Journal: Nature medicine
Article Title: Transgenic Expression of Human APOL1 Risk Variants in Podocytes Induces Kidney Disease in Mice
Figure Lengend Snippet: Mice with podocyte specific APOL1 risk allele expression show increased podocyte loss, autophagy block and increased inflammatory cell death ( a ) Representative immunohistochemistry images of WT1 (upper), nephrin (middle) and TUNEL staining (lower) in NPHS1-rtTA/TRE-APOL1-G0/G1/G2 mouse kidney sections. ( n > 5 per line) Scale bars, 10μm. ( b ) Left panel: Quantification of stained cells per glomerulus of WT1 (a podocyte marker), in transgenic mice. n = 11 analyzed images per condition. Student’s t- test, P =0.0036 compared to NPHS1-rtTA/TRE-APOL1-G0 mice and single transgenic control mice. Right panel: Quantification of stained cells per glomerulus of TUNEL stain from transgenic mice. n = 12 analyzed images per condition. Student’s t -test, P = 0.0011 compared to NPHS1-rtTA/TRE-APOL1-G0 mice and single transgenic control. ( c ) Representative transmission EM images of podocytes from transgenic mice showing increased numbers of MVB and autophagosomes. Inserts show multivesicular bodies (*) and amphisome-like structures (**). Scale bars, 500nm. ( d ) Quantification of autophagosomes (AP) and autolysosomes (AL) in transgenic mice. Note increased ratio of AP to AL in G1 and G2 mice compared to G0 mice. n > 50 analyzed organelles per each condition. Data are presented as means ± s.e.m. and Student’s t -test, P = 8.1816e-07 (left panel), 0.037 (right panel) compared to NPHS1-rtTA/TRE-APOL1-G0 mice. ( e ) Representative immunohistochemistry images of LC3II from transgenic mice. Note the increased podocyte stain in G1 and G2 mice compared to G0 mice, suggesting increase in autophagic vacuole content. ( n > 5 per line) Scale bars, 10μm. ( f–h ) Representative immunohistochemistry images of IL1β, NLRP3 and caspase1, showing increased stain of pyroptosis proteins in podocytes of G1 and G2 mice, compared to G0 mice. ( n > 5 per line) Scale bars, 10 μm. ( j ) Representative immunohistochemistry images of cleaved caspase3 from transgenic mice. Note lack of detectable stain, indicating apoptosis is not significantly induced in transgenic mice. ( n > 5 per line) Scale bars, 10 μm. ( i ) Differential expression of a set of genes by RNA-seq analysis in kidneys of NPHS1-rtTA/TRE-APOL1-G0/G1/G2 mice. Single transgenic littermates and NPHS1-rtTA/TRE-APOL1-G0 are controls.
Article Snippet: The following primary antibodies were used: GFP (immunohistochemistry (IHC), Clontech #632380, 1:500; western blot (WB), 1:3000), WT1 (IHC, Santa Cruz #M3561, 1:40), EEA1 (immunofluorescence (IF), BD transduction #610456, 1:100), Vamp8 (IF, Sigma #SAB1409943, 1:100), Rab7 (IF, Sigma #R8779, 1:100), Rab11 (IF, BD transduction #610656 1:100), LAMP2 (IF, BD transduction #555803, 1:100), LC3II (WB, Sigma #L7543, 1:1000; IF, cell signaling #27755, 1:100), STX17 (IF, Sigma #HPA001204, 1:100), APOL1 (WB, Sigma #HPA018885, 1:1000; IF, Sigma, 1:100), GM130 (IF, BD transduction #610822, 1:100), calnexin (IF, BD transduction #610523, 1:200), perilipin2 (IF, Sigma #611042, 1:500), Nephrin (IF, Fitzgerald Industries Intl Inc #20R-NP002, 1:100), cleaved-Caspase-3 (WB and IHC, cell signaling #9664, 1:1000), Caspase-1 (WB, Santa Cruz #SC515, 1:1000; IHC, Thermo Fisher #PA5-38099, 1:100), IL1β (WB, R & D #AF-201-NA, 1:1000; IHC, cell signaling #12242S, 1:100), NLRP3 (IHC, Novus #NBP2-12446, 1:100), α-tubulin (WB, Sigma #T6199, 1:3000).
Techniques: Mouse Assay, Expressing, Blocking Assay, Immunohistochemistry, TUNEL Assay, Staining, Marker, Transgenic Assay, Transmission Assay, RNA Sequencing Assay