The Expresssion Of Trpv5 In Renal Tissue Of Patients With Calcium-contained Renal Calculi

BackgroundUrinary stone is one of the most common urological diseases, and is also a global disease. The incidence of urinary stone is rising every year in China, southern China is one of the three major endemic area of urinary calculi in the world. Urolithiasis is easy to recur,and current preventive measures are not satisfactory. Studies have shown that: 10% of the males will recur a new calcium oxalate stone 1 year after the occurrence of calcium oxalate stones,and 50% will recur a new calculi in 10 years. It's necessary to study the etiology of urinary stone, so that the occurrence of urinary stones and its recurrence can be prevented effectively.The etiology of urolithiasis includes genetic,metabolic factors,environment, diet and so on.Metabolic factors are research hotspot in recent years.It has been proved that abnormal calcium metabolism is an important factor causing urinary calculi. There are many kinds of urinary stones,but at least more than 90% of which contain calcium, and about 1 / 3 of which have hypercalciuria. Studies have shown that the saturation of urinary calcium oxalate and calcium phosphate are associated with the concentration of calcium,so increased urinary calcium is easy to promote the formation of urinary stones. Urinary calcium depends on glomerular filtration and renal tubular reabsorption. 85% calcium are reabsorped in the proximal tubule and Henle loop in passive way,the remaining 15% are reabsorped in the renal distal tubules (DCT2) and the collecting duct (CNT) in active way. Transepithelial transport of Ca2+ is a three-step process. It initiates with influx of Ca2+ across the apical membrane mediated by TRPV5. Subsequently, entered Ca2+ is sequestered by the specialized intracellular carrier protein calbindin-D28K and diffuses to the basolateral membrane. Finally, transporter proteins, such as NCX1 and PMCA1b, extrude Ca2+ from the epithelial cell into the circulation .Active reabsorption is different from the passive re-absorption, it can be specially regulated by the factors like PTH,1, 25-(OH)2D3 and so on.It consumes energy,and therefore is an important factor affecting the level of urinary calcium.TRPV5 is an epithelial channel that affects the reabsorption of calcium, which expressed mainly in epithelial cells of renal distal convoluted tubules and collecting duct.It can regulate the level of urinary calcium by combinating the Ca2 + that reabsorped in active way,and thus maintain the body's calcium balance.Current studies show that decreased expression of TRPV5 may lead to higher urinary calcium.In this study, We collected the renal cortex specimens of patient with calcium-contained renal calcili(stone group)and patients without stones(control group) in our hospital ,5 cases of each group,and compared the expression of TRPV5 betweem the 2 groups .Immunohistochemistry,Western Blot and quantitative real time PCR were tested. Our purpose is to study the role of TRPV5 in the formation of calcium-containing renal calculi,and provide a theoretical basis for its prevention.ObjectiveComparing the different expression of TRPV5 in stone group and control group, study the role of TRPV5 in the formation of calcium-containing kidney stone ,and thus provide a theoretical basis for the preventive methods.Methods1.The source of specimensThe experiment is divided into two groups, 5 cases in each group.We chose patients with calcium-contained renal calculi(proved by postoperative stone composition analysis) whose urine calcium is higher than normal people (> 300mg/24h ,tested by 24H urine analysis)as stone group. 2 males and 3 females,with a mean age of 44 years old.We took a small amount of renal cortex during PCNL .As control group, 3 cases of male and 2 female, average age is 49 years, including 3 cases of UPJ obstruction, 2 cases of renal cell carcinoma.We also took a small amount of normal renal cortex during the open surgery.The 5 patients were diagnosed to be no renal stones by preoperative B ultrasound and IVP,and also no stone were found during surgery.The specimens were packed in EP tube with 1ml sample protective agent,and stored at -80℃. 2. H-E staining and ImmunohistochemistryThe specimens were cut into the size between 3*3mm and 3*5mm,and made to be paraffin sections.H-E staining was tested to make sure that the specimens were renal contex. The tissue from SD rat liver was used as negative control, which no TRPV5 was expressed.Routine immunohistochemical staining was also tested to compare the morphological differences between the two groups.3. Western BlotThe expression of TRPV5 protein between the two groups was compared by Western Blot.4. Quantitative real time PCRTotal RNA were extracted with TRIzol method. cDNA were acquired by RT reaction .Then we detect the expression of TRPV5mRNA in both groups from gene level by performing quantitative real time PCR.5. Statistical analysis.Results1. The results of H-E staining and immunohistochemistryWe can see a lot of renal corpuscle and renal tubules in all specimens by H-E staining,which confirmed to be renal contex.We find the renal distal tubules and collecting duct epithelial cell membrane of the 2 groups both have brown positive staining granules,but there are none in the negative control. Shedding, structural damage of the renal tubular epithelial cells also can be found in the stone group.The most important is the positive staining intensity of TRPV5 was lower than the control group significantly.2.Western Blot ResultThe expression of TRPV5 is similar in the 5 cases of control group.(153.91±3.44 vs 150.92±0.81 vs 148.70±2.31 vs 142.17±6.93, P> 0.05). The expression of TRPV5 is also similar in the 5 cases of stone group.(96.89±22.92 vs 104.51±12.14 vs 90.19±8.12 vs 93.75±10.14). However, the expression of TRPV5 protein in stone group is lower than control group by t-test. (t = 6.43> 3.355, p <0.01).3.The result of quantitative real time PCRAccording to the TRPV5 ct/GAPDH ct value,we find the expression of TRPV5mRNA in stone group is lower than control group by t-test.(t = 5.34> 2.306, p <0.05). According to the relative quantitative graph from the MXPro4.01 analysis software,we can calculate that TRPV5mRNA expression in stone group is 0.248 times of control group.ConclusionA. The stone group is different from the control group from morphological level.We can see shedding, structural damage in renal tubular epithelial cells,and the positive TRPV5 staining intensity was significantly lower than the control group.B. The expression of TRPV5 protein in stone group is lower than control group.C. The expression of TRPV5 mRNA in stone group is lower than control group.