Figure 6. OPN deficiency reduces DNM3-mediated cholesterol influx.

(A and B) Osteopontin (OPN) recombinant protein treatment causes increase of dynamin-3 (DNM3) protein expression in HK-2 cells. In panel B, n = 3–4 per group. Statistics based on ANOVA with Tukey’s post hoc test. (C and D) DNM3 was overexpressed by lentivirus in 293T and HK-2 cells and validated by qPCR. SCR, scrambled lentivirus. (E) DNM3 overexpression in HK-2 cells causes overexpression of LDL receptor (LDLR) protein as shown by Western blot. (F) Inhibition of dynamins by dynasore treatment in 30-minute TGF-β–stimulated HK-2 cells causes upregulation of PAI-1 and downregulation of LDLR protein levels 24 hours after treatment. (G) Inhibition of dynamins by dynasore treatment in 30-minute TGF-β–stimulated primary tubular epithelial cells (TECs) isolated from wild-type (WT) or Alport mice causes a decrease in LDLR protein levels 24 hours after treatment. In panels C–G, n = 2–4 per group. Statistics based on Student’s t test. n.d.u., normalized densitometry units. (H and I) LDLR protein expression is increased in Alport kidneys and normalized in Col4a3^–/– Opn^–/– kidneys. In panel I, n = 4–6 mice per group, and 4 images were quantified per mouse. Statistics based on ANOVA with Tukey’s post hoc test. Scale bars: 20 μm. (J) Infusion of 150 μg DiI-LDL cholesterol in Alport mice shows a significant 45.9-fold increase in LDL cholesterol uptake by Alport renal tubules after 2 hours. In panel J, n = 3 mice per group, and 3 images were quantified per mouse. Statistics based on Student’s t test. Scale bars: 20 μm. (K) Heterozygous deletion of LDLR in Alport mice tends to improve survival as shown by Kaplan-Meier curve. n = 8 Col4a3^–/– mice and n = 12 Col4a3^–/– Ldlr^–/– mice. All mice are littermates. For all, data are the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.