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Biomedical subjects

K Ienaga

Publications and source records attributed to K Ienaga.

At least 19 recordsLinked to original sources
PubMed2000

Crossline levels in serum and erythrocyte membrane proteins from patients with diabetic nephropathy.

Crossline is one of the structurally defined adducts of advanced glycation endproducts (AGEs) which has both a crosslink and fluorescence similar to AGE-protein in vivo. Crossline was measured in serum and erythrocyte membrane proteins (EMP) from 52 type 2 diabetic patients using a specific enzyme-linked immunosorbent assay system. Serum and EMP crossline levels in the diabetic patients were significantly higher than those in normal control. The patients with advanced diabetic nephropathy (serum creatinine levels of more than 1.2 mg/dl) had markedly elevated serum crossline levels compared to those with moderate diabetic nephropathy (clinical proteinuria) (180. 7+/-51.7 vs. 71.8+/-18.4 pmol/ml; P<0.01). On the other hand, there were no significant differences in EMP crossline levels between the two. EMP crossline levels in the patients with moderate diabetic nephropathy (8.8+/-2.9 pmol/mg protein) and those with advanced diabetic nephropathy (9.7+/-3.0 pmol/mg protein) were significantly higher than those without clinical proteinuria (6.4+/-1.9 pmol/mg protein; P<0.01). The present study demonstrated that EMP crossline levels were associated with the presence of nephropathy in patients with type 2 diabetes mellitus. Serum crossline levels were significantly influenced by remaining renal function. The measurement of crossline from a blood sample could provide us with important information for the study of clinical evaluation and pathogenesis of diabetic complications.

Aged↗
PubMed2000

The role of protein kinase C in the increased generation in isolated rat hepatocytes of the hydroxyl radical by puromycin aminonucleoside.

Puromycin aminonucleoside (PAN) has been known to induce proteinuria. The increased generation of reactive oxygen species (ROS) has been implicated in this toxicity of PAN. We have reported that PAN increases the synthesis of methylguanidine (MG) and creatol which are the products of the reaction of creatinine and the hydroxyl radical in isolated rat hepatocytes. However, the mechanism for the increased ROS induced by PAN is still unclear. In this paper, we investigate the role of protein kinase C (PKC) on the PAN induced reactive oxygen generation in isolated rat hepatocytes. Isolated hepatocytes were incubated in Krebs-Henseleit bicarbonate buffer containing 3% BSA, 16.6 mM creatinine and tested reagents. MG and creatol were determined by high-performance liquid chromatography using 9,10-phenanthrenequinone for the post-labeling. PAN increased MG and creatol synthesis in isolated rat hepatocytes by 60%. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a PKC inhibitor, at 10 and 100 microM significantly inhibited MG and creatol synthesis with or without PAN. The inhibition rate is dose dependent from 10 to 100 microM. H1004, a reagent used as control for H-7, did not affect (at 10 microM) or increased little (at 100 microM) the synthesis of MG and creatol. Ro31-8425, a potent PKC inhibitor, significantly inhibited (at 10 microM) MG synthesis in the presence of PAN. PKC in the membrane fraction, a marker of PKC activation, increased over the initial concentration by a factor of 1.65-fold at 60 min incubation and 2.16-fold at 120 min with PAN, while it changed little without PAN. These results indicate that PAN activates PKC resulting in increased hydroxyl radical generation in isolated rat hepatocytes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1999

A protein kinase C-beta-selective inhibitor ameliorates neural dysfunction in streptozotocin-induced diabetic rats.

Increased protein kinase C (PKC) activity has been implicated in the pathogenesis of diabetic retinopathy and nephropathy. However, the role of PKC in diabetic neuropathy remains unclear. The present study was conducted to compare the effect of PKC inhibition by a PKC-beta-selective inhibitor, LY333531 (LY), on diabetic nerve dysfunction with that of an aldose reductase inhibitor, NZ-314 (NZ). Streptozotocin-induced diabetic rats were treated with or without LY and/or NZ for 4 weeks, and motor nerve conduction velocity (MNCV), coefficient of variation of R-R interval (CVR-R), sciatic nerve blood flow (SNBF), peak latencies of oscillatory potentials on electroretinogram, PKC activities in membranous and cytosolic fractions of sciatic nerves, and polyol contents in the tail nerves were measured. Untreated diabetic rats demonstrated delayed MNCV, decreased CVR-R, reduced SNBF, and prolonged peak latencies of oscillatory potentials. Treatment with LY as well as NZ prevented all these deficits in diabetic rats. There were no significant differences in PKC activities in membranous or cytosolic fractions of sciatic nerves between normal and diabetic rats. Treatment with neither LY nor NZ altered PKC activities. Nerve myo-inositol depletion in diabetic rats was ameliorated not only by NZ, but also by LY. These observations suggest that inhibition of PKC-beta by LY may have a beneficial effect in preventing the development of diabetic nerve dysfunction, and that this effect may be mediated through its action on the endoneurial micro-vasculature.

Aldehyde Reductase↗
PubMed1998

Effect of neurotropin on the binding of high molecular weight kininogen and Hageman factor to human umbilical vein endothelial cells and the autoactivation of bound Hageman factor.

Bradykinin is generated by activation of the plasma kallikrein-kinin (K-K) cascade and contributes to the symptoms of allergic reactions and the perception of pain. Neurotropin is a biological material obtained from inflamed rabbit skin inoculated with vaccinia virus, which is widely used clinically in Japan as an effective agent for these disorders. Factor XII (FXII) and high molecular weight kininogen (HK), two critical constituents of the plasma K-K cascade, bind to endothelial cells, and bound FXII is autoactivated in the presence of zinc ions. We have investigated the effects of Neurotropin on the interactions of FXII and HK with endothelial cells. Neurotropin inhibited the binding of both proteins to cultured human umbilical vein endothelial cells (HUVEC) and inhibited autoactivation of FXII upon HUVEC in a concentration-dependent manner. These data suggest that the ameliorating effects of Neurotropin in allergic disorders and pain syndromes may be related to this ability to inhibit activation of the K-K cascade and, consequently, the formation of bradykinin.

Binding, Competitive↗
PubMed1998

Immunochemical quantification of crossline as a fluorescent advanced glycation endproduct in erythrocyte membrane proteins from diabetic patients with or without retinopathy.

Crossline is a novel advanced glycation endproduct (AGE) which has both a crosslink and fluorescence similar to AGE-protein in vivo. To assess the association of AGEs to the development of diabetic retinopathy we developed a sensitive and specific enzyme-linked immunosorbent assay (ELISA) for crossline in blood samples and investigated the association of the development of retinopathy and erythrocyte membrane protein (EMP)-crossline concentrations in patients with Type 2 diabetes mellitus (Type 2 DM). Crossline formation in EMP exceeded that in haemoglobin and was detectable in normal EMP samples without pretreatment by this ELISA system. Mean (+/-SE) EMP crossline levels were elevated 1.6-fold in diabetic patients without retinopathy (7.6 +/- 0.5 pmol mg(-1), p < 0.005), 2.2-fold in diabetic patients with non-proliferative retinopathy (10.5 +/- 0.6 pmol mg(-1), p < 0.001) and 2.6-fold in diabetic patients with proliferative retinopathy (12.0 +/- 0.6 pmol mg(-1), p < 0.001) compared with healthy control subjects (4.7 +/- 0.5 pmol mg(-1)). Type 2 DM patients with retinopathy had significantly higher EMP-crossline levels than those without retinopathy (p < 0.005). Our data suggest that elevated EMP-crossline concentrations are associated with the presence of retinopathy in patients with Type 2 DM and EMP-crossline measured by our ELISA may provide a useful marker for assessing the role of glycation in the development of diabetic retinopathy.

Aged↗
PubMed1998

Naturally occurring 2'-O-methylpurine nucleosides with hypotensive properties.

2'-O-Methylinosine (1) has been isolated for the first time and shown to be an intrinsic hypotensive principle. Its probable in vivo precursor, 2'-O-methyladenosine (3), showed stronger and even orally potent hypotensive activity. Resistance of the methyladenosine (3) against adenosine deaminase is thought to contribute to its long-lasting activity. The effect of both nucleosides (1 and 3) was not accompanied with any significant change in heart rate, which is often observed with adenosine.

Adenosine↗
PubMed1998

Neurotropin induces antinociceptive effect by enhancing descending pain inhibitory systems involving 5-HT3 and noradrenergic alpha2 receptors in spinal dorsal horn.

Neurotropin, a non-protein extract from the inflamed skin of rabbits inoculated with vaccinia virus, has been clinically used as an analgesic drug in Japan. Its analgesic effect has been demonstrated by reduced mechano-nociception in hyperalgesic rats exposed to SART-stress (a repeated cold stress) for 5 days. In order to clarify the mechanism of the analgesic effect of neurotropin at the spinal cord level, we examined the effects of several neurotransmitter receptor antagonists given by intrathecal (i.t.) injection on the antinociceptive effect of intraperitoneally (i.p.) injected neurotropin [100 and 200 Neurotropin Unit (NU)/kg]. The analgesic effect of neurotropin was significantly inhibited not only by methysergide (100 nmol/rat, i.t.), a non-selective antagonist against serotonin (5-HT), but also MDL 72222 (30 nmol/rat, i.t.), a selective 5-HT3 antagonist, but not influenced by ketanserin (100 nmol/rat, i.t.), a 5-HT2A antagonist. The antinociceptive effect of neurotropin (200 NU/kg, i. p.) was significantly inhibited also by yohimbine (30 nmol/rat, i.t.), a noradrenergic alpha2 antagonist. However, the analgesic effect of neurotropin (100 and 200 NU/kg, i.p.) was not influenced by naloxone (30 nmol/rat, i.t.), an opioid antagonist. These results suggest that the mechanism of the antinociceptive effect of neurotropin is via enhancement of endogenous descending pain inhibitory pathways of the serotonergic and noradrenergic systems, especially involving 5-HT3 and noradrenergic alpha2 receptors in spinal dorsal horn in which these neurons terminate. No influence of opioid receptors at the spinal cord level is indicated.

Adrenergic Antagonists↗
PubMed1998

Synthesis of creatol, a hydroxyl radical adduct of creatinine and its increase by puromycin aminonucleoside in isolated rat hepatocytes.

Creatol is a hydroxyl radical adduct of creatinine and the precursor of methylguanidine (MG), a uremic toxin. We investigate the synthesis of creatol and MG from creatinine and the effect of substances that affect the hydroxyl radical in isolated rat hepatocytes. In the presence of increasing concentrations of creatinine, rising level of creatol were found after 2 h incubation in Krebs-Henseleit bicarbonate buffer. However, further increase of creatol was not observed after 4 and 6h incubations. On the other hand, MG after 2 h incubation achieved a level of about 50% that of creatol and increased depending on both the creatinine concentration and the incubation period. DMSO, a hydroxyl radical scavenger decreased the generation of creatol and MG by about 50% at 2.5 mM and the inhibition depended on DMSO concentration. Puromycin amino-nucleoside (PAN) increased both by about 170%. These findings demonstrated that hepatocytes synthesize creatol prior to MG and are inhibited by a hydroxyl] radical scavenger. They also show that PAN increased hydroxyl radical generation in tissue cells.

Animals↗
PubMed1997

Advanced glycation end products induce expression of vascular endothelial growth factor by retinal Muller cells.

Recent studies have suggested that advanced glycation end products (AGEs) are involved in the development of diabetic complications. To assess the pathogenic role of AGEs and vascular endothelial growth factor (VEGF) in the development of retinal neovascularization in diabetic retinopathy, we investigated the effect of AGEs on induction of VEGF by retinal Muller cells and measured AGE and VEGF concentrations in the vitreous of patients with proliferative diabetic retinopathy (PDR) and nondiabetic patients. The expression of VEGF mRNA and the production of VEGF protein by cultured Muller cells were enhanced by the presence of AGEs. The vitreous concentrations of AGEs and VEGF were both elevated in patients with PDR compared with patients without diabetes (P < 0.01). There was a moderate positive correlation between the levels of crossline and VEGF (r=0.698, P < 0.01). Elevation of AGEs in the vitreous may promote intraocular neovascularization in diabetic retinopathy through production of VEGF from Muller cells.

Aged↗
PubMed1997

Acid-stable fluorescent advanced glycation end products: vesperlysines A, B, and C are formed as crosslinked products in the Maillard reaction between lysine or proteins with glucose.

Vesperlysines A and B, 6-hydroxy-1,4-di{6-(L-norleucyl)}-1H-pyrrolo[3,2-b]pyridinium and its 5-methyl derivative, respectively, and Vesperlysine C, 5-hydroxy-methyl-1,6-di{6-(L-norleucyl)}-1H-pyrrolo[3,4-b] pyridinium, are isolated as major fluorescent advanced glycation end products (AGEs) from hydrochloric acid hydrolysis of AGE-BSA, bovine serum albumin (BSA) modified by the Maillard reaction with glucose. These fluorophores are glycation products and not artifacts of hydrolysis, since they are also detected in the reaction mixture of lysine and glucose prior to hydrolysis. Vesperlysines are crosslinked products from two lysine side-chains in proteins and are considered to be generated from lysines and the oxidative degradation of glucose, because the six carbon skeleton of glucose in its original form was not incorporated into each structure. These compounds are most likely glycoxidation products like pentosidine. This reasoning is supported by the formation of the same compounds in the Maillard reactions in which ascorbic acid or other sugars with shorter carbon chains are used.

Chromatography, High Pressure Liquid↗
PubMed1997

Changes in serum levels of creatol and methylguanidine in renal injury induced by lipid peroxide produced by vitamin E deficiency and GSH depletion in rats.

A novel creatinine metabolite, creatol (5-hydroxycreatinine), is a key precursor in the synthesis of the uremic toxin methylguanidine (MG). Creatinine is converted to creatol within the mammalian body and this conversion is mediated specifically by hydroxyl radicals. We investigated the production of creatol and MG from creatinine in rats with renal failure induced by the lipid peroxide produced as a consequence of vitamin E deficiency and depletion of the reduced form of glutathione (GSH). In addition, we examined serum levels of other guanidino compounds, namely guanidinoacetic acid (GAA) and guanidinosuccinic acid (GSA). The injury to kidneys induced by the depletion of GSH in combination with vitamin E deficiency caused markedly elevated serum levels of creatol, MG and GSA and decreased serum GAA. The molar ratio of creatol to creatinine in the serum, which should be an index of the oxygen stress mediated by hydroxyl radicals, increased with time. Therefore, the enhanced production of creatol in vitamin-E-deficient rats that have been depleted of GSH might be due to the enhanced production of oxygen radicals in this system.

Acute Kidney Injury↗
PubMed1996

Formation of crossline as a fluorescent advanced glycation end product in vitro and in vivo.

Crossline is one of the major advanced glycation end products resulting the reaction mixture of free amino group(s) such as epsilon-one in lysine with D-glucose in vitro. To study crossline formation on proteins in vitro and in vivo, polyclonal antiserum to the crossline hapten was prepared. This antiserum reacted with bovine and human serum albumin that had been modified by prolonged incubation with glucose as well as with crossline itself. Antisera did not react with unmodified serum albumin or the other Maillard-related compounds. Crossline was formed in a time-dependent manner when a mixture of six different proteins was incubated with glucose at pH 7.2 or 9.0. Crossline levels could be measured in rat lens proteins and the levels increased with age. The crossline content of lens proteins in diabetic rats was more than two-fold higher than that of age-matched controls. Results of this study suggest that most proteins containing advanced glycation end products have crossline-like structures. Measurement of crossline-like structures in biological specimens may provide an index of aging and of the development of diabetic complications.

Animals↗
PubMed1996

Highly selective aldose reductase inhibitors. 1. 3-(Arylalkyl)-2,4,5-trioxoimidazolidine-1-acetic acids.

A series of 3-(arylalkyl)-2,4,5-trioxoimidazolidine-1-acetic acids (1) was prepared and tested for aldose reductase (AR) and aldehyde reductase (ALR) inhibitory activities. These compounds showed strong inhibitory activity against AR without significant inhibitory activity for ALR. The ratio of IC50(ALR)/IC50(AR) was > 1000 in some compounds. On the basis of pharmacological tests such as the recovery of reduced motor nerve conduction velocity and toxicological profile, 3-(3-nitrobenzyl)-2,4,5-trioxoimidazolidine-1-acetic acid (NZ-314) was selected as the candidate for clinical development.

Acetates↗
PubMed1996

Diabetic renal failure and serum accumulation of the creatinine oxidative metabolites creatol and methylguanidine.

Diabetic patients (n = 23) with chronic renal failure (CRF) accumulate the creatinine (Cr) oxidative metabolites creatol (CTL) and methylguanidine (MG; a uremic toxin) in their sera. Analysis of serum CTL, a key intermediate in mammalian Cr catabolism into MG, is shown to offer some useful diagnostic information on CRF, especially in the determination of an initial stage of pathological renal failure. The sera of all diabetic (n = 23) and nondiabetic (n = 20) patients with CRF (s-Cr > 1.25 mg/dl) contained s-CTL (> 2 micrograms/dl), whereas those from normal subjects (n = 18) and diabetic patients (n = 18) without CRF contained no detectable s-CTL. A similar accumulation of s-MG was observed, but only when s-Cr was higher than 2.0 mg/dl. Although each s-CTL (Y: microgram/dl, Y': mol/l) and s-MG level (Z: microgram/dl, Z': mol/l) is highly correlated with s-Cr (X: mg/dl, X': mol/l) in a normal equation, Y or Z = AX + B, an alternative correlation in a second-order equation, Y or Z = alpha X2 + beta X, could also fit well. Since the quadratic equation can be convertible to Y/X or Z/X = alpha X + beta [Y'/X' or Z'/X' = alpha' X+ beta'] and active oxygen species, especially hydroxyl radicals, convert Cr into CTL, Y/X, Y'/X', Z/X and Z'/X' values which might be a kind of indices for oxygen stress (oxidative stress) increased in proportion to the increased severity of CRF in such patients. Although its meaning and interpretation are still debatable, diabetic CRF patients had a significantly higher alpha' value (2.2) than that (0.89) of nondiabetic CRF patients. All serum values for Cr, CTL and MG were measured with HPLC.

Creatinine↗
PubMed1995

Crosslines, fluorophores in the AGE-related cross-linked proteins.

We can summarize our results as follows: (1) A pair of fluorescent crosslines were isolated from the Maillard reaction mixture; (2) AGE-proteins contained crossline-like structures, and (3) crossline-like immunoreactivities were accumulated in renal tissues of diabetic rats. From these results we concluded that fluorophores in AGE proteins have crossline-like structures and we had the first indication that XLs could be markers for renal disorders.

Animals↗
PubMed1994

Creatol, a creatinine metabolite, as a useful determinant of renal function.

Analysis of creatol (CTL, 5-hydroxycreatinine), an oxidative creatinine (Cr) metabolite, in serum and urine of human subjects has indicated that CTL is a useful determinant of renal function. The existence itself of serum CTL (s-CTL) could be a diagnostic sign for chronic renal failure (CRF): in all normal subjects, s-CTL was undetectable, but s-CTL was detectable in sera of all patients with CRF (s-Cr: > 2.0 mg/dl). And the s-CTL values increased in proportion to the severity of CRF in such patients. Furthermore, the molar ratio (CTL/Cr) in both urine and serum increased significantly in proportion to the severity of CRF. Our results indicated not only hyperproduction of CTL but also higher oxygen stress in patients according to the progression of CRF. The diagnostic importance of the CTL value and the CTL/Cr ratio are discussed.

Calibration↗