title: Effects of high glucose and TGF-beta1 on the expression of collagen IV and vascular endothelial growth factor in mouse podocytes.
abstract: Effects of high glucose and TGF-beta1 on the expression of collagen IV and vascular endothelial growth factor in mouse podocytes.
  
  
  BACKGROUND
  The podocyte takes center stage in the pathogenesis of glomerular basement membrane (GBM) thickening and proteinuria in diabetic glomerulopathy. In part, GBM thickening may occur when the podocyte synthesizes increased amounts of collagen IV. Proteinuria may develop if the podocyte secretes excessive amounts of vascular endothelial growth factor (VEGF), which may increase the glomerular permeability to macromolecules. The augmented production of collagen IV and VEGF may be caused by metabolic mediators of diabetes such as hyperglycemia and transforming growth factor-beta (TGF-beta).
  
  
  METHODS
  The effects of high glucose and exogenous TGF-beta1 were examined on a mouse podocyte cell line that retains its differentiated phenotype. The gene expression and protein production of certain alpha chains of collagen IV, the major isoforms of VEGF, and components of the TGF-beta system were assayed. An inhibitor of TGF-beta signaling was used to determine whether some of the high glucose effects might be mediated by the TGF-beta system.
  
  
  RESULTS
  Compared with normal glucose (5.5 mmol/L), high glucose (HG, 25 mmol/L) for 14 days stimulated [3H]-proline incorporation, a measure of collagen production, by 1.8-fold, and exogenous TGF-beta1 (2 ng/mL) for 24 hours stimulated proline incorporation by 2.4-fold. Northern analysis showed that exposure to HG for 14 days increased the mRNA level of alpha1(IV) collagen by 51% and alpha5(IV) by 90%, whereas treatment with TGF-beta1 (2 ng/mL) for 24 hours decreased the mRNA level of alpha1(IV) by 36% and alpha5(IV) by 40%. Consistent with these effects on mRNA expression, Western blotting showed that HG increased alpha1(IV) protein by 44% and alpha5(IV) by 28%, while TGF-beta1 decreased alpha1(IV) protein by 29% and alpha5(IV) by 7%. In contrast to their opposing actions on alpha1 and alpha5(IV), both HG and exogenous TGF-beta1 increased alpha3(IV) collagen and VEGF, with TGF-beta1 having the greater effect. An inhibitor of the TGF-beta type I receptor (ALK5) was able to prevent the stimulation of alpha3(IV) and VEGF proteins by HG. Unlike in other renal cell types, HG did not increase TGF-beta1 mRNA or protein in the podocyte, but HG did induce the expression of the ligand-binding TGF-beta type II receptor (TbetaRII). Because HG had up-regulated TbetaRII after two weeks, the addition of physiological-dose TGF-beta1 (0.010 ng/mL) for 24 hours stimulated the production of alpha3(IV) and VEGF proteins to a greater extent in high than in normal glucose. Up-regulation of TbetaRII in the podocyte was corroborated by immunohistochemistry of the kidney cortex in the db/db mouse, a model of type 2 diabetes.
  
  
  CONCLUSIONS
  High glucose and exogenous TGF-beta1 exert disparate effects on the expression of alpha1 and alpha5(IV) collagen. However, high glucose and TGF-beta1 coordinately induce the production of alpha3(IV) collagen and VEGF in the podocyte. The HG-induced increases in alpha3(IV) collagen and VEGF proteins are mediated by the TGF-beta system. By increasing the expression of TbetaRII, high glucose may augment the response of the podocyte to ambient levels of TGF-beta1.
authors.affiliation: Renal-Electrolyte and Hypertension Division, Department of Medicine, University of Pennsylvania, 700 Clinical Research Building, 415 Curie Boulevard, Philadelphia, PA 19104-4218, USA.
authors.forename: M Carmen
authors.initials: MC
authors.lastname: Iglesias-de la Cruz
authors.forename: Fuad N
authors.initials: FN
authors.lastname: Ziyadeh
authors.forename: Motohide
authors.initials: M
authors.lastname: Isono
authors.forename: Martine
authors.initials: M
authors.lastname: Kouahou
authors.forename: Dong Cheol
authors.initials: DC
authors.lastname: Han
authors.forename: Raghu
authors.initials: R
authors.lastname: Kalluri
authors.forename: Peter
authors.initials: P
authors.lastname: Mundel
authors.forename: Sheldon
authors.initials: S
authors.lastname: Chen
mesh_terms: D030201:Activin Receptors, Type I / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D002454:Cell Differentiation; D002478:Cells, Cultured; D024141:Collagen Type IV / Q000235:genetics*; D003928:Diabetic Nephropathies / Q000209:etiology / Q000503:physiopathology; D004149:Dioxoles / Q000494:pharmacology; D004357:Drug Synergism; D016228:Endothelial Growth Factors / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology*; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics*; D007678:Kidney Glomerulus / Q000737:chemistry / Q000166:cytology / Q000187:drug effects*; D008222:Lymphokines / Q000235:genetics*; D051379:Mice; D011392:Proline / Q000493:pharmacokinetics; D017346:Protein Serine-Threonine Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
chemical_list: C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; D024141:Collagen Type IV; D004149:Dioxoles; D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D018125:Receptors, Transforming Growth Factor beta; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D011392:Proline; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; C000625842:Tgfbr1 protein, mouse; D005947:Glucose