| Similar to wound healing, renal fibrosis probably initiates as a beneficial response to injury. If an injurious condition is sustained, which seems to be the case in most progressive renal diseases, pathological fibrosis results in glomerulosclerosis, tubular atrophy and dilation, tubulointerstitial fibrosis and rarefaction of the glomerular, as well as peritubular capillaries。Fibroblasts are stellate shaped cells with a stellate nucleus and abundant rough endoplasmatic reticulum (ER), collagen-secreting granules, actin filaments。Myofibroblasts differ from fibroblasts in that they contain microfilaments with focal densities (stress fibers), A key feature of myofibroblasts is their contractile function, which is aided by the expression of a-smooth muscle actin (a-SMA). Other myofibroblast markers include vimentin and fibronectin splice variant ectodomain A。The profibrotic role of monocytes and/or macrophages in mice with UUO。A large number of in vivo and in vitro studies have demonstrated that renal tubular cells undergo EMT in response to leukocyte infiltration, stress signals and various mediators (in particular TGF-β). Interestingly, many EMT regulatory genes contribute to cell survival and thus EMT, at least initially, may represent an adaptive response to cellular stress. EMT is induced via three major, converging signaling pathways, namely the TGF-β/Smad, integrin/ILK, and Wnt/β-catenin pathways transforming growth factor-β1(TGF-β1) has been recognized as a key mediator in the pathogenesis of renal fibrosis。TGF-β is a founding member of the TGF-β superfamily including activins, inhibins, growth and differentiation factors, and bone morphogenetic proteins. TGF-β1and its isoforms (TGF-β2and TGF-β3) are synthesized by a variety of cells including all cell types of the kidneyIt is now well established that the binding of TGF-β1to its receptor II (TβRII) can activate the TGF-β receptor type I (TβRI)-kinase, resulting in phosphorylation of Smad2and Smad3, two receptor-associated Smads (R-Smads). Subsequently, phosphorylated Smad2and Smad3bind to the common Smad4and form the Smad complex, which translocates into the nucleus to regulate the target gene transcription, including Smad7. Smad7is an inhibitory Smad that negatively regulates Smad2and Smad3activation and functions by targeting the TβRI and Smads for degradation via the ubiquitin proteasome degradation mechanisms In the fibrotic kidney, a multitude of mechanisms lead to a hyperactive TGF-β/Smad signaling。Tremendous effort has been made to inhibit TGF-β action in an attempt to hamper the progression of renal fibrosis. Over the years, many novel approaches have been tested, which include antisense inhibition of TGF-β expression, neutralizing anti-TGF-β antibody, soluble TGF-β receptor, or blockade of TGF-β activation by decorin. A new strategy in development is to use specific small molecule TGF-β receptor inhibitors. It poses a great challenge to achieve a meaningful antisense inhibition of TGF-β expression in the human kidney using current technology. But more importantly, we still do not know with great certainty whether long-term inhibition of TGF-β is an optimal approach to the therapy of renal fibrosis, in light of the fact that TGF-β is also an anti-inflammatory cytokine. The profibrotic and anti-inflammatory properties of TGF-β pose a dilemma for the therapeutic application of TGF-β inhibition. To circumvent this problem, several strategies are being developed to target the TGF-β downstream effectors or the signaling that mediates its fibrogenic action. In this area, suppression of connective tissue growth factor expression or activity is one option. Another approach includes inhibition of Smad signaling through the delivery of inhibitory Smad7.It is now well accepted that Smad2and Smad3are two critical downstream mediators responsible for the biological effects of TGF-β1. In the context of renal fibrosis, Smad2and Smad3are strongly activated in both experimental and human kidney diseases, including diabetic nephropathy obstructive kidney diseases, remnant kidney disease, hypertensive nephropathy, drug-associated nephropathy, and immunologically-mediated glomerulonephritis. Many fibrogenic genes, such as (ColIal, Colla2, ColIIIal, ColVa2, ColVIal, and ColVIa3) and tissue inhibitor of MMP-1(TIMP-1) are the downstream targets of TGF-β/Smad3signaling, suggesting that Smad3may be a critical mediator of TGF-β/Smad signaling in fibrosis. An essential role for Smad3in fibrogenesis is confirmed by the findings that deletion of Smad3from mice suppresses fibrogenesis in a number of rodent models, including diabetic nephropathy, obstructive nephropathy, and drug toxicity-related nephropathy. Furthermore, the use of a Smad3inhibitor to inhibit endothelial-myofibroblast transition and renal fibrosis in a type-1diabetic kidney disease demonstrates a therapeutic potential for kidney disease by targeting Smad3signalingThe model of unilateral ureteral obstruction (UUO) in the rodent generates progressive renal fibrosis. Surgically created UUO can be experimentally manipulated with respect to timing, severity, and duration, while reversal of the obstruction permits the study of recovery. The use of genetically engineered mice has greatly expanded the utility of the model in studying molecular mechanisms underlying the renal response to UUO. Ureteral obstruction results in marked renal hemodynamic and metabolic changes, followed by tubular injury and cell death by apoptosis or necrosis, with interstitial macrophage infiltration. Proliferation of interstitial fibroblasts with myofibroblast transformation leads to excess deposition of the extracellular matrix and renal fibrosis. Phenotypic transition of resident renal tubular cells, endothelial cells, and pericytes has also been implicated in this process. Technical aspects of the UUO model are discussed in this review, including the importance of rodent species or strain, the age of the animal, surgical procedures, and histological methods. The UUO model is likely to reveal useful biomarkers of progression of renal disease, as well as new therapies, which are desperately needed to allow intervention before the establishment of irreversible renal injury.In the past years, many promising targets for the treatment of renal fibrosis have been validated in various animal models, and even more new targets have been identified. For several of the targets reviewed, substances/drugs have already been developed, are being tested, or are already being therapeutically employed in patients with non-renal indications. Renal fibrosis, in contrast, remains a largely uncharted territory in clinical trials. The reasons for this are certainly multifactorial and may include long study durations if hard endpoints,the lack of non-invasive markers or diagnostic tools to assess kidney scarring, and thus, monitor therapy. However, the industry has noted the enormous potential market, given the possibility of developing antifibrotic therapy that might be of benefit in many different types of organ fibrosis. Furthermore, there is hope that with a large consortia search for biomarkers and advancing ultrasound, or through MR-based or molecular-imaging techniquesPart I Effects of GQ-5on tububulointerstital fibrosisObjective:In this study, we evaluated the efficacy of GQ-5for the treatment of renal fibrosis in a rat unilateral ureteral obstruction (UUO) model Method:The animals were anesthetized with an intraperitoneal (IP) injection. A midline incision was made in the abdominal wall, the left ureter was dissected out and ligated with4.0silk at two points along its length. The abdominal wound was approximated with the same silk suture. Three groups of rats comprising6animals each (total=18) were included as follows.(1) Sham group:(2) UUO+Propylene glycol group:the rats received IP injection of Propylene glycol and underwent unilateral ureteral ligation.(3) UUO+GQ-5group:these rats in addition those in group II received IP injection of GQ-5at a dose of40mg/kg a day. The rats were killed on day7after UUO or Sham operation, and the kidneys were harvested for various biochemical and morphological studies. Immunohistochemical analyses and Western Blot were carried out by using anti-phospho-Smad2, anti-phospho-Smad3,anti-fibronectin, anti-collagen-I and anti-a-SMA.Result:Histology stained with HE detected that GQ-5could make UUO rat renal interstitial inflammatory cells significantly reduced, fibroblasts was also reduced, renal tubular cell morphology had been restored. Western blot analysis showed that GQ-5decreased alpha SMA expression,(F=296.589, P=0.000)suggesting that activation of Myofibroblasts reduced. Similar findings were also demonstrated at the protein level by Immunohistochemically analysis.Western Blot and immunohistochemistry confirmed that GQ-5decreased Fibronectin and Collagen-I expression.(F=27.147, P=0.000) This experiment with Western Blot and immunohistochemistry revealed that GQ-5significantly inhibited Smad3phosphorylation levels,(F=178.644, P=0.000) but had no obvious effect on the levels of Smad2phosphorylation, immunohistochemistry demonstrated that GQ-5attenuated autoinduction of TGF-β1Summary1. Rat UUO model was successfully established, the pathological feature of rat UUO model was tububulointerstital fibrosi2. GQ-5inhibits tububulointerstital fibrosis In Vitro3. The disruption of TGF beta/Smad3Signaling Is a Mechanism by which GQ-5inhibits renal tissue fibrosisPart II Effects of GQ-5on the Activation of NRK52Einduced by TGF-β1Objective:To confirm the results found in the UUO kidney,we evaluated the efficacy of GQ-5on the activation of NRK52E induced by TGF-β1MethodCell culture. NRK52E were grown in DMEM/F12containing10%fetal bovine serum. When60-80%cells were stick on the well, they were cultured forl2h in free serum medium. Immunofluorescence analyses and Western Blot were carried out by using anti-phospho-Smad2, anti-phospho-Smad3,anti-fibronectin, anti-E-cadherin and anti-a-SMA. Cell proliferation was assessed using the MTT cell migration were studiedResult:MTT assay indicated that Y-27632did not show an inhibitory effect on proliferation of NRK52E cells. GQ-5abrogated TGF-β1-induced NRK52E cells migration (F=108.506, P=0.000). Exogenous TGF-β1induced downregulatin of E-cadherin in cultured NRK52E, and GQ-5increased the E-cadherin expression in a dose-dependent manner(F=7.25, P=0.002) Exogenous TGF-β1induced the α-SMA expression in cultured NRK52E, and GQ-5decreased the a-SMA expression in a dose-dependent manner.(F=26.182, P=0.000) GQ-5reduced the up-regulated expression of fibronectin protein(F=39.675, P=0.000) as well as a-SMA in cultured NRK52E. The pretreatment with1μM GQ-5reduced phosphorylation levels of Smad3induced by TGF-β(F=383.611, P=0.000),However, the phosphorylated levels of Smad2and the protein expression levels of Smad4and Smad7, the inhibitory Smad, were not affected by this reagent in the presence of TGF-β1. GQ-5did not affect the phosphorylation of other signaling pathways, such as MAPK/p38, ERK or phosphoinositide3-kinaseSummary1. GQ-5did not suppress proliferation of NRK52E2. GQ-5abrogated TGF-P1induced NRK52E cells migration3. GQ-5Restores Myofibroblast Differentiation of NRK52E4. GQ-5attenuated the TGF-P1-induced phosphorylation of Smad35. GQ-5Reduces the TGF-P1-induced ECM ExpressionIn ClonclutionTogether, in vivo and in vitro studies show that, GQ-5can block excessive extracellular matrix deposition, decreased TGF-β1autocrine.. In this study, we showed that specific inhibitor of Smad3(GQ-5), a new inhibitor of TGF-β signaling, expressed its effects via the selective suppression of Smad3phosphorylation.GQ-5can be used in the treatment of renal interstitial fibrosis... |
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