Recessive, gain-of-function toxicity in an APOL1 BAC transgenic mouse model mirrors human APOL1 kidney disease.
ABSTRACT: People of recent sub-Saharan African ancestry develop kidney failure much more frequently than other groups. A large fraction of this disparity is due to two coding sequence variants in the APOL1 gene. Inheriting two copies of these APOL1 risk variants, known as G1 and G2, causes high rates of focal segmental glomerulosclerosis (FSGS), HIV-associated nephropathy and hypertension-associated end-stage kidney disease. Disease risk follows a recessive mode of inheritance, which is puzzling given the considerable data that G1 and G2 are toxic gain-of-function variants. We developed coisogenic bacterial artificial chromosome (BAC) transgenic mice harboring either the wild-type (G0), G1 or G2 forms of human APOL1. Expression of interferon gamma (IFN-γ) via plasmid tail vein injection results in upregulation of APOL1 protein levels together with robust induction of heavy proteinuria and glomerulosclerosis in G1/G1 and G2/G2 but not G0/G0 mice. The disease phenotype was greater in G2/G2 mice. Neither heterozygous (G1/G0 or G2/G0) risk variant mice nor hemizygous (G1/-, G2/-) mice had significant kidney injury in response to IFN-γ, although the heterozygous mice had a greater proteinuric response than the hemizygous mice, suggesting that the lack of significant disease in humans heterozygous for G1 or G2 is not due to G0 rescue of G1 or G2 toxicity. Studies using additional mice (multicopy G2 and a non-isogenic G0 mouse) supported the notion that disease is largely a function of the level of risk variant APOL1 expression. Together, these findings shed light on the recessive nature of APOL1-nephropathy and present an important model for future studies.
Project description:<h4>Background</h4>Among African-Americans, genome wide association revealed a strong correlation between the G1 and G2 alleles of APOL1 (apolipoproteinL1, also called trypanolytic factor) and kidney diseases including focal and segmental glomerulosclerosis, HIV-associated nephropathy and hypertensive nephrosclerosis. In the prevailing hypothesis, heterozygous APOL1 G1 and G2 alleles increase resistance against Trypanosoma that cause African sleeping sickness, resulting in positive selection of these alleles, but when homozygous the G1 and G2 alleles predispose to glomerulosclerosis. While efforts are underway to screen patients for G1 and G2 alleles and to better understand "APOL1 glomerulopathy," no data prove that these APOL1 sequence variants cause glomerulosclerosis. G1 and G2 correlate best with glomerulosclerosis as recessive alleles, which suggests a loss of function mutation for which proof of causality is commonly tested with homozygous null alleles. This test cannot be performed in rodents as the APOL gene cluster evolved only in primates. However, there is a homozygous APOL1 null human being who lives in a village in rural India. This individual and his family offer a unique opportunity to test causality between APOL1 null alleles and glomerulosclerosis.<h4>Methods and findings</h4>We obtained clinical data, blood and urine from this APOL1 null patient and 50 related villagers. Based on measurements of blood pressure, BUN, creatinine, albuminuria, genotyping and immunoblotting, this APOL1 null individual does not have glomerulosclerosis, nor do his relatives who carry APOL1 null alleles.<h4>Conclusions</h4>This small study cannot provide definitive conclusions but the absence of glomerulosclerosis in this unique population is consistent with the possibility that African-American glomerulosclerosis is caused, not by loss of APOL1 function, but by other mechanisms including a subtle gain of function or by the "genetic hitchhiking" of deleterious mutations in a gene linked to APOL1 G1 and G2.
Project description: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.
Project description:Apolipoprotein L1 (APOL1) genetic variants G1 and G2, compared to the common allele G0, are major risk factors for non-diabetic kidney disease in African descent populations. APOL1 is a minor protein component of HDL, as well as being expressed in podocytes and vascular cells. Reverse cholesterol transport involves the transport of cholesterol to HDL by cellular ATP-binding cassette; ABCA1 and ABCG1 with subsequent delivery from peripheral tissues to the liver. With impaired reverse cholesterol transport, lipid accumulation occurs and macrophages morphologically transform into foam cells, releasing inflammatory factors. We asked whether the APOL1 risk variants alter peripheral cholesterol metabolism and specifically affect macrophage cholesterol efflux. Tissues and bone marrow (BM)-derived monocytes were isolated from wild-type mice (WT) and from BAC/APOL1 transgenic (APOL1-G0, APOL1-G1, and APOL1-G2) mice, which carry a bacterial artificial chromosome that contains the human APOL1 genomic region. Monocytes were differentiated into macrophages using M-CSF, and then polarized into M1 and M2 macrophages. Cholesterol content, cholesterol efflux, and ABCA1 and ABCG1 mRNA expression were measured. Kidney, spleen, and bone marrow-derived macrophages from APOL1-G1 and -G2 mice showed increased cholesterol accumulation and decreased ABCA1 and ABCG1 mRNA levels. BM-derived macrophages from APOL1-G1 and -G2 mice showed significantly reduced cholesterol efflux compared to WT or APOL1-G0 macrophages. Taken together, the evidence suggests that APOL1-G1 and -G2 risk variants impaired reverse cholesterol transport through decreased expression of cholesterol efflux transporters suggesting a possible mechanism to promote macrophage foam cell formation, driving inflammation in the glomerulus and renal interstitium.
Project description:Development of higher rates of nondiabetic glomerulosclerosis (GS) in African Americans has been attributed to two coding sequence variants (G1 and G2) in the APOL1 gene. To date, the cellular function and the role of APOL1 variants (Vs) in GS are still unknown. In this study, we examined the effects of overexpressing wild-type (G0) and kidney disease risk variants (G1 and G2) of APOL1 in human podocytes using a lentivirus expression system. Interestingly, G0 inflicted podocyte injury only at a higher concentration; however, G1 and G2 promoted moderate podocyte injury at lower and higher concentrations. APOL1Vs expressing podocytes displayed diffuse distribution of both Lucifer yellow dye and cathepsin L as manifestations of enhanced lysosomal membrane permeability (LMP). Chloroquine attenuated the APOL1Vs-induced increase in podocyte injury, consistent with targeting lysosomes. The chloride channel blocker DIDS prevented APOL1Vs- induced injury, indicating a role for chloride influx in osmotic swelling of lysosomes. Direct exposure of noninfected podocytes with conditioned media from G1- and G2-expressing podocytes also induced injury, suggesting a contributory role of the secreted component of G1 and G2 as well. Adverse host factors (AHFs) such as hydrogen peroxide, hypoxia, TNF-?, and puromycin aminonucleoside augmented APOL1- and APOL1Vs-induced podocyte injury, while the effect of human immunodeficiency virus (HIV) on podocyte injury was overwhelming under conditions of APOLVs expression. We conclude that G0 and G1 and G2 APOL1 variants have the potential to induce podocyte injury in a manner which is further augmented by AHFs, with HIV infection being especially prominent.
Project description:African Americans have a heightened risk of developing chronic and end-stage kidney disease, an association that is largely attributed to two common genetic variants, termed G1 and G2, in the APOL1 gene. Direct evidence demonstrating that these APOL1 risk alleles are pathogenic is still lacking because the APOL1 gene is present in only some primates and humans; thus it has been challenging to demonstrate experimental proof of causality of these risk alleles for renal disease. Here we generated mice with podocyte-specific inducible expression of the APOL1 reference allele (termed G0) or each of the risk-conferring alleles (G1 or G2). We show that mice with podocyte-specific expression of either APOL1 risk allele, but not of the G0 allele, develop functional (albuminuria and azotemia), structural (foot-process effacement and glomerulosclerosis) and molecular (gene-expression) changes that closely resemble human kidney disease. Disease development was cell-type specific and likely reversible, and the severity correlated with the level of expression of the risk allele. We further found that expression of the risk-variant APOL1 alleles interferes with endosomal trafficking and blocks autophagic flux, which ultimately leads to inflammatory-mediated podocyte death and glomerular scarring. In summary, this is the first demonstration that the expression of APOL1 risk alleles is causal for altered podocyte function and glomerular disease in vivo.
Project description:African polymorphisms in the gene for Apolipoprotein L1 (APOL1) confer a survival advantage against lethal trypanosomiasis but also an increased risk for several chronic kidney diseases (CKD) including HIV-associated nephropathy (HIVAN). APOL1 is expressed in renal cells, however, the pathogenic events that lead to renal cell damage and kidney disease are not fully understood. The podocyte function of APOL1-G0 versus APOL1-G2 in the setting of a known disease stressor was assessed using transgenic mouse models. Transgene expression, survival, renal pathology and function, and podocyte density were assessed in an intercross of a mouse model of HIVAN (Tg26) with two mouse models that express either APOL1-G0 or APOL1-G2 in podocytes. Mice that expressed HIV genes developed heavy proteinuria and glomerulosclerosis, and had significant losses in podocyte numbers and reductions in podocyte densities. Mice that co-expressed APOL1-G0 and HIV had preserved podocyte numbers and densities, with fewer morphologic manifestations typical of HIVAN pathology. Podocyte losses and pathology in mice co-expressing APOL1-G2 and HIV were not significantly different from mice expressing only HIV. Podocyte hypertrophy, a known compensatory event to stress, was increased in the mice co-expressing HIV and APOL1-G0, but absent in the mice co-expressing HIV and APOL1-G2. Mortality and renal function tests were not significantly different between groups. APOL1-G0 expressed in podocytes may have a protective function against podocyte loss or injury when exposed to an environmental stressor. This was absent with APOL1-G2 expression, suggesting APOL1-G2 may have lost this protective function.
Project description:Two coding sequence variants (G1 and G2) of Apolipoprotein L1 (APOL1) gene have been implicated as a higher risk factor for chronic kidney diseases (CKD) in African Americans when compared with European Americans. Previous studies have suggested that the APOL1 G1 and G2 variant proteins are more toxic to kidney cells than the wild-type APOL1 G0, but the underlying mechanisms are poorly understood. To determine whether endoplasmic reticulum (ER) stress contributes to podocyte toxicity, we generated human podocytes (HPs) that stably overexpressed APOL1 G0, G1, or G2 (Vec/HPs, G0/HPs, G1/HPs, and G2/HPs). Propidium iodide staining showed that HP overexpressing the APOL1 G1 or G2 variant exhibited a higher rate of necrosis when compared with those overexpressing the wild-type G0 counterpart. Consistently, the expression levels of nephrin and podocin proteins were significantly decreased in the G1- or G2-overexpressing cells despite the maintenance of their mRNA expressions levels. In contrast, the expression of the 78-kDa glucose-regulated protein ((GRP78), also known as the binding Ig protein, BiP) and the phosphorylation of the eukaryotic translation initiation factor 1 (eIF1) were significantly elevated in the G1/HPs and G2/HPs, suggesting a possible occurrence of ER stress in these cells. Furthermore, ER stress inhibitors not only restored nephrin protein expression, but also provided protection against necrosis in G1/HPs and G2/HPs, suggesting that APOL1 risk variants cause podocyte injury partly through enhancing ER stress.
Project description:Soluble urokinase plasminogen activator receptor (suPAR) independently predicts chronic kidney disease (CKD) incidence and progression. Apolipoprotein L1 (APOL1) gene variants G1 and G2, but not the reference allele (G0), are associated with an increased risk of CKD in individuals of recent African ancestry. Here we show in two large, unrelated cohorts that decline in kidney function associated with APOL1 risk variants was dependent on plasma suPAR levels: APOL1-related risk was attenuated in patients with lower suPAR, and strengthened in those with higher suPAR levels. Mechanistically, surface plasmon resonance studies identified high-affinity interactions between suPAR, APOL1 and ?v?3 integrin, whereby APOL1 protein variants G1 and G2 exhibited higher affinity for suPAR-activated avb3 integrin than APOL1 G0. APOL1 G1 or G2 augments ?v?3 integrin activation and causes proteinuria in mice in a suPAR-dependent manner. The synergy of circulating factor suPAR and APOL1 G1 or G2 on ?v?3 integrin activation is a mechanism for CKD.
Project description:Apolipoprotein L1 (APOL1) wild type (G0) plays a role in the metabolism of sphingolipids, glycosphingolipids, sphingomyelin and ceramide, which constitute bioactive components of the lipid rafts (DRM). We asked whether APOL1 variants (APOL1-Vs) G1 and G2 carry the potential to alter the metabolism of sphingolipids in human podocytes. The sphingolipid pattern in HPs overexpressing either APOL1G0 or APOL1-Vs was analysed by using a thin mono- and bi-dimensional layer chromatography, mass-spectrometry and metabolic labelling with [1-3H]sphingosine. HP G0 and G1/G2-Vs exhibit a comparable decrease in lactosylceramide and an increase in the globotriaosylceramide content. An analysis of the main glycohydrolases activity involved in glycosphingolipid catabolism showed an overall decrease in the activeness of the tested enzymes, irrespective of the type of APOL1-Vs expression. Similarly, the high throughput cell live-based assay showed a comparable increased action of the plasma membrane glycosphingolipid-glycohydrolases in living cells independent of the genetic APOL1 expression profile. Importantly, the most significative modification of the sphingolipid pattern induced by APOL1-Vs occurred in DRM resulted with a drastic reduction of radioactivity associated with sphingolipids. G1/G2-Vs present a decrease amount of globotriaosylceramide and globopentaosylceramide compared to G0. Additionally, ceramide at the DRM site and lactosylceramide in general, showed a greatest fall in G1/G2 in comparison with G0. Additionally, the levels of glucosylceramide decreased only in the DRM of human podocytes overexpressing G1/G2-Vs. These findings suggest that altered sphingolipidsprofiles may contribute to the deranged functionality of the plasma membrane in APOL1 risk milieu.
Project description:APOL1 risk variants are associated with kidney disease in blacks, but the mechanisms of renal injury associated with APOL1 risk variants are unknown. Because APOL1 is unique to humans and some primates, we created transgenic (Tg) mice using the promoter of nephrin-encoding Nphs1 to express the APOL1 reference sequence (G0) or the G2 risk variant in podocytes, establishing Tg lines with a spectrum of APOL1 expression levels. Podocytes from Tg-G0 and Tg-G2 mice did not undergo necrosis, apoptosis, or autophagic cell death in vivo, even in lines with highly expressed transgenes. Further, Tg-G0 and Tg-G2 mice did not develop kidney pathology, proteinuria, or azotemia as of 300 days of age. However, by 200 days of age, Tg-G2 mice had significantly lower podocyte density than age-matched WT and Tg-G0 mice had, a difference that was not evident at weaning. Notably, a pregnancy-associated phenotype that encompassed eclampsia, preeclampsia, fetal/neonatal deaths, and small litter sizes occurred in some Tg-G0 mice and more severely in Tg-G2 mice. Similar to human placenta, placentas of Tg mice expressed APOL1. Overall, these results suggest podocyte depletion could predispose individuals with APOL1 risk genotypes to kidney disease in response to a second stressor, and add to other published evidence associating APOL1 expression with preeclampsia.