| Objective:Nephrolithiasis is a common disease in urology,and hypercalciuria is one of the main risk factors for nephrolithiasis.Claudin14 reabsorbs about 25%of the Ca2+in the filtrate in the thick ascending branch of the loop through a paracellular pathway involving Claudin14,Claudin16 and Claudin19.Several studies have shown that Quercetin can regulate the metabolism of Ca2+by acting on multiple members of the Claudin family,including Claudin2 and 3.This study will further explore the relationship between quercetin and Claudin14 expression,urinary calcium excretion and renal stone formation through in vitro and in vivo experiments,and provide a theoretical basis for stone treatment and drug development.Methods:1.To study the effect of quercetin on Claudin14 in renal tubular epithelial cells and its possible mechanism（1）NRK-52E cells（a rat renal tubular epithelial cell line）were treated with quercetin,and the expression of Claudin14 was detected by WB.（2）The NRK-52E cells were treated with quercetin,and the expression of Claudin14,16,19 m RNA was detected by Real-time PCR.2.To study the effect of quercetin on the formation of kidney stones in rats（1）Construct a rat kidney stone model and detect the formation of kidney stones in each group:Kidney CT and Pizzolatto,s staining were used to detect the deposition of calcium crystals in the rat kidney tissue,and the differences between the groups were compared;the urine of the rats was collected.,observe the formation of crystals in urine under a microscope.3.Analysis of the effects of quercetin on renal function and metabolism in rats:（1）Collect blood and urine samples of rats in each group to detect biochemical indicators such as creatinine,blood urea nitrogen,uric acid,calcium,magnesium,potassium,sodium,and urine volume,and compare the differences between groups.4.Analysis of the effect of quercetin on the expression of Claudin14,16,19 in rat kidney tissue（1）WB detection of Claudin14 protein expression（2）Real-time PCR was used to detect the expression of Claudin14,16,19 m RNA.（3）Immunohistochemistry to detect the expression of Claudin14,16,19.Results:1.The expressions of Claudin14 protein and Claudin14 m RNA in the renal tubular epithelial cells of rats treated with quercetin decreased gradually with the increase of administration concentration;the expressions of Claudin16 and 19 m RNA gradually increased with the increase of administration concentration;2.The formation of calcium oxalate stones in kidney and crystal formation in urine of rats in quercetin-treated group were significantly reduced compared with those in lithogenic group,and the urinary calcium excretion of rats in quercetin-treated group was decreased compared with lithogenic group;3.The expressions of Claudin14 protein and Claudin14 m RNA in the kidneys of the quercetin-treated rats were lower than those of the control group and the lithogenic group,and the Claudin16 m RNA was increased compared with the lithogenic group and the control group;4.The results of immunohistochemistry showed that the expression of Claudin14decreased in the quercetin-treated group,while the expression of Claudin16 increased,and the expression of Claudin19 did not change significantly.Conclusions:1.Quercetin can reduce the expression of Claudin14 and increase the expression of Claudin16 and 19 in rat renal tubular epithelial cells;2.Quercetin can reduce the expression of Claudin14 in rat kidney tissue,enhance the expression of Claudin16,and reduce the excretion of urinary calcium in rats;3.Quercetin can inhibit the formation of calcium oxalate stones in rat kidneys;... |
