APOL1-Mediated Kidney Disease.

This JAMA Insights reviews the origin of APOL1 high-risk genetic variants, defines APOL1-mediated kidney disease, and discusses recommendations for screening and management.

APOL1-mediated monovalent cation transport contributes to APOL1-mediated podocytopathy in kidney disease.

Two coding variants of apolipoprotein L1 (APOL1), called G1 and G2, explain much of the excess risk of kidney disease in African Americans. While various cytotoxic phenotypes have been reported in experimental models, the proximal mechanism by which G1 and G2 cause kidney disease is poorly understood. Here, we leveraged 3 experimental models and a recently reported small molecule blocker of APOL1 protein, VX-147, to identify the upstream mechanism of G1-induced cytotoxicity. In HEK293 cells, we demonstrated that G1-mediated Na+ import/K+ efflux triggered activation of GPCR/IP3-mediated calcium release from the ER, impaired mitochondrial ATP production, and impaired translation, which were all reversed by VX-147. In human urine-derived podocyte-like epithelial cells (HUPECs), we demonstrated that G1 caused cytotoxicity that was again reversible by VX-147. Finally, in podocytes isolated from APOL1 G1 transgenic mice, we showed that IFN-γ-mediated induction of G1 caused K+ efflux, activation of GPCR/IP3 signaling, and inhibition of translation, podocyte injury, and proteinuria, all reversed by VX-147. Together, these results establish APOL1-mediated Na+/K+ transport as the proximal driver of APOL1-mediated kidney disease.

Use of Race in Kidney Function Estimation: Lessons Learned and the Path Toward Health Justice.

In 2020, the nephrology community formally interrogated long-standing race-based clinical algorithms used in the field, including the kidney function estimation equations. A comprehensive understanding of the history of kidney function estimation and racial essentialism is necessary to understand underpinnings of the incorporation of a Black race coefficient into prior equations. We provide a review of this history, as well as the considerations used to develop race-free equations that are a guidepost for a more equity-oriented, scientifically rigorous future for kidney function estimation and other clinical algorithms and processes in which race may be embedded as a variable.

Etiology of Persistent Microalbuminuria in Nigeria (P_MICRO study): protocol and study design.

BACKGROUND: Microalbuminuria is an independent risk factor for cardiovascular and kidney disease and a predictor of end organ damage, both in the general population and in persons with HIV (PWH). Microalbuminuria is also an important risk factor for mortality in PWH treated with antiretroviral therapy (ART). In the ongoing Renal Risk Reduction (R3) study in Nigeria, we identified a high prevalence of microalbuminuria confirmed by two measurements 4-8 weeks apart in ART-experienced, virologically suppressed PWH. Although Stage 1 or 2 hypertension and exposure to potentially nephrotoxic antiretroviral medications were common in R3 participants, other traditional risk factors for albuminuria and kidney disease, including diabetes, APOL1 high-risk genotype, and smoking were rare. Co-infection with endemic pathogens may also be significant contributors to albuminuria, but co-infections were not evaluated in the R3 study population. METHODS: In Aim 1, we will cross-sectionally compare the prevalence of albuminuria and established kidney disease risk factors in a cohort of PWH to age- and sex-matched HIV-negative adults presenting for routine care at the Aminu Kano Teaching Hospital in Kano, Nigeria. We will leverage stored specimens from 2500 R3 participants and enroll an additional 500 PLWH recently initiated on ART (≤ 24 months) and 750 age- and sex-matched HIV-negative adults to determine the contribution of HIV, hypertension, and other comorbid medical conditions to prevalent albuminuria. In Aim 2, we will follow a cohort of 1000 HIV-positive, ART-treated and 500 HIV-negative normoalbuminuric adults for 30 months to evaluate the incidence and predictors of albuminuria. DISCUSSION: The findings from this study will support the development of interventions to prevent or address microalbuminuria in PWH to reduce kidney and cardiovascular morbidity and mortality. Such interventions might include more intensive monitoring and treatment of traditional risk factors, the provision of renin-angiotensin aldosterone system or sodium-glucose cotransporter-2 inhibitors, consideration of changes in ART regimen, and screening and treatment for relevant co-infections.

JAK inhibitor blocks COVID-19 cytokine-induced JAK/STAT/APOL1 signaling in glomerular cells and podocytopathy in human kidney organoids.

COVID-19 infection causes collapse of glomerular capillaries and loss of podocytes, culminating in a severe kidney disease called COVID-19-associated nephropathy (COVAN). The underlying mechanism of COVAN is unknown. We hypothesized that cytokines induced by COVID-19 trigger expression of pathogenic APOL1 via JAK/STAT signaling, resulting in podocyte loss and COVAN phenotype. Here, based on 9 biopsy-proven COVAN cases, we demonstrated for the first time, to the best of our knowledge, that APOL1 protein was abundantly expressed in podocytes and glomerular endothelial cells (GECs) of COVAN kidneys but not in controls. Moreover, a majority of patients with COVAN carried 2 APOL1 risk alleles. We show that recombinant cytokines induced by SARS-CoV-2 acted synergistically to drive APOL1 expression through the JAK/STAT pathway in primary human podocytes, GECs, and kidney micro-organoids derived from a carrier of 2 APOL1 risk alleles, but expression was blocked by a JAK1/2 inhibitor, baricitinib. We demonstrate that cytokine-induced JAK/STAT/APOL1 signaling reduced the viability of kidney organoid podocytes but was rescued by baricitinib. Together, our results support the conclusion that COVID-19-induced cytokines are sufficient to drive COVAN-associated podocytopathy via JAK/STAT/APOL1 signaling and that JAK inhibitors could block this pathogenic process. These findings suggest JAK inhibitors may have therapeutic benefits for managing cytokine-induced, APOL1-mediated podocytopathy.

APOL1-associated kidney disease in northern Nigerians with treated HIV infection.

Apolipoprotein L1 (APOL1) high-risk genotypes strongly associate with HIV-associated nephropathy, and antiretroviral therapy reduces the incidence of HIV-associated nephropathy and progression to end-stage kidney disease. Wudil et al. report cross-sectional APOL1 associations with proteinuria and estimated glomerular filtration rate in a northern Nigerian sample with HIV infection on antiretroviral therapy. Multiple ethnic groups with different APOL1 risk variant frequencies were included. Overall, APOL1 was associated with proteinuric chronic kidney disease; however, relationships with underlying causes of nephropathy and progression rates require further study.

Diagnosis, Education, and Care of Patients with APOL1-Associated Nephropathy: A Delphi Consensus and Systematic Review.

BACKGROUND: APOL1 variants contribute to the markedly higher incidence of ESKD in Blacks compared with Whites. Genetic testing for these variants in patients with African ancestry who have nephropathy is uncommon, and no specific treatment or management protocol for APOL1-associated nephropathy currently exists. METHODS: A multidisciplinary, racially diverse group of 14 experts and patient advocates participated in a Delphi consensus process to establish practical guidance for clinicians caring for patients who may have APOL1-associated nephropathy. Consensus group members took part in three anonymous voting rounds to develop consensus statements relating to the following: (1) counseling, genotyping, and diagnosis; (2) disease awareness and education; and (3) a vision for management of APOL1-associated nephropathy in a future when treatment is available. A systematic literature search of the MEDLINE and Embase databases was conducted to identify relevant evidence published from January 1, 2009 to July 14, 2020. RESULTS: The consensus group agreed on 55 consensus statements covering such topics as demographic and clinical factors that suggest a patient has APOL1-associated nephropathy, as well as key considerations for counseling, testing, and diagnosis in current clinical practice. They achieved consensus on the need to increase awareness among key stakeholders of racial health disparities in kidney disease and of APOL1-associated nephropathy and on features of a successful education program to raise awareness among the patient community. The group also highlighted the unmet need for a specific treatment and agreed on best practice for management of these patients should a treatment become available. CONCLUSIONS: A multidisciplinary group of experts and patient advocates defined consensus-based guidance on the care of patients who may have APOL1-associated nephropathy.

Kidney Disease-Associated APOL1 Variants Have Dose-Dependent, Dominant Toxic Gain-of-Function.

BACKGROUND: Two coding renal risk variants (RRVs) of the APOL1 gene (G1 and G2) are associated with large increases in CKD rates among populations of recent African descent, but the underlying molecular mechanisms are unknown. Mammalian cell culture models are widely used to study cytotoxicity of RRVs, but results have been contradictory. It remains unclear whether cytotoxicity is RRV-dependent or driven solely by variant-independent overexpression. It is also unknown whether expression of the reference APOL1 allele, the wild-type G0, could prevent cytotoxicity of RRVs. METHODS: We generated tetracycline-inducible APOL1 expression in human embryonic kidney HEK293 cells and examined the effects of increased expression of APOL1 (G0, G1, G2, G0G0, G0G1, or G0G2) on known cytotoxicity phenotypes, including reduced viability, increased swelling, potassium loss, aberrant protein phosphorylation, and dysregulated energy metabolism. Furthermore, whole-genome transcriptome analysis examined deregulated canonical pathways. RESULTS: At moderate expression, RRVs but not G0 caused cytotoxicity in a dose-dependent manner that coexpression of G0 did not reduce. RRVs also have dominant effects on canonical pathways relevant for the cellular stress response. CONCLUSIONS: In HEK293 cells, RRVs exhibit a dominant toxic gain-of-function phenotype that worsens with increasing expression. These observations suggest that high steady-state levels of RRVs may underlie cellular injury in APOL1 nephropathy, and that interventions that reduce RRV expression in kidney compartments may mitigate APOL1 nephropathy.

The Role of Vitamin D and Oxidative Stress in Chronic Kidney Disease.

Chronic kidney disease (CKD) is a major non-communicable disease associated with high rates of premature morbidity and mortality. The prevalence of hypovitaminosis D (deficiency of 25(OH)D or 25D) is greater in racial/ethnic minorities and in patients with CKD than the general population. Low 25D is associated with bone and mineral disorders as well as immune, cardiometabolic and cardiovascular (CV) diseases. Thus, it has been suggested that low 25D contributes to the poor outcomes in patients with CKD. The prevalence of hypovitaminosis D rises progressively with advancing severity of kidney disease with over 30% of patients with CKD stage 3 and 70% patients with CKD stage 5 estimated to have low levels of 25D. This report describes several of the abnormal physiologic and counter-regulatory actions related to low 25D in CKD such as those in oxidative stress and inflammatory systems, and some of the preclinical and clinical evidence, or lack thereof, of normalizing serum 25D levels to improve outcomes in patients with CKD, and especially for the high risk subset of racial/ethnic minorities who suffer from higher rates of advanced CKD and hypovitaminosis D.

APOL1 Nephrotoxicity: What Does Ion Transport Have to Do With It?

Apolipoprotein L1 (APOL1) protein is the human serum factor that protect human beings against Trypanosoma brucei brucei, the cause of trypanosomiasis. Subspecies of T b brucei that cause human sleeping sickness-T b gambiense and T b rhodesiense evolved molecular mechanisms that enabled them to evade killing by APOL1. Sequence changes (termed G1 and G2) in the APOL1 gene that restored its ability to kill T b rhodesiense also increase the risk of developing glomerular diseases and accelerate progression to end-stage kidney disease. To lyse trypanosome parasites, APOL1 forms pores in the trypanosome endolysosomal and mitochondrial membranes, resulting in rapid membrane depolarization. However, the molecular mechanism underlying APOL1 nephropathy is unknown. Recent experimental evidence has shown that aberrant efflux of intracellular potassium is an early event in APOL1-induced death of human embryonic kidney cells. Here, we discuss the possibility that abnormal efflux of cellular potassium or other cations may be relevant to the pathogenesis of APOL1 nephropathy.

From man to fish: What can Zebrafish tell us about ApoL1 nephropathy?

BACKGROUND: Risk variant Apolipoprotein L1 (G1/G2) are strongly associated with a spectrum of kidney disease in people of recent African descent. The mechanism of ApoL1 nephropathy is unknown. Podocytes and/or endothelial cells are the presumed target kidney cells. Given the close homology in structure and function of zebrafish (ZF) pronephros and human nephron, we studied the effect of podocyte-specific or endothelium-specific expression of ApoL1 (G0, G1, or G2) on the structure and function of ZF pronephros. METHODS: Wild type (G0) or risk variant ApoL1 (G1/G2) were expressed in podocyte-specific or endothelium-specific under podocin/Flk promoters, respectively, using Gal4-UAS system. Structural pronephric changes were studied with light and electron microscopy (EM). Proteinuria was assayed by measuring renal excretion of GFP-vitamin D binding protein. Puromycin aminonucleoside (PAN) was used as inducer of podocyte injury. RESULTS: Endothelial-specific transgenic expression of G1/G2 is associated with endothelial injury indicated by endothelial cell swelling, segmental early double contours, and loss of endothelium fenestrae. Podocyte specific expression of G1 is associated with segmental podocyte foot process effacement and irregularities relative to G0. Despite the histological changes, the expression of G1/G2 alone in podocyte or endothelium compartment is not associated with edema, proteinuria, or gross whole fish phenotype. Moreover, PAN produced equal pericardial edema in all transgenic fish as well as nontransgenic controls. CONCLUSIONS: Transgenic expression human ApoL1 (G1/G2) is associated with histologic abnormalities in ZF glomeruli but is insufficient to cause quantifiable renal dysfunction. This finding supports the necessity of a "second hit" in the pathogenesis/progression of ApoL1-associated nephropathy.

APOL1 kidney disease risk variants cause cytotoxicity by depleting cellular potassium and inducing stress-activated protein kinases.

Two specific genetic variants of the apolipoprotein L1 (APOL1) gene are responsible for the high rate of kidney disease in people of recent African ancestry. Expression in cultured cells of these APOL1 risk variants, commonly referred to as G1 and G2, results in significant cytotoxicity. The underlying mechanism of this cytotoxicity is poorly understood. We hypothesized that this cytotoxicity is mediated by APOL1 risk variant-induced dysregulation of intracellular signaling relevant for cell survival. To test this hypothesis, we conditionally expressed WT human APOL1 (G0), the APOL1 G1 variant, or the APOL1 G2 variant in human embryonic kidney cells (T-REx-293) using a tetracycline-mediated (Tet-On) system. We found that expression of either G1 or G2 APOL1 variants increased apparent cell swelling and cell death compared with G0-expressing cells. These manifestations of cytotoxicity were preceded by G1 or G2 APOL1-induced net efflux of intracellular potassium as measured by X-ray fluorescence, resulting in the activation of stress-activated protein kinases (SAPKs), p38 MAPK, and JNK. Prevention of net K(+) efflux inhibited activation of these SAPKs by APOL1 G1 or G2. Furthermore, inhibition of SAPK signaling and inhibition of net K(+) efflux abrogated cytotoxicity associated with expression of APOL1 risk variants. These findings in cell culture raise the possibility that nephrotoxicity of APOL1 risk variants may be mediated by APOL1 risk variant-induced net loss of intracellular K(+) and subsequent induction of stress-activated protein kinase pathways.

Ablation of calcineurin Aß reveals hyperlipidemia and signaling cross-talks with phosphodiesterases.

Insulin resistance, hyperlipidemia, and cardiovascular complications are common dysregulations of metabolic syndrome. Transplant patients treated with immunosuppressant drugs such as cyclosporine A (CsA), an inhibitor of calcineurin phosphatase, frequently develop similar metabolic complications. Although calcineurin is known to mediate insulin sensitivity by regulating ß-cell growth and adipokine gene transcription, its role in lipid homeostasis is poorly understood. Here, we examined lipid homeostasis in mice lacking calcineurin Aß (CnAß(-/-)). We show that mice lacking calcineurin Aß are hyperlipidemic and develop age-dependent insulin resistance. Hyperlipidemia found in CnAß(-/-) mice is, in part, due to increased lipolysis in adipose tissues, a process mediated by ß-adrenergic G-protein-coupled receptor signaling pathways. CnAß(-/-) mice also exhibit additional pathophysiological phenotypes caused by the potentiated GPCR signaling pathways. A cell autonomous mechanism with sustained cAMP/PKA activation is found in CnAß(-/-) mice or upon CsA treatment to inhibit calcineurin. Increased PKA activation and cAMP accumulation in CnAß(-/-) mice, however, are sensitive to phosphodiesterase inhibitor. Indeed, we show that calcineurin regulates degradation of phosphodiesterase 3B, in addition to phosphodiesterase 4D. These results establish a role for calcineurin in lipid homeostasis. These data also indicate that potentiated cAMP signaling pathway may provide an alternative molecular pathogenesis for the metabolic complications elicited by CsA in transplant patients.

Assay for protein modification by poly-ADP-ribose in vitro.

The enzymatic function of poly(adenosine diphosphate (ADP)-ribose) polymerase (PARP) is central to many of its function as a component of DNA repair machinery, modulator of gene transcription, and cell differentiation. While the auto-modification domain of PARP has been shown to be a primary acceptor site of poly-ADP ribose (pADPr), other DNA binding nuclear proteins are also modified by pADPr. It is -generally accepted that pADPr polymer is built upon the carboxyl side chain of specific Glu, Asp, and/or Lys residues within the target protein. Identification of the unique amino acid acceptors of pADPr in these nuclear proteins is an active area of study. Because of the heterogeneity of pADPr chain on modified -protein targets, the resulting modified proteins have unpredictable final masses, making it difficult to -identify acceptor amino acids. Using recombinant proteins, in vitro pADP ribosylation assay and mass spectrometry, we have been able to identify conserved Glu residue in transcription factor NFAT that is enzymatically modified in vitro with pADPr by PARP-1. We discuss this protocol here as a model approach for identifying pADPr acceptors in other nuclear proteins.

Evolutionarily conserved role of calcineurin in phosphodegron-dependent degradation of phosphodiesterase 4D.

Calcineurin is a widely expressed and highly conserved Ser/Thr phosphatase. Calcineurin is inhibited by the immunosuppressant drug cyclosporine A (CsA) or tacrolimus (FK506). The critical role of CsA/FK506 as an immunosuppressant following transplantation surgery provides a strong incentive to understand the phosphatase calcineurin. Here we uncover a novel regulatory pathway for cyclic AMP (cAMP) signaling by the phosphatase calcineurin which is also evolutionarily conserved in Caenorhabditis elegans. We found that calcineurin binds directly to and inhibits the proteosomal degradation of cAMP-hydrolyzing phosphodiesterase 4D (PDE4D). We show that ubiquitin conjugation and proteosomal degradation of PDE4D are controlled by a cullin 1-containing E(3) ubiquitin ligase complex upon dual phosphorylation by casein kinase 1 (CK1) and glycogen synthase kinase 3beta (GSK3beta) in a phosphodegron motif. Our findings identify a novel signaling process governing G-protein-coupled cAMP signal transduction-opposing actions of the phosphatase calcineurin and the CK1/GSK3beta protein kinases on the phosphodegron-dependent degradation of PDE4D. This novel signaling system also provides unique functional insights into the complications elicited by CsA in transplant patients.

Regulation of transcription factor NFAT by ADP-ribosylation.

ADP-ribosylation is a reversible posttranslational modification mediated by poly-ADP-ribose polymerase (PARP). The results of recent studies demonstrate that ADP-ribosylation contributes to transcription regulation. Here, we report that transcription factor NFAT binds to and is ADP-ribosylated by PARP-1 in an activation-dependent manner. Mechanistically, ADP-ribosylation increases NFAT DNA binding. Functionally, NFAT-mediated interleukin-2 (IL-2) expression was reduced in T cells upon genetic ablation or pharmacological inhibition of PARP-1. Parp-1(-/-) T cells also exhibit reduced expression of other NFAT-dependent cytokines, such as IL-4. Together, these results demonstrate that ADP-ribosylation mediated by PARP-1 provides a molecular switch to positively regulate NFAT-dependent cytokine gene transcription. These results also imply that, similar to the effect of calcineurin inhibition, PARP-1 inhibition may be beneficial in modulating immune functions.