The Protective Roles of Estrogen Receptor β in Renal Calcium Oxalate Crystal Formation via Reducing the Liver Oxalate Biosynthesis and Renal Oxidative Stress-Mediated Cell Injury

Figure 2

Depletion of renal ERβ made renal epithelial cells more vulnerable to oxalate-induced ROS production and cell injury. (a) HK-2 and HKC-8 cells were transduced with lentiviral shLuc (control) or shERα or shERβ and treated with normal DMEM media or DMEM media containing 0.75 mM oxalate for 6 hr, and the ROS production in the cells was detected by dihydroethidium (DHE) staining under a fluorescence microscope. The images are representative of typical staining. (b) Digital scans of DHE-stained cells were quantified using ImageJ software by comparing cells with shERα or shERβ to shLuc. One-way ANOVA was applied for statistic analysis. (c) Western blots show ERα or ERβ knockdown efficiency in HK-2 and HKC-8 cells. (d) Detection of H₂O₂ levels in culture media of the shERα or shERβ or control (shLuc) renal tubular epithelial cells after challenge with 0.75 mM oxalate for 6 hr; Student’s tests, compared to the shLuc group. (e) LDH release measurement in the ERα or ERβ knocked-down renal tubular epithelial cells treated with 0.75 mM oxalate for 6 hr. (f) The qRT-PCR analysis of inflammation-related gene expression in HK-2 and HKC-8 cells with/without sh ERβ after 0.75 mM oxalate treatment for 6 hr. (g–j) Effect of ER antagonists on oxalate induced oxidative stress and cell injury. HK-2 cells were exposed to DMSO (Ctrl), 0.75 mM oxalate, oxalate + 10 nM E2, oxalate + 10 nM E2 + 10 μM ICI, oxalate + 10 nM E2 + 10 μM MPP, or oxalate + 10 nM E2 + 10 μM PHTPP for 6 hr. (g) The ROS production in the cells was detected by DHE staining under a fluorescence microscope. The images are representative of typical staining. (h) Digital scans of DHE-stained cells were quantified using ImageJ software; one-way ANOVA, compared to the control group. (i) Detection of H₂O₂ levels in culture media of each group. (j) LDH release measurement in cells with different treatments. For (b)–(f) and (h)–(j), data are presented as . ns = not significant. , , , and .

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)