Cell Biochem Biophys DOI 10.1007/s12013-014-9939-y
ORIGINAL PAPER
Protein Expression of Urate Transporters in Renal Tissue of Patients with Uric Acid Nephrolithiasis Weihua Fu • Qianwei Li • Jiwei Yao • Ji Zheng • Lang Lang • Weibing Li • Junan Yan
Ó Springer Science+Business Media New York 2014
Abstract URAT1 and GLUT9 are two primary urate transporters involved in the renal urate handling. Renal urate underexcretion was reported in uric acid stone formers (UASF) in previous clinical studies. The aim of this study was to investigate the clinical features and possible impact of protein expression of URAT1 and GLUT9 in renal tissues of patients with uric acid (UA) nephrolithiasis. 23 UASF, 27 patients with calcium oxalate (CaOx) stones, and 22 normal controls were enrolled in this study. Clinical data revealed that older age of onset, high plasma UA concentration, low urinary PH, and relative renal urate underexcretion were associated with UASF. By immunohistochemical or western blotting analysis, a significant increase in the relative expression quantity of URAT1 in renal tissue of UASF was found compared to patients with CaOx nephrolithiasis and normal controls. In conclusion, our results suggested that upregulated URAT1 protein expression might contribute to the relative urate underexcretion from the kidney of UASF. Keywords GLUT9
Uric acid stone Nephrolithiasis URAT1
W. Fu Q. Li J. Yao J. Zheng L. Lang W. Li (&) J. Yan (&) Center of Urology, Southwest Hospital, Third Military Medical University, 30, GaoTanYan, Chongqing 400038, People’s Republic of China e-mail: [email protected]; [email protected] J. Yan e-mail: [email protected]
Introduction Urolithiasis is a common urologic disease, with prevalence ranging from 5 to 15 % in the western hemisphere [1]. Among them, uric acid (UA) nephrolithiasis accounts for approximately 10 % of all urinary calculi in the United States [2], and UA stone formers (UASF) are more common in the eastern and southern regions, such as Japan (16 %) and Israel (40 %) [3, 4]. Furthermore, increased prevalence is identified in patients with type 2 diabetes mellitus, obesity. or other metabolic syndrome [5]. Previous studies have also indicated that several risk factors are implicated in the development of UA stones, including genetic variations, dietary indiscretion, environmental, and racial factors [6]. Three major urinary abnormalities contribute to UA stones formation: acidic urine, hyperuricosuria, and low urine volume. However, the underlying pathogenesis of UA stone formation is heterogeneous and complex, which is not still incompletely understood. UA, a weak acid, is the end production of purine degradation in humans. The kidney plays a crucial role in UA homeostasis; more than 70 % of urate is excreted from the kidney [7]. UA is freely filtered through the glomerulus, followed sequentially by almost complete reabsorption, secretion, and post-secretory reabsorption via urate transporter proteins located in the proximal tubule. Renal urate handling is a complicated physiological process. In recent decade, research in this field has made exciting progress. One of the breakthroughs is that urate transporter 1 (URAT1, encoded by SLC22A12) is identified as the urateanion exchanger on the luminal membrane of the proximal tubule, which primarily mediates renal urate reabsorption into endothelial cells [8, 9]. Subsequently, glucose transporter 9 (Glut9, encoded by SLC2A9) transports urate into the blood on the basolateral membrane of epithelial cells
123
Cell Biochem Biophys
[10]. Renal net urate excretion is primarily determined by the function of urate reabsorption [11]. Molecular genetic studies confirmed that mutations in either URAT1 or GLUT9 were associated with renal hypouricemia and hyperuricosuria by impairing urate reabsorption [10, 12, 13]. Although, hyperuricosuria is recognized as a risk factor for the propensity toward formation of urate crystals and UA stones, which results from genetic variations of URAT1 or GLUT9. Hyperuricemia or gout has long been associated with UA stone formation [14]. Clinical gout caused by hyperuricemia can lead to urate crystals in the joints, and intuitively an increase in uric acid excretion from the kidney. However, renal urate underexcretion is found in approximately 90 % of patients with hyperuricemia and gout [15]. A recent study also suggested that impairment in urate excretion was associated with some UASF [16]. But the pathologic mechanism of urate underexcretion is still unclear. Whether the altered renal protein expression of urate transporters is implicated in the pathogenesis of UA nephrolithiasis needs further research. The aim of this study was to investigate the clinical features and impact of protein expression of urate transporters in renal tissues of patients with UA nephrolithiasis.
renal disorders. Moreover, all participants did not receive any medications that may affect calcium or UA metabolism before this study.
Study Protocol Data of demographic characteristics, clinical history, blood routine biochemistry (group IV excepted), and 24-h urine analysis (group III excepted) of the participants were collected at the beginning of this study. All urine and blood tests were performed under regular diet without medical interventions. Stone composition analysis was performed with LIIR-20 infrared spectroscopy (LANMODE, China). The removed renal tissues were regarded as normal controls in this study. A tissue sample of every patient with stones was obtained through percutaneous kidney biopsy with ultrasound guidance. Guide wire and series of Amplatz dilators were introduced through the aspirating needle, and the operative access of PCNL was built. These samples were randomly assigned to measure the expression of urate transporters by immunohistochemical or western blotting analysis.
Immunohistochemical Analysis Materials and Methods Study Participants The study was approved by the ethics committee of Southwest Hospital Affiliated to Third Military Medical University, and all participants provided informed consent voluntarily. All participants were male adult. Subjects involved in assessing the protein expression of urate transporters were divided into three groups: patients with UA or urate containing stones (group I, n = 23), patients with calcium oxalate (CaOx) stones (group II n = 27), and normal controls (group III n = 7). Patients with unilateral stones were recruited from inpatients of our center of urology from September 2012 to October 2013, who underwent percutaneous nephrolithotomy (PCNL) for stone removal. Patients with recurrent urinary stones were excluded. Seven controls were enrolled from our Center and Urology of Emergency Medical Center of Chongqing, China from March 2010 to October 2013, who received emergency partial or radical nephrectomy owing to acute renal trauma, and were healthy before accidents. In order to investigate metabolic characteristics of UASF, 15 healthy volunteers (group IV) were recruited from Medical Examination Center of our hospital to receive 24-h urine analysis. Cases were excluded for subjects with documented familial inherited, gastrointestinal, liver, or other
123
A total of 23 renal samples were measured in this section of experiment (7, 9, and 7 sample of group I, II, and III, respectively). The tissue was fixed with 4 % paraformaldehyde for 1 h at 4 °C after rinsing in 0.1 M phosphate buffered saline (PBS, pH 7.4), then dehydrated in a graded ethanol series, and then embedded in paraffin wax. Serial sections (4 lm in thickness) were cut and mounted on glass slides. The sections were dewaxed in xylene, rehydrated in a graded ethanol series, rinsed in 0.01 M PBS, incubated with endogenous peroxidase blocking solution (Maixin Bio, China) for 10 min at room temperature, and rinsed in 0.01 M PBS (3 9 10 min). After a blocking step with 10 ng/mL normal goat serum (Maixin Bio, China) for 15 min, the sections were, respectively, incubated with an anti-URAT1 primary antibody (dilution 1:400; HPA024575, Sigma, St. Louis, MO, USA) or an antiGLUT9 primary antibody (dilution 1:400; AV43974, Sigma, St. Louis, MO, USA) overnight at 4 °C. On the next day, samples were incubated with goat anti-rabbit peroxidase-conjugated Affinipure secondary antibody (dilution 1:200; ZSGB-Bio, China) for 30 min at room temperature. After nucleus stained with hematoxylin and mounted with neutral balsam, the sections were examined under a BX-51 light microscope (Olympus, Japan), and photomicrographs were taken using a DP-70 diGI tractal microphotography system (Olympus, Japan).
Cell Biochem Biophys
Western Blotting A total of 41 renal samples were analyzed in this section of experiment (16, 18, and 7 sample of group I, II, and III, respectively). Renal cortical tissues were homogenized with a Teflon-glass tissue homogenizer in a RIPA lysis buffer (Beyotime Biotechnology, China), containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 0.1 % SDS, 1 % Triton X-100, 1 % sodium deoxycholate, 2 mM EDTA, 1 mM Na3VO4, 5 mM NaF, and 2 lM leupeptin. The homogenates were centrifuged at 12,0009g for 15 min at 4 °C to collect total proteins. Protein concentrations were determined by the Bradford assay. The protein samples (20 lg) were separated by SDS-PAGE gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with in PBS containing 10 mM sodium phosphate, 0.5 mM NaCl, 10 % bovine serum albumin, 0.05 % Tween-20, and 5 % nonfat dry milk for 1 h, and then incubated with an anti-URAT1 antibody (dilution 1:1,000; SAB2103811, Sigma, St. Louis, MO, USA) or an antiGLUT9 antibody (dilution 1:1,000; AV43974, Sigma, St. Louis, MO, USA) or an anti-b-actin antibody (dilution 1:1,000; AV40173, Sigma, St. Louis, MO, USA) overnight at 4 °C, and finally incubated with a peroxidase-conjugated Affinipure second antibody (dilution 1:1,000; ZSGB-Bio, China) for 2 h at room temperature. The reactive bands were detected using chemiluminescent reagents (Pierce Biotechnology, USA) and an image analysis system (BioRad, USA). The relative protein expression quantities of URAT1 and GLUT9 in the renal samples were performed with the Quantity One software (Bio-Rad, USA). Statistical Analysis Continuous data were expressed as mean ± SD and categorical data in percentage. Statistical analysis was performed with the software of SPSS 15.0 (SPSS Inc, Chicago, IL, USA). Differences in the relative expression quantities of the urate transporter and the clinical data [including age, body mass index (BMI) and parameters of blood biochemistry and 24-h urinalysis] among four groups were estimated with t-test, and p \ 0.05 was considered statistically significant.
suggested the mean age, and BMI of UASF was significantly higher than other three groups (p \ 0.05), but they did not differ significantly among other three groups. Moreover, Obesity (BMI [ 30), overweight (BMI 25–30), diabetes, hypertension, and hyperuricemia were more prevalent in UASF group compared with other groups. According to the blood biochemical examination, higher plasma concentrations of creatinine and UA were observed in UASF group than other two groups (p \ 0.05). The results of 24-h urinalysis revealed that urine pH of UASF was significantly lower than patients with CaOx stones or normal controls (p \ 0.05), but statistical difference of 24-h urine volume and uric acid excretion was not found between UASF and healthy controls, similar results as 24-h calcium and oxalate excretion, which was significant more in group of CaOx stones than UA group and normal group (p \ 0.05). Expression of URAT1 and GLUT9 in the Renal Samples The expression level of URAT1 and GLUT9 protein in 57 renal tissue samples was measured by immunohistochemistry or western blotting analysis. Immunohistochemical results showed that the two transporters mainly localized in the renal tubular endothelium near glomerulus in the cortex. Stronger immunoreactive staining of URAT1 was observed in UASF group compared with the other two groups. However, the immunoreactive intensity of GLUT9 was not significantly different among renal samples of three groups in our study (Fig. 1). Experimental results of western blotting analysis supported this finding. Three immunopositive bands were observed at 55–60, 50–55, and 40–45 kDa which suggested URAT1, GLUT9, and b-actin protein, respectively. Semi-quantitative expression levels of URAT1 and GLUT9 relative to b-actin in the same sample were obtained to statistical analysis. The results also revealed that the relative expression quantity of URAT1 was significantly higher in renal samples of UASF group than of other two groups. The protein expression of GLUT9 was increased in renal tissues of UASF, but statistical differences among the three study groups were not found (Fig. 2).
Results
Discussion
Clinical Characteristics of Participants
In the present study, we investigated the clinical characteristics of UASF, in comparison to patients with CaOx stones and normal controls. Furthermore, the altered protein expression of URAT1 and GLUT9 in UASF was determined for the first time by immunohistochemistry and
This study included 50 patients with urolithiasis and 22 normal controls. Clinical characteristics of the participants are summarized in Table 1. The demographic data
123
Cell Biochem Biophys Table 1 Clinical characteristics of the participants in this study
Variable
Group I (n = 23)
Group II (n = 27)
Group III (n = 7)
Group III (n = 15)
57.21 ± 10.82*m
43.66 ± 12.59
42.87 ± 11.54
43.72 ± 10.59
BMI (kg/m )
25.28 ± 4.13*m
23.42 ± 3.34
23.57 ± 3.59
23.51 ± 3.77
Obesity, n (%)
5 (21.7)
1 (3.7)
0
0
Overweight, n (%)
8 (34.8)
8 (29.6)
1 (14.3)
2 (13.3)
Diabetes, n (%)
Demographic data Age (years) 2
3 (13.0)
0
0
0
Hyperuricemia, n (%)
12 (52.2)
8 (29.6)
0
0
Hypertension, n (%)
4 (17.4)
2 (7.4)
0
0
92 ± 25
68 ± 12
–
Blood biochemistry Creatinine (lmol/L)
134 ± 70*m
Uric acid (lmol/day)
437 ± 121*
Calcium (mmol/L)
2.23 ± 0.21
m
370 ± 103
319 ± 87
–
2.24 ± 0.23
2.32 ± 0.28
–
24-h urinalysis parameters
BMI body mass index * p \ 0.05 versus patients with CaOx stone (group II); m p \ 0.05 versus the normal controls (group III)
Urine volume (L)
1.73 ± 0.82
1.82 ± 0.76
–
1.69 ± 0.58
pH Uric acid (mg)
5.76 ± 0.43*m 689 ± 187
6.35 ± 0.46 714 ± 216
– –
6.22 ± 0.54 659 ± 149
Calcium (mg)
172 ± 91*
233 ± 117
–
159 ± 78
Oxalate (mg)
38.6 ± 21.1*
43.7 ± 18.7
–
37.4 ± 16.5
Creatinine (g)
1.73 ± 0.62
1.63 ± 0.52
–
1.79 ± 0.57
Fig. 1 Immunohistological detection of URAT1 and GLUT9 in renal tissues. Stronger immunoreactive staining of URAT1 was observed in samples of uric acid stone formers (UASF) (a) compared to calcium
oxalate stone formers (CaOxSF) (b) and normal controls (NC) (c). Significant difference in immunoreactive staining of GLUT9 was not observed in renal samples of three groups (d–f). 9400 magnification
western blotting. All participants are adult male in this study, in order to avoid the potential impact of gender differences, which are observed in the expression of urate transporters in mice [17]. The demographic data of patients with urolithiasis suggested that UASF were associated with older age of onset than patients with CaOx stone. Other studies have also pointed out the epidemiologic characteristic of UA stone [18, 19]. Goldfarb et al. [20] suggested that lower
urinary supersaturations resulting from abnormal urinary pH would contribute to the high incidence of UA stones in older population. In addition, metabolic syndrome is more common in older people, which is a recognized risk factor [21]. A series of studies have reported the correlation between metabolic factors and UA stone formation, for example, obesity, diabetes mellitus, and hypertension [22– 24]. In our study, it was also revealed that UASF was more often accompanied by metabolic disorders.
123
Cell Biochem Biophys
Fig. 2 Protein expression of URAT1 and GLUT9 in renal tissues was determined by Western blotting (a). b-actin served as an internal loading control for estimating relative protein expression quantities, which were presented as mean ± SE (b). Results of semi-quantitative
analysis suggested higher relative protein expression quantity of URAT1 in renal samples of uric acid stone formers (UASF) than that of calcium oxalate stone formers (CaOxSF) and normal controls (NC) (b). * p \ 0.05
24-h urinalysis plays an important role in evaluation of metabolic characteristics. Furthermore, variations in 24-h urinary parameters are associated with the specific types of stone [25, 26]. In our study, lower urinary pH was observed in UASF group, which supported low urinary pH as a major urinary risk factor for UA stone formation, and was consistent with the recent report [26]. However, we did not find significant difference in 24-h urine volume and UA excretion between UASF group and other two study groups, even though higher plasma UA concentration was found in UASF. Other investigators have noted the similar finding [16, 26]. It seems to be indicated that low urine volume and high renal UA excretion are of less importance than other risk factors in UA stone formation, although hyperuricosuria, due to genetic disorder of the renal urate transporters as mentioned above, is a recognized risk factor for UA stones. URAT1 and GLUT9, as the primary urate transporters, play important roles in modulating the renal urate handling, which has been confirmed through much genetic research, as mentioned above. However, few studies have concerned about the roles of urate transporters protein in renal tissues in pathological conditions. In this study, we proposed the hypotheses that altered protein expression of URAT1 and GLUT9 was involved in the unexpected renal UA excretion, which seemed to be rather increased due to high plasma UA concentration. Our findings suggested that protein expression of URAT1 was significantly increased in renal samples of UASF compared to patients with CaOx stones and normal controls. Chen et al. [27] reported that total saponin of Dioscorea increased renal UA excretion by inhibiting renal expression of URAT1 protein in chronic hyperuricemia rats. A recent study suggested that increased expression of GLUT9 protein could decrease urate
excretion from the kidney in the mouse [28]. Base on these findings, it was deduced that upregulated URAT1 protein might lead to the relative urate underexcretion from the kidney in UASF, despite of high plasma UA concentration. Altogether, our study showed the clinical characteristics of patients with UA nephrolithiasis. Older age of onset, high plasma UA concentration, low urinary PH, and relative renal urate underexcretion were associated with patients with UA stones. Furthermore, a significant increase in the relative expression quantity of URAT1 in renal tissue of UASF was revealed by immunohistochemical or western blotting analysis, compared to patients with CaOx stones and normal controls. We purpose that upregulated URAT1 protein expression might contributes to the relative urate underexcretion from the kidney of UASF. Whether other synergistic urate transporters are involved in the pathophysiologic mechanisms needs further research. Acknowledgments We thank Urology of Emergency Medical Center of Chongqing, China for providing renal samples. This study was supported by the Natural Science Foundation of Chongqing Science and Technology Commission (Chongqing, China; Grant No. 2011BA5011).
References 1. Ramello, A., Vitale, C., & Marangella, M. (2000). Epidemiology of nephrolithiasis. Journal of Nephrology, 13, S65–S70. 2. Mandel, N. S., & Mandel, G. S. (1989). Urinary tract stone disease in the United States veteran population II. Geographical analysis of variations in composition. Journal of Urology, 1989(142), 1516–1521. 3. Knoll, T., Schubert, A. B., Fahlenkamp, D., Leusmann, D. B., Wendt-Nordahl, G., & Schubert, G. (2011). Urolithiasis through
123
Cell Biochem Biophys
4.
5. 6.
7.
8.
9.
10.
11. 12.
13.
14.
15.
the ages: data on more than 200,000 urinary stone analyses. Journal of Urology, 185(4), 1304–1311. Shekarriz, B., & Stoller, M. L. (2002). Uric acid nephrolithiasis: Current concepts and controversies. Journal of Urology, 2002(168), 1307–1314. Maalouf, N. M. (2011). Metabolic syndrome and the genesis of uric acid stones. Journal of Renal Nutrition, 21(1), 128–131. Maalouf, N. M., Cameron, M. A., Moe, O. W., & Sakhaee, K. (2004). Novel insights into the pathogenesis of uric acid nephrolithiasis. Current Opinion in Nephrology and Hypertension, 13(2), 181–189. Maesaka, J. K., & Fishbane, S. (1998). Regulation of renal urate excretion: A critical review. American Journal of Kidney Diseases, 1998(32), 917–933. Enomoto, A., Kimura, H., Chairoungdua, A., Shigeta, Y., Jutabha, P., Cha, S. H., et al. (2002). Molecular identification of a renal urate anion exchanger that regulates blood urate levels. Nature, 417(6887), 447–452. Hediger, M. A., Johnson, R. J., Miyazaki, H., & Endou, H. (2005). Molecular physiology of urate transport. Physiology (Bethesda), 2005(20), 125–133. Preitner, F., Bonny, O., Laverrie`re, A., Rotman, S., Firsov, D., Da Costa, A., et al. (2009). Glut9 is a major regulator of urate homeostasis and its genetic inactivation induces hyperuricosuria and urate nephropathy. Proceedings of the National Academy of Sciences of the United States of America, 2009(106), 15501–15506. Lipkowitz, M. S. (2012). Regulation of uric acid excretion by the kidney. Current Rheumatology Reports, 14(2), 179–188. Ichida, K., Hosoyamada, M., Hisatome, I., Enomoto, A., Hikita, M., Endou, H., et al. (2004). Clinical and molecular analysis of patients with renal hypouricemia in Japan-influence of URAT1 gene on urinary urate excretion. Journal of the American Society of Nephrology, 2004(15), 164–173. Matsuo, H., Chiba, T., Nagamori, S., Nakayama, A., Domoto, H., Phetdee, K., et al. (2008). Mutations in glucose transporter 9 gene SLC2A9 cause renal hypouricemia. American Journal of Human Genetics, 2008(83), 744–751. Yu¨, T., & Gutman, A. B. (1967). Uric acid nephrolithiasis in gout. Predisposing factors. Annals of Internal Medicine, 67(6), 1133–1148. Terkeltaub, R., Bushinsky, D. A., & Becker, M. A. (2006). Recent developments in our understanding of the renal basis of hyperuricemia and the development of novel antihyperuricemic therapeutics. Arthritis Research & Therapy, 8(Suppl 1), S4.
123
16. Negri, A. L., Spivacow, R., Del Valle, E., Pinduli, I., Marino, A., Fradinger, E., et al. (2007). Clinical and biochemical profile of patients with ‘‘pure’’ uric acid nephrolithiasis compared with ‘‘pure’’ calcium oxalate stone formers. Urological Research, 35(5), 247–251. 17. Sekine, T., Miyazaki, H., & Endou, H. (2006). Molecular physiology of renal organic anion transporters. American Journal of Physiology-Renal Physiology, 290(2), F251–F261. 18. Knoll, T., Schubert, A. B., Fahlenkamp, D., Leusmann, D. B., Wendt-Nordahl, G., & Schubert, G. (2011). Urolithiasis through the ages: data on more than 200,000 urinary stone analyses. Journal of Urology, 185(4), 1304–1311. 19. Krambeck, A. E., Lieske, J. C., Li, X., Bergstralh, E. J., Melton, L. J, 3rd, & Rule, A. D. (2013). Effect of age on the clinical presentation of incident symptomatic urolithiasis in the general population. Journal of Urology, 189(1), 158–164. 20. Goldfarb, D. S., Parks, J. H., & Coe, F. L. (1998). Renal stone disease in older adults. Clinics in Geriatric Medicine, 14(2), 367–381. 21. Reynolds, K., & He, J. (2005). Epidemiology of the metabolic syndrome. American Journal of the Medical Sciences, 330(6), 273–279. 22. Kadlec, A. O., Greco, K., Fridirici, Z. C., Hart, S. T., Vellos, T., & Turk, T. M. (2012). Metabolic syndrome and urinary stone composition: What factors matter most? Urology, 80(4), 805–810. 23. Cho, S. T., Jung, S. I., Myung, S. C., & Kim, T. H. (2013). Correlation of metabolic syndrome with urinary stone composition. International Journal of Urology, 20(2), 208–213. 24. Daudon, M., Lacour, B., & Jungers, P. (2006). Influence of body size on urinary stone composition in men and women. Urological Research, 34(3), 193–199. 25. Parvin, M., Shakhssalim, N., Basiri, A., Miladipour, A. H., Golestan, B., Mohammadi Torbati, P., et al. (2011). The most important metabolic risk factors in recurrent urinary stone formers. Urology Journal, 8(2), 99–106. 26. Moreira, D. M., Friedlander, J. I., Hartman, C., Elsamra, S. E., Smith, A. D., & Okeke, Z. (2013). Using 24-hour urinalysis to predict stone type. Journal of Urology, 190(6), 2106–2111. 27. Chen, G. L., Zhu, L. R., Na, S., & Li, L. (2013). Effect of total saponin of dioscorea on chronic hyperuricemia and expression of URAT1 in rats. Zhongguo Zhong Yao Za Zhi, 38(14), 2348–2353. 28. Kimura, T., Amonpatumrat, S., Tsukada, A., Fukutomi, T., Jutabha, P., Thammapratip, T., et al. (2011). Increased expression of SLC2A9 decreases urate excretion from the kidney. Nucleosides, Nucleotides & Nucleic Acids, 30(12), 1295–1301.
ORIGINAL PAPER
Protein Expression of Urate Transporters in Renal Tissue of Patients with Uric Acid Nephrolithiasis Weihua Fu • Qianwei Li • Jiwei Yao • Ji Zheng • Lang Lang • Weibing Li • Junan Yan
Ó Springer Science+Business Media New York 2014
Abstract URAT1 and GLUT9 are two primary urate transporters involved in the renal urate handling. Renal urate underexcretion was reported in uric acid stone formers (UASF) in previous clinical studies. The aim of this study was to investigate the clinical features and possible impact of protein expression of URAT1 and GLUT9 in renal tissues of patients with uric acid (UA) nephrolithiasis. 23 UASF, 27 patients with calcium oxalate (CaOx) stones, and 22 normal controls were enrolled in this study. Clinical data revealed that older age of onset, high plasma UA concentration, low urinary PH, and relative renal urate underexcretion were associated with UASF. By immunohistochemical or western blotting analysis, a significant increase in the relative expression quantity of URAT1 in renal tissue of UASF was found compared to patients with CaOx nephrolithiasis and normal controls. In conclusion, our results suggested that upregulated URAT1 protein expression might contribute to the relative urate underexcretion from the kidney of UASF. Keywords GLUT9
Uric acid stone Nephrolithiasis URAT1
W. Fu Q. Li J. Yao J. Zheng L. Lang W. Li (&) J. Yan (&) Center of Urology, Southwest Hospital, Third Military Medical University, 30, GaoTanYan, Chongqing 400038, People’s Republic of China e-mail: [email protected]; [email protected] J. Yan e-mail: [email protected]
Introduction Urolithiasis is a common urologic disease, with prevalence ranging from 5 to 15 % in the western hemisphere [1]. Among them, uric acid (UA) nephrolithiasis accounts for approximately 10 % of all urinary calculi in the United States [2], and UA stone formers (UASF) are more common in the eastern and southern regions, such as Japan (16 %) and Israel (40 %) [3, 4]. Furthermore, increased prevalence is identified in patients with type 2 diabetes mellitus, obesity. or other metabolic syndrome [5]. Previous studies have also indicated that several risk factors are implicated in the development of UA stones, including genetic variations, dietary indiscretion, environmental, and racial factors [6]. Three major urinary abnormalities contribute to UA stones formation: acidic urine, hyperuricosuria, and low urine volume. However, the underlying pathogenesis of UA stone formation is heterogeneous and complex, which is not still incompletely understood. UA, a weak acid, is the end production of purine degradation in humans. The kidney plays a crucial role in UA homeostasis; more than 70 % of urate is excreted from the kidney [7]. UA is freely filtered through the glomerulus, followed sequentially by almost complete reabsorption, secretion, and post-secretory reabsorption via urate transporter proteins located in the proximal tubule. Renal urate handling is a complicated physiological process. In recent decade, research in this field has made exciting progress. One of the breakthroughs is that urate transporter 1 (URAT1, encoded by SLC22A12) is identified as the urateanion exchanger on the luminal membrane of the proximal tubule, which primarily mediates renal urate reabsorption into endothelial cells [8, 9]. Subsequently, glucose transporter 9 (Glut9, encoded by SLC2A9) transports urate into the blood on the basolateral membrane of epithelial cells
123
Cell Biochem Biophys
[10]. Renal net urate excretion is primarily determined by the function of urate reabsorption [11]. Molecular genetic studies confirmed that mutations in either URAT1 or GLUT9 were associated with renal hypouricemia and hyperuricosuria by impairing urate reabsorption [10, 12, 13]. Although, hyperuricosuria is recognized as a risk factor for the propensity toward formation of urate crystals and UA stones, which results from genetic variations of URAT1 or GLUT9. Hyperuricemia or gout has long been associated with UA stone formation [14]. Clinical gout caused by hyperuricemia can lead to urate crystals in the joints, and intuitively an increase in uric acid excretion from the kidney. However, renal urate underexcretion is found in approximately 90 % of patients with hyperuricemia and gout [15]. A recent study also suggested that impairment in urate excretion was associated with some UASF [16]. But the pathologic mechanism of urate underexcretion is still unclear. Whether the altered renal protein expression of urate transporters is implicated in the pathogenesis of UA nephrolithiasis needs further research. The aim of this study was to investigate the clinical features and impact of protein expression of urate transporters in renal tissues of patients with UA nephrolithiasis.
renal disorders. Moreover, all participants did not receive any medications that may affect calcium or UA metabolism before this study.
Study Protocol Data of demographic characteristics, clinical history, blood routine biochemistry (group IV excepted), and 24-h urine analysis (group III excepted) of the participants were collected at the beginning of this study. All urine and blood tests were performed under regular diet without medical interventions. Stone composition analysis was performed with LIIR-20 infrared spectroscopy (LANMODE, China). The removed renal tissues were regarded as normal controls in this study. A tissue sample of every patient with stones was obtained through percutaneous kidney biopsy with ultrasound guidance. Guide wire and series of Amplatz dilators were introduced through the aspirating needle, and the operative access of PCNL was built. These samples were randomly assigned to measure the expression of urate transporters by immunohistochemical or western blotting analysis.
Immunohistochemical Analysis Materials and Methods Study Participants The study was approved by the ethics committee of Southwest Hospital Affiliated to Third Military Medical University, and all participants provided informed consent voluntarily. All participants were male adult. Subjects involved in assessing the protein expression of urate transporters were divided into three groups: patients with UA or urate containing stones (group I, n = 23), patients with calcium oxalate (CaOx) stones (group II n = 27), and normal controls (group III n = 7). Patients with unilateral stones were recruited from inpatients of our center of urology from September 2012 to October 2013, who underwent percutaneous nephrolithotomy (PCNL) for stone removal. Patients with recurrent urinary stones were excluded. Seven controls were enrolled from our Center and Urology of Emergency Medical Center of Chongqing, China from March 2010 to October 2013, who received emergency partial or radical nephrectomy owing to acute renal trauma, and were healthy before accidents. In order to investigate metabolic characteristics of UASF, 15 healthy volunteers (group IV) were recruited from Medical Examination Center of our hospital to receive 24-h urine analysis. Cases were excluded for subjects with documented familial inherited, gastrointestinal, liver, or other
123
A total of 23 renal samples were measured in this section of experiment (7, 9, and 7 sample of group I, II, and III, respectively). The tissue was fixed with 4 % paraformaldehyde for 1 h at 4 °C after rinsing in 0.1 M phosphate buffered saline (PBS, pH 7.4), then dehydrated in a graded ethanol series, and then embedded in paraffin wax. Serial sections (4 lm in thickness) were cut and mounted on glass slides. The sections were dewaxed in xylene, rehydrated in a graded ethanol series, rinsed in 0.01 M PBS, incubated with endogenous peroxidase blocking solution (Maixin Bio, China) for 10 min at room temperature, and rinsed in 0.01 M PBS (3 9 10 min). After a blocking step with 10 ng/mL normal goat serum (Maixin Bio, China) for 15 min, the sections were, respectively, incubated with an anti-URAT1 primary antibody (dilution 1:400; HPA024575, Sigma, St. Louis, MO, USA) or an antiGLUT9 primary antibody (dilution 1:400; AV43974, Sigma, St. Louis, MO, USA) overnight at 4 °C. On the next day, samples were incubated with goat anti-rabbit peroxidase-conjugated Affinipure secondary antibody (dilution 1:200; ZSGB-Bio, China) for 30 min at room temperature. After nucleus stained with hematoxylin and mounted with neutral balsam, the sections were examined under a BX-51 light microscope (Olympus, Japan), and photomicrographs were taken using a DP-70 diGI tractal microphotography system (Olympus, Japan).
Cell Biochem Biophys
Western Blotting A total of 41 renal samples were analyzed in this section of experiment (16, 18, and 7 sample of group I, II, and III, respectively). Renal cortical tissues were homogenized with a Teflon-glass tissue homogenizer in a RIPA lysis buffer (Beyotime Biotechnology, China), containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 0.1 % SDS, 1 % Triton X-100, 1 % sodium deoxycholate, 2 mM EDTA, 1 mM Na3VO4, 5 mM NaF, and 2 lM leupeptin. The homogenates were centrifuged at 12,0009g for 15 min at 4 °C to collect total proteins. Protein concentrations were determined by the Bradford assay. The protein samples (20 lg) were separated by SDS-PAGE gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with in PBS containing 10 mM sodium phosphate, 0.5 mM NaCl, 10 % bovine serum albumin, 0.05 % Tween-20, and 5 % nonfat dry milk for 1 h, and then incubated with an anti-URAT1 antibody (dilution 1:1,000; SAB2103811, Sigma, St. Louis, MO, USA) or an antiGLUT9 antibody (dilution 1:1,000; AV43974, Sigma, St. Louis, MO, USA) or an anti-b-actin antibody (dilution 1:1,000; AV40173, Sigma, St. Louis, MO, USA) overnight at 4 °C, and finally incubated with a peroxidase-conjugated Affinipure second antibody (dilution 1:1,000; ZSGB-Bio, China) for 2 h at room temperature. The reactive bands were detected using chemiluminescent reagents (Pierce Biotechnology, USA) and an image analysis system (BioRad, USA). The relative protein expression quantities of URAT1 and GLUT9 in the renal samples were performed with the Quantity One software (Bio-Rad, USA). Statistical Analysis Continuous data were expressed as mean ± SD and categorical data in percentage. Statistical analysis was performed with the software of SPSS 15.0 (SPSS Inc, Chicago, IL, USA). Differences in the relative expression quantities of the urate transporter and the clinical data [including age, body mass index (BMI) and parameters of blood biochemistry and 24-h urinalysis] among four groups were estimated with t-test, and p \ 0.05 was considered statistically significant.
suggested the mean age, and BMI of UASF was significantly higher than other three groups (p \ 0.05), but they did not differ significantly among other three groups. Moreover, Obesity (BMI [ 30), overweight (BMI 25–30), diabetes, hypertension, and hyperuricemia were more prevalent in UASF group compared with other groups. According to the blood biochemical examination, higher plasma concentrations of creatinine and UA were observed in UASF group than other two groups (p \ 0.05). The results of 24-h urinalysis revealed that urine pH of UASF was significantly lower than patients with CaOx stones or normal controls (p \ 0.05), but statistical difference of 24-h urine volume and uric acid excretion was not found between UASF and healthy controls, similar results as 24-h calcium and oxalate excretion, which was significant more in group of CaOx stones than UA group and normal group (p \ 0.05). Expression of URAT1 and GLUT9 in the Renal Samples The expression level of URAT1 and GLUT9 protein in 57 renal tissue samples was measured by immunohistochemistry or western blotting analysis. Immunohistochemical results showed that the two transporters mainly localized in the renal tubular endothelium near glomerulus in the cortex. Stronger immunoreactive staining of URAT1 was observed in UASF group compared with the other two groups. However, the immunoreactive intensity of GLUT9 was not significantly different among renal samples of three groups in our study (Fig. 1). Experimental results of western blotting analysis supported this finding. Three immunopositive bands were observed at 55–60, 50–55, and 40–45 kDa which suggested URAT1, GLUT9, and b-actin protein, respectively. Semi-quantitative expression levels of URAT1 and GLUT9 relative to b-actin in the same sample were obtained to statistical analysis. The results also revealed that the relative expression quantity of URAT1 was significantly higher in renal samples of UASF group than of other two groups. The protein expression of GLUT9 was increased in renal tissues of UASF, but statistical differences among the three study groups were not found (Fig. 2).
Results
Discussion
Clinical Characteristics of Participants
In the present study, we investigated the clinical characteristics of UASF, in comparison to patients with CaOx stones and normal controls. Furthermore, the altered protein expression of URAT1 and GLUT9 in UASF was determined for the first time by immunohistochemistry and
This study included 50 patients with urolithiasis and 22 normal controls. Clinical characteristics of the participants are summarized in Table 1. The demographic data
123
Cell Biochem Biophys Table 1 Clinical characteristics of the participants in this study
Variable
Group I (n = 23)
Group II (n = 27)
Group III (n = 7)
Group III (n = 15)
57.21 ± 10.82*m
43.66 ± 12.59
42.87 ± 11.54
43.72 ± 10.59
BMI (kg/m )
25.28 ± 4.13*m
23.42 ± 3.34
23.57 ± 3.59
23.51 ± 3.77
Obesity, n (%)
5 (21.7)
1 (3.7)
0
0
Overweight, n (%)
8 (34.8)
8 (29.6)
1 (14.3)
2 (13.3)
Diabetes, n (%)
Demographic data Age (years) 2
3 (13.0)
0
0
0
Hyperuricemia, n (%)
12 (52.2)
8 (29.6)
0
0
Hypertension, n (%)
4 (17.4)
2 (7.4)
0
0
92 ± 25
68 ± 12
–
Blood biochemistry Creatinine (lmol/L)
134 ± 70*m
Uric acid (lmol/day)
437 ± 121*
Calcium (mmol/L)
2.23 ± 0.21
m
370 ± 103
319 ± 87
–
2.24 ± 0.23
2.32 ± 0.28
–
24-h urinalysis parameters
BMI body mass index * p \ 0.05 versus patients with CaOx stone (group II); m p \ 0.05 versus the normal controls (group III)
Urine volume (L)
1.73 ± 0.82
1.82 ± 0.76
–
1.69 ± 0.58
pH Uric acid (mg)
5.76 ± 0.43*m 689 ± 187
6.35 ± 0.46 714 ± 216
– –
6.22 ± 0.54 659 ± 149
Calcium (mg)
172 ± 91*
233 ± 117
–
159 ± 78
Oxalate (mg)
38.6 ± 21.1*
43.7 ± 18.7
–
37.4 ± 16.5
Creatinine (g)
1.73 ± 0.62
1.63 ± 0.52
–
1.79 ± 0.57
Fig. 1 Immunohistological detection of URAT1 and GLUT9 in renal tissues. Stronger immunoreactive staining of URAT1 was observed in samples of uric acid stone formers (UASF) (a) compared to calcium
oxalate stone formers (CaOxSF) (b) and normal controls (NC) (c). Significant difference in immunoreactive staining of GLUT9 was not observed in renal samples of three groups (d–f). 9400 magnification
western blotting. All participants are adult male in this study, in order to avoid the potential impact of gender differences, which are observed in the expression of urate transporters in mice [17]. The demographic data of patients with urolithiasis suggested that UASF were associated with older age of onset than patients with CaOx stone. Other studies have also pointed out the epidemiologic characteristic of UA stone [18, 19]. Goldfarb et al. [20] suggested that lower
urinary supersaturations resulting from abnormal urinary pH would contribute to the high incidence of UA stones in older population. In addition, metabolic syndrome is more common in older people, which is a recognized risk factor [21]. A series of studies have reported the correlation between metabolic factors and UA stone formation, for example, obesity, diabetes mellitus, and hypertension [22– 24]. In our study, it was also revealed that UASF was more often accompanied by metabolic disorders.
123
Cell Biochem Biophys
Fig. 2 Protein expression of URAT1 and GLUT9 in renal tissues was determined by Western blotting (a). b-actin served as an internal loading control for estimating relative protein expression quantities, which were presented as mean ± SE (b). Results of semi-quantitative
analysis suggested higher relative protein expression quantity of URAT1 in renal samples of uric acid stone formers (UASF) than that of calcium oxalate stone formers (CaOxSF) and normal controls (NC) (b). * p \ 0.05
24-h urinalysis plays an important role in evaluation of metabolic characteristics. Furthermore, variations in 24-h urinary parameters are associated with the specific types of stone [25, 26]. In our study, lower urinary pH was observed in UASF group, which supported low urinary pH as a major urinary risk factor for UA stone formation, and was consistent with the recent report [26]. However, we did not find significant difference in 24-h urine volume and UA excretion between UASF group and other two study groups, even though higher plasma UA concentration was found in UASF. Other investigators have noted the similar finding [16, 26]. It seems to be indicated that low urine volume and high renal UA excretion are of less importance than other risk factors in UA stone formation, although hyperuricosuria, due to genetic disorder of the renal urate transporters as mentioned above, is a recognized risk factor for UA stones. URAT1 and GLUT9, as the primary urate transporters, play important roles in modulating the renal urate handling, which has been confirmed through much genetic research, as mentioned above. However, few studies have concerned about the roles of urate transporters protein in renal tissues in pathological conditions. In this study, we proposed the hypotheses that altered protein expression of URAT1 and GLUT9 was involved in the unexpected renal UA excretion, which seemed to be rather increased due to high plasma UA concentration. Our findings suggested that protein expression of URAT1 was significantly increased in renal samples of UASF compared to patients with CaOx stones and normal controls. Chen et al. [27] reported that total saponin of Dioscorea increased renal UA excretion by inhibiting renal expression of URAT1 protein in chronic hyperuricemia rats. A recent study suggested that increased expression of GLUT9 protein could decrease urate
excretion from the kidney in the mouse [28]. Base on these findings, it was deduced that upregulated URAT1 protein might lead to the relative urate underexcretion from the kidney in UASF, despite of high plasma UA concentration. Altogether, our study showed the clinical characteristics of patients with UA nephrolithiasis. Older age of onset, high plasma UA concentration, low urinary PH, and relative renal urate underexcretion were associated with patients with UA stones. Furthermore, a significant increase in the relative expression quantity of URAT1 in renal tissue of UASF was revealed by immunohistochemical or western blotting analysis, compared to patients with CaOx stones and normal controls. We purpose that upregulated URAT1 protein expression might contributes to the relative urate underexcretion from the kidney of UASF. Whether other synergistic urate transporters are involved in the pathophysiologic mechanisms needs further research. Acknowledgments We thank Urology of Emergency Medical Center of Chongqing, China for providing renal samples. This study was supported by the Natural Science Foundation of Chongqing Science and Technology Commission (Chongqing, China; Grant No. 2011BA5011).
References 1. Ramello, A., Vitale, C., & Marangella, M. (2000). Epidemiology of nephrolithiasis. Journal of Nephrology, 13, S65–S70. 2. Mandel, N. S., & Mandel, G. S. (1989). Urinary tract stone disease in the United States veteran population II. Geographical analysis of variations in composition. Journal of Urology, 1989(142), 1516–1521. 3. Knoll, T., Schubert, A. B., Fahlenkamp, D., Leusmann, D. B., Wendt-Nordahl, G., & Schubert, G. (2011). Urolithiasis through
123
Cell Biochem Biophys
4.
5. 6.
7.
8.
9.
10.
11. 12.
13.
14.
15.
the ages: data on more than 200,000 urinary stone analyses. Journal of Urology, 185(4), 1304–1311. Shekarriz, B., & Stoller, M. L. (2002). Uric acid nephrolithiasis: Current concepts and controversies. Journal of Urology, 2002(168), 1307–1314. Maalouf, N. M. (2011). Metabolic syndrome and the genesis of uric acid stones. Journal of Renal Nutrition, 21(1), 128–131. Maalouf, N. M., Cameron, M. A., Moe, O. W., & Sakhaee, K. (2004). Novel insights into the pathogenesis of uric acid nephrolithiasis. Current Opinion in Nephrology and Hypertension, 13(2), 181–189. Maesaka, J. K., & Fishbane, S. (1998). Regulation of renal urate excretion: A critical review. American Journal of Kidney Diseases, 1998(32), 917–933. Enomoto, A., Kimura, H., Chairoungdua, A., Shigeta, Y., Jutabha, P., Cha, S. H., et al. (2002). Molecular identification of a renal urate anion exchanger that regulates blood urate levels. Nature, 417(6887), 447–452. Hediger, M. A., Johnson, R. J., Miyazaki, H., & Endou, H. (2005). Molecular physiology of urate transport. Physiology (Bethesda), 2005(20), 125–133. Preitner, F., Bonny, O., Laverrie`re, A., Rotman, S., Firsov, D., Da Costa, A., et al. (2009). Glut9 is a major regulator of urate homeostasis and its genetic inactivation induces hyperuricosuria and urate nephropathy. Proceedings of the National Academy of Sciences of the United States of America, 2009(106), 15501–15506. Lipkowitz, M. S. (2012). Regulation of uric acid excretion by the kidney. Current Rheumatology Reports, 14(2), 179–188. Ichida, K., Hosoyamada, M., Hisatome, I., Enomoto, A., Hikita, M., Endou, H., et al. (2004). Clinical and molecular analysis of patients with renal hypouricemia in Japan-influence of URAT1 gene on urinary urate excretion. Journal of the American Society of Nephrology, 2004(15), 164–173. Matsuo, H., Chiba, T., Nagamori, S., Nakayama, A., Domoto, H., Phetdee, K., et al. (2008). Mutations in glucose transporter 9 gene SLC2A9 cause renal hypouricemia. American Journal of Human Genetics, 2008(83), 744–751. Yu¨, T., & Gutman, A. B. (1967). Uric acid nephrolithiasis in gout. Predisposing factors. Annals of Internal Medicine, 67(6), 1133–1148. Terkeltaub, R., Bushinsky, D. A., & Becker, M. A. (2006). Recent developments in our understanding of the renal basis of hyperuricemia and the development of novel antihyperuricemic therapeutics. Arthritis Research & Therapy, 8(Suppl 1), S4.
123
16. Negri, A. L., Spivacow, R., Del Valle, E., Pinduli, I., Marino, A., Fradinger, E., et al. (2007). Clinical and biochemical profile of patients with ‘‘pure’’ uric acid nephrolithiasis compared with ‘‘pure’’ calcium oxalate stone formers. Urological Research, 35(5), 247–251. 17. Sekine, T., Miyazaki, H., & Endou, H. (2006). Molecular physiology of renal organic anion transporters. American Journal of Physiology-Renal Physiology, 290(2), F251–F261. 18. Knoll, T., Schubert, A. B., Fahlenkamp, D., Leusmann, D. B., Wendt-Nordahl, G., & Schubert, G. (2011). Urolithiasis through the ages: data on more than 200,000 urinary stone analyses. Journal of Urology, 185(4), 1304–1311. 19. Krambeck, A. E., Lieske, J. C., Li, X., Bergstralh, E. J., Melton, L. J, 3rd, & Rule, A. D. (2013). Effect of age on the clinical presentation of incident symptomatic urolithiasis in the general population. Journal of Urology, 189(1), 158–164. 20. Goldfarb, D. S., Parks, J. H., & Coe, F. L. (1998). Renal stone disease in older adults. Clinics in Geriatric Medicine, 14(2), 367–381. 21. Reynolds, K., & He, J. (2005). Epidemiology of the metabolic syndrome. American Journal of the Medical Sciences, 330(6), 273–279. 22. Kadlec, A. O., Greco, K., Fridirici, Z. C., Hart, S. T., Vellos, T., & Turk, T. M. (2012). Metabolic syndrome and urinary stone composition: What factors matter most? Urology, 80(4), 805–810. 23. Cho, S. T., Jung, S. I., Myung, S. C., & Kim, T. H. (2013). Correlation of metabolic syndrome with urinary stone composition. International Journal of Urology, 20(2), 208–213. 24. Daudon, M., Lacour, B., & Jungers, P. (2006). Influence of body size on urinary stone composition in men and women. Urological Research, 34(3), 193–199. 25. Parvin, M., Shakhssalim, N., Basiri, A., Miladipour, A. H., Golestan, B., Mohammadi Torbati, P., et al. (2011). The most important metabolic risk factors in recurrent urinary stone formers. Urology Journal, 8(2), 99–106. 26. Moreira, D. M., Friedlander, J. I., Hartman, C., Elsamra, S. E., Smith, A. D., & Okeke, Z. (2013). Using 24-hour urinalysis to predict stone type. Journal of Urology, 190(6), 2106–2111. 27. Chen, G. L., Zhu, L. R., Na, S., & Li, L. (2013). Effect of total saponin of dioscorea on chronic hyperuricemia and expression of URAT1 in rats. Zhongguo Zhong Yao Za Zhi, 38(14), 2348–2353. 28. Kimura, T., Amonpatumrat, S., Tsukada, A., Fukutomi, T., Jutabha, P., Thammapratip, T., et al. (2011). Increased expression of SLC2A9 decreases urate excretion from the kidney. Nucleosides, Nucleotides & Nucleic Acids, 30(12), 1295–1301.
