牛磺酸对肾NRK-52E细胞缺氧/复氧损伤的保护及初步机制

目的观察牛磺酸(Tau)对NRK-52E细胞缺氧/复氧(H/R)损伤的保护作用及初步机制。方法用NRK-52E细胞缺氧8 h复氧12 h培养建立了H/R模型,流式细胞仪检测凋亡率和胞内游离钙离子浓度,RT-PCR和Western blot检测GRP78、Caspase-12、Caspase-3 mRNA和蛋白表达。结果与对照组比较,H/R组细胞凋亡率上升,GRP78、Caspase-12及Caspase-3 mRNA和蛋白表达均明显增高(P<0.01),胞质游离钙离子浓度也升高(P<0.01);与H/R组比较,各浓度牛磺酸处理组的细胞Caspase-3活性和凋亡率明显下降,胞质游离钙和GRP78、Caspase-12及Caspase-3 mRNA和蛋白表达均有明显降低(P<0.01)。结论牛磺酸对NRK-52E细胞H/R损伤有较好的抑制作用,且呈一定的剂量依赖性,其机制可能是调节胞内钙稳态、抑制GRP78、Caspase-12及Caspase-3表达,减少了细胞凋亡。     

Cadmium toxicity is caused by accumulation of p53 through the down-regulation of Ube2d family genes in vitro and in vivo

Cadmium (Cd) causes renal dysfunction with damage to kidney proximal tubule cells; however, the precise mechanisms of the toxicity remain unclear. Previously, we found that the expression of Ube2d4 gene, which is a member of the ubiquitin-conjugating enzyme Ube2d family, is suppressed by Cd in NRK-52E rat renal tubular epithelial cells. To investigate the mechanisms of Cd-induced renal toxicity, we examined the effects of Cd on the ubiquitin-proteasome system, particularly the expression and function of Ube2d family members in the NRK-52E cells and mice. Cd markedly decreased the expression of Ube2d1, Ube2d2, Ube2d3 and Ube2d4 prior to the appearance of cytotoxicity in the NRK-52E cells. Cd also dramatically increased p53 protein levels in the cells, without stimulation of p53 gene expression or inhibition of proteasome activity. In addition, Cd induced phosphorylation of p53 and caused apoptosis in the NRK-52E cells. In vivo, we examined the effect of orally administrated Cd for 12 months on the expression of Ube2d genes and accumulation of p53 in the mouse kidney. Chronic Cd exposure also caused suppression of Ube2d genes expression and accumulation of p53. Cd did not induce severe kidney injury, but caused apoptosis in the renal tubules. These results suggest that the Cd-induced accumulation of p53 may be due to inhibition of p53 degradation through the down-regulation of Ube2d family genes, and that Cd induces p53-dependent apoptosis in renal tubular cells. Moreover, Ube2d family members may be one of the critical targets of renal toxicity caused by Cd.     

Cadmium induces cell cycle arrest in rat kidney epithelial cells in G2/M phase

Cadmium (Cd) has been reported to cause cell cycle arrest in various cell types by p53-dependent and -independent mechanisms. This study was designed to investigate cell cycle progression in kidney cells that are the target of chronic Cd toxicity. Rat renal proximal tubular epithelial cells, NRK-52E, were treated with up to 20 microM CdCl2 in DMEM containing 10% calf serum for up to 24 h. Flow cytometric analysis revealed time- and concentration-dependent increases in cells in G2/M phase of the cell cycle. As compared to the control cells, the cells exposed to 20 microM Cd showed a doubling of the number of cells in this phase after 24 h. The cell cycle arrest was associated with a decrease in protein levels of both cyclins A and B. Further investigation into the mechanism revealed that Cd treatment led to down-modulation of cyclin-dependent kinases, Cdk1 and Cdk2, apparently by elevating the expression of cyclin kinase inhibitors, KIP1/p27 and WAF1/p21. Furthermore, the wild-type p53 DNA-binding activity was up-regulated. Based on these observations, it appears that Cd causes G2/M phase arrest in NRK-52E cells via elevation of p53 activity, increasing the expression of cyclin kinase inhibitors p27 and p21, and decreasing the expression of cyclin-dependent kinases Cdk1 and 2, and of cyclins A and B.     

激活素A及卵泡抑素对肾小管上皮细胞转分化的作用

目的探讨激活素A(ACTA)对大鼠肾小管上皮细胞(NRK-52E)转分化的影响及卵泡抑素(FS)的干预作用。方法 NRK-52E细胞生长于达氏修正伊氏培养基(DMEM)/F12培养基。实验A:将培养的NRK-52E细胞按rh-ACTA浓度不同分为4组:ACTA浓度分别为0,1,10,100ng/ml。实验B:分为4组:对照组;ACTA组(A组),rh-ACTA浓度10ng/ml;rh-FS组(F组),rh-FS浓度为100ng/ml;ACTA+FS组(A+F组):rh-ACTA浓度10ng/ml,rh-FS浓度为100ng/ml。24h后收集各组细胞及细胞上清液。蛋白印迹法检测各组细胞α-平滑肌肌动蛋白(α-SMA)和纤维连接蛋白(FN)的表达;酶联免疫吸附法(ELISA)检测各组细胞上清液转化生长因子-β(TGF-β)的含量。结果 ACTA能剂量依赖性刺激肾小管上皮细胞α-SMA及FN的表达(P<0.05),而对TGF-β的表达没有影响。FS能抑制ACTA的上述效应,而单纯FS对肾小管上皮细胞α-SMA及FN的表达无影响(P>0.05)。结论 ACTA能刺激NRK-52E细胞转分化及FN的合成;FS可通过阻断ACTA的上述效应产生有效的肾脏保护作用。     

Long-term leptin treatment exerts a pro-apoptotic effect on renal tubular cells via prostaglandin E2 augmentation

Adipokine leptin reportedly acts on the kidney in pathophysiological states. However, the influence of leptin on renal tubular epithelial cells is still unclear. Gentamicin, a widely used antibiotic for the treatment of bacterial infection, can cause nephrotoxicity. This study aims to investigate the influence of long-term leptin treatment on gentamicin-induced apoptosis in rat renal tubular cells (NRK-52E) and mice. We monitored apoptosis and molecular mechanisms using annexin V/ propidium iodide staining and small interfering RNA transfection. In NRK-52E cells, leptin reduced gentamicin-induced apoptosis at 24h, but significantly increased apoptosis at 48 h. Long-term treatment of leptin decreased Bcl-x(L) expression and increased caspase activity in gentamicin-treated NRK-52E cells. Leptin also increased the expression of cyclooxygenase-2 (COX-2) and its product, prostaglandin E(2) (PGE(2)), in a dose-dependent manner. The COX-2 inhibitor, NS398 (N-[2-(Cyclohexyloxy)-4- nitrophenyl]methanesulfonamide), blocked PGE(2) augmentation and the pro-apoptotic effects of leptin. The addition of PGE(2) recovered the pro-apoptotic effect of leptin in NS398-treated NRK-52E cells. In a mouse animal model, a 10 day leptin treatment significantly increased gentamicin-induced apoptotic cells in proximal tubules. NS398 treatment inhibited this in vivo pro-apoptotic effect of leptin. Results reveal that long-term elevation of leptin induces COX-2-mediated PGE(2) augmentation in renal tubular cells, and then increases these cells' susceptibility to gentamicin-induced apoptosis.     

Necroptosis contributes to the cyclosporin A-induced cytotoxicity in NRK-52E cells

Cyclosporin A (CsA) induces renal tubular epithelial cells apoptosis and necrosis following in vitro exposure. The mechanisms of CsA-induced apoptosis have been studied intensively, whereas the mechanisms of necrosis remain to be elucidated. Necroptosis has been described as programmed necrosis. This study investigated the ability of CsA to induce necroptosis in the rat tubular cell line NRK-52E. The NRK-52E cells were incubated with CsA for 24 hours with or without necrostatin-1 (Nec-1). The majority of the NRK-52E cells died of necrosis as indicated by LDH leakage, Hoechst 33342/PI staining, and flow cytometry analysis. Cell death was significantly reduced by Nec-1 pretreated before CsA exposure. CsA-induced apoptosis and necrosis were also compared in NRK-52E cells with or without knockdown of receptor interaction protein 3 (RIP3) expression using small interfering RNA. Moreover, the role of reactive oxygen species (ROS) in CsA-induced cell death was also attempted. The result suggests that necroptosis contributes to the CsA-induced cytotoxicity in NRK-52E cells. Meanwhile, RIP3 and ROS are involved in CsA-induced necroptosis. To our knowledge, this is the first report on necroptosis in CsA-induced renal tubular cell death pathways, which might offer a novel protective target for CsA nephrotoxicity.     

Ganoderma Lucidum polysaccharide peptides protect kidney from renal ischemia reperfusion injury via counteracting oxidative stress

OBJECTIVE Ganoderma lucidum polysaccharide peptides(GLPP)have an anti-oxidant activity.The oxidative stress implicates in the pathogenesis of renal ischemia-reperfusion injury(RIRI).The objective of this study was to determine whether GLPP could attenuate RIRI via counteracting the oxidative stress.METHODS Mice subjected to uninephrectomy with the right kidney ischemia for 35 min and reperfusion for 24 hwere used to explore the protective activity of GLPP against RIRI.In GLPP-treated group,100mg·kg-1·d-1 of GLPP were intraperitoneally injected for 7dbefore the procedure.In vitro,NRK-52 Ecells subjected to hypoxia-reoxygenation(H/R)and tunicamycin were used to explore the protective effect of GLPP against oxidative stress.The mechanisms in which GLPP protected kidney from RIRI were studied using a series of physiological and molecular biological methods.RESULTS Kidneys undergone ischemia-reperfusion showed renal dysfunction and characteristic morphological changes including cellular necrosis,brush border loss,cast formation,vacuolization and tubular dilatation while these damages were significantly attenuated by GLPP treatment.The abnormal levels of MPO,MDA and SOD caused by renal ischemia-reperfusion were significantly reversed by GLPP treatment.More apoptotic cells were found in the renal ischemia-reperfusion group than the sham group whereas GLPP reduced apoptotic cells in the ischemia-reperfusion mice by21.75%(P<0.01).The GLPPs(25-1μg·mL)alleviated H/R induced cell viability loss by 20.12%(P<0.01)andΔφm dissipation by 27.3%(P<0.01)in vitro as well and its pretreatment dramatically reduced H/R and tunicamycin induced cell injury.CONCLUSION Our study found that GLPP had a protective effect on RIRI via its anti-oxidative capacity,which suggests that GLPP may be developed as a candidate drug for preventing acute kidney injury.     

Cell growth inhibition and apoptosis by SDS-solubilized single-walled carbon nanotubes in normal rat kidney epithelial cells

Carbon nanotubes (CNTs), promising novel nanomaterials, have been applied to drug delivery and bio-imaging; however, their potential harmful effects on human health and environment have gained much attention recently. In the present study, we investigated cytotoxic effect of solubilized single-walled CNTs (SWCNTs), which were dispersed in water by sodium dodesyl sulfate (SDS), in normal rat kidney epithelial cells (NRK-52E). SDS-SWCNT (0.125–10 μg/mL)-treated NRK-52E cells showed decreased cell viability and enhanced cytotoxicity marker levels following 24–48 h incubation. In addition, SDS-SWCNT treatment evoked the cell growth inhibition: 8 μg/mL SDS-SWCNT induced the growth arrest at G₀/G₁ phase and levels of cell cycle-related proteins such as CDK2, CDK6 and phosphorylated-retinoblastoma (pRB) were significantly reduced by CNT. Whereas, at higher concentration of SDS-SWCNT, the percentage of cell numbers in apoptotic sub-G1 phase was substantially increased. Along with these changes, SDS-SWCNT treatment elevated protein levels for p53 and p21 with a concomitant increase in the single strand DNA breakage. Taken together, these results suggest that SDS-solubilized SWCNTs exert genotoxic effect in renal epithelial cells, and p53-dependent signaling can be associated with the growth arrest and apoptosis events upon CNT-induced DNA damage.     

高迁移率族蛋白1在醛固酮诱导肾小管上皮细胞自噬中的作用 #br#

目的 探讨高迁移率族蛋白 1（HMGB1）在醛固酮（ALDO）诱导大鼠肾小管上皮细胞自噬中的作用。方 法 取对数生长期的大鼠肾小管上皮细胞（NRK-52E）,分为 Control组、ALDO组（10 nmol/L）、HMGB1抗体组（1 mg/ L）、IgG组（1 mg/L）和 ALDO+HMGB1抗体组（1 mg/L HMGB1抗体预处理 1 h,再加入 10 nmol/L ALDO刺激 24 h）和阳 性对照组（Rosup,100 µmol/L）,处理24 h后利用荧光探针DCFH-DA标记后使用流式细胞仪检测NRK-52E细胞内活 性氧（ROS）水平的变化。另外,将培养的 NRK-52E 细胞分为 Control 组、ALDO 组（10 nmol/L）、N-乙酰半胱氨酸组 （NAC,50 µmol/L）和 NAC+ALDO组（50 µmol/L NAC预处理 1 h,再加入 10 nmol/L ALDO）,处理 24 h后 Western blot法 检测白细胞介素（IL）-1β蛋白的表达水平。观察10 nmol/L ALDO刺激24 h后,HMGB1蛋白表达的变化。最后,予以 1 mg/L HMGB1抗体预处理（1 mg/L HMGB1抗体处理 1 h后,再加入 10 nmol/L ALDO刺激 24 h）,Western blot法检测 LC3-Ⅱ、Beclin-1和p62蛋白的表达水平。结果 流式细胞仪检测结果显示,与Control组相比,ALDO组NRK-52E细 胞内 ROS 的产生明显增加（P＜0.05）;与 ALDO 组相比,ALDO+HMGB1 抗体组细胞内 ROS 的水平显著下降（P＜ 0.05）。Western blot结果显示,与Control组相比,ALDO组IL-1β、HMGB1、LC3-Ⅱ和Beclin-1蛋白的表达上调,p62蛋 白的表达下调（P＜0.05）;与 ALDO组相比,ALDO+NAC组 IL-1β蛋白的表达下调（P＜0.05）,ALDO+HMGB1抗体组 LC3-Ⅱ蛋白的表达降低、p62蛋白的表达增加（P＜0.05）,Beclin-1蛋白的表达差异无统计学意义。结论 ALDO通 过促进NRK-52E细胞内ROS的产生,增加HMGB1的分泌,刺激IL-1β的释放,进而诱导自噬的发生;抑制HMGB1的 释放可逆转这一现象,本研究为慢性肾脏病的防治提供了新靶点和新思路。     

Effect of Sedum sarmentosum Bunge Extract on Aristolochic Acid– Induced Renal Tubular Epithelial Cell Injury

Aristolochic acid (AA) is known as a potent mutagen that induces significant cytotoxic and mutagenic effects on renal tubular epithelial cells. Clinically, the persistent injury of AA results in the infiltration of inflammatory cells, epithelial-to-mesenchymal transition (EMT), and renal tubulointerstitial fibrosis. There are no truly effective pharmaceuticals. In this study, we investigated the potential role of the extract of Sedum sarmentosum Bunge (SSB), a traditional Chinese herbal medicine, on rat tubuloepithelial (NRK-52E) cells after AA injury in vitro. Evidence revealed that AA induced mitochondrial-pathway–mediated cellular apoptosis, accompanied by cell proliferation in a feedback mechanism. Treatment with SSB also induced cells to enter early apoptosis, but inhibited cell proliferation. In cultured NRK-52E cells, AA induced the imbalance of MMP-2/TIMP-2 and promoted EMT and ECM accumulation. SSB treatment significantly alleviated AA-induced NRK-52E cells fibrosis-like appearance, inhibited the induction of EMT, and deposition of ECM. SSB also decreased the activity of the NF-κB signaling pathway, resulting in down-regulated expression of NF-κB–controlled chemokines and pro-inflammatory cytokines, including MCP-1, MIF, and M-CSF, which may regulate the macrophage-mediated inflammatory reaction during renal fibrosis in vivo. Therefore, these findings suggest that SSB exerts protective effects against AA-induced tubular epithelial cells injury through suppressing the synthesis of inflammatory factors, EMT, and ECM production.     

Cellular energetics and glutathione status in NRK-52E cells: toxicological implications

Cellular energetics and redox status were evaluated in NRK-52E cells, a stable cell line derived from rat proximal tubules. To assess toxicological implications of these properties, susceptibility to apoptosis induced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a well-known mitochondrial and renal cytotoxicant, was studied. Cells exhibited high activities of several glutathione (GSH)-dependent enzymes, including gamma-glutamylcysteine synthetase, GSH peroxidase, glutathione disulfide reductase, and GSH S-transferase, but very low activities of gamma-glutamyltransferase and alkaline phosphatase, consistent with a low content of brush-border microvilli. Uptake and total cellular accumulation of [14C]alpha-methylglucose was significantly higher when cells were exposed at the basolateral as compared to the brush-border membrane. Similarly, uptake of GSH was nearly 2-fold higher across the basolateral than the brush-border membrane. High activities of (Na(+)+K(+))-ATPase and malic dehydrogenase, but low activities of other mitochondrial enzymes, respiration, and transport of GSH and dicarboxylates into mitochondria were observed. Examination of mitochondrial density by confocal microscopy, using a fluorescent marker (MitoTracker Orange), indicated that NRK-52E cells contain a much lower content of mitochondria than rat renal proximal tubules in vivo. Incubation of cells with DCVC caused time- and concentration-dependent ATP depletion that was largely dependent on transport and bioactivation, as observed in the rat, on induction of apoptosis, and on morphological damage. Comparison with primary cultures of rat and human proximal tubular cells suggests that the NRK-52E cells are modestly less sensitive to DCVC. In most respects, however, NRK-52E cells exhibited functions similar to those of the rat renal proximal tubule in vivo.     

Pathways of proximal tubular cell death in bismuth nephrotoxicity

Colloidal bismuth subcitrate (CBS), a drug for treatment of peptic ulcers, has been reported in the literature to be nephrotoxic in humans when taken in high overdoses. To investigate the mechanism of bismuth nephropathy, we developed an animal model by feeding rats single doses of CBS containing 3.0 mmol Bi/kg body weight. Terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling assay, immunostaining for active caspase-3, and electron microscopy showed that proximal tubular epithelial cells die by necrosis and not by apoptosis within 3 h after CBS administration. Exposure of the renal epithelial cell lines NRK-52E and LLC-PK1 to Bi(3+) in citrate buffer served as an in vitro model of bismuth nephropathy. NRK-52E cells exposed to 100 microM Bi(3+) or more died by necrosis, as was demonstrated by nuclear staining with Hoechst 33258 and flow cytometry using Alexa(488)-labeled Annexin-V and the vital nuclear dye TOPRO-3. Bismuth-induced cell death of NRK-52E cells was not prevented by the caspase-3 inhibitor z-VAD-fmk, whereas this inhibitor did prevent cisplatinum-induced apoptosis. Mitochondrial dysfunction and induction of free radicals were shown not to be involved in bismuth nephrotoxicity. The early time point of damage induction in vitro as well as in vivo and the early displacement of N-cadherin, as found in previous studies, suggest that bismuth induces cell death by destabilizing the cell membrane. In conclusion, we showed that high overdose of bismuth induced cell death by necrosis in vivo as well as in vitro, possibly by destabilization of the cell membrane.     

Effects of hydroxyl radical scavenging on cisplatin-induced p53 activation, tubular cell apoptosis and nephrotoxicity

Nephrotoxicity is a major side effect of cisplatin, a widely used cancer therapy drug. Recent work has suggested a role of p53 in renal cell injury by cisplatin. However, the mechanism of p53 activation by cisplatin is unclear. This study determined the possible involvement of oxidative stress in p53 activation under the pathological condition using in vitro and in vivo models. In cultured renal proximal tubular cells, cisplatin at 20 microM induced an early p53 phosphorylation followed by protein accumulation. Cisplatin also induced reactive oxygen species (ROS), among which hydroxyl radicals showed a rapid and drastic accumulation. Dimethylthiourea (DMTU) and N-acetyl-cysteine (NAC) attenuated hydroxyl radical accumulation, and importantly, diminished p53 activation during cisplatin treatment. This was accompanied by the suppression of PUMA-alpha, a p53-regulated apoptotic gene. Concomitantly, mitochondrial cytochrome c release and apoptosis were ameliorated. Notably, DMTU and NAC, when added post-cisplatin treatment, were also inhibitory to p53 activation and apoptosis. In C57BL/6 mice, cisplatin at 30 mg/kg induced p53 phosphorylation and protein accumulation, which was also abrogated by DMTU. DMTU also ameliorated tissue damage, tubular cell apoptosis and cisplatin-induced renal failure. Collectively, this study has suggested a role of oxidative stress, particularly hydroxyl radicals, in cisplatin-induced p53 activation, tubular cell apoptosis and nephrotoxicity.     

Berberine protects HK-2 cells from hypoxia/reoxygenation induced apoptosis via inhibiting SPHK1 expression

Renal ischemia reperfusion injury (RIRI) refers to the irreversible damage for renal function when blood perfusion is recovered after ischemia for an extended period, which is common in clinical surgeries and has been regarded as a major risk for acute renal failures (ARF) that is accompanied with unimaginably high morbidity and mortality. Hypoxia during ischemia followed by reoxygenation via reperfusion serves as a major event contributing to cell apoptosis, which has been widely accepted as the vital pathogenesis in RIRI. Preventing apoptosis in renal tubular epithelial cell has been considered as effective method for blocking RIRI. In this paper, we established a hypoxia/reoxygenation (H/R) injury model in human proximal tubular epithelial HK-2 cells. Here, we found increased SPHK1 levels in H/R injured HK-2 cells, which could be significantly down regulated after berberine treatment. Berberine has been reported to exert a protective effect on H/R-induced apoptosis of HK-2 cells. So, in our present study, we planned to investigate whether SPHK1 participated in the anti-apoptosis process of berberine in H/R injured HK-2 cells. Our study confirmed the protective effect of berberine against H/R-induced apoptosis in HK-2 cells through promoting cells viability, inhibiting cells apoptosis, and down-regulating p-P38, caspase-3, caspase-9 as well as SPHK1, while up regulating the ratio of Bcl-2/Bax. However, SPHK1 overexpression in HK-2 cells induced severe apoptosis, which can be significantly ameliorated with additional berberine treatment. We concluded that berberine could remarkably prevent H/R-induced apoptosis in HK-2 cells through down-regulating SPHK1 expression levels, and the mechanisms included the suppression of p38 MAPK activation and mitochondrial stress pathways.