Renal Medullary MicroRNAs in Dahl Salt-Sensitive Rats
miR-29b Regulates Several Collagens and Related Genes
MicroRNAs are endogenous repressors of gene expression. We examined microRNAs in the renal medulla of Dahl salt-sensitive rats and consomic SS-13BN rats. Salt-induced hypertension and renal injury in Dahl salt-sensitive rats, particularly medullary interstitial fibrosis, have been shown previously to be substantially attenuated in SS-13BN rats. Of 377 microRNAs examined, 5 were found to be differentially expressed between Dahl salt-sensitive rats and consomic SS-13BN rats receiving a high-salt diet. Real-time PCR analysis demonstrated that high-salt diets induced substantial upregulation of miR-29b in the renal medulla of SS-13BN rats but not in SS rats. miR-29b was predicted to regulate 20 collagen genes, matrix metalloproteinase 2 (Mmp2), integrin β1 (Itgb1), and other genes related to the extracellular matrix. Expression of 9 collagen genes and Mmp2 was upregulated by a high-salt diet in the renal medulla of SS rats, but not in SS-13BN rats, an expression pattern opposite to miR-29b. Knockdown of miR-29b in the kidneys of SS-13BN rats resulted in upregulation of several collagen genes. miR-29b reduced expression levels of several collagen genes and Itgb1 in cultured rat renal medullary epithelial cells. Moreover, miR-29b suppressed the activity of luciferase when the reporter gene was linked to a 3′-untranslated segment of collagen genes Col1a1, Col3a1, Col4a1, Col5a1, Col5a2, Col5a3, Col7a1, Col8a1, Mmp2, or Itgb1 but not Col12a1. The result demonstrated broad effects of miR-29b on a large number of collagens and genes related to the extracellular matrix and suggested involvement of miR-29b in the protection from renal medullary injury in SS-13BN rats.
A mammalian genome encodes several hundred microRNAs (miRNAs). Each miRNA is computationally predicted to regulate dozens to hundreds of protein-coding genes.1,2 In most cases, miRNAs decrease the expression level of target genes by suppressing translation or reducing mRNA abundance.3–5 miRNAs have been shown in recent years to be important regulators of several physiological processes and diseases in human and mammalian model organisms.
Evidence is beginning to emerge that suggests a role for miRNAs in the development of hypertension and renal injury.6 For example, the miRNA miR-155 has been shown to be involved in angiotensin II signaling.7,8 miR-192 and miR-377 may contribute to diabetic glomerular injury.9,10 miRNAs in general appears to be critical for the development of podocytes.11–13
The role of miRNAs in salt-sensitive hypertension and renal injury remains unknown. The Dahl salt-sensitive (SS) rat is the most widely used genetic model of human salt-sensitive forms of hypertension and renal injury.14–16 SS-13BN rats are consomic rats in which chromosome 13 has been introgressed from Brown Norway rats into the SS genome.17 Salt-induced hypertension and renal injury are substantially attenuated in SS-13BN rats.17–20 Comparison of SS and SS-13BN rats has led to the discovery of several pathways, such as renal medullary oxidative stress,20 local glucocorticoid excess,21 and deficiencies in fumarate metabolism,22,23 that may contribute to salt-induced hypertension and renal injury. It appears that a tree-like regulatory network might be underlying the disease phenotypes in SS rats.18,21 An exciting possibility is that miRNAs could play an important role in this complex regulatory network.6,24
In the present study, we found that an miRNA, miR-29b, was elevated in the renal medulla of SS-13BN rats by a high-salt diet but not in SS rats. We discovered evidence suggesting that miR-29 might be a master regulator of a large number of collagens and other genes related to the extracellular matrix, which might be relevant to the development of prominent medullary interstitial fibrosis in SS rats.
Methods and Materials
Inbred SS and consomic SS-13BN rats, produced at the Medical College of Wisconsin,17 were maintained on the AIN-76A diet containing 0.4% NaCl (Dyets), as described previously.17,21,23,25,26 Male rats were used for experiments at ≈6 weeks of age. The animal study was approved by the institutional animal care and use committee at the Medical College of Wisconsin.
miRNA Isolation and Expression Profiling
Expression levels of 377 miRNAs were analyzed with a custom-made miRNA microarray, as described previously.27 The renal medullas of SS and SS-13BN rats on the 0.4% NaCl diet or after 3 days on a 4% NaCl diet were examined with a total of 16 microarrays (n=4). Signal intensity data were processed and normalized as described previously.27 Criteria of differential expression were set according to the permutation-based false-positive rate, as described previously.19,27,28
Measurement of Individual miRNAs Using Modified Real-Time PCR
Expression levels of individual miRNAs were quantified using modified real-time PCR with TaqMan (Applied Biosystems) chemistry, as described previously.27 Total RNA samples used for the real-time PCR analysis were independent of those used in the microarray experiment. 5S rRNA was used as an internal normalizer.
miRNA target predictions were retrieved from 3 commonly used algorithms, TargetScan (http://www.targetscan.org/), PicTar (http://pictar.mdc-berlin.de/), and miRanda (http://microrna.sanger.ac.uk/targets/v4/). Sequence analysis of Col8a1 was performed using DNASTAR.
3′-Untranslated Region Reporter Constructs
Reporter gene constructs containing the 3′-untranslated region (UTR) of an miRNA target gene were generated using molecular cloning techniques that we described previously.21,23,29,30 Briefly, a segment, typically a few hundred bases, of the 3′-UTR that included the binding sites for miR-29b was amplified from DNA samples extracted from rat tissues using PCR primers containing restriction endonuclease recognition sequences. Locations of the segments and primer sequences are shown in Table S1 (please see the online Supplemental Database at http://hyper.ahajournals.org). The PCR product with flanking restriction nuclease sites was double digested and inserted into the multiple cloning site of the pMIR-REPORT luciferase plasmid (Ambion) downstream from a firefly luciferase gene. The chimeric 3′-UTR luciferase reporter construct was transformed into competent Escherichia coli (Invitrogen). Colonies that were confirmed by PCR to contain an insert of the expected size were cultured. The plasmids were extracted using a QIAprep Spin Miniprep kit (Qiagen) and sequenced to verify the insertion of the 3′-UTR region. Transfection-quality plasmids were prepared using a PureLink HiPure Plasmid Filter Purification kit (Invitrogen).
Transfection and Analysis of 3′-UTR Reporter Constructs
HeLa cells were cultured in 96-well CELLSTAR white μClear plates (Greiner Bio-One) with DMEM plus 5% FBS. Cells at 80% to 90% confluence were cotransfected with a 3′-UTR reporter construct (100 ng per well), a pMIR-REPORT β-galactosidase plasmid (50 ng per well), and control pre-miR oligonucleotides or the miR-29b mimic pre-miR-29b (10 pmol per well, from Ambion), using Lipofectamine 2000 (Invitrogen) following the Invitrogen protocol.21–23,29,30 At 24 hours after the transfection, luciferase and β-galactosidase activities in each well were measured using the Dual-Light system (ABI) following the manufacturer’s protocol for microplate assay. The light signals were acquired using the high-throughput Analyst HT 96.384 microplate reader (Molecular Devices). β-Galactosidase activity was used to normalize luciferase activity to control for transfection efficiency and cell density.
Culture and Transfection of Rat Medullary Thick Ascending Limb Cells
Rat medullary thick ascending limb cells were cultured as described previously.31 Transfection of the miR-29b mimic pre-miR-29b or the control pre-miR (Ambion) was performed using Lipofectamine 2000 (Invitrogen) as described previously.21–23,29,30,32
Real-time PCR using the SYBR Green chemistry was performed as described previously.28 18S rRNA was used as the internal normalizer. Sequences of the primers used were shown in Table S2.
Western blot was performed as we described previously.21–23,27,29,30 Antibodies were from Santa Cruz Biotechnology. The density of a specific band was normalized by Coomassie blue staining of the entire lane.
In Vivo Administration of Locked Nucleic Acid–Modified Anti-miR in Chronically Instrumented Rats
Locked nucleic acid (LNA)-modified anti-miR has been reported recently to efficiently knock down specific miRNAs in mice and monkeys.33 We adopted the method for use in rats. SS-13BN rats were instrumented for chronic infusion through the femoral vein and blood pressure measurement through the femoral artery, as we described previously.21–23,29 After recovery from surgery and stable baseline recording of blood pressure, LNA anti–miR-29b or LNA-scrambled anti-miR was injected intravenously (10 mg/kg of body weight, dissolved in 2 mL of normal saline, and injected >3 minutes). The rats were switched to the 4% NaCl diet after the injection and tissue collected 14 days later for expression analysis.
Data were analyzed using Student t test or multiple-group ANOVA. P<0.05 was considered significant unless otherwise indicated. Data are shown as mean±SEM.
miRNA Expression Profiles in the Renal Medulla of SS and SS-13BN Rats
The abundance of 377 miRNAs in the renal medulla of 6-week–old SS and SS-13BN rats was examined with miRNA microarrays. Five miRNAs were considered differentially expressed between the 2 rat strains after 3 days on the 4% NaCl diet (Figure 1A). Rno-miR-29b and hsa-miR-302d were expressed at lower levels in SS rats, whereas rno-miR-365, hsa-miR-211, and rno-miR-15b were higher in SS rats. The criteria of differential expression were P<0.05, signal difference >150, and fold change >50%. The overall, permutation-based, false-positive rate was 20%.
In rats maintained on the 0.4% NaCl diet, no criteria for differential expression could be set that would yield an overall, array-wide false-positive rate of <50%. Interestingly, an miRNA located on rat chromosome 13, miR-214, was readily detectable in SS rats but barely so in SS-13BN rats. Signal intensity data from all 16 of the microarrays, before any normalization or data processing, are shown in Table S3.
miR-29 Was Predicted to Regulate 20 Collagens and Several Related Genes
miR-29b, one of the miRNAs expressed at lower levels in the renal medulla of SS rats than in SS-13BN rats, is a member of the miR-29 family that also includes miR-29a and miR-29c. The mature sequences of the 3 isoforms differ at nucleotide positions 10, 18, 21, 22, or 23 but have identical seed regions in nucleotides 2 to 8. Consequently, the 3 isoforms have largely overlapping sets of predicted target genes.
As shown in the Table, a highly prominent group of predicted targets of miR-29 included 20 collagens and several other genes related to the extracellular matrix, such as Mmp2, Itgb1, Eln, Fbn1, Lamc1, and Sparc. The large number of collagens as predicted targets is unique to miR-29, because no other miRNA was predicted to target >11 of the 20 collagen genes.
The predicted role of miR-29b in the regulation of collagens and other genes related to the extracellular matrix is very interesting in the context of SS rats. Extracellular matrix formation and renal medullary interstitial fibrosis are prominent features of SS rats, which are substantially attenuated in SS-13BN rats.17,19,20 The subsequent studies focused on assessing changes in collagens and related genes in SS rats and the interaction between miR-29b and those genes.
Reciprocal Expression of miR-29b and Several Collagens and Related Genes in the Medulla
Real-time PCR analysis confirmed that the renal medullary abundance of miR-29b in SS-13BN rats was increased by >4-fold after 3 days on the 4% NaCl diet (Figure 1B). miR-29b levels in SS rats were not significantly altered by the high-salt intake. Similar changes were observed when rats were exposed to an 8% NaCl diet for 3 days (1.1±0.2-fold of 0.4% NaCl for SS, n=4, P value not significant; 2.1±0.3-fold for SS-13BN, n=4, P<0.05). Response of miR-29a or miR-29c in the renal medulla to 3 days of the 8% NaCl diet was not different between SS and SS-13BN rats (n=4; P value not significant in any of the following comparisons). miR-29a levels after 3 days of the 8.0% NaCl diet, compared with the 0.4% NaCl diet, were 1.2±0.2-fold in SS rats and 1.3±0.2-fold in SS-13BN rats. miR-29c levels were 1.8±0.3-fold in SS rats and 1.7±0.3-fold in SS-13BN rats.
In the renal cortex, miR-29b was not significantly altered by 3 days of an 8% NaCl diet in SS rats (1.2±0.4-fold of 0.4% NaCl; n=6; P value not significant) or in SS-13BN rats (0.8±0.2-fold of 0.4% NaCl; n=3; P value not significant).
TaqMan real-time PCR was performed to analyze the renal outer medullary mRNA abundance of 12 collagens, as well as Mmp2 and Itgb1, which were predicted to be miR-29 targets, and 4 collagens not predicted to be miR-29 targets. Of the predicted targets, Col1a1, Col3a1, Col4a1, Col5a1, Col5a2, Col5a3, Col7a1, and Mmp2 were found to be upregulated in SS rats, but not in SS-13BN rats, after 3 or 7 days on the 8% NaCl diet (Figure 2A). Col1a2, Col4a2, Col6a3, Col15a1, Col16a1, and Itgb1 were not significantly altered, although several of them tended to be upregulated in SS rats. Of the genes not predicted to be targets of miR-29b, Col8a1 and Col12a1 were significantly upregulated in SS rats after 7 days on the 8% NaCl diet, whereas Col6a1 and Col6a2 were not (Figure 2A). See the 3′-UTR Reporter Analysis section for additional information about a target site in Col8a1.
Exposure to the 4%, instead of 8%, NaCl diet significantly increased the renal medullary mRNA levels of Col1a2 (1.7±0.2-fold; 7 days), Col4a1 (1.6±0.2-fold; 3 days), and Col7a1 (2.3±0.3-fold; 7 days) in SS rats (n=3 to 6; P<0.05 versus 0.4% NaCl diet) but did not significantly affect these genes in SS-13BN rats. The 4% NaCl diet for 3 or 7 days increased Col1a1 mRNA in both SS and SS-13BN rats and did not cause significant changes in Col3a1, Col4a2, Col5a1, Col5a2, Col5a3, Col6a3, Col15a1, Col16a1, Mmp2, Igtb1, Col6a1, Col6a2, Col8a1, or Col12a1 in either rat strain. Western blot analysis showed that the renal medullary protein abundance of Col1a1 was significantly higher in SS rats than in SS-13BN rats after 7 days of exposure to the 4% NaCl diet (Figure 2B).
Knockdown of miR-29b Upregulated Several Extracellular Matrix Genes in the Kidneys of SS-13BN Rats
Intravenous administration of LNA anti–miR-29b (10 mg/kg of body weight) in SS-13BN rats resulted in substantial and significant knockdown of miR-29b by 76% and 63% in the renal cortex and the renal inner medulla, respectively, compared with rats treated with a scrambled LNA anti-miR (Figure 3A). miR-29b levels in the outer medulla, however, were not significantly altered (1.1±0.2-fold compared with rats treated with the scrambled anti-miR; n=4 to 7; P value not significant). In addition, LNA anti–miR-29b did not significantly suppress miR-29a (1.1±0.4-fold in the cortex and 0.8±0.1-fold in the inner medulla compared with the scrambled) or miR-29c (0.9±0.3-fold in the cortex and 0.8±0.2-fold in the inner medulla; n=4 to 7; P value not significant).
mRNA levels of Col1a1, Col3a1, Col4a1, Col4a2, Col5a1, Col5a2, and Col5a3 were examined. Knockdown of miR-29b was associated with significant upregulation of Col3a1, Col4a1, Col4a2, and Col5a1 in the renal cortex and Col3a1 in the inner medulla (Figure 3B, 3C). Col5a2 in the cortex and Col1a1 in the inner medulla tended to be upregulated but did not reach statistical significance (P=0.071 and 0.088, respectively). The upregulation of ECM genes was not secondary to changes in arterial blood pressure, because the treatment with LNA anti–miR-29b did not significantly affect mean arterial blood pressure (Figure 3D).
Suppression of Collagens and Related Genes by miR-29b in Cultured Cells
Cultured rat medullary thick ascending limb cells were treated with control pre-miR oligonucleotides or the miR-29b mimic pre–miR-29b. mRNA levels of 18 collagen genes, Mmp2, and Itgb1 were measured with TaqMan real-time PCR 48 hours later. The medullary thick ascending limb cells were used in this analysis because they were from the renal medulla of rats, although other cell types may also contribute to medullary interstitial fibrosis. The result showed that miR-29b, compared with control pre-miR, significantly reduced mRNA levels of Col3a1, Col4a1, Col4a2, Col5a1, Col5a2, Col5a3, Col7a1, and Itgb1 (Figure 4). miR-29b did not affect the mRNA level of Col18a1, which did not contain an miR-29b binding site and could be viewed as a negative control. miR-29b did not significantly affect the mRNA level of Col16a1, although Col16a1 has a predicted miR-29b binding site. mRNA levels of Col1a1, Col1a2, Col6a1, Col6a2, Col6a3, Col12a1, Col14a1, Col15a1, Col17a1, and Mmp2 were not readily detectable in these cultured cells.
3′-UTR Reporter Analysis
3′-UTR reporter analysis (Figure 5A) was performed to directly examine any 3′-UTR–mediated interactions between miR-29b and 9 collagen genes, as well as Mmp2 and Itgb1. The miR-29b mimic, compared with a control miRNA mimic, significantly reduced luciferase activity when the reporter gene was linked to a 3′-UTR segment of Col1a1, Col3a1, Col4a1, Col5a1, Col5a2, Col5a3, Col7a1, Col8a1, Mmp2, or Itgb1 (Figure 5B).
Col8a1 is not a predicted target for miR-29b according to TargetScan, PicTar, or miRanda (Table). A 3′-UTR segment of 183 bases was used for rat Col8a1 in those prediction algorithms. A segment of 2655 bases, however, was used for mouse Col8a1. Bioinformatic analysis of rat genomic DNA indicated the presence of a putative 3′-UTR segment largely homologous to the mouse segment. Importantly, the 8-base sequence in the mouse 3′-UTR that is complementary to the seed region of miR-29b is also present in the putative rat 3′-UTR. Primers (Table S1) were designed to clone a 673-base segment of the predicted 3′-UTR from rat Col8a1 mRNA, which was used in the 3′-UTR reporter analysis. The result indicated that Col8a1 was a target of miR-29b (Figure 5B).
Col12a1 does not have a predicted binding site for miR-29b. The 3′-UTR analysis confirmed that Col12a1 was not a target of miR-29b (Figure 5B). The result of Col12a1 could be viewed as a negative control for the 3′-UTR analysis.
miR-29 as a “Master” Regulator of Collagens and Related Genes
The Table summarized the data supporting a master regulatory role of miR-29 in the expression of collagens and other genes related to the extracellular matrix in the renal medulla of SS and SS-13BN rats. It included computationally predicted interactions, upregulation of collagens and Mmp2 in the renal medulla in SS rats (as opposed to the upregulation of miR-29b in SS-13BN rats) in response to the 8% NaCl diet, suppression of collagens and Itgb1 in cultured renal medullary cells by a miR-29b mimic, and miR-29b target interactions demonstrated by 3′-UTR reporter analysis.
The present study discovered the first miRNA possibly involved in the renal injury in SS rats and demonstrated a broad role of miR-29b in the regulation of a large number of collagens and other genes related to the extracellular matrix. Renal medullary interstitial fibrosis is a prominent pathological feature of SS rats, especially when the rats are on a high salt diet. Medullary interstitial fibrosis, along with salt-sensitive hypertension, is substantially attenuated in consomic SS-13BN rats.17–20 Upregulation of miR-29b may be an important part of the molecular mechanism underlying the attenuation of medullary fibrosis in SS-13BN rats. The notion is supported by reciprocal expression of miR-29b and a large number of collagens and Mmp2 in vivo, as well as upregulation of several collagens in vivo after miR-29b knockdown and suppression of several collagens by miR-29b in vitro. Several collagens targeted by miR-29b are components of, or associated with, the basement membrane or intercellular matrix.34 Matrix metallopeptidase 2 might be profibrotic in the kidney.35 Moreover, an antifibrotic role for miR-29b in the renal medulla would be consistent with the recently reported antifibrotic role of miR-29b in a mouse model of cardiac fibrosis.36
A previous study of miRNA expression in SS rats did not find differential expression of any miRNAs between SS and Lewis rats.37 A subset of miRNAs was examined in that study using whole kidney extracts and mostly pooled samples. Although the present study also indicated that the differential expression of miRNAs associated with SS rats was limited, we were able to identify miRNAs that appeared relevant to the disease phenotypes in SS rats. This may be attributed to the use of consomic rats as the control, a focus specifically on the renal medulla, a more complete coverage of miRNAs, or different techniques of expression analysis.
An important question that remains to be addressed is why miR-29b is upregulated in SS-13BN rats but not in SS rats. One copy of the miR-29b gene, miR-29b-2, is located on chromosome 13 of the rat genome, the only chromosome in SS-13BN that is derived from the Brown Norway rat. This could suggest that genetic differences might exist between SS and Brown Norway genomes in the regulatory sequence for the miR-29b-2 gene. However, the miR-29b-2 gene is separated on the chromosome from miR-29c only by 448 bp. The other copy of the miR-29b gene, miR-29b-1, is located on chromosome 4, separated from miR-29a by 292 bp. But miR-29c or miR-29a did not seem to follow the same expression pattern as miR-29b. Together, this suggests that the differential regulation of miR-29b in SS and SS-13BN rats could involve posttranscriptional processing, in addition to any transcriptional changes.
The regulation of extracellular matrix genes by miR-29b shown by the present study and other recent studies36,38,39 is one of the first experimentally proven cases in which one miRNA acts as a master regulator of a large group of related genes. Each miRNA has been predicted in silico to regulate a large number of protein-coding genes.1,2 It has led to the exciting theory that miRNAs may serve as hubs in complex regulatory networks by fine-tuning multiple components of a network.6,24 But experimental evidence supporting the theory has been sparse. Recent studies of cardiac tissue, bone cells, and tumors have led to the findings that miR-29b targets Col1a1, Col1a2, Col3a1, Col4a2, Col5a3, elastin, and fibrillin 136,39 and that miR-29c targets COL1A1, COL1A2, COL3A1, COL4A1, COL4A2, COL15A1, laminin-γ1, and osteonectin.38 The present study, which was focused on the renal medulla of SS rats, has shown that miR-29b targets Col5a1, Col5a2, Col7a1, Col8a1, Mmp2, and Itgb1, in addition to confirming the targeting of Col1a1, Col3a1, Col4a1, and Col5a3. Together, these findings (see the Table) support a master role for miR-29 in the regulation of extracellular matrix gene expression, which might be important in a number of diseases, including hypertensive renal injury.
miRNAs may suppress target expression through translational repression or induction of mRNA degradation. miR-29 decreased the mRNA abundance of collagens and other extracellular matrix genes, as shown in the present study, as well as previous publications.36,38 It appears that induction of mRNA degradation is a major mode of action for miR-29b.
Despite the broad effect of miR-29 on collagens and related genes, it is clear from the Table that miR-29 is not the only regulator of these genes in the medulla of SS and SS-13BN rats. Collagen expression and medullary fibrosis in the SS model are likely determined by a number of antagonistic forces. miR-29b appears to be one of the forces opposing fibrosis, whereas other mechanisms may drive fibrosis. This may explain why upregulation of miR-29b or knockdown of miR-29b in vivo can be associated with a mosaic of target extracellular matrix gene expression, depending on the state of the rest of the regulatory network. For example, Col1a2, Col4a2, and Col15a1 are targets of miR-29 in vitro on the basis of the present study and/or previous publications.36,38,39 However, these genes were not significantly upregulated in SS rats within 7 days of 8% NaCl exposure. Col12a1 was upregulated in SS rats, but it is not a target of miR-29. Knockdown of miR-29b in the renal cortex of SS-13BN rats was associated with upregulation of several collagen genes, although miR-29b was not differentially regulated in the renal cortex, as it was in the medulla, suggesting that baseline levels of miR-29b may already be important in controlling the expression of extracellular matrix genes. LNA anti–miR-29b remarkably suppressed miR-29b in the cortex and the inner medulla, but not in the outer medulla, preventing us from directly assessing the in vivo role of outer medullary miR-29b. The reason for the regional differences in the efficiency of knock down is not clear. In most cases, the in vivo and in vitro data were consistent, because Col1a1, Col3a1, Col4a1, Col5a1, Col5a2, Col5a3, Col7a1, Col8a1, and Mmp2 are targets of miR-29 in vitro and also upregulated in SS rats in vivo. However, even in these cases, the lack of miR-29b upregulation in SS rats likely played a permissive role in the upregulation of those genes instead of being a primary stimulus.
The findings of the present study suggest that the function and regulation of miRNA may contribute to the molecular mechanisms underlying the development of tissue injury associated with hypertension. In addition, the master role of miR-29 as a regulator of collagens and genes related to the extracellular matrix has significant implications for understanding the general biology of miRNAs.
Sources of Funding
The study was supported by National Institutes of Health grants HL077263, HL085267, DK084405 (to M.L.), HL-029587, and HL-082798 (to A.W.C.).
- Received October 2, 2009.
- Revision received October 20, 2009.
- Accepted January 29, 2010.
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