The Effect Of Testosterone On The Nephrolithiasis

BackgroundsThe prevalence of renal stone in men was much higher than in the women, but the reason why the gender disparity exist is always unclear. Some studies had found that the urinary citrate level of females is much higher than the males. In the recent years, the study on the kidey stone is still focus on the urinary Supersaturation like calcium, oxalate, and the insufficient urinary inhibitors for formation of stone like Citrate, Osteopontin, Magnesium. Actually, in addition to urine constituents, the renal tubule epithelial cells play a role in the formation of stone. Previous studies have found that lethal renal epithelial cellular injury promotes crystal nucleation, aggregation and retention. Another study revealed that the adherence of crystals to pericellular matrixes may encompass more than their simple fixation to the polysaccharide Hyaluronan (HA). Calcium oxalate crystal retention is not prevented by coating crystals with urinary constituents such as glycoproteins and, therefore, may depend primarily on the surface properties of the renal tubular epithelium. However, the relation of the androgen and kidney stone is not still clear, and the effect of androgen on the renal tubular epithelial cell is unknown, either. Thus we speculate that perhaps androgen cytotoxic to the renal tubule epithelial cell, which induce the formation of kidney stone.Objective1. The purpose of this study was to determine whether testosterone would contribute to the nephrolithiasis.2. To evaluate the effects of testosterone at different levels on human renal tubular epithelial cells (HK-2) and whether the testosterone has the effect of pro-apoptosis on the renal tubular epithelial cells3. To evaluate the expression of clusterin in the renal tubular epithelial cell of the patients with renal stone, and identify the differently expressed proteins of two isoforms of clusterin in HK-2 cells treated with testosterone at different levels, so as to find the exact effect of the clusterin in the HK-2 cells treated with testosterone and provide new ways for further kidney stone formation.Methods1. Patients with renal calculi undergoing percutaneous nephrolithotomy (PCNL) were included, whose stones were analysed with a transform-infrared (FT-IR) spectrometer, but all the patients participated in this study were excluded the urinary tract infection and hydronephrosis, at the end of procedure ultrasound-guided puncture biopsy was performed to acquire the kidney tissue, while kidney tissues from autopsy archived in pathology department were obtained as the control. The expression of androgen receptor and clusterin in renal tubular epithelial cell were evaluated by immunohistochemistry. Besides this, the blood of all the subjects was collected into a tube containing 2% heparin in the morning, and the total and free serum testosterone levels were measured for each person by enzyme linked immunosorbent assay(ELISA).2. Normal HK-2 cells were cultured in vitro and the culture medium was changed with serum-free medium after cell growth to confluence. Testosterone with different concentrations or testosterone combined flutamide were then added and the cells were incubated for 6h,24h,48h or 72h, respectively, then the ratio of cell viability was measured by MTT, while cell apoptosis was measured by annexin V/propidium iodide (PI) staining.3. The expression two activation isoforms of clusterin were analyzed by western blotting, and the mRNA level of clusterin was measured by RT-PCR, after the HK-2 cells incubated with different levels testosterone or incubated with both testosterone and flutamide. Then the results were analyzed to clarity the function of clusterin in the apoptosis of HK-2 cell induced by testosterone.Results1. All the calculi included calcium oxalate monohydrate and calcium oxalate dihydrate, and a small quantity of calculi also contained the protein or uric acid. The mean serum levels of total testosterone and free testosterone in patients with kidney stone were 13.29±4.79ng/ml,28.58±4.70 pg/ml; and the levels of total testosterone, free testosterone in control group was7.30±0.82 ng/ml,24.91±4.47 pg/ml. There was significant difference of the hormones level between patients and healthy men, respectively (P<0.01). Moreover, there was a significant correlation between the total testosterone levels and free testosterone levels (r=0.824, p<0.0001). The AR expression in renal tissues was localized to cell nuclei of the renal distal tubule epithelial cell. Immunohistochemistry assessment showed up-regulation of AR in patients with kidney stones (P<0.05). Immunohistochemistry assessment showed the expression of clusterin was located in the cytoplasm of renal tubular epithelial cell, and the expression in the cells of patients was higher than in the controls significantly(P<0.05). 2. The results showed that the ratio of cell viability inhibition of HK-2 cells increased as the testosterone concentration increased and as the time increased. Maximal inhibition of proliferation was achieved after 72 h of cell exposure to testosterone 1000nM,72h, and this inhibition was time- and dose-dependent. Cells undergoing early apoptosis were usually characterized by the degree of phosphatidylserine exposure with the testosterone. HK-2 cells treated with testosterone exhibited a significant increase, which indicated that testosterone induced apoptosis in HK-2 cells in a dose- and time-dependent manner but did not cause obvious necrosis.3. HK-2 cells treated with testosterone were analyzed by western blotting that detected the expression of clusterin. The result showed that testosterone activated the expression of N-clusterin. We also found that the down-regulation of S-clusterin in the cells exposed to testosterone, and the expression of S-clu was also shown, which was much weaker than the N-clu. The mRNA expression levels of clusterin in the HK-2 after exposure to testosterone was measured by quantitative RT-PCR.Conclusions1. We found both serum total testosterone and free testosterone in the male patients with renal calculi were higher in comparison with healthy men, and the expression of androgen receptor in kidney of patients was also stronger than the controls. This study indicates that total and free testosterone may play an important role in the formation of kidney calclui in male patients.2. The HK-2 cells incubated with testosterone showed a significant dose- and time-dependent decrease in viability, and the testosterone also could induced the HK-2 to apoptosis. Therefore, the effect of pro-apoptosis on the renal tubular epithelial cell could play an important role in formation of kidney stone.3. Our findings showed that HK-2 cell exposure to testosterone resulted in a marked accumulation of N-clusterin, which is considered an apoptotic death trigger in target cells, so we speculate that the effect of testosterone could be mediated through both the up-regulation of N-clusterin and the down-regulation of S-clusterin.4. This study evaluated the effect of androgen on the formation of kidney stone, the the mechanism of promotion the stone, the effect of testosterone on the renal tubular epithelial cell and the expression of clusterin in the renal tubular epithelial cell of patients with renal stone. Therefore, our study provides an important knowledge of testosterone-treated HK-2 cells in vitro and should be helpful for the further elucidation of the molecular mechanisms involved in the interaction between androgen and renal epithelial cells and kidney stone formation, and provide new methods and for seeking effective precautionary measures for the recurrence of renal calculi.