Apolipoprotein L1 (APOL1) cation current in HEK-293 cells and in human podocytes.

Two heterozygous missense variants (G1 and G2) of Apolipoprotein L1 (APOL1) found in individuals of recent African ancestry can attenuate the severity of infection by some forms of Trypanosoma brucei. However, these two variants within a broader African haplotype also increase the risk of kidney disease in Americans of African descent. Although overexpression of either variant G1 or G2 causes multiple pathogenic changes in cultured cells and transgenic mouse models, the mechanism(s) promoting kidney disease remain unclear. Human serum APOL1 kills trypanosomes through its cation channel activity, and cation channel activity of recombinant APOL1 has been reconstituted in lipid bilayers and proteoliposomes. Although APOL1 overexpression increases whole cell cation currents in HEK-293 cells, the ion channel activity of APOL1 has not been assessed in glomerular podocytes, the major site of APOL1-associated kidney diseases. We characterize APOL1-associated whole cell and on-cell cation currents in HEK-293 T-Rex cells and demonstrate partial inhibition of currents by anti-APOL antibodies. We detect in primary human podocytes a similar cation current inducible by interferon-γ (IFNγ) and sensitive to inhibition by anti-APOL antibody as well as by a fragment of T. brucei Serum Resistance-Associated protein (SRA). CRISPR knockout of APOL1 in human primary podocytes abrogates the IFNγ-induced, antibody-sensitive current. Our novel characterization in HEK-293 cells of heterologous APOL1-associated cation conductance inhibited by anti-APOL antibody and our documentation in primary human glomerular podocytes of endogenous IFNγ-stimulated, APOL1-mediated, SRA and anti-APOL-sensitive ion channel activity together support APOL1-mediated channel activity as a therapeutic target for treatment of APOL1-associated kidney diseases.

DGAT2 Inhibition Potentiates Lipid Droplet Formation To Reduce Cytotoxicity in APOL1 Kidney Risk Variants.

BACKGROUND: Two variants in the gene encoding apolipoprotein L1 (APOL1) that are highly associated with African ancestry are major contributors to the large racial disparity in rates of human kidney disease. We previously demonstrated that recruitment of APOL1 risk variants G1 and G2 from the endoplasmic reticulum to lipid droplets leads to reduced APOL1-mediated cytotoxicity in human podocytes. METHODS: We used CRISPR-Cas9 gene editing of induced pluripotent stem cells to develop human-derived APOL1G0/G0 and APOL1G2/G2 kidney organoids on an isogenic background, and performed bulk RNA sequencing of organoids before and after treatment with IFN-γ. We examined the number and distribution of lipid droplets in response to treatment with inhibitors of diacylglycerol O-acyltransferases 1 and 2 (DGAT1 and DGAT2) in kidney cells and organoids. RESULTS: APOL1 was highly upregulated in response to IFN-γ in human kidney organoids, with greater increases in organoids of high-risk G1 and G2 genotypes compared with wild-type (G0) organoids. RNA sequencing of organoids revealed that high-risk APOL1G2/G2 organoids exhibited downregulation of a number of genes involved in lipogenesis and lipid droplet biogenesis, as well as upregulation of genes involved in fatty acid oxidation. There were fewer lipid droplets in unstimulated high-risk APOL1G2/G2 kidney organoids than in wild-type APOL1G0/G0 organoids. Whereas DGAT1 inhibition reduced kidney organoid lipid droplet number, DGAT2 inhibition unexpectedly increased organoid lipid droplet number. DGAT2 inhibition promoted the recruitment of APOL1 to lipid droplets, with associated reduction in cytotoxicity. CONCLUSIONS: Lipogenesis and lipid droplet formation are important modulators of APOL1-associated cytotoxicity. Inhibition of DGAT2 may offer a potential therapeutic strategy to attenuate cytotoxic effects of APOL1 risk variants.

Recessive, gain-of-function toxicity in an APOL1 BAC transgenic mouse model mirrors human APOL1 kidney disease.

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.

Recruitment of APOL1 kidney disease risk variants to lipid droplets attenuates cell toxicity.

Two coding variants in the apolipoprotein L1 (APOL1) gene (termed G1 and G2) are strongly associated with increased risk of nondiabetic kidney disease in people of recent African ancestry. The mechanisms by which the risk variants cause kidney damage, although not well-understood, are believed to involve injury to glomerular podocytes. The intracellular localization and function of APOL1 in podocytes remain unclear, with recent studies suggesting possible roles in the endoplasmic reticulum (ER), mitochondria, endosomes, lysosomes, and autophagosomes. Here, we demonstrate that APOL1 also localizes to intracellular lipid droplets (LDs). While a large fraction of risk variant APOL1 (G1 and G2) localizes to the ER, a significant proportion of wild-type APOL1 (G0) localizes to LDs. APOL1 transiently interacts with numerous organelles, including the ER, mitochondria, and endosomes. Treatment of cells that promote LD formation with oleic acid shifted the localization of G1 and G2 from the ER to LDs, with accompanying reduction of autophagic flux and cytotoxicity. Coexpression of G0 APOL1 with risk variant APOL1 enabled recruitment of G1 and G2 from the ER to LDs, accompanied by reduced cell death. The ability of G0 APOL1 to recruit risk variant APOL1 to LDs may help explain the recessive pattern of kidney disease inheritance. These studies establish APOL1 as a bona fide LD-associated protein, and reveal that recruitment of risk variant APOL1 to LDs reduces cell toxicity, autophagic flux, and cell death. Thus, interventions that divert APOL1 risk variants to LDs may serve as a novel therapeutic strategy to alleviate their cytotoxic effects.

Tbo-Filgrastim versus Filgrastim during Mobilization and Neutrophil Engraftment for Autologous Stem Cell Transplantation.

There are limited data available supporting the use of the recombinant granulocyte colony-stimulating factor (G-CSF), tbo-filgrastim, rather than traditionally used filgrastim to mobilize peripheral blood stem cells (PBSC) or to accelerate engraftment after autologous stem cell transplantation (ASCT). We sought to compare the efficacy and cost of tbo-filgrastim to filgrastim in these settings. Patients diagnosed with lymphoma or plasma cell disorders undergoing G-CSF mobilization, with or without plerixafor, were included in this retrospective analysis. The primary outcome was total collected CD34(+) cells/kg. Secondary mobilization endpoints included peripheral CD34(+) cells/µL on days 4 and 5 of mobilization, adjunctive use of plerixafor, CD34(+) cells/kg collected on day 5, number of collection days and volumes processed, number of collections reaching 5 million CD34(+) cells/kg, and percent reaching target collection goal in 1 day. Secondary engraftment endpoints included time to neutrophil and platelet engraftment, number of blood product transfusions required before engraftment, events of febrile neutropenia, and length of stay. A total of 185 patients were included in the final analysis. Patients receiving filgrastim (n = 86) collected a median of 5.56 × 10(6) CD34(+) cells/kg, compared with a median of 5.85 × 10(6) CD34(+) cells/kg in the tbo-filgrastim group (n = 99; P = .58). There were no statistically significant differences in all secondary endpoints with the exception of apheresis volumes processed (tbo-filgrastim, 17.0 liters versus filgrastim, 19.7 liters; P < .01) and mean platelet transfusions (tbo-filgrastim, 1.7 units versus filgrastim, 1.4 units; P = .04). In conclusion, tbo-filgrastim demonstrated similar CD34(+) yield compared with filgrastim in mobilization and post-transplantation settings, with no clinically meaningful differences in secondary efficacy and safety endpoints. Furthermore, tbo-filgrastim utilization was associated with cost savings of approximately $1406 per patient utilizing average wholesale price.