| Background: Ischemia-reperfusion injury (I/RI) is a major cause of renal dysfunctionin both native kidneys and renal allografts, miRNAs have emerged as importantregulators of gene expression and function in various diseases, but there seems tobe little understanding of how miRNAs play a role in renal ischemia-reperfusion injury.MicroRNA-24(miR-24) may have a significant regulating effect on renal ischemia-reperfusion injury. Here, we investigated the dynamic expression profiles of miR-24and target mRNA (GATA-2) in renal I/RI in Rat, and discussed the potential regulatingeffect of miR-24and its mechanism in renal I/RI.Methods: To understand the potential regulating effect of miR-24involved in renal IRI,Firstly, We had successfully established a rat model of renal I/RI. Preoperative andpostoperative changes of serum creatinine (SCR) and urea nitrogen (BUN) levelswere measured as markers of renal injury using an Autolab Analyzer(Hitachi Japan),HE staining was used to observe the renal histopathological changes. And then weperformed real-time quantification RT-PCR to detect the expression of miR-24andtarget mRNA (GATA-2) at baseline,4,24,48,72h after reperfusion in the rat modelof renal I/RI，and Immunohistochemistry Staining was conducted to detect GATA-2protein expression levels of distinct groups in renal I/RI of rats.Results：1. Successful model was necessary for measuring the dynamic expression profiles ofmiR-24and target mRNA (GATA-2) in renal I/RI research, which was assessed bypreoperative and postoperative renal function levels and histopathological changes ofdifferent ischemia-reperfusion groups in rats. The average SCR level (234±50.52umol/L) of rats in the experimental group of24h after reperfusion were the highest andits pathological damages were the most serious, and there were significant differencesin all I/RI groups,compared with the sham group and normal group (P <0.01). 2. Real-time PCR with miR-24: the real-time PCR assay used for the detection of theexpression of miR-24in a rat model of renal I/RI showed that the post-ischemic ratkidneys performed different degrees of up-regulated trend of miR-24at baseline,4,24,48,72h after reperfusion, miR-24expression ratio of the sham control is1.31±0.49(X±s,n=6), which had no significant difference compared with normalgroups (1.11±0.27)(P>0.05), and miR-24expression ratio of the ischemia groups atthe4,24,48,72h after reperfusion respectively were3.13±0.88,1.76±0.17,3.13±0.97and6.27±0.83, of which the72h groups exhibited the highest level. The experimentalgroups of24h and72h after reperfusion had significant difference compared with thesham group (P<0.01), and other ischemia groups had statistical significance comparedwith the sham group (P<0.05).3. Real-time PCR with GATA-2: the real-time PCR assay used for the detection of theexpression of GATA-2in a rat model of renal I/RI showed that the post-ischemic ratkidneys performed different degrees of down-regulated trend of GATA-2at baseline,4,24,48,72h after reperfusion, the average GATA-2expression ratio of the shamcontrols is0.91±0.18(X±s,n=6), which had no significant difference compared withnormal groups (0.97±0.20)(P>0.05), and GATA-2expression ratio of the ischemiagroups at the4,24,48,72h after reperfusion respectively were0.54±0.10,0.65±0.13,0.30±0.08and0.21±0.11, of which the72h groups exhibited the lowest level, Thesham controls had no significant difference compared with normal groups (P>0.05),but all I/RI groups had significant difference compared with the sham group (P<0.01).4. Immunohistochemistry Staining with GATA-2: Immunohistochemistry Stainingwas conducted to detect GATA-2protein expression levels of distinct groups in renalI/RI of rats. The result showed that GATA-2is mainly expressed in the nucleus ofrenal tubular epithelial cells (yellowish-brown), but the nucleus of glomerularendothelial cell also had visible expression in small quantities. The total expression level of GATA-2at baseline,4,24,48,72h after reperfusion demonstrates adown-regulated current, of which the72h groups exhibited the lowest level. Thesham controls had no significant difference compared with normal groups (P>0.05),but all I/RI groups had significant difference compared with the normal group andsham group (P<0.01).Conclusion:The expression profiles of miR-24were significantly up-regulated in renalischemia-reperfusion injury,but its target mRNA (GATA-2) showed significantdown-regulated current; GATA-2, as major miR-24target mRNA,may be involvedin the regulation process of angiogenesis in kidney repair process via inhibiting theexpression activity of VEGF and VEGF2. |
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