Human Renal Tubular Cell Line Research Articles

SARS-CoV-2 viral protein ORF3A injures renal tubules by interacting with TRIM59 to induce STAT3 activation

Acute kidney injury occurs frequently in COVID-19 patients infected by the coronavirus SARS-CoV-2, and infection of kidney cells by this virus has been reported. However, little is known about the direct impact of the SARS-CoV-2 infection upon the renal tubular cells. We report that SARS-CoV-2 activated signal transducer and activator of transcription 3 (STAT3) signaling and caused cellular injury in the human renal tubular cell line. Mechanistically, the viral protein ORF3A of SARS-CoV-2 augmented both NF-κB and STAT3 signaling and increased the expression of kidney injury molecule 1. SARS-CoV-2 infection or expression of ORF3A alone elevated the protein level of tripartite motif-containing protein 59 (TRIM59), an E3 ubiquitin ligase, which interacts with both ORF3A and STAT3. The excessive TRIM59 in turn dissociated the phosphatase TCPTP from binding to STAT3 and hence inhibited the dephosphorylation of STAT3, leading to persistent STAT3 activation. Consistently, ORF3A induced renal injury in zebrafish and mice. In addition, expression of TRIM59 was elevated in the kidney autopsies of COVID-19 patients with acute kidney injury. Thus, the aberrant activation of STAT3 signaling by TRIM59 plays a significant role in the renal tubular cell injury caused by SARS-CoV-2, which suggests a potential targeted therapy for the renal complications of COVID-19.

Loss of sphingosine kinase 2 protects against cisplatin-induced kidney injury.

Cisplatin is an established chemotherapeutic drug for treatment of solid-organ cancers and is the primary drug used in the treatment of head and neck cancer; however, cisplatin-induced nephrotoxicity largely limits its clinical use. Inhibition of sphingosine kinase 2 (SphK2) has been demonstrated to alleviate various kidney diseases. Therefore, we hypothesized that inhibition of SphK2 could also protect against cisplatin-induced nephrotoxicity. Results from the present study showed that the SphK2 inhibitor ABC294640 or knockdown of SphK2 by siRNA blocked the cisplatin-induced increase of cellular injury markers (neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, and cleaved caspase-3) by Western blot analysis in HK-2 cells, a human renal tubular cell line. In addition, SphK2 inhibition blocked cisplatin-induced activation of NF-κB by Western blot analysis and immunostaining analysis. Furthermore, SphK2 inhibition suppressed cisplatin-induced increases of proinflammatory markers (NLR family pyrin domain containing 3, interleukin-1β, and interleukin-6). Genetic deletion of the SphK2 gene in mice further confirmed that inhibition of SphK2 protected against cisplatin-induced kidney damage in vivo. Compared with wild-type mice, SphK2 knockout mice exhibited less renal dysfunction and reduced promotion of kidney injury markers, inflammatory factors, tubular morphology damage, and fibrotic staining. At the same time, the SphK2 inhibitor ABC294640 failed to interfere with the activity of cisplatin or radiation in two cell culture models of head and neck cancer. It is concluded that inhibition of Sphk2 protects against cisplatin-induced kidney injury. SphK2 may be used as a potential therapeutic target for the prevention or treatment of cisplatin-induced kidney injury.NEW & NOTEWORTHY The present study provides new findings that sphingosine kinase 2 (SphK2) is highly expressed in renal tubules, cisplatin treatment increases the expression of SphK2 in proximal tubular cells and kidneys, and inhibition of SphK2 alleviates cisplatin-induced kidney injury by suppressing the activation of NF-κB, production of inflammatory factors, and apoptosis. SphK2 may serve as a potential therapeutic target for the prevention or treatment of cisplatin-induced nephrotoxicity.

Blockade of Autophagy Prevents the Progression of Hyperuricemic Nephropathy Through Inhibiting NLRP3 Inflammasome-Mediated Pyroptosis.

Hyperuricemia has become a common metabolic disease, and is a risk factor for multiple diseases, including chronic kidney disease. Our recent study indicated that following persistent uric acid stimulation, autophagy was activated in rats of hyperuricemic nephropathy (HN) and facilitated the development of renal fibrosis. Nevertheless, the potential mechanism by which autophagy promoted the progression of HN is still not fully elucidated. Thus, in the current study, we investigated the mechanisms of autophagy inhibition on the development of HN. Our data showed that autophagy was activated in human renal tubular cell lines (HK-2) exposure to uric acid. Inhibition of autophagy with 3-methyladenine (3-MA) and transfected with Beclin-1 siRNA prevented uric acid-induced upregulation of α-SMA, Collagen I and Collagen III in HK-2 cells. Moreover, uric acid upregulated autophagy via promoting the p53 pathway. In vivo, we showed that hyperuricemic injury induced the activation of NLRP3 inflammasome and pyroptosis, as evidenced by cleavage of caspase-1 and caspase-11, activation of gasdermin D (GSDMD) and the release of IL-1β and IL-18. Treatment with autophagy inhibitor 3-MA alleviated aforementioned phenomenon. Stimulation with uric acid in HK-2 cells also resulted in NLRP3 inflammasome activation and pyroptotic cell death, however treatment with 3-MA prevented all these responses. Mechanistically, we showed that the elevation of autophagy and degradation of autophagolysosomes resulted in the release of cathepsin B (CTSB), which is related to the activation of NLRP3 inflammasome. CTSB siRNA can inhibit the activation of NLRP3 inflammasome and pyroptosis. Collectively, our results indicate that autophagy inhibition protects against HN through inhibiting NLRP3 inflammasome-mediated pyroptosis. What’s more, blockade the release of CTSB plays a crucial role in this process. Thus, inhibition of autophagy may be a promising therapeutic strategy for hyperuricemic nephropathy.

LncRNA OIP5-AS1 reduces renal epithelial cell apoptosis in cisplatin-induced AKI by regulating the miR-144-5p/PKM2 axis

BackgroundThe abnormal expression of long non-coding RNA (lncRNA) Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) has been observed in many human cancers and the underlying mechanisms have been well studied. However, the function of OIP5-AS1 in acute kidney injury (AKI) remains unclear. MethodsTo explore the role of OIP5-AS1 in the progression of AKI, the cisplatin-induced AKI mouse and cell model were established. To confirm the potential protective effect of OIP5-AS1 during cisplatin-induced AKI, rescue experiments were performed. Targetscan was used to predict the potential targets of miR-144-5p. To further determine whether the effect of miR-144-5p during cisplatin-induced AKI was mediated by PMK2, the recuse experiments using PMK2 overexpressing vector was applied. ResultsOIP5-AS1 was significantly downregulated both in cisplatin-induced AKI mice and human renal tubular cell line HK-2 cells. Moreover, overexpression of OIP5-AS1 efficiently promoted cell growth and reduced cisplatin-induced apoptosis of HK-2 cells. Furthermore, OIP5-AS1 was identified as a sponge of miR-144-5p, and upregulation of miR-144-5p could significantly reverse overexpression of OIP5-AS1-induced protective effect on the damage of cisplatin to HK-2 cells. In addition, pyruvate kinase M2 (PKM2) was found to be a direct target of miR-144-5p, and overexpression of PKM2 efficiently reversed the effect of miR-144-5p mimics on the damage in cisplatin-stimulated HK-2 cells. ConclusionsOIP5-AS1 reduced the apoptosis of cisplatin-stimulated renal epithelial cells by targeting the miR-144-5p/PKM2 axis, which extended the regulatory network of lncRNAs in cisplatin-induced AKI and also provided a novel therapeutic target for AKI treatment.

Phloretin ameliorates hyperuricemia-induced chronic renal dysfunction through inhibiting NLRP3 inflammasome and uric acid reabsorption

BackgroundHyperuricemia (HUA) is an important risk factor for renal diseases and contributes to renal fibrosis. It has been proved that phloretin has antioxidant and anti-inflammatory properties and could inhibit uric acid (UA) uptake in vitro. However, whether phloretin has a renal protective role in vivo remains unknown. PurposeThis study aims to evaluate the therapeutic effect of phloretin on HUA-induced renal injury in mice and to reveal its underlying mechanism. MethodsMice were induced hyperuricemic by oral gavage of adenine/potassium oxonate. The effects of phloretin on renal function, fibrosis, oxidative stress, inflammation, and UA metabolism in HUA mice were evaluated. The effect of phloretin on NLRP3 pathway was analyzed in human renal tubular cell lines (HK-2). ResultsHUA mice showed renal dysfunction with increased renal fibrosis, inflammation and mitochondrial stress. By contrast, phloretin reduced the level of serum blood urea nitrogen (BUN), urinary albumin to creatinine ratio (UACR), tubular necrosis, extracellular matrix (ECM) deposition and interstitial fibroblasts in HUA mice. The renal infiltration of inflammatory cells, cytokines such as NOD-like receptor family pyrin domain containing 3 (NLRP3) and interleukin-1β (IL-1β) release, mitochondrial reactive oxygen species (ROS) and morphological lesions in HUA mice also decreased. Furthermore, phloretin partly inhibited renal glucose transporter 9 (GLUT9) and promoted urinary UA excretion in HUA mice. In vitro, phloretin suppressed the NLPR3 pathway under LPS or UA stimulation in HK-2 cells. ConclusionsPhloretin could effectively attenuate UA-induced renal injury via co-inhibiting NLRP3 and UA reabsorption, and thus it might be a potential therapy to hyperuricemia-related renal diseases.

Tanshinone IIA Attenuates Contrast-Induced Nephropathy via Nrf2 Activation in Rats

Background/Aims: Tanshinone IIA is a chemical compound extracted from Salvia miltiorrhiza Bunge, a perennial plant also known as red sage used in traditional Chinese medicine. Tanshinone IIA has been shown to protect against various organ injuries. In this study, we hypothesized that Tanshinone IIA could play an anti-oxidative role in contrast-induced nephropathy (CIN) through enhancing Nrf2/ARE activation. Methods: To test whether Tanshinone IIA can attenuate CIN, oxidative stress, and apoptosis, we utilized two models: an in vivo Sprague-Dawley rat model of ioversol-induced CIN and an in vitro cell model of oxidative stress in which HK2 cells, a human renal tubular cell line, are treated with hydrogen peroxide (H₂O₂). Rats were randomly assigned to 4 groups (n = 6 per group): control group, ioversol group (ioversol-induced CIN), vehicle group (ioversol-induced CIN rats pretreated with vehicle), and Tanshinone IIA group (ioversol-induced CIN rats pretreated with 25mg/kg Tanshinone IIA). Renal functions, renal injuries and apoptosis were evaluated by using serum creatinine, histological scoring, and TUNEL staning respectively. Malondialdehyde, 8-hydroxy-2’ –deoxyguanosine, and intracellular reactive oxygen species were used for oxidative stress assessment. Levels of Nrf2 and heme oxygenase-1 (HO-1) were measured in vivo and in vitro. Results: Tanshinone IIA attenuated renal tubular necrosis, apoptosis and oxidative stress in rats and oxidative stress in HK2 cells. Furthermore, Tanshinone IIA activated Nrf2, and up-regulated HO-1 expression in vivo and in vitro, resulting in a reduction in oxidative stress. Conclusion: Tanshinone IIA may protect against CIN through enhancing Nrf2/ARE activation.

Inhibitory effect of transforming growth factor-β1 on expression of N-myc downstream regulatory gene 2 in human renal tubular epithelial cell line HK-2 and its significance

Objective To investigate the effect of transforming growth factor-β1 (TGF-β1) on the expression of N-myc downstream regulatory gene 2 (NDRG2) in human renal tubular epithelial cell line HK-2. Methods The control group and the experimental group were set up according to whether or not the TGF-β1 was incubated. Western blotting and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) were applied to detect the expression of E-cadherin, α-smooth muscle actin (α-SMA), Vimentin and Snail protein in epithelial cells. The expression of NDRG2 in human renal tubular epithelial cells (HK-2) treated with different concentrations of TGF-β1 was detected by Western blotting and RT-qPCR. Results The results showed that the protein expression level expression of E-cadherin in the control group and the experimental group was (1.0±0.2) vs. (0.3±0.2) (t=4.850, P=0.008), that of α-SMA was (1.0±0.1) vs. (2.6±0.4) (t=6.721, P=0.003), that of Vimentin was (1.0±0.1) vs. (2.6±0.4) (t=6.721, P=0.003), and that of Snail was (1.0±0.1) vs. (2.5±0.2) (t=11.620, P=0.000). In the control group and the experimental group, the mRNA expression level of E-cadherin was (1.0±0.2) vs. (0.2±0.1) (t=7.686, P=0.002), that of α-SMA was (1.0±0.3) vs. (2.4±0.3) (t=5.715, P=0.005), that of Vimentin was (1.1±0.15) vs. (2.6±0.5) (t=4.977, P=0.008), and that of Snail was (0.9±0.2) vs. (1.9±0.2) (t=6.124, P=0.004). At the same time, after incubation with TGF-β1, NDRG2 in HK-2 cells was significantly down-regulated at both protein and mRNA levels. The levels of protein expression in each group were (1.00±0.04 vs. 0.80±0.03 vs. 0.50±0.05 vs. 0.30±0.03 vs. 0.20±0.02) (P=0.019), and mRNA expression in each group were (1.00±0.05 vs. 0.70±0.03 vs. 0.50±0.04 vs. 0.30±0.02 vs. 0.20±0.05) (P=0.021). Especially when incubated with TGF-β1 concentration greater than 10 ng/ml, the difference was statistically significant. Conclusion TGF-β1 can promote the process of EMT in HK-2 cells, and TGF-β1 can down-regulate the expression of NDRG2 in protein and RNA in HK-2 cells. Key words: N-myc downstream regulatory gene 2; Transforming growth factor-β1; Renal tubular epithelial cells; Epithelial-mesenchymal transition

14-3-3ζ-Mediated Stimulation of Oxidative Phosphorylation Exacerbates Oxidative Damage Under Hypothermic Oxygenated Conditions in Human Renal Tubular Cells (HK-2)

Cellular survival and death are at least partially regulated by the phosphorylation of proteins. A chaperon protein, 14-3-3ζ, regulates the activity of many proteins by covering the phosphorylation site within a 14-3-3 binding motif. Therefore, regulation of 14-3-3ζ activity may affect the fate of cells subjected to cold preservation and/or hypothermic oxygenated conditions. The present study assessed whether 14-3-3ζ protects cells from hypothermic oxygenation-induced injury and clarified its role in mitochondrial functions. Human renal tubular cell line HK-2 or 14-3-3ζ-overexpressed HK-2 (ζHK-2) cells were subjected to 72 hours of normoxic cold preservation in UW solution with or without antioxidants and hydroperoxides. Cellular death, adenosine triphosphate (ATP) content, and MTT catabolism were evaluated. Deferoxamine treatment reduced cellular death and augmented ATP content in both cell types. These indices were higher in ζHK-2, regardless of deferoxamine treatment. Exposure to hydroperoxides did not affect cellular death in either cell type, whereas hydroperoxide supplementation significantly reduced ATP content, except for low-dose hydrogen peroxide in HK-2 cells. MTT assay at normal state showed higher values in ζHK-2 cells, whereas it was impaired by hydroperoxides in both cell types. These results suggest that accumulation of hydroperoxides as a byproduct of the augmented oxidative phosphorylation by 14-3-3ζ overexpression causes mitochondrial dysfunction. In conclusion, despite possessing many potentially protective functions, 14-3-3ζ exacerbates cellular injury under hypothermic oxygenated conditions. 14-3-3ζ accelerates mitochondrial functions together with iron-dependent oxidative damage. Although further investigations are necessary, upregulation of 14-3-3ζ could be a method to maintain mitochondrial function under hypothermic oxygenated conditions, as shown in hypothermic machine preservation of renal grafts, when appropriate antioxidant treatment is administered.

Research of Anti-Cancer Components in Traditional Chinese Medicine on Hollow Fibre Cell Fishing and Hollow Fibre Liquid Phase Microextraction

Hollow fibre cell fishing with HPLC (HFCF-HPLC) based on the human ovarian cancer cell line SKOV-3, human renal tubular cell line ACHN or hepatoma cell line HepG-2 was employed to screen active groups of coumarin and volatile oil in Radix angelicae sinensis, Radix angelicae dahuricae and Fructus citri sarcodactylis. Simultaneously, hollow fibre liquid phase microextraction with HPLC (HFLPME-HPLC) was conducted to enrich and determine the contents of active components in the same sample solution. Before application, for HFCF-HPLC, cells growth states and survival rates on the fibre, effect of ethanol concentration in the extract of samples on cell survival rates, non-specific binding between fibre active centres and the target components, positive and negative controls and repeatabilities were validated; for HFLPME, extraction solvent, sample phase pH, agitation speed, extraction time and sample phase volume were investigated. Many active components were screened from three medicines. Some of them, such as scoparone, psoralen, bergapten, oxypeucedanin, imperatorin, ligustilide, were identified by MS. The target fishing factors of active components and the cell apoptosis rates of three cells under the medicines effect were researched. The binding sites of active groups on HepG-2 cells were preliminarily determined. The results demonstrated that HFCF-HPLC, coupled with HFLPME-HPLC, is a simple and universal approach to find bioactive components at the cellular level, determine their content and research traditional Chinese medicines (TCMs) entirety effect of multi-component and multi-target. The approach may provide us a new and good solution to clarify the material basis of anti-cancer effect and conduct personalized quality control for the components associated with efficacy in TCMs.

The study of cytotoxic effect to creatinine metabolite on HK-2 cells in vitro

Objective To investigate the effect of methylguanidine and 1-methylhydantoin on cells cytotoxicity, apoptosis in human renal tubular epithelial cell line (HK-2). Methods Human PTEC cell line HK-2 was used in this study. HK-2 was cultured and divided into 3 groups: Norma1 control group (A), methylguanidine group(B) and 1-methylhydantoin group (C). The cell inhibitory rate of HK-2 was detected by MTT method. The cytotoxicity of methylguanidine to HK-2 was determined by NAG release test. Cell apoptosis was evaluated by using Hoechst stain and FACS with Annexin-V/PI. Results The OD value and NAG concentration of creatinine, methylguanidine and 1-methylhydantoin group were compared with normal control group. OD value decreased and NAG concentration significantly increased(0.188±0.011, 0.176±0.010 vs 0.545±0.021, F＝1557.74, P＜0.01; 20.488±0.473, 22.225±0.565 vs 5.125±0.198, F＝3848.22, P＜0.01). By Hoechst stain, pycnosis and apoptotic body could be found when HK-2 was cultivated in methylguanidine 1-methylhydantoin group. In methylguanidine, 1-methylhydantoin group apoptotic HK-2 apparently increased, compared with that in control group (18.23±1.1581, 20.22±1.1433 vs 2.473±0.321, F＝526.06, P＜0.01). Compared with group B, the OD value in group C decreased significantly (0.176±0.010 vs 0.188±0.011,t＝2.26, P＜0.05), NAG concentration increased significantly (22.225±0.565 vs 20.488±0.473,t＝-6.67, P＜0.01), and apoptotic rate in-creased significantly (20.22±1.1433 vs 18.23±1.1581,t＝-2.762, P＜0.05). Conclusions 1-methylhydantoin has more powerful cytotoxic effect to renal tubular epithelial cells than that of Methylguanidine. Key words: Methylguanidine/AE; Creatinine/AE; Kidney tubules/CY; Epithelial cells/DE/PA

NF–κB activation in renal tubular epithelial cells by oxalate stimulation

The transcription factor nuclear factor-kappaB (NF-kappaB) is involved in inflammatory and immune responses through the induction of various cytokines and growth factors. Recently, the coordinated action of NF-kappaB and activator protein-1 was reported in osteopontin (OPN) expression. In the present study, we demonstrated that oxalate induces OPN expression by activating NF-kappaB in renal tubular cells. Furthermore, we investigated the inhibitory effect of N-acetyl-L-cysteine (NAC) on NF-kappaB activation in the human renal tubular cell line. All of the experiments were carried out using human kidney-2 cells, which are human proximal tubular epithelial cells immortalized by transduction with the human papillomavirus 16E6/E7 gene. The time-dependent extraction of total protein was performed after the uptake of 0.5 mM oxalate by the cells. The NF-kappaB activation and OPN expression were examined by western blotting and immunocytochemistry. As a result of oxalate stimulation, the amount of p65 subunit in the nucleus increased significantly (P < 0.05), and NAC significantly inhibited the translocation of p65 into the nucleus (P < 0.05). These observations indicate that NAC can be used as a drug to prevent stone formation.

Influence of endothelin on local aldosterone production synthesized by human renal tubular cells

To observe whether local aldosterone (ALDO) may be synthesized by human renal proximal tubular cell lines (HKC) and investigate whether endothelin-1 (ET-1) has effects on the production of local ALDO. The following tests were performed: (1) stimulative tests: after the HKC cells were incubated with ET-1 in different concentration (0, 10(-11), 10(-9), 10(-7) mol/L) or for different time (0, 6, 12, 24, 48 h), the levels of ALDO and angiotensin II (AngII) in the media were measured by radioimmunoassay. (2) inhibitive test: after the HKC cells were incubated with different concentration of angiotensin converting enzyme inhibitor (ACEI) (0, 10(-11), 10(-9), 10(-7), 10(-5) mol/L) and ET-1 (10(-7) mol/L), or incubated with ACEI (10(-5) mol/L) and ET-1 (10(-7) mol/L) for different time (0, 6, 12, 24, 48 h), the levels of ALDO and AngII in the media were detected by radioimmunoassay. (1) Both ALDO and AngII at a basic quantity were produced by HKC cells without any stimulants. After stimulation with ET-1, the production of ALDO and AngII was increased with a dose and time dependent manner. When the HKC cells were incubated with 10(-9) and 10(-7) mol/L ET-1 for 48 h, the levels of ALDO and AngII were significantly higher than those with 0 mol/L ET-1 (P < 0.05 or P < 0.01). When the HKC cells were incubated with 10(-7) mol/L ET-1 in different time, the levels of ALDO and AngII in 12, 24 and 48 h were significantly higher than those in 0 h (P < 0.05 or P < 0.01). (2) when the HKC cells were incubated with 10(-7) mol/L ET-1 and ACEI in different concentration for 48 h, the production of AngII and ALDO was significantly decreased in 10(-9), 10(-7) and 10(-5) mol/L ACEI (vs 0 mol/L ACEI, P < 0.01). Although the AngII production was completely blocked by these concentration of ACEI (vs the basic quantity of AngII, P > 0.05), the ALDO production was not (vs the basic quantity of ALDO, P < 0.05). When the HKC cells were incubated with 10(-7) mol/L ET-1 and 10(-5) mol/L ACEI for different time, the production of AngII and ALDO was completely blocked in 6 h and 12 h (vs 0 h, P > 0.05). Although the AngII production was still completely blocked in 24 h and 48 h (vs 0 h, P > 0.05), the ALDO production was not (vs 0 h, P < 0.05). Local aldosterone can be synthesized by human renal proximal tubular cells. Its production can be up-regulated by ET-1. This effect is partially mediated by AngII.

Raft.1, A Monoclonal Antibody Raised Against the Raft Microdomain, Recognizes G-Protein β1 and 2, Which Assemble Near Nucleus After Shiga Toxin Binding to Human Renal Cell Line

Raft microdomains are glycolipid-enriched microdomain scaffolding molecules involved in signal transduction. The binding of Shiga toxin to globotriaosyl ceramide in raft microdomains of the human renal tubular cell line ACHN causes temporal activation of Src-kinase Yes. To study the downstream signaling mechanism proceeding to the activation of Yes, we raised monoclonal antibodies (MAbs) against raft microdomains. The MAbs were screened on the basis of, first, binding to raft microdomains with dot-blot immunostaining, second, intracellular localization of the epitope by flowcytometry after permeabilization, and third, translocation of the antigen molecules after Stx treatment by immunohistochemical staining. Raft.1 MAb bound to the molecules that accumulated to the particular region near the nucleus after Stx treatment. Two-dimensional Western blotting and matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis revealed that the antigen molecule is GTP binding protein β subunits 1 and 2 (Gβ1 and 2). That Raft.1 recognized Gβ1 and 2 was further confirmed by the reactivity to recombinant Gβ1 and 2 proteins. To our knowledge, this is the first report of production of a MAb recognizing Gβ1 and 2. Because Gβ1 and 2 are highly conserved all through organisms and are deeply involved in signal transduction, Raft.1 is expected to be utilized frequently in research.

The Effect of Shiga Toxin Binding to Globotriaosylceramide in Rafts of Human Kidney Cells and Burkitt's Lymphoma Ramos Cells

Shiga toxin, an enterotoxin produced from Shigella dysenteriae serotype and enterohemorrhagic Escherichia coli binds specifically to globotriaosylceramide, Gb3, on the cell surface and causes cell death. Stx is shown to induce apoptosis in several cell lines, including human renal tubular cell line ACHN and Burkitt's lymphoma Ramos cells. In order to study the early signal transduction after Stx addition, Gb3-enriched microdomains were prepared from these cells by sucrose density gradient centrifugation of Triton X-100 lysate as buoyant, detergent-insoluble microdomain, rafts. Gb3 was only recovered in rafts and associated with Src family kinase Yes or Lyn. Tyrosine residues of raft proteins were hyperphosphorylated in 10min and turned to resting level in 30min after Stx addition. Yes was thought to be responsible for hyperphosphorylation observed in raft proteins, because Yes activity increased in 3-10min after Stx addition. Unexpectedly, however, all of the Yes activity was obtained in high-density, detergent-soluble fraction, but not in rafts. Stx was known to be internalized as complex with Gb3 via retrograde endocytosis. Therefore, Yes was also thought to enter into intracellular space with Stx/Gb3 complex, where Yes was activated and increased solubility for Triton X-100. Since Stx B subunit could alone induce Yes activation, the binding of Stx to Gb3 might cause temporal activation of Yes in DS in the process of apoptosis.

Prominent immunogenicity of monosialosyl galactosylgloboside, carrying a stage-specific embryonic antigen-4 (SSEA-4) epitope in the ACHN human renal tubular cell line-a simple method for producing monoclonal antibodies against detergent-insoluble microdomains/raft.

The binding of Shiga toxin (Stx) to Gb3Cer in detergent-insoluble microdomains (DIM)/raft of the ACHN human renal tubular cell line causes the temporal activation of the Src-family kinase Yes [1]. As a strategy for examining signaling mechanisms in DIM/raft, monoclonal antibodies (MAbs) are reliable tools for characterizing the constituent molecules in these microdomains. Thus, we employed DIM/raft suspensions of ACHN cells as an immunogen to develop MAbs. Simply subcutaneous injections of ACHN DIM/raft could elevate the serum titer after several boosts. The first screening was performed using dot-blot immunostaining with culture supernatants on a polyvinylidene difluoride (PVDF) membrane, on which DIM/raft or their chloroform/methanol (C/M) (2:1, v/v) extracts were dot-blotted. The next screening was performed by flowcytometric analysis of ACHN cells treated with or without a permeabilizing reagent. Many of the clones (21/31 clones=68%) thus obtained were also found to recognize to lipid fractions of the DIM/raft. Strikingly, all of the 21 clones that reacted to the lipid fraction were found to recognize monosialosyl galactosylgloboside (MSGG) or GL7, which carries the SSEA-4 epitope. Using DIM/raft as immunogens may enable us to easily obtain MAbs for glycolipids.

Activation of Src Family Kinase Yes Induced by Shiga Toxin Binding to Globotriaosyl Ceramide (Gb3/CD77) in Low Density, Detergent-insoluble Microdomains

Shiga toxin (Stx) is an enterotoxin produced by Shigella dysenteriae serotype 1 and enterohemorrhagic Escherichia coli, which binds specifically to globotriaosylceramide, Gb3, on the cell surface and causes cell death. We previously demonstrated that Stx induced apoptosis in human renal tubular cell line ACHN cells (Taguchi, T., Uchida, H., Kiyokawa, N., Mori, T., Sato, N., Horie, H., Takeda, T and Fujimoto, J. (1998) Kidney Int. 53, 1681-1688). To study the early signal transduction after Stx addition, Gb3-enriched microdomains were prepared from ACHN cells by sucrose density gradient centrifugation of Triton X-100 lysate as buoyant, detergent-insoluble microdomains (DIM). Gb3 was only recovered in DIM and was associated with Src family kinase Yes. Phosphorylation of tyrosine residues of proteins in the DIM fraction increased by 10 min and returned to the resting level by 30 min after the addition of Stx. Since the kinase activity of Yes changed with the same kinetics, Yes was thought to be responsible for the hyperphosphorylation observed in DIM proteins. Unexpectedly, however, all of the Yes kinase activity was obtained in the high density, detergent-soluble fraction. Yes was assumed to be activated and show increased Triton X-100 solubility in the early phase of retrograde endocytosis of Stx-Gb3 complex. Since Yes activation by the Stx addition was suppressed by filipin pretreatment, Gb3-enriched microdomains containing cholesterol were deeply involved in Stx signal transduction.