No abstract

Connective tissue growth factor (CTGF) is one of the candidate factors that are thought to mediate the downstream profibrotic action of TGF-␤. However, its precise role in renal interstitial fibrogenesis has not yet been clarified. It was demonstrated previously that CTGF was expressed in tubular epithelial cells that had been engulfed by interstitial fibrosis in the remnant kidney of the subtotal nephrectomy (SNx) model. In the present study, co-cultures of tubular epithelial cells (mProx24) and tubulointerstitial fibroblasts (TFB) that mimic the subepithelial mesenchyme in the kidney were used to study the profibrotic effects of TGF-␤1-induced CTGF. In these co-cultures, TGF-␤1 treatment resulted in significantly increased mRNA levels of type I collagen and fibronectin in the TFB. These effects were both direct and indirect, with the latter being mediated by CTGF derived from the co-cultured mProx24. C onnective tissue growth factor (CTGF) is a 38-kD cysteine-rich peptide that belongs to the emerging CCN (CTGF, cyr 61/cef 10, nov) family of multifunctional growth factors (1-4). Murine CTGF, which is also called fisp-12, promotes chemotaxis, migration, adhesion, proliferation, and differentiation or formation of the extracellular matrix (ECM), depending on whether the target cell is a fibroblast, chondrocyte, or vascular endothelial cell (2). CTGF is induced exclusively by TGF-␤ and is thought to mediate the latter's profibrotic effects by modulating fibroblast cell growth and ECM protein synthesis (1,4). CTGF expression has been shown to increase in a variety of human diseases and experimental disease models that are characterized by fibrosis, including those that affect the kidney, skin, blood vessels, lung, and liver (1,2). In the case of the kidney, CTGF mRNA was shown to be expressed primarily in glomerular mesangial, epithelial, and endothelial cells in IgA nephropathy, focal and segmental glomerulosclerosis, and diabetic nephropathy (5,6). Moreover, CTGF mRNA overexpression was found in tubular epithelial cells and interstitial cells at sites of chronic interstitial damage (5,6). In the remnant kidney of the subtotal nephrectomy (SNx) model and in the kidneys with ureteral obstruction, tubular CTGF was shown to be expressed in response to renal interstitial fibrosis (7,8). These findings strongly suggest that renal interstitial fibrogenesis is mediated by CTGF that is expressed in the tubular epithelial cells. However, whether CTGF plays a direct role in vivo in renal interstitial fibrogenesis remains to be elucidated. In the present study, we demonstrated, using neutralization protocols, that tubular CTGF directly and significantly contributed to TGF-␤1-dependent renal interstitial fibrogenesis. Materials and Methods CTGF Antisense OligodeoxynucleotidesPhosphorothioate-capped oligodeoxynucleotides (ODN) were synthesized by an automated synthesizer. After deprotection, ODN were dissolved in water, extracted with phenol/chloroform/isoamyl alco-

Abstract. A number of experimental and clinical investigations support the notion that angiotensin-converting enzyme inhibitor (ACEi) and angiotensin II type 1 receptor blocker (ARB) compounds attenuate renal fibrosis. Fibrosis can be attenuated by either suppressing matrix formation or facilitating matrix degradation. In this study, drugs of ACEi and ARB classes were tested for their ability to facilitate matrix degradation in the kidney. A murine model system in which cyclosporin A (CsA) treatment for a specified period caused interstitial matrix deposition in the kidney was used. CsA was then discontinued, and experimental procedures were initiated to investigate matrix degradation. Benazepril, an ACEi, facilitated matrix degradation via the bradykinin (BK) B2 receptor on tubular epithelial cells in the kidney, whereas CGP-48933, an ARB, did not. In this murine model of CsA nephropathy under ACE blockade, plasminogen activator inhibitor-1 (PAI-1) expression was decreased in tubular epithelial cells, possibly leading to conversion of plasminogen to plasmin by plasminogen activator and subsequent activation of matrix metalloproteinases. These findings were confirmed in this study by measurements of plasmin activity, collagenolytic activity, and matrix metalloproteinase activities in the kidneys. In tubular epithelial cells stimulated in vitro, BK suppressed PAI-1 gene expression. All of these results suggest that ACEi can decrease PAI-1 expression via BK, thereby facilitating matrix degradation via activation of degradative enzymes to reduce interstitial matrix deposition.

Connective tissue growth factor (CTGF), a downstream mediator of transforming growth factor-␤1, mediates mesangial cell/fibroblast proliferation and extracellular matrix production by renal cells. Here, we show that renal tubular epithelial cells from patients with minimal change nephritic syndrome produced CTGF after glucocorticoid treatment. In addition, the glucocorticoid dexamethasone (DEX) increased CTGF mRNA levels in the kidneys of C57B6 but not SJL mice and produced intermediate CTGF mRNA levels in the kidneys of F1 (C57B6 ؋ SJL) mice, midway between the levels found for parental strains. DEX also increased CTGF mRNA levels in cultured tubular epithelial cells derived from C57B6 (mProx24) but not SJL (MCT) mice via transcriptional up-regulation of CTGF mRNA. Transient transfection experiments using luciferase reporter constructs bearing CTGF promoter fragments revealed that the ؊897-to ؊628-bp fragment contained DEX-responsive positive regulatory elements, which were active in mProx24 but not MCT cells. Long-term DEX treatment resulted in fibronectin deposition in the kidneys of C57B6 but not SJL mice, and this effect was inhibited by co-administration of CTGF anti-sense oligodeoxynucleotides. Thus, glucocorticoid-induced renal fibrogenesis seems to be influenced by genetic background, with the critical DEX-responsive elements in the ؊897-to ؊628-bp region of the CTGF promoter.

We previously reported that angiotensin II type 1 receptor (AT 1 R) blockade attenuates renal inflammation/fibrogenesis in immune-mediated glomerulonephritis via angiotensin II type 2 receptor (AT 2 R). In the present study, further in vivo experiments revealed that AT 2 R was expressed in tubular epithelial cells of nephritic kidneys in mice, and feedback activation of the renin-angiotensin system during AT 1 R blockade significantly reduced p-ERK, but not intranuclear nuclear factor-B, levels via AT 2 R. This led to reduction in mRNA levels of the proinflammatory mediator monocyte chemoattractant protein-1 and overall interstitial inflammation and subsequent fibrogenesis. Specific blockade of ERK expression in tubular epithelium by anti-sense oligodeoxynucleotides also attenuated interstitial inflammation, mimicking the anti-inflammatory action of AT 2 R in nephritic kidneys. Alternatively, we succeeded in confirming such an AT 2 R function by demonstrating that AT 1 R blockade did not confer renoprotection in nephritic, The potent vasoactive peptide angiotensin II (Ang II) binds two main subtypes of receptors, ie, type 1 and 2 receptors (AT 1 R and AT 2 R) that belong to the superfamily of G protein-coupled receptors but that display primarily opposite biological and physiological effects.1 Animals with anti-glomerular basement membrane (anti-GBM) nephritis, protein-overload nephropathy, subtotal nephrectomy, unilateral ureteral obstruction, or diabetic nephropathy that were treated with AT 1 R blockers, as well as AT 1A R gene-deficient mice with such kidney diseases, exhibited renoprotection.2-7 Although the anti-inflammatory and anti-fibrotic effects of AT 1 R blockade have been reported in the cardiovascular system, 8,9 similar effects have also been found in the liver.10 These findings suggest that AT 1 R mediates not only hemodynamic but also proinflammatory/fibrogenic functions. The effects of AT 1 R blockers, however, cannot be attributed solely to the action of AT 1 R. In most instances, feedback activation of the renin-angiotensin system may result in increased stimulation of AT 2 Rs that are freed from AT 1 R blockers. 2In contrast to AT 1 R, the pathophysiological role of AT 2 R remains controversial and has not yet been fully elucidated.11 AT 2 R activates unconventional signaling pathways that generally do not involve coupling to classical regulatory G proteins. In many cell types, AT 2 R is capable of activating protein tyrosine phosphatases