Urinary miR-16 transactivated by C/EBPß reduces kidney function after ischemia/reperfusion-induced injury.

Ischemia-reperfusion (I/R) induced acute kidney injury (AKI) is regulated by transcriptional factors and microRNAs (miRs). However, modulation of miRs by transcriptional factors has not been characterized in AKI. Here, we found that urinary miR-16 was 100-fold higher in AKI patients. MiR-16 was detected earlier than creatinine in mouse after I/R. Using TargetScan, the 3'UTR of B-cell lymphoma 2 (BCL-2) was found complementary to miR-16 to decrease the fluorescent reporter activity. Overexpression of miR-16 in mice significantly attenuated renal function and increased TUNEL activity in epithelium tubule cells. The CCAAT enhancer binding protein beta (C/EBP-ß) increased the expression of miR-16 after I/R injury. The ChIP and luciferase promoter assay indicated that about -1.0 kb to -0.5 kb upstream of miR-16 genome promoter region containing C/EBP-ß binding motif transcriptionally regulated miR-16 expression. Meanwhile, the level of pri-miR-16 was higher in mice infected with lentivirus containing C/EBP-ß compared with wild-type (WT) mice and overexpression of C/EBP-ß in the kidney of WT mice reduced kidney function, increased kidney apoptosis, and elevated urinary miR-16 level. Our results indicated that miR-16 was transactivated by C/EBP-ß resulting in aggravated I/R induced AKI and that urinary miR-16 may serve as a potential biomarker for AKI.

Exosomal ATF3 RNA attenuates pro-inflammatory gene MCP-1 transcription in renal ischemia-reperfusion.

Transcriptional repressor activating transcription factor 3 (ATF3) is induced by various stress stimuli, including inflammation-induced renal injury. In addition, ATF3 also down-regulates adhesion molecules like intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), and monocyte chemotactic protein-1 (MCP-1). However, the relation between up-regulated ATF3 after renal ischemia/reperfusion (I/R) injury and MCP-1 is not completely understood. In this study, we demonstrated that, in renal I/R induced inflammation, induction of adhesion molecules (interleukin-6, P-selectin, E-selectin, ICAM, VCAM, and MCP-1) was higher in ATF3-knockout mice than in wild-type animals. Molecular and biochemical analyses revealed that ATF3 binds to the ATF/CRE sites in the MCP-1 promoter and inhibits the secretion of MCP-1 from renal epithelial cells after I/R injury. Urinary exosome containing ATF3 RNA was 60-fold higher in patients with acute kidney injury than in normal controls, but no difference in total urinary ATF3 RNA levels was found. In addition, in vitro study showed that exosome containing ATF3 RNA derived from epithelial cells also inhibits MCP-1 expression in the epithelial cells and macrophage migration. Furthermore, direct administration of the epithelium-derived exosomal ATF3 RNA attenuates I/R induced kidney injury. Together, our studies reveal a novel regulatory mechanism of MCP-1 expression mediated by the exosomal ATF3 RNA under renal I/R insult and suggest a potential targeted therapy for I/R induced acute kidney injury.

Thromboxane A2 mediates iron-overload cardiomyopathy in mice through calcineurin-nuclear factor of activated T cells signaling pathway.

BACKGROUND: Recent studies demonstrated that iron overload could enhance the production of arachidonic acid and prostanoid, suggesting a causal connection between these signals and iron-overload cardiomyopathy. However, information regarding the downstream signaling is limited. Because thromboxane A2 (TXA2) and prostacyclin are the 2 major prostanoids in the cardiovascular system, and TXA2 plays a major role in vascular atherosclerosis and has pro-inflammatory characteristics, we intended to elucidate the role of TXA2 in iron-overload cardiomyopathy. METHODS AND RESULTS: A 4-week iron loading protocol was instituted for both TXAS gene-deleted (TXAS(-/-)) mice and wild-type (WT) mice, with less severe cardiac fibrosis and preserved normal left ventricular contraction in the TXAS(-/-) mice. Inflammatory profiles, including MCP-1, TNF-α, IL-6, ICAM-1, and myeloperoxidase activity were also lower in the TXAS(-/-) as compared with WT littermates. TXAS supplement to the iron-injured TXAS(-/-) mice re-aggravated cardiac inflammation. Using a TXA2 analog, U46619, for NFAT reporter luciferase activity on cardiomyoctes, and intraperitonal injection of U46619 into nuclear factor of activated T cells (NFAT)-luciferase transgenic mice demonstrated that U46619 increase NFAT expression, and this expression, as well as TNF-α expression, can be blocked by TXA2 receptor antagonist (SQ29548), NFAT-SiRNA, calcineurin inhibitor, or calcium chelator. Finally, intraperitoneal injection of the TNF-α antibody, infliximab, into iron-injured mice decreased TXAS expression and attenuated cardiac fibrosis. CONCLUSIONS: TXA2 mediates iron-overload cardiomyopathy through the TNF-α-associated calcineurin-NFAT signaling pathway.

MicroRNA-494 reduces ATF3 expression and promotes AKI.

MicroRNA-494 mediates apoptosis and necrosis in several types of cells, but its renal target and potential role in AKI are unknown. Here, we found that microRNA-494 binds to the 3'UTR of activating transcription factor 3 (ATF3) and decreases its transcription. In mice, overexpression of microRNA-494 significantly attenuated the level of ATF3 and induced inflammatory mediators, such as IL-6, monocyte chemotactic protein-1, and P-selectin, after renal ischemia/reperfusion, exacerbating apoptosis and further decreasing renal function. Activation of NF-κB mediated this proinflammatory response. In this ischemia/reperfusion model, urinary levels of microRNA-494 increased significantly before the rise in serum creatinine. In humans, urinary microRNA-494 levels were 60-fold higher in patients with AKI than normal controls. In conclusion, upregulation of microRNA-494 contributes to inflammatory or adhesion molecule-induced kidney injury after ischemia/reperfusion by inhibiting expression of ATF3. Furthermore, microRNA-494 may be a specific and noninvasive biomarker for AKI.

Protective effects of adiponectin against renal ischemia-reperfusion injury via prostacyclin-PPARα-heme oxygenase-1 signaling pathway.

Adiponectin (APN), a circulating adipose-derived hormone that regulates inflammation and energy metabolism, has beneficial effects on the cardiovascular disorders. Serum APN levels are lower in patients with coronary artery disease and higher in patients with chronic kidney disease. However, the precise role of APN in acute reno-vascular disease is not clear. Results of the present study show that serum APN concentration decreased after renal ischemia reperfusion (I/R) injury in mice. In addition, I/R-induced renal dysfunction (elevated serum creatinine and urea levels), inflammation (number of infiltrating neutrophils, myeloperoxidase activity), and apoptotic responses (apoptotic cell number and caspase-3 activation) were attenuated in APN-treated compared to control mice. Molecular and biochemical analysis revealed that APN up-regulates heme oxygenase-1 (HO-1) via peroxisome-proliferator-activated-receptor-α (PPARα) dependent pathway which is mediated through the enhancement of COX-2 and 6-keto PGF1α expression. Chromatin immune-precipitation assay demonstrated that APN increases the binding activity of PPARα to PPRE region of HO-1 promoter. Furthermore, APN induced HO-1 expression was only found in wild-type but not in PPARα gene deleted mice. This provides in vivo evidence that APN mediated HO-1 expression depends on PPARα regulation. In conclusion, our results provide a novel APN mediated prostacyclin-PPARα-HO-1 signaling pathway in protecting renal I/R injury.

BDNF mediated TrkB activation is a survival signal for transitional cell carcinoma cells.

Pathologically, >90% of bladder cancer is transitional cell carcinoma (TCC). Previously, brain-derived neurotrophic factor (BDNF) but not tropomyosin-related kinase B (TrkB) was found in normal urothelium. TrkB activation by BDNF has been shown to promote the progression of several cancers, however, the existence and functional roles of both BDNF and TrkB in TCC have not been elucidated. In this study, three human TCC cell lines, BFTC905, TSGH8301, and T24 were used for the investigation. Both BDNF and TrkB but not TrkA or TrkC identified by RT-PCR and Western blotting were found in these cell lines. Immunostaining demonstrated the cytosolic expression of BDNF and TrkB, as well as membranous expression of TrkB in these cells. BDNF released from three cell lines was also detected in culture medium by ELISA. The proliferation of BFTC905 cells was enhanced by recombinant human BDNF (rhBDNF) in vitro, which was associated with increased phospho-TrkB and phospho-ERK levels. In contrast, TrkB-Fc chimeric protein served as BDNF scavenger eliciting cytotoxicity. Addition of rhBDNF in these cell lines cultured in poly-HEMA [Poly(2-hydroxyethyl methacrylate)] coated dishes for 48 h did not confer resistance to anoikis. Increased phospho-Akt expression was observed transiently within an hour after rhBDNF administration but disappeared 24 h later. Weekly injections of 100 ng rhBDNF into the cancer cell-loading site for 6 weeks promoted BFTC905 xenograft growth in SCID mice. Daily injection of 5 microg TrkB-Fc chimeric protein into the tumor 2 weeks after tumor cell implantation delayed tumor growth concomitant with phospho-TrkB suppression in xenografts. These results indicate that BDNF binding to TrkB receptor is a survival signal for TCC cells. Drugs that block BDNF or TrkB may provide a new and potential approach for TCC therapy.