APOL1 Variants Research Articles

Establishing the value of genomics in medicine: the IGNITE Pragmatic Trials Network

PurposeA critical gap in the adoption of genomic medicine into medical practice is the need for the rigorous evaluation of the utility of genomic medicine interventions. MethodsThe Implementing Genomics in Practice Pragmatic Trials Network (IGNITE PTN) was formed in 2018 to measure the clinical utility and cost-effectiveness of genomic medicine interventions, to assess approaches for real-world application of genomic medicine in diverse clinical settings, and to produce generalizable knowledge on clinical trials using genomic interventions. Five clinical sites and a coordinating center evaluated trial proposals and developed working groups to enable their implementation. ResultsTwo pragmatic clinical trials (PCTs) have been initiated, one evaluating genetic risk APOL1 variants in African Americans in the management of their hypertension, and the other to evaluate the use of pharmacogenetic testing for medications to manage acute and chronic pain as well as depression. ConclusionIGNITE PTN is a network that carries out PCTs in genomic medicine; it is focused on diversity and inclusion of underrepresented minority trial participants; it uses electronic health records and clinical decision support to deliver the interventions. IGNITE PTN will develop the evidence to support (or oppose) the adoption of genomic medicine interventions by patients, providers, and payers.

APOL1 genotyping in kidney transplantation: to do or not to do, that is the question? (contra)

Discussions regarding genetic testing for variants in apolipoprotein L1 (APOL1), a gene located on chromosome 22 whose risk variants are strongly associated with end-stage kidney disease (ESKD), in the context of kidney transplantation must address donors (deceased and living) and recipients. In addition, these discussions must occur in the context of known genetic epidemiology regarding APOL1. Specifically, APOL1 risk variants are only found among individuals of African descent, as these variants are derived from exposure to African sleeping sickness that is largely confined to countries along the west coast of Africa. 1 Genovese G. Friedman D.J. Ross M.D. et al. Association of trypanolytic ApoL1 variants with kidney disease in African Americans. Science. 2010; 329: 841-845 Crossref PubMed Scopus (1388) Google Scholar In other words, not every individual who self identifies as Black or African American will even be at risk for carrying these genetic variants, and in fact, 87% of African Americans do not actually carry 2 APOL1 renal risk variants. 2 Foster M.C. Coresh J. Fornage M. et al. APOL1 variants associate with increased risk of CKD among African Americans. J Am Soc Nephrol. 2013; 24: 1484-1491 Crossref PubMed Scopus (180) Google Scholar More important, among the 13% of African Americans who do carry 2 APOL1 renal risk variants, the presence of these variants alone is insufficient to cause ESKD. 2 Foster M.C. Coresh J. Fornage M. et al. APOL1 variants associate with increased risk of CKD among African Americans. J Am Soc Nephrol. 2013; 24: 1484-1491 Crossref PubMed Scopus (180) Google Scholar We question the rationale for routine testing of all African American donors and recipients. Those in favor would likely remind us of the Hippocratic Oath—first do no harm—and that it is our duty to assign and avoid such risk. Paternalistic strategies such as this, although good intentioned, often overlook the broader context of how such dogmas may eliminate patient autonomy in decision making and even exacerbate existing disparities. In the following paragraphs, we will lay the foundation for the case against routine APOL1 testing in kidney transplantation with the hope of encouraging a more balanced approach to the care of donors and recipients alike. APOL1 genotyping in kidney transplantation: a look into the futureKidney InternationalVol. 100Issue 1PreviewChronic kidney disease (CKD) and end-stage kidney disease disproportionately affect individuals with African ancestry.1 African Americans have a lower rate of referral for kidney transplantation and lower graft survival than other ethnic groups.2 The risk of end-stage kidney disease is higher in Black living donors compared with White counterparts.3 Variants in the apolipoprotein L1 (APOL1) gene (G1: S342G and I384M; and G2: del N388 and Y389) explain most of this disparate risk for nondiabetic kidney disease, with the magnitude of risk varying depending on the clinical phenotype. Full-Text PDF

APOL1 genotyping in kidney transplantation: to do or not to do, that is the question? (pro)

Dogma, a principle laid down by authorities as incontrovertibly true, is difficult to change once proven incorrect. Considering chronic kidney disease (CKD) in African Americans, the dogma that nearly 40% have mild-moderate hypertension as the cause of nephropathy, low-level proteinuria excludes glomerular disease, and focal segmental glomerulosclerosis is a nonspecific pathology was suddenly struck down with discovery of association between the apolipoprotein L1 gene (APOL1) and nondiabetic CKD. 1 Freedman B.I. Cohen A.H. Hypertension-attributed nephropathy: what's in a name?. Nat Rev Nephrol. 2016; 12: 27-36 Crossref PubMed Scopus (51) Google Scholar ,2 Skorecki K.L. Wasser W.G. Hypertension-misattributed kidney disease in African Americans. Kidney Int. 2013; 83: 6-9 Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar APOL1 G1 and G2 kidney-risk variants (KRVs) display the strongest genetic association in complex disease; G0 is nonrisk. KRVs are found virtually only in populations with recent African ancestry and provide protection from African sleeping sickness. 3 Genovese G. Friedman D.J. Ross M.D. et al. Association of trypanolytic ApoL1 variants with kidney disease in African Americans. Science. 2010; 329: 841-845 Crossref PubMed Scopus (1388) Google Scholar Possessing 2 KRVs, autosomal recessive inheritance (G1G1/G2G2/G1G2, 1 KRV from each parent) defines high-risk genotypes with markedly increased risk for CKD. Approximately 13% of African Americans possess 2 KRVs, and 87% have APOL1 low-risk genotypes (∼39% G0G1/G0G2; ∼48% G0G0). 3 Genovese G. Friedman D.J. Ross M.D. et al. Association of trypanolytic ApoL1 variants with kidney disease in African Americans. Science. 2010; 329: 841-845 Crossref PubMed Scopus (1388) Google Scholar APOL1 genotyping in kidney transplantation: a look into the futureKidney InternationalVol. 100Issue 1PreviewChronic kidney disease (CKD) and end-stage kidney disease disproportionately affect individuals with African ancestry.1 African Americans have a lower rate of referral for kidney transplantation and lower graft survival than other ethnic groups.2 The risk of end-stage kidney disease is higher in Black living donors compared with White counterparts.3 Variants in the apolipoprotein L1 (APOL1) gene (G1: S342G and I384M; and G2: del N388 and Y389) explain most of this disparate risk for nondiabetic kidney disease, with the magnitude of risk varying depending on the clinical phenotype. Full-Text PDF

310 A Case Report of Thin Basement Membrane with Proteinuria Associated with Complement 5 and Apolipoprotein L1 Variants in the Heterozygous State

310 A Case Report of Thin Basement Membrane with Proteinuria Associated with Complement 5 and Apolipoprotein L1 Variants in the Heterozygous State

Apolipoprotein L1 and mechanisms of kidney disease susceptibility.

Allelic variants in the gene for apolipoprotein L1 (APOL1), found only in individuals of African ancestry, explain a majority of the excess risk of kidney disease in African Americans. However, a clear understanding how the disease-associated APOL1 variants cause kidney injury and the identity of environmental stressors that trigger the injury process have not been determined. Basic mechanistic studies of APOL1 biochemistry and cell biology, bolstered by new antibody reagents and inducible pluripotent stem cell-derived cell systems, have focused on the cytotoxic effect of the risk variants when APOL1 gene expression is induced. Since the APOL1 variants evolved to alter a key protein-protein interaction with the trypanosome serum resistance-associated protein, additional studies have begun to address differences in APOL1 interactions with other proteins expressed in podocytes, including new observations that APOL1 variants may alter podocyte cytoskeleton dynamics. A unified mechanism of pathogenesis for the various APOL1 nephropathies still remains unclear and controversial. As ongoing studies have consistently implicated the pathogenic gain-of-function effects of the variant proteins, novel therapeutic development inhibiting the synthesis or function of APOL1 proteins is moving toward clinical trials.

Podocyte density is reduced in kidney allografts with high-risk APOL1 genotypes at transplantation.

Variants in apolipoprotein L1 (APOL1) gene are associated with nondiabetic kidney diseases in black subjects and reduced kidney transplant graft survival. Living and deceased black kidney donors (n=107) were genotyped for APOL1 variants. To determine whether allografts from high-risk APOL1 donors have reduced podocyte densities contributing to allograft failure, we morphometrically estimated podocyte number, glomerular volume, and podocyte density. We compared allograft loss and eGFR trajectories stratified by APOL1 high-risk and low-risk genotypes. Demographic characteristics were similar in high-risk (n=16) and low-risk (n=91) donors. Podocyte density was significantly lower in high-risk than low-risk donors (108±26 vs 127±40 podocytes/106 um3 , P=.03). Kaplan-Meier graft survival (high-risk 61% vs. low-risk 91%, p-value=0.049) and multivariable Cox models (hazard ratio=2.6; 95% CI, 0.9-7.8) revealed higher graft loss in recipients of APOL1 high-risk allografts over 48months. More rapid eGFR decline was seen in recipients of high-risk APOL1 allografts (P<.001). At 60months, eGFR was 27 vs. 51mL/min/1.73min2 in recipients of APOL1 high-risk vs low-risk kidney allografts, respectively. Kidneys from high-risk APOL1 donors had worse outcomes versus low-risk APOL1 genotypes. Lower podocyte density in kidneys from high-risk APOL1 donors may increase susceptibility to CKD from subsequent stresses in both the recipients and donors.

APOL1 risk variants affect podocyte lipid homeostasis and energy production in focal segmental glomerulosclerosis.

Lipotoxicity was recently reported in several forms of kidney disease, including focal segmental glomerulosclerosis (FSGS). Susceptibility to FSGS in African Americans is associated with the presence of genetic variants of the Apolipoprotein L1 gene (APOL1) named G1 and G2. If and how endogenous APOL1 may alter mitochondrial function by the modifying cellular lipid metabolism is unknown. Using transgenic mice expressing the APOL1 variants (G0, G1 or G2) under endogenous promoter, we show that APOL1 risk variant expression in transgenic mice does not impair kidney function at baseline. However, APOL1 G1 expression worsens proteinuria and kidney function in mice characterized by the podocyte inducible expression of nuclear factor of activated T-cells (NFAT), which we have found to cause FSGS. APOL1 G1 expression in this FSGS-model also results in increased triglyceride and cholesterol ester contents in kidney cortices, where lipid accumulation correlated with loss of renal function. In vitro, we show that the expression of endogenous APOL1 G1/G2 in human urinary podocytes is associated with increased cellular triglyceride content and is accompanied by mitochondrial dysfunction in the presence of compensatory oxidative phosphorylation (OXPHOS) complexes elevation. Our findings indicate that APOL1 risk variant expression increases the susceptibility to lipid-dependent podocyte injury, ultimately leading to mitochondrial dysfunction.

The Relationship between APOL1 Structure and Function: Clinical Implications.

Common variants in the APOL1 gene are associated with an increased risk of nondiabetic kidney disease in individuals of African ancestry. Mechanisms by which APOL1 variants mediate kidney disease pathogenesis are not well understood. Amino acid changes resulting from the kidney disease-associated APOL1 variants alter the three-dimensional structure and conformational dynamics of the C-terminal α-helical domain of the protein, which can rationalize the functional consequences. Understanding the three-dimensional structure of the protein, with and without the risk variants, can provide insights into the pathogenesis of kidney diseases mediated by APOL1 variants.

Kidney-disease-associated variants of Apolipoprotein L1 show gain of function in cation channel activity

Variants in Apolipoprotein L1 (ApoL1) are known to be responsible for increased risk of some progressive kidney diseases among people of African ancestry. ApoL1 is an amphitropic protein that can insert into phospholipid membranes and confer anion- or cation-selective permeability to phospholipid membranes depending on pH. Whether these activities differ among the variants or whether they contribute to disease pathogenesis is unknown. We used assays of voltage-driven ion flux from phospholipid vesicles and of stable membrane association to assess differences among ApoL1 isoforms. There is a significant (approximately twofold) increase in the cation-selective ion permease activity of the two kidney-disease-associated variants compared with the reference protein. In contrast, we find no difference in the anion-selective permease activity at low pH among the isoforms. Compared with the reference sequence, the two disease-associated variants show increased stable association with phospholipid vesicles under conditions that support the cation permease activity, suggesting that the increased activity may be due to more efficient membrane association and insertion. There is no difference in membrane association among isoforms under optimal conditions for the anion permease activity. These data support a model in which enhanced cation permeability may contribute to the progressive kidney diseases associated with high-risk ApoL1 alleles.

Personalized Medicine and Chronic Kidney Disease in Sub-Saharan Africa: Advances and Challenges

Abstract Chronic kidney disease is increasing in prevalence sub-Saharan Africa, largely driven by the growing burden of hypertension, obesity, diabetes, and HIV infection. Underlying common and rare genetic variants may add to this risk at both the individual and population levels. Here we explore the advances and challenges in the translation of genetic discovery to personalized medicine for chronic kidney disease (CKD) in children and adults living in sub-Saharan Africa. The review discusses monogenic and polygenic causes of CKD with a focus on the African-specific APOL1 and NPHS2 variants. In summary, advances in genomics research capacity herald improvement in health outcomes through personalized medicine, precision molecular diagnosis of diseases, and through public health initiatives targeting high-risk populations.

APOL1 risk variants and the development of HIV-associated nephropathy.

HIV-associated nephropathy (HIVAN) remains a concern among untreated HIV patients, notably of African descent, as patients can reach end-stage renal disease within 3years. Two variants (G1 and G2) of the APOL1 gene, common in African populations to protect against African sleeping sickness, have been associated with an increased risk of several glomerular disorders including HIVAN, hypertension-attributed chronic kidney disease, and idiopathic focal segmental glomerulosclerosis and are accordingly named renal risk variants (RRVs). This review examines the mechanisms by which APOL1 RRVs drive glomerular injury in the setting of HIV infection and their potential application to patient management. Innate antiviral mechanisms activated by chronic HIV infection, especially those involving type 1 interferons, are of particular interest as they have been shown to upregulate APOL1 expression. Additionally, the downregulation of miRNA 193a (a repressor of APOL1) is also associated with the upregulation of APOL1. Interestingly, glomerular damage affected by APOL1 RRVs is caused by both loss- and gain-of-function changes in the protein, explicitly characterizing these effects. Their intracellular localization offers a further understanding of the nuances of APOL1 variant effects in promoting renal disease. Finally, although APOL1 variants have been recognized as a critical genetic player in mediating kidney disease, there are significant gaps in their application to patient management for screening, diagnosis, and treatment.

APOL1 renal risk variants exacerbate podocyte injury by increasing inflammatory stress

BackgroundApolipoprotein L1, APOL1, is a trypanosome lytic factor present in human and certain other primates. APOL1 gene variants, present in individuals of recent sub-Saharan African descent, increase risk for glomerular disease and associate with the disease progression, but the molecular mechanisms have not been defined.ObjectivesWe focus on the mechanism how APOL1 variant proteins enhance podocyte injury in the stressed kidney.MethodsFirst, we investigated the expression of APOL1 protein isoform and the localization of APOL1 protein in the kidney. Next, we examined the role of APOL1 in the podocyte stress and the inflammatory signaling in the kidney after hemi-nephrectomy.ResultsWe identified a novel RNA variant that lacks a secretory pathway signal sequence and we found that the predicted APOL1-B3 protein isoform was expressed in human podocytes in vivo and by BAC-APOL1 transgenic mice. APOL1-B3-G2 transgenic mice, carrying a renal risk variant, manifested podocyte injury and increased pro-IL-1β mRNA in isolated glomeruli and increased IL-1β production in the remnant kidney after uninephrectomy. APOL1-B3 interacted with NLRP12, a key regulator of Toll-like receptor signaling.ConclusionsThese results suggest a possible mechanism for podocyte injury by which one of the APOL1 protein isoforms, APOL1-B3 and its renal risk variants, enhances inflammatory signaling.

Podocytopathies.

Podocytopathies are kidney diseases in which direct or indirect podocyte injury drives proteinuria or nephrotic syndrome. In children and young adults, genetic variants in >50podocyte-expressed genes, syndromal non-podocyte-specific genes and phenocopies with other underlying genetic abnormalities cause podocytopathies associated with steroid-resistant nephrotic syndrome or severe proteinuria. A variety of genetic variants likely contribute to disease development. Among genes with non-Mendelian inheritance, variants in APOL1 have the largest effect size. In addition to genetic variants, environmental triggers such as immune-related, infection-related, toxic and haemodynamic factors and obesity are also important causes of podocyte injury and frequently combine to cause various degrees of proteinuria in children and adults. Typical manifestations on kidney biopsy are minimal change lesions and focal segmental glomerulosclerosis lesions. Standard treatment for primary podocytopathies manifesting with focal segmental glomerulosclerosis lesions includes glucocorticoids and other immunosuppressive drugs; individuals not responding with a resolution of proteinuria have a poor renal prognosis. Renin-angiotensin system antagonists help to control proteinuria and slow the progression of fibrosis. Symptomatic management may include the use of diuretics, statins, infection prophylaxis and anticoagulation. This Primer discusses a shift in paradigm from patient stratification based on kidney biopsy findings towardspersonalized management based on clinical, morphological and genetic data as well as pathophysiological understanding.

Apolipoprotein L1-Specific Antibodies Detect Endogenous APOL1 inside the Endoplasmic Reticulum and on the Plasma Membrane of Podocytes.

APOL1 is found in human kidney podocytes and endothelia. Variants G1 and G2 of the APOL1 gene account for the high frequency of nondiabetic CKD among African Americans. Proposed mechanisms of kidney podocyte cytotoxicity resulting from APOL1 variant overexpression implicate different subcellular compartments. It is unclear where endogenous podocyte APOL1 resides, because previous immunolocalization studies utilized overexpressed protein or commercially available antibodies that crossreact with APOL2. This study describes and distinguishes the locations of both APOLs. Immunohistochemistry, confocal and immunoelectron microscopy, and podocyte fractionation localized endogenous and transfected APOL1 using a large panel of novel APOL1-specific mouse and rabbit monoclonal antibodies. Both endogenous podocyte and transfected APOL1 isoforms vA and vB1 (and a little of isoform vC) localize to the luminal face of the endoplasmic reticulum (ER) and to the cell surface, but not to mitochondria, endosomes, or lipid droplets. In contrast, APOL2, isoform vB3, and most vC of APOL1 localize to the cytoplasmic face of the ER and are consequently absent from the cell surface. APOL1 knockout podocytes do not stain for APOL1, attesting to the APOL1-specificity of the antibodies. Stable re-transfection of knockout podocytes with inducible APOL1-G0, -G1, and -G2 showed no differences in localization among variants. APOL1 is found in the ER and plasma membrane, consistent with either the ER stress or surface cation channel models of APOL1-mediated cytotoxicity. The surface localization of APOL1 variants potentially opens new therapeutic targeting avenues.

AKI and Collapsing Glomerulopathy Associated with COVID-19 and APOL 1 High-Risk Genotype.

Kidney involvement is a feature of COVID-19 and it can be severe in Black patients. Previous research linked increased susceptibility to collapsing glomerulopathy, including in patients with HIV-associated nephropathy, to apo L1 (APOL1) variants that are more common in those of African descent. To investigate genetic, histopathologic, and molecular features in six Black patients with COVID-19 presenting with AKI and de novo nephrotic-range proteinuria, we obtained biopsied kidney tissue, which was examined by in situ hybridization for viral detection and by NanoString for COVID-19 and acute tubular injury-associated genes. We also collected peripheral blood for APOL1 genotyping. This case series included six Black patients with COVID-19 (four men, two women), mean age 55 years. At biopsy day, mean serum creatinine was 6.5 mg/dl and mean urine protein-creatinine ratio was 11.5 g. Kidney biopsy specimens showed collapsing glomerulopathy, extensive foot process effacement, and focal/diffuse acute tubular injury. Three patients had endothelial reticular aggregates. We found no evidence of viral particles or SARS-CoV-2 RNA. NanoString showed elevated chemokine gene expression and changes in expression of genes associated with acute tubular injury compared with controls. All six patients had an APOL1 high-risk genotype. Five patients needed dialysis (two of whom died); one partially recovered without dialysis. Collapsing glomerulopathy in Black patients with COVID-19 was associated with high-risk APOL1 variants. We found no direct viral infection in the kidneys, suggesting a possible alternative mechanism: a "two-hit" combination of genetic predisposition and cytokine-mediated host response to SARS-CoV-2 infection. Given this entity's resemblance with HIV-associated nephropathy, we propose the term COVID-19-associated nephropathy to describe it.

Association of preeclampsia with infant APOL1 genotype in African Americans

BackgroundBlack women in the United States and Africa are at an increased risk for preeclampsia. Allelic variants in the gene for apolipoprotein LI, APOL1, are found only in populations of African ancestry, and have been shown to contribute significant risk for kidney disease. Recent studies suggest these APOL1 variants also may contribute risk for preeclampsia.MethodsThe association of preeclampsia with carriage of APOL1 risk alleles was evaluated in a case-control study of deliveries from black women at a single center in Cleveland, Ohio that included gross and histopathologic evaluations of placental tissues (395 cases and 282 controls). Using logistic regression models, associations between fetal APOL1 genotype and preeclampsia were evaluated using several case definitions based on prematurity and severity of preeclampsia, with uncomplicated term pregnancies as controls. Associations between APOL1 genotype and pathological features were also examined.ResultsThe infant APOL1 genotype was significantly associated with preeclampsia in a dominant inheritance pattern with odds ratio of 1.41 (P=0.029, 95% CI 1.037, 1.926). Stratifying preeclampsia cases by preterm birth, significant associations were detected for both recessive (O.R.=1.70, P=0.038) and additive (O.R.=1.33, P=0.028) inheritance patterns. APOL1 genotype, however, was not significantly associated with pathological changes or other perinatal observations.ConclusionsPreeclampsia appears to be another disease associated with APOL1 variants, however, further studies are needed to increase confidence in the mode of inheritance. By understanding the association of APOL1 variants with preeclampsia, genetic screening tests for APOL1 may be useful to predict at-risk pregnancies and targeted interventions may be developed to improve pregnancy outcomes.

Association of Race and Risk of Graft Loss among Kidney Transplant Recipients in the US Military Health System.

Racial disparities in kidney transplant outcomes are well documented and are attributed to biologic differences ( e.g. , gene variants in APOL1), immunologic factors, and other barriers, including lower socioeconomic status (SES), nonadherence to immunosuppressive medications, reduced access to care

Hypertension Does Not Enhance Proteinuria in African Green Monkeys that Exhibit One or More Copies of an APOL1‐Like Gene Insertion

Historically, people of African descent have increased instances of early onset kidney disease which can be correlated with genetic variants at the Apolipoprotein L‐1 (APOL1) gene locus. APOL1 is a component of high density lipoprotein (HDL) that serves as a serum trypanolytic factor to protect against T. brucei, which causes African sleeping sickness. Genetic variants in APOL1 elicit some resistance to T. brucei, and have been linked to increased risk of early‐onset end‐stage renal disease (ESRD). A translational animal model is key to understanding the pathogenesis of APOL1‐mediated ESRD. The African Green Monkey (AGM) develops spontaneous hypertension with renal glomerular pathologies. AGMs exhibit a genetic insert at the APOL1 locus homologous to that in the human genome that is associated with HTN in homozygous AGMs. APOL1‐mediated ESRD typically develops in response to a second hit stressor such as HTN. We hypothesized that hypertensive (HT) AGMs with at least one copy of the APOL1‐like insertion (I) will have altered kidney function compared to HT AGMs without the insertion (NI). Systolic (SBP) and diastolic (DBP) blood pressures and heart rates were measured with forearm plethysmography. AGMs with SBP ≤ 120 were normotensive (NT) and SBP ≥ 140 were HT. Daily urine output and water intake was collected from single‐housed AGMs for three consecutive days. During urine collections, all animals were fed a standard, nonhuman primate chow with ad libitum water. Urinary protein and plasma creatinine were measured using standard colorimetric techniques. SBP was not different between HT NI AGMs (163 ± 3.8 mm Hg, n = 12) and HT I AGMs (158 ± 7.3mm Hg, n = 7, p &gt; 0.05) nor was SBP different between NT NI AGMs (94.3 ± 4.0 mm Hg, n = 17) and NT I AGMs (103 ± 6.3 mm Hg, n = 6, p &gt; 0.05). Body weight was used to approximate age and was not different between NI and I AGMs (p &gt; 0.05). Protein excretion was higher for HT NI AGMs (416.3 ± 44.8 mg/day, n = 10) than for HT I AGMS (289.2 ± 33.0 mg/day, n = 6, p &lt; 0.05) and higher for NT NI AGMs (450.7 ± 43.3 mg/day, n = 15) than for NT I AGMs (227.0 ± 83.6 mg/day, n = 4, p &lt; 0.05). Plasma creatinine concentration was not different between NT NI and NT I AGMs (p &gt; 0.05) as well as HT NI and HT I AGMs (p &gt; 0.05). Plasma creatinine concentration and protein excretion rates were not different between HT and NT NI AGMs (p &gt; 0.05) nor were they different between HT and NT I AGMs (p &gt; 0.05). Thus, at this age, differences in plasma creatinine concentration and protein excretion were likely due to insertion status and not BP phenotype. These results indicate that in the AGM a single copy of the APOL1‐like insertion may not be sufficient to cause progression to ESRD. In humans, the high‐risk genotypes for APOL1‐mediated ESRD have two copies of the variant, therefore future studies should aim to assess more homozygous animals. The AGM as a model organism will allow investigation of links between HTN and APOL1‐like genetic variants that could result in novel treatments and targets for patients with APOL1‐mediated renal dysfunction.Support or Funding InformationUndergraduate Summer Research Fellowship, American Physiological Society; Summer Research and Creativity Fellowship, University of Kentucky Office of Undergraduate Research

Quality over quantity: A qualitative, targeted bottom-up proteomics approach to genotyping apolipoprotein L1

A targeted, bottom-up proteomic assay was developed for the qualitative detection of apolipoprotein L1 (ApoL1) protein variants (G0, G1, and G2) in blood plasma for identification of high and low renal risk genotypes. Following trypsin digestion of liquid or dry plasma, surrogate peptides specific to each ApoL1 variant were detected by liquid chromatography-tandem mass spectrometry along with two surrogate peptides common among all variants which served as internal (positive) controls to verify correct sample processing. Using isotopically labeled peptide internal standards, the presence or absence of each surrogate peptide was determined using multiple objective metrics including: 1) retention time confirmation relative to its internal standard, 2) comparison of the internal standard-normalized response relative to pre-established thresholds for confident detection, and 3) ion ratio monitoring. Based on the pattern of variant-specific surrogate peptides detected, the genotype was accurately inferred. The final, optimized method was fully validated for liquid plasma specimens, as well as dry plasma specimens collected on a laminar flow blood separation device. For both specimen types, the latter which can be self-collected for remote or in-home sampling, the assay was shown to be reproducible, interference-free with the exception of gross hemolysis, and accurate relative to Sanger sequencing (100% agreement). This targeted, qualitative bottom-up proteomic assay for detection of ApoL1 variants in blood provides a high-throughput, cost-effective alternative to molecular methods and has potential implications to improve organ allocation by facilitating screening kidney donors for high-risk ApoL1 genotypes, but could be applicable for genotyping other clinically relevant blood proteins variants.

Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics.

DNA variants in APOL1 associate with kidney disease, but the pathophysiologic mechanisms remain incompletely understood. Model organisms lack the APOL1 gene, limiting the degree to which disease states can be recapitulated. Here we present single-cell RNA sequencing (scRNA-seq) of genome-edited human kidney organoids as a platform for profiling effects of APOL1 risk variants in diverse nephron cell types. We performed footprint-free CRISPR-Cas9 genome editing of human induced pluripotent stem cells (iPSCs) to knock in APOL1 high-risk G1 variants at the native genomic locus. iPSCs were differentiated into kidney organoids, treated with vehicle, IFN-γ, or the combination of IFN-γ and tunicamycin, and analyzed with scRNA-seq to profile cell-specific changes in differential gene expression patterns, compared with isogenic G0 controls. Both G0 and G1 iPSCs differentiated into kidney organoids containing nephron-like structures with glomerular epithelial cells, proximal tubules, distal tubules, and endothelial cells. Organoids expressed detectable APOL1 only after exposure to IFN-γ. scRNA-seq revealed cell type-specific differences in G1 organoid response to APOL1 induction. Additional stress of tunicamycin exposure led to increased glomerular epithelial cell dedifferentiation in G1 organoids. Single-cell transcriptomic profiling of human genome-edited kidney organoids expressing APOL1 risk variants provides a novel platform for studying the pathophysiology of APOL1-mediated kidney disease.