Xialian Xu

Delayed ischemic preconditioning contributes to renal protection by upregulation of miR-21.

Delayed ischemic preconditioning effectively protects kidneys from ischemia-reperfusion injury but the mechanism underlying renal protection remains poorly understood. Here we examined the in vivo role of microRNA miR-21 in the renal protection conferred by delayed ischemic preconditioning in mice. A 15 minute renal ischemic preconditioning significantly increased the expression of miR-21 by 4 hours and substantially attenuated ischemia-reperfusion injury induced 4 days later. A locked nucleic acid-modified anti-miR-21 given at the time of ischemic preconditioning knocked down miR-21 and significantly exacerbated subsequent ischemia-reperfusion injury in the mouse kidney. Knockdown of miR-21 resulted in significant upregulation of programmed cell death protein 4, a pro-apoptotic target gene of miR-21, and substantially increased tubular cell apoptosis. Hypoxia inducible factor-1α in the kidney was activated after ischemic preconditioning and blockade of its activity with a decoy abolished the up-regulation of miR-21 in cultured human renal epithelial cells treated with the inducer cobalt chloride. In the absence of ischemic preconditioning, knockdown of miR-21 alone did not significantly affect ischemia-reperfusion injury in the mouse kidney. Thus, upregulation of miR-21 contributes to the protective effect of delayed ischemic preconditioning against subsequent renal ischemia-reperfusion injury.

MicroRNA: a new frontier in kidney and blood pressure research

MicroRNA (miRNA) has emerged rapidly as a major new direction in many fields of research including kidney and blood pressure research. A mammalian genome encodes several hundred miRNAs. These miRNAs potentially regulate the expression of thousands of proteins. miRNA expression profiles differ substantially between the kidney and other organs as well as between kidney regions. miRNAs may be functionally important in models of diabetic nephropathy, podocyte development, and polycystic disease. miRNAs may be involved in the regulation of arterial blood pressure, including possible involvement in genetic elements of hypertension. Studies of miRNAs could generate diagnostic biomarkers for kidney disease and new mechanistic insights into the complex regulatory networks underlying kidney disease and hypertension. Further progress in the understanding of miRNA biogenesis and action and technical improvements for target identification and miRNA manipulation will be important for studying miRNAs in renal function and blood pressure regulation.

miR-29c is downregulated in renal interstitial fibrosis in humans and rats and restored by HIF-α activation.

Treatment with L-mimosine, which activates hypoxia-inducible factor-α (HIF-α), attenuates renal tubulointerstitial injury and improves renal function in a rat remnant kidney model. The miR-29 family of microRNAs directly targets a large number of extracellular matrix genes and reduces renal interstitial fibrosis. We analyzed microRNA expression profiles in rat remnant kidneys with or without treatment with L-mimosine. The expression of miR-29c was downregulated in rat remnant kidneys compared with sham control and significantly restored by the L-mimosine treatment. In cultured human kidney epithelial HK2 cells, cobalt chloride activated HIF-α and upregulated miR-29c expression. The upregulation of miR-29c expression was significantly attenuated by knockdown of HIF-1α or HIF-2α. Downregulation of miR-29c was associated with significant increases in interstitial fibrosis, collagen type II α1 (COL2A1) protein, and tropomyosin 1α (TPM1) protein in rat remnant kidneys and in kidneys from IgA nephropathy patients. The increases in rat remnant kidneys were attenuated by the L-mimosine treatment. COL2A1 and TPM1 were confirmed to be new, direct targets of miR-29c. In conclusion, miR-29c, an antifibrotic microRNA, is upregulated by HIF-α activation. MiR-29c is downregulated in renal interstitial fibrosis in humans and rats and restored by activation of HIF-α that attenuates fibrosis.

miR-21 in ischemia/reperfusion injury: a double-edged sword?

MicroRNAs (miRNAs or miRs) are endogenous, small RNA molecules that suppress expression of targeted mRNA. miR-21, one of the most extensively studied miRNAs, is importantly involved in divergent pathophysiological processes relating to ischemia/reperfusion (I/R) injury, such as inflammation and angiogenesis. The role of miR-21 in renal I/R is complex, with both protective and pathological pathways being regulated by miR-21. Preconditioning-induced upregulation of miR-21 contributes to the protection against subsequent renal I/R injury through the targeting of genes such as the proapoptotic gene programmed cell death 4 and interactions between miR-21 and hypoxia-inducible factor. Conversely, long-term elevation of miR-21 may be detrimental to the organ by promoting the development of renal interstitial fibrosis following I/R injury. miR-21 is importantly involved in several pathophysiological processes related to I/R injury including inflammation and angiogenesis as well as the biology of stem cells that could be used to treat I/R injury; however, the effect of miR-21 on these processes in renal I/R injury remains to be studied.

Renal Protection Mediated by Hypoxia Inducible Factor-1α Depends on Proangiogenesis Function of mir-21 by Targeting Thrombospondin 1

Background Angiogenesis contributes to the repair process after renal ischemia/reperfusion (I/R) injury. In the present study, we tested the role of miR-21 in the angiogenesis induced by hypoxia inducible factor (HIF)-1&agr; through inhibiting a predicted target gene thrombospondin 1 (TSP-1). Methods To stabilize HIF-1&agr;, hypoxia (1% O2 for 24 hours) was performed in human umbilical vein endothelial cells and cobalt chloride (CoCl2) was pretreated intraperitoneally 24 hours before renal I/R in mice. Locked nucleic acid modified anti-miR-21 and scrambled control was transfected with hypoxic cells or delivered into the mice via tail vein 1 hour before CoCl2 injection. The kidneys and blood were collected at 24 hours after reperfusion. Results HIF-1&agr; induced by hypoxia and CoCl2 upregulated vascular endothelial growth factor and miR-21, and increased angiogenesis. It was found that expression of TSP-1 was inversely related with miR-21 in vitro and in vivo. Targeting of TSP-1 by miR-21 was further confirmed in vitro. Furthermore, HIF-1&agr; improved renal function, accompanied with increased angiogenesis after I/R injury in mice. The protective effect of HIF-1&agr; was attenuated by inhibition of miR-21. Conclusions HIF-1&agr; induced angiogenesis by upregulating not only vascular endothelial growth factor but also miR-21 via inhibiting a novel target gene TSP-1. Both of them may contribute to the protective effect of HIF-1&agr; on renal I/R injury.

Augmented O-GlcNAc signaling via glucosamine attenuates oxidative stress and apoptosis following contrast-induced acute kidney injury in rats

ABSTRACT Contrast‐induced acute kidney injury (CI‐AKI) is an iatrogenic renal injury and associated with substantial morbidity and mortality in susceptible individuals. Despite extensive study of a variety of agents for renal protection, limited strategies have been shown to be effective in the reduction of CI‐AKI. O‐linked &bgr;‐N‐acetylglucosamine (O‐GlcNAc) is a post‐translational regulatory modification of intracellular proteins and governs the function of numerous proteins, both cytosolic and nuclear. Increasing evidence suggests that O‐GlcNAc levels are increased in response to stress and that acute augmentation of this reaction is cytoprotective. However, the underlying mechanisms by which augmented OGlcNAc signaling provides renoprotection against contrast media insults is still unknown. Here, we investigated the effect of augmented O‐GlcNAc signaling via glucosamine on CI‐AKI and explored the underlying molecular mechanisms, particularly its relationship with PI3‐kinase (PI3K)/Akt signaling. We used a novel and reliable CI‐AKI model consisting of 5/6 nephrectomized (NE) rats, and a low‐osmolar contrast media (iohexol, 10 mL/kg, 3.5gI) injected via the tail vein after dehydration for 48 h. The results showed that augmented O‐GlcNAc signaling by glucosamine prevented the kidneys against iohexol‐induced injury characterized by the attenuation of renal dysfunction, tubular damage, apoptosis and oxidative stress. Furthermore, this renoprotection was blocked by treatment with alloxan, an O‐GlcNAc transferase inhibitor. Augmented O‐GlcNAc signaling also increased the protein expression levels of phospho‐Akt (Ser473, but not Thr308 and Thr450), phospho‐GSK‐3&bgr;, Nrf2, and Bcl‐2, and decreased the levels of Bax and cleaved caspase‐3. Both alloxan and specific inhibitors of PI3K (Wortmannin and LY294002) blocked the protection of glucosamine via inhibiting Akt signaling pathway. We further identified O‐GlcNAcylated Akt through immunoprecipitation and western blot. We confirmed that Akt was modified by O‐GlcNAcylation, and glucosamine pretreatment increased the O‐GlcNAcylation of Akt. Collectively, the results demonstrate that glucosamine induces renoprotection against CI‐AKI through augmented O‐GlcNAc and activation of PI3K/Akt signaling, making it a promising strategy for preventing CI‐AKI. HighlightsIncreasing evidence suggests that O‐linked &bgr;‐N‐acetylglucosamine (O‐GlcNAc) levels are increased in response to stress and that acute augmentation of this reaction is cytoprotective.We used a novel and reliable contrast‐induced acute kidney injury (CI‐AKI) model and the results showed that augmented O‐GlcNAc signaling by glucosamine prevented the kidneys against iohexol‐induced injury by the attenuation of apoptosis and oxidative stress.Furthermore, both alloxan and specific inhibitors of PI3K (Wortmannin and LY294002) blocked the protection of glucosamine via inhibiting Akt signaling pathway.

miR‐21 contributes to renal protection by targeting prolyl hydroxylase domain protein 2 in delayed ischemic preconditioning

Upregulation of miR‐21 in renal ischaemic preconditioning (IPC) was associated with increased hypoxia inducible factor (HIF)‐1α expression. Hypoxic induction of HIF‐1α is mediated by inhibition of prolyl hydroxylase domain protein 2 (PHD2) .We hypothesized that miR‐21 regulated HIF‐1α by targeting PHD2 in the renal IPC.

Role of miR‑21 on vascular endothelial cells in the protective effect of renal delayed ischemic preconditioning

Vascular endothelial cells may serve crucial roles in the development of acute kidney injury (AKI). microRNA (miR)-21, which possesses a renal protective function has been found on vascular endothelial cells. The present study aimed to test the hypothesis that miR-21 may protect vascular endothelial cells against injury, which may contribute to the protective effects of renal delayed ischemic preconditioning (IPC). Preconditioned (15 min ischemia) or Sham mice (not clamped) were subjected to 35 min occlusion of bilateral renal pedicles 4 days following preconditioning or Sham treatment. Human umbilical vein endothelial cells (HUVECs) were treated with cobalt(II) chloride (CoCl2) to establish an in vitro hypoxia model. Locked nucleic acid-modified anti-miR-21 or scrambled control oligonucleotides were transfected into cells or delivered into mice via tail vein injection <1 h prior to IPC. Following 24 h of reperfusion or hypoxia, morphological and functional parameters, apoptosis and miR-21 and programmed cell death 4 (PDCD4) expression were assessed in vivo and in vitro. Treatment of HUVECs with CoCl2 led to an upregulation of miR-21 expression, a downregulation of PDCD4 protein expression and attenuation of apoptosis. Inhibition of miR-21 expression led to increased expression levels of PDCD4 protein and apoptosis in HUVECs. IPC attenuated renal IR injury in mice. The protective effect of IPC appeared to be dependent on upregulated miR-21 expression. IPC-induced upregulation of miR-21 expression also occurred in HUVECs, and IPC also led to reduced PDCD4 expression and vascular permeability in mouse kidneys. The effects of IPC were attenuated by the inhibition of miR-21; miR-21 expression attenuated damage in vascular endothelial cells, which may contribute to the protective effects of delayed IPC on renal IR injury. The present study suggested a novel target for the prevention and repair of AKI in the future.

The effect of long noncoding RNA GAS5 on apoptosis in renal ischemia/reperfusion injury: The effect of lncRNA GAS5 on apoptosis in renal I/R injury

Long non‐coding RNA (lncRNAs) have been shown to play a critical role in a variety of pathophysiological processes, such as cell proliferation, apoptosis and migration. However, there were few studies addressing the function of lncRNAs in renal ischaemia/reperfusion (I/R) injury. Apoptosis is an important pathogenesis during I/R injury. Here, we identified the effect of hypoxia‐responsive lncRNA growth arrest‐specific 5 (GAS5) on apoptosis in renal I/R injury.

miR-21 Protects Against Ischemia/Reperfusion-Induced Acute Kidney Injury by Preventing Epithelial Cell Apoptosis and Inhibiting Dendritic Cell Maturation

Renal tubular injury and innate immune responses induced by hypoxia contribute to acute kidney injury. Accumulating evidence suggests that miR-21 overexpression protects against kidney ischemia injury. Additionally, miR-21 emerges as a key inhibitor in dendritic cell maturation. Thus, we hypothesized that miR-21 protects the kidney from IR injury by suppressing epithelial cell damage and inflammatory reaction. In this study, we investigated effects of miR-21 and its signaling pathways (PTEN/AKT/mTOR/HIF, PDCD4/NFκ-B) on kidney ischemia/reperfusion (IR) injury in vitro and in vivo. The results revealed that IR increased miR-21, HIF1α, and 2α expression in vivo and in vitro. MiR-21 interacted with HIF1α and 2α through the PTEN/AKT/mTOR pathway. Moreover, inhibition of miR-21 activated PDCD4/NFκ-B pathways, which are critical for dendritic cell maturation. Renal IR triggers local inflammation by inducing the dendritic cell maturation and promoting the secretion of IL-12, IL-6, and TNF-α cytokines. Knockdown of miR-21 intensified the effect of IR on tubular epithelial cell apoptosis and dendritic cell maturation. Our results suggested that IR-inducible miR-21 protects epithelial cells from IR injury via a feedback interaction with HIF (PTEN/AKT/mTOR/HIF/miR-21) and by inhibiting maturation of DCs through the PDCD4/NF-κB pathway. These findings highlight new therapeutic opportunities in AKI.