Solute-linked carrier 26 gene family 6 (SLC26A6), which is mainly expressed in intestines and kidneys, is a multifunctional anion transporter crucial in the transport of oxalate anions. This study aimed to investigate the role of kidney SLC26A6 in urolithiasis.
Patients were divided into two groups: stone formers and nonstone formers. Samples were collected from patients following nephrectomy. Lentivirus with Slc26a6 (lentivirus-Slc26a6) sequence and lentivirus with siRNA-Slc26a6 (lentivirus-siRNA-Slc26a6) sequence were transfected into rats’ kidneys respectively and Slc26a6 expression was detected using Western blot and immunohistochemical analyses. After administering ethylene glycol, oxalate concentration and prevalence of stone formation between the transgenic and control groups were measured using 24-h urine analysis and Von Kossa staining, respectively.
Immunohistochemical and Western blot analyses indicated that stone formers had a significantly higher level of expression of SLC26A6 in the kidney compared with the control group. After lentivirus infection, the urinary oxalate concentration and rate of stone formation in lentivirus-Slc26a6-tranfected rats increased remarkably, while lentivirus-siRNA-Slc26a6-transfected rats showed few crystals.
The results showed that high expression levels of renal SLC26A6 may account for kidney stone formation. Downregulating the expression of SLC26A6 in the kidney may be a potential therapeutic target to prevent or treat urolithiasis.
Nephrolithiasis is one of the most common urological conditions. An increase in both prevalence and incidence of the disease has been observed over the last several decades (
The Solute-linked carrier 2
This study was performed at the Tongji Hospital, and the ethical approval was given by the Medical Ethics Committee at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (TJ-C20141225; Wuhan, China). All patients signed the informed consent form to participate. All methods were performed in full compliance with the Declaration of Helsinki.
Ten patients with calcium oxalate stones (stone group) and 10 patients with nonstone diseases, including tumor or tuberculosis (control group), between November 2015 and October 2016 in Tongji Hospital were included in this study. The patients in the stone group had undergone nephrectomy for severe hydronephrosis leading to the loss of renal function (
Subject | Age (year)/Gender | Dx | Procedure | Stone analysis |
---|---|---|---|---|
S1 | 58/M | Kidney stone (R) | Nephrectomy | CaC2O4, carbonate apatite |
S2 | 65/M | Kidney stone (R) | Nephrectomy | CaC2O4, carbonate apatite |
S3 | 60/F | Upper ureteral stone (L) | Nephrectomy | CaC2O4, carbonate apatite |
S4 | 59/F | Kidney stone (R) | Nephrectomy | CaC2O4, carbonate apatite |
S5 | 54/M | Kidney stone (R) | Nephrectomy | CaC2O4, carbonate apatite |
S6 | 54/M | Kidney stone (L) | Nephrectomy | CaC2O4, carbonate apatite |
S7 | 48/F | Kidney stone (R) | Nephrectomy | CaC2O4, carbonate apatite |
S8 | 61/F | Kidney stone (L) | Nephrectomy | CaC2O4, carbonate apatite |
S9 | 56/M | Kidney stone (R) | Nephrectomy | CaC2O4, carbonate apatite |
S10 | 49/M | Kidney stone (R) | Nephrectomy | CaC2O4, carbonate apatite |
C1 | 27/F | Renal tuberculosis (R) | Nephrectomy | NA |
C2 | 69/F | Kidney tumor (R) | Nephrectomy | NA |
C3 | 43/M | Kidney tumor (R) | Nephrectomy | NA |
C4 | 49/F | Renal tuberculosis (L) | Nephrectomy | NA |
C5 | 76/F | Kidney tumor (R) | Nephrectomy | NA |
C6 | 58/F | Renal tuberculosis (L) | Nephrectomy | NA |
C7 | 54/M | Kidney tumor (R) | Nephrectomy | NA |
C8 | 68/F | Kidney tumor (R) | Nephrectomy | NA |
C9 | 61/M | Kidney tumor (R) | Nephrectomy | NA |
C10 | 43/M | Kidney tumor (L) | Nephrectomy | NA |
S, stone former group; C, control group; F, female; M, male; Dx, diagnosis; R, right; L, left; NA, none.
About 1.5 mg of stones were taken out and ground into powder. It was then mixed with 250 mg potassium bromide. This mixture was dried and subjected to a pressure of 20 MPa to make tablets with a thickness of 0.3–0.5 mm. Then, the spectral characteristic peaks of tablets were measured using an LIIR20 infrared spectrum analyzer (Lambda, Tianjin, China). The components were evaluated by comparing with the standard image.
The standard protocol was followed to collect 24-h urine collected from the patients. After measuring the pH of each sample, 500 μL of 6 mol/L hydrochloric acid was added into each 10 mL fresh urine sample (
Renal cortex tissue was obtained using sterilized scissors following nephrectomy. Kidney tissue far away from a primary lesion was chosen in patients with tuberculosis and tumors who underwent nephrectomy and served as the control group. The tissues were stored at –80 °C and fixed with 4% paraformaldehyde (Boster, Wuhan, China) at room temperature for further analysis.
Samples were dissociated using radio immunoprecipitation assay and phenylmethanesulfonyl fluoride. The protein (40 µg/lane) was electrophoresed on 10% sodium dodecyl sulfate–polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Immobilon-P Transfer Membrane; Millipore Corporation, Burlington, MA, USA). The protein samples were equally mixed, and the expression of SLC26A6 in the two groups was analyzed. The primary antibody was goat anti-SLC26A6 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse β-actin (1:500; Boster, Wuhan, China). The secondary antibody was rabbit anti-goat (1:5000; Boster, Wuhan, China) or goat anti-mouse (1:5000; Boster, Wuhan, China). After incubation with the secondary antibody at room temperature for 2 h, proteins were detected with the Bio-Rad Clarity Western Enhanced Chemiluminescence (ECL) Substrate (Bio-Rad Laboratories, Hercules, CA, USA) and ECL detection system (1705061; Bio-Rad Laboratories, Hercules, CA, USA). The immunohistochemical (IHC) assays were performed according to the standard protocol, and the dilution rate of anti-SLC26A6 primary antibody was 1:100. Microscopy (BX53; Olympus, Tokyo, Japan) was used to observe the expression of SLC26A6. Fluorescence intensities were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
The experimental protocol was conducted in accordance with the institutional ethical committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology according to the “Guidelines for Experimental Animal Ethical Committee of Huazhong University of Science and Technology.” This study was approved by the Ethical Committee (TJ-A 20141219).
A total of 40 male Sprague–Dawley rats (275–300 g) were obtained from the Animal Center of Tongji Medical College, Huazhong University of Science and Technology. They were reacclimatized to 12-h light/dark cycles at 23 °C for one week prior to the start of experiments in a specific pathogen-free animal house with a relative humidity of 45–55%. They were maintained on a diet consisting of standard laboratory chow and had free access to food. They were randomly divided into four groups (10 rats in each group): negative control, lentivirus (lv-SLC26A6), siRNA-lentivirus (siRNA-SLC26A6), and vector groups.
The
The rats were placed in a prone position and anesthetized with sodium pentobarbital (40 mg/kg). An amount of 2 × 107 Tu per rat lentivirus were applied to subcapsular renal injection (2–3 points per kidney). Lentivirus subcapsular renal infection was successfully accomplished according to the previously published protocol (
A total of two weeks after the successful infection of lentivirus into the kidneys of rats, the kidneys and duodenal segments of four rats from different groups (
Ethylene glycol (EG) was dissolved in water to titrate 1% EG. The remaining animals (
Data were presented as mean ± standard deviation. The statistical analysis was performed using two-way analysis of variance. Differences were considered significant if the
From 2015 to 2016, a total of 20 subjects (10 stone formers and 10 nonstone formers) were recruited in this study (
After recruiting idiopathic stone formers, 24-h urine was collected and the urinary oxalate, calcium, citrate, magnesium, phosphorus, and pH were measured (
(A) 24-h urinary analysis of stone formers and nonstone formers.
After successful nephrectomy, IHC and Western blot analyses were performed to detect the expression of SLC26A6 in the renal tissue (
Lentiviruses (
(A) The Slc26a6 sequence was inserted into a lentiviral vector as pWSLV-05-Slc26a6. And ds-siRNA anti-Slc26a6 sequence was inserted into a lentiviral vector as pLenti-siRNA (Slc26a6)-RFP. (B) After successful lentivirus insertion into the kidneys of rats, frozen sections of kidneys and duodenums were observed under a fluorescence microscope. Cell nucleus were stained with DAPI. RFP was used as a marker to show successful insertion. Compared with the control group, transfection in the kidneys of the experimental groups (lv-Slc26a6 and siRNA-Slc26a6 groups) was successful as seen under a fluorescence microscope (magnification ×200). No difference of RFP expression was observed in the duodenum tissue between the control group and the experimental groups. The data are expressed as means ± SD (
Western blot and IHC analyses were used after successful lv-Slc26a6 and siRNA-Slc26a6 infection to demonstrate the variation in the expression of Slc26a6 in the kidneys and duodenum of rats (
(A) Western analysis of duodenal tissue showed no difference in the expression of SLC26A6 among lv-Slc26a6, siRNA-Slc26a6, control, and vector groups. The data are expressed as means ± SD (
The rate of stone formation and urinary oxalate concentration in rats with lv-Slc26a6 (Slc26a6 group) increased remarkably, whereas the rate in those with siRNA-Slc26a6 (siRNA group) decreased after lentivirus infection. The supplementation of drinking water with 1.0% EG induced hyperoxaluria, and the subsequent oxalate excretion was measured. The urinary oxalate concentration of rats was 71.90 ± 17.21, 32.80 ± 12.20, 47.73 ± 10.50, and 53.24 ± 14.97 µmol/24 h, in the lv-Slc26a6, siRNA-Slc26a6, control, and vector groups, respectively (
(A) Supplementation of the drinking water with 1.0% EG induced hyperoxaluria; 24-h urine was collected by putting the rats in a metabolic cage. The oxalate level in urine was measured by ion chromatography, and the result showed that Slc26a6-lentivirus-transfected rats had significantly higher urinary oxalate excretion compared with the vector and control groups. siRNA-Slc26a6-transfected rats had less urinary oxalate compared with the control group. The data are expressed as means ± SD (
Oxalate secretion in proximal tubules is associated with the expression of both basolateral SLC26A1 and apical SLC26A6 in the kidneys (
Tissue samples were collected from patients with renal stones (stone former group) and patients suffering from renal tuberculosis or tumors (control group) to reveal the role of renal SLC26A6 in stone formers. Western blot and IHC analysis results showed a direct relationship between kidney stone formation and expression of SLC26A6. However, whether the increased expression of SLC26A6 in the stone group is the cause or result of the disease was difficult to predict.
Furthermore, infection of lentivirus with Slc26a6 sequence and lentivirus with siRNA anti-Slc26a6 into the kidneys of rats was achieved. The renal subcapsular injection technique was used to ensure the organ-specific expression of the virus and avoid expression in other organs especially in the intestinal tract (
In the renal proximal tubule cells, oxalate transport is associated with Slc26a1 expressed on basolateral membrane and Slc26a6 expressed on apical membrane. Slc26a1 mediates the uptake of oxalate in exchange for reabsorbed sulfate (or Cl− or HCO3−). The high expression of Slc26a6 mediates more secretion by oxalate–Cl− exchange and reabsorption by sulfate–oxalate exchange. According to the results, oxalate–Cl− exchange possessed a dominant position resulting in the enhanced net secretion of oxalate.
The animal experiment results showed that Slc26a6 was one of the causes of kidney stone formation. The potential mechanism might be that Slc26a6 led to the secretion and reabsorption of oxalate, resulting in higher oxalate concentration in urine and interstitium (
This study showed that the increased expression of renal Slc26a6 caused higher oxalate concentration in urine, leading to an increased crystal deposition. Moreover, reducing the expression of renal Slc26a6 by siRNA injection attenuated stone formation. However, the study had several limitations. First, due to the small cohort size, future studies with a larger number of patients are needed to confirm the results. Second, Slc26a6 was an important but not the only oxalate transporter; therefore, measuring oxalate flux mediated by other proteins and oxalate concentration in other areas help better understanding of oxalate transport. Third, all experimental procedures were subject to ethical approval. Patients with renal tuberculosis and cancer were selected as the control group because it was nearly impossible to acquire a sample of normal human renal tissue for this study. Moreover, the results of previous studies including samples from patients with cancer as the control group have been widely accepted (
In conclusion, the present study demonstrated that the overexpression of Slc26a6 in the kidneys increased oxalate excretion and urinary oxalate concentration, contributing to an increase in the prevalence of stone formation. Downregulating the expression of SLC26A6 in the kidneys might be a potential therapeutic target to prevent or treat urolithiasis.
Kidney tissues from stone formers were stained by IHC assay to detected SLC26A6 expression. The brown part represents the expression of SLC26A6 (magnification: X200).
Kidney tissues from non-stone formers were stained by IHC assay to detected SLC26A6 expression. The brown part represents the expression of SLC26A6 (magnification: X200). Compare to the
The authors declare that they have no competing interests.
The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):
This study was performed at the Tongji Hospital, and the ethical approval was given by the Medical Ethics Committee at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China; TJ-C20141225).
The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):
The experimental protocol was conducted in accordance with the institutional ethical committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology according to the “Guidelines for Experimental Animal Ethical Committee of Huazhong University of Science and Technology.”
The following information was supplied regarding data availability:
The raw data are provided in the