Advanced glycation end product-induced apoptosis and overexpression of vascular endothelial growth factor and monocyte chemoattractant protein-1 in human-cultured mesangial cells

Advanced glycation end products (AGE) have been implicated in the pathogenesis of glomerulosclerosis in diabetes. However, their involvement in the development of the early phase of diabetic nephropathy has not been fully elucidated. We investigated the effects of AGE on growth and on vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) expression in human cultured mesangial cells. We prepared three immunochemically distinct AGE by incubating bovine serum albumin (BSA) with glucose, glyceraldehyde, or glycolaldehyde. When human mesangial cells were cultured with various types of AGE-BSA, viable cell numbers as well as DNA syntheses were significantly decreased. All of the AGE-BSA were found to significantly increase p53 and Bax protein accumulations and subsequently induce apoptotic cell death in mesangial cells. An antioxidant, N-acetylcysteine, significantly prevented the AGE-induced apoptotic cell death in mesangial cells. Human mesangial cells stimulated prostacyclin production by co-cultured glomerular endothelial cells. Furthermore, various types of AGE-BSA were found to up-regulate the levels of mRNAs for VEGF and stimulate the secretion of VEGF and MCP-1 proteins in mesangial cells. The results suggest that AGE disturbed glomerular homeostasis by inducing apoptotic cell death in mesangial cells and elicited hyperfiltration and microalbuminuria by stimulating the secretion of VEGF and MCP-1 proteins, thereby being involved in the pathogenesis of the early phase of diabetic nephropathy.     

Inhibition of MCP-1/CCR2 pathway ameliorates the development of diabetic nephropathy

Monocyte chemoattractant protein (MCP-1) is an important mediator for macrophage recruitment in atherosclerosis and various glomerulonephritis. However, the role of MCP-1 and its receptor CCR2 in the progression of diabetic nephropathy remains unknown. Using a type 1 diabetic nephropathy model that shows noticeable glomerulosclerosis, we examined the role of MCP-1/CCR2 by propagermanium (Pro; CCR2 antagonist) treatment, and confirmed it by transfection of plasmids carrying the 7ND (a mutant of MCP-1) gene. We measured the mesangial matrix expansion, type IV collagen (Col4), transforming growth factor (TGF)-beta1 positive area, and macrophage infiltration in glomeruli after 12 weeks. Mesangial matrix expansion and macrophage infiltration were increased in diabetic mice and inhibited by Pro or 7ND-treatment. Increased glomerular expression of Col4 and TGF-beta1 in diabetic mice was also ameliorated. Thus blocking the MCP-1/CCR2 pathway ameliorated glomerulosclerosis, indicating that the MCP-1/CCR2 pathway plays a crucial role in the progression of diabetic nephropathy.     

Involvement of glomerular SREBP-1c in diabetic nephropathy

The role of glomerular SREBP-1c in diabetic nephropathy was investigated. PEPCK-promoter transgenic mice overexpressing nuclear SREBP-1c exhibited enhancement of proteinuria with mesangial proliferation and matrix accumulation, mimicking diabetic nephropathy, despite the absence of hyperglycemia or hyperlipidemia. Isolated transgenic glomeruli had higher expression of TGFβ-1, fibronectin, and SPARC in the absence of marked lipid accumulation. Gene expression of P47phox, p67phox, and PU.1 were also activated, accompanying increased 8-OHdG in urine and kidney, demonstrating that glomerular SREBP-1c could directly cause oxidative stress through induced NADPH oxidase. Similar changes were observed in STZ-treated diabetic mice with activation of endogenous SREBP-1c. Finally, diabetic proteinuria and oxidative stress were ameliorated in SREBP-1-null mice. Adenoviral overexpression of active and dominant-negative SREBP-1c caused consistent reciprocal changes in expression of both profibrotic and oxidative stress genes in MES13 mesangial cells. These data suggest that activation of glomerular SREBP-1c could contribute to emergence and/or progression of diabetic nephropathy.     

SPARC regulates cell cycle progression in mesangial cells via its inhibition of IGF-dependent signaling

Glomerular mesangial cells both synthesize and respond to insulin-like growth factor-1 (IGF-1). Increased activity of the IGF signaling pathway has been implicated as a major contributor to renal enlargement and subsequent development of diabetic nephropathy. Secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, has been shown to modulate the interaction of cells with growth factors and extracellular matrix. We have reported that primary glomerular mesangial cells derived from SPARC-null mice exhibit an accelerated rate of proliferation and produce substantially decreased levels of transforming growth factor beta1 (TGF-beta1) in comparison to their wild-type counterparts (Francki et al. [1999] J. Biol. Chem. 274: 32145-32152). Herein we present evidence that SPARC modulates IGF-dependent signaling in glomerular mesangial cells. SPARC-null mesangial cells produce increased amounts of IGF-1 and -2, as well as IGF-1 receptor (IGF-1R) in comparison to wild-type cells. Addition of recombinant SPARC to SPARC-null cells inhibited IGF-1-stimulated mitogen activated protein kinase (MAPK) activation and DNA synthesis. We also show that the observed accelerated rate of basal and IGF-1-stimulated proliferation in mesangial cells derived from SPARC-null animals is due, at least in part, to markedly diminished levels of cyclin D1 and the cyclin-dependent kinase (cdk) inhibitors p21 and p27. Since expression of SPARC in the glomerulus is especially prominent during renal injury, our findings substantiate previous claims that SPARC is involved in glomerular remodeling and repair, a process commonly associated with mesangioproliferative glomerulonephritis and diabetic nephropathy.     

Rho-kinase mediates TNF-α-induced MCP-1 expression via p38 MAPK signaling pathway in mesangial cells

Macrophage accumulation has been implicated in the pathogenesis of inflammatory glomerular disease. Monocyte chemoattractant protein-1 (MCP-1) plays a central role in recruiting monocytes to the glomeruli. Tumor necrosis factor-α (TNF-α) has been shown to induce MCP-1 expression in mesangial cells, although the precise mechanisms remain unclear. We previously demonstrated that RhoA and its effector, Rho-kinase (Rho-associated coiled-coil containing protein kinase, ROCK), are involved in the pathogenesis of diabetic nephropathy. However, its role in MCP-1 induction by TNF-α has not been elucidated. In the present study, we investigated whether the Rho/Rho-kinase signaling pathway regulates the TNF-α-mediated induction of MCP-1 in mesangial cells. Exposure of mouse mesangial cells (MES-13) to TNF-α resulted in an increase of MCP-1 expression (by RT-PCR) and secretion into the medium (by ELISA). Pull down and Western blot analysis revealed that TNF-α activated RhoA and Rho-kinase. Based on these observations, we speculated that the Rho/Rho-kinase signaling pathway may be involved in MCP-1 induction by TNF-α. In agreement with this concept, Y-27632, a specific Rho-kinase inhibitor, attenuated TNF-α-mediated induction of MCP-1. We demonstrated that Y-27632 inhibited TNF-α-mediated monocyte migration and attenuated TNF-α-mediated p38 MAPK activation. Based on these data we infer that Y-27632 inhibits TNF-α-induced MCP-1 expression, secretion and function through inhibition of Rho-kinase and p38 MAPK activity. Our study suggests that Rho/Rho-kinase is an important therapeutic target of monocyte recruitment and accumulation within the glomerulus in inflammatory renal disease.     

The Kinetics of Glomerular Deposition of Nephritogenic IgA

Whether IgA nephropathy is attributable to mesangial IgA is unclear as there is no correlation between intensity of deposits and extent of glomerular injury and no clear mechanism explaining how these mesangial deposits induce hematuria and subsequent proteinuria. This hinders the development of a specific therapy. Thus, precise events during deposition still remain clinical challenge to clarify. Since no study assessed induction of IgA nephropathy by nephritogenic IgA, we analyzed sequential events involving nephritogenic IgA from IgA nephropathy-prone mice by real-time imaging systems. Immunofluorescence and electron microscopy showed that serum IgA from susceptible mice had strong affinity to mesangial, subepithelial, and subendothelial lesions, with effacement/actin aggregation in podocytes and arcade formation in endothelial cells. The deposits disappeared 24-h after single IgA injection. The data were supported by a fluorescence molecular tomography system and real-time and 3D in vivo imaging. In vivo imaging showed that IgA from the susceptible mice began depositing along the glomerular capillary from 1 min and accumulated until 2-h on the first stick in a focal and segmental manner. The findings indicate that glomerular IgA depositions in IgAN may be expressed under the balance between deposition and clearance. Since nephritogenic IgA showed mesangial as well as focal and segmental deposition along the capillary with acute cellular activation, all glomerular cellular elements are a plausible target for injury such as hematuria.     

Nicorandil as a novel therapy for advanced diabetic nephropathy in the eNOS-deficient mouse

Nicorandil is an orally available drug that can act as a nitric oxide donor, an antioxidant, and an ATP-dependent K channel activator. We hypothesized that it may have a beneficial role in treating diabetic nephropathy. We administered nicorandil to a model of advanced diabetic nephropathy (the streptozotocin-induced diabetes in mice lacking endothelial nitric oxide synthase, eNOSKO); controls included diabetic eNOS KO mice without nicorandil and nondiabetic eNOS KO mice treated with either nicorandil or vehicle. Mice were treated for 8 wk. Histology, blood pressure, and renal function were determined. Additional studies involved examining the effects of nicorandil on cultured human podocytes. Here, we found that nicorandil did not affect blood glucose levels, blood pressure, or systemic endothelial function, but significantly reduced proteinuria and glomerular injury (mesangiolysis and glomerulosclerosis). Nicorandil protected against podocyte loss and podocyte oxidative stress. Studies in cultured podocytes showed that nicorandil likely protects against glucose-mediated oxidant stress via the ATP-dependent K channel as opposed to its NO-stimulating effects. In conclusion, nicorandil may be beneficial in diabetic nephropathy by preserving podocyte function. We recommend clinical trials to determine whether nicorandil may benefit diabetic nephropathy or other conditions associated with podocyte dysfunction.     

Growth arrest-specific gene 6 is involved in glomerular hypertrophy in the early stage of diabetic nephropathy

Nephropathy is one of the most common complications of diabetes mellitus. Glomerular hypertrophy is a hallmark in the early phase of the nephropathy. The mechanism of glomerular hypertrophy, however, remains incompletely understood. We have reported that Gas6 (growth arrest-specific gene 6) and its receptor, Axl, play a key role in the development of glomerulonephritis. Here we show the important role of Gas6/Axl in the pathogenesis of diabetic glomerular hypertrophy. In streptozotocin (STZ)-induced diabetic rats, mesangial and glomerular hypertrophy and an increase in the glomerular filtration rate (GFR) and albuminuria were observed after 12 weeks of STZ injection. The glomerular expression of Gas6 and Axl was increased in those rats. Administration of warfarin inhibited mesangial and glomerular hypertrophy and the increase in GFR and albuminuria in STZ rats. Moreover, we found less mesangial hypertrophy in STZ-treated Gas6 knockout mice than control mice. In vitro we found that stimulation of mesangial cells with Gas6 resulted in mesangial cell hypertrophy. Thus we have found a novel mechanism of glomerular hypertrophy through the Gas6/Axl-mediated pathway in the development of diabetic nephropathy. Inhibition of the Gas6/Axl pathway in diabetic patients might be beneficial to slow down the progression of diabetic nephropathy.     

Exacerbation of Diabetic Renal Alterations in Mice Lacking Vasohibin-1

Vasohibin-1 (VASH1) is a unique endogenous inhibitor of angiogenesis that is induced in endothelial cells by pro-angiogenic factors. We previously reported renoprotective effect of adenoviral delivery of VASH1 in diabetic nephropathy model, and herein investigated the potential protective role of endogenous VASH1 by using VASH1-deficient mice. Streptozotocin-induced type 1 diabetic VASH1 heterozygous knockout mice (VASH1^+/−) or wild-type diabetic mice were sacrificed 16 weeks after inducing diabetes. In the diabetic VASH1^+/− mice, albuminuria were significantly exacerbated compared with the diabetic wild-type littermates, in association with the dysregulated distribution of glomerular slit diaphragm related proteins, nephrin and ZO-1, glomerular basement membrane thickning and reduction of slit diaphragm density. Glomerular monocyte/macrophage infiltration and glomerular nuclear translocation of phosphorylated NF-κB p65 were significantly exacerbated in the diabetic VASH1^+/− mice compared with the diabetic wild-type littermates, accompanied by the augmentation of VEGF-A, M1 macrophage-derived MCP-1 and phosphorylation of IκBα, and the decrease of angiopoietin-1/2 ratio and M2 macrophage-derived Arginase-1. The glomerular CD31⁺ endothelial area was also increased in the diabetic VASH1^+/− mice compared with the diabetic-wild type littermates. Furthermore, the renal and glomerular hypertrophy, glomerular accumulation of mesangial matrix and type IV collagen and activation of renal TGF-β₁/Smad3 signaling, a key mediator of renal fibrosis, were exacerbated in the diabetic VASH1^+/− mice compared with the diabetic wild-type littermates. In conditionally immortalized mouse podocytes cultured under high glucose condition, transfection of VASH1 small interfering RNA (siRNA) resulted in the reduction of nephrin, angiopoietin-1 and ZO-1, and the augmentation of VEGF-A compared with control siRNA. These results suggest that endogenous VASH1 may regulate the development of diabetic renal alterations, partly via direct effects on podocytes, and thus, a strategy to recover VASH1 might potentially lead to the development of a novel therapeutic approach for diabetic nephropathy.     

Comparative investigation of kidney mesangial cells from increased oxidative stress-induced diabetic rats by using different microscopy techniques

High glucose and increased oxidative stress levels are the known important mediators of diabetic nephropathy. However, the effects of these mediators on tissue damage basically due to extracellular matrix expansion in mesangial cells have yet to be fully examined within the context of early stage diabetic nephropathy. In this study, we attempted to characterize changes in mesangial cells of streptozotocin-induced diabetic rats with a comparative investigation of kidney tissue by using different microscopy techniques. The serum levels of urea and creatinine of diabetic rats, as biomarkers of kidney degeneration, decreased significantly compared to those of age-matched controls. In diabetic rats, there are increased malondialdehyde and oxidized-glutathione levels as well as reduced-glutathione and glutathione-peroxidase activity levels in renal tissue compared to those of the controls. By using light and electron microscopies, we showed that there were marked thickening in Bowman’s membrane and glomerular capillary wall, increased amount of extracellular matrix often occupying Bowman’s space, degenerations in tubules, an increased number of mesangial cells in the network of glomerular capillary walls, and increased amount of lipid accumulation in proximal tubules in the renal tissue of diabetic rats. Our confocal microscopy data confirmed also the presence of irregularity and widened in glomerular capillaries, their attachment to the Bowman’s capsule, degenerated heterochromatin, thickening in foci of glomerular basement membrane, and marked increase in mesangial cells. These results suggest that a detailed structural investigation of kidney tissue provides further information on the important role of mesangial cells in pathogenesis of diabetic nephropathy.     

Vascular Endothelial Growth Factor-Receptor 1 Inhibition Aggravates Diabetic Nephropathy through eNOS Signaling Pathway in db/db Mice

The manipulation of vascular endothelial growth factor (VEGF)-receptors (VEGFRs) in diabetic nephropathy is as controversial as issue as ever. It is known to be VEGF-A and VEGFR2 that regulate most of the cellular actions of VEGF in experimental diabetic nephropathy. On the other hand, such factors as VEGF-A, -B and placenta growth factor bind to VEGFR1 with high affinity. Such notion instigated us to investigate on whether selective VEGFR1 inhibition with GNQWFI hexamer aggravates the progression of diabetic nephropathy in db/db mice.While diabetes suppressed VEGFR1, it did increase VEGFR2 expressions in the glomerulus. Db/db mice with VEGFR1 inhibition showed more prominent features with respect to, albuminuria, mesangial matrix expansion, inflammatory cell infiltration and greater numbers of apoptotic cells in the glomerulus, and oxidative stress than that of control db/db mice. All these changes were related to the suppression of diabetes-induced increases in PI3K activity and Akt phosphorylation as well as the aggravation of endothelial dysfunction associated with the inactivation of FoxO3a and eNOS-NOx. In cultured human glomerular endothelial cells (HGECs), high-glucose media with VEGFR1 inhibition induced more apoptotic cells and oxidative stress than did high-glucose media alone, which were associated with the suppression of PI3K-Akt phosphorylation, independently of the activation of AMP-activated protein kinase, and inactivation of FoxO3a and eNOS-NOx pathway. In addition, transfection with VEGFR1 siRNA in HGECs also suppressed PI3K-Akt-eNOS signaling.In conclusion, the specific blockade of VEGFR1 with GNQWFI caused severe renal injury related to profound suppression of the PI3K-Akt, FoxO3a and eNOS-NOx pathway, giving rise to the oxidative stress-induced apoptosis of glomerular cells in type 2 diabetic nephropathy.     

过敏毒素受体(C3aR)在db/db糖尿病肾病小鼠肾脏中的表达及病理意义分析

利用半定量RT-PCR、免疫组化和Western blotting的方法,同时从mRNA水平和蛋白质水平对过敏毒素受体(C3aR)在不同病理阶段的2型糖尿病肾病模型小鼠——db/db小鼠肾脏中的表达情况进行了较为系统的分析．结果发现：a．在糖尿病前的db/db小鼠(4周龄的db/db小鼠),C3aR与作为正常对照的db/m小鼠相比没有明显差异．随着肥胖的加剧,高血糖、蛋白尿的发生和发展,C3aR在db/db小鼠肾脏中的表达显著升高．b．免疫组化分析显示,C3aR广泛地表达于db/m和db/db小鼠肾脏的皮质和髓质,分布于肾脏的上皮细胞中(包括肾小管上皮细胞、肾小球中的脏层上皮细胞(足细胞)和壁层上皮细胞)．从部位来看,皮髓交界处的肾小管中C3aR表达量明显要比其他部位的多．在肾小球,C3aR特异地存在于足细胞部位．在db/m小鼠,不同周龄小鼠肾脏中C3aR的表达量并没有明显变化,但在db/db小鼠,从8周龄开始,分布在db/db小鼠肾小管上皮细胞和小球足细胞中的C3aR均随小鼠周龄的增加而增加,至少在时间上,与小鼠糖尿病肾病的发生发展相关,其中尤以足细胞中和皮髓交界处肾小管上皮细胞中的变化最为明显． c．在糖尿病肾病小鼠中高表达C3aR的肾小管上皮细胞常有空泡变性的情况．上述工作印证了先前对2型糖尿病肾病患者肾小球基因表达谱的分析结果,更加明确了C3aR与糖尿病肾病的相关性,同时揭示了C3aR在正常小鼠和糖尿病肾病小鼠肾脏中的表达、分布和变化规律,有利于进一步揭示C3aR的功能及其在糖尿病肾病发生、发展过程中的可能作用,探讨糖尿病肾病的分子机制．     

IL-6 and its role in IgA nephropathy development

IL-6 is considered one of the well characterized cytokines exhibiting homeostatic, pro- and anti-inflammatory activities, depending on the receptor variant and the induced intracellular cis- or trans-signaling responses. IL-6-activated pathways are involved in the regulation of cell proliferation, survival, differentiation, and cell metabolism changes.Deviations in IL-6 levels or abnormal response to IL-6 signaling are associated with several autoimmune diseases including IgA nephropathy (IgAN), one of most frequent primary glomerulonephritis worldwide. IgAN is associated with increased plasma concentration of IL-6 and increased plasma concentration of aberrantly galactosylated IgA1 immunoglobulin (Gd-IgA1). Gd-IgA1 is specifically recognized by autoantibodies, leading to the formation of circulating immune complexes (CIC) with nephritogenic potential, since CIC deposited in the glomerular mesangium induce mesangial cells proliferation and glomerular injury. Infection of the upper respiratory or digestive tract enhances IL-6 production and in IgAN patients is often followed by the macroscopic hematuria.This review recapitulates general aspects of IL-6 signaling and summarizes experimental evidences about IL-6 involvement in the etiopathogenesis of IgA nephropathy through the production of Gd-IgA1 and regulation of mesangial cell proliferation.     

LncRNA H2k2促进高糖培养的肾小球系膜细胞增殖的研究

该研究主要探讨lncRNA H2k2对高糖培养的肾小球系膜细胞增殖的影响,采用qRTPCR检测lncH2k2在正常及糖尿病肾病小鼠肾脏组织中的表达,以及高低糖培养的系膜细胞中的表达;FISH与qRT-PCR检测lncH2k2的亚细胞定位;qRT-PCR检测lncH2k2过表达质粒及siRNA的转染效率;EdU检测转染lncH2k2过表达质粒或siRNA后系膜细胞增殖的变化。结果表明,lncH2k2在糖尿病肾病小鼠肾脏组织及高糖培养的系膜细胞中的表达升高,且lncH2k2主要分布于系膜细胞的细胞质中。在低糖培养的系膜细胞中转染lncH2k2过表达质粒后,与低糖培养的系膜细胞相比,过表达lncH2k2的低糖培养的系膜细胞增殖能力显著提高,并且将qRT-PCR检测筛选出的一条lncH2k2 siRNA转染到高糖培养的系膜细胞内,与高糖培养的系膜细胞相比,敲低lncH2k2后系膜细胞增殖能力显著降低。研究结果揭示,lncRNA H2k2在糖尿病肾病小鼠肾脏组织及系膜细胞中表达显著,lncRNA H2k2促进了系膜细胞增殖,这些结果表明,lncRNA H2k2可能参与了糖尿病肾病的发生发展。     

Cellular Cholesterol Transport Proteins in Diabetic Nephropathy

Background

Lipid accumulation has been shown to accelerate renal injury, and the intracellular accumulation of lipids may be caused by alterations in synthesis as well as lipid uptake and efflux. We have investigated the role of cellular cholesterol transport proteins including adenosine triphosphate binding cassette transporter A1 (ABCA1), G1 (ABCG1) and scavenger receptor class B type I (SR-BI) in diabetic nephropathy.

Methods

Protein expression and the ability to mediate cholesterol efflux of ABCA1, ABCG1 and SR-BI was determined in human renal mesangial cells and proximal tubular epithelial cells cultured under normal or high glucose conditions. Renal expression of these cholesterol transporters was examined in a murine model of streptozotocin-induced type 1 diabetes.

Results

ABCA1, ABCG1 and SR-BI were expressed in both human renal mesangial cells and proximal tubular epithelial cells, and mediated cholesterol efflux to apolipoprotein AI and HDL. In vitro, hyperglycemia reduced the expression and the ability to mediate cholesterol efflux of all three cholesterol transporters (p<0.05). In vivo studies showed that intra-renal accumulation of lipids was increased in diabetic mice, particularly in mice with nephropathy. This was associated with a significant reduction in the expression of ABCA1, ABCG1 and SR-BI in the kidneys. These changes were already seen in diabetic mice without nephropathy and preceded the development of nephropathy. Diabetic mice with nephropathy had the lowest level of these cholesterol transporters.

Conclusion

Inducing diabetes with streptozotocin significantly reduced renal expression of ABCA1, ABCG1 and SR-BI. Defects in cholesterol export pathway in renal cells could therefore promote cholesterol accumulation and might contribute to the development of diabetic nephropathy.     

FOG2 Protein Down-regulation by Transforming Growth Factor-β1-induced MicroRNA-200b/c Leads to Akt Kinase Activation and Glomerular Mesangial Hypertrophy Related to Diabetic Nephropathy

Glomerular hypertrophy is a hallmark of diabetic nephropathy. Akt kinase activated by transforming growth factor-β1 (TGF-β) plays an important role in glomerular mesangial hypertrophy. However, the mechanisms of Akt activation by TGF-β are not fully understood. Recently, miR-200 and its target FOG2 were reported to regulate the activity of phosphatidylinositol 3-kinase (the upstream activator of Akt) in insulin signaling. Here, we show that TGF-β activates Akt in glomerular mesangial cells by inducing miR-200b and miR-200c, both of which target FOG2, an inhibitor of phosphatidylinositol 3-kinase activation. FOG2 expression was reduced in the glomeruli of diabetic mice as well as TGF-β-treated mouse mesangial cells (MMC). FOG2 knockdown by siRNAs in MMC activated Akt and increased the protein content/cell ratio suggesting hypertrophy. A significant increase of miR-200b/c levels was detected in diabetic mouse glomeruli and TGF-β-treated MMC. Transfection of MMC with miR-200b/c mimics significantly decreased the expression of FOG2. Conversely, miR-200b/c inhibitors attenuated TGF-β-induced decrease in FOG2 expression. Furthermore, miR-200b/c mimics increased the protein content/cell ratio, whereas miR-200b/c inhibitors abrogated the TGF-β-induced increase in protein content/cell. In addition, down-regulation of FOG2 by miR-200b/c could activate not only Akt but also ERK, which was also through PI3K activation. These data suggest a new mechanism for TGF-β-induced Akt activation through FOG2 down-regulation by miR-200b/c, which can lead to glomerular mesangial hypertrophy in the progression of diabetic nephropathy.     

Diabetes induces changes of catecholamines in primary mesangial cells

Diabetes mellitus is a frequent cause of kidney function damage with diabetic nephropathy being predominantly related to glomerular dysfunction. Diabetes is capable of interfering with distinct hormonal systems, as well as catecholamine metabolism. Since mesangial cells, the major constituent of renal glomerulus, constitute a potential site for catecholamine production, the present study was carried out to investigate alterations in catecholamine metabolism in cultured mesangial cells from the nonobese diabetic mouse, a well-established model for type I diabetes. We evaluated mesangial cells from normoglycemic and hyperglycemic nonobese diabetic mice, as well as cells from normoglycemic Swiss mice as control. Mesangial cells from normoglycemic mice presented similar profiles concerning all determinations. However, cells isolated from hyperglycemic animals presented increased dopamine and norepinephrine production/secretion. Among the studied mechanisms, we observed an upregulation of tyrosine hydroxylase expression accompanied by increased tetrahydrobiopterin consumption, the tyrosine hydroxylase enzymatic cofactor. However, this increase in synthetic pathways was followed by decreased monoamine oxidase activity, which corresponds to the major metabolic pathway of catecholamines in mesangial cells. In addition, whole kidney homogenates from diabetic animals also presented increased dopamine and norepinephrine levels when compared to normoglycemic animals. Thus, our results suggest that diabetes alters catecholamine production by interfering with both synthesizing and degrading enzymes, suggesting a possible role of catecholamine in the pathogenesis of acute and chronic renal complications of diabetes mellitus.     

Hyaluronan structures synthesized by rat mesangial cells in response to hyperglycemia induce monocyte adhesion

Mesangial expansion, the principal glomerular lesion in diabetic nephropathy, is preceded by a phenotypic activation and transient proliferation of the glomerular mesangial cells and by a prominent glomerular infiltration of monocytes and macrophages. Because this infiltration seems to play a key role in the subsequent mesangial matrix expansion, we tested the response of cultures of rat mesangial cells (RMCs) for monocyte adhesion in response to hyperglycemia. Increasing the medium glucose concentration from 5.6 mm (normal) to 25.6 mm (hyperglycemic) significantly increased hyaluronan in the cell matrix, with a concurrent 3- to 4-fold increase in adhesion of U937 monocytic leukemic cells to cultures of near confluent RMCs. These responses were attributed directly to the high glucose concentration and not to increased extracellular osmolality. The monocytes primarily bind directly to hyaluronan-based structures in vitro. Abnormal deposits of hyaluronan were found in glomeruli of kidney sections from diabetic rats 1 week after streptozotocin treatment, often with closely associated monocytes/macrophages, suggesting that similar structures are relevant in vivo. The monocyte adhesion response to high glucose concentration required growth stimulation of RMCs by serum and activation of protein kinase C, and was inhibited by prior passage of the RMCs in the presence of heparin. These results suggest that the response may be cell growth state and protein kinase C-dependent. When incubated with the viral mimetic, poly I:C, in the presence of normal glucose, heparin-passaged RMCs still increased cell-associated hyaluronan and exhibited hyaluronan-mediated adhesion of monocytes, indicating that the two stimuli, high glucose and viral mimetic, induce the production of the hyaluronan structures that promote monocyte adhesion by distinctly different intracellular signaling mechanisms.