miR-210 Protects Renal Cell Against Hypoxia-induced Apoptosis by Targeting HIF-1 Alpha
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The kidney is vulnerable to hypoxia-induced injury. One of the mechanisms underlying this phenomenon is cell apoptosis triggered by hypoxia-inducible factor-1- α (HIF-1α) activation. MicroRNA-210 (miR-210) is known to be induced by HIF-1α and can regulate various pathological processes, but its role in hypoxic kidney injury remains unclear. Here, in both rat systemic hypoxia and local kidney hypoxia models, we found miR-210 levels were upregulated significantly in injured kidney, especially in renal tubular cells. A similar increase was observed in hypoxia-treated human renal tubular HK-2 cells. We also verified that miR-210 can directly suppress HIF-1α expression by targeting the 3′ untranslated region of HIF-1α mRNA in HK-2 cells in severe hypoxia. Accordingly, miR-210 overexpression caused significant inhibition of the HIF-1α pathway and attenuated apoptosis caused by hypoxia, while miR-210 knockdown exerted the opposite effect. Taken together, our findings verify that miR-210 is involved in the molecular response in hypoxic kidney lesions in vivo and attenuates hypoxia-induced renal tubular cell apoptosis by targeting HIF-1α directly and suppressing HIF-1α pathway activation in vitro.
Oxygen is a vital microenvironmental substrate for sustaining tissue homeostasis. Many physiological and pathological conditions cause insufficiency in oxygen availability, or hypoxia, which can eventually lead to apoptosis if the hypoxia is severe (1,2). Despite constituting merely 0.4%–0.5% of a person’s total body weight, the kidney is responsible for almost 7% of the body’s oxygen consumption (3). The high sensitivity of the kidney makes it prone to hypoxic injury and even renal tubular damage and diffuse kidney lesions (4). In the clinic, many diseases induce systemic hypoxia, such as high-altitude disease, postoperative hypoxemia, adult respiratory distress syndrome and shock. Systemic hypoxia drives multiorgan damage, especially kidney injury (5, 6, 7). Another kind of kidney hypoxic injury is caused by local hypoxia, in which oxygen sufficiency usually originates from a decreased renal or intrarenal blood supply (8). Both kinds of kidney hypoxic lesion can lead to acute kidney injury (AKI), which is a common cause of in-hospital mortality (9). As for the mechanism of hypoxic kidney injury, the excessive and sustaining activation of hypoxia-inducible factor-1-α (HIF-1α) was deemed a critical event (10, 11, 12).
Hypoxia-inducible factors (HIFs) are the key transcriptional factors in the cell to induce hypoxia response. Three isoforms of HIF are found in mammals: HIF-1α, HIF-2α and HIF-3α. Only HIF-1α is found in all cell types, and it acts as a core regulator (1). In addition to transcribing adaptive genes, HIF-1α also transcribes some proapoptotic genes, such as Bcl-2 interacting protein 3 (BNIP3) and Bcl-2 interacting protein 3-like (NIX) (13), and stabilizes the tumor suppressor p53 (14) to induce apoptosis. Therefore, HIF-1α has been reported as a risk factor for renal disease (15). HIF-1α is controlled by a series of complex and elaborate mechanisms because of its abovementioned vital function. Several studies have reported an interesting resolution pattern of HIF-1α, which showed that HIF-1α is not continuous, but undergoes a transient increase followed by a decline under hypoxic conditions (16,17). But canonical oxygen-dependent protein degradation mediated by prolyl hydroxylase domain-containing enzymes (PHDs) cannot competently explain this phenomenon (18). Other regulatory mechanisms of HIF-1α resolution, such as microRNA (miRNA)-mediated regulation, have attracted interest in recent years (16).
miRNAs are small non-coding RNAs that were discovered in recent decades. They regulate target genes primarily by silencing mRNA at the post-transcriptional level through interacting with mRNA untranslated regions (UTRs). Hypoxia regulates a cluster of specific miRNAs, which are termed hypoxamiRs (19). microRNA-210 (miR-210) is known as the master hypoxamiR, as it is ubiquitously expressed in a wide range of cell types, is induced by HIF-1α and regulates a variety of responses to cope with hypoxia-induced stress (20,21). However, to date, the role of miR-210 in hypoxic renal injury is unknown. Moreover, the relationship between miR-210 and HIF-1α has never been studied in the kidney before. A small number of studies, the results of which were controversial, focused on the impact of miR-210 on HIF-1α. One study showed that miR-210 regulates HIF-1α via indirect positive feedback (22), whereas another group observed that miR-210 regulates HIF-1α via negative feedback in differentiated T cells (23).
In this study, we established a systemic hypoxia rat model using a hypobaric chamber (7500 m, 7 h) and a local kidney hypoxia rat model using a microvascular clamp to induce hypoxic injury. We observed that both hypoxic conditions led to kidney lesions accompanied by obvious HIF-1α pathway activation. Simultaneously, miR-210 was also found to be significantly upregulated in both kinds of kidney lesions and connected to injury severity. Using the human renal tubular epithelial HK-2 cell line, we proved that miR-210 protects against apoptosis induced by severe hypoxia. Subsequently, we verified that HIF-1α is a target gene of miR-210 in HK-2 cells, and HIF-1α activation can thus be suppressed by miR-210 to attenuate hypoxia-induced apoptosis. Collectively, these results reveal that increased miR-210 expression in hypoxia-induced kidney lesions attenuates cell injury and apoptosis by targeting HIF-1α in severe hypoxia.
Materials and Methods
Animal Maintenance, Systemic Hypoxia and Local Kidney Hypoxia Models
Six-to-eight-week-old male Sprague-Dawley rats with body weights of 180–220 g were housed in a pathogen-free and temperature-controlled room (22°C ± 2°C) with a 12 h light and 12 h dark cycle. Food and water were available ad libitum. For the systemic hypoxia model, the rats chosen randomly for the experimental group were put in a hypobaric chamber (Fenglei) to mimic an elevated altitude of 7500 m (9.0% O2) within 10 min, and the rats in the control group were placed in the corresponding normoxia condition. An acute kidney injury model was used to induce local kidney hypoxia. In brief, Sprague-Dawley rats were anesthetized with sodium pentobarbital (30 mg/kg intraperitoneally; Sigma) under aseptic conditions. The bilateral renal arteries were then exposed and occluded using a microvascular nontraumatic bulldog clamp via a flank incision. Following warm ischemia of 60 min, the rats were euthanized and kidney tissue were removed for further study. The sham group rats were only anesthetized and their kidneys were exposed under the same conditions, without clamping. All animal experimental procedures complied fully with the regulations of the Institute of Basic Medical Sciences.
Cell Culture, Hypoxia and Transfection
HK-2 cells were cultured in Dulbecco’s modified Eagle’s medium/Ham’s Nutrient Mixture F-12 (Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco), 25 U/mL penicillin (Hyclone) and 25 µg/mL streptomycin (Hyclone) at 37°C in a humidified incubator containing 5% (v/v) CO2. The hypoxic environment of cultured cells was realized in a hypoxia glove box (Coy), in which nitrogen gas was infused to obtain an oxygen concentration of 0.3% and to maintain a CO2 concentration of 5% at a temperature of 37°C. Cells were transfected with 100 nM miR-210 mimic, inhibitor, HIF-1α siRNA and negative control (Ribo) and 1ug plasmids using TurboFect Reagent (Thermo), according to the manufacturer’s protocols.
RNA Extraction and Real-Time Polymerase Chain Reaction
Total RNA was extracted from tissue, cell, serum and medium using RNAiso plus (Takara), according to the manufacturer’s instructions. Reverse transcription (RT) was performed using an RT kit (Takara) with miRNA-specific primers (Ribo) or oligo dT (Takara), respectively. pri-miR-210 reverse transcriptional primer: 5′-CACAGATCAGCCGCTGTCAC-3′. Real-time RNA quantification was conducted on an ABI StepOne Plus Detection System (Applied Biosystems) using Power SYBR Green PCR Master Mix (Applied Biosystems) and specific primers. The primer sequences are shown in Supplementary Table S1. The miR-210 primer, RNU6 primer and miR-39 primer were purchased from Ribo. Spike-in miR-39 miRNA (Ribo) was used for quantification of extracellular miR-210 levels.
Detection of Cell Apoptosis by Flow Cytometry
The HK-2 cells were trypsinized, collected and then labeled using an Annexin V-PI apoptosis kit (Dijindo). The cells were incubated with fluorescein isothiocyanate (FITC)-labeled Annexin V and propidium iodide (PI) solution in the dark for 15 min. Flow cytometry was conducted using FACS Calibur FL1 (530 nm) bandpass filters (Becton Dickinson), and the data were analyzed using CellQuest software (BD).
Western Blotting and Analysis
HK-2 cells were lysed using radioim-munoprecipitation assay (RIPA) buffer (Pierce) supplemented with protease inhibitor cocktail (Roche). The protein samples were subjected to routine sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane and incubated with primary antibodies overnight. The primary antibodies against HIF-1α (H1α 67), FAS (A-20) and β-actin (I-19), were purchased from Santa Cruz Biotechnology. Antibodies against NIX and BNIP3 (EPR4034) were purchased from Abcam. Antibody against p53 was purchased from Cell Signaling Technology. Antibody against HIF-2α was purchased from Novus Biologicals. Antibody against glucose transporter-1 (GLUT-1) was purchased from Proteintech Group. A horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) and enhanced chemiluminescence (Pierce) were applied for protein detection. The relative expression of the protein bands was quantified by densitometric scanning using Image Pro Plus (Media Cybernetics) and standardized for densitometric analysis to β-actin levels.
Kidney Periodic Acid-Schiff Staining and TdT-Mediated dUTP Nick-End Labeling Assay
Kidney tissues were fixed with 10% neutral buffered formalin for 48 h and then embedded in paraffin and cut into 3–5 µm sections using routine methods. The kidney tissue samples were stained with periodic acid-Schiff (PAS) (Sigma), and histologic structures were examined under a light microscope. Kidney TdT-mediated dUTP Nick-end Labeling (TUNEL) assay was performed using an In Situ Cell Death Detection Kit (Roche), according to the manufacturer’s instructions. Three images per kidney (eight kidneys per group) were acquired, and the positive cells were counted individually.
Luciferase Reporter Assay
The predicted seed sequence of miR-210 in the 3′UTR of human HIF-1α mRNA (length: 220 bp) and the corresponding mutant sequence (length: 220 bp) were constructed into modified pGL3-Luc control luciferase reporter plasmids (Promega). These plasmids were co-transfected into HK-2 cells with a miR-210 mimic or a negative control, as indicated. After 48 h of transfection, the cells were lysed with passive lysis buffer (Promega). Luciferase activity was measured with a dual luciferase assay system (Promega), in accordance with the manufacturer’s protocol.
Primary Glomerular and Tubular Cell isolation
The kidneys of rats were removed, then cortical tissue was separated from the medulla and minced into a paste. The cortical paste was pressed through a 106 µm metal sieve to collect large tubular fragments and suspended in phosphate-buffered saline. This suspension was then triturated and poured over a 75 µm sieve. The glomeruli were trapped on the sieve, and the tubules were filtered through the sieve. Isolated glomerular and tubular cells were used for detection of RNA and protein expression.
Data are shown as the mean ± standard deviation (SD) and were analyzed using Student t test, except in special cases. P < 0.05 was considered statistically significant.
All supplementary materials are available online at https://doi.org/www.molmed.org.
Systemic Hypoxia and Local Kidney Hypoxia Led to Rat Renal Lesions Accompanied by HiF-1αActivation
Levels of miR-210 Significantly increased in Rat Kidney and Circulation after Systemic or Local Kidney Hypoxia
Severe Hypoxia-induced HiF-1α Pathway Activation and Apoptotic Gene Expression Accompanied by miR-210 Elevation in Human Renal Tubular HK-2 Cells
As most of the apoptotic cells were localized in the renal tubules, where miR-210 was remarkably upregulated in vivo, we conducted an in depth study in vitro involving human renal tubular epithelial HK-2 cells.
In summary, severe hypoxia induced HIF-1α and apoptosis pathway activation in renal tubular cells and increased miR-210 expression in vitro.
miR-210 Alleviated Hypoxia-Induced Cell injury and Apoptosis in HK-2 Cells
miR-210 Targeted 3′UTR of HIF-1α mRNA Directly in Hypoxia
After knockdown of HIF-1α, miR-210 upregulation in hypoxia-treated HK-2 cells was significantly inhibited, which indicates that HIF-1α is necessary for miR-210 induction in renal tubular epithelial cells (Figure 6B). HIF-2α knockdown had no such impact on miR-210 (Figure 6C) and other gene (Supplementary Figure S3) expressions. Subsequently, we successfully found the seed sequence of miR-210-3p in the 3′UTR of human HIF-1α mRNA via using the Memorial Sloan-Kettering Cancer Center miRNA database (https://doi.org/www.microrna.org/). This sequence is highly conserved in several mammals, as reported by other groups (23) (Figure 6D). We inserted this seed sequence (wildtype) and the corresponding mutated sequence (mutant-type) into a luciferase reporter vector to examine whether miR-210 binds the 3′UTR of human HIF-1α mRNA directly in HK-2 cells (Figure 6E). As shown in Figure 6F, the miR-210 mimic sharply decreased the activity of the wild-type 3′UTR of HIF-1α mRNA but had no significant impact on the mutant-type 3′UTR.
These results indicate that, in renal tubular cells, HIF-1α is necessary for miR-210 induction, and miR-210 can target the 3′UTR of HIF-1α mRNA reversely, which implies that miR-210 may contribute to the prevention of excessive accumulation of HIF-1α in the above-mentioned hypoxic renal injury.
miR-210 Attenuated Hypoxic Apoptosis by Suppressing HiF-1α Activation
Previous studies have shown that HIF-1α activation is a risk factor for kidney disease (10,26,27) and that activated HIF-1α may transcribe some pro-apoptotic genes, such as BNIP3 and NIX, or stabilize p53, leading to apoptosis. We explored how the HIF-1α and apoptosis pathways are altered by miR-210 overexpression or knockdown, since miR-210 targeted 3′UTR of HIF-1α mRNA directly.
In summary, these findings show that, in hypoxic renal tubular cell injury, HIF-1α is a target gene of miR-210, and miR-210 can attenuate cell apoptosis by blocking HIF-1α pathway activation.
AKI is a syndrome characterized by rapid deterioration of kidney function within several hours to several days, is a common disease in critically ill patients and is often diagnosed in the context of other acute illnesses (28). Among the primary causes of AKI are renal hypoxia and energy supply failure after kidney ischemia. Expanding our understanding of the hypoxic pathophysiology of AKI will provide useful therapeutic methods in the clinic. In kidneys, miRNAs play an indispensable role in maintaining renal homeostasis and are involved in various kidney diseases (29).
Many studies have demonstrated that miR-210 is the most consistently upregulated miRNA induced by hypoxia (20,30) and HIF-1α (21,22,31). Indeed, miR-210 was highly increased in both kidney (32) and serum (33) in clear cell renal cell carcinoma patients, which is associated with a mutation of von Hippel-Lindau-induced HIF-1α accumulation. But its expression in hypoxic kidneys is unknown. In our work, we observed HIF-1α pathway activation, apoptosis formation and robust miR-210 upregulation simultaneously occurring in the rat kidney after hypoxia. In recent years, circulating miRNAs were found (34) and regarded as novel biomarkers for some diseases, and our study observed that serum miR-210 was increased only in the local hypoxia group. The reason for the serum miR-210 remaining unchanged in systemic hypoxia may partly be because the kidney hypoxia was mild and early in this situation. Similar results were observed in HK-2 cells in hypoxia. We also found that the expression levels of pro-apoptotic target genes of HIF-1α, such as BNIP3 and NIX, were remarkably increased. Using TUNEL assay, we observed that the numbers of apoptotic cells increased notably in the kidney, especially in the renal tubular region, where HIF-1α is predominantly expressed (24). Compared with other vital organs, the kidney, especially the renal tubule, expresses high levels of miR-210. In summary, we found that miR-210 and HIF-1α are closely related with respect to their expression and localization in hypoxic kidney lesions in vivo. We also observed elevated serum Cr, UA levels and declined eGFR in hypoxia-treated rats, which is further support of the harmfulness of hypoxia.
Although it is known that miR-210 can be induced directly by HIF-1α (21,22,31), no consensus has been reached regarding how miR-210 impacts HIF-1α. In HK-2 cells, we observed increases in HIF-1α and miR-210 expression in the early phase of hypoxia, similar to our results in vivo. Interestingly, in sustained hypoxia, HIF-1α mRNA and protein expression tended to decrease after 24 h, whereas miR-210 expression continued to increase. HIF-1α protein stability is primarily negatively regulated by PHD-dependent hydroxylation in normoxia, which results in ubiquitination and proteasome degradation (35). In hypoxia, PHD activity is inhibited, leading to increased HIF-1α protein levels (36). However, this mechanism does not sufficiently explain our observation of resolution of HIF-1α expression in prolonged hypoxia, which has also been reported by others (17,37). miRNA-mediated regulation has become a new theory illustrating the resolution of HIF-1α (16). Several studies have demonstrated that HIF-1α can be regulated by microRNAs, including miR-210 (23,38, 39, 40, 41, 42). However, one group reported that miR-210 negatively regulated HIF-1α in mouse T cells (23), while another group found that miR-210 overexpression indirectly resulted in HIF-1α accumulation and created a positive feedback loop in hypoxia (22). In our study, we found a potential miR-210 binding sequence in the 3′UTR of human HIF-1α mRNA. Furthermore, using a luciferase reporter assay, we verified that HIF-1α was a target gene of miR-210 in HK-2 cells. Based on the collective findings, we concluded that a negative feedback loop involving miR-210 and HIF-1α exists in hypoxic human kidney, as well as the mouse immune system. Though HIF-2α was reported to have overlapping functions with HIF-1α, in our study, knockdown of HIF-2α had no obvious effect on miR-210 and other genes, which further supports that HIF-1α was the dominant regulator for miR-210 induction (31,43). More importantly, we found that miR-210 contributes to HIF-1α resolution in prolonged hypoxia.
miR-210 was proven to have beneficial effect on ischemic diseases such as acute peripheral ischemia (51), cerebral ischemia (52) and ischemic heart disease (53). It has been reported that plasma miR-210 was upregulated (54) and serum miR-210 was decreased (55) in AKI patients. However, the biologic function of miR-210 in kidney ischemia remains unclear. Our results were obtained immediately after renal ischemia in vivo, which is normally prior to the manifestation of clinical AKI. And the study in vitro showed for the first time that increased miR-210 has a renoprotective function in kidney hypoxic injury. miRNAs are regarded as sensitive biomarkers in different diseases and at different scales. Our findings imply that miR-210 has the possibility to be a promising biomarker and potential therapeutic target for early detection and treatment of hypoxic kidney injury.
This study reveals that both HIF-1α and its target miR-210 are significantly upregulated in hypoxic kidney injury in vivo and in vitro. Increased miR-210 reversely targets HIF-1α directly to suppress its expression in renal tubular cells. This negative feedback loop contributes to HIF-1α resolution in severe hypoxia and further inhibits the downstream pro-apoptotic gene expression of HIF-1α, thereby attenuating hypoxic kidney damage. Considering the complexity and poor outcome of AKI, our results provide an expanded understanding of AKI mechanism and points to a potential role for miR-210 in developing novel diagnosis and intervention of hypoxic kidney injury in the future.
The authors declare they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This work was supported by the grants from the Natural Science Foundation of China (Nos. 81430044 and 31271205) and the National Basic Research Program of China (Nos. 2011CB910800 and 2012CB518200).
- 3.Valtin H. (1983) Renal Function: Mechanisms Preserving Fluid and Solute Balance in Health. Boston: Little Brown.Google Scholar
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