Ca²⁺ Promotes Adhesion Molecules Expression Via ROS Dependent NF?B Pathway In The Development Of Urolithiasis

PART L Integrative microRNA-gene expression network analysis in genetic hypercalciuric stone-forming rat kidneyPurpose. To identify rat hypercalciuria-related miRNAs and their target mRNAs.Methods. A multi-step approach combining microarray miRNA and mRNA expression profile and bioinformatics analysis was adopted to analyze dysregulated miRNAs and genes in genetic hypercalciuric stone-forming (GHS) rat kidneys, using normal Sprague-Dawley (SD) rats as controls. We identified significantly differentially expressed Gene Ontology (GO) terms and KEGG pathways that were significantly enriched in GHS rats. In addition, we successfully constructed the miRNA-gene network and signal-net in GHS rat kidneys.Results. We identified 2418 mRNAs and 19 miRNAs as significantly differentially expressed, over 700 GO terms and 83 KEGG pathways that were significantly enriched in GHS rats. In addition, we constructed an miRNA-gene network that suggested that rno-miR-674-5p, rno-miR-672-5p, rno-miR-138-5p and rno-miR-21-3p may play important roles in the regulatory network, meanwhile, Sema5a, Lpin2, Gcntl, Masp1, Olrl and TraB were the most common miRNA targets. Furthermore, signal-net analysis suggested that NF-kappaB likely plays a crucial role in hypercalciuria urolithiasis.Conclusions. This study presents a global view of mRNA and miRNA expression in GHS rat kidneys, and suggests that miRNAs may be important in the regulation of hypercalciuria. The data provide valuable insights for future research, which should aim at validating the role of the genes featured here in the pathophysiology of hypercalciuria.PART ?. Ca2+ promotes adhesion molecules expression via ROS dependent NF?B pathwayPurpose. To identify the mechanism of Ca2+ promotes adhesion molecules expression in renal tubular epithelial cell and calcium oxalate crystals adhesion on cell surface.Methods. ICAM-1, VCAM-1 and IL-6 were detected by qRT-PCR, western blot and immunohistochemical staining in kidneys of GHS rat and SD rat, and serum levels of ICAM-1, VCAM-1 and IL-6 were detected by ELISA. The phosphorylation levels of p65 and I?B? were also examined by western blot. Von-kossal staining was used to investigated the crystals deposition in GHS rat and SD rat kidneys. The primary renal tubular epithelial cells of GHS rat was separated and cultured. The immunophenotypes (CK-18, CK19 and E-cadherin) were identified by flow cytometry. Intracellular Ca2+ concentration and ROS were measured in both primary renal tubular epithelial cells of GHS rat and NRK-52E cell line after Ca2+ treatment. qRT-PCR and western blot were used to detected the expression levels of ICAM-1,VCAM-1,IL-6 and the phosphorylation levels of p65 and I?B? Cel ultrastructure was observed with transmission electron microscopy. N-acetyl-L-cysteine (NAC), a reactive oxygen species (ROS) scavenger, and BAY117082, a NF?B inhibitor were pretreated NRK-52E cell line before Ca2+ treatment, the levels of ICAM-1,VCAM-1, IL-6 and the phosphorylation levels of p65 and I?B? were identified. Meanwhile, calcium oxalate crystals adhesion on the cell surface was detected by both microscope and ion chromatograph.Results. The mRNA and protein levels of ICAM-1, VCAM-1 and IL-6 were significantly increased in GHS rat kidney tissues than SD rat. The phosphorylation levels of p65 and I?B? were also increased. Von-kossal staining showed more crystals deposition in GHS rat kidneys. Ca2+ induce intracellular Ca2+ concentration and ROS production in both primary renal tubular epithelial cells of GHS rat and NRK-52E cell line. Ca2+induced ROS production was significantly inhibited by NAC. Furthermore, Ca2+ activated the NF?B signaling pathway by inducing phosphorylation of p65 and I?B? and increasing p65 nuclear translocation.Conclusion. These results provide evidence that Ca2+ induces ROS production formation which leads to NF?B signaling pathway activation and downstream adhesion molecules expression.Furthermore, the adhesion molecules promote the calcium oxalate crystals adhesion on the surface of renal tubular epithelial cell.