APOL1 genotype modulates lipid accumulation in macrophages.

(A) Biopsy of kidney allograft from a donor with high-risk APOL1 genotype reveals endocapillary foamy macrophages within the glomerulus (PAS, scale bar = 40 μm). (B) This glomerulus contains representative collapsing lesions seen in individuals with high-risk APOL1 genotype. Endocapillary foamy macrophages are captured in this view (Masson’s trichrome, scale bar = 40 μm). (C) Western blot of APOL1 protein expression in BMDMs over a 24-hour time course of 5 ng/mL IFNγ treatment. (D) Oil Red O (ORO) stained images of BMDMs in the presence or absence of 5ng/mL IFNγ and 50 μg/mL oxidized LDL (oxLDL) for 72 hours. (E) Quantification of ORO staining. (F) Relative Abca1 and (G) Arg1 gene expression levels in BMDMs treated with IFNγ and oxLDL. Experiments were performed in BMDMs from 4-8 mice per genotype per group across 2 independent experiments, with both sexes represented. Data are expressed as mean + SD. *p<0.05, **p<0.01 ***p<0.005 ****p>0.001. 2-way ANOVA with Tukey’s Multiple Comparison Test (D) and unpaired t-test (G).

G1 APOL1 promotes higher levels of sustained inflammation.

BMDMs treated with 5ng/mL IFNγ, 10 pg/mL LPS, and 10 ng/mL IL-4 for 8 hours were analyzed with flow cytometry to measure the surface expression of (A) CD86 and CD206. Histogram of (B) CD86 and (C) CD206 expression. (D) qPCR array of immune-related genes was completed with BMDMs treated with 5ng/mL IFNγ and 10 ng/mL IL-10. n=2 mice per genotype per treatment. (E) Dot blot measuring cytokines in iPSDM treated with 25ng/mL IFNγ and 10 pg/mL of LPS for 24 hours. Experiment was performed in n=2 iPSDM per genotype. (F, G) Gene expression of Casp1, Nlrp3 in BMDMs treated with 5ng/mL IFNγ for 8 hours. (H) Autophagy measurement of BMDMs treated with IFNγ and oxLDL. Experiments were performed in BMDMs from 4-6 mice per genotype per group across 2 independent experiments, with both sexes represented (A-C, F-G). Data are expressed as mean + SD. 2-way ANOVA with Tukey’s Multiple Comparison Test (B, H) Unpaired t-test (F-G), *p<0.05

APOL1 localizes to the ER and mitochondria in macrophages and modulates mitochondrial morphology.

(A) Representative images from confocal fluorescent microscopy of BMDMs stained with anti-APOL1, anti-Calnexin, and anti-Tom20 antibodies and DAPI. BMDMs were treated with 5ng/mL IFNγ for 8 hours. Experiment was performed in BMDMs from 4 mice per genotype per group. (Scale bar = 20 μm) (B) Representative transmission electron microscopy images of mitochondria in APOL1 BMDMs treated with 5 ng/mL IFNγ. (Scale bar = 500 nm) (C) Quantification of the area and circularity of BMDM mitochondria. Experiment was performed in BMDMs from 4 mice per genotype per group across 2 independent experiments, with both sexes represented. Measurements were performed in at least 100 mitochondria per sample. Data are expressed as median and interquartile ranges. Unpaired t-test, ****p>0.001

G1 and G2 APOL1 increase respiratory capacity

(A) Oxygen consumption rate of BMDMs treated with 5 ng/mL IFNγ or control measured by Seahorse Mito Stress Test. (B) Basal respiration, maximal respiration, and spare respiratory capacity of BMDMs from Mito Stress Test. (C) Proton efflux rate of BMDMs treated with 5 ng/mL IFNγ or control measured by Seahorse Glycolytic Rate Assay. (D) Basal and compensatory glycolysis measurement from Glycolytic Rate assay. Experiments were performed in BMDMs from 8 mice per genotype per group across 3 independent experiments, with both sexes represented. Data are expressed as mean + SEM. 2-way ANOVA with Tukey’s Multiple Comparison Test, *p<0.05, **p<0.01

Metabolomics analysis of APOL1 BMDMS shows modified steady state of polyamines

(A) Volcano plot of significantly regulated metabolites in IFNγ treated G0 vs G1 BMDMs. (B) Most impactful pathways enriched in metabolomics analysis in G0 vs G1 BMDMs. (C) Volcano plot of significantly regulated metabolites in IFNγ treated G0 vs G2 BMDMs. (D) Most impactful pathways enriched in metabolomics analysis in G0 vs G2 BMDMs. (E) Fold change of metabolites in spermidine and spermine biosynthesis pathway. Experiments were performed in BMDMs from 3 mice per genotype per group across 2 independent experiments, with both sexes represented. ND = Not Detected

Polyamine inhibitor α-difluoromethylornithine (DFMO) decreases lipid toxicity and inflammation in BMDMs

(A) Mechanism of DFMO inhibition of polyamine synthesis pathway. (B) Representative images of BMDMs treated with IFNγ (5 ng/mL), oxLDL (50 μg/mL), and DFMO (200 μM) for 72 hours and stained with ORO. (C) Quantification of ORO stain. (Scale bar = 50 μm) (D-G) Gene expression of Tnf, Ccl2, Casp1, and Nlrp3 in APOL1 BMDMs treated with IFNγ (5 ng/mL) and DFMO (200 μM) for 24 hours. Experiments were completed in BMDMS from 4 mice per genotype per group, with both sexes represented. Data are expressed as mean ± SD. Unpaired t-test, *p<0.05

APOL1 expression in BMDMs and iPSDM is increased with IFNγ.

(A) Gene expression of APOL1 measured with qRT-PCR in combined sex, female, and male BMDMs. (B) Quantification of APOL1 protein expressed measured with western blot in combined, female, and male BMDMs. (C) Flow cytometric analysis of CD11b, CD64, CD14, and CD16 surface expression on G1 iPSDM and G1 iPSCs. (D) Gene expression of APOL1 measured in G0 and G1 iPSDMs treated with IFNγ. (E) western blot of APOL1 in G0 and G1 iPSDMs treated with IFNγ. BMDM experiments were performed in BMDMs from 3-6 mice per genotype per group, with both sexes represented. (F) Gene expression of TNA, IL1B, and CCR7 measured with qRT-PCR in G0 and G1 iPSDMs treated with IFNγ (25 ng/mL), LPS (10 pg/mL), and IL-4 (10 ng/mL) for 24 hours. BMDM experiments in (A, B) were completed in BMDMs from 4 mice from each genotype and each sex. iPSDM experiments in (C, D) were performed in 2 separate differentiations across 2 independent experiments. Experiments in (E) were performed with 3 separate wells of iPSDM. Data are expressed as mean + SD. *p<0.05, **p<0.01 ***p<0.005 ****p>0.001. 2-way ANOVA with Tukey’s Multiple Comparison Test

G2 BMDMs from the C57Bl/6J background increase lipid accumulation with oxLDL incubation.

(A) Oil Red O (ORO) stained images of BMDMs in the presence or absence of 5ng/mL IFNγ and 50 μg/mL oxidized LDL (oxLDL) for 72 hours. BMDMs were generated from G0 and G2 mice from the C57Bl/6J background. (B) Quantification of the Oil Red O stain. Experiments were completed in BMDMs from 2-3 mice per genotype per group, with both sexes represented. Data are expressed as mean ± SD.

Cell viability of BMDMs treated with IFNγ is slightly decreased at 24 hours.

(A-C) Cell viability of BMDMs treated with 0-100 ng/mL IFNγ for 4, 8, and 24 hours measured with CellTiter-Glo. Experiments were performed using BMDMs from 8 mice across 2 independent experiments, with both sexes represented. Data are expressed as mean + SD.

qPCR array of immune genes in female and male BMDMs.

(A-B) qPCR array of immune-related genes in female and male BMDMs treated with IFNγ (5 ng/mL) and IL-10 (10 ng/mL). Experiment was completed in BMDMs from 2 mice per genotype per group.

NLRP3 inflammasome proteins are not activated with IFNγ treatment in APOL1 BMDMs.

(A) Representative western blot of NLRP3, AIM2, cleaved IL-1β, cleaved Caspase-1, and ACS in G0, G1 and G2 BMDMs treated with IFNγ (5 ng/mL) or control. Experiments were performed in BMDMs from 4 mice per genotype per group, with both sexes represented.

ER stress proteins and genes are not expressed in APOL1 BMDMs with IFNγ treatment.

Representative western blot of ATF4 (A), phospho-PERK (C), phospho-IRE1α (D) and RT-PCR of Spliced Xbp1 (B) in G0, G1 and G2 BMDMs treated with IFNγ (5 ng/mL). Experiments were completed in BMDMs from 4 mice per genotype per group, with both sexes represented.

Mitochondrial area is increased in G1 iPSDM compared to G0 G0 and G1 iPSDMs were treated with 20 ng/mL IFNγ for 8 hours.

TEM images were collected of at least 20 cells and over 100 mitochondria were quantified for area and circularity. (A) Representative images of mitochondria in control and treated iPSDM. (B, C) Quantification of mitochondrial area in iPSDM. (D, E) Quantification of mitochondrial circularity in iPSDM. Experiments were performed in iPSDM from 4 separate differentiations across 2 independent experiments. Data are expressed as median and interquartile range. Unpaired t test. ****p <0.001

Thallium flux in APOL1 BMDMs is unchanged with IFNγ and VX-147 treatment.

(A) Diagram of Thallium flux assay. (B) Slope of fluorescence after thallium addition to G0, G1 and G2 BMDMs treated with IFNγ (5 ng/mL), VX-147(1 μM) or DMSO control. Experiments were performed in BMDMs from 4 mice per genotype per group, with both sexes represented. Data are expressed as mean + SD. Unpaired t test. *p <0.05