Most chronic kidney injuries inevitably progress to irreversible renal fibrosis. Tubular epithelial-to-mesenchymal transition (EMT) is recognized to play pivotal roles in the process of renal fibrosis. However, a comprehensive understanding of the pathogenesis of renal scar formation and progression remains an urgent task for renal researchers. The endogenously produced microRNAs (miRNAs), proved to play important roles in gene regulation, probably regulate most genes involved in EMT. In this study, we applied microarray analysis to investigate the expression profiles of miRNA in murine interstitial fibrotic kidneys induced by unilateral ureteral obstruction (UUO). It was found that miR-200a and miR-141, two members of the miR-200 family, were downregulated at the early phase of UUO. In TGF-β1-induced tubular EMT in vitro, it was also found that the members of the miR-200 family were downregulated in a Smad signaling-dependent manner. It was demonstrated that the miR-200 family was responsible for protecting tubular epithelial cells from mesenchymal transition by target suppression of zinc finger E-box-binding homeobox (ZEB) 1 and ZEB2, which are E-cadherin transcriptional repressors. The results suggest that downregulation of the miR-200 family initiates the dedifferentiation of renal tubules and progression of renal fibrosis, which might provide important targets for novel therapeutic strategies.
- renal fibrosis
despite various primary diseases, most kidney injuries inevitably progress to irreversible renal fibrosis (21, 33). A comprehensive understanding of the pathogenesis of renal scar formation and progression remains an urgent task for renal researchers. Tubular epithelial-to-mesenchymal transition (EMT) is recognized to play critical roles in renal fibrosis (12, 20, 27). There has been a growing amount of evidence that many genes are involved in tubular EMT (18, 19, 28, 31, 32). However, the underlying molecular mechanisms of specific phenotypic conversion, especially the key regulator that triggers the cascade of progression toward mesenchymal transition, remain to be determined.
MicroRNAs (miRNAs) are a family of short, noncoding RNAs ∼22 nucleotides in length. These endogenously produced transcripts have been proved to play important roles in gene regulation. It is reported that up to 30% of mammalian protein-encoding genes are the targets of miRNAs (1, 4, 10, 11, 13–16, 25, 26). Given the complexity of tubular EMT, it is conceivable that miRNAs probably regulated most genes involved in EMT. Recent investigations demonstrated that the miR-200 family regulated cancer cell EMT and migration (3, 9, 17, 22, 23). We also found that miR-200a and miR-141, two members of the miR-200 family, were downregulated at the early phase of tubular interstitial fibrosis induced by unilateral ureteral obstruction (UUO), which was in accordance with recent findings demonstrated that miR-200a, miR-200b, and miR-141 were downregulated in day 7 UUO kidneys (7). The miR-200 family, composed of miR-200a, miR-200b, miR-200c, miR-141, and miR-429, was previously reported to posttranscriptionally repress zinc finger E-box-binding homeobox (ZEB) 1 and ZEB2, which are E-cadherin transcriptional repressors (9, 17, 22).
In this study, we found that the members of the miR-200 family were downregulated in transforming growth factor (TGF)-β1-induced tubular EMT in a Smad signaling-dependent manner. We demonstrated that the miR-200 family was responsible for protecting tubular epithelial cells from mesenchymal transition by targeted suppression of ZEB1 and ZEB2. Our results suggest that downregulation of the miR-200 family initiates the dedifferentiation of renal tubule and progression of renal fibrosis.
MATERIALS AND METHODS
Male Sprague-Dawley rats, weighing 180–200 g, and male CD1 mice, weighing 18–20 g, were obtained from the Shanghai Experimental Animal Center (Shanghai, China). They were housed in the animal facilities of the Experimental Animal Center at Nanjing Medical University with free access to food and water. Animals were treated humanely in accordance with NMAC guidelines and approved protocols of the Nanjing Medical University Institutional Animal Use and Care Committee. Animals were randomly assigned to five groups (with 5 mice/group): a sham control group and groups with UUO for 1, 3, 7, and 14 days. UUO was performed using an established procedure (30). Briefly, under general anesthesia, complete ureteral obstruction was performed by double ligation of the left ureter using 4-0 silk after a midline abdominal incision. Sham controls had their ureters exposed and manipulated but not ligated. Animals were euthanized after surgery at different time points as indicated, and the obstructed kidneys were harvested. Kidneys were snap-frozen in liquid nitrogen and stored at −80°C.
Cell culture and treatment.
Rat renal proximal tubule epithelial cells (NRK-52E) were acquired from the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Cells were cultured in DMEM supplemented with 10% fetal bovine serum (Invitrogen, San Diego, CA). For TGF-β1 treatment, NRK-52E cells were seeded at 80% confluence in complete medium containing 10% fetal bovine serum. Twenty-four hours later, the cells were changed to serum-free medium and incubated for 16 h thereafter, and TGF-β1 (Sigma) was added at a concentration of 5 ng/ml for various periods of time as indicated. The cells were then collected for further characterization.
The procedure was carried out as previously described (6). The complementary probes (in triplicate) against miRNAs were designed based on miRBase release 12.0. RNA labeling, microarray hybridization, and array scanning were performed as follows. Briefly, 25 μg of total RNA was used to isolate the low-molecular-weight RNA using polyethylene glycol solution precipitation. Subsequently, low-molecular-weight RNAs were labeled with Cy3 and hybridized with an miRNA microarray (CapitalBio, Beijing, China). Finally, hybridization signals were detected and quantified. Four independent adult amphioxus RNA samples were hybridized with an miRNA microarray separately. Hybridization intensity values from individual amphioxus samples were filtered, and the global median was normalized. We considered candidate miRNAs with a signal >3,000 and P < 0.001 from a Student's t-test (compared with blank spotting solution) to be positive.
Quantitative RT-PCR of mature miRNAs.
Assays to quantify the mature miRNAs were conducted as previously described with minor modification (5). The expression levels of target miRNAs were normalized to U6. Total RNA was prepared using a TRIzol RNA isolation system according to the instructions specified by the manufacturer (Invitrogen). The first strand of cDNA was synthesized using 1 μg of RNA in 20 μl of reaction buffer using miScript RT buffer (Qiagen). This allowed for creation of an miRNA cDNA library. The mix was incubated at 37°C for 60 min, and 95°C for 5 min. Subsequently, real-time quantification was performed using an Applied Biosystems 7300 Sequence Detection system. The 20-μl PCR reaction included 1 μl RT product, 2 μl 10× miScript Universal Primer, 2 μl 10× miScript Primer Assay, 10 μl 2× QuantiTect SYBR Green PCR Master Mix, and RNase-free water (Qiagen). The reaction was incubated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. All reactions were run in triplicate. After reactions, the CT data were determined using default threshold settings, and the mean CT was determined from the duplicate PCRs. The ratio of UUO miRNAs to sham control miRNA was calculated by using the equation 2−ΔCT, in which ΔCT = CT treatment − CT sham control. All the primers were acquired from Qiagen.
Total RNA was prepared using the TRIzol RNA isolation system according to the instructions specified by the manufacturer (Invitrogen). The first strand of cDNA was synthesized using 2 μg of RNA in 20 μl of reaction buffer using Moloney leukemia virus-RT (Promega, Madison, WI) and random primers at 42°C for 30 min. PCR was performed using a standard PCR kit on 1-μl aliquots of cDNA and HotStarTaq polymerase (Promega, Madison, WI) with specific primer pairs. The sequences of primer pairs were as follows: Smad7 (forward) 5′-CCA TCA AGG CTT TTG ACT ATG AAA-3′ and (reverse) 5′-CGG TGA AGC CCG TCC AT-3′; ZEB1 (forward) 5′-TGG GAA AGC GTT CAA GTA CAA A-3′ and (reverse) 5′-TTG GTT TAC AGA AAG CGG TTC TT-3′; ZEB2 (forward) 5′-AGC CAA GGA ATG CTA CCA A-3′ and (reverse) 5′-GGC CCC AGA GCA TCA TAA TC-3′; and β-actin (forward) 5′-CAG CTG AGA GGG AAA TCG TG-3′ and (reverse) 5′-CGT TGC CAA TAG TGA TGA CC-3′. The PCR products were size-fractionated on a 1.0% agarose gel and detected by NA-green (D0133, Beyotime) staining.
miR-200 family mimics or inhibitors and their negative control RNA (purchased from Qiagen), as well as ZEB1 and ZEB2 small interfering (si) RNA (Ambion), were transfected into NRK-52E cells using Liptofectamine 2000 reagent (Invitrogen) following the protocols provided by the manufacturer. After transfection, cells were incubated at 37°C in a CO2 incubator for 24–48 h until they were ready for gene expression assay or further treatment.
Smad7 expression plasmid and the empty expression plasmid vector pcDNA3 were obtained from Invitrogen. Plasmids were transfected into cells using Lipofectamine 2000 transfection reagent (Invitrogen) and the protocols provided by the manufacturer. After transfection, the cells were incubated at 37°C in a CO2 incubator for 24 h until they were ready for gene transfection assay and further treatment.
Western immunoblot analysis.
NRK-52E cells were lysed with SDS sample buffer (62.5 mM Tris·HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, and 0.1% bromophenol blue). Kidney tissues were homogenized with a polytron homogenizer (Brinkmann Instruments, Westbury, NY) in RIPA lysis buffer (1% NP-40, 0.1% SDS, 100 μg/ml phenylmethylsulfonyl fluoride, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 2 μg/ml aprotinin, 2 μg/ml antipain, and 2 μg/ml leupeptin in PBS) on ice. The supernatants were collected after centrifugation at 13,000 g at 4°C for 20 min. Protein concentration was determined using a BCA protein assay kit (Sigma), and whole-tissue lysates were mixed with an equal amount of 2× SDS loading buffer (125 mM Tris·HCl, 4% SDS, 20% glycerol, 100 mM DTT, and 0.2% bromophenol blue). Samples were heated at 100°C for ∼5–10 min before loading and were separated on precasted 10 or 5% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA). Detection of protein expression by Western blotting was completed according to the established protocols described previously (30). The primary antibodies used were as follows: anti-E-cadherin (610182, BD Transduction Laboratories), anti-α-smooth muscle actin (SMA; A5691, Sigma-Aldrich), anti-fibronectin (F3648, Sigma-Aldrich), anti-ZEB1 (SAB2500097, Sigma-Aldrich), anti-ZEB2 (SAB2102760, Sigma-Aldrich), and anti-actin (A5385, Sigma-Aldrich). Quantification was performed by measurement of the intensity of the signals with the use of National Institutes of Health image-analysis software.
Indirect immunofluorescence staining was performed according to an established procedure (30). Briefly, cells cultured on coverslips were washed twice with cold PBS and fixed with cold methanol/acetone (1:1) for 10 min at −20°C. Following three extensive washings with PBS, the cells were blocked with 0.1% Triton X-100 and 2% normal donkey serum in PBS buffer for 40 min at room temperature and then incubated with the specific primary antibodies described above, followed by staining with FITC- or TRITC-conjugated secondary antibody. Cells were double stained with 4,6-diamidino-2-phenylindole to visualize the nuclei. Slides were viewed with a Nikon Eclipse 80i Epi-fluorescence microscope equipped with a digital camera (DS-Ri1, Nikon). In each experimental setting, immunofluorescence images were captured with identical light exposure parameters.
miRNA in situ hybridization.
miRNA in situ hybridization (ISH) was performed using a mercury LNA microRNA ISH optimization kit (Exiqon) for formalin-fixed, paraffin-embedded kidney samples according to the protocol by the manufacturer. Briefly, 10-μm-thick sections were prepared, followed by deparaffinization in xylene and ethanol. The slides were incubated with 15 mg/ml of proteinase K (Exiqon) for 20 min at 37°C. After being washed and dehydrated, the slides were hybridized with a double digoxygenin (DIG)-labeled LNA miR-200 probe, LNA scrambled miRNA probe, LNA U6 snRNA probe, and a LNA miR-126 probe (positive control, Exiqon) for 1 h at 55°C. The slides were washed with SSC buffer and then incubated with blocking solution for 15 min, followed by incubation with anti-DIG reagent for 60 min, AP substrate for 2 h at 30°C, and KTBT buffer for 10 min. The slides were mounted with mounting medium, and analysis results were by light microscopy (Nikon Eclipse 80i).
Animals were randomly assigned to control and treatment groups. Statistical analysis was performed using SigmaStat software (Jandel Scientific Software, San Rafael, CA). Comparisons between groups were made using one-way ANOVA, followed by Student's t-test. P < 0.05 was considered significant.
miR-200 family members were downregulated during renal tubular EMT both in vivo and in vitro.
In this study, chronic renal diseases were induced by UUO. As shown in Fig. 1A, there are dramatic changes in renal tubular epithelial cell phenotypes after UUO, with loss of E-cadherin expression and de novo expression of α-SMA and upregulation of fibronectin. Microarray assays were performed to screen mature miRNA expression profiles in five groups of UUO and sham control kidney samples. After analysis, a list of as many as 45 miRNAs was generated, which were expressed differently in UUO samples compared with sham control samples. Among them, 26 miRNAs were downregulated (Fig. 1C), while 19 were upregulated (data not shown). To further illustrate the microarray results, Fig. 1C depicts the average fold-changes obtained with this method after being log2-transformed and normalized to sham controls. miRNAs included in this figure were those downregulated >50% (value less than −1 after log2-transformed and normalized to sham control), which could reflect a significant change in at least one time point after UUO. Among those downregulated miRNAs, miR-200a and miR-141, two members of the miR-200 family, were included. The miR-200 family consists of miR-200a, miR-200b, miR-200c, miR-141, and miR-429, which can be divided into two closely related subfamilies (Fig. 1D). There are several identical positions among all five family members including the seed sequences (Fig. 1D). To warrant the accuracy of the microarray assay, quantitative (q) RT-PCR was carried out using the same RNA preparations as in the microarray assay. The expressions of five members of the miR-200 family were all downregulated in the obstructed kidney, which were concordant with the results of microarray analysis (data not shown). ISH was applied to localize miR-200a in the kidney. As shown in Fig. 1E, miR-200a was expressed in proximal tubular cells of normal kidneys. The expression of miR-200a was markedly decreased in proximal tubules of obstructed kidneys after UUO (Fig. 1F). ISH assay using probes with a scrambled sequence (Fig. 1, G and H) suggested that the staining with miR-200a probes was specific for miR-200a. Similar results were obtained using probes specific for miR-200b, miR-200c, miR-141, and miR-429.
q-PCR was performed to investigate miR-200 family expression in rat renal tubular epithelial cells (NRK-52E) after TGF-β1 treatment. Figure 2, A–H, shows the phenotypic conversion of NRK-52E cells after 5 ng/ml of TGF-β1 incubation. The NRK-52E cells lost E-cadherin; however, they expressed an abundance of α-SMA and fibronectin. As shown in Fig. 2I, a time-dependent study revealed that all five members of the miR-200 family were significantly downregulated as early as 3 h of TGF-β1 treatment, which is in accordance with the results in vivo.
Downregulation of miR-200 family expression by TGF-β1 depends on the Smad signaling pathway.
We next investigated the possible signaling pathways involved in miR-200 family depression by TGF-β1 in NRK-52E cells. As revealed previously, TGF-β1 activates Smad2/3, p38 MAPK, Akt kinase, etc. However, pretreatment with specific pharmacological inhibitors of p38 MAPK and Akt kinase failed to reverse the downregulation of the miR-200 family by TGF-β1 (data not shown). To study the potential involvement of the Smad pathway, miR-200 family expression in NRK-52E cells overexpressing inhibitory Smad7 was examined. As shown in Fig. 3A, compared with pcDNA3, transfection of the Smad7 expression plasmid dramatically increased the Smad7 mRNA expression in NRK-52E cells, and therefore nearly totally blocked the TGF-β1-mediated miR-200 family depression. Therefore, downregulation of the miR-200 family by TGF-β1 probably depends on the intracellular Smad signaling pathway.
Ectopic expression of mir-200 family confers resistance to TGF-β1-induced renal tubular EMT in vitro.
A reverse strategy was adopted to investigate the important role of the miR-200 family in EMT by examining the responsiveness of renal tubular cells (NRK-52E) to TGF-β1 after forced expression of the exogenous miR-200 family. As shown in Fig. 4, ectopic expression of miR-200b by transfection of its mimics conferred resistance to TGF-β1-induced EMT in vitro, as demonstrated by suppression of E-cadherin, de novo induction of α-SMA, and expression of fibronectin. TGF-β1 (5 ng/ml) induced phenotypic conversion in control NRK-52E cells transfected with empty vector pcDNA3. However, the same concentration of TGF-β1 was unable to induce EMT in NRK-52E cells overexpressing miR-200b. Similar results were obtained when NRK-52E cells were transfected with miR-200a, miR200c, or miR-141 mimic. Thus ectopic expression of the miR-200 family can override TGF-β1-induced EMT in renal tubular epithelial cells.
Downregulation of miR-200 family induces tubular EMT in vitro.
To further confirm the important role of the miR-200 family in the process of EMT, the impacts of downregulated miR-200 family expression on tubular EMT by transfection of their inhibitors were tested. As shown in Fig. 5, compared with NRK-52E cells with negative control siRNA transfection, NRK-52E cells transfected with an miR-200b inhibitor displayed a phenotypic conversion, as demonstrated by loss of E-cadherin and induction of α-SMA and fibronectin, which is similar to TGF-β1 stimulation. Similar results were observed after miR-200a, miR-200c, or miR-141 inhibitor transfection. This suggests that downregulation of the miR-200 family enhances the phenotypic conversion of tubular epithelial to mesenchymal cells.
E-cadherin transcriptional repressors ZEB1 and ZEB2 are induced rapidly during TGF-β1-induced EMT.
ZEB1 and ZEB2 are E-cadherin transcriptional repressors, which are upregulated during the pathogenesis of cancer EMT (3, 9, 22). To investigate ZEB1 and ZEB2 expression during tubular EMT, we used the RT-PCR approach to identify whether ZEB1 and ZEB2 expression was altered in the initial stage of tubular EMT induced by TGF-β1. As shown in Fig. 6, A and B, mRNAs of ZEB1 and ZEB2 were induced more than 5- and 12-fold at 1 h in NRK-52E cells after treatment with 5 ng/ml TGF-β1, respectively. Western blot analysis confirmed the induction of ZEB1 and ZEB2 protein expression in NRK-52E cells that were treated with TGF-β1 (Fig. 6C).
miR-200 family targets ZEB1 and ZEB2.
By searching the TargetScan data base, several probable targets of the miR-200 family were identified, among which the E-cadherin transcriptional repressors ZEB1 and ZEB2 have already been proved by luciferase assay (17) in cancer cell lines. To verify the targeting of endogenous ZEB1 and ZEB2 by the miR-200 family in tubular epithelial cells, Western blot analysis of the protein expression levels of ZEB1 and ZEB2 in NRK-52E cells after transfection with miR-200 family mimics or inhibitors was performed. As shown in Fig. 7A, overexpression of each of the miR-200 family members (miR-200a, miR-200b, miR200c, and miR-141) by transfection of their mimics significantly suppressed the expression of ZEB1 and ZEB2. Meanwhile, the protein expression of ZEB1 and ZEB2 was significantly increased after downregulation of the miR-200 family by transfection of their inhibitors (Fig. 7C). Further RT-PCR analysis was conducted to examine the TGF-β1-induced changes in ZEB1 and ZEB2 mRNA expression after modulating the expression of the miR-200 family by transfection of their mimics or inhibitors. As shown in Fig. 8A, forced expression of the miR-200 family by transfection of their mimics markedly suppressed TGF-β1-induced ZEB1 and ZEB2 gene expression. Similarly, downregulation of the miR-200 family by transfecting their inhibitors significantly increased ZEB1 and ZEB2 gene expression (Fig. 8C). Thus we hypothesized that the miR-200 family regulated tubular EMT through reducing the expression of the transcriptional repressor of E-cadherin by targeting ZEB1 and ZEB2.
Downregulation of ZEB1 or ZEB2 blocks depression of mir-200 family-induced tubular EMT.
To assess whether ZEB1 or ZEB2 induction is necessary for miR-200 family depression-induced tubular EMT, Western blot analysis was carried out to detect the phenotypic conversion after downregulation of ZEB1 or ZEB2 by transfecting siRNA individually or simultaneously in NRK-52E cells. As shown in Fig. 9, A and B, specific siRNA transfection resulted in a considerable inhibition of ZEB1 and ZEB2 expression, respectively. Figure 9C shows the phenotypic conversion was demonstrated by loss of E-cadherin and expression of α-SMA in control or miR-200 family-depressed NRK-52E cells after transient transfection with negative control or ZEB siRNA. The miR-200 family was downregulated by transfection of its inhibitor. Transfection of ZEB siRNA partially restored E-cadherin expression and attenuated α-SMA induction after downregulation of the miR-200 family compared with negative control siRNA. Hence ZEB1 and ZEB2 seem to be required for mediating downregulation of miR-200 family-induced tubular EMT stimulated by TGF-β1 treatment.
Renal fibrosis is an inevitable outcome of most all progressive chronic kidney disease (21, 33). Tubular EMT, a series of highly regulated pathological events, has already emerged as one of the major mechanisms leading to renal fibrosis (12, 20, 27, 30). However, the underlying mechanism of EMT remains poorly understood. Recent studies suggested that all five members of the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) were markedly downregulated in breast cancer cells (9), each of which alone was sufficient to prevent EMT and cell migration. In addition, Chung et al. (7) recently demonstrated that downregulation of miR-200a, miR-200b, and miR-141, three members of the miR-200 family, was evident in day 7 UUO kidneys (7). In our microarray study of UUO kidneys, we found that miR-200a and miR-141 were significantly downregulated as early as day 1 and this persisted until day 14 after UUO (Fig. 1C). Meanwhile, q-PCR analysis revealed that the miR-200 family was also downregulated in TGF-β1-induced tubular EMT in vitro (Fig. 2I). Ectopic expression of the miR-200 family resisted TGF-β1-induced tubular EMT (Fig. 4). On the contrary, downregulation of the miR-200 family by RNA interference directly suppressed E-cadherin and induced α-SMA expression in renal tubular epithelial cells (Fig. 5). Therefore, the miR-200 family seems to be responsible for protecting tubular epithelial cells from dedifferentiation and mesenchymal transition.
TGF-β1 is a profibrotic agent in renal cells. To date, Smads have been implicated in many aspects of TGF-β1-induced EMT (2, 24, 29). A recent report suggested that the processing of primary transcripts of miR-21 into precursor miR-21 by the DROSHA complex was enhanced by Smad protein phosphorylation induced by TGF-β1 action (8). Our observations clearly established that inhibition of the Smad signaling pathway by transfection of inhibitory Smad7 completely blocked the TGF-β1-mediated miR-200 family downregulation. Therefore, the TGF-β1-induced EMT by suppression of the miR-200 family seems to act via the Smad signaling pathway.
Target screening and previous luciferase assays have linked the miR-200 family with ZEB1 and ZEB2 (22). Expression of ZEB1 and ZEB2 has been found to be negatively correlated with the miR-200 family during embryonic development (3, 17). In addition, our study found that ZEB1 and ZEB2 were implicated in the initial stage of tubular EMT (Fig. 6). Alteration of miR-200 family expression by RNA transfection demonstrated that mRNA and protein expression of ZEB1 and ZEB2 of tubular epithelial cells were negatively regulated by the miR-200 family (Figs. 7 and 8). Repression of ZEB by miR-200 family member regulation of EMT has been previously demonstrated in cancer cells (3, 17, 22, 23). A recent investigation also demonstrated that the miR-200 family regulated EMT of renal distal tubular cells by targeting ZEB1 (9). In this study, it suggests that the miR-200 family, through directly targeting of ZEB1 and ZEB2, play an essential role in renal proximal tubular EMT.
In our present study, we found that ectopic expression of the miR-200 family hindered progression of EMT in TGF-β1-treated NRK-52E cells by downregulation of ZEB1 and ZEB2 expression, resulting in maintaining a high expression level of E-cadherin. Directly keeping low expression levels of ZEB1 and ZEB2 by RNA interference blocked the downregulation of miR-200 family cluster-induced tubular EMT (Fig. 9). Since ZEB1 and ZEB2 have been implicated in the progression of EMT by suppression of E-cadherin expression, they might provide important targets for therapeutic strategies.
Our findings in this study suggested that members of the miR-200 family were downregulated during the cascade of destructive events of renal tubular dilation, degeneration, and fibrosis in obstructed kidneys. This not only reveals a close correlation between miR-200 family expression level and epithelial phenotypic conversion but also implicates a potential role of the miR-200 family in the regulation of tubular dedifferentiation, which is a common pathogenesis in tubular interstitial fibrosis in many types of chronic kidney disease. Evidently, more studies are needed to further characterize the role of the miR-200 family in the pathogenesis of EMT as well as to depict detailed molecular pathways that are involved in EMT and kidney fibrosis after chronic renal injury.
This work was supported by National Science Foundation of China Grants 30470800/30771010/30871201, the “973” Science Program of the Ministry of Science and Technology, China (2006CB503909), and Jiangsu Province's Outstanding Medical Academic Leader Program (SWKJ 2006-50) to J. W. Yang.
No conflicts of interest, financial or otherwise, are declared by the authors.
Author contributions: M.X., L.J., W.Q., L.F., and P.W. performed experiments; M.X., L.J., Y.Z., R.T., and J.Y. analyzed data; Y.Z. and J.Y. prepared figures; Y.Z. drafted manuscript; R.T. and J.Y. interpreted results of experiments; J.Y. provided conception and design of research; J.Y. edited and revised manuscript; J.Y. approved final version of manuscript.
Part of this work was presented as an abstract at the annual meeting of the American Society of Nephrology in November 2009.
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