Loss of homotypic epithelial cell adhesion by selective N-cadherin displacement in bismuth nephrotoxicity

The nephrotoxicity of single high doses of bismuth (Bi)-containing therapeutic drugs is characterized morphologically by detachment of proximal tubular epithelial cells (PTECs) from each other, followed by cell death. We investigated whether Bi nephrotoxicity is mediated by changes in the distribution of proteins involved in intercellular adhesion. A nephrotoxic dose of colloidal bismuth subcitrate (CBS; 3.0 mmol Bi/kg) was orally administrated to 10 female Wistar rats. After 1 h, N-cadherin had disappeared from the adherence junctions of vital PTECs, whereas ZO-1, a tight junction marker, remained present at the cell-cell border until cell death occurred after 3 h. E-Cadherin, absent in PTECs, remained absent. Exposure of the renal epithelial cell lines NRK-52E and LLC-PK1 to 400 microM Bi(3+) also resulted in the disappearance of N-cadherin expression after 1 h, whereas ZO-1, E-cadherin, and Desmoplakin expression did not resolve before cell death at 24 h, thus confirming in vivo results. Our results are the first to indicate that Bi-induced death of PTECs is preceded by redistribution of N-cadherin and the disruption of homotypic cell adhesion.     

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.     

Renal epithelial gene expression profile and bismuth-induced resistance against cisplatin nephrotoxicity

Nephrotoxicity is the most important dose-limiting factor in cisplatin based anti-neoplastic treatment. Pretreatment with bismuth salts, used as pharmaceuticals to treat gastric disorders, has been demonstrated to reduce cisplatin-induced renal cell death in clinical settings and during in vivo and in vitro animal experiments. To investigate the genomic basis of this renoprotective effect, we exposed NRK-52E cells, a cell line of rat proximal tubular epithelial origin, to 33 microM Bi3+ for 12 hours, which made them resistant to cisplatin-induced apoptosis. Differentially expressed genes in treated and untreated NRK-52E cells were detected by subtraction PCR and microarray techniques. Genes found to be down regulated (0.17-0.31-times) were cytochrome c oxidase subunit I, BAR (an apoptosis regulator), heat-shock protein 70-like protein, and three proteins belonging to the translation machinery (ribosomal proteins S7 and L17, and S1, a member of the elongation factor 1-alpha family). The only up-regulated gene was glutathione S-transferase subunit 3A (1.89-times). Guided by the expression levels of these genes, it may be possible to improve renoprotective treatments during anti-neoplastic therapies.     

Omi/HtrA2短发夹RNA在大鼠肾小管上皮细胞凋亡中的作用及机制

目的探讨Omi/HtrA2短发夹RNA(shRNA)对缺氧/复氧诱导大鼠肾小管上皮细胞(NRK-52E)凋亡的作用及机制。方法细胞分为5组:正常组(常规培养),模型组(缺氧/复氧组)、HK组(转染重组质粒Pgenesil-1/HK)、shRNA1组(转染Pgenesil-1/Omi/HtrA2shRNA1)、shRNA2组(转染Pgenesil-1/Omi/HtrA2shRNA2)。用荧光显微镜观察荧光蛋白的表达,Westernblot检测各组Omi/HtrA2、半胱氨酰天冬氨酸特异性蛋白酶(caspase)-3/-9蛋白表达,比色法测定caspase-3/-9的活性。结果在荧光显微镜下,转染组细胞均可见绿色荧光,未转染组未见绿色荧光。与模型组相比,shRNA1组和shRNA2组Omi/HtrA2、caspase-3/-9蛋白表达明显减少(P<0.01)。模型组和HK组、shRNA1组和shRNA2组之间Omi/HtrA2、caspase-3/-9蛋白表达差异无统计学意义。与正常组相比,模型组Omi/HtrA2、caspase-3/-9蛋白表达明显增强(P<0.01)。结论Pgenesil-1/Omi/HtrA2shRNA1和Pgenesil-1/Omi/HtrA2shRNA2能明显减少缺氧/复氧诱导的NRK-52E中Omi/HtrA2蛋白的表达。通过抑制procaspase-9的活化,进而减少了下游procaspase-3的激活,最终减轻了缺氧/复氧诱导的NRK-52E的凋亡程度。     

Oxalate toxicity in renal epithelial cells: characteristics of apoptosis and necrosis

Studies in various tissues, including the kidney, have demonstrated that toxins elicit apoptosis under certain conditions and necrosis under others. The nature of the response has important consequences for the injured tissue in that necrotic cells elicit inflammatory responses, whereas apoptotic cells do not. Thus, there has been considerable interest in defining the mode of cell death elicited by known cytotoxins. The present studies examined the response of renal epithelial cells to oxalate, a metabolite excreted by the kidney that produces oxidant stress and death of renal cells at pathophysiological concentrations. These studies employed LLC-PK1 cells, a renal epithelial cell line from pig kidney and NRK-52E (NRK) cells, a line from normal rat kidney, and compared the effects of oxalate with those of known apoptotic agents. Changes in cellular and nuclear morphology, in nuclear size, in ceramide production, and in DNA integrity were assessed. The ability of bcl-2, an anti-apoptotic gene product, to attenuate oxalate toxicity was also assessed. These studies indicated that oxalate-induced death of renal epithelial cells exhibits several features characteristic of apoptotic cell death, including increased production of ceramide, increased abundance of apoptotic bodies, and marked sensitivity to the level of expression of the anti-apoptotic gene bcl-2. Oxalate-induced cell death also exhibits several characteristics of necrotic cell death in that the majority of the cells exhibited cellular and nuclear swelling after oxalate treatment and showed little evidence of DNA cleavage by TUNEL assay. These results suggest that toxic concentrations of oxalate trigger both forms of cell death in renal epithelial cells.     

Bismuth biokinetics and kidney histopathology after bismuth overdose in rats

Bismuth induced nephrotoxicity has been reported to occur after acute overdoses of Bi-containing therapeutic drugs. We studied the development of bismuth induced nephropathy and bismuth biokinetics in rats. Bismuth nephropathy was induced in 33 young adult female Wistar rats weighing ca. 175 g by feeding them a single overdose of colloidal bismuth subcitrate containing 3.0 mmol Bi/kg at (t = 0). Control animals (n = 7) were fed the vehicle only. The animals were sacrificed after 1-48 h. Plasma creatinine increased from 51 +/- 6 micromol/l at t = 0 to 550 +/- 250 micromol/l after 48 h in the experimental group. The S3 segment of the proximal tubule showed epithelial cell vacuolation after 1 h and necrosis after 3 h. Cells of the S1/S2 segment demonstrated vacuolation after 6 h and necrosis after 12 h. Biokinetics of bismuth in blood could best be described with a one-compartment model characterized by an absorption half-life of 0.32 h and an elimination halflife of 16 h. The peak concentration of about 7.0 mg Bi/l was reached after 2 h. In conclusion, cells of the S3 segment of the proximal tubule necrotized first after an oral colloidal bismuth subcitrate overdose and biokinetics of Bi in blood was best described by a one-compartment model.     

Uranium induces apoptosis and is genotoxic to normal rat kidney (NRK-52E) proximal cells.

Uranium (U) is a heavy metal used in the nuclear industry and for military applications. U compounds are toxic. Their toxicity is mediated either by their radioactivity or their chemical properties. Mammalian kidneys and bones are the main organs affected by U toxicity. Although the most characteristic response to U exposure is renal dysfunction, little information is available on the mechanisms of its toxicity at the molecular level. This report studied the genotoxicity of U. Apoptosis induction in normal rat kidney (NRK-52(E)) proximal cells was investigated as a function of exposure time or concentrations (0-800microM). In parallel, DNA damage was evaluated by several methods. In order to distinguish between the intrinsic and the extrinsic pathways of apoptosis, caspases-8, -9, -10 assays were conducted and the mitochondrial membrane potential was measured. Three methods were selected for their complementarities in the detection of genetic lesions. The comet assay was used for the detection of primary lesions of DNA. gamma-H2AX immunostaining was achieved to detect DNA double-strand breaks. The micronucleus assay was used to detect chromosomic breaks or losses. DNA damage and apoptosis were observed in a concentration-dependent manner. This study demonstrated that U is genotoxic from 300microM and induces caspase-dependent apoptosis cell death from 200microM mainly through the intrinsic pathway in NRK-52(E) cells. These results suggest that the DNA damage caused by U is reversible at low concentration (200-400microM) but becomes irreversible and leads to cell death for higher concentrations (500-800microM).     

Silymarin augments human cervical cancer HeLa cell apoptosis via P38/JNK MAPK pathways in serum-free medium

Silymarin was proved to have a protective effect of UV-induced A375-S2 cell apoptosis in our previous research. In this study, its pro-apoptotic and anti-apoptotic activities on human cervical cancer (HeLa) cells in vitro were investigated. Silymarin induced HeLa cell death through both apoptotic and necrotic pathways. At low doses (below 80 micromol l-1), it induced cell apoptosis, but caused necrosis at high dose (160 micromol l-1). Silymarin induced typical chromatin condensation and nuclear fragmentation as a hallmark of apoptosis. In this case, mitochondrial Bcl-2 family, Bcl-2 and Bax, were not involved in apoptotic effects; however, silymarin-induced cell death was regulated by the activation of p38 and JNK MAPKs. We also found that pan-caspase inhibitor and caspase-3 inhibitor could not antagonise silymarin-induced apoptosis. Therefore, silymarin induced and augmented HeLa cell apoptosis through p38/JNK MAPKs in the serum-free medium.     

Cisplatin-treated murine peritoneal macrophages induce apoptosis in L929 cells: role of Fas-Fas ligand and tumor necrosis factor-tumor necrosis factor receptor 1

Cisplatin [cis-diamminedichloroplatinum (II)]-treated murine peritoneal macrophages interact with L929 cells in vitro in a sequential manner, resulting in the formation of contact between the two cells. This interaction leads to the death of L929 cells by the process of apoptosis. The detailed investigations have suggested the involvement of two different pathways in macrophage-mediated L929 cell apoptosis. It is observed that the induction of apoptosis in L929 cells by cisplatin-treated macrophages is contact dependent and is mediated through Fas-Fas ligand and tumor necrosis factor-tumor necrosis factor receptor 1 pathways. This conclusion was based on the Western blot and immunoprecipitation analysis of Fas-Fas ligand, tumor necrosis factor-tumor necrosis factor receptor 1, Fas-associated death domain and tumor necrosis factor receptor-associated death domain. The Fas-Fas ligand interaction between macrophages and L929 cells increased the expression of Fas-associated death domain, and tumor necrosis factor-tumor necrosis factor receptor 1 interaction between macrophages and L929 cells increased the expression of tumor necrosis factor receptor-associated death domain in L929 cells. The induction of apoptosis in L929 cells was investigated by DNA fragmentation, Annexin V staining and Western blot analysis of Bax, Bcl-2, Bid, cytochrome c, poly(ADP ribose) polymerase, CAD, caspase-8 and caspase-3.     

Epithelial barrier characteristics and expression of cell adhesion molecules in proximal tubule-derived cell lines commonly used for in vitro toxicity studies.

Recent studies indicate that the actions of several nephrotoxic substances involve alterations in the function of cell adhesion molecules and changes in the paracellular permeability of the proximal tubule. In light of these findings, there is a need for appropriate in vitro model systems to study these phenomenae in greater detail. In the present study, the transepithelial resistance (TER), paracellular permeability of 14C-mannitol and immunofluorescent labeling of cell adhesion molecules (E-cadherin, N-cadherin, ZO-1, occludin, and claudins-2 and -7) were evaluated in several proximal tubule-derived cell lines that have been commonly used as model systems for in vitro toxicity studies. The cell lines studied included: LLC-PK1, OK, NRK-52E and HK-2, along with commercially available primary cultures of human renal proximal tubule epithelial cells (HRPTE). LLC-PK1 cells developed the highest TER followed by the OK cells and NRK-52E cells. The other cell lines failed to develop a TER even after 2 weeks in culture. There was a direct correlation between TER and ability to restrict paracellular movement of 14C-mannitol. Labeling studies showed that the LLC-PK1 and NRK-52E cells expressed high levels of E-cadherin while the HRPTE cells expressed lower levels. OK cells expressed moderate levels of N-cadherin while LLC-PK1 and NRK-52E cells expressed lower levels in isolated patches of cells. All cell lines expressed moderate-high levels of ZO-1. LLC-PK1 also expressed the tight-junction proteins occludin and claudin-7; OK cells also expressed moderate levels of occludin. All other cell lines had weak claudin-7 and occludin labeling. None of the cell lines expressed claudin-2. These results show that the LLC-PK1, OK and NRK-52E cell lines exhibit characteristics that most closely resembled those of the proximal tubule in vivo, and they indicate that these cell lines would be appropriate models for studying the effects of toxicants on cell-cell junctions and cell adhesion molecules.     

Arsenic trioxide-induced apoptosis in H9c2 cardiomyocytes: implications in cardiotoxicity

Arsenic trioxide (As(2)O(3)) achieved dramatic remissions in patients with acute promyelocytic leukaemia. Clinical reports have shown that treatment was associated with cardiotoxicity. We investigated the toxic mechanisms of As(2)O(3) in H9c2 cardiomyocytes. Clinically relevant concentrations of As(2)O(3) (2-10 microM) reduced the viability of H9c2 cells in a concentration-dependent manner. The decreased cell viability was because As(2)O(3) induced cell apoptosis (cell shrinkage, nuclear alterations and caspase-3 activation), or even necrosis at higher concentrations. Inhibition of caspase-3 with a specific inhibitor, Ac-DEVD-CHO, suppressed apoptosis induced by As(2)O(3). In addition, reactive oxygen species formation and cellular Ca(2+) overload were observed in H9c2 cells exposed to As(2)O(3), which was partly inhibited by vitamin E and verapamil. These results suggest that As(2)O(3)-induced cardiotoxicity is mediated, at least in part, by activation of caspase-3 pathway, which may be triggered by reactive oxygen species formation and intracellular Ca(2+) overload.     

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.     

Urothelial cells malignantly transformed by exposure to cadmium (Cd(+2)) and arsenite (As(+3)) have increased resistance to Cd(+2) and As(+3)-induced cell death.

This laboratory has shown that both Cd(+2) and As(+3) can malignantly transform human urothelial cells. The present study examined metal resistance and the mechanism of cell death when the parental and malignantly transformed UROtsa cells were exposed to Cd(+2) and As(+3). It was shown that the malignantly transformed UROtsa cells were more resistant to the toxic effects of both metals. The assessment of the mode of cell death demonstrated that the parental UROtsa cells died by both apoptosis and necrosis when exposed to either metal. It was shown that apoptosis was the more prominent mechanism of cell death, accounting for over 50% of cell death. Apoptotic cell death was determined by the observation of fragmented nuclei using 4',6-diamidino-2-phenylindole staining, the formation of a DNA ladder, and the detection of cleaved caspase-3 and caspase-9 products in the cell lysates. Necrotic cell death was determined by measuring the release of lactate dehydrogenase into the growth medium. It was determined that the extent of apoptosis of the malignantly transformed UROtsa cells was decreased and that the extent of necrosis was increased compared to the parental UROtsa cells. These observations are consistent with in vivo studies which suggest that As(+3) can act as a tumor promoter during the regeneration of the bladder urothelium. The present in vitro studies suggest that As(+3)-induced cytotoxicity could set the stage for tissue repair due to its own inherent toxicity to normal urothelium, and then subsequently act as a tumor promoter during the regeneration process through the stimulation of the regrowth of cells that have gained increased resistance to As(+3).     

Cadmium induces caspase-mediated cell death: suppression by Bcl-2

Apoptosis is a process of active cell death and is characterized by activation of caspases, DNA fragmentation, and biochemical and morphological changes. To better understand apoptosis, we have characterized the dose- and time-dependent toxic effects of cadmium in Rat-1 fibroblasts. Staining of cells with phosphatidylserine (PS)-annexin V, Hoechst 33258 or Rhodamine 123 and Tunel assays showed that incubating cells with 10 microM cadmium induced a form of cell death exhibiting typical characteristics of apoptosis, including cell shrinkage, externalization of PS, loss of mitochondria membrane potential, nuclear condensation and DNA fragmentation. Expression of Bcl-2 or CrmA each suppressed cadmium-induced cell death although Bcl-2 was somewhat more effective than CrmA. In vitro assay of caspase activity carried out using poly(ADP-ribose) polymerase (PARP) as a substrate as well as intracellular caspase assays using a fluorigenic caspase-3 substrate confirmed that caspase-3 is activated in Rat-1 cells undergoing cadmium-induced apoptosis. Both Asp-Glu-Val-Asp-aldehyde (DEVD-cho) and Tyr-Val-Ala-Asp-chloromethylketone (YVAD-cmk), selective inhibitors of caspase-3 and caspase-1, respectively, suppressed significantly cadmium-induced cell death. However, the nonselective caspase inhibitor, z-Val-Ala-Asp-floromethylketone (zVAD-fmk), was the most efficacious agent, almost completely blocking cadmium-induced cell death. Taken together, these results demonstrate that as in other forms of apoptosis, caspases play a central role in cadmium-induced cell death.