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Toshimitsu Niwa

Publications and source records attributed to Toshimitsu Niwa.

At least 37 records · Page 2Linked to original sources
PubMed2003

Interactions of human organic anion as well as cation transporters with indoxyl sulfate.

Various uremic toxicants including indoxyl sulfate exert a number of biological effects on uremic patients. In order to elucidate the molecular mechanisms for the pharmacokinetics of indoxyl sulfate in human, we examined the interactions of human organic anion transporters (human-OATs) and human organic cation transporters (human-OCTs) with indoxyl sulfate using stable transfectants. Indoxyl sulfate inhibited human-OAT1, human-OAT3 and human-OAT4, but not human-OAT2, human-OCT1 and human-OCT2. Kinetic analysis revealed that the K(i) values for human-OAT1, human-OAT3 and human-OAT4 were 22.7, 168.7 and 181.3 microM, respectively. Human-OAT1 and human-OAT3 mediated the uptake of indoxyl sulfate and human-OAT4 mediated not only the uptake but also the efflux of indoxyl sulfate. In conclusion, by comparing the K(i) values with the plasma concentration of unbound indoxyl sulfate, it was predicted that human-OAT1 and human-OAT3 mediate the transport of indoxyl sulfate in vivo. In addition, it was suggested that human-OAT1 and human-OAT3 are involved in the urinary excretion of indoxyl sulfate, the exacerbation of renal dysfunction and the induction of uremic encephalopathy by indoxyl sulfate.

Animals↗
PubMed2003

Relative quantification of N(epsilon)-(Carboxymethyl)lysine, imidazolone A, and the Amadori product in glycated lysozyme by MALDI-TOF mass spectrometry.

The nonenzymatic glycation of proteins by reducing sugars, also known as the Maillard reaction, has received increasing recognition from nutritional science and medical research. In this study, we applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to perform relative and simultaneous quantification of the Amadori product, which is an early glycation product, and of N(epsilon)-(carboxymethyl)lysine and imidazolone A, two important advanced glycation end products. Therefore, native lysozyme was incubated with d-glucose for increasing periods of time (1, 4, 8, and 16 weeks) in phosphate-buffered saline pH 7.8 at 50 degrees C. After enzymatic digestion with endoproteinase Glu-C, the N-terminal peptide fragment (m/z 838; amino acid sequence KVFGRCE) and the C-terminal peptide fragment (m/z 1202; amino acid sequence VQAWIRGCRL) were used for relative quantification of the three Maillard products. Amadori product, N(epsilon)-(carboxymethyl)lysine, and imidazolone A were the main glycation products formed under these conditions. Their formation was dependent on glucose concentration and reaction time. The kinetics were similar to those obtained by competitive ELISA, an established method for quantification of N(epsilon)-(carboxymethyl)lysine and imidazolone A. Inhibition experiments showed that coincubation with N(alpha)-acetylargine suppressed formation of imidazolone A but not of the Amadori product or N(epsilon)-(carboxymethyl)lysine. The presence of N(alpha)-acetyllysine resulted in the inhibition of lysine modifications but in higher concentrations of imidazolone A. o-Phenylenediamine decreased the yield of the Amadori product and completely inhibited the formation of N(epsilon)-(carboxymethyl)lysine and imidazolone A. MALDI-TOF-MS proved to be a new analytical tool for the simultaneous, relative quantification of specific products of the Maillard reaction. For the first time, kinetic data of defined products on specific sites of glycated protein could be measured. This characterizes MALDI-TOF-MS as a valuable method for monitoring the Maillard reaction in the course of food processing.

Enzyme-Linked Immunosorbent Assay↗
PubMed2003

Increased levels of advanced glycation end products in human cataractous lenses.

PURPOSE: To investigate the occurrence of advanced glycation end products (AGEs) formed oxidatively (pentosidine and N(epsilon)-carboxymethyl lysine [CML]) or nonoxidatively (imidazolone) in human lenses and the relation of AGEs to lens coloration, cataract type, and patients' diabetic state. SETTING: Departments of Ophthalmology and Internal Medicine III, University of Jena, Jena, Germany. METHODS: Pentosidine, CML, and imidazolone concentrations were measured in the water-soluble protein fraction of 44 cataractous lenses (from 24 nondiabetic and 20 diabetic donors) and 6 noncataractous control lenses. RESULTS: Pentosidine, CML, and imidazolone were higher in cataractous lenses than in control lenses (pentosidine, 3.7 pmol/mg +/- 5.3 (SD) and 1.9 +/- 1.7 pmol/mg, respectively; CML, 3.0 +/- 2.2 nmol/mg and 1.3 +/- 0.7 nmol/mg, respectively; imidazoline, 80.4 +/- 93.3 AU/mg and 19.6 +/- 18.5 AU/mg, respectively). Among the cataractous lenses, the highest AGE concentrations were found in mature cataracts, with a statistically significant increase in CML. The AGE content increased relative to the intensity of brown coloration of the lens; the brown coloration also indicated the highest rise of imidazolone compared to pentosidine and CML. Lenses from diabetic donors had generally similar pentosidine values and elevated CML and imidazolone levels compared to lenses from nondiabetic donors. The pentosidine, CML, and imidazolone levels in the lenses correlated significantly with one another but not with patient age. CONCLUSION: Advanced glycation end products formed oxidatively and nonoxidatively occurred to a higher degree in cataractous lenses than in noncataractous lenses. The strong relationship between the lenses' AGE content, color/opacity, and the state of the cataract may indicate that advanced glycation plays a pivotal role in cataract formation.

Aged↗
PubMed2003

Immunohistochemical detection of an AGE, a ligand for macrophage receptor, in peritoneum of CAPD patients.

BACKGROUND: In patients on long-term continuous ambulatory peritoneal dialysis (CAPD), ultrafiltration (UF) capacity of peritoneal membrane may be impaired due to accumulation of advanced glycation end products (AGEs). This study aimed to elucidate the characteristics of a novel anti-AGE antibody, ODI-GLC19, and to demonstrate AGE accumulation in the peritoneum of CAPD patients using the antibody. METHODS: A monoclonal anti-AGE antibody (ODI-GLC19) was prepared by immunizing female balb/c mice using D-glucose-modified keyhole limpet hemocyanin. The characteristics of ODI-GLC19 were determined by enzyme-linked immunosorbent assay and receptor binding inhibition assay. Immunohistochemistry using ODI-GLC19 was performed to detect AGE in peritoneal tissues obtained from patients with nonrenal disease, and CAPD patients with normal and low UF. RESULTS: ODI-GLC19 reacted with glycolaldehyde-modified BSA (GA-BSA) and glucose-modified BSA (GLC-BSA), but not with imidazolone or N epsilon-(carboxymethyl)lysine. GA-BSA and GLC-BSA strongly bound to cultured macrophages. Time-dependent recognition of newly formed GA-BSA by ODI-GLC19 was similar to that by macrophages. The binding of GA-BSA to macrophages was inhibited by ODI-GLC19 in a dose-dependent manner. Immunohistochemical studies revealed that ODI-GLC19-positive AGE was exclusively detected in peritoneal cells including macrophages, and its staining intensity was more prominent in the peritoneum of CAPD patients, especially with low UF, than in patients with nonrenal disease. CONCLUSIONS: A novel monoclonal anti-AGE antibody, ODI-GLC19, recognizes a ligand for an AGE receptor on macrophages. Incorporation of AGE into peritoneal cells including macrophages may be involved in progressive peritoneal dysfunction in CAPD patients.

Animals↗
PubMed2003

Effects of oral adsorbent on gene expression profile in uremic rat kidney: cDNA array analysis.

BACKGROUND: An oral adsorbent, AST-120, is effective in removing such uremic toxins as indoxyl sulfate and delays the progression of chronic renal failure. To elucidate the molecular mechanisms underlying the renoprotective effects of AST-120, the complementary DNA (cDNA) array method was used to survey the alteration in gene expression profiles of uremic rat kidneys in response to AST-120. METHODS: Six weeks after five-sixth nephrectomy, 10 uremic rats were divided into two groups: those administered AST-120 and control uremic rats. Rats subjected to sham operation also were included as normal rats. After administration of AST-120 for 18 weeks, renal tissues were analyzed by cDNA array. RESULTS: Among the 343 genes selected as expressed, control uremic rats showed significantly increased levels of 139 genes and significantly decreased levels of 45 genes compared with normal rats. AST-120 treatment attenuated expression levels of 43 of the 139 upregulated renal genes and 21 of the 45 downregulated renal genes in uremic rat kidney. Those genes could be subdivided into several functional categories, including cytokine (transforming growth factor-beta1 [TGF-beta1], etc), intracellular signaling, transcription, translation, channel and transporter (organic anion transporter 1 [OAT1], etc), metabolism, and protease and its inhibitor. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that AST-120 significantly increased OAT1 renal expression and tended to decrease TGF-beta1, tissue inhibitor of metalloproteinase-1 (TIMP-1), and endothelin-1 renal expression in uremic rats. There were statistically significant positive correlations between cDNA array-based and RT-PCR-based gene expression levels of TGF-beta1, TIMP-1, and endothelin-1. CONCLUSION: The cDNA array method determines changes in the gene expression profile in uremic rat kidney in response to AST-120 and provides new insights into elucidation of the molecular mechanism underlying the renoprotective effects of AST-120.

Administration, Oral↗
PubMed2003

Are advanced glycation end products cardiovascular risk factors in patients with CRF?

BACKGROUND: Chronic renal failure (CRF) is characterized by enhanced formation and accumulation of advanced glycation end products (AGEs), which are involved in the pathogenesis of vascular damage. Their role as risk factors for cardiovascular complications is still unknown. This study aims to investigate whether elevated serum levels of the AGEs pentosidine, N(epsilon)-carboxymethyllysine (CML), and the 3-deoxyglucosone-derived imidazolone involve a greater risk for cardiovascular events (CVEs) and left ventricular hypertrophy (LVH). METHODS: Patients with CRF (n = 99), on maintenance hemodialysis (HD) therapy (n = 84), and renal transplant recipients (RTRs; n = 50) were included. Pentosidine was measured by high-performance liquid chromatography, and CML and imidazolone, by enzyme-linked immunosorbent assays. Statistical analyses were performed using Mann-Whitney U test, logistic regression analysis, and Cox proportional hazards model. RESULTS: At baseline in all investigated groups, patients with a history of CVEs or LVH showed greater mean serum AGE levels. By retrospective data analysis, significant odds ratios for increases in CML and imidazolone levels were calculated for LVH in HD patients, as well as for increases in CML levels for CVEs in RTRs, respectively. By prospective data analysis, serum AGE levels could not be evaluated as independent risk factors for CVEs in all investigated groups. CONCLUSION: From these preliminary results, serum AGE levels could not be identified as independent risk factors for CVEs or LVH in patients with CRF. Prospective studies are needed to answer this question.

Adult↗
PubMed2003

Formation of advanced glycation end products during CAPD.

Advanced glycation end products (AGEs) are formed during the nonenzymatic reaction of sugars with proteins. Conventional peritoneal dialysis fluids (PDFs) lead to the formation of AGEs in the peritoneal membrane that are associated with histopathologic changes and loss of ultrafiltration. PDFs may cause AGE formation because of a high glucose concentration or reactive glucose degradation products (GDPs), which are formed during heat sterilization of PDFs. This formation of GDPs is strongly pH dependent, which is exploited in newly developed double-chamber bag PDFs. Accordingly, 3-deoxyglucosone levels in double-chamber bag PDFs are reduced by approximately 80%, and levels of the GDPs acetaldehyde, formaldehyde, and methylglyoxal are less than the detection limit. Using an in vitro model that mimics regular changes in PDFs during continuous ambulatory peritoneal dialysis treatment, the contribution of high glucose versus GDP concentrations to AGE formation was investigated. The latter was determined by measuring protein bound N(epsilon)-(carboxymethyl)-lysine (CML) and imidazolone by enzyme-linked immunosorbent assay. In this model, more than 85% of imidazolone and more than 70% of CML were formed by GDPs, whereas only a minor part resulted from a high glucose concentration per se. New in vivo investigations suggest that GDPs from PDFs also can exert systemic effects after absorption into the blood circulation. Imidazolone levels in blood serum decrease significantly after switching from single- to double-chamber PDFs. In summary, the use of double-chamber PDFs may decrease not only local, but also systemic AGE formation.

Glucose↗
PubMed2003

Role of advanced glycation end products and growth factors in peritoneal dysfunction in CAPD patients.

High levels of glucose degradation products in peritoneal dialysis fluids are believed to cause excess accumulation of advanced glycation end products (AGEs) in the peritoneum during continuous ambulatory peritoneal dialysis (CAPD) treatment, resulting in functional and structural changes in the peritoneal membrane of CAPD patients. In this study, we investigated whether AGEs, the receptor for AGE (RAGE), and growth factors are involved in deteriorating ultrafiltration (UF) capacity of the peritoneal membrane in patients on CAPD therapy. Immunohistochemical staining showed that ODI-GLC19, a novel monoclonal anti-AGE antibody, was localized exclusively in peritoneal cells, in contrast to imidazolone, localized mostly in peritoneal degenerative collagen. Numbers of ODI-GLC19- and RAGE-positive cells in the peritoneum were increased significantly in CAPD patients, even before a decrease in UF capacity, compared with patients with nonrenal disease. Cells positive for ODI-GLC19 were identified as myofibroblasts and RAGE-positive cells and partly as CD68-positive macrophages in the peritoneum. The peritoneal membrane was thickened significantly in CAPD patients, especially patients with low UF. The number of blood vessels was increased significantly in CAPD patients with low UF. Transforming growth factor-beta1, macrophage colony-stimulating factor, and vascular endothelial growth factor were recognized in the peritoneum of CAPD patients, especially those with low UF, where imidazolone was deposited. Focal hepatocyte growth factor expression was noted in the peritoneum of patients with low UF in moderate intensity, specifically in the area without severe structural changes. In conclusion, progressive accumulation of AGEs in the peritoneum may promote peritoneal expression of various growth factors and subsequently deteriorate UF capacity in CAPD patients.

Antibodies, Monoclonal↗
PubMed2003

An inhibitor of advanced glycation end product formation reduces N epsilon-(carboxymethyl)lysine accumulation in glomeruli of diabetic rats.

BACKGROUND: An inhibitor of advanced glycation, OPB-9195, retards the progression of nephropathy in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of non-insulin-dependent diabetes mellitus. The aim of this study is to evaluate histologically the role of N(epsilon)-(carboxymethyl)lysine (CML) in the development of diabetic nephropathy and investigate whether inhibition of CML accumulation by OPB-9195 is associated directly with the prevention of glomerular lesions in OLETF rats. METHODS: Kidneys of OLETF and Long-Evans Tokushima Otsuka rats were obtained at ages 7, 20, 50, and 68 weeks after collecting their blood and urine samples. OPB-9195 had been administered to the rats from age 24 weeks to the end of the experiments. CML in kidneys was detected by using a monoclonal antibody against CML according to an indirect immunofluorescence technique. CML-positive glomerular area was measured using NIH Image software (Research Services Branch of NIMH, Bethesda, MD). Hyalinized and/or sclerotic areas in glomeruli and mesangial and glomerular volume were measured using a point-counting technique. RESULTS: CML-positive area in glomeruli correlated closely not only with urinary albumin excretion (r = 0.912; P = 0.001), but also with volumes of mesangium and hyalinized and/or sclerotic lesions (r = 0.859; P = 0.0019 and r = 0.833; P = 0.0027, respectively). Treatment with OPB-9195 reduced CML-positive area and prevented the increase in mesangial volume, with no significant change in glomerular volume at age 68 weeks. The volume of hyalinized and/or sclerotic lesions also decreased by treatment with OPB-9195 in three of four rats at age 68 weeks. CONCLUSION: CML is a major advanced glycation end product contributing to the development of diabetic nephropathy, and inhibition of its accumulation by OPB-9195 results in amelioration of glomerular lesions in OLETF rats.

Age Factors↗
PubMed2003

Bifidobacterium in gastro-resistant seamless capsule reduces serum levels of indoxyl sulfate in patients on hemodialysis.

BACKGROUND: Intestinal microflora is deranged in hemodialysis (HD) patients as increased aerobacteria such as Escherichia coli, and decreased anaerobacteria such as bifidobacteria. Indole, a precursor of indoxyl sulfate, is produced by E coli but not by bifidobacteria. The serum levels of indoxyl sulfate are increased markedly in HD patients and cannot be reduced efficiently by HD because of its albumin binding. METHODS: To compare the effect of oral administration of Bifidobacterium longum in gastro-resistant seamless capsule (Bifina) on indoxyl sulfate levels with that of Bifidobacteria in powder formulation (Lac B), Bifina was administered to 11 HD patients for 5 weeks, and Lac B to another group of 11 HD patients for 5 weeks. The authors measured the serum level of indoxyl sulfate by using high-performance liquid chromatography. RESULTS: The pre-HD serum levels of indoxyl sulfate significantly decreased in Bifina-treated patients (before, 4.9 +/- 1.7 mg/dL, 4.5 mg/dL, mean +/- SD, median, after 5 weeks, 3.5 +/- 1.3 mg/dL, 3.8 mg/dL; P < 0.005). However, they did not decrease in the Lac B-treated patients (before, 4.8 +/- 1.4 mg/dL, 4.5 mg/dL, after 5 weeks, 5.2 +/- 2.0 mg/dL, 5.1 mg/dL). CONCLUSION: Oral administration of Bifina to HD patients is effective in reducing the serum levels of indoxyl sulfate by correcting the intestinal microflora. Gastro-resistant seamless capsule prevents bifidobacteria from its inactivation in acidic gastric juice, and allows it to be actived in the intestine.

Administration, Oral↗
PubMed2002

Gas chromatographic-mass spectrometric determination of erythrocyte 3-deoxyglucosone in diabetic patients.

To determine if the erythrocyte levels of 3-deoxyglucosone (3-DG) are increased in diabetic patients, and if they correlate with glycemic status, they were measured in diabetic patients without renal disease as well as in healthy subjects. The erythrocyte levels of 3-DG were measured by a selected ion monitoring method of gas chromatography-chemical ionization mass spectrometry using [(13)C(6)]-3-DG as an internal standard. The erythrocyte levels of 3-DG were significantly higher in diabetic patients than in healthy subjects. The erythrocyte concentration of 3-DG was significantly and positively correlated with HbA1c (r=0.84, P<0.001). However, no significant correlation could be found between erythrocyte 3-DG and age, onset age of diabetes, or duration of diabetes in our group of diabetic patients. In diabetes, the production of 3-DG in the erythrocytes is increased via the polyol pathway and/or the Maillard reaction due to hyperglycemia.

Adolescent↗
PubMed2002

Characterization of antibody affinities using an AGE-modified dipeptide spot library.

Recent immunological approaches have greatly helped understanding the significance of advanced glycation end products (AGEs) in age-related diseases. However, immunohistochemical localization of AGEs in tissues or their quantitative determination in biological fluids sometimes yields inconsistent results among different investigators. Since these differences might be caused by the heterogeneity of the AGE antibodies, a systematic mapping of their epitope recognition pattern would help in the evaluation of these different results. For this purpose, an N-terminally acetylated combinatorial dipeptide library with 400 positionally defined dipeptides was modified by glucose to yield AGEs, which are thought to be present on the protein side chains. Using this library, we have characterized six different AGE antibodies in respect to their immunoreactivity towards AGE-modified dipeptides. All antibodies predominantly recognized only one AGE-modified amino acid side chain (and not a particular dipeptide). In most cases, arginine- and lysine- derived AGEs, but also some epitopes on asparagine and on heterocyclic amino acids were recognized. Very interestingly, the immunization of different animals with the same antigen produced AGE antibodies with a totally different epitope recognition pattern. Defining the antigen recognition pattern with this method might help in the quality control of polyclonal AGE antibodies for immunodetection. In addition, defined AGE antibodies (as neutralizing antibodies) could also help to define "signal-active" AGEs in terms of specific AGE-mediated cellular responses.

Antibody Affinity↗
PubMed2002

Molecular identification of a novel carnitine transporter specific to human testis. Insights into the mechanism of carnitine recognition.

l-Carnitine is an essential component of mitochondrial fatty acid beta-oxidation and plays a pivotal role in the maturation of spermatozoa within the male reproductive tract. Epididymal plasma contains the highest levels of l-carnitine found in the human body, and initiation of sperm motility occurs in parallel to l-carnitine increase in the epididymal lumen. Using a specific carrier, epididymal epithelium secretes l-carnitine into the lumen by an active transport mechanism; however, the structure-activity relationship comprising the carnitine-permeation pathway is poorly understood. We discovered a novel carnitine transporter (CT2) specifically located in human testis. Analyzing the primary structure of CT2 revealed that it is phylogenetically located between the organic cation transporter (OCT/OCTN) and anion transporter (OAT) families. Hence, CT2 represents a novel transporter family. When expressed in Xenopus oocytes, CT2 mediates the high affinity transport of l-carnitine but does not accept mainstream OCT/OCTN cationic or OAT anionic substrates. We synthesized and tested various carnitine-related compounds and investigated the physicochemical properties of substrate recognition by semi-empirical computational chemistry. The data suggest that the quaternary ammonium cation bulkiness and relative hydrophobicity be the most important factors that trigger CT2-substrate interactions. Immunohistochemistry showed that the CT2 protein is located in the luminal membrane of epididymal epithelium and within the Sertoli cells of the testis. The identification of CT2 represents an interesting evolutionary link between OCT/OCTNs and OATs, as well as provides us with an important insight into the maturation of human spermatozoa.

Amino Acid Sequence↗
PubMed2002

Molecular identification of a renal urate anion exchanger that regulates blood urate levels.

Urate, a naturally occurring product of purine metabolism, is a scavenger of biological oxidants implicated in numerous disease processes, as demonstrated by its capacity of neuroprotection. It is present at higher levels in human blood (200 500 microM) than in other mammals, because humans have an effective renal urate reabsorption system, despite their evolutionary loss of hepatic uricase by mutational silencing. The molecular basis for urate handling in the human kidney remains unclear because of difficulties in understanding diverse urate transport systems and species differences. Here we identify the long-hypothesized urate transporter in the human kidney (URAT1, encoded by SLC22A12), a urate anion exchanger regulating blood urate levels and targeted by uricosuric and antiuricosuric agents (which affect excretion of uric acid). Moreover, we provide evidence that patients with idiopathic renal hypouricaemia (lack of blood uric acid) have defects in SLC22A12. Identification of URAT1 should provide insights into the nature of urate homeostasis, as well as lead to the development of better agents against hyperuricaemia, a disadvantage concomitant with human evolution.

Amino Acid Sequence↗
PubMed2002

Effect of protein-bound uraemic toxins on the thermodynamic characteristics of human albumin.

The ability of albumin to bind drugs and other lipophilic organic acids is decreased in chronic renal failure by the accumulation of albumin-bound uraemic toxins such as hippuric acid, indoxyl sulphate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF). This furan acid is the most highly bound and is not removed by haemodialysis. The inhibitory effects of these three uraemic toxins on the interaction of three marker ligands sodium octanoate (for medium chain fatty acids), salicylic acid and phenol red (bilirubin site/site I) with albumin have been investigated by differential scanning microcalorimetry and flow microcalorimetry. CMPF was the most potent inhibitor and its binding site coincided with that of bilirubin (site I). Indoxyl sulphate binds to the site for medium-chain fatty acids and tryptophan (site II) and hippuric acid, the weakest inhibitor, inhibited binding to the salicylic acid site.

Albumins↗
PubMed2002

Signal transduction of beta2m-induced expression of VCAM-1 and COX-2 in synovial fibroblasts.

BACKGROUND: beta2 microglobulin (beta2m) amyloidosis is a destructive articular disease affecting dialysis patients. We have demonstrated that beta2m increases the expression of vascular cell adhesion molecule (VCAM-1) and cyclooxygenase-2 (COX-2) in human osteoarthritic synovial fibroblasts (SFLs). METHODS: To determine the cell signaling pathways, SFLs were incubated with beta2m in the presence or absence of various inhibitors for 24 hours. Intracellular calcium ([Ca2+]i) was measured by fluorometric techniques and vascular cell adhesion molecule-1 (VCAM-1) and cyclooxygenase-2 (COX-2) expression was determined by immunohistochemistry and Western blotting. RESULTS: beta2m increased [Ca2+]i levels in a dose dependent manner (P < 0.05) in SFLs. BAPTA-AM, a [Ca2+]i chelator, completely inhibited beta2m-induced expression of VCAM-1 and COX-2. U73122 [phospholipase C (PLC) inhibitor] or 2-APB [specific inhibitor of inositol 1,4,5-trisphosphate (IP3)-induced [Ca2+]i release] completely blocked the beta2m-induced increase in [Ca2+]i and the up-regulation of VCAM-1 and COX-2. However, pretreatment with staurosporin, a protein kinase C inhibitor, had no effect. Disruption of the actin cytoskeleton by treatment with cytochalasin D or latrunculin A blocked beta2m up-regulation of VCAM-1 and COX-2. Finally, cells treated with phosphatidylinositol-3 kinase (PI-3 kinase) inhibitors wortmannin or LY294002 also failed to express VCAM-1 and COX-2. CONCLUSIONS: These results demonstrate that IP3-mediated [Ca2+]i release, PI-3 kinase, and actin cytoskeleton reorganization are involved in beta2m-induced expression of VCAM-1 and COX-2 in human SFLs. Understanding the potential pathways by which beta2m exerts its inflammatory-like effects may lead to the development of future therapies.

Actins↗
PubMed2002

Role of organic anion transporters in the tubular transport of indoxyl sulfate and the induction of its nephrotoxicity.

In uremic patients, various uremic toxins are accumulated and exert various biologic effects on uremia. Indoxyl sulfate (IS) is one of uremic toxins that is derived from dietary protein, and serum levels of IS are markedly increased in both uremic rats and patients. It has been previously reported that the accumulation of IS promotes the progression of chronic renal failure (CRF). This study demonstrates the role of rat organic anion transporters (rOATs) in the transport of IS and the induction of its nephrotoxicity. The administration of IS to 5/6-nephrectomized rats caused a faster progression of CRF, and immunohistochemistry revealed that IS was detected in the proximal and distal tubules where rOAT1 (proximal tubules) and/or rOAT3 (proximal and distal tubules) were also shown to be localized. In in vitro study, the proximal tubular cells derived from mouse that stably express rOAT1 (S2 rOAT1) and rOAT3 (S2 rOAT3) were established. IS inhibited organic anion uptake by S2 rOAT1 and S2 rOAT3, and the Ki values were 34.2 and 74.4 microM, respectively. Compared with mock, S2 rOAT1 and S2 rOAT3 exhibited higher levels of IS uptake, which was inhibited by probenecid and cilastatin, organic anion transport inhibitors. The addition of IS induced a decrease in the viability of S2 rOAT1 and S2 rOAT3 as compared with the mock, which was rescued by probenecid. These results suggest that rOAT1 and rOAT3 play an important role in the transcellular transport of IS and the induction of its nephrotoxicity.

Animals↗