FOXO3a Protects against Kidney Injury in Type II Diabetic Nephropathy by Promoting Sirt6 Expression and Inhibiting Smad3 Acetylation.

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease. Although numerous reports have demonstrated a correlation between epithelial-mesenchymal transition (EMT) and renal fibrosis, how these processes lead to tubular dysfunction remains unclear. Here, we show that FOXO3a protects kidneys from injury in type II DN by increasing Sirt6 expression, which deacetylates Smad3 and inhibits its transcriptional activity. The results showed that progressive EMT in the kidneys from db/db mice is associated with Sirt6 downregulation and involved in tubular injury and dysfunction. The reduction of Sirt6 levels in db/db mice resulted in progressive kidney injury, indicating the protective role of Sirt6. Furthermore, Sirt6 was shown to directly bind to Smad3, a key downstream mediator of TGF-ß, and could deacetylate it to inhibit its nuclear accumulation and transcriptional activity in HK2 cells. Besides, we demonstrate that FOXO3a activates Sirt6 expression by binding to its promoter. shRNA-induced FOXO3a knockdown in the kidneys of db/db mice exacerbated tubular injury and renal function loss. Mechanistically, FOXO3a protects against kidney injury in type II DN through the Sirt6/Smad3 axis. Thus, the pharmacological targeting of FOXO3a-mediated Sirt6/Smad3 signaling pathways may provide a novel strategy for treating type II DN.

Ameliorative effects of miR-186 on cisplatin-triggered acute kidney injury via targeting ZEB1.

Cisplatin is a commonly used chemotherapy drug in cancers, which can lead to acute kidney injury (AKI). AKI can occur in almost one third of tumor patients, who receive cisplatin treatment. microRNAs (miRNAs) are significant tools in regulating the expression of crucial factors in multiple diseases, but little is known about their biological roles in AKI. As exhibited, miR-186 has been observed to be down-regulated in tumors. Our study concentrated on the function of miR-186 in cisplatin-triggered AKI. Here, we reported miR-186 was considerably decreased in the serum samples from AKI patients compared with those from the healthy controls. Additionally, we found in NRK-52E cells exposed to 6 mM cisplatin, miR-186 was greatly decreased time-dependently. Meanwhile, an AKI model in rats was successfully set in our study. Levels of serum creatinine and blood urea nitrogen were significantly induced by cisplatin exposure. In AKI rat models, miR-186 exhibited a rapid decrease in both the serum and the kidney tissues. Then, miR-186 overexpression improved NRK-52E cell proliferation and protected NRK-52E cells against cisplatin-triggered apoptosis. Furthermore, ZEB1 was identified and confirmed as a target gene of miR-186. It has been demonstrated that ZEB1 exerts crucial roles in the development of AKI. As evidenced in our current study, ZEB1 was remarkably elevated in AKI patients and AKI rat models. Moreover, ZEB1 was induced by indicated doses of cisplatin in different time periods in NRK-52E cells. ZEB1 inhibition rescued the reduced proliferation and increased apoptosis of NRK-52E cells. In conclusion, loss miR-186 expression contributed to cisplatin-induced AKI, partly through targeting ZEB1. miR-186 might be provided as an effective biomarker for AKI via targeting ZEB1.

Circ_LARP4 regulates high glucose-induced cell proliferation, apoptosis, and fibrosis in mouse mesangial cells.

The current study aimed to investigate the role and underlying mechanisms of circ_LARP4 in diabetic nephropathy (DN). Here, mouse mesangial cells (SV40-MES13) were cultured with 30 mM glucose to establish a DN cellular model. The qRT-PCR results indicated that circ_LARP4 expression was downregulated in the DN cellular model compared to that in the control cells. As determined by an MTT assay, circ_LARP4 overexpression via the circ_LARP4 overexpression (OE) plasmids inhibited the cell proliferation rate. As determined by an Annexin V/PI kit and flow cytometry, circ_LARP4 overexpression increased the cell apoptosis rate. As measured by Western blot, circ_LARP4 overexpression enhanced BAX expression but reduced Bcl-2 expression, also suggesting an enhancement of cell apoptosis. Moreover, regarding cell fibrosis, circ_LARP4 overexpression reduced the mRNA levels of fibrosis markers, including fibronectin, collagen I and collagen IV. Interestingly, miR-424 was found to be reduced in the DN cellular model after transfection with the circ_LARP4 OE plasmids. In addition, restoration of miR-424 expression with the miR-424 mimics reversed the negative effects of circ_LARP4 overexpression on cell proliferation and fibrosis. In conclusion, circ_LARP4 was lower in the DN cellular model than in normal cells, and circ_LARP4 overexpression resulted in decreased cell proliferation and cell fibrosis but increased cell apoptosis in the DN cellular model by sponging miR-424.

LINC00052 ameliorates acute kidney injury by sponging miR-532-3p and activating the Wnt signaling pathway.

Acute kidney injury (AKI) is a complex renal disease. Long non-coding RNAs (lncRNAs) have frequently been associated with AKI. In the present study, we aimed to investigate the molecular mechanism(s) of LINC00052 in AKI. We found that LINC00052 expression was significantly decreased in AKI patient serum. In addition, in a hypoxic AKI cell model, LINC00052 expression was strongly elevated. In an I/R-triggered AKI rat model, the expression of TNF-α, IL-6 and IL-1ß mRNA was strongly elevated. Moreover, we predicted miR-532-3p to be targeted by LINC00052 in AKI. Overexpression of LINC00052 increased hypoxia-induced inhibition of NRK-52E cell proliferation and reversed hypoxia-triggered apoptosis. Furthermore, we found that induction of TNF-α, IL-6 and IL-1ß was repressed by overexpression of LINC00052. LINC00052 decreased hypoxia-induced ROS and MDA accumulation in vitro and increased SOD activity. Decreased levels of c-myc and cyclin D1 were observed in renal tissues of AKI rats. Lastly, Wnt/ß-catenin signaling was inactivated in NRK-52E cells experiencing hypoxia, and LINC00052 upregulation reactivated Wnt/ß-catenin signaling by sponging miR-532-3p. Taken together, these results suggest that LINC00052 ameliorates AKI by sponging miR-532-3p and activating Wnt signaling.

Exosomal circ_DLGAP4 promotes diabetic kidney disease progression by sponging miR-143 and targeting ERBB3/NF-κB/MMP-2 axis.

Diabetic kidney disease (DKD) is closely associated with the high risk of cardiovascular disease and mortality. Exosomal circRNAs can exert significant roles in the pathology of various diseases. Nevertheless, the role of exosomal circRNAs in DKD progression remains barely known. Circular RNA DLGAP4 has been reported to be in involved in acute ischemic stroke. In our study, we found exosomal circ_DLGAP4 was increased in the exosomes isolated from HG-treated mesangial cells (MCs), DKD patients, and DKD rat models compared with the corresponding normal subjects. Then, we observed that exo-circ_DLGAP4 significantly promoted proliferation and fibrosis of MCs cells. Moreover, to study the underlying mechanism of circ_DLGAP4 in regulating DKD, bioinformatics method was consulted and miR-143 was predicted as its target. The direct correlation between miR-143 and circ_DLGAP4 was validated in MCs. MCs proliferation and fibrosis were increased by circ_DLGAP4, which could be decreased by mimic-miR-143. Next, elevated expression of Erb-b2 receptor tyrosine kinase 3 (ERBB3) is involved in various diseases. However, the function of ERBB3 in DKD development remains poorly known. Next, ERBB3 was predicted as the downstream target for miR-143. It was displayed that circ_DLGAP4 promoted proliferation and fibrosis of MCs by sponging miR-143 and regulating ERBB3/NF-κB/MMP-2 axis. Meanwhile, the loss of exo-circ_DLGAP4 induced miR-143 and repressed ERBB3/NF-κB/MMP-2 expression in MCs. Subsequently, in vivo assays were performed and it was proved that overexpression of circ_DLGAP4 markedly promoted DKD progression in vivo via modulating miR-143/ERBB3/NF-κB/MMP-2. In conclusion, we indicated that exosomal circ_DLGAP4 could prove a novel insight for DKD development.

Loss of lncRNA MIAT ameliorates proliferation and fibrosis of diabetic nephropathy through reducing E2F3 expression.

Diabetic nephropathy (DN) is a serious kidney disease resulted from diabetes. Dys-regulated proliferation and extracellular matrix (ECM) accumulation in mesangial cells contribute to DN progression. In this study, we tested expression level of MIAT in DN patients and mesangial cells treated by high glucose (HG). Up-regulation of MIAT was observed in DN. Then, functional assays displayed that silence of MIAT by siRNA significantly repressed the proliferation and cycle progression in mesangial cells induced by HG. Meanwhile, we found that collagen IV, fibronectin and TGF-ß1 protein expression was obviously triggered by HG, which could be rescued by loss of MIAT. Then, further assessment indicated that MIAT served as sponge harbouring miR-147a. Moreover, miR-147a was decreased in DN, which exhibited an antagonistic effect of MIAT on modulating mesangial cell proliferation and fibrosis. Moreover, bioinformatics analysis displayed that E2F transcription factor 3 (E2F3) could act as direct target of miR-147a. We demonstrated that E2F3 was greatly increased in DN and the direct binding association between miR-147a and E2F3 was evidenced using luciferase reporter assay. In summary, our data explored the underlying mechanism of DN pathogenesis validated that MIAT induced mesangial cell proliferation and fibrosis via sponging miR-147a and regulating E2F3.

Correlation of TNF-α and IL-10 gene polymorphisms with primary nephrotic syndrome.

The study explored the correlations of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) gene polymorphisms with susceptibility and the condition of primary nephrotic syndrome. A total of 200 patients with primary nephrotic syndrome in Qilu hospital were collected as disease group, and 200 healthy people were selected as control group. Genomic deoxyribonucleic acids (DNAs) of nucleated cells in the peripheral blood were extracted to detect the gene polymorphisms of TNF-α (rs1799724 and rs1800629) and IL-10 (rs1800872 and rs141219090). Allele distributions at rs1799724 (P=0.003) and rs1800629 (P=0.011) of TNF-α gene and at rs1800872 (P=0.033) of IL-10 gene in disease group were different from those in control group. In the disease group, allele C frequency at rs1799724 and allele A frequency at rs1800629 of TNF-α gene and allele T frequency at rs1800872 of IL-10 gene were higher. There were differences between rs1799724 (P=0.007) and rs1800629 (P=0.002) of TNF-α gene. In addition, there was a difference in the frequency of the dominant model of TNF-α gene rs1800629 between disease group and control group (P=0.035), and the frequency of dominant model GG+GA was remarkably lower in the disease group. Additionally, TT genotype at rs1799724 of TNF-α gene was obviously related to the plasma TNF-α level (P<0.05), and the plasma TNF-α level was significantly increased in disease group. AA genotype at rs141219090 of IL-10 gene had a notable correlation with the plasma IL-10 level (P<0.05), and the plasma IL-10 level in disease group was markedly raised. Additionally, CT genotype at rs1799724 of TNF-α gene was related to the 24-h urine protein level (P=0.035), GG genotype at rs1800872 of IL-10 gene was associated with the plasma albumin level (P=0.031), and GG genotype at rs141219090 was related to the serum creatinine level (P=0.047). TNF-α and IL-10 gene polymorphisms are predominantly correlated with the susceptibility and the condition of primary nephrotic syndrome.

Circ-AKT3 inhibits the accumulation of extracellular matrix of mesangial cells in diabetic nephropathy via modulating miR-296-3p/E-cadherin signals.

Diabetic nephropathy is a leading cause of end-stage renal disease globally. The vital role of circular RNAs (circRNAs) has been reported in diabetic nephropathy progression, but the molecular mechanism linking diabetic nephropathy to circRNAs remains elusive. In this study, we investigated the significant function of circ-AKT3/miR-296-3p/E-cadherin regulatory network on the extracellular matrix accumulation in mesangial cells in diabetic nephropathy. The expression of circ-AKT3 and fibrosis-associated proteins, including fibronectin, collagen type I and collagen type IV, was assessed via RT-PCR and Western blot analysis in diabetic nephropathy animal model and mouse mesangial SV40-MES13 cells. Luciferase reporter assays were used to investigate interactions among E-cadherin, circ-AKT3 and miR-296-3p in mouse mesangial SV40-MES13 cells. Cell apoptosis was evaluated via flow cytometry. The level of circ-AKT3 was significantly lower in diabetic nephropathy mice model group and mouse mesangial SV40-MES13 cells treated with high-concentration (25 mmol/L) glucose. In addition, circ-AKT3 overexpression inhibited the level of fibrosis-associated protein, such as fibronectin, collagen type I and collagen type IV. Circ-AKT3 overexpression also inhibited the apoptosis of mouse mesangial SV40-MES13 cells treated with high glucose. Luciferase reporter assay and bioinformatics tools identified that circ-AKT3 could act as a sponge of miR-296-3p and E-cadherin was the miR-296-3p direct target. Moreover, circ-AKT3/miR-296-3p/E-cadherin modulated the extracellular matrix of mouse mesangial cells in high-concentration (25 mmol/L) glucose, inhibiting the synthesis of related extracellular matrix protein. In conclusion, circ-AKT3 inhibited the extracellular matrix accumulation in diabetic nephropathy mesangial cells through modulating miR-296-3p/E-cadherin signals, which might offer novel potential opportunities for clinical diagnosis targets and therapeutic biomarkers for diabetic nephropathy.

Downregulation of XIST ameliorates acute kidney injury by sponging miR-142-5p and targeting PDCD4.

Acute kidney injury (AKI) is a common kidney disease that markedly affects public health. To date, the roles of long noncoding RNA XIST in AKI are poorly understood. Here, we investigated the biological functions of XIST in AKI. We observed that XIST expression increased in patients with AKI and HK-2 cells stimulated by CoCl2 . In addition, a rat AKI model induced by ischemia-reperfusion was established. Tumor necrosis factor-α, interleukin-6, and cyclooxygenase-2 messenger RNA expression were induced in vivo; moreover, XIST expression was upregulated. Knockdown of XIST significantly repressed CoCl2 -triggered injury in HK-2 cells. However, microRNA (miR)-142-5p, a downstream target of XIST, was downregulated in AKI. miR-142-5p was repressed by XIST and miR-142-5p could inhibit CoCl2 -induced injury in HK-2 cells. Moreover, PDCD4 expression was significantly increased in AKI. PDCD4 was predicted to be the target of miR-142-5p. Subsequently, loss of PDCD4 was able to retard injury in HK-2 cells exposed to CoCl2. Thus, we suggest that XIST regulates miR-142-5p and PDCD4, and it has the potential to function as a biomarker in therapeutic strategies for AKI.

Long noncoding RNA Gm6135 functions as a competitive endogenous RNA to regulate toll-like receptor 4 expression by sponging miR-203-3p in diabetic nephropathy.

We aim to explore the relationship between Gm6135 and diabetic nephropathy. We detected the relative expression levels of Gm6135 and toll-like receptor 4 (TLR4) in diabetic nephropathy mice and high-glucose-cultured mouse mesangial cells SV40-MES-13 by the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot detection. Cell proliferation and apoptosis were detected after small interfering RNA (siRNA) interference or plasmid overexpression of Gm6135/TLR4, and bioinformatics method was used to predict and screen miR-203 as an intermediate factor. Through dual-luciferase reporter gene, RNA pull-down, qRT-PCR, and western blot, the binding relationship between Gm6135, miR-203-3p, and TLR4 was confirmed. The possibility of the competing endogenous RNA mechanism was demonstrated by cell localization assays and rip assays. Finally, the proliferation of mouse mesangial cells SV40-MES-13 was detected after mimics and inhibitor of microRNA, which were reversed with TLR4 overexpression and siRNA. The results showed that the relative expression levels of Gm6135 and TLR4 in the kidney and high-glucose-cultured mouse mesangial cells of diabetic nephropathy mice increased significantly. Overexpression or downregulation of Gm6135/TLR4 significantly affected the proliferation and apoptosis of mouse mesangial cells. Gm6135 upregulates TLR4 by competitively binding to miR-203-3p.

LncRNA NEAT1 promotes extracellular matrix accumulation and epithelial-to-mesenchymal transition by targeting miR-27b-3p and ZEB1 in diabetic nephropathy.

Diabetic nephropathy (DN) is a kind of microvascular complications of diabetes. Long noncoding RNAs (lnRNAs) can participate in the development of various diseases, including DN. However, the function of lncRNA NEAT1 is unclear. In our present study, we reported that NEAT1 was significantly increased in streptozotocin-induced DN rat models and high-glucose-induced mice mesangial cells. We observed that knockdown of NEAT1 greatly inhibited renal injury of DN rats. Meanwhile, downregulation of NEAT1-modulated extracellular matrix (ECM) proteins (ASK1, fibronectin, and TGF-ß1) expression and epithelial-mesenchymal transition (EMT) proteins (E-cadherin and N-cadherin) in vitro. Previously, miR-27b-3p has been reported to be involved in diabetes. Here, miR-27b-3p was decreased in DN rats and high-glucose-induced mice mesangial cells. The direct correlation between NEAT1 and miR-27b-3p was validated using the dual-luciferase reporter assay and RNA immunoprecipitation experiments. In addition, zinc finger E-box binding homeobox 1 (ZEB1), which has been identified in the process of EMT clearly contributes to EMT progression. ZEB1 was predicted as a target of miR-27b-3p and overexpression of miR-27b-3p dramatically repressed ZEB1 expression. Therefore, our data implied the potential role of NEAT1 in the fibrogenesis and EMT in DN via targeting miR-27b-3p and ZEB1.

Suppression of CIP4/Par6 attenuates TGF-ß1-induced epithelial-mesenchymal transition in NRK-52E cells.

Transforming growth factor-ß (TGF-ß) induces epithelial-mesenchymal transition (EMT) primarily via a Smad­dependent mechanism. However, there are few studies available on TGF-ß-induced EMT through the activation of non­canonical pathways. In this study, the Cdc42-interacting protein-4 (CIP4)/partitioning-defective protein 6 (Par6) pathway was investigated in TGF-ß1­stimulated NRK-52E cells. Rat NRK-52E cells were obtained and stimulated with TGF-ß1. The expression levels of E-cadherin, α-smooth muscle actin (α-SMA) and CIP4 were then examined by western blot analyses. Rat NRK-52E cells were transfected with Par6 or CIP4 small interfering RNA (siRNA), and scrambled siRNA as controls. The cells were incubated with 20 ng/ml of TGF-ß1 for 72 h in order to observe the effects of Par6 and CIP4 silencing. Confocal fluorescence microscopy was also applied to reveal the expression and distribution of E-cadherin, α-SMA, Par6 and CIP4. The results demonstrated that E-cadherin expression was decreased, and α-SMA expression was increased in the TGF-ß1­stimulated cells. Simultaneously, the increased expression of CIP4 and p-Par6 was confirmed by western blot analyses. The results of confocal fluorescence microscopy revealed that rat CIP4 exhibited cluster formations located adjacent to the cell periphery; however, as for the protein expression and distribution of Par6, there was no obvious difference between the control cells and cells exposed to TGF-ß1. siRNA molecules capable of CIP4 and Par6 knockdown were used to demonstrate reversed TGF-ß1­induced EMT. Moreover, CIP4 loss of function reversed the increase in p-Par6 protein expression in the TGF-ß1­stimulated NRK-52E cells. A similar result was observed with the decreased CIP4 protein expression due to Par6 loss of function. Our data thus suggest that the CIP4/Par6 complex plays an important role in the occurrence of EMT in TGF-ß1-stimulated NRK-52E cells. The underlying mechanisms are mediated, at least in part, through the upregulation of CIP4, which occurrs due to stimulation with TGF-ß1; subsequently, CIP4 increases the phosphorylation of Par6, which accelerates the process of EMT.

Cdc42-interacting protein-4 promotes TGF-Β1-induced epithelial-mesenchymal transition and extracellular matrix deposition in renal proximal tubular epithelial cells.

Cdc42-interacting protein-4 (CIP4) is an F-BAR (Fer/CIP4 and Bin, amphiphysin, Rvs) family member that regulates membrane deformation and endocytosis, playing a key role in extracellular matrix (ECM) deposition and invasion of cancer cells. These processes are analogous to those observed during the initial epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells. The role of CIP4 in renal tubular EMT and renal tubulointerstitial fibrosis was investigated over the course of the current study, demonstrating that the expression of CIP4 increased in the tubular epithelia of 5/6-nephrectomized rats and TGF-ß1 treated HK-2 cells. Endogenous CIP4 evidenced punctate localization throughout the cytosol, with elevated levels observed in the perinuclear region of HK-2 cells. Subsequent to TGF-ß1 treatment, CIP4 expression increased, forming clusters at the cell periphery that gradually redistributed into the cytoplasm. Simultaneously, EMT induction in cells was confirmed by the prevalence of morphological changes, loss of E-cadherin, increase in α-SMA expression, and secretion of fibronectin. Overexpression of CIP4 promoted characteristics similar to those commonly observed in EMT, and small interfering RNA (siRNA) molecules capable of CIP4 knockdown were used to demonstrate reversed EMT. Cumulatively, results of the current study suggest that CIP4 promotes TGF-ß1-induced EMT in tubular epithelial cells. Through this mechanism, CIP4 is capable of inducing ECM deposition and exacerbating progressive fibrosis in chronic renal failure.

Erbin inhibits TGF-ß1-induced EMT in renal tubular epithelial cells through an ERK-dependent pathway.

Epithelial-to-mesenchymal transition (EMT) plays a crucial role in the progression of renal interstitial fibrosis, which finally leads to renal failure. Erbin, a member of LAP family, is recently reported to inhibit Smads and ERK pathway which are two important types of intracellular signaling involved in TGF-ß1-induced EMT. However, the role of Erbin in the regulation of EMT and the underlying mechanisms remain to be fully understood. To that end, we aimed to evaluate the expression of Erbin in renal interstitial fibrosis and the potential role of Erbin in tubular EMT stimulated by TGF-ß1. In this study we demonstrated that the expression of Erbin was upregulated in the tubular epithelia of 5/6-nephrectomized rats. We also showed here that TGF-ß1 upregulated Erbin expression in NRK52E cells during their EMT phenotype acquisition. Importantly, elevated expression of Erbin inhibited ERK signaling and partial reversed EMT stimulated by TGF-ß1. In the mean time, reducing Erbin expression enhanced ERK phosphorylation, promoted the E-cadherin suppression, and induced α-SMA expression and fibronection secretion in response to TGF-ß1, which could be rescued if cells were treated with the inhibitor of MEK1/2 U0126. However, in the absence of TGF-ß1, Erbin failed to affect ERK activation and EMT process. These results suggest that Erbin is a negative feedback molecule induced by TGF-ß1 and inhibits TGF-ß1-induced EMT via ERK signaling pathway.

Upregulated DJ-1 promotes renal tubular EMT by suppressing cytoplasmic PTEN expression and Akt activation.

Recently, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is suggested as a new agent in the fighting against fibrogenesis. In tumor, DJ-1 is identified as a negative regulator of PTEN. But the expression of DJ-1 and the regulation of PTEN in fibrosis are unclear. Renal fibrosis was induced in 5/6 subtotal nephrectomy rat model. Human proximal tubular epithelial cells (HKC) were treated with transforming growth factor-beta 1 (TGF-ß1), or transfected with DJ-1 or PTEN. Confocal microscope was used to investigate the localization of DJ-1 and PTEN. The selective phosphoinositide-3 kinase (PI3K) inhibitor, LY294002, was administered to inhibit PI3K pathway. The DJ-1 and PTEN expression, markers of epithelial-mesenchymal transition (EMT) and Akt phosphorylation were measured by RT-PCR, Western blotting or immunocytochemistry. In vitro, after HKC cells were stimulated with 10 ng/mL TGF-ß1 for 72 h, the expression of DJ-1 was increased, and that of PTEN was decreased. In vivo, the same results were identified in 5/6-nephrectomized rats. In normal HKC cells, most of DJ-1 protein localized in cytoplasm, and little in nucleus. TGF-ß1 upregulated DJ-1 expression in both cytoplasma and nuclei. In contrary, TGF-ß1 emptied cytoplasmic PTEN protein into nucleus. Overexpression of DJ-1 decreased the expression of PTEN, promoted the activation of Akt and the expression of vimentin, and also led to the loss of cytoplasmic PTEN. Contrarily, overexpression of PTEN protected HKC cells from TGF-ß1-induced EMT. In conclusion, DJ-1 is upregulated in renal fibrosis and DJ-1 mediates EMT by suppressing cytoplasmic PTEN expression and Akt activation.

Role of Sema4C in TGF-ß1-induced mitogen-activated protein kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells.

BACKGROUND: The p38 mitogen-activated protein kinase (p38 MAPK) is an important intracellular signal transduction pathway involved in TGF-ß1-induced epithelial-mesenchymal transition (EMT). Sema4C, a member of the semaphorin family, was found to be essential for the activation of p38 MAPK. However, the role of Sema4C in promoting TGF-ß1-induced EMT is unclear. METHODS: Renal fibrosis was induced by 5/6 subtotal nephrectomy rat model. In vitro, Sema4C was induced in human proximal tubular epithelial cells (HKC) by treatment with TGF-ß1, or was inhibited by siRNA or was over-expressed by Sema4C transfection. The selective p38 MAPK inhibitor, SB203580, was administered to inhibit the p38 pathway. The expression of Sema4C, the markers of EMT, p38 phosphorylation and fibronectin secretion were measured by western blotting, immunohistochemistry, immunocytochemistry or enzyme-linked immunosorbent assay. RESULTS: The expression of Sema4C increased in HKC cells that were treated with TGF-ß1. Knockdown of Sema4C potently inhibited phosphorylation of p38 MAPK and reversed TGF-ß1-induced EMT. Over-expression of Sema4C via Sema4C transfection elicited p38 MAPK phosphorylation and promoted EMT. The effects of Sema4C during EMT were blocked by a p38-specific inhibitor. In vivo, the expression of Sema4C increased in the tubular epithelia of 5/6-nephrectomized rats and human fibrotic renal tissue, and similar localization of phosphorylated p38 and Sema4C was demonstrated by immunohistochemistry on serial sections. CONCLUSIONS: Our findings suggest that Sema4C plays an important role in TGF-ß1-induced EMT through activation of p38 MAPK in proximal tubular epithelial cells.