- Email: [email protected]
The renal cortical fibroblast in renal tubulointerstitial fibrosis
The International Journal of Biochemistry & Cell Biology 38 (2006) 1–5
Cells in focus
The renal cortical fibroblast in renal tubulointerstitial fibrosis Weier Qi a , Xinming Chen a , Philip Poronnik b , Carol A. Pollock a,∗ a
Department of Medicine, University of Sydney, Kolling Institute, Level 3, Wallace Freeborn Professorial Block, Royal North Shore Hospital, St. Leonards, NSW 2065, Australia b School of Biomedical Sciences, University of Queensland, Queensland, Australia Received 8 July 2005; received in revised form 22 August 2005; accepted 5 September 2005
Abstract Renal cortical fibroblasts have key roles in mediating intercellular communication with neighboring/infiltrating cells and extracellular matrix (ECM) and maintenance of renal tissue architecture. They express a variety of cytokines, chemokines, growth factors and cell adhesion molecules, playing an active role in paracrine and autocrine interactions and regulating both fibrogenesis and the interstitial inflammatory response. They additionally have an endocrine function in the production of epoetin. Tubulointerstitial fibrosis, the common pathological consequence of renal injury, is characterized by the accumulation of extracellular matrix largely due to excessive production in parallel with reduced degradation, and activated fibroblasts characterized by a myofibroblastic phenotype. Fibroblasts in the kidney may derive from resident fibroblasts, from the circulating fibroblast population or from haemopoetic progenitor or stromal cells derived from the bone marrow. Cells exhibiting a myofibroblastic phenotype may derive from these sources and from tubular cells undergoing epithelial to mesenchymal transformation in response to renal injury. The number of interstitial myofibroblasts correlates closely with tubulointerstitial fibrosis and progressive renal failure. Hence inhibiting myofibroblast formation may be an effective strategy in attenuating the development of renal failure in kidney disease of diverse etiology. © 2005 Elsevier Ltd. All rights reserved. Keywords: Cortical fibroblasts; Myofibroblasts; Extracellular matrix accumulation; Tubulointerstitial fibrosis
Cell facts • Renal cortical fibroblasts are derived from the resident renal fibroblast population, from the circulating fibroblast population or from haemopoetic progenitor cells or stromal cells derived from the bone marrow. • Renal cortical fibroblasts are responsible for the regulation of extracellular matrix production and turnover, the maintenance of tissue architecture, contribute to the inflammatory cell population in the renal interstitium and produce epoetin. • In response to renal injury fibroblasts become activated, exhibiting a myofibroblastic phenotype. Myofibroblasts may also derive from tubular cells in a process known as epithelial to mesenchymal transformation • Myofibroblasts correlate with tubulointerstitial fibrosis and progressive renal failure in kidney disease due to diverse etiologies.
∗
Corresponding author. Tel.: +61 2 9926 7126; fax: +61 2 9436 3719. E-mail address: [email protected] (C.A. Pollock).
1357-2725/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2005.09.005
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W. Qi et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1–5
1. Introduction The tubulointerstitium of the kidney encompasses the tubular epithelium, vascular structures, interstitial cells, extracellular matrix (ECM) and interstitial fluid, accounting for more than 90% of the kidney volume. The interstitial cells in the kidney are a heterogeneous population including fibroblasts, dendritic, inflammatory and haematopoetic progenitor cells. Cortical fibroblasts are the prototypic mesenchymal cell type. They display a typical spindle shaped elongated cellular phenotype with extensive branching and often sheet-like processes, forming with their cytoplasmic leaflets a complex net of intercellular contacts. Subcellularly they have large nuclei, dense rough endoplasmic reticulum and an extensive Golgi apparatus. Mitochondria are sparse and scattered randomly in the cytoplasm. They are characterized by a thick mat of actin filaments under the plasma membrane (Norman, Orphanides, Garcia, & Fine, 1999). They exhibit a high content of microfibrils and adhere to adjacent tubules, parietal cells of Bowman’s capsule, capillaries, dendritic cells, all types of migrating cells and nerve terminals by specific attachment plaques. Hence they provide a scaffold-like structure to support the architecture of the kidney (Grupp & Muller, 1999; Zeisberg, Strutz, & Muller, 2000). Ecto-5 nucleotidase, platelet-derived growth factor (PDGF)-␣ and  receptors, nerve growth factor (NGF), fibroblast-specific protein-1 (FSP-1) and CD90 are putative markers for renal cortical fibroblasts. Fibroblasts in culture exhibit distinct morphologic and biochemical features depending on their origin, state of differentiation and culture conditions. They are differentiated in culture from other interstitial cell types by the absence of desmin, cytokeratin and factor VIII (Zeisberg et al., 2000). In diseased kidneys, fibroblasts become activated, proliferating dramatically and producing excessive matrix. During this process fibroblasts undergo functional and phenotypic changes acquiring a myofibroblastic phenotype, which ultimately correlates with the development of tubulointerstitial fibrosis (Zeisberg et al., 2000). Myofibroblats are characterized by bundles of microfilaments, well-developed rough endoplasmic reticulum, intercellular attachments, hemidesmosomes and the occasional presence of an incomplete layer of basement membrane on the surface. Myofibroblasts are large with long processes and they functionally resemble fibroblasts and smooth muscle cells (Ina et al., 2002). Alpha-smooth muscle actin (␣-SMA) is a putative marker for myofibroblasts. In normal kidney, only a very small number of cells with a myofibroblast-like
appearance are detected. Increasing number of ␣-SMA positive cells is an indicator of progression of renal disease. This review highlights the important roles of renal cortical fibroblasts and how they mediate tubulointerstitial fibrosis in the kidney. Recent developments for the treatment of tubulointerstitial fibrosis are also addressed. 2. Cell origin and plasticity Bohman was the first to classify the cell types in the interstitium—fibroblast-like cells (type I cells), monocytes and macrophages (type II cells) and pericytes (type III cells) (Grupp & Muller, 1999). In the kidney fibroblasts may be derived from resident renal fibroblasts, from the circulating fibroblast population or from haemopoetic progenitor or stromal cells derived from the bone marrow. Cells exhibiting a myofibroblastic phenotype may derive from these sources and from tubular cells undergoing epithelial to mesenchymal transformation in response to renal injury. Bohman originally classified renal fibroblasts into ‘fibroblast-like’ cells in the renal cortex and ‘lipidladen’ cells in the inner medulla. Since then several classifications have been developed which subdivide renal fibroblasts into groups based on their mitotic and matrix-synthesizing activities and their differentiation state (Grupp & Muller, 1999; Zeisberg et al., 2000). These classification systems to date have not recognized that subpopulations exist with differing functional properties, including the fibroblasts that synthesise and secrete epoetin in response to renal hypoxia. Additionally it is recognized that in the normal adult kidney, cortical fibroblasts are a relatively quiescent population with a low turnover rate, but they retain the capacity to proliferate in response to a variety of stimuli. Hence such classifications do not take into account subpopulations of fibroblasts with specialized functions and the plasticity of the phenotype of the fibroblast depending on prevailing local and systemic factors. More recent evidence suggests that during renal injury, tubular epithelial cells are capable of transdifferentiation into fibroblast/myofibroblasts in a process known as tubular epithelial–mesenchymal transdifferentiation (EMT) (Lan, 2003). EMT is the process whereby the adult phenotype undergoes regression to the tubule’s embryonic, metanephric/mesenchymal phenotype in response to injury. In addition to transforming growth factor 1 (TGF-1) activating fibroblasts into myofibroblasts, it plays a key role in the initiation and maintenance of EMT (Liu, 2004). Tubular epithelial cells lose their apical-basal polarity and become elongated, migrate into the peritubular interstitium through
W. Qi et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1–5
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the damaged basement membrane and proliferate exuberantly resulting in tubulointerstitial fibrosis. The process of EMT consists of four key steps: 1. Loss of epithelial cell adhesion; 2. De-novo ␣-SMA expression and actin reorganization; 3. Disruption of tubular basement; 4. Enhanced cell migration and invasion. 3. Functions Renal cortical fibroblasts have key roles in mediating intercellular communication with neighboring/infiltrating cells and ECM and maintenance of renal tissue architecture. They produce a variety of cytokines, chemokines and growth factors under basal and pathological conditions (Qi et al., 2005). Conversely, they express many different growth factor receptors, integrins, including ␣1, ␣4, ␣5 and 1 (Norman et al., 1999), cell adhesion molecules and non-integrin matrix receptors, such as discoidin domain receptors. Hence they are susceptible to a variety of different stimuli and in turn communicate with ECM proteins such as collagen, fibronectin and proteoglycans. Moreover, they have an endocytic and antigen-presenting capacity, facilitating their role in mediating inflammatory processes (Zeisberg et al., 2000). A subset of renal cortical fibroblasts has recently been demonstrated to produce epoetin, a heavily glycosylated protein which stimulates the division and differentiation of erythroid precursors in the bone marrow. Hence the cortical fibroblasts have a role in both renal and systemic physiology, and functional as well as phenotypic differences occur within the population. Cortical fibroblasts are able to respond to a variety of autocrine and paracrine factors released by cells infiltrating the renal tissue or by resident renal cells. In a co-culture model of human cortical fibroblasts and proximal tubule cells, we have demonstrated that human renal fibroblasts modulate proximal tubule cell growth and transport via the insulin growth factor-1 (IGF-1) axis. Conversely proximal tubule cells modulate the biological behavior of cortical fibroblasts in the human kidney through paracrine mechanisms, which include the production and release of PDGF-AB and TGF-1 (Johnson, Saunders, Baxter, Field, & Pollock, 1998). More recently we have demonstrated that CTGF facilitates TGF-1 to induce the cortical fibroblast to increase ECM production (Qi et al., 2005) (Fig. 1). Cortical fibroblasts play a role in the maintenance and turnover of ECM but under pathological conditions may participate in the fibrotic response in the setting of
Fig. 1. The integrated actions of TGF-1 and CTGF in cortical fibroblasts. TGF-1 signals through TGF- receptors to induce CTGF. However, CTGF-induced ECM requires TGF- and signals through TGF- receptors and its Smad signaling in renal cortical fibroblasts (Qi et al., 2005).
renal injury (Zeisberg et al., 2000). They are responsible for production of the extracellular material, fibers and ground substance. The well developed rough endoplasmic reticulum in these cells reflects a high rate of protein synthesis for collagenous and non-collagenous extracellular proteins (Qi et al., 2005), and for the secretion of factors known to regulate matrix degradation such as matrix metalloproteinases (MMPs), tissue inhibitor of MMPs (TIMP-1, -2 and -3) and plasminogen activator inhibitor-1 (PAI-1) (Eddy, 1996; Qi et al., 2005). The transformation from a quiescent to an activated population of fibroblasts can be initiated by four distinct mechanisms. 1. In response to autocrine or paracrine growth factor production (including TGF-1, connective tissue growth factor (CTGF), fibroblast growth factor (FGF) and platelet derived growth factor (PDGF). 2. By direct cell–cell contact (leucocytes and macrophages). 3. By ECM–integrin interaction (␣1 and 1). 4. Following exposure to environmental stimuli such as hypoxia, high glucose, complement proteins, advanced glycation endproducts and oxidative stress. Activated fibroblasts produce significant amount of matrix proteins, with fibronectin being initially produced. This adhesive glycoprotein is thought to form a scaffold for the deposition of other protein and function as a fibroblast chemoattractant to amplify the fibrotic response. TGF-1, which is known to activate fibroblasts and initiate EMT is also known to inhibit matrix
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W. Qi et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1–5
Fig. 2. The roles of fibroblasts in tubulointerstitial fibrosis. Fibroblasts mediates renal fibrosis in three stages: (1) fibroblasts are stimulated and chemoattractants released which promote the migration of mononuclear cells; (2) fibroblasts produce excessive matrix proteins which resemble a wound healing process; (3) fibroblasts autonomously proliferate, undergo phenotype changes and transform to myfibroblasts. MMPs activities decrease and TIMPs and PAI-1 expression are upregulated. ECM turnover balance is in favor of ECM accumulation, thus fibrosis occurs.
degradation by upregulation of TIMPs and induction of PAI-1, resulting in ECM accumulation and tubulointerstitial fibrosis (Eddy, 2002). Angiotensin II has also been reported to induce EMT. It stimulates TGF-1 secretion which leads to increased ECM and decreased degradation of matrix proteins. 4. Associated pathologies Renal fibrogenesis is a common response to diverse renal insults resulting in renal failure. It occurs in three distinct phases: induction, matrix synthesis due to inflammation and matrix synthesis independent of the inciting injury. In the phase of induction, fibroblasts are stimulated and chemoattractants released which promote the migration of mononuclear cells. In the second phase, fibroblasts produce excessive matrix proteins which resemble a wound healing process. These functional changes are reversible. However in the third phase, fibroblasts undergo autonomous proliferation and matrix synthesis. Fibroblasts undergo phenotype changes and transform to myfibroblasts. This phase is independent of the primary insult resulting in irreversible fibrosis (Kuncio, Neilson, & Haverty, 1991; Zeisberg et al., 2000) (Fig. 2). The importance of tubulointerstitial fibrosis in determining the ultimate prognosis of the kidney has been confirmed for a diverse range of renal disease, including glomerulonephritis, diabetic nephropathy, polycystic kidney disease, interstitial nephritis, hypertension and primary vascular disease. Because of the centrality of TGF-1 in inducing activation of renal fibroblasts and inhibiting matrix degradation, inhibition of TGF-1 as a therapeutic strategy in
the treatment of renal fibrosis has been well studied in a variety of models using varied targeted strategies including the use of TGF-1 neutralizing antibodies, decorin, Smad-7, bone morphogenetic protein 7 (BMP-7) and anti-sense oligonucleotides against thrombospondin-1 (TSP-1) (Daniel et al., 2003; Lan et al., 2003; Schaefer et al., 2002; Wang & Hirschberg, 2003; Ziyadeh et al., 2000). Using TGF-1 neutralizing antibodies, Ziyadeh and colleagues have successfully prevented the glomerulosclerosis and renal insufficiency resulting from type 2 diabetes in db/db mice (Ziyadeh et al., 2000). Schaefer et al. has demonstrated decorin exerts beneficial effects on tubulointerstitial fibrosis in a model of renal obstruction (Schaefer et al., 2002). TGF-1 inhibition by overexpression of Smad-7 delivered by gene transfer has been shown to inhibit renal fibrosis in a rat model of ureteric obstruction (Lan et al., 2003). Similarly, BMP-7 has been demonstrated to antagonize TGF--dependent fibrogenesis in mesangial cells (Wang & Hirschberg, 2003). Anti-sense oligonucleotide against TSP-1 has also been reported to inhibit the activation of TGF- in an in vivo model (Daniel et al., 2003). However, the systemic effects of TGF-1 blockade have limited its therapeutic potential. We have recently demonstrated that tranilast attenuates TGF-1-induced fibronectin in renal cortical fibroblasts through mechanisms that are likely to include interruption of the downstream Smad signalling pathway (Mifsud et al., 2003 & unpublished data). Because of the systemic effects of TGF-1 blockade, subsequent therapies have targeted the downstream mediator CTGF, with several investigators suggesting that blockade of CTGF using antisense oligodeoxynucleotide or small interference RNA (siRNA) is a more effective anti-fibrotic approach (Okada et al., 2005; Wang, Olson, Ma, Brigstock, & Hart, 2004). Recent studies have targeted interruption of EMT as a key therapeutic strategy. Liu et al. have demonstrated that hepatocyte growth factor (HGF) can completely prevent fibroblast to myofibroblastic activation and EMT in the diseased kidney (Liu, 2004). BMP-7 has also been successfully used in prevention of EMT (Postlethwaite, Shigemitsu, & Kanangat, 2004). Furthermore, the ROCK inhibitor, Y-27632 has been shown to prevent tubulointerstitial fibrosis in mouse model of obstructive nephropathy (Nagatoya et al., 2002). Integrin-linked kinase (ILK) acts as a major intermediate signaling molecule that couples TGF-/Smad signaling and tubular EMT, thus blockade of ILK signaling is now being exploited as a novel therapeutic target to prevent EMT (Liu, 2004). Clearly the developments of strategies to specifically interrupt the development of myofibroblasts in the kidney have the promise of therapeutic potential.
W. Qi et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1–5
Acknowledgements These studies were supported by the National Health and Medical Research Council of Australia and the Juvenile Diabetes Research Foundation. Ms. W. Qi is supported by National Health Medical Research Council Scholarship. References Daniel, C., Takabatake, Y., Mizui, M., Isaka, Y., Kawashi, H., Rupprecht, H., et al. (2003). Antisense oligonucleotides against thrombospondin-1 inhibit activation of tgf-beta in fibrotic renal disease in the rat in vivo. Am. J. Pathol., 163, 1185–1192. Eddy, A. A. (1996). Molecular insights into renal interstitial fibrosis. J. Am. Soc. Nephrol., 7, 2495–2508. Eddy, A. A. (2002). Plasminogen activator inhibitor-1 and the kidney. Am. J. Physiol. Renal Physiol., 283, F209–F220. Grupp, C., & Muller, G. A. (1999). Renal fibroblast culture. Exp. Nephrol., 7, 377–385. Ina, K., Kitamura, H., Tatsukawa, S., Takayama, T., Fujikura, Y., & Shimada, T. (2002). Transformation of interstitial fibroblasts and tubulointerstitial fibrosis in diabetic nephropathy. Med. Electron Microsc., 35, 87–95. Johnson, D. W., Saunders, H. J., Baxter, R. C., Field, M. J., & Pollock, C. A. (1998). Paracrine stimulation of human renal fibroblasts by proximal tubule cells. Kidney Int., 54, 747–757. Kuncio, G. S., Neilson, E. G., & Haverty, T. (1991). Mechanisms of tubulointerstitial fibrosis. Kidney Int., 39, 550–556. Lan, H. Y. (2003). Tubular epithelial–myofibroblast transdifferentiation mechanisms in proximal tubule cells. Curr. Opin. Nephrol. Hypertens, 12, 25–29. Lan, H. Y., Mu, W., Tomita, N., Huang, X. R., Li, J. H., Zhu, H. J., et al. (2003). Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound–microbubble system in rat UUO model. J. Am. Soc. Nephrol., 14, 1535–1548. Liu, Y. (2004). Epithelial to mesenchymal transition in renal fibrogenesis: pathologic significance, molecular mechanism, and therapeutic intervention. J. Am. Soc. Nephrol., 15, 1–12. Mifsud, S., Kelly, D. J., Qi, W., Zhang, Y., Pollock, C. A., WilkinsonBerka, J. L., et al. (2003). Intervention with tranilast attenuates
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renal pathology and albuminuria in advanced experimental diabetic nephropathy. Nephron. Physiol., 95, 83–91. Nagatoya, K., Moriyama, T., Kawada, N., Takeji, M., Oseto, S., Murozono, T., et al. (2002). Y-27632 prevents tubulointerstitial fibrosis in mouse kidneys with unilateral ureteral obstruction. Kidney Int., 61, 1684–1695. Norman, J. T., Orphanides, C., Garcia, P., & Fine, L. G. (1999). Hypoxia-induced changes in extracellular matrix metabolism in renal cells. Exp. Nephrol., 7, 463–469. Okada, H., Kikuta, T., Kobayashi, T., Inoue, T., Kanno, Y., Takigawa, M., et al. (2005). Connective tissue growth factor expressed in tubular epithelium plays a pivotal role in renal fibrogenesis. J. Am. Soc. Nephrol., 16, 133–143. Postlethwaite, A. E., Shigemitsu, H., & Kanangat, S. (2004). Cellular origins of fibroblasts: possible implications for organ fibrosis in systemic sclerosis. Curr. Opin. Rheumatol., 16, 733– 738. Qi, W., Twigg, S., Chen, X., Polhill, T. S., Poronnik, P., Gilbert, R. E., et al. (2005). Integrated actions of transforming growth factor{beta}1 and connective tissue growth factor in renal fibrosis. Am. J. Physiol. Renal Physiol., 288, F800–F809. Schaefer, L., Macakova, K., Raslik, I., Micegova, M., Grone, H. J., Schonherr, E., et al. (2002). Absence of decorin adversely influences tubulointerstitial fibrosis of the obstructed kidney by enhanced apoptosis and increased inflammatory reaction. Am. J. Pathol., 160, 1181–1191. Wang, J. F., Olson, M. E., Ma, L., Brigstock, D. R., & Hart, D. A. (2004). Connective tissue growth factor siRNA modulates mRNA levels for a subset of molecules in normal and TGF-beta 1-stimulated porcine skin fibroblasts. Wound Repair Regen., 12, 205–216. Wang, S., & Hirschberg, R. (2003). BMP7 antagonizes TGF-beta dependent fibrogenesis in mesangial cells. Am. J. Physiol. Renal Physiol., 284, F1006–F1013. Zeisberg, M., Strutz, F., & Muller, G. A. (2000). Role of fibroblast activation in inducing interstitial fibrosis. J. Nephrol., 13(Suppl 3), S111–S120. Ziyadeh, F. N., Hoffman, B. B., Han, D. C., Iglesias-De La Cruz, M. C., Hong, S. W., Isono, M., et al. (2000). Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-beta antibody in db/db diabetic mice. Proc. Natl. Acad. Sci. U.S.A., 97, 8015–8020.
Cells in focus
The renal cortical fibroblast in renal tubulointerstitial fibrosis Weier Qi a , Xinming Chen a , Philip Poronnik b , Carol A. Pollock a,∗ a
Department of Medicine, University of Sydney, Kolling Institute, Level 3, Wallace Freeborn Professorial Block, Royal North Shore Hospital, St. Leonards, NSW 2065, Australia b School of Biomedical Sciences, University of Queensland, Queensland, Australia Received 8 July 2005; received in revised form 22 August 2005; accepted 5 September 2005
Abstract Renal cortical fibroblasts have key roles in mediating intercellular communication with neighboring/infiltrating cells and extracellular matrix (ECM) and maintenance of renal tissue architecture. They express a variety of cytokines, chemokines, growth factors and cell adhesion molecules, playing an active role in paracrine and autocrine interactions and regulating both fibrogenesis and the interstitial inflammatory response. They additionally have an endocrine function in the production of epoetin. Tubulointerstitial fibrosis, the common pathological consequence of renal injury, is characterized by the accumulation of extracellular matrix largely due to excessive production in parallel with reduced degradation, and activated fibroblasts characterized by a myofibroblastic phenotype. Fibroblasts in the kidney may derive from resident fibroblasts, from the circulating fibroblast population or from haemopoetic progenitor or stromal cells derived from the bone marrow. Cells exhibiting a myofibroblastic phenotype may derive from these sources and from tubular cells undergoing epithelial to mesenchymal transformation in response to renal injury. The number of interstitial myofibroblasts correlates closely with tubulointerstitial fibrosis and progressive renal failure. Hence inhibiting myofibroblast formation may be an effective strategy in attenuating the development of renal failure in kidney disease of diverse etiology. © 2005 Elsevier Ltd. All rights reserved. Keywords: Cortical fibroblasts; Myofibroblasts; Extracellular matrix accumulation; Tubulointerstitial fibrosis
Cell facts • Renal cortical fibroblasts are derived from the resident renal fibroblast population, from the circulating fibroblast population or from haemopoetic progenitor cells or stromal cells derived from the bone marrow. • Renal cortical fibroblasts are responsible for the regulation of extracellular matrix production and turnover, the maintenance of tissue architecture, contribute to the inflammatory cell population in the renal interstitium and produce epoetin. • In response to renal injury fibroblasts become activated, exhibiting a myofibroblastic phenotype. Myofibroblasts may also derive from tubular cells in a process known as epithelial to mesenchymal transformation • Myofibroblasts correlate with tubulointerstitial fibrosis and progressive renal failure in kidney disease due to diverse etiologies.
∗
Corresponding author. Tel.: +61 2 9926 7126; fax: +61 2 9436 3719. E-mail address: [email protected] (C.A. Pollock).
1357-2725/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2005.09.005
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W. Qi et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1–5
1. Introduction The tubulointerstitium of the kidney encompasses the tubular epithelium, vascular structures, interstitial cells, extracellular matrix (ECM) and interstitial fluid, accounting for more than 90% of the kidney volume. The interstitial cells in the kidney are a heterogeneous population including fibroblasts, dendritic, inflammatory and haematopoetic progenitor cells. Cortical fibroblasts are the prototypic mesenchymal cell type. They display a typical spindle shaped elongated cellular phenotype with extensive branching and often sheet-like processes, forming with their cytoplasmic leaflets a complex net of intercellular contacts. Subcellularly they have large nuclei, dense rough endoplasmic reticulum and an extensive Golgi apparatus. Mitochondria are sparse and scattered randomly in the cytoplasm. They are characterized by a thick mat of actin filaments under the plasma membrane (Norman, Orphanides, Garcia, & Fine, 1999). They exhibit a high content of microfibrils and adhere to adjacent tubules, parietal cells of Bowman’s capsule, capillaries, dendritic cells, all types of migrating cells and nerve terminals by specific attachment plaques. Hence they provide a scaffold-like structure to support the architecture of the kidney (Grupp & Muller, 1999; Zeisberg, Strutz, & Muller, 2000). Ecto-5 nucleotidase, platelet-derived growth factor (PDGF)-␣ and  receptors, nerve growth factor (NGF), fibroblast-specific protein-1 (FSP-1) and CD90 are putative markers for renal cortical fibroblasts. Fibroblasts in culture exhibit distinct morphologic and biochemical features depending on their origin, state of differentiation and culture conditions. They are differentiated in culture from other interstitial cell types by the absence of desmin, cytokeratin and factor VIII (Zeisberg et al., 2000). In diseased kidneys, fibroblasts become activated, proliferating dramatically and producing excessive matrix. During this process fibroblasts undergo functional and phenotypic changes acquiring a myofibroblastic phenotype, which ultimately correlates with the development of tubulointerstitial fibrosis (Zeisberg et al., 2000). Myofibroblats are characterized by bundles of microfilaments, well-developed rough endoplasmic reticulum, intercellular attachments, hemidesmosomes and the occasional presence of an incomplete layer of basement membrane on the surface. Myofibroblasts are large with long processes and they functionally resemble fibroblasts and smooth muscle cells (Ina et al., 2002). Alpha-smooth muscle actin (␣-SMA) is a putative marker for myofibroblasts. In normal kidney, only a very small number of cells with a myofibroblast-like
appearance are detected. Increasing number of ␣-SMA positive cells is an indicator of progression of renal disease. This review highlights the important roles of renal cortical fibroblasts and how they mediate tubulointerstitial fibrosis in the kidney. Recent developments for the treatment of tubulointerstitial fibrosis are also addressed. 2. Cell origin and plasticity Bohman was the first to classify the cell types in the interstitium—fibroblast-like cells (type I cells), monocytes and macrophages (type II cells) and pericytes (type III cells) (Grupp & Muller, 1999). In the kidney fibroblasts may be derived from resident renal fibroblasts, from the circulating fibroblast population or from haemopoetic progenitor or stromal cells derived from the bone marrow. Cells exhibiting a myofibroblastic phenotype may derive from these sources and from tubular cells undergoing epithelial to mesenchymal transformation in response to renal injury. Bohman originally classified renal fibroblasts into ‘fibroblast-like’ cells in the renal cortex and ‘lipidladen’ cells in the inner medulla. Since then several classifications have been developed which subdivide renal fibroblasts into groups based on their mitotic and matrix-synthesizing activities and their differentiation state (Grupp & Muller, 1999; Zeisberg et al., 2000). These classification systems to date have not recognized that subpopulations exist with differing functional properties, including the fibroblasts that synthesise and secrete epoetin in response to renal hypoxia. Additionally it is recognized that in the normal adult kidney, cortical fibroblasts are a relatively quiescent population with a low turnover rate, but they retain the capacity to proliferate in response to a variety of stimuli. Hence such classifications do not take into account subpopulations of fibroblasts with specialized functions and the plasticity of the phenotype of the fibroblast depending on prevailing local and systemic factors. More recent evidence suggests that during renal injury, tubular epithelial cells are capable of transdifferentiation into fibroblast/myofibroblasts in a process known as tubular epithelial–mesenchymal transdifferentiation (EMT) (Lan, 2003). EMT is the process whereby the adult phenotype undergoes regression to the tubule’s embryonic, metanephric/mesenchymal phenotype in response to injury. In addition to transforming growth factor 1 (TGF-1) activating fibroblasts into myofibroblasts, it plays a key role in the initiation and maintenance of EMT (Liu, 2004). Tubular epithelial cells lose their apical-basal polarity and become elongated, migrate into the peritubular interstitium through
W. Qi et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1–5
3
the damaged basement membrane and proliferate exuberantly resulting in tubulointerstitial fibrosis. The process of EMT consists of four key steps: 1. Loss of epithelial cell adhesion; 2. De-novo ␣-SMA expression and actin reorganization; 3. Disruption of tubular basement; 4. Enhanced cell migration and invasion. 3. Functions Renal cortical fibroblasts have key roles in mediating intercellular communication with neighboring/infiltrating cells and ECM and maintenance of renal tissue architecture. They produce a variety of cytokines, chemokines and growth factors under basal and pathological conditions (Qi et al., 2005). Conversely, they express many different growth factor receptors, integrins, including ␣1, ␣4, ␣5 and 1 (Norman et al., 1999), cell adhesion molecules and non-integrin matrix receptors, such as discoidin domain receptors. Hence they are susceptible to a variety of different stimuli and in turn communicate with ECM proteins such as collagen, fibronectin and proteoglycans. Moreover, they have an endocytic and antigen-presenting capacity, facilitating their role in mediating inflammatory processes (Zeisberg et al., 2000). A subset of renal cortical fibroblasts has recently been demonstrated to produce epoetin, a heavily glycosylated protein which stimulates the division and differentiation of erythroid precursors in the bone marrow. Hence the cortical fibroblasts have a role in both renal and systemic physiology, and functional as well as phenotypic differences occur within the population. Cortical fibroblasts are able to respond to a variety of autocrine and paracrine factors released by cells infiltrating the renal tissue or by resident renal cells. In a co-culture model of human cortical fibroblasts and proximal tubule cells, we have demonstrated that human renal fibroblasts modulate proximal tubule cell growth and transport via the insulin growth factor-1 (IGF-1) axis. Conversely proximal tubule cells modulate the biological behavior of cortical fibroblasts in the human kidney through paracrine mechanisms, which include the production and release of PDGF-AB and TGF-1 (Johnson, Saunders, Baxter, Field, & Pollock, 1998). More recently we have demonstrated that CTGF facilitates TGF-1 to induce the cortical fibroblast to increase ECM production (Qi et al., 2005) (Fig. 1). Cortical fibroblasts play a role in the maintenance and turnover of ECM but under pathological conditions may participate in the fibrotic response in the setting of
Fig. 1. The integrated actions of TGF-1 and CTGF in cortical fibroblasts. TGF-1 signals through TGF- receptors to induce CTGF. However, CTGF-induced ECM requires TGF- and signals through TGF- receptors and its Smad signaling in renal cortical fibroblasts (Qi et al., 2005).
renal injury (Zeisberg et al., 2000). They are responsible for production of the extracellular material, fibers and ground substance. The well developed rough endoplasmic reticulum in these cells reflects a high rate of protein synthesis for collagenous and non-collagenous extracellular proteins (Qi et al., 2005), and for the secretion of factors known to regulate matrix degradation such as matrix metalloproteinases (MMPs), tissue inhibitor of MMPs (TIMP-1, -2 and -3) and plasminogen activator inhibitor-1 (PAI-1) (Eddy, 1996; Qi et al., 2005). The transformation from a quiescent to an activated population of fibroblasts can be initiated by four distinct mechanisms. 1. In response to autocrine or paracrine growth factor production (including TGF-1, connective tissue growth factor (CTGF), fibroblast growth factor (FGF) and platelet derived growth factor (PDGF). 2. By direct cell–cell contact (leucocytes and macrophages). 3. By ECM–integrin interaction (␣1 and 1). 4. Following exposure to environmental stimuli such as hypoxia, high glucose, complement proteins, advanced glycation endproducts and oxidative stress. Activated fibroblasts produce significant amount of matrix proteins, with fibronectin being initially produced. This adhesive glycoprotein is thought to form a scaffold for the deposition of other protein and function as a fibroblast chemoattractant to amplify the fibrotic response. TGF-1, which is known to activate fibroblasts and initiate EMT is also known to inhibit matrix
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W. Qi et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1–5
Fig. 2. The roles of fibroblasts in tubulointerstitial fibrosis. Fibroblasts mediates renal fibrosis in three stages: (1) fibroblasts are stimulated and chemoattractants released which promote the migration of mononuclear cells; (2) fibroblasts produce excessive matrix proteins which resemble a wound healing process; (3) fibroblasts autonomously proliferate, undergo phenotype changes and transform to myfibroblasts. MMPs activities decrease and TIMPs and PAI-1 expression are upregulated. ECM turnover balance is in favor of ECM accumulation, thus fibrosis occurs.
degradation by upregulation of TIMPs and induction of PAI-1, resulting in ECM accumulation and tubulointerstitial fibrosis (Eddy, 2002). Angiotensin II has also been reported to induce EMT. It stimulates TGF-1 secretion which leads to increased ECM and decreased degradation of matrix proteins. 4. Associated pathologies Renal fibrogenesis is a common response to diverse renal insults resulting in renal failure. It occurs in three distinct phases: induction, matrix synthesis due to inflammation and matrix synthesis independent of the inciting injury. In the phase of induction, fibroblasts are stimulated and chemoattractants released which promote the migration of mononuclear cells. In the second phase, fibroblasts produce excessive matrix proteins which resemble a wound healing process. These functional changes are reversible. However in the third phase, fibroblasts undergo autonomous proliferation and matrix synthesis. Fibroblasts undergo phenotype changes and transform to myfibroblasts. This phase is independent of the primary insult resulting in irreversible fibrosis (Kuncio, Neilson, & Haverty, 1991; Zeisberg et al., 2000) (Fig. 2). The importance of tubulointerstitial fibrosis in determining the ultimate prognosis of the kidney has been confirmed for a diverse range of renal disease, including glomerulonephritis, diabetic nephropathy, polycystic kidney disease, interstitial nephritis, hypertension and primary vascular disease. Because of the centrality of TGF-1 in inducing activation of renal fibroblasts and inhibiting matrix degradation, inhibition of TGF-1 as a therapeutic strategy in
the treatment of renal fibrosis has been well studied in a variety of models using varied targeted strategies including the use of TGF-1 neutralizing antibodies, decorin, Smad-7, bone morphogenetic protein 7 (BMP-7) and anti-sense oligonucleotides against thrombospondin-1 (TSP-1) (Daniel et al., 2003; Lan et al., 2003; Schaefer et al., 2002; Wang & Hirschberg, 2003; Ziyadeh et al., 2000). Using TGF-1 neutralizing antibodies, Ziyadeh and colleagues have successfully prevented the glomerulosclerosis and renal insufficiency resulting from type 2 diabetes in db/db mice (Ziyadeh et al., 2000). Schaefer et al. has demonstrated decorin exerts beneficial effects on tubulointerstitial fibrosis in a model of renal obstruction (Schaefer et al., 2002). TGF-1 inhibition by overexpression of Smad-7 delivered by gene transfer has been shown to inhibit renal fibrosis in a rat model of ureteric obstruction (Lan et al., 2003). Similarly, BMP-7 has been demonstrated to antagonize TGF--dependent fibrogenesis in mesangial cells (Wang & Hirschberg, 2003). Anti-sense oligonucleotide against TSP-1 has also been reported to inhibit the activation of TGF- in an in vivo model (Daniel et al., 2003). However, the systemic effects of TGF-1 blockade have limited its therapeutic potential. We have recently demonstrated that tranilast attenuates TGF-1-induced fibronectin in renal cortical fibroblasts through mechanisms that are likely to include interruption of the downstream Smad signalling pathway (Mifsud et al., 2003 & unpublished data). Because of the systemic effects of TGF-1 blockade, subsequent therapies have targeted the downstream mediator CTGF, with several investigators suggesting that blockade of CTGF using antisense oligodeoxynucleotide or small interference RNA (siRNA) is a more effective anti-fibrotic approach (Okada et al., 2005; Wang, Olson, Ma, Brigstock, & Hart, 2004). Recent studies have targeted interruption of EMT as a key therapeutic strategy. Liu et al. have demonstrated that hepatocyte growth factor (HGF) can completely prevent fibroblast to myofibroblastic activation and EMT in the diseased kidney (Liu, 2004). BMP-7 has also been successfully used in prevention of EMT (Postlethwaite, Shigemitsu, & Kanangat, 2004). Furthermore, the ROCK inhibitor, Y-27632 has been shown to prevent tubulointerstitial fibrosis in mouse model of obstructive nephropathy (Nagatoya et al., 2002). Integrin-linked kinase (ILK) acts as a major intermediate signaling molecule that couples TGF-/Smad signaling and tubular EMT, thus blockade of ILK signaling is now being exploited as a novel therapeutic target to prevent EMT (Liu, 2004). Clearly the developments of strategies to specifically interrupt the development of myofibroblasts in the kidney have the promise of therapeutic potential.
W. Qi et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1–5
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