内因性抗エリスロポエチン受容体抗体の機能解析

金沢大学附属病院当研究室では、貧血の病態の1つとして知られているエリスロポエチン(erythropoietin ; EPO)低反応性に関与する可能性のある新規因子として、貧血患者の血清中に内因性の抗EPO受容体抗体を同定した。また、前年度までの臨床的検討から、ELISA法による抗EPO受容体抗体の吸光度の大きさと患者骨髄中の赤芽球や網赤血球数と相関を認めたことより、同抗体が貧血の病態形成に関与することが示唆された。そこで今年度は、抗EPO受容体抗体の生物学的特性を明らかにする目的で、さらなる生化学的検討に取り組んだ。1)血清からの抗体の精製はじめに、健常血清、疾患コントロール血清および抗EPO受容体抗体陽性の患者血清の精製を行った。血清をろ過した後に、IgGおよびIgM画分をアフィニティクロマトグラフィーで精製した。さらに、精製した低濃度の抗体は膜濃縮器を用いて濃縮した。2)抗体の特異性の検証上記により精製した抗体画分を用い、抗EPO受容体抗体と膜結合EPO受容体の特異的結合を検討した。まずEPO受容体を発現しているヒト白血病細胞株であるAS-E2の膜画分のライセートを作成した。次に精製したIgGまたはIgM抗体を加え、Protein A-sepharoseまたは抗ヒトIgM-sepharoseを用いて免疫沈降を行った。引き続きウェスタンブロッティングで確認した。その結果、抗EPO受容体抗体が陽性の検体では、ヒトFPO受容体の分子量に相当する場所にバンドを認めた。この結果より、抗EPO受容体抗体は細胞膜上のEPO受容体と結合することが示唆された。3)EPO依存性細胞株の増殖抑制試験次に、抗EPO受容体抗体のEPO依存性の細胞株の増殖に及ぼす影響を検討した。ここで用いた細胞はEPOおよびSCFで増殖することが知られているAS-E2細胞である。AS-E2細胞をEPOまたはSCF、および精製した抗体存在下に培養し、培養後の生細胞数を測定した。その結果、抗EPO受容体抗体が陽性の血清から精製したIgGおよびIgM検体はEPO依存性のAS-E2細胞の増殖を容量依存性に抑制することが判明した。なお、正常対照や疾患コントロールの抗体はAS-E2の増殖を抑制しなかった。以上の結果より、抗EPO受容体抗体の生物学的活性が示唆された。以上より、ELISAで検出された抗EPO受容体抗体のEPO低反応性を伴う貧血との関連が示唆された。研究課題/領域番号:23790935, 研究期間(年度):2011出典：研究課題「内因性抗エリスロポエチン受容体抗体の機能解析」課題番号23790935（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-23790935/）を加工して作

Blockade of VEGF accelerates proteinuria, via decrease in nephrin expression in rat crescentic glomerulonephritis

取得学位 : 博士（医学）, 学位授与番号 : 医博甲第1809号, 学位授与年月日 : 平成18年9月28日, 学位授与大学 : 金沢大学, 主査教授 : 多久和 陽, 副査教授 : 並木 幹夫, 中尾 眞

Clinical impact of albuminuria in diabetic nephropathy

金沢大学医薬保健研究域医学系Patients suffering from diabetic nephropathy, resulting in end-stage renal failure, are increasing in number. The pathophysiology of diabetic nephropathy remains to be fully investigated. In the clinical setting, the presence of albuminuria/overt proteinuria and a low glomerular filtration rate may predict poor renal prognosis, but the prognosis of the normoalbuminuric renally insufficient diabetic patient remains controversial. In addition to the measurement of urinary albumin excretion, biomarker studies to detect diabetic nephropathy more specifically at the early stage have been performed worldwide. There is a growing body of evidence for remission and/or regression of diabetic nephropathy, which may be an indicator for cardiovascular and renal risk reduction. Deeper insights into the pathological characteristics as well as the clinical impact of albuminuria on renal and cardiovascular outcome are required. © 2011 Japanese Society of Nephrology

The Impacts of Albuminuria and Low eGFR on the Risk of Cardiovascular Death, All-Cause Mortality, and Renal Events in Diabetic Patients: Meta-Analysis

Background:Precise effects of albuminuria and low estimated glomerular filtration rate (eGFR) on cardiovascular mortality, all-cause mortality, and renal events in diabetic patients are uncertain.Materials and Methods:A systematic review was conducted of the literature through MEDLINE, EMBASE, and CINHAL from 1950 to December 2010. Cohort studies of diabetic patients providing adjusted relative risk (RR) of albuminuria and eGFR for risks of cardiovascular mortality, all-cause mortality, and renal events were selected. Two reviewers screened abstracts and full papers of each study using standardized protocol.Results:We identified 31 studies fulfilling the criteria from 6546 abstracts. With regard to the risk of cardiovascular mortality, microalbuminuria (RR 1.76, 95%CI 1.38-2.25) and macroalbuminuria (RR 2.96 95%CI 2.44-3.60) were significant risk factors compared to normoalbuminuria. The same trends were seen in microalbuminuria (RR 1.60, 95%CI 1.42-1.81), and macroalbuminuria (RR 2.64, 95%CI 2.13-3.27) for the risk of all-cause mortality, and also in microalbuminuria (RR 3.21, 95%CI 2.05-5.02) and macroalbuminuria (RR 11.63, 95%CI 5.68-23.83) for the risk of renal events. The magnitudes of relative risks associated with low eGFR along with albuminuria were almost equal to multiplying each risk rate of low eGFR and albuminuria. No significant factors were found by investigating potential sources of heterogeneity using subgroup analysis.Conclusions:High albuminuria and low eGFR are relevant risk factors in diabetic patients. Albuminuria and low eGFR may be independent of each other. To evaluate the effects of low eGFR, intervention, or race, appropriately designed studies are needed. © 2013 Toyama et al

Relationship between serum uric acid levels and chronic kidney disease in a Japanese cohort with normal or mildly reduced kidney function

Background: Some observational studies have shown the relationships between hyperuricemia and chronic kidney disease (CKD); however, the threshold of serum uric acid (SUA) for deterioration of kidney function and the association between SUA and kidney injury by baseline kidney function remains unclear. This study aimed to clarify the relationships between SUA and reduced kidney function. Methods: We analyzed a historical cohort of male Japanese individuals who underwent medical checkup between 1998 and 2007. Participants with baseline data and who were followed up for at least one year were included and stratified according to baseline kidney function. Kidney function was classified as normal [estimated glomerular filtration rate (eGFR) ≥ 90 ml/min/1.73 m2] or mildly reduced (eGFR 60-89 ml/min/1.73 m2). The outcome measured was kidney impairment defined as a decrease in eGFR to < 60 ml/min/1.73 m2. Associations between SUA and risk for outcome and eGFR slopes were assessed. Results: A total of 41632 subjects with mean age 45.4 years were included. During a mean follow-up of four years, 3186 (7.6%) subjects developed kidney dysfunction. Subjects with SUA ≥ 6.0 mg/dL had a significantly increased risk for kidney impairment compared with subjects with SUA of 4-4.9 mg/dL. SUA threshold levels were different according to baseline kidney function; SUA ≤ 7.0 and ≤ 6.0 mg/dL for normal and mildly reduced kidney function, respectively. Approximately the same trends were observed for eGFR slopes. Conclusion: In the general population, hyperuricemia appears to be a risk factor for kidney impairment in males. For participants with mild kidney dysfunction, even a slight elevation of SUA can be a risk factor. Copyright: © 2015 Toyama et al.This article has a supplementary figure. Please see the last page of the text

Combined pure red cell aplasia and autoimmune hemolytic anemia in systemic lupus erythematosus with anti-erythropoietin autoantibodies

金沢大学医薬保健研究域医学系A 42-year-old woman with systemic lupus erythematosus was admitted to our hospital because of severe anemia. Her bone marrow was almost normocellular and erythroblasts were nearly absent. Laboratory data showed elevated levels of lactate dehydrogenase and positive findings on Coombs\u27 tests. On the basis of these findings, her anemia was diagnosed as the overlap of pure red cell aplasia with autoimmune hemolytic anemia. Radioimmunoprecipitation assay revealed that her serum was positive for anti-erythropoietin antibodies before therapy. Furthermore, the autoantibodies inhibited proliferation of an erythropoietin-dependent cell line in a dose-dependent manner. Immunosuppressive treatment improved the anemia accompanied with disappearance of the autoantibodies. © 2008 Wiley-Liss, Inc