Fig 1.
APOL1 protein and mRNA expression in human kidney.
A. APOL1 protein expression by immunofluorescence microscopy using validated anti-APOL1 antibodies, with co-immunostaining with GLEPP1 to identify podocytes and DAPI as a nuclear stain. B. APOL1 mRNA expression by in situ hybridization. Expression was evident in podocytes (“P”) and vascular endothelia of glomerular capillaries (“E”), peritubular capillaries (“1”, arrows), and larger vessel (“2”) epithelia, but not tubular epithelia. Scale bar = 40μm.
Fig 2.
APOL1 protein expression in BAC-APOL1 transgenic mouse kidneys.
A. Immunofluorescent staining for APOL1, nephrin (to identify podocytes), and CD31 (to identify endothelia cells) in the BAC-APOL1 transgenic mouse kidney (G1 mouse is shown). B. Comparison of APOL1 expression patterns in all three APOL1 transgenic lines. Proximal tubules were identified by labeling with fluorescent Lotus tetragonolobus (LT) lectin. APOL1 was present in vascular endothelia (arrow heads), in podocytes (arrows), and trapped in vascular spaces (*), but not tubular epithelia. Scale bar = 40μm.
Fig 3.
APOL1 protein in circulation in the BAC-APOL1 transgenic mouse models.
Plasma APOL1 protein levels were measured by immunoassay in transgenic mice compared to age-matched wild-type (WT) littermates (WT, n = 10; G0, n = 6; G1, n = 7; G2, n = 10; all animals were male, approximately 21 weeks of age). APOL1 expression levels in each transgenic line were all significantly different than WT (P<0.0001). Data are mean±SD, one measurement per animal, with significance determined by one-way ANOVA.
Fig 4.
APOL1 mRNA expression in the BAC-APOL1 transgenic mouse models.
Duplex mRNA in situ hybridization for (A-D) APOL1 and nephrin (Nphs1) gene expression and (E-K) APOL1 and CD31 (Pecam1) expression to identified podocytes or endothelial cells respectively. APOL1 expression was detected in peritubular capillaries (“1” arrows), arterioles (“2” arrows), glomerular capillaries (“3” arrows), podocytes (“4” arrows), and larger (interlobular) arteries (“5”) and veins (“6”) but not proximal tubules (“PT”) or any other tubular segment. Images shown are from BAC-APOL1-G0 and BAC-APOL1-G1 mice.
Fig 5.
BAC-APOL1 transgenic mouse models developed similar proteinuria when intercrossed with the HIVAN mouse model.
A. Example of proteinuria in single and dual transgenic mice assessed by gel electrophoresis of urine (Coomassie stain). Arrowhead marks albumin, and additional low molecular weight urinary proteins (asterisk) are a normal finding in mice. B. Western blot of mouse serum in the same single and dual transgenic mice showing maintenance of high levels of serum APOL1 protein in the setting of proteinuria. APOA1 Western blot as a control for a common serum protein that is freely filtered. Representative blot is shown; number of animal examined in each genotype group were: wildtype, n = 3; HIVAN4, n = 6; G0 x HIVAN4, n = 6; G1 x HIVAN4, n = 3; G2 x HIVAN4, n = 4.
Fig 6.
APOL1 does not appear in proximal tubules in proteinuric BAC-APOL1 transgenic mice.
A. Control immunostaining in non-transgenic (wildtype) and proteinuric HIVAN4 mice for APOL1 along with fluorescent Lotus tetragonolobus (“LT”) lectin binding to demarcate the proximal tubule brush border. Since wildtype and HIVAN4 mice do not have APOL1, the staining observed in parietal cells (arrows) is artifact. B. Immunostaining for APOL1 in proteinuric BAC transgenic mice of each APOL1 genotype (representative images are shown, number of animal examined in each genotype group were the same as for Fig 5). Images show proximal tubules at the transition with Bowman capsule. The boxed region is magnified below each panel along with the isolated fluorescent channels shown in black and white. C. Positive control immunostaining for a filtered lipoprotein, APOA1, and fluorescently-labelled Lotus tetragonolobus (“LT”) lectin. An APOL1-G0 mouse and an APOL1-G0 x HIVAN4 dual transgenic mouse with proteinuria is shown; below each respective color panel is the individual fluorescent channels (in black and white) of the boxed region for either LT lectin or APOA1. White arrows mark glomerular capillaries containing circulating APOA1 protein within capillary lumens, red arrows denote APOA1 in protein reabsorption droplets at the brush border of proximal tubules. Scale bar = 40μm.