Fig 1: APOL1 protein levels and zygosity influence toxicity; the presence of the APOL1 allele G0 does not provide protection from G1 toxicity. (A,B) Comparison of G2multi/G2multi and G2/G2 mice were compared regarding proteinurea (A) and survival (B). Quantification of the urinary albumin (Alb) to creatinine (Cre) ratio in mice before (BL) and 1 day after (D1) injection with pCpG-Mu? (A). Plotted are the mean values ±s.d. with individual data points. ****P<0.0001, two-way ANOVA with Tukey's multiple comparisons test comparing all means. (B) Plotted is the rate of survival for individual mouse lines as indicated in weekly increments up to week 8 (W8) post injection. CTRL, FVB control. Log-rank test P value <0.0001. A subset of the G2/G2 data are also shown in Fig. 3. (C,D) Albuminuria in G1 (C) and G2 (D) heterozygous (G0/G1 or G0/G2) and hemizygous (G1/- or G2/-) mice, with homozygous (G0/G0, G1/G1 or G2/G2) and control FVB mice (CTRL) for comparison. Individual data points are shown with splines modeling the data trends. †P<0.05 is different from G0/G0 and hemizygous mice; *P<0.05, **P<0.01, ***P<0.001, compared to G0/G0, hemizygous, and heterozygous mice; mixed-effects model with Tukey's multiple comparisons test. No differences were seen in heterozygous mice containing the G0 allele comprising both S342G and I384M versus those containing G0I384M, i.e. comprising only the I384M part of this allele (data not shown). (E) Albuminuria in G1/G1 and G1/G1/G0alt mice; the Alb:Cre ratio for G0alt/- mice is shown as a reference. No significant differences were observed between G1/G1 and G1/G1/G0alt at any time point; mixed-effect model with Tukey's multiple comparisons test. Individual data points are plotted with a smoothing spline to visualize the trend over time. Baseline measurements are shown on the y-axis.
Fig 2: G1 and G2 mice show evidence of podocyte loss. G0I384M/G0I384M, G1/G1 and G2/G2 mice were injected with pCpG-Muy for sustained levels of IFN-?. (A,B) Representative immunofluorescence images showing kidney tissue at week 1 and 3 post injection, immunostained for nephrin and APOL1 (Abcam) (A), and WT1 and APOL1 (Proteintech). (B). Asterisks indicate glomeruli without staining. Scale bars: 20 µM. (C) Quantification of WT1-stained cells in glomeruli of mice (as indicated) 1 and 3 weeks after injection. Each symbol represents one mouse (n=3 mice per genotype at each time point). Counts are the average number of WT1-positive cells in 35 glomeruli. **P<0.005, ****P<0.0001. At 3 weeks, G1/G1 and G2/G2 are significantly different from G0/G0, G1/G1 and G2/G2 mice at 1 week, and from G0/G0 at 3 weeks.
Fig 3: APOL1 transgenic mouse development and baseline proteinuria. (A) APOL1 transgenic mice were created using a 104 kb BAC that contains APOL1 (G0) and parts of APOL2 (exons 1-5) and MYH9 (exons 4-41). (B) APOL1 transgenic mice were originally created using a BAC that contains APOL1 (p.E150K haplotype). This BAC contained G0 APOL1; G1 and G2 variants were generated using site-directed mutagenesis. The BACs were injected into FVB/NJ oocytes resulting in three mouse lines, i.e. G0alt, G1E150K and G2multi. Of these, the G0alt mice were unable to breed homozygous and G2multi had multiple copies of the BAC. These two lines were used only used to address questions regarding APOL1 amounts and G0 rescue. The third line, G1, was further mutated using CRISPR, ultimately generating four additional lines: G0I384M and its follow-up line G0, as well as the G1 and G2 lines, all of which have a single copy of APOL1 integrated into the same chromosome 4 locus. These four mouse lines were used to assess differences between the APOL1 genotypes. Amino acids at the variable sites are shown for each mouse and the names used for each mouse line are indicated in the colored boxes. (C/D) Quantification of albuminuria in control and transgenic mice at 9-13 (C) and 38-42 weeks of age (D). Shown is the means±s.d. of individual data. No significant differences were found (one-way ANOVA with Tukey's multiple comparisons).
Fig 4: APOL1 expression is induced in podocytes following pCpG-Mu? injection. (A) APOL1 expression 1 day after hydrodynamic injection of pCpG-EV or pCpG-Mu? in G1 mice. Kidney tissue was co-stained for APOL1 (Abcam), the podocyte slit diaphragm marker nephrin and the endothelial cell marker endomucin. Scale bars: 50 µM. (B) SIM images of CTRL, G0/G0, G1/G1, and G2/G2 glomeruli 1 day after injection with pCpG-Mu?, co-stained with APOL1 (Abcam), nephrin, and endomucin. Scale bars: 5 µM.
Fig 5: Injection of pCpG-Mu? induces expression of APOL1 in glomeruli. Mice were hydrodynamically injected with 0.3 µg of pCpG-Mu? (IFN-?) for sustained levels of IFN-? or with 0.3 µg pCpG-EV (EV) as control. (A) Serum IFN-? levels measured using ELISA following injection of pCpG-Mu?. Individual data points for all four transgenic genotypes plus non-transgenic FVB controls (CTRL) are shown, n numbers are shown in the figure legend. IFN-? levels before-injection (day 0) were undetectable (not shown). (B) Western blots, showing APOL1 levels (Abcam) in G0I384M kidney, liver and plasma 24 h after injection of EV (n=3), IFN-? (n=3), or without injection (No Inj., n=2). Loading controls were GAPDH (kidney), vinculin (liver) and transferrin (plasma). (C) Levels of APOL1 (Sigma-Aldrich) in glomeruli of G1 mice 24 h and 7 days after injection of pCpG-Mu? (IFN) or pCpG-EV (EV); n=2. (D) Levels of glomerular APOL1 (Sigma-Aldrich) at baseline and 24-h after pCpG-Mu? injection (G0/G0 here is G0I384M/G0I384M); n=2 for G0/G0, n=3 for G1/G1 and G2/G2). In C and D, nephrin is shown as a control for glomerular preparation. (E) Chromatin immunoprecipitation was performed on podocytes derived from G0/G0 mice using antibody against IRF1 (D5E4, Cell Signaling Technology) or Rabbit IgG (2729S, Cell signaling Technology). Fold-enrichment over IgG is shown at baseline or at 6 h after treatment with 10 ng/ml IFN?. n=3, ***-P<0.0005, Student's t-test. (F) In situ hybridization of APOL1 in kidney of G1/G1 mice 24-h after pCpG-Mu? injection. Images of FVB control and non-injected G1/G1 mice are shown in Fig. S7.
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