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 5

In vivo mouse model data confirm that ERβ deficiency promotes renal CaOx crystal deposition via increasing the hepatic oxalate biosynthesis and renal oxidative stress. (a) Diagram describing the injection schedule for mock control, a selective ERβ antagonist PHTPP, and glyoxylate (upper panel). Crystal staining (lower panels) showing the deposition of CaOx crystals in kidney tissues of the PHTPP-treated and mock-control mice. CaOx crystal formation was detected by Pizzolato staining. Crystallization in each kidney section was quantified by calculating the ratio (percent) of the area containing crystals to the entire kidney section using ImageJ software. (b) Staining (left panels) and quantification (right panels) results for CaOx crystal deposition as detected by Pizzolato staining in kidney tissues of the ERβKO and their WT female littermate mice. (c) Detection of 24 hr oxalate excretion in urine samples collected from the ERβKO and WT female mice prior to sacrifice. (d) Western blot of 60 μg liver protein from ERβKO and WT mice probed with affinity-purified rabbit antibody raised against recombinant mouse AGT1 shows lower expression of AGT1 in the ERβKO mice. (e) Fresh-frozen renal tissues were stained for 30 min with DHE. Representative sections from the ERβKO and their WT female littermate mice are shown (left panels). Right bar graphs show quantification of superoxide generation. (f) H₂O₂ concentration in urine sample from ERβKO and WT female mice. (g) Representative micrographs of NOX2 immunostaining in renal tissue from the ERβKO and their WT littermate mice. The NOX2 protein was mainly located in renal tubular cells. Quantification of NOX2 is shown in the right panel. Scores were classified as 0 to 3, based on the intensity of staining and the percentage of positive cells. (h) Inhibitory effect of apocynin treatment in mice. CaOx crystal formation in ERβKO mice was significantly decreased after in vivo apocynin treatment. (i) Apocynin treatment reduced H₂O₂ concentration in urine samples from ERβKO mice. For (a)–(c) and (e)–(i), data are presented as . and .

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