Formin mDia1 contributes to migration and epithelial-mesenchymal transition of tubular epithelial cells exposed to TGF-ß1.

Renal tubular epithelial cells may undergo epithelial-mesenchymal transition (EMT) in response to stimuli, such as transforming growth factor (TGF)-ß1, leading to myofibroblast activation and renal fibrosis. The formin mDia1 is required for nucleation and polymerization of actin and the microtubule cytoskeleton. The present study sought to explore the role of mDia1 in EMT of tubular epithelial cells. A rat model of unilateral ureteral obstruction (UUO) was established. The expression of TGF-ß1, collagen I, collagen III, and mDia1 in the kidneys was examined at day 7 after surgery. The effect of mDia1 on EMT was explored in NRK-52E cells by exposing them to TGF-ß1. Increased expression of TGF-ß1, collagen I, collagen III, and mDia1 was found in obstructive kidneys of UUO model rats. Exposing rat tubular epithelial cells to TGF-ß1 promoted collagen I and collagen III expression but had no effect on mDia1 expression. Silencing mDia1 expression impeded epithelial cell migration as well as reduced TGF-ß1, collagen, and Profilin1 expression, whereas mDia1 overexpression exerted an opposite effect. Furthermore, mDia1 regulated the expression of vimentin, α-smooth muscle actin, and E-cadherin and focal adhesion-kinase (FAK)/Src activation through Profilin1. Inhibition of the mDia1 activator RhoA by fasudil reversed EMT, and FAK/Src activation induced by mDia1. In conclusion, mDia1 regulated tubular epithelial cell migration, collagen expression, and EMT in NRK-52E cells exposed to TGF-ß1. Thus, suppression of mDia1 activation might be a strategy to counteract renal fibrosis.

Novel functional role of heat shock protein 90 in ATP-sensitive K+ channel-mediated hypoxic preconditioning.

AIMS: ATP-sensitive K+ (K ATP) channels are implicated in the protective effect of ischaemic preconditioning (IPC). Kir6.2 has been shown to be involved in the cardioprotection of IPC. However, the mechanism by which Kir6.2-containing K ATP channels protect the heart is still largely unknown. The present study was designed to explore the potential mechanism involved in K ATP channel-mediated cardioprotection. METHODS AND RESULTS: Cellular models of hypoxic preconditioning (HP) from rat heart-derived H9c2 cells and adult rat cardiomyocytes were employed. Dominant negative and small interfering RNA (siRNA) technology were utilized in combination with biochemical, immunofluorescent, and cell viability assays. The cell viability study revealed that HP significantly increased the viable cells after prolonged hypoxia and reoxygenation. This protective effect was prevented by expression of dominant negative Kir6.2AAA, siRNA targeting Kir6.2, or the K ATP channel inhibitor 5-hydroxydecanoate. Further, our data showed that inhibiting heat shock protein 90 (HSP90) function with the HSP90 inhibitor geldanamycin or HSP90 expression with siRNA completely inhibited the protection of HP. We found that HSP90 was associated with Kir6.2 and its activity was linked to mitochondrial targeting of Kir6.2. CONCLUSION: We demonstrate that Kir6.2 is critical in HP of cardiomyocytes. Importantly, we show that HSP90 is involved in K ATP-mediated cytoprotection, possibly by promoting mitochondrial targeting of Kir6.2.

ANEPIII, a new recombinant neurotoxic polypeptide derived from scorpion peptide, inhibits delayed rectifier, but not A-type potassium currents in rat primary cultured hippocampal and cortical neurons.

A new recombinant neurotoxic polypeptide ANEPIII (BmK ANEPIII) derived from Scorpion peptide, which was demonstrated with antineuroexcitation properties in animal models, was examined for its action on K+ currents in primary cultured rat hippocampal and cortical neurons using the patch clamp technique in the whole-cell configuration. The delayed rectifier K+ current (I(k)) was inhibited by externally applied recombinant BmK ANEPIII, while the transient A-current (I(A)) remained virtually unaffected. BmK ANEPIII 3 microM, reduced the delayed rectifier current by 28.2% and 23.6% in cultured rat hippocampal and cortical neurons, respectively. The concentration of half-maximal block was 155.1 nM for hippocampal neurons and 227.2 nM for cortical neurons, respectively. These results suggest that BmK ANEPIII affect K+ currents, which may lead to a reduction in neuronal excitability.

Indapamide induces apoptosis of GH3 pituitary cells independently of its inhibition of voltage-dependent K+ currents.

Indapamide blocks multiple voltage-dependent K+ currents (Kv) in the heart and Kv have an important role in cell proliferation and apoptosis, so the aim of this work was to study the effects of indapamide on Kv and the viability of GH3 cells. Indapamide inhibited Kv of GH3 cells and the inhibition was irreversible after a 10-min washout when more than 250 microM indapamide was used. Indapamide reduced the viability of GH3 cells in a concentration-dependent manner. The decreased cell viability was because indapamide induced cell apoptosis, or even necrosis at higher concentrations. HepG2 cells, which express no apparent Kv, were used to determine the association between inhibition of Kv and the apoptotic action of indapamide. Indapamide had a similar action on cell viability and apoptosis of HepG2 cells. 4-Aminopyridine, the voltage-dependent K+ channel blocker, inhibited Kv of GH3 cells but did not induce the cell apoptosis. We concluded that while indapamide inhibited Kv and induced apoptosis of GH3 cells, the apoptotic action of indapamide was not associated with its inhibition of Kv.

[The cardioprotective effect and mechanism of lumbrokinase].

AIM: To investigate the protective effect of lumbrokinase against myocardial ischemia and to further explore its underlying mechanisms. METHODS: The effect of lumbrokinase on myocardial ischemia was observed by a model of acute myocardial infarction due to permanent ligation of the left anterior descending coronary artery in rats. Patch-clamp technique and laser scanning confocal microscopy were utilized to study the action of lumbrokinase on L-type calcium current (ICa-L) and intracellular calcium concentration ([Ca2+]i). RESULTS: Lumbrokinase decreased the infarct size of myocardium in a dose-dependent manner. The inhibitory rate of lumbrokinase at the dose of 20, 40 and 80 mg x kg(-1) was 7.7%, 34.6% and 46.2%, respectively. The electrophysiological studies displayed that, at + 10 mV, the ICa-L was markedly reduced from (-14.42 +/- 1.53) pA/pF to (-11.33 +/- 1.40) pA/pF (decreased by 21.4%, P <0.01) and (-9.92 +/- 1.31) pA/pF (decreased by 36.5%, P <0.01) by lumbrokinase (10 and 50 micromol x L(-1)), respectively. Confocal experiments showed that 10 micromol x L(-1) lumbrokinase showed no obvious effects on [Ca2+]i at resting states (P > 0.05). However, the increase of [Ca2+]i induced by 60 mmol x L(-1) KCl was distinctly limited by 10 micromol x L(-1) lumbrokinase (P <0.01). Within 240 s, the no obvious peak value of fluorescent intensity (FI) was shown. CONCLUSION: Lumbrokinase showed protective action against myocardial infarction in rats. The possible mechanisms of anti-ischemia could be attributed to decreasing ICa-L and [Ca2+] of ventricular myocytes in rats.

[Inhibitory effects of chloride channel blockers NPPB on proliferation of human glomerular mesangial cells].

AIM: To investigate the effects of NPPB, a chloride channel blocker, on proliferation of mesangial cells. METHODS: Cell proliferation was determined by measuring cell number and 3H-thymidine incorporation. The LDH activity released from these cells was measured as evaluation of cell viability. The phase of cell cycle was detected by flow cytometry. RESULTS: Cell proliferation assays showed that treatment with both NPPB (50 and 25 micromol x L(-1)) and in hypertonic media (100% increased osmolarity with D-mannitol ) significantly reduced the number of human MC and 3H-thymidine incorporation in a dose-dependent manner. But the LDH activity was not significantly altered in the treatment with 50 micromol x L(-1) NPPB. Flow cytometry experiments showed that 50 and 25 micromol x L(-1) NPPB arrested (84.2 +/- 2.4) % and (80.8 +/- 2.9) % of cells at G0/G1 stage, versus (70.5 +/- 1.4) % of control cells. Conclusion NPPB suppresses cell proliferation and produces growth arrest at G0/G1 phase in human MC by a mechanism probably associated with changes in cell volume.

M3-R/IK(M3)--a new target of antiarrhythmic agents.

AIM: To investigate the relationship between M3-R/IK(M3) and arrhythmia in order to find a new target for antiarrhythmic agents. METHODS: Using the acute ischemic model of rats and patch-clamp techniques, the effects of the M3 receptor on the occurrence of arrhythmias and its possible mechanisms were studied. RESULTS: In acute ischemic model of rats, the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine-methiodide (4DAMP) increased the occurrence of arrhythmias, and the M3 receptor agonist choline suppressed the onset and the development of arrhythmias (P < 0. 01). No change was observed after treatment with other receptor antagonists (M1, M2, and M4). With patch-clamp techniques, it was found that choline induced K+ current could be inhibited by 4DAMP. Antagonists toward M1, M2, and M4 receptors all failed to alter the current. CONCLUSION: Choline modulates the cellular electrical properties of the heart, probably by activating a K+ current via stimulation of the M3 receptor. M3-R/IK(M3) may act as a new target for antiarrhythmic agents.

[Establishment of a novel arrhythmia model in rats].

AIM: To establish a novel arrhythmia model in rats. METHODS: Coronary artery occlusion was produced in hyperlipidemic rats after the animals were fed a high fat and cholesterol chow for 15 days. The incidence, duration and score of arrhythmias were determined 1 hour after coronary occlusion. RESULTS: The incidence, duration and score of arrhythmia induced by coronary artery occlusion increased significantly in hyperlipidemic rats compared with those in normal rats (P < 0.05). In normal rats, pretreatment with amiodarone 60 mg x kg(-1) or verapamil 25 mg x kg(-1) 3 days before coronary artery occlusion did not influence the incidence, duration and score of arrhythmia (P > 0.05). In hyperlipidemic rats, amiodarone 60 mg x kg(-1) decreased the incidence, duration and score of arrhythmia (P < 0.05), but not verapamil 25 mg x kg(-1) (P > 0.05). CONCLUSION: The novel arrhythmia model induced by coronary artery occlusion in hyperlipidemic rats is reliable and similar to the pathophysiological state in human being.

Ulinastatin attenuates renal interstitial inflammation and inhibits fibrosis progression in rats under unilateral ureteral obstruction.

The aim of the present study was to examine the protective effects of the urinary trypsin inhibitor ulinastatin (UTI) on renal interstitial inflammation and fibrosis in rats subjected to unilateral ureteral obstruction (UUO). A total of 24 male Wistar rats were randomly divided into the three groups; the sham operation (SOR) group (n=8), the UUO group (n=8) and the UUO+UTI group (post­UUO UTI treatment, n=8). UUO was performed with complete ligation of the left ureter. As a medical intervention, saline (4 ml kg­1 d­1) and UTI (40000 units kg­1 d­1) were injected, respectively, into the animals of the corresponding groups on day one following surgery. The rats in all three groups were euthanized on day seven post surgery. Blood samples were harvested for blood urea nitrogen (BUN) and serum creatinine (Scr) content measurements. The degree of interstitial pathological changes in the tissues from the obstructed kidneys were observed through hematoxylin and eosin (H&E) and Masson staining. The CD68+ macrophage amount, tumor necrosis factor­α (TNF­α), interleukin 1ß (IL­1ß), nuclear factor­κB (NF­κB), transforming growth factor­ß1 (TGF­ß1) and type I collagen (Col­I) levels were examined immunohistochemically. The protein expression levels of NF­κB were examined using western blot analysis. Total superoxide dismutase (SOD) activity and malondialdehyde (MDA) content of homogenates were measured spectrophotometrically. The results revealed that ulinastatin had no statistically significant effect on the BUN and Scr levels (P>0.05). However, in comparison with the SOR group, the UUO group exhibited significantly more severe renal interstitial pathological injury in terms of tubular dilation, epithelial atrophy, renal interstitial inflammatory cell infiltration and proliferation of fibrous tissues, as well as significantly elevated levels of interstitial CD68+ macrophages, IL­1ß, TNF­α, NF­κB, TGF­ß1 and Col­I (P<0.01). UTI treatment significantly reduced UUO­induced renal interstitial damage with reduced levels of interstitial CD68+ macrophages, IL­1ß, TNF­α, NF­κB, TGF­ß1 and Col­I and MDA (P<0.05), and increased SOD levels (P<0.05). In conclusion, the present study indicated that UTI is able to effectively inhibit UUO­side renal interstitial inflammatory reaction and fibrosis in UUO­inflicted rats.

Effect of ANEPIII, a novel recombinant neurotoxic polypeptide, on sodium channels in primary cultured rat hippocampal and cortical neurons.

Previous studies have shown that the recombinant neurotoxic polypeptide BmK ANEP (ANEPIII) displayed good anti-neuroexcitation activity as demonstrated by pharmacological tests of the blockade of chemical-induced convulsive seizures. In order to search for further anticonvulsant mechanism of action of ANEPIII, the effects of ANEPIII on sodium channels were assessed using the whole-cell patch clamp recordings in primary cultures of rat hippocampal and cortical neurons. ANEPIII decreased the sodium currents in a voltage-dependent manner, which appeared as a shift of the current-voltage relation to positive potentials. The effect was reversible after washing. The concentration-responsiveness measured in hippocampal and cortical neurons revealed an IC(50) value of 124.6 nM and 192.7 nM, respectively. Furthermore, ANEPIII 1000 nM significantly shifted the activation curves of sodium current in hippocampal and cortical neurons to more positive potentials and the recovery from inactivation of sodium current was significantly slower. Voltage-dependent inactivation curves of sodium channels in hippocampal and cortical neurons did not change in the presence of 1000 nM ANEPIII. Thus, our results demonstrated that ANEPIII in submicromolar concentrations was a voltage-dependent, reversible blocker of sodium current in hippocampal and cortical neurons. It is concluded that these phenomena may explain, at least in part, the anti-neuroexciting properties of this peptide.

Volume-sensitive outwardly rectifying chloride channels are involved in oxidative stress-induced apoptosis of mesangial cells.

Volume-sensitive outwardly rectifying (VSOR) Cl- channels have been electrophysiologically identified in human and mouse mesangial cells, but the functional role of VSOR Cl- channels in mesangial cell apoptosis is not clear. The aim of the present study was to demonstrate the role of VSOR Cl- channels in oxidative stress-induced mesangial cell apoptosis. H2O2-induced Cl- currents showed phenotypic properties of VSOR Cl- channels, including outward rectification, voltage-dependent inactivation at more positive potentials, sensitivity to hyperosmolarity, and inhibition by VSOR Cl- channel blockers. Moreover, blockage of VSOR Cl- channels by DIDS (100 microM), NPPB (10 microM) or niflumic acid (10 microM) rescued mesangial cell apoptosis induced by H2O2. Treatment with 150 microM H2O2 for 2h resulted in significant reduction of cell volume, in contrast, nuclear condensation and/or fragmentation were not observed and the caspase-3 activity was also not increased. The early-phase alterations in cell volume were markedly abolished by pretreatment with VSOR Cl- channel blockers. We conclude that VSOR Cl- channels are involved in H2O2-induced apoptosis in cultured mesangial cells and its mechanism is associated with apoptotic volume decrease processes.