CircRTN4 aggravates mesangial cell dysfunction by activating the miR-513a-5p/FN axis in lupus nephritis.

Circular RNAs (circRNAs) are regulators of gene expression that can regulate cell proliferation and programmed cell death and serve as biomarkers in renal diseases. However, the specific traits and underlying mechanisms of circRNAs in the progression of lupus nephritis (LN) have not been elucidated. In the present study, we clarified that hsa_circ_0054595 (circRTN4) was upregulated in human renal mesangial cells (HRMCs). In cultured HRMCs, circRTN4 could enhance FN expression by directly interacting with miR-513a-5p. High circRTN4 expression in monocytes disseminated into HRMCs in an exosomal manner, thereby accelerating cell proliferation and extracellular matrix deposition. In addition, knockdown of circRTN4 in the kidney or peripheral blood alleviated renal damage in MRL/lpr and BALB/c mice. Clinically, high levels of circRTN4 were found in peripheral blood mononuclear cells and kidney tissues of LN patients, hence serving as an effective biomarker for LN detection and a novel therapeutic target. Our findings indicated that circRTN4 exacerbates mesangial cell dysfunction by activating the miR-513a-5p/FN axis in lupus nephritis.

TRIM27 contributes to glomerular endothelial cell injury in lupus nephritis by mediating the FoxO1 signaling pathway.

Tripartite motif-containing 27 (TRIM27) belongs to the triple motif (TRIM) protein family, which plays a role in a variety of biological activities. Our previous study showed that the TRIM27 protein was highly expressed in the glomerular endothelial cells of patients suffering from lupus nephritis (LN). However, whether TRIM27 is involved in the injury of glomerular endothelial cells in lupus nephritis remains to be clarified. Here, we detected the expression of the TRIM27 protein in glomerular endothelial cells in vivo and in vitro. In addition, the influence of TRIM27 knockdown on endothelial cell damage in MRL/lpr mice and cultured human renal glomerular endothelial cells (HRGECs) was explored. The results revealed that the expression of TRIM27 in endothelial cells was significantly enhanced in vivo and in vitro. Downregulating the expression of TRIM27 inhibited the breakdown of the glycocalyx and the injury of endothelial cells via the FoxO1 pathway. Moreover, HRGECs transfected with the WT-FoxO1 plasmid showed a reduction in impairment caused by LN plasma. Furthermore, suppression of the protein kinase B (Akt) pathway could attenuate damage by mediating the expression of TRIM27. Thus, the present study showed that TRIM27 participated in the injury of glomerular endothelial cells and served as a potential therapeutic target for the treatment of lupus nephritis.

HMGB1 protein promotes glomerular mesangial matrix deposition via TLR2 in lupus nephritis.

Lupus nephritis (LN) is the most common complication of systemic lupus erythematosus. Patients with LN mostly die of sclerosing glomerulonephritis and renal failure. The inhibition of glomerular mesangial matrix deposition is an efficient method to restrict the progress of renal injury. By recognizing and binding extracellular and intracellular ligands, Toll-like receptor 2 (TLR2) contributes to the pathogenesis of most immune diseases. However, the relationship between TLR2 and LN is still unknown. Our previous studies confirmed that high-mobility group box 1 (HMGB1), an important ligand of TLR2, promotes the progression of LN by inducing the proliferation of glomerular mesangial cells. However, whether or not HMGB1 participates in the pathogenesis of glomerular mesangial matrix deposition in LN remains unknown. In this study, we observed the upregulated expression of TLR2 in the glomeruli of LN patients and MRL/lpr mice. The inhibition of either TLR2 or HMGB1 inhibited the release of fibronectin and the activation of the MyD88/NF-κB pathway in mesangial cells cultured with LN plasma. In addition, both TLR2- and HMGB1-deficient mice showed reduced 24 hr urine protein levels and improved glomerular histological changes and sclerosis levels. These results indicate that TLR2 regulates glomerular mesangial matrix deposition in LN through the activation of the MyD88/NF-κB pathway by binding to HMGB1.

Knockdown of TRIM27 expression suppresses the dysfunction of mesangial cells in lupus nephritis by FoxO1 pathway.

TRIM27 (tripartite motif-containing 27) is a member of the TRIM (tripartite motif) protein family and participates in a variety of biological processes. Some research has reported that TRIM27 was highly expressed in certain kinds of carcinoma cells and tissues and played an important role in the proliferation of carcinoma cells. However, whether TRIM27 takes part in the progression of lupus nephritis (LN) especially in cells proliferation remains unclear. Our study revealed that the overexpression of TRIM27 was observed in the kidneys of patients with LN, lupus mice and mesangial cells exposed to LN plasma which correlated with the proliferation of mesangial cells and ECM (extracellular matrix) deposition. Downregulation of TRIM27 expression suppressed the proliferation of mesangial cells and ECM accumulation in MRL/lpr mice and cultured human mesangial cells (HMCs) by regulating the FoxO1 pathway. Furthermore, the overexpression of FoxO1 remarkably decreased HMCs proliferation level and ECM accumulation in LN plasma-treated HMCs. In addition, the protein kinase B (Akt) signal pathway inhibitor LY294002 significantly reduced the expression of TRIM27 and inhibited the dysfunction of mesangial cells. These above data suggested that TRIM27 mediated abnormal mesangial cell proliferation in kidney of lupus and might be the potential target for treating mesangial cell proliferation of lupus nephritis.

Nudel involvement in the high-glucose-induced epithelial-mesenchymal transition of tubular epithelial cells.

Nudel is a newly discovered factor related to cell migration. The tubular epithelial-mesenchymal transition (EMT) includes four steps: the loss of the adhesive properties of epithelial cells, the acquisition of a mesenchymal cell phenotype, the destruction of the tubular basal membrane, and the migration into the renal interstitium. The purpose of this study was to investigate the role of Nudel in the high-glucose-induced EMT of tubular epithelial cells. Human renal proximal tubular epithelial cells (HKCs) were treated with Nudel shRNA to clarify the role and mechanism of Nudel in tubular EMT induced by high glucose. We found that Nudel was expressed at a high level in high-glucose-stimulated HKCs, and the expression of Nudel was associated with the activation of signal transducer and activator of transcription 3. After transfection with Nudel shRNA, we detected the expression levels of E-cadherin, α-smooth muscle actin (α-SMA), and the Wiskott-Aldrich syndrome family of proteins (including WASP, N-WASP, WAVE1, WAVE2, and WAVE3) via assay. Cell migration was analyzed by the scratching method. The results showed that high glucose downregulated E-cadherin expression, upregulated α-SMA expression, and promoted the migration of HKCs. The expression levels of N-WASP, WAVE1, and WAVE2 were also elevated in HKCs treated with high glucose. All changes induced by high glucose were ameliorated by Nudel depletion. We conclude that Nudel participates in the transition and the migration of tubular epithelial cells via the regulation of WASP family proteins.

S3I-201 ameliorates tubulointerstitial lesion of the kidneys in MRL/lpr mice.

It is high incidence of tubulointerstitial lesion (TIL) in lupus nephritis (LN) and TIL can affect the prognosis of patients with LN. Signal transducer and activator of transcription (STAT) 3 was activated in LN and STAT3 inhibition could delay the onset of LN. Here, we evaluated the role of a well-known STAT3 inhibitor, S3I-201, on TIL in lupus nephritis. STAT3 was activated in MRL/lpr mice (a mouse model of lupus nephritis), and treatment with S3I-201 inhibited the activation of it. The level of 24-h urine protein and nitrogen urea increased in MRL/lpr mice and adminstration of S3I-201 reduced the level of urinary protein. In addition, S3I-201 attenuated the expression of α-smooth muscle actin (α-SMA), Fibronectin (FN) proteins, as well as the expression of monocyte chemotactic factor-1 (MCP-1) and intercellular adhesion molecule (ICAM-1). However, the expression of E-cadherin improved when treatment with S3I-201. These results revealed that the activation of STAT3 mediates tubulointerstitial lesion in mice with LN. S3I-201, by suppressing STAT3 activity, has therapeutic effect in lupus nephritis.

Anti-OSM Antibody Inhibits Tubulointerstitial Lesion in a Murine Model of Lupus Nephritis.

The purpose of this study was to investigate the role of oncostatin M (OSM) in tubulointerstitial lesion (TIL) in lupus nephritis (LN). We found that OSM was highly expressed in the renal tissue of LN mice. OSM is one of the interleukin-6 cytokine family members. In order to clarify the role and mechanism of OSM in LN, mice with LN were treated with anti-OSM antibody or isotype antibody. We evaluated the tubular epithelial-mesenchymal transdifferentiation (EMT) by detecting the E-cadherin, α-smooth muscle actin (α-SMA), and fibronectin (FN) expression. We analyzed the inflammation by observing the monocyte chemotactic factor-1 (MCP-1) and intercellular adhesion molecule (ICAM-1) expression and calculated the tubulointerstitial fibrosis area by Masson staining. The results showed that anti-OSM antibody, rather than isotype antibody, improved EMT, inflammation, and tubulointerstitial fibrosis. In addition, the signal transducer and activator of transcription (STAT) 1 and STAT3 signaling was activated by tyrosine phosphorylation in LN mouse renal tissue, indicating that the phosphorylated STAT1 (p-STAT1) and p-STAT3 were involved in kidney injury. Moreover, decreased p-STAT3 instead of p-STAT1 has been observed after anti-OSM antibody injection. Thus, we concluded that OSM is associated with TIL in lupus nephritis, which may be connected with the activation of STAT3 rather than that of STAT1.

PTEN Regulates Renal Extracellular Matrix Deposit via Increased CTGF in Diabetes Mellitus.

Extracellular matrix accumulation and fibrosis are the features of diabetic nephropathy. PI3K (phosphatidylinositol 3-kinase)/Akt (protein kinase B) signal pathway and its inhibitor PTEN (phosphatase and tensin homolog deleted on chromosome 10) are revealed to modulate renal fibrosis. However, the exact mechanism is still not well known. In the present study we found that compared with normal mice, diabetic mice showed decreased PTEN, increased phospho-Akt (Ser 473), phospho-Akt (Thr 308), CTGF (connective tissue growth factor), α-SMA (α-smooth muscle actin), and matricellular protein in kidney. Knocking down of PTEN caused an increase in phospho-Akt (Ser 473), phospho-Akt (Thr 308), CTGF, secreted fibronectin, and secreted Col 3 in HKC cells (human renal tubular epithelial cells). Again, in vitro experiment revealed 1.89, 2.18, 1.92, 3.06, 2.06-fold increases of phospho-Akt (Ser 473), phospho-Akt (Thr 308), CTGF, secreted fibronectin, and secreted Col 3 in high glucose-stimulated HKC cells in comparison with normal control cells. Furthermore, knocking down of CTGF reversed increased secreted fibronectin and Col 3 in high glucose-treated HKC cells. Moreover, transfection of PTEN expression vector prevented high glucose-caused these changes in HKC cells. Especially, CTGF expression, secretion of fibronectin and Col 3 were, respectively, decreased by 38.81, 53.85, and 39.12%. The treatment of LY294002 inhibited phospho-Akt (Ser 473) and phospho-Akt (Thr 308) expression followed by decreased CTGF, secretory fibronectin and secretory Col 3 in high glucose-treated HKC cells. In the end our study suggests that PTEN regulates renal extracellular matrix production via activated Akt and increased CTGF in diabetes mellitus.

Co-regulation of SREBP-1 and mTOR ameliorates lipid accumulation in kidney of diabetic mice.

SREBP-1 and mTOR have been proved to involve in renal lipid metabolism of diabetes mellitus. In the present study, we investigated the effect of co-regulation of SREBP-1 and mTOR on renal lipid metabolism using diabetic mice and cultured renal tubular cells. The results showed that compared with those in high glucose-stimulated HKC cells single transfected with shRNA-SREBP-1 vector, the level of SREBP-1 protein were significantly reduced by 64.1% followed by decreased FASN mRNA, ACC mRNA, ADRP protein and lipid droplets in HKC cells co-transfected with shRNA-SREBP-1 vector and kinase-dead mTOR vector. Furthermore, diabetic mice co-injected with shRNA-SREBP-1 vector and kinase-dead mTOR vector showed that renal SREBP-1 protein, FASN mRNA and ACC mRNA were respectively decreased by 34.6%, 45.9%, 22.0% in comparison with those in diabetic mice single injected with shRNA-SREBP-1 vector accompanied by reduced ADRP protein and triglyceride content. In the end our study suggests that co-regulation of SREBP-1 and mTOR in kidney of diabetic mice is more effective in lowering renal lipogenesis than only regulation of SREBP-1.

PTEN Inhibits High Glucose-Induced Phenotypic Transition in Podocytes.

Accumulating evidence has suggested that podocytes undergo epithelial-mesenchymal transition (EMT) in diabetic nephropathy (DN). However, the underlying mechanisms of EMT in podocyte are not well understood. PI3K/Akt pathway is involved in the progression of DN. In the present study, we demonstrated that PI3K/Akt pathway was activated in podocytes exposed to high glucose conditions, accompanied by down-regulation of the podocalyxin (PCX) and nephrin expression and up-regulation of the desmin and α-smooth muscle actin (α-SMA) expression. Inhibition of PI3K/Akt pathway by chemical LY294002 or Phosphase and tensin homology deleted on chromosome ten (PTEN) prevented the phenotypic transition. These findings indicate that PTEN/PI3K/Akt pathway mediates high glucose-induced phenotypic transition in podocytes.

The PTEN/PI3K/Akt signaling pathway mediates HMGB1-induced cell proliferation by regulating the NF-κB/cyclin D1 pathway in mouse mesangial cells.

Our previous experiment confirmed that high-mobility group box chromosomal protein 1 (HMGB1) was involved in the pathogenesis of Lupus nephritis (LN) by upregulating the proliferation of the mouse mesangial cell line (MMC) through the cyclin D1/CDK4/p16 system, but the precise mechanism is still unknown. Therefore, in the present study, we demonstrated that HMGB1 induced the proliferation of MMC cells in a time- and concentration-dependent manner, downregulated phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression, increased the level of Akt serine 473 phosphorylation, and induced p65 subunit nuclear translocation. The overexpression of PTEN prevented the upregulation of HMGB1-induced proliferation by blocking the activation of Akt. The knockdown of Akt by siRNA technology and blocking the nuclear factor-κB (NF-κB) pathway using pyrrolidine dithiocarbamate (PDTC) and SN50, inhibitors of NF-κB, both attenuated the HMGB1-induced proliferation by counteracting the activation of the cyclin D1. In addition, while sh-Akt partly blocked the nuclear translocation of the p65 subunit, PDTC did not affect the activation of the Akt induced by HMGB1 in MMC cells. These findings indicate that HMGB1 induced the proliferation of MMC cells by activating the PTEN/phosphoinositide-3-kinase (PI3K)/Akt/NF-κB signaling pathway.

Phosphorylation of PRAS40-Thr246 involved in renal lipid accumulation of diabetes.

Lipid accumulation of kidney is a threat to renal physiological function of diabetes. The previous studies on diabetic nephropathy have demonstrated that activated Akt was involved in renal lipogenesis through enhancing transcription factor SREBP-1. PRAS40 is one of the downstream targets of activated Akt that was reported to involve in lipid metabolism in hepatic cells. However, it is still not clear whether PRAS40 is also involved in the renal lipogenesis of diabetes. Our study revealed that phosphorylation of PRAS40-Thr246 known as inactivated style increased in renal tubular cells of diabetic rats accompanied with over-expression of phospho-Akt, SREBP-1, and ADRP. In addition, in vitro experiment also found that high glucose enhanced expression of phospho-PRAS40-Thr246 followed by increased SREBP-1 and lipid droplets in HKC cells. After treated with LY294002, high glucose-induced HKC cells showed decreased phospho-PRAS40-Thr246, phospho-Akt-Ser473, and SREBP-1. Furthermore, wild type PRAS40 vector-caused increased phospho-PRAS40-Thr246 exaggerated lipid deposits in high glucose-treated HKC cells, which was effectively prevented in cells transfected with mutant PRAS40 vector (T246A). These above data suggested that phosphorylation of PRAS40-Thr246 mediated abnormal lipid metabolism in kidney of diabetes and might be the potential target for treating lipogenesis of diabetic nephropathy.

Interplay between the Notch and PI3K/Akt pathways in high glucose-induced podocyte apoptosis.

Podocyte apoptosis contributes to the pathogenesis of diabetic nephropathy (DN). However, the mechanisms that mediate high glucose (HG)-induced podocyte apoptosis remain poorly understood. Conditionally immortalized mouse podocytes were cultured in HG medium. A chemical inhibitor or a specific short-hairpin RNA (shRNA) vector was used to inhibit the activation of the Notch pathway and the PI3K/Akt pathway in HG-treated podocytes. Western blotting and real-time PCR were used to evaluate the levels of Notch, PI3K/Akt, and apoptotic pathway signaling. The apoptosis rate of HG-treated podocytes was assessed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling and annexin V/propidium iodide staining. In HG-treated podocytes, PI3K/Akt pathway activation prevented podocyte apoptosis in the early stage of HG stimulation and Notch pathway-induced podocyte apoptosis in the late stage of HG stimulation. The inhibition of the Notch pathway or the activation of the PI3K/Akt pathway prevented cell apoptosis in HG-treated podocytes. These findings suggest that the Notch and PI3K/Akt pathways may mediate HG-induced podocyte apoptosis.

Phospho-mTOR: a novel target in regulation of renal lipid metabolism abnormality of diabetes.

The activation of Akt has been proved to involve in the lipogenesis of diabetic nephropathy. However, it's still not clear whether mTOR, another main gene in PI3K/Akt pathway, is also involved in the renal lipogenesis of diabetes. In the present study, it was revealed that the phosphorylation of mTOR was up-regulated in the renal tubular cells of diabetic rats, followed by the over-expression of SREBP-1, ADRP and lipogenesis. Again, high glucose increased the expression of phospho-mTOR accompanied with SREBP-1 and ADRP up-regulation and lipid accumulation in HKC cells. Rapamycin, known as mTOR inhibitor, was used to inhibit the activation of mTOR, which prevented effectively high glucose-induced SREBP-1 up-regulation and lipogenesis in HKC cells. Furthermore, high glucose-stimulated HKC cells transfected with wild-type mTOR vector showed the enhanced SREBP-1 and lipid droplets, however, TE mTOR vector (kinase dead)-transfected HKC cells presented resistance to high glucose and decreased SREBP-1 expression and lipogenesis. These above data suggested that phospho-mTOR mediated lipid accumulation in renal tubular cells of diabetes and might be the potential targets for treating lipogenesis of diabetic nephropathy.

Notch pathway is involved in high glucose-induced apoptosis in podocytes via Bcl-2 and p53 pathways.

Recent studies have shown that Notch pathway plays a key role in the pathogenesis of diabetic nephropathy (DN), however, the exact mechanisms remain elusive. Here we demonstrated that high glucose (HG) upregulated Notch pathway in podocytes accompanied with the alteration of Bcl-2 and p53 pathways, subsequently leading to podocytes apoptosis. Inhibition of Notch pathway by chemical inhibitor or specific short hairpin RNA (shRNA) vector in podocytes prevented Bcl-2- and p53-dependent cell apoptosis. These findings suggest that Notch pathway mediates HG-induced podocytes apoptosis via Bcl-2 and p53 pathways.

Role of SUMOylation of STAT1 in tubular epithelial­mesenchymal transition induced by high glucose.

Tubulointerstitial fibrosis (TIF) is an important pathological change that occurs during the development of diabetic kidney disease. The epithelial­mesenchymal transition (EMT) of renal tubular epithelial cells is a manifestation of TIF. STAT1, a member of the STAT family of transcription factors, can be modified by the small ubiquitin­related modifier (SUMO), thus affecting the activity of STAT1. The present study investigated the role of STAT1 SUMOylation in high glucose­induced tubular EMT by western blotting, immunocytochemistry, immunofluorescence, co­immunoprecipitation and dual luciferase reporter analysis. The results indicated that in the process of high glucose­induced EMT, STAT1 activation protected the cells from EMT. However, high glucose also increased the SUMOylation of STAT1, which prevented STAT1 from exerting an effective protective role by inhibiting its activity.

PI3K/Akt pathway mediates high glucose-induced lipid accumulation in human renal proximal tubular cells via spliced XBP-1.

In the present study, we investigated the effect of X-box-binding protein-1 (XBP-1) splicing on lipogenesis in high glucose-stimulated human renal proximal tubular cell line (HKC). The results revealed that high glucose promoted the splicing of XBP-1, concomitant with up-regulation of lipogenic genes including fatty acid synthase, acetyl-CoA carboxylase, adipocyte differentiation-related protein, and cellular triglyceride. Again, silence of XBP-1 with shRNA vector inhibited high glucose-caused increased lipogenesis. Furthermore, we confirmed that the inhibition of phosphotidyl inositol 3-kinase (PI3K)/Akt pathway with LY294002 or Akt shRNA vector blocked the effect of high glucose on XBP-1 splicing and cellular triglyceride. These above data suggest that spliced XBP-1 mediates high glucose-induced lipid accumulation in HKC cells and PI3K/Akt pathway may be involved in high glucose-caused XBP-1 splicing.

Nestin protects mouse podocytes against high glucose-induced apoptosis by a Cdk5-dependent mechanism.

Podocyte apoptosis contributes to the pathogenesis of diabetic nephropathy (DN). However, the mechanisms that mediate hyperglycemia-induced podocyte apoptosis remain poorly understood. Recent findings indicate that the disruption of the cytoskeleton is related to the podocyte apoptosis. In the present study, we investigated the involvement of nestin, an important cytoskeleton-associated class VI intermediate filament (IF) protein, in the high glucose (HG)-induced podocyte apoptosis. Our data showed that HG decreased the expression level of nestin, either mRNA or protein, in a time-dependent manner in cultured podocytes. Also, through knockdown of nestin expression by miRNA interference, the HG-induced podocyte apoptotic rate was significantly increased. The expression of cleaved caspase-3 was also markedly elevated. Considering that nestin is a substrate of cyclin-dependent kinase 5 (Cdk5), we further assessed the expression of Cdk5 in HG-treated podocytes. The results showed that HG stimulation increased the protein and mRNA expression of Cdk5 in a time-dependent manner in cultured mouse podocytes. The protein activator of Cdk5, p35, was also increased in a time-dependent manner by HG stimulation, and downregulation of Cdk5 by miRNA interference attenuated the nestin reduction in HG-treated podocytes; the HG-induced podocyte apoptosis, the increased cleaved caspase-3 expression and the Bax/Bcl-2 ratio were all effectively attenuated. These data suggested that nestin, which is dependent on Cdk5 regulation, plays a cytoprotective role in HG-induced podocyte apoptosis.

HMGB1 mediates IFN-γ-induced cell proliferation in MMC cells through regulation of cyclin D1/CDK4/p16 pathway.

Previous studies have revealed the elevated serum levels of High-mobility group box-1(HMGB1) and the interferon-γ (IFN-γ)-induced proliferation of renal mesangial cells in patients or experimental animals with systemic lupus erythematosus (SLE). However, it is still not elucidated whether HMGB1 involves in the pathogenesis of lupus nephritis (LN) and mediates IFN-γ-induced mesangial cell proliferation. Therefore, in the present study we demonstrated HMGB1 mRNA and protein levels were increased in the glomeruli of LN patients and BXSB mice. HMGB1 increased the proliferation index of mouse mesangial cells (MMC) that was accompanied with the up-regulation of cyclin D1, CDK4 and the down-regulation of p16, subsequently promoting the transition from the G0/G1 to S stage. Inhibition of HMGB1 by a specific short hairpin RNA vector prevented cyclin D1/CDK4/p16 up-regulation and attenuated IFN-γ-induced MMC cell proliferation and PCNA (proliferating cell nuclear antigen, PCNA) expression. These findings indicate that HMGB1 mediates IFN-γ-induced cell proliferation in MMC cells through regulation of cyclin D1/CDK4/p16 pathway and promoting the cell cycle transition from G1 to S stage.