Figure 7 ∣. G2-APOL1–dependent cell death is blunted by pharmacologic inhibition of endoplasmic reticulum (ER) stress.

(a–d) Terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) labeling of garland cell nephrocytes from third instar larvae is shown for G1-APOL1 (a,b) and G2-APOL1 (c,d). Animals were raised in liquid for 24 hours containing 4 mM 4-phenylbutyric acid (4-PBA; b,d) or vehicle alone (H₂O; a,c). TUNEL positivity is indicated by an intranuclear “+.” Outlines of nuclei are shown by dotted circles based on Hoechst 33342 staining (data not shown). Note that TUNEL-positive cells become less abundant on 4-PBA treatment. Bar = 10 μm. (e) Quantitation of data analogous to (a–d); the fraction of TUNEL-positive cells is shown (percentage); n = 10 to 13 animals per genotype and condition. P < 0.0001 for G1-APOL1 and P > 0.05 for G2-APOL1 comparing 4-PBA with control treatment. (f–g″) Larvae expressing G2-APOL1 under control of dKlf15-GAL4 were fed with yeast with addition of vehicle alone (H₂O; f–f″) or 4 mM 4-PBA (g–g”) for ~48 hours before reaching puparium. After eclosure, adult animals were dissected to stain adult nephrocytes for slit diaphragm protein polychaetoid (pyd) and HA tag. After mock treatment (H₂O), nephrocyte numbers were reduced on G2-APOL1 expression (compare Supplementary Figure S2E and F) and surviving nephrocytes mostly showed downregulation of the transgene (the typical situation with exclusively nonexpressing nephrocyte is shown in f–f″). In contrast, treatment with 4-PBA increased the number of surviving nephrocytes, further resulting in a higher number of cells expressing G2-APOL1 (g–g″). Bars = 10 μm. (h) Quantitation of data analogous to (f–g″) for the total number of nephrocytes per adult animal shows significant increase after attenuation of ER stress using 4-PBA (n = 8–12 per condition; P < 0.01). (i) Quantitation of data analogous to (f–g″) for the number of nephrocytes positive for HA signal, indicating transgene expression per adult animal shows significant increase after attenuation of ER stress using 4-PBA (n = 9–12 per condition; P < 0.05). (j–k‴) Larvae expressing G2-APOL1 using ptc-GAL4 in the wing disc were exposed to ER stress inhibitor 4-PBA in liquid food for 24 hours. Confocal imaging of wing disc after treatment reveals mild residual upregulation of protein disulfide-isomerase (PDI) and strongly reduced induction of apoptosis indicated by cleaved caspase-3 (cl. casp3). Pharmacologic inhibition of ER stress induction thus partially blocks induction of apoptosis associated with G2-APOL1 (j–j‴). Larvae expressing G2-APOL1 under ptc-GAL4 after treatment with vehicle alone (H₂O) in liquid food for 24 hours show robust upregulation of PDI and numerous cells that are positive for cleaved caspase-3 (j–k‴). Nuclei are marked by Hoechst 33342 in blue here. Bar = 50 μm. (l) Quantitation of data in (j–k‴) for fluorescence intensity of cleaved caspase-3 staining in ratio to the expression strength indicated by the HA signal shows significant reduction of cleaved caspase-3 signal (n = 6 per condition; P < 0.05). NS, not significant. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.