The Molecule Mechanism Of Hypoxia-induced Microrna On Regulating Renal Angiogenesis After Ischemic Injury

Renal Ischemia/Reperfusion injury (IRI) is an inevitable issue that occurs in major kidney resection and kidney transplantation. In recent decades, extensive studies have revealed a variety of angiogenic factors and their receptors, including vascular endothelial growth factor (VEGF)-VEGFRs, Angiopoietin-Tie, Ephrin-EphRs and Delta-Notch to be the very important regulators of angiogenesis in vertebrates. Neovascularization is a major physiological response to ischemia that involves a sequence of events resulting in development of new capillaries from endothelial cells or preexisting vessels. Despite these phenomenal findings, the precise protective mechanisms involved in renal ischemic injury and angiogenesis remains to be undetermined. VEGF signaling pathway may be the most potent stimulator of renal angiogenesis. But the relation of Notch and renal angiogenesis is unclear. miRNAs are a family of endogenous noncoding RNAs of 22 to 24 nucleotides, which have emerged as important regulators of various physiologic and pathologic processes in eukaryotes, such as development, differentiation, metabolism, growth, proliferation and apoptosis. Other reports displayed miRNAs also play an important role during angiogenesis after ischemic injury. Our question is that whether renal miRNAs are involved in this effect remains completely unknown. To address this issue, we employed miRNA microarray to investigate potential involvement of microRNAs in mouse kidney subjected to IRI, to identify some of the microRNAs possibly associated with renal angiogenesis, then to explore the molecule mechanism of renal angiogenesis after ischemic injury.Male Balb/c mice were subjected to a standard renal I/R. Mice were allowed to recover for 4h,24h and 72h post ischemia or sham-surgery. The serum creatinine (SCr), urea nitrogen (BUN) level were dramatically increased in the mice subjected to I/R 24h compared to sham-operated mice, hematoxylin and eosin (HE) staining showed that there was significant damage at 24 h following ischemic injury, which manifested primarily as cytoplasmic vacuolization, cell necrosis of the proximal convoluted tubule, and tubular lumen obstruction and impairments, which is consistent with serum SCR and BUN levels of kidney ischemic injury. Taken together, we confirmed that renal I/R injury model succeed based on biochemistry measurement and pathological level. To examine ischemia-induced angiogenesis in renal ischemia on the capillary rarefaction changes, we quantified renal microvascular density following the ischemic insult in our mouse model of renal ischemia We utilized the endothelial cell marker CD31 to identify renal capillary rarefaction by immunohistochemistry staining, and used stereological method to analyze number of microvessels and found a significant increase of microvessels rarefaction in renal ischemic region compared to control group regardless of 24 or 72 hours after I/R injury, indicating that angiogenesis indeed appeared in kidney after ischemia injury. To identify whether the change of renal ischemia is related to miRNA, We performed microarray analysis and identified miRNAs changes relatived with angiogenesis. we found that seventy-six microRNAs exhibiting more than twofold differences were identified in the kidney I/R injury. Among them,40 miRNAs were down-regulated and 36 miRNAs up-regulated. On the basis of documents previously reported and computation analysis, we verificated the representative miRNAs expression (miR-210, miR-126 and miR-320) associated with renal angiogenesis. We also put human umbilical vein endothelial cells(HUVEC) under hypoxia for 12h to establish hypoxic model. The expression of HIF-1, VEGF, VEGFR2, notchl and its target gene were increased in renal I/R and hypoxic cells at both the mRNA and protein levels which detected by qRT-PCR, Western Blot, immunofluorescence staining and flow cytometry. These results display that HIF-VEGF-Notch signaling pathway is involved in angiogenesis after renal I/R injury. To define whether miR-210 may regulate VEGF-Notch pathway and involve in angiogenesis after renal I/R injury, we infected supernatant containing miR-210 into HUVE-12 cell line and made stable cell line which consistently expresses miR-210.80% cell expressed miR-210 with GFP. The over-expression of miR-210 in cells was confirmed using qRT-PCR and GFP expression observed under fluorescence microscope at 72 hours after infection. VEGF, VEGFR2, notchl after infection of LV-miR-210-GFP were up-regulated which detected by qRT-PCR, Western Blot, and flow cytometry. Taken all together, this finding indicates that VEGF-Notch signaling pathway is involved in formation of capillary under miR-210 over-expression.In summary, our results imply that miRNAs may be involved in targeting HIF-VEGF-notch pathway signaling to regulate angiogenesis after renal I/R injury. The specific miRNAs as key regulators of the response to renal ischemia has provided novel insights into the angiogenesis mechanism of renal I/R injury and opened clinical avenues.