIV, and vitamin D receptor agonists downregulate miR-193a expression in PECs (55); these agents not only induced APOL1 expression in PECs but also stimulated their transition (Fig. 2). However, PECs expressing AOL1 RRVs displayed attenuation of this property (unpublished observations). On that account, PECs expressing APOL1 RRVs are not able to renew injured podocytes in adverse milieus, resulting in an increased occurrence of FSGS.

ROLE OF APOL1 IN STEM CELL DIFFERENTIATION

There is not enough data on the expression of APOL1 protein, but APOL1 mRNA has been found to be low in stem cells or progenitor cells compared with their transited or differentiated stages. Musah et al. (75) studied the directed differentiation of human induced pluripotent stem cells into matured kidney podocytes and reported the expression of APOL1 along with WT1 as an acquired podocyte differentiation maker. van de Leemput et al. (98), in their study of cerebral cortical development of embryonic stem cells, observed that cellular differentiation was associated with an increase in APOL1 mRNA; on day 77, expression of APOL1 mRNA was fourfold higher compared with embryonic stem cells on day 1. Similarly, H9 embryonic cells showed increased expression of APOL1 mRNA during their differentiation to human intestinal organoids (23, 25). Both interferon-γ and PMA stimulated expression of APOL1 mRNA in THP-1 polarization experiments (93). As expected, reversal of the differentiated cells such as fibroblasts to a fibroblast-induced pluripotent stem cell phenotype was associated with decreased APOL1 mRNA expression (68). Since induced pluripotent stem cells were not the replica of stem cells, expression of APOL1 was also higher in fibroblast-induced pluripotent stem cells compared with embryonic stem cells. A developmental study (101) in zebrafish also demonstrated a 10-fold increase in the expression of APOL1 mRNA on the 5th larval day compared with the zygote. RNA analysis from human purified intestinal epithelial samples and organoids derived from fetal and pediatric small and large intestines showed increased expression of APOL1 mRNA in the postnatal compared with fetal stage (data set 2015; http://www.ebi.ac.uk/gxa/experiments/E-MTAB-3704). Unfortunately, APOL1 protein levels were not measured in the majority of studies, but we expect them to correlate with mRNA levels. Nonetheless, APOL1 seems to play a role in the differentiation of progenitor cells in other organs.

CROSS-TALK OF APOL1 WITH LIPIDS

The development of both FSGS and atherosclerosis are considered to be analogous in regard to their pathophysiological mechanisms (19). Macrophages laden with lipids (foam cells) characterize atherosclerosis and are also noted in glomerular sclerotic lesions in patients with FSGS. However, the involved mechanism of the generation of foam cells in FSGS is far from clear. In a recent report, Ryu et al. (85) showed that expression of APOL1 RRVs diminishes cholesterol efflux in macrophages, resulting in the alteration of their phenotype. These investigators harvested monocytes from the kidney, spleen, and bone marrow from wild-type, APOL1 G0, APOL1 G1, and APOL1 G2 transgenic mice. Differentiated and polarized macrophages (M1 and M2 molecular phenotypes) from APOL1 G1 and APOL1 G2 mice displayed enhanced cholesterol accumulation but decreased expression of cholesterol transporters (ABCA1 and ABCG1 mRNAs). Bone marrow-derived macrophages from APOL1 G1 and APOL1 G2 mice exhibited significantly reduced cholesterol efflux compared with wild-type or APOL1 G0 macrophages. These findings indicated that expression of APOL1 RRVs in macrophages impaired reverse cholesterol transport through decreased expression of cholesterol transporters, resulting in the development of the foam cell phenotype. In the past, the presence of foam cells in glomerular sclerotic lesions was considered to be a secondary event in response to ongoing hyperlipidemia (as a consequence of proteinuria) in patients with FSGS; however, the findings of Ryu et al. (85) suggested that in patients carrying APOL1 RRVs, the presence of foam cells in kidney tissues also contributed to decreased expression of cholesterol transporters.

Recent reports have suggested that translocation of APOL1 RRVs to lipid droplets prevents toxic activities of APOL1 RRVs in the endoplasmic reticulum. Normally, a significant pool of APOL1 RRVs localizes to the endoplasmic reticulum, whereas a large fraction of APOL1 NR localizes to intracellular lipid droplets (17). In experimental conditions, APOL1 RRVs were observed to be minimally colocalized with the lipid droplet marker perilipin 2 but found to be in high proximity with late endosomes and autophagosomes in an experimental APOL1 cellular localization study (10, 35). APOL1 NR and RRVs interacted with several organelles, including mitochondria, endosomes, and the endoplasmic reticulum, but they differed in their organelle-specific translocation and retention (66). APOL1 NR was associated predominantly with lipid droplets, whereas APOL1 RRVs were translocated to the endoplasmic reticulum and contributed to cellular toxicity. Treatment of HEK T-REx-293 cells with oleic acid translocated APOL1 RRVs from the endoplasmic reticulum to lipid droplets, resulting in downregulation of autophagic flux and associated cellular injury. Similarly, coexpression of APOL1 NR with APOL1 RRVs enhanced import of APOL1 RRVs to lipid droplets from the endoplasmic reticulum, resulting in the downregulation of autophagic flux and cellular damage. This diverted translocation of APOL1 RRVs by APOL1 NR or oleic acid could rescue cells from impaired mitochondrial functions like altered membrane potential, respiration capacity, and the NAD biosynthesis pathway, which are highly downregulated in the presence of APOL1 RRVs in mitochondria (17). The property of APOL1 NR to import RRVs to lipid droplets explains the recessive pattern of kidney disease inheritance. Nonetheless, these findings also suggest the development of podocyte toxicity as a consequence of loss of function by APOL1 RRVs.

APOL1 TRANSGENIC ANIMAL MODELS

Nonhuman models are used extensively and have proven to be a useful tool to establish a causal relationship between adverse milieu and the development of the disease. The high allelic frequency and association with disease trait at a high odds ratio necessitate the development of animal models for APOL1 NR and APOL1 RRVs. However, the construction of an appropriate animal model for APOL1 has been challenging because of its presence only in humans and some primates. Therefore, several investigators attempted to develop functional models to study APOL1-mediated diseases (2, 10, 13). Knockin of the human APOL1 gene in an animal that lacks it in its genome and evolved without this gene may not lead to the development of the expected phenotype. In these models, APOL1 can face animal-specific cellular resistance and an immunological response. Nonetheless, insertion of a protein that doesn't exist in a genome could also provide the relevant answers about its functional properties. At this time, 10 mouse, 3 zebrafish, and 2 Drosophila models have been created to study APOL1 function and its association with disease (2). Details of these animal models are shown in Table 3.

Table 3.

APOL1 NR and APOL1 RRV animal models

Investigators	Year	APOL1 Variant Type	Severe Phenotype	Organelle Damaged	Transgenic Animal
Mouse models
Molina-Portela et al. (73)	2008	G0	Not tested	Not tested	Mouse
Lecordier et al. (62)	2009	G0	Not tested	Not tested	Mouse
Thomson et al. (96)	2014	G0	G1 > G2 > G0	Liver damage	Mouse
Cheng et al. (16)	2015	G0	G1 > G2 > G0	Not tested	Mouse
Bruggeman et al. (13)	2016	G0 and G2	G2 > G0	Kidney disease(>200 days old); preeclampsia	Mouse
Hayek et al. (39)	2016	G0, G1, and G2	G1/G2 > G0	Kidney damage	Mouse
Beckerman et al. (10)	2017	G0, G1, and G2	G1/G2 > G0	Podocyte loss	Mouse
Kumar et al. (55)	2018	G0-human immunodeficiency virus		Parietal epithelial cell transition	Mouse
Kumar et al. (53)	2019	G0, G1, and G2	G1 > G0	Destabilization of the adherens complex	Mouse
Ryu et al. (85)	2019	G0, G1, and G2	G1 and G2 > G0	Cholesterol accumulation	Mouse
Zebrafish models
Anderson et al. (2)	2015	G0, G1, and G2	G0 mRNA protective	Nephrocytes	Zebrafish
Olabisi et al. (80)	2016	G0, G1, and G2	G1/G2 > G0	No kidney damage	Zebrafish
Kotb et al. (49)	2016	G0	G0 knockout	Podocyte loss	Zebrafish
Drosophila melanogaster models
Fu et al. (32)	2017	G0, G1, and G2	G1 and G2 > G0	Nephrocyte damage	Drosophila melanogaster
Kruzel-Davila et al. (51)	2017	G0, G1, and G2	G1 and G2 > G0	Nephrocyte damage	Drosophila melanogaster Saccharomyces cerevisiae

APOL1, apolipoprotein L1; NR, nonrisk; RRV, renal risk variant.

Mouse Models

Thomson et al. (95) were the first to report APOL1-associated organelle damage in vivo in Swiss-Webster mice, where G1 mice developed severe liver injury compared with G2 mice. In contrast, B6D2F1/J mice displaying liver-specific increased expression of APOL1 NR under the apolipoprotein E promoter did not show any organelle-specific damage. Before these reports, Molina-Portela et al. (73) and Lecordier et al. (62) did not observe any organelle damage in mice transfected with APOL1 NR transgene. These studies showed that organelle damage was associated with APOL1 RRV expression only; interestingly, none of the studies revealed any endocrine effects of circulating APOL1 in the kidneys. Thus, it appears that kidney injury was predominantly mediated through local cellular expression of APOL1 RRVs. To validate the effects of cellular expression of APOL1, transgenic mouse models having podocyte-specific APOL1 expression were generated; the two APOL1 transgenic models, expressing nephrin-controlled APOL1 NR and RRVs, were created by two different groups. The first group of transgenic mouse models constitutively expressed either G0 or G2. G2 mice showed an altered podocyte phenotype (podocytopenia) after 200 days; nonetheless, both G0 and G2 mice had eclampsia and preeclampsia (13). In the second group of transgenic models, expression of G0, G1, and G2 was doxycycline inducible (10); these mice developed progressive proteinuria and focal and global glomerulosclerosis that were correlated with APOL1 expression. To study the nonspecific toxicity of APOL1 RRVs to podocytes, the same group also created Pax8-driven tubular cell-specific APOL1 transgenic mice (10). Taken together, these studies indicated that APOL1 toxicity restricted predominantly to podocytes expressing APOL1RRVs. However, G1 was more toxic compared with G2. Similar observations were reported by other groups in their APOL1 transgenic mouse models (39, 53, 55, 85).

Injection of APOL1 RRV DNA in mice induced proteinuria, focal lesions, and foot effacement in podocytes (39). In another study (53), APOL1 G1 transgenic mice displayed nuclear import of dendrin in podocytes and focal segmental glomerular lesions. Expression of APOL1 RRVs has also been demonstrated to enhance accumulation of cholesterol in macrophages through attenuation of the transcription of cholesterol transporters in APOL1 RRV transgenic mice (85). Kumar et al. (54) also created APOL1 NR-expressing Tg26 (NL4-3 transgene) mice and demonstrated increased transdifferentiation of PECs to podocytes, which provided mechanistic insights into the accumulation of PECs in Bowman's space in HIVAN.

Zebrafish

Zebrafish have endogenous expression for APOL1 protein and have been exploited for disease association (2, 49, 80). In two different studies, knockdown of APOL1 was associated with podocyte loss and a diminished glomerular filtration barrier, which could be restored with injection of APOL1 NR (2) and nephrin mRNAs (49); conversely, the administration of APOL1 RRVs failed to do so. Interestingly, podocyte-specific expression of APOL1 RRVs in zebrafish needed a second hit to induce renal abnormalities (80).

Drosophila Melanogaster

This model was created to study the mechanism of APOL1 toxicity; G1 induced greater phenotypic severity, reduced function, and accelerated cell death (32). These investigators concluded that APOL1 RRV-mediated toxicity was mediated by perturbations in endosomal trafficking and acidification. Kruzel et al. (51) showed that transgenic expression of APOL1 RRVs in Saccharomyces cerevisiae was more cytotoxic compared with APOL1 NR. Additionally, these investigators introduced systemic expression of APOL1 in various mutant yeast strains, identified defective organelle acidification, and demonstrated that a strain defective in endosomal trafficking but not in autophagy flux was more sensitive to APOL1-induced cytotoxicity (51).

APOL1 NEPHROPATHIES

APOL1 RRVs have been reported to be associated with a spectrum of diseases related to FSGS; these include solidified glomerulosclerosis with minimal to absent proteinuria, collapsing glomerulopathy, severe lupus nephritis, sickle cell nephropathy, and more rapid failure of renal allografts based on the kidney donors' genotype (28, 29, 47, 60). Thus, it appears that African American individuals who had hypertension with progressive kidney disease may have primary kidney disease, and its progression may not be related to hypertension. The odds ratios for developing major CKDs in carriers of two APOL1 RRVs are as follows: HIVAN (United States: 29 and South Africa: 89) (44, 46), focal segmental glomerulosclerosis (United States: 17) (46), arterionephrosclerosis (United States: 7) (59); lupus nephritis (United States: 2.57) (59), and deceased donor kidney transplant (United States: >2.0) (26). These high odds ratios were reported in only case-control studies. In contrast, longitudinal cohort studies revealed the effects of APOL1 on the progression of kidney disease, with odds ratios of <2.

Focal Segment Glomerulosclerosis

APOL1 FSGS is the most common form of genetic FSGS in countries with substantial African descent populations; furthermore, APOL1 FSGS carries a worse prognosis in terms of the rate of progression to ESKD (47). It contributes to significant proteinuria in populations of sub-Saharan African descent. In the United States, ~40% of ESRD attributed to FSGS occurs in black individuals, and of this, 72% is associated with APOL1 RRVs (29). In an analysis of 94 patients with FSGS with nephrotic syndrome, 27 patients carried APOL1 risk alleles (47); these patients were older, with a lower baseline estimated glomerular filtration rate (eGFR).

Collapsing glomerulopathy is considered to be a part of FSGS classification, but it carries a significant morphological differentiation. The majority of African American individuals with HIV infections and two APOL1 RRVs develop collapsing glomerulopathy (as a manifestation of HIVAN) if not treated with antiviral therapy (46). African American individuals manifesting collapsing glomerulopathy in response to interferon therapy were also reported to carry two APOL1 RRVs (70). Finally, the development of collapsing lesions in African American individuals suffering from lupus nephritis was also reported to carry two APOL1 RRVs (60). Therefore, the association of collapsing glomerulopathy with two APOL1 RRVs stands out as remarkable.

In initial studies, the abundance of proliferating cells in Bowman’s space was considered to be podocytes in response to ongoing massive podocyte injury in patients with HIVAN (9). However, subsequent studies indicated that these cells were of PEC lineage (20). Since PECs are considered to be progenitor cells for the lost podocytes, their proliferation seems to be in response to accelerated podocyte loss indicated by collapsed capillaries (a consequence of loss of capillary support). Since PECs carrying APOL1 RRVs are not able to transit to podocytes (unpublished observations), they accumulate in Bowman’s space. On that account, collapsing glomerulopathy is a manifestation of failed PEC transition and could be considered a characteristic phenotype of APOL1 RRV-associated loss of function.

HIV-Associated Nephropathy

In the 1970s, heroin nephropathy was considered a disease of adult men of African ancestry; however, with the emergence of HIV in 1980s, heroin nephropathy completely disappeared in this population, and HIVAN manifested with high odds ratios (31, 46). Although there are no available data on APOL1 polymorphism in patients with heroin nephropathy, it appears that there could be a genetic association between these two entities.

Among all CKDs, HIVAN is the most strongly associated with APOL1 RRVs, with odds ratios of 29 in African American individuals and 89 in South African individuals (44, 46); it suggests an interaction between APOL1 and HIV-1. Direct HIV infection of PECs and podocytes has been reported (12). HIV induces oxidative stress, apoptosis, and pyroptosis in podocytes (37, 41). Cytokines that are in abundance in HIV milieu have been reported to enhance APOL1 transcription in kidney cells (71); moreover, renal biopsy specimens in patients with HIVAN confirmed the occurrence of increased APOL1 transcripts in kidney cells (10). In an in vitro study (71), APOL1 NR restricted HIV survival in human podocytes compared with podocytes expressing APOL1 RVVs. Similarly, restriction of HIV replication was demonstrated in macrophages and differentiated monocytes expressing APOL1 NR (94). APOL1 NR facilitated degradation of HIV-1 Gag and depletion of HIV-1 Vif, resulting in APOBEC3G-mediated HIV replication restriction in lysosomes (94). However, it was not clear whether APOL1 RRV milieu would facilitate HIV replication. In a recent report (1), the role of APOL1 RRVs on viral load was evaluated using the conditional logistic regression test in African American individuals enrolled in the acquired immunodeficiency syndrome (AIDS)-linked IntraVenous Experience HIV natural history cohort. These investigators did not observe any evidence that APOL1 RRVs were associated with host susceptibility to HIV-1 acquisition, setpoint HIV-1 viral load, or time to incident AIDS. These findings suggest that APOL1 RRVs do not influence HIV infection or progression in individuals with African ancestry. However, because of the alteration of the macrophage differentiation profile in APOL1 RRV milieu, an accelerated progression of tubulointerstitial disease remains a possibility in patients with HIVAN (63).

Historically, the majority of patients with HIV infection carrying two APOL1 RRVs developed HIVAN if not treated with antiviral agents; however, the incidence of HIVAN has significantly decreased in the postantiviral era (103). Although the renal risk is largely recessive, requiring two APOL1 RRVs, in certain situations (e.g., HIV-positive South African individuals), a single copy of an RRV (G1) has a significant association with HIVAN (7, 59). Interestingly, further analysis revealed that a significant number of cases of HIVAN were associated with APOL1 NR in individuals with African ancestry (7). In contrast, the rare occurrence of HIVAN in Caucasian individuals suggests that there may be other renal risk gene(s) besides APOL1 RRVs in individuals with African ancestry that make them susceptible to develop HIVAN.

Hypertension Attributed to Kidney Disease

In the African American Study of Kidney Disease and Hypertension (AASK) trial, patients randomized to the aggressive blood pressure control arm did not show any benefit in slowing the rate of progression of kidney failure compared with the conventional control of blood pressure arm (102). Six hundred seventy-five AASK patients with hypertension had APOL1 genotyping and CKD. The investigators concluded that the progression of kidney disease in AASK participants with nondiabetic CKD was strongly associated with APOL1 RRVs, and systemic blood pressure had no effect. Similarly, in the Systolic Blood Pressure Intervention Trial and posthoc analysis, all African American individuals with hypertension who had cardiovascular disease events were found to be nondiabetic (36). In African American individuals with APOL1 RRVs, baseline systolic and diastolic blood pressures were similar to those of African American individuals who had zero or one APOL1 RRV. However, African American individuals with two APOL1 RRVs had a lower baseline eGFR and significantly more proteinuria. No significant relationship was observed between APOL1 RRVs and the composite primary cardiovascular disease end point.

The APOL1 genotype, hypertension, and family history of ESKD were analyzed in 93 pediatric and young adult African American individuals with severe hypertension or FSGS (3). APOL1 RRVs showed an association with CKD and family history of ESKD. Sixty-six percent of patients with a family history of ESKD and 83% of children with previously diagnosed with “hypertension-attributed CKD” had two APOL1 RRVs. On the other hand, African American children with hypertension with a normal eGFR, no proteinuria, and no family history of ESKD did not carry two APOL1 RRVs.

The cohort of African American living kidney donors also showed similar predonation and postdonation blood pressures irrespective of APOL1 genotype (21). Donors with APOL1 RRVs showed lower predonation and postdonation eGFRs. Both cohort studies supported the hypothesis that hypertension was primarily a manifestation of underlying CKD and did not initiate nephropathy.

On the contrary, analysis of hypertension and APOL1 RRV associations in Mount Sinai BioMe Biobank, Vanderbilt BioVU, and Northwestern Nugen Biobank participants revealed that 14–16% of African American individuals had an additive effect on systolic blood pressure and age at diagnosis of hypertension (76). African American individuals carrying two APOL1 RRVs were diagnosed with hypertension 2−5 yr earlier than those with zero or one RRV and showed a decline in GFR 10 yr after the diagnosis of hypertension, indicating that hypertension predated and contributed to the decline in eGFR. These investigators proposed that the earliest effect of APOL1 RRVs might be on hypertension and not on CKD. However, one may argue that available biomarkers to diagnose CKD are not as sensitive as the detection of blood pressure. For example, if we have to diagnose hypertension by analyzing data on retinopathy or left ventricular hypertrophy, we may conclude that a decrease in GFR predates hypertension.

Systemic Lupus Erythematosus

Larsen et al. (60) reported an association between two APOL1 RRVs and collapsing glomerulopathy in patients with systemic lupus erythematosus. Similarly, Freedman et al. (26) reported an accelerated progression of renal failure with a shortened time to start dialysis in patients with lupus nephritis with two APOL1 RRVs. Expression of APOL1 RRVs is also frequent in patients with lupus nephritis-associated ESKD with phenotypic presentation as membranous and focal and diffuse proliferative glomerulonephritis (28).

Kidney Donor Graft Loss

The presence of two APOL1 RRVs in African American deceased kidney donors has been shown to contribute to shorter allograft survival after transplantation irrespective of the APOL1 genotype of recipients (27, 30, 84). In a single-center study (84), renal histology in the majority of failed allografts with APOL1 RRV kidneys displayed APOL1 nephropathies that were uncommon in failed allografts with APOL1 NR donors. Multivariate analyses of studies from 113 transplant recipients showed that donor APOL1 RRV genotypes correlated with shorter renal allograft survival; additionally, the age of the donor and recipient played a role in graft survival. At 6 yr posttransplantation, the recipients of two RRV kidneys displayed a twofold increase in allograft failure (84).

Rates of postdonation ESKD have been reported to be higher in African American living kidney donors than in European American living kidney donors (107). The National Institute of Diabetes and Digestive and Kidney Diseases-sponsored APOL1 Long Term Kidney Transplantation Outcomes (APOLLO) study has been being conducted for the last couple of years. The possible outcomes of this project have also been analyzed. In one study (43), donor race (African American) has been replaced with APOL1 genotype in the kidney donor risk index to refine the estimate of allograft survival. In another study (64), the likelihood of CKD in a cohort of young adults aged 18–30 yr old was projected, meeting criteria to be kidney donors. The outcome of the APOLLO study will provide guidelines to establish criteria for the selection of African American individuals carrying one or two RRVs as kidney donors.

APOL1 and Sickle Cell Kidney Disease

Epidemiological studies have identified associations between APOL1 gene polymorphisms and the risk of sickle cell kidney disease in the African American population (6, 77, 87–89). In one study (6), two APOL1 RRVs were associated with proteinuria (as measured by urine dipstick) and a lower eGFR; in two other studies (86, 88), patients with two APOL1 RRVs displayed albuminuria, hemoglobinuria, and a lower eGFR. In children with sickle cell kidney disease, the G1 variant was found to be associated with albuminuria (48, 89). Korman et al. (48) analyzed 152 patients with sickle cell kidney disease mainly of sub‐Saharan African ancestry. Homozygous or double‐heterozygous APOL G1 and G2 genotypes were strongly associated with ESKD and worse kidney disease.

CONCLUSIONS

The evolution of APOL1 NR in certain primates has been presumed to occur to facilitate podocyte longevity in an increasingly challenging environment. The discovery of a relationship between two RRVs and the development and progression of CKD provided some answers to the disparity in patients with African ancestry. However, the story is not complete, and other gene(s) that are also contributing to this disparity will be identified shortly. Meanwhile, the understanding of APOL1 function is evolving, and, being part of the AMA, APOL1 plays a vital role in the maintenance of the podocyte molecular phenotype and podocyte renewal in adverse milieus. Since the expression of APOL1 RRVs compromises downregulation of miR-193a in podocytes, it is a loss of one of APOL1’s properties; on the other hand, cellular toxicity inflicted by escalated miR-193a levels in APOL1 RRV milieu could be considered as a gain of function. Similar arguments could be put forward for several other reported APOL1 RRV-associated cellular manifestations, including K⁺ depletion, mitochondrial dysfunction, autophagy blockade, protein kinase receptor activation, ubiquitin D degradation, and a reduction in cholesterol efflux. The major manifestations of APOL1 RRV toxicity have been reported in terms of cell death in multiple morphological modes, including necrosis, apoptosis, and pyroptosis. At present, these effects have been reported in APOL1 RRV-overexpressing cells only and in predominantly in vitro studies. Therefore, these findings need to be validated in both experimental animal and human kidney disease models. Nonetheless, it is beyond doubt that advancement in the understanding of mechanistic aspects of APOL1 NR and APOL1 RRV functioning would help to develop therapeutic strategies to prevent, slow down the progression, and provide resolution of CKDs.

GRANTS

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-118017 (to P. C. Singhal).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

P.C.S. conceived and designed research; V.K. analyzed data; V.K. prepared figures; V.K. and P.C.S. drafted manuscript; V.K. and P.C.S. edited and revised manuscript; V.K. and P.C.S. approved final version of manuscript.