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Biomedical subjects

R Inagi

Publications and source records attributed to R Inagi.

At least 19 recordsLinked to original sources
PubMed2006

Albumin induces endoplasmic reticulum stress and apoptosis in renal proximal tubular cells.

Chronic proteinuria appears to be a key factor in tubulointerstitial damage. Recent studies have emphasized a pathogenic role of endoplasmic reticulum (ER) stress which is induced by the accumulation of misfolded proteins in ER, extracellular stress, etc. In the present study, we investigated ER stress and ER stress-induced apoptosis in proximal tubular cells (PTCs). Immortalized rat PTCs (IRPTCs) were cultured with bovine serum albumin (BSA). The viability of IRPTCs decreased proportionately with BSA overload in a time-dependent manner. Quantitative real-time polymerase chain reaction analysis revealed that 40 mg/ml BSA increases mRNA of ER stress markers by 7.7- and 4.6-fold (glucose-regulated protein 78 (GRP78) and oxygen-regulated protein 150 (ORP150), respectively) as compared to control. The increased expression of ORP150 and GRP78 in IRPTCs with albumin overload was detected by Western blot and immunofluorescence study. These in vitro observations were supported by in vivo studies, which demonstrated that ER stress proteins were upregulated at PTCs in experimental proteinuric rats. Furthermore, increased ER stress-induced apoptosis and activation of caspase-12 were observed in IRPTCs with albumin overload and kidneys of experimental proteinuric rats. We confirmed that apoptotic cell death was attenuated by co-incubation with caspase-3 inhibitor or calpain inhibitors. These results indicate that the ER stress-induced apoptosis pathway contributed to the insult of tubular cells by proteinuria. In conclusion, renal tubular cells exposed to high protein load suffer from ER stress. ER stress may subsequently lead to tubular damage by activation of caspase-12.

Acrylates↗
PubMed2003

Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded vMIP-I and vMIP-II induce signal transduction and chemotaxis in monocytic cells.

Kaposi's sarcoma-associated herpesvirus (KSHV)/ Human herpesvirus 8 encodes three chemokines, which are called viral macrophage inflammatory protein (vMIP)-I, -II, and -III. Here, we expressed the KSHV vMIP-I and vMIP-II proteins and analyzed their biological functions. Both vMIP-I and vMIP-II had an apparent molecular mass of 7.8 kDa and were localized to the cytoplasm in a body cavity-based lymphoma cell line BC-3, stimulated with phorbol ester. We next treated a human monocytic leukemia cell line, THP-1, with purified recombinant vMIP-I and vMIP-II, or vMIP-I and vMIP-II fused with alkaline phosphatase to study Ca(2+) signalling and in vitro chemotaxis in response to these proteins. Calcium mobilization was induced by both vMIP-I and vMIP-II. Furthermore, vMIP-I and vMIP-II induced Ca(2+) mobilization in K562 cells expressing the CC chemokine receptor 5 (CCR5), suggesting that both may be agonistic for CCR5. Additionally, vMIP-I induced Ca(2+) mobilization through the intermediary of CCR8. These viral MIPs were also capable of chemotactically activating the THP-1 cells. These results imply that vMIP-I and vMIP-II may play important roles in the propagation of KS and primary effusion lymphoma by inducing the chemotaxis of CCR5-expressing monocytes.

Calcium↗
PubMed2001

Specific tissue distribution of megsin, a novel serpin, in the glomerulus and its up-regulation in IgA nephropathy.

Mesangial cells play critical roles in maintaining a structure and function of the glomerulus. We previously cloned a novel mesangium-predominant gene, megsin, a new serine protease inhibitor. To clarify localization and roles of megsin protein, we raised polyclonal antibodies to megsin. By immunohistochemistry, megsin protein was specifically identified in the mesangial area. The amount of megsin protein was increased in glomeruli of patients with IgA nephropathy than in those of normal individuals and of patients with minimal change nephrotic syndrome or membranous nephropathy, suggesting a pathophysiological role of megsin as a functional modulator of mesangial functions in situ.

Amino Acid Sequence↗
PubMed2001

Glyoxal and methylglyoxal trigger distinct signals for map family kinases and caspase activation in human endothelial cells.

Carbonyl compounds with diverse carbon skeletons may be differentially related to the pathogenesis of vascular diseases. In this study, we compared intracellular signals delivered into cultured human umbilical vein endothelial cells (HUVECs) by glyoxal (GO) and methylglyoxal (MGO), which differ only by a methyl group. Depending on their concentrations, GO and MGO promoted phosphorylations of ERK1 and ERK2, which were blocked by the protein-tyrosine kinase (PTK) inhibitors herbimycin A and staurosporine, thereby being PTK-dependent. GO and MGO also induced phosphorylations of JNK, p38 MAPK, and c-Jun, either PTK-dependently (GO) or -independently (MGO). Next, we found that MGO, but not GO, induced degradation of poly(ADP-ribose) polymerase (PARP) as the intracellular substrate of caspase-3. Curcumin and SB203580, which inhibit JNK and p38 MAPK signaling pathways, but not herbimycin A/staurosporine, prevented the MGO-induced PARP degradation. We then found that MGO, but not GO, reduced the intracellular glutathione level, and that cysteine, but not cystine, inhibited the MGO-mediated activation of ERK, JNK, p38 MAPK, or c-Jun more extensively than did lysine or arginine. In addition, all the signals triggered by GO and MGO were blocked by amino guanidine (AG), which traps carbonyls. These results demonstrated that GO and MGO triggered two distinct signal cascades, one for PTK-dependent control of ERK and another for PTK-independent redox-linked activation of JNK/p38 MAPK and caspases in HUVECs, depending on the structure of the carbon skeleton of the chemicals.

Caspases↗
PubMed2001

Glyoxalase I deficiency is associated with an unusual level of advanced glycation end products in a hemodialysis patient.

BACKGROUND: Advanced glycation of proteins and their attendant advanced glycation end products (AGEs) contribute to the complications associated with diabetes mellitus or uremia. Regulatory mechanisms of AGE formation in vivo remain an issue of particular interest. We investigated a role of the glyoxalase detoxification system of precursor reactive carbonyl compounds (RCOs) in the in vivo AGE formation. METHODS: Plasma levels of AGEs [pentosidine and Nepsilon-carboxymethyllysine (CML)], their RCO precursors, d-lactate (the final product resulting from the glyoxalase detoxification pathway), as well as of various compounds known to generate AGE precursors and surrogate markers for oxidative stress (antioxidant enzymes and glutathione), were measured in both hemodialysis (HD) patients and normal subjects. The activity and protein expression of glyoxalase I, an enzyme essential for the detoxification of alpha-oxoaldehydes, in red blood cells (RBC) were also examined. RESULTS: In one 69-year-old lady who had been on hemodialysis (HD) for three years and had suffered from recurrent cardiovascular complications despite the absence of significant risk factors, plasma levels of pentosidine (77.3 +/- 2.4 pmol/mg protein) and CML (330.8 +/- 8.2 pmol/mg protein) were markedly elevated as compared to other HD patients (N = 20: 26.6 +/- 11.8 pmol/mg protein for pentosidine and 224.4 +/- 51.7 pmol/mg protein for CML). The plasma level of RCO precursors for pentosidine and CML was also higher in this patient than in other HD patients. Further investigation disclosed a very low activity in RBC of glyoxalase I (1.5 +/- 0.4 mU/106 RBC), as compared to other HD patients (3.9 +/- 0.6 mU/106 RBC) or normal subjects (4.0 +/- 0.6 mU/106 RBC). The glyoxalase I protein level, assessed in RBC by immunoblot analysis with a specific antibody, was markedly lower than that observed in HD patients and normal subjects. The causes of this deficiency remain unknown. Nucleotide sequencing of the products of reverse transcription-polymerase chain reaction from the patient's mononuclear cells revealed no genetic mutation within the coding region of the glyoxalase I gene. Plasma d-lactate level was also in the lower range (0.18 +/- 0.03 mg/dL) of the values measured in the other HD patients (0.27 +/- 0.09 mg/dL) and normal subjects (0.35 +/- 0.12 mg/dL). The plasma levels of various compounds known to generate AGE precursors (glucose, lipids and ascorbic acid) were either normal or low. The surrogate markers for oxidative stress such as antioxidant enzymes (glutathione peroxidases and superoxide dismutase) and glutathione were all within the range observed in the other HD patients. CONCLUSION: The unusually high levels of AGEs in this patient implicate a deficient glyoxalase detoxification of RCO precursors. The present clinical observation implicates, to our knowledge for the first time, the glyoxalase detoxification system and, in particular, glyoxalase in the actual level of AGEs in a uremic patient.

Aged↗
PubMed2001

Cloning of rodent megsin revealed its up-regulation in mesangioproliferative nephritis.

BACKGROUND: We recently cloned a new human mesangium-predominant gene, megsin. Megsin is a novel member of the serine protease inhibitor (serpin) superfamily. To elucidate functional roles of this gene, we cloned megsin in rodents and investigated its role in a rat nephritis model. METHODS: Megsin homologues were cloned from cultured rat and mouse mesangial cDNAs utilizing polymerase chain reaction (PCR) with degenerative primers. Expression of megsin in three different types of resident glomerular cells was investigated by PCR. Levels of megsin mRNA expression at various time points in the anti-Thy1 rat nephritis model were studied by semiquantitative PCR and Northern blotting analysis. In order to investigate megsin protein expression in anti-Thy1 nephritis rats, we raised antibody against rat megsin-specific synthetic peptide, with which immunohistochemical studies were performed. RESULTS: Rat and mouse megsins were composed of 380 amino acids, which revealed 75.3 and 73.9% identity, respectively, with human megsin at the amino acid level. Characteristic features as an inhibitory serpin were conserved in both rat and megsin megsins. PCR analysis revealed expression of megsin in cultured mesangial cells but not in glomerular epithelial or endothelial cells. In anti-Thy1 nephritis rats, semiquantitative PCR and Northern blotting showed that expression of megsin mRNA was up-regulated in glomeruli at day 8. Immunohistochemical studies demonstrated the prominent accumulation of megsin in glomeruli at the same time point. Megsin was mainly localized in mesangial area. The megsin expression level returned to the basal level at day 28. CONCLUSION: Sequences of megsin were well conserved among different species. Rat megsin was also predominantly expressed in mesangial cells. Expression of megsin was up-regulated at the peak of hypercellularity and matrix accumulation in the mesangioproliferative glomerulonephritis model, suggesting that megsin may participate in the process of glomerulosclerosis by modulating extracellular matrix deposition or cell survival.

Amino Acid Sequence↗
PubMed2000

Reactivation of human herpesvirus 6 by infection of human herpesvirus 7.

We have attempted to reactivate human herpesvirus 6 (HHV-6) by infection with HHV-7 using childhood exanthem subitum patients in vitro. Peripheral blood mononuclear cells (PBMCs) were collected from children who had a history of exanthem subitum(ES) by HHV-6 and were infected by human herpesvirus 7 (HHV-7) in vitro. The antigen positive rate to HHV-6 started to increase 7 days after the infection and reached a maximum by Day 15 using an immunofluorescence antibody test. The copy number of HHV-6 DNA also increased in the samples in 10 days after infection in vitro. No antigen or increase in DNA was detected in PBMCs, that were mock-infected or infected with supernatant of stock virus after ultracentrifugation, suggesting that an infection by HHV-7 is necessary to reactivate HHV-6. In the paired sera samples during the acute and the convalescent phases of ES, seven to ten bands, that were specific for HHV-6, were recognized in samples from the acute phase, and at least 5 dominant polypeptides were found more intensively after HHV-7 infection.

Blotting, Southern↗
PubMed2000

Human herpesvirus 8-encoded interleukin-6 homologue (viral IL-6) induces endogenous human IL-6 secretion.

We found that human herpesvirus 8-encoded IL-6 (vIL-6) induced endogenous human IL-6 (huIL-6) secretion from various cell lines (MT-4, THP-1, U937, Raji, and CESS) including patients with multicentric Castleman's disease. Especially, in MT-4 cells, huIL-6 was enhanced with vIL-6 by 30-fold compared with that of control. In addition, reverse transcriptase-polymerase chain reaction confirmed that vIL-6 induced huIL-6 expression in MT-4 cells. Our novel finding of the huIL-6 induction by vIL-6 indicates that vIL-6 may play a significant role in the pathogenesis of HHV-8 associated diseases.

Animals↗
PubMed2000

Effect of dwell time on carbonyl stress using icodextrin and amino acid peritoneal dialysis fluids.

BACKGROUND: Deterioration of the peritoneal membrane limits the technical survival of peritoneal dialysis (PD). Advanced glycation of the membrane has been incriminated in this evolution. Advanced glycation end products (AGEs) develop under the influence of glucose and of its degradation products, mainly reactive carbonyl compounds (RCOs) such as glyoxal (GO), methylglyoxal (MGO), and 3-deoxyglucosone (3-DG). The present study was undertaken to evaluate the impact of recently developed glucose-free PD fluids on AGE generation. METHODS: Recently developed glucose-free PD fluids containing either icodextrin or amino acids were investigated. GO, MGO, and 3-DG [high-performance liquid chromatography (HPLC)] and total RCOs (spectrophotometry) were measured in fresh solutions and in effluents after various dwell duration. The AGE formation potential of PD fluids and effluents was assessed by incubation at 37 degrees C, for one week, with bovine serum albumin and by the eventual measurement of pentosidine (HPLC) and Nepsilon-carboxymethyllysine (CML; gas chromatography/mass spectrometry). RESULTS: GO, MGO, and 3-DG (P < 0. 001) as well as total RCOs levels (P < 0.01) were significantly lower in icodextrin and amino acid PD fluid than in commercial, heat-sterilized, 1.36% glucose PD fluid. Pentosidine and CML generation were also significantly lower (P < 0.001) in icodextrin and amino acid PD fluid than in conventional 1.36% glucose PD fluid. The levels of total RCOs, however, increased in icodextrin and amino acid PD fluid effluents with dwell time. AGE formation potential rose accordingly, as demonstrated by a parallel increase in the generation of pentosidine and CML during incubation of PD effluents. CONCLUSION: The present data demonstrate lower RCO contents and AGE formation potential in fresh icodextrin and amino acid PD fluids than in fresh heat-sterilized glucose PD fluids. However, this difference decreases progressively during dwell time, mainly as a result of the influx of total RCOs.

Aged↗
PubMed2000

Nitric oxide inhibits the formation of advanced glycation end products.

BACKGROUND: Advanced glycation end products (AGEs) are elevated in renal failure and have been implicated in the pathogenesis of several uremic complications. Their formation is closely associated with oxidative stress. The recent observation that nitric oxide (NO) has an antioxidant effect led us to examine the possible role of NO in the generation of AGEs. METHODS: We examined the effect of NO donors, 2, 2'-(hydroxynitrosohydrazono)bis-ethanamine (NOC18) and S-nitroso-N-acetyl-DL-penicillamine (SNAP), on the in vitro formation of pentosidine, which was used as a surrogate marker for AGEs. Bovine serum albumin was incubated under air at 37 degrees C in a medium containing either several AGE precursors or uremic plasma. To elucidate further the mechanism of the NO effect on AGE formation, we examined the generation of free radicals and carbonyls in pentose-driven pentosidine formation. RESULTS: NO donors significantly inhibit the formation of pentosidine in a dose-dependent manner. The effect is abolished by the addition of a NO scavenging agent, 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO). The inhibitory effect results from NO but not from the NO donor molecule. It is best explained by the ability of NO to scavenge carbon-centered radicals, hydroxyl radical, and carbonyl compounds. CONCLUSIONS: NO inhibits pentosidine formation by scavenging free radicals and by inhibiting carbonyl compound formation. NO might be implicated in the atherogenic and inflammatory effects of AGEs: Reduced NO production and increased oxidative stress associated with atherosclerotic lesions may accelerate AGE formation and, thus, exacerbate endothelial dysfunction and accelerate the development of atherosclerosis in uremia.

Adult↗
PubMed2000

Identification and analysis of the K5 gene of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8 (HHV-8), belongs to the gammaherpesvirus subfamily and encodes approximately 80 open reading frames (ORFs). Among them are a few candidates for immediate-early genes (e.g., K5). We developed a monoclonal antibody (MAb), 328C7, against the K5 antigen. This MAb reacted with the K5 gene product by immunoscreening of a cDNA library from BCBL-1 cells, and this result was confirmed by transfection of the K5 ORF into Cos-7 cells. After induction of lytic infection by treatment with 12-O-tetradecanoylphorbol-13-acetate, MAb 328C7 reacted with an antigen in the cytoplasm of BCBL-1 and BC-3 cells as early as after 4 h of induction. Immunoelectron microscopy showed that the K5 antigen was situated mainly in the endoplasmic reticulum but was not present on the virion or in the nucleus. Northern blotting with a K5-specific probe revealed a single transcript of 1.2 kb, while Western blotting showed the antigen to be a 36-kDa polypeptide. The 5' and 3' ends were then determined by rapid amplification of cDNA, followed by sequencing of RACE products, and a splice was revealed upstream of the K5 ORF. K5 expression was unaffected by the respective DNA and protein synthesis inhibitors phosphonoformic acid and cycloheximide plus actinomycin D, confirming its immediate-early nature. Transient-transfection assays showed that the K5 promoter was transactivated by ORF 50 (KSHV Rta), a homolog of Epstein-Barr virus Rta, but the K5 gene product exhibited no transregulation of its own promoter or those of DNA polymerase and the human immunodeficiency virus type 1 long terminal repeat. This is the first such analysis of an immediate-early gene product; determination of its specific biological function requires further investigation.

Animals↗
PubMed1999

Glucose degradation product methylglyoxal enhances the production of vascular endothelial growth factor in peritoneal cells: role in the functional and morphological alterations of peritoneal membranes in peritoneal dialysis.

Peritoneal membrane permeability deteriorates in peritoneal dialysis (PD) patients. We test whether glucose degradation products (GDPs) in PD fluids, glyoxal, methylglyoxal and 3-deoxyglucosone, stimulate the production of vascular endothelial growth factor (VEGF), a factor known to enhance vascular permeability and angiogenesis. VEGF increased in cultured rat mesothelial and human endothelial cells exposed to methylglyoxal, but not to glyoxal or 3-deoxyglucosone. VEGF also increased in peritoneal tissue of rats given intraperitoneally methylglyoxal. VEGF and carboxymethyllysine (CML) (formed from GDPs) co-localized immunohistochemically in mesothelial layer and vascular walls of the peritoneal membrane of patients given chronic PD. By contrast, in the peritoneum of non-uremic subjects, VEGF was identified only in vascular walls, in the absence of CML. VEGF production induced by GDPs may play a role in the progressive deterioration of the peritoneal membrane.

Animals↗
PubMed1999

2-Isopropylidenehydrazono-4-oxo-thiazolidin-5-ylacetanilide (OPB-9195) treatment inhibits the development of intimal thickening after balloon injury of rat carotid artery: role of glycoxidation and lipoxidation reactions in vascular tissue damage.

We have pursued the hypothesis that the carbonyl modification of proteins by glycoxidation and lipoxidation reactions plays a role in atherogenesis. Human atherosclerotic tissues with fatty streaks and uremic arteriosclerotic tissues were examined, with specific antibodies, to detect protein adducts formed with carbonyl compounds by glycoxidation or lipoxidation reactions, i.e. advanced glycation end products (AGEs) or glycoxidation products, such as carboxymethyllysine (CML) and pentosidine, and lipoxidation products, such as malondialdehyde (MDA)-lysine and 4-hydroxy-nonenal (HNE)-protein adduct. All the four adducts were identified in the proliferative intima and in macrophage-rich fatty streaks. If the carbonyl modification is not a mere result but is a contributor to atherogenesis, inhibition of glycoxidation and lipoxidation reactions might prevent vascular tissue damage. We tested this hypothesis in rats following balloon injury of their carotid arteries, a model exhibiting a remarkable intimal thickening, which are stained positive for all the four adducts. Oral administration of 2-isopropylidenehydrazono-4-oxo-thiazolidin-5-ylacetanili de (OPB-9195), an inhibitor of both glycoxidation and lipoxidation reactions, in rats following balloon injury effectively prevented the intimal thickening. These data suggest a role for the carbonyl modification of proteins by glycoxidation and lipoxidation reactions in most, if not all, types of vascular tissue damage ('carbonyl stress'), and the usefulness of inhibitors of carbonyl reactions for the treatment of vascular tissue damage.

Aged↗
PubMed1999

Increased carbonyl modification by lipids and carbohydrates in diabetic nephropathy.

BACKGROUND: In diabetic nephropathy (DN), possible mediators of untoward effects of hyperglycemia include the advanced glycation end products (AGEs). Indeed, an AGE, carboxymethyllysine (CML), accumulates in expanded mesangial matrix and nodular lesions. An advanced lipoxidation end product (ALE), malondialdehyde-lysine (MDA-lysine), generated on proteins during lipid peroxidation also accumulates in these lesions. As both ALEs and AGEs are formed by carbonyl amine chemistry between protein and carbonyl compounds derived from autoxidation of lipids and carbohydrates, their colocalization suggests an increased carbonyl modification of proteins. METHODS: To address this hypothesis, human diabetic renal tissues were examined to characterize carbonyl modification of proteins by lipids and carbohydrates: (a) ALEs, MDA-lysine and 4-hydroxynonenal (HNE) protein adduct, derived from lipids, and (b) AGEs, pentosidine and CML, derived from carbohydrates. Furthermore, to elucidate the biological effect of carbonyl modification on primary cultured human and rat mesangial cells, the intracellular protein phosphorylation was examined in the presence of various kinds of carbonyl compounds. RESULTS: The ALE and AGE adducts examined were identified in expanded mesangial matrix and nodular lesions. The exposure of cultured mesangial cells to carbonyl compounds resulted in phosphorylation of tyrosine residues of a number of intracellular proteins. CONCLUSIONS: These data suggest a broad derangement in nonenzymatic biochemistry involving both lipids and carbohydrates exists in diabetic glomerular lesions ("carbonyl stress").

Animals↗
PubMed1999

Primary infection of human herpesvirus 6 in children with vertical infection of human immunodeficiency virus type 1.

The role of human herpesvirus 6 (HHV-6) infection in 227 children born to human immunodeficiency virus (HIV)-seropositive mothers was investigated. Of 41 HIV-uninfected infants, 3 (7%) were positive for HHV-6 DNA in the first month of life, suggesting possible intrauterine infection. The cumulative infection rates of HHV-6 at 6 and 12 months of age were significantly lower in HIV-infected children (11% and 33%, respectively) than in uninfected children (28% and 78%, respectively; P<.001). There was an association between high CD4+ cell numbers (>15%) before HHV-6 infection and high HHV-6 infection rate. Twenty-two infants with HIV classed as Centers for Disease Control and Prevention stages N1 or N2 were studied for an association of HHV-6 infection with progression of HIV disease. Ten of the infants had HHV-6, and 12 did not. In 5 of the infants without HHV-6 (42%), HIV disease had not progressed by 1 year of age; however, HIV disease had progressed in all 10 children with HHV-6 infection. These results suggest an association of HHV-6 infection and progression of HIV disease in the study children with vertical HIV-1 infection (P<.05).

CD4 Lymphocyte Count↗
PubMed1999

A sensitive and specific ELISA for plasma pentosidine.

BACKGROUND: Advanced glycation end products are formed by non-enzymatic glycation and oxidation reaction. Pentosidine is a well-known and characterized structure among them, and has been implicated in the pathogenesis of complications associated with chronic renal failure and long-term dialysis, such as dialysis-related amyloidosis and atherosclerosis. METHODS: We established a highly sensitive and specific competitive enzyme-linked immunosorbent assay (ELISA) for plasma pentosidine and applied it to large numbers of plasma samples including haemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) patients. We compared their plasma pentosidine levels determined by the competitive ELISA with those determined by high-performance liquid chromatographic (HPLC) assay currently used. RESULTS: The plasma pentosidine levels determined by the ELISA were correlated well with those determined by sophisticated instrumental HPLC assay, both in non-diabetic and diabetic dialysis patients. Both analyses yielded comparable results, with over 8-fold higher plasma pentosidine levels in HD and CAPD patients, irrespective of the presence or absence of diabetes, as compared to normal subjects and non-uraemic diabetic patients. CONCLUSIONS: The competitive ELISA will provide a rapid and convenient determination of plasma pentosidine content and thus be useful to assess the carbonyl stress in uraemic patients.

Adult↗