Urinary Oxalate Excretion Decreased in Androgen Receptor-Knockout Mice by Suppressing Oxalate Synthesis in the Liver ()
1. Introduction
The incidence of idiopathic calcium oxalate (CaOx) renal stone disease in men is 2.5 - 3 times higher than that in women [1] [2] . Although the reason for the gender disparity remains unclear, the several studies have demonstrated that urinary excretion of calcium, oxalate, and uric acid is higher, and that of citrate is lower, in males than in females [3] [4] . Among these substances, urinary oxalate is a strong dicarboxylic organic acid that forms insoluble salt with calcium, and is one of the most important factors in calcium oxalate kidney stone formation [5] [6] . The sources of urinary oxalate are considered to be hepatic synthesis and the diet. In humans, the absorption of dietary oxalate constitutes 30% - 50% of urinary oxalate excretion [7] [8] , and hepatic synthesis is thus considered important. In the liver, oxalate is produced from glycolate via glyoxylate in the hepatic peroxysomes, and this oxidation is catalyzed by glycolate oxidase (GO), lactate dehydrogenase and xanthine oxidase [9] -[11] . Meanwhile, glyoxylate is metabolized to glycine by serine pyruvate aminotransferase/alanine: glyoxylate amino transferase (SPT/AGT), which is thought to inhibit oxalate synthesis [12] .
Several experimental studies have been conducted to explain gender differences in the prevalence of urolithiasis [13] -[19] . Lee et al. demonstrated that urolithiasis was generated in intact male rats, but not female rats, after being fed ethylene glycol (EG), the precursor of oxalate [13] . They also demonstrated that the castrated male rats fed EG dramatically decreased the incidence of renal stones. In addition, they evaluated the contribution of sex hormones in urolithiasis by subcutaneously implanting testosterone into intact and castrated male and oophorectomized female rats, and found that exogenous testosterone could restore kidney stone forming tendency in EG-fed castrated rats and enhance stone formation in oophorectomized female rats [14] . Furthermore, Yoshihara et al. showed that increased urinary oxalate excretion is associated with testosterone-promoted enhanced GO activity [15] . On the other hand, estrogen inhibits urinary oxalate excretion [16] -[19] . These studies suggested that androgens are associated with renal CaOx stone formation by increasing urinary oxalate excretion through hepatic synthesis. Androgens are essential for the differentiation and growth of male reproductive organs as well as for various biological effects in the kidney, brain, liver, muscle, bone, and skin. In males, testosterone is the major circulating androgen, and 95% of it is bound by sex hormone-binding protein [20] . The most biologically active form of endogenous androgens is 5α-dihydrotestosterone (5α-DHT), which is converted from its precursor, testosterone, by the action of a P450 family member enzyme, 3-oxo-5α-steroid 4-dehydrogenase, commonly known as 5α-reductase [21] . Testosterone is approximately 100-fold less biologically active than 5α-DHT [21] . Free testosterone (1% - 2% of total testosterone) diffuses into target cells, is converted to 5α-DHT and binds to androgen recdeptor (AR), a nuclear receptor, which regulates androgen signaling [22] . Although the androgens are play an important role in renal CaOx stone formation, the role of AR in oxalate metabolism has not been well studied.
In the present study, we used the androgen receptor (AR)―knockout (KO) mice generated by means of Cre-loxP system [23] . To assess how AR is related to oxalate metabolisms, we evaluated urinary oxalate levels and the expression of hepatic GO and AGT mRNA during EG loading in ARKO mice. In addition, we also assessed the urinary excretion of other kidney stone-related substances, including calcium, inorganic phosphorus, citrate, and uric acid, and calculated the ion-actibity products of calcium oxalate (APCaOx) in mouse, AP(CaOx)- indexMOUSE index, to evaluate the risk of in vivo crystallization of urinary calcium oxalate in both control mice and ARKO mice.
2. Materials and Methods
2.1. Generating ARKO Mice
The genomic AR DNA fragment was isolated from a TT2 embryonic stem (ES) cell genomic library using human AR A/B domain cDNA as a probe. The targeting vector consisted of a 7.6-kb 5′ region containing exon-1, a 1.3-kb 3′ homologous region, a single loxP site, and a neo cassette with two loxP sites [24] . Targeted clones (FB-18 and FC-61) were aggregated with single eight-cell embryos from CD-1 mice to generate chimeras, as described previously [23] [25] , and floxed AR mice (C57BL/6) were obtained. The floxed AR mice were then crossed with CMV-Cre transgenic mice provided by Pierre Chambon et al. [26] . The chromosomal sex of each pup was determined by genomic polymerase chain reaction (PCR) analysis of the Y chromosome Sry gene, which is expressed in male urogenital ridge at a time consistent with its having a role in testis determination [27] . The genotype of animals was then confirmed by Southern blotting. We mated male ARL+/Y mouse with female CMV-Cre-ARL−/+ mouse. We gained AR+/Y, CMV-Cre AR+/Y, ARL−/Y, CMV-Cre ARL−/Y, ARL−/L+, CMV-Cre ARL−/L−, ARL+/+, and CMV-Cre ARL−/+ [28] . ARL−/Y mice are infertile; therefore, we mated ARL+/Y mouse with ARL−/L+ mouse and gained ARL−/Y and ARL+/Y mice. ARL−/Y mice were used as ARKO mice, and male floxed AR mice (ARL+/Y) breeding pairs of the same genetic background were used as controls.
2.2. Animal Groups, Treatment and Urine Collection
All mice were fed a standard commercial diet (Rodent Diet CE-2; CLEA Japan, Tokyo, Japan) and given distilled water. At 14 weeks of age, five ARL−/Y and five ARL+/Y mice were weighed, and administered 0.5% ethylene glycol (EG) as drinking water for 28 days because glycolic acid oxidase is involoved in the metabolism of ethylene glycol to oxlate, and the activity may be enhanced by testosterone [16] . Using a metabolic cage, 24-h urine samples were collected from each mouse at three time points: before the administration of EG, after 14 days of treatment, and after 28 days of treatment. Urine samples were acidified with concentrated HCl to a pH < 3.0 for measurement of urinary oxalate. Next, groups of five ARL+/Y and ARL−/Y mice underwent orchiectomy (ORX) or sham operation, and were then subcutaneously implanted with 60-day sustained release dihydrotestosterone (DHT) pellets (10 mg; Innovative Research). These orchiectomized DHT-implanted ARL+/Y mice (ARL+/Y-ORX-DHT), and orchiectomized DHT-implanted ARL−/Y mice (ARL−/Y-ORX-DHT) were given a normal diet and distilled water for 7 days. The distilled water was then replaced with 0.5% EG for 28 days. Afterward, 24-h urine samples were collected and acidified with concentrated HCl at three points; before the administration of EG, after 14 days of treatment, and after 28 days of treatment.
2.3. Blood Collection, Removal of Kidney and Liver, Determination of Uriary Chemistry, Serum Testosterone and Luteinizing Hormone (LH)
Urinary oxalate levels were determined at all points using high-performance liquid chromatography (HPLC) [29] . Urinary citrate levels were also measured by HPLC before EG treatment and after 15 days of treatment. Urinary calcium, uric acid, and inorganic phosphorus levels were measured before EG treatment and 14 days after by the o-cresolphthalein complexone method [30] , uricase-POD method, and molybdic acid method, respectively. To evaluate the risk of in vitro crystallization of calcium oxalate in urine, AP(CaOx)-indexMOUSE index was then calculated on the basis of the urinary data obtained before and after 15 days of EG treatment, using the formula described by Tiselius et al. [31] ;
AP(CaOx)-indexMOUSE = 0.7 × [Calcium]1.05 × [Oxalate]0.95 (0.9 − 0.0225 × [Citrate]) + (6.6 × 10−8 × [Citrate]3.98), where [Calcium], [Oxalate], and [Citrate] are the measured urinary levels (mmol/L).
Mice were sacrificed after 28 days of EG treatment. Immediately after blood samples were obtained, both the kidneys and the liver were removed. The serum levels of testosterone and luteinizing hormone (LH) in ARL−/Y and ARL+/Y mice were measured by electrochemiluminescent immunoassay by using Eclusys testosterone kit® and Architect LH kit ® respectively. Serum creatinine concentration levels were determined using the alkalinel picrate method (SRL Ltd., Tokyo, Japan). One side of kidneys was cut longitudinally, fixed in 4% paraformaldehyde, and embedded in paraffin. For the observation of renal morphology, crystals or stones in renal pelvis, the paraffin-embedded kidneys were cut into 8-μm sections, stained with hematoxylin and eosin, and mounted on slides. The other kidney was used to assess calcium content. Briefly, the kidneys were minced in a beaker, and then 2 ml of 0.5-N nitric acid was added. The beaker was then heated until the liquid become transparent, and up to 5 ml of distilled water was added to the solvent extract, with stirring. After calibration using a standard calcium solution, the calcium content was determined by atomic absorption spectroscopy [32] . For measuring the mRNA expression of GO and AGT, the livers were placed under liquid nitrogen, and were stored at −70˚C until analysis.
All studies were conducted in an accredited animal care facility, and all protocols were approved by the Ethics Committee of the University of Tokyo, which is in accordance with the National Institute of Health guidelines for the care and use of laboratory animals. Carbon dioxide inhalation was used for the euthanasia of the mice.
2.4. Northern Blot Analysis
Frozen liver samples were homogenized in ISOGEN (Nippon gene, Japan), and total RNA was extracted following the manufacturer’s instructions. Oligo-dT-primed cDNA was synthesized from 1 µg of liver RNA from a wild-type male mouse (C57BL/6) using SuperScript reverse transcriptase (Gibco BRL, Gaithersburg, MD) in a 20-µl reaction volume. The cDNA probe templates were PCR-amplified from the cDNA. Primers were designed from cDNA sequences of mouse GAPDH, GO, and AGT using the primer pairs 5′-ACCACAGTCCATGCCAT CAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′ (437 bp), 5′-ATGTTGCCTCCGACTGGTCTG-3′ and 5′-TC AFATCTTGGAAACAGCCAAAGG-3′ (1113 bp), and 5′-AACCFFTATTGCTGTTCCTG-3′ and 5′-CTCCAG CACATAGCTGACGA-3′ (581 bp), respectively. The annealing temperatures were 60, 58, and 57 degrees Celsius, respectively. Digoxigenin (DIG)-labeled probes were created using a DIG DNA Labeling Mix (Roche Diagnostic K.K., Japan). Extracted total RNA was transferred onto nitrocellulose membranes, and membranes were pre-hybridized with DIG easy Hyb solution at 50˚C. Membranes were then hybridized overnight with each DIG labeled probe at 50˚C, washed, blocked using the DIG Wash and Block Buffer Set, incubated with Anti-DIG-AP, Fab fragments (Roche Diagnostic K.K), and detected using CDP-Star Reagent (New England Biolabs). RNA bands were quantified using the IMAGEJ software package and normalized by GAPDH [33] [34] .
2.5. Statistical Analyses
Statistical analysis was performed using unpaired Student’s t tests to evaluate the differences in serum LH, testosterone and urinary levels of substances between control and ARKO mice, one-way repeated measurement analysis of variance (ANOVA) to evaluate the effect of the ethylene glycol treatment within each group using the JMP computer statistical software. All values are expressed as mean ± standard errors.
3. Results
3.1. Renal Morphology and Function of ARKO Male Mice
ARL−/Y mice exhibited a female-typical external appearance. We confirmed that atrophic testes in 14-week-old ARL−/Y resulted in lower levels of testosterone (p = 0.052, statistically marginal significant) and higher levels of LH (p = 0.046) (Figure 1(a) and Figure 1(b)), as previously described for seven to twenty-week-old mice [35] . There were no significant differences between body weight, 24-h fluid intake, and urine pH between ARL−/Y and ARL+/Y at 14 week of age (Table 1). Kidneys and livers appeared normal macroscopically (data not shown). Histological analysis revealed no significant abnormality in the glomeruli, proximal tubules and collecting ducts (Figure 1(c)). In addition, the 24-h urine volume and the serum creatinine levels after 14 days of EG treatment was comparable between ARL−/Y and ARL+/Y (Table 1). This suggests that the kidney function of the ARL−/Y mice was in the normal range. As shown in Table 1, amount of urinary excretion of calcium, uric acid, inorganic phosphorus and citrate in ARL−/Y mice also did not differ from ARL+/Y mice, either before treatment or after 15 days of treatment.
3.2. Urinary Oxalate Excretion Was Suppressed in ARKO Mice
The 24-h urinary excretion of oxalate in ARL−/Y mice was less than that in ARL+/Y mice, but the difference was not statistically significant (p = 0.09, unpaired t-test) (Table 2). After treatment with EG for 14 days and 28 days, the excretion of oxalate was increased between 2.5 and 4.8 times in both ARL−/Y and ARL+/Y (p < 0.001, one way ANOVA) (Table 2). The amount of urinary oxalate excreted by ARL+/Y was significantly higher than urinary oxalate excreted by ARL−/Y (p < 0.05 at 14 days and 28 days unpaired t-test) (Table 2).
3.3. Urinary Oxalate Excretion Was Suppressed in ARKO Mice by ORX and DHT
Serum testosterone concentrations were decreased due to atrophic testes in ARL−/Y mice. To address this issue, we orchiectomized ARL+/Y or sham operated ARL−/Y mice, and then implanted DHT pellets subcutaneously. In mice fed a normal diet and distilled water, the level of urinary oxalate was 1.7 times higher in ARL+/Y-ORX-DHT mice than that in ARL−/Y-ORX-DHT mice but the difference was not statistically significant (p = 0.209, unpaired
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Figure 1. (a) Serum testosterone levels in the ARL+/Y mice and ARL−/Y mice; n = 5. Testosterone level was 8.58 times higher in the ARL+/Y mice; however, the difference was not statistically significant; (b) Serum luteinizing hormone levels in the ARL+/Y mice and ARL−/Y mice; n = 5. Luteinizing hormone level was significantly higher in the ARL−/Y mice (*, p < 0.05); (c) Representative microscopic photographs of hematoxylin and eosin stained proximal tubules or collecting ducts of the ARL+/Y mice and ARL−/Y mice. Pathologically, there were no remarkable differences between the ARL+/Y mice and ARL−/Y mice.
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Table 1. Body weight, urinary pH, serum and urinary chemistry of mice.
Unit of Body Weight: g. Unit of Serum: 10−6 mol/L, Urinary Creatinine: 10−3 mol/L. Unit of Urinary Volume and Fluid Intake: ml/24hour. Units of Urinary Calcium, Inorganic Phosphorus, Uric Acid and Citrate: 10−4 mol/24hour/kg. *T-test.
t-test) (Table 2). After 14 days and 28 days of EG treatment, the urinary excretion of oxalate was increased within each group (p < 0.001, one way ANOVA). ARL+/Y-ORX-DHT mice excreted oxalate 3.9 times more than ARL−/Y ORX-DHT mice after 14 days of treatment and 2.6 times more after 28 days of treatment (p < 0.005, unpaired t-test) (Table 2).
3.4. AP(CaOx)-IndexMOUSE Was Decreased in ARKO Mice
The AP(CaOx)-indexMOUSE index was calculated to evaluate the risk of in vivo crystallization of calcium oxalate in urine. ARL+/Y and AR+/Y-ORX-DHT exhibited significantly increased values of the AP(CaOx)-indexMOUSE index compared with ARL−/Y and ARL−/Y-ORX-DHT, respectively (p < 0.05, unpaired t-test) (Table 1). These results suggest that ARKO mice have less risk of crystallization of calcium oxalate by EG loading.
3.5. GO mRNA Levels Were Decreased in ARKO Mice
GO and AGT mRNA levels were assessed in livers by Northern blot hybridization. As shown in Figure 2, GO mRNA expression increased significantly in ARL+/Y-ORX-DHT mice compared with ARL−/Y-ORX-DHT mice (p < 0.05, unpaired t-test). However, the expression of AGT was comparable between ARL+/Y-ORX-DHT mice and ARL−/Y-ORX-DHT mice.
3.6. Calcium Deposits
We could not observe any crystals in the kidneys in both groups. We then assessed calcium deposits in the kidneys. Although we expected to observe greater amounts of calcium deposits in floxed AR mice than in ARKO mice, the difference was not significant (Figure 3).
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Table 2. Urinary Oxalate Excretion (10−6 mol/24hour/kg) before and after EG treatment.
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(a) (b)
Figure 2. (a) Northern blotting of RNA from livers of the ARL+/Y-ORX-DHT and ARL−/Y-ORX-DHT mice after ethylene glycol treatment. (b) Levels of AGT and GO relative to GAPDH in livers from the ARL+/Y-ORX-DHT and ARL−/Y-ORX-DHT mice, quantified from (a). There was no significant difference of AGT expression between ARL+/Y-ORX-DHT and ARL−/Y-ORX-DHT mice. GO expression level was significantly higher in ARL+/Y-ORX-DHT mice compared to ARL−/Y-ORX-DHT mice (*, p < 0.05).
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Figure 3. There was no significant difference between the levels of calcium deposits in kidneys after 28 days of EG treatment.
4. Discussion
In this study, we measured levels of urinary oxalate in the ARL+/Y and ARL−/Y mice fed normal diet and water, and found that urinary oxalate excretion was higher in the ARL+/Y mice compared with that in ARL−/Y mice, and treatment with EG exacerbated this difference. As the previous studies demonstrated [35] [36] , the testes of the ARL−/Y mice were atrophic, and their serum testosterone levels were significantly lower than serum testosterone levels in the AR+/Y mice. To equalize the serum androgen levels between the ARL+/Y and ARL−/Y, the ARL+/Y mice were ochiectomised, whereas the ARL−/Y mice were given sham operation because the testes of ARL−/Y mice were too atrophic to detect, and DHT pellets were then implanted. Even under the same DHT levels, urinary oxalate excretion was significantly higher in AR+/Y-ORX-DHT than in ARL−/Y-ORX-DHT mice after EG loading. This suggests that AR may play an important role in oxalate metabolism.
GOmRNA expression was increased significantly in the floxed AR mice compared to ARKO mice. These results suggest that DHT-activated GO may contribute to oxalate production and increased urinary oxalate excretion via AR signaling.
Liang et al. reported that by using the cre-loxP system to selectively knock out AR in glyoxylate-induced calcium oxalate (CaOx) crystal mouse models, they found that the mice lacking hepatic AR had less oxalate biosynthesis, which might lead to lower CaOx crystal formation, and also demonstrated that the mice lacking kidney proximal or distal epithelial AR also had lower CaOx crystal formation. They detected an AR biding site on the androgen response element (ARE) in the −626 to −611 bp of the 5’ promoter region of GO and performed promoter assay. They found that AR could induce GO expression at the transcriptional level [37] .
There are several other studies which indicate the function of stone-related protein using knockout mice, in which they reported that the administration of 1% ethylene glycol in free drinking or plus vitamin D3 could induce stone formation in knockout mice kidney, but not in wild-type mice [38] [39] . Okada et al. demonstrated that intraperitoneal injection of glyoxylate could induce kidney stone formation in wild-type mice [40] . In our study, we decided that intraperitoneal injection of glyoxylate may be too traumatic for the mice. Therefore, we chose administration of EG by drinking. Although the ARL+/Y and ARL+/Y-ORX-DHT mice exhibited increased urinary oxalate excretion by EG loading, the formation of kidney stones was not detected in these mice. Animal experiments to study the risk of in vivo crystallization of calcium oxalate in urine should be undertaken with the knowledge of iron-activity products. Recently Tiselius et al. described a simplified calculation to determine the AP(CaOx)-indexMOUSE index in mice [31] .
AP(CaOx)-indexMOUSE index of the ARL+/Y and ARL+/Y-ORX-DHT mice treated with EG were significantly increased than that of the ARL−/Y and ARL−/Y-ORX-DHT mice respectively. This finding suggests that ARKO mice are at less risk for the formation of CaOx urolithiasis than control mice.
The present study demonstrated that urinary calcium excretion did not differ between control and ARKO mice. However, gender difference in the renal handling of calcium has been previously reported. Male mice had higher urinary calcium excretion than female mice [41] [42] , and their renal calcium transporters were expressed at lower levels. In addition, androgen-deficient mice after orchiectomy also showed lower urinary calcium excretion than sham-operated male mice, but hypocalciuria was normalized by testosterone replacement therapy [43] . Interestingly, androgen deficiency increased the mRNA and protein expression of the renal luminal transient receptor potential vanilloid-subtype 5 (TRPV5) and intracellular calbindin-D28k transporters, which were then suppressed by testosterone treatment. However, in the present study urinary calcium excretion did not differ between the ARL+/Y and ARL−/Y, also ARL+/Y-ORX-DHT and ARL−/Y-ORX-DHT mice. No previous studies using ARKO mice to evaluate urinary calcium excretion have been reported. We previously used ARKO mice to demonstrate the suppressive function of the AR, which may accelerate calcium release from bone and potentially increase urinary calcium excretion [35] . Furthermore, it is unclear whether activated AR directly or indirectly decreases the expression of TRPV5 in mouse kidneys, because DHT did not modulate TRPV5 promoter activity in a luciferase assay [43] . An experimental study of renal calcium handling in ARKO mice would therefore be intriguing.
The exact mechanisms by which sex hormones have influence on the formation of renal stones are still unclear. Experimental studies using AR knockout mice may contribute to the elucidation of mechanisms of sex difference in morbidity of urolithiasis.
NOTES
*Corresponding author.