Advanced glycation end-products: Implications for diabetic and non-diabetic nephropathies

Glycation is the process whereby sugars bind to the free amine residues of proteins. These newly formed modified molecular species are known as ‘advanced glycation end-products’, or AGEs. AGE toxicity may occur through at least three mechanisms: interaction with the receptor for AGEs (RAGE); tissue deposition; and in situ glycation. AGEs trigger proinflammatory, profibrotic and procoagulant cellular responses that are capable of damaging tissues, often targeting particular organs. In diabetic patients, the conditions needed to promote AGE formation are all present, and are further accentuated by accompanying renal failure. The aim of this review is to outline the involvement of AGEs in the various forms of renal pathology associated with diabetic and non-diabetic nephropathies. AGEs are present in all renal compartments in diabetic patients, including the vessels, glomeruli, tubules and interstitium. Many cell types may be activated—specifically, endothelial, tubular and mesangial cells, and podocytes. AGEs play a major role in the accumulation of extracellular matrix, as occurs in diabetic glomerulosclerosis, and are also involved in most diabetic (renovascular, microangiopathic and glomerular) and non-diabetic renal injury associated with progressive glomerulosclerosis and ageing.     

Connective tissue growth factor: A new and important player in the pathogenesis of fibrosis

Connective tissue fibrosis is the final common pathogenic process for almost all forms of chronic tissue injury. Whether caused by vascular dysfunction, inflammation, metabolic injury, trauma, or environmental agents, once initiated the fibrogenic process results in the progressive replacement of the normal tissue architecture with fibrotic lesions that eventually lead to organ compromise and failure. Fibrosis can be considered as a dysregulation in the normal tissue repair mechanism, resulting in severe tissue scarring. Fibrosis appears to be a consequence of linked processes, including the proliferation of resident fibroblast cell types, the increased production and deposition of extracellular matrix components, and the transition of fibroblasts into cells exhibiting a myofibroblast phenotype. Although transforming growth factor-beta (TGFβ) has long been regarded as a pivotal growth factor in the formation and maintenance of connective tissues and as a major driving influence in many progressive fibrotic diseases, attention has focused recently on the role of connective tissue growth factor (CTGF) in fibrosis. CTGF is selectively and rapidly induced in mesenchymally derived cells by the action of TGFβ. CTGF expression is increased in many fibrosing diseases. In addition, increasing evidence from in vivo and in vitro models of tissue remodeling and fibrosis suggest that CTGF may represent a downstream effector molecule of the profibrotic activities of TGFβ in the maintenance and repair of connective tissues and within fibrotic disease settings.     

Cytokine directed therapy in scleroderma: rationale,current status,and the future

The hallmark of scleroderma is cutaneous and visceral fibrosis characterized and by increased biosynthesis of multiple matrix proteins by interstitial fibroblasts. Studies over recent years have delineated pathways involved in promoting matrix synthesis and elucidated the molecular pathways of regulation. Central to the regulation of fibrosis are extracellular mediators, called cytokines, which are elaborated by a variety of cells, including those in the immune system, vascular cells, and fibroblasts themselves. The concept that inhibiting or promoting the action of these naturally occurring profibrotic or antifibrotic molecules, respectively, is a rational therapeutic approach to treating scleroderma and other fibrotic diseases finds support in animal studies and anticytokine therapy conducted in relation to rheumatoid arthritis and other disorders. This review looks at cytokines known or thought to play a role in scleroderma and/or other fibrotic states and at potential therapy directed at these mediators. Potential targets for therapy include transforming growth factor beta (TGF-beta), connective tissue growth factor (CTGF), IL-4, IL-13, MCP-1, and endothelin, among others.     

Contribution of activin receptor-like kinase 5 (transforming growth factor beta receptor type I) signaling to the fibrotic phenotype of scleroderma fibroblasts

OBJECTIVE: To use a specific transforming growth factor beta receptor type I (TGFbetaRI; activin receptor-like kinase 5 [ALK-5]) kinase inhibitor (SD208) to determine the role of activation of the TGFbetaRI kinase (ALK-5) in maintaining the profibrotic phenotype of dermal fibroblasts in systemic sclerosis (SSc). METHODS: The effect of SD208 on the expression of key biochemical markers of the fibrotic phenotype was compared in fibroblasts cultured from clinically involved (lesional) and clinically uninvolved skin of patients with diffuse cutaneous SSc (dcSSc) and in fibroblasts from healthy controls matched for age, sex, and anatomic site. Protein expression was compared together with the ability of fibroblasts to adhere to the extracellular matrix and to remodel and contract a free-floating fibroblast-populated type I collagen lattice. RESULTS: Inhibiting TGFbetaRI kinase reduced the expression of a cohort of fibrotic markers by dermal fibroblasts from patients with dcSSc, including type I collagen and beta1 integrin. Moreover, inhibition also attenuated the elevated adhesive and contractile abilities of dcSSc fibroblasts. CONCLUSION: Our data suggest that some of the key profibrotic features of lesional SSc fibroblasts are dependent upon ALK-5 activity. Thus, TGFbetaRI kinase-mediated signaling may contribute to dermal fibrosis in dcSSc.     

Advanced glycation end products up-regulate gene expression found in diabetic glomerular disease.

Several lines of evidence suggest that the excessive accumulation of extracellular matrix in the glomeruli of diabetic kidneys may be due to reactive intermediates forming between glucose and matrix proteins called advanced glycation end products (AGEs). Normal mice received AGE-modified mouse serum albumin i.p. for 4 weeks, and glomerular extracellular matrix, growth factor mRNA levels, and morphology were examined. We found that AGE induced an increase in glomerular extracellular matrix alpha 1(IV) collagen, laminin B1, and transforming growth factor beta 1 mRNA levels, as measured by competitive PCR, as well as glomerular hypertrophy. The AGE response was specific because the coadministration of an AGE inhibitor, aminoguanidine, reduced all these changes. We conclude that AGEs affected expression of genes implicated in diabetic kidney disease and may play a major role in nephropathy.     

The rapamycin analogue SDZ RAD attenuates bleomycin-induced pulmonary fibrosis in rats.

Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix proteins within the pulmonary interstitium. The new macrolide immunosuppressant SDZ RAD, a rapamycin analogue, inhibits growth-factor dependent proliferation of mesenchymal cells and might therefore be of therapeutic interest for the treatment of fibrotic lung disease. In this study the effect of SDZ RAD on lung-collagen accumulation in the bleomycin model of pulmonary fibrosis in rats was investigated. SDZ RAD (2.5 mg x kg(-1) x day(-1)) or drug vehicle were administered orally by daily gavage. Successful dosing was confirmed by measuring splenic weight. Total lung-collagen content was measured by high-performance liquid chromatographic quantitation of hydroxyproline. In animals given bleomycin and drug vehicle, total lung collagen was increased by 182+/-11% (mean+/-SEM) compared with saline controls at 14 days (p<0.001). The increase in lung-collagen accumulation was reduced by 75+/-12% (p<0.01) in animals given SDZ RAD and was accompanied by a concomitant 56+/-6% (p<0.001) reduction in lung weight. SDZ RAD is currently in clinical trials for the prevention of solid organ graft rejection, another condition characterized by excessive extracellular matrix production. The authors propose that SDZ RAD warrants evaluation as a novel therapeutic agent for fibrotic lung disease.     

Myostatin promotes a fibrotic phenotypic switch in multipotent C3H 10T1/2 cells without affecting their differentiation into myofibroblasts

Tissue fibrosis, the excessive deposition of collagen/extracellular matrix combined with the reduction of the cell compartment, defines fibroproliferative diseases, a major cause of death and a public health burden. Key cellular processes in fibrosis include the generation of myofibroblasts from progenitor cells, and the activation or switch of already differentiated cells to a fibrotic synthetic phenotype. Myostatin, a negative regulator of skeletal muscle mass, is postulated to be involved in muscle fibrosis. We have examined whether myostatin affects the differentiation of a multipotent mesenchymal mouse cell line into myofibroblasts, and/or modulates the fibrotic phenotype and Smad expression of the cell population. In addition, we investigated the role of follistatin in this process. Incubation of cells with recombinant myostatin protein did not affect the proportion of myofibroblasts in the culture, but significantly upregulated the expression of fibrotic markers such as collagen and the key profibrotic factors transforming growth factor-beta1 (TGF-beta1) and plasminogen activator inhibitor (PAI-1), as well as Smad3 and 4, and the pSmad2/3. An antifibrotic process evidenced by the upregulation of follistatin, Smad7, and matrix metalloproteinase 8 accompanied these changes. Follistatin inhibited TGF-beta1 induction by myostatin. Transfection with a cDNA expressing myostatin upregulated PAI-1, whereas an shRNA against myostatin blocked this effect. In conclusion, myostatin induced a fibrotic phenotype without significantly affecting differentiation into myofibroblasts. The concurrent endogenous antifibrotic reaction confirms the view that phenotypic switches in multipotent and differentiated cells may affect the progress or reversion of fibrosis, and that myostatin pharmacological inactivation may be a novel therapeutic target against fibrosis.     

T cells in systemic sclerosis: a reappraisal

SSc is an autoimmune disease characterized by inflammation and extracellular matrix deposition that ultimately leads to loss of organ function. T cells appear to play a prominent role in its pathogenesis. The evidence for this comes from their being at the site of fibrosis, their activated phenotype and alteration in their number and frequency in peripheral blood. This review examines the role of T cells in the pathogenesis of SSc and specifically examines the key soluble profibrotic mediators (IL-4, IL-6, IL-13) secreted by Th2 cells and their interactions with fibroblasts that deposit excess extracellular matrix leading to fibrosis. We finally examine possible therapeutic options in targeting T-cell mediators to disrupt the cellular interactions between T cells and fibroblasts that serve to drive the fibrotic response. One of the factors driving fibrosis is IL-6 and this can be neutralized in vivo not only to limit IL-6-driven tissue fibrosis but concomitantly to suppress switching of Tregs to Th17 T cells that will provide more IL-6, thus perpetuating the fibrosis. Taken together, these data implicate the role of T cells in SSc and suggest that Th2-polarized T cells and the fibrotic mediators subsequently released directly induce fibrosis. Targeting such cytokines may be therapeutic not only in SSc but more generally in diseases where fibrosis is directed by inflammatory signals.     

The pathogenesis of fibrosis and renal disease in scleroderma: Recent insights from glomerulosclerosis

Acute and chronic renal diseases remain common complications of systemic sclerosis. Although treatment for acute scleroderma renal crisis may arrest the rapid progression of renal disease, many patients develop persistent renal dysfunction. Based on recent insights gained from progressive renal diseases of diverse etiologies, novel approaches to understanding the pathobiology of scleroderma renal disease may be applicable. Key factors involved in progression of renal disease include accumulation of extracellular matrix in the glomerular and tubulointerstitial compartments, epithelial to mesenchymal transformation, and vascular changes. The relevant factors mediating these events include the reninangiotensin system, the profibrotic growth factors, transforming growth factor-beta and connective tissue growth factor, and reactive oxygen species. Much of the molecular details of the role of these factors have been revealed and promise to alter the practice of therapy of progressive renal disease.     

Mechanical aspects of lung fibrosis: a spotlight on the myofibroblast

Contractile myofibroblasts are responsible for the irreversible alterations of the lung parenchyma that hallmark pulmonary fibrosis. In response to lung injury, a variety of different precursor cells can become activated to develop myofibroblast features, most notably formation of stress fibers and expression of α-smooth muscle actin. Starting as an acute and beneficial repair process, myofibroblast secretion of collagen and contraction frequently becomes excessive and persists. The result is accumulation of stiff scar tissue that obstructs and ultimately destroys lung function. In addition to being a consequence of myofibroblast activities, the stiffened tissue is also a major promoter of the myofibroblast. The mechanical properties of scarred lung and fibrotic foci promote myofibroblast contraction and differentiation. One essential element in this detrimental feed-forward loop is the mechanical activation of the profibrotic growth factor transforming growth factor-β1 from stores in the extracellular matrix. Interfering with myofibroblast contraction and integrin-mediated force transmission to latent transforming growth factor-β1 and matrix proteins are here presented as possible therapeutic strategies to halt fibrosis.     

Endothelin is a downstream mediator of profibrotic responses to transforming growth factor beta in human lung fibroblasts

OBJECTIVE: Fibrosis is excessive scarring caused by the accumulation and contraction of extracellular matrix proteins and is a common end pathway in many chronic diseases, including scleroderma (systemic sclerosis [SSc]). Indeed, pulmonary fibrosis is a major cause of death in SSc. Transforming growth factor beta (TGFbeta) induces endothelin 1 (ET-1) in human lung fibroblasts by a Smad-independent, JNK-dependent mechanism. The goal of this study was to assess whether ET-1 is a downstream mediator of the profibrotic effects of TGFbeta in lung fibroblasts. METHODS: We used a specific endothelin receptor antagonist to determine whether ET-1 is a downstream mediator of TGFbeta responses in lung fibroblasts, using microarray technology, real-time polymerase chain reaction, and Western blot analyses. RESULTS: The ability of TGFbeta to induce the expression of a cohort of profibrotic genes, including type I collagen, fibronectin, and CCN2, and to contract a collagen gel matrix, depends on ET-1. CONCLUSION: ET-1 contributes to the ability of TGFbeta to promote a profibrotic phenotype in human lung fibroblasts, consistent with the notion that endothelin receptor antagonism may be beneficial in controlling fibrogenic responses in lung fibroblasts.     

The impact of TGF-β on lung fibrosis: from targeting to biomarkers

Transforming growth factor-β (TGF-β) is extensively involved in the development of fibrosis in different organs. Overproduction or potentiation of its profibrotic effects leads to an aberrant wound healing response during the initiation of fibrotic processes. Idiopathic pulmonary fibrosis (IPF) is a chronic, devastating disease, in which TGF-β\x{2013}induced disturbances of the homeostatic microenvironment are critical to promote cell activation, migration, invasion, or hyperplastic changes. In addition, excess extracellular matrix production contributes in a major way to disease pathogenesis. For this reason, this review will focus on discussing novel data and highlight growing interest in deepening the understanding of the profibrotic role of TGF-β and its direct or indirect targeting for disease modulation.     

Uridine triphosphate (UTP) induces profibrotic responses in cardiac fibroblasts by activation of P2Y2 receptors

Cardiac fibroblasts (CFs) play a key role in response to injury and remodeling of the heart. Nucleotide (P2) receptors regulate the heart but limited information is available regarding such receptors in CFs. We thus sought to determine if extracellular nucleotides regulate fibrotic responses (e.g., proliferation, migration and expression of profibrotic markers) of CFs in primary culture. UTP increased rat CF migration 3-fold (p < 0.001), proliferation by 30% (p < 0.05) and mRNA expression of profibrotic markers: alpha smooth muscle actin (α-SMA), plasminogen activator inhibitor-1 (PAI-1), transforming growth factor beta, soluble ST2, interleukin-6 and monocyte chemoattractant protein-1 (MCP-1) by 3.0-, 15-, 2.0-, 7.6-, 11-, and 6.1-fold, respectively (p < 0.05). PAI-1 protein expression induced by UTP was dependent on protein kinase C (PKC) and extracellular signal-regulated kinase (ERK), based on blockade by the PKC inhibitor Ro-31-8220 and the ERK inhibitor U0126, respectively. The rank order for enhanced expression of PAI-1 and α-SMA by nucleotides (UTPγS> > UDPβS> > ATPγS), the expression of P2Y2 receptors as the most abundantly expressed P2Y receptor in rat CFs and a blunted response to UTP in P2Y2^-/- mice all implicate P2Y2 as the predominant P2Y receptor that mediates nucleotide-promoted profibrotic responses. Additional results indicate that P2Y2 receptor-promoted profibrotic responses in CFs are transient, perhaps as a consequence of receptor desensitization. We conclude that P2Y2 receptor activation is profibrotic in CFs; thus inhibition of P2Y2 receptors may provide a novel means to diminish fibrotic remodeling and turnover of extracellular matrix in the heart.     

Pediatric lung disease: from proteinases to pulmonary fibrosis

One distinctive outcome of interstitial lung diseases in childhood is the abnormal accumulation of pulmonary extracellular matrix. The clinical consequence of such excessive connective tissue accumulation is known as pulmonary fibrosis. While numerous aspects of its pathogenesis have become familiar, many key events involved in its inception and progression still remain unclear. There is now compelling evidence that lung damage due to uncontrolled proteolysis may help drive critical processes that regulate fibrotic matrix remodeling. In this regard, a number of proteinases have been implicated in promoting both the initial lung injury and the fibroproliferative repair that follows. This review summarizes the knowledge of how different matrix-targeting enzymes may act to influence the development of pediatric pulmonary fibrosis. Understanding the scientific basis of this complex process may highlight opportunities to limit unwanted proteolysis and the intensity of its fibrotic sequelae.     

Targeting fibrosis for the treatment of heart failure: a role for transforming growth factor-β

Chronic heart failure (CHF) is a growing health problem in developed nations. The pathological accumulation of extracellular matrix is a key contributor to CHF in both diabetic and nondiabetic states, resulting in progressive stiffening of the ventricular walls and loss of contractility. Proinflammatory disease processes, including inflammatory cytokine activation, contribute to accumulation of extracellular matrix in the heart. Transforming growth factor-β is a key profibrotic cytokine mediating fibrosis. Current therapeutic strategies do not directly target the profibrotic inflammatory processes occurring in the heart and hence there is a clear unmet clinical need to develop new therapeutic agents targeting fibrosis. Accordingly, strategies that inhibit proinflammatory cytokine activation and pathological accumulation of extracellular matrix (ECM) provide a potential therapeutic target for prevention of heart failure. This review focuses on the therapeutic targeting of TGF-β in the prevention of pathological fibrosis in the heart.     

Insight on the pathogenesis of diabetic nephropathy from the study of podocyte and mesangial cell biology

Diabetic nephropathy is characterised by increased glomerular permeability to proteins, thickening of the glomerular basement membrane, and excessive extracellular matrix accumulation in the mesangium. Both mesangial cells and podocytes play a pivotal role in the pathogenesis of these alterations. Recent studies have cast light on both the mediators and the intracellular signalling molecules whereby high glucose and stretch, mimicking glomerular capillary hypertension, induce an abnormal extracellular matrix deposition. Furthermore, they have provided a better understanding of the mechanisms by which multiple pathways of hyperglycaemia- and hypertension-induced damage may converge at the cellular level. Glomerulosclerosis only partially explains the development of proteinuria and in recent years there has been a growing interest on the potential role of podocytes. The discovery of nephrin, a key molecule of the slit-diaphragm, has stressed the importance of podocytes in maintaining the glomerular size-selective barrier. Nephrin is lost in both human and experimental diabetic nephropathy and studies on cultured podocytes have shown that insults relevant to diabetes, such as high glucose, AGE, angiotensin II, and stretch, have important deleterious effects on podocyte survival and adhesion. This review focuses on the most significant advances in understanding the pathophysiology of both mesangial cells and podocytes, and their potential impact on diabetic nephropathy future treatments.     

Tissue inhibitor of metalloproteinase-3 is up-regulated by transforming growth factor-beta1 in vitro and expressed in fibroblastic foci in vivo in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by fibroblast expansion and extracellular matrix accumulation. However, the mechanisms involved in matrix remodeling have not been elucidated. In this study, the authors aimed to evaluate the expression of the tissue inhibitors of matrix metalloproteinases (TIMPs) in human fibroblasts and whole tissues from IPF and normal lungs. They also determined the role of mitogen-activated protein kinase (MAPK) in TIMP3 expression. TIMP1, TIMP2, and TIMP3 were highly expressed in lung fibroblasts. Transforming growth factor (TGF)-beta1, a profibrotic mediator, induced strong up-regulation of TIMP3 at the mRNA and protein levels. The authors examined whether the MAPK pathway was involved in TGF-beta1-induced TIMP3 expression. TGF-beta1 induced the phosphorylation of p38 and extracellular signal-regulated kinase (ERK)1/2. Biochemical blockade of p38 by SB203580, but not of the ERK MAPK pathway, inhibited the effect of this factor. The effect was also blocked by the tyrosine kinase inhibitor genistein and by antagonizing TGF-beta1 receptor type I (activin-linked kinase [ALK5]). In IPF tissues TIMP3 gene expression was significantly increased and the protein was localized to fibroblastic foci and extracellular matrix. Our findings suggest that TGF-beta1-induced TIMP3 may be an important mediator in lung fibrogenesis.     

Increased expression of selectins in kidneys of patients with diabetic nephropathy

Summary In diabetic nephropathy leukocytes, mainly composed of monocytes/macrophages, which accumulate in the glomeruli and the interstitium, play an important part in the progression of glomerulosclerosis. The infiltration of leukocytes into inflammatory tissues or atherosclerotic lesions is mediated by adhesion molecules, which are expressed on the vascular endothelial cells, although little is known about the mechanism of leukocyte infiltration into diabetic renal tissues. P- and E-selectin are leukocyte adhesion molecules, which are expressed on the vascular endothelial cells and promote the adhesion of leukocytes to the endothelium. We investigated the expression of P- and E-selectin in the kidney tissue of patients with diabetic nephropathy and compared it with that of patients with other glomerular diseases (minimal change nephrotic syndrome, membranous nephropathy, IgA nephropathy, mesangioproliferative glomerulonephritis, and lupus nephritis). Expression of P- and E-selectin were both significantly increased in the glomeruli and the interstitium of patients with diabetic nephropathy as compared with those with other glomerular diseases. P- and E-selectin were both expressed along the glomerular capillaries and the peritubular capillaries in the interstitium. Neither P- nor E-selectin were correlated with the number of infiltrated leukocytes in the glomeruli, however, interestingly the E-selectin expression on peritubular capillaries was correlated with the number of infiltrated CD14 positive cells in the interstitium. These results suggest that E-selectin may play a key role in leukocyte infiltration into the renal interstitium in patients with diabetic nephropathy. [Diabetologia (1998) 41: 185–192] Received: 22 April 1997 and in final revised form: 29 September 1997     

Potential roles of CCL2/monocyte chemoattractant protein-1 in the pathogenesis of cutaneous sclerosis

Scleroderma is a connective tissue disease of unknown etiology characterized by the excessive deposition of extracellular matrix in the skin. Cellular infiltrates of certain immune cells and pro-inflammatory mediators are suggested to play a crucial role in cutaneous fibrosis, forming complicated networks between fibroblasts and immune cells and/or cell-cell communications. Tissue-selective trafficking of leukocytes is mediated by combinations of adhesion molecules and chemokines. Although chemokines and their receptors are considered to be mediators of inflammation and fibrosis in scleroderma, their pathophysiological role remains incompletely understood. Recent studies suggest that CCL2/monocyte chemoattractant protein-1 plays an important role in the fibrotic process, including liver fibrosis, pulmonary fibrosis, and scleroderma. This review summarizes recent findings of the potential roles of CCL2 in cutaneous sclerosis in experimental animal models of scleroderma as well as human scleroderma.     

The role of the extracellular matrix in tissue distribution of macromolecules in normal and pathological tissues: potential therapeutic consequences

The interstitial space is a dynamic microenvironment that consists of interstitial fluid and structural molecules of the extracellular matrix, such as glycosaminoglycans (hyaluronan and proteoglycans) and collagen. Macromolecules can distribute in the interstitium only in those spaces unoccupied by structural components, a phenomenon called interstitial exclusion. The exclusion phenomenon has direct consequences for plasma volume regulation. Early studies have assigned a major role to collagen as an excluding agent that accounts for the sterical (geometrical) exclusion. More recently, it has been shown that the contribution of negatively charged glycosaminoglycans might also be significant, resulting in an additional electrostatical exclusion effect. This charge effect may be of importance for drug uptake and suggests that either the glycosaminoglycans or the net charge of macromolecular substances to be delivered may be targeted to increase the available volume and uptake of macromolecular therapeutic agents in tumor tissue. Here, we provide an overview of the structural components of the interstitium and discuss the importance the sterical and electrostatical components have on the dynamics of transcapillary fluid exchange.