Protease‐activated receptor‐1 contributes to renal injury and interstitial fibrosis during chronic obstructive nephropathy

End‐stage renal disease, the final stage of all chronic kidney disorders, is associated with renal fibrosis and inevitably leads to renal failure and death. Transition of tubular epithelial cells (TECs) into mesenchymal fibroblasts constitutes a proposed mechanism underlying the progression of renal fibrosis and here we assessed whether protease‐activated receptor (PAR)‐1, which recently emerged as an inducer of epithelial‐to‐mesenchymal transition (EMT), aggravates renal fibrosis. We show that PAR‐1 activation on TECs reduces the expression of epithelial markers and simultaneously induces mesenchymal marker expression reminiscent of EMT. We next show that kidney damage was reduced in PAR‐1‐deficient mice during unilateral ureter obstruction (UUO) and that PAR‐1‐deficient mice develop a diminished fibrotic response. Importantly, however, we did hardly observe any signs of mesenchymal transition in both wild‐type and PAR‐1‐deficient mice suggesting that diminished fibrosis in PAR‐1‐deficient mice is not due to reduced EMT. Instead, the accumulation of macrophages and fibroblasts was significantly reduced in PAR‐1‐deficient animals which were accompanied by diminished production of MCP‐1 and TGF‐β. Overall, we thus show that PAR‐1 drives EMT of TECs in vitro and aggravates UUO‐induced renal fibrosis although this is likely due to PAR‐1‐dependent pro‐fibrotic cytokine production rather than EMT.     

依帕司他对单侧输尿管梗阻大鼠肾间质纤维化的干预作用及其机制

目的:观察依帕司他(EPS)对单侧输尿管梗阻(UUO)大鼠间质纤维化的保护作用及其机制。方法:实验设假手术组(Sham)组、UUO、UUO+EPS(50 mg/kg)及UUO+EPS(100 mg/kg)剂量组,每组n=8。左侧输尿管结扎制备UUO大鼠模型。造模后连续灌胃给药3周,sham和UUO组给予等体积的羟甲基纤维素钠。HE和Masson染色观察肾组织病理变化及胶原沉积情况。免疫组化法观察肾组织醛糖还原酶(AR)表达情况,分别采用real-time PCR和(或) Western blot检测肾脏I型胶原(collagen I)、III型胶原(collagen III)、α-平滑肌肌动蛋白(α-SMA)、成纤维细胞特异蛋白-1(FSP-1)、纤连蛋白(FN)、E-钙粘蛋白(E-cadherin)、转化生成因子-β1(TGF-β1)和AR mRNA及蛋白表达。结果:与Sham组相比,UUO组大鼠小管上皮细胞萎缩、空泡样变性,肾间质成纤维细胞及肌成纤维细胞大量增殖并伴大量炎症细胞浸润,胶原沉积明显增加,collagen I、collagen III、TGF-β1和AR mRNA及蛋白表达水平明显升高(P<0.01),同时EMT标志性蛋白α-SMA、FSP-1、FN mRNA及蛋白表达水平明显升高(P<0.01),而E-cadherin mRNA及蛋白表达水平明显降低。与UUO组相比,经EPS治疗3周后,肾间质纤维化程度明显减轻,胶原沉积明显减少,collagen I、collagen III、TGF-β1和AR mRNA及蛋白表达水平明显降低(P<0.01或P<0.05),另外α-SMA、FSP-1、FN mRNA及蛋白表达水平明显降低(P<0.01或P<0.05),而E-cadherin mRNA及蛋白表达水平明显升高(P<0.01或P<0.05),而且100 mg/kg剂量组上述指标的改变均好于低剂量组(P<0.05,P<0.01)。结论:依帕司他对肾间质纤维化具有一定的改善作用,其机制可能与其抑制TGF-β1介导的AR表达、进而抑制大鼠肾小管上皮细胞EMT有关。     

miR-200b precursor can ameliorate renal tubulointerstitial fibrosis

Members of the miR-200 family of micro RNAs (miRNAs) have been shown to inhibit epithelial-mesenchymal transition (EMT). EMT of tubular epithelial cells is the mechanism by which renal fibroblasts are generated. Here we show that miR-200 family members inhibit transforming growth factor-beta (TGF-beta)-induced EMT of tubular cells. Unilateral ureter obstruction (UUO) is a common model of EMT of tubular cells and subsequent tubulointerstitial fibrosis. In order to examine the role of miR-200 family members in tubulointerstitial fibrosis, their expression was investigated in the kidneys of UUO mice. The expression of miR-200 family miRNAs was increased in a time-dependent manner, with induction of miR-200b most pronounced. To clarify the effect of miR-200b on tubulointerstitial fibrosis, we injected miR-200b precursor intravenously. A single injection of 0.5 nM miR-200b precursor was sufficient to inhibit the increase of collagen types I, III and fibronectin in obstructed kidneys, and amelioration of fibrosis was confirmed by observation of the kidneys with Azan staining. miR-200 family members have been previously shown to inhibit EMT by reducing the expression of ZEB-1 and ZEB-2 which are known repressors of E-cadherin. We demonstrated that expression of ZEB-1 and ZEB-2 was increased after ureter obstruction and that administration of the miR-200b precursor reversed this effect. In summary, these results indicate that miR-200 family is up-regulated after ureter obstruction, miR-200b being strongly induced, and that miR-200b ameliorates tubulointerstitial fibrosis in obstructed kidneys. We suggest that members of the miR-200 family, and miR-200b specifically, might constitute novel therapeutic targets in kidney disease.     

The renal cortical fibroblast in renal tubulointerstitial fibrosis

Renal cortical fibroblasts have key roles in mediating intercellular communication with neighboring/infiltrating cells and extracellular matrix (ECM) and maintenance of renal tissue architecture. They express a variety of cytokines, chemokines, growth factors and cell adhesion molecules, playing an active role in paracrine and autocrine interactions and regulating both fibrogenesis and the interstitial inflammatory response. They additionally have an endocrine function in the production of epoetin. Tubulointerstitial fibrosis, the common pathological consequence of renal injury, is characterized by the accumulation of extracellular matrix largely due to excessive production in parallel with reduced degradation, and activated fibroblasts characterized by a myofibroblastic phenotype. Fibroblasts in the kidney may derive from resident fibroblasts, from the circulating fibroblast population or from haemopoetic progenitor or stromal cells derived from the bone marrow. Cells exhibiting a myofibroblastic phenotype may derive from these sources and from tubular cells undergoing epithelial to mesenchymal transformation in response to renal injury. The number of interstitial myofibroblasts correlates closely with tubulointerstitial fibrosis and progressive renal failure. Hence inhibiting myofibroblast formation may be an effective strategy in attenuating the development of renal failure in kidney disease of diverse etiology.     

Snai1-induced partial epithelial–mesenchymal transition orchestrates p53–p21-mediated G2/M arrest in the progression of renal fibrosis via NF-κB-mediated inflammation

Renal fibrosis is the common feature of all progressive kidney diseases and exerts great burden on public health worldwide. The maladaptive repair mechanism of tubular epithelial cells, an important mediator of renal fibrogenesis, manifests with partial epithelial–mesenchymal transition (EMT) and cell cycle arrest. The aim of this study is to investigate the possible correlation between partial EMT and cell cycle arrest, and elucidate the underlying mechanism. We examined human kidney allograft samples with interstitial fibrosis and three mice renal fibrosis models, unilateral ureter obstruction (UUO), ischemia–reperfusion injury, and Adriamycin nephropathy. The partial EMT process and p53–p21 axis were elevated in both human allograft with interstitial fibrosis, as well as three mice renal fibrosis models, and showed a time-dependent increase as fibrosis progressed in the UUO model. Snai1 controlled the partial EMT process, and led to parallel changes in renal fibrosis, G2/M arrest, and inflammation. p53–p21 axis arrested cell cycle at G2/M, and prompted partial EMT and fibrosis together with inflammation. NF-κB inhibitor Bay11-7082 disrupted the reciprocal loop between Snai1-induced partial EMT and p53–p21-mediated G2/M arrest. We demonstrated the reciprocal loop between partial EMT and G2/M arrest of TECs during renal fibrogenesis and revealed NF-κB-mediated inflammatory response as the underlying mechanism. This study suggests that targeting NF-κB might be a plausible therapeutic strategy to disrupt the reciprocal loop between partial EMT and G2/M arrest, therefore alleviating renal fibrosis.Subject terms: Cell-cycle exit, Mechanisms of disease     

Galectin-3 preserves renal tubules and modulates extracellular matrix remodeling in progressive fibrosis

Renal tubular cell apoptosis is a critical detrimental event that leads to chronic kidney injury in association with renal fibrosis. The present study was designed to investigate the role of galectin-3 (Gal-3), an important regulator of multiple apoptotic pathways, in chronic kidney disease induced by unilateral ureteral obstruction (UUO). After UUO, Gal-3 expression significantly increased compared with basal levels reaching a peak increase of 95-fold by day 7. Upregulated Gal-3 is predominantly tubular at early time points after UUO but shifts to interstitial cells as the injury progresses. On day 14, there was a significant increase in TdT-mediated dUTP nick end labeling-positive cells (129%) and cytochrome c release (29%), and a decrease in BrdU-positive cells (62%) in Gal-3-deficient compared with wild-type mice. The degree of renal damage was more extensive in Gal-3-deficient mice at days 14 and 21, 35 and 21% increase in total collagen, respectively. Despite more severe fibrosis, myofibroblasts were significantly decreased by 58% on day 14 in the Gal-3-deficient compared with wild-type mice. There was also a corresponding 80% decrease in extracellular matrix synthesis in Gal-3-deficient compared with wild-type mice. Endo180 is a recently recognized receptor for intracellular collagen degradation that is expressed by interstitial cells during renal fibrogenesis. Endo180 expression was significantly decreased by greater than 50% in Gal-3-deficient compared with wild-type mice. Taken together, these results suggested that Gal-3 not only protects renal tubules from chronic injury by limiting apoptosis but that it may lead to enhanced matrix remodeling and fibrosis attenuation.     

Validation of highly selective sphingosine kinase 2 inhibitors SLM6031434 and HWG-35D as effective anti-fibrotic treatment options in a mouse model of tubulointerstitial fibrosis

Renal fibrosis is characterized by chronic inflammation and excessive accumulation of extracellular matrix and progressively leads to functional insufficiency and even total loss of kidney function. In this study we investigated the anti-fibrotic potential of two highly selective and potent SK2 inhibitors, SLM6031434 and HWG-35D, in unilateral ureter obstruction (UUO), a model for progressive renal fibrosis, in mice. In both cases, treatment with SLM6031434 or HWG-35D resulted in an attenuated fibrotic response to UUO in comparison to vehicle-treated mice as demonstrated by reduced collagen accumulation and a decreased expression of collagen-1 (Col1), fibronectin-1 (FN-1), connective tissue growth factor (CTGF), and α-smooth muscle actin (α-SMA). Similar to our previous study in Sphk2^−/− mice, we found an increased protein expression of Smad7, a negative regulator of the pro-fibrotic TGFβ/Smad signalling cascade, accompanied by a strong accumulation of sphingosine in SK2 inhibitor-treated kidneys. Treatment of primary renal fibroblasts with SLM6031434 or HWG-35D dose-dependently increased Smad7 expression and ameliorated the expression of Col1, FN-1 and CTGF.In summary, these data prove the anti-fibrotic potential of SK2 inhibition in a mouse model of renal fibrosis, thereby validating SK2 as pharmacological target for the treatment of fibrosis in chronic kidney disease.     

Overexpression of Heme Oxygenase-1 Prevents Renal Interstitial Inflammation and Fibrosis Induced by Unilateral Ureter Obstruction

Renal fibrosis plays an important role in the onset and progression of chronic kidney diseases. Many studies have demonstrated that heme oxygenase-1 (HO-1) is involved in diverse biological processes as a cytoprotective molecule, including anti-inflammatory, anti-oxidant, anti-apoptotic, antiproliferative, and immunomodulatory effects. However, the mechanisms of HO-1 prevention in renal interstitial fibrosis remain unknown. In this study, HO-1 transgenic (TG) mice were employed to investigate the effect of HO-1 on renal fibrosis using a unilateral ureter obstruction (UUO) model and to explore the potential mechanisms. We found that HO-1 was adaptively upregulated in kidneys of both TG and wild type (WT) mice after UUO. The levels of HO-1 mRNA and protein were increased in TG mice compared with WT mice under normal conditions. HO-1 expression was further enhanced after UUO and remained high during the entire experimental process. Renal interstitial fibrosis in the TG group was significantly attenuated compared with that in the WT group after UUO. Moreover, overexpression of HO-1 inhibited the loss of peritubular capillaries. In addition, UUO-induced activation and proliferation of myofibroblasts were suppressed by HO-1 overexpression. Furthermore, HO-1 restrained tubulointerstitial infiltration of macrophages and regulated the secretion of inflammatory cytokines in UUO mice. We also found that high expression of HO-1 inhibited reactivation of Wnt/β-catenin signaling, which could play a crucial role in attenuating renal fibrosis. In conclusion, these data suggest that HO-1 prevents renal tubulointerstitial fibrosis possibly by regulating the inflammatory response and Wnt/β-catenin signaling. This study provides evidence that augmentation of HO-1 levels may be a therapeutic strategy against renal interstitial fibrosis.     

Akt2 Is Involved in Loss of Epithelial Cells and Renal Fibrosis following Unilateral Ureteral Obstruction

Obstructive nephropathy is an aggressive form of chronic kidney disease (CKD), which is characterized by an epithelial-to-mesenchymal transition (EMT) and interstitial fibrosis. However, the molecular mechanisms of EMT and fibrosis are complex and not fully understood. In this study, we investigated the contribution of Akt2 to experimental renal EMT and fibrosis using the well-established model of unilateral ureteral obstruction (UUO). We found that Akt2 and phosphor (p)-Akt protein levels were increased in the obstructed kidneys. UUO induced activation of transforming growth factor-β1 (TGF-β1) signaling. Importantly, knockout of Akt2 suppressed UUO-induced EMT, kidney fibrosis, increased GSK3β activity, and decreased expression of Snail and β-catenin. Inhibition of GSK3β with LiCl (the inhibitor of GSK3β) increased the expression of Snail and β-catenin in cultured kidney epithelial cells. Our findings suggest that Akt2 partially contributes to interstitial fibrosis following UUO and that inhibition of this signaling pathway may provide a novel approach of prevent progression of renal fibrosis.     

The AMPK Agonist AICAR Inhibits TGF-β1 Induced Activation of Kidney Myofibroblasts

Activation of interstitial myofibroblasts and excessive production of extracellular matrix proteins are common pathways that contribute to chronic kidney disease. In a number of tissues, AMP-activated kinase (AMPK) activation has been shown to inhibit fibrosis. Here, we examined the inhibitory effect of the AMPK activator, 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR), on renal fibrosis in vivo and TGF-β1-induced renal fibroblasts activation in vitro. A unilateral ureteral obstruction (UUO) model was induced in male BALB/c mice. Mice with UUO were administered AICAR (500 mg/Kg/day) or saline intraperitoneally 1 day before UUO surgery and daily thereafter. Both kidneys were harvested 7 days after surgery for further analysis. For the in vitro studies, NRK-49F rat fibroblasts were pre-incubated with AICAR before TGF-β1 stimulation. The inhibitory effects of AICAR on signaling pathways down-stream of TGF-β1 were analyzed. In UUO model mice, administration of AICAR attenuated extracellular matrix protein deposition and the expression of α-smooth muscle actin (α-SMA), type I collagen and fibronectin. Pre-incubation of NRK-49F cells with AICAR inhibited TGF-β1-induced myofibroblast activation. Silencing of AMPKα1 by siRNA or by blocking AMPK activation with Compound C diminished the inhibitory effect of AICAR. Moreover, the inhibitory effects of AICAR on TGF-β1-mediated myofibroblast activation were associated with down-regulation of ERK 1/2 and STAT3. Our results suggest that AICAR reduces tubulointerstitial fibrosis in UUO mice and inhibits TGF-β1-induced kidney myofibroblast activation. AMPK activation by AICAR may have therapeutic potential for the treatment of renal tubulointerstitial fibrosis.     

Bortezomib attenuates renal interstitial fibrosis in kidney transplantation via regulating the EMT induced by TNF‐α‐Smurf1‐Akt‐mTOR‐P70S6K pathway

Allograft interstitial fibrosis was characterized by massive extracellular matrix deposition caused by activated fibroblasts and myofibroblasts. Epithelial‐mesenchymal transition (EMT) is recognized as an important source of myofibroblasts contributing to the pathogenesis of allograft interstitial fibrosis. Smad ubiquitination regulatory factor 1 (Smurf1) has been recently reported to be involved in the progression of EMT. Our study was to detect the effect of Bortezomib and Smurf1 in the EMT and allograft interstitial fibrosis. Biomarkers of EMT, as well as Smurf1, were examined in human proximal tubular epithelial cells (HK‐2) treated with tumour necrosis factor‐alpha (TNF‐α) in various doses or at various time points by Western Blotting or qRT‐PCR. We knockdown or overexpressed Smurf1 in HK‐2 cells. Furthermore, rat renal transplant model was established and intervened by Bortezomib. Allograft tissues from human and rats were also collected and prepared for HE, Masson's trichrome, immunohistochemical staining and western blotting assays. As a result, we found that TNF‐α significantly promoted the development of EMT in a time‐dependent and dose‐dependent manner through Smurf1/Akt/mTOR/P70S6K signalling pathway. More importantly, Bortezomib alleviated the progression of EMT and allograft interstitial fibrosis in vivo and in vitro by inhibiting the production of TNF‐α and expression of Smurf1. In conclusion, Smurf1 plays a critical role in the development of EMT induced by TNF‐α. Bortezomib can attenuate the Sumrf1‐mediated progression of EMT and renal allograft interstitial fibrosis, which could be suggested as a novel choice for the prevention and treatment of renal allograft interstitial fibrosis.     

ICAM-1 interactions in the renal interstitium: a novel activator of fibroblasts during nephritis.

Chronic renal diseases often degenerate towards end-stage failure, requiring replacement renal therapy. The progressive decline of such diseases is a highly complex, multi-factorial process, which is poorly understood. Indeed, not all chronic conditions take on a progressive course, some may recover to regain normal function, while others may remain functionally impaired yet stable. The structural features of progressive decline, however, show common histological features, despite the diverse nature of the primary injury. These aberrant structural alterations are characterised essentially by a dramatic expansion of the tubulointerstitium, with accompanying tubular atrophy, resulting from interstitial fibrosis. These changes are thought to be a uniform response to prolonged inflammation which may originate in the glomerulus, the vasculature or the interstitial space (Strutz et al., 1995). A histomorphometric analysis of renal diseases, initially performed by Risdon et al. (1968), and supported by Bohle et al. (1987) and others (Eknoyan et al., 1990), revealed that the severity of abnormal glomerular pathology did not always correlate directly with impaired renal function. The extent of interstitial inflammation and the degree of interstitial fibrosis, however, were both shown to be more accurate predictors of renal function (Bohle et al., 1992). Furthermore there was a high probability of irreversible functional decline, in the presence of interstitial fibrotic lesions and tubular atrophy. Interstitial fibrosis is therefore considered an important histological marker for end stage renal failure, and is believed to be functionally more significant than primary changes within the glomerulus. In most tissues, resident fibroblasts are believed to be the cells principally responsible for the synthesis and breakdown of extracellular matrix (ECM) within connective tissues. Indeed in fibrotic diseases of lung and skin, the resident fibroblast has been identified as the most important cell responsible for the abnormal deposition of ECM components during the disease process (Phan et al., 1985). In the kidney, there are probably several sources of matrix components during fibrosis including tubular epithelial cells, inflammatory macrophages (Vaage and Linbland, 1990) as well as interstitial fibroblasts. Although the precise cellular source of the bulk of this matrix requires clarification, there is mounting evidence supporting a significant contribution from resident or infiltrating fibroblasts (Rodemann and Muller, 1990, 1991a,b; Strutz and Muller, 1995).     

Effect of beraprost sodium (BPS) in a new rat partial unilateral ureteral obstruction model

Unilateral ureteral obstruction (UUO) is a representative model for investigating the common mechanism of decreasing renal function in chronic renal failure. In this study, we present a new partial UUO model in adult rats and evaluated the effect of beraprost sodium (BPS: stable prostaglandin I₂ (PGI₂) analog). We could make reproductive and uniform partial UUO by ligating the left ureter together with a 0.5 mm diameter stainless steel wire with nylon thread, and withdrawing the stainless wire. One week later, the ureteral obstruction was released. After 3 weeks from the release of UUO, all animals of control group, without BPS administration, developed basophilic degeneration of tubular epithelium, tubular dilatation and interstitial fibrosis. The areas of tubular degeneration and fibrosis were significantly reduced in the BPS group, orally administered BPS 300 μg/kg twice a day from the next day of the release of obstruction, than in control group.In conclusion, we can established the adult rat partial UUO-release model and revealed that BPS can inhibit renal tubular damage and tubulointerstitial fibrosis.     

兔慢性肾功能衰竭模型的建立与评价

目的建立兔慢性肾功能衰竭模型,为干细胞移植治疗和相关研究奠定基础。方法普通级大耳白兔随机分为正常对照组和单侧输尿管结扎（unilateral ureteral obstruction,UUO）组。UUO组于输尿管结扎后2、4、6、8周进行血生化肾功能指标检测,并取肾组织观察肾脏病理学改变,通过SPECT动态观察肾小球滤过率的变化,采用免疫组织化学方法观察肾组织转化生长因子-β1（TGF-β1）的表达情况。结果①UUO组术后第2周,出现明显的血肌酐升高,尿素氮术后第8周开始升高（P〈0.01）。②UUO组术后第4周,肾脏组织出现了早期间质纤维化的病理改变,术后第8周肾小球开始出现硬化,间质纤维化明显,皮质明显变薄。术后第12周,肾小球硬化比例增加,肾小管玻璃样变性,间质纤维化进一步加重（P〈0.05）。③SPECT动态观察肾小球滤过率,UUO组第4周GFR值比正常对照组降低,到第8周时,GFR值进一步下降,结扎侧肾脏功能降低甚至丧失。④免疫组织化学染色显示,TGF-β1在术后第4、8、12周均明显增强,并且各时间点表达均有显著差异（P〈0.05）。结论单侧输尿管结扎法成功制作比较稳定的慢性肾功能不全模型,UUO后第8周符合肾脏间质纤维化模型标准。