Long Zheng

Baicalin Ameliorates H2O2 Induced Cytotoxicity in HK-2 Cells through the Inhibition of ER Stress and the Activation of Nrf2 Signaling

Renal ischemia-reperfusion injury plays a key role in renal transplantation and greatly affects the outcome of allograft. Our previous study proved that Baicalin, a flavonoid glycoside isolated from Scutellaria baicalensis, protects kidney from ischemia-reperfusion injury. This study aimed to study the underlying mechanism in vitro. Human renal proximal tubular epithelial cell line HK-2 cells were stimulated by H2O2 with and without Baicalin pretreatment. The cell viability, apoptosis and oxidative stress level were measured. The expression of endoplasmic reticulum (ER) stress hallmarks, such as binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP), were analyzed by western blot and real-time PCR. NF-E2-related factor 2 (Nrf2) expression was also measured. In the H2O2 group, cell viability decreased and cell apoptosis increased. Reactive Oxygen Species (ROS) and Glutathione/Oxidized Glutathione (GSH/GSSG) analysis revealed increased oxidative stress. ER stress and Nrf2 signaling also increased. Baicalin pretreatment ameliorated H2O2-induced cytotoxicity, reduced oxidative stress and ER stress and further activated the anti-oxidative Nrf2 signaling pathway. The inducer of ER stress and the inhibitor of Nrf2 abrogated the protective effects, while the inhibitor of ER stress and the inducer of Nrf2 did not improve the outcome. This study revealed that Baicalin pretreatment serves a protective role against H2O2-induced cytotoxicity in HK-2 cells, where the inhibition of ER stress and the activation of downstream Nrf2 signaling are involved.

A novel cytoprotective peptide protects mesenchymal stem cells against mitochondrial dysfunction and apoptosis induced by starvation via Nrf2/Sirt3/FoxO3a pathway

BackgroundMesenchymal stem cell (MSC) has been widely explored in the past decade as a cell-based treatment for various diseases. However, poor survival of adaptively transferred MSCs limits their clinical therapeutic potentials, which is largely ascribed to the nutrient starvation. In this study, we determined whether a novel kidney protective peptide CHBP could protect MSCs against starvation and invested the underlying mechanisms.MethodsMSCs were subjected to serum deprivation and CHBP of graded concentrations was administered. Cell viability and apoptosis were detected by CCK-8, Annexin V/PI assay and Hoechst staining. ROS generation, mitochondrial membrane potential indicated by JC-1 and mitochondrial mass were measured by flow cytometry. The location of cytochrome c within cells was observed under fluorescence microscopy. Expressions of Nrf2, Sirt3, and FoxO3a were analyzed by western blot. In addition, preconditioning MSCs with CHBP was applied to test the possible protection against starvation. Finally, the effect of CHBP on the differentiation and self-renewal capacity of MSCs was also examined.ResultsCHBP improved cell viability and suppressed apoptosis in a dose dependent manner. Starvation resulted in the mitochondrial dysfunction and treatment of CHBP could alleviate mitochondrial stress by diminishing oxidative injury of ROS, restoring mitochondrial membrane potential and maintaining mitochondrial membrane integrity. Importantly, Nrf2/Sirt3/FoxO3a pathway was activated by CHBP and Sirt3 knockdown partially abolished the protection of CHBP. Moreover, MSCs pretreated with CHBP were more resistant to starvation. Under normal condition, CHBP exerted little effects on the differential and self-renewal capacity of MSCs.ConclusionsThe present study demonstrated the efficient protection of CHBP upon MSCs against starvation-induced mitochondrial dysfunction and apoptosis and indicated possible involvement of Nrf2/Sirt3/FoxO3a pathway in the protective effect.

The mTOR signal regulates myeloid-derived suppressor cells differentiation and immunosuppressive function in acute kidney injury

The mammalian target of rapamycin (mTOR) signal controls innate and adaptive immune response in multiple immunoregulatory contexts. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid cells of potent immunosuppressive capacity. In this study, we aimed to investigate the role of MDSCs in the protection of acute kidney injury (AKI) and the regulation of mTOR signal on MDSCs protective role in this context. In mice AKI model, rapamycin administration was associated with improved renal function, restored histological damage and decreased CD4+ and CD8+ T-cell infiltration in kidney tissue. MDSCs, especially CD11b+Ly6G+Ly6Clow G-MDSCs were recruited to the injured kidney following the interaction of CXCL1, CXCL2 and their receptor CXCR2 after inhibiting mTOR signal with rapamycin treatment. The adoptive transfer of rapamycin-treated MDSCs into the mice with AKI significantly improved the renal function, ameliorated histologic damages and limited the infiltration of T cells in kidney tissue. In addition, the expression of pro-inflammatory cytokines IL-1β and IFN-γ mRNA was downregulated while the expression of TGF-β1 and Foxp3 mRNA was upregulated in kidney tissue after transferring rapamycin-treated MDSCs. Adoptive transfer of rapamycin-treated MDSCs also downregulated the serum levels of IL-1β, IL-6 and IFN-γ and upregulated the serum levels of TGF-β1 compared with the IR group and PBS-treated MDSC group. In in vitro study, inhibiting mTOR signal regulated the induction of MDSC towards the CD11b+Ly6G+Ly6Clow G-MDSC subset. The ability to suppress T-cell proliferation of both bone marrow–derived CD11b+Ly6G+Ly6Clow G-MDSCs and CD11b+Ly6G-Ly6Chigh M-MDSCs was enhanced by mTOR signal inhibition via upregulating the expression of Arginase-1 and iNOS. Accordingly, both G-MDSCs and M-MDSCs presented downregulated runx1 gene expression after rapamycin treatment. Taken together, our results demonstrated that MDSCs ameliorated AKI and the protective effect was enhanced by mTOR signal inhibition via promoting MDSCs recruitment, regulating the induction of MDSCs and strengthening their immunosuppressive activity.

Resveratrol Alleviates Inflammatory Responses and Oxidative Stress in Rat Kidney Ischemia-Reperfusion Injury and H 2 O 2 -Induced NRK-52E Cells via the Nrf2/TLR4/NF-κB Pathway

Background: Ischemia-reperfusion injury (IRI) is one of the major causes of postoperative renal allograft dysfunction, which is mainly the result of proinflammatory reactions including inflammatory responses, oxidative stress, and metabolic disorders. Resveratrol (RSV) plays an important role in protecting various organs in IRI because it reduces oxidative stress, lessens the inflammatory response, and exerts anti-apoptotic effects. The aim of this study was to demonstrate the renoprotective effect of RSV in inhibiting inflammatory responses, reducing oxidative stress, and decreasing cell apoptosis in vivo and in vitro. Methods: RSV was administered before renal ischemia and H2O2 induction. Serum and kidneys were harvested 24 h after reperfusion and NRK-52E cells were collected 4 h after H2O2 stimulation. Serum creatinine and blood urea nitrogen were used to assess renal function. Hematoxylin and eosin staining was performed to assess histological injury. Quantitative real-time PCR and enzyme-linked immunosorbent assay were used to assess proinflammatory cytokine expression. Oxidative stress–related proteins, such as Nrf2 and TLR4, were evaluated by western blot. Terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end labeling assay was used to detect apoptotic cells in tissues, and western blot was used to evaluate the expression of caspase-3, -8, and -9 in this study. Results: RSV inhibited inflammatory responses and improved renal function after renal IRI. Additionally, RSV decreased oxidative stress and reduced cell apoptosis by upregulating Nrf2 expression, downregulating the TLR4/NF-κB signaling pathway, and by decreasing caspase-3 activity and caspase cascades. Conclusion: Our study demonstrated the mechanisms underlying RSV renoprotection. We found that RSV exerts its greatest effects by blocking inflammatory responses, lowering oxidative stress, and reducing apoptosis via the Nrf2/TLR4/NF-κB pathway.

Erythropoietin protects against rhabdomyolysis-induced acute kidney injury by modulating macrophage polarization

Erythropoietin (EPO) is a well-known hormone that is clinically used for the treatment of anemia. Very recently, an increasing body of evidence showed that EPO could still regulate bioactivities of macrophages. However, the details about the immunomodulatory effect of EPO on macrophages are not fully delineated, particularly in the setting of renal damages. Therefore, in the present study, we determined whether EPO could exert an impact on the dynamics of macrophages in a well-established model of rhabdomyolysis-induced acute kidney injury and explored the potential mechanisms. EPO was found to ameliorate kidney injuries by reducing macrophages recruitment and promoting phenotype switch toward M2 macrophages in vivo. It was also confirmed that EPO could directly suppress pro-inflammatory responses of M1 macrophages and promote M2 marker expression in vitro. Data indicated the possible involvement of Jak2/STAT3/STAT6 pathway in the augmentation of EPO on M2 polarization. These results improved the understanding of the immunoregulatory capacity of EPO on macrophages, which might optimize the therapeutic modalities of EPO.

Endothelin Receptor Down-Regulation Mediated Ligand Regulation Mechanisms Protect Against Cellular Hypoxia Injury in Rat Vascular Endothelial Cells

Objective: Investigation of the effect of endothelin receptor A (ETaR)-targeting small interfering RNA (siRNA) on rat vascular endothelial cellular hypoxia injury, as well as its underlying mechanism. Methods: An in vitro rat vascular smooth muscle cells - endothelial cells co-culture model was established and transfected with ETaR siRNA before hypoxia treatment. Cell culture supernatant, cellular protein and RNA were collected and examined at 0.5hrs, 1hrs, 2hrs, 4hrs, 8hrs, 16hrs, 24hrs and 48hrs of hypoxia with 1% oxygen. The time point at which the best silencing effect was achieved was chosen, eNOS inhibitor L-NAME was added, and post hypoxia cell culture supernatant, cellular protein and RNA was collected for further examination. Results: After hypoxic treatment, endothelial-1 (ET-1) and ETaR expression levels gradually increased as oxygen deprivation extended. ET-1 and ETaR expression levels were significantly lower in the ETaR siRNA group compared with the Hypoxia group (P<0.001). Such difference peaked at 4hrs of hypoxia. ELISA examination of cell culture supernatant revealed that the amount of ET-1 and TGF-βin the ETaR siRNA group were significantly lower compared to the Hypoxia group at all times, while the amount of NO and eNOS was higher. After 4 hrs of hypoxia, Smad2, Smad3, HIF-1, TNF-α, IFN-γ, IL-6, MCP-1, NF-κb, ET-1 and ANG II mRNA expression in endothelial cells and ETaR mRNA expression in A-10 cells of the ETaR siRNA group were lower than those of the Hypoxia siRNA group, while such results were much higher in the L-NAME group. Western Blot results showed lower expression of ETaR in the ETaR siRNA group compared with the hypoxia and negative siRNA groups, as well as significantly higher ETaR expression in the L-NAME group compared with the ETaR siRNA group. PI3K and p-AKT expression levels were mildly elevated after mild oxygen deprivation, and ETaR siRNA was able to enhance such elevation induced by hypoxia. In the L-NAME group, PI3K and p-AKT expression was much higher than the ETaR siRNA group. PKG and sGC expression levels significantly descended after mild oxygen deprivation. While such levels were higher in the ETaR siRNA group, compared with the hypoxia and negative siRNA groups, the L-NAME group had lower levels of PKG and sGC compared with the ETaR siRNA group. Conclusion: ETaR siRNA is capable of down-regulating the expression of inflammatory and transcription factors among endothelial cells treated with hypoxia. Down-regulation of ET-1 is triggered by altered nucleus transcription factor activity through the sGC/PKG signal pathway, and results in enhanced eNOS activity through the PI3K/Akt signal pathway. We suspect this to be the mechanism of the protective effect of ETaR siRNA.

Baicalin ameliorates renal fibrosis via inhibition of transforming growth factor β1 production and downstream signal transduction

Previous studies have demonstrated the potential antifibrotic effects of baicalin in vitro, via examination of 21 compounds isolated from plants. However, its biological activity and underlying mechanisms of action in vivo remain to be elucidated. The present study aimed to evaluate the effect of baicalin on renal fibrosis in vivo, and the potential signaling pathways involved. A unilateral ureteral obstruction (UUO)-induced renal fibrosis model was established using Sprague-Dawley rats. Baicalin was administrated intraperitoneally every 2 days for 10 days. The degree of renal damage and fibrosis was investigated by histological assessment, and detection of fibronectin and collagen I mRNA expression levels. Epithelial-mesenchymal transition (EMT) markers, transforming growth factor-β1 (TGF-β1) levels and downstream phosphorylation of mothers against decapentaplegic 2/3 (Smad2/3) were examined in vivo and in an NRK-52E rat renal tubular cell line in vitro. Baicalin was demonstrated to markedly ameliorate renal fibrosis and suppress EMT, as evidenced by reduced interstitial collagen accumulation, decreased fibronectin and collagen I mRNA expression levels, upregulation of N- and E-cadherin expression levels, and downregulation of α-smooth muscle actin and vimentin expression. Furthermore, baicalin decreased TGF-β1 expression levels in serum and kidney tissue following UUO, and suppressed Smad2/3 phosphorylation in rat kidney tissue. In vitro studies identified that baicalin may inhibit the phosphorylation of Smad2/3 under the same TGF-β1 concentration. In conclusion, baicalin may protect against renal fibrosis, potentially via inhibition of TGF-β1 production and its downstream signal transduction.

Protective effects of cyclic helix B peptide on aristolochic acid induced acute kidney injury

BACKGROUND Aristolochic acid (AA) injuries remain a serious condition associated with acute renal dysfunction. Herein, the effect and mechanism of a novel tissue protective peptide, cyclic helical B-peptide (CHBP) derived from erythropoietin, were investigated in a mice model. METHODS Mice were randomly divided into four groups, receiving the following treatments (1: saline; 2: AA 10mg/kg; 3: AA 10mg/kg +CHBP 4nmol/kg; 4: AA 10mg/kg +CHBP 8nmol/kg). RESULTS Blood urea nitrogen and serum creatinine was increased by AA but decreased by CHBP in a dose-dependent fashion. CHBP also significantly improved renal tubular injury and inflammatory infiltration, which was gradually increased by AA. Apoptotic cells, infiltrating inflammatory cells, and active caspase-3+ cells were greatly reduced by CHBP. In addition, CHBP inhibited caspase-3, 9 and improved bcl-2, bcl-xl protein expression in vivo. CONCLUSION Taken together, we demonstrated, for the first time, that CHBP effectively improved renal function and tissue damage caused by AA, which maybe through reducing caspase-3 activation, apoptosis, and inflammation.

GC/MS-based urine metabolomics analysis of renal allograft recipients with acute rejection

BackgroundAcute renal allograft rejection is a common complication after renal transplantation that often leads to chronic rejection and ultimate graft loss. While renal allograft biopsy remains the gold standard for diagnosis of acute rejection, the possibility of biopsy-associated complications cannot be overlooked. The development of noninvasive methods for accurate detection of acute renal allograft rejection is thus of significant clinical importance.MethodsGas chromatography–mass spectrometry (GC/MS) was employed for analysis of urine metabolites in 15 renal allograft recipients with acute rejection and 15 stable renal transplant recipients. Partial least squares (PLS) regression and leave-one-out analyses were performed to ascertain whether the metabolites identified could be exploited to distinguish acute rejection from stable groups as well as their sensitivity and specificity.ResultsOverall, 14 metabolites were significantly altered in the acute rejection group (11 and 3 metabolites displayed higher and lower levels, respectively) relative to the stable transplant group. Data from PLS and leave-one-out analyses revealed that the differential metabolites identified not only distinguished acute rejection from stable transplant recipients but also showed high sensitivity and specificity for diagnosis of renal allograft recipients with acute rejection.ConclusionUrine metabolites identified with GC/MS can effectively distinguish acute rejection from stable transplant recipients, supporting the potential utility of metabolome analysis in non-invasive diagnosis of acute rejection.

Transplantation of Telocytes Attenuates Unilateral Ureter Obstruction-Induced Renal Fibrosis in Rats

Background/Aims: Previous studies imply that telocytes may have a protective effect on fibrosis in various organs, including the liver, colon, and heart. The effect of telocytes on renal fibrosis remains unknown. Herein, this study was designed to investigate the effect of telocytes on renal fibrosis and the potential mechanisms involved. Methods: In a unilateral ureteral obstruction (UUO)-induced renal fibrosis model, telocytes were injected via the tail vein every other day for 10 days. The degree of renal damage and fibrosis was determined using histological assessment. The expression of collagen I, fibronectin, epithelial-mesenchymal transition markers, and Smad2/3 phosphorylation was examined by western blot analyses. Real-time PCR and enzyme-linked immunosorbent assay were performed in vivo to detect the levels of transforming growth factor (TGF)-β1 and various growth factors. Results: Telocytes attenuated renal fibrosis, as evidenced by reduced interstitial collagen accumulation, decreased expression of fibronectin and collagen I, upregulation of E-cadherin, and downregulation of α-smooth muscle actin. Furthermore, telocytes decreased serum TGF-β1 levels, suppressed Smad2/3 phosphorylation, and increased the expression of hepatocyte growth factor (HGF) in rat kidney tissue following UUO. Blockage of HGF counteracted the protective effect of telocytes on UUO-treated kidneys. Through the detection of HGF mRNA levels in vitro, we found that telocytes had no effect on HGF expression compared with renal fibroblasts. Conclusion: Telocytes attenuated UUO-induced renal fibrosis in rats, likely through enhancing the expression of HGF in an indirect manner.