Possibility of culturing the early developing kidney cells by utilizing simulated microgravity environment.

In recent years, the successful construction of tissues derived from established iPSCs has been disclosed, but it has been reported that the constructed tissues encounter problems of internal necrosis when their size increases. To solve this problem, a simulated microgravity device is used. However, the culture of early developing kidney cells using this device has not yet been reported. This study investigated whether developing kidney cells cultured in a simulated microgravity environment can differentiate into glomerular cells and renal epithelial cells. The results showed that both mouse developing kidney cells cultured in simulated microgravity and static environment formed kidney spheroids. In static culture, ureteric bud and glomerular structures were not found. While ureteric buds, podocytes, PECAM-1 positive cell aggregates, and primordial vascular plexus were formed in the kidney spheroids in simulated microgravity culture. Moreover, the expression level of the PECAM-1 gene was significant in simulated microgravity culture as compared to that of static culture. These results indicate that simulated microgravity is effective for the differentiation of developing kidney cells.

Change in Renal Glomerular Collagens and Glomerular Filtration Barrier-Related Proteins in a Dextran Sulfate Sodium-Induced Colitis Mouse Model.

Renal disease is not rare among patients with inflammatory bowel disease (IBD) and is gaining interest as a target of research. However, related changes in glomerular structural have rarely been investigated. This study was aimed at clarifying the changes in collagens and glomerular filtration barrier (GFB)-related proteins of glomeruli in a dextran sulfate sodium (DSS)-induced colitis mouse model. Acute colitis was induced by administering 3.5% DSS in Slc:ICR strain mice for eight days. Histological changes to glomeruli were examined by periodic acid-Schiff (PAS) and Masson's trichrome staining. Expressions of glomerular collagens and GFB-related proteins were analyzed by immunofluorescent staining and Western blot analysis. DSS-colitis mice showed an elevated disease activity index (DAI), colon shortening, massive cellular infiltration and colon damage, confirming that DSS-colitis mice can be used as an IBD animal model. DSS-colitis mice showed increased glycoprotein and collagen deposition in glomeruli. Interestingly, we observed significant changes in glomerular collagens, including a decrease in type IV collagen, and an increment in type I and type V collagens. Moreover, declined GFB-related proteins expressions were detected, including synaptopodin, podocalyxin, nephrin and VE-cadherin. These results suggest that renal disease in DSS-colitis mice might be associated with changes in glomerular collagens and GFB-related proteins. These findings are important for further elucidation of the clinical pathological mechanisms underlying IBD-associated renal disease.

A novel autoantibody against moesin in the serum of patients with MPO-ANCA-associated vasculitis.

BACKGROUND: Antineutrophil cytoplasmic autoantibody (ANCA) directed against myeloperoxidase (MPO), a diagnostic criterion in MPO-ANCA-associated vasculitis (MPO-AAV), does not always correlate with disease activity. Here, we detected autoantibodies against moesin, which was located on the surface of stimulated endothelial cells, in the serum of patients. METHODS: The anti-moesin autoantibody titer was evaluated by ELISA. Seventeen kinds of cytokines/chemokines were measured by a Bio-Plex system. RESULTS: Serum creatinine in the anti-moesin autoantibody-positive group was higher than that in the negative group. Additionally, interferon (IFN)-γ, macrophage chemotactic peptide-1 (MCP-1), interleukin (IL)-2, IL-7, IL-12p70, IL-13, granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor were significantly higher in the positive group. Furthermore, IL-7 and IL-12p70 levels correlated with the anti-moesin autoantibody titer. Based on these findings and the binding of anti-moesin IgG to neutrophils and monocytes, we detected the secretion of cytokines/chemokines such as IFN-γ, MCP-1 and GM-CSF from these cells. CONCLUSIONS: The anti-moesin autoantibody existed in the serum of patients with MPO-AAV and was associated with the production of inflammatory cytokines/chemokines targeting neutrophils with a cytoplasmic profile, which suggests that the anti-moesin autoantibody has the possibility to be a novel autoantibody developing vasculitis via neutrophil and endothelial cell activation.

Type V collagen fibrils in mouse metanephroi.

Type V collagen (Col V) molecule, a minor component of kidney connective tissues, was found in adult cornea, and has been considered as a regulatory fibril-forming collagen that emerges into type I collagen to trigger the initiation of Col I fiber assembly. Col V was also found in injured, wound healing tissues or placenta, and was considered as a dysfunctional extracellular matrix (ECM). Reconstituted Col V fibril was characterized as an ECM to detach cells in vitro, and our previous study showed that the reconstituted Col V fibril facilitated the migration of glomerular endothelial cells and induced ECM remodeling, whereas Col V molecules stabilized cells. These facts suggest that not only the structure but also the function of Col V fibril are different from Col V molecule. Recently, Col V molecule has been reported existing in various developing tissues such as bone and lung, but Col V fibril has not been reported yet. In this study, we firstly explored the existence of Col V fibril in metanephroi, and found it distributed in the immature kidney tissues whereas disappeared when the tissues reached mature. It is likely that Col V fibril may form a prototype of pericellular microenvironment and the transient existence of Col V fibril may play a role as the pioneering ECM during metanephric tissue morphogenesis.

Direct activation of glomerular endothelial cells by anti-moesin activity of anti-myeloperoxidase antibody.

BACKGROUND: Glomerular neutrophil infiltration has been thought to be a key pathological event in the development of myeloperoxidase (MPO)-specific anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis involving glomerulonephritis. Accordingly, we sought to explore the molecules responsible for glomerular neutrophil accumulation. METHODS: Glomerular neutrophil infiltration and renal chemokine expression in mice treated with anti-MPO IgG were evaluated. Chemokine expression in vitro induced by anti-MPO IgG was measured in the primary mouse glomerular endothelial cells (mGEC). The target molecule reacted with anti-MPO IgG on the mGEC was determined by peptide mass fingerprint analysis. RESULTS: A significant glomerular neutrophil infiltration was observed in the mice administered with anti-MPO IgG. The expressions of CXC chemokines, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2), were significantly increased in the renal cortex, indicating that these chemokines contribute to the neutrophil infiltration. Based on the previous findings of upregulation of adhesion molecule expression in mGEC treated with anti-MPO IgG, we examined whether mGEC secrete these chemokines in response to anti-MPO IgG. Indeed, anti-MPO IgG induced secretion of KC and MIP-2, leading to neutrophil chemotaxis in vitro. Furthermore, complete depletion of MPO in mGEC and serum using MPO-deficient mice showed an upregulation of intercellular adhesion molecule-1, indicating cross-reactive molecule(s) were existing on mGEC. We identified the molecule as moesin by a proteomic approach. CONCLUSIONS: The endothelial CXC chemokines, KC and MIP-2, contribute to infiltration of neutrophils in MPO-ANCA-associated vasculitis involving glomerulonephritis. The activation of glomerular endothelial cells by anti-MPO IgG appeared to directly involve a signaling through moesin.

[Development of novel cell culture systems utilizing the advantages of collagen vitrigel membrane].

The white of an egg, rendered opaque by boiling, can be converted into a thin, transparent and rigid material like glass by evaporating the moisture. This phenomenon is known as the vitrification of heat-denatured proteins. We applied vitrification technology to a collagen gel and converted it into a rigid glass-like material. We attempted to rehydrate the glass-like material and succeeded in preparing a novel stable state of collagen gel that was a thin and transparent membrane with excellent gel strength and protein permeability. We called it "collagen vitrigel" because it was produced from the vitrification process of a traditional hydrogel. Further, a framework-embedded collagen vitrigel membrane that can be easily turned inside out with tweezers was prepared by inserting a nylon membrane ring in the collagen sol prior to the gelation, thereby allowing the membrane to function as a removable cell culture substratum. Different types of anchorage-dependent cells could be cultured on both surfaces of the substratum by the manipulation of two-dimensional cultures, and consequently a three-dimensional crosstalk model with paracrine effects from each cell type was reconstructed. Also, the collagen vitrigel membrane containing a bioactive molecule provided a drug delivery system (DDS) with sustainable release. In this review, we summarize the recent progress of applied studies using the collagen vitrigel membrane as follows: a corneal model for eye irritant and permeability tests, a skin model for sensitization test, a renal glomerular model for evaluating blood filtration, an endometrial model for developing a new treatment and a DDS of hepatocyte growth factor for improving liver disorder.

The role of type V collagen fibril as an ECM that induces the motility of glomerular endothelial cells.

Although type V collagen (Col V) is present in developing and mature connective tissues of glomeruli, its primary function has not been elucidated yet. The purpose of this study was to elucidate the role of Col V fibrils in glomerular cells. We isolated primary cells from porcine kidney and cultured them on Col V fibrils reconstructed from purified Col V molecules extracted from porcine cornea. Time-lapse observation showed that Col V fibrils induce dynamic movement of glomerular endothelial cells (GEC) by stimulating them to extend long filopodial protrusions and wide lamellipodia. Col V signaling mediated through beta1 integrin activated phosphorylation of paxillin at tyrosine 118 (paxillin-pY118) and of focal adhesion kinase at tyrosine 861 (FAKpY861) at the cell periphery; a second Col V signal was mediated through neuroglycan 2 and activated FAKpY397. FAKpY861 was present in loose attachment points between Col V fibrils and GEC, allowing the cells to migrate easily. Activation of FAKpY397 induced incomplete focal adhesion at the centers of cells and caused cell movement. Therefore both signaling pathways facilitated cell motility, which was inhibited by the addition of antibodies to beta1 integrin, NG2, and Col V. We suggest that Col V fibrils activate 'outside-in' signaling in GEC and induce their dynamic motility.

Anionic C-terminal proregion of nematode antimicrobial peptide cecropin P4 precursor inhibits antimicrobial activity of the mature peptide.

Recently, an anionic proregion was found to be conserved at the C terminus of the antimicrobial peptide, nematode cecropin. Our results suggest that the antimicrobial activity of mature peptide is suppressed by the proregion in its precursor and is released from inhibition after processing. Inhibition is not likely to be due to direct suppression of membrane disruption.

Pentylenetetrazol modulates redox system by inducing addicsin translocation from endoplasmic reticulum to plasma membrane in NG108-15 cells.

Addicsin (Arl6ip5) is a multifunctional physiological and pathophysiological regulator that exerts its effects by readily forming homo- and hetero-complexes with various functional factors. In particular, addicsin acts as a negative modulator of neural glutamate transporter excitatory amino acid carrier 1 (EAAC1) and participates in the regulation of intracellular glutathione (GSH) content by negatively modulating EAAC1-mediated cysteine and glutamate uptake. Addicsin is considered to play a crucial role in the onset of neurodegenerative diseases including epilepsy. However, the molecular dynamics of addicsin remains largely unknown. Here, we report the dynamics of addicsin in NG108-15 cells upon exposure to pentylenetetrazol (PTZ), a representative epileptogenic agent acting on the gamma-Aminobutyric acid A (GABAA) receptor. Fluorescent immunostaining analysis demonstrated that addicsin drastically changed its localization from the endoplasmic reticulum (ER) to the plasma membrane within 1 h of PTZ exposure in a dose-dependent manner. Moreover, addicsin was co-localized with the plasma membrane markers EAAC1 and Na+/K+ ATPase alpha-3 upon PTZ stimulation. This translocation was significantly inhibited by a non-competitive GABAA receptor antagonist, picrotoxin, but not by a competitive GABAA receptor antagonist, bicuculline. Furthermore, lactate dehydrogenase (LDH) assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay showed that PTZ-induced addicsin translocation was accompanied by a decrease of radical-scavenging activity and an increase of cytotoxicity in a PTZ dose-dependent manner. These findings suggest that PTZ induces the translocation of addicsin from the ER to the plasma membrane and modulates the redox system by regulating EAAC1-mediated GSH synthesis, which leads to the activation of cell death signaling.

Detection of initial angiogenesis from dorsal aorta into metanephroi and elucidation of its role in kidney development.

Reconstruction of blood vessels is considered the most difficult part for the complicated organs, therefore, blood vessel construction is regarded as a key point for kidney regeneration in vitro. Vasculogenesis and angiogenesis are the two mechanisms to form blood vessels in embryonic organs, and most studies resided in vaculogenesis. Angiogenesis resided mostly in adult diseases such as wound healing, growth of tumors, and psoriasis diseases. However, renal angiogenesis is simply attributed to the sprouting of pre-existing blood vessel from dorsal aorta into metanephroi, and its occurrence is considered to be at a late stage of metanephric development. Since no techniques are available for delicate detection, the initial angiogenesis from dorsal aorta into metanephroi as well as its role in kidney development still remained unclear. In this study, we developed a method to detect the initial angiogenesis of dorsal aorta into metanephroi, and firstly clarified that dorsal aorta angiogenesis occurred at an early stage of metanephric development. We also elucidated the role of dorsal aorta angiogenesis in promoting the early blood vessel formation, tubule formation and glomeruli maturation. It is suggested that blood flow and dynamic circulation of various factors at the early developing stage may be prerequisite to a successful construction of blood vessels in the complicated organs either in vitro or in vivo. These findings contribute to a better understanding of dorsal aorta angiogenesis during kidney development and shed light on its significant value for the application of tissue engineering to complicated organs.

Poly(N-isopropylacrylamide)-graft-polypropylene membranes containing adsorbed antibody for cell separation.

We developed a novel selective cell-separation method based on using a poly(N-isopropylacrylamide)-graft-polypropylene (PNIPAAm-g-PP) membrane containing adsorbed monoclonal antibody specific to the target cell. This membrane was prepared by plasma-induced polymerization and soaking in an antibody solution at 37 degrees C. Poly(N-isopropylacrylamide) has a thermoresponsive phase transition: at 32 degrees C water-insoluble (hydrophobic) and water-soluble (hydrophilic) states interconvert. Adsorption of antibody onto PNIPAAm-g-PP membrane at 37 degrees C and its desorption at 4 degrees C was verified by fluorescence-microscopy of the PNIPAAm-g-PP membrane after soaking it in fluorescein-conjugated goat anti-mouse IgG in phosphate-buffered saline. PNIPAAm-g-PP membranes containing adsorbed anti-mouse CD80 monoclonal antibody preferentially captured mouse-CD80 transfected cells at 37 degrees C compared with membranes lacking antibody or containing anti-mouse CD86 monoclonal antibody. Detachment of captured cells from PNIPAAm-g-PP membranes was facilitated by washing at 4 degrees C because of the thermoresponsive phase transition of PNIPAAm. With this method, mouse CD80- or mouse CD86-transfected cells were enriched from a 1:1 cell suspension to 72% or 66%, simply and with high yield.

Reconstruction of renal glomerular tissue using collagen vitrigel scaffold.

The construction of renal glomerular tissue has provided an important tool not only for the understanding of renal physiology and pathology in blood ultrafiltration and cell dysfunction, but also in the application of tissue engineering to glomeruli regeneration and nephritic therapy. In this study, a novel method to reconstruct glomerular tissue combining cultured cells on a collagen vitrigel scaffold is described. The method consists of two newly developed techniques, one to isolate glomerular epithelial and mesangial cells rapidly from kidney, which facilitates the prolongation of cell population doublings and allows a long-term cell culture without losing cellular features, and another to prepare a stable and thin transparent collagen gel membrane termed collagen vitrigel that can facilitate three-dimensional cultures for reconstructing an epithelial-mesenchymal model. By combining the two methods, we cocultured glomerular epithelial and mesangial cells on both surfaces of the collagen vitrigel by the manipulation of two-dimensional cultures, resulting in the successful reconstruction of a three-dimensional glomerular organoid. The coculture results showed that the collagen vitrigel maintains cell growth and cell viability for more than 1 month, and surprisingly, the epithelial layer demonstrated polarity formation, which usually appears in in vivo normal epithelial cells existing at the glomerular basement membrane, but seldom appears in epithelial cells cultured in vitro. Moreover, the coculture results showed that fibronectin, an extracellular matrix component, and integrin beta1, a receptor of fibronectin, were detected in high amounts on both cells, suggesting our collagen vitrigel can provide a suitable environment for cell-cell interactions that stabilize the cell structure and may contribute to the polarity formation of epithelial cells.

Generation of stomach tissue from mouse embryonic stem cells.

Successful pluripotent stem cell differentiation methods have been developed for several endoderm-derived cells, including hepatocytes, ß-cells and intestinal cells. However, stomach lineage commitment from pluripotent stem cells has remained a challenge, and only antrum specification has been demonstrated. We established a method for stomach differentiation from embryonic stem cells by inducing mesenchymal Barx1, an essential gene for in vivo stomach specification from gut endoderm. Barx1-inducing culture conditions generated stomach primordium-like spheroids, which differentiated into mature stomach tissue cells in both the corpus and antrum by three-dimensional culture. This embryonic stem cell-derived stomach tissue (e-ST) shared a similar gene expression profile with adult stomach, and secreted pepsinogen as well as gastric acid. Furthermore, TGFA overexpression in e-ST caused hypertrophic mucus and gastric anacidity, which mimicked Ménétrier disease in vitro. Thus, in vitro stomach tissue derived from pluripotent stem cells mimics in vivo development and can be used for stomach disease models.

Targeting Mortalin by Embelin Causes Activation of Tumor Suppressor p53 and Deactivation of Metastatic Signaling in Human Breast Cancer Cells.

Embelin, a natural quinone found in the fruits of Embelia ribes, is commonly used in Ayurvedic home medicine for a variety of therapeutic potentials including anti-inflammation, anti-fever, anti-bacteria and anti-cancer. Molecular mechanisms of these activities and cellular targets have not been clarified to-date. We demonstrate that the embelin inhibits mortalin-p53 interactions, and activates p53 protein in tumor cells. We provide bioinformatics, molecular docking and experimental evidence to the binding affinity of embelin with mortalin and p53. Binding of embelin with mortalin/p53 abrogates their complex resulted in nuclear translocation and transcriptional activation function of p53 causing growth arrest in cancer cells. Furthermore, analyses of growth factors and metastatic signaling using antibody membrane array revealed their downregulation in embelin-treated cells. We also found that the embelin causes transcriptional attenuation of mortalin and several other proteins involved in metastatic signaling in cancer cells. Based on these molecular dynamics and experimental data, it is concluded that the anticancer activity of embelin involves targeting of mortalin, activation of p53 and inactivation of metastatic signaling.

Effect of stimulators such as GLP-1, PACAP, and nicotinamide on glucose-stimulated insulin secretion from porcine pancreatic endocrine cells in long-term culture.

INTRODUCTION: Transplantation of glucose-responsive insulin-secreting cells has the potential to result in a cure for diabetes. AIM: To report the development of a model of adult porcine pancreatic endocrine cells (PE cells) exhibiting glucose-stimulated insulin secretion during a long-term culture period, in vitro. METHODOLOGY: The PE cells were prepared by non-enzymatic digestion and purified by modifying a technique developed in our laboratory. The cells were first cultured for 7 days in RPMI-1640 medium containing 10% fetal bovine serum and 10 mM nicotinamide. On adhesion to the culture flasks, cells were collected by trypsinization, and then cultured in tissue culture dishes in medium with or without stimulators such as glucagon-like peptide-1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP), and nicotinamide. The ability of the cells to respond to glucose-stimulated insulin secretion was also observed with and without stimulators. The immunocytochemical studies demonstrated pancreatic islets with well-preserved insulin and glucagon-containing cells. The morphologic integrity of cultured porcine cells was observed for up to 5-6 weeks after the purification. RESULTS: At a concentration of 3.3 mM glucose, PACAP and nicotinamide did not affect glucose-dependent insulin secretion, whereas 10 nM GLP-1 stimulated insulin secretion significantly. However, when glucose concentration was increased to 20 mM, 10 nM GLP-1 had no effect on insulin secretion. We also demonstrated that GLP-1 and PACAP could maintain insulin secretion better than control in the culture up to 5 weeks. Also GLP-1 and PACAP increased the number of insulin-secreting cells in culture. CONCLUSION: These results demonstrate that GLP-1 and PACAP increased the number of pancreatic beta-cells in culture.

Cytotoxicity for porcine islet cells by complement of six animal species.

Complement-mediated cytotoxicity for porcine islet cells (PICs) was evaluated using sera of six animal species. Then soluble complement receptor type-1 (sCR1) as an anti-complement agent was added to those sera, and the changes in 50% hemolytic unit of complement serum (CH50) and cytotoxic effect of those sera on PICs were examined. All the sera except for that of pig showed cytotoxicity. However, the extent of toxicity was considerably different between species. In the rat and human serum, sCR1 significantly reduced CH50 and cytotoxicity, however in the dog serum, sCR1 had no suppressive effects. These results may suggest that complement contribute to humoral cytotoxicity for PICs as a main factor, and the compatibility of complement with PICs differs between animal species.

Prohibitin 2 regulates the proliferation and lineage-specific differentiation of mouse embryonic stem cells in mitochondria.

BACKGROUND: The pluripotent state of embryonic stem (ES) cells is controlled by a network of specific transcription factors. Recent studies also suggested the significant contribution of mitochondria on the regulation of pluripotent stem cells. However, the molecules involved in these regulations are still unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we found that prohibitin 2 (PHB2), a pleiotrophic factor mainly localized in mitochondria, is a crucial regulatory factor for the homeostasis and differentiation of ES cells. PHB2 was highly expressed in undifferentiated mouse ES cells, and the expression was decreased during the differentiation of ES cells. Knockdown of PHB2 induced significant apoptosis in pluripotent ES cells, whereas enhanced expression of PHB2 contributed to the proliferation of ES cells. However, enhanced expression of PHB2 strongly inhibited ES cell differentiation into neuronal and endodermal cells. Interestingly, only PHB2 with intact mitochondrial targeting signal showed these specific effects on ES cells. Moreover, overexpression of PHB2 enhanced the processing of a dynamin-like GTPase (OPA1) that regulates mitochondrial fusion and cristae remodeling, which could induce partial dysfunction of mitochondria. CONCLUSIONS/SIGNIFICANCE: Our results suggest that PHB2 is a crucial mitochondrial regulator for homeostasis and lineage-specific differentiation of ES cells.

Novel cell surface genes expressed in the stomach primordium during gastrointestinal morphogenesis of mouse embryos.

The mechanisms of gastrointestinal morphogenesis in mammals are not well understood. This is partly due to the lack of appropriate markers that are expressed with spatiotemporal specificity in the gastrointestinal tract during development. Using mouse embryos, we surveyed markers of the prospective stomach region during gastrointestinal morphogenesis. The initiation of organ bud formation occurs at E10.5 in mice. These primordia for the digestive organs protrude from a tube-like structured endoderm and have their own distinct morphogenesis. We identified 3 cell surface genes -Adra2a, Fzd5, and Trpv6 - that are expressed in the developing stomach region during gastrointestinal morphogenesis using a microarray-based screening. These novel genes will be useful in expanding our understanding of the mechanisms of gastrointestinal development.

Development of a human corneal epithelium model utilizing a collagen vitrigel membrane and the changes of its barrier function induced by exposing eye irritant chemicals.

The brief TEER (trans-epithelial electrical resistance) assay after exposing chemicals to corneal epithelium in vivo is known as a suitable method for evaluating corneal irritancy and permeability quantitatively and continuously. A collagen vitrigel membrane we previously developed is a thin (about 20 µm thick) and transparent membrane composed of high density collagen fibrils equivalent to connective tissues in vivo, e.g. corneal Bowman's membrane. To develop such a TEER assay system in vitro utilizing a human corneal epithelial model, HCE-T cells (a human corneal epithelial cell line) were cultured on the collagen vitrigel membrane substratum prepared in a Millicell chamber suitable for TEER measurement. Human corneal epithelium model possessing 5-6 cell layers sufficient for TEER assay was successfully reconstructed on the substratum in the Millicell chamber by culturing the cells in monolayer for 2 days and subsequently in air-liquid interface for 7 days. The exposure of chemicals to the model induced the time-dependent relative changes of TEER in response to the characteristic of each chemical within a few minutes. These results suggest that the TEER assay using the human corneal epithelial model is very useful for an ocular irritancy evaluation as an alternative to the Draize eye irritation test.

Determination of lead(II) in fly ash leachate using a newly developed simple spectrophotometric method.

A new simple method for the spectrophotometric determination of Pb(II) in fly ash leachates was developed. These leachates tend to contain a large amount of Ca(II) and Zn(II); this interferes with spectrophotometric determination of Pb(II) when conventional colorimetric agents are used. A copolymer consisting of protoporphyrin IX disodium salt and acrylamide was synthesized as a colorimetric agent. A measuring reagent containing ethylenediamine-N,N'-dipropionic acid (EDDP) as a masking agent for Zn(II) and an appropriate amount of Ca(II) together with the copolymer was applied to determine Pb(II). The temporal change in the absorption spectrum of the measuring reagent was acquired with a newly developed portable spectrophotometer for this method. The composition of EDDP and Ca(II) in the measuring reagent was optimized to measure leachates contaminated with Ca(II) and Zn(II). The detection limit and relative standard deviation of Pb(II) measured using the optimized method were 0.05 mg L(-1) and 2.3%, respectively. The tolerance limits for Ca(II) and Zn(II) contaminants, where errors of less than 10% were allowed at a concentration of 0.5 mg L(-1) Pb(II), were 4000 and 4 mg L(-1), respectively. The determination of Pb(II) in various samples of actual leachates from incinerator fly ash was examined with this method. The obtained values correlated well with those obtained by flame atomic absorption spectroscopy.