Yanqiu Wang

Epigallocatechin-3-Gallate Attenuates Oxidative Stress and Inflammation in Obstructive Nephropathy via NF-κB and Nrf2/HO-1 Signalling Pathway Regulation.

Oxidative stress and inflammation contribute importantly to the pathogenesis of chronic kidney disease (CKD). Epigallocatechin‐3‐gallate (EGCG), which is the most abundant and most active catechin polyphenol extracted from green tea, has been proved to have many bioactivities. In this study, the renoprotective effect of EGCG was evaluated in a widely used kidney disease model, the unilateral ureteral obstruction (UUO) mice model. After 14 days of EGCG administration, mean arterial blood pressure, body‐weight and obstructed kidney weight were measured. Levels of blood urea nitrogen (BUN) and creatinine (CR) and activities of glutamic–pyruvic transaminase (GPT) and lactate dehydrogenase (LDH) in serum were estimated as indicators of renal function. Periodic acid–Schiff (PAS) staining was performed to observe the pathological changes of the obstructed kidney. Antioxidant enzymes and pro‐inflammatory cytokine production were estimated to reflect the oxidative stress and inflammatory state in the obstructed kidney. Finally, the main proteins in the NF‐κB and Nrf2 signalling pathway and DNA binding activity of NF‐κB and Nrf2 were measured to investigate the effect of EGCG on these two pathways. The results demonstrated that EGCG could restore UUO‐induced kidney weight loss and renal dysfunction. In addition, UUO‐induced oxidative stress and inflammatory responses in the obstructed kidney were also prevented by EGCG. Furthermore, EGCG could induce both NF‐κB and Nrf2 nuclear translocation in the UUO kidney and promote heme oxygenase‐1 (HO‐1) production. These results indicated that the renoprotective effect of EGCG might be through its NF‐κB and Nrf2 signalling pathway regulations.

Epigallocatechin-3-gallate attenuates transforming growth factor-β1 induced epithelial-mesenchymal transition via Nrf2 regulation in renal tubular epithelial cells.

Transforming growth factor-β1 (TGF-β1) induced epithelial-mesenchymal transition (EMT) plays an important role in renal fibrotic process regulation. Epigallocatechin-3-gallate (EGCG) exerts a protective effect against acute renal damage through its anti-oxidative effect by activating the Nrf2 signaling pathway. This study aims to investigate whether EGCG prevents TGF-β1 induced EMT and whether this effect acts via the Nrf2-mediated suppression of TGF-β1 signaling. MTT was used for cytotoxicity of EGCG examination and Western blotting and immunofluorescence were used for protein expression analysis. Results showed that EGCG prevented TGF-β1 mediated EMT and Smad 2 and Smad 3 phosphorylation in a dose dependent manner in NRK-52E cells. In addition, EGCG increased Nrf2 nuclear accumulation. Overexpression of Nrf2 blocked the phosphorylation of Smad 2 and Smad 3 mediated by TGF-β1 and decreased protein expression of plasminogen activator inhibitor 1 (PAI-1) and α-smooth muscle actin (α-SMA). Furthermore, siRNA-mediated knockdown of Nrf2 gene completely blocked the effects of EGCG, indicated by the reduced expressions of type I collagen (Col-I) and α-SMA were restored. In summary, EGCG inhibits TGF-β1 induced EMT and fibrotic proteins expression by Nrf2 activation. This study reveals a possible underlying mechanism of the renal protective effects of EGCG, and may provide a potential candidate to renal fibrosis therapy.

Epigallocatechin-3-gallate attenuates unilateral ureteral obstruction-induced renal interstitial fibrosis in mice.

The severity of tubulointerstitial fibrosis is regarded as an important determinant of renal prognosis. Therapeutic strategies targeting tubulointerstitial fibrosis have been considered to have potential in the treatment of chronic kidney disease. This study aims to evaluate the protective effects of (-)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol, against renal interstitial fibrosis in mice. EGCG was administrated intraperitoneally for 14 days in a mouse model of unilateral ureteral obstruction (UUO). The results of our histological examination showed that EGCG alleviated glomerular and tubular injury and attenuated renal interstitial fibrosis in UUO mice. Furthermore, the inflammatory responses induced by UUO were inhibited, as represented by decreased macrophage infiltration and inflammatory cytokine production. Additionally, the expression of type I and III collagen in the kidney were reduced by EGCG, which indicated an inhibition of extracellular matrix accumulation. EGCG also caused an up-regulation in α-smooth muscle actin expression and a down-regulation in E-cadherin expression, indicating the inhibition of epithelial-to-mesenchymal transition. These changes were found to be in parallel with the decreased level of TGF-β1 and phosphorylated Smad. In conclusion, the present study demonstrates that EGCG could attenuate renal interstitial fibrosis in UUO mice, and this renoprotective effect might be associated with its effects of inflammatory responses alleviation and TGF-β/Smad signaling pathway inhibition.

HBx inhibits proliferation and induces apoptosis via Fas/FasL upregulation in rat renal tubular epithelial cells.

BACKGROUND The hepatitis B virus X protein (HBx) regulates numerous signaling pathways, including those that modulate apoptosis. However, previous research has indicated different effect of HBx on apoptosis, possibly because of differences in the experimental conditions and cell types. The purpose of this study was to investigate the mechanism of HBx-induced apoptosis of rat renal tubular epithelial (NRK-52E) cells. METHODS An HBx expression vector (pc-DNA3.1(+)-HBx) was used to transfect NRK-52E cells to establish an HBx overexpression model. One control group was not transfected and the other control group was transfected with plasmid lacking the HBx-encoding insert. Cell proliferation was measured using the MTT assay, and the rate of apoptosis was determined by flow cytometry and fluorescence microscopy. The expressions of Fas, FasL, Bcl-2 and Bax were determined by Western blotting and reverse transcription polymerase chain reaction, and the activity of caspase-8 was measured by spectrophotometry.
 RESULTS Transfection of NRK-52E cells with pc-DNA3.1(+)-HBx led to inhibition of proliferation and increased apoptosis relative to the controls. Transfected cells had increased mRNA and protein expression of Fas, FasL and Bax and decreased mRNA and protein expression of Bcl-2 relative to the controls. In addition, transfected cells had increased caspase-8 activity relative to the controls. CONCLUSIONS Our results suggest that HBx induces apoptosis in NRK-52E cells, at least in part through activation of the Fas/FasL pathway. The activation of caspase-8 appears to mediate the induction of apoptosis.

The role of CD44-hyaluronic acid interaction in exogenous mesenchymal stem cells homing to rat remnant kidney.

Background/Aims: The aim of our study was to reveal the role of CD44-Hyaluronic acid (HA) in the homing and improving renal function of systemically transplanted MSCs in chronic renal failure. Methods: First, a remnant kidney model was established in rats and the expression of HA was determined using immunohistochemistry (IHC) and western blotting. Next, chemotaxis assay using flow cytometry, and cell migration assay of MSCs were performed in vitro. Then, MSCs were transplanted into rats, thus, sprague-Dawley (SD) rats were randomly divided into sham group, 5/6 nephrectomy (5/6 Nx) group, MSC group and MSC/Anti-CD44 group (n = 8 for all groups). Migration of MSCs to the kidney in these rats was assessed by using cell tracking experiments, and tissue damage was evaluated by morphological analysis using Massons trichrome staining and periodic acid Schiff staining. Results: HA was significantly observed in 5/6 Nx group, but not in sham group. Meanwhile, HA was discovered induced MSCs migration remarkably (p < 0.05) and anti-CD44 antibody inhibited the migration significantly (p < 0.05) in vitro. In vivo, the GFP-MSCs were observed in MSC group and the cells reduced in MSC/Anti-CD44 groups, especially, in the tubulointerstitium. Conclusion: Our findings reveal that CD44-HA has the potential to induce MSCs homing to injured tissue, while its effect on the ability of MSCs, improving tissue function, is not significant.

Gut microbial metabolite TMAO contributes to renal dysfunction in a mouse model of diet-induced obesity

Emerging evidence shows that obesity induces renal injury and is an independent risk factor for the development of chronic kidney disease (CKD), even without diabetes or hyperglycemia. Although multiple metabolic factors have been suggested to account for obesity-associated renal injury, the precious underlying mechanisms are not completely understood. Recent study shows that increased trimethylamine N-Oxide (TMAO), a gut microbiota-generated metabolite, directly contributes to renal interstitial fibrosis and dysfunction. Circulating TMAO is elevated in high-fat diets (HFD)-induced obese animals. Here we tested the hypothesis that elevated TMAO might play a contributory role in the development of renal dysfunction in a mouse model of HFD-induced obesity that mimics human obesity syndrome. Male C57BL/6 mice received either a low-fat diet (LFD) or a HFD, without or with 3,3-Dimethyl-1-butanol (DMB, a trimethylamine formation inhibitor) for 16 weeks. Compared with mice fed a LFD, mice fed a HFD developed obesity and metabolic disorders, and exhibited significantly elevated plasma TMAO levels at the end of the experiment. Molecular and morphological studies revealed that renal interstitial fibrosis, phosphorylation of SMAD3 (a key regulator of renal fibrosis), expression of kidney injury molecule-1 and plasma cystatin C were significantly increased in mice fed a HFD, compared with mice fed a LFD. Additionally, expression of NADPH oxidase-4 and pro-inflammatory cytokines tumor necrosis factor-α and interleukin-1 β was also augmented in mice fed a HFD as compared to mice fed a LFD. These molecular and morphological alterations observed in mice fed a HFD were prevented by concomitant treatment with DMB, which reduced plasma TMAO levels. Furthermore, elevated circulating TMAO levels were positively correlated with increased renal interstitial fibrosis and expression of kidney injury molecule-1. Notable, there was no difference in blood pressure among groups, and DMB treatment had no effects on body weight and metabolic parameters. These data suggest that HFD-induced obesity leads to elevations in gut microbiota-generated metabolite TMAO in the circulation, which contributes to renal interstitial fibrosis and dysfunction by promoting renal oxidative stress and inflammation. These findings may provide new insights into the mechanisms underlying obesity-associated CKD. Targeting TMAO may be a novel strategy for prevention and treatment of CKD in patients with obesity.

Hepatitis B Virus X Protein Modulates Apoptosis in NRK-52E Cells and Activates Fas/FasL Through the MLK3-MKK7-JNK3 Signaling Pathway.

Background/Aims: The hepatitis B virus X protein (HBx) contributes to HBV-induced injury of renal tubular cells and induces apoptosis via Fas/FasL up-regulation. However, the mechanism of Fas/FasL activation is unknown. Recent studies indicated that HBx induction of apoptosis in hepatic cells depends on activating the MLK3-MKK7-JNKs signaling module, which then up-regulates FasL expression. In this study, we used NRK-52E cells transfected an HBx expression vector to examine the role of the MLK3-MKK7-JNKs signaling pathway on HBx-induced renal tubular cell injury. Methods: NRK-52E cells were transfected with pc-DNA3.1(+)-HBx to establish an HBx over-expression model, and with pc-DNA3.1(+)-HBx and pSilencer3.1-shHBx to establish an HBx low expression model. One control group was not transfected and another control group was transfected with an empty plasmid. Cell proliferation was determined by the formazan dye method (Cell Counting Kit-8) and apoptosis was measured by flow cytometry and fluorescence microscopy. Western blotting was used to measure the expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins. The activity of caspase-8 was measured by spectrophotometry. Results: Transfection of NRK-52E cells with pc-DNA3.1(+)-HBx inhibited cell proliferation and increased apoptosis and caspase-8 activity. The expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins were also greater in the pc-DNA3.1(+)-HBx group, but lower in RNAi group. Furthermore, the activity of MLK3-MKK7-JNKs signaling pathway, expression of Fas/FasL, and apoptosis were significantly lower in the pc-DNA3.1(+)-HBx group when treated with K252a, a known inhibitor of MLK3. Conclusions: Our results show that HBx induces apoptosis in NRK-52E cells and activates Fas/FasL via the MLK3-MKK7-JNK3-c-Jun signaling pathway.

Epigallocatechin-3-gallate reduces tubular cell apoptosis in mice with ureteral obstruction

BACKGROUND Tubular cell apoptosis plays a crucial role in different kinds of renal diseases. Epigallocatechin-3-gallate (EGCG), a polyphenol extracted from green tea, has been shown to inhibit renal fibrosis in unilateral ureteral obstruction (UUO) mice, but its role in preventing tubular cell apoptosis and the underlying signaling mechanisms still remains unclear. MATERIALS AND METHODS Mice subjected to UUO were intraperitoneally administered EGCG (5 mg/kg) for 14 d. Normal rat kidney proximal tubular epithelial cell line NRK-52E was induced by transforming growth factor β1 (TGF-β1). Periodic acid-schiff and Massons trichrome staining was used for histologic study. TUNEL, Hoechst staining, and flow cytometry analysis were used to measure the apoptotic status of tubular cells. Western blotting was used to determine the expression of apoptotic-associated proteins and mitogen-activated protein kinase pathway proteins. RESULTS EGCG significantly attenuated tubular injury and renal tubulointerstitial fibrosis in the obstructed kidneys of UUO mice. In addition, EGCG prevented UUO and TGF-β1-induced tubular apoptosis in a dose-dependent manner. In parallel, protein expression of B-clell lymphoma-2 (Bcl-2) was upregulated and protein expressions of Bcl-2 accosiated X protein (Bax), cleaved caspase 3, and cleaved poly ADP-ribose polymerase (PARP) were downregulated by EGCG. Furthermore, UUO and TGF-β1-stimulated phosphorylation of mitogen-activated protein kinase was inhibited by EGCG. CONCLUSIONS EGCG effectively reduces tubular cell apoptosis induced by UUO and may have potential as a clinical treatment in patients with chronic kidney disease.

Hepatitis B Virus X Protein Reduces Podocyte Adhesion via Downregulation of α3β1 Integrin

Background/Aims: Hepatitis B virus (HBV)-associated glomerulonephritis (HBV-GN) is characterized by a reduced number of podocytes due to apoptosis and shedding from the basement membrane. However, the pathological mechanism of HBV-GN is unclear. We previously showed that hepatitis B virus X protein (HBx) promotes apoptosis in tubular epithelial cells. In this study, we transfected podocytes with HBx and examined the effects on adhesion and apoptosis of these cells. Methods: Podocytes were transfected with pc-DNA3.1 (+)-HBx. One control group was not transfected and another control group was transfected with empty plasmids. Podocyte adhesion was assessed by a fluorescence assay, apoptosis was measured by flow cytometry and fluorescence microscopy, and expression of α3β1 integrin was determined by western blotting and the reverse transcription polymerase chain reaction (RT-PCR). Activity of caspase-8 was measured by a spectrophotometric assay. Results: Relative to controls, podocytes with pc-DNA3.1(+)-HBx had reduced cell adhesion, increased apoptosis, reduced expression of α3β1 integrin, and increased caspase-8 activity. β1 integrin blockage reduced podocyte adhesion, but increased apoptosis and caspase-8 activity. Treatment of transfected podocytes with a caspase-8 inhibitor (Z-IETD-FMK) had no effect on the HBx-mediated integrin downregulation and reduced podocyte adhesion, suggesting that α3β1 integrin downregulaton is sufficient to alter cell adhesion. Conclusions: Our in vitro results indicate that HBx reduced podocyte adhesion and expression of α3β1 integrin, and increased apoptosis. Moreover, HBx-mediated downregulation of α3β1 integrin expression is sufficient to reduce podocyte adhesion. HBx-induced apoptosis of podocytes may contribute to HBV-GN.

Telmisartan attenuates peritoneal fibrosis via peroxisome proliferator‐activated receptor‐γ activation in rats

Peritoneal dialysis (PD) is an effective treatment for patients with end‐stage renal diseases, but long‐term continuous PD causes peritoneal fibrosis (PF). This study aims to evaluate the anti‐fibrotic effect of telmisartan on a rat model of PF and to investigate the underlying mechanisms. Five‐sixths kidney nephrectomy and PD were used to establish the PF rat model. Glucose (2.5%) was used to establish an in vitro model in rat peritoneal mesothelial cells (PMC). Haematoxylin–eosin staining was used to examine the structural alterations. Massons trichrome staining was used to observe the tissue fibrosis in peritoneal membrane of rats. Real‐time polymerase chain reaction was used to measure messenger RNA expressions of profibrotic factors. Western blotting was used to determine protein expressions of profibrotic factors, peroxisome proliferator‐activated receptor‐γ, and mitogen‐activated protein kinases (MAPK). Results demonstrated that administration of telmisartan dose‐dependently attenuated the thickening of the peritoneal membrane and the fibrosis induced by long‐term PD fluid exposure in rats. In addition, telmisartan treatment inhibited the upregulation of profibrotic factors induced by PD in the peritoneum of rats and by high‐concentration glucose in PMC. Telmisartan was also effective in inhibiting PD and high‐concentration, glucose‐induced phosphorylation of MAPK in the peritoneum and PMC. Furthermore, peroxisome proliferator‐activated receptor‐γ (PPARγ) inhibitor GW9662 blocked these protective effects of telmisartan in PMC. The results suggest that telmisartan is effective in attenuating PD‐induced PF, and this effect may be associated with the inhibition of profibrotic factor expression and MAPK phosphorylation via PPARγ activation.