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.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/29119 |
| Date | 11 1900 |
| Creators | O'Neil, Kian |
| Contributors | Krepinsky, Joan, Medical Sciences (Blood and Cardiovascular) |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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