京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19222号 / 医博第4021号 / 新制||医||1010(附属図書館) / 32221 / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 長澤 丘司, 教授 伊達 洋至 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20276号 / 医博第4235号 / 新制||医||1021(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 齊藤 博英, 教授 楠見 明弘, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21639号 / 医博第4445号 / 新制||医||1034(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 濵﨑 洋子, 教授 小池 薫, 教授 清水 章 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第24504号 / 医博第4946号 / 新制||医||1064(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 溝脇 尚志, 教授 浅野 雅秀, 教授 鈴木 実 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease of unknown etiology, in which excessive accumulation of scar tissue in the interstitial spaces of the lung obstruct normal pulmonary function. Currently, the only curative treatment is lung transplantation. While pharmaceutical therapeutics have been recently approved for use in IPF in 2014, they are still unable to provide a truly curative treatment. While genetic risk factors have been identified, the most commonly occurring mutation is only detected in approximately 38% of IPF patients, leaving an uncertainty in the very existence of a common genetic factor in IPF. Cigarette smoke and other environmental particulates have been significantly linked to the diagnosis of IPF, implicating an initial immunological response to trigger the pathogenesis of IPF. Nintedanib, a potent tyrosine kinase receptor inhibitor was first developed in 1998 as a candidate for cancer treatment. Investigation of its effects in fibrosis in the past few decades has led to a significant discovery of its application in IPF. Nintedanib significantly inhibits the fibrotic activity of fibrotic myofibroblasts in the lungs by inhibiting signaling cascades necessary for cell proliferation and progression of the disease. However, nintedanib falls short in that it cannot fully inhibit the advancement of the disease and mortality rates of IPF still remain high. Pirfenidone, the other currently available pharmaceutical therapeutic, was discovered in 1976 as a potent inhibitor of inflammation. Subsequent experiments further reviled its potency as an anti-fibrotic drug. After decades of research, pirfenidone’s mechanism of antifibrotic characteristics were revealed as a potent inhibitor of fibrocyte recruitment and chemotaxis, and as an inhibitor of transcription growth factor beta (a growth factor heavily implicated in the activity of myofibroblasts) mediated pathways. However, like nintedanib, pirfenidone fails as a curative treatment, only delaying the progression of the disease. In the search for new molecular targets for pharmaceutical therapy, forkhead box M1 (FOXM1), programmed cell death protein-1 (PD-1), and prostaglandin E2, have been identified to play a mediatory role in many of the pathways involved in myofibroblast activity. Many of these targets have also been identified in other disease models such as cancer and immunological inflammatory disease. Avasimibe has been recently identified as a potent inhibitor of aldo-ketoreductase through a FOXM1 mediated pathway. Its molecular mechanism in osteosarcoma cancer disease model may prove to be a novel pharmaceutical therapeutic for IPF. BI 853250, a novel focal adhesion kinase (FAK) inhibitor also demonstrates potential to be a new pharmaceutical therapeutic for IPF patients. Exploring signaling pathways that involve these newly found targets and collaborative research with cancer and immunological diseases shows promise in providing steps to cure IPF in the future.

ATP-citrate lyase (ACLY), upregulated in chronic kidney disease (CKD), catalyzes the synthesis of acetyl-coA from citrate. Acetyl-CoA is a vital precursor for lipid/cholesterol synthesis and histone acetylation that regulates gene expression. In renal cells, ACLY regulates fibrogenic, lipogenic and inflammatory gene expression; its inhibition reduced fibrosis in the unilateral ureteral obstruction (UUO) model. The ACLY metabolic by-product malonyl-coA is also an important inhibitor of fatty acid oxidation (FAO), and defective FAO in proximal tubular epithelial cells (PTEC) is now established as a major contributor to fibrosis. Here we tested the efficacy of a novel ACLY inhibitor on reducing fibrosis and its potential role in improving FAO in UUO. 8-week-old male C57BL/6J mice underwent UUO surgery and were treated orally with an ACLY inhibitor (EVT0185, Espervita Therapeutics) for 10 days. Kidneys were assessed by immunohistochemistry, immunoblotting, and RNAseq. Effects of ACLY inhibition were tested on the HK2 PTEC cell line and primary renal fibroblast responses to TGFβ1 (5ng/ml, 48h), a cytokine known to promote fibrosis and reduce FAO. Lipid accumulation was assessed by Oil Red O staining and LC/MS analysis. ACLY inhibition significantly and dose-dependently decreased fibrosis in the UUO model determined by trichrome, PSR, fibronectin, and α-smooth muscle actin (SMA) expression. ACLY inhibition decreased macrophage (F4/80) infiltration including that of the profibrotic M2 phenotype marked by CD206. RNAseq analysis showed upregulation of FAO-related hallmark pathways and reduction in inflammation pathways with ACLY inhibition. Defective FAO is known to result in PTEC apoptosis and lipid accumulation. ACLY inhibition reduced both apoptosis, as assessed by the presence of cleaved caspase 3, as well as lipid accumulation, with a particular decrease in cholesteryl esters. In HK2 cells and renal fibroblasts, TGFβ1-induced fibrotic protein expression was inhibited by ACLY inhibition, and lipid accumulation was reduced in PTECs. ACLY inhibition reduced renal fibrosis, apoptosis, and lipid accumulation in UUO mice. ACLY inhibition also prevented profibrotic responses to TGFβ1 in PTECs and fibroblasts. Current studies are ongoing to confirm beneficial effects on restoring FAO. / Thesis / Master of Science (MSc) / Chronic kidney disease (CKD) is the leading cause of kidney failure in Canada, affecting 4 million Canadians. There is no cure for CKD and current treatments are only able to slow down disease progression. CKD is caused by scarring in the kidney. The kidney requires a lot of energy to do its job filtering our blood and creating urine, and with CKD the ability to create and use energy is reduced. The protein ATP-citrate lyase (ACLY) that is present in the kidney contributes to CKD. Research has shown that people and mice with CKD have higher levels of this protein than healthy individuals. ACLY creates a molecule called acetyl-coA that is likely to cause our kidneys to produce less energy. This study will test if ACLY is causing the kidneys to produce and use less energy. This will be done by using mice with CKD and blocking the activity of ACLY using a drug to see if this will help the kidney create more energy for itself. The kidneys of the mice will be tested to see if the drug worked in increasing energy levels and if it prevented kidney scarring. A type of cell in the kidney, called tubular cells, makes up most of the kidney and requires a lot of energy to function. We performed experiments with tubular cells and gave them stressors, like those found in CKD, and ACLY-blockers to test if the energy levels are restored and if scarring was reduced. This study is important because there is no cure for CKD and many patients will eventually develop end-stage kidney disease, requiring dialysis or transplant. Research needs to be done to create new medications for those suffering from CKD. Current studies are testing ACLY-blocking drugs to treat heart disease. If our study is successful, this drug is well-positioned to be developed into a new treatment for CKD.

Cystic fibrosis (CF) is one of the most common genetic diseases in Europe and North America. Chronic bacterial infections with Pseudomonas aeruginosa (P. aeruginosa) are common among CF patients and are associated with increased disease progression among patients. While inflammation is considered to be a key driver of lung function decline, the precise mechanisms at play have remained unclear. The objective of this thesis was to evaluate the role of inflammatory signalling components that result in host cell death during respiratory infections observed in CF. First, I investigated the differences in inflammatory mechanisms and cytokine expression induced by P. aeruginosa isolated from early versus chronic infections in CF. I found that early respiratory isolates of P. aeruginosa from CF patients induced inflammasome signalling, cell death, and IL-1β expression by THP-1 macrophages, yet little expression of other proinflammatory cytokines. However, P. aeruginosa isolates from chronic infections induced relatively less THP-1 macrophage inflammasome signalling, cell death, and IL-1β expression but greater production of other cytokines. Using laboratory reference strains and various mutants of P. aeruginosa, I validated how due to their inability to induce early and extensive host cell death, isolates from chronic infections are able to induce sustained levels of proinflammatory cytokines, which may contribute to the pathogenesis observed in CF. I then investigated one specific virulence factor identified among clinical P. aeruginosa isolates, the effector protein ExoU. ExoU is known to induce rapid host cell death and has previously been described to be an inhibitor of caspase-1, limiting IL-1β secretion in immune cells. Using relevant laboratory reference strains, I have shown that ExoU is able to induce IL-1β expression at lower multiplicities of infection or at earlier time points than described in previous reports when infecting THP-1 macrophages and NuLi-1 bronchial epithelial cells. Through immunoblotting and the use of relevant inhibitors, it was found that this observed difference could be partially dependent on the activation of various caspases, including ones that induced canonical and non-canonical inflammasome activation. Overall, this described work adds to our understanding of respiratory infections observed among CF patients and could shed light on possible therapeutic options to reduce disease progression.