| Kidney stone is a complex disease caused by environmental and dietary factors and genetic factors.It is characterized by abnormal crystalline deposition of renal tubules or renal interstitium.The clinical manifestations are usually renal function damage and urinary obstruction.The rate of incidence exceeds 6-12% and recurrence is 50%,meanwhile,it can be repeated several times in life.The mechanism of formation of kidney stone is complex.Up to now,the molecular pharmacological mechanism of kidney stone remains unclear.There is only the temporary relief of symptoms by lithotripsy or surgery because of the lack of effective treatment and prevention methods in clinic.However,these treatments can not provide effective drug suppression and recurrence prevention based on the pathogenesis of renal stone formation.The kidney can maintain and regulate the balance of body's water and salt,and can eliminate metabolic waste,it is also prone to become salt supersaturation in the renal tubules.Crystals are deposited in the renal tubules or interstitium under common metabolic conditions such as high oxaluria and high calcium urine risk,leading to calcification of the kidney,continuous nucleation,aggregation and growth of crystallization,and ultimately the formation of kidney stone.In this process,the kidney expresses and secretes some macromolecular proteins through active or passive biological behaviors to participate in and influence the formation of stone.Matrix glutamic acid protein(MGP)is a vitamin K-dependent secretory protein.Some of studies have shown that MGP is associated with vascular calcification and inhibitor of vascular calcification.Our previous study has found that there was no crystal in the kidney where the MGP protein was highly expressed in hyperoxalamate rats,suggesting that MGP may also be related to the pathogenesis of kidney stone.I n order to explore the specific mechanism of MGP in the formation of kidney stone,we elucidated the effects of MGP on the formation of calcium oxalate stones and analysis their effects on cell damage at the cellular and animal level.First,we study the effect of the change of MGP gene expression on renal tubular epithelial cell injury induced by calcium oxalate crystallography,we constructed a recombinant adenovirus(Ad-siMGP)expressing small interfering RNA sequences targeting the MGP gene and recombinant adenovirus overexpressing the MGP gene(Ad-MGP).We used these two kinds of adenoviruses to study the effect of regulation of the expression of intracellular MGP genes on the formation of calcium oxalate crystals.The results of quantitative real-time PCR showed that after the transfection of HK-2 cells with Ad-MGP and Ad-siMGP for 48 h,the levels of MGP m RNA were significantly increased or decreased.The atomic absorption spectroscopy results of our preliminary experiments showed that the high expression of MGP gene induced by Ad-MGP can significantly reduced the adhesion between calcium oxalate crystals and HK-2,suggesting that expression of MGP can play a protective role in renal tubular epithelial cells by reducing cell adhesion.Next,we used calcium oxalate crystals to stimulate renal tubular epithelial cells(HK2)to study the effects of changes in the activity of MGP proteins on crystallization as well as on injury and apoptosis of HK-2.It is was reported that vitamin K1 can significantly increase the activity of MGP,and warfarin can inhibit the activity of MGP by inhibiting the synthesis of vitamin K1.Therefore,vitamin K1 and warfarin were used to upregulate or downregulate the activity of MGP in this study.The calcium oxalate crystals combined with vitamin K1 and warfarin were treated to stimulated HK-2 The results obtained from MTT assay and DAPI staining showed that calcium oxalate crystals could induce cell injury and death,the use of warfarin increased the effect of crystallization on cell injury and apoptosis in the case of calcium oxalate crystals induced injury and apoptosis of HK-2,and the combination of vitamin K1 and warfarin rescued the HK-2 cells from the role of warfarin.This result suggests that MGP plays a cytoprotective role in cell injury and apoptosis induced by calcium oxalate crystals.Theatomic absorption spectroscopy was then performed to detect the effect of calcium oxalate crystals on cell adhesion.The results showed that vitamin K1 added cells showed a significant decrease in cell adhesion,suggesting that activation of MGP can play a protective role in renal tubular epithelial cells by reducing cell adhesion.The down-regulation of MGP gene induced by Ad-siMGP also reversed the protective effect of vitamin K1 on crystallization-induced injury of HK-2.This result demonstrates that the protective effect of vitamin K1 on renal tubular epithelial cells against calcium oxalate crystals by enhancing the activity of MGP.Finally,we investigated the possible effects of MGP on the formation of renal crystallization at the animal level.The rats used in the experiment were randomly divided into three groups: blank control group(-),model control group(Normal saline group,calcium oxalate kidney stone model control group injected with saline),and vitamin K1 group(Vitamin K1 group,injection Vitamin K1 Calcium Oxalate Kidney Stone Model Group).The results of immunohistochemistry combined with polarized light microscopy showed that compared with the blank control group,the kidneys of the model control rats showed obvious crystallization,which proved that the model of kidney stone caused by ethylene glycol was successful.Compared with the model control group,the kidney tissue of the vitamin K1 group was significantly smaller and the number was significantly reduced,demonstrating that the addition of vitamin K1 reduced the formation of calcium oxalate crystals.This result also suggests that vitamin K1 supplementation can become a new way of preventing and treating kidney stones.In summary,the effect of MGP on the formation of calcium oxalate stones was preliminary studied at the cellular and animal level.It was demonstrated that decrease in the activity of MGP protein can facilitate the injury and apoptosis of renal tubular epithelial cellsinduced by the crystallization.At the same time,the enhanced activity and up-regulation of gene expression of MGP protein can significantly reduce the adhesion between HK-2 and calcium oxalate crystals.Vitamin K1 significantly reduces the formationof calcium oxalate crystals in rats.These results demonstrate that the MGP protein plays a role in inhibiting the formation of kidney stone and protecting the injury of kidney epithelial cells by reducing crystallization and cell adhesion.Our results enriched the theory of kidney stone formation,and laid foundations for the intensive study of the molecular mechanism of kidney stone.Futhermore our data work also provided new ideas for the prevention and treatment of clinical kidney stones. |
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