Study On The Effect Of ATP1A1 In Kidney Stone Formation Based On Big Data Mining

Nephrolithiasis(or kidney stone)is a common problem in the world caused by environmental and genetic factors,with a high incidence and recurrence rate,bringing great physical and economic burdens for patients.The majority of nephrolithiasis is idiopathic calcium stones,which are primarily composed of calcium oxalate(Ca Ox)and often admixed with some calcium phosphate as the initial nidus of the stone.The pathogenesis of nephrolithiasis is complex and unclear,but the crystal-cell adhesion is recognized as the key initiation process for crystal retention and stone formation in the kidney.Evidences revealed that damaged renal tubular epithelial cells could increase the affinity for crystal attachment,and crystal deposition also aggravated cell injury and inflammatory response by producing excessive reactive oxygen species(ROS).However,the key genes involved in this process have not been fully understood so far.With the development of high-throughput detection technology,a variety of omics technologies have emerged,and multi-omics integrated analysis provides an effective method for discovering key susceptibility genes of diseases and exploring the molecular mechanism of complex diseases.Although several kidney stone-related omics studies have been reported,the multiomics meta-analysis is not common.Therefore,In the present research,integrated analysis of the gene expression profiles and the whole exome sequencing(WES)data from calcium stone patients was performed to search key genes for nephrolithiasis.The correlation of the key gene and the risk of stone formation was investigated by expanding the samples.Then the function and mechanism of the candidate key gene were investigated in vivo and in vitro by using the methods of cell biology and molecular biology.Firstly,the gene expression profiles(GSE73680)of calcium stone patients were downloaded from the Gene Expression Omnibus(GEO).1.5-fold differentially expressed genes were screened and the weighted correlation network analysis(WGCNA)was performed.A total of 400 1.5-fold differentially expressed genes were obtained and one gene module related to kidney stone was identified(including 434 genes).GO and KEGG enrichment analysis of these two data sets showed that several genes were significantly enriched in “sodium: potassium-exchanging ATPase complex”(Na/K-ATPase,NKA)and the ion reabsorption pathway involved by NKA,suggesting that NKA may play an important role in kidney stone formation.Secondly,peripheral blood DNA from 28 patients with Ca Ox patients was extracted for WES sequencing,and a total of 76 single nucleotide polymorphisms(SNPs),which were associated with the risk of kidney stone and included31 missense SNPs,30 coding-synonymous SNPs,7 SNPs in the 5’-untraslated region(5’-UTR)and 8 SNPs in 3’-UTR,were obtained(p < 0.005).These SNPs were mapped to 69 genes,which were integrated with the differentially expressed genes from GSE73680 for Venn analysis,finding that ATP1A1 was the only gene in the intersection of the three sets of data.These results suggested an important role of ATP1A1 in calcium stone formation,which may not only contribute to the risk of kidney stones at the genetic level,but also play an important role in the pathogenesis of kidney stones through changing its expression level.In order to further study the genetic contribution of ATP1A1 gene to the pathogenesis of kidney stones,we continued to collect peripheral blood genomic DNA from 214 calcium stone patients and 232 controls to detect the genotype of SNPrs11540947 in the 5’-UTR of ATP1A1 with high resolution melting curve analysis.Statistical analysis showed that the T allele of rs11540947 was associated with a higher risk of stone formation(p = 0.0311,OR = 1.537,95% IC 1.034-2.277),and this association remained significant in men patients alone(p = 0.0336,OR = 1.731,95% CI 1.056-2.87).The dual luciferase reporter gene system experiments showed that the T allele of rs11540947 reduced the promoter activity of ATP1A1 in vitro,suggesting that the genetic variation of rs11540947 in ATP1A1 may be involved in stone formation by regulating the ATP1A1 expression.In order to further explore the manner and mechanism of ATP1A1 in the kidney stone formation,we conducted a series of cellular and molecular biology studies on renal tubular epithelial cells HK2 and Ca Ox stone rat models.We found that calcium oxalate monohydrate(COM)stimulation significantly inhibited the NKA activity and reduced the m RNA and protein levels of ATP1A1 in HK2 cells.With the decrease of ATP1A1 expression,the phosphorylation levels of Src,p38 and JNK as well as the intracellular ROS level were also significantly increased.However,the recombinant adenovirus-mediated ATP1A1 overexpression significantly inhibited the activation of these signaling molecules and reduced the intracellular ROS accumulation,demonstrating that COM activated ATP1A1/Src/ROS/MAPKs signaling system by inhibiting the expression of ATP1A1.Further experiments showed that ATP1A1 overexpression also inhibited COM-induced NF-κB/Nrf2 inflammatory response and apoptosis.ATP1A1 is a multifunctional protein with both NKA enzyme activity and inhibitory activity of Src/ROS signaling pathway.In order to explore its specific pathway of cell protection,we synthesized p Na Ktide,a specific inhibitor of ATP1A1/Src/ROS signaling pathway.It is a polypeptide sequence from the Nterminal of ATP1A1 that binds to the kinase domain of Src,thus simulating the interaction between ATP1A1 and Src without the enzyme activity of ATP1A1.Similar to ATP1A1 overexpression,p Na Ktide also significantly inhibited the ATP1A1/Src/ROS/MAPKs signaling system,and alleviated COM-induced NF-κB/Nrf2 inflammatory response and cell apoptosis,suggestting that the cell protection mediated by ATP1A1 overexpression was mainly achieved through inhibition of the Src/ROS related signaling pathways.Crystal-cell adhesion is a key initial process of the stone formation.We found that ATP1A1 overexpression or p Na Ktide treatment inhibited the crystal-cell adhesion,and p Na Ktide also promoted the expression of calcification-inhibiting protein MGP.Next,we explored the molecular mechanism of the down-regulation of ATP1A1 induced by COM,and found that COM exposure significantly upregulated the m RNA and protein levels of DNA methylation transferases including DNMT1,DNMT3 a and DNMT3 b.The specific inhibitor of DNA methylation transferase 5Aza-2dc significantly prevented the decrease of ATP1A1 m RNA level and protein level induced by COM stimulation,proving that the increase of DNA methylation level was the main way of the COM-induced ATP1A1 downregulation.Consistent with in vitro experiments,in vivo studies also showed that the expression of ATP1A1 in the kidney of the Ca Ox stone model rats was significantly decreased,which activated the Src/ROS/MAPKs/NF-κB signaling system.However,p Na Ktide inhibited the formation of renal crystals and urinary oxalic acid excretion by inhibiting this signaling system,and 5Aza-2dc also significantly inhibited the decrease of ATP1A1 expression.These results demonstrated that crystallization deposition can inhibit the expression of ATP1A1 through DNA methylation,thereby activating the Src/ROS/MAPKs signaling system,and inhibiting the activation of this signaling system can alleviate the crystal-induced inflammatory response,apoptosis,crystal-cell adhesion,and promote the expression of calcification inhibitor MGP,thus inhibiting the kidney stone formation.Kidney stone formation is similar to vascular calcification in that extracellular matrix proteins are involved.MGP,a vitamin K-dependent extracellular matrix protein,is a key molecular in the inhibition of vascular and kidney calcification.Our experiments also showed MGP was a key downstream protein of ATP1A1/Src/ROS signaling pathway,but its specific mechanism in stone formation remains unclear.Therefore,we used vitamin K1(VK1),an activator of MGP to study the role of MGP in the stone formation.In vivo experiments showed that VK1 significantly inhibited the formation of renal crystals and the m RNA expressions of inflammatory factor MCP-1 as well as the renal fibrosis marker Collagen I.In vitro studies showed that VK1 could alleviate HK2 cell injury and apoptosis induced by warfarin,reduce crystal-cell adhesion,and promote the expression of MGP in vitro and in vivo.Overexpression of MGP inhibited crystal-cell adhesion,while interference of MGP enhanced crystal-cell adhesion and eliminated the improvement of VK1 on crystal-cell adhesion,suggesting that MGP protein was necessary for VK1 to inhibit the crystal-cell adhesion.These findings suggested that the expression and activity of MGP played an important role in the kidney stone formation,and VK1 may be a potential drug for the treatment and prevention of stone formation.In conclusion,the present study demonstrated,for the first time,that ATP1A1 played an important role in the kidney stone formation as both a genetic risk factor and an environmental induction factor by integrating transcriptome data and WES sequencing data and combining genetic,cellular,molecular and other multidisciplinary biotechnology.We also revealed that DNA methylation was a novel gene expression mechanism of Ca Ox stones,and found,for the first time,that vitamin K1 inhibited the crystal formation in vivo through the MGP protein.The present study enriched the theory of kidney stone formation and suggested that both ATP1A1 and MGP may be potential therapeutic targets for kidney stones,which has important guiding significance for the development of new strategies for kidney stone treatment and prevention of recurrence.