Human antigen R regulates hypoxia-induced mitophagy in renal tubular cells through PARKIN/BNIP3L expressions.

Mitochondrial dysfunction contributes to the pathophysiology of acute kidney injury (AKI). Mitophagy selectively degrades damaged mitochondria and thereby regulates cellular homeostasis. RNA-binding proteins (RBPs) regulate RNA processing at multiple levels and thereby control cellular function. In this study, we aimed to understand the role of human antigen R (HuR) in hypoxia-induced mitophagy process in the renal tubular cells. Mitophagy marker expressions (PARKIN, p-PARKIN, PINK1, BNIP3L, BNIP3, LC3) were determined by western blot analysis. Immunofluorescence studies were performed to analyze mitophagosome, mitolysosome, co-localization of p-PARKIN/TOMM20 and BNIP3L/TOMM20. HuR-mediated regulation of PARKIN/BNIP3L expressions was determined by RNA-immunoprecipitation analysis and RNA stability experiments. Hypoxia induced mitochondrial dysfunction by increased ROS, decline in membrane potential and activated mitophagy through up-regulated PARKIN, PINK1, BNIP3 and BNIP3L expressions. HuR knockdown studies revealed that HuR regulates hypoxia-induced mitophagosome and mitolysosome formation. HuR was significantly bound to PARKIN and BNIP3L mRNA under hypoxia and thereby up-regulated their expressions through mRNA stability. Altogether, our data highlight the importance of HuR in mitophagy regulation through up-regulating PARKIN/BNIP3L expressions in renal tubular cells.

Exosomal tumor necrosis factor-α from hepatocellular cancer cells (Huh-7) promote osteoclast differentiation.

Bone is the common extra-hepatic site for cancer metastasis. Hepatic cancer is associated with a higher incidence of pathological fracture. However, this important regulatory mechanism remains unexplored. Thus, exosome-mediated cell-cell communication between hepatocellular cancer and bone might be key to osteolytic bone destruction. Huh-7 exosomes were characterized for size and exosome marker expressions (CD63, Alix). Exosome mediated osteoclast differentiation in the RAW 264.7 cells was monitored from day 1 to 6 and multinucleated osteoclast formation and bone resorption activity were analyzed. The osteoclastogenic factor expressions in the exosomes and osteoclast differentiation markers such as tumor necrosis factor receptor 6 (TRAF6), nuclear factor κB (NF-κB), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), and cathepsin K (CTSK) were analyzed using western blot. Exosomes released by liver cancer cells (Huh-7) promoted osteoclast differentiation in RAW 264.7 cells. Analysis of osteoclastogenic factors in the exosomes showed that exosomes were specifically enriched with tumor necrosis factor α (TNF-α). Huh-7 exosomes promoted osteoclast differentiation by significantly increasing the number of TRAP-positive multi nucleated osteoclasts and resorption pits. Importantly, exosomes upregulated osteoclast markers TRAF6, NF-κB, and CTSK expressions. Further, neutralizing exosomal TNF-α reverted exosome-mediated osteoclast differentiation in RAW 264.7 cells. Collectively, our findings show that cellular communication of exosomal TNF-α from hepatocellular cancer cells (Huh-7) regulates osteoclast differentiation through NF-κB/CTSK/TRAP expressions. Thus, exosomal TNF-α might act as an important therapeutic target to prevent hepatocellular cancer mediated pathological bone disease.

The functional interplay of lncRNA EGOT and HuR regulates hypoxia-induced autophagy in renal tubular cells.

Autophagy, an important cellular homeostatic mechanism regulates cell survival under stress and protects against acute kidney injury. However, the role of long noncoding RNA (lncRNA) in autophagy regulation in renal tubular cells (HK-2) is unclear. The study was aimed to understand the importance of lncRNA in hypoxia-induced autophagy in HK-2 cells. LncRNA eosinophil granule ontogeny transcript (EGOT) was identified as autophagy-associated lncRNA under hypoxia. The lncRNA EGOT expression was significantly downregulated in renal tubular cells during hypoxia-induced autophagy. Gain- and loss-of-EGOT functional studies revealed that EGOT overexpression reduced autophagy by downregulation of ATG7, ATG16L1, LC3II expressions and LC 3 puncta while EGOT knockdown reversed the suppression of autophagy. Importantly, RNA-binding protein, (ELAVL1)/Hu antigen R (HuR) binds and stabilizes the EGOT expression under normoxia and ATG7/16L1 expressions under hypoxia. Furthermore, HuR mediated stabilization of ATG7/16L1 expressions under hypoxia causes a decline in EGOT levels and thereby promotes autophagy. Altogether, the study first reveals the functional interplay of lncRNA EGOT and HuR on the posttranscriptional regulation of the ATG7/16L1 expressions. Thus, the HuR/EGOT/ATG7/16L1 axis is crucial for hypoxia-induced autophagy in renal tubular cells.

RISK OF AGE-RELATED MACULAR DEGENERATION IN PATIENTS WITH PERIODONTITIS: A Nationwide Population-Based Cohort Study.

PURPOSE: Periodontitis is an inflammatory disease that results in loss of connective tissue and bone support. Evidence shows a possible relationship between periodontitis and age-related macular degeneration (AMD). METHODS: This population-based cohort study was conducted using data from the National Health Insurance Research Database in Taiwan, with a 13-year follow-up, to investigate the risk of AMD in patients with periodontitis. The periodontitis cohort included patients with newly diagnosed periodontitis between 2000 and 2012. The nonperiodontitis cohort was frequency-matched with the periodontitis cohort by age and sex, with a sample size of 41,661 in each cohort. RESULTS: Patients with periodontitis had an increased risk of developing AMD compared with individuals without periodontitis (5.95 vs. 3.41 per 1,000 person-years, adjusted hazard ratio = 1.58 [95% confidence interval, 1.46-1.70]). The risk of developing AMD remained significant after stratification by age (adjusted hazard ratio = 1.48 [1.34-1.64] for age <65 years and 1.76 [1.57-1.97] for age ≥65 years), sex (adjusted hazard ratio = 1.40 [1.26-1.55] for women and 1.82 [1.63-2.04] for men), and presence of comorbidity (adjusted hazard ratio = 1.52 [1.40-1.66] for with comorbidity and 1.92 [1.63-2.26] for without comorbidity). In addition, patients with periodontitis showed an increased incidence for both nonexudative type AMD (5.43 vs. 3.13 per 1,000 person-years) and exudative type AMD (0.52 vs. 0.28 per 1,000 person-years). CONCLUSION: People with periodontitis could be at a greater risk of developing AMD than those without periodontitis. However, we need more evidence to support this association.

RNA-binding protein, human antigen R regulates hypoxia-induced autophagy by targeting ATG7/ATG16L1 expressions and autophagosome formation.

Autophagy, a prosurvival mechanism offers a protective role during acute kidney injury. We show novel findings on the functional role of RNA binding protein, HuR during hypoxia-induced autophagy in renal proximal tubular cells-2 (HK-2). HK-2 cells showed upregulated expressions of HuR and autophagy-related proteins such as autophagy related 7 (ATG7), autophagy related 16 like 1 (ATG16L1), and LC3II under hypoxia. Increased autophagosome formation was visualized as LC3 puncta in hypoxic cells. Further, short hairpin-RNA-mediated loss of HuR function in HK-2 cells significantly decreased ATG7 and ATG16L1 protein expressions. Bioinformatics prediction revealed HuR motif binding on the coding region of ATG7 and AU-rich element at 3'UTR ATG16L1 messnger RNA (mRNA). The RNA immunoprecipitation study showed that HuR was predominantly associated with ATG7 and ATG16L1 mRNAs under hypoxia. In addition, HuR enhanced autophagosome formation by regulating LC3II expressions. These results show that HuR regulates ATG7 and ATG16L1 expressions and thereby mediate autophagy in HK-2 cells. Importantly, HuR knockdown cells underwent apoptosis during hypoxia as observed through the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Collectively, these findings show the crucial role of HuR under hypoxia by regulating autophagy and suppressing apoptosis in renal tubular cells.

The association between Type 1 diabetes mellitus and periodontal diseases.

BACKGROUND/PURPOSE: Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease affecting oral health. Evidence shows possible association between T1DM and periodontal diseases (PDs). We conducted a nationwide population-based study in Taiwan, with a 14-year follow-up to investigate the risk of PDs in T1DM patients. METHODS: We used data from the National Health Insurance Research Database in Taiwan. The T1DM cohort was identified with newly diagnosed T1DM from 1998 to 2011. The non-T1DM cohort was frequency matched with the T1DM cohort. Participants comprised 4248 patients in the T1DM cohort and 16992 persons in the non-T1DM cohort. RESULTS: The T1DM patients showed an increased risk of PDs compared to non-T1DM individuals [adjusted hazard ratio (aHR) = 1.45]. T1DM patients who visited the emergency room more than twice per year had a higher aHR of 13.0 for developing PDs. The aHR for PDs was 13.2 in the T1DM patients who had been hospitalized more than twice per year. CONCLUSION: T1DM patients are at higher risk of developing PDs than non-T1DM individuals. Our results further showed that the number of T1DM interventions; that is, annual emergency visits and hospitalizations were associated with increased the risk of developing PDs.

Periodontitis and the subsequent risk of glaucoma: results from the real-world practice.

Periodontitis is a multifactorial inflammatory disease that can cause tooth loss and contribute to systemic inflammation. It is suggested that periodontitis may be associated with the development of glaucoma. Based on data from Taiwan's National Health Insurance Research Database, a retrospective cohort study was conducted to investigate the risk of developing glaucoma in patients with periodontitis. The periodontitis cohort consisted of newly diagnosed adult patients (n = 194,090, minimum age = 20 years) between 2000 and 2012. The comparison group included age-, gender-, and diagnosis date-matched people without periodontitis (n = 194,090, minimum age = 20 years). Incident glaucoma was monitored until the end of 2013. Hazard ratios (HRs) with confidence intervals (CIs) were established based on the Cox proportional hazard models. The risk of developing glaucoma was higher in patients with periodontitis than those without periodontitis (31.2 vs. 23.3 patients per 10,000 person-years, with an adjusted HR of 1.26 [95% CI 1.21-1.32]). A high risk was evident even after stratifying by age (adjusted HRs = 1.34 [1.26-1.44] for ages 20-49, 1.24 [1.13-1.36] for ages ≥ 65, and 1.20 [1.12-1.29] for ages 50-64 years), sex (adjusted HRs = 1.33 [1.24-1.41] and 1.21 [1.14-1.28] for men and women, respectively), presence of comorbidity (adjusted HRs = 1.38 [1.29-1.47] and 1.18 [1.12-1.25] for without and with comorbidity, respectively), and corticosteroid use (adjusted HRs = 1.27 [1.21-1.33] and 1.21 [1.08-1.35] for without and with corticosteroid use, respectively). Specifically, patients with periodontitis exhibited a significantly high risk of primary open-angle glaucoma (adjusted HR = 1.31 [1.21-1.32]) but not for primary closed-angle glaucoma (adjusted HR = 1.05 [0.94-1.17]). People with periodontitis are at a greater risk of glaucoma than individuals without periodontitis. Ocular health should be emphasized for such patients, and the underlying mechanisms need further investigation.

Plasma exosomal miR-223 expression regulates inflammatory responses during cardiac surgery with cardiopulmonary bypass.

Cardiopulmonary bypass (CPB) induces inflammatory responses, and effective endogenous homeostasis is important for preventing systemic inflammation. We assessed whether plasma exosomal microRNAs in patients undergoing cardiac surgery with CPB are involved in the regulation of inflammatory responses. Plasma samples were isolated from CPB patients (n = 21) at 5 specified time points: pre-surgery, pre-CPB and 2 hours (h), 4 h and 24 h after CPB began. Plasma TNF-α expression was increased after CPB began compared to that in the pre-surgery samples. Plasma IL-8 and IL-6 expression peaked at 4 h after CPB began but was downregulated at 24 h. The number of plasma exosomes collected at 2 h (55.1 ± 8.3%), 4 h (63.8 ± 10.1%) and 24 h (83.5 ± 3.72%) after CPB began was significantly increased compared to that in the pre-CPB samples (42.8 ± 0.11%). These exosomes had a predominantly parental cellular origin from RBCs and platelets. Additionally, the plasma exosomal miR-223 levels were significantly increased after CPB began compared to those in the pre-CPB samples. Further, exosomal miR-223 from plasma collected after CPB began downregulated IL-6 and NLRP3 expression in the monocytes. Here, we present the novel findings that increased plasma exosomal miR-223 expression during cardiac surgery with CPB might play homeostatic roles in downregulating inflammatory responses through intercellular communication.

Reciprocal regulation of γ-globin expression by exo-miRNAs: Relevance to γ-globin silencing in ß-thalassemia major.

Induction of fetal hemoglobin (HbF) is a promising strategy in the treatment of ß-thalassemia major (ß-TM). The present study shows that plasma exosomal miRNAs (exo-miRs) are involved in γ-globin regulation. Exosomes shuttle miRNAs and mediate cell-cell communication. MiRNAs are regulators of biological processes through post-transcriptional targeting. Compared to HD (Healthy Donor), ß-TM patients showed increased levels of plasma exosomes and the majority of exosomes had cellular origin from CD34+ cells. Further, HD and ß-TM exosomes showed differential miRNA expressions. Among them, deregulated miR-223-3p and miR-138-5p in ß-TM exosomes and HD had specific targets for γ-globin regulator and repressor respectively. Functional studies in K562 cells showed that HD exosomes and miR-138-5p regulated γ-globin expression by targeting BCL11A. ß-TM exosomes and miR-223-3p down regulated γ-globin expression through LMO2 targeting. Importantly, miR-223-3p targeting through sponge repression resulted in γ-globin activation. Further, hnRNPA1 bound to stem-loop structure of pre-miR-223 and we found that hnRNPA1 knockdown or mutagenesis at miR-223-3p stem-loop sequence resulted in less mature exo-miR-223-3p levels. Altogether, the study shows for the first time on the important clinical evidence that differentially expressed exo-miRNAs reciprocally control γ-globin expressions. Further, the hnRNPA1-exo-miR-223-LMO2 axis may be critical to γ-globin silencing in ß-TM.

RANTES mediates kidney ischemia reperfusion injury through a possible role of HIF-1α and LncRNA PRINS.

RANTES (Regulated on activation, normal T-cell expressed and secreted), recruits circulating leukocytes and augments inflammatory responses in many clinical conditions. Inflammatory responses in ischemia-reperfusion injury (IRI) significantly affect the unfavorable outcomes of acute kidney injury (AKI), and that infiltrating immune cells are important mediators of AKI. However, the significance of RANTES in AKI and whether hypoxia-induced LncRNAs are involved in the regulatory process of AKI are not known. Here we show that, in the kidney IRI mice model, significant RANTES expression was observed in renal tubular cells of wild type mice. RANTES deficient (RANTES(-/-)) mice showed better renal function by reducing the acute tubular necrosis, serum creatinine levels, infiltration of inflammatory cells and cytokine expressions compared to wild type. In vitro, we found that RANTES expression was regulated by NF-κB. Further, renal tubular cells showed deregulated LncRNA expression under hypoxia. Among HIF-1α dependent LncRNAs, PRINS (Psoriasis susceptibility-related RNA Gene Induced by Stress) was significantly up regulated in hypoxic conditions and had specific interaction with RANTES as confirmed through reporter assay. These observations show first evidence for RANTES produced by renal tubular cells act as a key chemokine in AKI and HIF-1α regulated LncRNA-PRINS might be involved in RANTES production.

Eicosapentaenoic acid prevents TCDD-induced oxidative stress and inflammatory response by modulating MAP kinases and redox-sensitive transcription factors.

BACKGROUND AND PURPOSE: Oxidative stress and subsequent activation of inflammatory responses is a widely accepted consequence of exposure to environmental toxins. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), a well-known environmental toxin, exerts its toxicity through many signalling mechanisms, with liver being the principal organ affected. However, an effective antidote to TCDD-induced toxicity is unknown. The present study evaluated the effect of eicosapentaenoic acid (EPA), an n3 fatty acid, on TCDD-induced toxicity. EXPERIMENTAL APPROACH: In cultures of HepG2 cells, the EPA/AA ratio was determined using gas chromatography, oxidative stress and inflammatory responses through reactive oxygen species (ROS) levels, antioxidant status, [Ca(2+) ]i , nuclear migration of two redox-sensitive transcription factors, NF-κB p65 and Nrf-2, expression of MAP kinase (p-Erk, p-p38), NF-κB p65, COX-2 and Nrf-2. Cellular changes in ΔΨm, acidic vesicular organelle formation, cell cycle analysis and scanning electron microscopy analysis were performed. KEY RESULTS: EPA offered significant cytoprotection by increasing EPA/AA ratios in cell membranes, inhibiting ROS generation, enhancing antioxidant status and modulating nuclear translocation of redox-sensitive transcription factors (NF-κB p65 and Nrf-2) and expression of NF-κB p65, COX-2 and Nrf-2. Furthermore, TCDD-induced upstream events of MAPK phosphorylation, the increase in [Ca(2+) ]i levels and cell surface changes in microvilli were significantly inhibited by EPA. EPA treatment maintained ΔΨm and prevented formation of acidic vesicular organelles. CONCLUSION AND IMPLICATIONS: The present study demonstrates for the first time some underlying molecular mechanisms of cytoprotection exerted by EPA against TCDD-induced oxidative stress and inflammatory responses.