Nadia Abdel Wahab

Extracellular Matrix Metabolism in Diabetic Nephropathy

Diabetic nephropathy is characterized by excessive deposition of extracellular matrix proteins in the mesangium and basement membrane of the glomerulus and in the renal tubulointerstitium. This review summarizes the main changes in protein composition of the glomerular mesangium and basement membrane and the evidence that, in the mesangium, these are initiated by changes in glucose metabolism and the formation of advanced glycation end products. Both processes generate reactive oxygen species (ROS). The review includes discussion of how ROS may activate intracellular signaling pathways leading to the activation of redox-sensitive transcription factors. This in turn leads to change in the expression of genes encoding extracellular matrix proteins and the protease systems responsible for their turnover.

CTGF Mediates TGF-β–Induced Fibronectin Matrix Deposition by Upregulating Active α5β1 Integrin in Human Mesangial Cells

ABSTRACT. Excessive deposition of fibronectin in the glomerular mesangium in diabetic nephropathy (DN) is partly due to the induction of transforming growth factor-β (TGF-β) by high glucose. TGF-β induces its downstream mediator connective tissue growth factor (CTGF), which stimulates fibronectin matrix synthesis, a process that requires the presence of α5β1 integrin. Although TGF-β has been shown to upregulate α5β1 integrin expression in human mesangial cells (HMC), little is known about the effect of CTGF on levels of this receptor. This study tested whether CTGF modulates α5β1 expression by HMC in culture and whether changes induced by TGF-β are mediated through the induction of CTGF. FACS analysis showed that both TGF-β and CTGF significantly increased cell-surface α5β1 levels compared with basal conditions. RT-PCR indicated that the changes were at the level of transcription. Treatment of cells with TGF-β and antisense CTGF oligonucleotides significantly reduced the TGF-β–induced increases in α5β1 levels. CTGF and TGF-β also significantly increased levels of ligand-occupied cell-surface β1 integrins and cell adhesion to fibronectin, the main α5β1 substrate. Antisense CTGF significantly reduced the number of adherent cells from TGF-β–stimulated cultures. Finally, α5β1 blocking antibodies inhibited HMC fibronectin matrix deposition, confirming the importance of this receptor for this process. Taken together, these data provide evidence that CTGF controls α5β1 expression by HMC in vitro . Alterations in α5β1 levels induced by TGF-β are mediated at least in part through the induction of CTGF, and specific targeting of either α5β1 or CTGF could be useful in controlling excessive fibronectin matrix production in DN. E-mail roger.mason@ic.ac.uk

The differential role of Smad2 and Smad3 in the regulation of pro-fibrotic TGFβ1 responses in human proximal-tubule epithelial cells

In chronic renal diseases, progressive loss of renal function correlates with advancing tubulo-interstitial fibrosis. TGFbeta1-Smad (transforming growth factor-beta1-Sma and Mad protein) signalling plays an important role in the development of renal tubulo-interstitial fibrosis. Secretion of CTGF (connective-tissue growth factor; CCN2) by PTECs (proximal-tubule epithelial cells) and EMT (epithelial-mesenchymal transdifferentiation) of PTECs to myofibroblasts in response to TGFbeta are critical Smad-dependent events in the development of tubulo-interstitial fibrosis. In the present study we have investigated the distinct contributions of Smad2 and Smad3 to expression of CTGF, E-cadherin, alpha-SMA (alpha-smooth-muscle actin) and MMP-2 (matrix-metalloproteinase-2) in response to TGFbeta1 treatment in an in vitro culture model of HKC-8 (transformed human PTECs). RNA interference was used to achieve selective and specific knockdown of Smad2 and Smad3. Cellular E-cadherin, alpha-SMA as well as secreted CTGF and MMP-2 were assessed by Western immunoblotting. TGFbeta1 treatment induced a fibrotic phenotype with increased expression of CTGF, MMP-2 and alpha-SMA, and decreased expression of E-cadherin. TGFbeta1-induced increases in CTGF and decreases in E-cadherin expression were Smad3-dependent, whereas increases in MMP-2 expression were Smad2-dependent. Increases in alpha-SMA expression were dependent on both Smad2 and Smad3 and were abolished by combined knockdown of both Smad2 and Smad3. In conclusion, we have demonstrated distinct roles for Smad2 and Smad3 in TGFbeta1-induced CTGF expression and markers of EMT in human PTECs. This can be of therapeutic value in designing targeted anti-fibrotic therapies for tubulo-interstitial fibrosis.

Connective Tissue Growth Factor and Regulation of the Mesangial Cell Cycle: Role in Cellular Hypertrophy

Connective tissue growth factor (CTGF) is now considered to be one of the important driver molecules for the pathogenesis of diabetic nephropathy (DN) and possibly many other fibrotic disorders. However, the molecular mechanisms by which CTGF functions remain to be established. In an attempt to define these mechanisms, this study was designed to investigate whether CTGF has any effect on the cell cycle of human mesangial cells (HMC), which are known to undergo hypertrophy in DN. This report provides the first evidence that CTGF is a hypertrophic factor for HMC. CTGF stimulates HMC to actively enter the G(1) phase from G(0), but they do not then progress further through the cell cycle. The molecular mechanisms underlying this G(1) phase arrest appear to be due to the induction of the cyclin-dependent kinase inhibitors (CDKI) p15(INK4), p21(Cip1), and p27(Kip1), which are known to bind and inactivate cyclinD/CDK4/6 and the cyclin E/CDK2 kinase complexes. This could account for the maintenance of pRb protein in a non- or very low-phosphorylated state, preventing cell cycle progression. Using CTGF antisense oligonucleotides, the results also indicate that the previously identified transforming growth factor-beta (TGF-beta)-induced hypertrophy in mesangial cells is CTGF-dependent. Mesangial cell hypertrophy is one of the earliest abnormalities of diabetic nephropathy; therefore, therapeutic strategies targeting CTGF may be beneficial in controlling DN.

Glomerular expression of thrombospondin-1, transforming growth factor beta and connective tissue growth factor at different stages of diabetic nephropathy and their interdependent roles in mesangial response to diabetic stimuli.

Aims/hypothesisWe quantified the glomerular expression of thrombospondin-1 (THBS1, also known as TSP-1), transforming growth factor beta 1 (TGFB1, also known as TGF-β1) and connective tissue growth factor (CTGF) at each stage of diabetic nephropathy. We also examined the roles of THBS1 and CTGF in mediating high-glucose- and glycated-albumin-induced synthesis of the matrix protein, fibronectin, by mesangial cells.MethodsTHBS1, latent and active TGFB1, and CTGF, were detected by immunohistochemistry and in situ hybridisation in biopsies from 19 insulin-dependent diabetic patients with incipient, manifest and advanced diabetic nephropathy, and in 11 control kidneys. Findings were quantified by image analysis. Human mesangial cells were cultured with normal or high glucose, albumin or glycated albumin (Amadori product), +/−THBS1 or CTGF antisense oligonucleotides, or with peptide W, an inhibitor of TGFB1 bioactivation by THBS1. Proteins were measured by western blot analysis or ELISA.ResultsIn glomeruli of normal kidneys, mRNA and protein levels for THBS1, latent-TGFB1 and CTGF were low. They were increased in the incipient stage of diabetic nephropathy, predominantly in mesangial areas, with further increases at later stages of the disease. Little or no active TGFB1 immunostaining was detected prior to manifest diabetic nephropathy. In contrast to high-glucose conditions, increases in fibronectin synthesis that were stimulated by glycated albumin were not dependent on THBS1 activation of latent TGFB1. However, increased fibronectin synthesis in both conditions required CTGF.Conclusions/interpretationIncreased glomerular expression of all three factors occurs from the earliest stage of diabetic nephropathy. In contrast to THBS1, CTGF is required for mesangial synthesis of fibronectin stimulated by high glucose or glycated albumin, and is thus a potential therapeutic target.

Uptake and intracellular transport of the connective tissue growth factor: a potential mode of action

Connective tissue growth factor (CTGF) is a secreted cysteine-rich protein now considered as an important effector molecule in both physiological and pathological processes. An increasing amount of evidence indicates that CTGF plays a key role in the pathogenesis of different fibrotic disorders including diabetic nephropathy. However, the molecular mechanisms by which CTGF exerts its effects are not known. Here we provide the first evidence for the existence of an intracellular transport pathway for the growth factor in human mesangial cells. Our results demonstrate that CTGF is internalized from the cell surface in endosomes and accumulates in a juxtanuclear organelle from which the growth factor is then translocated into the cytosol. In the cytosol CTGF is phosphorylated by protein kinase C and PMA treatment can enhance this phosphorylation. Phosphorylated CTGF may have an important role in the cytosol, but it is also translocated into the nucleus where it may directly affect transcription.

TGF-β1-Induced Connective Tissue Growth Factor (CCN2) Expression in Human Renal Proximal Tubule Epithelial Cells Requires Ras/MEK/ERK and Smad Signalling

Background: Connective tissue growth factor (CTGF, CCN2) plays a fundamental role in the development of tissue fibrosis by stimulating matrix deposition and mediating many of the pro-fibrotic effects of transforming growth factor (TGF)-β. CCN2 induction by TGF-β in renal proximal tubule epithelial cells (PTECs) is likely to play an important role in the development of tubulointerstitial fibrosis. In this study, we investigated the induction of CCN2 by TGF-β1 and the possible mechanisms of this induction in human PTECs. Methods: Experiments were performed on primary and transformed (human kidney cell (HKC)-clone 8) human PTECs. Induction of CCN2 in response to TGF-β1 was studied at the gene promoter level by reporter gene assay, mRNA by semi-quantitative RT-PCR and protein by immunoblotting. While chemical inhibitors were used to assess the role of Ras/MEK/ERK1,2 signalling, an HKC cell line over-expressing Smad7 was used to assess the role of Smad signalling in induction of CCN2 by TGF-β1. Results: TGF-β1 induced CCN2 promoter activity, mRNA and protein in human PTECs. TGF-β1-dependent CCN2 promoter activity was reduced by inhibiting Ras and MEK activation. MEK inhibition also resulted in inhibition of the TGF-β1-induced secreted CCN2 protein. There was no significant increase in CCN2 gene promoter activity or protein by TGF-β1 in Smad7 over-expressing HKCs. Conclusions: TGF-β1 induces the expression of CCN2 in human PTECs. This induction is dependent on Ras/MEK/ERK and Smad signalling. Inhibiting TGF-β induced CCN2 by targeting Smad and/or Ras/MEK/ERK1,2 signalling pathways could be of therapeutic value in renal fibrosis.

A critical look at growth factors and epithelial-to-mesenchymal transition in the adult kidney. Interrelationships between growth factors that regulate EMT in the adult kidney.

In the adult kidney, the cellular phenotypes are maintained by a strict balance of growth factors. Epithelial-to-mesenchymal transition (EMT) is a program whereby injured epithelial cells that function as ion and fluid transporters become matrix remodelling mesenchymal cells. This process requires either transcriptional repression of genes that maintain the epithelial phenotype and transcriptional activation, or relieved repression of genes needed for functional myofibroblasts. The transcriptional regulators are controlled by several integrated signalling pathways which are triggered by growth factors. Emerging evidence indicates that the growth factors TGFβ/CTGF and BMP-7/HGF are the main determinants that maintain the two cellular phenotypes. Both TGFβ and BMP-7 counteract the activity of each other by cross-inducing their respective inhibitory Smads. Both growth factors may also induce the expression of other factors that can change the cellular environment and enhance their function. Chronic kidney diseases (regardless of the aetiology of the disease) are associated with increased TGFβ and CTGF expression levels which, in turn, have an inverse effect on the activity level of BMP-7 and HGF, leading to an EMT of injured tubular epithelial cells and a progression of the disease. A detailed understanding of the complex interrelationship between these growth factors may lead to the development of novel drugs.

Connective tissue growth factor and renal diseases: some answers, more questions.

Purpose of reviewConnective tissue growth factor (CCN2) has recently received much attention as a possible key determinant of progressive renal fibrosis. However, the mechanism(s) by which this growth factor functions is not known. The purpose of this review is to summarize and discuss the recent findings regarding the possible mechanisms involved. Recent findingsEmerging evidence from in-vitro studies of renal cells indicates that connective tissue growth factor is a crucial mediator for transforming growth factor-β-induced cellular dysfunction, manifest by increased cellular hypertrophy, synthesis of extracellular matrix proteins and their deposition and assembly around the cells. Indeed, recent evidence suggests that the interrelationship between connective tissue growth factor and transforming growth factor-β is stronger than first thought. While transforming growth factor-β induces the expression of connective tissue growth factor, the latter plays a key role in both bioactivation of latent transforming growth factor-β and the promotion of its Smad signalling activity. SummaryConnective tissue growth factor is clearly implicated in the pathogenesis of progressive renal disease. Although there is much to learn about the production, function, and mechanism of action of connective tissue growth factor, some progress has been made in understanding the molecular basis of its relationship with transforming growth factor-β. Elucidating the signal transduction pathways activated by connective tissue growth factor will also definitely help to clarify other actions of connective tissue growth factor which may be independent of transforming growth factor-β. Because of the inflammatory and immunosuppressive properties of transforming growth factor-β, connective tissue growth factor seems to be an attractive alternative therapeutic target for combating renal fibrosis.

Connective tissue growth factor (CTGF) promotes activated mesangial cell survival via up-regulation of mitogen-activated protein kinase phosphatase-1 (MKP-1)

Activated mesangial cells are thought to play a pivotal role in the development of kidney fibrosis under chronic pathological conditions, including DN (diabetic nephropathy). Their prolonged survival may enhance the development of the disease since they express increased amounts of growth factors and extracellular matrix proteins. CTGF (connective tissue growth factor) is one of the growth factors produced by activated mesangial cells and is reported to play a key role in the pathogenesis of DN. Previous studies have shown that addition of exogenous CTGF to HMCs (human mesangial cells) rapidly activates ERK1/2 (extracellular-signal-regulated kinase 1/2) MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase) MAPK, but not the p38 MAPK, despite the activation of the upstream kinases, MKK3/6 (MAPK kinase 3/6). The aim of the present study was to investigate whether the lack of phosphorylated p38 MAPK by CTGF has an anti-apoptotic effect on activated HMCs. We show that in HMC CTGF induces the rapid transcriptional activation and synthesis of MKP-1 (MAPK phosphatase-1), a dual specificity phosphatase that dephosphorylates p38 MAPK. This in turn prevents the anti-apoptotic protein, Bcl-2, from being phosphorylated and losing its function, leading to the survival of the cells. Knockout of MKP-1 protein in mesangial cells treated with CTGF, using siRNA (small interfering RNA) or antisense oligonucleotides, allows p38 MAPK activation and induces mesangial cell death.