Rhubarb Protect Against Tubulointerstitial Fibrosis by Inhibiting TGF-?/Smad Pathway and Improving Abnormal Metabolome in Chronic Kidney Disease.
ABSTRACT: Tubulointerstitial fibrosis is the final common pathway for all kidney diseases leading to chronic kidney disease (CKD). TGF-?/Smad signaling pathway plays a key role in renal fibrosis. Previous studies have revealed that rhubarb extracts attenuated the increase of transforming growth factor-? 1 (TGF-?1) in CKD rats. To gain an in-depth insight into the mechanism of the anti-fibrotic activities of the rhubarb extracts, we investigated the influence of rhubarb extracts on TGF-?/Smad signaling pathway and the influence on metabolome in a rat model of CKD with adenine-induced chronic tubulointerstitial nephropathy. Male Sprague-Dawley rats were divided into four groups, including control, CKD, CKD + petroleum ether extract, CKD + ethyl acetate extract, and CKD + n-butanol extract groups. Kidneys harvested on the week three were evaluated for renal fibrosis, the expression of proteins in TGF-?/Smad signaling pathway and metabolomic study. We found rhubarb extracts suppressed TGF-?/Smad3-mediated renal fibrosis by reducing the TGF-?1, transforming growth factor-? receptor I (TGF-? RI), transforming growth factor-? receptor II (TGF-? RII), Smad2, p-Smad2, Smad3, p-Smad3, and Smad4, meanwhile increased Smad7. In addition, rhubarb extracts mitigated renal injury and dysfunction, and either fully or partially reversed the abnormalities of tissue metabolites. Thus, rebalancing the disorder of TGF-?/Smad signaling and metabolic dysfunction by treatment with rhubarb extracts may represent as an effective therapy for CKD associated with fibrosis.
Project description:TGF-? (transforming growth factor-?) is well identified as a central mediator in renal fibrosis. TGF-? initiates canonical and non-canonical pathways to exert multiple biological effects. Among them, Smad signaling is recognized as a major pathway of TGF-? signaling in progressive renal fibrosis. During fibrogenesis, Smad3 is highly activated, which is associated with the down-regulation of an inhibitory Smad7 via an ubiquitin E3-ligases-dependent degradation mechanism. The equilibrium shift between Smad3 and Smad7 leads to accumulation and activation of myofibroblasts, overproduction of ECM (extracellular matrix), and reduction in ECM degradation in the diseased kidney. Therefore, overexpression of Smad7 has been shown to be a therapeutic agent for renal fibrosis in various models of kidney diseases. In contrast, another downstream effecter of TGF-?/Smad signaling pathway, Smad2, exerts its renal protective role by counter-regulating the Smad3. Furthermore, recent studies demonstrated that Smad3 mediates renal fibrosis by down-regulating miR-29 and miR-200 but up-regulating miR-21 and miR-192. Thus, overexpression of miR-29 and miR-200 or down-regulation of miR-21 and miR-192 is capable of attenuating Smad3-mediated renal fibrosis in various mouse models of chronic kidney diseases (CKD). Taken together, TGF-?/Smad signaling plays an important role in renal fibrosis. Targeting TGF-?/Smad3 signaling may represent a specific and effective therapy for CKD associated with renal fibrosis.
Project description:Overexpression of transforming growth factor beta (TGF-beta) has been shown to play pathogenic roles in progression of renal fibrosis, and the severity of tubulointerstitial fibrosis correlates better with renal function than the severity of glomerulosclerosis. Smad proteins are signaling transducers downstream from TGF-beta receptors. Three families of Smad proteins have been identified: receptor-regulated Smad2 and Smad3, common partner Smad4, and inhibitory Smad7 (part of a negative-feedback loop). We investigated Smad-mediated TGF-beta signaling pathway and regulatory mechanisms of inhibitory Smad7 in unilateral ureteral obstruction (UUO) kidneys in mice, a model of progressive tubulointerstitial fibrosis. Compared with sham-operated kidneys, the level of Smad7 protein, but not mRNA, decreased progressively in UUO kidneys, whereas immunoreactivity for nuclear phosphorylated Smad2 and Smad3 and renal fibrosis were inversely increased. Furthermore, we demonstrated that both the degradation and ubiquitination activity of Smad7 protein were increased markedly in UUO kidneys compared with sham-operated ones. We also found that both Smurf1 and Smurf2 (Smad ubiquitination regulatory factors), which are E3 ubiquitin ligases for Smad7, were increased and that they interacted with Smad7 in UUO kidneys. Our results suggest that the reduction of Smad7 protein resulting from enhanced ubiquitin-dependent degradation plays a pathogenic role in progression of tubulointerstitial fibrosis.
Project description:Inflammation and fibrosis are two pathological features of chronic kidney disease (CKD). Transforming growth factor-? (TGF-?) has been long considered as a key mediator of renal fibrosis. In addition, TGF-? also acts as a potent anti-inflammatory cytokine that negatively regulates renal inflammation. Thus, blockade of TGF-? inhibits renal fibrosis while promoting inflammation, revealing a diverse role for TGF-? in CKD. It is now well documented that TGF-?1 activates its downstream signaling molecules such as Smad3 and Smad3-dependent non-coding RNAs to transcriptionally and differentially regulate renal inflammation and fibrosis, which is negatively regulated by Smad7. Therefore, treatments by rebalancing Smad3/Smad7 signaling or by specifically targeting Smad3-dependent non-coding RNAs that regulate renal fibrosis or inflammation could be a better therapeutic approach. In this review, the paradoxical functions and underlying mechanisms by which TGF-?1 regulates in renal inflammation and fibrosis are discussed and novel therapeutic strategies for kidney disease by targeting downstream TGF-?/Smad signaling and transcriptomes are highlighted.
Project description:Transforming growth factor-?1 (TGF-?1)-induced epithelial-to-mesenchymal transition (EMT) contributes to the pathophysiological development of kidney fibrosis. Although it was reported that TGF-?1 enhances ?(1) integrin levels in NMuMG cells, the detailed molecular mechanisms underlying TGF-?1-induced ?(1) integrin gene expression and the role of ?(1) integrin during EMT in the renal system are still unclear. In this study, we examined the role of ?(1) integrin in TGF-?1-induced EMT both in vitro and in vivo. TGF-?1-induced augmentation of ?(1) integrin expression was required for EMT in several epithelial cell lines, and knockdown of Smad3 inhibited TGF-?1-induced augmentation of ?(1) integrin. TGF-?1 triggered ?(1) integrin gene promoter activity as assessed by luciferase activity assay. Both knockdown of Smad3 and mutation of the Smad-binding element to block binding to the ?(1) integrin promoter markedly reduced TGF-?1-induced ?(1) integrin promoter activity. Chromatin immunoprecipitation assay showed that TGF-?1 enhanced Smad3 binding to the ?(1) integrin promoter. Furthermore, induction of unilateral ureteral obstruction triggered increases of ?(1) integrin in both renal epithelial and interstitial cells. In human kidney with chronic tubulointerstitial fibrosis, we also found a concomitant increase of ?(1) integrin and ?-smooth muscle actin in tubule epithelia. Blockade of ?(1) integrin signaling dampened the progression of fibrosis. Taken together, ?(1) integrin mediates EMT and subsequent tubulointerstitutial fibrosis, suggesting that inhibition of ?(1) integrin is a possible therapeutic target for prevention of renal fibrosis.
Project description:Tubulointerstitial fibrosis is the final common result of a variety of progressive injuries leading to chronic renal failure. Transforming growth factor-beta (TGF-beta) is reportedly upregulated in response to injurious stimuli such as unilateral ureteral obstruction (UUO), causing renal fibrosis associated with epithelial-mesenchymal transition (EMT) of the renal tubules and synthesis of extracellular matrix. We now show that mice lacking Smad3 (Smad3ex8/ex8), a key signaling intermediate downstream of the TGF-beta receptors, are protected against tubulointerstitial fibrosis following UUO as evidenced by blocking of EMT and abrogation of monocyte influx and collagen accumulation. Culture of primary renal tubular epithelial cells from wild-type or Smad3-null mice confirms that the Smad3 pathway is essential for TGF-beta1-induced EMT and autoinduction of TGF-beta1. Moreover, mechanical stretch of the cultured epithelial cells, mimicking renal tubular distention due to accumulation of urine after UUO, induces EMT following Smad3-mediated upregulation of TGF-beta1. Exogenous bone marrow monocytes accelerate EMT of the cultured epithelial cells and renal tubules in the obstructed kidney after UUO dependent on Smad3 signaling. Together the data demonstrate that the Smad3 pathway is central to the pathogenesis of interstitial fibrosis and suggest that inhibitors of this pathway may have clinical application in the treatment of obstructive nephropathy.
Project description:Chronic renal failure (CRF) is a major public health problem worldwide. Earlier studies have revealed salutary effects of rhubarb extracts in CRF. In this study, we employed lipidomic and metabolomic approaches to identify the plasma biomarkers and to determine the effect of treatment with petroleum ether, ethyl acetate and n-butanol extracts of rhubarb in a rat model of CRF with adenine-induced chronic tubulointerstitial nephropathy. In addition, clinical biochemistry, histological evaluation and pro-fibrotic protein expression were analyzed. Significant changes were found between the CRF and control groups representing characteristic phenotypes of rats with CRF. Treatment with the three rhubarb extracts improved renal injury and dysfunction, either fully or partially reversed the plasma metabolites abnormalities and attenuated upregulation of pro-fibrotic proteins including TGF-?1, ?-SMA, PAI-1, CTGF, FN and collagen-1. The nephroprotective effect of ethyl acetate extract was better than other extracts. The differential metabolites were closely associated with glycerophospholipid, fatty acid and amino acid metabolisms. The results revealed a strong link between renal tubulointerstitial fibrosis and glycerophospholipid metabolism and L-carnitine metabolism in the development of CRF. Amelioration of CRF with the three rhubarb extracts was associated with the delayed development and/or reversal the disorders in key metabolites associated with adenine-induced CRF.
Project description:Chronic kidney disease (CKD) is often accompanied with colon mucosal barrier damage and gut microbiota disturbance, which strongly associate with up-regulated inflammation and kidney tubulointerstitial fibrosis. However, few interventions could protect the damaged barrier effectively. Rheum palmatum L or rhubarb is a common herbal medicine which is widely used to protect the colon mucosal barrier. In previous studies, we found that rhubarb intervention may reduce renal inflammation and tubulointerstitial fibrosis, via gut microbiota modification. However, whether intestinal barrier function could be improved by rhubarb intervention and the relationship with intestinal flora are still unknown. Therefore, we investigated the effects of rhubarb enema on intestinal barrier, and further analyzed the relationship with gut microbiota in 5/6 nephrectomy rats. Results indicated that rhubarb enema improved the intestinal barrier, regulated gut microbiota dysbiosis, suppressed systemic inflammation, and alleviated renal fibrosis. More specifically, rhubarb enema treatment inhibited the overgrowth of conditional pathogenic gut bacteria, including Akkermansia, Methanosphaera, and Clostridiaceae in CKD. The modification of gut microbiota with rhubarb intervention displayed significant correlation to intestinal barrier markers, TLR4-MyD88-NF-?B inflammatory response, and systemic inflammation. These results revealed that rhubarb enema could restore intestinal barrier by modifying several functional enteric bacteria, which may further explain the renal protection mechanism of the rhubarb enema.
Project description:Background:Acute kidney injury (AKI) is one of the major risk factors for progression to chronic kidney disease (CKD) and renal fibrosis. However, effective therapies remain poorly understood. Here, we examined the renoprotective effects of melatonin and poricoic acid A (PAA) isolated from the surface layer of Poria cocos, and investigated the effects of combined therapy on the interaction of TGF-?/Smad and Wnt/?-catenin in a rat model of renal ischemia-reperfusion injury (IRI) and hypoxia/reoxygenation (H/R) or TGF-?1-induced HK-2 cells. Methods:Western blot and immunohistochemical staining were used to examine protein expression, while qRT-PCR was used to examine mRNA expression. Coimmunoprecipitation, chromatin immunoprecipitation, RNA interference, and luciferase reporter gene analysis were employed to explore the mechanisms of PAA and melatonin's renoprotective effects. Results:PAA and combined therapy exhibited renoprotective and antifibrotic effects, but the underlying mechanisms were different during AKI-to-CKD continuum. Melatonin suppressed Smad-dependent and Smad-independent pathways, while PAA selectively inhibited Smad3 phosphorylation through distrupting the interactions of Smad3 with TGF?RI and SARA. Further studies demonstrated that the inhibitory effects of melatonin and PAA were partially depended on Smad3, especially PAA. Melatonin and PAA also inhibited the Wnt/?-catenin pathway and its profibrotic downstream targets, and PAA performed better. We further determined that IRI induced a nuclear Smad3/?-catenin complex, while melatonin and PAA disturbed the interaction of Smad3 and ?-catenin, and supplementing with PAA could enhance the inhibitory effects of melatonin on the TGF-?/Smad and Wnt/?-catenin pathways. Conclusions:Combined melatonin and PAA provides a promising therapeutic strategy to treat renal fibrosis during the AKI-to-CKD continuum.
Project description:Renal tubulointerstitial fibrosis is a common feature of the final stage of nearly all cause types of chronic kidney disease. Although classic peroxisome proliferator-activated receptor ? (PPAR?) agonists have a protective effect on diabetic nephropathy, much less is known about their direct effects in renal fibrosis. This study aimed to investigate possible beneficial effects of lobeglitazone, a novel PPAR? agonist, on renal fibrosis in mice.We examined the effects of lobeglitazone on renal tubulointerstitial fibrosis in unilateral ureteral obstruction (UUO) induced renal fibrosis mice. We further defined the role of lobeglitazone on transforming growth factor (TGF)-signaling pathways in renal tubulointerstitial fibrosis through in vivo and in vitro study.Through hematoxylin/eosin and sirius red staining, we observed that lobeglitazone effectively attenuates UUO-induced renal atrophy and fibrosis. Immunohistochemical analysis in conjunction with quantitative reverse transcription polymerase chain reaction and Western blot analysis revealed that lobeglitazone treatment inhibited UUO-induced upregulation of renal Smad-3 phosphorylation, ?-smooth muscle actin, plasminogen activator inhibitor 1, and type 1 collagen. In vitro experiments with rat mesangial cells and NRK-49F renal fibroblast cells suggested that the effects of lobeglitazone on UUO-induced renal fibrosis are mediated by inhibition of the TGF-?/Smad signaling pathway.The present study demonstrates that lobeglitazone has a protective effect on UUO-induced renal fibrosis, suggesting that its clinical applications could extend to the treatment of non-diabetic origin renal disease.
Project description:OBJECTIVE:Increasing evidence shows that TGF-?1 is a key mediator in diabetic nephropathy (DN) and induces renal fibrosis positively by Smad3 but negatively by Smad7. However, treatment of DN by blocking the TGF-?/Smad pathway remains limited. The present study investigated the anti-fibrotic effect of a traditional Chinese medicine, Chaihuang-Yishen granule (CHYS), on DN. RESEARCH DESIGN AND METHODS:Protective role of CHYS in DN was examined in an accelerated type 1 DN induced by streptozotocin in uninephrectomized Wistar rats. CHYS, at a dose of 0.56 g/kg body weight, was administered by a daily gastric gavage for 20 weeks and the therapeutic effect and potential mechanisms of CHYS on diabetic kidney injury were examined. RESULTS:Treatment with CHYS attenuated diabetic kidney injury by significantly inhibiting 24-h proteinuria and progressive renal fibrosis including glomerulosclerotic index, tubulointerstitial fibrosis index, and upregulation of extracellular matrix (collagen I, IV, and fibronectin), despite the same levels of blood glucose. Further studies revealed that inhibition of renal fibrosis in CHYS-treated diabetic rats was associated with inhibition of TGF-?1/Smad3 signaling as demonstrated by upregulation of Smad7 but downregulation of TGF-?1, TGF-? receptors, activation of Smad3, and expression of miRNA-21. CONCLUSIONS:CHYS may be a therapeutic agent for DN. CHYS attenuates DN by blocking TGF-?/Smad3-mediated renal fibrosis.