Androgen Receptor Enhances Kidney Stone-CaOx Crystal Formation via Modulation of Oxalate Biosynthesis & Oxidative Stress

Males develop kidney stones far more frequently than females with a ratio of 2–3:1, suggesting that androgen receptor (AR) signaling might play a key role in the development of nephrolithiasis. Using the cre-loxP system to selectively knock out AR in glyoxylate-induced calcium oxalate (CaOx) crystal mouse models, we found that the mice lacking hepatic AR had less oxalate biosynthesis, which might lead to lower CaOx crystal formation, and that the mice lacking kidney proximal or distal epithelial AR also had lower CaOx crystal formation. We found that AR could directly up-regulate hepatic glycolate oxidase and kidney epithelial NADPH oxidase subunit p22-PHOX at the transcriptional level. This up-regulation might then increase oxalate biosynthesis and oxidative stress that resulted in induction of kidney tubular injury. Targeting AR with the AR degradation enhancer ASC-J9 led to suppression of CaOx crystal formation via modulation of oxalate biosynthesis and oxidative stress in both in vitro and in vivo studies. Taken together, these results established the roles of AR in CaOx crystal formation.


RNA Extraction and qPCR Analysis
Total RNAs were isolated from cells or tissues using Trizol reagent (Invitrogen, Grand Island, NY) according to the manufacturer's instructions. 1 µg of total RNA was subjected to reverse transcription using Superscript III transcriptase (Invitrogen, Grand Island, NY). Quantitative real-time PCR (qRT-PCR) was conducted using a Bio-Rad CFX96 system with SYBR green to determine the level of mRNA expression of a gene of interest. Expression levels were normalized to the expression of GAPDH mRNA.

Serum testosterone concentration detection
Mice were killed at the indicated time points, drew 0.6-0.8 mL of blood from the inferior cava vein, serum was seperated and immediately assayed for serum testosterone level using the Coat-ACount Total Testosterone radioimmunoassay (Diagnostic Automation, Inc, Calabasas, CA). A 50 µl sample in Triplicate was used for the assay. The procedure entails soli-phase redioimmunoassay based on hormone specific antibody immobilized to the wall of a polypropylene tube. 125I-labeled testosterone competes for a fixed time with the specific hormone in the given sample for antibody sites. The tube is then decanted to separate bound from free and is then counted in a Cobra gamma counter.

hour mice urine collection
Experimental mice were separated into one metabolism cage (3 mice per cage). The mice have the free right for drinking and chowing. Urine was collected by the plastic tubes (under cages) from 8:00 p.m. to 8:00 a.m. covered with mineral oil.

Supplemental Figure 1 Positive control and negative control for the IHC staining of AR in the health and kidney stone patients.
IHC staining of positive control and negative control for the AR staining. Orthotropic tumors tissues of LNCap cells were used as positive control, no primary antibody control were used as negative control. Quantitation at right. (T: tumor, P: mouse prostate, G: glomerulus). Figure 2 High power view for CaOx crystal formation. H&E and Pizzolato staining showed the intratubular deposition of calcium oxalate crystal in the C57/B6 WT mouse kidney (400×). Glyoxylate solution (100 mg/kg) was i.p. injected to the C57/B6 WT mice every day for 7 days, followed with H&E and Pizzolato staining.

Supplemental
Supplemental Figure 3 Strategies for establishing different kinds of ARKO mice (General, liver specific, kidney proximal and distal/collecting tubular specific knockout mice). a, Breeding strategies. b, Genotyping results of the ARKO mice developed. c, AR expressions in liver and kidney tissues obtained from the ARKO mice and their WT littermate control mice. d, IHC staining of AR in liver and kidney tissues obtained from the ARKO mice and their WT littermate control mice(100×). (arrow p: proximal tubular , arrow d: distal tubular). e, Serum total testosterone measurements of the ARKO mice generated and WT littermate control mice.

Supplemental Figure 4. AR and downstream gene expression in human kidney stone patients.
The expression of AR and downstream gene (GO and p22-PHOX) in kidney tissues of male kidney stone patient and health males (H: health male; S: Kidney stone patient). Figure 5 Effect of ASC-J9 ® on serum testosterone concentration. ELISA assay analyzing serum testosterone concentration in the ASC-J9 ® treated mice.