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. Author manuscript; available in PMC: 2023 Mar 30.
Published in final edited form as: Kidney Int. 2022 Feb 1;101(6):1216–1231. doi: 10.1016/j.kint.2021.12.031

Figure 4 ∣. Endoplasmic reticulum (ER) resident APOL1 risk variants induce ER stress in nephrocytes.

Figure 4 ∣

(a–d”) Shown are equatorial cross-sections of garland cell nephrocytes from third instar larvae expressing various APOL1 transgenes under control of dKlf15-GAL4. Cells are costained for HA to visualize the tagged transgenes and the ER marker/ER stress response target gene PDI. All transgenes show complete colocalization with the ER marker, including ΔBH3-G2-APOL1 (a–a″), G0-APOL1 (b–b″), G1-APOL1 (c–c″), and G2-APOL1 (d–d″). The renal risk variants reveal an enhanced signal from the protein disulfide-isomerase (PDI) antibody, suggesting a greater abundance of PDI as a result of ER stress induction. All flies were raised at 29 °C. Bar = 10 μm, and nuclei are marked by Hoechst 33342 in blue here and throughout the figure. (e) Quantitation of fluorescence intensity of signal derived from PDI staining is shown for confocal images analogous to (a–d). For quantitation, the fluorescence intensity of PDI was measured within the area of the cell that was strongly hemagglutinin (HA) positive. Wild-type and renal risk variants are compared with the control transgene ΔBH3-G2-APOL1 (n = 7–9; P > 0.05 for G0-APOL1, P < 0.001 for G1-APOL1, and P < 0.0001 for G2-APOL1). (f,g) Shown is fluorescein isothiocyanate (FITC)–albumin endocytosis after 30 seconds exposure as readout of nephrocyte function for garland cell nephrocytes from control animals exposed to ER stress inducer tunicamycin or vehicle control (dimethylsulfoxide). Exposure to ER stress inducer tunicamycin for 5 hours results in increased uptake of FITC-albumin (g) compared with treatment with vehicle alone (f). (h) Quantitation of data in (f) through (g) (mean fluorescence per animal in ratio to control experiment is shown; n = 8 animals per condition; P < 0.001). (i–i″) Equatorial cross-section of wild-type garland cell nephrocytes dissected from a larva treated with tunicamycin for 5 hours shows regular staining pattern of the slit diaphragm proteins Sns (green) and Kirre (red). Insets show a magnified detail of slit diaphragms (left) and tangential sections revealing the regular fingerprint-like staining pattern (right). Bar = 10 μm, and nuclei are marked by Hoechst 33342 in blue here. Flies were raised at 29 °C. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.