Orientador: Priscylla Tatiana Chalfun Guimarães-Okamoto / Resumo: A doença renal crônica (DRC) em cães apresenta grande morbidade e alta taxa de mortalidade, sendo considerada a alteração renal mais comum, tendo a nefropatia glomerular como a alteração de maior prevalência. Este estudo tem por objetivo avaliar a prevalência das alterações glomerulares em conjunto com avaliação da razão proteína creatinina urinárias (RPC), dos níveis séricos de albumina e creatinina e pressão arterial sistólica comparando os parâmetros clínicos e laboratoriais entre o grupo de animais com a lesão predominante e o grupo abrangendo os demais tipos de lesões glomerulares que foram encontrados. Foram utilizados 24 cães com doença renal crônica que morreram. As lesões glomerulares foram classificadas segundo Cianciolo et al. (2013) por meio da análise histopatológica de tecidos renais corados pelas técnicas histoquímicas de hematoxilina e eosina, tricrômico de Masson, ácido periódico de Schiff e vermelho Congo. Foram inclusos animais com exames de urina com sedimento inativo, RPC, creatinina e albumina sérica. Para análise estatística foram utilizados os últimos valores obtidos antes do óbito, não excedendo o limite de quatro semanas. Não foi observado diferenças entre RPC, albumina e creatinina séricas e pressão arterial sistólica entre os animais com alteração membranosa e o grupo contendo os demais tipos de lesão glomerular encontrados em cães com DRC. Na DRC, independentemente do tipo de lesão glomerular, os parâmetros clínicos e laboratoriais avaliados neste... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

The characteristics of cement kiln dusts (CKDs) and their effects as partial replacement of Portland Cement (PC) were studied in this research program. The materials used in this study were two different types of PC (normal and moderate sulfate resistant) and seven CKDs. The CKDs used in this study were selected to provide a representation of those available in North America from the three major types of cement manufacturing processes: wet, long-dry, and preheater/precalciner. Two fillers (limestone powder and quartz powder) were also used to compare their effects to that of CKDs at an equivalent replacement of PC. It was found that CKDs can contain significant amounts of amorphous material (>30%) and clinker compounds (>20%) and small amounts of slag and/or flyash (<5%) and calcium langbeinite (<5%). The study found that CKDs from preheater/precalciner kilns have different effects on workability and heat evolution than CKDs from wet and long-dry kilns due to the presence of very reactive and high free lime contents (>20%). The blends with the two CKDs from preheater/precalciner plants had higher paste water demand, lower mortar flows, and higher heat generation during initial hydrolysis in comparison to all other CKD-PC blends and control cements. The hardened properties of CKD as a partial substitute of PC appear to be governed by the sulfate content of the CKD-PC blend (the form of the CKD sulfate is not significant). According to analysis of the ASTM expansion in limewater test results, the CKD-PC blend sulfate content should be less than ~0.40% above the optimum sulfate content of the PC. It was also found that the sulfate contribution of CKD behaves similar to gypsum. Therefore, CKD-PC blends could be optimized for sulfate content by using CKD as a partial substitute of gypsum during the grinding process to control the early hydration of C3A. The wet and long-dry kiln CKDs contain significant amounts of calcium carbonate (>20%) which could also be used as partial replacement of limestone filler in PC.

The characteristics of cement kiln dusts (CKDs) and their effects as partial replacement of Portland Cement (PC) were studied in this research program. The materials used in this study were two different types of PC (normal and moderate sulfate resistant) and seven CKDs. The CKDs used in this study were selected to provide a representation of those available in North America from the three major types of cement manufacturing processes: wet, long-dry, and preheater/precalciner. Two fillers (limestone powder and quartz powder) were also used to compare their effects to that of CKDs at an equivalent replacement of PC. It was found that CKDs can contain significant amounts of amorphous material (>30%) and clinker compounds (>20%) and small amounts of slag and/or flyash (<5%) and calcium langbeinite (<5%). The study found that CKDs from preheater/precalciner kilns have different effects on workability and heat evolution than CKDs from wet and long-dry kilns due to the presence of very reactive and high free lime contents (>20%). The blends with the two CKDs from preheater/precalciner plants had higher paste water demand, lower mortar flows, and higher heat generation during initial hydrolysis in comparison to all other CKD-PC blends and control cements. The hardened properties of CKD as a partial substitute of PC appear to be governed by the sulfate content of the CKD-PC blend (the form of the CKD sulfate is not significant). According to analysis of the ASTM expansion in limewater test results, the CKD-PC blend sulfate content should be less than ~0.40% above the optimum sulfate content of the PC. It was also found that the sulfate contribution of CKD behaves similar to gypsum. Therefore, CKD-PC blends could be optimized for sulfate content by using CKD as a partial substitute of gypsum during the grinding process to control the early hydration of C3A. The wet and long-dry kiln CKDs contain significant amounts of calcium carbonate (>20%) which could also be used as partial replacement of limestone filler in PC.

A doença renal crônica (DRC) é uma afecção frequente em cães idosos, de alta morbidade e mortalidade, sendo definida como uma injúria renal morfo-funcional irreversível, de um ou ambos os rins, que está intrinsecamente associada à degeneração celular. Seu tratamento é paliativo, sendo que nos estágios mais avançados, o animal pode necessitar de hemodiálise ou transplante renal, prática dificultada e muitas vezes inviável na medicina veterinária. As células-tronco mesenquimais derivadas do tecido amniótico caracterizam-se por ser uma população de células de alta plasticidade e de grande potencial imunomodulador, sendo capazes de se diferenciar e produzir diferentes tipos celulares necessários num processo de reparação. Os avanços nos estudos das células-tronco podem tornar a terapia celular uma forma viável de tratamento alternativo ou adjuvante dessa doença, uma vez que estas poderiam restaurar a funcionalidade e manter a integridade do rim. O objetivo deste estudo foi avaliar se o tratamento experimental com células-tronco mesenquimais derivadas do âmnio (CTMAs) canino podem reduzir ou estabilizar a taxa de progressão e o quadro clínico da DRC em cães. Para tanto, células-tronco provenientes da membrana amniótica foram cultivadas até a segunda passagem (P2) e criopreservadas para posterior aplicação. Onze cães domésticos, machos e/ou fêmeas, acometidos pela DRC adquirida em graus II ou III segundo classificação da IRIS e sem outra afecção adjacente receberam duas aplicações de CTMAs nos dias D0 e D30, por via endovenosa. Para avaliar a progressão ou estabilização do quadro clínico foram colhidas amostras de sangue total, soro sanguíneo e urina para exames de hemograma, bioquímica sérica e urinaria e urinálise em quatro momentos: D0, D7, D30 e D60. A análise estatística foi realizada através da aplicação do teste ANOVA, para comparação de médias nas diferentes fases de tratamento, seguida pelo teste de Tukey, para comparação das médias entre os grupos. Do ponto de vista clínico, dois animais apresentaram melhora e se mantiveram estáveis durante todo o período de acompanhamento, dois animais apresentaram melhora nos primeiros 30 dias, apresentando novamente sintomatologia da doença após esse período e os demais apresentaram melhora nos primeiros sete dias de tratamento, havendo uma piora geral do quadro após esse período. Contudo, os exames laboratoriais em todos os casos não revelaram uma melhora significativa com o tratamento. Aparentemente, a utilização de células-tronco de origem amniótica não influencia de forma relevante na melhora da doença devido à extensa lesão renal que cães apresentam. / Chronic kidney disease (CKD) is a common condition in older dogs with high morbidity and mortality and is defined as an irreversible morpho-functional renal injury of one or both kidneys, which is intrinsically associated with cell degeneration. Its treatment is palliative, and in the more advanced stages, the animal may need dialysis or kidney transplantation, a practice that is difficult and often not feasible in veterinary medicine. The mesenchymal stem cells derived from amniotic tissue characterized by being a population of high plasticity and high cell immunomodulatory potential, being able to differentiate and produce different cell types required in a repair process. Advances in stem cell studies may make cell therapy a viable alternative or adjunctive treatment for this disease, since it could restore functionality and maintain kidney integrity. The objective of this study was to evaluate if the experimental treatment with the canine amnio- derived mesenchymal stem cells (AMSCs) can reduce or stabilize the rate of progression, and clinical condition of CKD in dogs. For this purpose, stem cells from the amniotic membrane were grown until the second pass (P2) and cryopreserved for later use. Eleven domestic male and / or female dogs, affected by the CKD acquired in grades II or III according to IRIS classification and without another adjacent disease, received two applications on days D0 and D30 intravenously. To evaluate the progression or stabilization of the clinical condition, whole blood, blood serum and urine samples were collected for hemogram, serum and urinary biochemistry and urinalysis at four moments: D0, D7, D30 and D60. Statistical analysis was performed using the ANOVA test, to compare means in the different treatment phases, followed by the Tukey test, to compare the means between the groups. From a clinical point of view, two animals showed improvement and remained stable throughout the follow-up period, two animals showed improvement in the first 30 days, showing again symptoms of the disease after this period and the other showed improvement in the first seven days of treatment, with a general worsening of the condition after this period. However, laboratory tests in all cases showed no significant improvement with treatment. Apparently, the use of stem cells of amniotic origin does not influence in a relevant way the improvement of the pathology due the extensive kidney lesion presented by dogs.

Das Ziel dieser Arbeit war die Identifikation und Assoziation nierenpathogener Gen-Varianten mit dem klinischen Phänotyp einer chronischen Niereninsuffizienz (CKD) durch chronisch tubulo-interstitielle Nephritis (CIN) unbekannter Ursache bei Erwachsenen. Die Auswahl der Studienkohorte erfolgte anhand festgelegter histologischer und klinischer Kriterien (prädominante CIN unklarer Ursache), die sich in 52 von 785 Biopsie-Befunden aus den Jahren 1983 bis 2013 finden ließen. Insbesondere aufgrund fehlender Kontaktierbarkeit infolge des langen Beobachtungszeitraums konnten letztlich 10 der 52 Probanden rekrutiert und in die genetische Analyse inkludiert werden. Nach der Durchführung einer Exom-Sequenzierung wurden die Rohdatensätze jedes einzelnen Probanden auf ausschließlich seltene Varianten gefiltert. Der verbliebene Datensatz wurde dann auf seltene Varianten in 199 bekannten mit hereditären Nephropathien assoziierten Gene hin untersucht (virtuelles CKD-Genpanel). Zunächst konzentrierten wir uns auf Gene die mit tubulo-interstitiellen Nephropathien assoziiert sind (NPHP-RC, ARPKD, ADTKD). Im weiteren Verlauf inkludierten wir Gene für glomeruläre Nephropathien (FSGS und COL4-Nephropathie/Alport-Syndrom) und kongenitale Nierenanomalien (CAKUT). Die dabei detektierten genetischen Varianten wurden auf mögliche Pathogenität anhand der ACMG-Klassifizierung final bewertet. Daraus resultierten bei vier Probanden insgesamt fünf wahrscheinlich pathogene Varianten der ACMG-Klasse 4 und 5 (pathogenic, likely pathogenic) in den Genen NPHS2, COL4A4, COL4A3 und DSTYK. Die diagnostisch bedeutsamen Varianten wurden in der Literatur bereits im Zusammenhang mit hereditäre FSGS, Kollagen-IV-Nephropathie und CAKUT beschrieben. Bei einem Probanden (A5204) mit pathogenen Varianten im NPHS2-Gen konnten wir mittels Segregationsanalyse der Eltern den klinischen Phänotyp eindeutig zuordnen. Des Weiteren konnten wir 12 Varianten unklarer Signifikanz (VUS) bei insgesamt sechs Probanden ausfindig machen, welche sich teilweise in funktionell bedeutsamen Genregionen befinden. Hier zu nennen sind insbesondere die gefundenen VUS-Varianten in den Genen TNXB und FN1, die eine Pathogenität nahelegen, deren Bedeutung aber für die Entwicklung einer chronischen Niereninsuffizienz in der vorliegenden Studie nicht abschließend beurteilt werden konnte. Zusammenfassend demonstriert diese Arbeit die häufig fehlende Übereinstimmung von histologischen Diagnosen (vordergründig tubulo-interstitielle Nephropathie) und genetischen Diagnosen (vordergründig glomeruläre Nephropathie) in der Abklärung einer chronischen Niereninsuffizienz. Insbesondere zeigt sich in der vorliegenden Arbeit der Wert einer breiten genetischen Analyse zur genaueren ätiologischen Abklärung von CKD-Patienten mit unspezifischen nephropathologischen Veränderungen, wie der der chronisch-interstitiellen Nephritis.:1 Inhaltsverzeichnis ........................................................................................................................... 1 2 Abkürzungsverzeichnis ................................................................................................................... 3 3 Einführung ....................................................................................................................................... 6 3.1 Chronische Niereninsuffizienz (CKD) ...................................................................................... 6 3.1.1 Bedeutung der Nierenbiospie ......................................................................................... 8 3.1.2 CIN – chronische interstitielle Nephritis .......................................................................... 9 3.2 Hereditäre chronische Nephropathien .................................................................................. 9 3.2.1 Hereditäre tubulo-interstitielle Nephropathien ............................................................ 11 3.2.1.1 Nephronophthise (NPHP) / Nephronophthise-assoziierte Ziliopathien .................... 11 3.2.1.2 ADPKD – autosomal dominante polyzystische Nierenerkrankung ........................... 14 3.2.1.3 ARPKD – autosomal rezessive polyzystische Nierenerkrankung ............................... 15 3.2.1.4 ADTKD – autosomal dominante tubulo-interstitielle Nierenerkrankung .................. 15 3.2.2 Hereditäre Glomerulopathien ....................................................................................... 16 3.2.2.1 Alport-Syndrom / Syndrom der dünnen Basalmembran (TBMD, TBMN) ................. 16 3.2.2.2 FSGS – fokal segmentale Glomerulosklerose ............................................................ 17 3.2.3 CAKUT ............................................................................................................................ 17 4 Aufgabenstellung .......................................................................................................................... 18 5 Material und Methoden ............................................................................................................... 19 5.1 Studienaufbau ....................................................................................................................... 19 5.1.1 Nierenbiopsien und histologischer Befund ................................................................... 19 5.1.2 Auswahl und Rekrutierung der Probanden ................................................................... 19 5.2 Exom-Sequenzierung ............................................................................................................ 20 5.2.1 Definition und Bedeutung ............................................................................................. 20 5.2.2 Durchführung des WES .................................................................................................. 21 5.2.3 Zu untersuchende Gene ................................................................................................ 21 5.3 Filterung und Analyse genetischer Varianten...................................................................... 24 5.3.1 Filterkriterien für Rohdatensätze .................................................................................. 24 5.3.2 Prädiktionstools für genetische Varianten .................................................................... 27 5.3.3 Mutationsdatenbank (HGMD) ....................................................................................... 28 5.3.4 ACMG-Klassifizierungssystem ....................................................................................... 28 6 Ergebnisse ..................................................................................................................................... 29 6.1 WES-Datenqualität ............................................................................................................... 29 6.2 Ergebnisse anhand der ACMG-Klassifizierung ..................................................................... 31 6.2.1 Genetische Varianten der Klasse 4 und 5 (pathogen und wahrscheinlich pathogen) .. 31 6.2.2 Genetische Varianten der Klasse 3 (VUS) ...................................................................... 36 7 Diskussion ..................................................................................................................................... 43 8 Zusammenfassung ........................................................................................................................ 49 9 Literaturverzeichnis ...................................................................................................................... 51 10 Anlagen ......................................................................................................................................... 57 10.1 Abbildungsverzeichnis ........................................................................................................... 57 10.2 Tabellenverzeichnis ............................................................................................................... 57 11 Selbstständigkeitserklärung ......................................................................................................... 58 12 Lebenslauf 13 Danksagung

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21008号 / 医博第4354号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 小川 修, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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This systematic review aims to examine the relationship between the social determinants of health that significantly impact the prevalence and progression of chronic kidney disease (CKD) amongst Black Americans. The Black American population has the highest prevalence of CKD in the United States, while concurrently possessing prominent genetic risk factors for this chronic disease. The social determinants: healthcare quality and access (extended to account for health behaviors), social and community context, and economic stability were specifically assessed in this review. Key terms were utilized to search electronic databases PubMed and Web of Science, which yielded 470 unduplicated articles. Twenty-nine articles met the inclusion criteria for this systematic review, with three articles being applicable to the three selected social determinants, six being applicable to social and community context, six being applicable to economic stability, and fourteen being applicable to healthcare quality and access. Major contributors towards CKD incidence and progression amongst Black Americans were identified to be: exposure to discrimination, expectations of discrimination and prejudice, low routine medical care, limited health literacy, distrust of health providers, being of low socioeconomic status, and a lack of engagement in functional health behaviors (fruit/vegetable consumption and CKD screening). Each social determinant was seldom observed to be operating exclusively as a contributing factor towards CKD, exemplifying how the intersectionality of these factors contributes to increased CKD risk and progression. Findings from this systematic review highlight the need for targeted healthcare initiatives for Black Americans to remedy the CKD endemic.

Metabolic syndrome is a significant worldwide public health challenge and is inextricably linked to adverse renal and cardiovascular outcomes. The inhibition of the transient receptor potential cation channel subfamily C member 6 (TRPC6) has been found to ameliorate renal outcomes in the unilateral ureteral obstruction (UUO) of accelerated renal fibrosis. Therefore, the pharmacological inhibition of TPRC6 could be a promising therapeutic intervention in the progressive tubulo-interstitial fibrosis in hypertension and metabolic syndrome. In the present study, we hypothesized that the novel selective TRPC6 inhibitor SH045 (larixyl N-methylcarbamate) ameliorates UUO-accelerated renal fibrosis in a New Zealand obese (NZO) mouse model, which is a polygenic model of metabolic syndrome. The in vivo inhibition of TRPC6 by SH045 markedly decreased the mRNA expression of pro-fibrotic markers (Col1a1, Col3a1, Col4a1, Acta2, Ccn2, Fn1) and chemokines (Cxcl1, Ccl5, Ccr2) in UUO kidneys of NZO mice compared to kidneys of vehicle-treated animals. Renal expressions of intercellular adhesion molecule 1 (ICAM-1) and -smooth muscle actin (-SMA) were diminished in SH045- versus vehicle-treated UUO mice. Furthermore, renal inflammatory cell infiltration (F4/80+ and CD4+) and tubulointerstitial fibrosis (Sirius red and fibronectin staining) were ameliorated in SH045-treated NZO mice. We conclude that the pharmacological inhibition of TRPC6 might be a promising antifibrotic therapeutic method to treat progressive tubulo-interstitial fibrosis in hypertension and metabolic syndrome.

Chronic kidney disease (CKD) is growing global public health problem affecting 1 in 10 adults in developed countries and recognised as an important risk factor for cardiovascular disease (CVD) development. CVD is the main cause of death among CKD patients. Endothelial injury and dysfunction are critical steps in atherosclerosis, a major CVD. Oxidative stress (increased level of reactive oxygen species, ROS) has been associated with CVD development. Intravenous (IV) iron preparations are widely used in the management of CKD mediated anaemia, and have been associated with increased oxidative stress and cellular dysfunction. This study examined the effect of pharmacologically-relevant concentrations of IV Venofer (iron sucrose) or IV Ferinject (Ferric carboxymaltose, FCM) on primary human umbilical vein endothelial cell (HUVEC) activation/damage and on intracellular ROS generation as well as studying the potential mechanisms responsible. Data from TUNEL assay and Annexin V-FITC/PI staining showed that, IV FCM had no effect, but IV iron sucrose increased HUVEC apoptosis at 24hr. IV iron sucrose inhibited cell proliferation and reduced cell viability. Both compounds induced EC activation through sustained activation of p38 MAPK and up-regulation of ICAM-1 and VCAM-1. Additionally, the compounds induced significant increase in total ROS and superoxide anion production, which was attenuated by the anti-oxidant N-acetylcysteine (NAC). P38 MAPK showed up-regulation of pro-apoptotic protein Bax and down-regulation of antiapoptotic Bcl-2 protein in HUVEC treated with IV iron sucrose and p38 inhibition reversed these effects. In summary, these results suggest that IV iron sucrose causes more severe EC injury than IV FCM. However, both IV iron preparations induced intracellular ROS and superoxide anion generation in HUVEC leading to EC activation/dysfunction, providing a potential explanation for vascular damage in CKD patients.