TRPV4 ion channel as important cell sensors

This review provides a summary of the physiological significance of the TRPV4 ion channel. Although TRPV4 was initially characterized as an osmosensor, we found that TRPV4 can also act as a thermosensor or a mechanosensor in brain neurons or epithelial cells in the urinary bladder. Here, we summarize the newly characterized functions of TRPV4, including the research progress that has been made toward our understanding of TRPV4 physiology, and discuss other recent data pertaining to TRPV4. It is thought that TRPV4 may be an important drug target based on its broad expression patterns and important physiological functions. Possible associations between diseases and TRPV4 are also discussed.     

Chemokines as therapeutic targets in renal disease

Increased understanding of the fundamental importance of the role of chemokines and their receptors in inflammation, together with the demonstration of their involvement in human and experimental inflammatory renal disease, make these molecules potential therapeutic targets. A number of recent studies using genetically deficient mice and chemokine receptor antagonists in animal models have demonstrated that chemokine inhibition can attenuate experimental renal injury. Because there is simultaneous expression of multiple chemokines and receptors in disease, strategies that are aimed at antagonizing multiple chemokines receptor interactions are likely to be more effective than therapies that target a single chemokine. It is also now recognized that chemokines are involved in normal immune development and immune regulation. These observations, together with the results of studies that have demonstrated deleterious effects of chemokine receptor antagonism in experimental renal disease, highlight the need for thorough understanding of the role of individual chemokines in the pathogenesis of different types of renal disease before optimal therapeutic interventions may be achieved.     

ROCK1 as a potential therapeutic target in osteosarcoma

Osteosarcoma is the most common primary malignancy of bone. Patients with localized disease are routinely treated with surgery and chemotherapy. Unfortunately, many of these patients eventually relapse even after high‐dose pre‐ and postoperative chemotherapy. Upon recurrence of the tumor locally or distantly, they have limited treatment options that are usually unsuccessful. Our prior studies screening lentiviral shRNA libraries, searching for kinases involved in osteosarcoma cell growth and proliferation have identified the Rho‐associated coiled‐coil containing protein kinase 1 (ROCK1) as a possible hit. We show in this study that ROCK1 is highly expressed in various tumor cell lines and tumor tissues from osteosarcoma patients. ROCK1 knockdown by synthetic siRNA decreases cell proliferation, viability and induces apoptosis in osteosarcoma cell lines KHOS and U‐2OS. Finally, we established the relationship between expression levels of ROCK1 and clinical prognosis in osteosarcoma patients by using immunohistochemistry. There were significant differences in overall survival between cohorts of patients with ROCK1 levels categorized as high‐staining, moderate‐staining, and low‐staining. High levels of ROCK1 were associated with poor outcomes in clinical osteosarcoma. These findings suggest that knockdown of ROCK1 inhibits proliferation and induces apoptosis in osteosarcoma cell lines. ROCK1 may be a promising therapeutic target for the treatment of osteosarcoma patients. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1259–1266, 2011     

Effect of ion channel modulators on the membrane potential of human chondrocytes

As all living cell systems, human chondrocytes are provided with a membrane potential. For its origin the existence of ion channels at the cell membrane is an essential prerequisite. In non-human chondrocytes, different ion channels could already be identified. A connection between the potassium channel activity and the proliferation has been detected in different human cell systems. Whereas, the proof of a connection between ion channel activity of human chondrocytes and the proliferation has yet to be established. In this study the concentration-dependent influence of the ion channel modulators tetraethylammonium (TEA), 4-amino-pyridin (4-AP), 4',4'diisothiocyanatostilbene-2,2'disulfonic acid (DIDS), 4-acetamido-4'isothiocyano-2,2'disulfonic acid stilbene (SITS) and verapamil on the membrane potential and the proliferation of human chondrocytes was investigated using flow cytometry. The results show an effect of the used ion channel modulators causing a change of the membrane potential of human chondrocytes. The decrease of the membrane potential by 18% was measured with 0.25 mmol/l verapamil meaning the maximal measurable effect compared with a control group. When measuring DNA distribution, it became apparent that the human chondrocytes are diploid cells with a very low proliferation tendency. These results allow the conclusion of an influence of ion channel modulators on chondrocyte proliferation.     

Inhibitors of vascular calcification as potential therapeutic targets

Vascular calcification is frequent in the general population. Its incidence increases with age. It contributes to cardiovascular morbidity and mortality in patients with advanced atherosclerosis, in the presence or absence of diabetes mellitus and chronic kidney disease (CKD). Both diabetes and CKD aggravate its degree of severity and accelerate its progression. Vascular calcification is the result of both passive and active processes of calcium phosphate deposition in the arterial wall. These processes are more or less successfully opposed by inhibitory proteins and nonpeptidic factors. In the present overview we discuss the roles of several among these vascular calcification inhibitors which represent potential therapeutic targets.     

L-arginine as a therapeutic tool in kidney disease

Infusion of L-arginine in experimental animals increases renal plasma flow (RPF) and glomerular filtration rate (GFR). It is likely that a component of these hemodynamic changes are mediated by nitric oxide (NO) as suggested by studies with specific antagonists of L-arginine metabolism. L-arginine administration ameliorates the infiltration of the renal parenchyma by macrophages in rats with obstructive nephropathy or rats with puromycin-induced nephrotic syndrome. L-arginine administration also blunts the increase in interstitial volume, collagen IV, and alpha-smooth muscle actin. Rats with a remnant kidney given 1% L-arginine in the drinking water had a greater GFR and RPF. L-arginine administration also decreased proteinuria. Diabetic rats given L-arginine had significantly lower excretion of protein and cyclic guanosine monophosphate than diabetic rats not receiving L-arginine. Despite persistent hyperglycemia, the administration of L-arginine prevented the development of hyperfiltration and ameliorated proteinuria in diabetic rats. In the setting of ischemic acute renal failure, the administration of L-arginine had a beneficial effect on GFR and RPF, decreased O2- production, diminished up-regulation of soluble guanylate cyclase, and prevented up-regulation of inducible NO synthase (iNOS). The pharmacokinetics of L-arginine indicate that side effects are rare and mostly mild and dose dependent.     

Mesenchymal stem cells: immunobiology and therapeutic potential in kidney disease

SUMMARY: Mesenchymal stem cells (MSC) are non‐haematopoietic cells that are prevalent in the adult bone marrow but can also be isolated from a variety of other postnatal tissues. MSC are non‐immunogenic and are immunosuppressive, with the ability to inhibit maturation of dendritic cells and suppress the function of naïve and memory T cells, B cells and NK cells. In addition to their immunomodulatory properties, MSC are capable of differentiating into various tissues of mesenchymal and non‐mesenchymal origin and migrating to sites of tissue injury and inflammation to participate in tissue repair. A number of studies in animal models of cardiac injury, stroke and ischaemic renal injury have demonstrated the clinical potential of MSC in tissue regeneration and repair. MSC are currently being evaluated in various preclinical and clinical studies in humans and offer significant potential as a novel cellular therapy for tissue regeneration and immunological conditions. The present review focuses on the unique immunomodulatory and regenerative properties of MSC and their potential role in the treatment of kidney disease.     

Hypoxic preconditioning in coronary microarteries: role of EDHF and K+ channel openers

BACKGROUND: Hypoxic preconditioning may provide a useful method of myocardial protection in cardiac operations. The present study was designed to investigate the possible mechanisms of preconditioning regarding endothelium-derived hyperpolarizing factor (EDHF) and the effect of a potassium channel opener KRN4884 on the porcine coronary microartery in mimicking hypoxic preconditioning. METHODS: Porcine coronary microartery rings (diameter 200 to 500 microm) studied in a myograph were divided into seven groups: (1) control group; (2) hypoxia-reoxygenation group (hypoxia for 60 minutes followed by reoxygenation for 30 minutes); (3) preconditioning group (hypoxia for 5 minutes followed by reoxygenation for 10 minutes before hypoxia reoxygenation); (4) KRN4884 pretreatment group (KRN4884 was added into the myograph chamber 20 minutes before hypoxia reoxygenation); (5) 5-hydroxydecanoate + KRN group (5-hydroxydecanoate was given 20 minutes before KRN4884 pretreatetment); (6) glibenclamide (GBC) + KRN group (GBC was added 20 minutes before KRN4884 pretreatment); and (7) endothelium denuded group (the endothelium was removed). The endothelium-derived hyperpolarizing factor-mediated relaxation to bradykinin was studied in the rings precontracted with U46619 in the presence of N(omega)-nitro-L-arginine and indomethacin. RESULTS: The maximal relaxation induced by bradykinin was reduced in hypoxia reoxygenation (40.7% +/- 2.8% vs 66.9% +/- 2.5% in control, p = 0.000). This reduced relaxation was recovered in either preconditioning (64.6% +/- 4.6%, p = 0.002), or KRN4884 pretreatment (67.1% +/- 3.6%, p = 0.000). The 5-hydroxydecanoate, but not GBC pretreatment abolished the effect of KRN44884 pretreatment (67.1% +/- 3.6% vs 42.9% +/- 3%, p = 0.001). CONCLUSIONS: Hypoxia reoxygenation reduces the relaxation mediated by endothelium-derived hyperpolarizing factor in the coronary microartery. This function can be restored by either hypoxic preconditioning or the K(ATP) channel opener KRN4884, and therefore K(ATP) channel openers may provide similar effect as preconditioning. The mechanism is mainly related to the mitochondrial ATP-sensitive K+ channels.     

Potassium channels and human corporeal smooth muscle cell tone: diabetes and relaxation of human corpus cavernosum smooth muscle by adenosine triphosphate sensitive potassium channel openers

PURPOSE: Sustained contraction of human corporeal smooth muscle depends on continuous transmembrane calcium flux through voltage gated calcium channels. K channels modulate corporeal smooth muscle membrane potential and, thus, ultimately affect transmembrane calcium flux. Therefore, we characterized relaxation responses elicited by the K channel modulators pinacidil and levcromakalim on isolated human corporeal tissue strips. We also evaluated the possibility that there may be alterations in adenosine triphosphate sensitive K channel pharmacology/function related to the presence of diabetes mellitus. MATERIALS AND METHODS: A total of 215 isolated human corporeal tissue strips obtained from 57 male patients with organic erectile dysfunction were investigated. Cumulative concentration-response curves were constructed at half log increments for steady state relaxation responses elicited by pinacidil and levcromakalim on equivalently phenylephrine pre-contracted (to approximately 75% of maximum) isolated corporeal tissue strips. Potassium currents were measured using the cell attached whole cell patch clamp technique on freshly isolated corporeal smooth muscle cells. RESULTS: A concentration dependent, glibenclamide sensitive relaxation response of phenylephrine pre-contracted corporeal tissue strips was observed for pinacidil and levcromakalim. Consistent with such observations, electrophysiological recordings on freshly isolated myocytes revealed that pinacidil (10 microM.) and levcromakalim (10 microM.) induced whole cell potassium currents that were blocked by glibenclamide (10 microM.). In addition, statistical analysis revealed that phenylephrine pre-contracted corporeal tissue strips from patients without diabetes were more sensitive to relaxation by both compounds than corporeal tissue strips excised from those with diabetes. Furthermore, relaxation responses elicited by pinacidil and levcromakalim were not affected by charybdotoxin or 4-aminopyridine but were completely reversed by KCl or tetraethylammonium chloride. CONCLUSIONS: These data indicate that the adenosine triphosphate sensitive K channel subtype is likely to have an important role in the relaxation of isolated corporeal tissue strips and, moreover, they are the molecular target for the K channel modulators/openers levcromakalim and pinacidil. Such observations are consistent with the supposition that alterations in the structure/function/activity of these potassium channels may underlie at least some aspects of observed diabetes related differences in tissue sensitivity to K channel modulators.     

Sphingosine-1-phosphate receptors: biology and therapeutic potential in kidney disease

The major sphingolipid metabolite, sphingosine-1-phosphate (S1P), has important biological functions. S1P is the ligand for a family of five G-protein-coupled receptors with distinct signaling pathways that regulate angiogenesis, vascular maturation, immunity, chemotaxis, and other important biological pathways. Recently, clinical trials have targeted S1P receptors (S1PRs) for autoimmune diseases and transplantation and have generated considerable interest in developing additional, more selective compounds. This review summarizes current knowledge on the biology of S1P and S1PRs that forms the basis for future drug development and the treatment of kidney disease.     

Neural stem cells. Biological features and therapeutic potential in Parkinson's disease

AIM: Neural stem cells (NSC) are clonogenic cells, capable of self-renewal and multilineage differentiation, since, under the appropriated experimental conditions, they proliferate indefinitely as undifferentiated neurospheres or differentiate in neurons, astrocytes and oligodendrocytes. Parkinson's disease is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons. METHODS: Here we investigated the suitability of recently identified and characterized neuronal progenitor cells at eliciting functional recovery in unilateral 6HODA-lesioned mice. We describe herein that intrastriatal engraftment of stem cell-derived neurons isolated from the olfactory bulb to give rise dopaminergic-like neurons results in long lasting functional recovery in 6OHDA-injured mice. RESULTS: Unilateral injection of 6OHDA resulted in a progressive neurodegeneration of the nigro-striatal pathway. Likewise, the systemic administration of L-DOPA in these mice elicited a marked contralateral turning which was evident 1 week post, increased during the following week and than stabilize throughout the time of the experiment. Conversely, the intrastriatal implantation of partially differentiated stem cells at 14 days postlesion, resulted in a profound decrease in L-DOPA-induced circling behavior; interestingly, the effect was evident 1 week after the engraftment and was retained during the following 9 weeks. Detailed biochemical and immunohistochemical evaluation is currently under investigation in our laboratory. Conclusion. Our observation opens new perspectives for the treatment of neurodegeneration in Parkinson's disease.     

Thiopental and propofol impair relaxation produced by ATP-sensitive potassium channel openers in the rat aorta

ATP-sensitive potassium channel openers are used as vasodilators in the treatment of cardiovascular disorders. The effects of i.v. anaesthetics on arterial relaxation induced by ATP-sensitive potassium channel openers have not been studied. Therefore, in this study, we have examined if thiopental (thiopentone) and propofol affect the vascular response to the ATP-sensitive potassium channel openers, cromakalim and pinacidil, in the isolated rat aorta. Rings of rat thoracic aortas without endothelium were suspended for isometric force recording. Concentration-response curves were obtained in a cumulative manner. During submaximal contractions with phenylephrine 0.3 microgramsmol litre-1, relaxation after cromakalim 0.1-30 microgramsmol litre-1, pinacidil 0.1-30 microgramsmol litre-1 and papaverine 0.1-300 microgramsmol litre-1 was demonstrated. Thiopental 30-300 microgramsmol litre-1, propofol 10-100 microgramsmol litre-1, 10% Intralipid 45 microliters or glibenclamide 5 microgramsmol litre-1 were applied 15 min before addition of phenylephrine. During contractions with phenylephrine, cromakalim and pinacidil induced concentration-dependent relaxation. A selective ATP-sensitive potassium channel antagonist, glibenclamide 5 microgramsmol litre-1, abolished this relaxation, whereas it did not affect relaxation produced by papaverine. Thiopental concentrations > 30 microgramsmol litre-1 significantly impaired relaxation produced by cromakalim or pinacidil. Propofol concentrations > 10 microgramsmol litre-1 also significantly reduced relaxation produced by cromakalim or pinacidil, whereas Intralipid was ineffective. Thiopental 300 microgramsmol litre-1 and propofol 100 microgramsmol litre-1 did not alter relaxation produced by papaverine. These results suggest that the i.v. anaesthetics, thiopental and propofol, impaired vasodilatation mediated by ATP-sensitive potassium channels in vascular smooth muscle cells.      

The brain erythropoietin system and its potential for therapeutic exploitation in brain disease

The discovery of the broad neuroprotective potential of erythropoietin (EPO), an endogenous hematopoietic growth factor, has opened new therapeutic avenues in the treatment of brain diseases. EPO expression in the brain is induced by hypoxia. Practically all brain cells are capable of production and release of EPO and expression of its receptor. EPO exerts multifaceted protective effects on brain cells. It protects neuronal cells from noxious stimuli such as hypoxia, excess glutamate, serum deprivation or kainic acid exposure in vitro by targeting a variety of mechanisms and involves neuronal, glial and endothelial cell functions. In rodent models of ischemic stroke, EPO reduces infarct volume and improves functional outcome, but beneficial effects have also been observed in animal models of subarachnoid hemorrhage, intracerebral hemorrhage, traumatic brain injury, and spinal cord injury. EPO has a convenient therapeutic window upon ischemic stroke and favorable pharmacokinetics. Results from first therapeutic trials in humans are promising, but will need to be validated in larger trials. The safety profile and effectiveness of EPO in a wide variety of neurologic disease models make EPO a candidate compound for a potential first-line therapeutic for neurologic emergencies.