Activation Of Hypoxia Signaling Research Articles

Sirt3 deficiency promotes endothelial dysfunction and aggravates renal injury.

Sirtuin 3 (SIRT3), the main deacetylase of mitochondria, modulates the acetylation levels of substrates governing metabolism and oxidative stress. In the kidney, we showed that SIRT3 affects the proper functioning of high energy-demanding cells, such as tubular cells and podocytes. Less is known about the role of SIRT3 in regulating endothelial cell function and its impact on the progression of kidney disease. Here, we found that whole body Sirt3-deficient mice exhibited reduced renal capillary density, reflecting endothelial dysfunction, and VEGFA expression compared to wild-type mice. This was paralleled by activation of hypoxia signaling, upregulation of HIF-1α and Angiopietin-2, and oxidative stress increase. These alterations did not result in kidney disease. However, when Sirt3-deficient mice were exposed to the nephrotoxic stimulus Adriamycin (ADR) they developed aggravated endothelial rarefaction, altered VEGFA signaling, and higher oxidative stress compared to wild-type mice receiving ADR. As a result, ADR-treated Sirt3-deficient mice experienced a more severe injury with exacerbated albuminuria, podocyte loss and fibrotic lesions. These data suggest that SIRT3 is a crucial regulator of renal vascular homeostasis and its dysregulation is a predisposing factor for kidney disease. By extension, our findings indicate SIRT3 as a pharmacologic target in progressive renal disease whose treatments are still imperfect.

The association between renal accumulation of pancreatic amyloid-forming amylin and renal hypoxia.

Chronic kidney disease (CKD) is increasing worldwide and is associated with diabetic states (obesity, prediabetes and type-2 diabetes mellitus). The kidney is intrinsically susceptible to low oxygen (hypoxia) and renal hypoxia plays a vital role in the progression of CKD. Recent studies suggest an association between CKD and renal deposition of amyloid-forming amylin secreted from the pancreas. Renal accumulation of amyloid-forming amylin is associated with hypertension, mitochondrial dysfunction, increased production of reactive oxygen species (ROS) and activation of hypoxia signaling in the kidney. In this review we will discuss potential associations between renal amylin amyloid accumulation, hypertension, and mechanism of hypoxia-induced kidney dysfunction, including activation of hypoxia-inducible factors (HIFs) and mitochondrial dysfunction.

Different subpopulations of kidney interstitial cells produce erythropoietin and factors supporting tissue oxygenation in response to hypoxia in vivo

Genetic induction of hypoxia signaling by deletion of the von Hippel-Lindau (Vhl) protein in mesenchymal PDGFR-β+ cells leads to abundant HIF-2 dependent erythropoietin (EPO) expression in the cortex and outer medulla of the kidney. This rather unique feature of kidney PDGFR-β+ cells promote questions about their special characteristics and general functional response to hypoxia. To address these issues, we characterized kidney PDGFR-β+ EPO expressing cells based on additional cell markers and their gene expression profile in response to hypoxia signaling induced by targeted deletion of Vhl or exposure to low oxygen and carbon monoxide respectively, and after unilateral ureteral obstruction. CD73+, Gli1+, tenascin C+ and interstitial SMMHC+ cells were identified as zonally distributed subpopulations of PDGFR-β+ cells. EPO expression could be induced by Vhl deletion in all PDGFR-β+ subpopulations. Under hypoxemic conditions, recruited EPO+ cells were mostly part of the CD73+ subpopulation. Besides EPO production, expression of adrenomedullin and regulator of G-protein signaling 4 was upregulated in PDGFR-β+ subpopulations in response to the different hypoxic stimuli. Thus, different kidney interstitial PDGFR-β+ subpopulations exist, capable of producing EPO in response to different stimuli. Activation of hypoxia signaling in these cells also induces factors likely contributing to improved kidney interstitial tissue oxygenation.

RESTORATION OF HYPOXIA SIGNALING IMPROVES AGING-ASSOCIATED LOSS OF SKELETAL MUSCLE REGENERATIVE POTENTIAL

Skeletal muscle retains the ability to regenerate throughout life, but this decreases significantly with aging. The present study investigates whether aging-associated loss of muscle hypoxia signaling limits regenerative potential. Utilizing young (3 months) and old (22-24 months) mice, skeletal muscle from old mice exhibited a 40% decline in the cross-sectional area (CSA) of newly regenerating fibers following cryoinjury at day 10 (p < 0.01) post-injury as compared to young. Focused PCR array demonstrated a greater than 3-fold decline in expression of the majority of hypoxia signaling genes. In particular, aryl hydrocarbon receptor nuclear translocator (ARNT), which is required for downstream hypoxia signaling and the transcription of hypoxia response genes, is 5-fold lower for both gene expression (p < 0.01) and protein levels (p < 0.01) in old versus young mice. To determine the effects of ARNT on muscle regeneration, we utilized a genetically modified mouse which results in an 80% decrease in ARNT gene expression following activation, specifically in skeletal muscle. Compared to littermate controls, mice with a muscle specific knockdown of ARNT (mKO ARNT) exhibit a 30% decline in regenerating fiber sizes at day 10 (p < 0.01) following cryoinjury, without any loss of regenerative potential in FACS isolated satellite cells ex vivo. Administration of a pharmacologic hypoxia activator, ML228, induced a 30% increase in regenerating fiber CSA in both old mice and mKO ARNT mice (p < 0.01) as compared to treatment with vehicle control. These data suggest hypoxia signaling declines with aging in skeletal muscle and activation of hypoxia signaling may promote regeneration.

FGF9 inhibits browning program of white adipocytes and associates with human obesity

Browning of white adipose tissue has been proven to be a potential target to fight against obesity and its metabolic commodities, making the exploration of molecules involved in browning process important. Among those browning agents reported recently, FGF21 play as a quite promising candidate for treating obesity for its obvious enhancement of thermogenic capacity in adipocyte and significant improvement of metabolic disorders in both mice and human. However, whether other members of fibroblast growth factor (FGF) family play roles in adipose thermogenesis and obese development is still an open question. Here, we examined the mRNA expression of all FGF family members in three adipose tissues of male C57BL/6 mice and found that FGF9 is highly expressed in adipose tissue and decreased under cold stress. Furthermore, FGF9 treatment inhibited thermogenic genes in the process of beige adipocytes differentiation from stromal vascular fraction (SVF) in a dose-dependent manner. Similar results were obtained with FGF9 overexpression. Consistently, knockdown of FGF9 in SVF cells by using lentiviral shRNA increased thermogenic genes in differentiated beige adipocytes. RNA sequencing analysis revealed a significant increment of hypoxia-inducible factor (HIF) pathway in the early stage of beige adipocytes differentiation under FGF9 treatment, which was validated by real-time PCR. FGF9 expression was increased in subcutaneous WAT of obese human and mice. This study shows that adipose-derived FGF9 play as an inhibitory role in the browning of white adipocytes. Activation of hypoxia signaling at early stage of adipose browning process may contribute to this anti-thermogenic effect of FGF9.

The Evolving Role of Succinate in Tumor Metabolism: An 18F-FDG-Based Study.

In recent years, inherited and acquired mutations in the tricarboxylic acid (TCA) cycle enzymes have been reported in diverse cancers. Pheochromocytomas and paragangliomas often exhibit dysregulation of glucose metabolism, which is also driven by mutations in genes encoding the TCA cycle enzymes or by activation of hypoxia signaling. Pheochromocytomas and paragangliomas associated with succinate dehydrogenase (SDH) deficiency are characterized by high 18F-FDG avidity. This association is currently only partially explained. Therefore, we hypothesized that accumulation of succinate due to the TCA cycle defect could be the major connecting hub between SDH-mutated tumors and the 18F-FDG uptake profile. Methods: To test whether succinate modifies the 18F-FDG metabolic profile of tumors, we performed in vitro and in vivo (small-animal PET/CT imaging and autoradiography) experiments in the presence of succinate, fumarate, and phosphate-buffered saline (PBS) in different cell models. As a control, we also evaluated the impact of succinate on 18F-fluorocholine uptake and retention. Glucose transporter 1 (GLUT1) immunohistochemistry was performed to assess whether 18F-FDG uptake correlates with GLUT1 staining. Results: Intratumoral injection of succinate significantly increased 18F-FDG uptake at 24 h on small-animal PET/CT imaging and autoradiography. No effect of succinate was observed on cancer cells in vitro, but interestingly, we found that succinate caused increased 18F-FDG uptake by human umbilical vein endothelial cells in a concentration-dependent manner. No significant effect was observed after intratumoral injection of fumarate or PBS. Succinate, fumarate, and PBS have no effect on cell viability, regardless of cell lineage. Intramuscular injection of succinate also significantly increases 18F-FDG uptake by muscle when compared with either PBS or fumarate, highlighting the effect of succinate on connective tissues. No difference was observed between PBS and succinate on 18F-fluorocholine uptake in the tumor and muscle and on hind limb blood flow. GLUT1 expression quantification did not significantly differ between the study groups. Conclusion: The present study shows that succinate stimulates 18F-FDG uptake by endothelial cells, a finding that partially explains the 18F-FDG metabotype observed in tumors with SDH deficiency. Although this study is an 18F-FDG-based approach, it provides an impetus to better characterize the determinants of 18F-FDG uptake in various tumors and their surrounding microenvironment, with a special emphasis on the role of tumor-specific oncometabolites.

Activation of Hypoxia Signaling in Stromal Progenitors Impairs Kidney Development

Intrauterine hypoxia is a reason for impaired kidney development. The cellular and molecular pathways along which hypoxia exerts effects on nephrogenesis are not well understood. They are likely triggered by hypoxia-inducible transcription factors (HIFs), and their effects appear to be dependent on the cell compartment contributing to kidney formation. In this study, we investigated the effects of HIF activation in the developing renal stroma, which also essentially modulates nephron development fromthe metanephric mesenchyme. HIF activation was achieved by conditional deletion of the von Hippel-Lindau tumor suppressor (VHL) protein in the forkhead box FOXD1 cell lineage, from which stromal progenitors arise. The resulting kidneys showed maturation defects associated with early postnatal death. In particular, nephron formation, tubular maturation, and the differentiation of smooth muscle, renin, and mesangial cells were impaired. Erythropoietin expression was strongly enhanced. Codeletion of VHL together with HIF2A but not with HIF1A led to apparently normal kidneys, and the animals reached normal age but were anemic because of low erythropoietin levels. Stromal deletion of HIF2A or HIF1A alone did not affect kidney development. These findings emphasize the relevance of sufficient intrauterine oxygenation for normal renal stroma differentiation, suggesting that chronic activity of HIF2 in stromal progenitors impairs kidney development. Finally, these data confirm the concept that normal stroma function is essential for normal tubular differentiation.

SLC30A10 Deficiency: A Novel Cause of Polycythemia and Hepcidin Deficiency

Like iron, manganese is an essential yet potentially toxic metal. While inherited forms of iron excess were discovered long ago, the first disease of inherited manganese excess was only recently reported in 2012. Mutations in a metal transport protein, SLC30A10, were described in patients with manganese excess, polycythemia, liver failure and Parkinsonism. Manganese excess in these patients is attributed to an essential role for SLC30A10 in hepatobiliary manganese excretion. Polycythemia, liver failure and Parkinsonism are attributed to manganese excess although the underlying mechanisms have yet to be established. To explore the role of SLC30A10 in metal biology and disease, we have generated Slc30a10-deficient mice. Characterization of these mice indicates that murine Slc30a10 deficiency recapitulates the human disease. Given that manganese and iron share some transport pathways, we interrogated iron homeostasis in these mutant mice. Intriguingly, mice display hepcidin deficiency and tissue iron excess. While hepcidin deficiency may explain the tissue iron excess, the cause of hepcidin deficiency is unclear. Based on expression profiling of hypoxia-regulated genes, we propose that hepcidin deficiency reflects aberrant activation of hypoxia signaling by manganese excess in the absence of true hypoxia. Overall, this data suggests novel interactions between iron and manganese with implications for our understanding of the mechanisms by which aberrant metal homeostasis leads to human disease. Disclosures No relevant conflicts of interest to declare.

Erythropoietin Synthesis in Renal Myofibroblasts Is Restored by Activation of Hypoxia Signaling

Erythropoietin (Epo) is produced by renal Epo-producing cells (REPs) in a hypoxia-inducible manner. The conversion of REPs into myofibroblasts and coincident loss of Epo-producing ability are the major cause of renal fibrosis and anemia. However, the hypoxic response of these transformed myofibroblasts remains unclear. Here, we used complementary in vivo transgenic and live imaging approaches to better understand the importance of hypoxia signaling in Epo production. Live imaging of REPs in transgenic mice expressing green fluorescent protein from a modified Epo-gene locus revealed that healthy REPs tightly associated with endothelium by wrapping processes around capillaries. However, this association was hampered in states of renal injury-induced inflammation previously shown to correlate with the transition to myofibroblast-transformed renal Epo-producing cells (MF-REPs). Furthermore, activation of hypoxia-inducible factors (HIFs) by genetic inactivation of HIF-prolyl hydroxylases (PHD1, PHD2, and PHD3) selectively in Epo-producing cells reactivated Epo production in MF-REPs. Loss of PHD2 in REPs restored Epo-gene expression in injured kidneys but caused polycythemia. Notably, combined deletions of PHD1 and PHD3 prevented loss of Epo expression without provoking polycythemia. Mice with PHD-deficient REPs also showed resistance to LPS-induced Epo repression in kidneys, suggesting that augmented HIF signaling counterbalances inflammatory stimuli in regulation of Epo production. Thus, augmentation of HIF signaling may be an attractive therapeutic strategy for treating renal anemia by reactivating Epo synthesis in MF-REPs.

Fumarate Hydratase Deficiency and Cancer: Activation of Hypoxia Signaling?

Molecular genetic analysis of hereditary leiomyomatosis and renal cell cancer (HLRCC) unexpectedly revealed germline defects in the gene encoding the Krebs cycle enzyme fumarate hydratase (FH), stimulating great interest in the underlying mechanism of oncogenesis. It has been proposed that the associated accumulation of fumarate competitively inhibits the 2-oxoglutarate-dependent dioxygenases that regulate hypoxia-inducible factor (HIF), thus activating oncogenic hypoxia pathways. In this issue of Cancer Cell, Pollard and colleagues describe a genetic mouse model of FH deficiency that recapitulates aspects of the human disease, including HIF activation and renal cysts, enabling further insights into this unusual cancer syndrome.