Podocyte depletion, one of the common phenomena in chronic kidney diseases (CKDs) such as focal segmental glomerulosclerosis (FSGS), diabetic nephropathy (DN), membranous glomerulopathy, and lupus nephritis, may be attributed to the defects of the podocytes, glomerular basement membrane (GBM), endothelial cells, and/or negatively charged proteins present on the three layers [1
]. Podocytes are formed by interdigitating cellular extensions, which are bridged by slit diaphragms, which is similar to an adherence-like intercellular junction. Slit diaphragms of podocytes select the size of proteins that freely infiltrate the filtration barrier, exclusive water and small solutes. Recent studies have found that many molecular structures of the slit diaphragm have functional work in its integrity [2
], such as zonula occludens-1 (ZO-1), nephrin, and P-cadherin.
The injury of podocytes would destroy the structure and function of the slit diaphragms, and might undergo epithelial-to-mesenchymal transition (EMT) [5
], a reversal of embryogenesis presented to tubulointerstitial fibrosis and diabetic kidney. EMT induces three primary changes in cellular phenotype [6
]. Morphology of cells converts from cobblestone-like epithelial cells to spindle-shaped mesenchymal cells. Alteration of functions connects with the changes of settled cells to migratory cells. Marker gene expression varies, including loss of ZO-1 and E-cadherin as markers of epithelial cells, and upregulation of Collagen I and Fibronectin as markers for mesenchymal cells. Furthermore, markers of proteins switch from cytokeratin to vimentin. Moreover, EMT is considered to contribute to renal fibrogenesis and defined as a source of the phenotype and functional properties of transformation of epithelial cells into mesenchymal fibroblasts. Transforming growth factor beta (TGFβ) is an essential factor for EMT and fibrosis of tubular epithelial cells in Diabetic kidney disease [8
]. More interestingly, nuclear factor of activated T cells (NFAT) also plays an important role in EMT. NFATc2/c3/c4 activates EMT in the myocardium of embryonic mouse by repressing VEGF expression [9
]. Moreover, NFAT inducing activation of the fibronectin promoter and upregulation of fibronectin in podocytes, dependent on the calcium/calmodulin pathway and Rho kinase, finally, leads to podocyte injury and proteinuria [10
]. These results strongly support that EMT is a key step in embryogenesis and chronic kidney diseases, especially the podocytes in response to various injuries and diseases. However, increased efforts must be made to understand the latent mechanisms of EMT in podocyte injury.
MicroRNAs (miRNAs) are a large family of endogenous, single stranded, small non-coding RNAs with 18-25 nucleotides in length, which downregulate target gene expression by binding to the 3ʹ-untranslated region (3ʹUTR) of target mRNAs leading to mRNA degradation or translational inhibition. Multiple studies have demonstrated that miRNAs regulate most biological processes of cells, including proliferation, differentiation, senescence and apoptosis, and that severe diseases can be attributed to abnormal expression of miRNA. Recent studies indicated miRNAs can affect various gene expression in kidney diseases. For instance, miR-195 could facilitate mouse podocytes apoptosis by targeting BCL2 under high-glucose conditions [11
]. Another example is miR-93 that modulates the pathogenesis of diabetic nephropathy through repression of VEGF expression and its downstream signaling [12
]. Meanwhile, accumulating studies have demonstrated that miRNAs are also linked to EMT through dysregulation of EMT-related genes. MiR-34a, decreased in hypoxia-induced renal tubular epithelial cells, could promote EMT by directly targeting Notch1 and Jagged1 [13
]. Another study showed miRNA-200b causes EMT in kidney proximal tubular cells by targeting TGF-β1 [14
]. All these studies demonstrate that miRNAs play indispensable roles in the process of glomerular diseases, especially the EMT-associated disharmonies. However, the underlying mechanisms of miRNAs on EMT remain largely unknown.
MiR-30a is specifically expressed in collecting duct cells and podocytes [15
] and is identified as a biomarker in the urine of FSGS patients [16
]. In addition, miR-30a was reportedly associated with EMT in peritoneal fibrosis [17
] and several cancers, including non-small cell lung cancer (NSCLC) [18
], gastric cancer [20
] and hepatocellular carcinoma (HCC) [21
]. Despite these findings, the molecular mechanisms of miR-30a in the EMT of podocyte injury need further investigation. A large number of targets for miR-30a were predicted using bioinformatic analyses including: miRwalk (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/
), miRanda (http://microrna.org
), and Target Scan release 6.2 (http://www.targetscan.org
). Among these targets, NFATc3 has been revealed to be involved in EMT as previously described [9
]. In our study, we examined the role and potential mechanism of miR-30a in podocyte EMT. We found that miR-30a was repressed in patients with FSGS and a mouse model of podocyte injury. It is also downregulated in cultured podocytes that have been treated with Adriamycin (ADR) [22
]. We further demonstrated that ectopic expression miR-30a inhibits EMT of podocytes by targeting NFATc3, and downregulation of miR-30a results in promotion of EMT. Thus, we identified miR-30a as having a novel role in the process of podocyte EMT, which may provide a new understanding of molecular mechanisms about podocyte injury and provide a new potential target for the treatment of podocytopathy.
Podocyte dysfunction, as found in hypertrophy, dedifferentiation, detachment, and apoptosis, plays a central role in numerous kidney diseases [28
], such as FSGS and diabetic nephropathy. In this study, we have shown that miR-30a was significantly decreased in both ADR-induced glomeruli of mice and cultured podocytes, and the same result in glomeruli of FSGS patients. The fact suggests that miR-30a may be a clinically relevant biomarker for glomerulus and podocyte injury. Multiple factors could account for the podocyte injury [30
], and the mechanisms underlying podocyte injury remain ambiguous. In our study, we focused on investigating the function of miR-30a putative target NFATc3, an important transcription factor of the non-canonical Wnt signaling pathway, in podocyte injury. The downregulation of NFATc3 is associated with the concomitant overexpression of miR-30a in podocytes, suggesting that miR-30a might be a novel beneficial factor for podocytes and protect podocytes from impairment through targeting NFATc3.
Podocyte depletion often appears in incipient-stage renal diseases accompanied with prominent proteinuria. Despite a recent study indicating that miR-30s could protect the podocyte cytoskeleton from damage and apoptosis [32
], early-stage change after podocyte injury was not observed. However, EMT could be a primary reason leading to podocyte dysfunction, proteinuria, and glomerulosclerosis [33
]. Our study validates EMT as a bona fide stage after podocyte injury and demonstrates that the mesenchymal markers of EMT are activated in ADR-treated podocytes. In this study, we found that the alteration of epithelial markers was upregulated in the presence of miR-30a mimics and reduced when cells are transfected with the miR-30a inhibitor. On the contrary, the mesenchymal markers of EMT in podocytes was suppressed in the presence of miR-30a mimics and enhanced when cells are transfected with miR-30a inhibitor. All these results suggest that the suppression of epithelial-mesenchymal transition in podocytes is associated with enhanced expression of miR-30a, thus demonstrating that miR-30a may be a potential mechanism to prevent EMT in podocytes. In addition, the findings of this study show that miR-30a is downregulated in ADR-treated podocytes, however the underlying mechanisms of ADR on the regulation of miR-30a in podocytes remain unclear and requires further research.
MiRNAs are a large family of short endogenous noncoding RNAs that regulate EMT through targeting genes by translational silencing or mRNA degradation. Using bioinformatics software, we found that miR-30a could potentially interact with NFATc3. NFAT signaling is known to maintain podocyte function in areas such as protein synthesis [34
], metabolism [35
], cytoskeleton regulation [36
] and degradation [37
], and via regulation of the transcription of important genes for podocytes. Altered NFAT signaling can induce podocyte dysfunction, ultimately resulting in glomerulosclerosis [24
] and the activation of fibronectin protein [10
]. In the present study, we found that NFATc3 had higher expression than any other member of the NFAT family and was visibly enhanced after ADR treatment, suggesting that NFATc3 may play an important role in podocyte injury. Unequivocal evidence of whether or how miR-30a downregulates NFATc3 is still not available. However, this study shows that miR-30a downregulates NFATc3 by suppressing its nuclear translocation. The overexpression of miR-30a in podocytes enhances the prevention of cytoskeleton disorder or rearrangement of the actin cytoskeleton, a main factor of the complex architecture for podocyte function, suggesting that augmented miR-30a levels in podocytes inhibit the nuclear translocation of NFATc3 to protect the architecture and function of podocytes.
In summary, based on the observation that miR-30a is downregulated in podocyte injury models and glomeruli of FSGS patients, the present study demonstrates for the first time that miR-30a reduces EMT to protect podocyte function by targeting NFATc3. Thus, miR-30a might be a potential therapeutic target for inhibiting podocytopathy, and inhibition of it can be a potential diagnostic biomarker for podocyte injury.
4. Experimental Section
4.1. Human Samples
One sample was taken from a patient with FSGS at Department of Nephrology, the First Affiliated Hospital, Chongqing Medical University (Chongqing, China) and was identified by renal biopsy [25
]. The control sample was taken from one patient with kidney rupture due to violence. Isolation of glomeruli from renal tissues by standard mechanical sieving technique was performed as described previously [38
], snap-frozen in liquid nitrogen for RNA preparation, followed by cDNA synthesis and qPCR. Informed consent was acquired from all participants. This study was approved by the Chongqing Medical University Ethics Committee (Chongqing, China).
4.2. Animals and Treatment
All the experiments of animals in this study were approved by the Chongqing Medical University Animal Ethics Committee according to the “China Code of Practice for the Care and Use of Animals for Scientific Purposes”. BALB/c mice (6–7 weeks old) were used to generate adriamycin (ADR)-induced podocyte injury model [22
]. All mice were purchased from the Animal Center Management, Chongqing Medical University, China. Three BALB/c mice per group were intravenously injected with Adriamycin (ADR, 10.5 mg/kg; Sigma, St. Louis, MO, USA). The control group was injected with an equivalent volume of normal saline (NS). At 4, 7, 11, 15 and 20 days post-injection, the mice were sacrificed and glomeruli were isolated by standard mechanical sieving technique as described before [38
] and stored in liquid nitrogen for the next experiment.
4.3. Cell Culture and Transfection
Media and fetal bovine serum (FBS) were purchased from Invitrogen/ Gibco (Gibco, BRL Co., Ltd., Grand Island, NY, USA). HEK293T cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS, at 37 °C with 5% CO2
. Conditionally immortalized mouse podocytes line 5 (MPC5) were cultured as described before [39
] at 33 °C, 5% CO2
in RPMI 1640 medium supplemented with 10% FBS and recombinant mouse interferon gamma (10 U/mL; Peprotech, London, UK). MPC5 cells were maintained under nonpermissive conditions, at 37 °C in the absence of the recombinant mouse interferon gamma to induce differentiation, after at least 14 days, cells were treated with different concentrations of ADR at different time point depending on the next experimental setups.
For transfection, MPC5 and 293T cells were transfected with miR-30a mimics/inhibitor (50 nM) (RiboBio, Guangzhou, China) or pCDNA3.1(+)-Luc-NFATc3-3ʹUTR and pRL-CMV vectors by Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY, USA).
4.4. Constructs and Luciferase Assays
Genomic DNA fragments containing 3ʹUTR of NFATc3 were obtained by PCR and inserted into pCDNA3.1-Luc reporter vector. Two miR-30a complementary sites with NFATc3-3ʹUTR were mutated with site directed mutagenesis. All the primer sequences were listed in Table 1
. To normalize difference of transfection efficiency, pRL-CMV vector was applied and co-transfected for each well. 293T and MPC5 cells were seeded in 24-well plates and co-transfected with appropriate reporter and Renila plasmid and miR-30a mimics or the negative control of miRNA mimic (miR-NC) by Lipofectamine 2000 reagent. Thirty-six hours later, the dual-luciferase assay system (Promega, Madison, WI, USA) was used according to previous instructions [40
The sequences of the PCR primers for constructing NFATc3-3ʹUTR.
The sequences of the PCR primers for constructing NFATc3-3ʹUTR.
|Name||Primer Sequences||Product Size|
|WT-NFATc3-3ʹUTR||Sense: TTTGCCCACCACGGACTG||2450 bp|
4.5. RNA Extraction and Real Time PCR Analysis
Total RNA in MPC5 cells and isolated glomeruli were extracted with TRIzol reagent (Ambion, Austin, TX, USA) following the manufacturer’s instructions. Quantitative real time PCR of miR-30a was amplified by internal reference primers (U6) and miR-30a specific primers (RiboBio, Guangzhou, China) with Revert Aid First Strand cDNA Synthesis kit (Fermentas, Burlington, ON, Canada) and SYBR Premix Ex Taq TM II (Takara, Dalian, China) according to the manufacturers’ protocols. The first-strand cDNA of protein-encoding genes was synthesized with random primers from Revert Aid First Strand cDNA Synthesis kit. miRNAs’ quantification were determined by Ultra SYBR Mixture (CWBIO, Beijing, China) with relevant primers of protein-encoding genes according to the manufacturers’ protocols. All the sequences of real time PCR primers were listed in Table 2
. The comparative cycle threshold (Ct
) method was adopted to calculate the relative abundance of miRNA compared with U6 RNA. Levels of miR-30a and other mRNAs were respectively normalized to U6 RNA and 18s and calculated by using comparative cycle threshold (Ct
The sequences of the real time PCR primers.
The sequences of the real time PCR primers.
|Gene||Gene Bank Association Number||Sense||Anti-Sense||Product Size|
|Collagen I||NM_007742||AGCACGTCTGGTTTGGAGAG||GACATTAGGCGCAGGAAGGT||112 bp|
4.6. Protein Extraction and Western Blot Analysis
Cells were lysed with RIPA lysis buffer (Beyotime, Haimen, China). Protein concentration of each sample was measured by BCA reagent kit (Merck, Darmstadt, Germany). Cellular proteins (40 µg) was separated by SDS-PAGE gels electrophoresis, electro-transferred to PVDF membranes (Millipore Corporation, Billerica, MA, USA) and subsequently was blocked with 5% (w/v) no fat milk in TBST for 1 h at room temperature. The blots were probed with rabbit polyclonal antibody against desmin (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:1000), nephrin (Abcam, Cambridge, MA, USA; 1:2000), E-cadherin (Santa Cruz Biotechnology; 1:1000), NFATc3 (Bioss, Beijing, China; 1:500) and mouse monoclonal anti-beta-tubulin (Santa Cruz Biotechnology; 1:3000, anti-beta-actin (Santa Cruz Biotechnology; 1:3000) overnight at 4 °C. The membranes were then incubated with goat Ig anti-rabbit IgG-HRP (Santa Cruz Biotechnology) and goat Ig anti-mouse IgG-HRP (Santa Cruz Biotechnology) for 1 h at room temperature the next day. Western Blot chemiluminescent HRP substrate Reagent (Millipore Corporation) was used to detect Western Blotting antibody. Normalization of proteic expression was used of internal control (β-tubulin or β-actin).
4.7. Immunofluorescence Staining and F-Actin Cytoskeleton Staining
After treatment by transfecting with miR-30a or miR-NC, cells growing on glass coverslips were fixed with pre-cold acetone for 15 min at −20 °C. Subsequently, cells were permeabilized and blocked respectively with 1% Triton X-100 (in PBS) and 5% BSA (in PBS) for 1 h at room temperature. For immunofluorescence staining, the rabbit polyclonal antibody against NFATc3 (Santa Cruz Biotechnology; 1:100) was used to probe NFATc3 overnight at 4 °C and goat anti-rabbit IgG-CFL 488 (Santa Cruz Biotechnology; 1:2000) was used to detect rabbit IgG for 1 h at room temperature. For F-actin cytoskeleton staining, cells were incubated in rhodamine-labeled phalloidin (Sigma; diluted in PBS containing 5% BSA, 1:1000) overnight at 4 °C.
Finally, nuclei were stained by DAPI (Santa Cruz Biotechnology; 1:5000) at room temperature for 5 min. All the coverslips were washed with PBS for three times and then softly rinsed with water as following. The coverslips were immobilized on the glass slides by 50% glycerol in PBS and viewed under a fluorescence microscope (ECLIPSE Ti-s, Nikon, Tokyo, Japan); and relevant images were taken with a SPOT Diagnostic (Sterling Heights, MI, USA) CCD camera.
4.8. Statistical Analysis
All the data were presented as the mean ± SD. Student’s t-test was employed for the statistical analysis of two independent groups by GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). For all tests, p < 0.05 (*) or p < 0.01 (**) was considered statistically significant. All experiments were performed at least three times.