Figure 3.

GalNAc Conjugation Attenuates the Cytotoxicity Potential of AONs in Renal Tubular Cells

(A) Test AONs and their respective targets and in vivo toxicity scores. (B and C) Measurement of intracellular ATP of PTECs (B) and accumulation of EGF in PTEC supernatants (C) showing concentration-dependent cytotoxicity of nephrotoxic AON-B and AON-C when applied in their naked form for 6 and 9 days, respectively, whereas their GalNAc-conjugated form showed flat EGF and ATP profiles, indicative of reduced cytotoxicity. Innocuous AON-A was used as a control and remained inert in both forms. Data represent means and SD (n = 3). Statistical significance over control was observed at 10, 30, and 100 μM for naked AON-B and AON-C (p < 0.0001) and at 30 and 100 μM for GalNac AON-C (p < 0.001); ANOVA with Dunnett’s multiple comparisons test. (D and E) Morphological alterations induced by 9 days of exposure to 100 μM of AONs are mitigated by GalNAc conjugation. In (D), representative high-content images are shown of AON-treated PTECs stained for actin (phalloidin, green) and nuclei (DRAQ5, red). Scale bars, 50 μm. In (E), quantification of AON-induced changes in RPTEC morphology expressed as cells retaining normal, vehicle-like morphology, nine fields per condition, is shown; data represent mean and SD (n = 3). Statistical significance over control was observed at 10 μM (p < 0.008), 30 μM, and 100 μM (p < 0.0001) for naked AON-B and AON-C and at 100 μM for GalNac AON-C (p < 0.0001); ANOVA with Dunnett’s multiple comparisons test.