Epithelial-mesenchymal Transition In HK-2 Cells Research Articles

2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1, 4-Dione isolated from Averrhoa carambola L. root inhibits high glucose-induced EMT in HK-2 cells through targeting the regulation of miR-21–5p/Smad7 signaling pathway

Objective2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1, 4-Dione (DMDD) isolated from Averrhoa carambola L. root, has been proven therapeutic effects on diabetic kidney disease (DKD). This research aims to assess DMDD’s effects on DKD and to investigate its underlying mechanisms, to establish DMDD as a novel pharmaceutical agent for DKD treatment. MethodsThe human renal tubular epithelial (HK-2) cells were induced by high glucose (HG) to mimic DKD and followed by DMDD treatment. The cytotoxicity of DMDD was assessed using the Cell Counting Kit-8 (CCK-8) assay. The migratory capacity of HK-2 cells was evaluated through transwell and scratch-wound assays. To investigate the effect of Smad7 and miR-21–5p, lentiviral transfection was employed in HK-2 cells. Additionally, the expression of proteins related to epithelial-mesenchymal transition (EMT) and TGFβ1/Smad2/3 pathway was checked by QRT-PCR, Western blot, and immunofluorescence techniques. ResultsThis study has shown that DMDD significantly suppresses cell migration and the expression of Vimentin, α-SMA, TGFβ1, and p-Smad2/3 in HK-2 cells under HG conditions. Concurrently, DMDD enhances the protein expression of E-cadherin and Smad7. Intriguingly, the therapeutic effect of DMDD was abrogated upon Smad7 silencing. Further investigations revealed that DMDD effectively inhibits miR-21–5p expression, which is upregulated by HG. Downregulation of miR-21–5p inhibits the activation of the TGFβ1/Smad2/3 pathway and EMT induced by HG. In contrast, overexpression of miR-21–5p negates DMDD’s therapeutic benefits. ConclusionDMDD mitigates EMT in HG-induced HK-2 cells by modulating the miR-21–5p/Smad7 pathway, thereby inhibiting renal fibrosis in DKD. These findings suggest that DMDD holds promise as a potential therapeutic agent for DKD.

Colquhounia root tablet improves diabetic kidney disease by regulating epithelial-mesenchymal transition via the PTEN/PI3K/AKT pathway.

Diabetic kidney disease (DKD) is a severe microvascular complication of diabetes mellitus that can lead to end-stage renal disease. Colquhounia root tablet (CRT) has shown therapeutic potential in treating DKD, but its efficacy and underlying mechanisms remain to be elucidated. A randomized controlled clinical trial was conducted on 61 DKD patients. The treatment group received CRT in addition to standard therapy, while the control group received standard therapy alone. Treatment efficacy and adverse events were evaluated after 3months. Additionally, in vitro experiments using human renal tubular epithelial cells (HK-2) were performed to investigate the effect of CRT on high glucose (HG)-induced epithelial-mesenchymal transition (EMT) and the involvement of the PTEN/PI3K/AKT signaling pathway. CRT treatment significantly improved proteinuria and increased the effective treatment rate in DKD patients compared to the control group, with no significant difference in adverse events. Moreover, CRT reversed HG-induced EMT in HK-2 cells, as evidenced by the downregulation of α-SMA and upregulation of E-cadherin at both mRNA and protein levels. Mechanistically, CRT increased PTEN expression and inhibited the PI3K/AKT pathway, similar to the effects of the PI3K inhibitor LY29400. The combination of CRT and LY29400 further enhanced PTEN mRNA expression under HG conditions. CRT effectively improves proteinuria in DKD patients and ameliorates HG-induced EMT in HK-2 cells. The underlying mechanism may involve the upregulation of PTEN and subsequent inhibition of the PI3K/AKT signaling pathway. These findings provide new insights into the therapeutic potential of CRT for DKD treatment.

Sweroside plays a role in mitigating high glucose-induced damage in human renal tubular epithelial HK-2 cells by regulating the SIRT1/NF-κB signaling pathway.

Sweroside is a natural monoterpene derived from Swertia pseudochinensis Hara. Recently, studies have shown that sweroside exhibits a variety of biological activities, such as anti-inflammatory, antioxidant, and hypoglycemic effects. However, its role and mechanisms in high glucose (HG)-induced renal injury remain unclear. Herein, we established a renal injury model in vitro by inducing human renal tubular epithelial cell (HK-2 cells) injury by HG. Then, the effects of sweroside on HK-2 cell activity, inflammation, reactive oxygen species (ROS) production, and epithelial mesenchymal transition (EMT) were observed. As a result, sweroside treatment ameliorated the viability, inhibited the secretion of inflammatory cytokines (TNF-α, IL-1β, and VCAM-1), reduced the generation of ROS, and inhibited EMT in HK-2 cells. Moreover, the protein expression of SIRT1 was increased and the acetylation of p65 NF-kB was decreased in HK-2 cells with sweroside treatment. More importantly, EX527, an inhibitor of SIRT1, that inactivated SIRT1, abolished the improvement effects of sweroside on HK-2 cells. Our findings suggested that sweroside may mitigate HG-caused injury in HK-2 cells by promoting SIRT1-mediated deacetylation of p65 NF-kB.

Liuwei Dihuang Decoction Drug-containing Serum Attenuates Transforming Growth Factor-β1-induced Epithelial-Mesenchymal Transition in HK-2 cells by Inhibiting NF-κB/Snail Signaling Pathway.

The nuclear factor-κB (NF-κB) signaling pathway plays an important role in regulating tubular epithelial-mesenchymal transition (EMT), an indispensable cellular programme for driving organ fibrosis and tumor progression. Liuwei Dihuang Decoction (LWD) is an effective Chinese formula for treating chronic renal failure. First, by using morphological examination, immunofluorescence staining assay, RT-qPCR, and Western blot analysis, in vitro experiments were designed to analyze NF-κB and EMT markers (including Snail, α-SMA, and E-cadherin) in transforming growth factor-β1 (TGF-β1) induced renal tubular epithelial cells (HK-2) and to detect the expression levels of LWD-CS co-treatment. Then, the recombinant lentiviral vector was overexpressed and knocked down by NF-κB and transfected into HK-2 cells. Cells were treated with TGF-β1 (10ng/ml) with blank serum or LWD-containing serum, respectively, and the expression of these molecules in the NF-κB/Snail signaling pathway was further evaluated. Our results confirmed that TGF-β1 could induce EMT, nuclear translocation of NF-κB p65, and activate the NF-κB/Snail signaling pathway in HK-2 cells. Furthermore, NF-κB knocked-down dramatically increases the TGF-β1-induced mRNA and protein expression level of E-cadherin and reduces the level of Snail and α-SMA; this is reversed by NF-κB overexpression. LWD can decrease the EMT levels through the NF-κB/Snail signaling activation in TGF-β1-induced EMT of HK-2 cells. The present study provides evidence suggesting a novel mechanism that LWD exerts anti-fibrosis effects through inhibiting activation of the NF-κB/Snail signaling pathway and consequently downregulating the TGF-β1-induced EMT in renal tubular epithelial cell.

N-methyl-d-aspartate receptor 1 activation mediates cadmium-induced epithelial-mesenchymal transition in proximal tubular cells

Cadmium (Cd) is a commonly found environmental pollutant and is known to damage multiple organs with kidneys being the most common one. N-methyl-d-aspartate receptor 1 (NMDAR1) is a ligand-gated ion channel that is highly permeable to calcium ion (Ca2+). Because Cd2+ and Ca2+ have structural and physicochemical similarities, whether and how Cd could interfere NMDAR1 function to cause renal epithelial cells dysfunction remains unknown. In this study, we investigated the role of NMDAR1 in Cd-induced renal damage and found that Cd treatment upregulated NMDAR1 expression and promoted epithelial-mesenchymal transition (EMT) in mouse kidneys in vivo and human proximal tubular epithelial HK-2 cells in vitro, which were accompanied with activation of the inositol-requiring enzyme 1 (IRE-1α) / spliced X box binding protein-1 (XBP-1s) pathway, an indicative of endoplasmic reticulum (ER) stress. Mechanistically, NMDAR1 upregulation by Cd promoted Ca2+ channel opening and Ca2+ influx, resulting in ER stress and subsequently EMT in HK-2 cells. Inhibition of NMDAR1 by pharmacological antagonist MK-801 significantly attenuated Cd-induced Ca2+ influx, ER stress, and EMT. Pretreatment with the IRE-1α/XBP-1s pathway inhibitor STF-083010 also restored the epithelial phenotype of Cd-treated HK-2 cells. Therefore, our findings suggest that NMDAR1 activation mediates Cd-induced EMT in proximal epithelial cells likely through the IRE-1α/XBP-1s pathway, supporting the idea that NMDAR1 could be a potential therapeutic target for Cd-induced renal damage.

#6827 DI(2-ETHYLHEXYL) PHTHALATE INDUCES RENAL PROXIMAL TUBULAR CELLS TO UNDERGO EPITHELIAL-MESENCHYMAL TRANSITION VIA AHR SIGNALING

Abstract Background and Aims Environmental factors account for the majority of identified risks associated with CKD of uncertain etiology (CKDu). Among the various environmental factors, plasticizer di(2-ethylhexyl) phthalate (DEHP) is widely used in modern life. The exposure to DEHP is extensive but mostly underestimated in the general population. The link between DEHP exposure and renal injuries has been scarcely reported but the underlying mechanisms are largely unknown. This study aimed to examine the mechanisms by which DEHP causes renal tubular injuries. Method Human proximal tubular cells (PTC), HK2 cells, were used as a cellular model to test the central hypothesis of this study. Cells were treated with various concentrations of DEHP at different time points. WST-1 assay was used to examine the impacts of DEHP on cell viability. Bright field microscopy and immunofluorescence were used to analyze the morphological evidence of epithelial-mesenchymal transition (EMT). Western blots were used to investigate the changes in the expression of EMT-related proteins and arylhydrocarbon receptor (AhR) signaling. Inhibitors of AhR signaling were employed to investigate the crucial roles of AhR signaling in DEHP-induced EMT in HK2 cells. Results By using WST-1 assay, we found that DEHP did not affect cell viability when the examined concentration was below 50 μM, which resembled the low-level environmental exposures. However, when the concentration was higher than 100 μM, DEHP reduced the cell viability in a dose-dependent manner. In response to DEHP, HK-2 cells changed from cuboid to spindle shape when cultured with DEHP (25 μM). This morphological evidence became evident when the treatment time was longer than 48 hours. At the protein levels, DEHP resulted in down-regulation of e-cadherin and upregulation of vimentin and α-SMA. DEHP also lead to upregulation of AhR. The inhibitor of AhR signaling, CQDP (chloroquine diphosphate) reversed the DEHP-induced EMT, which were evidenced by morphology and by the expression patterns of EMT-related proteins. Conclusion At cellular level, our results suggested that low-level environmental exposures of DEHP led to EMT in renal PTC via AhR signaling.

Shen Qi Wan attenuates renal interstitial fibrosis through upregulating AQP1.

Renal interstitial fibrosis (RIF) is the crucial pathway in chronic kidney disease (CKD) leading to the end-stage renal failure. However, the underlying mechanism of Shen Qi Wan (SQW) on RIF is not fully understood. In the current study, we investigated the role of Aquaporin 1 (AQP1) in SQW on tubular epithelial-to-mesenchymal transition (EMT). A RIF mouse model induced by adenine and a TGF-β1-stimulated HK-2 cell model were etablished to explore the involvement of AQP 1 in the protective effect of SQW on EMT in vitro and in vivo. Subsequently, the molecular mechanism of SQW on EMT was explored in HK-2 cells with AQP1 knockdown. The results indicated that SQW alleviated kidney injury and renal collagen deposition in the kidneys of mice induced by adenine, increased the protein expression of E-cadherin and AQP1 expression, and decreased the expression of vimentin and α-smooth muscle actin (α-SMA). Similarly, treatmement with SQW-containing serum significantly halted EMT process in TGF-β1 stimulated HK-2 cells. The expression of snail and slug was significantly upregulated in HK-2 cells after knockdown of AQP1. AQP1 knockdown also increased the mRNA expression of vimentin and α-SMA, and decreased the expression of E-cadherin. The protein expression of vimentin increased, while the expression of E-cadherin and CK-18 significantly decreased after AQP1 knockdown in HK-2 cells. These results revealed that AQP1 knockdown promoted EMT. Furthermore, AQP1 knockdown abolished the protective effect of SQW-containing serum on EMT in HK-2 cells. In sum, SQW attentuates EMT process in RIF through upregulation of the expression of AQP1.

MicroRNA-10 Family Promotes the Epithelial-to-Mesenchymal Transition in Renal Fibrosis by the PTEN/Akt Pathway.

Renal fibrosis (RF) is a common reason for renal failure, and epithelial-mesenchymal transition (EMT) is a vital mechanism that promotes the development of RF. It is known that microRNA-10 (miR-10) plays an important role in cancer EMT; however, whether it takes part in the EMT process of RF remains unclear. Therefore, we established an in vivo model of unilateral ureteral obstruction (UUO), and an in vitro model using TGF-β1, to investigate whether and how miR-10a and miR-10b take part in the EMT of RF. In addition, the combinatorial effects of miR-10a and miR-10b were assessed. We discovered that miR-10a and miR-10b are overexpressed in UUO mice, and miR-10a, miR-10b, and miRs-10a/10b knockout attenuated RF and EMT in UUO-treated mouse kidneys. Moreover, miR-10a and miR-10b overexpression combinatorially promoted RF and EMT in TGF-β1-treated HK-2 cells. Inhibiting miR-10a and miR-10b attenuated RF and EMT induced by TGF-β1. Mechanistically, miR-10a and miR-10b suppressed PTEN expression by binding to its mRNA3'-UTR and promoting the Akt pathway. Moreover, PTEN overexpression reduced miR-10a and miR-10b effects on Akt phosphorylation (p-Akt), RF, and EMT in HK-2 cells treated with TGF-β1. Taken together, miR-10a and miR-10b act combinatorially to negatively regulate PTEN, thereby activating the Akt pathway and promoting the EMT process, which exacerbates RF progression.

Exosomes derived from mesenchymal stem cells attenuate diabetic kidney disease by inhibiting cell apoptosis and epithelial-to-mesenchymal transition via miR-424-5p.

Diabetic kidney disease (DKD) is well-acknowledged as one of the most common complications in diabetes mellitus. Recent studies have demonstrated the promising role of mesenchymal stem cell-derived exosomes (MSC-exos) as a cell-free treatment strategy for DKD. The present study sought to investigate the therapeutic potential and the underlying mechanisms of MSC-exos in DKD. The authentication of MSC-exos was validated by western blot, transmission electron microscope (TEM), and nanosight tracking analysis (NTA). Apoptosis was detected by western blot, TUNEL staining, and flow cytometry. Epithelial-to-mesenchymal transition (EMT) was evaluated by western blot and immunofluorescence. The relationship between miR-424-5p and Yes-associated protein 1 (YAP1) was revealed by dual luciferase reporter assay. We observed that MSC-exos could attenuate DKD by decreasing cell apoptosis and inhibiting epithelial-to-mesenchymal transition (EMT) in diabetic kidneys in db/db mice. Besides, we documented that MSC-exos could reverse high glucose-induced apoptosis and EMT in HK2 cells. Interestingly, miR-424-5p derived from MSC-exos could inhibit YAP1 activation in HK2 cells, resulting in alleviation of high glucose-induced cell apoptosis and EMT. Our study provides novel insights into MSC-exos-mediated protective effect in DKD. MSC-exos could inhibit high glucose-induced apoptosis and EMT through miR-424-5p targeting of YAP1.

Mesenchymal Stem Cell-Derived Exosomes Attenuate Epithelial-Mesenchymal Transition of HK-2 Cells.

Renal fibrosis (RF) predisposes patients to an increased risk of progressive chronic kidney disease, and effective treatments remain elusive. Mesenchymal stem cell (MSC)-derived exosomes are considered a new treatment for tissue damage. Our study aimed to investigate the in vitro effects of bone marrow MSC-derived exosomes (BM-MSC-Exs) on transforming growth factor-β1 (TGF-β1)-induced fibrosis in renal tubular epithelial cells (HK-2 cells) and the associated mechanisms. Herein, we found BM-MSC-Exs could inhibit TGF-β1-induced epithelial-mesenchymal transition (EMT) in HK-2 cells, and may involve autophagy activation of BM-MSC-Exs. Moreover, we first reported that after ceria nanoparticles (CeNPs) treatment, the improvements induced by BM-MSC-Ex on EMT were significantly enhanced by upregulating the expression of Nedd4Lof MSCs and promoting the secretion of exosomes, which contained Nedd4L. In addition, Nedd4L could activate autophagy in HK-2 cells. In conclusion, BM-MSC-Ex prevents the TGF-β1-induced EMT of renal tubular epithelial cells by transporting Nedd4L, which activates autophagy. The results of this in vitro experiment may extend to RF, whereby BM-MSC-Ex may also be used as a novel treatment for improving RF. Impact statement Renal fibrosis (RF) is an important pathological change in chronic kidney disease that ultimately leads to end-stage renal failure, and effective treatments remain elusive. In this study, there are two contributions. First, our results suggest that bone marrow mesenchymal stem cell-derived exosomes (BM-MSC-Exs) can prevent transforming growth factor-β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells through Nedd4L trafficking, which activates autophagy. Second, the improvement effects of BM-MSC-Ex on TGF-β1-induced HK-2 EMT can be enhanced by ceria nanoparticles (CeNPs). The findings in this study may be extended to RF: BM-MSC-Exs may be used as a novel treatment to improve RF.

CUX1 promotes epithelial-mesenchymal transition (EMT) in renal fibrosis of UUO model by targeting MMP7

Epithelial-to-mesenchymal transition (EMT) displays a critical role in the development of renal fibrosis, an important pathological process of chronic kidney disease (CKD). Transcription factor Cut-like homeobox 1 (CUX1) has shown profound effects on several kidney diseases. However, its role in CKD has not been understood yet. In this study, unilateral ureteric obstruction (UUO) surgery was performed on male C57BL/6 mice to simulate CKD in vivo. Renal fibrosis was further induced in human proximal tubular epithelial cell (HK-2) by TGF-β1 stimulation. CUX1 and MMP7 were found to be over-expressed in renal tissue of UUO mice. Renal functional analyses and histological assessment indicated that CUX1 knockdown alleviated renal injury in UUO mice. Mitochondrial dysfunction was determined in UUO group and improved after CUX1 silencing. Besides, CUX1 knockdown suppressed EMT in UUO mice and TGF-β1 treated HK-2 cells, as evidenced by reduced expressions of α-SMA, vimentin, fibronectin and augmented abundance of E-cadherin. Furthermore, CUX1 knockdown decreased MMP7 expression by targeting at its promoter region. MMP7 was responsible for the inhibitory effect of CUX1 knockdown on EMT in HK-2 cells. In summary, our findings suggest that CUX1 promotes EMT in CKD by targeting MMP7, and highlight the crucial role of CUX1 in CKD pathogenesis.

The ILEI/LIFR complex induces EMT via the Akt and ERK pathways in renal interstitial fibrosis

BackgroundChronic kidney disease (CKD) is characterized by high morbidity and mortality and is difficult to cure. Renal interstitial fibrosis (RIF) is a major determinant of, and commonly occurs within, CKD progression. Epithelial mesenchymal transition (EMT) has been identified as a crucial process in triggering renal interstitial fibrosis (RIF). Interleukin‐like EMT inducer (ILEI) is an important promotor of EMT; this study aims to elucidate the mechanisms involved.MethodsMale C57BL6/J mouse were randomly divided into 6 groups: sham (n = 10), sham with negative control (NC) shRNA (sham + NC, n = 10), sham with ILEI shRNA (sham + shILEI, n = 10), unilateral ureteral obstruction (UUO, n = 10), UUO with NC (UUO + NC, n = 10) and UUO with ILEI shRNA (UUO + shILEI, n = 10). Hematoxylin and eosin (H&E), Masson, and immunohistochemical (IHC) staining and western blotting (WB) were performed on murine kidney tissue to identify the function and mechanism of ILEI in RIF. In vitro, ILEI was overexpressed to induce EMT in HK2 cells and analyzed via transwell, WB, real-time PCR, and co-immunoprecipitation. Finally, tissue from 12 pediatric CKD patients (seven with RIF and five without RIF) were studied with H&E, Masson, and IHC staining.ResultsOur in vitro model revealed that ILEI facilitates RIF in the UUO model via the Akt and ERK pathways. Further experiments in vivo and in vitro revealed that ILEI promotes renal tubular EMT by binding and activating leukemia inhibitory factor receptor (LIFR), in which phosphorylation of Akt and ERK is involved. We further find markedly increased expression levels of ILEI and LIFR in kidneys from pediatric CKD patients with RIF.ConclusionOur results indicate that ILEI may be a useful biomarker for renal fibrosis and a potential therapeutic target for modulating RIF.

Etoposide-induced protein 2.4 ameliorates high glucose-induced epithelial-mesenchymal transition by activating adenosine monophosphate-activated protein kinase pathway in renal tubular cells.

Epithelial-mesenchymal transition (EMT), known as the transition of tubular epithelial cells into fibroblasts, is one of the potential mechanisms of renal fibrosis, which promotes the development of diabetic kidney disease (DKD). Etoposide-induced protein 2.4 (EI24) is known as an endoplasmic reticulum (ER)-localized Bcl-2-binding transmembrane protein with various functions that can affect autophagy, apoptosis and differentiation. However, whether EI24 is involved in EMT of renal tubular epithelial cells and the exact mechanism is still not known. In this study, we first reported that EI24 expression was significantly downregulated in the kidneys of diabetic mice and in high glucose-stimulated HK2 cells. Knockdown of EI24 led to EMT of HK2 cells, as indicated by decreased E-cadherin and increased α-smooth muscle actin (α-SMA). Meanwhile, overexpression of EI24 ameliorated high glucose-induced EMT of HK2 cells via activation of the adenosine monophosphate-activated protein kinase (AMPK) pathway. Then, DNA methyltransferase (DNMT) inhibitor 5-Aza-2'-deoxycytidine (5-Aza) treatment enhanced EI24 expression and alleviated EMT in high glucose-treated HK2 cells and the kidneys of diabetic mice. Furthermore, DNMT1 and DNMT3a upregulation were found to be involved in the decrease of EI24 in high glucose-stimulated HK2 cells. Silencing of DNMT1 and DNMT3a effectively reversed high glucose-induced downregulation of EI24 and aggravation of EMT. Our findings demonstrate that the DNA methyltransferase-regulated EI24 affects EMT of renal tubular cells via AMPK signaling pathway. It is suggested that EI24 may be a potential therapeutic target for diabetic renal injury.

Arginyl-fructosyl-glucose from red ginseng alleviates TGF-β1-induced epithelial-mesenchymal transition of renal tubular epithelial cells.

This study aims to explore the effect and mechanism of arginyl-fructosyl-glucose (AFG) on TGF-β1-induced epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells. HK-2 cells were induced by TGF-β1 and then co-cultured with AFG at different concentrations (0, 25, 50, and 100 μmol/l) for 48 h. The morphology of HK-2 cells was observed under an inverted microscope and the expressions of α-SMA, Vimentin, and E-cadherin were assessed by qRT-PCR, Western blot, and immunofluorescence. The mRNA expressions of ERK and STAT3 were also examined by qRT-PCR, and the protein levels of ERK, STAT3, p-ERK, and p-STAT3 were measured by Western blot. Finally, CCK-8 and transwell assays were used to detect cell proliferation and invasion. TGF-β1 treatment significantly induced EMT in HK-2 cells. The expressions of p-ERK and p-STAT3 were signally increased after TGF-β1 induction, while Mogrol treatment inhibited p-ERK, p-STAT3, α-SMA, and Vimentin expression levels, enhanced E-cadherin expression, and suppressed cell proliferation and invasion. AFG exposure could also inhibit p-ERK, p-STAT3, α-SMA, and Vimentin expressions, promote E-cadherin expression, and markedly inhibit HK-2 cell proliferation and invasion. AFG inhibited TGF-β1-induced EMT of renal tubular epithelial cells by regulating phosphorylation of ERK and STAT3.

Tanshinone IIA attenuates high glucose-induced epithelial-to-mesenchymal transition in HK-2 cells through VDR/Wnt/β-catenin signaling pathway.

The progression of diabetic kidney disease (DKD) is closely related to renal tubular epithelial- to-mesenchymal transition (EMT) and tubulointerstitial fibrosis. Tanshinone IIA (TSIIA), extracted from a traditional Chinese medicine named Salvia miltiorrhiza, has been proved to have anti-fibrosis effects. The aim of this study was to investigate the effect of TSIIA on high glucose-induced EMT in human proximal tubular cells (HK-2 cells) and its possible mechanism. The proliferation of cells exposed to different concentrations of glucose was measured by light microscopy and CCK-8 test. The cells were stimulated with 30 mM glucose and different concentrations of TSIIA (5 μM or 10 μM) for 48 h. Vitamin D receptor (VDR)-siRNA was used to transfect cells, and high glucose and TSIIA treatment were further used to treat cells. The expression of alpha smooth muscle actin (a-SMA) mRNA was detected by qPCR to ensure successful induction of EMT, and the expression of VDR mRNA was detected by qPCR to ensure successful transfection of VDR-siRNA. Protein expression of a-SMA, E-cadherin, VDR, b-catenin and glycogen synthase kinase 3b (GSK-3b) was detected by Western blot analysis. The results showed that high glucose concentration inhibited cell proliferation and promoted EMT in HK-2 cells. TSIIA could reverse high glucose-induced EMT by increasing the level of VDR protein and inhibiting the levels of b-catenin and GSK-3b proteins suggestive of a negative correlation between VDR and the Wnt/b-catenin pathway. After VDR-siRNA transfection and incubation of cells at high glucose concentration, the inhibitory effect of VDR on the expression of b-catenin and GSK-3b of Wnt pathway was suppressed and the b-catenin pathway was activated. When VDR level was restored by TSIIA, the inhibitory effect of VDR on the pathway was also restored and the activation of the pathway was suppressed. TSIIA was able to attenuate high glucose-induced EMT in HK-2 cells by up-regulating VDR levels, which might be related to the inhibitory effect of VDR on the Wnt pathway.

Involvement of Mechanosensitive Channel Piezo1 in Renal Fibrosis

Renal fibrosis is the final common pathway of numerous progressive kidney diseases, characterized by deposition of extracellular matrix in the tubulointerstitial areas, leading to scar formation and increased tissue stiffness of kidney. The underlying mechanisms of renal fibrosis are not fully identified. Piezo1 is known as a mechanosensitive nonselective cation channel, playing an important role in many physiological and pathological processes. The purpose of the current study is to investigate the role of Piezo1 in renal fibrosis and potential molecular mechanism. In human fibrotic kidneys, Piezo1 protein expression was markedly upregulated. The abundance of Piezo1 protein in kidneys of mice was instantly increased after 30 minutes of unilateral ureteral obstruction (UUO), and persistently increased at the 3rd and the 7th days after UUO. Inhibition of Piezo1 with GsMTx4 dramatically ameliorated UUO-induced renal fibrosis. Mechanical stretch induced Piezo1 activation and epithelial-mesenchymal transition (EMT) in human proximal tubular HK2 cells, as seen in increased abundance of a fibrotic marker fibronectin and decreased expression of an epithelial marker E-cadherin, which were greatly reversed by inhibition or silence of Piezo1. Similarly, in HK2 cells, TGF-β1-induced EMT was significantly inhibited by GsMTx4, whereas a specific Piezo1 agonist Yoda1 markedly induced EMT. In addition, the increased expression of Piezo1 protein was found when HK2 cells were cultured in stiff polyacrylamide hydrogels, mimicking the natural accumulation of ECM. Stimulation of Piezo1 by Yoda1 caused calcium influx in association with activation of calpain2 and integrinβ1. Also, Yoda1 promoted an interaction between ECM and integrinβ1 in HK2 cells. In conclusion, activation of Piezo1 is involved in the development of renal fibrosis and EMT in HK2 cells, likely through calcium-calpain2-integrinβ1 pathway.

Carvacrol Ameliorates Transforming Growth Factor-β1-Induced Extracellular Matrix Deposition and Reduces Epithelial-Mesenchymal Transition by Regulating The Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway In Hk-2 Cells

Transforming growth factor-β1 promotes excessive extracellular matrix deposition and epithelial-mesenchymal transition of tubular epithelial cells, thus stimulating the progression of renal fibrosis. Carvacrol has been shown to alleviate cardiac and liver fibrosis and attenuate renal injury. However, the role of carvacrol on renal fibrosis has not been examined. First, measurements using Cell Counting Kit-8 showed that carvacrol reduced cell viability of tubular epithelial cell line HK-2 in a dose-dependent fashion. Second, transforming growth factor-β1 induced excessive extracellular matrix deposition in HK-2 cells with enhanced collagen I, collagen IV, and fibronectin expression. However, carvacrol decreased the expression of collagen I, collagen IV in a dose-dependent manner and fibronectin to attenuate the extracellular matrix deposition in HK-2. Third, carvacrol attenuated TGF-β1-induced decrease of E-cadherin and increase of snail, vimentin, and alpha-smooth muscle actin in HK-2 cells. Transforming growth factor-β1-induced increase in PI3K and AKT phosphorylation in HK-2 were also reversed by carvacrol. Collectively, carvacrol ameliorates renal fibrosis through inhibition of transforming growth factor-β1-induced extracellular matrix deposition and epithelial-mesenchymal transition of HK-2 cells, providing potential therapy for the treatment of renal fibrosis.

Regulation of connective tissue growth factor expression by miR-133b for the treatment of renal interstitial fibrosis in aged mice with unilateral ureteral obstruction

IntroductionRenal interstitial fibrosis, an important pathological feature of kidney aging and chronic renal failure, is regulated by mesenchymal stem cells (MSCs). We have previously demonstrated low expression of miR-133b in MSC-derived extracellular vesicles (MSC-EVs) in aged rats. However, miR-133b can mediate the inhibition of epithelial-mesenchymal transition (EMT) of renal tubules induced by transforming growth factor-β1 (TGF-β1). We investigated the effect of miR-133b for the treatment of geriatric renal interstitial fibrosis and evaluated its target genes.MethodsWe performed real-time polymerase chain reaction to detect miR-133b expression induced during EMT of HK2 cells by TGF-β1 at different concentrations (0, 6, 8, and 10 ng/mL) and at different time points (0, 24, 48, and 72 h). The target genes of miR-133b were validated using the dual-luciferase reporter assay. In vitro experiments were performed to evaluate mRNA and protein expression of miR-133b targets, E-cadherin, α-smooth muscle actin (SMA), fibronectin, and collagen 3A1 (Col3A1), in HK2 cells transfected with miR-133b under TGF-β1 stimulation. A 24-month-old unilateral ureteral obstruction (UUO) mouse model was established and injected with transfection reagent and miR-133b into the caudal vein. The target gene of miR-133b and other parameters mentioned above such as mRNA and protein expression levels and renal interstitial fibrosis were detected at 7 and 14 days.ResultsmiR-133b expression gradually decreased with an increase in TGF-β1 concentration and treatment time, and the miR-133b mimic downregulated connective tissue growth factor (CTGF) expression. The dual-luciferase reporter assay confirmed CTGF as a direct target of miR-133b. Transfection of the miR-133b mimic inhibited TGF-β1-induced EMT of HK2 cells; this effect was reversed by CTGF overexpression. miRNA-133b expression significantly increased (approximately 70–100 times) in mouse kidney tissues after injection of the miRNA-133b overexpression complex, which significantly alleviated renal interstitial fibrosis in mice with UUO.ConclusionmiR-133b exerted targeted inhibitory effects on CTGF expression, which consequently reduced TGF-β1-induced EMT of HK2 cells and renal interstitial fibrosis in aged mice with UUO.

Protective effect of miRNA-containing extracellular vesicles derived from mesenchymal stromal cells of old rats on renal function in chronic kidney disease

IntroductionMesenchymal stromal cells (MSCs) play an important role in the prevention of cell and tissue fibrosis. Senescence may decrease the function of MSCs during recovery from tissue and organ damage. Extracellular vesicles (EVs) released from MSCs contribute to the repair of kidney injury. We explored the influence of senescence on EVs derived from MSCs (MSC-EVs) and detected the protective effects of MSC-EVs expressing low levels of miR-294/miR-133 derived from old rats against chronic kidney disease (CKD).MethodsThe effects of MSC-EVs derived from 3-month-old and 18-month-old male Fisher 344 rats on renal fibrosis were explored in a unilateral ureteral obstruction (UUO) model. pLV-miR-294/pLV-miR-133 mimic/inhibitor were injected into young and old rats before UUO to detect the effects of miR-294/miR-133, which were decreased in MSC-EVs and sera from old rats, on renal function in CKD. Transforming growth factor-β1 (TGF-β1)-induced human renal proximal tubular epithelial (HK2) cells were used to imitate the pathological process of renal fibrosis in vitro. Western blotting was used to assess the expression of epithelial/mesenchymal markers and phosphorylation of proteins in HK2 cells.ResultsThe inhibition of UUO-induced CKD by MSC-EVs was weaker in old rats than in young rats. Downregulation of miRNAs (miR-294 and miR-133) in both MSC-EVs and sera from old rats obviously attenuated UUO-induced renal injury in old rats. miR-294 and miR-133 overexpression mitigated TGF-β1-mediated epithelial-mesenchymal transition (EMT) in HK2 cells, and the obvious increase in the phosphorylation of both SMAD2/3 and ERK1/2 induced by TGF-β1 was prevented in miR-294- and miR-133-overexpressing HK2 cells.ConclusionsThe ability of MSC-EVs to inhibit renal fibrosis decreased with age. miR-294/miR-133 in MSC-EVs and sera had an important effect on renal fibrosis in old rats and on EMT in HK2 cells. Furthermore, miR-294/miR-133 overexpression prevented SMAD2/3 and ERK1/2 phosphorylation in HK2 cells during TGF-β1-mediated EMT. These findings show that miR-294/miR-133 may be therapeutic in renal fibrosis and related renal dysfunction in elderly individuals.

Dexmedetomidine Attenuates High Glucose-induced HK-2 Epithelial-mesenchymal Transition by Inhibiting AKT and ERK

ObjectiveTo explore the protective effects of dexmedetomidine (Dex) against high glucose-induced epithelial-mesenchymal transition in HK-2 cells and relevant mechanisms. MethodsHK-2 cells were exposed to either glucose or glucose+Dex for 6 h. The production of ROS, morphology of HK-2 cells, and cell cycle were detected. Moreover, the expression of AKT, p-AKT, ERK, p-ERK, PI3K, E-Cadherin, Claudin-1, and α-SMA were determined and compared between HK-2 cells exposed to glucose and those exposed to both glucose and Dex with or without PI3K/AKT pathway inhibitor LY294002 and ERK pathway inhibitor U0126. ResultsCompared with HK-2 cells exposed to high level of glucose, the HK-2 cells exposed to both high level of glucose and Dex showed: (1) lower level of ROS production; (2) cell morphology was complete; (3) more cells in G1 phase; (4) lower expression of p-AKT, p-ERK and α-SMA, higher expression of E-Cadherin and Claudin-1. PI3K/AKT inhibitor LY294002 and ERK inhibitor U0126 decreased the expression of p-AKT, p-ERK and α-SMA, and increased the expression of E-Cadherin and Claudin-1. ConclusionDex can attenuate high glucose-induced HK-2 epithelial-mesenchymal transition by inhibiting AKT and ERK.