The Role Of MGP And Urinary Exosomes In The Formation Of Kidney Stones

Part Ⅰ.The effect of high concentration of calcium on MGP expression in NRK-52 E cellsObjective: To explore the effect of high concentration of calcium on MGP expression in NRK-52 E cells and address whether MGP can inhibit mineralization in NRK-52 E cells under high concentration of calcium.Methods: NRK-52 E cells were treated with high concentration of calcium.The viability and apoptosis of cells were detected by cell counting kit-8 and flow cytology respectively.RT-PCR,Western blotting and immunofluorescence analysis were conducted to detect the expression of MGP.Cells were transfected with plasmid-MGP or si RNA-MGP for up-or down-regulation the expression of MGP respectively.Rat recombinant MGP was also used as supplementation of exogenous MGP.Alizarin red staining was conducted to detect the adherent and deposition of calcium salt.Results: High concentration of calcium suppressed MGP expression in NRK-52 E cells.There was significant mineralization when NRK-52 E cells were treated with high concentration of calcium.Supplementation with exogenous rat recombinant MGP and overexpression of endogenous MGP both decreased the adherent and deposition of calcium salt to NRK-52 E cells,while silence of MGP showed reverse results.Conclusions: MGP plays an inhibitory role in the stone formation.However,high concentration of calcium significantly inhibits the expression of MGP and then promotes mineralization in NRK-52 E cells.Part Ⅱ.The effect of carboxylation status of MGP on kidney stone formationObjective: To study the effect of regulating GGCX,Vit K and warfarin on MGP carboxylation status and explore how MGP carboxylation status influences the stone formation in hypercalciuric rats.Methods: For effect of GGCX on MGP carboxylation in NRK-52 E cells: GGCX interfered or over-expressed NRK-52 E cells+normal or high calcium medium.For effect of Vit K or warfarin on MGP carboxylation in NRK-52 E cells,control group: NRK-52 E cells+normal or high calcium medium;Vit K1 group: NRK-52 E cells+Vit K1+normal or high calcium medium;Vit K2 group: NRK-52 E cells+Vit K2+normal or high calcium medium;Warfarin group: NRK-52 E cells+warfarin+normal or high calcium medium;Solvent group: NRK-52 E cells+DMSO+normal or high calcium medium.Elisa analysis was conducted to detect the level of ucMGP and MGP in the supernatant.Western blot was performed to detect the expression of MGP,BMP2,OPN and Runx2 in cells.Calcium ion probe was used to detect the calcium influx into cells.Animal experiment was grouped as follows.Control group: normal diet and drinking water;1,25(OH)2D3 group: intraperitoneal injection of 1,25(OH)2D3 every other day;Vit K2 group: intraperitoneal injection of Vit K2+1,25(OH)2D3 every other day;Warfarin group: intraperitoneal injection of warfarin+1,25(OH)2D3 every other day;Hydrochlorothiazide group: intraperitoneal injection of hydrochlorothiazide+1,25(OH)2D3 every other day.Body weight was measured every two weeks.In the end of twelfth week,24 h urine,blood and kidney tissue samples from rats were collected.Urine volume was recorded and level of urinary calcium and phosphorus was measured.Coagulation function,liver and kidney function,serum calcium and phosphorus,plasma ucMGP and Vit K2 level was also measured.MGP,BMP2 and OPN expression in kidney tissues was detected by immunohistochemistry.Von Kossa staining was performed to detect the calcium salt deposition.Tunel staining was used to detect tissue apoptosis.Results: When treated with normal medium,regulation of GGCX in NRK-52 E cells did not affect the expression of MGP but inhibition of GGCX could increase the level of ucMGP.When treated with high calcium medium,up-regulation of GGCX could decrease the ucMGP level and inhibit the expression of BMP2 and OPN in NRK-52 E cells.In contrast,down-regulation of GGCX promoted the expression of BMP2 and OPN.Vit K1,Vit K2 and warfarin supplementation had no effect on MGP expression in NRK-52 E cells.Vit K1 and Vit K2 supplementation could decrease the ucMGP level and inhibit the BMP2,OPN expression in NRK-52 E cells.In addition,the effect of Vit K2 was more significant.The level of ucMGP in NRK-52 E cells significantly increased when treated with warfarin.In addition,Vit K could inhibit the influx of calcium ion into NRK-52 E cells.Compared with 1,25(OH)2D3 group,Vit K2 and hydrochlorothiazide group showed less calcium salt deposition,tissue apoptosis and OPN expression in kidney tissues.Plasma ucMGP level and renal BMP2 expression in the Vit K2 group were also lower than in 1,25(OH)2D3 group.However the hydrochlorothiazide group showed no significant difference with 1,25(OH)2D3 group in terms of plasma ucMGP level and renal BMP2 expression.Warfarin supplementation increased the plasma ucMGP level,calcium deposition,tissue apoptosis,and the renal expression of BMP2 and OPN compared with 1,25(OH)2D3 group.Conclusion: Up-regulating GGCX or supplementing Vit K can promote the carboxylation of MGP,which inhibits the activation of BMP2 signaling pathway and calcium salt deposition in renal tissues.Part Ⅲ.The correlation between plasma ucMGP level and recurrence of kidney stonesObjective: To detect the plasma ucMGP level in kidney stone patients and investigate the correlation between plasma ucMGP level and recurrence of kidney stones.Methods: Kidney stone patients who came to our department were invited to participate in this research.Those with fever,hematuria,nephrostomy tube or urinary tube,urinary system deformity or urinary tuberculosis were excluded.In addition,patients with a history of taking Vit K or warfarin within one month were excluded.According to the recurrence history,patients were divided into the initial group and the recurrence group.Clinical data of patients,including age,gender,height,weight,history of hypertension and diabetes,smoking and drinking history,history of stone recurrence,family history of stones,urine routine and blood biochemical data were collected.Plasma ucMGP level was measured using Elisa kit.24 h urine from patients was collected.The urine volume was recorded and the level of urine calcium and oxalate was measured.Clinical data were compared between two groups.Logistic regression analysis was conducted to evaluate the correlation between age,BMI,history of hypertension and diabetes,urine volume,24-hour oxalate and calcium excretion,plasma ucMGP and the recurrence of kidney stones.Results: A total of 91 kidney stone patients were included in this study,involving 48 initial patients and 43 recurrence patients.There was no significant difference in terms of gender,age,BMI,serum calcium,phosphorus,magnesium and uric acid level,history of hypertension and diabetes,family history of stones,history of smoking and drinking between two groups.In addition,there was no significant difference in 24 h urine oxalate and calcium excretion between two groups.We found that the level of plasma ucMGP in patients with recurrence kidney stones was significantly higher than in initial kidney stone patients(2946.06ng/m L vs 2250.78ng/m L,P<0.05).Multivariate logistic regression analysis showed that ucMGP≥2400ng/m L and 24 h urinary oxalate excretion≥25mg are risk factors for recurrence of kidney stones.The risk of stone recurrence in patients with plasma ucMGP≥2400ng/m L was 3.035 times higher than those with ucMGP<2400ng/m L(OR 3.035,95% CI 1.115-8.859,P=0.03),and the risk of stone recurrence in patients with 24 h urinary oxalate excretion≥25mg were 3.586 times higher than those with oxalate excretion<25mg(OR 3.586,95% CI 1.092-11.779,P=0.035).Conclusion: Plasma ucMGP≥2400ng/m L and 24 h urinary oxalate excretion≥25mg are risk factors for recurrence of stones.Part Ⅳ.Quantitative proteomic analysis of urinary exosomes in kidney stone patientsObjective: Increased urinary exosomes are associated with kidney stones but how they work is unknown.In this study,we aim to identify dysregulated proteins in urinary exosomes from kidney stone patients and to explore the potential role of exosomal proteins in nephrolithiasis.Methods: Morning urine samples were collected from six patients with kidney stones and six age-/sex-matched healthy controls.Urinary exosomes were isolated via ultracentrifugation.Label free liquid chromatography-tandem mass spectrometry was performed to analyze the proteome of urine exosomes.Bioinformatics analysis was conducted to identify dysregulated proteins related to stone formation.Results of proteomic analysis were verified by Western blotting.Results: 960 proteins were identified in urinary exosomes,of which 831 were identified in the control group and 879 in the stone group.16 proteins in urinary exosomes were found most significantly different between kidney stone patients and healthy controls.Gene ontology analysis showed that these differential proteins were mainly enriched in innate immune response,defense response to bacterium and calcium-binding.S100A8,S100A9 and S100A12 were common in above three GO terms and were chosen for further study.Western blotting confirmed that the expression of these three S100 proteins was higher in urinary exosomes from kidney stone patients.In addition,S100 proteins were aggregated in urinary exosome vesicles and it was hard to detect them in urine.Conclusions: Urinary exosomes from kidney stone patients are rich in S100 proteins and play a role in innate immune response,defense response to bacterium and calcium-binding.