| Part Ⅰ The role and mechanism of MMP-9 in kidney stone formation under high calcium microenvironmentObjective:To study the expression level of matrix metalloprotease 9(MMP-9)in renal tubular epithelial cells in a high calcium microenvironment,and to further explore the role and potential mechanism of MMP-9 in kidney stone formation.Methods:MMP-9 and other molecules related to kidney stone disease in the renal microarray of genetic hypercalciuric stone-forming rats were analyzed by the limma package in R software.The model of hypercalciuric stone-forming rats was constructed by intraperitoneal injection of 1,25(OH)2D3 every other day,and the expression of MMP-9 was blocked with MMP-9 Inhibitor I.Rat renal tubular epithelial cells(NRK-52E)were treated with medium containing high concentration of calcium,and MMP-9 was up-or downregulated by transfection with MMP-9 overexpression or interference plasmids,respectively.The 24-hour urine total volume of the rats in each group was recorded.The concentrations of 24-hour urinary calcium and serum MMP-9 were detected in each group of rats.The intracellular Ca2+ concentration and apoptosis level were detected by flow cytometry.The cell proliferation activity was detected by cell counting kit-8.The reverse-transcription quantitative real-time polymerase chain reaction(RT-qPCR)and western blot were used to detect the mRNA and protein expression levels of MMP-9,osteopontin(OPN),runt-related transcription factor 2(RUNX2),bone morphogenetic protein 2(BMP2),and E-cadherin protein(E-cadherin)in NRK-52E cells,respectively.Immunohistochemistry was used to detect the expression levels of MMP-9,OPN,RUNX2,BMP2,and E-cadherin in rat kidney tissue.Silver nitrate staining and alizarin red staining were used to detect calcium crystal deposition in kidney tissue and cells,respectively.The morphological changes of kidney tissue were detected by hematoxylin-eosin staining.The level of apoptosis in kidney tissue was detected by TUNEL staining.Ultrastructure of cells was observed by using transmission electron microscopy.The expression intensity of MMP-9 in cells was detected by immunofluorescence assay.The fluorescence intensity of osteogenic marker factor(RUNX2)and epithelial phenotype marker factor(E-cadherin)were detected by immunofluorescence double-label staining.Results:The results of microarray analysis showed that the expression of MMP-9 was significantly increased in kidney tissue of stone-forming rats.High concentration of calcium could increase the intracellular Ca2+ concentration,inhibit the proliferation activity of cells,and promote their apoptosis.High concentration of calcium could promote the expression of MMP-9,RUNX2,and OPN in cells,but inhibit the expression of E-cadherin.After upregulating or down-regulating the expression of MMP-9,the effect of high concentration of calcium was significantly enhanced or weakened.Up-regulation or down-regulation of MMP-9 expression could increase or decrease high concentration of calcium-induced calcium salt deposition in cells,respectively.Down-regulation of MMP-9 expression ameliorated the cellular microstructural changes induced by high concentration of calcium,whereas the effect of high concentration of calcium was significantly enhanced after upregulation of MMP-9 expression.No significant difference was found in the total 24-hour urine volume between the groups of rats.However,rats in the 1,25(OH)2D3 group had higher 24-hour urinary calcium and serum MMP-9 concentrations compared with the control group.In the 1,25(OH)2D3 group,the protein expressions of MMP-9,BMP2,OPN,and RUNX2 were significantly increased,and E-cadherin was significantly decreased,after supplementation of MMP-9 Inhibitor I,the effect of 1,25(OH)2D3 was significantly inhibited.A large amount of calcium salt deposition appeared in the kidneys of rats in the 1,25(OH)2D3 group,which was significantly reduced after MMP-9 Inhibitor I supplementation.The supplementation of MMP-9 Inhibitor I could significantly improve 1,25(OH)2D3-induced renal pathological injury and apoptosis in rats.After supplementation of MMP-9 Inhibitor I,compared with the 1,25(OH)2D3 group,the fluorescence signal intensities of RUNX2 and E-cadherin were significantly attenuated and enhanced,respectively.Conclusion:High concentration of calcium activates the BMP2 signaling pathway by promoting the expression of MMP-9 in renal tubular epithelial cells,thereby inducing the transdifferentiation of cells into osteoblast-like cells,which eventually leads to the deposition of calcium salts in cells and the formation of kidney stones.Part Ⅱ The regulatory mechanism of high concentration of calcium on the expression of MMP-9 in renal tubular epithelial cellsObjective:The high-glucose medium containing high concentration of calcium was used to simulate the high-calcium microenvironment of cells,and the regulatory mechanism of high concentration of calcium on the expression of MMP-9 in renal tubular epithelial cells was investigated at the cellular level.Methods:After high concentration of calcium treatment,the levels of lactate dehydrogenase(LDH),malondialdehyde(MDA),and superoxide dismutase(SOD)in NRK-52E cells were determined by colorimetry.The production levels of intracellular reactive oxygen species(ROS)were detected by flow cytometry.The expression levels of nuclear factor kappa-light-chain-enhancer of activated B cells(NFκB)signaling pathwayrelated proteins were detected by western blot.The cells were pretreated with the ROS inhibitor N-acetylcysteine(NAC)or the NFκB pathway inhibitor BAY11-7082,and then treated with high concentration of calcium,RT-qPCR and western blot were used to detect the mRNA and protein expression levels of MMP-9,OPN,RUNX2,and E-cadherin in cells,respectively.The fluorescence signal intensity of high concentration of calcium-induced pp65 in NRK-52E cells was detected by immunofluorescence in the presence or absence of ROS inhibitor NAC or NFκB pathway inhibitor BAY11-7082 pretreatment.Results:After high concentration of calcium treatment,MDA and LDH levels in NRK-52E cells were significantly increased,while SOD levels were significantly decreased,and this effect showed a calcium concentration dependent trend.High concentration of calcium treatment could promote the increase of intracellular ROS levels.High concentration of calcium treatment could activate the NFκB signaling pathway in NRK-52E cells.Pretreatment with the ROS inhibitor NAC or the NFκB pathway inhibitor BAY11-7082 could significantly inhibit the increased mRNA and protein expression of MMP-9,OPN,and RUNX2,and the decreased mRNA and protein expression of E-cadherin in cells induced by high concentration of calcium.Immunofluorescence results showed that BAY11-7082 pretreatment could significantly inhibit the activation of NFκB signaling pathway in cells induced by high concentration of calcium.Western blot and immunofluorescence results showed that pretreatment with ROS inhibitor NAC could significantly inhibit the activation of NFκB signaling pathway in cells induced by high concentration of calcium.Conclusion:The results of in vitro experiments show that high concentration of calcium can promote the expression of MMP-9 by activating the ROS/NFκB signaling pathway,thereby leading to the formation of kidney stones.Part Ⅲ Correlation between serum MMP-9 level and stone recurrence in patients with kidney stonesObjective:This study started with samples from newly admitted patients with kidney stones,by detecting the concentration of serum MMP-9,to explore its correlation with the recurrence of kidney stones,and to reveal the clinical value of serum MMP-9 in predicting the recurrence of kidney stones.Methods:Newly admitted patients with kidney stones were inquired their medical history.Patients with hematuria,fever,urinary system malformation,urinary catheter or nephrostomy tube,and urinary system tuberculosis were excluded.The general clinical characteristics(including gender,age,weight,height,history of heart disease,history of diabetes,history of hypertension,smoking history,drinking history,family history of kidney stones,and history of stone recurrence),blood biochemical data(blood potassium,blood sodium,blood chloride,blood calcium,blood phosphorus,blood magnesium,blood urea,blood creatinine,blood uric acid,blood bicarbonate,and eGFR),and urine test data(urine pH,urine specific gravity,and urine crystals)were recorded.The patients’ 24-hour urine and blood samples were then collected.Urinary calcium,urinary oxalate,and serum MMP9 concentrations were detected respectively.The patients were divided into two groups based on the history of stone recurrence(initial stone and recurrence stone groups).Differences in general clinical characteristics were compared between the two groups of patients,followed by subgroup analysis.Then,multivariate Logistic regression analysis was performed according to the relevant factors affecting stone recurrence.Finally,a nomogram was constructed to predict the risk of stone recurrence in patients with kidney stones.Results:A total of 99 patients with kidney stones were collected,of which 46 were initial stones and 53 were recurrent stones.The analysis of general clinical characteristics,blood biochemical data,and urine test data between the two groups of patients showed that there were no significant differences in gender,age,body mass index,history of heart disease,history of diabetes,history of hypertension,smoking history,drinking history,family history of kidney stones,urine pH,urine specific gravity,urine crystals,urine calcium concentration,urine oxalate concentration,24-hour urine volume,24-hour urine oxalate,blood chloride,blood sodium,blood potassium,blood phosphorus,blood calcium,blood magnesium,blood creatinine,blood urea,blood uric acid,blood bicarbonate,and eGFR between the two groups.However,the 24-hour urinary calcium(P=0.027)and serum MMP-9 levels(P=0.003)in the patients with recurrence stone group were significantly higher than those in the patients with initial stone group.Further subgroup analysis showed that the number of patients with serum MMP-9≥1324 μg/L(P=0.002),24-hour urinary calcium≥200 mg(P=0.020),and 24-hour urinary oxalate≥22 mg(P=0.007)in the recurrence stone group were significantly more than that in the initial stone group.The following multivariate Logistic regression analysis showed that hyperuricemia(OR=3.098,95%CI:1.021-9.406,P=0.046),serum MMP-9≥1324 μg/L(OR=3.244,95%CI:1.086-9.689,P=0.035),24-hour urinary calcium≥200 mg(OR=3.859,95%CI:1.190-12.516,P=0.024),and 24-hour urinary oxalate ≥22 mg(OR=3.859,95%CI:1.397-65.008,P=0.024)were independent risk factors for stone recurrence.Finally,a nomogram for predicting the risk of stone recurrence in patients with kidney stones was constructed.The calibration curve and decision curve analysis showed that the nomogram had better prediction accuracy and clinical utility.Conclusion:The analysis results of clinical samples show that hyperuricemia,serum MMP9≥1324 μg/L,24-hour urinary calcium≥200 mg,and 24-hour urinary oxalate≥22 mg are independent risk factors for stone recurrence in patients with kidney stones. |
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