- Background: To explore long non-coding RNA (lncRNA), mRNA and circular RNA (circRNA) expression profiles and their biological functions in the pathogenesis of kidney stones in ethylene glycol-induced urolithiasis rats.. - Results: The expression of 1440 lncRNAs, 2455 mRNAs and 145 circRNAs was altered in the kidneys of urolithiasis rats. - mRNAs coexpressed with lncRNAs were involved in many kidney diseases, e.g., Ephb6 was associated with the reabsorption ability of the kidney. - Arl5b was associated with the dynamic changes in the podocyte foot process in podocyte injury. - Conclusion: The expression profile provided a systematic perspective on the potential functions of lncRNAs and circRNAs in the pathogenesis of kidney stones. - Differentially expressed lncRNAs and circRNAs might serve as treatment targets for kidney stones.. - Kidney stone patients usually present with pain and urinary tract infection, which can lead to chronic renal disease and even kidney function loss [3, 4]. - Approximately 80% of kidney stones are composed of calcium oxalate, calcium phosphate, or both [5]. - Circular RNAs may bind RNA-binding proteins or even base pair with RNAs, resulting in the forma- tion of large RNA-protein complexes. - To date, little is known about the functions of lncRNAs and circRNAs in the pathological processes of kidney stones. - Full list of author information is available at the end of the article. - 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - in rats with kidney stones. - Our findings might illuminate a novel mechan- ism of nephrolithiasis pathogenesis and provide new tar- gets for the prevention and treatment of kidney stones.. - Kidney stone modeling experiments. - The kidney stone group received drinking water with 1%. - Food and water were available in the cages during experimentation. - The specific gravity, pH, and concentrations of calcium, citrate, so- dium, potassium, uric acid, creatinine and urea nitrogen of the urine were measured with an Auto Analyzer (Hitachi, 7170A, Japan).. - To evaluate the aggregation of CaOx deposits, 5 slides were selected randomly and observed in the microscopic field with a magnification of 10*40. - The total RNA of the kidney stone group samples and control group samples was extracted using TRIZOL reagent (Invitrogen, NY, USA). - Co-expression of lncRNAs/mRNAs and function prediction The function of lncRNAs is forecasted according to an- notations of the function of the coexpressed mRNAs. - A DElncRNAs-DEmRNA co-expression network was con- structed to explore the connection between lncRNAs and mRNAs in the pathogenesis of kidney stones. - Genomic localizations of the paired lncRNAs and mRNAs were identified for cis prediction. - Analysis of the circRNA-miRNA interaction network The networks among circRNAs and miRNAs were pre- dicted based on miRanda, with a maximum binding free energy of − 20. - As shown in Table 2, the specific gravity of the kidney stone group was significantly decreased compared to the normal group. - How- ever, urinary output significantly increased in the kidney stone group. - There was no difference in the concentration of urinary sodium, potassium, and phosphate. - Urinary cal- cium, uric acid, urine creatinine and urea nitrogen were significantly increased in the kidney stone group.. - As shown in Table 2, the serum potassium, chlorine, phosphorus, and magnesium were similar in the two groups. - However, the concentrations of sodium and cal- cium were significantly increased in the kidney stone group. - Figure 1 suggests that in the 10× magnification and 40×. - magnification of the light microscope field, the renal paren- chyma of normal rat had integrity, and there was no CaOx crystal formation. - However, in the kidney stone group, the structure of the renal parenchyma was destroyed by abun- dant calcium oxalate crystals, which were colorless, with high refractivity (Arrow).. - Kidney tissues obtained from the kidney stone group and control group were applied for RNA sequencing. - and clean reads were obtained from four samples from the kidney stone group, and and clean reads were gener- ated from four corresponding control tissues (Additional file 1:. - More than 88% of the raw reads per sample were clean reads. - In the present study, 1440 lncRNAs, 2455 mRNAs and 145 circRNAs were identified as remarkably differentially expressed, with |fold change| ≥2.0, P <. - There were 58 up-regulated circRNAs and 87 down-regulated circRNAs in four kidney stone tissues compared with the controls. - Hierarchical clustering of the expression of the lncRNA, circRNA and mRNA showed obvious discrimination in kidney tissues between kidney stone rats and control rats (Fig. - Validation of deregulated lncRNAs and mRNAs. - Three lncRNAs and 3 mRNAs were chosen to verify the RNA-sequencing results in 4 pairs of samples by quanti- tative real-time PCR. - Meanwhile, of the 3 target mRNAs, ENSRNOT was up-regulated, and ENSRN OT and ENSRNOT were down-regulated in kidney stone tissues compared with controls (Fig. - Hence, the finding provides valid evi- dence that these lncRNAs and mRNAs could be impli- cated in the pathogenesis of kidney stones.. - Figure 2 Circos plot showing lncRNAs and mRNAs on rat chromosomes. - The outermost layer of the circos plot is a chromosome map of the rat genome. - The increased or decreased lncRNAs have been marked with red or green bars, respectively, and bar heights in the larger inner circle represent numbers of differently expressed lncRNAs. - Increased or decreased lncRNAs are marked with red or green bars, respectively, and bar heights in the smallest inner circle represent numbers of differently expressed lncRNAs. - a GO enrichment analysis for up-regulated mRNAs. - b GO enrichment analysis for down-regulated mRNAs. - c GO enrichment analysis for up-regulated circRNAs. - d GO enrichment analysis for down-regulated circRNAs. - Figure 6 KEGG pathway enrichment analysis of the differentially expressed mRNAs with the twenty highest enrichment scores. - a KEGG pathway enrichment analysis for up-regulated mRNAs. - b KEGG pathway enrichment analysis for down-regulated mRNAs. - GO enrichment analysis of the cir- cRNAs showed that they were mainly related to biological processes, and only a few differentially expressed genes were associated with cellular components and molecular function. - In the up-regulated circRNAs, the GO terms for BP, CC, and MF were correlated with endocytosis, tran- scription factor complex, and SH3 domain binding. - In the down-regulated circRNAs, the GO terms for BP, CC, and MF were correlated with L-amino acid transport, inter- mediate filament cytoskeleton, and L-amino acid trans- membrane transporter activity (Fig. - Co-expression of lncRNAs/mRNAs and function prediction To explore the molecular mechanisms of the pathogenesis of kidney stones, a co-expression network was built based on the expression levels of DElncRNAs and DEmRNAs. - A total of 129 DEmRNAs and 223 DElncRNAs were in- volved in the network, and it consisted of 352 nodes and 500 edges (Fig. - LncRNAs and their potential trans-regulated genes are shown in Fig. - During the pathogenesis of kidney stones, there is a core circRNA-miRNA regu- lation network. - Oral administration of ethylene glycol to rats for 4 weeks was reported to promote the deposition of crystals in the kidneys [12]. - In our study, according to the results of urine parameters, serum parameters and histology, we conclude that 1% EG with drinking water successfully led to CaOx kidney stones and renal hypofunction. - Figure 7 Construction of the lncRNA-mRNA co-expression network. - b LncRNAs and their potential cis-regulated nearby genes. - c LncRNAs and their potential trans-regulated genes. - comprehensive analyses of the profiles of differentially expressed lncRNAs and circRNAs in kidney stones have not yet been studied. - To explore the potential function of lncRNAs and circRNAs in kidney stones, we performed expression profiles genome-wide for kidney stone and matched control tissues using RNA sequencing and bio- informatics analysis.. - In our study, a total of 711 and 58 up-regulated, and 717 and 87 down-regulated lncRNAs and circRNAs, re- spectively, were identified to reveal the significant differ- ential expression in kidney stones. - Accordingly, 1732 up-regulated and 723 down-regulated mRNAs were iden- tified in kidney stone tissues compared with controls.. - Among them, Ephb6 which located in the tubules of the outer medulla and cortex regulated cytoarchitecture of medullary tubule cells may affect the reabsorption ability of the kidney [14].. - Figure 8 Construction of the circRNA-miRNA co-expression network. - Co-expression networks of lncRNAs/mRNAs and circRNAs/miRNAs were constructed to predict the function of lncRNAs and circRNAs in kidney stone rats. - Thus, our study provided a comprehensive understanding of the functions of lncRNAs and circRNAs in ethylene glycol-induced kidney stone rats. - our findings could help determine the effects of lncRNAs and circRNAs on kidney stone pathology.. - In the present study, we found dysregulated lncRNAs in the kidney tissues of CaOx rats and predicted their corresponding mRNAs through cis- and trans-targeting.. - Rnf2 was increased in the diabetic rat kidney and may play roles in the development of type 1 diabetes-induced renal fibrosis [20]. - The function of Rnf2 in regulating basal and aldosterone-stimulated transcription of the α-ENaC gene (which was related to salt balance) was found in the duct cell line [21]. - Xirp1 is a newly identified vitamin D recep- tor interacting protein and has an influence on VDR ac- tivity in the heart [23]. - Xirp1 might play an important role in the pathology of kidney stones. - However, few studies focus on the role of lncRNAs in kidney stones.. - In our study, most DElncRNAs in the co-expression net- work have not yet been annotated. - It is worthwhile to perform further studies to reveal the underlying link be- tween these lncRNAs and the pathomechanism of kid- ney disease.. - GO enrichment analysis on the circRNAs showed that in the development of kidney stones, circRNAs were mainly related to biological processes. - regulation network was present during the pathogenesis of kidney stones. - Some of the predicted co-expressed miR- NAs have been proven to be functional in hypercalciuria urolithiasis, such as rno-miR-138-5p co-expressed with circRNA_1297 and rno-miR-672-5p co-expressed with circRNA_0528 and circRNA_1620 [24].. - We did notdid not select areas of the rat kidney for RNA isolation, RT-PCR and sequencing analyses. - In conclusion, we found a profile of dysregulated lncRNAs, circRNAs and mRNAs that might be prospect- ive clinical markers associated with the development of kidney stones. - Our data laid a foundation for further po- tentially functional research into the lncRNAs and cir- cRNAs involved in kidney stones. - These results revealed that specific lncRNAs and circRNAs could be valuable for the diagnosis and therapy of kidney stones and may be of biological importance.. - The expression profile provided a systematic perspective on the potential functions of lncRNAs and circRNAs in the pathogenesis of nephrolithiasis and new targets for the prevention and treatment of kidney stones.. - DElncRNAs: Differentially expressed lncRNAs. - Prevalence of kidney stone in China: an ultrasonography based cross-sectional study. - Kidney stone and kidney function loss: a cohort study. - Kidney stone disease. - Differential gene and lncRNA expression in the lower thoracic spinal cord following ischemia/reperfusion-induced acute kidney injury in rats. - EphB2 and ephrin-B1 expressed in the adult kidney regulate the cytoarchitecture of medullary tubule cells through Rho family GTPases.. - Arl5b is a Golgi-localised small G protein involved in the regulation of retrograde transport. - Physical and functional interaction of Rnf2 with Af9 regulates basal and aldosterone-stimulated transcription of the alpha- ENaC gene in a renal collecting duct cell line. - Expression of oxidative stress and antioxidant defense genes in the kidney of inbred mice after intestinal ischemia and reperfusion
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