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Crucial role of serum response factor in renal tubular epithelial cell epithelial-mesenchymal transition in hyperuricemic nephropathy.

ABSTRACT: OBJECTIVE:To explore the regulation and function of serum response factor (SRF) in epithelial-mesenchymal transition (EMT) in renal tubular epithelial cells (TECs) in hyperuricemic nephropathy (HN). RESULTS:In NRK-52E cells treated with UA and renal medulla tissue samples from hyperuricemic rats, SRF, fibronectin, ?-SMA and FSP-1 expression was upregulated, while ZO-1 and E-cadherin expression was downregulated. SRF upregulation in NRK-52E cells increased slug expression. Blockade of SRF by an SRF-specific siRNA or CCG-1423 reduced slug induction and protected TECs from undergoing EMT both in vitro and in vivo. CONCLUSION:Increased SRF activity promotes EMT and dysfunction in TECs in HN. Targeting SRF with CCG-1423 may be an attractive therapeutic strategy in HN. METHODS:The expression of SRF, mesenchymal markers (fibronectin, ?-SMA, and FSP-1), epithelial markers (ZO-1 and E-cadherin) and was examined in rat renal TECs (NRK-52E cells) or renal medulla tissue samples following uric acid (UA) treatment. SRF overexpressed with pcDNA-SRF plasmid and suppressed by CCG-1423 (a small molecule inhibitor of SRF) to study how SRF influences EMT in TECs in HN. Oxonic acid (OA) was used to establish HN in rats.

SUBMITTER: Zhao L

PROVIDER: S-EPMC6914413 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Crucial role of serum response factor in renal tubular epithelial cell epithelial-mesenchymal transition in hyperuricemic nephropathy.

Zhao Long L Li Chenyu C Zhou Bin B Luo Congjuan C Wang Yanfei Y Che Lin L Zhao Jun J Xu Yan Y

Aging 20191127 22

<h4>Objective</h4>To explore the regulation and function of serum response factor (SRF) in epithelial-mesenchymal transition (EMT) in renal tubular epithelial cells (TECs) in hyperuricemic nephropathy (HN).<h4>Results</h4>In NRK-52E cells treated with UA and renal medulla tissue samples from hyperuricemic rats, SRF, fibronectin, α-SMA and FSP-1 expression was upregulated, while ZO-1 and E-cadherin expression was downregulated. SRF upregulation in NRK-52E cells increased slug expression. Blockade ...[more]

PMID: 31774735

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Project description:Epithelial-mesenchymal transition (EMT) occurs in several disease states, including renal fibrosis and carcinogenesis. Myofibroblasts produced from EMT of renal tubular cells are responsible for the deposition of extracellular matrix components in a large portion of renal interstitial fibrosis. Transforming growth factor-beta (TGF-beta) plays an essential role in the EMT of renal tubular cells, but the molecular mechanism governing this process remains largely unknown. In this study, we found that RGC-32 (response gene to complement 32) is critical for TGF-beta-induced EMT of human renal proximal tubular cells (HPTCs). RGC-32 is not normally expressed in the HPTCs. However, TGF-beta stimulation markedly activates RGC-32 while inducing an EMT, as shown by the induction of smooth muscle alpha-actin (alpha-SMA) and extracellular matrix proteins collagen I and fibronectin, as well as the reduction of epithelial marker E-cadherin. TGF-beta function is mediated by several signaling pathways, but RGC-32 expression in HPTCs appears to be mainly regulated by Smad. Functionally, RGC-32 appears to mediate TGF-beta-induced EMT of HPTCs. Blockage of RGC-32 using short hairpin interfering RNA significantly inhibits TGF-beta induction of myofibroblast marker gene alpha-SMA while repressing the expression of E-cadherin. In contrast, overexpression of RGC-32 induces alpha-SMA expression while restoring E-cadherin. RGC-32 also inhibits the expression of another adherens junction protein, N-cadherin, suggesting that RGC-32 alone induces the phenotypic conversion of renal epithelial cells to myofibroblasts. Additional studies show that RGC-32 stimulates the production of extracellular matrix components fibronectin and collagen I. Mechanistically, RGC-32 induces EMT via the activation of other transcription factors such as Snail and Slug. RGC-32 knockdown inhibits the expression of Snail and Slug during TGF-beta-induced EMT. Taken together, our data demonstrate for the first time that RGC-32 plays a critical role in TGF-beta-induced EMT of renal tubular cells.

Project description:BACKGROUND:The use of microRNAs in the therapy of kidney disease is hampered by the difficulties in their effective delivery. Micro-vesicles (MVs) are known as natural carriers of small RNAs. Our prior research has demonstrated that MVs isolated from mesenchymal stem cells (MSCs) are capable of attenuating kidney injuries induced by unilateral ureteral obstruction and 5/6 sub-total nephrectomy in mice. The present study aimed to evaluate the effects of miR-34a-5p (miR-34a)-modified MSC-MVs on transforming growth factor (TGF)-?1-induced fibrosis and apoptosis in vitro. METHODS:Bone marrow MSCs were modified by lentiviruses over-expressing miR-34a, from which MVs were collected for the treatment of human Kidney-2 (HK-2) renal tubular cells exposed to TGF-?1 (6?ng/mL). The survival of HK-2 cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and Annexin V-Light 650/propidium iodide (PI) assays. The expression levels of epithelial markers (tight junction protein 1 [TJP1] and E-cadherin) and mesenchymal markers (smooth muscle actin alpha (?-SMA) and fibronectin) in HK-2 cells were measured using Western blot analysis and an immunofluorescence assay. In addition, changes in Notch-1/Jagged-1 signaling were analyzed using Western blotting. Data were analyzed using a Student's t test or one-way analysis of variance. RESULTS:MiR-34a expression increased three-fold in MVs generated by miR-34a-modified MSCs compared with that expressed in control MVs (P?<?0.01, t?=?16.55). In HK-2 cells, TJP1 and E-cadherin levels decreased to 31% and 37% after treatment with TGF-?1, respectively, and were restored to 62% and 70% by miR-34a-enriched MSC-MVs, respectively. The expression of ?-SMA and fibronectin increased by 3.9- and 5.0-fold following TGF-?1 treatment, and decreased to 2.0- and 1.7-fold after treatment of HK-2 cells with miR-34a-enriched MSC-MVs. The effects of miR-34a-enriched MSC-MVs on epithelial-mesenchymal transition (EMT) markers were stronger than control MSC-MVs. The effects of miR-34a-enriched MSC-MVs on these EMT markers were stronger than control MSC-MVs. Notch-1 receptor and Jagged-1 ligand, two major molecules of Notch signaling pathway, are predicted targets of miR-34a. It was further observed that elevation of Notch-1 and Jagged-1 induced by TGF-?1 was inhibited by miR-34a-enriched MSC-MVs. In addition, TGF-?1 exposure also induced apoptosis in HK-2 cells. Although miR-34a-mofidied MSC-MVs were able to inhibit TGF-?1-triggered apoptosis in HK-2 cells, the effects were less significant than control MSC-MVs (control:TGF-?1: miR-nc-MV:miR-34a-MV?=?1.3:0.6:1.1:0.9 for MTT assay, 1.8%:23.3%:9.4%:17.4% for apoptosis assay). This phenomenon may be the result of the pro-apoptotic effects of miR-34a. CONCLUSIONS:The present study demonstrated that miR-34a-over-expressing MSC-MVs inhibit EMT induced by pro-fibrotic TGF-?1 in renal tubular epithelial cells, possibly through inhibition of the Jagged-1/Notch-1 pathway. Genetic modification of MSC-MVs with an anti-fibrotic molecule may represent a novel strategy for the treatment of renal injuries.

Project description:BackgroundEverolimus (EVE) is a drug widely used in several renal transplant protocols. Although characterized by a relatively low nephrotoxicity, it may induce several adverse effects including severe fibro-interstitial pneumonitis. The exact molecular/biological mechanism associated to these pro-fibrotic effects is unknown, but epithelial to mesenchymal transition (EMT) may have a central role. Additionally, heparanase, an enzyme recently associated with the progression of chronic allograft nephropathy, could contribute to activate this machinery in renal cells.MethodsSeveral biomolecular strategies (RT-PCR, immunofluorescence, zymography and migration assay) have been used to assess the capability of EVE (10, 100, 200 and 500 nM) to induce an in vitro heparanase-mediated EMT in wild-type (WT) and Heparanase (HPSE)-silenced immortalized human renal epithelial proximal tubular cells (HK-2). Additionally, microarray technology was used to find additional biological elements involved in EVE-induced EMT.ResultsBiomolecular experiments demonstrated a significant up-regulation (more than 1.5 fold increase) of several genes encoding for well known EMT markers [(alpha-smooth muscle actin (?-SMA), Vimentin (VIM), Fibronectin (FN) and matrix metalloproteinase-9 (MMP9)], enhancement of MMP9 protein level and increment of cells motility in WT HK2 cells treated with high concentrations of EVE (higher than 100 nM). Similarly, immunofluorescence analysis showed that 100 nM of EVE increased ?-SMA, VIM and FN protein expression in WT HK2 cells. All these effects were absent in both HPSE- and AKT-silenced cell lines. AKT is a protein having a central role in EMT. Additionally, microarray analysis identified other 2 genes significantly up-regulated in 100 nM EVE-treated cells (p?<?0.005 and FDR?<?5%): transforming growth factor beta-2 (TGF?2) and epidermal growth factor receptor (EGFR). Real-time PCR analysis validated microarray.ConclusionsOur in vitro study reveals new biological/cellular aspects of the pro-fibrotic activity of EVE and it demonstrates, for the first time, that an heparanase-mediated EMT of renal tubular cells may be activated by high doses of this drug. Additionally, our results suggest that clinicians should administer the adequate dosage of EVE in order to increase efficacy and reduce adverse effects. Finally heparanase could be a new potential therapeutic target useful to prevent/minimize drug-related systemic fibrotic adverse effects.

Dataset Information

Crucial role of serum response factor in renal tubular epithelial cell epithelial-mesenchymal transition in hyperuricemic nephropathy.

Publications

Crucial role of serum response factor in renal tubular epithelial cell epithelial-mesenchymal transition in hyperuricemic nephropathy.

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