Mesangial Cells In Diabetic Nephropathy Research Articles

LncRNA SNHG14 silencing attenuates the progression of diabetic nephropathy via the miR-30e-5p/SOX4 axis.

Diabetic nephropathy (DN) is a diabetic complication. LncRNAs are reported to participate in the pathophysiology of DN. Here, the function and mechanism of lncRNA small nucleolar RNA host gene 14 (SNHG14) in DN were explored. Streptozotocin (STZ)-induced DN mouse models and high glucose (HG)-treated human mesangial cells (MCs) were used to detect SNHG14 expression. SNHG14 silencing plasmids were applied to examine the function of SNHG14 on proliferation and fibrosis in HG-treated MCs. Potential targets of SNHG14 were predicted using bioinformatics tools and verified by luciferase reporter, RNA pulldown, and northern blotting assays. The functional role of SNHG14 in DN in vivo was detected by injection with adenoviral vector carrying sh-SNHG14 into DN mice. Serum creatinine, blood urea nitrogen, blood glucose, 24-h proteinuria, relative kidney weight, and renal pathological changes were examined in DN mice. SNHG14 expression was elevated in the kidneys of DN mice and HG-treated MCs. SNHG14 silencing inhibited proliferation and fibrosis of HG-stimulated MCs. SNHG14 bound to miR-30e-5p to upregulate SOX4 expression. In rescue assays, SOX4 elevation diminished the effects of SNHG14 silencing in HG-treated MCs, and SOX4 silencing reversed the effects of SNHG14 overexpression. In in vivo studies, SNHG14 downregulation significantly ameliorated renal injuries and renal interstitial fibrosis in DN mice. SNHG14 silencing attenuates kidney injury in DN mice and reduces proliferation and fibrotic phenotype of HG-stimulated MCs via the miR-30e-5p/SOX4 axis.

Macrophage-derived exosomes promote activation of NLRP3 inflammasome and autophagy deficiency of mesangial cells in diabetic nephropathy

Dysfunction of mesangial cells plays a significant role in the glomerular lesions and is implicated in the pathophysiology of diabetic nephropathy (DN). Macrophages infiltration is the main pathological feature of DN, which can ultimately lead to renal inflammation. Recent studies suggest that the crosstalk between kidney resident cells and inflammatory cells influences the development of DN, and that controlling this crosstalk may help treat DN. Here, we found that DN mice appeared renal pathological damage, including dilation of mesangial matrix and significant infiltration of macrophages, accompanied by increased inflammatory response, NLRP3 inflammasome activation and autophagy deficiency. Additionally, mesangial cells internalized exosomes from high glucose (HG) treated macrophage, resulting the activation of inflammatory cytokines and NLRP3 inflammasome and deficiency of autophagy in vitro and in vivo. Moreover, C57BL/6 mice injected HG-stimulated macrophages-derived exosomes exhibited renal dysfunction and mesangial matrix expansion. Taken together, the present study demonstrated that mesangial cells responded to HG treated macrophage-derived exosomes by promoting the activation of NLRP3 inflammasome and autophagy deficiency, thereby participating in the development of DN.

Tenascin-C promotes the proliferation and fibrosis of mesangial cells in diabetic nephropathy through the β-catenin pathway.

To mechanistically assess the involvement of tenascin-C (TNC) in diabetic nephropathy (DN). Renal specimens from DN patients were histopathologically examined, and their TNC expression patterns were evaluated via immunohistochemistry. Additionally, the hereditarily diabetic C57BL/KsJ db/db mice were induced to develop DN via adaptive feeding, and then their renal levels of TNC and β-catenin were assessed via western blotting and immunohistochemistry. Furthermore, the TNC and β-catenin levels in primary rat mesangial cells (RMCs) cultured with high glucose levels were assessed via western blotting. In parallel, RMCs cultured with TNC in the presence or absence of the β-catenin blocker ICG-001 were analyzed for their fibronectin and collagen I levels via immunostaining, and for their fibronectin, α-SMA, vimentin, PDGFR-β, PCNA, and β-catenin levels via western blotting. The TNC levels in the specimens were associated with the pathological classification. In these DN specimens, TNC protein was highly detected in the MCs and slightly in the tubulointerstitium. Renal TNC (P < 0.05) and β-catenin (P < 0.001) were upregulated in db/db vs. db/m mice. High-glucose treatment upregulated TNC (P < 0.01) and β-catenin (P < 0.05) in RMCs. TNC treatment upregulated fibronectin (P < 0.05), α-SMA (P < 0.01), vimentin (P < 0.05), PCNA (P < 0.05), and β-catenin (P < 0.05) in RMCs, as assessed via western blotting. Immunohistochemical analysis confirmed the fibronectin upregulation and showed collagen I upregulation. Western-blot results also showed that levels of fibronectin (P < 0.001), α-SMA (P < 0.01), vimentin (P < 0.001), PCNA (P < 0.05), PDGFR-β (P < 0.05), and β-catenin (P < 0.01) were lower in RMCs co-treated with TNC and ICG-001 than in TNC-treated cells. Immunofluorescence analysis confirmed the decreased fibronectin level and showed that the collagen I level was also decreased by ICG-001. TNC is upregulated in DN and induces MC proliferation and fibrosis through the β-catenin pathway.

ENSMUST00000147869 regulates proliferation and fibrosis of mesangial cells in diabetic nephropathy by interacting with Hspa9.

Our previous study showed that ENSMUST00000147869 was abnormally low expressed in the early stage of diabetic nephropathy (DN). ENSMUST00000147869 could inhibit the fibrosis and proliferation of mouse mesangial cells (MMCs), but the mechanism is still unclear. This study aims to explore the specific mechanism underline ENSMUST00000147869 regulates the proliferation and fibrosis of MMCs in DN. Nucleocytoplasmic fractionation was applied to define the location of ENSMUST00000147869 in MMCs. RNA-protein pulldown, RNA immunoprecipitation and mass spectrometry were used to identify upregulated Hspa9 directly interacting with ENSMUST00000147869. SiRNA and lentivirus packaging were used to clarify the role of Hspa9 downregulated by ENSMUST00000147869 in promoting proliferation and fibrosis in MMCs. CHX and MG132 were used to clarify the regulatory role of ENSMUST00000147869 to Hspa9. Immunoprecipitation confirmed the binding of Hspa9 and HMGB1. HSPA9 was a direct binding protein of ENSMUST00000147869, and ENSMUST00000147869 could inhibit proliferation and fibrosis of MMCs by down-regulating HSPA9 through ubiquitination process. HMGB1 was the downstream binding protein of Hspa9, and ENSMUST00000147869 could inhibit the interaction between Hspa9 and HMGB1. Our data showed that ENSMUST00000147869 regulates Hspa9 through the ubiquitin proteasome pathway and inhibits the binding of Hspa9 and HMGB1. The ENSMUST00000147869/Hspa9/HMGB1 axis may act as a diagnostic molecular marker and an effective therapeutic target for DN.

Flavonoids of Coreopsis tinctoria Nutt alleviate the oxidative stress and inflammation of glomerular mesangial cells in diabetic nephropathy via RhoA/ROCK signaling

• FC improved glucolipid metabolic disorder, abnormal renal function, and renal pathological injury in db/db DN mice. • FC could exert a protective effect on DN by regulating oxidative stress mediated by RhoA/ROCK signaling both in vivo and in vitro. • FC down-regulated inflammatory responses by NF-κB/TGF-β1/Smad signaling both in vivo and in vitro. Diabetic nephropathy (DN) is one of the serious diabetic microvascular complications, and oxidative stress and inflammation may play a vital role in its initiation and development. Previous studies indicated that Coreopsis tinctoria Nutt had anti-inflammatory and antioxidant activities. This study aimed to examine the protective effects of flavonoids from Coreopsis tinctoria Nutt (FC) on DN. In vivo, db/db mice were treated with FC (150, 300 mg/kg), FC150 + Metformin. DN model in vivo of high glucose and palmitic acid-induced glomerular mesangial cell (HBZY-1) was established and treated with FC . The results showed that treatment of FC, especially in the dose of 150 mg/kg and FC150 mg/kg plus metformin significantly improved glucolipid metabolic disorder, abnormal renal function, and pathological renal injury in db/db mice. Both in vivo and in vitro , FC intervention reduced the levels of renal oxidative stress-related factors, inflammatory cytokines, and fibrotic factors. Additionally, FC down-regulated the expressions of proteins related to Ras homolog gene family member (RhoA)/Rho-associated protein kinase (ROCK), nuclear factor kappa-B (NF-κB), and transforming growth factor (TGF-β)/Smad signaling pathways. In conclusion, FC might protect against diabetic renal injury by inhibiting renal oxidation, inflammation, and fibrosis via RhoA/ROCK and NF-κB/TGF-β/Smad signalings.

O-linked N-acetylglucosaminyltransferase OGT inhibits diabetic nephropathy by stabilizing histone methyltransferases EZH2 via the HES1/PTEN axis

BackgroundO-linked N-acetylglucosaminyltransferase (OGT) is involved in diabetes-related diseases including diabetic nephropathy (DN), and responsible for O-GlcNAcylation. Moreover, O-GlcNAcylation and OGT could be induced by high glucose. Thus, we sought to explore the molecular mechanism of OGT in DN. MethodsLoss- and gain-functions were conducted to determine the roles of OGT, enhancer of zeste homolog 2 (EZH2), hairy and enhancer of split 1 (HES1) and phosphatase and tensin homolog (PTEN) in the viability, cell cycle and fibrosis of mesangial cells (MCs), followed by the assessment using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, and Western blot assay (fibrosis-related proteins). The interaction between OGT and EZH2 and the effect on EZH2 glycosylation were verified by chromatin immunoprecipitation (ChIP) and glutathione S-transferase (GST) pull-down assays. EZH2 stability was checked by treatment with cycloheximide. ResultsExpression of OGT was repressed in the DN mice and high glucose-treated MCs. Elevated OGT suppressed viability of high glucose-treated MCs, blocked proliferation characterized by repressed cyclin D1, but enhanced p21 levels, and inhibited fibrosis evidenced by reduced levels of fibronectin (FN) and collagen-4 (col-4). OGT interacted with EZH2 and promoted its glycosylation thus stabilizing the EZH2. EZH2 overexpression enhanced the enrichment of EZH2 and histone H3 Lys27 trimethylation (H3K27me3) in the HES1 promoter. HES1 was upregulated and PTEN was downregulated in DN mice. Transduction of lentivirus vector containing overexpression (oe)-OGT alleviated renal injury in DN mice. ConclusionsCollectively, OGT stabilizes histone methyltransferases EZH2 to regulate HES1/PTEN thus inhibiting DN.

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.

Metformin inhibits extracellular matrix accumulation, inflammation and proliferation of mesangial cells in diabetic nephropathy by regulating H19/miR-143-3p/TGF-β1 axis.

Metformin (MET) has protective effect on diabetic nephropathy (DN). This study aims to demystify the mechanism of MET function in DN. Mouse glomerular membrane epithelial cell line SV40-MES-13 was treated with normal or high glucose combined with or without MET. The relationships among H19, miR-143-3p and TGF-β1 were evaluated by luciferase reporter assay. MTT assay was performed to detect cell proliferation. The levels of inflammatory factors were investigated by enzyme-linked immunosorbent assay. Quantitative real-time PCR and western blot were performed to examine gene and protein expression. H19 was up-regulated in the SV40-MES-13 cells after treated with high glucose, which was effectively repressed by MET treatment. MET promoted extracellular matrix accumulation, inflammation and proliferation in the SV40-MES-13 cells after treated with high glucose. These influences conferred by MET were abolished by H19 overexpression. H19 regulated TGF-β1 expression by sponging miR-143-3p. Furthermore, MET inhibited extracellular matrix accumulation, inflammation and proliferation by regulating H19/miR-143-3p/TGF-β1 axis. Our studies demonstrated that the protective effect of MET on DN was attributed to the inhibition of proliferation, inflammation and ECM accumulation in mesangial cells via H19/miR-143-3p/TGF-β1 axis, which suggested that the H19/miR-143-3p/TGF-β1 axis could be a valuable target for DN therapies.

LncRNA TUG1 inhibits the proliferation and fibrosis of mesangial cells in diabetic nephropathy via inhibiting the PI3K/AKT pathway.

To elucidate the potential function of long non-coding RNA (lncRNA) TUG1 in the progression of diabetic nephropathy (DN) and the underlying mechanism. Rat diabetes mellitus (DM) model was established by streptozocin (STZ) administration. In vivo levels of TUG1 and relative genes in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway in DM rats and control rats were determined by the quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Moreover, levels of kidney weight, 24 h-urine protein, blood urea nitrogen and serum creatinine in DM rats and controls were detected. Mesangial cells were subjected to induction of high-level glucose. Relative levels of TUG1 and relative genes in the PI3K/AKT pathway in mesangial cells were determined as well. Through Cell Counting Kit-8 (CCK-8) and 5-Ethynyl-2'-deoxyuridine (EdU) assay, the regulatory effect of TUG1 on the proliferative ability of mesangial cells induced with high-level glucose was evaluated. Finally, expression changes in the PI3K/AKT pathway and extracellular matrix (ECM)-related genes in mesangial cells were determined. TUG1 was downregulated in DM rats and mesangial cells induced with high-level glucose. Compared with controls, DM rats presented higher levels of kidney weight, 24 h-urine protein, blood urea nitrogen and serum creatinine, which were markedly reduced after TUG1 overexpression in vivo. Moreover, overexpression of TUG1 downregulated TGF-β1, FN, and COL-IV, and inhibited the activation of the PI3K/AKT pathway. TUG1 is downregulated in DN. The overexpression of TUG1 could suppress the proliferation and ECM accumulation of mesangial cells via inhibiting the PI3K/AKT pathway.

Long non-coding RNA Rpph1 promotes inflammation and proliferation of mesangial cells in diabetic nephropathy via an interaction with Gal-3

Diabetic nephropathy (DN) is one of the most significant complications of diabetes and is the primary cause of end-stage kidney disease. Cumulating evidence has shown that renal inflammation plays a role in the development and progression of DN, but the exact cellular mechanisms are unclear. Irregular expression of long non-coding RNAs (lncRNAs) is present in many diseases, including DN. However, the relationship between lncRNAs and inflammation in DN is unclear. In this study, we identified differentially expressed lncRNAs in DN using RNA-sequencing. Among these lncRNAs, we identified seven DN-related lncRNAs in vivo and in vitro using quantitative real-time PCR. One lncRNA in particular, Rpph1 (ribonuclease P RNA component H1), exhibited significantly increased expression. Further, over-expression or knockdown of Rpph1 was found to regulate cell proliferation and the expression of inflammatory cytokines in mesangial cells (MCs). The results revealed that Rpph1 directly interacts with the DN-related factor galectin-3 (Gal-3). Further, over-expression of Rpph1 promoted inflammation and cell proliferation through the Gal-3/Mek/Erk signaling pathway in MCs under low glucose conditions, while knockdown of Rpph1 inhibited inflammation and cell proliferation through the Gal-3/Mek/Erk pathway in MCs under high glucose conditions. These results provide new insight into the association between Rpph1 and the Gal-3/Mek/Erk signaling pathway during DN progression.

Rapeseed protein-derived antioxidant peptide RAP alleviates renal fibrosis through MAPK/NF-&amp;kappa;B signaling pathways in diabetic nephropathy

IntroductionKidney fibrosis is the main pathologic change in diabetic nephropathy (DN), which is the major cause of end-stage renal disease. Current therapeutic strategies slow down but cannot reverse the progression of renal dysfunction in DN. Plant-derived bioactive peptides in foodstuffs are widely used in many fields because of their potential pharmaceutical and nutraceutical benefits. However, this type of peptide has not yet been studied in renal fibrosis of DN. Previous studies have indicated that the peptide YWDHNNPQIR (named RAP), a natural peptide derived from rapeseed protein, has an antioxidative stress effect. The oxidative stress is believed to be associated with DN. The aim of this study was to evaluate the pharmacologic effects of RAP against renal fibrosis of DN and high glucose (HG)-induced mesangial dysfunction.Materials and methodsDiabetes was induced by streptozotocin and high-fat diet in C57BL/6 mice and these mice were treated by subcutaneous injection of different doses of RAP (0.1 mg/kg and 0.5 mg/kg, every other day) or PBS for 12 weeks. Later, functional and histopathologic analyses were performed. Parallel experiments verifying the molecular mechanism by which RAP alleviates DN were carried out in HG-induced mesangial cells (MCs).ResultsRAP improved the renal function indices, including 24-h albuminuria, triglyceride, serum creatinine, and blood urea nitrogen levels, but did not lower blood glucose levels in DN mice. RAP also simultaneously attenuated extracellular matrix accumulation in DN mice and HG-induced MCs. Furthermore, RAP reduced HG-induced cell proliferation, but it showed no toxicity in MCs. Additionally, RAP inhibited the mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways.ConclusionRAP can attenuate fibrosis in vivo and in vitro by antagonizing the MAPK and NF-κB pathways.

Gremlin Regulates Podocyte Apoptosis via Transforming Growth Factor-β (TGF-β) Pathway in Diabetic Nephropathy.

BackgroundGremlin has been reported to be up-regulated in glomerular mesangial cells in diabetic nephropathy (DN). However, the regulation of gremlin in podocytes is still rarely reported. This study aimed to investigate the underlying mechanisms by which gremlin mediates the pathogenesis of DN via transforming growth factor-β (TGF-β) signaling pathways.Material/MethodsLentiviral and RNAi transfection were performed to increase and decrease gremlin expression in high-glucose conditions. Expression at the mRNA and protein level was detected by RT-qPCR and Western blotting.ResultsThe expression of gremlin was significantly higher in high-glucose (HG, 30 mM) than normal-glucose (NG, 5.5 mM) conditions. The gremlin overexpression significantly suppressed the expression of nephrin and synaptopodin. The phosphorylation of canonical TGF-β signaling pathway components, including Smad2/3 and MKK, was increased in the gremlin-overexpressing group. In addition, the expression levels of Bax and cleaved caspase-3 were also higher in the gremlin-overexpressing group. TGF-β pathway inhibitor (SB505124) significantly inhibited TGF-β pathway activity and enhanced the expression of nephrin and synaptopodin.ConclusionsThese results indicate that gremlin can aggravate podocyte lesions through the TGF-β signaling pathway, providing a novel therapeutic target for DN.

The mTOR promotes oxidative stress-induced apoptosis of mesangial cells in diabetic nephropathy.

Glomerular mesangial cell (MC) apoptosis is one of the important mechanisms of glomerulosclerosis, which induces an increased severity of albuminuria and promotes the development of diabetic nephropathy (DN). However, the mechanism by which high glucose (HG) induces MCs apoptosis is not fully understood. In the present study, we investigated the effects of mTOR signalling on apoptosis in cultured MCs exposed to HG and in type I diabetes, and tried to clarify the specific mechanisms underlying these effects. Invitro, exposure of MCs to HG stimulated ROS production, decreased the antioxidant enzyme superoxide dismutase (SOD) activity and glutathione (GSH) level, increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, upregulated P53 expression and Bax/Bcl-2 ratio and enhanced cleavage of caspase 3, resulting in an increase in programmed cell death. Pretreatment of the cells with rapamycin ameliorated oxidative stress, reduced the number of apoptotic cells induced by HG and caused the downstream effects of mTOR activation. Invivo, compared with control rats, diabetic rats had more apoptotic cells in glomeruli. Induction of diabetes increased the level of MDA and NADPH oxidase activity, decreased the SOD activity and GSH level, elevated the Bax/Bcl ratio and P53 expression and activated caspase 3. mTOR inhibitor rapamycin treatment prevented these changes further alleviated albuminuria and improved renal function. Taken together, our data suggest that mTOR plays a key role in mediating ROS-induced MC apoptosis in diabetic nephropathy, and these effects have been associated with the promotion of ROS production by upregulating the antioxidant enzyme and downregulating the NADPH oxidase activity.

The effect of FoxO1 on the proliferation of rat mesangial cells under high glucose conditions

Hyperproliferation of glomerular mesangial cells (MCs) is a major pathological characteristic in the early stage of diabetic nephropathy (DN). We have previously confirmed that forkhead transcription factor O1 (FoxO1) was significantly downregulated in both the renal cortex of DN rats and MCs cultured under high-glucose (HG) conditions, but the effects and mechanisms of FoxO1 involved in the hyperproliferation of MCs are still unclear. This study aims to investigate whether FoxO1 regulates the hyperproliferation of MCs induced under high-glucose conditions, through modulating the cyclin-dependent kinase inhibitor (CKI), p27. Lentiviral vectors of LV-constitutively active FoxO1 (CA-FoxO1) and LV-small interfering RNA (siRNA)-FoxO1 were constructed to up- and downregulate FoxO1. Similarly, LV-NC-FoxO1 was used as negative control (NC). Rat MCs were cultured in normal glucose (5.6 mM) medium, HG (30 mM) medium, HG with LV-NC-FoxO1, HG with LV-CA-FoxO1 and HG with LV-siRNA-FoxO1 for 72 h. Cell proliferation, cell cycle progression, messenger RNA and protein expression of FoxO1, p27, cyclin D1 and CDK4 were detected by methyl thiazolyl tetrazolium assay, flow cytometry, quantitative real-time polymerase chain reaction and western blotting, respectively. MCs exposed to HG medium triggered hyperproliferation of MCs. Nevertheless, overexpression of FoxO1 caused by LV-CA-FoxO1 promoted cell cycle arrest at the G0/G1 phase and attenuated proliferation, which was associated with upregulation of p27 and downregulation of cyclin D1 and CDK4. Moreover, specific degradation of FoxO1 by LV-siRNA-FoxO1 caused a decrease of p27, increase of cyclin D1 and CDK4, overrode the limited cell cycle and stimulated proliferation of MCs. Overexpression of FoxO1 caused upregulation of p27, which promoted cell cycle arrest and inhibited hyperproliferation of MCs induced by HG. Degradation of FoxO1 caused an increase in p27 and stimulated MC proliferation. These findings unveil part of the molecular mechanism of FoxO1 regulation of MC hyperproliferation induced by HG.

High Glucose-induced O-GlcNAcylated Carbohydrate Response Element-binding Protein (ChREBP) Mediates Mesangial Cell Lipogenesis and Fibrosis: THE POSSIBLE ROLE IN THE DEVELOPMENT OF DIABETIC NEPHROPATHY

Carbohydrate response element-binding protein (ChREBP) is a transcription factor responsible for carbohydrate metabolism in the liver. However, the role of ChREBP in diabetic nephropathy has not been elucidated. Thus, we investigated the role of ChREBP in mesangial cells in diabetic nephropathy. Treatment with 25 mM glucose (high glucose; HG) increased cellular O-GlcNAc and O-GlcNAcylated ChREBP in mesangial cells compared with normal 5.5 mM glucose. O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenylcarbamate (PUGNAc), a drug that increases O-GlcNAc, augmented the expression of ChREBP targets, whereas DON, a drug that decreases O-GlcNAc and O-GlcNAcase overexpression, mitigated the increase with HG. O-GlcNAc augmented the protein stability, transcriptional activity, and nuclear translocation of ChREBP. HG treatment also stimulated lipid accumulation and the contents of triglyceride and cholesterol in mesangial cells. In addition, HG triggered expression of hypoxia-inducible factor 1-α, vascular endothelial growth factor, and extracellular matrix components related to nephrosclerosis. The ChREBP mutant, W130A, did not exhibit HG-induced lipid accumulation and fibrotic proteins, suggesting that the Trp-130 residue in the MCR3 domain is important in the development of glomerulosclerosis. O-GlcNAcylated ChREBP was elevated in mesangium cells of streptozotocin-induced diabetic rats. In conclusion, HG increased the O-GlcNAcylated ChREBP level, which resulted in lipid accumulation and up-regulation of fibrotic proteins in mesangial cells. These effects may lead mesangial cells to an ultimately pathological state.

Changes in the gene expression programs of renal mesangial cells during diabetic nephropathy

BackgroundDiabetic nephropathy is the leading cause of end stage renal disease. All three cell types of the glomerulus, podocytes, endothelial cells and mesangial cells, play important roles in diabetic nephropathy. In this report we used Meis1-GFP transgenic mice to purify mesangial cells from normal mice and from db/db mice, which suffer diabetic nephropathy. The purpose of the study is to better define the unique character of normal mesangial cells, and to characterize their pathogenic and protective responses during diabetic nephropathy.MethodsComprehensive gene expression states of the normal and diseased mesangial cells were defined with microarrays. By comparing the gene expression profiles of mesangial cells with those of multiple other renal cell types, including podocytes, endothelial cells and renal vesicles, it was possible to better define their exceptional nature, which includes smooth muscle, phagocytic and neuronal traits.ResultsThe complete set of mesangial cell expressed transcription factors, growth factors and receptors were identified. In addition, the analysis of the mesangial cells from diabetic nephropathy mice characterized their changes in gene expression. Molecular functions and biological processes specific to diseased mesangial cells were characterized, identifying genes involved in extracellular matrix, cell division, vasculogenesis, and growth factor modulation. Selected gene changes considered of particular importance to the disease process were validated and localized within the glomuerulus by immunostaining. For example, thrombospondin, a key mediator of TGFβ signaling, was upregulated in the diabetic nephropathy mesangial cells, likely contributing to fibrosis. On the other hand the decorin gene was also upregulated, and expression of this gene has been strongly implicated in the reduction of TGFβ induced fibrosis.ConclusionsThe results provide an important complement to previous studies examining mesangial cells grown in culture. The remarkable qualities of the mesangial cell are more fully defined in both the normal and diabetic nephropathy diseased state. New gene expression changes and biological pathways are discovered, yielding a deeper understanding of the diabetic nephropathy pathogenic process, and identifying candidate targets for the development of novel therapies.

Scleraxis Modulates Bone Morphogenetic Protein 4 (BMP4)-Smad1 Protein-Smooth Muscle α-Actin (SMA) Signal Transduction in Diabetic Nephropathy

Activation of mesangial cells (MCs), which is characterized by induction of smooth muscle α-actin (SMA) expression, contributes to a key event in various renal diseases; however, the mechanisms controlling MC differentiation are still largely undefined. Activated Smad1 induced SMA in a dose-dependent manner in MCs. As a direct regulating molecule for SMA, we identified and characterized scleraxis (Scx) as a new phenotype modulator in advanced glycation end product (AGE)-exposed MCs. Scx physically associated with E12 and bound the E-box in the promoter of SMA and negatively regulated the AGE-induced SMA expression. Scx induced expression and secretion of bone morphogenetic protein 4 (BMP4), thereby controlling the Smad1 activation in AGE-treated MCs. In diabetic mice, Scx was concomitantly expressed with SMA in the glomeruli. Inhibitor of differentiation 1 (Id1) was further induced by extended treatment with AGE, thereby dislodging Scx from the SMA promoter. These data suggest that Scx and Id1 are involved in the BMP4-Smad1-SMA signal transduction pathway besides the TGFβ1-Smad1-SMA signaling pathway and modulate phenotypic changes in MCs in diabetic nephropathy.

Expression patterns of connective tissue growth factor and of TGF-β isoforms during glomerular injury recapitulate glomerulogenesis

Transforming growth factor (TGF)-beta(1), -beta(2), and -beta(3) are involved in control of wound repair and development of fibrosis. Connective tissue growth factor (CTGF) expression is stimulated by all TGF-beta isoforms and is abundant in glomerulosclerosis and other fibrotic disorders. CTGF is hypothesized to mediate profibrotic effects of TGF-beta(1) or to facilitate interaction of TGF-beta(1) with its receptor, but its interactions with TGF-beta isoforms in nonpathological conditions are unexplored so far. Tissue repair and remodeling may recapitulate gene transcription at play in organogenesis. To further delineate the relationship between CTGF and TGF-beta, we compared expression patterns of CTGF and TGF-beta isoforms in rat and human glomerulogenesis and in various human glomerulopathies. CTGF mRNA was present in the immediate precursors of glomerular visceral and parietal epithelial cells in the comma- and S-shaped stages, but not in earlier stages of nephron development. During the capillary loop and maturing glomerular stages and simultaneous with the presence of TGF-beta(1), -beta(2), and -beta(3) protein, CTGF mRNA expression was maximal and present only in differentiating glomerular epithelial cells. CTGF protein was also present on precursors of mesangium and glomerular endothelium, suggesting possible paracrine interaction. Concomitant with the presence of TGF-beta(2) and -beta(3) protein, and in the absence of TGF-beta(1), CTGF mRNA and protein expression was restricted to podocytes in normal adult glomeruli. However, TGF-beta(1) and CTGF were again coexpressed, often with TGF-beta(2) and -beta(3), in particular in podocytes in proliferative glomerulonephritis and also in mesangial cells in diabetic nephropathy and IgA nephropathy (IgA NP). Coordinated expression of TGF-beta isoforms and of CTGF may be involved in normal glomerulogenesis and possibly in maintenance of glomerular structure and function at adult age. Prolonged overexpression of TGF-beta(1) and CTGF is associated with development of severe glomerulonephritis and glomerulosclerosis.

Proteomic Identification of 14-3-3ζ as an Adapter for IGF-1 and Akt/GSK-3β Signaling and Survival of Renal Mesangial Cells

Recently we demonstrated that IGF-1 expression is increased in the diabetic kidney and that it may involve in renal hypertrophy and extracellular matrix protein (ECM) accumulation in mesangial cells as seen in diabetic glomerulopathy. The present study investigates the molecular mechanism(s) of IGF-1 and Akt/glycogen synthase kinase-3beta (GSK-3beta) signaling pathway in the regulation of fibronectin and cyclin D1 expression and survival of renal mesangial cells. A proteomic approach is also employed to identify protein targets of IGF-1 signaling via GSK-3beta inhibition in mesangial cells. We show that IGF-1 (100 ng/ml) significantly increases the protein kinase Akt/PKB activity (1.5-2-fold, p<0.05) within 1-5 minutes, which is completely blocked by the presence of 100 nM Wortmannin (phosphatidyl-inositol 3-kinase inhibitor). Akt activation is coupled with Ser9 phosphorylation and inactivation of its down-stream target GSK-3beta. IGF-1 increases the cyclic AMP-responsive element (CRE) binding transcription factor CREB phosphorylation at Ser 133 and CRE-binding activity in mesangial cells, which parallels cyclin D1 and fibronectin expressions. Both proteins are known to have CRE-sequences in their promoter regions upstream of the transcription start site. Suppression of GSK-3beta by SB216763 (100 nM) increases CREB phosphorylation, cyclin D1 and fibronectin levels. Two dimensional gel electrophoresis followed by MALDI-TOF mass spectrometric analysis of mesangial proteins reveals that IGF-1 treatment or an inhibition of GSK-3beta increases the expression of the phosphorylated Ser/Thr binding signal adapter protein 14-3-3zeta. Immuno-precipitation of 14-3-3zeta followed by Western blotting validates the association of phosphorylated GSK-3beta with 14-3-3zeta in renal mesangial cells. Stable expression of a constitutively active GSK-3beta(Ser9Ala) induces cell death while overexpression of HA-tagged 14-3-3zeta increases cell viability as measured by MTT assays. These results indicate that the Akt/GSK-3beta pathway and the adapter protein 14-3-3zeta may play an important role in IGF-1 signaling and survival of mesangial cells in diabetic nephropathy.

Overview of glucose signaling in mesangial cells in diabetic nephropathy.

Clinical studies such as the Diabetes Control and Complications Trial (DCCT) in subjects with type 1 diabetes ([1][1]), the UK Prospective Diabetes Study (UKPDS) in subjects with type 2 diabetes ([2][2]), and the Kumamoto Study in Japanese subjects with type 2 diabetes ([3][3]) clearly link