Anisodamine ameliorates ischemia/reperfusion-induced renal injury in rats through activation of the extracellular signal-regulated kinase (ERK) pathway and anti-apoptotic effect.

Anisodamine exerts significant protective effect on ischemia/reperfusion (I/R) injury in various organs. However, little is known about the mechanisms of anisodamine in renal I/R injury. Activation of extracellular regulated protein kinases (ERK) pathway promotes the repair of renal epithelial cells following oxidant injury. The present study investigated whether the renoprotective role of anisodamine against renal I/R injury in rats was associated with the activation of ERK signaling pathway. Male Sprague-Dawley (SD) rats were separated into the following groups: Sham-operated group, I/R group, anisodamine-treated group, PD98059 (MEK-1/ERK inhibitor)-treated group and anisodamine plus PD98059-treated group. A rat model of renal I/R was established by excising the right kidney and then clamping the left renal pedicle for 45 min followed by reperfusion for 24 h. Serum and renal tissue samples were obtained for assays of the associated morphological, molecular and biochemical parameters. Treatment with anisodamine ameliorated renal I/R injury, as evidenced by improvements of renal histology and kidney function, a decrease in paller's score and apoptosis index. Anisodamine also upregulated the phosphorylation levels of ERK1/2 and its downstream targets, including 90 ribosomal S6 kinase (p90rsk) and Bad, as well as the expression of antiapoptotic Bcl-2 protein, downregulated the expression levels of proapoptotic proteins Bax and cleaved-caspase-3, whereas these effects were greatly abolished by administration of PD98059. In conclusion, the results suggest that anisodamine prevents renal I/R injury in rats as a result of an activation of the ERK signaling pathway and anti-apoptotic properties.

Metformin attenuates renal interstitial fibrosis through upregulation of Deptor in unilateral ureteral obstruction in rats.

Renal interstitial fibrosis (RIF) is a common pathological process that accompanies chronic kidney disease (CKD) and that progresses to end-stage renal failure (ESRD). Accumulating evidence has revealed that persistent mammalian target of rapamycin (mTOR) activation in kidneys is closely associated with the occurrence and progression of CKD. The DEP domain-containing mTOR interacting protein (Deptor) is an endogenous negative regulator of mTOR. Metformin can attenuate renal fibrosis in an animal model of diabetic nephropathy. Previous studies demonstrated that metformin can attenuate renal fibrosis in several models of CKD. However, the precise mechanisms of this effect are not well understood. The present study aimed to examine the mechanism of action of metformin on unilateral ureteral obstruction (UUO)-induced RIF in rats in vivo. Sprague-Dawley rats were randomly divided into a sham-operated group, three UUO groups examined at different time points (3, 7 and 14 days after UUO surgery), and three metformin-treated groups, treated with three different concentrations of metformin. The metformin-treated groups were administered metformin orally every day for 14 consecutive days following surgery. The protein expression levels of Deptor, α-smooth muscle actin (α-SMA), phosphorylated (p-)mTOR, p-ribosomal protein S6 kinase (p-p70S6K) and CD68 were assessed. The present results suggested that, following UUO, there was a significant reduction of Deptor expression, and an increase in collagen deposition in the extracellular matrix over time, accompanied by an increased expression of several proteins including CD68, α-SMA, p-mTOR and p-p70S6K. Notably, metformin treatment reversed these effects. In conclusion, the present results suggested that metformin attenuated RIF of UUO rats, and the mechanism of action was found to be associated with the increase in Deptor expression and inhibition of the mTOR/p70S6K pathway in the kidneys of UUO rats.

Febuxostat inhibits TGF­ß1­induced epithelial­mesenchymal transition via downregulation of USAG­1 expression in Madin­Darby canine kidney cells in vitro.

Our previous study demonstrated that febuxostat, a xanthine oxidase inhibitor, can alleviate kidney dysfunction and ameliorate renal tubulointerstitial fibrosis in a rat unilateral ureteral obstruction (UUO) model; however, the underlying mechanisms remain unknown. Increasing evidence has revealed that epithelial­mesenchymal transition (EMT) is one of the key mechanisms mediating the progression of renal tubulointerstitial fibrosis in chronic kidney disease (CKD). Uterine sensitization­associated gene­1 (USAG­1), a kidney­specific bone morphogenetic protein antagonist, is involved in the development of numerous types of CKDs. The present study aimed to investigate the role of febuxostat in the process of EMT in Madin­Darby canine kidney (MDCK) cells in vitro. Western blotting, reverse transcription­semiquantitative polymerase chain reaction analysis and immunofluorescence staining were used to evaluate the expression levels of bone morphogenetic protein 7, USAG­1, α­smooth muscle actin (α­SMA) and E­cadherin, respectively. The results demonstrated that the expression of USAG­1 and α­SMA increased, and that of E­cadherin decreased significantly in MDCK cells following treatment with transforming growth factor­ß1 (TGF­ß1). The application of small interfering RNA­USAG­1 potently inhibited TGF­ß1­induced EMT. Subsequently, the effects of febuxostat on TGF­ß1­induced EMT was investigated. The results demonstrated that febuxostat downregulated the expression of USAG­1, and reversed TGF­ß1­induced EMT in MDCK cells. Furthermore, pretreatment with febuxostat significantly restored the decreased expression levels of phosphorylated Smad1/5/8 induced by TGF­ß1 in MDCK cells. The results of the present study suggested that USAG­1 may be involved in the EMT process of MDCK cells induced by TGF­ß1, and febuxostat inhibited EMT by activating the Smad1/5/8 signaling pathway via downregulating the expression of USAG­1 in MDCK cells.

Co-treatment with BEZ235 enhances chemosensitivity of A549/DDP cells to cisplatin via inhibition of PI3K/Akt/mTOR signaling and downregulation of ERCC1 expression.

The activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling and upregulation of excision repair cross complementation group 1 (ERCC1) are the two most important factors that confer resistance to cisplatin (DDP) therapy in non-small-cell lung cancer (NSCLC). Therefore, inhibition of the PI3K/Akt/mTOR signaling pathway and ERCC1 expression is a potential approach for the treatment of patients with advanced NSCLC. In the present study, whether combined treatment with DDP and BEZ235, a dual PI3K/mTOR inhibitor, could provide a synergistic antitumor effect in A549/DDP cells was investigated, and the possible mechanisms involved were explored. The half-maximal inhibitory concentration (IC50) values were calculated in A549/DDP cells. Synergistic interaction of BEZ235 and DDP was evaluated by combination index (CI) analysis. The levels of phosphorylated Akt (p-Akt), phosphorylated mTOR (p-mTOR), apoptosis-related proteins and ERCC1 were detected by western blot analysis. Apoptotic cells were quantified by flow cytometry and Hoechst 33342 staining. The migration and invasion abilities of A549/DDP cells were evaluated by wound healing and Transwell assays, respectively. It was observed that the dose reduction index (DRI) of BEZ235 was 13.82 and for DDP it was 13.58, and the CI of combination was <1 over a wide range of doses. In addition, the levels of p-Akt, p-mTOR and ERCC1 were significantly elevated by DDP treatment, and were reduced by co-administration of BEZ235 and DDP. Furthermore, the combination treatment significantly induced apoptotic cell death, decreased migration and invasion abilities compared with those treated with either BEZ235 or DDP alone. In conclusion, the combination of BEZ235 with DDP had synergistic antitumor effects in A549/DDP cells as reflected by reduced proliferation, increased apoptosis, and suppression of the migration and invasion abilities of A549/DDP cells, and the mechanism mediating these effects may be associated with the inhibition of PI3K/Akt/mTOR signaling and down-regulation of ERCC1 expression.

Role of artesunate in TGF­ß1­induced renal tubular epithelial­mesenchymal transdifferentiation in NRK­52E cells.

The implications of epithelial­mesenchymal transdifferentiation (EMT) have extended beyond the confines of renal fibrosis to renal tubulointerstitial fibrosis. It has been proposed that EMT may be one of the mechanisms involved in the pathogenesis of renal fibrosis. However, the underlying mechanisms remain unknown. Transforming growth factor (TGF)­ß1 is considered to be an important cytokine which regulates the transdifferentiation of tubular epithelial cells into myofibroblasts in renal tubulointerstitial fibrosis. In the present study, normal rat kidney tubular epithelial cells (NRK­52E) were treated for 48 h with TGF­ß1 (5 ng/ml) and different concentrations of artesunate (ART; 0.01, 0.1 and 1 µg/ml). Western blotting, reverse transcription­semi quantitative polymerase chain reaction analysis and immunofluorescence staining were used to evaluate the expression of bone morphogenetic protein (BMP)­7, uterine sensitization­associated gene (USAG)­1, E­cadherin, α­smooth muscle actin (α­SMA) and extracellular matrix collagen type I (Col I) mRNA. ART was able to attenuate renal injury in a unilateral ureteral obstruction model. However, its anti­fibrotic effect remains to be elucidated. In the present study, it was observed that ART was able to ameliorate the TGF­ß1­induced alterations in cellular morphology. In addition, ART inhibited the TGF­ß1­induced USAG­1 increase and the decrease in BMP­7. Treatment with ART markedly attenuated the TGF­ß1­induced upregulation of α­SMA and downregulation of E­cadherin. Additionally, ART was able to significantly attenuate the deposition of interstitial collagens, including Col I. The results of the present study further verified the therapeutic efficacy of ART in TGF­ß1­induced renal interstitial fibrosis. These findings indicated that ART may hold the potential to prevent chronic kidney diseases via the suppression of USAG­1 expression or by increasing BMP­7 expression.

Artesunate attenuates unilateral ureteral obstruction-induced renal fibrosis by regulating the expressions of bone morphogenetic protein-7 and uterine sensitization-associated gene-1 in rats.

PURPOSE: Kidney fibrosis is the most common final stage of progressive renal disease. Bone morphogenetic protein-7 (BMP-7) has been shown to be important in both preservation of kidney function and resistance to injury. Recently, it has been realized that uterine sensitization-associated gene-1 (USAG-1) functions as a kidney-specific BMP antagonist. Because of the reported anti-fibrotic effects of artesunate (Art), this study was designed to investigate the effects of Art on renal fibrosis in unilateral ureteral obstruction (UUO) rats and to explore the underlying mechanisms. METHODS: Thirty male Sprague-Dawley rats were randomly divided into five groups: Sham group, UUO group, low-dose Art-treated (Art-L), middle-dose Art-treated (Art-M), and high-dose Art-treated (Art-H) groups. The UUO rat model was established by ligating the left ureter. Fourteen days later, interstitial collagen deposition, expression of USAG-1, BMP-7, E-cadherin, α-smooth muscle actin (α-SMA), fibronectin (FN), collagen I, as well as the inflammatory infiltration levels in the kidneys were assessed. RESULTS: Art treatment significantly attenuated the deposition of interstitial collagens in the UUO rats' kidneys and exhibited the ability to improve renal function, followed by the up-regulated expression of BMP-7 and E-cadherin and the down-regulated expression of USAG-1 and α-SMA. In addition, increased macrophages infiltration in the kidneys of the UUO rats were also attenuated by the administration of Art. CONCLUSIONS: These results indicate that Art is able to improve the renal function decline and renal fibrosis induced by UUO, which may be associated with the up-regulation of BMP-7 and down-regulation of USAG-1. Accordingly, Art may become a potential preventive or therapeutic agent for chronic kidney diseases.

Febuxostat Prevents Renal Interstitial Fibrosis by the Activation of BMP-7 Signaling and Inhibition of USAG-1 Expression in Rats.

BACKGROUND: Renal interstitial fibrosis (RIF) is a common pathology associated with end-stage renal diseases. The activation of bone morphogenetic protein-7 (BMP-7)-Smad1/5/8 pathway seems to alleviate RIF. Uterine sensitization-associated gene-1 (USAG-1), a kidney-specific BMPs antagonist, is associated with the development and prognosis of several renal diseases. Febuxostat is a xanthine oxidase inhibitor that can attenuate the renal dysfunction of patients. The purpose of this study was to investigate the effects of febuxostat on renal fibrosis and to clarify the mechanisms underlying these effects. METHODS: Rats were randomly divided into 6 groups termed a sham-operated group, a unilateral ureteral obstruction (UUO) group, 3 doses of febuxostat groups (low, intermediate and high doses) and a sham group treated with high-dose febuxostat. After 14 days, renal function, relative kidney weight, accumulation of glycogen and collagens were examined by different methods. Expression of α-SMA, transforming growth factor-ß1 (TGF-ß1), BMP-7 and USAG-1 was detected by western blotting and RT-PCR, respectively. The phosphorylation level of Smad1/5/8 was also quantified by western blotting. RESULTS: The renal function was declined, and large amounts of glycogen and collagens were deposited in the kidneys of UUO rats compared with the rats in the sham group. Besides, expression of α-SMA and USAG-1 in these kidneys was elevated, and the TGF-ß1 was also activated, while the BMP-7-Smad1/5/8 pathway was inhibited. Febuxostat reversed the changes stated earlier, exhibiting protective effects on RIF induced by UUO. CONCLUSION: Febuxostat was able to attenuate RIF caused by UUO, which was associated with the activation of BMP-7-Smad1/5/8 pathway and the inhibition of USAG-1 expression in the kidneys of UUO rats.

Cardioprotective effect of betulinic Acid on myocardial ischemia reperfusion injury in rats.

Objectives. This study aims to investigate the effect of betulinic acid (BA) on myocardial ischemia reperfusion/injury in an open-chest anesthetized rat model. Methods. The model was induced by 30 minutes left anterior descending occlusion followed by 2 hours reperfusion. There are six groups in our present study: sham operation group, ischemia/reperfusion group, low-dosage BA group, medium-dosage BA group, high-dosage BA group, and fosinopril sodium group. Rats in the latter four groups were administrated with BA (50, 100, and 200 mg/kg, i.g.) or fosinopril sodium (10 mg/kg, i.g.) once a day for 7 days before operation, respectively. Rats in the former two groups were given the same volume of vehicle (0.5% CMC-Na, i.g.). During the operation, cardiac function was continuously monitored. Serum LDH and CK were measured with colorimetric assays. The expression of Bcl-2 and Bax and the apoptosis of cardiomyocytes were investigated with western blot and TUNEL assay, respectively. Results. Pretreatment with BA improved cardiac function and attenuated LDH and CK activities compared with IR group. Further investigation demonstrated that the expression of Bcl-2 and Bax and TUNEL assay was in line with the above results. Conclusion. BA may reduce the release of LDH and CK, prevent cardiomyocytes apoptosis, and eventually alleviate the extent of the myocardial ischemia/reperfusion injury.

Roles of MAPKAPK-2 and HSP27 in the reduction of renal ischemia-reperfusion injury by ischemic postconditioning in rats.

PURPOSE: Ischemic postconditioning is a procedure during which intermittent reperfusions are performed in the early phase of reperfusion to protect organs from ischemia/reperfusion injury. And in this study, we mainly investigated the injury-alleviative role of mitogen-activated protein kinase-activating protein kinase-2 (MAPKAPK-2) and heat shock protein 27 (HSP27) in renal ischemic reperfusion injury during the procedure of ischemic postconditioning. METHODS: Sprague-Dawley rats were randomly divided into four groups. The injury models were prepared by clipping the left renal pedicle of rats after ligating the right renal pedicle for 60 min. In the ischemic postconditioning group, sequential reperfusions were done for 10 s and another ischemia for 10 s for six cycles after kidney ischemia for 60 min. In addition, the specific inhibitor SB203580 was injected through caudal vein before ischemia. Serum creatinine, blood urea nitrogen and the expression of HSP27 and MAPKAPK-2 were detected 1, 3, 6 and 24 h later after reperfusion. Furthermore, phosphorylation of HSP27 and MAPKAPK-2 protein contents, histological changes and apoptosis were compared 24 h later after reperfusion. RESULTS: Our data showed that ischemic postconditioning attenuated the renal dysfunction and cell apoptosis induced by I/R and increased phosphorylation of MAPKAPK-2 and HSP27. The results indicated that ischemic postconditioning decreased apoptosis and improved renal function. CONCLUSIONS: Taken together, it is suggested that the renal protective effect may be related to the levels of HSP27 and MAPKAPK-2 activation.

Naloxone Postconditioning Alleviates Rat Myocardial Ischemia Reperfusion Injury by Inhibiting JNK Activity.

To investigate the alteration of c-Jun N-terminal kinase (JNK) activity after myocardial ischemia reperfusion injury (MIRI) and further explore the effect of naloxone postconditioning on MIRI. Forty male Sprague Dawley rats were randomly divided into five groups: sham operation (sham, n=8); ischemia reperfusion (IR, n=8); IR+naloxone 0.5 mg/kg (Nal L, n=8); IR+naloxone 1.0 mg/kg (Nal M, n=8); IR+naloxone 2.0 mg/kg (Nal H, n=8). Pathological changes of myocardial tissue were visualized by HE staining. The expression of p-JNK, and the apoptosis of cardiomyocytes were investigated with Western blotting and the TUNEL assay, respectively. Irregular arrangement and aberrant structure of myocardial fibers, cardiomyocytes with granular or vacuolar degeneration, and inflammatory cells infiltrating the myocardial interstitial regions characterized MIRI in the IR group. Signs of myocardial injury and inflammatory infiltration were less prominent in the Nal-treated groups. The expression of p-JNK in the sham group and in all Nal-treated groups was significantly lower than that in the IR group (p<0.01). The apoptosis index of cardiomyocytes in the IR group was significantly higher than in the sham group (p< 0.01). The apoptosis indices of cardiomyocytes in all Nal-treated groups were significantly reduced to 55.4%, 26.2%, and 27.6%, respectively, of the IR group (p< 0.01). This study revealed that Naloxone postconditioning before reperfusion inhibits p-JNK expression and decreases cell apoptosis, thus alleviating MIRI.

Protective effect of edaravone against renal ischemia/reperfusion injury and compared with ischemic postconditioning in rats.

The aim of this study is to clarify whether edaravone postconditioning had protective effect against renal ischemia/reperfusion injury and to compare the protective effect between ischemic postconditioning and edaravone postconditioning. Rats were subjected to 45 min ischemia followed by 24 h reperfusion. The rats were randomly assigned to seven groups: a sham-operated control group, an ischemia/reperfusion group, an ischemic postconditioning group, a normal saline vehicle postconditioning group and an edaravone postconditioning (1, 3, and 6 mg x kg(-1)) group. Renal function was assessed by serum creatinine and BUN concentration, while histological damage of renal tissue was assessed with HE staining. MDA content and SOD activity of renal tissue were determined. TUNEL staining was performed to analyze the apoptosis of the tubular epithelial cells, the protein level of Bcl-2 and Bax in renal tissue was examined by Western blotting. Compared to the ischemia/reperfusion group, edaravone postconditioning significantly decreased serum creatinine and BUN concentration, and ameliorated histological damage of renal tissue. MDA was less after 24 h reperfusion in the edaravone postconditioning group than that in the ischemia/reperfusion group, consistent with an increase in SOD activity. In addition, edaravone postconditioning decreased TUNEL-positive cells and Bax expression, and increased Bcl-2 expression. Results detected in the edaravone postconditioning group showed no significant difference from the ischemic postconditioning group. Edaravone administered during the last 3 min of ischemia, prior to reperfusion induces a pharmacological postconditioning in vivo against renal ischemia/reperfusion injury in rats. This protection is similar to that observed with ischemic postconditioning.