Associations between metabolic profiles and incident CKD in the Chinese population aged 45-85 years.

PURPOSE: The roles of metabolic indices in predicting chronic kidney disease (CKD) were lacking. This study aimed to examine the concomitant impact of metabolic and novel anthropometric indices on incident CKD in the Chinese populations. METHODS: This prospective cohort study included 1825 males and 2218 females aged between 45 and 85 years, derived from the ongoing prospectively cohort of China Health and Retirement Longitudinal Study (CHARLS), from 2011 to 2015. The outcome was defined as an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2. RESULTS: During the 5-years follow-up period, 3.0% (55/1825) of males and 4.1% (90/2218) of the females developed CKD. After multivariable adjustment, elevated triglyceride (TG), low high-density lipoprotein cholesterol (HDL-C), serum uric acid (sUA), elevated visceral fat index (VFI), elevated body shape index (BSI) and elevated body roundness index (BRI) in males, and sUA, and BRI in females were the independent predictors for CKD. Composite scores, composed of sUA, history of cardiovascular disease (CVD), waist circumstance (WC), HDL-C, and BRI in males and sUA, hypertension, and BRI in females were constructed that could accurately predict CKD. CONCLUSION: Our study found that elevated levels of TG, sUA, BSI, BRI, and diminished HDL in males and elevated levels of sUA, and BRI in females, are indicative of the incident CKD. The composite score, integrating a history of disease, metabolic indices, and noval anthropometric indices, could accurately differentiate individuals with and without incident CKD, proving useful for CKD care and management.

Serum Insulin-Like Growth Factor-Binding Protein 7 Deriving from Spleen and Lung Could Be Used for Early Recognition of Cardiac Surgery-Associated Acute Kidney Injury.

INTRODUCTION: The utility of arithmetic product of urinary tissue metalloproteinase inhibitor 2 (TIMP2) and insulin-like growth factor-binding protein 7 (IGFBP7) concentrations has been widely accepted on early diagnosis of acute kidney injury (AKI). However, which organ is the main source of those two factors and how the concentration of IGFBP7 and TIMP2 changed in serum during AKI still remain to be defined. METHODS: In mice, gene transcription and protein levels of IGFBP7/TIMP2 in the heart, liver, spleen, lung, and kidney were measured in both ischemia-reperfusion injury (IRI)- and cisplatin-induced AKI models. Serum IGFBP7 and TIMP2 levels were measured and compared in patients before cardiac surgery and at inclusion (0 h), 2 h, 6 h, and 12 h after intensive care unit (ICU) admission, and compared with serum creatinine (SCr), blood urea nitrogen (BUN), estimated glomerular filtration rate (eGFR), and serum uric acid (UA). RESULTS: In mouse IRI-AKI model, compared with the sham group, the expression levels of IGFBP7 and TIMP2 did not change in the kidney, but significantly upregulated in the spleen and lung. Compared with patients who did not develop AKI, the concentration of serum IGFBP7 at as early as 2 h after ICU admission (sIGFBP7-2 h) was significantly higher in patients who developed AKI. The relationships between sIGFBP7-2 h in AKI patients and log2 (SCr), log2 (BUN), log2 (eGFR), and log2 (UA) were statistically significant. The diagnostic performance of sIGFBP7-2 h measured by the macro-averaged area under the receiver operating characteristic curve was 0.948 (95% CI, 0.853-1.000; p < 0.001). CONCLUSION: The spleen and lung might be the main source of serum IGFBP7 and TIMP2 during AKI. The serum IGFBP7 value demonstrated good predictive accuracy for AKI following cardiac surgery within 2 h after ICU admission.

DNA binding protein YB-1 is a part of the neutrophil extracellular trap mediation of kidney damage and cross-organ effects.

Open-heart surgery is associated with high morbidity, with acute kidney injury (AKI) being one of the most commonly observed postoperative complications. Following open-heart surgery, in an observational study we found significantly higher numbers of blood neutrophils in a group of 13 patients with AKI compared to 25 patients without AKI (AKI: 12.9±5.4 ×109 cells/L; non-AKI: 10.1±2. 9 ×109 cells/L). Elevated serum levels of neutrophil extracellular trap (NETs) components, such as dsDNA, histone 3, and DNA binding protein Y-box protein (YB)-1, were found within the first 24 hours in patients who later developed AKI. We could demonstrate that NET formation and hypoxia triggered the release of YB-1, which was subsequently shown to act as a mediator of kidney tubular damage. Experimentally, in two models of AKI mimicking kidney hypoperfusion during cardiac surgery (bilateral ischemia/reperfusion (I/R) and systemic lipopolysaccharide (LPS) administration), a neutralizing YB-1 antibody was administered to mice. In both models, prophylactic YB-1 antibody administration significantly reduced the tubular damage (damage score range 1-4, the LPS model: non-specific IgG control, 0.92±0.23; anti-YB-1 0.65±0.18; and in the I/R model: non-specific IgG control 2.42±0.23; anti-YB-1 1.86±0.44). Even in a therapeutic, delayed treatment model, antagonism of YB-1 ameliorated AKI (damage score, non-specific IgG control 3.03±0.31; anti-YB-1 2.58±0.18). Thus, blocking extracellular YB-1 reduced the effects induced by hypoxia and NET formation in the kidney and significantly limited AKI, suggesting that YB-1 is part of the NET formation process and an integral mediator of cross-organ effects.

Proteinuria is a risk factor for acute kidney injury after cardiac surgery in patients with stages 3-4 chronic kidney disease: a case control study.

BACKGROUND: Acute kidney injury (AKI) is a common complication after cardiac surgery, and preoperative renal dysfunction is an important risk factor. Proteinuria indicates renal structural damage, but there are few studies on proteinuria and the risk of AKI after cardiac surgery in patients with renal dysfunction. This study aimed to elucidate whether proteinuria can predict AKI after cardiac surgery in patients with renal dysfunction. METHODS: Patients with stages 3-4 chronic kidney disease (CKD) who underwent cardiac surgery were included in this retrospective study. AKI was defined according to the KDIGO criteria. The association between proteinuria and AKI in patients with CKD stages 3-4 was investigated. RESULTS: The incidence of AKI in the entire cohort (n = 1546) was 53.55%. The in-hospital mortality of patients with was higher than patients without AKI (AKI vs. no AKI, 4.7 vs. 0.8%, P < 0.001). Multivariate logistic regression analysis showed that proteinuria was an independent risk factor for AKI (trace to 1+ OR 2.37; 2+ -3+ OR 5.16) and AKI requiring renal replacement therapy (AKI-RRT) (trace to 1+ OR 3.64; 2+-3+ OR 5.71). Mild proteinuria (trace to 1+ OR 2.59) was also an independent risk factor for in-hospital death. In patients with diabetes mellitus, mild proteinuria (OR 1.925), instead of severe proteinuria (2-3+), was a risk factor of AKI in patients with kidney dysfunction and diabetes. CONCLUSIONS: In the population of patients with renal dysfunction, the incidence of AKI was high, which significantly compromised renal and overall prognosis. As a simple and inexpensive routine test, preoperative proteinuria still has value in predicting AKI in patients with impaired renal function.

TWIK-related acid-sensitive K+ channel 2 promotes renal fibrosis by inducing cell-cycle arrest.

TWIK-related acid-sensitive K+ channel-2 (TASK-2, encoded by Kcnk5) is essential in cell biological processes, by regulating transmembrane K+ balance. In the present study, we aimed to clarify the role of TASK-2 in renal fibrosis and explore the underlying mechanism. We found that TASK-2 level was elevated in the renal tubular UUO- and UIR-induced renal fibrosis as well as in patients with renal tubulointerstitial fibrosis. Knockout of Kcnk5 or inhibition of TASK-2 in renal tubules attenuated G2/M cell-cycle arrest and alleviated renal fibrosis. Mechanistically, demethylase fat mass and obesity-associated protein (FTO) reduced N6-adenosine methylation (m6A) of Kcnk5 mRNA following renal fibrosis. FTO deficiency attenuated the upregulation of TASK-2 and renal fibrosis. The results demonstrated the crucial role of TASK-2 in renal fibrosis, which is conducive to a better understanding of the pathogenesis of renal fibrosis. TASK-2 may be a potential treatment strategy to alleviate the development of renal fibrosis.

Suppressing Syndecan-1 Shedding to Protect Against Renal Ischemia/Reperfusion Injury by Maintaining Polarity of Tubular Epithelial Cells.

ABSTRACT: Syndecan-1 (SDC-1), a type of heparan sulfate proteoglycan on the surface of epithelial cells, is involved in maintaining cell morphology. Loss of cell polarity constitutes the early stage of ischemic acute kidney injury (AKI). This study investigated the role of SDC-1 shedding in I/R-induced AKI and the underlying mechanisms. Levels of the shed SDC-1 in the serum were measured with ELISA 12 and 24 h after reperfusion in renal I/R model mice. Na+/K+-ATPase-α1 expression was evaluated using western blotting in vivo and immunofluorescence in hypoxia/reoxygenation (H/R) cysts. Renal tubular epithelial cell apoptosis was measured using TUNEL in vivo and flow cytometry in vitro. Furthermore, plasma syndecan-1 (pSDC-1) levels were measured in patients at the time of anesthesia resuscitation after cardiac surgery. We found that shed SDC-1 levels increased and Na+/K+-ATPase-α1 expression decreased after H/R in the three-dimensional (3D) tubular model, and this state was exacerbated with extended period of hypoxia. After the inhibition of SDC-1 shedding by GM6001, SDC-1 and Na+/K+-ATPase-α1 expression was restored, while H/R-induced apoptosis was decreased. In vivo, SDC-1 shedding was induced by renal I/R and was accompanied with a loss of renal tubular epithelial cell polarity and increased apoptosis. GM6001 pretreatment protected against I/R injury by alleviating the disruption of cell polarity and apoptosis. pSDC-1 levels were significantly higher in AKI patients than in non-AKI patients. ROC curve showed that the accuracy of pSDC-1 for AKI prediction was 0.769. In conclusion, inhibition of I/R-induced SDC-1 shedding could contribute to renal protection by restoring the loss of cell polarity and alleviating apoptosis in tubular epithelial cells.

Social Isolation Is Associated With Rapid Kidney Function Decline and the Development of Chronic Kidney Diseases in Middle-Aged and Elderly Adults: Findings From the China Health and Retirement Longitudinal Study (CHARLS).

Background: There is limited evidence on the relationship between social isolation and renal outcomes. To address this gap, this study estimated the prospective relationship of social isolation with rapid kidney function decline and the development of chronic kidney disease (CKD) in middle-aged and elderly Chinese with normal kidney function. Methods: We analyzed data from 3,031 participants aged ≥ 45 years with baseline estimated glomerular filtration rates (eGFR) ≥ 60 ml/min/1.73 m2. All data were obtained from the 2011 and 2015 waves of the Chinese Longitudinal Study of Health and Retirement (CHARLS). eGFR was estimated based on a combination of serum creatinine and cystatin C. The primary outcome was rapid decline in renal function, as defined by an eGFR decrease of > 5 ml/min/1.73 m2 per year, while the secondary outcome was the development of CKD, as defined by an eGFR decrease to a level < 60 ml/min/1.73 m2. Results: During the follow-up of 4 years, 258 (8.5%) participants experienced a rapid decline in renal function, while 87 (2.9%) developed CKD. In the fully adjusted model, high social isolation was significantly related to an increased risk of experiencing a rapid decline in renal function (OR 1.805, 95% CI 1.310-2.487) and CKD onset (OR 1.842, 95% CI 1.084-3.129). Among the five components of social isolation, being unmarried, not participating in social activities, and living alone independently predicted declined renal function. Conclusions: Social isolation is significantly associated with the risk of rapid eGFR decline and CKD onset in middle-aged and older adults with normal kidney function in China.

Association Between Muscle Strength and Cystatin C-Based Estimated Glomerular Filtration Rate Among Middle-Aged and Elderly Population: Findings Based on the China Health and Retirement Longitudinal Study (CHARLS), 2015.

BACKGROUND: Patients with chronic kidney disease (CKD) are prone to muscle strength degeneration. However, the relationship between mild-to-moderate renal insufficiency and low muscle strength remains unclear. As cystatin C is not subject to muscular conditions and is a sensitive serum marker in preclinical renal disease, we aimed to investigate the association between estimated glomerular filtration rate (eGFR) based on cystatin C and muscle strength in the Chinese population. METHODS: This was a cross-sectional study enrolling 12,398 Chinese participants aged above 45 years (5762 men and 6636 women) from the 2015 China Health and Retirement Longitudinal Study. Handgrip strength (HGS) was used to assess muscle strength. Locally weighted scatterplot smoothing (LOWESS) curves were employed to visualize the relationships between eGFR and HGS. Multivariable logistic regression was conducted to analyze the correlation between kidney function and low muscle strength. RESULTS: Significant differences in HGS by CKD stage were observed in both sexes after adjusting for age and body mass index. LOWESS curves demonstrated concomitant decreases in HGS and kidney function at eGFR levels below 120 mL/min/1.73 m2 in both sexes. According to multivariate logistic regression, participants with CKD stages 2 (odds ratio [OR]: 1.256, 95% confidence interval [CI]: 1.120-1.409), 3 (OR: 2.725, 95% CI: 2.2585-3.288), and 4-5 (OR: 3.069, 95% CI 1.747-5.392) had higher risk of low muscle strength than those who were normal or had CKD stage 1 after adjusting for demographic and clinical variables. CONCLUSION: Our study illustrated that CKD stage was independently associated with low muscle strength in Chinese middle-aged and elderly populations.

Erythrocyte transfusion limits the role of elevated red cell distribution width on predicting cardiac surgery associated acute kidney injury.

BACKGROUND: Acute kidney injury (AKI) is one of the more serious complications after cardiac surgery. Elevated red cell distribution width (RDW) was reported as a predictor for cardiac surgery associated acute kidney injury (CSAKI). However, the increment of RDW by erythrocyte transfusion makes its prognostic role doubtful. The aim of this study is to elucidate the impact of erythrocyte transfusion on the prognostic role of elevated RDW for predicting CSAKI. METHODS: A total of 3207 eligible patients who underwent cardiac surgery during 2016-2017 were enrolled. Changes of RDW was defined as the difference between preoperative RDW and RDW measured 24 h after cardiac surgery. The primary outcome was CSAKI which was defined by the Kidney Disease: Improving Global Outcomes Definition and Staging (KDIGO) criteria. Univariate and multivariate analysis were performed to identify predictors for CSAKI. RESULTS: The incidence of CSAKI was 38.07% and the mortality was 1.18%. CSAKI patients had higher elevated RDW than those without CSAKI (0.65% vs. 0.39%, p < 0.001). Multivariate regression showed that male, age, New York Heat Association classification 3-4, elevated RDW, estimated glomerular filtration rate < 60 mL/min/1.73 m2, cardiopulmonary bypass time > 120 min and erythrocyte transfusion were associated with CSAKI. Subgroup analysis showed elevated RDW was an independent predictor for CSAKI in the non-transfused subset (adjusted odds ratio: 1.616, p < 0.001) whereas no significant association between elevated RDW and CSAKI was found in the transfused patients (odds ratio: 1.040, p = 0.497). CONCLUSIONS: Elevated RDW is one of the independent predictors of CSAKI in the absence of erythrocyte transfusion, which limits the prognostic role of the former on predicting CSAKI.

Upregulation of miR-382 contributes to renal fibrosis secondary to aristolochic acid-induced kidney injury via PTEN signaling pathway.

Acute kidney injury (AKI) has a critical role in the development of chronic kidney disease (CKD). Building on our previous findings, we explored the role of miR-382 in facilitating the transition of AKI to CKD using the Aristolochic acid (AA) nephropathy model, which was induced by intraperitoneal injection of aristolochic acid I salt (10 or 20 mg/kg). The effects of genetic depletion, pharmacologic inhibition, or overexpression of miR-382 on the PTEN/AKT signaling pathway were examined in vivo and in vitro. Changes in renal pathology and renal epithelial polarity were evaluated. A luciferase reporter assay was performed to investigate the reciprocal suppression relationship between miR-382 and PTEN. Renal fibrosis developed 14 d after AA exposure in a dose- and time-dependent manner. Renal abundance of miR-382 was upregulated following AA treatment, while genetic depletion or pharmacological inhibition of miR-382 partially reversed renal tubulointerstitial fibrosis. Expression of PTEN, a target of miR-382, was downregulated and subsequently its downstream AKT signaling pathway was activated during AKI to CKD transition induced by AA. Inhibition of PTEN in vitro resulted in the acquisition of the EMT phenotypes. Furthermore, upregulation of miR-382 in renal epithelial cells was partially mediated by the activation of NF-kB signaling, with a substantial elevation of proinflammatory cytokines. An in vivo study revealed that either miR-382 knockdown or miR-382 knockout was pivotal for inflammatory suppression, while an in vitro experiment confirmed that upregulation of miR-382 in cultured MTEC cells under AA exposure was remarkably reversed by NF-kB siRNA. These data indicated a novel role for the NF-κB/miR-382/PTEN/AKT axis in the pathogenesis of tubulointerstitial fibrosis following AA-induced acute renal tubular epithelial injury. Targeting miR-382 may lead to a potential novel therapeutic approach for retarding the AKT to CKD transition.

RacGAP1 ameliorates acute kidney injury by promoting proliferation and suppressing apoptosis of renal tubular cells.

BACKGROUND: Acute kidney injury (AKI) remains correlated with high mortality. Novel therapeutic strategies are urgently needed for AKI patients. Rac GTPase-activating protein 1 (RacGAP1) regulates the activity of RhoGTPase and acts as a predictive biomarker in several types of malignant tumor but the role of RacGAP1 in AKI has not been revealed. METHODS: Animal models of AKI induced by renal ischemia-reperfusion (I/R) and cisplatin treatment were generated in C57BL/6 mice. Hypoxia/reoxygenation (H/R) and cisplatin treatment were practiced in human renal tubular epithelial (HK-2) and renal tubular duct epithelial cells of rat (NRK-52E) cells. The role of RacGAP1 in cell proliferation and apoptosis was estimated using western bolting, immunocytochemistry and flow cytometry. Verteporfin was used to activate the Hippo pathway to show whether the protective effects of RacGAP1 on cell growth and survival in renal tubular cells were dependent on the activation of YAP. RESULTS: The expression of RacGAP1 was significantly increased in mice kidneys after I/R or cisplatin treatment, combined with increased expression of RacGAP1 in H/R or cisplatin challenged cells. Overexpression of RacGAP1 protected HK2 and NRK-52E cells by promoting proliferation and decreasing apoptosis. We also disclosed that RacGAP1 exerted its function through activation of YAP. CONCLUSION: The present study provides evidence that RacGAP1 is involved in AKI. It promotes proliferation and limits apoptosis of tubular epithelial cells via stimulating activation and nuclear translocation of YAP. Consequently, RacGAP1 may be a novel therapeutic target for AKI.

LncRNA GAS5 promotes apoptosis as a competing endogenous RNA for miR-21 via thrombospondin 1 in ischemic AKI.

Mounting evidence has indicated that long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) played important roles in renal ischemia/reperfusion (I/R) injury. However, the involvement of lncRNA growth arrest specific 5 (GAS5) in acute kidney injury (AKI) remained largely unexplored. This study aimed to determine possible mechanisms of GAS5 in the renal I/R process. We found that GAS5, noticeably upregulated by renal I/R injury, was further suppressed by delayed IPC while knockdown of miR-21 in vivo before IPC could significantly increased the GAS5 levels. Concurrently, TSP-1 was negatively regulated by miR-21 in vivo and vitro. Additionally, Reciprocal repression of GAS5 and miR-21 was identified. Knockdown of miR-21 in H6R0.5 treated HK-2 cells promoted apoptosis. Co-transfection of miR-21 mimic and pcDNA-GAS5 or pcDNA-Vector were performed, results of which showed that inhibition of miR-21 on TSP-1 could be rescued by overexpression of GAS5. This study suggested that GAS5 facilitated apoptosis by competitively sponging miR-21, which negatively regulated TSP-1 in renal I/R injury. This novel regulatory axis could act as a therapeutic target for AKI in the future.

Transforming growth factor-ß1-overexpressing mesenchymal stromal cells induced local tolerance in rat renal ischemia/reperfusion injury.

BACKGROUND: Regulatory T cells (Tregs) suppress excessive immune responses and play a crucial protective role in acute kidney injury (AKI). The aim of this study was to examine the therapeutic potential of transforming growth factor (TGF)-ß1-overexpressing mesenchymal stromal cells (MSCs) in inducing local generation of Tregs in the kidney after ischemia/reperfusion (I/R) injury. METHODS: MSCs were transduced with a lentiviral vector expressing the TGF-ß1 gene; TGF-ß1-overexpressing MSCs (designated TGF-ß1/MSCs) were then transfused into the I/R-injured kidney via the renal artery. RESULTS: MSCs genetically modified with TGF-ß1 achieved overexpression of TGF-ß1. Compared with green fluorescent protein (GFP)/MSCs, TGF-ß1/MSCs markedly improved renal function after I/R injury and reduced epithelial apoptosis and subsequent inflammation. The enhanced immunosuppressive and therapeutic abilities of TGF-ß1/MSCs were associated with increased generation of induced Tregs and improved intrarenal migration of the injected cells. Futhermore, the mechanism of TGF-ß1/MSCs in attenuating renal I/R injury was not through a direct canonical TGF-ß1/Smad pathway. CONCLUSION: TGF-ß1/MSCs can induce a local immunosuppressive effect in the I/R-injured kidney. The immunomodulatory activity of TGF-ß1-modified MSCs appears to be a gateway to new therapeutic approaches to prevent renal I/R injury.

Evaluation of three hormonal protocols for anovulatory lactating cows under regulations restricting the use of estrogenic compounds.

When European Union regulations restricted the use of estrogenic compounds in food-producing animals, refined hormonal protocols were no longer applicable for anovulatory cows. However, Ovsynch and its adaptations are routinely and uniformly applied to all cows regardless of ovarian function. To evaluate their efficacy on anovulatory cows, 143, 147 and 144 anovulatory cows received Ovsynch, Presynch and G6G protocols, respectively. In comparison, 150 cyclic cows were bred without using a synchronized protocol. Results showed that cows in the Presynch group had luteolysis responding to the last prostaglandin F2α (PGF2α ) injection greater than the Ovsynch group. The serous progesterone levels at the first gonadotropin-releasing hormone of Ovsych and the last PGF2α injection was greater in the G6G group than the other two hormonal treatment groups. Concentrations of Ca2+ and total protein in cervical mucus in all three hormone-treated groups before artificial insemination (AI) were significantly different from the controls. The G6G group obtained a greater pregnancy rate compared with Ovsynch and Presynch, but significantly less than the controls. For open cows in the Ovsynch group, estrus rate within 24 days after the first AI was significantly less than the controls. In conclusion, the G6G treatment resulted to better reproductive performance in anovulatory cows.

Acetylation promotes TyrRS nuclear translocation to prevent oxidative damage.

Tyrosyl-tRNA synthetase (TyrRS) is well known for its essential aminoacylation function in protein synthesis. Recently, TyrRS has been shown to translocate to the nucleus and protect against DNA damage due to oxidative stress. However, the mechanism of TyrRS nuclear localization has not yet been determined. Herein, we report that TyrRS becomes highly acetylated in response to oxidative stress, which promotes nuclear translocation. Moreover, p300/CBP-associated factor (PCAF), an acetyltransferase, and sirtuin 1 (SIRT1), a NAD+-dependent deacetylase, regulate the nuclear localization of TyrRS in an acetylation-dependent manner. Oxidative stress increases the level of PCAF and decreases the level of SIRT1 and deacetylase activity, all of which promote the nuclear translocation of hyperacetylated TyrRS. Furthermore, TyrRS is primarily acetylated on the K244 residue near the nuclear localization signal (NLS), and acetylation inhibits the aminoacylation activity of TyrRS. Molecular dynamics simulations have shown that the in silico acetylation of K244 induces conformational changes in TyrRS near the NLS, which may promote the nuclear translocation of acetylated TyrRS. Herein, we show that the acetylated K244 residue of TyrRS protects against DNA damage in mammalian cells and zebrafish by activating DNA repair genes downstream of transcription factor E2F1. Our study reveals a previously unknown mechanism by which acetylation regulates an aminoacyl-tRNA synthetase, thus affecting the repair pathways for damaged DNA.

Roles of interferon-stimulated gene 15 protein in bovine embryo development.

Interferon (IFN)-stimulated gene 15 (ISG15) is one of several proteins induced by conceptus-derived Type I or II IFNs in the uterus, and is implicated as an important factor in determining uterine receptivity to embryos in ruminants. But little is known about the role the ISG15 gene or gene product plays during embryo development. In the present study, both the expression profile and function of ISG15 were investigated in early bovine embryos in vitro. ISG15 mRNA was detectable in Day 0, 2, 6 and 8 bovine embryos, but IFN-τ (IFNT) mRNA only appeared from Day 6. This means that embryonic expression of ISG15 on Days 0 and 2 was not induced by embryonic IFNT. However, ISG15 mRNA expression paralleled the expression of IFNT mRNA in Day 6 and 8 embryos. ISG15-lentivirus interference plasmid (ISG15i) was injected into 2-cell embryos to knockdown ISG15 expression. This resulted in decreases in the proportion of hatching blastocysts, the diameter of blastocysts and cell number per diameter of blastocysts compared with control embryos. In addition, ISG15i inhibited IFNT, Ets2 (E26 oncogene homolog 2) mRNA and connexion 43 protein expression in Day 8 blastocysts, whereas exogenous IFNT treatment (100ngmL-1, from Day 4 to Day 8) improved ISG15 mRNA and connexion 43 protein expression. In conclusion, it appears that ISG15 is involved in early bovine embryo development and that it regulates IFNT expression in the blastocyst.

Lysosomes are involved in induction of steroidogenic acute regulatory protein (StAR) gene expression and progesterone synthesis through low-density lipoprotein in cultured bovine granulosa cells.

Progesterone is an important steroid hormone in the regulation of the bovine estrous cycle. The steroidogenic acute regulatory protein (StAR) is an indispensable component for transporting cholesterol to the inner mitochondrial membrane, which is one of the rate-limiting steps for progesterone synthesis. Low-density lipoprotein (LDL) supplies cholesterol precursors for progesterone formation, and the lysosomal degradation pathway of LDL is essential for progesterone biosynthesis in granulosa cells after ovulation. However, it is currently unknown how LDL and lysosomes coordinate the expression of the StAR gene and progesterone production in bovine granulosa cells. Here, we investigated the role of lysosomes in LDL-treated bovine granulosa cells. Our results reported that LDL induced expression of StAR messenger RNA and protein as well as expression of cholesterol side-chain cleavage cytochrome P-450 (CYP11A1) messenger RNA and progesterone production in cultured bovine granulosa cells. The number of lysosomes in the granulosa cells was also significantly increased by LDL; whereas the lysosomal inhibitor, chloroquine, strikingly abolished these LDL-induced effects. Our results indicate that LDL promotes StAR expression, synthesis of progesterone, and formation of lysosomes in bovine granulosa cells, and lysosomes participate in the process by releasing free cholesterol from hydrolyzed LDL.

Imidazole inhibits autophagy flux by blocking autophagic degradation and triggers apoptosis via increasing FoxO3a-Bim expression.

Imidazole, an organic alkaloid, is an important pharmacophore in drug discovery. Anti-neoplastic potential of imidazole derivatives has been documented in several studies; however, mechanisms by which tumor cells respond to these stimuli remain to be elucidated. Autophagy and apoptosis have key roles in tumorigenesis and tumor treatment. In this study, we systematically examined autophagic events induced by imidazole in HEC-1B cells. Accumulation of autophagic vacuoles in imidazole-treated cells was verified by conversion of LC3 protein, as well as confocal and transmission electron microscopy. Furthermore, imidazole blocked autophagic degradation by impairing maturation of autophagosomes into autolysosomes. Concurrently, imidazole treatment induced apoptosis in HEC-1B cells, accompanied by activation of caspase 9 and 3. The proapoptotic effect was mediated by increased Bim expression. Moreover, imidazole upregulated the protein level of Foxo3a and induced its increased nuclear localisation. In addition, siRNA-mediated silencing of FoxO3a effectively attenuated imidazole-induced Bim upregulation and cell death, indicating direct involvement of this pathway in the imidazole-induced apoptosis. Taken together, our data provided a molecular link between imidazoles and anticancer therapies; understanding of these properties of imidazole is essential for development of effective cancer therapeutics using imidazoles.

Recombinant bovine interferon-τ enhances in vitro development of bovine embryos by upregulating expression of connexin 43 and E-cadherin.

Interferon-τ (IFNT), produced in ruminants by embryonic trophoblastic cells before implantation, is involved in the maternal recognition of pregnancy. It is a pleiotropic molecule that alters the synthesis of endometrial proteins and inhibits the proliferation of some cells. The present study investigated the effects of recombinant bovine IFNT on the development of early-stage bovine embryos and the molecular mechanism underlying this effect. This study demonstrated that expression of mRNA encoding type I IFN receptor subunits was detectable from d 4 to 8 in in vitro fertilized (IVF) bovine embryos. A considerable number of IVF (n = 1,941) and parthenogenetic activated (n = 1,552) bovine embryos demonstrated that supplementing the culture medium with IFNT (100 ng/mL) produced a greater percentage of blastocysts, and the total cell number within the resulting blastocysts was higher. In addition, IFNT upregulated the expression levels of both mRNA and protein for connexin 43 (GJA1) and E-cadherin (CDH1) and expression levels for granulocyte-macrophage colony-stimulating factor and insulin-like growth factor 2 mRNA but not for their proteins in d 8 embryos. However, IFNT inhibited mRNA expression for leukemia inhibitory factor (LIF), LIF receptor α, and the sodium/potassium-transporting ATPase subunit ß-1. We concluded that IFNT promoted the development of bovine embryos by upregulating the expression of GJA1 and CDH1. Thus, supplementing embryo cultures or transfer medium with IFNT may stimulate embryo development and improve embryo transfer efficiency.

A novel role of transient receptor potential mucolipin1 (TRPML1) in protecting against imidazole-induced cytotoxicity.

Lysosomotropic amines cause serious side effects such as cytoplasmic vacuolation and cell death. TRPML1 (also known as mucolipin1), a member of the transient receptor potential (TRP) protein family, may regulate fusion/fission of vesicles along the endocytic pathway and some aspects of lysosomal ion homeostasis. Nevertheless, it is still unknown whether TRPML1 is involved in death of mammalian cells induced by lysosomotropic agents. In this study, imidazole was used as a model to investigate the role of TRPML1 in the cytotoxicity of lysosomotropic agents. Overexpression of wild-type TRPML1 inhibited imidazole-induced vacuole formation and cell death in human endometrial adenocarcinoma (HEC-1B) cells. In contrast, siRNA-mediated TRPML1 knockdown increased the cell death induced by imidazole. Bafilomycin A1 raises the pH of acidic organelles and therefore suppresses accumulation of weak bases in them. Similarly, lysosomal pH was raised in TRPML1-overexpressing cells; therefore, we inferred that TRPML1 protected against imidazole toxicity by regulating the pH of acidic organelles. We concluded that TRPML1 had a novel role in protecting against lysosomotropic amine toxicity.