Effects and mechanism of miR-23b on glucose-mediated epithelial-to-mesenchymal transition in diabetic nephropathy

MicroRNAs (miRNAs) play important roles in epithelial-to-mesenchymal transition (EMT). Moreover, hyperglycaemia induces damage to renal tubular epithelial cells, which may lead to EMT in diabetic nephropathy. However, the effects of miRNAs on EMT in diabetic nephropathy are poorly understood. In the present study, we found that the level of microRNA-23b (miR-23b) was significantly decreased in high glucose (HG)-induced human kidney proximal tubular epithelial cells (HK2) and in kidney tissues of db/db mice. Overexpression of miR-23b attenuated HG-induced EMT, whereas knockdown of miR-23b induced normal glucose (NG)-mediated EMT in HK2 cells. Mechanistically, miR-23b suppressed EMT in diabetic nephropathy by targeting high mobility group A2 (HMGA2), thereby repressing PI3K-AKT signalling pathway activation. Additionally, HMGA2 knockdown or inhibition of the PI3K-AKT signalling pathway with LY294002 mimicked the effects of miR-23b overexpression on HG-mediated EMT, whereas HMGA2 overexpression or activation of the PI3K-AKT signalling pathway with BpV prevented the effects of miR-23b on HG-mediated EMT. We also confirmed that overexpression of miR-23b alleviated EMT, decreased the expression levels of EMT-related genes, ameliorated renal morphology, glycogen accumulation, fibrotic responses and improved renal functions in db/db mice. Taken together, we showed for the first time that miR-23b acts as a suppressor of EMT in diabetic nephropathy through repressing PI3K-AKT signalling pathway activation by targeting HMGA2, which maybe a potential therapeutic target for diabetes-induced renal dysfunction.     

MicroRNA-194 overexpression protects against hypoxia/reperfusion-induced HK-2 cell injury through direct targeting Rheb

Renal ischemia-reperfusion injury, a major cause of renal failure, always leads to acute kidney injury and kidney fibrosis. MicroRNAs (miRs) have been reported to be associated with renal ischemia-reperfusion injury. miR-194 was downregulated following renal ischemia-reperfusion injury; however, the function and mechanism of miR-194 in renal ischemia-reperfusion injury have not yet been fully understood. In the present study, we constructed renal ischemia-reperfusion injury model in vitro through treatment of human kidney proximal tubular epithelial cells HK-2 by hypoxia/reperfusion (H/R). We observed that miR-194 was decreased in H/R-induced HK-2 cells. miR-194 mimic increased H/R-induced HK-2 cell survival, whereas miR-194 inhibitor further strengthened H/R- inhibited HK-2 cell survival. Also, we observed that miR-194 overexpression suppressed oxidative stress markers, including malondialdehyde, glutathione, and secretion of pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α; however, miR-194 inhibitor showed the reverse effects. Results from dual-luciferase analysis confirmed that Ras homology enriched in brain (Rheb) was a direct target of miR-194. Finally, we corroborated that miR-194 affected cell growth, oxidative stress, and inflammation through targeting Rheb in H/R-induced HK-2 cells. In conclusion, our results suggested that miR-194 protect against H/R-induced injury in HK-2 cells through direct targeting Rheb.     

Hypoxia-induced down-regulation of microRNA-34a promotes EMT by targeting the Notch signaling pathway in tubular epithelial cells

Background

Hypoxia-induced renal tubular cell epithelial–mesenchymal transition (EMT) is an important event leading to renal fibrosis. MicroRNAs (miRNAs) are small non-coding RNA molecules that bind to their mRNA targets, thereby leading to translational repression. The role of miRNA in hypoxia-induced EMT is largely unknown.

Methodology/Principal Findings

miRNA profiling was performed for the identification of differentially expressed miRNAs in HK-2 cells under normal and low oxygen, and the results were then verified by quantitative real time RT-PCR (qRT-PCR). The function of miRNAs in hypoxia-induced renal tubular cell EMT was assessed by the transfection of specific miRNA inhibitors and mimics. Luciferase reporter gene assays and western blot analysis were performed to validate the target genes of miR-34a. siRNA against Jagged1 was designed to investigate the role of the miR-34a-Notch pathway in hypoxia induced renal tubular cell EMT. miRNA-34a was identified as being downregulated in hypoxic renal tubular epithelial cells. Inhibition of miR-34a expression in HK-2 cells, which highly express endogenous miR-34a, promoted a mesenchymal phenotype accompanied by reduced expression of the epithelial marker Z0-1, E-cadherin and increased expression of the mesenchymal markers α-SMA and vimentin. Conversely, miR-34a mimics effectively prevented hypoxia-induced EMT. Transfection of miRNA-34a in HK-2 cells under hypoxia abolished hypoxia-induced expression of Notch1 and Jagged1 as well as Notch downstream signals, such as snail. Western blot analysis and luciferase reporter gene assays showed direct evidence for miR-34a targeting Notch1 and Jagged1. siRNAs against Jagged1 or Notch1 effectively prevented miR-34a inhibitor-induced tubular epithelial cell EMT.

Conclusions/Significance

Our study provides evidence that the hypoxia-induced decrease of miR-34a expression could promote EMT in renal tubular epithelial cells by directly targeting Notch1 and Jagged1, and subsequently, Notch downstream signaling.     

Empagliflozin reduces high glucose-induced oxidative stress and miR-21-dependent TRAF3IP2 induction and RECK suppression,and inhibits human renal proximal tubular epithelial cell migration and epithelial-to-mesenchymal transition

Proximal tubular epithelial cells (PTEC) in the S1 segment of the kidney abundantly express sodium-glucose co-transporters (SGLT) that play a critical role in whole body glucose homeostasis. We recently reported suppression of RECK (Reversion Inducing Cysteine Rich Protein with Kazal Motifs), a membrane anchored endogenous MMP inhibitor and anti-fibrotic mediator, in the kidneys of db/db mice, a model of diabetic kidney disease (DKD), as well as in high glucose (HG) treated human kidney proximal tubule cells (HK−2). We further demonstrated that empagliflozin (EMPA), an SGLT2 inhibitor, reversed these effects. Little is known regarding the mechanisms underlying RECK suppression under hyperglycemic conditions, and its rescue by EMPA. Consistent with our previous studies, HG (25 mM) suppressed RECK expression in HK-2 cells. Further mechanistic investigations revealed that HG induced superoxide and hydrogen peroxide generation, oxidative stress-dependent TRAF3IP2 upregulation, NF-κB and p38 MAPK activation, inflammatory cytokine expression (IL-1β, IL-6, TNF-α, and MCP-1), miR-21 induction, MMP2 activation, and RECK suppression. Moreover, RECK gain-of-function inhibited HG-induced MMP2 activation and HK-2 cell migration. Similar to HG, advanced glycation end products (AGE) induced TRAF3IP2 and suppressed RECK, effects that were inhibited by EMPA. Importantly, EMPA treatment ameliorated all of these deleterious effects, and inhibited epithelial-to-mesenchymal transition (EMT) and HK-2 cell migration. Collectively, these findings indicate that hyperglycemia and associated AGE suppress RECK expression via oxidative stress/TRAF3IP2/NF-κB and p38 MAPK/miR-21 induction. Furthermore, these results suggest that interventions aimed at restoring RECK or inhibiting SGLT2 have the potential to treat kidney inflammatory response/fibrosis and nephropathy under chronic hyperglycemic conditions, such as DKD.     

Inhibition of lncRNA MEG3 protects renal tubular from hypoxia-induced kidney injury in acute renal allografts by regulating miR-181b/TNF-α signaling pathway

Early damage to transplanted organs initiates excess inflammation that deteriorates existing injury, which is a leading cause of graft loss. Long noncoding RNAs (lncRNAs) are recently thought to play a significant role in cellular homeostasis during pathological process of kidney diseases. The aim of this study was to assess the function and mechanism of lncRNA, maternally expressed gene 3 (MEG3), on early renal allografts pathogenesis. Real-time polymerase chain reaction (RT-PCR) analysis found that the levels of MEG3 and miR-181b-5p were increased and decreased respectively in grafted kidney. The Western blot assay showed that TNF-alpha was upregulated in the kidney and in HK-2 cells. Administering MEG3-specific small interfering RNA (siRNA) in mice silenced MEG3 expression and protected kidney renal allograft from injury. Bioinformatical analysis and luciferase assay indicated that MEG3 is a target of miR-181b-5p. MEG3 inhibition and overexpression promoted and suppressed miR-181b-5p levels respectively. In addition, Western blot and immunohistochemical staining suggested that decreased TNF-alpha expression was observed in the kidney. In contrary to MEG3, miR181b overexpression attenuated hypoxia-induced HK-2 cell apoptosis, as well as suppressed hypoxia-induced TNF-alpha upregulation. In luciferase reporter assay, we confirmed that miR-181b directly bound to the 3′-untranslated region (3′-UTR) of TNF-alpha, thereby negatively regulating the TNF-alpha expression. Our data suggested that MEG3 functions as a competing endogenous RNA for miR-181b to regulate the TNF-alpha expression in hypoxia-induced kidney injury in acute renal allografts.     

The interaction of S100A16 and GRP78 actives endoplasmic reticulum stress-mediated through the IRE1α/XBP1 pathway in renal tubulointerstitial fibrosis

Recent studies have indicated that the development of acute and chronic kidney disease including renal fibrosis is associated with endoplasmic reticulum (ER) stress. S100 calcium-binding protein 16 (S100A16) as a novel member of the S100 family is involved in kidney disease; however, few studies have examined fibrotic kidneys for a relationship between S100A16 and ER stress. In our previous study, we identified GRP78 as a protein partner of S100A16 in HK-2 cells. Here, we confirmed a physical interaction between GRP78 and S100A16 in HK-2 cells and a markedly increased expression of GRP78 in the kidneys of unilateral ureteral occlusion mice. S100A16 overexpression in HK-2 cells by infection with Lenti-S100A16 also induced upregulation of ER stress markers, including GRP78, p-IRE1α, and XBP1s. Immunofluorescence staining demonstrated that the interaction between S100A16 and GRP78 predominantly occurred in the ER of control HK-2 cells. By contrast, HK-2 cells overexpressing S100A16 showed colocalization of S100A16 and GRP78 mainly in the cytoplasm. Pretreatment with BAPTA-AM, a calcium chelator, blunted the upregulation of renal fibrosis genes and ER stress markers induced by S100A16 overexpression in HK-2 cells and suppressed the cytoplasmic colocalization of GRP78 and S100A16. Co-immunoprecipitation studies suggested a competitive binding between S100A16 and IRE1α with GRP78 in HK-2 cells. Taken together, our findings demonstrate a significant increase in S100A16 expression in the cytoplasm following renal injury. GRP78 then moves into the cytoplasm and binds with S100A16 to promote the release of IRE1α. The subsequent phosphorylation of IRE1α then leads to XBP1 splicing that activates ER stress.Subject terms: Stress signalling, Experimental models of disease     

MiR-92d-3p suppresses the progression of diabetic nephropathy renal fibrosis by inhibiting the C3/HMGB1/TGF-β1 pathway

The pathogenesis of diabetic nephropathy (DN) has not been fully elucidated. MicroRNAs (miRNAs) play an important role in the onset and development of DN renal fibrosis. Thus, the present study aimed to investigate the effect of miR-92d-3p on the progression of DN renal fibrosis. We used qRT-PCR to detect the expression levels of miR-92d-3p in the kidneys of patients with DN. Then, after transfecting lentiviruses containing miR-92d-3p into the kidneys of a DN mouse model and HK-2 cell line, we used qRT-PCR to detect the expression levels of miR-92d-3p, C3, HMGB1, TGF-β1, α-SMA, E-cadherin, and Col I. The expression levels of interleukin (IL) 1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in the HK-2 cells were detected through enzyme-linked immunosorbent assay (ELISA), and Western blotting and immunofluorescence were used in detecting the expression levels of fibronectin, α-SMA, E-cadherin, and vimentin. Results showed that the expression levels of miR-92d-3p in the kidney tissues of patients with DN and DN animal model mice decreased, and C3 stimulated HK-2 cells to produce inflammatory cytokines. The C3/HMGB1/TGF-β1 pathway was activated, and epithelial-to-interstitial transition (EMT) was induced in the HK-2 cells after human recombinant C3 and TGF-β1 protein were added. miR-92d-3p inhibited inflammatory factor production by C3 in the HK-2 cells and the activation of the C3/HMGB1/TGF-β1 pathway and EMT by C3 and TGF-β1. miR-92d-3p suppressed the progression of DN renal fibrosis by inhibiting the activation of the C3/HMGB1/TGF-β1 pathway and EMT.     

Up-regulation of the human-specific CHRFAM7A gene protects against renal fibrosis in mice with obstructive nephropathy

Renal fibrosis is a major factor in the progression of chronic kidney diseases. Obstructive nephropathy is a common cause of renal fibrosis, which is also accompanied by inflammation. To explore the effect of human-specific CHRFAM7A expression, an inflammation-related gene, on renal fibrosis during obstructive nephropathy, we studied CHRFAM7A transgenic mice and wild type mice that underwent unilateral ureteral obstruction (UUO) injury. Transgenic overexpression of CHRFAM7A gene inhibited UUO-induced renal fibrosis, which was demonstrated by decreased fibrotic gene expression and collagen deposition. Furthermore, kidneys from transgenic mice had reduced TGF-β1 and Smad2/3 expression following UUO compared with those from wild type mice with UUO. In addition, the overexpression of CHRFAM7A decreased release of inflammatory cytokines in the kidneys of UUO-injured mice. In vitro, the overexpression of CHRFAM7A inhibited TGF-β1-induced increase in expression of fibrosis-related genes in human renal tubular epithelial cells (HK-2 cells). Additionally, up-regulated expression of CHRFAM7A in HK-2 cells decreased TGF-β1-induced epithelial-mesenchymal transition (EMT) and inhibited activation f TGF-β1/Smad2/3 signalling pathways. Collectively, our findings demonstrate that overexpression of the human-specific CHRFAM7A gene can reduce UUO-induced renal fibrosis by inhibiting TGF-β1/Smad2/3 signalling pathway to reduce inflammatory reactions and EMT of renal tubular epithelial cells.     

MicroRNA-7 Inhibits Tumor Metastasis and Reverses Epithelial-Mesenchymal Transition through AKT/ERK1/2 Inactivation by Targeting EGFR in Epithelial Ovarian Cancer

Epidermal growth factor receptor (EGFR) overexpression and activation result in increased proliferation and migration of solid tumors including ovarian cancer. In recent years, mounting evidence indicates that EGFR is a direct and functional target of miR-7. In this study, we found that miR-7 expression was significantly downregulated in highly metastatic epithelial ovarian cancer (EOC) cell lines and metastatic tissues, whereas the expression of, EGFR correlated positively with metastasis in both EOC patients and cell lines. Overexpression of miR-7 markedly suppressed the capacities of cell invasion and migration and resulted in morphological changes from a mesenchymal phenotype to an epithelial-like phenotype in EOC. In addition, overexpression of miR-7 upregulated CK-18 and β-catenin expression and downregulated Vimentin expression, accompanied with EGFR inhibition and AKT/ERK1/2 inactivation. Similar to miR-7 transfection, silencing of EGFR with this siRNA in EOC cells also upregulated CK-18 and β-catenin expression and downregulated Vimentin expression, and decreased phosphorylation of both Akt and ERK1/2, confirming that EGFR is a target of miR-7 in reversing EMT. The pharmacological inhibition of PI3K-AKT and ERK1/2 both significantly enhanced CK-18 and β-catenin expression and suppressed vimentin expression, indicating that AKT and ERK1/2 pathways are required for miR-7 mediating EMT. Finally, the expression of miR-7 and EGFR in primary EOC with matched metastasis tissues was explored. It was showed that miR-7 is inversely correlated with EGFR. Taken together, our results suggested that miR-7 inhibited tumor metastasis and reversed EMT through AKT and ERK1/2 pathway inactivation by reducing EGFR expression in EOC cell lines. Thus, miR-7 might be a potential prognostic marker and therapeutic target for ovarian cancer metastasis intervention.     

ER stress contributes to renal proximal tubule injury by increasing SREBP-2-mediated lipid accumulation and apoptotic cell death

Renal proximal tubule injury is induced by agents/conditions known to cause endoplasmic reticulum (ER) stress, including cyclosporine A (CsA), an immunosuppressant drug with nephrotoxic effects. However, the underlying mechanism by which ER stress contributes to proximal tubule cell injury is not well understood. In this study, we report lipid accumulation, sterol regulatory element-binding protein-2 (SREBP-2) expression, and ER stress in proximal tubules of kidneys from mice treated with the classic ER stressor tunicamycin (Tm) or in human renal biopsy specimens showing CsA-induced nephrotoxicity. Colocalization of ER stress markers [78-kDa glucose regulated protein (GRP78), CHOP] with SREBP-2 expression and lipid accumulation was prominent within the proximal tubule cells exposed to Tm or CsA. Prolonged ER stress resulted in increased apoptotic cell death of lipid-enriched proximal tubule cells with colocalization of GRP78, SREBP-2, and Ca(2+)-independent phospholipase A(2) (iPLA(2)β), an SREBP-2 inducible gene with proapoptotic characteristics. In cultured HK-2 human proximal tubule cells, CsA- and Tm-induced ER stress caused lipid accumulation and SREBP-2 activation. Furthermore, overexpression of SREBP-2 or activation of endogenous SREBP-2 in HK-2 cells stimulated apoptosis. Inhibition of SREBP-2 activation with the site-1-serine protease inhibitor AEBSF prevented ER stress-induced lipid accumulation and apoptosis. Overexpression of the ER-resident chaperone GRP78 attenuated ER stress and inhibited CsA-induced SREBP-2 expression and lipid accumulation. In summary, our findings suggest that ER stress-induced SREBP-2 activation contributes to renal proximal tubule cell injury by dysregulating lipid homeostasis.     

Lefty A attenuates the TGF-β1-induced epithelial to mesenchymal transition of human renal proximal epithelial tubular cells

The epithelial to mesenchymal transition (EMT) is a crucial event for renal fibrosis that can be elicited by TGF-β1/Smads signaling and its downstream mediator connective tissue growth factor (CTGF). As a distinct member of the TGF-β superfamily, Lefty A has been shown to be significantly downregulated in the kidneys of patients with severe ureteral obstruction, suggesting its role in renal fibrosis induced by obstructive nephropathy. In order to determine whether Lefty A prevents TGF-β1-induced EMT, human proximal tubule epithelial cells (HK-2) were stably transfected with Lefty A or control vectors and stimulated with 10 ng/ml TGF-β1 for 48 h. The results show that stimulation with TGF-β1 led to EMT including cell morphology changes, Smad2/3 signaling pathway activation, increased α-SMA, collagen type I, and CTGF expression, and decreased E-cadherin expression in mock-transfected HK-2 cells. Overexpression of Lefty A efficiently blocked p-Smad2/3 activation and attenuated all these EMT changes induced by TGF-β1. This finding suggests that Lefty A may serve as a potential new therapeutic target to inhibit or even reverse EMT during the process of renal fibrosis.     

A bioinformatics and network pharmacology approach to the mechanisms of action of Shenxiao decoction for the treatment of diabetic nephropathy

BackgroundThe epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells is the main pathological alteration in diabetic nephropathy (DN). Traditional Chinese medicine (TCM) has been used for the treatment of DN in clinical practice and has been proven to be effective.PurposeThis aim of this study was to shed light on the efficacy of Shenxiao decoction (SXD) on the EMT of renal tubular epithelial cells and the molecular mechanisms of SXD in mice with DN, as well as on the high glucose (HG)- and TGF-β1-induced EMT of NRK-52E and HK-2 cells.Study design and methodsA bioinformatics and network pharmacology method were utilized to construct the active ingredient-target networks of SXD that were responsible for the beneficial effects against DN. The effects of RUNX3 were validated in HG- and TGF-β1-induced EMT processes in NRK-52E and HK-2 cells.ResultsBioinformatics analysis revealed that 122 matching targets were closely associated with the regulation of cell migration and the AGE-RAGE signaling pathway in diabetic complications. The results also revealed that, relative to the mice with DN, the mice in the treatment group had an improved general state and reduced blood glucose levels. The degradation of renal function was ameliorated by SXD. Moreover, the protective effects of SXD were also observed on renal structural changes. Furthermore, SXD suppressed the activation of the transforming growth factor (TGF)-β1/Smad pathway and upregulated the RUNX3 and E-cadherin levels and downregulated the extracellular matrix (ECM) protein levels in mice with DN. SXD was further found to prevent the HG- and TGF-β1-induced EMT processes in NRK-52E and HK-2 cells. Additionally, the overexpression of RUNX3 markedly inhibited the EMT and TGF-β1/Smad pathway induced by HG and TGF-β1 in NRK-52E and HK-2 cells.ConclusionTaken together, these results suggest that SXD maybe alleviate EMT in DN via the inhibition of the TGF-β1/Smad/RUNX3 signaling pathway under hyperglycemic conditions.     

TDAG51 mediates epithelial-to-mesenchymal transition in human proximal tubular epithelium

Epithelial-to-mesenchymal transition (EMT) contributes to renal fibrosis in chronic kidney disease. Endoplasmic reticulum (ER) stress, a feature of many forms of kidney disease, results from the accumulation of misfolded proteins in the ER and leads to the unfolded protein response (UPR). We hypothesized that ER stress mediates EMT in human renal proximal tubules. ER stress is induced by a variety of stressors differing in their mechanism of action, including tunicamycin, thapsigargin, and the calcineurin inhibitor cyclosporine A. These ER stressors increased the UPR markers GRP78, GRP94, and phospho-eIF2α in human proximal tubular cells. Thapsigargin and cyclosporine A also increased cytosolic Ca(2+) concentration and T cell death-associated gene 51 (TDAG51) expression, whereas tunicamycin did not. Thapsigargin was also shown to increase levels of active transforming growth factor (TGF)-β1 in the media of cultured human proximal tubular cells. Thapsigargin induced cytoskeletal rearrangement, β-catenin nuclear translocation, and α-smooth muscle actin and vinculin expression in proximal tubular cells, indicating an EMT response. Subconfluent primary human proximal tubular cells were induced to undergo EMT by TGF-β1 treatment. In contrast, tunicamycin treatment did not produce an EMT response. Plasmid-mediated overexpression of TDAG51 resulted in cell shape change and β-catenin nuclear translocation. These results allowed us to develop a two-hit model of ER stress-induced EMT, where Ca(2+) dysregulation-mediated TDAG51 upregulation primes the cell for mesenchymal transformation via Wnt signaling and then TGF-β1 activation leads to a complete EMT response. Thus the release of Ca(2+) from ER stores mediates EMT in human proximal tubular epithelium via the induction of TDAG51.     

The interaction of LFA-1 on mononuclear cells and ICAM-1 on tubular epithelial cells accelerates TGF-β1-induced renal epithelial-mesenchymal transition

The epithelial-mesenchymal transition (EMT) of renal epithelial cells (RTECs) has pivotal roles in the development of renal fibrosis. Although the interaction of lymphocyte function-associated antigen 1 (LFA-1) on leukocytes and its ligand, intracellular adhesion molecule 1 (ICAM-1), plays essential roles in most inflammatory reactions, its pathogenetic role in the EMT of RTECs remains to be clarified. In the present study, we investigated the effect of the interaction of LFA-1 on peripheral blood mononuclear cells (PBMCs) and ICAM-1 on HK-2 cells after stimulation with TGF-β(1) on the EMT of RTECs. ICAM-1 was highly expressed in HK-2 cells. After TGF-β(1) stimulation, the chemokines CCL3 and CXCL12 increased on HK-2 cells. After co-culture of PBMCs and HK-2 cells pre-stimulated with TGF-β(1) (0.1 ng/ml) (HK-2-TGF-β(1) (0.1)), the expression of the active form of LFA-1 increased on PBMCs; however, total LFA-1 expression did not change. The expression of the active form of LFA-1 on PBMCs did not increase after co-culture with not CCL3 but CXCL12 knockdown HK-2-TGF-β(1) (0.1). The expression of epithelial cell junction markers (E-cadherin and occludin) further decreased and that of mesenchymal markers (vimentin and fibronectin) further increased in HK-2-TGF-β(1) (0.1) after co-culture with PBMCs for 24 hrs (HK-2-TGF-β(1) (0.1)-PBMCs). The phosphorylation of ERK 1/2 but not smad2 and smad3 increased in HK-2-TGF-β(1) (0.1)-PBMCs. The snail and slug signaling did not increase HK-2-TGF-β(1) (0.1)-PBMCs. Although the migration and invasion of HK-2 cells induced full EMT by a high dose (10.0 ng/ml) and long-term (72-96 hrs) TGF-β(1) stimulation increased, that of HK-2-TGF-β(1) (0.1)-PBMCs did not increase. These results suggested that HK-2 cells stimulated with TGF-β(1) induced conformational activation of LFA-1 on PBMCs by increased CXCL12. Then, the direct interaction of LFA-1 on PBMCs and ICAM-1 on HK-2 cells activated ERK1/2 signaling to accelerate the part of EMT of HK-2 cells induced by TGF-β(1).