Akihiko Kimura

Wakayama Medical University, Wakayama, Wakayama, Japan

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Publications (71)239.98 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: We immunohistochemically examined the expression of IFNγ, TNFα, and TNF receptor p55 (TNF-Rp55) in a stasis-induced venous thrombus murine model. IFNγ(+), TNFα(+), and TNF-Rp55(+) cells could be first detected 7, 3, and 3 days after inferior vena cava (IVC) ligation. Thereafter, the numbers of these positive cells increased with time after IVC ligation. Double-color immunofluorescence analyses demonstrated that each molecule was expressed by intrathrombotic macrophages. In all samples with postligation intervals of 7 days or less, IFNγ/macrophage ratio (IFNγ/Mϕ), TNFα/macrophage ratio (TNFα/Mϕ), and TNF-Rp55/macrophage ratio (TNF-Rp55/Mϕ) were <0.2, <0.2, and <0.4, respectively. In contrast, IFNγ/Mϕ and TNFα/Mϕ were greater than 0.2 and 0.3 at ≥10 days after the IVC ligation, respectively. These observations suggested that IFNγ/Mϕ ratios of >0.2 and TNFα/Mϕ ratios of >0.3 indicated thrombus age of ≥10 days. Moreover, TNF-Rp55/Mϕ ratios of >0.7 strongly suggested thrombus age of >14 days. The present study demonstrated that the immunohistochemical detection of IFNγ, TNFα, and TNF-Rp55 was suitable to estimate the age of venous thrombi. However, the variation in TNF-Rp55/Mϕ ratios is wider than those of the other two markers. Thus, IFNγ and TNFα could be more practical markers.
    Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin 05/2013; · 2.69 Impact Factor
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    ABSTRACT: We report a case of sudden unexpected death due to late onset neonatal group B streptococcal sepsis. A male neonate weighing 2731g was born at 35week gestational age, and discharged at the age of 4days after the birth. At 6days after the discharge (10days after the birth), because of consciousness loss and hypothermia, the neonate was conveyed to an emergency hospital, eventually followed by his death. Forensic autopsy revealed neither severe trauma nor cardiac anomaly. Both lungs were edematous. Histopathologically, a lot of bacterial clusters were found in the lungs and intracerebral vessels. Cerebrospinal fluid contained a lot of leukocytes. Streptococcus agalactiae was detected in the specimens from the feces and the blood. Collectively, we diagnosed that the cause of the neonate's death was late onset group B streptococcal sepsis. In autopsy cases of neonates, careful macroscopic and microscopic observations and bacteriological/virological examination should be performed.
    Legal Medicine 03/2013; · 1.08 Impact Factor
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    ABSTRACT: The chemotherapeutic agent cisplatin often causes severe renal dysfunction; however, the molecular mechanism causing renal injury remains unclear. In wild-type mice, intrarenal interferon (IFN)-γ gene expression was found to be enhanced while CD3(+) T cells and Ly-6G neutrophils were the main cellular source of IFN-γ following cisplatin injection. Compared to wild-type mice, cisplatin-treated IFN-γ-deficient (IFN-γ(-/-)) mice exhibited exaggerated histopathological changes with higher blood urea nitrogen and creatinine levels. Cisplatin-induced apoptosis was associated with enhanced caspase-3 activation in renal proximal tubular epithelial cells, with effects suppressed by IFN-γ resulting in increased cell viability. IFN-γ significantly reduced the levels of the autophagic markers LC3-II and p62, and enhanced cathepsin D expression in cisplatin-treated renal proximal tubule epithelial cells, implying that IFN-γ can accelerate autophagic flux. Tubular cell apoptosis was more evident with enhanced caspase-3 activation in IFN-γ-deficient compared to wild-type mice. Elevated intrarenal LC3-II and increased p62 accumulation were associated with reduced cathepsin D activation in IFN-γ-deficient mice, implying that the absence of IFN-γ suppressed autophagic flux. Thus, IFN-γ can accelerate autophagic flux by augmenting cathepsin D levels and reciprocally increasing the viability of renal tubular cells, thereby attenuating cisplatin-induced acute renal injury.Kidney International advance online publication, 11 July 2012; doi:10.1038/ki.2012.240.
    Kidney International 07/2012; · 8.52 Impact Factor
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    ABSTRACT: We report a case of fatal intoxication caused by the ingestion of an organophosphate pesticide, methidathion (DMTP). An 80-year-old male was found dead in his bed. Forensic autopsy revealed no remarkable morphological changes. However, in a toxicological screening test, methidathion was qualitatively detected in extracts of stomach contents. Concentrations of methidathion (μg/g) in body fluids and organ tissues, determined by gas chromatography-mass spectrometry, were as follows; 66.2 in heart blood, 8.33 in peripheral blood, 8.80 in urine, 2000 in the brain (frontal lobe), 4800 in the left lung, 810 in the liver, 150 in the left kidney, and 64,000 in the stomach contents (total 1.9 g). These results strongly suggested that the victim orally ingested methidathion. Additionally, xylene was determined in body fluids and organ tissues. From the toxicological data together with autopsy findings, the cause of his death was diagnosed as acute poisoning by an emulsion of methidathion.
    Legal Medicine 06/2012; 14(5):263-6. · 1.08 Impact Factor
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    ABSTRACT: Immunohistochemical study combined with morphometry was carried out to examine the expression of cyclooxygenase-2 (COX-2) using 60 human skin wounds of different ages: group I, 0-4 h (n = 11); II, 8 h-2 days (n = 21); III, 3-9 days (n = 14); and IV, 12-21 days (n = 14). In wound specimens aged 2 h to 2 days, anti-myeloperoxidase-positive neutrophils observed at the wound site expressed immunopositive reaction to COX-2. In wound specimens of more than 3 days, CD68-positive macrophages as well as neutrophils were positively immunostained with anti-COX-2. In group II, all 21 wound samples had COX-2-positive ratios of >40 %, and 15 out of them showed >50 %. In group III, only three wound samples with the postinfliction intervals of 3 days showed positive ratios of 40-50 % and the remaining 11 cases less than 40 %. In groups I and IV, all 25 wound specimens had COX-2-positive ratio of <40 %. With regard to the practical applicability with forensic safety, these observations suggested that a COX-2-positive ratio of >40 % indicated a wound age of 8 h to 3 days. Moreover, COX-2-positive ratios, considerably exceeding a ratio of 50 %, indicate a wound age of 8 h to 2 days. Collectively, COX-2 would be a useful marker for the determination of early wound age.
    Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin 03/2012; 126(3):435-40. · 2.69 Impact Factor
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    ABSTRACT: We immunohistochemically examined the expression of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), and plasminogen-activator inhibitor type-1 (PAI-1) using venous thrombi developed by ligation of the inferior vena cava (IVC) in mice. The uPA-, tPA- and PAI-1-positive cells could be firstly detected 5, 7, and 3 days, respectively, after IVC ligation. Morphometrically, the number of PAI-1-positive cells was significantly higher than those of uPA- and tPA-positive cells at later than 7 days. In all of the thrombus samples aged 10-21 days, the uPA/PAI-1 and tPA/PAI-1 ratios were >0.1 and >0.2, respectively. In contrast, all of the thrombus samples aged 1-7 days had uPA/PAI-1 of <0.1 and tPA/PAI-1 ratios of <0.2. These findings implied that uPA/PAI-1 of >0.1 and tPA/PAI-1 of >0.2 indicated an age of 10 days or more. Moreover, in four of five samples aged 10 days, uPA/PAI-1 ratios were <0.3, and the remaining one had uPA/PAI-1 of 0.32. All thrombi aged 14-21 days showed values greater than 0.3. Thus, uPA/PAI-1 ratios, markedly exceeding 0.3, strongly indicated an age of more than 14 days. The present study demonstrated that the immunohistochemical detection of uPA, tPA, and PAI-1 was suitable to estimate the age of venous thrombi.
    Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin 02/2012; 126(3):421-5. · 2.69 Impact Factor
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    ABSTRACT: BM-derived endothelial progenitor cells (EPCs) are critical and essential for neovascularization in tissue repair and tumorigenesis. EPCs migrate from BM to tissues via the bloodstream, but specific chemotactic cues have not been identified. Here we show in mice that the absence of CCR5 reduced vascular EPC accumulation and neovascularization, but not macrophage recruitment, and eventually delayed healing in wounded skin. When transferred into Ccr5-/- mice, Ccr5+/+ BM cells, but not Ccr5-/- cells, accumulated in the wound site, were incorporated into the vasculature, and restored normal neovascularization. Consistent with these observations, CCL5 induced in vitro EPC migration in a CCR5-dependent manner. Moreover, expression of VEGF and TGF-β was substantially diminished at wound sites in Ccr5-/- mice, which suggests that EPCs are important not only as the progenitors of endothelial cells, but also as the source of growth factors during tissue repair. Taken together, these data identify the CCL5/CCR5 interaction as what we believe to be a novel molecular target for modulation of neovascularization and eventual tissue repair.
    The Journal of clinical investigation 01/2012; 122(2):711-21. · 15.39 Impact Factor
  • International journal of cardiology 11/2011; 158(2):e23-5. · 6.18 Impact Factor
  • Cytokine 10/2011; 56(1):79-79. · 2.52 Impact Factor
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    ABSTRACT: Deep vein thrombosis (DVT) is a major cause of pulmonary thromboembolism, a leading cause of death in individuals with DVT. Several lines of evidence indicate proinflammatory cytokines such as TNF-α are involved in thrombus formation and resolution, but the roles of IFN-γ remain unclear. To address this issue, we performed ligation of the inferior vena cava to induce DVT in WT and IFN-γ-deficient (Ifng-/-) mice. In WT mice, intrathrombotic IFN-γ levels were elevated progressively as the postligation interval was extended. Thrombus size was substantially smaller at 10 and 14 days in Ifng-/- mice than in WT mice. Intrathrombotic collagen content was remarkably reduced at more than 10 days after the ligation in Ifng-/- mice compared with WT mice. The expression and activity of MMP-9, but not MMP-2, was higher at the late phase in Ifng-/- mice than in WT mice. Moreover, intrathrombotic recanalization was increased in Ifng-/- mice, with enhanced Vegf gene expression, compared with that in WT mice. Activation of the IFN-γ/Stat1 signal pathway suppressed PMA-induced Mmp9 and Vegf gene expression in peritoneal macrophages. Furthermore, administration of anti-IFN-γ mAbs accelerated thrombus resolution in WT mice. Collectively, these findings indicate that IFN-γ can have detrimental roles in thrombus resolution and may be a good molecular target for the acceleration of thrombus resolution in individuals with DVT.
    The Journal of clinical investigation 06/2011; 121(7):2911-20. · 15.39 Impact Factor
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    ABSTRACT: The injection of Clostridium difficile toxin A into the ileal loops caused fluid accumulation with the destruction of intestinal epithelial structure and the recruitment of neutrophils and macrophages. Concomitantly, intraileal gene expression of CX3CL1/fractalkine (FKN) and its receptor, CX3CR1, was enhanced. When treated with toxin A in a similar manner, CX3CR1-deficient (CX3CR1(-/-)) mice exhibited exaggerated fluid accumulation, histopathological alterations, and neutrophil recruitment, but not macrophage infiltration. Mice reconstituted with CX3CR1(-/-) mouse-derived bone marrow cells exhibited exacerbated toxin A-induced enteritis, indicating that the lack of the CX3CR1 gene for hematopoietic cells aggravated toxin A-induced enteritis. A heme oxygenase-1 (HO-1) inhibitor, tin-protoporphyrin-IX, markedly increased fluid accumulation in toxin A-treated wild-type mice, indicating the protective roles of HO-1 in this situation. HO-1 expression was detected mainly in F4/80-positive cells expressing CX3CR1, and CX3CR1(-/-) mice failed to increase HO-1 expression after toxin A treatment. Moreover, CX3CL1/FKN induced HO-1 gene expression by isolated lamina propria-derived macrophages or a mouse macrophage cell line, RAW264.7, through the activation of the ERK signal pathway. Thus, CX3CL1/FKN could induce CX3CR1-expressing macrophages to express HO-1, thereby ameliorating toxin A-induced enteritis.
    The Journal of Immunology 01/2011; 186(1):423-31. · 5.52 Impact Factor
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    ABSTRACT: The biological clock may stop at the time of death in a dead body. Therefore, the biological clock seems useful for estimating the time of death. In this study, we tried to read the biological clock in tissues from dead bodies to estimate the time of death using molecular biological techniques. At first, we examined real-time RT-PCR analysis of gene expression for mPer2 and mBmal1, which constitutes a feedback loop in the oscillation system, in the kidney, liver, and heart of mice. We could detect circadian oscillation of these gene expressions in mouse tissues even at <48 h after death. Thus, the ratio of mPer2/mBmal1 was found to be useful for estimating the time of death. We next applied this method to the liver, kidney, and heart obtained from forensic autopsy cases with less than 72 h of postmortem interval. Significant circadian oscillation of hPer2/hBmal1 ratio could be detected in these autopsy samples. We further examined gene expression for hRev-Erbα, a component of another feedback loop. The ratios of hRev-Erbα/hBmal1 showed higher amplitude of oscillation than those of hPer2/hBmal1 and are considered more suitable for estimating the time of death. In particular, a hRev/hBmal1 ratio of >50 indicated the time of death as 0200-0900 hours, and a hRev/hBmal1 ratio that considerably exceeded 75 indicated the time of death as 0200-0800 hours. On the other hand, a hRev/hBmal1 ratio of less than 25 strongly indicated the time of death as 1000-2300 hours. Taken together, these findings indicate that gene expression analyses of the biological clock could be powerful methods for estimation of the time of death.
    Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin 11/2010; 125(3):385-91. · 2.69 Impact Factor
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    ABSTRACT: Human brain samples were collected from 70 autopsy cases including 22 freshwater drowning (FWD), 26 saltwater drowning (SWD), and 22 non-drowning cases as controls. Then, immunohistochemical study combined with morphometry was carried out in order to examine the differential expression of AQP1 and AQP4 in the brain samples. Immunohistochemically, star-shaped cells bearing highly branched processes, often surrounding blood vessels, showed positive reactions for AQP1 and AQP4 in FWD, SWD, as well as control groups. Additionally, with double-color immunofluorescence analysis, AQP1- or AQP4-positive cells could be identified as GFAP-positive astrocytes. Moreover, AQP1-positive reaction was also observed in blood vessels. Morphometrically, there were no significant differences in AQP1 expression in astrocytes or in blood vessels among the three groups. In contrast, the average value of AQP4-positive astrocytes was significantly higher in FWD cases than in SWD and control groups. Moreover, AQP4 expression was significantly lower in SWD than in the control group (p < 0.05). Moreover, there was no significant correlation between post-submerged interval and AQP expression in drowning cases. Therefore, immunohistochemical analysis of intracerebral AQP4 expression would be forensically useful for differentiation between FWD and SWD.
    Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin 11/2010; 125(1):59-65. · 2.69 Impact Factor
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    ABSTRACT: We immunohistochemically examined the expression of matrix metalloproteinase (MMP)-2 and MMP-9 using venous thrombi developed by ligation of the inferior vena cava (IVC) in mice. Both MMP-2- and MMP-9- positive cells could be detected in the whole course of thrombus formation after IVC ligation. Morphometrically, their number was greatest 14 days after IVC ligation and thereafter, gradually decreased at 21 days. The number of MMP-9-positive cells was significantly higher than that of MMP-2-positive cells at 1 to 7 days. The average ratio of MMP-9 to MMP-2 (MMP-9/MMP-2 ratio) was >2.0 in all thrombus samples at 1-5 days. After 7 days, the MMP-9/MMP-2 ratio was less than 2.0. These observations implied that an MMP-9/MMP-2 ratio markedly exceeding 2.0 strongly indicates an age of 5 days or less. Furthermore, an MMP-9/MMP-2 ratio of <2.0 probably indicates an age of more than 7 days. The present study demonstrated that the immunohistochemical detection of intrathrombotic MMP-2 and MMP-9 was suitable to estimate the age of venous thrombi.
    Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin 09/2010; 124(5):439-44. · 2.69 Impact Factor
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    ABSTRACT: We report a case of fatal intoxication caused by the ingestion of the herbicide Gesapax (an emulsion type), which consists of 25% ametryn (ametryne, ametrin, ametrine, and ametrina), and 75% other components (xylene and cyclohexanone). A female in her 70's was found dead in her bed. Forensic autopsy revealed no remarkable injury or morphological changes. In a gas chromatography-mass spectrometry (GC-MS) screening test, ametryn was qualitatively detected in her stomach content. Then, the other components of xylene and cyclohexanone were detected by headspace GC-MS in the stomach content. The amount of ametryn (microg/g) extract from each body fluid or organ tissue was determined by GC-MS as follows: 6.69 (heart blood), 3.50 (peripheral blood), 0.085 (urine), 17.2 (brain frontal lobe), 71.9 (right lung), 23.9 (liver), 19.1 (right kidney), and 74,200 (stomach contents). The other components, xylene and cyclohexanone, were also determined in the body fluids and organ tissues. These data strongly suggest that the female orally ingested the herbicide. From these toxicological data together with autopsy findings, the cause of her death was determined to be an acute herbicide poisoning.
    Journal of analytical toxicology 06/2010; 34(5):287-91. · 2.11 Impact Factor
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    ABSTRACT: Using histochemical and immunohistochemical techniques, we examined the intrathrombotic collagen contents and the appearance of hemosiderin-positive cells, neovessels, and myofibroblasts in a stasis-induced venous thrombosis model. The intrathrombotic collagen deposition area occupied about 20% at 5 days, and exceeded 80% at 21 days after ligation of the inferior vena cava (IVC). Hemosiderin-positive cells in the thrombus first appeared at 3 days in only one of the five samples, and positive cells were constantly detected in all thrombi at 5 days or later. CD31-positive neovessels in the thrombus first appeared at 5 days in one of five samples and were detected in all samples after 10 days. At 7 days, alphaSMA-positive myofibroblasts at the periphery of the thrombus first appeared in three of five samples, and were detected and enhanced time-dependently in all samples after 10 days. These observations demonstrated that these markers would be applicable for thrombus age determination.
    Forensic science international 02/2010; 195(1-3):143-7. · 2.10 Impact Factor
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    ABSTRACT: Carbamazepine (CMZ) and its metabolites, carbamazepine-10,11-epoxide (CMZepo) and 2,2’-iminostilbene (Imi), were simultaneously assayed by gas chromatography-mass spectrometry (GC-MS) in body fluids and organ tissues taken from victims in five autopsy cases. Clomipramine-HCl was added to each specimen as internal standard (IS); target compounds and IS were simultaneously extracted with an Extrelut NT column before analysis by GC-MS. The concentrations of CMZ were generally much higher in organ tissues than in blood and urine, and were higher in the liver than in the lung in three cases. The concentrations of metabolites CMZepo and Imi were lower than those of CMZ in all body fl uid and organ tissue specimens; the Imi concentrations in the brain were higher than CMZepo concentrations in four cases. Imi concentrations in the kidney, however, were lower than those of CMZepo in four cases. In the lung and liver, the concentrations of Imi did not show a consistent trend; those of Imi were higher or lower than those of CMZepo, but both metabolites were not at negligible levels. To our knowledge, this is the fi rst demonstration of the distribution of Imi among human body fluids and tissues. KeywordsCarbamazepine-Carbamazepine-10,11-epoxide-Iminostilbene-GC-MS-Tissue distribution-Human
    Forensic Toxicology 01/2010; 28(2):124-128. · 3.19 Impact Factor
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    ABSTRACT: Sodium arsenite (NaAs)-induced autophagic cell death (ACD) of a mouse renal tubular epithelial cell line (mProx24), which expresses enhanced levels of interleukin-6 (IL-6), was reduced by the suppression of autophagy by 3-methyladenine or Atg7 knockdown. The inhibition of the IL-6/signal transducer and activator of transcription 3 (STAT3) signal pathway by anti-IL-6 antibody or a Jak2 inhibitor (AG490) exaggerated ACD of mProx24 cells after NaAs challenge, attenuating STAT3 activation and reciprocally enhancing extracellular signal-regulated kinase (ERK) phosphorylation. In contrast, an ERK inhibitor, PD98059, reduced NaAs-induced ACD in mProx24 cells. Subcutaneous injection of NaAs (12.5 mg/kg) into BALB/c (wild-type) mice enhanced intrarenal expression of IL-6, mainly produced by tubular cells, and caused severe renal injury characterized by hemorrhages, acute tubular necrosis, cast formation, and brush border disappearance, with increases in serum urea nitrogen (blood urea nitrogen) and creatinine levels. In addition, IL-6-deficient (IL-6(-/-)) mice exhibited exaggerated histopathological changes with higher blood urea nitrogen and creatinine levels. Moreover, in IL-6(-/-) mice treated with NaAs, ACD in renal tubular cells was significantly augmented, along with diminished STAT3 activation and reciprocal enhancement of ERK signaling, compared with wild-type mice. Finally, the administration of exogenous IL-6 into wild-type mice significantly reduced NaAs-induced ACD along with diminished ERK activation and eventually alleviated acute renal dysfunction. Thus, IL-6/STAT3 signal pathway could inhibit ERK activation, a crucial step for ACD, eventually attenuating NaAs-induced renal dysfunction.
    American Journal Of Pathology 12/2009; 176(1):40-50. · 4.60 Impact Factor
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    ABSTRACT: Sepsis is a systemic inflammatory disease with high mortality. In the present study, we immunohistochemically examined CCR2 and CX3CR1 expression in sepsis-induced lung injury, and discussed its availability for the postmortem diagnosis of sepsis. Lung samples were obtained from different lung lobes of nine sepsis and eight control cases with postmortem intervals between 12 and 48h. Immunohistochemically, mononuclear cells recruited into the lungs expressed CCR2 and CX3CR1 in both sepsis and non-septic groups. In double-color immunofluorescence analysis, CCR2- or CX3CR1-positive cells could be identified as CD68-positive macrophages. Moreover, most of CD68-positive macrophages expressed both CCR2 and CX3CR1. Morphometrically, the average of CCR2- and CX3CR1-positive macrophages was significantly increased in sepsis group, compared with control group (sepsis vs. control: 41.6+/-1.3% vs. 8.0+/-0.4% in CCR2; 36.2+/-1.3% vs. 9.2+/-0.4% in CX3CR1). These observations implied that CCR2- or CX3CR1-positive macrophages were recruited into the lungs under several pathological conditions. In particular, their recruitment might be more evident in sepsis. Moreover, from the forensic aspects, immunohistochemical detection of CCR2 and CX3CR1 expression in the lungs can be considered as valuable diagnostic tools for the postmortem diagnosis of sepsis.
    Forensic science international 10/2009; 192(1-3):e21-5. · 2.10 Impact Factor
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    ABSTRACT: Aquaporins (AQPs) are a family of homologous water channel proteins. In this study, the expressions of AQP1, 2, and 4 were immunohistochemically examined in kidney samples to evaluate their forensic applicability to differentiate between freshwater drowning (FWD) and saltwater drowning (SWD). Kidney samples were obtained from 51 drowning cases (23 FWD and 28 SWD) and 19 non-drowning cases. AQP1 was expressed in the proximal tubules and glomeruli, and AQP4 was localized in the collecting ducts. However, there were no significant differences in AQP1 and AQP4 expressions among FWD, SWD, and control groups. Immunohistochemically, AQP2 was predominantly expressed in the apical plasma membrane of the collecting duct principal cells in all kidney samples of FWD and SWD. Morphometrically, AQP2 expression at the apical plasma membrane of collecting ducts was significantly enhanced in SWD group, compared with FWD and control groups. On the other hand, AQP-2 expression was significantly lower in FWD group than in control group. Moreover, in drowning cases, there was no correlation between post-submersion intervals and AQP expression. From a forensic aspect, immunohistochemical detection of AQP2 in the kidney can be considered a valuable marker to differentiate between FWD and SWD.
    Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin 10/2009; 124(2):99-104. · 2.69 Impact Factor

Publication Stats

686 Citations
239.98 Total Impact Points

Institutions

  • 1991–2013
    • Wakayama Medical University
      • • Department of Forensic Medicine
      • • Department of Legal Medicine
      Wakayama, Wakayama, Japan
  • 2004–2012
    • Kanazawa University
      • • Department of Forensic and Social Environmental Medicine
      • • Division of Molecular Bioregulation
      Kanazawa, Ishikawa, Japan