Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis

Connective tissue growth factor (CTGF/CCN2) is one of the candidate factors mediating fibrogenic activity of TGF-beta. It was shown previously that the blockade of CTGF by antisense oligonucleotide (ODN) inhibits TGF-beta-induced production of fibronectin and type I collagen in cultured renal fibroblasts. The in vivo contribution of CTGF in renal interstitial fibrosis, however, remains to be clarified. With the use of a hydrodynamics-based gene transfer technique, the effects of CTGF antisense ODN are investigated in rat kidneys with unilateral ureteral obstruction (UUO). FITC-labeled ODN injection via the renal vein showed that the ODN was specifically introduced into the interstitium. At day 7 after UUO, the gene expression of CTGF, fibronectin, fibronectin ED-A, and alpha1(I) collagen in untreated or control ODN-treated obstructed kidneys was prominently upregulated. CTGF antisense ODN treatment, by contrast, markedly attenuated the induction of CTGF, fibronectin, fibronectin ED-A, and alpha1(I) collagen genes, whereas TGF-beta gene upregulation was not affected. The antisense treatment also reduced interstitial deposition of CTGF, fibronectin ED-A, and type I collagen and the interstitial fibrotic areas. The number of myofibroblasts determined by the expression of alpha-smooth muscle actin was significantly decreased as well. Proliferation of tubular and interstitial cells was not altered with the treatment. These findings indicate that CTGF expression in the interstitium plays a crucial role in the progression of interstitial fibrosis but not in the proliferation of tubular and interstitial cells during UUO. CTGF may become a potential therapeutic target against tubulointerstitial fibrosis.     

Application of gene therapy to kidney diseases

There is little doubt that molecular biological intervention therapy has come of age, and tremendous excitement has emerged from its potential. A gene transfer technique, the HVJ liposome method, is now applicable to analysis of the molecular aspects of the pathophysiology of renal diseases and is a tool for gene therapy. We demonstrated that overexpressed transforming growth factor-β (TGF-β) in the normal rat glomeruli by gene transfer of TGF-β cDNA leads to glomerulosclerosis. Inhibition of TGF-β action by direct introduction of antisense oligodeoxynucleotides (ODNs) into glomeruli could suppress glomerular matrix accumulation in experimental glomerulonephritis. In addition, continuous delivery of decorin or chimeric soluble TGF-β receptor from gene-transferred skeletal muscle also inhibits extracellular matrix expansion in experimental glomerulonephritis. We examined whether in vivo gene transfer of antisense ODNs for TGF-β into interstitial fibroblasts can suppress the progression of interstitial fibrosis in unilateral ureteral obstruction model rats. Introduction of TGF-β1 antisense ODNs significantly reduced levels of TGF-β1 and type I collagen mRNA expression in obstructed kidneys and consequently suppress the progression of interstitial fibrosis following ureteral obstruction. Taken together with clinical observations that upregulation of TGF-β plays an important role in the formation of various kidney fibrosis, manipulation of the overexpression of TGF-β may provide a novel therapeutic intervention to ameliorate the progression of renal disease. Received: June 7, 1999 / Accepted: June 21, 1999     

Y-27632 prevents tubulointerstitial fibrosis in mouse kidneys with unilateral ureteral obstruction

BACKGROUND: The small GTPase Rho is involved in cell-to-substratum adhesion and cell contraction. These actions of Rho mediated by downstream Rho effectors such as Rho-associated coiled-coil forming protein kinase (ROCK) may be partly responsible for the progression of renal interstitial fibrosis. METHODS: The anti-fibrosis effects of Y-27632, a specific ROCK inhibitor, were studied both in vivo (unilateral ureteral obstruction; UUO) and in vitro. To investigate the therapeutic efficacy of Y-27632 in UUO kidneys, smooth muscle alpha actin (SMalphaA) expression, macrophage infiltration and fibrosis in the obstructed kidneys were studied. SMalphaA, transforming growth factor beta (TGF-beta), alpha1 (I) collagen, osteopontin, macrophage chemoattractant peptide-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) gene expression were examined by Northern blotting. To elucidate the mechanism linking the Rho-ROCK pathway with renal fibrosis, the effects of Y-27632 on in vitro cell proliferation and cell migration were studied. RESULTS: In vivo analysis showed that Y-27632 suppressed SMalphaA expression, macrophage infiltration and interstitial fibrosis, and that Y-27632 suppressed SMalphaA, TGF-beta and alpha1 (I) collagen mRNA expression. In vitro analysis showed that Y-27632 did not suppress proliferation of renal fibroblasts but suppressed migration of macrophages. CONCLUSIONS: The Rho-ROCK system may play an important role in the development of tissue fibrosis, and the Rho-ROCK signaling pathway may be a new therapeutic target for preventing interstitial fibrosis in progressive renal disease.     

Obstructive uropathy in the mouse: role of osteopontin in interstitial fibrosis and apoptosis.

BACKGROUND: Osteopontin is a macrophage adhesive protein that is expressed by renal tubules in tubulointerstitial disease. METHODS: To investigate the function of OPN, we induced tubulointerstitial disease in OPN null mutant (OPN-/-) and wild-type (OPN+/+) mice by unilateral ureteral ligation. Tissue was analyzed for macrophages (ED-1), types I and IV collagen deposition, TGF-beta expression, and for tubular and interstitial cell apoptosis. RESULTS: Obstructed kidneys from both OPN-/- and OPN+/+ mice developed hydronephrosis, tubular atrophy, interstitial inflammation and fibrosis. OPN was absent in OPN-/- kidneys but was increased in obstructed OPN+/+ kidneys. Macrophage influx, measured by computer-assisted quantitative immunostaining, was less in OPN-/- mice compared to OPN+/+ mice at day 4 (threefold, P < 0.02), day 7 (fivefold, P < 0.02), but not at day 14. Interstitial deposition of types I and IV collagen were also two- to fourfold less in obstructed OPN-/- kidneys (P < 0.02). There was also a reduction of TGF-beta mRNA expression in the interstitium at day 7 (by in situ hybridization) and a near significant 34% reduction in cortical TGF-beta activity (P = 0.06) compared to obstructed OPN+/+ kidneys at day 14. Obstructed kidneys from OPN-/- mice also had more interstitial and tubular apoptotic cells (TUNEL assay) compared to obstructed OPN+/+ mice at all time points. The ability of OPN to act as a cell survival factor was also documented by showing that the apoptosis of serum-starved NRK52E renal epithelial cells was markedly enhanced in the presence of neutralizing anti-OPN antibody. CONCLUSION: OPN mediates early interstitial macrophage influx and interstitial fibrosis in unilateral ureteral obstruction. OPN may also function as a survival factor for renal tubulointerstitial cells.     

Obstructive nephropathy in the mouse: progressive fibrosis correlates with tubulointerstitial chemokine expression and accumulation of CC chemokine receptor 2- and 5-positive leukocytes

The infiltration of leukocytes plays a major role in mediating tubulointerstitial inflammation and fibrosis in chronic renal disease. CC chemokines participate in leukocyte migration and infiltration into inflamed renal tissue. Because CC chemokine-directed leukocyte migration is mediated by target cell expression of a group of CC chemokine receptors, this study examined the expression of CC chemokines and their receptors during initiation of tubulointerstitial fibrosis after unilateral ureteral obstruction in C57BL/6 mice. Obstructed kidneys developed hydronephrosis, tubular cell damage, interstitial inflammation, and fibrosis. From days 2 to 10, a progressive interstitial influx of F4/80+ macrophages and CD3+ lymphocytes occurred (macrophages, 4-fold; lymphocytes, 20-fold at day 10, compared with contralateral control kidneys). In parallel, the number of activated fibroblast-specific protein 1+ fibroblasts and interstitial collagen IV accumulation increased from days 2 to 10. The mRNA expression of CC chemokines (predominantly monocyte chemoattractant protein-1 [MCP-1]/CCL2, RANTES/CCL5) and their receptors CCR1, CCR2, CCR5 increased progressively from days 2 to 10. By in situ hybridization, a prominent interstitial mRNA expression of MCP-1 and RANTES and their receptors CCR2 and CCR5 localized to interstitial mononuclear cell infiltrates. MCP-1 and RANTES expression was also seen in tubular epithelial cells. Fluorescence-activated cell sorter analysis of single-cell suspensions from obstructed kidneys revealed a prominent expression of CCR2 and CCR5 by infiltrating macrophages, whereas most lymphocytes expressed CCR5 only. These data demonstrate an increased expression of MCP-1/CCL2 and RANTES/CCL5 at sites of tubulointerstitial damage and progressive fibrosis during unilateral ureteral obstruction that correlates with simultaneous accumulation of interstitial macrophages and T lymphocytes expressing the respective surface receptors CCR2 and CCR5. The chemokine receptor-mediated leukocyte influx into the tubulointerstitium could offer a new potential target for therapeutic intervention in progressive renal tubulointerstitial fibrosis.     

Antibody to transforming growth factor-beta ameliorates tubular apoptosis in unilateral ureteral obstruction

BACKGROUND: Unilateral ureteral obstruction (UUO) is characterized by progressive renal atrophy, renal interstitial fibrosis, an increase in renal transforming growth factor-beta (TGF-beta), and renal tubular apoptosis. The present study was undertaken to determine the effect of a monoclonal antibody to TGF-beta (1D11) in UUO. METHODS: Mechanical stretch was applied to tubular epithelial cells (NRK-52E) by a computer-assisted system. Three doses of 1D11 (either 0.5, 2, or 4 mg/rat) were administered to rats one day prior to UUO and every two days thereafter, and kidneys were harvested at day 13. Fibrosis was assessed by measuring tissue hydroxyproline and mRNA for collagen and fibronectin. Apoptosis was assessed with the terminal deoxy transferase uridine triphosphate nick end-labeling assay. TGF-beta levels were determined by bioassay. Western blot and immunostaining were used to identify proliferating cell nuclear antigen (PCNA), p53, bcl-2, and inducible nitric oxide synthase (iNOS). RESULTS: Stretch significantly induced apoptosis in NRK-52E cells, which was accompanied by an increased release of TGF-beta; 1D11 (10 microg/mL) totally inhibited stretch-induced apoptosis. Control obstructed kidney contained 20-fold higher TGF-beta as compared with its unobstructed kidney; 1D11 neutralized tissue TGF-beta of the obstructed kidney. Control obstructed kidney exhibited significantly more fibrosis and tubular apoptosis than its unobstructed counterpart, which was blunted by 1D11. In contrast, 1D11 significantly increased tubular proliferation. p53 immunostaining was localized to renal tubular nuclei of control obstructed kidney and was diminished by 1D11. In contrast, bcl-2 was up-regulated in the 1D11-treated obstructed kidney. Total NOS activity and iNOS activity of the obstructed kidney were increased by 1D11 treatment. CONCLUSION: The present study strongly suggests that an antibody to TGF-beta is a promising agent to prevent renal tubular fibrosis and apoptosis in UUO.     

Effects of suppressing intrarenal angiotensinogen on renal transforming growth factor-beta1 expression in acute ureteral obstruction

BACKGROUND: Angiotensin II (Ang II) mediates the up-regulation of fibrogenic factors such as transforming growth factor-beta1 (TGF-beta1) in chronic renal diseases. In addition, it has been proposed that the intrarenal renin-angiotensin system (RAS) is as important as the systemic RAS in kidney disease progression. METHODS: We suppressed angiotensinogen (AGT) gene expression in the kidney by transferring recombinant adenoviral vectors carrying a transgene expressing AGT antisense mRNA, and determined the effect of the local inhibition of the RAS on TGF-beta1 synthesis in the kidneys of rats with unilateral ureteral obstruction (UUO). Immediately after UUO, recombinant adenovirus vectors were injected intraparenchymally into the cortex of obstructed kidneys. RESULTS: beta-galactosidase (beta-gal)-stained kidney sections revealed the efficient transduction of the recombinant adenoviral vectors into tubular epithelial cells. Kidney cortex injected with AGT antisense showed significantly lower native AGT mRNA and protein expressions than control UUO kidneys at 24 hours and 5 days post-UUO. TGF-beta1 was significantly up-regulated in the renal cortex 24 hours and 5 days post-UUO, whereas AGT antisense-injected UUO rats showed significantly reduced TGF-beta1 expression compared to control UUO rats. Both fibronectin and collagen type I expressions were increased 24 hours and 5 days post-UUO, and these augmentations were considerably reduced by AGT antisense RNA treatment. CONCLUSION: This study demonstrates that the suppression of intrarenal RAS prevents the formation of renal cortical TGF-beta1, and of related fibrogenic factors, in early UUO.     

Chemokine receptor CCR1 but not CCR5 mediates leukocyte recruitment and subsequent renal fibrosis after unilateral ureteral obstruction

As chemokine receptor CCR1 and CCR5 expression on circulating leukocytes is thought to contribute to leukocyte recruitment during renal fibrosis, the authors examined the effects of unilateral ureteral obstruction (UUO) in mice deficient for CCR1 or CCR5. Analysis of UUO kidneys from CCR1-deficient mice revealed a reduction of interstitial macrophages and lymphocytes (35% and 55%, respectively) compared with wild-type controls. CCR1-deficient mice had reduced CCR5 mRNA levels in UUO kidneys, which correlated with a reduction of CCR5+ T cell infiltrate as determined by flow cytometry. Interstitial fibroblasts, renal TGF-beta1 mRNA expression, interstitial volume, and collagen I deposits were all significantly reduced in CCR1-deficient mice. In contrast, renal leukocytes and fibrosis were unaffected in CCR5-deficient mice with UUO. However, if treated with the CCR1 antagonist BX471, CCR5-deficient mice showed a similar reduction of renal leukocytes and fibrosis as CCR1-deficient mice. To determine the underlying mechanism labeled macrophages and T cells isolated from either wild-type, CCR1-deficient, or CCR5-deficient mice were injected into wild-type mice with UUO. Three hours later, renal cell recruitment was reduced for CCR1-deficient cells or cells pretreated with BX471 compared with CCR5-deficient or wild-type cells. Thus, CCR1 but not CCR5 is required for leukocyte recruitment and fibrosis after UUO in mice. Therefore, CCR1 is a promising target for therapeutic intervention in leukocyte-mediated fibrotic tissue injury, e.g. progressive renal fibrosis.     

Connective tissue growth factor expressed in tubular epithelium plays a pivotal role in renal fibrogenesis

Connective tissue growth factor (CTGF) is one of the candidate factors that are thought to mediate the downstream profibrotic action of TGF-beta. 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-beta1-induced CTGF. In these co-cultures, TGF-beta1 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. Then TGF-beta1 transgenic mice were subtotally nephrectomized and treated with CTGF antisense oligodeoxynucleotide, and their kidneys were analyzed for fibrosis. Intravenous administration of CTGF antisense oligodeoxynucleotide significantly blocked CTGF expression in the proximal tubular epithelial cells in the remnant kidney of these animals despite the sustained level of TGF-beta1 mRNA. This reduction in CTGF mRNA level paralleled a reduction in mRNA levels of matrix molecules as well as proteinase inhibitors plasminogen activator inhibitor-1 and tissue inhibitor of metalloproteinase-1, suppressing renal interstitial fibrogenesis. In conclusion, tubular CTGF acts as a downstream mediator of the profibrotic effects of TGF-beta1 in the remnant kidney, which is a promising target for antifibrotic drugs designed to treat TGF-beta1-dependent interstitial fibrosis.     

Tranilast ameliorates renal tubular damage in unilateral ureteral obstruction

PURPOSE: We determined whether tranilast, the anti-allergic agent N-(3, 4-dimethoxyciannamoyl)-anthranilic acid, would diminish renal transforming growth factor-beta (TGF-beta) levels in unilateral ureteral obstruction and concomitantly affect renal tubular apoptosis and proliferation in that condition. MATERIALS AND METHODS: Tranilast (150 mg./kg.) was administered to rats 1 day before unilateral ureteral obstruction and each day thereafter. Kidneys were harvested day 14 after unilateral ureteral obstruction. Tissue TGF-beta was measured by bioassay using mink lung epithelial cells. Renal tubular proliferation and apoptosis were detected by immunostaining proliferating cell nuclear antigen and the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay, respectively. Fibrosis was assessed by measuring collagen deposition with trichrome stained slides. RESULTS: TGF-beta bioassay showed that obstructed kidneys in controls contained significantly higher mean TGF-beta plus or minus standard deviation than unobstructed kidneys in controls (73.7 +/- 13.6 versus 14.1 +/- 5.5 pg./mg. tissue) and tranilast significantly decreased tissue TGF-beta in obstructed kidneys (15.9 +/- 4.8 pg./mg. tissue). The terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay demonstrated that obstructed kidneys in controls had significantly more mean tubular apoptosis than the unobstructed counterparts (36.6 +/- 6.7 versus 5.8 +/- 5.5 nuclei per high power field) and tranilast significantly decreased mean renal tubular apoptosis in obstructed kidneys (16.2 +/- 1.7 nuclei per high power field). In addition, immunostaining proliferating cell nuclear antigen showed that obstructed kidneys in controls had significantly more mean renal tubular proliferation than unobstructed kidneys (20.7 +/- 3.4 versus 6.2 +/- 2.1 per high power field) and tranilast significantly increased proliferating renal tubules in obstructed and unobstructed kidneys (26.5 +/- 8.3 and 14.5 +/- 3.4 per high power field, respectively). Control obstructed kidneys exhibited significantly more fibrosis, which was also blunted by tranilast. CONCLUSIONS: Tranilast significantly decreases tissue TGF-beta, resulting in a reduction in tubular apoptosis and an increase in tubular proliferation. This finding suggests that tranilast is a promising agent for preventing renal tubular damage in unilateral ureteral obstruction.     

Does cyclooxygenase-2 inhibitor prevent renal tissue damage in unilateral ureteral obstruction?

PURPOSE: We determined whether the cyclooxygenase-2 inhibitor etodolac affects renal tubular damage and interstitial fibrosis in unilateral ureteral obstruction. MATERIALS AND METHODS: Etodolac (10 mg./kg.) was administered to rats 1 day before unilateral ureteral obstruction and every day thereafter. Kidneys were harvested at day 14 after unilateral ureteral obstruction. Tissue transforming growth factor-beta and prostaglandin E2 were measured by bioassay using mink lung epithelial cells and enzyme linked immunosorbent-sandwich assay. Renal tubular proliferation and apoptosis were detected by immunostaining with proliferating cellular nuclear antigen and by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling, respectively. Cyclooxygenase-2 expression was detected by immunohistochemistry. Fibrosis was assessed by measuring collagen deposition in trichrome stained slides. RESULTS: Bioassay showed that in the control group obstructed kidneys contained significantly higher mean transforming growth factor-beta1 than unobstructed kidneys (79.1 +/- 8.3 versus 33.6 +/- 4.2 ng./gm. tissue) and etodolac significantly decrease the mean value in obstructed kidneys (46.2 +/- 10.0 ng./gm. tissue). Assay demonstrated that obstructed control kidneys had significantly more mean tubular apoptosis than their unobstructed counterparts (26.6 +/- 5.4 versus 2.2 +/- 1.4 nuclei per high power field) and etodolac significantly decreased mean renal tubular apoptosis in the obstructed kidneys (16.2 +/- 1.9 nuclei per high power field). In addition, immunostaining with proliferating cellular nuclear antigen showed that obstructed kidneys in the control group had significantly more mean renal tubular proliferation than unobstructed kidneys (9.8 +/- 3.4 versus 3.9 +/- 0.1 per high power field) and etodolac significantly increased mean proliferating renal tubule in the obstructed kidneys (24.9 +/- 4.3 per high power field). Control obstructed kidneys had significantly more fibrosis and prostaglandin E2 production, which were also significantly blunted by etodolac. CONCLUSIONS: The cyclooxygenase-2 inhibitor etodolac significantly reduces tissue transforming growth factor-beta, resulting in decreased tubular damage and interstitial fibrosis. This finding suggests that etodolac is a promising agent for preventing renal tissue damage in unilateral ureteral obstruction.     

mTOR通路调控肾间质成纤维细胞活化的机制

目的 探讨mTOR信号通路在肾间质成纤维细胞增生活化过程中的调控作用,并研究其抑制剂在抗肾纤维化治疗中的可行性.方法 用8周龄雌性C57BL/6小鼠构建单侧输尿管结扎(UUO)肾间质纤维化动物模型(n=30),按数字随机法分为雷帕霉素组(n=15)及UUO组(n=15).雷帕霉素组术前1d开始腹腔注射雷帕霉素(2 mg·kg-1·d-1)至实验结束;UUO组注射生理盐水.分别于术后1、3、7、14 d处死小鼠(n=3),留肾组织进行相关检测.同时,体外实验评估雷帕霉素对TGF-β诱导鼠成纤维细胞株(NIH3T3细胞)活化的干预作用.结果 UUO小鼠肾组织中活化的肌成纤维细胞[α肌动蛋白(α-SMA)阳性]高表达mTOR通路下游效应因子pS6K.雷帕霉素显著抑制pS6K表达及肾间质中肌成纤维细胞的活化,改善肾小管间质损伤及纤维化程度.实时荧光定量PCR结果提示雷帕霉素组小鼠肾皮质组织中成纤维细胞特异蛋白1 (FSP1)、转化生长因子β(TGF-β)、结缔组织因子(CTGF)及Ⅳ型胶原蛋白基因α1 (Col 4A1)的mRNA水平显著下降.体外实验结果示TGF-β诱导小鼠成纤维细胞株( NIH3T3)的mTOR通路显著活化,并大量合成α-SMA.雷帕霉素能够明显抑制mTOR通路活性,降低细胞的纤维化活性.结论 肾间质纤维化过程中成纤维细胞内的mTOR信号通路高度活化.抑制mTOR通路能够显著降低成纤维细胞的活性,改善肾间质纤维化程度.     

Fluvastatin suppresses oxidative stress and fibrosis in the interstitium of mouse kidneys with unilateral ureteral obstruction

BACKGROUND: Recently, we demonstrated increased oxidative stress in the interstitium of ureteral obstructed kidneys based on the increased expression of heme oxygenase-1 and immunohistochemical detection of advanced glycation end products (AGE) in the interstitium. Antioxidant therapy may have a therapeutic potential toward interstitial fibrosis of unilateral ureteral obstruction (UUO) kidneys. Fluvastatin is an HMG-CoA reductase inhibitor and has been demonstrated to have an antioxidant activity in vitro. METHODS: The effects of fluvastatin on UUO kidneys from the viewpoints of antioxidant action in vivo and antifibrosis action were studied. To investigate the antioxidant action and its therapeutic efficacy of fluvastatin in UUO kidneys, AGE accumulation and fibrosis in the obstructed kidneys was compared among vehicle-, pravastatin-, or fluvastatin-treated (10 or 40 mg/kg/day) groups. RESULTS: Tubulointerstitial fibrosis was significantly attenuated in fluvastatin-treated animals. Fluvastatin significantly suppressed the degree of immunostaining of AGE in UUO kidneys. CONCLUSIONS: These results provide evidence for the antioxidant action of fluvastatin in vivo. The decreased interstitial fibrosis along with a decreased oxidative stress marker in the interstitial lesion strongly suggests the existence of a causal relationship between them. Fluvastatin may have therapeutic value in slowing or preventing interstitial fibrosis in progressive renal disease.     

Hepatocyte growth factor gene therapy and angiotensin II blockade synergistically attenuate renal interstitial fibrosis in mice

Tubulointerstitial fibrosis is considered to be common endpoint result of many forms of chronic renal diseases. Except for renal replacement, chronic renal fibrosis is presently incurable. This study demonstrates that the combination of hepatocyte growth factor (HGF) gene therapy with inhibition of the renin-angiotensin system produced synergistic beneficial effects leading to dramatic attenuation of renal tubulointerstitial fibrosis in obstructive nephropathy in mice. The combined treatment with human HGF gene and losartan, an angiotensin II (AngII) type I receptor blocker, preserved renal mass and gross morphology of the obstructed kidneys. Although HGF gene therapy alone inhibited the expression of alpha-smooth muscle actin (alpha SMA) by approximately 54% and 60% at day 7 and day 14 after surgery, respectively, its combination with losartan almost completely abolished alpha SMA induction in the obstructed kidneys. The combined therapy also synergistically inhibited the accumulation of interstitial matrix components, such as fibronectin and collagen I, and suppressed renal expression of transforming growth factor-beta1 (TGF-beta1) and its type I receptor. In vitro studies revealed that AngII by itself did not induce alpha SMA, but it drastically potentiated TGF-beta1-initiated alpha SMA expression in tubular epithelial cells. Furthermore, HGF abrogated de novo alpha SMA expression induced by TGF-beta1 plus AngII. These results suggest that many factors are implicated in the pathogenesis of renal interstitial fibrosis; therefore, a combined therapy aimed at simultaneously targeting multiple pathologic pathways may be necessary for halting the progression of chronic renal diseases. These findings may provide the basis for designing future therapeutic regimens for blocking progressive renal fibrosis in patients.     

CXCL16 recruits bone marrow-derived fibroblast precursors in renal fibrosis

Although fibroblasts are responsible for the production and deposition of extracellular matrix in renal fibrosis, their origin is controversial. Circulating fibroblast precursors may contribute to the pathogenesis of renal fibrosis, but the signaling mechanisms underlying the recruitment of bone marrow-derived fibroblast precursors into the kidney in response to injury are incompletely understood. Here, in the unilateral ureteral obstruction model of renal fibrosis, tubular epithelial cells upregulated the chemokine CXCL16 in obstructed kidneys, and circulating fibroblast precursors expressed the CXCL16 receptor, CXCR6. Compared with wild-type mice, CXCL16-knockout mice accumulated significantly fewer bone marrow-derived fibroblast precursors in obstructed kidneys. CXCL16-knockout mice also exhibited significantly fewer CD45-, collagen I-, and CXCR6-triple-positive fibroblast precursors in injured kidneys. Furthermore, targeted deletion of CXCL16 inhibited myofibroblast activation, reduced collagen deposition, and suppressed expression of collagen I and fibronectin. In conclusion, CXCL16 contributes to the pathogenesis of renal fibrosis by recruiting bone marrow-derived fibroblast precursors.     

Activin A is a potent activator of renal interstitial fibroblasts

The present study was conducted to examine the involvement of the activin-follistatin system in the fibrotic process of the kidney. Immunoreactive activin A was upregulated in tubular cells in the kidneys with unilateral ureteral obstruction but not in normal and contralateral kidneys. Activin A promoted cell proliferation, enhanced the expression of type I collagen mRNA, and induced the production of alpha-smooth muscle actin in a rat kidney fibroblast cell line (NRK-49F cells) as well as in primary cultured renal interstitial fibroblasts. In contrast, activin A did not affect the expressions of alpha-smooth muscle actin and type I collagen in renal epithelial tubular cell lines LLC-PK1, and MDCK. Follistatin, an antagonist of activin A, significantly inhibited cell proliferation in NRK-49F cells. Blockade of activin signaling by overexpression of truncated type II activin receptor, which lacked the intracellular kinase domain, decreased cell proliferation and reduced the expression level of type I collagen mRNA in NRK-49F cells. The expression of activin A was induced by TGF-beta 1 or activin A itself. Induction of type I collagen expression by TGF-beta 1 was reduced by follistatin or by overexpression of truncated type II activin receptor. These results suggest that activin A produced by tubular cells acts as a paracrine factor that activates renal interstitial fibroblasts during the fibrotic processes of the kidney.     

Time course of cytokine mRNA expression in kidneys of rats with unilateral ureteral obstruction

The development of renal interstitial fibrosis (RIF) is related to the expression and excretion of cytokines and growth factors. Thus, we investigated the time course of mRNA expression of cytokines known as causative factors in a model of RIF in rats before and on day 10 after unilateral ureteral obstruction (UUO), when first signs of fibrosis were visible, as well as during progressive RIF. UUO causes a fivefold increase in mRNA expression of monocyte chemoattractant protein 1 15 days after surgery as compared with contralateral kidneys. The level remains elevated about three-fold up to day 25. The mRNA of the fibrogenic cytokine transforming growth factor beta 1 (TGF-beta1) is increased two- to threefold during the time course, whereas the mRNAs of platelet-derived growth factor B chain (PDGF-B) and its receptor beta (PDGF-Rbeta) increase after UUO, reaching their maxima on days 10-15. PDGF-B mRNA increase up to day 15, marking the onset of fibrosis, and decreases thereafter, whereas the expression of the PDGF-Rbeta mRNA remains elevated more than threefold over the entire study period. Incubation of cultured renal fibroblasts with TGF-beta1 and/or PDGF-B suggests that their specific action on cell growth and proliferation is maintained even when they are used in combination. The sustained elevation of TGF-beta1 and PDGF-B/PDGF-Rbeta mRNA levels confirms the assumption of a particular involvement of these cytokines in the pathogenesis of RIF. The mRNA expression of the gap junctional protein connexin 43 in ureteral ligated kidneys is increased sixfold already 5 days after UUO. In this way, the increased connexin 43 mRNA levels indicate a possible function in the remodeling of the kidney tissue after tubular damage and fibrosis.