Effects of long-term tubular HIF-2α overexpression on progressive renal fibrosis in a chronic kidney disease model.

Renal fibrosis is the final manifestation of chronic kidney disease (CKD) regardless of etiology. Hypoxia-inducible factor-2 alpha (HIF-2α) is an important regulator of chronic hypoxia, and the late-stage renal tubular HIF-2α activation exerts protective effects against renal fibrosis. However, its specific role in progressive renal fibrosis remains unclear. Here, we investigated the effects of the long-term tubular activation of HIF-2α on renal function and fibrosis, using in vivo and in vitro models of renal fibrosis. Progressive renal fibrosis was induced in renal tubular epithelial cells (TECs) of tetracycline-controlled HIF-2α transgenic (Tg) mice and wild-type (WT) controls through a 6-week adenine diet. Tg mice were maintained on doxycycline (DOX) for the diet period to induce Tg HIF-2α expression. Primary TECs isolated from Tg mice were treated with DOX (5 µg/ml), transforming growth factor-ß1 (TGF-ß1) (10 ng/ml), and a combination of both for 24, 48, and 72 hr. Blood was collected to analyze creatinine (Cr) and blood urea nitrogen (BUN) levels. Pathological changes in the kidney tissues were observed using hematoxylin and eosin, Masson's trichrome, and Sirius Red staining. Meanwhile, the expression of fibronectin, E-cadherin and α-smooth muscle actin (α-SMA) and the phosphorylation of p38 mitogenactivated protein kinase (MAPK) was observed using western blotting. Our data showed that serum Cr and BUN levels were significantly lower in Tg mice than in WT mice following the adenine diet. Moreover, the protein levels of fibronectin and E-cadherin and the phosphorylation of p38 MAPK were markedly reduced in the kidneys of adenine-fed Tg mice. These results were accompanied by attenuated fibrosis in Tg mice following adenine administration. Consistent with these findings, HIF-2α overexpression significantly decreased the expression of fibronectin in TECs, whereas an increase in α-SMA protein levels was observed after TGF-ß1 stimulation for 72 hr. Taken together, these results indicate that long-term HIF-2α activation in CKD may inhibit the progression of renal fibrosis and improve renal function, suggesting that long-term renal HIF-2α activation may be used as a novel therapeutic strategy for the treatment of CKD. [BMB Reports 2023; 56(3): 196-201].

Therapeutic effects of three human-derived materials in a mouse corneal alkali burn model.

Purpose: To compare the therapeutic effects of human derivatives in a mouse alkali burn model. Methods: The right eyes of mice were injured using NaOH. After alkali injury, one of the following agents was topically administered for 7 d: human amniotic membrane (hAM) suspension, human umbilical cord serum (hUCS), and human peripheral blood serum (hPBS), or saline. The epithelial defect areas on days 1, 2, and 3 degrees of opacity on days 2, 3, and 7, and corneal neovascularization (NV) areas on day 7 were evaluated. Histologic examination and mRNA expression levels of tumour necrosis factor (TNF)-α, interleukin (IL)-6, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, MMP-8, and MMP-9 were also evaluated on day 7. Results: The epithelial defect areas in the hUCS group were smaller than those in the control and hPBS groups on day 3 (p < .05, respectively). The epithelial defect areas in the hAM suspension group showed smaller than those in the control and hPBS groups on days 1 and 2 (p < .05, respectively). The degrees of opacity were lower in all treatment groups than that of the saline control group on day 7 (p < .05, respectively). Corneal NV areas were not different among groups on day 7 (p = 0.20). The expression levels of TNF-α, IL-6, MMP-8, and MMP-9 mRNA and the infiltration of the inflammatory cells in all treatment groups were lesser than those in the control group on day 7 (p< .05, respectively). Conclusions: All treatments reduced inflammatory reactions and corneal opacity development. Corneal reepithelialization was faster in the hUCS group.

Sirt1-hypoxia-inducible factor-1α interaction is a key mediator of tubulointerstitial damage in the aged kidney.

Although it is known that the expression and activity of sirtuin 1 (Sirt1) decrease in the aged kidney, the role of interaction between Sirt1 and hypoxia-inducible factor (HIF)-1α is largely unknown. In this study, we investigated whether HIF-1α could be a deacetylation target of Sirt1 and the effect of their interaction on age-associated renal injury. Five-week-old (young) and 24-month-old (old) C57Bl/6J mice were assessed for their age-associated changes. Kidneys from aged mice showed increased infiltration of CD68-positive macrophages, higher expression of extracellular matrix (ECM) proteins, and more apoptosis than young controls. They also showed decreased Sirt1 expression along with increased acetylated HIF-1α. The level of Bcl-2/adenovirus E1B-interacting protein 3, carbonic anhydrase 9, Snail, and transforming growth factor-ß1, which are regulated by HIF-1α, was significantly higher in aged mice suggesting that HIF-1α activity was increased. In HK-2 cells, Sirt1 inhibitor sirtinol and siRNA-mediated knockdown of Sirt1 enhanced apoptosis and ECM accumulation. During hypoxia, Sirt1 was down-regulated, which allowed the acetylation and activation of HIF-1α. Resveratrol, a Sirt1 activator, effectively prevented hypoxia-induced production of ECM proteins, mitochondrial damage, reactive oxygen species generation, and apoptosis. The inhibition of HIF-1α activity by Sirt1-induced deacetylation of HIF-1α was confirmed by Sirt1 overexpression under hypoxic conditions and by resveratrol treatment or Sirt1 overexpression in HIF-1α-transfected HK-2 cells. Finally, we confirmed that chronic activation of HIF-1α promoted apoptosis and fibrosis, using tubular cell-specific HIF-1α transgenic mice. Taken together, our data suggest that Sirt1-induced deacetylation of HIF-1α may have protective effects against tubulointerstitial damage in aged kidney.

Selective tubular activation of hypoxia-inducible factor-2α has dual effects on renal fibrosis.

Hypoxia-inducible factor (HIF) is a key transcriptional factor in the response to hypoxia. Although the effect of HIF activation in chronic kidney disease (CKD) has been widely evaluated, the results have been inconsistent until now. This study aimed to investigate the effects of HIF-2α activation on renal fibrosis according to the activation timing in inducible tubule-specific transgenic mice with non-diabetic CKD. HIF-2α activation in renal tubular cells upregulated mRNA and protein expressions of fibronectin and type 1 collagen associated with the activation of p38 mitogen-activated protein kinase. In CKD mice, activation of HIF-2α at the beginning of CKD significantly aggravated renal fibrosis, whereas it did not lead to renal dysfunction. However, activation at a late-stage of CKD abrogated both renal dysfunction and fibrosis, which was associated with restoration of renal vasculature and amelioration of hypoxia through increased renal tubular expression of VEGF and its isoforms. As with tubular cells with HIF-2α activation, those under hypoxia also upregulated VEGF, fibronectin, and type 1 collagen expressions associated with HIF-1α activation. In conclusion, late-stage renal tubular HIF-2α activation has protective effects on renal fibrosis and the resultant renal dysfunction, thus it could represent a therapeutic target in late stage of CKD.

Calpain-mediated cleavage of DARPP-32 in Alzheimer's disease.

Toxicity induced by aberrant protein aggregates in Alzheimer's disease (AD) causes synaptic disconnection and concomitant progressive neurodegeneration that eventually impair cognitive function. cAMP-response element-binding protein (CREB) is a transcription factor involved in the molecular switch that converts short-term to long-term memory. Although disturbances in CREB function have been suggested to cause memory deficits in both AD and AD animal models, the mechanism of CREB dysfunction is still unclear. Here, we show that the dopamine- and cAMP-regulated phosphoprotein 32 kDa (DARPP-32), a key inhibitor of protein phosphate-1 (PP-1) that regulates CREB phosphorylation, is cleaved by activated calpain in both AD brains and neuronal cells treated with amyloid-ß or okadaic acid, a protein phosphatase-2A inhibitor that induces tau hyperphosphorylation and neuronal death. We found that DARPP-32 is mainly cleaved at Thr(153) by calpain and that this cleavage of DARPP-32 reduces CREB phosphorylation via loss of its inhibitory function on PP1. Our results suggest a novel mechanism of DARPP-32-CREB signalling dysregulation in AD.

Vancomycin blocks autophagy and induces interleukin-1ß release in macrophages.

Systemic inflammatory response syndrome (SIRS) is a serious condition that can cause organ failure as an exaggerated immunoresponse to the infection or other causes. Recently, autophagy was reported as a key process that regulates inflammatory responses in macrophages. Vancomycin is one of the most commonly prescribed antibiotics for sepsis treatment or following surgery. However, there are no studies on how vancomycin affects autophagy or inflammation. Here, we treated macrophage cell lines with vancomycin and lipopolysaccharides and found that vancomycin blocks autophagy and increases inflammatory responses. This finding suggests that vancomycin should be more cautiously administered in order to prevent unwanted SIRS during sepsis.

Resveratrol inhibits the proliferation of neural progenitor cells and hippocampal neurogenesis.

Resveratrol is a phytoalexin and natural phenol that is present at relatively high concentrations in peanuts and red grapes and wine. Based upon studies of yeast and invertebrate models, it has been proposed that ingestion of resveratrol may also have anti-aging actions in mammals including humans. It has been suggested that resveratrol exerts its beneficial effects on health by activating the same cellular signaling pathways that are activated by dietary energy restriction (DR). Some studies have reported therapeutic actions of resveratrol in animal models of metabolic and neurodegenerative disorders. However, the effects of resveratrol on cell, tissue and organ function in healthy subjects are largely unknown. In the present study, we evaluated the potential effects of resveratrol on the proliferation and survival of neural progenitor cells (NPCs) in culture, and in the hippocampus of healthy young adult mice. Resveratrol reduced the proliferation of cultured mouse multi-potent NPCs, and activated AMP-activated protein kinase (AMPK), in a concentration-dependent manner. Administration of resveratrol to mice (1-10 mg/kg) resulted in activation of AMPK, and reduced the proliferation and survival of NPCs in the dentate gyrus of the hippocampus. Resveratrol down-regulated the levels of the phosphorylated form of cyclic AMP response element-binding protein (pCREB) and brain-derived neurotrophic factor (BDNF) in the hippocampus. Finally, resveratrol-treated mice exhibited deficits in hippocampus-dependent spatial learning and memory. Our findings suggest that resveratrol, unlike DR, adversely affects hippocampal neurogenesis and cognitive function by a mechanism involving activation of AMPK and suppression of CREB and BDNF signaling.

Capsaicin impairs proliferation of neural progenitor cells and hippocampal neurogenesis in young mice.

Capsaicin (N-vanillyl-8-methyl-1-nonenamide) is a major pungent ingredient in hot peppers and induces apoptosis in malignant carcinoma cell lines. However, the adverse effects of capsaicin on neuronal development have not been fully explored. The aim of this study was to determine whether capsaicin affected murine-derived cerebellar multi-potent neural progenitor cells (NPC) or adult hippocampal neurogenesis in vivo. Capsaicin dose-dependently suppressed NPC proliferation, and higher concentrations were cytotoxic. Capsaicin decreased the activation of extracellular signal-regulated kinases (ERK) without markedly affecting p38 kinases. Capsaicin reduced the number of newly generated cells in the dentate gyrus of the hippocampus but did not significantly alter learning and memory performance in young adult mice. Interestingly, capsaicin decreased ERK activation in the hippocampus, suggesting that reduced ERK signaling may be involved in the capsaicin-mediated regulation of hippocampal neurogenesis.

Acrylamide induces cell death in neural progenitor cells and impairs hippocampal neurogenesis.

Acrylamide (ACR) is a well-known neurotoxin in mammalian species that causes neuropathy characterized by ataxia and skeletal muscle weakness. Therefore, ACR-mediated axon damage in the central and peripheral nervous systems is considered to be central-peripheral axonopathy. However, the molecular mechanisms underlying ACR's toxicity to neural progenitor cells are unknown. This study investigated the adverse effects of ACR on mouse multipotent neural progenitor cells and adult hippocampal neurogenesis. ACR significantly reduced the proliferation of neural progenitor cells, and high ACR concentrations induced apoptotic and necrotic cell death. We found that elevated intracellular levels of reactive oxygen species were involved in ACR-mediated cytotoxicity. Interestingly, the administration of ACR to young mice resulted in a significant decrease in the number of newly generated cells in the dentate gyrus of the hippocampus, suggesting an impairment of adult neurogenesis. These results suggest that ACR's deleterious effects on the central nervous system are due to the death of neural progenitor cells and impaired adult neurogenesis.

Risk assessment for the combinational effects of food color additives: neural progenitor cells and hippocampal neurogenesis.

In 2006, the Korea Food and Drug Administration reported that combinations of dietary colors such as allura red AC (R40), tartrazine (Y4), sunset yellow FCF (Y5), amaranth (R2), and brilliant blue FCF (B1) are widely used in food manufacturing. Although individual tar food colors are controlled based on acceptable daily intake (ADI), there is no apparent information available for how combinations of these additives affect food safety. In the current study, the potencies of single and combination use of R40, Y4, Y5, R2, and B1 were examined on neural progenitor cell (NPC) toxicity, a biomarker for developmental stage, and neurogenesis, indicative of adult central nervous system (CNS) functions. R40 and R2 reduced NPC proliferation and viability in mouse multipotent NPC, in the developing CNS model. Among several combinations tested in mouse model, combination of Y4 and B1 at 1000-fold higher than average daily intake in Korea significantly decreased numbers of newly generated cells in adult mouse hippocampus, indicating potent adverse actions on hippocampal neurogenesis. However, other combinations including R40 and R2 did not affect adult hippocampal neurogenesis in the dentate gyrus. Evidence indicates that single and combination use of most tar food colors may be safe with respect to risk using developmental NPC and adult hippocampal neurogenesis. However, the response to excessively high dose combination of Y4 and B1 is suggestive of synergistic effects to suppress proliferation of NPC in adult hippocampus. Data indicated that combinations of tar colors may adversely affect both developmental and adult hippocampal neurogenesis; thus, further extensive studies are required to assess the safety of these additive combinations.

Selenium Inhibits Metastasis of Murine Melanoma Cells through the Induction of Cell Cycle Arrest and Cell Death.

BACKGROUND: Melanoma is the most fatal form of skin cancer due to its rapid metastasis. Recently, several studies reported that selenium can induce apoptosis in melanoma cells. However, the precise mechanism remains to be elucidated. In this study, we investigated the effect of selenium on cell proliferation in murine melanoma and on tumor growth and metastasis in C57BL/6 mice. METHODS: Cell proliferation was measured by MTT assay in selenium-treated melanoma cells. Cell cycle distribution was analysized by staining DNA with propidum iodide (PI). mRNA and protein expression related to cell cycle arrest was measured by reverse transcription PCR and western blot. Tumor growth and metastasis was measured by in vivo model. RESULTS: Selenium was suppressed the proliferation of melanoma cells in a dose dependent manner. The growth inhibition of melanoma by selenium was associated with an arrest of cell cycle distribution at G0/G1 stage. The mRNA and protein level of CDK2/CDK4 was suppressed by treatment with selenium in a time-dependent manner. In vivo, tumor growth was not suppressed by selenium; however tumor metastasis was suppressed by selenium in mouse model. CONCLUSION: These results suggest that selenium might be a potent agent to inhibit proliferative activity of melanoma cells.