Inhibition of asparagine synthetase effectively retards polycystic kidney disease progression.

Polycystic kidney disease (PKD) is a genetic disorder characterized by bilateral cyst formation. We showed that PKD cells and kidneys display metabolic alterations, including the Warburg effect and glutaminolysis, sustained in vitro by the enzyme asparagine synthetase (ASNS). Here, we used antisense oligonucleotides (ASO) against Asns in orthologous and slowly progressive PKD murine models and show that treatment leads to a drastic reduction of total kidney volume (measured by MRI) and a prominent rescue of renal function in the mouse. Mechanistically, the upregulation of an ATF4-ASNS axis in PKD is driven by the amino acid response (AAR) branch of the integrated stress response (ISR). Metabolic profiling of PKD or control kidneys treated with Asns-ASO or Scr-ASO revealed major changes in the mutants, several of which are rescued by Asns silencing in vivo. Indeed, ASNS drives glutamine-dependent de novo pyrimidine synthesis and proliferation in cystic epithelia. Notably, while several metabolic pathways were completely corrected by Asns-ASO, glycolysis was only partially restored. Accordingly, combining the glycolytic inhibitor 2DG with Asns-ASO further improved efficacy. Our studies identify a new therapeutic target and novel metabolic vulnerabilities in PKD.

Metabolic Reprogramming and Reconstruction: Integration of Experimental and Computational Studies to Set the Path Forward in ADPKD.

Metabolic reprogramming is a key feature of Autosomal Dominant Polycystic Kidney Disease (ADPKD) characterized by changes in cellular pathways occurring in response to the pathological cell conditions. In ADPKD, a broad range of dysregulated pathways have been found. The studies supporting alterations in cell metabolism have shown that the metabolic preference for abnormal cystic growth is to utilize aerobic glycolysis, increasing glutamine uptake and reducing oxidative phosphorylation, consequently resulting in ADPKD cells shifting their energy to alternative energetic pathways. The mechanism behind the role of the polycystin proteins and how it leads to disease remains unclear, despite the identification of numerous signaling pathways. The integration of computational data analysis that accompanies experimental findings was pivotal in the identification of metabolic reprogramming in ADPKD. Here, we summarize the important results and argue that their exploitation may give further insights into the regulative mechanisms driving metabolic reprogramming in ADPKD. The aim of this review is to provide a comprehensive overview on metabolic focused studies and potential targets for treatment, and to propose that computational approaches could be instrumental in advancing this field of research.

Metabolic reprogramming and the role of mitochondria in polycystic kidney disease.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a slowly progressive disease characterized by the relentless growth of renal cysts throughout the life of affected individuals. Early evidence suggested that the epithelia lining the cysts share neoplastic features, leading to the definition of PKD as a "neoplasm in disguise". Recent work from our and other laboratories has identified a profound metabolic reprogramming in PKD, similar to the one reported in cancer and consistent with the reported increased proliferation. Multiple lines of evidence suggest that aerobic glycolysis (a Warburg-like effect) is present in the disease, along with other metabolic dysfunctions such as an increase in the pentose phosphate pathway, in glutamine anaplerosis and fatty acid biosynthesis, while fatty acid oxidation and oxidative phosphorylation (OXPHOS) are decreased. In addition to glutamine, other amino acid-related pathways appear altered, including asparagine and arginine. The precise origin of the metabolic alterations is not entirely clear, but two hypotheses can be formulated, not mutually exclusive. First, the polycystins have been recently shown to regulate directly mitochondrial function and structure either by regulating Ca2+ uptake in mitochondria at the Mitochondria Associated Membranes (MAMs) of the Endoplasmic Reticulum, or by a direct translocation of a small fragment of the protein into the matrix of mitochondria. One alternative possibility is that metabolic and mitochondrial dysfunctions in ADPKD are secondary to the de-regulation of proliferation, driven by the multiple signaling pathways identified in the disease, which include mTORC1 and AMPK among the most relevant. While the precise mechanisms underlying these novel alterations identified in ADPKD will need further investigation, it is evident that they offer a great opportunity for novel interventions in the disease.

Metabolism and mitochondria in polycystic kidney disease research and therapy.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common, potentially lethal, monogenic diseases and is caused predominantly by mutations in polycystic kidney disease 1 (PKD1) and PKD2, which encode polycystin 1 (PC1) and PC2, respectively. Over the decades-long course of the disease, patients develop large fluid-filled renal cysts that impair kidney function, leading to end-stage renal disease in ~50% of patients. Despite the identification of numerous dysregulated pathways in ADPKD, the molecular mechanisms underlying the renal dysfunction from mutations in PKD genes and the physiological functions of the polycystin proteins are still unclear. Alterations in cell metabolism have emerged in the past decade as a hallmark of ADPKD. ADPKD cells shift their mode of energy production from oxidative phosphorylation to alternative pathways, such as glycolysis. In addition, the polycystins seem to play regulatory roles in modulating mechanisms and machinery related to energy production and utilization, including AMPK, PPARα, PGC1α, calcium signalling at mitochondria-associated membranes, mTORC1, cAMP and CFTR-mediated ion transport as well as the expression of crucial components of the mitochondrial energy production apparatus. In this Review, we explore these metabolic changes and discuss in detail the relationship between energy metabolism and ADPKD pathogenesis and identify potential therapeutic targets.

MacroH2A1 isoforms are associated with epigenetic markers for activation of lipogenic genes in fat-induced steatosis.

The importance of epigenetic changes in the development of hepatic steatosis is largely unknown. The histone variant macroH2A1 under alternative splicing gives rise to macroH2A1.1 and macroH2A1.2. In this study, we show that the macroH2A1 isoforms play an important role in the regulation of lipid accumulation in hepatocytes. Hepatoma cell line and immortalized human hepatocytes transiently transfected or knocked down with macroH2A1 isoforms were used as in vitro model of fat-induced steatosis. Gene expressions were analyzed by quantitative PCR array and Western blot. Chromatin immunoprecipitation analysis was performed to check the association of histone H3 lysine 27 trimethylation (H3K27me3) and histone H3 lysine 4 trimethylation (H3K4me3) with the promoter of lipogenic genes. Livers from knockout mice that are resistant to lipid deposition despite a high-fat diet were used for histopathology. We found that macroH2A1.2 is regulated by fat uptake and that its overexpression caused an increase in lipid uptake, triglycerides, and lipogenic genes compared with macroH2A1.1. This suggests that macroH2A1.2 is important for lipid uptake, whereas macroH2A1.1 was found to be protective. The result was supported by a high positivity for macroH2A1.1 in knockout mice for genes targeted by macroH2A1 (Atp5a1 and Fam73b), that under a high-fat diet presented minimal lipidosis. Moreover, macroH2A1 isoforms differentially regulate the expression of lipogenic genes by modulating the association of the active (H3K4me3) and repressive (H3K27me3) histone marks on their promoters. This study underlines the importance of the replacement of noncanonical histones in the regulation of genes involved in lipid metabolism in the progression of steatosis.

An N-ethyl-N-nitrosourea induced corticotropin-releasing hormone promoter mutation provides a mouse model for endogenous glucocorticoid excess.

Cushing's syndrome, which is characterized by excessive circulating glucocorticoid concentrations, may be due to ACTH-dependent or -independent causes that include anterior pituitary and adrenal cortical tumors, respectively. ACTH secretion is stimulated by CRH, and we report a mouse model for Cushing's syndrome due to an N-ethyl-N-nitrosourea (ENU) induced Crh mutation at -120 bp of the promoter region, which significantly increased luciferase reporter activity and was thus a gain-of-function mutation. Crh(-120/+) mice, when compared with wild-type littermates, had obesity, muscle wasting, thin skin, hair loss, and elevated plasma and urinary concentrations of corticosterone. In addition, Crh(-120/+) mice had hyperglycemia, hyperfructosaminemia, hyperinsulinemia, hypercholesterolemia, hypertriglyceridemia, and hyperleptinemia but normal adiponectin. Crh(-120/+) mice also had low bone mineral density, hypercalcemia, hypercalciuria, and decreased concentrations of plasma PTH and osteocalcin. Bone histomorphometry revealed Crh(-120/+) mice to have significant reductions in mineralizing surface area, mineral apposition, bone formation rates, osteoblast number, and the percentage of corticoendosteal bone covered by osteoblasts, which was accompanied by an increase in adipocytes in the bone marrow. Thus, a mouse model for Cushing's syndrome has been established, and this will help in further elucidating the pathophysiological effects of glucocorticoid excess and in evaluating treatments for corticosteroid-induced osteoporosis.

Immunopositivity for histone macroH2A1 isoforms marks steatosis-associated hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Prevention and risk reduction are important and the identification of specific biomarkers for early diagnosis of HCC represents an active field of research. Increasing evidence indicates that fat accumulation in the liver, defined as hepatosteatosis, is an independent and strong risk factor for developing an HCC. MacroH2A1, a histone protein generally associated with the repressed regions of chromosomes, is involved in hepatic lipid metabolism and is present in two alternative spliced isoforms, macroH2A1.1 and macroH2A1.2. These isoforms have been shown to predict lung and colon cancer recurrence but to our knowledge, their role in fatty-liver associated HCC has not been investigated previously. METHODS: We examined macroH2A1.1 and macroH2A1.2 protein expression levels in the liver of two murine models of fat-associated HCC, the high fat diet/diethylnistrosamine (DEN) and the phosphatase and tensin homolog (PTEN) liver specific knock-out (KO) mouse, and in human liver samples of subjects with steatosis or HCC, using immunoblotting and immunohistochemistry. RESULTS: Protein levels for both macroH2A1 isoforms were massively upregulated in HCC, whereas macroH2A1.2 was specifically upregulated in steatosis. In addition, examination of human liver samples showed a significant difference (p<0.01) in number of positive nuclei in HCC (100% of tumor cells positive for either macroH2A1.1 or macroH2A1.2), when compared to steatosis (<2% of hepatocytes positive for either isoform). The steatotic areas flanking the tumors were highly immunopositive for macroH2A1.1 and macroH2A1.2. CONCLUSIONS: These data obtained in mice and humans suggest that both macroH2A1 isoforms may play a role in HCC pathogenesis and moreover may be considered as novel diagnostic markers for human HCC.

Redox homeostasis and epigenetics in non-alcoholic fatty liver disease (NAFLD).

Non-alcoholic fatty liver disease (NAFLD), an accumulation of intra-hepatic triglycerides that is often considered the hepatic manifestation of insulin resistance, is the most common cause of chronic liver disease in the Western countries with up to one third of the population affected. NAFLD is a spectrum of disturbances that encompasses various degrees of liver damage ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). NASH is characterized by hepatocellular injury/inflammation with or without fibrosis. The individuals with NAFLD develop NASH in 10% of the cases, and are also at risk of developing hepatocellular carcinoma (HCC). Epigenetic mechanisms of nuclear chromatin remodeling, such as DNA methylation, post-translational modifications of histones, and incorporation of histone variants into the chromatin are increasingly recognized as crucial factors in the pathophysiology of NAFLD. NAFLD is often accompanied by oxidative stress: reactive oxygen species (ROS) are implicated in altered reduction/oxidation (redox) reactions that attack cellular macromolecules and are detected in the liver of patients and animal models of NAFLD. In this review, we summarize recent knowledge advancements in the hepatic epigenetic and redox mechanisms, and their possible links, involved in the pathogenesis and treatment of NAFLD.

The critical role of histone H2A-deubiquitinase Mysm1 in hematopoiesis and lymphocyte differentiation.

Stem cell differentiation and lineage specification depend on coordinated programs of gene expression, but our knowledge of the chromatin-modifying factors regulating these events remains incomplete. Ubiquitination of histone H2A (H2A-K119u) is a common chromatin modification associated with gene silencing, and controlled by the ubiquitin-ligase polycomb repressor complex 1 (PRC1) and H2A-deubiquitinating enzymes (H2A-DUBs). The roles of H2A-DUBs in mammalian development, stem cells, and hematopoiesis have not been addressed. Here we characterized an H2A-DUB targeted mouse line Mysm1(tm1a/tm1a) and demonstrated defects in BM hematopoiesis, resulting in lymphopenia, anemia, and thrombocytosis. Development of lymphocytes was impaired from the earliest stages of their differentiation, and there was also a depletion of erythroid cells and a defect in erythroid progenitor function. These phenotypes resulted from a cell-intrinsic requirement for Mysm1 in the BM. Importantly, Mysm1(tm1a/tm1a) HSCs were functionally impaired, and this was associated with elevated levels of reactive oxygen species, γH2AX DNA damage marker, and p53 protein in the hematopoietic progenitors. Overall, these data establish a role for Mysm1 in the maintenance of BM stem cell function, in the control of oxidative stress and genetic stability in hematopoietic progenitors, and in the development of lymphoid and erythroid lineages.