Transgenic overexpression of GLUT1 in mouse glomeruli produces renal disease resembling diabetic glomerulosclerosis

Previous work identified an important role for hyperglycemia in diabetic nephropathy (The Diabetes Control and Complications Trial Research Group. N Engl J Med 329: 977–986, 1993; UK Prospective Diabetes Study Group. Lancet 352: 837–853, 1998), and increased glomerular GLUT1 has been implicated. However, the roles of GLUT1 and intracellular glucose have not been determined. Here, we developed transgenic GLUT1-overexpressing mice (GT1S) to characterize the roles of GLUT1 and intracellular glucose in the development of glomerular disease without diabetes. GLUT1 was overexpressed in glomerular mesangial cells (MC) of C57BL6 mice, a line relatively resistant to diabetic nephropathy. Blood pressure, blood glucose, glomerular morphometry, matrix proteins, cell signaling, transcription factors, and selected growth factors were examined. Kidneys of GT1S mice overexpressed GLUT1 in glomerular MCs and small vessels, rather than renal tubules. GT1S mice were neither diabetic nor hypertensive. Glomerular GLUT1, glucose uptake, mean capillary diameter, and mean glomerular volume were all increased in the GT1S mice. Moderately severe glomerulosclerosis (GS) was established by 26 wk of age in GT1S mice, with increased glomerular type IV collagen and fibronectin. Modest increases in glomerular basement membrane thickness and albuminuria were detected with podocyte foot processes largely preserved, in the absence of podocyte GLUT1 overexpression. Activation of glomerular PKC, along with increased transforming growth factor-β1, VEGFR1, VEGFR2, and VEGF were all detected in glomeruli of GT1S mice, likely contributing to GS. The transcription factor NF-κB was also activated. Overexpression of glomerular GLUT1, mimicking the diabetic GLUT1 response, produced numerous features typical of diabetic glomerular disease, without diabetes or hypertension. This suggested GLUT1 may play an important role in the development of diabetic GS.

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O-linked β-N-acetylglucosamine supports p38 MAPK activation by high glucose in glomerular mesangial cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00108.2011 ◽

2011 ◽

Vol 301 (4) ◽

pp. E713-E726 ◽

Cited By ~ 50

Author(s):

Howard Goldberg ◽

Catharine Whiteside ◽

I. George Fantus

Keyword(s):

Diabetic Nephropathy ◽

P38 Mapk ◽

High Glucose ◽

Transforming Growth Factor ◽

Kinase Inhibitor ◽

Mesangial Cells ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Glomerular Mesangial Cells ◽

Matrix Accumulation

Hyperglycemia augments flux through the hexosamine biosynthetic pathway and subsequent O-linkage of single β- N-acetyl-d-glucosamine moieties to serine and threonine residues on cytoplasmic and nuclear proteins ( O-GlcNAcylation). Perturbations in this posttranslational modification have been proposed to promote glomerular matrix accumulation in diabetic nephropathy, but clear evidence and mechanism are lacking. We tested the hypothesis that O-GlcNAcylation enhances profibrotic signaling in rat mesangial cells. An adenovirus expressing shRNA directed against O-GlcNAc transferase (OGT) markedly reduced basal and high-glucose-stimulated O-GlcNAcylation. Interestingly, O-GlcNAc depletion prevented high-glucose-induced p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase phosphorylation. Downstream of p38, O-GlcNAc controlled the expression of plasminogen activator inhibitor-1, fibronectin, and transforming growth factor-β, important factors in matrix accumulation in diabetic nephropathy. Treating mesangial cells with thiamet-G, a highly selective inhibitor of O-GlcNAc-specific hexosaminidase ( O-GlcNAcase), increased O-GlcNAcylation and p38 phosphorylation. The high-glucose-stimulated kinase activity of apoptosis signal-regulating kinase 1 (ASK1), an upstream MAPK kinase kinase for p38 that is negatively regulated by Akt, was inhibited by OGT shRNA. Akt Thr308 and Ser473 phosphorylation were enhanced following OGT shRNA expression in high-glucose-exposed mesangial cells, but high-glucose-induced p38 phosphorylation was not attenuated by OGT shRNA in cells pretreated with the phosphatidylinositol 3-kinase inhibitor LY-294002. OGT shRNA also reduced high-glucose-stimulated reactive oxygen species (ROS) formation. In contrast, diminished O-GlcNAcylation caused elevated ERK phosphorylation and PKCδ membrane translocation. Thus, O-GlcNAcylation is coupled to profibrotic p38 MAPK signaling by high glucose in part through Akt and possibly through ROS.

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Suppressions of chronic glomerular injuries and TGF-β1 production by HGF in attenuation of murine diabetic nephropathy

AJP Renal Physiology ◽

10.1152/ajprenal.00199.2003 ◽

2004 ◽

Vol 286 (1) ◽

pp. F134-F143 ◽

Cited By ~ 69

Author(s):

Shinya Mizuno ◽

Toshikazu Nakamura

Keyword(s):

Diabetic Nephropathy ◽

Growth Factor ◽

Renal Dysfunction ◽

Transforming Growth Factor ◽

Mesangial Cells ◽

Renal Diseases ◽

Glomerular Mesangial Cells ◽

Diabetic Mice

Diabetic nephropathy is now the leading cause of end-stage renal diseases, and glomerular sclerotic injury is an initial event that provokes renal dysfunction during processes of diabetes-linked kidney disease. Growing evidence shows that transforming growth factor-β1 (TGF-β1) plays a key role in this process, especially in eliciting hypertrophy and matrix overaccumulation. Thus it is important to find a ligand system to antagonize the TGF-β1-mediated pathogenesis under high-glucose conditions. Herein, we provide evidence that hepatocyte growth factor (HGF) targets mesangial cells, suppresses TGF-β1 production, and minimizes glomerular sclerotic changes, using streptozotocin-induced diabetic mice. In our murine model, glomerular sclerogenesis (such as tuft area expansion and collagen deposition) progressed between 6 and 10 wk after the induction of hyperglycemia, during a natural course of diabetic disease. Glomerular HGF expression levels in the diabetic kidney transiently increased but then declined below a basal level, with manifestation of glomerular sclerogenesis. When anti-HGF IgG was injected into mice for 2 wk (i.e., from weeks 4 to 6 after onset of hyperglycemia), these glomerular changes were significantly aggravated. When recombinant HGF was injected into the mice for 4 wk (i.e., between 6 and 10 wk following streptozotocin treatment), the progression of glomerular hypertrophy and sclerosis was almost completely inhibited, even though glucose levels remained unchanged (>500 mg/dl). Even more important, HGF repressed TGF-β1 production in glomerular mesangial cells even under hyperglycemic conditions both in vitro and in vivo. Consequently, not only albuminuria but also tubulointerstitial fibrogenesis were attenuated by HGF. Overall, HGF therapy inhibited the onset of renal dysfunction in the diabetic mice. On the basis of these findings, we wish to emphasize that HGF plays physiological and therapeutic roles in blocking renal fibrogenesis during a course of diabetic nephropathy.

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Tubular overexpression of Gremlin in transgenic mice aggravates renal damage in diabetic nephropathy

AJP Renal Physiology ◽

10.1152/ajprenal.00023.2015 ◽

2015 ◽

Vol 309 (6) ◽

pp. F559-F568 ◽

Cited By ~ 24

Author(s):

Vanessa Marchant ◽

Alejandra Droguett ◽

Graciela Valderrama ◽

M. Eugenia Burgos ◽

Daniel Carpio ◽

...

Keyword(s):

Diabetic Nephropathy ◽

Renal Damage ◽

Transforming Growth Factor ◽

Mesangial Cells ◽

Interstitial Fibrosis ◽

Smooth Muscle Actin ◽

Periodic Acid ◽

Kidney Tissue ◽

Renal Tubules ◽

Periodic Acid Schiff

Diabetic nephropathy (DN) is currently a leading cause of end-stage renal failure worldwide. Gremlin was identified as a gene differentially expressed in mesangial cells exposed to high glucose and in experimental diabetic kidneys. We have described that Gremlin is highly expressed in biopsies from patients with diabetic nephropathy, predominantly in areas of tubulointerstitial fibrosis. In streptozotocin (STZ)-induced experimental diabetes, Gremlin deletion using Grem1 heterozygous knockout mice or by gene silencing, ameliorates renal damage. To study the in vivo role of Gremlin in renal damage, we developed a diabetic model induced by STZ in transgenic (TG) mice expressing human Gremlin in proximal tubular epithelial cells. The albuminuria/creatinuria ratio, determined at week 20 after treatment, was significantly increased in diabetic mice but with no significant differences between transgenic (TG/STZ) and wild-type mice (WT/STZ). To assess the level of renal damage, kidney tissue was analyzed by light microscopy (periodic acid-Schiff and Masson staining), electron microscopy, and quantitative PCR. TG/STZ mice had significantly greater thickening of the glomerular basement membrane, increased mesangial matrix, and podocytopenia vs. WT/STZ. At the tubulointerstitial level, TG/STZ showed increased cell infiltration and mild interstitial fibrosis. In addition, we observed a decreased expression of podocin and overexpression of monocyte chemoattractant protein-1 and fibrotic-related markers, including transforming growth factor-β1, Col1a1, and α-smooth muscle actin. Together, these results show that TG mice overexpressing Gremlin in renal tubules develop greater glomerular and tubulointerstitial injury in response to diabetic-mediated damage and support the involvement of Gremlin in diabetic nephropathy.

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Inhibitor of myogenic differentiation family isoform a, a new positive regulator of fibronectin production by glomerular mesangial cells

AJP Renal Physiology ◽

10.1152/ajprenal.00508.2019 ◽

2020 ◽

Vol 318 (3) ◽

pp. F673-F682

Author(s):

Parisa Yazdizadeh Shotorbani ◽

Sarika Chaudhari ◽

Yu Tao ◽

Leonidas Tsiokas ◽

Rong Ma

Keyword(s):

Diabetic Nephropathy ◽

High Glucose ◽

Protein Level ◽

Transforming Growth Factor ◽

Mesangial Cells ◽

Myogenic Differentiation ◽

Mrna Levels ◽

Dependent Manner ◽

Glomerular Mesangial Cells ◽

Significant Difference

Overproduction of extracellular matrix proteins, including fibronectin by mesangial cells (MCs), contributes to diabetic nephropathy. Inhibitor of myogenic differentiation family isoform a (I-mfa) is a multifunctional cytosolic protein functioning as a transcriptional modulator or plasma channel protein regulator. However, its renal effects are unknown. The present study was conducted to determine whether I-mfa regulated fibronectin production by glomerular MCs. In human MCs, overexpression of I-mfa significantly increased fibronectin abundance. Silencing I-mfa significantly reduced the level of fibronectin mRNA and blunted transforming growth factor-β1-stimulated production of fibronectin. We further found that high glucose increased I-mfa protein content in a time course (≥48 h) and concentration (≥25 mM)-dependent manner. Although high glucose exposure increased I-mfa at the protein level, it did not significantly alter transcripts of I-mfa in MCs. Furthermore, the abundance of I-mfa protein was significantly increased in the renal cortex of rats with diabetic nephropathy. The I-mfa protein level was also elevated in the glomerulus of mice with diabetic kidney disease. However, there was no significant difference in glomerular I-mfa mRNA levels between mice with and without diabetic nephropathy. Moreover, H2O2 significantly increased I-mfa protein abundance in a dose-dependent manner in cultured human MCs. The antioxidants polyethylene glycol-catalase, ammonium pyrrolidithiocarbamate, and N-acetylcysteine significantly blocked the high glucose-induced increase of I-mfa protein. Taken together, our results suggest that I-mfa, increased by high glucose/diabetes through the production of reactive oxygen species, stimulates fibronectin production by MCs.

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High Glucose and Lipopolysaccharide Prime NLRP3 Inflammasome via ROS/TXNIP Pathway in Mesangial Cells

Journal of Diabetes Research ◽

10.1155/2016/6973175 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 48

Author(s):

Hong Feng ◽

Junling Gu ◽

Fang Gou ◽

Wei Huang ◽

Chenlin Gao ◽

...

Keyword(s):

Diabetic Nephropathy ◽

Nlrp3 Inflammasome ◽

High Glucose ◽

Mesangial Cells ◽

Central Component ◽

Dependent Manner ◽

Glomerular Mesangial Cells ◽

Interacting Protein

While inflammation is considered a central component in the development in diabetic nephropathy, the mechanism remains unclear. The NLRP3 inflammasome acts as both a sensor and a regulator of the inflammatory response. The NLRP3 inflammasome responds to exogenous and endogenous danger signals, resulting in cleavage of procaspase-1 and activation of cytokines IL-1β, IL-18, and IL-33, ultimately triggering an inflammatory cascade reaction. This study observed the expression of NLRP3 inflammasome signaling stimulated by high glucose, lipopolysaccharide, and reactive oxygen species (ROS) inhibitor N-acetyl-L-cysteine in glomerular mesangial cells, aiming to elucidate the mechanism by which the NLRP3 inflammasome signaling pathway may contribute to diabetic nephropathy. We found that the expression of thioredoxin-interacting protein (TXNIP), NLRP3, and IL-1βwas observed by immunohistochemistry in vivo. Simultaneously, the mRNA and protein levels of TXNIP, NLRP3, procaspase-1, and IL-1βwere significantly induced by high glucose concentration and lipopolysaccharide in a dose-dependent and time-dependent manner in vitro. This induction by both high glucose and lipopolysaccharide was significantly inhibited by N-acetyl-L-cysteine. Our results firstly reveal that high glucose and lipopolysaccharide activate ROS/TXNIP/ NLRP3/IL-1βinflammasome signaling in glomerular mesangial cells, suggesting a mechanism by which inflammation may contribute to the development of diabetic nephropathy.

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MiR-153-3p reduces extracellular matrix accumulation in high glucose-stimulated human glomerular mesangial cells via targeting PAQR3 in diabetic nephropathy

Endocrinología Diabetes y Nutrición ◽

10.1016/j.endinu.2021.03.005 ◽

2021 ◽

Author(s):

Hongli Yang ◽

Xingxing Fang ◽

Yan Shen ◽

Wubin Yao ◽

Dongmei Chen ◽

...

Keyword(s):

Extracellular Matrix ◽

Diabetic Nephropathy ◽

High Glucose ◽

Mesangial Cells ◽

Glomerular Mesangial Cells ◽

Human Glomerular Mesangial Cells ◽

Matrix Accumulation

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Store-operated calcium entry suppressed the TGF-β1/Smad3 signaling pathway in glomerular mesangial cells

AJP Renal Physiology ◽

10.1152/ajprenal.00483.2016 ◽

2017 ◽

Vol 313 (3) ◽

pp. F729-F739 ◽

Cited By ~ 8

Author(s):

Sarika Chaudhari ◽

Weizu Li ◽

Yanxia Wang ◽

Hui Jiang ◽

Yuhong Ma ◽

...

Keyword(s):

Signaling Pathway ◽

Transforming Growth Factor ◽

Nuclear Translocation ◽

Mesangial Cells ◽

Underlying Mechanism ◽

Glomerular Mesangial Cells ◽

Critical Pathway ◽

Smad3 Phosphorylation ◽

Tgf Β1

Our previous study demonstrated that the abundance of extracellular matrix proteins was suppressed by store-operated Ca2+entry (SOCE) in mesangial cells (MCs). The present study was conducted to investigate the underlying mechanism focused on the transforming growth factor-β1 (TGF-β1)/Smad3 pathway, a critical pathway for ECM expansion in diabetic kidneys. We hypothesized that SOCE suppressed ECM protein expression by inhibiting this pathway in MCs. In cultured human MCs, we observed that TGF-β1 (5 ng/ml for 15 h) significantly increased Smad3 phosphorylation, as evaluated by immunoblot. However, this response was markedly inhibited by thapsigargin (1 µM), a classical activator of store-operated Ca2+channels. Consistently, both immunocytochemistry and immunoblot showed that TGF-β1 significantly increased nuclear translocation of Smad3, which was prevented by pretreatment with thapsigargin. Importantly, the thapsigargin effect was reversed by lanthanum (La3+; 5 µM) and GSK-7975A (10 µM), both of which are selective blockers of store-operated Ca2+channels. Furthermore, knockdown of Orai1, the pore-forming subunit of the store-operated Ca2+channels, significantly augmented TGF-β1-induced Smad3 phosphorylation. Overexpression of Orai1 augmented the inhibitory effect of thapsigargin on TGF-β1-induced phosphorylation of Smad3. In agreement with the data from cultured MCs, in vivo knockdown of Orai1 specific to MCs using a targeted nanoparticle small interfering RNA delivery system resulted in a marked increase in abundance of phosphorylated Smad3 and in nuclear translocation of Smad3 in the glomerulus of mice. Taken together, our results indicate that SOCE in MCs negatively regulates the TGF-β1/Smad3 signaling pathway.

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Long Non-Coding RNA NEAT1 Regulates Pyroptosis in Diabetic Nephropathy via Mediating the miR-34c/NLRP3 Axis

Kidney & Blood Pressure Research ◽

10.1159/000508372 ◽

2020 ◽

Vol 45 (4) ◽

pp. 589-602 ◽

Cited By ~ 1

Author(s):

Jin-Feng Zhan ◽

Hong-Wei Huang ◽

Chong Huang ◽

Li-Li Hu ◽

Wen-Wei Xu

Keyword(s):

Diabetic Nephropathy ◽

Target Gene ◽

Molecular Mechanisms ◽

Mesangial Cells ◽

Receptor Protein ◽

Luciferase Reporter ◽

Glomerular Mesangial Cells ◽

Non Coding Rna ◽

Vitro Model

Introduction: Diabetic nephropathy (DN) is a serious complication of diabetes mellitus and is considered to be a sterile inflammatory disease. Increasing evidence suggest that pyroptosis and subsequent inflammatory response play a key role in the pathogenesis of DN. However, the underlying cellular and molecular mechanisms responsible for pyroptosis in DN are largely unknown. Methods: The rat models of DN were successfully established by single 65 mg/kg streptozotocin treatment. Glomerular mesangial cells were exposed to 30 mmol/L high glucose media for 48 h to mimic the DN environment in vitro. Gene and protein expressions were determined by quantitative real-time PCR and Western blot. Cell viability and pyroptosis were measured by MTT assay and flow cytometry analysis, respectively. The relationship between lncRNA NEAT1, miR-34c, and Nod-like receptor protein-3 (NLRP3) was confirmed by luciferase reporter assay. Results: We found that upregulation of NEAT1 was associated with the increase of pyroptosis in DN models. miR-34c, as a target gene of NEAT1, mediated the effect of NEAT1 on pyroptosis in DN by regulating the expression of NLRP3 as well as the expressions of caspase-1 and interleukin-1β. Either miR-34c inhibition or NLRP3 overexpression could reverse the accentuation of pyroptosis and inflammation by sh-NEAT1 transfection in the in vitro model of DN. Conclusions: Our findings suggested NEAT1 and its target gene miR-34c regulated cell pyroptosis via mediating NLRP3 in DN, providing new insights into understanding the molecular mechanisms of pyroptosis in the pathogenesis of DN.

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Targeting NF-κB by the Cell-Permeable NEMO-Binding Domain Peptide Improves Albuminuria and Renal Lesions in an Experimental Model of Type 2 Diabetic Nephropathy

International Journal of Molecular Sciences ◽

10.3390/ijms21124225 ◽

2020 ◽

Vol 21 (12) ◽

pp. 4225

Author(s):

Lucas Opazo-Ríos ◽

Anita Plaza ◽

Yenniffer Sánchez Matus ◽

Susana Bernal ◽

Laura Lopez-Sanz ◽

...

Keyword(s):

Diabetic Nephropathy ◽

Binding Domain ◽

Inflammatory Factors ◽

Preclinical Model ◽

Membrane Thickness ◽

Basement Membrane Thickness ◽

Nemo Binding Domain ◽

Nbd Peptide

Diabetic nephropathy (DN) is a multifactorial disease characterized by hyperglycemia and close interaction of hemodynamic, metabolic and inflammatory factors. Nuclear factor-κB (NF-κB) is a principal matchmaker linking hyperglycemia and inflammation. The present work investigates the cell-permeable peptide containing the inhibitor of kappa B kinase γ (IKKγ)/NF-κB essential modulator (NEMO)-binding domain (NBD) as therapeutic option to modulate inflammation in a preclinical model of type 2 diabetes (T2D) with DN. Black and tan, brachyuric obese/obese mice were randomized into 4 interventions groups: Active NBD peptide (10 and 6 µg/g body weight); Inactive mutant peptide (10 µg/g); and vehicle control. In vivo/ex vivo fluorescence imaging revealed efficient delivery of NBD peptide, systemic biodistribution and selective renal metabolization. In vivo administration of active NBD peptide improved albuminuria (>40% reduction on average) and kidney damage, decreased podocyte loss and basement membrane thickness, and modulated the expression of proinflammatory and oxidative stress markers. In vitro, NBD blocked IKK-mediated NF-κB induction and target gene expression in mesangial cells exposed to diabetic-like milieu. These results constitute the first nephroprotective effect of NBD peptide in a T2D mouse model that recapitulates the kidney lesions observed in DN patients. Targeting IKK-dependent NF-κB activation could be a therapeutic strategy to combat kidney inflammation in DN.

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