{"created":"2023-06-19T07:18:26.207244+00:00","id":3796,"links":{},"metadata":{"_buckets":{"deposit":"1a97e367-7718-419e-8775-c8e88f3edc7f"},"_deposit":{"created_by":4,"id":"3796","owners":[4],"pid":{"revision_id":0,"type":"depid","value":"3796"},"status":"published"},"_oai":{"id":"oai:az.repo.nii.ac.jp:00003796","sets":["370:15:391"]},"author_link":["17670"],"item_10006_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2009-03-15"}]},"item_10006_degree_grantor_9":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"麻布大学"}]}]},"item_10006_degree_name_8":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士(獣医学)"}]},"item_10006_description_22":{"attribute_name":"Abstract","attribute_value_mlt":[{"subitem_description":"Chronic glomerular diseases, regardless of primary disease, lead to tubulointerstitial injury (TII) as a common complication. Close pathological analysis has revealed that functional impairment of the kidney correlated better with the degree of tubulointerstitial damage than with that of glomerular injury. Therefore, inhibition of TII is important treatment strategy for renoprotection. TII is histologically included tubular atrophy, mononuclear cell infiltration in tubulointerstitium and interstitial fibrosis. Recently, a clear relationship between interstitial inflammation and fibrogenesis has been demonstrated.\n Recently, proteinuria, develops in chronic renal failure, themselves may elicit proinflammatory and pro-fibrotic effects that directly contribute to chronic tubulointerstitial damage. Therefore, proteinuria becomes the focus of attention as the important factor pathobiologically.\n Osborne-Mendel rat (OM) develops glomerulopathy with overt proteinuria in early life and the renal disease progresses into chronic nephropathy earlier than that in other laboratory rats. We assumed that the OM might be a suitable model for studying mechanism of progression of chronic renal failure from nephrotic syndrome. In chronic renal diseases, TII more significantly contributes to decreasing renal function than glomerular injury. There is no investigation about the pathogenesis of TII in OM.\n In the present study, the author investigated the pathological features of TII and its relationship with glomerular injury or proteinuria on a progressive nephritis in OM. Furthermore, the author hypothesized that the pathogenesis of TII may be associated with proteinuria and two experiments were performed to investigate the relationship between proteinuria and TII.\n\nChapter 1: Pathological investigation of progressive nephropathy in OM rat\n Male OM at various ages (5-20 weeks) were used in this study. Urinalysis using 24-hour pooled urine revealed that the level of total protein significantly increased more than that found in the age-matched females. In sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of urine, a band in the albumin (Alb) region revealed at 5 weeks of age. The band was obviously thickened and other bands of higher molecular weight appeared at 10 weeks of age. Serum creatinine (Cre) level was significantly increased compared with that found in the age-matched female at 20 weeks of age. Histologically, there were hyaline droplets in glomerular podocytes at 5 weeks of age. Furthermore, adhesion of the visceral epithelium to Bowman's capsule and segmental glomerular sclerosis were seen at 7 weeks of age. These lesions were age-related and obviously progressed at 13 weeks of age. No inflammation was occurred in the glomeruli during the observation. Hyaline droplets in tubular epithelial cell (TEC) cytoplasm were recognized at 5 weeks of age. Then, deposition of hyaline droplets in TEC cytoplasm and dilation of tubules were aggravated with age. TEC showed swelling and vacuolar degeneration at 13 weeks of age. At 5 weeks of age, mononuclear cells were scattered in tubulointerstitium. The number of mononuclear cells in the interstitium significantly increased at 10 weeks of age. Diffuse TEC injury and interstitial inflammation accompanied with fibrosis were recognized at 20 weeks of age. Immunohistologically, TII was semi-quantified by a marker of TEC injury (osteopontin: OPN), mononuclear cell infiltration (ED-1, CD3, CD45RA) and fibrosis (alpha-smooth muscle actin: α-SMA). OPN were expressed in TEC at 10 weeks of age prior to development of morphological changes and the positive TEC significantly increased at 13 weeks of age. In addition, hypertrophic epithelial cells of Bowman's capsule were OPN-positive but podocytes and mesangial cells were negative. Mononuclear cells in tubulointerstitium consisted of ED-1-positive cells, CD3-positive cells and CD45RA-positive cells at 5 weeks of age. Infiltrating cells were mainly T cells and influx of these cells was significantly increased at 10 weeks of age. α-SMA positive cell appeared around the dilated tubuli and Bowman's capsules at 13 weeks of age. Local mRNA expression of monocyte chemoattractant protein 1 (MCP-1), one of the most powerful mononuclear and dendritic cell attractants, and OPN, a macrophage chemotactic protein, in tubulointerstitial area was examined using dissected tissues by the laser microdissection system. OPNmRNA was expressed at 5 weeks of age. Meanwhile, MCP-1mRNA was expressed at 10 weeks of age. Then, both expression levels of mRNA increased with age. OPN expression means TEC transdifferentiation which might be caused by excess reabsorption of the urinary protein recognized as hyaline droplets in TEC cytoplasm. In in vitro study, transdifferentiated TEC expressed interleukin-1 and tumor necrosis factor alpha which were induced by OPN and major histocompatibility complex (MHC) class II. It was speculated that these cytokine and MHC antigen expression were associated with mononuclear cells infiltration in addition to OPN and MCP-l. In conclusion, the occurrence and development of TII might be associated with the stimulation of the tubular cells by protein in the urine.\n\nChapter 2: The alteration of tubulointerstitial injury by suppression of proteinuria using angiotensin-converting enzyme inhibitor in OM rat\n From the conclusion of chapter 1, the author hypothesized that an inhibition of proteinuria reduces TII in OM. Therefore, the author examined the effect of anti-proteinuric treatment on glomerular injury and TII by administration an angiotensin-converting enzyme inhibitor (ACEi: lisinopril) in drinking water to OM from 3 weeks of age to 13 or 20 weeks of age. ACEi significantly reduced systolic blood pressure and proteinuria. In urine SDS-PAGE of the ACEi-treated group, a band in the Alb region appeared weakly. However, there were no bands of higher molecular weight. The serum Cre level of the treated group was significantly low compared to untreated group. Histologically, severe hyaline droplet formation in podocytes and adhesion of the visceral epithelium to Bowman's capsule were inhibited in ACEi-treated group. There were a few hyaline droplets in TEC cytoplasm and small number of macrophages and T cells were scattered in tubulointerstitial area in ACEi-treated group. No morphological changes and OPN positive cells were recognized. OPNmRNA was slightly expressed in tubulointerstitial area of ACEi-treated group, however, OPNmRNA expression was significantly reduced compared with that found in the age-matched untreated group. Furthermore, MCP-1mRNA was slightly expressed in tubulointerstitial area at 20 weeks of age of ACEi-treated group. There was no expression of OPNmR.NA and MCP-1mRNA in the glomeruli of ACEi-treated group. As the inhibition of proteinuria by ACEi treatment coincided with alleviation of TII, we expected that the cytokine expression such as OPN and MCP-l was induced by proteinuria due to glomerular injury and these cytokines might induce mononuclear cells infiltration in tubulointerstitium in OM.\n\nChapter 3: The alteration of tubulointerstitial injury by protein-overload proteinuria in OM rat\n To confirm the hypothesis that leaked urinary protein may play an important role in the pathogenesis of TII in OM, the author therefore injected OM with bovine serum albumin (BSA) to aggravate glomerular injury and proteinuria to see the modification of TII.\n OM were assigned to three groups at 4 weeks of age; intraperitoneal injection (IP) of 3.5mL (1.0g/ 100g) of BSA (BSA group); 3.5mL (1.0g/ 100g) of BSA with 75mg/ L of ACEi in drinking water (BSA+ACEi group); IP of 3.5mL of saline (control group). All groups daily received the treatment for 3 weeks. BSA group developed overt proteinuria compared with control group. The level of urinary TP of BSA+ACEi group was lower compared with BSA group but the level was higher than that in control group. In urine SDS-PAGE, a band in the Alb region was stained weakly, however, there were no bands of higher molecular weight in control group. In BSA group, a band in the Alb region was obviously thicker compared with that in control group. Furthermore, the pattern of other band higher than Alb was resembled to that found in the age- matched serum. The bands in BSA+ACEi group were obviously thinner compared with that found in BSA group. There was no significant difference of serum Cre level among three groups. Histologically, control group developed few glomerular injuries, however, BSA group had severe hyaline droplet formation in podocytes and adhesion of the visceral epithelium to Bowman's capsule. In BSA+ACEi group, hyaline droplets in podocytes were recognized but there was rare adhesion of the visceral epithelium to Bowman's capsule. In addition, there was no inflammation in the glomeruli. In tubulointerstitium, control group showed only hyaline droplet formation in TEC. In BSA group, however, there were severe dilation of tubular lumens and vacuolation of TEC. Furthermore, multifocal mononuclear cell infiltration and fibrosis were found in tubulointerstitium. These lesions in BSA+ACEi group were milder compared with BSA group. Immunologically, macrophages, T cells and B cells were infiltrated in tubulointerstitium in all groups. However, the infiltrating cells were mainly macrophages in BSA and BSA+ACEi groups. In control and BSA+ACEi groups, OPNmRNA slightly expressed in tubulointerstitial area, however MCP- 1mRNA was not expressed. In BSA group, OPNmRNA expression was significantly higher than that in other groups and MCP- 1mRNA was distinctly expressed in tubulointerstitial area. In conclusion, as protein-overload induced more severe glomerular injury, leaking larger amount of protein from the glomerular and severe TII than spontaneous injuries, urinary proteins might closely related to development of TII.\n\n The results of this study suggest that TII in OM might be result from proteinuria due glomerular injury. The severity of proteinuria might be related to TEC transdifferentiation, tubular injury and cytokine expression such as OPN and MCP-1 in tubulointerstitial area. Influx of mononuclear cells in the interstitium might be related to the cytokine expression from the TEC by stimulation of urinary protein.\n","subitem_description_type":"Other"}]},"item_10006_description_7":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"慢性糸球体疾患では、腎機能の低下やその予後は糸球体障害よりも尿細管間質障害(TII)と強く相関することが示唆されており、TIIの抑制が腎機能保護にとって重要な治療戦略と考えられている。TIIは組織学的に尿細管の萎縮、間質への単核細胞浸潤、間質の線維化を包含し、腎組織の退行性変化とともに浸潤細胞、尿細管上皮細胞(TEC)、筋線維芽細胞の相互作用によって間質線維化が進行する。糸球体病変に伴うTIIは、従来糸球体の機能廃絶の結果、付随する尿細管が萎縮し、周囲に二次的な炎症が引き起こされると考えられてきたが、近年では蛋白尿自体がTIIの原因または悪化因子として腎不全進行を促進させることが示唆されており、病理生物学的に重要な因子として蛋白尿が注目されている。\n　Osborne-Mendel系ラット(OM)は、若齢より大量の蛋白尿を伴う糸球体障害を発症し、他のラットに認める自然発症性慢性腎症に比べて進行がかなり早く、進行すると慢性腎不全となることから、ヒトのネフローゼ症候群の慢性腎不全進行モデルとして期待されているが、現在のところ尿細管間質病変の病理発生に関する研究は行われていない。\n　本研究は、OMの糸球体病変に続くTIIの進行メカニズムと、糸球体病変および尿細管間質病変の進展と蛋白尿との関連を探求することを目的とし、OMの進行性腎障害の病理学的検索を行った。また、OMのTIIの発生・進展には蛋白尿が関連していると仮定し、アンジオテンシン変換酵素阻害薬(ACEi)、ウシ血清アルブミン(BSA)の投与により蛋白尿を軽減ないし増悪させ、尿細管間質障害の変化について考察した。\n\n第1章　OMにおける進行性腎障害の病理学的検索\n　OMの病変の糸球体病変および尿細管間質病変は、進行の早いオスの5週齢から20週齢を用いて病理学的に検索した。\n　24時間蓄尿による尿検査では、総蛋白質量(TP)は7週齢以降、同週齢のメスよりも有意に上昇した。尿のSDS-PAGEでは、5週齢からアルブミン(Alb)の分子量と一致したバンドを認め、10週齢でバンドは明らかに太くなり、またAlbよりも高分子のバンドを形成し、その後これらのバンドは加齢性に太さを増した。血清クレアチニン(Cre)値は20週齢で雌雄間に有意差を認めた。組織学的に、糸球体は5週齢から足細胞の細胞質内に硝子滴の沈着が認められた。また、ボーマン嚢と血管係蹄との癒着、糸球体硬化といった硬化性病変は7週齢頃から観察され、その後加齢性に増加した。なお、糸球体に炎症反応は認められなかった。尿細管間質では5週齢からTECの細胞質内に硝子滴が認められ、13週齢で大小の硝子滴が大量に沈着してTECが膨化するほか、空胞変性などの重度の病変が散見された。間質には5週齢から単核細胞が確認されたが、著明な細胞浸潤は、10週齢より観察された。その後、これらの病変は間質の線維化を伴いながら加齢性に進展し、20週齢でびまん性に病変を形成した。免疫組織化学的に検索したところ、尿細管は形態学的な変化に先行して10週齢よりマクロファージ(mφ)の誘導因子であるとともに、TECの障害マーカーとして知られるosteopontin(OPN)に陽性を示し、13週齢でOPN陽性の尿細管数は有意に増加した。また、糸球体では肥厚したボーマン嚢上皮細胞が陽性を示したが、足細胞、メサンギウム細胞は陰性であった。浸潤細胞は5週齢からmφ、T細胞、B細胞のすべてが散在性に浸潤しており、浸潤した細胞の主体はT細胞で、10週齢以降顕著に増加した。また、間質線維化の際に線維芽細胞から形質転換する筋線維芽細胞について、α-smooth muscle actin(α-SMA)の抗体を用いて検索したところ、陽性細胞は13週齢から拡張した尿細管、ボーマン嚢周囲に認められた。次に、TEC障害と間質に浸潤した単核細胞の関連を検索するために、最も強力な単核細胞のケモカインである monocyte chemoattractant protein 1 (MCP-1)と、mφを誘導するサイトカインであるOPNについて、Laser microdissection 法を用いて切片から尿細管間質領域のみを採取し、リアルタイムPRCによりOPN、MCP-1のmRNA発現の定量解析を行ったところ、OPNmRNAは5週齢、MCP-1mRNAは10週齢から発現を認め、これらは加齢性に増加した。\n　以上の結果をまとめると、OMは観察を開始した5週齢で蛋白尿を発症しており、間質にはT細胞を主体とした単核細胞の浸潤が起こっていることが確認された。尿細管間質領域ではこれらの単核細胞を誘導するOPNmRNAが5週齢で発現しており、明らかに細胞浸潤が増加した10週齢ではOPN陽性TECを認め、さらにMCP-1mRNAも発現していた。TECにおけるOPN発現はTECの形質転換を意味し、これは硝子滴の存在から尿蛋白質の過剰な再吸収によることが示唆された。形質転換したTECからはOPNやMCP-1のほかに、OPNの発現との関連が報告されているinterleukin 1 (IL-1)や腫瘍壊死因子(TNF-α)、また主要組織適合抗原(MHC-II)などが発現することがin vitroの実験によって示されており、これらも単核細胞浸潤に関連していることが推察された。以上のことより、OMのTIIの発症・進展には、蛋白尿によるTECの刺激が関連している可能性が示唆された。\n\n第2章　ACEi投与による蛋白尿抑制時における尿細管間質の変化\n　第1章の結果から、蛋白尿を抑制すると尿細管間質病変は軽減されるという仮説を立て、OMにACEi (Lisinopril)を3週齢より飲水に混ぜて投与し、13、20週齢の糸球体および尿細管間質病変を検索した。\n　ACEi投与により糸球体高血圧ないし過剰濾過の抑制を期待したところ、OMの収縮期血圧は低下し、尿中のTPが有意に低下した。尿のSDS-PAGEではAlbの分子量にバンドが認められたが、それ以上の高分子蛋白質のバンドは形成されず、未処置群に比べて有意に抑制された。投与群の血清Cre値は未処置群に比べて有意に減少した。組織学的に、ACEiの投与によって未処置群にみられた足細胞における硝子滴の高度な沈着、ボーマン嚢上皮細胞への癒着は有意に抑制された。また、TECの細胞質内に硝子滴が認められたが、著明な形態学変化はなくOPN抗体にも陰性であったが、間質にはmφ、T細胞がわずかに浸潤していた。尿細管間質領域におけるOPNmRNAの発現は未処置群に比べて有意に抑制されたが、13、20週齢ともわずかに発現していた。また、20週齢ではMCP-1mRNAが発現していた。糸球体では、投与群はどちらの週齢もOPNmRNA、MCP-1mRNAの発現はなかった。\n　以上、蛋白尿の抑制によるTIIの変化から、尿細管間質領域におけるOPN、MCP-1といったサイトカインの発現が蛋白尿によって誘導され、間質への細胞浸潤を惹起することが予想された。\n\n第3章　BSA投与による蛋白質負荷下における尿細管間質の変化\n　次に、蛋白尿を増悪させることを目的として、BSAを投与して糸球体に蛋白質負荷を加え、自然発症よりも早期かつ重度に蛋白尿を発症させ、TIIが発症、増悪するかを調べた。BSAは4週齢より3.5mL(1.0g/100g)を3週間腹腔内投与した。また、同時にBSAの腹腔内投与とACEi(Lisinopril)の自由飲水による経口投与を併用した群(併用群)、生理食塩液3.5mLを腹腔内投与した群(対照群)を設定し、これらの病変と比較した。\n　BSAのみを投与した群(BSA群)の尿中TPは対照群に比べて顕著に増加した。併用群ではBSA群と比べて有意に抑制されたが、対照群より高い値を示した。尿のSDS-PAGEでは、対照群はAlbの分子量付近にバンドを形成したが、これより高分子のバンドは認められなかったのに対し、BSA群ではAlbの分子量付近に血清よりも太いバンドが認められ、また血清と類似した高分子のバンドを形成した。併用群ではBSA群と比べて高分子のバンドは明らかに細くなったが、対照群と比べて太いAlbのバンドを形成した。血清Cre値は3群間に有意差はなかった。組織学的には、対照群の糸球体にはほとんど病変はなかったが、BSA群で足細胞の高度な硝子滴沈着と、ボーマン嚢上皮細胞と血管係蹄の癒着像を多数認めた。併用群では硝子滴の沈着した足細胞が散見されたが、ボーマン嚢との癒着はほとんど認められなかった。なお、BSA投与による糸球体の炎症反応は観察されなかった。尿細管間質では対照群ではTEC内に硝子滴を認めるほかに病変はなかったが、BSA群では尿細管の高度な拡張やTECの空胞変性が散見され、また間質に単核細胞の集簇、線維化を多巣状性に認められた。併用群ではBSA群よりも病変は軽減し、間質には散在性に単核細胞の浸潤を認めたが、集簇巣や線維化はほとんど観察されなかった。また、間質に浸潤した単核細胞について免疫組織化学的に検索した結果、いずれの群においてもmφ、T細胞、B細胞の浸潤が確認されたが、BSA群、併用群では浸潤細胞の割合が対照群とは異なっており、mφが最も多く浸潤していた。尿細管間質におけるサイトカイン発現の検索では、対照群はOPNmRNAのみわずかに発現していたが、BSA群ではOPNmRNAの高度な発現に加えてMCP-1mRNAが発現していた。併用群のOPNmRNAの発現量はBSA群に比べて減少したが、対照群より高い値を示した。また、MCP-1mRNAの発現は認められなかった。\n　以上の結果から、自然発症よりも強い糸球体障害を惹起し、より大量に糸球体から蛋白尿が漏出すると、高度なTIIが引き起こされることが示された。\n\n　以上、OMの糸球体および尿細管間質の自然発症病変の病理学的検索と、蛋白尿の軽減、増悪による腎組織病変の変化の解析から、OMのTIIの発現・進展には糸球体障害による蛋白尿が関連し、蛋白尿の加齢性の悪化と一致して尿細管障害、単核細胞浸潤は進行することが示された。また、間質への細胞浸潤は尿細管間質領域で発現するOPN、MCP-1といったサイトカインの発現量の変化に関連し、これらの病変およびサイトカインの発現は、蛋白尿を増減させることにより変化することが示された。このことから、OMのTIIでは、糸球体障害に起因する蛋白尿がTECの形質転換、サイトカインの発現を誘導し、間質への細胞浸潤を惹起すると結論づけられた。\n","subitem_description_type":"Abstract"}]},"item_10006_dissertation_number_12":{"attribute_name":"学位授与番号","attribute_value_mlt":[{"subitem_dissertationnumber":"甲第120号"}]},"item_10006_version_type_18":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_ab4af688f83e57aa","subitem_version_type":"AM"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"馬場, 智成"}],"nameIdentifiers":[{"nameIdentifier":"17670","nameIdentifierScheme":"WEKO"}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2014-04-16"}],"displaytype":"detail","filename":"diss_dv_kou0120.pdf","filesize":[{"value":"25.5 MB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"diss_dv_kou0120","url":"https://az.repo.nii.ac.jp/record/3796/files/diss_dv_kou0120.pdf"},"version_id":"9d648b60-d5eb-4e23-8d40-eef30e5b785d"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2014-08-19"}],"displaytype":"detail","filename":"diss_dv_kou0120_jab&rev.pdf","filesize":[{"value":"432.9 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