Connective Tissue Growth Factor Expressed in Tubular Epithelium Plays a Pivotal Role in Renal Fibrogenesis

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-

The C-terminal module IV of connective tissue growth factor is a novel immune modulator of the Th17 response

“…13,14 Therapeutic approaches that selectively block endogenous CCN2 activity have proven beneficial effects in fibrotic-related diseases, including experimental lung, liver, vascular, and renal diseases, and some authors have suggested that CCN2 could be a therapeutic target for fibrosis. [12][13][14][15][16] However, cardiac CCN2 overexpression conferred cardioprotection in Angiotensin II-infused mice and in ischemia-reperfusion injury, 17,18 showing that CCN2 exerts protective effects in some pathological settings. These data suggest that before using CCN2 blockers in humans, it is necessary to fully understand the in vivo biological functions of this protein and its fragments.…”

Poly(ADP-Ribose) Polymerase-1 Enhances Transcription of the Profibrotic CCN2 Gene

“…We previously demonstrated that specific CCN2 mRNA knockdown in tubular epithelial cells significantly attenuated renal fibrosis. 4 These results provide a new insight into the pathologic processes in fibrotic kidney diseases and suggest new targets against which to develop new, selective antifibrotic strategies against a potent profibrotic growth factor, CCN2.…”

“…36 We also reported that when a given antisense ODN was injected intravenously into rodents, it was absorbed into the proximal tubular epithelium, where it was retained for nearly 48 h, during which time it could block transcription of a target gene. 3,4,35 Therefore, for groups of male, 5-to 6-wk-old C57BL/6J mice (n ϭ 5, respectively) were purchased from Charles River Laboratories Japan (Yokohama, Japan), and two groups were administered an intravenous injection of the PARP-1 antisense ODN and the others of mutated-antisense ODN (as the negative control) at a concentration of 1.0 mg/kg twice at 48 and 6 h before ureter ligation. At 48 and 96 h after ureter ligation, mice were killed, and obstructed and unobstructed kidney tissues were sampled for RNA extraction, protein extraction, and paraformaldehyde fixation for paraffin blocks.…”

“…Previously, we showed that tubular epithelial cells also express CCN2, which played a pivotal role in renal fibrosis. [2][3][4] A better understanding of the control of CCN2 expression in the tubular epithelial cells should facilitate the development of novel antifibrotic therapies targeting renal fibrosis. The expression of CCN2 has been studied in detail in fibroblasts, and TGF-␤1 is a major stimulator of CCN2 gene expression in most mesenchymal cells.…”

“…5,7,9 In addition, we and others also demonstrated that TGF-␤ is an important inducer of CCN2 in epithelial cells. [2][3][4][5]10 Although several consensus gene regulatory motifs, including SBE, BCE-1, Ets1, and Sp1, have been identified in the murine CCN2 promoter, their functional relevance in renal tubular epithelial cells has not yet been demonstrated; therefore, in this study, we investigated the mechanism underlying CCN2 transcription in tubular epithelial cells.…”