Human podocyte injury in the early course of hypertensive renal injury.

BACKGROUND: Hypertension is prevalent in the general population and is regarded as the second leading cause of renal damage and dysfunction, outnumbered only by diabetes. However, the mechanisms remain unclear. AIM: To investigate podocyte injury induced by hypertension in the early course without massive proteinuria or renal dysfunction. METHODS: The hypertension group comprised 18 patients with hypertension accompanied by microalbuminuria, diagnosed with hypertensive renal injury according to biopsy results. For a comparison of pathological changes in renal tissue, control group 1 comprised 10 healthy volunteers, and control group 2 comprised 16 patients who underwent surgery for renal trauma. RESULTS: The hypertension group had significantly higher blood pressure (P = 0.000) and microalbuminuria (P = 0.000) compared with control group 1. In the hypertension group, urinary podocytes were detected following positive staining of podocyte-specific nephrin and/or CD2-associated protein (CD2AP) in urine sediment. Podocyte foot process fusion and a significant decrease in nephrin and/or CD2AP expression in glomeruli were observed in the hypertension group compared with control group 2. This indicated that hypertension caused podocyte injury and detachment from the glomerular basement membrane, which was consistent with urinary detection of podocytes. CONCLUSION: Our results suggest that podocyturia appears early in the course of hypertensive renal injury, and may be a sensitive marker for early prediction of hypertensive renal injury.

CT-707, a Novel FAK Inhibitor, Synergizes with Cabozantinib to Suppress Hepatocellular Carcinoma by Blocking Cabozantinib-Induced FAK Activation.

Hepatocellular carcinoma is among the leading causes of cancer-related deaths worldwide, and the development of new treatment regimens is urgently needed to improve therapeutic approach. In our study, we found that the combination of a Met inhibitor, cabozantinib, and a novel FAK inhibitor, CT-707, exerted synergistic antitumor effects against hepatocellular carcinoma in vitro and in vivo Interestingly, further studies showed that therapeutic concentrations of cabozantinib increased the phosphorylation of FAK, which might attenuate the antitumor activity of cabozantinib. The simultaneous exposure to CT-707 effectively inhibited the activation of FAK that was induced by cabozantinib, which contributes to the synergistic effect of the combination. Furthermore, cabozantinib increased the mRNA and protein levels of integrin α5, which is a canonical upstream of FAK, and the introduction of cilengitide to block integrin function could abrogate FAK activation by cabozantinib, indicating that cabozantinib upregulated the phosphorylation of FAK in an integrin-dependent manner. Similar synergy was also observed on PHA-665752, another selective MET inhibitor, indicating that this observation might be a common characteristic of MET-targeting strategies. Our findings not only favor the development of the novel FAK inhibitor CT-707 as a therapeutic agent against hepatocellular carcinoma but also provide a new strategy of combining MET and FAK inhibitors to potentiate the anticancer activities of these two types of agents for treating hepatocellular carcinoma patients. Mol Cancer Ther; 15(12); 2916-25. ©2016 AACR.

MicroRNA-23a participates in estrogen deficiency induced gap junction remodeling of rats by targeting GJA1.

Increased incidence of arrhythmias in women after menopause has been widely documented, which is considered to be related to estrogen (E2) deficiency induced cardiac electrophysiological abnormalities. However, its molecular mechanism remains incompletely clear. In the present study, we found cardiac conduction blockage in post-menopausal rats. Thereafter, the results showed that cardiac gap junctions were impaired and Connexin43 (Cx43) expression was reduced in the myocardium of post-menopausal rats. The phenomenon was also observed in ovariectomized (OVX) rats, which was attenuated by E2 supplement. Further study displayed that microRNA-23a (miR-23a) level was significantly increased in both post-menopausal and OVX rats, which was reversed by daily E2 treatment after OVX. Importantly, forced overexpression of miR-23a led to gap junction impairment and Cx43 downregulation in cultured cardiomyocytes, which was rescued by suppressing miR-23a by transfection of miR-23a specific inhibitory oligonucleotide (AMO-23a). GJA1 was identified as the target gene of miR-23a by luciferase assay and miRNA-masking antisense ODN (miR-Mask) assay. We also found that E2 supplement could reverse cardiac conduction blockage, Cx43 downregulation, gap junction remodeling and miR-23a upregulation in post-menopausal rats. These findings provide the evidence that miR-23a mediated repression of Cx43 participates in estrogen deficiency induced damages of cardiac gap junction, and highlights a new insight into molecular mechanism of post-menopause related arrhythmia at the microRNA level.

MicroRNA-23a mediates mitochondrial compromise in estrogen deficiency-induced concentric remodeling via targeting PGC-1α.

It is well known that menopause could worsen age-related ventricular concentric remodeling following estrogen (E2) deficiency. However the underlying mechanisms of such phenomena are not fully understood. Mitochondria, as the 'cellular power station' of hearts, play an important role in maintaining normal cardiac function and structure. Therefore, the present study aims to investigate whether mitochondrial compromise is responsible for E2 deficiency associated concentric remodeling and, if so, what is its underlying molecular mechanism. We found evident concentric remodeling pattern in both postmenopausal and ovariectomized (OVX) mice, which could be attenuated by E2 replacement. Further study showed mitochondrial structural damages and respiratory function impairment in myocardium of both postmenopausal and OVX mice and E2 supplement reversed mitochondrial dysfunction in OVX mice, suggesting that E2 deficiency could induce mitochondrial compromise in the heart. Then, peroxisome proliferator-activated receptor-γ co-activator 1-α (PGC-1α), a key mitochondrial function and biology regulator, was found significantly reduced in both postmenopausal and OVX mice. The reduction of PGC-1α protein level in OVX mice could be rescued by E2 delivery, indicating that E2 could positively regulate PGC-1α expression. Next, we found that microRNA-23a (miR-23a) could be negatively regulated by E2 in both myocardium and cultured cardiomyocytes. Moreover, miR-23a could directly downregulate PGC-1α expression in cardiomyocytes via binding to its 3'UTR which implied that miR-23a could be critical for the downregulation of PGC-1α under E2 deficiency. Overexpression of miR-23a was also found to damage mitochondria in cultured cardiomyocytes, ascribed to PGC-1α downregulation. Taken together, E2 deficiency may cause mitochondrial compromise through miR-23a-mediated PGC-1α downregulation, which may subsequently lead to the menopause-associated concentric remodeling.

Myocardial dysfunction in early diabetes patients with microalbuminuria: a 2-dimensional speckle tracking strain study.

The aim of this study was to assess myocardial dysfunction in primary diabetes patients with microalbuminuria by 2-dimensional speckle tracking strain. Sixty-two patients with diabetes with or without hypertension and 37 matched hypertension controls were consecutively recruited from January 2011 to 2013. Routine physical examinations, laboratory tests, and echocardiography were performed in all patients. Subjects enrolled were divided into three groups according to history and urine albumin/creatinine ratio (ACR): group I: patients with only hypertension and normoalbuminuria (ACR < 30 mg/g), group II: patients with both hypertension and diabetes and normoalbuminuria (ACR < 30 mg/g), and group III: patients with both hypertension and diabetes and microalbuminuria (ACR 30-300 mg/g). Echocardiographic images of three cardiac cycles were acquired for off-line analysis using the GE EchoPAC software. Indices of cardiac function, including longitudinal, radial and circumferential strains, torsion, and left ventricular ejection fraction (LVEF) were assessed. Statistical analysis was performed using SPSS 13.0. Finally, 56 subjects and 32 controls were included in the analyses. There was no significant difference in age, gender, heart rate, BMI, and LVEF among groups, except for the blood pressure, ACR, and HbA1c. E wave, A wave, EDT, E m, and E/E m in group III were different with those in group I. Mean longitudinal strain (mSL), average SL of six segments in 4-chamber apical view (SL4) decreased obviously. The peak circumferential strain decreased in group III, while the torsion was compensatively increased. ACR was negatively related to mSL, SL4, E/E m, and positively related to torsion. We deduced that ACR maybe a predictor for myocardial damage in primary diabetes.

[mTOR/p70s6k signaling pathway is involved in megakaryocyte polyploidization].

AIM: To investigate the mechanisms of megakaryocyte polyploid cell cycle control. METHODS: The expression and phosphorylation of mTOR/p70s6k pathway proteins was detected by western blot. Double-labeling techniques were used to investigate in which of the phase of the polyploid cell cycle S6K1 at Thr421/Ser424 are phosphorylated. RESULTS: Nocodazole induced a relatively synchronized polyploidization in Dami cells. The expression of mTOR and the phosphorylation of mTOR at Ser2448 increased when Dami cells begin to progress from G1 to S-phase in cell cycle. Analysis of flow cytometry showed that phosphorylation of S6K1 at Thr421/Ser424 increased at G2/M-phase. CONCLUSION: mTOR/S6K1 pathway is involved in megakaryocyte polyploid cell cycle control.

[Effects of ventricular demand and dual-chamber pacing models on the long-term clinical outcome and cardiac remodeling in patients with symptomatic bradycardia].

OBJECTIVE: To assess the effects of VVI (ventricular demand) and DDD (dual-chamber) pacing models on cardiac remodeling and the long-term clinical outcome of patients with symptomatic bradycardia. METHODS: All patients with DDD and VVI pacing models at our hospital from January 1991 to January 2003 were retrospectively analyzed. RESULTS: After a follow-up period of over 8 years in DDD and VVI groups (97 ± 27, 107 ± 44 months), left atrial diameter [(45 ± 12) mm vs (39 ± 12) mm, P < 0.01] and left ventricular end-diastolic diameter [(53 ± 11) mm vs (50 ± 9) mm, P = 0.01] in 57 patients with VVI pacing model were markedly enlarged than those at pre-implantation. And tricuspid regurgitation increased (42.4% vs 16.9%, P < 0.05). But in 59 patients with DDD pacing model, except for increased tricuspid regurgitation (42.1% vs 10.5%, P < 0.01), left atrial diameter [(37 ± 5) mm vs. (35 ± 5) mm, P = 0.07] and left ventricular end-diastolic diameter [(47 ± 7) mm vs (47 ± 5) mm, P = 0.32] were not significantly different. Mitral regurgitation significantly increased only in the VVI group (P < 0.01). The increases of left ventricular end-diastolic diameter (P = 0.04), mitral valve (P = 0.02) and tricuspid regurgitation (P < 0.01) were much more pronounced in the VVI group than those in the DDD group. Left ventricular ejection fraction (LVEF) showed no difference with that at pre-implantation (P = 0.11 in DDD group, P = 0.05 in VVI group). But the LVEF value was lower (P = 0.04) while the incidence of thrombosis was higher (P = 0.03) in the VVI group than those in the DDD group at post-implantation. However, the incidence of atrial fibrillation (P = 0.14), hospitalization (P = 0.08) and survival (P = 0.77) showed no significant difference between two groups. CONCLUSION: DDD pacing offers more benefits over VVI pacing through improving cardiac functions and arresting left ventricular remodeling. However, neither groups showed any difference in decreasing mortality rate and hospitalization. Moreover, both pacing modes fail to reverse cardiac electrical and anatomical remodeling. It is imperative to explore more physiological pacing site and rational atrioventricular (AV) interval to improve the prognosis of patients.

The modifier protein of glyceraldehyde-3-phosphate dehydrogenase reduces free radical-mediated renal damage in a rat model of acute kidney injury.

BACKGROUND: The modifier protein (MP) of glyceraldehyde-3-phosphate dehydrogenase has been shown to promote growth of renal epithelial cells in vitro. The aim of this study was to show the in vivo effects of MP in a rat model of gentamicin-induced acute kidney injury (AKI). METHOD: MP was purified from monkey renal tubular epithelial cell line BSC-1 and confirmed by amino acid sequencing. Male Sprague-Dawley rats were divided into the following groups: normal control, gentamicin-treated, epidermal growth factor (EGF) plus gentamicin-treated, and MP plus gentamicin-treated, as well as control groups for EGF and MP alone. Levels of serum creatinine (SCr), serum and tissue lipid peroxide, nitric oxide and glutathione-S-hydrogenase for each group were measured on the 7th and 14th days of treatment. Tissue sections were studied with light microscopy. RESULTS: The gentamicin-treated group showed a marked increase in SCr compared to the normal control group. Co-treatment of gentamicin with MP and/or EGF produced similar significant decreases preventing the increase in SCr. There were also significant reductions in serum and tissue homogenate levels of lipid peroxide and nitric oxide, accompanied by an increase in the level of glutathione-S-hydrogenase, in the MP co-treated groups compared to the gentamicin-treated group. AKI was confirmed histologically in the gentamicin-treated group, with damage to the tubular epithelium recorded. This was attenuated by MP co-treatment. There were also reductions in the expression of intercellular adhesion molecule-1 and proliferating cell nuclear antigen in the MP co-treated groups. CONCLUSION: Using a gentamicin model of AKI, MP was able to reduce free radical production in kidney tissue and in the circulation, thus preventing oxidant injury and minimizing damage in renal epithelial cells.

The leaf extract of Siberian Crabapple (Malus baccata (Linn.) Borkh) contains potential fatty acid synthase inhibitors.

The present work focused on the kinetics of the inhibitory effects of the leaf extract of Siberian Crabapple, named Shan jingzi in China, on chicken liver fatty acid synthase. The results showed that this extract had much stronger inhibitory ability on fatty acid synthase than that from green teas described in many previous reports. The inhibitory ability of this extract is closely related to the extracting solvent, and the time of extraction was also an important influencing factor. The inhibitory types of this extract on diffeerent substrates of chicken liver fatty acid synthase, acetyl-CoA, malonyl-CoA and NADPH, were found to be noncompetitive, uncompetitive and mixed, respectively. The studies here shed a new light on the exploration for inhibitors of fatty acid synthase.

A substitutive substrate for measurements of beta-ketoacyl reductases in two fatty acid synthase systems.

Bacterial beta-ketoacyl-ACP reductase (FabG) and the beta-ketoacyl reductase domain in mammalian fatty acid synthase (FAS) have the same function and both are rendered as the novel targets for drugs. Herein we developed a convenient method, using an available compound ethyl acetoacetate (EAA) as the substitutive substrate, to measure their activities by monitoring decrease of NADPH absorbance at 340 nm. In addition to the result, ethyl 3-hydroxybutyrate (EHB) was detected by HPLC analysis in the reaction system, indicating that EAA worked effectively as the substrate of FabG and FAS since its beta-keto group was reduced. Then, the detailed kinetic characteristics, such as optimal ionic strength, pH value and temperature, and kinetic parameters, for FabG and FAS with this substitutive substrate were determined. The Km and kcat values of FabG obtained for EAA were 127 mM and 0.30 s(-1), while those of this enzyme for NADPH were 10.0 microM and 0.59 s(-1), respectively. The corresponding Km and kcat values of FAS were 126 mM and 4.63 s(-1) for EAA; 8.7 microM and 4.09 s(-1) for NADPH. Additionally, the inhibitory kinetics of FabG and FAS, by a known inhibitor EGCG, was also studied.

The antibacterial efficacy of an aceraceous plant [Shantung maple (Acer truncatum Bunge)] may be related to inhibition of bacterial beta-oxoacyl-acyl carrier protein reductase (FabG).

Polyphenols, including flavonoids, are the major components of the extracts from aceraceous plants. They possess remarkable antibacterial and antitumour activity. Our study focused on whether the inhibition of the bacterial type II fatty acid synthesis system is the mechanism for the antibacterial effect of the related plant polyphenols. Extracts obtained from the fallen leaves of the Shantung maple (Acer truncatum Bunge) using different solvents, and the related pure compound PGG (1,2,3,4,6-penta-O-galloyl-beta-D-glucose), potently inhibited the FabG (beta-oxoacyl-ACP reductase) steps in the fatty-acid-elongation cycle with the IC(50) values between 0.9 and 7.2 microg/ml. An ethyl acetate extract appeared to inhibit FabG reductase in a mixed manner with NADPH, as did PGG with NADPH, demonstrating that they interfered with the binding of the cofactor to the enzyme. Gram-positive and Gram-negative bacteria and some fungi were used to evaluate the antibacterial abilities of different extract samples. The experiments showed that a higher polyphenol content of the extracts led to a more potent inhibitory capacity against FabG, thus enhancing the antibacterial efficacy.

Inactivation mechanism of the beta-ketoacyl-[acyl carrier protein] reductase of bacterial type-II fatty acid synthase by epigallocatechin gallate.

Epigallocatechin gallate (EGCG), a major compound from green tea, reversibly inhibits beta-ketoacyl-[acyl carrier protein] reductase (FabG) from Escherichia coli. In this study, we found that EGCG exhibited an atypical time-dependent inhibition of FabG, which possibly resulted from the EGCG-induced aggregation of FabG. It was observed that FabG inactivation and aggregation occurred nearly simultaneously, with a lag time that decreased with increasing EGCG concentration. These results suggest that some chemical reactions, required for aggregation and inactivation, occurred during the lag time. Since EGC was detected by HPLC after the incubation of EGCG with FabG, EGCG probably covalently modified FabG. These further results showed that 1 tetramer of FabG must be modified by several, possibly 4, EGCG molecules before the formation of FabG aggregates. FabG aggregation was a first-order reaction independent of protein concentration. Due to an initial lag time, the first-order rate of aggregation gradually increased, reaching a maximal and constant value. The effect of increasing concentration of EGCG on the first-order rate constant for aggregation indicated that EGCG bound to FabG by affinity labeling. Based on the results, we propose a mechanism for the interaction of EGCG with FabG:EGCG first binds reversibly to each subunit of FabG, followed by covalent modification and then aggregation of the 4 EGCG-modified subunits.

[The proliferation, phenotype change and anti-tumor activity of cytokine induced killer cells].

AIM: To study the growth regularity, the phenotype change and the cytotoxicity of CIK cells. METHODS: The number of CIK cells was counted by living cell counting in different culturing time to observe the growth of the CIK cells, and the phenotype change of the CIK cells was detected by flow cytometry. Meanwhile cytotoxicity of CIK cells to tumor cell lines was also detected by CytoTox96 non-radiated cytotoxicity kit. RESULTS: After stimulated by cytokines and anti-CD3 antibody, CIK cells can proliferate significantly. The cell number of CIK was increased to 473.28+/-27.53 fold in serum-free medium plus auto-serum, 218.24+/-16.86 fold in serum-free medium and only 11.52+/-1.04 fold in RPMI1640 plus fetal FCS, respectively. The CD3(+)+CD8(+), CD3(+)+CD56(+), CD226(+)+CD11a(+) and CD305(+)+CD11a(+) cells were increased with the progression of the cultural time and the CD3(+)+CD4(+) cells were decreased with the progression of cultural time. The cytotoxicity of CIK cells to tumor cell lines was significantly higher than that of LAK cells (P<0.01) and its cytotoxicity was increased with progression of the cultural time. CONCLUSION: CIK cells have strong proliferative ability and higher cytotoxicity to tumor cells in vitro, which could be used as a potential anti-tumor adoptive immunotherapy in clinic.