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miR-211 downregulation contributes to Candidemia-induced kidney injuries via derepression of Hmx1 expression
Life Sciences 102 (2014) 139–144
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miR-204/miR-211 downregulation contributes to Candidemia-induced kidney injuries via derepression of Hmx1 expression Xiao-Yue Li a,1, Ke Zhang b,1, Zhi-Yi Jiang c, Li-Hua Cai a,⁎ a b c
Department of Critical Care Medicine, Dongguan People's Hospital, Dongguan, Guangdong 523059, China Department of Infectious Diseases, PLA421 Hospital, Guangzhou, Guangdong 510318, China Department of Critical Care Medicine, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong 510080, China
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Article history: Received 26 December 2013 Accepted 6 March 2014 Available online 15 March 2014 Keywords: Candidemia Candida albicans Renal function Kidney injuries Hmx1 MicroRNA MiR-204 MiR-211 Gene expression regulation Renal glomerular filtration rate (GFR) Serum cystatin C (CysC) Serum β2-microglobulin (β2-MG)
a b s t r a c t Aims: This study was aimed to exploit the role of heme oxygenase Hmx1 and the potential miRNA mechanisms in the kidney injuries induced by urinary tract infection by Candida species/Candidemia. Main methods: We employed a mouse model of systemic Candidiasis by injection of the Candida albicans strain SC5314 into C57BL/6 mice. Kidney injuries were assessed by measuring serum cystatin C (CysC), serum β2-microglobulin (β2-MG) and blood urea nitrogen (BUN). Validation of miRNA target gene was conducted by luciferase reporter gene assay, Western blot analysis and real-time RT-PCR. Key findings: We showed here that Candidemia caused significant downregulation of microRNAs miR-204 and miR-211. In sharp contrast, Hmx1 expression was remarkably upregulated, particularly at the protein level. Computational analysis predicted Hmx1 as a target gene for both miR-204 and miR-211 that share the same seed site sequence. We then experimentally validated the targeting relationship between miR-204/miR-211 and Hmx1, which explains the reciprocal changes of expression of miR-204/miR-211 and Hmx1 in Candidemia. Administration of miR-204/miR-211 mimics substantially downregulated Hmx1 and mitigated the severity of the kidney injuries induced by Candidemia, as reflected by improved renal glomerular filtration rate (GFR) determined by serum cystatin C (CysC), serum β2-microglobulin (β2-MG) and blood urea nitrogen (BUN). Knockdown of miR-204/miR-211 worsened while forced expression of miR-204/miR-211 ameliorated kidney injuries in mice with systemic Candidiasis. Significance: Our findings indicate that miR-204/miR-211 downregulation accounts at least partially for the Hmx1 upregulation and the miR-204/miR-211–Hmx1 signaling axis may contribute to immune-suppression in the host thereby the Candidemia-induced kidney dysfunction. © 2014 Elsevier Inc. All rights reserved.
Introduction Urinary tract infection (UTI) by Candida species or Candidemia is unusual in healthy individuals, but common in patients with predisposing diseases and structural abnormalities of the kidney and collecting system (Fisher et al., 2011; Serefhanoglu et al., 2012; Sychev et al., 2009; Turner et al., 1985). Bloodstream infection with Candida species is a severe invasive fungal infection with high mortality, representing the fourth most common nosocomial blood-borne infection in immunocompromised patients. Candida albicans (C. albicans) is the leading cause of fungal UTI among various Candida species, which utilizes a repertoire of virulence factors to colonize tissue and disseminate. These events run in concert with altered expression of varying factors at different stages during a Candida infection. There is an urgent need to identify
⁎ Corresponding author. Tel.: +86 769 22222365. E-mail address: [email protected] (L.-H. Cai). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.lfs.2014.03.010 0024-3205/© 2014 Elsevier Inc. All rights reserved.
host factors that predispose to Candidemia in order to establish a welldefined predictive profile for patients at risk for developing infection. As the first step for the infection, Candida species are recognized by pattern recognition receptors such as Toll-like receptors (TLRs) (Plantinga et al., 2012). Activation of TLRs allows the fungi to adhere to endothelial or urothelial cells and colonizes the local area, afterwards evading the immune response and ultimately invading tissue or disseminating to distant sites within the body. Deficiency in the host immune system is the major predisposing factor to Candida infection (Fisher et al., 2011). The pathogenic yeast C. albicans encodes a heme oxygenase\Hmx1 that plays an important role in immune regulation by producing immunosuppressive carbon monoxide (CO) (Scapagnini et al., 2002; Otterbein et al., 2000; Overhaus et al., 2006). Hmx1 is specifically induced by the host protein hemoglobin after C. albicans infection, and is involved in the pathogenesis of disseminated bloodstream infections (Navarathna & Roberts, 2010; Kim et al., 2006; Pendrak et al., 2004). It has been shown that exposing mice to therapeutic levels of CO increases C. albicans virulence, whereas an Hmx1-null strain decreases virulence in murine disseminated Candidiasis (Navarathna &
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Roberts, 2010). Additionally, levels of several regulatory cytokines and chemokines are decreased in mice infected with the Hmx1-null strain, and initial lesions in the kidney are more rapidly cleared after polymorphonuclear leukocyte infiltration. In contrast, inhaled CO partially reverses the virulence defect of the null strain and restores several host cytokine responses to wild-type levels (Navarathna & Roberts, 2010). However, the precise mechanisms for how Hmx1 modulates C. albicans virulence remained poorly understood. Although C. albicans infect multiple organs, the kidney is the most heavily colonized organ, which can cause some loss of renal function (Leunk & Moon, 1979; Spellberg et al., 2005). It is known that in progressive renal infection initiated by the virulent strain, the fungus proliferated in the kidneys, accompanied by a massive transcriptional change, mostly genes associated with innate and adaptive immune responses, such as Hmx1. It was reported that glibenclamide improves kidney and heart structure and function partially by decreasing the expression of HO-1 (Diwan et al., 2014). On the other hand, adenine diet induced the characteristics of human chronic kidney disease accompanied by increased HO-1 expression (Diwan et al., 2013). Recently, a class of non-protein-coding mRNAs known as microRNAs (miRNAs) has emerged as a central player in fine-tune regulation of protein-coding genes. These 22–26 nucleotides molecules primarily repress the expression of protein-coding genes at the post-transcriptional level by complementarity of its 5′-end 2–8 nucleotides to the matched motifs in the 3′-untranslated region (3′UTR) of mRNAs. Intriguingly, it has been documented that either C. albicans infection or bacterial cellsurface lipopolysaccharide (LPS or endotoxin) treatment can cause upregulation of miRNAs such as miR-146, miR-155, miR-455 and miR125a in macrophages (Monk et al., 2010). This effect may play a role in inflammatory signaling tailoring the macrophage response to the appropriate pathogen. In light of these findings, we thought that miRNAs might be involved in the expression regulation of Hmx1 in kidney with C. albicans infection. This thought urged us to carry out preliminary analyses to search for the miRNAs that have the potential to target Hmx1. Utilizing computational prediction algorithms, we identified miR-204 and miR-211 that share the same seed site sequence and 91% overall sequence homology as candidate regulators of Hmx1 gene. This initial finding prompted us to set up the present study to experimentally validate the targeting relationship between miR-204/miR211 and Hmx1, and to exploit the role of this regulation in the kidney injuries induced by Candidemia. Materials & methods Animals C57BL/6 mice purchased from Charles River Laboratories (Wilmington, MA, USA) were used for the experiments. The animals received balanced diet and water ad libitum. All procedures involving animals were carried out in accordance with the guidelines set by and approved by the Institutional Animal Care and Use Committee of the Dongguan People's Hospital. Induction of systemic candidiasis The C. albicans strain SC5314 was obtained from a fungal suspension as described elsewhere (Venturini et al., 2009) and cultivated in YPD (1% yeast extract, 1% peptone, 2% dextrose) broth with overnight shaking at 30 °C. Yeast cells were washed with phosphate buffered saline (PBS) and resuspended in PBS at the desired concentration. Mice were inoculated through the lateral tail vein with a designated number of yeast colony-forming units (CFUs) via the retro-orbital venous plexus (FragaSilva et al., 2013 Mar 25). To confirm the fungal dose injected into mice, 100 ml of the yeast cell suspension was plated on YPD agar plates, and the number of CFU was determined after 24 h of incubation at 30 °C. The fungal concentration was adjusted to 5.0 × 105 viable C. albicans/ml. This model is commonly used for studying urinary tract infection and its
associated kidney injuries (Venturini et al., 2009; Fraga-Silva et al., 2013 Mar 25). Our pilot experiments had demonstrated that this inoculum size is not lethal in C57BL/6 mice and it reproduces a systemic fungal infection. Determination of fungal burden Mice were sacrificed and kidneys were removed aseptically at necropsy and washed with sterile PBS. The preparations were then weighted and placed in 1.5 ml sterile tissue lysis buffer (200 mM NaCl, 5 mM EDTA, 10 mM Tris, 10% glycerol, 1× protease inhibitor cocktail) (Roche Applied Science, Upper Bavaria, Germany) on ice. The samples were aseptically homogenized using a homogenizer and serial dilutions of homogenates were plated in triplicate on YPD plates containing ampicillin, tetracycline, and chloramphenicol. Colonies were counted after 1, 3 and 7 days of incubation at 30 °C. The fungal burden was calculated as colony forming units (CFUs) per gram of tissue. Measurement of glomerular filtration rate (GFR) To assess kidney tissue damage, glomerular filtration rate (GFR) was measured on day 7 after infection. For this purpose, serum cystatin C (CysC) and β2-microglobulin (Grubb, 2000; Herrero-Morín et al., 2007) were measured using RANDOX enzymatic creatinine assay (Randox Laboratories Limited, Crumlin, County Antrim, UK) with calibration traceable to the international standard reference material as recently recommended by the National Kidney Foundation (KDIGO 2012, Kidney International 2013). Additionally, blood urea nitrogen (BUN) levels were determined in serum samples by a routine veterinarian diagnostics laboratory (InVitro GmbH). Preparation of miRNA-mimics, antisense inhibitors, decoy oligodeoxynucleotides miR-204 and miR-211 mimics, mutant miR-204 (5′-UUaagaUUGU CAUCCUAUGCCU-3′), mutant miR-211 (5′-UUaagaUUGUCAUCCUUC GCCU-3′), antimiR-204 (antisense inhibitor of miR-204: 5′-AGGCAU AGGAUGACAAAGGGAA-3′), and mutant antimiR-204 (5′-AGGCAUAG GAUGACAAcuuuAA-3′) were all synthesized by Invitrogen (Carlsbad, CA, USA). Hmx1 siRNA was obtained from Santa Cruz Biotech (Cat#: sc-75269; Santa Cruz, CA, USA), and scrambled siRNA (5′-AACAGTCG CGTTTGCGACTGG-3′) that does not match any known mammalian GenBank sequence was synthesized by Invitrogen. For in vivo applications, all these above constructs were modified with locked nucleic acid (LNA) to acquire long-standing stability in the cells (Elmén et al., 2008a; Elmén et al., 2008b). These LNA constructs were injected via tail vein into mice at a dosage of 50 ng/ml once a day for 4 days prior to C. albicans infection. mRNA and miRNA quantification Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA). Both mRNA and miRNA levels were determined using the TaqMan quantitative real-time PCR (qRT-PCR) method (10 ng/assay) on an ABI 7500 Real-Time PCR System (Applied Biosystems, San Mateo County, CA, USA). Taqman Primer assays and the reagents for reverse transcriptase and qRT-PCR reactions were all provided by Applied Biosystems. Relative expression levels were calculated using the comparative cycle threshold (Ct) method (2−ΔΔCt) with mRNA levels normalized to GAPDH and miRNA levels to U6 RNA. Western blot analysis The isolated total protein samples (20 μg) were loaded to SDS-PAGE and then transferred to the membrane. The membrane was incubated with a 1:1000 dilution of primary antibody, followed by a 1:5000 dilution of horseradish peroxidase-conjugated secondary antibody. Rabbit
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polyclonal anti-Hmx1 (Abcam, Cambridge, MA, USA; Cat# ab28733) was used as the primary antibody. Rabbit anti-IgG (Alexa Fluor 350; Life Technologies, Carlsbad, CA, USA) was used as the secondary antibody. The intensity (area × density) of immunoblot bands was measured by densitometry (model GS-800, Imaging Densitometer; Bio-Rad). Relative protein levels were calculated by normalizing to GAPDH detected by anti-GAPDH antibody (Life Technologies). Immunochemistry for Hmx1 Animals were perfused with heparinized saline solution-4% buffered formalin. Kidneys were removed, halved longitudinally. Sections of 3-μm thick were cut and mounted on glass slides for immunohistochemical studies. The primary antibody was rabbit polyclonal anti-Hmx1 (Abcam, Cambridge, MA, USA; Cat# ab28733). Following washes in phosphate-buffered saline, sections were incubated with DAKO LSAB2 system + HRP (DAKO), and 3,3′-diaminobenzidine (BioGenex, San Ramon, CA, USA) was used as chromogen. Luciferase reporter gene assay The 3′UTR of Hmx1 was PCR synthesized and the fragment was subcloned into the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Beijing, China). PCR-based mutagenesis was made to the putative binding site for miR-204/miR-211 by base replacement to the site motif at the 3′UTR of Hmx1 mRNA (underlined lower case letters: 628-UUGCGUAUGGAAUAAAAcattcC-651) and was also cloned in the luciferase vector. HEK293 human embryonic kidney cell line (ATCC) in culture was cotransfected with the luciferase vector and varying constructs including miR-204 or miR-211 mimic, mutant miR-204 or miR-211, antimiR204, mutant antimiR-203, Hmx1 siRNA, or scrambled siRNA. The cells were cultured in DMEM supplemented with 10% fetal bovine serum. Prior to transfection, cells were starved to synchronize growth by incubating in serum- and antibiotics-free medium for 12 h. Forty-eight hours after transfection, luciferase activities were measured using the Dual Luciferase Reporter® Assay System (Promega) on a Luminoskan ™ Ascent Microplate Luminometer (Thermo Fisher Scientific, Waltham, MA, USA). Statistical methods Values are reported as the mean ± SEM with sample sizes indicated in each legend. Statistical comparisons among multiple groups were performed with one-way ANOVA, and intergroup differences were tested with Bonferroni inequality if one-way ANOVA yielded significance. All other results were analyzed by using a two-tailed Student's t-test between two groups. Significance was accepted at p-value b 0.05. All statistical analyses were performed using SPSS18.0 analysis software. Receiver operating characteristic (ROC) analysis was performed with Sensitivity plotted against 1-Specificity. Area under ROC curve (AUC) was estimated to assess the diagnostic accuracy.
Fig. 1. Reciprocal changes of expression of miR-204/miR-211 and Hmx1 protein in renal tissues of mice with systemic Candidiasis. (a) Downregulation of miR-204 and miR-211 in Candidiasis relative to healthy control mice, measured on day 7 after C. albicans infection using TaqMan real-time RT-PCR. (b) Upregulation of Hmx1 protein in Candidiasis relative to healthy control mice, measured on day 7 after C. albicans infection using Western blot analysis. (c) Comparison of Hmx1 mRNA levels between Candidiasis and healthy control mice. ***p b 0.001 Candidiasis vs healthy control, n = 12 for each group. Histological evaluation of C. albicans infection was performed on day 7 post-infection. Histological sections of kidney from the indicated mice were stained with H&E (upper panel) or PAS (lower panel). Kidney structure of C. albicans-infected mice was disturbed by infiltrating inflammatory cells (blue triangle) and red blood cells (white triangle). IL-17+ MSC infusion showed significant rescue of the kidney structures compared to bulk MSC and IL-17− MSC groups, as assessed by H&E staining. Infected mice showed the infiltration of C. albicans clusters (yellow triangle) in kidney, while the IL-17+ MSC infusion group showed a significantly more effective clearance of C. albicans infiltration compared to bulk and IL-17− MSC groups, as viewed by PAS staining. *p b 0.05; ** p b 0.01; and ***p b 0.001 (mean ± SD). Scale bars, 50 μm. The data are representative of three independent experiments.
On the other hand, Hmx1 was found robustly upregulated at both protein and mRNA levels despite that the changes were markedly greater for protein than for mRNA (Fig. 1b & c).
Results Hmx1 as a target gene for miR-204 and miR-211 Downregulation of miR-204 and miR-211 and upregulation of Hmx1 by Candidemia Since our preliminary analysis indicates miR-204 and miR-211 as candidate regulators of Hmx1 gene, we focused our experiments on the changes of expression of miR-204 and miR-211. As shown in Fig. 1a, in the mice with induction of systemic Candidiasis, miR-204 and miR-211 transcript levels in renal tissue were both found considerably decreased relative to healthy animals. As a negative control, miR210 was unaltered in its expression.
Our computational analysis using the Targetscan algorithm (http://www.targetscan.org/cgi-bin/vert_61/view_genetable.cgi? rs=NM_018942&taxid=9606&members=&showcnc=1&shownc= 1&sortText=cs) predicted Hmx1 as a target gene for both miR-204 and miR-211 that share the same seed site sequence, which is highly conserved across species (Fig. 2). To experimentally clarify if Hmx1 is indeed a cognate target for miR204/miR-211, we first carried out the luciferase activity assay in HEK293 cells. As shown in Fig. 3a, transfection with miR-204 or miR-211
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UUG GCG GUA AUG GGA AAU UAA AAA AAG GGG GAC C Po ositiion 62 28-634 4o of H HMX X1 3' UT TR 5'-U ||| ||||||| 3 3'-UC CCG GCU UUC CCU UAC CUG GUU UUC CCC CUU U hsa a-m miR R-21 11 UUG GCG GUA AUG GGA AAU UAA AAA AAG GGG GAC C Po ositiion 62 28-634 4o of H HMX X1 3' UT TR 5'-U ||| ||||||| 3 3'-UC CCG GUA AUC CCU UAC CUG GUU UUC CCC CUU U hsa a-m miR R-20 04
Fig. 2. Bioinformatics prediction of Hmx1 as a candidate target gene for miR-204 and miR211 based on the results from Targetscan miRNA database (http://www.targetscan.org). (a) Sequence alignment between miR-204/miR-211 and the 3′UTR of Hmx1 mRNA. Note the full complementarity between the seed sites of miR-204/miR-211 and the fragment containing the putative binding site for these miRNAs. (b) Sequence conservation across various species.
significantly diminished luciferase activity elicited by the luciferase vector carrying the complementary sequence for these miRNAs in the 3′ UTR of Hmx1 mRNA. The magnitude of decreases produced by miR204 or miR-211 was diminished by co-transfection with antimiR-204, but not by anti-miR-204 mutant. miR-204 or miR-211 mutant did not significantly affect the luciferase activities. Injection of LNA-miR-204 or miR-211 significantly reduced the renal protein level of Hmx1 in both healthy control (data not shown) and Candidiasis mice (Fig. 3b). On the contrary, injection of LNA-antimiR204 slightly increased Hmx1 protein level, indicating a relief of basal repression of Hmx1 by endogenous miR-204/miR-211. None of the mutant constructs produced any statistically significant changes of Hmx1 protein level. The renal mRNA level of Hmx1 was not significantly altered, although LNA-miR-204 or miR-211 tended to downregulate while LNAantimiR-204 tended to upregulate Hmx1 mRNA expression (Fig. 3c). The efficacy of the antimiR-204 to knockdown both miR-204 and miR-211 was confirmed by our results showing the 86.3 ± 7.5% (p b 0.001, n = 12) decrease in miR-204 level and 78.8 ± 6.9% (p b 0.001, n = 12) in renal tissues. miR-204/miR-211 relieves kidney injuries induced by Candidemia The fungal burden in mice with Candidemia was calculated as colony forming unit (CFU) per gram of renal tissue. The results in Fig. 4a demonstrate that LNA-miR-204 or LNA-miR-211 moderately but significantly decreased CFU in Candidemia mice, whereas LNA-antimiR-204 increased it. By comparison, the mutant constructs did not have significant effects on CFU. To connect these changes to kidney injuries, we compared the changes of the renal glomerular filtration rate (GFR) by measuring serum cystatin C (CysC; Fig. 4b), serum β2-microglobulin (Fig. 4b) and blood urea nitrogen (BUN; Fig. 4c) in the presence of varying constructs. Expectedly, all these measurements were enormously higher in Candidemia mice than in healthy control animals, indicating severe
Fig. 3. Validation of Hmx1 as a cognate target gene for miR-204 and miR-211. (a) Luciferase reporter gene assay for the direct repressive effects of miR-204 and miR-211 on Hmx1 in HEK293 cells. Note that the luciferase activities induced by the vector carrying the 3′UTR of Hmx1 were substantially repressed by transfection with either miR-204 or miR-211. **p b 0.01 miRNA vs control, +p b 0.05 and ++p b 0.01 antimiR vs miRNA; n = 5 for each group. (b) Effects of LNA-miR-204/LNA-miR-211 and LNA-antimiR-204 on Hmx1 protein level on day 7 after C. albicans infection. Lane 1: control; lane 2: miR-204; lane 3: mutant miR-204; lane 4: antimiR-204; lane 5: mutant antimiR-204. *p b 0.05 miRNA vs control, n = 12 for each group. (c) Lack of significant effects of LNA-miR-204/LNA-miR-211 and LNA-antimiR-204 on Hmx1 mRNA level on day 7 post C. albicans infection.
kidney injuries in C. albicans-infected animals. LNA-miR-204 or LNAmiR-211 significantly restored the increased CysC, β2-microglobulin and BUN in Candidemia mice, whereas LNA-antimiR-204 worsened the adverse changes. In healthy mice, LNA-antimiR-204 decreased all three measurements. By comparison, the mutant constructs did not have significant effects on any of these measurements. These results suggest that knockdown of miR-204 and miR-211 by LNA-antimiR204 caused kidney injuries while forced expression of these miRNAs by LNA-miR-204 or LNA-miR-211 administration produced protective effects against kidney injuries. We have shown that downregulation of miR-204/miR-211 is accompanied by upregulation of Hmx1 in Candidemia mice (Fig. 1) and Hmx1 is a cognate target for miR-204/ miR-211 (Fig. 3). It is conceivable that Hmx1 mediates deleterious effects of miR-204/miR-211 downregulation on kidney function. If this is true, then knockdown of Hmx1 itself should be able to relieve kidney injuries. Our experiments using Hmx1 siRNA indeed provided the evidence in support of this notion (Fig. 4a–d).
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Fig. 4. Knockdown of miR-204/miR-211 worsens and forced expression of miR-204/miR-211 ameliorates kidney injuries in mice with systemic Candidiasis. (a) Effects of miR-204/miR-211 on the fungal burden in mice with Candidemia, which was assessed as colony forming unit (CFU) per gram of renal tissue. Measurements were performed on days 1, 3 and 7 after C. albicans injection. Significant differences (p b 0.05) were found for LNA-miR-204, LNA-miR-211, LNA-antimiR-204, and Hmx1 siRNA at all three time points. (b)–(d) Effects of various constructs on renal glomerular filtration rate (GFR) as reflected by serum cystatin C (CysC), serum β2-microglobulin (β2-MG) and blood urea nitrogen (BUN). Note that LNA-miR-204 and LNA-miR-211 restored the damaged GFR, and so did Hmx1 siRNA; in contrast, LNA-antimiR-204 worsened the damage. ***p b 0.001 Candidiasis vs healthy control, +p b 0.05 & p b 0.001 vs Candidiasis; n = 12 for each group. (e) Immunostaining of Hmx1 (brown) in kidney sections verifying the presence of Hmx1 and the changes of its expression by Candidemia and antimiR-204 in kidney. Similar results were consistently observed in another three animals for each group.
We then further verified the presence of Hmx1 and the changes of its expression in kidney with immunochemistry. As shown in Fig. 4e, Hmx1 was substantially expressed in Candidemia, which was otherwise minimal in healthy controls. LNA-antimiR-204 eliminated the Hmx1 signal whereas the mutant antimiR failed to do so. Discussion Although Candidemia is a relatively unusual event in clinical practice, it is indeed a severe invasive fungal infection with high mortality (Fisher et al., 2011; Serefhanoglu et al., 2012; Sychev et al., 2009; Turner et al., 1985) and previous studies have documented that delays in treatment are associated with increased mortality in Candidemia patients (Taur et al., 2010). Because Candidemia is a rare complication, there is uncertainty about its optimal medical management. Therefore, there is a necessity to better understand host factors that predispose to Candidemia in order to establish a well-defined treatment. Here we have provided experimental observations on the roles of miRNAs miR-
204/miR-211 and Hmx1 which allow us to get some insight into the issue. First, we showed an inverse relationship between miR-204/miR211 and Hmx1 in terms of their expression levels in a mouse model of Candidemia: downregulation of miR-204 and miR-211 and upregulation of Hmx1. Second, we established Hmx1 as a cognate target gene for miR-204 and miR-211, which explains the reciprocal changes of expression of miR-204/miR-211 and Hmx1 in Candidemia. Third, miR204/miR-211 relieves kidney injuries induced by Candidemia: knockdown of miR-204/miR-211 worsened while forced expression of miR-204/miR-211 ameliorated kidney injuries in mice with systemic Candidiasis. These findings allow us to reach a conclusion that the miR-204/miR-211–Hmx1 signaling axis may contribute to the development of Candidemia and the associated kidney injuries and interventions with miR-204/miR-211 may be an approach for the management of Candidemia. Especially, our study represents the first to identify the role of miRNAs in this particular pathogenesis. Prior studies have shown that Hmx1 is critically involved in the pathogenesis of disseminated bloodstream infection by C. albicans
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(Navarathna & Roberts, 2010; Kim et al., 2006; Pendrak et al., 2004). Exposing mice to therapeutic levels of CO increases C. albicans virulence, suggesting Hmx1 as a host responsive and promoting factor for C. albicans infection (Navarathna & Roberts, 2010). Furthermore, evidence existing indicate that Hmx1 exaggerates kidney lesions (Navarathna & Roberts, 2010). However, the precise mechanisms for how Hmx1 is dysregulated by C. albicans infection to produce these deleterious effects remained incompletely understood. The present study sheds light on this issue. We first showed the upregulation of renal Hmx1 expression in response to C. albicans infection. We then demonstrated detrimental actions of Hmx1 kidney injuries in mice with Candidemia by showing the restoration of damaged kidney function by knocking down Hmx1 using siRNA. Our findings strongly suggest that Hmx1 is likely involved in the development of kidney injuries in Candidemia. We further unraveled that downregulation of miR-204 and miR-211 could lead to Hmx1 upregulation as a result of relief of basal repression of the gene. Recently, miRNAs have emerged as central regulators of human disease including kidney disease (Aguado-Fraile et al., 2013; Hou & Zhao, 2013; Gomez et al., 2013) and infection disease (Fritz et al., 2006; Martinez-Nunez et al., 2009). They act via targeting the proteincoding genes relevant to the specific pathological entities. In this study, we found that miR-204 and miR-211 that share identical seed site were both significantly downregulated in renal tissue from Candidemia mice relative to healthy control mice. This downregulation appears to be detrimental to kidney function as artificial knockdown of these miRNAs using their antisense inhibitor worsened the kidney injuries as reflected by the increased CFU, CysC, β2-MG and BUN, and administration of exogenous miR-204/miR-211 was able to efficiently restore and ameliorate the damaged kidney in Candidemia mice. This downregulation also likely accounts, at least partially, for the upregulation of Hmx1 as we have verified Hmx1 as a target gene for these two miRNAs. Artificial knockdown of miR-204/miR-211 increased, whereas forced expression of these two miRNAs decreased Hmx1 protein levels in mice with Candidemia. The fact that Hmx1 expression was upregulated to a significantly greater degree at the protein level than at its mRNA level indicates that in addition to the transcriptional activation by the host protein hemoglobin Hmx1 can be further upregulated via posttranscriptional derepression from miRNAs. It is known that miRNAs primarily act to inhibit protein translation with or without affecting the mRNA stability of their target genes, depending on whether any of the 5′-end 10–12 nucleotides is complementary to the sequence motif in the 3′-untranslated region of their target genes (mRNAs). It is noticed that there is a discrepancy between Hmx1 protein and mRNA expression, miR-204 and miR-211 significantly repressed Hmx1 protein without altering Hmx1 mRNA (Fig. 3b & c). This discrepancy reflects the canonical action of miRNAs: inhibition of protein translation. In our case, neither miR-204 nor miR-211 has complementarity to Hmx1 mRNA in the positions of 5′-end 10–12 nucleotides, and thus, neither of them produced significant changes of Hmx1 mRNA. Taking together previous results and the present findings, it is quite possible that when infected by C. albicans, the Hmx1 gene encoded by this pathogenic yeast is transcriptionally activated by the host protein hemoglobin. Meanwhile miR-204 and miR-211 are somehow downregulated and this downregulation further upregulates Hmx1. Hmx1 then produces detrimental effects on kidney function. Thus, our study identified the new miR-204/miR-211–Hmx1 axis in renal injuries induced by C. albicans infection. However, our study failed to elucidate the downstream components mediating the miR-204/miR-211–Hmx1 signaling and the upstream mechanisms for the downregulation of these two miRNAs. Additionally, the present study focused on miR-204 and miR211 and could not exclude the possible involvement of other miRNAs in the regulation of Hmx1 expression in the setting of Candidemia. Conflict of interest None.
Funding support This study is supported by the Techpool Research Foundation grant (01200909). References Aguado-Fraile E, Ramos E, Conde E, Rodríguez M, Liaño F, García-Bermejo ML. MicroRNAs in the kidney: novel biomarkers of acute kidney injury. Nefrologia 2013;33(6): 826–34. Diwan V, Mistry A, Gobe G, Brown L. Adenine-induced chronic kidney and cardiovascular damage in rats. J Pharmacol Toxicol Methods 2013;68:197–207. Diwan V, Gobe G, Brown L. Glibenclamide improves kidney and heart structure and function in the adenine-diet model of chronic kidney disease. Pharmacol Res 2014;79: 104–10. Elmén J, Lindow M, Schütz S, Lawrence M, Petri A, Obad S, et al. LNA-mediated microRNA silencing in non-human primates. Nature 2008a;452(7189):896–9. Elmén J, Lindow M, Silahtaroglu A, Bak M, Christensen M, Lind-Thomsen A, et al. Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver. Nucleic Acids Res 2008b;36(4):1153–62. Fisher JF, Kavanagh K, Sobel JD, Kauffman CA, Newman CA. Candida urinary tract infection: pathogenesis. Clin Infect Dis 2011;52(Suppl. 6):S437–51. Fraga-Silva TF, Venturini J, de Arruda MS. Trafficking of phagocytic peritoneal cells in hypoinsulinemic–hyperglycemic mice with systemic candidiasis. BMC Infect Dis 2013;13:147. (Mar 25). Fritz JH, Girardin SE, Philpott DJ. Innate immune defense through RNA interference. Sci STKE 2006;2006(339):e27. Gomez IG, Grafals M, Portilla D, Duffield JS. MicroRNAs as potential therapeutic targets in kidney disease. J Formos Med Assoc 2013;112(5):237–43. Grubb AO. Cystatin C—properties and use as diagnostic marker. Adv Clin Chem 2000;35: 63–99. Herrero-Morín JD, Málaga S, Fernández N, Rey C, Diéguez MA, Solís G, et al. Cystatin C and beta2-microglobulin: markers of glomerular filtration in critically ill children. Crit Care 2007;11:R59. Hou J, Zhao D. MicroRNA regulation in renal pathophysiology. Int J Mol Sci 2013;14(7): 13078–92. Kim D, Yukl ET, Moënne-Loccoz P, Montellano PR. Fungal heme oxygenases: functional expression and characterization of Hmx1 from Saccharomyces cerevisiae and CaHmx1 from Candida albicans. Biochemistry 2006;45:14772–80. Leunk RD, Moon RJ. Physiological and metabolic alterations accompanying systemic candidiasis in mice. Infect Immun 1979;26:1035–41. Martinez-Nunez RT, Louafi F, Friedmann PS, Sanchez-Elsner T. MicroRNA-155 modulates the pathogen binding ability of dendritic cells (DCs) by down-regulation of DCspecific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN). J Biol Chem 2009;284(24):16334–42. Monk CE, Hutvagner G, Arthur JS. Regulation of miRNA transcription in macrophages in response to Candida albicans. PLoS One 2010;5:e13669. Navarathna DH, Roberts DD. Candida albicans heme oxygenase and its product CO contribute to pathogenesis of candidemia and alter systemic chemokine and cytokine expression. Free Radic Biol Med 2010;49:1561–73. Otterbein LE, Bach FH, Alam J, Soares M, Lu HT, Wysk M, et al. Carbon monoxide has antiinflammatory effects involving the mitogen-activated protein kinase pathway. Nat Med 2000;6:422–8. Overhaus M, Moore BA, Barbato JE, Behrendt FF, Doering JG, Bauer AJ. Biliverdin protects against polymicrobial sepsis by modulating inflammatory mediators. Am J Physiol 2006;290:G695–703. Pendrak ML, Chao MP, Yan SS, Roberts DD. Heme oxygenase in Candida albicans is regulated by hemoglobin and is necessary for metabolism of exogenous heme and hemoglobin to alpha-biliverdin. J Biol Chem 2004;279:3426–33. Plantinga TS, Johnson MD, Scott WK, van de Vosse E, Velez Edwards DR, Smith PB, et al. Toll-like receptor 1 polymorphisms increase susceptibility to candidemia. J Infect Dis 2012;205(6):934–43. Scapagnini G, D'Agata V, Calabrese V, Pascale A, Colombrita C, Alkon D, et al. Gene expression profiles of heme oxygenase isoforms in the rat brain. Brain Res 2002;954:51–9. Serefhanoglu K, Timurkaynak F, Can F, Cagir U, Arslan H, Ozdemir FN. Risk factors for candidemia with non-albicans Candida spp. in intensive care unit patients with end-stage renal disease on chronic hemodialysis. J Formos Med Assoc 2012;111: 325–32. Spellberg B, Ibrahim AS, Edwards Jr JE, Filler SG. Mice with disseminated candidiasis die of progressive sepsis. J Infect Dis 2005;192:336–43. Sychev D, Maya ID, Allon M. Clinical outcomes of dialysis catheter-related candidemia in hemodialysis patients. Clin J Am Soc Nephrol 2009;4:1102–5. Taur Y, Cohen N, Dubnow S, Paskovaty A, Seo SK. Effect of antifungal therapy timing on mortality in cancer patients with candidemia. Antimicrob Agents Chemother 2010; 54(1):184–90. Turner RB, Donowitz LG, Hendley JO. Consequences of candidemia for pediatric patients. Am J Dis Child 1985;139:178–80. Venturini J, De Camargo MR, Félix MC, Vilani-Moreno FR, De Arruda MS. Influence of tumour condition on the macrophage activity in Candida albicans infection. Scand J Immunol 2009;13:10–7.
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miR-204/miR-211 downregulation contributes to Candidemia-induced kidney injuries via derepression of Hmx1 expression Xiao-Yue Li a,1, Ke Zhang b,1, Zhi-Yi Jiang c, Li-Hua Cai a,⁎ a b c
Department of Critical Care Medicine, Dongguan People's Hospital, Dongguan, Guangdong 523059, China Department of Infectious Diseases, PLA421 Hospital, Guangzhou, Guangdong 510318, China Department of Critical Care Medicine, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong 510080, China
a r t i c l e
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Article history: Received 26 December 2013 Accepted 6 March 2014 Available online 15 March 2014 Keywords: Candidemia Candida albicans Renal function Kidney injuries Hmx1 MicroRNA MiR-204 MiR-211 Gene expression regulation Renal glomerular filtration rate (GFR) Serum cystatin C (CysC) Serum β2-microglobulin (β2-MG)
a b s t r a c t Aims: This study was aimed to exploit the role of heme oxygenase Hmx1 and the potential miRNA mechanisms in the kidney injuries induced by urinary tract infection by Candida species/Candidemia. Main methods: We employed a mouse model of systemic Candidiasis by injection of the Candida albicans strain SC5314 into C57BL/6 mice. Kidney injuries were assessed by measuring serum cystatin C (CysC), serum β2-microglobulin (β2-MG) and blood urea nitrogen (BUN). Validation of miRNA target gene was conducted by luciferase reporter gene assay, Western blot analysis and real-time RT-PCR. Key findings: We showed here that Candidemia caused significant downregulation of microRNAs miR-204 and miR-211. In sharp contrast, Hmx1 expression was remarkably upregulated, particularly at the protein level. Computational analysis predicted Hmx1 as a target gene for both miR-204 and miR-211 that share the same seed site sequence. We then experimentally validated the targeting relationship between miR-204/miR-211 and Hmx1, which explains the reciprocal changes of expression of miR-204/miR-211 and Hmx1 in Candidemia. Administration of miR-204/miR-211 mimics substantially downregulated Hmx1 and mitigated the severity of the kidney injuries induced by Candidemia, as reflected by improved renal glomerular filtration rate (GFR) determined by serum cystatin C (CysC), serum β2-microglobulin (β2-MG) and blood urea nitrogen (BUN). Knockdown of miR-204/miR-211 worsened while forced expression of miR-204/miR-211 ameliorated kidney injuries in mice with systemic Candidiasis. Significance: Our findings indicate that miR-204/miR-211 downregulation accounts at least partially for the Hmx1 upregulation and the miR-204/miR-211–Hmx1 signaling axis may contribute to immune-suppression in the host thereby the Candidemia-induced kidney dysfunction. © 2014 Elsevier Inc. All rights reserved.
Introduction Urinary tract infection (UTI) by Candida species or Candidemia is unusual in healthy individuals, but common in patients with predisposing diseases and structural abnormalities of the kidney and collecting system (Fisher et al., 2011; Serefhanoglu et al., 2012; Sychev et al., 2009; Turner et al., 1985). Bloodstream infection with Candida species is a severe invasive fungal infection with high mortality, representing the fourth most common nosocomial blood-borne infection in immunocompromised patients. Candida albicans (C. albicans) is the leading cause of fungal UTI among various Candida species, which utilizes a repertoire of virulence factors to colonize tissue and disseminate. These events run in concert with altered expression of varying factors at different stages during a Candida infection. There is an urgent need to identify
⁎ Corresponding author. Tel.: +86 769 22222365. E-mail address: [email protected] (L.-H. Cai). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.lfs.2014.03.010 0024-3205/© 2014 Elsevier Inc. All rights reserved.
host factors that predispose to Candidemia in order to establish a welldefined predictive profile for patients at risk for developing infection. As the first step for the infection, Candida species are recognized by pattern recognition receptors such as Toll-like receptors (TLRs) (Plantinga et al., 2012). Activation of TLRs allows the fungi to adhere to endothelial or urothelial cells and colonizes the local area, afterwards evading the immune response and ultimately invading tissue or disseminating to distant sites within the body. Deficiency in the host immune system is the major predisposing factor to Candida infection (Fisher et al., 2011). The pathogenic yeast C. albicans encodes a heme oxygenase\Hmx1 that plays an important role in immune regulation by producing immunosuppressive carbon monoxide (CO) (Scapagnini et al., 2002; Otterbein et al., 2000; Overhaus et al., 2006). Hmx1 is specifically induced by the host protein hemoglobin after C. albicans infection, and is involved in the pathogenesis of disseminated bloodstream infections (Navarathna & Roberts, 2010; Kim et al., 2006; Pendrak et al., 2004). It has been shown that exposing mice to therapeutic levels of CO increases C. albicans virulence, whereas an Hmx1-null strain decreases virulence in murine disseminated Candidiasis (Navarathna &
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Roberts, 2010). Additionally, levels of several regulatory cytokines and chemokines are decreased in mice infected with the Hmx1-null strain, and initial lesions in the kidney are more rapidly cleared after polymorphonuclear leukocyte infiltration. In contrast, inhaled CO partially reverses the virulence defect of the null strain and restores several host cytokine responses to wild-type levels (Navarathna & Roberts, 2010). However, the precise mechanisms for how Hmx1 modulates C. albicans virulence remained poorly understood. Although C. albicans infect multiple organs, the kidney is the most heavily colonized organ, which can cause some loss of renal function (Leunk & Moon, 1979; Spellberg et al., 2005). It is known that in progressive renal infection initiated by the virulent strain, the fungus proliferated in the kidneys, accompanied by a massive transcriptional change, mostly genes associated with innate and adaptive immune responses, such as Hmx1. It was reported that glibenclamide improves kidney and heart structure and function partially by decreasing the expression of HO-1 (Diwan et al., 2014). On the other hand, adenine diet induced the characteristics of human chronic kidney disease accompanied by increased HO-1 expression (Diwan et al., 2013). Recently, a class of non-protein-coding mRNAs known as microRNAs (miRNAs) has emerged as a central player in fine-tune regulation of protein-coding genes. These 22–26 nucleotides molecules primarily repress the expression of protein-coding genes at the post-transcriptional level by complementarity of its 5′-end 2–8 nucleotides to the matched motifs in the 3′-untranslated region (3′UTR) of mRNAs. Intriguingly, it has been documented that either C. albicans infection or bacterial cellsurface lipopolysaccharide (LPS or endotoxin) treatment can cause upregulation of miRNAs such as miR-146, miR-155, miR-455 and miR125a in macrophages (Monk et al., 2010). This effect may play a role in inflammatory signaling tailoring the macrophage response to the appropriate pathogen. In light of these findings, we thought that miRNAs might be involved in the expression regulation of Hmx1 in kidney with C. albicans infection. This thought urged us to carry out preliminary analyses to search for the miRNAs that have the potential to target Hmx1. Utilizing computational prediction algorithms, we identified miR-204 and miR-211 that share the same seed site sequence and 91% overall sequence homology as candidate regulators of Hmx1 gene. This initial finding prompted us to set up the present study to experimentally validate the targeting relationship between miR-204/miR211 and Hmx1, and to exploit the role of this regulation in the kidney injuries induced by Candidemia. Materials & methods Animals C57BL/6 mice purchased from Charles River Laboratories (Wilmington, MA, USA) were used for the experiments. The animals received balanced diet and water ad libitum. All procedures involving animals were carried out in accordance with the guidelines set by and approved by the Institutional Animal Care and Use Committee of the Dongguan People's Hospital. Induction of systemic candidiasis The C. albicans strain SC5314 was obtained from a fungal suspension as described elsewhere (Venturini et al., 2009) and cultivated in YPD (1% yeast extract, 1% peptone, 2% dextrose) broth with overnight shaking at 30 °C. Yeast cells were washed with phosphate buffered saline (PBS) and resuspended in PBS at the desired concentration. Mice were inoculated through the lateral tail vein with a designated number of yeast colony-forming units (CFUs) via the retro-orbital venous plexus (FragaSilva et al., 2013 Mar 25). To confirm the fungal dose injected into mice, 100 ml of the yeast cell suspension was plated on YPD agar plates, and the number of CFU was determined after 24 h of incubation at 30 °C. The fungal concentration was adjusted to 5.0 × 105 viable C. albicans/ml. This model is commonly used for studying urinary tract infection and its
associated kidney injuries (Venturini et al., 2009; Fraga-Silva et al., 2013 Mar 25). Our pilot experiments had demonstrated that this inoculum size is not lethal in C57BL/6 mice and it reproduces a systemic fungal infection. Determination of fungal burden Mice were sacrificed and kidneys were removed aseptically at necropsy and washed with sterile PBS. The preparations were then weighted and placed in 1.5 ml sterile tissue lysis buffer (200 mM NaCl, 5 mM EDTA, 10 mM Tris, 10% glycerol, 1× protease inhibitor cocktail) (Roche Applied Science, Upper Bavaria, Germany) on ice. The samples were aseptically homogenized using a homogenizer and serial dilutions of homogenates were plated in triplicate on YPD plates containing ampicillin, tetracycline, and chloramphenicol. Colonies were counted after 1, 3 and 7 days of incubation at 30 °C. The fungal burden was calculated as colony forming units (CFUs) per gram of tissue. Measurement of glomerular filtration rate (GFR) To assess kidney tissue damage, glomerular filtration rate (GFR) was measured on day 7 after infection. For this purpose, serum cystatin C (CysC) and β2-microglobulin (Grubb, 2000; Herrero-Morín et al., 2007) were measured using RANDOX enzymatic creatinine assay (Randox Laboratories Limited, Crumlin, County Antrim, UK) with calibration traceable to the international standard reference material as recently recommended by the National Kidney Foundation (KDIGO 2012, Kidney International 2013). Additionally, blood urea nitrogen (BUN) levels were determined in serum samples by a routine veterinarian diagnostics laboratory (InVitro GmbH). Preparation of miRNA-mimics, antisense inhibitors, decoy oligodeoxynucleotides miR-204 and miR-211 mimics, mutant miR-204 (5′-UUaagaUUGU CAUCCUAUGCCU-3′), mutant miR-211 (5′-UUaagaUUGUCAUCCUUC GCCU-3′), antimiR-204 (antisense inhibitor of miR-204: 5′-AGGCAU AGGAUGACAAAGGGAA-3′), and mutant antimiR-204 (5′-AGGCAUAG GAUGACAAcuuuAA-3′) were all synthesized by Invitrogen (Carlsbad, CA, USA). Hmx1 siRNA was obtained from Santa Cruz Biotech (Cat#: sc-75269; Santa Cruz, CA, USA), and scrambled siRNA (5′-AACAGTCG CGTTTGCGACTGG-3′) that does not match any known mammalian GenBank sequence was synthesized by Invitrogen. For in vivo applications, all these above constructs were modified with locked nucleic acid (LNA) to acquire long-standing stability in the cells (Elmén et al., 2008a; Elmén et al., 2008b). These LNA constructs were injected via tail vein into mice at a dosage of 50 ng/ml once a day for 4 days prior to C. albicans infection. mRNA and miRNA quantification Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA). Both mRNA and miRNA levels were determined using the TaqMan quantitative real-time PCR (qRT-PCR) method (10 ng/assay) on an ABI 7500 Real-Time PCR System (Applied Biosystems, San Mateo County, CA, USA). Taqman Primer assays and the reagents for reverse transcriptase and qRT-PCR reactions were all provided by Applied Biosystems. Relative expression levels were calculated using the comparative cycle threshold (Ct) method (2−ΔΔCt) with mRNA levels normalized to GAPDH and miRNA levels to U6 RNA. Western blot analysis The isolated total protein samples (20 μg) were loaded to SDS-PAGE and then transferred to the membrane. The membrane was incubated with a 1:1000 dilution of primary antibody, followed by a 1:5000 dilution of horseradish peroxidase-conjugated secondary antibody. Rabbit
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polyclonal anti-Hmx1 (Abcam, Cambridge, MA, USA; Cat# ab28733) was used as the primary antibody. Rabbit anti-IgG (Alexa Fluor 350; Life Technologies, Carlsbad, CA, USA) was used as the secondary antibody. The intensity (area × density) of immunoblot bands was measured by densitometry (model GS-800, Imaging Densitometer; Bio-Rad). Relative protein levels were calculated by normalizing to GAPDH detected by anti-GAPDH antibody (Life Technologies). Immunochemistry for Hmx1 Animals were perfused with heparinized saline solution-4% buffered formalin. Kidneys were removed, halved longitudinally. Sections of 3-μm thick were cut and mounted on glass slides for immunohistochemical studies. The primary antibody was rabbit polyclonal anti-Hmx1 (Abcam, Cambridge, MA, USA; Cat# ab28733). Following washes in phosphate-buffered saline, sections were incubated with DAKO LSAB2 system + HRP (DAKO), and 3,3′-diaminobenzidine (BioGenex, San Ramon, CA, USA) was used as chromogen. Luciferase reporter gene assay The 3′UTR of Hmx1 was PCR synthesized and the fragment was subcloned into the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Beijing, China). PCR-based mutagenesis was made to the putative binding site for miR-204/miR-211 by base replacement to the site motif at the 3′UTR of Hmx1 mRNA (underlined lower case letters: 628-UUGCGUAUGGAAUAAAAcattcC-651) and was also cloned in the luciferase vector. HEK293 human embryonic kidney cell line (ATCC) in culture was cotransfected with the luciferase vector and varying constructs including miR-204 or miR-211 mimic, mutant miR-204 or miR-211, antimiR204, mutant antimiR-203, Hmx1 siRNA, or scrambled siRNA. The cells were cultured in DMEM supplemented with 10% fetal bovine serum. Prior to transfection, cells were starved to synchronize growth by incubating in serum- and antibiotics-free medium for 12 h. Forty-eight hours after transfection, luciferase activities were measured using the Dual Luciferase Reporter® Assay System (Promega) on a Luminoskan ™ Ascent Microplate Luminometer (Thermo Fisher Scientific, Waltham, MA, USA). Statistical methods Values are reported as the mean ± SEM with sample sizes indicated in each legend. Statistical comparisons among multiple groups were performed with one-way ANOVA, and intergroup differences were tested with Bonferroni inequality if one-way ANOVA yielded significance. All other results were analyzed by using a two-tailed Student's t-test between two groups. Significance was accepted at p-value b 0.05. All statistical analyses were performed using SPSS18.0 analysis software. Receiver operating characteristic (ROC) analysis was performed with Sensitivity plotted against 1-Specificity. Area under ROC curve (AUC) was estimated to assess the diagnostic accuracy.
Fig. 1. Reciprocal changes of expression of miR-204/miR-211 and Hmx1 protein in renal tissues of mice with systemic Candidiasis. (a) Downregulation of miR-204 and miR-211 in Candidiasis relative to healthy control mice, measured on day 7 after C. albicans infection using TaqMan real-time RT-PCR. (b) Upregulation of Hmx1 protein in Candidiasis relative to healthy control mice, measured on day 7 after C. albicans infection using Western blot analysis. (c) Comparison of Hmx1 mRNA levels between Candidiasis and healthy control mice. ***p b 0.001 Candidiasis vs healthy control, n = 12 for each group. Histological evaluation of C. albicans infection was performed on day 7 post-infection. Histological sections of kidney from the indicated mice were stained with H&E (upper panel) or PAS (lower panel). Kidney structure of C. albicans-infected mice was disturbed by infiltrating inflammatory cells (blue triangle) and red blood cells (white triangle). IL-17+ MSC infusion showed significant rescue of the kidney structures compared to bulk MSC and IL-17− MSC groups, as assessed by H&E staining. Infected mice showed the infiltration of C. albicans clusters (yellow triangle) in kidney, while the IL-17+ MSC infusion group showed a significantly more effective clearance of C. albicans infiltration compared to bulk and IL-17− MSC groups, as viewed by PAS staining. *p b 0.05; ** p b 0.01; and ***p b 0.001 (mean ± SD). Scale bars, 50 μm. The data are representative of three independent experiments.
On the other hand, Hmx1 was found robustly upregulated at both protein and mRNA levels despite that the changes were markedly greater for protein than for mRNA (Fig. 1b & c).
Results Hmx1 as a target gene for miR-204 and miR-211 Downregulation of miR-204 and miR-211 and upregulation of Hmx1 by Candidemia Since our preliminary analysis indicates miR-204 and miR-211 as candidate regulators of Hmx1 gene, we focused our experiments on the changes of expression of miR-204 and miR-211. As shown in Fig. 1a, in the mice with induction of systemic Candidiasis, miR-204 and miR-211 transcript levels in renal tissue were both found considerably decreased relative to healthy animals. As a negative control, miR210 was unaltered in its expression.
Our computational analysis using the Targetscan algorithm (http://www.targetscan.org/cgi-bin/vert_61/view_genetable.cgi? rs=NM_018942&taxid=9606&members=&showcnc=1&shownc= 1&sortText=cs) predicted Hmx1 as a target gene for both miR-204 and miR-211 that share the same seed site sequence, which is highly conserved across species (Fig. 2). To experimentally clarify if Hmx1 is indeed a cognate target for miR204/miR-211, we first carried out the luciferase activity assay in HEK293 cells. As shown in Fig. 3a, transfection with miR-204 or miR-211
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UUG GCG GUA AUG GGA AAU UAA AAA AAG GGG GAC C Po ositiion 62 28-634 4o of H HMX X1 3' UT TR 5'-U ||| ||||||| 3 3'-UC CCG GCU UUC CCU UAC CUG GUU UUC CCC CUU U hsa a-m miR R-21 11 UUG GCG GUA AUG GGA AAU UAA AAA AAG GGG GAC C Po ositiion 62 28-634 4o of H HMX X1 3' UT TR 5'-U ||| ||||||| 3 3'-UC CCG GUA AUC CCU UAC CUG GUU UUC CCC CUU U hsa a-m miR R-20 04
Fig. 2. Bioinformatics prediction of Hmx1 as a candidate target gene for miR-204 and miR211 based on the results from Targetscan miRNA database (http://www.targetscan.org). (a) Sequence alignment between miR-204/miR-211 and the 3′UTR of Hmx1 mRNA. Note the full complementarity between the seed sites of miR-204/miR-211 and the fragment containing the putative binding site for these miRNAs. (b) Sequence conservation across various species.
significantly diminished luciferase activity elicited by the luciferase vector carrying the complementary sequence for these miRNAs in the 3′ UTR of Hmx1 mRNA. The magnitude of decreases produced by miR204 or miR-211 was diminished by co-transfection with antimiR-204, but not by anti-miR-204 mutant. miR-204 or miR-211 mutant did not significantly affect the luciferase activities. Injection of LNA-miR-204 or miR-211 significantly reduced the renal protein level of Hmx1 in both healthy control (data not shown) and Candidiasis mice (Fig. 3b). On the contrary, injection of LNA-antimiR204 slightly increased Hmx1 protein level, indicating a relief of basal repression of Hmx1 by endogenous miR-204/miR-211. None of the mutant constructs produced any statistically significant changes of Hmx1 protein level. The renal mRNA level of Hmx1 was not significantly altered, although LNA-miR-204 or miR-211 tended to downregulate while LNAantimiR-204 tended to upregulate Hmx1 mRNA expression (Fig. 3c). The efficacy of the antimiR-204 to knockdown both miR-204 and miR-211 was confirmed by our results showing the 86.3 ± 7.5% (p b 0.001, n = 12) decrease in miR-204 level and 78.8 ± 6.9% (p b 0.001, n = 12) in renal tissues. miR-204/miR-211 relieves kidney injuries induced by Candidemia The fungal burden in mice with Candidemia was calculated as colony forming unit (CFU) per gram of renal tissue. The results in Fig. 4a demonstrate that LNA-miR-204 or LNA-miR-211 moderately but significantly decreased CFU in Candidemia mice, whereas LNA-antimiR-204 increased it. By comparison, the mutant constructs did not have significant effects on CFU. To connect these changes to kidney injuries, we compared the changes of the renal glomerular filtration rate (GFR) by measuring serum cystatin C (CysC; Fig. 4b), serum β2-microglobulin (Fig. 4b) and blood urea nitrogen (BUN; Fig. 4c) in the presence of varying constructs. Expectedly, all these measurements were enormously higher in Candidemia mice than in healthy control animals, indicating severe
Fig. 3. Validation of Hmx1 as a cognate target gene for miR-204 and miR-211. (a) Luciferase reporter gene assay for the direct repressive effects of miR-204 and miR-211 on Hmx1 in HEK293 cells. Note that the luciferase activities induced by the vector carrying the 3′UTR of Hmx1 were substantially repressed by transfection with either miR-204 or miR-211. **p b 0.01 miRNA vs control, +p b 0.05 and ++p b 0.01 antimiR vs miRNA; n = 5 for each group. (b) Effects of LNA-miR-204/LNA-miR-211 and LNA-antimiR-204 on Hmx1 protein level on day 7 after C. albicans infection. Lane 1: control; lane 2: miR-204; lane 3: mutant miR-204; lane 4: antimiR-204; lane 5: mutant antimiR-204. *p b 0.05 miRNA vs control, n = 12 for each group. (c) Lack of significant effects of LNA-miR-204/LNA-miR-211 and LNA-antimiR-204 on Hmx1 mRNA level on day 7 post C. albicans infection.
kidney injuries in C. albicans-infected animals. LNA-miR-204 or LNAmiR-211 significantly restored the increased CysC, β2-microglobulin and BUN in Candidemia mice, whereas LNA-antimiR-204 worsened the adverse changes. In healthy mice, LNA-antimiR-204 decreased all three measurements. By comparison, the mutant constructs did not have significant effects on any of these measurements. These results suggest that knockdown of miR-204 and miR-211 by LNA-antimiR204 caused kidney injuries while forced expression of these miRNAs by LNA-miR-204 or LNA-miR-211 administration produced protective effects against kidney injuries. We have shown that downregulation of miR-204/miR-211 is accompanied by upregulation of Hmx1 in Candidemia mice (Fig. 1) and Hmx1 is a cognate target for miR-204/ miR-211 (Fig. 3). It is conceivable that Hmx1 mediates deleterious effects of miR-204/miR-211 downregulation on kidney function. If this is true, then knockdown of Hmx1 itself should be able to relieve kidney injuries. Our experiments using Hmx1 siRNA indeed provided the evidence in support of this notion (Fig. 4a–d).
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Fig. 4. Knockdown of miR-204/miR-211 worsens and forced expression of miR-204/miR-211 ameliorates kidney injuries in mice with systemic Candidiasis. (a) Effects of miR-204/miR-211 on the fungal burden in mice with Candidemia, which was assessed as colony forming unit (CFU) per gram of renal tissue. Measurements were performed on days 1, 3 and 7 after C. albicans injection. Significant differences (p b 0.05) were found for LNA-miR-204, LNA-miR-211, LNA-antimiR-204, and Hmx1 siRNA at all three time points. (b)–(d) Effects of various constructs on renal glomerular filtration rate (GFR) as reflected by serum cystatin C (CysC), serum β2-microglobulin (β2-MG) and blood urea nitrogen (BUN). Note that LNA-miR-204 and LNA-miR-211 restored the damaged GFR, and so did Hmx1 siRNA; in contrast, LNA-antimiR-204 worsened the damage. ***p b 0.001 Candidiasis vs healthy control, +p b 0.05 & p b 0.001 vs Candidiasis; n = 12 for each group. (e) Immunostaining of Hmx1 (brown) in kidney sections verifying the presence of Hmx1 and the changes of its expression by Candidemia and antimiR-204 in kidney. Similar results were consistently observed in another three animals for each group.
We then further verified the presence of Hmx1 and the changes of its expression in kidney with immunochemistry. As shown in Fig. 4e, Hmx1 was substantially expressed in Candidemia, which was otherwise minimal in healthy controls. LNA-antimiR-204 eliminated the Hmx1 signal whereas the mutant antimiR failed to do so. Discussion Although Candidemia is a relatively unusual event in clinical practice, it is indeed a severe invasive fungal infection with high mortality (Fisher et al., 2011; Serefhanoglu et al., 2012; Sychev et al., 2009; Turner et al., 1985) and previous studies have documented that delays in treatment are associated with increased mortality in Candidemia patients (Taur et al., 2010). Because Candidemia is a rare complication, there is uncertainty about its optimal medical management. Therefore, there is a necessity to better understand host factors that predispose to Candidemia in order to establish a well-defined treatment. Here we have provided experimental observations on the roles of miRNAs miR-
204/miR-211 and Hmx1 which allow us to get some insight into the issue. First, we showed an inverse relationship between miR-204/miR211 and Hmx1 in terms of their expression levels in a mouse model of Candidemia: downregulation of miR-204 and miR-211 and upregulation of Hmx1. Second, we established Hmx1 as a cognate target gene for miR-204 and miR-211, which explains the reciprocal changes of expression of miR-204/miR-211 and Hmx1 in Candidemia. Third, miR204/miR-211 relieves kidney injuries induced by Candidemia: knockdown of miR-204/miR-211 worsened while forced expression of miR-204/miR-211 ameliorated kidney injuries in mice with systemic Candidiasis. These findings allow us to reach a conclusion that the miR-204/miR-211–Hmx1 signaling axis may contribute to the development of Candidemia and the associated kidney injuries and interventions with miR-204/miR-211 may be an approach for the management of Candidemia. Especially, our study represents the first to identify the role of miRNAs in this particular pathogenesis. Prior studies have shown that Hmx1 is critically involved in the pathogenesis of disseminated bloodstream infection by C. albicans
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(Navarathna & Roberts, 2010; Kim et al., 2006; Pendrak et al., 2004). Exposing mice to therapeutic levels of CO increases C. albicans virulence, suggesting Hmx1 as a host responsive and promoting factor for C. albicans infection (Navarathna & Roberts, 2010). Furthermore, evidence existing indicate that Hmx1 exaggerates kidney lesions (Navarathna & Roberts, 2010). However, the precise mechanisms for how Hmx1 is dysregulated by C. albicans infection to produce these deleterious effects remained incompletely understood. The present study sheds light on this issue. We first showed the upregulation of renal Hmx1 expression in response to C. albicans infection. We then demonstrated detrimental actions of Hmx1 kidney injuries in mice with Candidemia by showing the restoration of damaged kidney function by knocking down Hmx1 using siRNA. Our findings strongly suggest that Hmx1 is likely involved in the development of kidney injuries in Candidemia. We further unraveled that downregulation of miR-204 and miR-211 could lead to Hmx1 upregulation as a result of relief of basal repression of the gene. Recently, miRNAs have emerged as central regulators of human disease including kidney disease (Aguado-Fraile et al., 2013; Hou & Zhao, 2013; Gomez et al., 2013) and infection disease (Fritz et al., 2006; Martinez-Nunez et al., 2009). They act via targeting the proteincoding genes relevant to the specific pathological entities. In this study, we found that miR-204 and miR-211 that share identical seed site were both significantly downregulated in renal tissue from Candidemia mice relative to healthy control mice. This downregulation appears to be detrimental to kidney function as artificial knockdown of these miRNAs using their antisense inhibitor worsened the kidney injuries as reflected by the increased CFU, CysC, β2-MG and BUN, and administration of exogenous miR-204/miR-211 was able to efficiently restore and ameliorate the damaged kidney in Candidemia mice. This downregulation also likely accounts, at least partially, for the upregulation of Hmx1 as we have verified Hmx1 as a target gene for these two miRNAs. Artificial knockdown of miR-204/miR-211 increased, whereas forced expression of these two miRNAs decreased Hmx1 protein levels in mice with Candidemia. The fact that Hmx1 expression was upregulated to a significantly greater degree at the protein level than at its mRNA level indicates that in addition to the transcriptional activation by the host protein hemoglobin Hmx1 can be further upregulated via posttranscriptional derepression from miRNAs. It is known that miRNAs primarily act to inhibit protein translation with or without affecting the mRNA stability of their target genes, depending on whether any of the 5′-end 10–12 nucleotides is complementary to the sequence motif in the 3′-untranslated region of their target genes (mRNAs). It is noticed that there is a discrepancy between Hmx1 protein and mRNA expression, miR-204 and miR-211 significantly repressed Hmx1 protein without altering Hmx1 mRNA (Fig. 3b & c). This discrepancy reflects the canonical action of miRNAs: inhibition of protein translation. In our case, neither miR-204 nor miR-211 has complementarity to Hmx1 mRNA in the positions of 5′-end 10–12 nucleotides, and thus, neither of them produced significant changes of Hmx1 mRNA. Taking together previous results and the present findings, it is quite possible that when infected by C. albicans, the Hmx1 gene encoded by this pathogenic yeast is transcriptionally activated by the host protein hemoglobin. Meanwhile miR-204 and miR-211 are somehow downregulated and this downregulation further upregulates Hmx1. Hmx1 then produces detrimental effects on kidney function. Thus, our study identified the new miR-204/miR-211–Hmx1 axis in renal injuries induced by C. albicans infection. However, our study failed to elucidate the downstream components mediating the miR-204/miR-211–Hmx1 signaling and the upstream mechanisms for the downregulation of these two miRNAs. Additionally, the present study focused on miR-204 and miR211 and could not exclude the possible involvement of other miRNAs in the regulation of Hmx1 expression in the setting of Candidemia. Conflict of interest None.
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