Hypoxia Inducible Factor And Mir-21 Late In The Kidney Ischemic Preconditioning Mechanism

Part one:Protective effect of Delayed ischemic preconditioning on Renal ischemia/reperfusion injury via Induction of hypoxia inducible factorBackground and objective:Ischemia/reperfusion (I/R) injury is the major cause of acute renal injury (AKI) in the native as well as in the transplanted kidney. At present, there is no specific treatment for this devastating clinical syndrome with high morbidity and mortality. Brief and sublethal prior ischemia renders an organ more tolerant to subsequent prolonged I/R injury. This phenomenon is called "ischemic preconditioning" (IPC). Delayed IPC appears about 12-24 h after the preconditioning ischemia and the beneficial effects of delayed IPC require new protein synthesis and can sustain for days to weeks. Hypoxia is the earliest stage of the ischemic injury. The hypoxia-inducible factor (HIF) plays a key role in the regulation of cellular oxygen homeostasis. HIF-la and HIF-2a is two isoforms of HIF-a subunite. Previous studies have found that high expression of HIF-la plays an important protective role of delayed IPC in the heart, brain and other organs. The mechanism underlying the renal protection of HIF-1αand HIF-2a in the delayed IPC remains poorly understood. On basis of past research experience of delayed IPC in rats kidney, we will establish a mouse model of delayed IPC in the kidney by 15min ischemia-4d interval-30min re-ischemia, and observe the protective role of delayed IPC against renal I/R injury via induction of HIF.Materials and methods:Male C57/BL6N mice were randomly divided into three groups:sham operation group (Sham), I/R group (IR) and IPC group (IPC).30-min ischemia was induced by clamping renal bilateral pedicles followed by reperfusion in IR group. Sham mice underwent the same surgical procedures without the clamped renal pedicles.15-min pre-ischemia was induced 4 days before renal I/R in IPC group. Blood sample and kidneys from mice in the IPC and IR groups were collected at indicated times after the second ischemia (n=6 in each group at each time point). Serum creatinine (Scr) and blood urea nitrogen (BUN) levels, mortality, and histological change were observed throughout the study. Apoptosis and proliferation were confirmed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay and the immunohistochemistry of proliferating cell nuclear antigen (PCNA). Renal tissue MPO levels were detected by colorimetric. The expression and distribution of HIF-la and HIF-2a in renal tissue were evaluated by immunohistochemistry and Western blot analysis. The mRNA expression of vascular endothelial growth factor (VEGF) and glucose transporter-1 (Glut-1) was detected using real-time RT-PCR.Results:1) Survival rate of mice in the IR group was only 57%. The survival rate was up to 100% in the IPC and Sham groups. Scr and BUN increased significantly in the IR group, up to the peak at 24 h after reperfusion and gradually recovered. The Scr of mouse in the IPC group was significantly lower than the IR group at 2 h-2 w after reperfusion compared to the Sham group (P<0.05).2) Histological evaluation revealed severe tubulointerstitial damage from the IR group, histological injury score of the IPC group is significantly lower than that of the IR group at 2h,24h and 1w after reperfusion (P<0.05).3) renal tissue MPO levels from the IR group mice was higher than the IPC group with especially significant difference at 2h,6h and 24h after reperfusion(P<0.05).4) Extensive proximal tubular apoptosis was found at 24 h after renal I/R injury. Apoptotic cells in the kidney mainly concentrated in the corticomedullary junction and outer medulla, less in the glomerular. IPC significantly attenuated tubule cell apoptosis (12.3±1.5/HPF vs 38.2±2.0/HPF, P<0.01).5) Less cell proliferation (PCNA-positive cells) between dead renal tubular epithelial cells at 24h to 72h after reperfusion in the IR group. Cell proliferation was increased significantly by IPC, mainly in proximal renal tubular (63±5.48/HPF vs 32.6±7.06/HPF, P<0.01).6) Nuclear expression of HIF-la in the kidney from the IR and IPC groups increased from 2 h after reperfusion, reached the peak at 24h. After that, expression of HIF-la in the IPC group kept high level up to 48 h. HIF-la expression was significantly higher in the IPC group than that in the IR group from 6 h to 1 week after reperfusion (P<0.01). Expression trend of HIF-2a and HIF-1αis similar. HIF-2a expression in the mice kidneys from the IPC group was higher than the IR group at each time point after reperfusion, especially significant at 12h to 1w after reperfusion (P<0.01). Immunohistochemistry showed that expression of HIF-la was mainly in renal tubular epithelial cells, and HIF-2a was mainly in the renal interstitial cells and glomerular endothelial cells.7) Expression of VEGF mRNA in mouse kidney from 12 h after reperfusion significantly increased, to the peak at 48h, and the higher levels continue to 1 week after reperfusion. IPC significantly increased the expression of VEGF mRNA from 12h tolw after reperfusion (P<0.05). Expression of Glut-1 mRNA in mice kidneys from the IPC group gradually increased from 6h after reperfusion, reached the peak at 72h, then gradually decreased but remained at a higher level, with a higher expression than that of the IR group from 12 h to 2 weeks after reperfusion.Conclusions:1) Delayed ischemic preconditioning could improve kidney function, reduce the renal pathological damage and reduce I/R induced renal inflammatory response; 2) delayed IPC could reduce apoptosis of tubular epithelial cell and promote cell proliferation; 3) delayed IPC could induce overexpression of HIF-la and HIF-2a, increased renal tubular epithelial cells resistance to ischemia/hypoxia; expression trend of HIF-1αand HIF-2a is similar after I/R, but the cell distribution of them in the kidney is different; 4) the renal protective effect of HIF in the delayed IPC may be achieved through regulation of HIF downstream genes VEGF and Glut-1. In a word, delayed ischemic preconditioning can attenuate renal ischemia/reperfusion injury. This protective effect may be related to the increased expression of hypoxia inducible factor. Part two:miR-21 Contributes to Renal Protection Conferred by Delayed Ischemic Preconditioning and Regulation by Hypoxia inducible factorBackground and objective:MicroRNAs (miRNAs) are endogenous, small (18-22 nucleotides) RNA molecules that play an important and ubiquitous role in regulating gene expression for various biological processes. The role of miRNAs in renal I/R injury is not well understood. A series of hypoxia-related miRNAs (HRMs) have been found in cancer research. HIF is a key regulator of HRMs expression under the hypoxic condition. As one of HRMs, miR-21 has been shown to be a strong anti-apoptotic factor at least in part by targeting pro-apoptotic genes including programmed cell death protein 4 (PDCD4), but the regulative relationship between miR-21 and HIF has not been reported yet. We therefore utilized a mouse model of renal delayed IPC and tested the hypothesis that miR-21 might play an important role in the renal protective effects of delayed IPC. If the activation of HIF would mediate up-regulation of miR-21 and the role of miR-21 in the cell proliferation and apoptosis under hypoxic condition will be known through in vitro cell hypoxic model.Materials and methods:Bilateral renal pedicles were clamped for 15-min in male C57BL/6J mice (20-22g). The blood and kidneys in separate groups of mice were harvested at 4 h,24 h and 4 d after the surgery. Sham mice underwent the same surgical procedures except the renal pedicles were not clamped. For the delayed IPC and I/R model,4 d after IPC or sham surgeries, preconditioned mice were subjected to 30-min occlusion of bilateral renal pedicles, followed by 24 hours reperfusion. Locked nucleic acid (LNA) modified anti-scrambled or anti-miR-21 delivered into the tail vein less than 1 hour prior to ischemia surgery (n=5-6 in each group at each time point). Scr was measured using the improved Jaffe method. The kidney tissue morphology is stained by PAS. Apoptosis was confirmed by TUNEL assay. Expression levels of several miRNAs and mRNAs were quantified in total RNA using real-time PCR. The relative abundance of PDCD4 and HIF-la was analyzed using western blot. Primary human renal epithelial (HRE) cells were cultured. In vitro hypoxic model was established by cobalt chloride (CoCl2) chemical hypoxia and 2% O2 hypoxia. Double-stranded oligodeoxynucleotides carrying a hypoxia-responsive element were used as a decoy to block the activity of HIF. MTT test and Caspase-3/7 assay were used for detection of cell proliferation and apoptosis under hypoxia. Results:1) Renal abundance of miR-21 expression was increased in the IPC+I/R group by 179%±17%, compared to the Sham+I/R group (p<0.05). miR-21 target genes PDCD4 mRNA expression in the kidney from the IPC+I/R group significantly decreased after reperfusion, compared to the Sham+I/R group.2) Time-course analysis indicated that miR-21 was upregulated at 4 h after IPC compared to the Sham mice, and remained significantly higher 4 days after IPC (P<0.05). Although expression of other injury-related miRNAs such as miR-320, miR-214 and let-7e increased at 4 days after IPC, but the difference was not significant.3) Administration of LNA anti-miR-21 (10 mg/kg) at the time of IPC decreased the miR-21 level in the kidney by 98%(p<0.05), and significantly exacerbated renal functional and histological damage of subsequent I/R injury in the mouse kidney (Scr: 0.21±0.01 vs.0.31±0.02mg/dL, scrambled control vs.LNA anti-miR-21, P<0.01). In the absence of delayed IPC, knockdown of miR-21 did not significantly affect I/R injury in the mouse kidney. Knockdown of miR-21 resulted in significant upregulation of PDCD4 protein, a pro-apoptotic target gene of miR-21 (arbitrary unit,89±27 vs.20±10, P<0.05), and a substantial increase in tubular cell apoptosis in the mouse kidney as determined by TUNEL staining (proportion of positive cells,16%±3%vs.3%±1%, P<0.05).4) IPC activated the protein expression of HIF-la in the mouse kidney, with a significantly higher expression than that of the Sham group at the early phase (4h) and late phase (4 days) after 15min preconditioning (4h:134.36%±8.81% of Sham; 4 days:120.41%±5.25% of Sham, P<0.05).5) Treatment of the HRE cells with CoCl2 (300μM for 4h) or hypoxia (2% O2 for 24h), which are classical inducers of HIF activation, caused significant up-regulation of miR-21 level by 183%±9%(vs. control, P<0.05) and 168%±22%(vs. normoxia, P<0.05), respectively. Blockade of HIF activity with a decoy significantly reduced miR-21 level in the HRE cells treated with CoCl2 by 42%±13%, compared to the scrambled oligonucleotides (P<0.05).6) Hypoxic model also is established by 150μM CoCl2 treatment for 24h, LNA anti-miR-21 effectively inhibited the expression of miR-21 in the HRE cells under hypoxic condition(11.41%±2.26% of scrambled control, P<0.01). MTT test results showed that inhibition of miR-21 significantly reduced the cell growth (P<0.01). The inhibition of miR-21 slightly increased Caspase3/7 expression without significant statistical difference.Conclusions:1) Delayed IPC induced high expression of miR-21, accompanied by reduced expression of the pro-apoptotic target gene PDCD4.2) Administration of LNA anti-miR-21 at the time of IPC knocked down the miR-21 expression, resulting to significantly exacerbate subsequent I/R injury in the mouse kidney. Knockdown of miR-21 at the time of I/R injury in the absence of IPC, however, did not further exacerbate renal injury.3) miR-21 expression has been shown to be inversely correlated with PDCD4 expression and cellular apoptosis in mouse kidney.4) Cell hypoxia induced up-regulation of miR-21. HIF played an important role in the regulation of miR-21 expression.5) HIF-1αinduced by hypoxia maybe maintained the cell survival via miR-21 expression. Taken together, our results indicated that delayed IPC could protect renal against ischemic injury by increasing miR-21 expression with reduction of the target gene PDCD4 expression and renal tubular epithelial cells apoptosis, besides the direct regulation of VEGF and Glut-1, both of which was maybe related with HIF.