Gene Expression In Mesangial Cells Research Articles

Elevated glucose alters global gene expression and tenascin-C alternative splicing in mesangial cells

Mesangial cells are the major extracellular matrix (ECM)-producing cells in the kidney glomerulus and, when exposed to elevated glucose levels, they up-regulate assembly of fibronectin (FN) and other ECM proteins. Increases in glucose concentration are known to alter gene expression; here we investigated the connection between increased ECM production and changes in gene expression in mesangial cells. Comparison of mesangial cells grown in normal or high glucose conditions by RNA-sequencing showed significant expression changes in over 6000 genes and, when grouped by KEGG pathway analysis, identified the ECM-receptor interaction and focal adhesion pathways among the top 5 upregulated pathways. Of note was the significant increase in expression of tenascin-C (TN-C), a known regulator of FN matrix assembly. Mouse TN-C has multiple isoforms due to alternative splicing of 6 FNIII repeat exons. In addition to the transcriptional increase with high glucose, exon inclusion via alternative splicing was also changed resulting in production of higher molecular weight isoforms of TN-C. Mesangial cells grown in normal glucose secreted small isoforms with 1–2 variable repeats included whereas in high glucose large isoforms estimated to include 5 repeats were secreted. Unlike the smaller isoforms, the larger TN-C was not detected in the FN matrix. This change in TN-C isoforms may affect the regulation of FN matrix assembly and in this way may contribute to increased ECM accumulation under high glucose conditions.

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.

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.

Dysregulation of histone H3 lysine 27 trimethylation in transforming growth factor-β1–induced gene expression in mesangial cells and diabetic kidney

Transforming growth factor-β1 (TGF-β)-induced fibrotic and inflammatory genes in renal mesangial cells (MCs) play important roles in glomerular dysfunction associated with diabetic nephropathy (DN). TGF-β regulates gene expression in MCs by altering key chromatin histone modifications at target gene promoters. However, the role of the repressive histone H3 lysine 27 trimethylation (H3K27me3) modification is unclear. Here we show that TGF-β reduces H3K27me3 at the Ctgf, Serpine1, and Ccl2 gene promoters in rat MCs (RMCs) and reciprocally up-regulates the expression of these pro-fibrotic and inflammatory genes. In parallel, TGF-β down-regulates Enhancer of Zeste homolog 2 (Ezh2), an H3K27me3 methyltransferase, and decreases its recruitment at Ctgf and Ccl2 but not Serpine1 promoters. Ezh2 knockdown with siRNAs enhances TGF-β-induced expression of these genes, supporting its repressive function. Mechanistically, Ezh2 down-regulation is mediated by TGF-β-induced microRNA, miR-101b, which targets Ezh2 3'-UTR. TGF-β also up-regulates Jmjd3 and Utx in RMCs, suggesting a key role for these H3K27me3 demethylases in H3K27me3 inhibition. In RMCs, Utx knockdown inhibits hypertrophy, a key event in glomerular dysfunction. The H3K27me3 regulators are similarly altered in human and mouse MCs. High glucose inhibits Ezh2 and increases miR-101b in a TGF-β-dependent manner. Furthermore, in kidneys from rodent models of DN, fibrotic genes, miR-101b, and H3K27me3 demethylases are up-regulated, whereas Ezh2 protein levels as well as enrichment of Ezh2 and H3K27me3 at target genes are decreased, demonstrating in vivo relevance. These results suggest that H3K27me3 inhibition by TGF-β via dysregulation of related histone-modifying enzymes and miRNAs augments pathological genes mediating glomerular mesangial dysfunction and DN.

High glucose induces platelet-derived growth factor-C via carbohydrate response element-binding protein in glomerular mesangial cells.

Persistent high concentration of glucose causes cellular stress and damage in diabetes via derangement of gene expressions. We previously reported high glucose activates hypoxia‐inducible factor‐1α and downstream gene expression in mesangial cells, leading to an extracellular matrix expansion in the glomeruli. A glucose‐responsive transcription factor carbohydrate response element‐binding protein (ChREBP) is a key mediator for such perturbation of gene regulation. To provide insight into glucose‐mediated gene regulation in mesangial cells, we performed chromatin immunoprecipitation followed by DNA microarray analysis and identified platelet‐derived growth factor‐C (PDGF‐C) as a novel target gene of ChREBP. In streptozotocin‐induced diabetic mice, glomerular cells showed a significant increase in PDGF‐C expression; the ratio of PDGF‐C‐positive cells to the total number glomerular cells demonstrated more than threefold increase when compared with control animals. In cultured human mesangial cells, high glucose enhanced expression of PDGF‐C protein by 1.9‐fold. Knock‐down of ChREBP abrogated this induction response. Upregulated PDGF‐C contributed to the production of type IV and type VI collagen, possibly via an autocrine mechanism. Interestingly, urinary PDGF‐C levels in diabetic model mice were significantly elevated in a fashion similar to urinary albumin. Taken together, we hypothesize that a high glucose‐mediated induction of PDGF‐C via ChREBP in mesangial cells contributes to the development of glomerular mesangial expansion in diabetes, which may provide a platform for novel predictive and therapeutic strategies for diabetic nephropathy.

Olmesartan Blocks Advanced Glycation End Products-Induced VCAM-1 Gene Expression in Mesangial Cells by Restoring Angiotensin-Converting Enzyme 2 Level

Advanced glycation end products (AGEs) and their receptor (RAGE) system are involved in diabetic nephropathy. Angiotensin-converting enzyme 2 (ACE 2) plays a protective role against cardiovascular and renal injury by stimulating the production of angiotensin-(1-7) [Ang-(1-7)], an antagonist of angiotensin II (Ang II). However, effects of the AGEs-RAGE axis on ACE 2 expression in mesangial cells remain unknown. We examined here the role of ACE 2 in the AGEs-RAGE-induced mesangial cell damage and investigated whether olmesartan, one of the Ang II type 1 receptor blockers (ARB), prevented the deleterious effects of AGEs via restoration of ACE 2 and Ang-(1-7) level. AGEs significantly increased superoxide generation, upregulated RAGE mRNA level, and decreased ACE 2 gene expression and Ang-(1-7) production in mesangial cells, all of which were blocked by olmesartan, but not by a different type of ARB, azilsartan. An antioxidant, N-acetylcysteine or RAGE-antibodies also restored the decrease in ACE 2 mRNA level in AGEs-exposed mesangial cells. Moreover, olmesartan, but not azilsartan completely inhibited the AGEs-induced increase in vascular cell adhesion molecule-1 (VCAM-1) mRNA level in mesangial cells, which was abolished by the treatment with A-779, an antagonist of Ang-(1-7) receptor, Mas receptor. Our present study suggests that olmesartan could block the AGEs-induced VCAM-1 gene induction in mesangial cells by restoring the downregulated ACE 2 levels and subsequently stimulating the Ang-(1-7)-Mas receptor axis. Restoration of ACE 2 levels and blockade of renin-angiotensin system by olmesartan might be a promising strategy for the treatment of diabetic nephropathy.

Glucagon-like peptide-1 inhibits angiotensin II-induced mesangial cell damage via protein kinase A

There is a growing body of evidence that renin–angiotensin system plays a role in diabetic nephropathy. Recently, we have found that glucagon-like peptide-1 (GLP-1), one of the incretins, a gut hormone secreted from L cells in the intestine in response to food intake, inhibits advanced glycation end product-induced monocyte chemoattractant protein-1 gene expression in mesangial cells thorugh the interaction with the receptor of GLP-1. However, effects of GLP-1 on angiotensin II-exposed mesangial cells are unknown. This study investigated whether and how GLP-1 blocked the angiotensin II-induced mesangial cell damage in vitro. GLP-1 completely blocked the angiotensin II-induced superoxide generation, NF-κB activation, up-regulation of mRNA levels of intercellular adhesion molecule-1 and plasminogen activator inhibitor-1 in mesangial cells, all of which were prevented by the treatments with H-89, an inhibitor of protein kinase A. The present results demonstrated for the first time that GLP-1 blocked the angiotensin II-induced mesangial cell injury by inhibiting superoxide-mediated NF-κB activation via protein kinase C pathway. Our present study suggests that strategies to enhance the biological actions of GLP-1 may be a promising strategy for the treatment of diabetic nephropathy.

Glucagon-like peptide–1 suppresses advanced glycation end product–induced monocyte chemoattractant protein–1 expression in mesangial cells by reducing advanced glycation end product receptor level

Advanced glycation end products (AGE) and receptor for AGE (RAGE) interaction elicits reactive oxygen species (ROS) generation and inflammatory reactions, thereby being involved in the development and progression of diabetic nephropathy. Recently, we, along with others, found that glucagon-like peptide-1 (GLP-1), one of the incretins and a gut hormone secreted from L cells in the intestine in response to food intake, could have anti-inflammatory and antithrombogenic properties in cultured endothelial cells. However, the effects of GLP-1 on renal mesangial cells are largely unknown. Therefore, to elucidate the role of GLP-1 in diabetic nephropathy, this study investigated whether and how GLP-1 blocked AGE-induced monocyte chemoattractant protein-1 expression in human cultured mesangial cells. Gene and protein expression was analyzed by quantitative real-time reverse transcription polymerase chain reactions, Western blots, and enzyme-linked immunosorbent assay. The ROS generation was measured with dihydroethidium staining. Glucagon-like peptide-1 receptor (GLP-1R) was expressed in mesangial cells. Glucagon-like peptide-1 inhibited RAGE gene expression in mesangial cells, which was blocked by small interfering RNAs raised against GLP-1R. Furthermore, GLP-1 decreased ROS generation and subsequently reduced monocyte chemoattractant protein-1 gene and protein expression in AGE-exposed mesangial cells. An analogue of cyclic adenosine monophosphate mimicked the effects of GLP-1 on mesangial cells. Our present study suggests that GLP-1 may directly act on mesangial cells via GLP-1R and that it could work as an anti-inflammatory agent against AGE by reducing RAGE expression via activation of cyclic adenosine monophosphate pathway.

High glucose activates HIF-1-mediated signal transduction in glomerular mesangial cells through a carbohydrate response element binding protein

High glucose evokes a variety of signals in mesangial cells that alter cellular functions responsible for the development of diabetic glomerulopathy. The hypoxia-inducible factor-1alpha (HIF-1alpha) regulates cellular homeostasis under hypoxic conditions, but it also has pleiotropic effects in response to cellular stresses at normoxia. Here we determined whether HIF-1alpha has a role in the regulation of mesangial cells in hyperglycemia. In the streptozotocin-induced diabetic mouse model, glomerular mesangial cells had a significant increase in HIF-1alpha expression in the nucleus. In cultured mesangial cells, high glucose enhanced the expression of HIF-1alpha and its target genes known to be involved in the development of diabetic glomerulopathy. A glucose-responsive carbohydrate response element binding protein (ChREBP) was found to have a critical role in the transcriptional upregulation of HIF-1alpha and downstream gene expression in mesangial cells exposed to high glucose. Knockdown of HIF-1alpha or ChREBP in mesangial cells abrogated the high glucose-mediated perturbation of gene expression. Our results show that ChREBP and HIF-1alpha mediate gene regulation in mesangial cells. Further studies will be needed to find out whether these findings are relevant to the development of the diabetic nephropathy.

Pathogenic anti-DNA antibodies modulate gene expression in mesangial cells: Involvement of HMGB1 in anti-DNA antibody-induced renal injury

Although anti-DNA antibodies have been decisively linked to the pathogenesis of lupus nephritis, the mechanisms have not been conclusively determined. Recently, we reported that anti-DNA antibodies may contribute to kidney damage by upregulation of proinflammatory genes in mesangial cells (MC), a process involving both Fc receptor-dependent and independent pathways. In investigating the mechanism by which pathogenic anti-DNA antibodies modulate gene expression in MC, we found that the pathogenic anti-DNA antibody 1A3F bound to high mobility group binding protein 1 (HMGB1), an endogenous ligand for TLR2/4 and RAGE (receptor for advanced glycation end products). Interestingly, HMGB1 treatment of MC induced a similar pattern of genes as stimulation with 1A3F. Furthermore, HMGB1 and 1A3F exhibited a synergistic proinflammatory effect in the kidney, where increased expression of HMGB1 was found in lupus patients but not in patients with other types of renal disease. TLR2/Fc and RAGE/Fc inhibited the proinflammatory effects of 1A3F on MC. Finally, we found enhanced susceptibility of lupus prone MRL- lpr/ lpr (MRL/ lpr) as compared to normal BALB/c derived MC to pathogenic anti-DNA antibody and LPS stimulation (in particular enhanced chemokine synthesis), in addition to significantly increased expression of TLR4. Our results suggest that gene upregulation in MC induced by nephritogenic anti-DNA antibodies is TLR2/4 and RAGE-dependent. Finally, HMGB1 may act as a proinflammatory mediator in antibody-induced kidney damage in systemic lupus erythematosus (SLE).

Telmisartan, an Angiotensin II Type 1 Receptor Blocker, Inhibits Advanced Glycation End-product (AGE)-induced Monocyte Chemoattractant Protein-1 Expression in Mesangial Cells through Downregulation of Receptor for AGEs via Peroxisome Proliferator-activated Receptor-γ Activation

Interaction between advanced glycation end-products (AGEs) and their receptor (RAGE) plays a central role in diabetic nephropathy pathogenesis. Pathophysiological crosstalk between the AGEs-RAGE system and angiotensin II (Ang II) is also involved in this disease. This study investigated the role of proliferator-activated receptor-gamma (PPAR-gamma)-modulating activity on inhibition of monocyte chemoattractant protein (MCP-1) expression. Telmisartan, an Ang II type 1 receptor blocker, downregulated RAGE mRNA and inhibited superoxide generation and MCP-1 gene expression in mesangial cells; these processes were blocked by GW9662, a PPAR-gamma inhibitor. Candesartan, an Ang II type 1 receptor blocker, did not suppress AGEs-induced superoxide generation. Telmisartan and the antioxidant, N-acetylcysteine, completely inhibited AGEs-induced MCP-1 overproduction by mesangial cells. These results suggest that telmisartan inhibits AGEs-signalling to MCP-1 expression in mesangial cells by downregulating RAGE gene expression and subsequent oxidative stress generation via PPAR-gamma activation. This study has demonstrated a unique benefit of telmisartan in that it may function as an anti-inflammatory agent against AGEs via PPAR-gamma activation and may play a protective role in diabetic nephropathy.

Nitric oxide modulates expression of extracellular matrix genes linked to fibrosis in kidney mesangial cells

Mesangial cells are thought to be important mediators of glomerular inflammation and fibrosis. Studies have established a direct role for nitric oxide (NO) in the regulation of gene expression in mesangial cells. Representational difference analysis was used to investigate changes in gene expression elicited by the treatment of S-nitroso-L-glutathione in rat mesangial cells. Seven upregulated and 11 downregulated genes were identified. Four out of 11 downregulated genes (connective tissue growth factor, thrombospondin-1, collagen type I alpha1 and collagen type I alpha2) are known to be linked to inflammation and fibrosis. Results were verified across species in mesangial cells treated with a series of NO donors using Northern blot analysis, quantitative real-time PCR and protein analysis methods. Induction of endogenous NO production by cytokine stimulation also triggered regulation of the genes. One example gene, connective tissue growth factor, was studied at the promoter level. Promoter-reporter gene studies in mesangial cells demonstrated that NO acts at the transcriptional level to suppress gene expression. Our results reveal a complex role of NO in regulating gene expression in mesangial cells and suggest an antifibrotic potential for NO.

ERK contributes to the effects of Smad signaling on oxidized LDL‐induced PAI‐1 experssion in human mesangial cells

Hypercholesterolemia and lipoprotein abnormalities are risk factors for the progression of glomerular injury to glomerulosclerosis. Oxidized Low-Density Lipoprotein (Ox-LDL) stimulates plasminogen activator inhibitor-1 (PAI-1) expression in human mesangial cells mediated by transforming growth factor-beta (TGF-beta)/Smad signaling pathway. TGF-beta activates extracellular signal-regulated kinase (ERK) in mesangial cells, and ERK is involved in activation of Smad2/3. This study examines whether an interaction exists between Ox-LDL-induced TGF-beta/Smad signaling pathways and ERK activation leading to PAI-1 transcription in human mesangial cells. Ox-LDL (50 microg/mL) induced an acute increase in ERK activity within 15 min, which decreased to control value at 2 h. Incubation with anti-TGF-beta or SB-431542, an inhibitor of the TGF-beta type I receptor, along with Ox-LDL, inhibited the expected increase in ERK phosphorylation. Treatment with PD98059 or UO126, mitogen-activated ERK-activating kinase 1/2 inhibitors, significantly inhibited the Ox-LDL-induced increase in PAI-1 mRNA and nuclear Smad3 expression, DNA/protein complex formation, and PAI-1 promoter activity. These results suggest that phosphorylation of ERK is induced by Ox-LDL through the induction of the TGF-beta signaling pathway and that activated ERK, in turn, participates in the Ox-LDL-induced Smad3 activation and subsequent PAI-1 gene expression in mesangial cells. Thus, in patients with chronic renal disease and persistent hypercholesterolemia, ERK activation by Ox-LDL might contribute to mesangial ECM accumulation and subsequently renal fibrosis due to interaction between Ox-LDL and the Smad signaling pathway.

ERK contributes to the effects of Smad signaling on oxidized LDL-induced PAI-1 expression in human mesangial cells

Oxidized low-density lipoprotein (Ox-LDL) stimulates plasminogen activator inhibitor-1 (PAI-1) expression in human mesangial cells mediated by transforming growth factor-beta (TGF-beta)/Smad signaling pathway. TGF-beta activates extracellular signal-regulated kinase (ERK) in mesangial cells, and ERK is involved in activation of Smad2/3. This study examines whether an interaction exists between Ox-LDL-induced TGF-beta/Smad signaling pathways and ERK activation leading to PAI-1 transcription in human mesangial cells. Ox-LDL (50 microg/mL) induced an acute increase in ERK activity within 15 min, which decreased to control value at 2 h. Incubation with anti-TGF-beta or SB-431542, an inhibitor of the TGF-beta type I receptor, along with Ox-LDL, inhibited the expected increase in ERK phosphorylation. Treatment with PD98059 or UO126, mitogen-activated ERK-activating kinase 1/2 inhibitors, significantly inhibited the Ox-LDL-induced increase in PAI-1 mRNA and nuclear Smad3 expression, DNA/protein complex formation, and PAI-1 promoter activity. These results suggest that phosphorylation of ERK is induced by Ox-LDL through the induction of the TGF-beta signaling pathway and that activated ERK, in turn, participates in the Ox-LDL-induced Smad3 activation and subsequent PAI-1 gene expression in mesangial cells.

IGF-1 increases laminin, cyclin D1, and P21Cip1 expression in glomerular mesangial cells: An investigation of the intracellular signaling pathway and cell-cycle progression

Insulin-like growth factor (IGF)-1 is accumulated in the diabetic kidney and is considered to be involved in the development of glomerular sclerosis. Here, we investigate IGF-1 regulation of laminin, an extracellular matrix (ECM) component, and cyclin D1 and p21Cip1, cell-cycle progression factor, expressions in glomerular mesangial cells. We show that IGF-1 increases the level of laminin gamma1 and beta1 subunits approximately 1.5- and 2.5-fold, respectively, in a time-dependent manner. IGF-1 also stimulates protein kinase Akt/PKB phosphorylation at Thr 308, which correlates with its activity, up to 24 h. The Akt activation is coupled with Ser 9 phosphorylation of its downstream target, glycogen synthase kinase-3beta (GSK-3beta), which inhibits its kinase activity. Laminin beta1 is reduced significantly (P < 0.03) by inhibitors of Akt and p38MAPK whereas laminin gamma1 is not affected. Surprisingly, IGF-1 activates the expression of both cyclin D1 and cell-cycle arrest factor, p21Cip1 parallely. Pharmacological inhibition of calcineurin by cyclosporin A blocks IGF-1-induced cyclin D1 and p21Cip1expression significantly (P < 0.05). IGF-1 enhances cellular metabolic activity and viability of rat mesangial cells; however, they are arrested at the G1 phase of cell cycle as revealed by the FACS analysis. These results indicate that IGF-1 mediates mesangial cell-cycle progression, hypertrophy, and ECM protein synthesis. The Akt/GSK-3beta, p38MAPK, and calcineurin pathways may play an important role in IGF-1 signaling, cell-cycle regulation, and matrix gene expression in mesangial cells leading to the development of diabetic glomerulopathy.

Physiological genomics in PG and beyond: October to December 2005

physiological genomics ( PG ) publishes, on a quarterly basis, brief editorial perspectives entitled “Physiological genomics in PG and beyond.” These editorial perspectives provide brief, accessible highlights of the studies published in PG in the most recent quarter, and relate those studies to

Differential regulation of the rat heme oxygenase-1 expression by Ets oncoproteins in glomerular mesangial cells

The Ets-1 oncoprotein and the heme-catabolizing enzyme heme oxygenase (HO)-1 have been implicated in the pathogenesis of renal disease. We investigated the role of the putative Ets-binding sites (EBSs) in the transactivation of the proximal promoter of rat heme oxygenase 1 (hmox1) gene by the Ets oncoproteins Fli-1, Erg-2, and Ets-1 in mesangial cells. We examined several rat hmox1-chloramphenicol acetytransferase (CAT) constructs and EBS mutant constructs in an effort to assess the effect of ETS oncoproteins on transactivation of the rat hmox1 proximal promoter in renal glomerular mesangial cells. CAT assays demonstrated that the proximal promoter region (-1387 to -40) contains positive and negative regulatory regions and that the EBS-2, 3, and 4 play a role in basal promoter activity. Overexpression of Fli-1 and Erg-2 proteins showed a significant increase in promoter activity, whereas Ets-1 showed no effect on promoter activity. The Fli-1-induced transcriptional activation was not altered by mutation of EBSs, either independently or in combination. However, mutation of EBS-4 independently or a combined mutation of sites 3 and 4 led to a 50% reduction in Erg-2-induced transcriptional activation. Furthermore, mutation of EBS-2 and 4 completely abolished Erg-2-mediated promoter activation. Our results support a role for Ets transcription factors in the regulation of rat hmox-1 gene expression in mesangial cells.

Transforming growth factor-β1 regulation of laminin γ1 and fibronectin expression and survival of mouse mesangial cells

The transforming growth factor-beta (TGF-beta) 1 is a mediator of extracellular matrix (ECM) gene expression in mesangial cells and the development of diabetic glomerulopathy. Here, we investigate the effects of TGF-beta1 on laminin gamma1 and fibronectin polypeptide expression and cell survival in mouse mesangial cells (MES-13). TGF-beta1 (10 ng/ml) stimulates laminin-gamma1 and fibronectin expression approximately two-fold in a time-dependent manner (0-48 h). TGF-beta1 treatment also retards laminin-gamma1 mobility on SDS-gels, and tunicamycin, an inhibitor of the N-linked glycosylation, blocks the mobility shift. TGF-beta1 increases the binding of laminin gamma1 to WGA-agarose and the binding is abolished by tunicamycin suggesting that laminin gamma1 is modified by N-linked glycosylation. TGF-beta1 also elevates fibronectin glycosylation but its mobility is not altered. The degradation of laminin gamma1 and fibronectin proteins is reduced by their glycosylation. In addition, TGF-beta1 enhances mesangial cell viability and metabolic activities initially (0-24 h); however, eventually leads to cell death (24-48 h). TGF-beta1 elevates pro-apoptotic caspase-3 activity and decrease cell cycle progression factor cyclin D1 expression, which parallels cell death. These results indicate that TGF-beta1 plays an important role in ECM expression, protein glycosylation and demise of mesangial cells in the diabetic glomerular mesangium.

Interaction between the Polyol Pathway and Non-Enzymatic Glycation on Mesangial Cell Gene Expression

Background/Aims: Both activation of the polyol pathway and enhanced non-enzymatic glycation have been implicated in the pathogenesis of diabetic glomerulopathy. We investigated the interaction between these two pathways using normal mesangial cells (MCs) and transgenic (TG) MCs with elevated aldose reductase (AR) activity. Methods: TG mice with expression of the human AR (hAR) gene in kidney MCs were established. Mouse glomeruli and primary cultures of MCs from hAR TG and wild-type (WT) mice were studied regarding the changes in AR activity, transforming growth factor-β₁ (TGF-β₁) and type IV collagen mRNA and protein levels, in response to BSA modified by advanced glycation end-products (AGE-BSA). Results: Ex vivo addition of AGE-BSA increased AR activity, TGF-β₁ and type IV collagen mRNA levels in both WT and TG glomeruli, with greater rise in TG glomeruli. These increments were attenuated by zopolrestat, an AR inhibitor. In cultured MCs, AGE-BSA enhanced AR activity, TGF-β₁ and type IV collagen mRNA and protein levels both in WT and TG MCs, again with greater increases in TG MCs. The AGE-induced enhancement in TGF-β₁ and type IV collagen expression were suppressed by either zopolrestat or transfection with an AR antisense oligonucleotide. Conclusion: These data suggest that the activation of the polyol pathway by AGEs, more marked in genetic conditions with increased AR activity, may contribute to the pathogenesis of diabetic glomerulopathy, through enhancing mesangial cell expression of TGF-β₁ and type IV collagen.

Effect of high glucose on gene expression in mesangial cells: upregulation of the thiol pathway is an adaptational response.

Pathological alterations in glomerular mesangial cells play a critical role in the development of diabetic nephropathy, the leading cause of end-stage renal disease. Molecular mechanisms mediating such alterations, however, remain to be fully understood. The present study first examined the effect of high glucose on the mRNA expression profile in rat mesangial cells using cDNA microarray. Based on variation-weighted criteria and with a false discovery rate of 4.3%, 459 of 17,664 cDNA elements examined were found to be upregulated and 151 downregulated by exposure to 25 mM d-glucose for 5 days. A large number of differentially expressed genes belonged to several functional categories, indicating high glucose had a profound effect on mesangial cell proliferation, protein synthesis, energy metabolism, and, somewhat unexpectedly, protein sorting and the cytoskeleton. Interestingly, several thiol antioxidative genes (glutathione peroxidase 1, peroxiredoxin 6, and thioredoxin 2) were found by microarray and confirmed by real-time PCR to be upregulated by high glucose. These changes suggested that the oxidative stress known to be induced in mesangial cells by high glucose might be buffered by upregulation of the thiol antioxidative pathway. Upregulation of thiol antioxidative genes also occurred in high-glucose-treated human mesangial cells and in glomeruli isolated from rats after 1 wk of streptozotocin-induced diabetes, but not in human proximal tubule cells. High glucose slightly increased lipid peroxidation and decreased the amount of reduced thiols in rat and human mesangial cells. Disruption of the thiol antioxidative pathway by two different thiol-oxidizing agents resulted in a three- to fivefold increase in high-glucose-induced lipid peroxidation. In summary, the present study provided a global view of the short-term effect of high glucose on mesangial cells at the level of mRNA expression and identified the upregulation of the thiol antioxidative pathway as an adaptational response of mesangial cells to high glucose.

Transcriptional profiling of gene expression patterns during sphingosine 1-phosphate-induced mesangial cell proliferation

Sphingosine 1-phosphate (S1P) is known to regulate cell proliferation, apoptosis, and motility. Recently, we have reported that S1P and its analogue dihydro-S1P (DHS1P) promote proliferation of rat cultured mesangial cells. To investigate the signaling mechanisms underlying S1P- and DHS1P-induced mesangial cell proliferation, we performed cDNA microarray analysis of gene expression during mesangial cell proliferation. In terms of the overall pattern, gene expression waves induced by S1P and DHS1P were similar to those induced by a potent mesangial mitogen platelet-derived growth factor (PDGF), whereas we found several genes, such as two growth factors, connective tissue growth factor (CTGF) and heparin-binding EGF-like growth factor (HB-EGF), which were induced by the sphingolipids, but not by PDGF. Cluster analysis also identified calcium-dependent molecules highly expressed in DHS1P-stimulated cells compared to S1P-stimulated cells. Calcium mobilization analysis showed that DHS1P had higher magnitudes of intracellular calcium mobilization than S1P, suggesting that S1P and DHS1P differentially regulate intracellular calcium mobilization, possibly leading to different gene expression in mesangial cells. The large-scale monitoring of gene expression performed here allows us to identify S1P-induced transcriptional properties during mesangial cell proliferation.