Exosomal hsa_circ_0125310 promotes cell proliferation and fibrosis in diabetic nephropathy via sponging miR-422a and targeting the IGF1R/p38 axis.

Diabetic nephropathy (DN) is still on the rise worldwide, and millions of patients have to be treated through dialysis or transplant because of kidney failure caused by DN. Recent reports have highlighted circRNAs in the treatment of DN. Herein, we aimed to investigate the mechanism by which high glucose-induced exo-circ_0125310 promotes diabetic nephropathy progression. circ_0125310 is highly expressed in diabetic nephropathy and exosomes isolated from high glucose-induced mesangial cells (MCs). High glucose-induced exosomes promote the proliferation and fibrosis of MCs. However, results showed that the effects of exosomes on MCs can be reversed by the knockdown of circ_0125310. miR-422a, which targets IGF1R, was the direct target of circ_0125310. circ_0125310 regulated IGF1R/p38 axis by sponging miR-422a. Exo-circ_0125310 increased the luciferase activity of the WT-IGF1R reporter in the dual-luciferase reporter gene assays and upregulated the expression level of IGF1R and p38. Finally, in vivo research indicated that the overexpression of circ_0125310 promoted the diabetic nephropathy progression. Above results demonstrated that the high glucose-induced exo-circ_0125310 promoted cell proliferation and fibrosis in diabetic nephropathy via sponging miR-422a and targeting the IGF1R/p38 axis.

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.

Uric acid induced epithelial-mesenchymal transition of renal tubular cells through PI3K/p-Akt signaling pathway.

The phenotypic changes of tubular epithelial cell are hallmark features of renal diseases caused by abnormal uric acid levels. We hereby intend to investigate whether PI3K/p-Akt signaling plays a role in uric-acid induced epithelial-mesenchymal transition process. The normal rat kidney cell line (NRK-52E) was used as a proximal tubular cell model in this study. NRK-52E cells were exposed to different concentrations of uric acid, or PI3K inhibitor LY294002, or both, respectively. The effects of uric acid on cell morphology were examined by phase contrast microscopy, while molecular alternations were assessed by western blot analysis and immunofluorescence staining. We found that uric acid induced visible morphological alterations in NRK-52E cells accompanied by increased expression of α-smooth muscle actin and reduced expression of E-cadherin. Moreover, phosphorylation of Akt protein was obviously increased, whereas Akt level remained stable. Furthermore, the above effects were abolished when PI3K/p-Akt pathway was blocked by the PI3K inhibitor. These findings demonstrated that high uric acid could induce phenotypic transition of cultured renal tubular cells, which was probably via activating PI3K/p-Akt signaling pathway.

Association of vaspin rs2236242 gene variants with type 2 diabetes and obesity in a Chinese population: A prospective, single-center study.

AIM: Vaspin is an adipokine separated from visceral fat tissues of obese diabetic rats. This study was to investigate the association between vaspin rs2236242 gene polymorphism and type 2 diabetes mellitus (T2DM) or obesity in a Chinese population. MATERIALS AND METHODS: T2DM patients and nondiabetic controls were recruited from Qingpu Branch of Zhongshan Hospital Affiliated to Fudan University (Shanghai, China) from May 1, 2015 to June 30, 2017. Clinicopathologic characteristics were recorded and their blood samples were collected. Serum vaspin levels were detected by enzyme-linked immunosorbent assay and vaspin rs2236242 genotypes by tetra-amplification refractory mutation system-polymerase chain reaction. RESULTS: Two hundred and ninety-nine patients with T2DM and 311 controls were recruited at last. The vaspin genotypes of diabetic patients were distinct from nondiabetic controls (χ 2 = 54.611, p < 0.0001). Genotyping revealed that T2DM patients have a greater prevalence of A allele compared with controls (61.9% vs. 42.1%, p < 0.0001). A allele was associated with an increased risk of T2DM (odds ratio = 2.23, 95% confidence interval = 1.773-2.804, p < 0.0001) compared with T allele. The genotype distribution did not differ among nondiabetic subjects with or without obesity. The serum vaspin levels were higher in T2DM patients and obese controls than the nonobese controls, however, the rs2236242 was not found to be significantly related to serum vaspin levels. CONCLUSIONS: Our findings showed the association between vaspin rs2236242 gene variants with obesity and T2DM in a Chinese population. People with rs2236242 A allele had a 2.23-fold increased risk of T2DM. These findings suggest that vaspin rs2236242 may serve as a potential diagnostic and/or therapeutic targets for T2DM.

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.

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.

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.

Gypenosides inhibit renal fibrosis by regulating expression of related genes in rats with unilateral ureteral obstruction.

BACKGROUND: Transforming growth factor ß1 (TGF-ß1), connective tissue growth factor (CTGF) and Smad7 are potent components of fibrogenesis-related signal transduction pathways. Renal fibrosis is the major pathological change in the rat models with unilateral ureteral obstruction (UUO). Investigating the effects of gypenosides (GPs) on the expression of fibrogenesis-related genes in the UUO model may lead to the development of effective therapy for renal diseases. METHODS: Rats were randomly divided into 3 experimental groups: (i) sham operation rats treated with saline (sham group), (ii) UUO model rats treated with saline (control group) and (iii) UUO model rats treated with GPs (GPs group). Blood urea nitrogen and serum creatinine were detected as the measurement of renal function. UUO-treated kidney tissues were taken for assessment of renal damage index and determination of related gene expression through immunohistochemistry and RT-PCR. RESULTS: UUO-induced tubulointerstitial damage and fibrosis were attenuated by the application of GPs (day 3 and day 7, p<0.01; day 14, p<0.05). The expression of TGF-ß1 and CTGF was significantly reduced with GPs treatment (TGF-ß1, p<0.01; CTGF, p<0.05). Smad7 expression was elevated with GPs treatment at days 7 and 14 (p<0.01). GPs' protective effects on renal function were also demonstrated with this UUO model. CONCLUSIONS: These results suggest that UUO-induced tubulointerstitial fibrosis can be effectively attenuated by GPs application. GPs-mediated down-regulation of TGF-ß1 and CTGF and up-regulation of Smad7 are essential for their effects of antifibrogenesis.