There is increasing evidence demonstrating that the renoprotective effects of mineralocorticoid receptor (MR) blockade are independent of the effects exerted by renin-angiotensin inhibitors. MR is expressed not only in tubular cells but also in other renal cells including glomerular mesangial cells, podocytes, and renal interstitial fibroblasts. Animal experiments have shown that MR blockers prevent aldosterone-induced proteinuria, glomerular injury, and tubulointerstitial fibrosis. In vitro studies have also demonstrated that MR blockers inhibit aldosterone-induced renal cell damage. Recent clinical studies have shown that treatment with MR blockers attenuates the development of proteinuria in patients with chronic kidney disease (CKD) and hypertension, independent of changes in blood pressure. In some cases, MR blockers elicit potent renoprotective effects in conditions where aldosterone levels are not elevated. These data suggest that treatment with MR blockers may possibly present an effective therapeutic strategy for patients with CKD.
Accelerated cardiovascular disease (CVD) is a frequent complication of renal disease. Chronic kidney disease (CKD) develops hypertension and dyslipidemia, which in turn can contribute to the progression of renal failure. There is general agreement that endothelin-1 (ET-1), which acts through the two subtypes of receptor ETA and ETB, plays important physiological roles in the regulation of normal cardiovascular function and that excessive ET-1 production is linked to CVD and CKD. Although selective ETA or nonselective ETA/ETB receptor antagonisms have been recognized as a potential strategy for treatment of several cardiovascular disease, it remains unclear which of the antagonisms is suitable for the individuals with CKD because upregulation of the nitric oxide (NO) system via ETB receptor is responsible for renal function such as natriuresis, diuresis, and glomerular hemodynamics. Our findings clearly indicate that the blockade of ET receptors, in particular ETA-receptor antagonism, not only produces a potential renoprotective effect in CKD but also reduces the risk of CVD. In contrast, pharmacological blockade or genetic deficiency of ETB receptor seems to aggravate CKD and CVD in several experimental models of rats. Moreover, preliminary evidence in patients with CKD also suggests that both selective ETA- and nonselective ETA/ETB-receptor blockade decreases blood pressure but that selective ETA blockade has additional desirable effects on renal hemodynamics. Thus, at least in CKD, these findings support the notion that ETB receptor–mediated actions produce a renoprotective effect and that nonselective ETA/ETB-receptors blockade seem to offer no advantage over selective ETA antagonism, and if anything may potentially reduce the benefits.
Chronic kidney disease (CKD) is becoming a major public health problem worldwide. It is important to protect endothelial function in CKD treatment because injury of the endothelium is a critical event for the generation and progression of CKD. Recently, clinical studies showed that nifedipine, an antihypertensive drug, acts as a protective agent of endothelial cells (ECs). Nifedipine is reported to partially decompose to a nitrosonifedipine that has high reactivity against lipid-derived radicals in vitro. However, it is still unclear whether nitrosonifedipine is a biologically active agent against endothelial injury. We observed that nitrosonifedipine was converted to radical form by reaction with cultured ECs. The cumene hydroperoxide mediated cytotoxity was reduced by nitrosonifedipine in cultured human glomerular ECs (HGECs). Also nitrosonifedipine suppressed the expression of TNF-α–induced intercellular cell adhesion molecule-1 in HGECs. Chronic administration of Nω-nitro-L-arginine methyl ester (L-NAME) caused systemic arterial hypertension, endotherial injury, and renal dysfunction. In L-NAME–induced hypertensive rats, nitrosonifedipine treatment improved not only the acetylcholine-induced vasodilation of the aortic rings, but also renal dysfunction such as increasing the levels of serum creatinine and urinary protein excretion. Our preliminary data suggest that nitrosonifedipine is a new and useful drug for the treatment of CKD involving ameliorating effects on EC disorder.
Discovery of the (pro)renin receptor uncovered a novel function of renin/prorenin as the receptor ligands in addition to the enzyme and its precursor. The bindings of renin and prorenin to the (pro)renin receptor trigger two major pathways: the angiotensin II–dependent pathway as a result of the enzymatic activation of renin/prorenin and the angiotensin II–independent intracellular pathway involving hypertrophic, hyperplastic, and profibrotic signals. A specific blocker of the receptor was discovered through identification of the amino acid sequence of prorenin prosegment that binds to the receptor and leads to non-proteolytic conversion of prorenin to its active form. A peptide containing this sequence was found to block the binding of prorenin to its receptor. Its infusion in animal models of diabetes and low-renin hypertension significantly inhibited the development and progression of nephropathy, but (pro)renin receptor blockade had no benefit in the clipped kidney of 2K1C rats or rat models of high-renin hypertension. Since renin is still active without a (pro)renin receptor, (pro)renin-receptor blockade elicits a maximum benefit under low-renin conditions. Thus, (pro)renin-receptor blockade can be a useful therapy for chronic kidney disease with low renin levels in the plasma.
Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of an oxygen-dependent α-subunit and constitutively expressed β subunit, which plays a central role in cellular adaptation to hypoxia by transcriptionally upregulating its target genes involved in angiogenesis, erythropoiesis, glycolysis, and so on. Recent studies demonstrated that hypoxia in the tubulointerstitium is involved in the pathology of progressive renal diseases and that HIF, which is activated in experimental kidney diseases, may serve to protect tubulointerstitium from the ischemic insult. The expression of HIF α-chains is post-translationally regulated and hydroxylation at one or two of the conserved proline residues by prolyl-hydroxylase domains (PHDs) is a critical step for the oxygen-dependent recruitment of the von Hippel–Lindau gene product (pVHL), a recognition component of the E3 ubiquitin ligase complex, and degradation of HIF-α. Conversely, modalities to inhibit the enzymatic activities of PHDs have been shown to activate HIF irrespective of oxygenation status and are regarded as candidate targets of pharmacological approaches against chronic kidney diseases characterized by hypoxia.
Arginine vasopressin (AVP) activates three vasopressin receptors and it also has an agonistic activity on the oxytocin receptor. For an accurate description of the target receptor subtype(s) responsible for complex AVP and oxytocin actions, a careful evaluation of ligand specificity and receptor activities are required, particularly when these receptors are co-expressed in the central nervous system. Previous studies suggest that AVP plays a regulatory role in nociception through the direct activation of central vasopressin receptors and also through the receptors that reside in the peripheral tissues. Genetically altered rodent models, including the AVP-deficient mutant Brattleboro rat and gene knockout mice lacking an endogenous opioid peptide, advanced the understanding of the interactions between the pain perception process and AVP system. This report reviews previous findings in this important field and reconciles them with the findings of recent gene knockout/knockdown studies.
Arginine vasopressin (AVP) receptors have been classified into V1a, V1b, and V2 subtypes. Recent studies have demonstrated the involvement of AVP in anti-nociception and in morphine-induced anti-nociception. However, the roles of individual AVP-receptor subtypes have not been fully elucidated. Here, we have summarized the role of V1-receptor subtypes in behavioral responses to noxious stimuli and to morphine. In this review, we focus on studies using mice lacking the V1a receptor (V1a−/− mice) and the V1b receptor (V1b−/− mice).
Arginine vasopressin (AVP) is a neurohypophyseal peptide best known as an antidiuretic hormone. AVP receptors have been classified into three subtypes: V1a, V1b, and V2 receptors. V1a receptor (V1aR) and V1b receptor (V1bR) are widely distributed in the central nervous system, including the septum, cortex, hippocampus, and hypothalamus. Clinical studies have demonstrated an involvement of AVP in psychiatric disorders. In the present study, we examined the performance of V1aR or V1bR knockout (KO) mice compared to wild-type (WT) mice in behavioral tests. V1aR and V1bR KO mice exhibited deficits of social behavior and prepulse inhibition in comparison to WT mice. Moreover, V1aR KO mice exhibited reduced anxiety-like behavior and impairment of spatial learning. These results suggest that V1aR and V1bR play an important role in psychological and cognitive functions.
The neurohypophyseal peptide [Arg8]-vasopressin (AVP) is involved in diverse functions such as the regulation of body fluid homeostasis, metabolism, and hormone secretion. In this study, we analyzed the functional roles of AVP in hormone release and metabolisms of glucose, fat, and protein in mutant mice lacking the V1a (V1aR-KO) or V1b receptor (V1bR-KO). Our study suggests that antagonists for the receptors could affect the hormone secretions and metabolisms.
Hyponatremia is the most common electrolyte disorder in hospitalized patients and is associated with the risk of intractable seizures and death. The effectiveness of conventional therapies for hyponatremia is inconsistent, and the rapid correction of plasma sodium levels is thought to result in the occurrence of neurological complications. Arginine vasopressin (AVP) is the primary regulator of renal electrolyte-free water reabsorption via AVP-receptor type 2 (V2-R), and inappropriate or excessive AVP secretion independent of serum osmolality frequently causes excessive water retention, which is the etiological basis of hyponatremia. Therefore, the use of V2-R antagonists as anti-hyponatremic drugs in the clinical setting is anticipated to be reliable and safe. Conivaptan hydrochloride (YM087) is a novel dual AVP–R antagonist for AVP-R types 1a (V1a) and V2-R. In vitro studies have shown that it possesses high affinity for V1a-R and V2-R without any species differences. It also potently inhibited AVP-induced intracellular signaling through human V2 and V1a receptors with no agonistic activity. Conivaptan hydrochloride improved the plasma sodium concentration and plasma osmolality in hyponatremic rats, and its effectiveness was demonstrated in hyponatremic patients. This drug has been approved for use in the United States, which will bring relief to patients with hyponatremia.
The organotin trimethyltin (TMT) is known to cause neuronal degeneration in the central nervous system. A systemic injection of TMT produced neuronal damage in the cerebral frontal cortex of mice. To elucidate the mechanism(s) underlying the toxicity of TMT toward neurons, we prepared primary cultures of neurons from the cerebral cortex of mouse embryos for use in this study. Microscopic observations revealed that a continuous exposure to TMT produced neuronal damage with nuclear condensation in an incubation time–dependent manner up to 48 h. The neuronal damage induced by TMT was not blocked by N-methyl-D-aspartate receptor channel–blocker MK-801. The exposure to TMT produced an elevation of the phosphorylation level of c-Jun N-terminal kinase (JNK)p46, but not JNKp54, prior to neuronal death. Under the same conditions, a significant elevation was seen in the phosphorylation level of stress-activated protein kinase 1, which activates JNKs. Furthermore, TMT enhanced the expression and phosphorylation of c-Jun during a continuous exposure. The JNK inhibitor SP600125 was effective in significantly but only partially attenuating the TMT-induced nuclear condensation and accumulation of lactate dehydrogenase in the culture medium. Taken together, our data suggest that the neuronal damage induced by TMT was independent of excitotoxicity but that at least some of it was dependent on the JNK cascades in primary cultures of cortical neurons.
Antinociceptive effects of systemically administered midazolam remain controversial. The present study was performed to investigate its antinociceptive effects on different types of nociception in mice. Four different doses of midazolam (1, 3, 10, and 30 mg/kg) were administered intraperitoneally (i.p.). Saline was used as a control. The hot plate test, tail pressure test, acetic acid writhing test, the running wheel test, and the balance beam test were performed following the drug administration. In the hot plate test and tail pressure test, i.p. midazolam produced significant antinociceptive effects with the 50% effective dose (ED50) of 3.46 mg/kg [confidence interval (CI), 1.99 – 6.01 mg/kg] and 3.52 mg/kg (CI, 2.77 – 4.47 mg/kg), respectively. In the acetic acid writhing test, i.p. midazolam also produced significant antinociceptive effects. In the running wheel test, no mice stopped running after saline or midazolam at 1, 3, or 10 mg/kg, but all mice stopped running 30 and 45 min after i.p. administration of midazolam at 30 mg/kg. In the balance beam test, 30 min after i.p. administration of saline or midazolam at 1, 3, and 10 mg/kg, all mice were able to stay on the beam for 90 s, none of them could with midazolam at 30 mg/kg. In conclusion, systemically administered midazolam had antinociceptive effects on acute thermal, acute mechanical, and acute inflammatory-induced nociception in mice. The antinociceptive potency of midazolam was the same for both acute thermal-induced nociception and mechanical-induced nociception.
Total glucosides of paeony (TGP), extracted from the root of Paeonia lactiflora pall, has been shown to have ant-inflammatory and antioxidative actions. The aims of this study were to elucidate the renoprotective effect of TGP and its mechanism in experimental diabetes. Streptozotocin-induced diabetic rats were treated with TGP for 8 weeks. Treatment with TGP at 50, 100, and 200 mg/kg significantly lowered 24-h urinary albumin excretion rate in diabetic rats. TGP treatment in all doses markedly attenuated glomerular volume, and treatment with TGP at 100 and 200 mg/kg markedly reduced indices for tubulointerstitial injury in diabetic rats. Western blot analysis showed that the expressions of 1α (IV) collagen, intercellular adhesion molecule (ICAM)-1, interleukin (IL)-1, tumor necrosis factor (TNF)-α, NF-κB p65, and 3-nitrotyrosine (3-NT) protein were increased in the kidneys of diabetic rats; the increases in these proteins were all dose-dependently and significantly inhibited by TGP treatment. The expression of nephrin protein was significantly reduced in the kidneys from diabetic rats and markedly increased by TGP treatment. The expression of transforming growth factor (TGF)-β1 protein in the kidney was also significantly increased in diabetic rats, which was significantly inhibited by treatment with TGP at all doses. Our data suggest that TGP treatment ameliorates early renal injury via the inhibition of expression of ICAM-1, IL-1, TNF-α, and 3-NT in the kidneys of diabetic rats.
Accumulating evidence indicates a pivotal role for neuroinflammation in ischemic and excitotoxic brain injury. Cytokine-induced neutrophil chemoattractant-1 (CINC-1) is a CXC chemokine implicated in the infiltration of inflammatory cells into the brain parenchyma. In this study, we investigated the effect of N-methyl-D-aspartate (NMDA)-induced neuronal injury on CINC-1 production in the organotypic cortico-striatal slice cultures. Treatment with 50 μM NMDA for 3 – 4 h caused devastating neuronal damage and increased CINC-1 production. Immunohistochemical analysis revealed that the CINC-1 immunoreactivity was predominantly detected in astrocytes. NMDA failed to induce CINC-1 production in enriched astrocyte cultures or neuron-depleted slice cultures, suggesting that NMDA acted on neuronal cells to induce astrocytic CINC-1 production. NMDA-induced CINC-1 mRNA expression was significantly inhibited by U0126, a mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor. These results suggest that NMDA-evoked neuronal injury induced astrocytic CINC-1 production via a MEK/ERK signaling pathway. Manipulation of this signaling pathway may serve as a target for suppressing neuroinflammation and, thereby, treating ischemic brain injury.
3-Hydroxy-3-methylglutaryl CoA reductase inhibitors (statins) are safe and well-tolerated therapeutic drugs. However, they occasionally induce myotoxicity such as myopathy and rhabdomyolysis. Here, we investigated the mechanism of statin-induced myotoxicity in L6 fibroblasts and in rats in vivo. L6 fibroblasts were differentiated and then treated with pravastatin, simvastatin, or fluvastatin for 72 h. Hydrophobic simvastatin and fluvastatin decreased cell viability in a dose-dependent manner via apoptosis characterized by typical nuclear fragmentation and condensation and caspase-3 activation. Both hydrophobic statins transferred RhoA localization from the cell membrane to the cytosol. These changes induced by both hydrophobic statins were completely abolished by the co-application of geranylgeranylpyrophosphate (GGPP). Y27632, a Rho-kinase inhibitor, mimicked the hydrophobic statin-induced apoptosis. Hydrophilic pravastatin did not affect the viability of the cells. Fluvastatin was continuously infused (2.08 mg/kg at an infusion rate of 0.5 mL/h) into the right internal jugular vein of the rats in vivo for 72 h. Fluvastatin infusion significantly elevated the plasma CPK level and transferred RhoA localization in the skeletal muscle from the cell membrane to the cytosol. In conclusion, RhoA dysfunction due to loss of lipid modification with GGPP is involved in the mechanisms of statin-induced skeletal muscle toxicity.
Choline is essential for synthesis of the major membrane phospholipid phosphatidylcholine. Moreover, it serves as a precursor for synthesis of the neurotransmitter acetylcholine (ACh). Keratinocytes of the epidermis synthesize and release ACh. The uptake of choline is the rate-limiting step in both ACh synthesis and choline phospholipid metabolism, and it is a prerequisite for keratinocyte proliferation. However, the nature of the choline transport system in keratinocytes is poorly understood. In this study, we examined the molecular and functional characterization of choline uptake into cultured human keratinocytes. Choline uptake into keratinocytes was independent of extracellular Na+, saturable, and mediated by a single transport system with an apparent Michaelis-Menten constant of 12.3 μM. Choline uptake was reduced when the keratinocyte membrane potential was depolarized by high K+. These results provide evidence that the choline transport activity is potential-sensitive. Various organic cations inhibit the choline transport system. RT-PCR demonstrated that keratinocytes expressed mRNA for choline transporter-like protein 1 (CTL1), mainly the CTL1a subtype. The present biochemical and pharmacological data suggest that CTL1a is functionally expressed in human keratinocytes and is responsible for the uptake of choline and organic cations in these cells.
We investigated the effect of serofendic acid, a neuroprotective substance derived from fetal calf serum, on the morphological changes in cultured cortical astrocytes. Cultured astrocytes developed a stellate morphology with several processes following exposure to dibutylyl cAMP (dbcAMP), a membrane-permeable cAMP analog; 8-Br-cGMP, a membrane-permeable cGMP analog; or phorbol-12-myristate-13-acetate (PMA), a protein kinase C activator. Serofendic acid significantly accelerated the stellation induced by dbcAMP- and 8-Br-cGMP. In contrast, the PMA-induced stellation was not affected by serofendic acid. Next, we attempted to elucidate the mechanism underlying the dbcAMP-induced stellation and explore the site of action of serofendic acid. Both the stellation induced by dbcAMP and the promotional effect of serofendic acid were partially inhibited by KT5720, a specific protein kinase A (PKA) inhibitor. Furthermore, serofendic acid failed to facilitate the stellation induced by Y-27632, an inhibitor of Rho-associated kinase (ROCK). These results indicate that serofendic acid promotes dbcAMP- and 8-Br-cGMP-induced stellation and the promotional effect on dbcAMP-induced stellation is mediated at least partly by the regulation of PKA activity and not by controlling ROCK activity.
The hepatoprotective effects of ACTIValoe®N-931 complex, a mixture of Aloe vera and Silybum marianum, against acute and chronic carbon tetrachloride (CCl4)-induced liver injuries were investigated. Acute hepatotoxicity was induced by intraperitoneal injection of CCl4 (50 μl/kg), and ACTIValoe®N-931 complex at 85, 170, and 340 mg/kg was administered orally 48, 24, and 2 h before and 6 h after injection of CCl4. Hepatotoxicity was assessed 24 h after CCl4 treatment. Liver fibrosis was induced by intraperitoneal injection of CCl4 for 8 weeks (0.5 ml/kg, twice per week), and mice were treated with ACTIValoe®N-931 complex at 85, 170, or 340 mg/kg once a day (p.o.). In both acute hepatotoxicity and liver fibrosis, serum aminotransferase levels and lipid peroxidation were increased and the hepatic glutathione content was decreased. These changes were prevented by ACTIValoe®N-931 complex. The ACTIValoe®N-931 complex attenuated the increase in tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), mRNA expressions in acute hepatotoxicity. In antifibrotic experiments, tissue inhibitor of metalloprotease-1 (TIMP-1) mRNA expression was attenuated by treatment with ACTIValoe®N-931 complex. The ACTIValoe®N-931 complex decreased the hepatic hydroxyproline content and the transforming growth factor-beta1 levels. Our results suggest that the ACTIValoe®N-931 complex has hepatoprotective effects in both acute and chronic liver injuries induced by CCl4.
Nicotine has been shown to reduce both tone and muscular activity in the human colon by releasing nitric oxide (NO) from nerves. To our knowledge, however, the effect of nicotine on mouse colon has not been elucidated, and the response in tissue from ulcerative colitis (UC) has not been investigated. We examined nicotine-induced responses in colon from control mice and mice with dextran sodium sulfate (DSS)-induced UC. In controls, bath application of nicotine caused a transient relaxation in longitudinal preparations from the transverse and distal colons but not from the rectum. The response was observed in the presence of bethanechol, abolished by treatment with tetrodotoxin and hexamethonium, and mediated partially (>50%) by the NO pathway. In longitudinal preparations of the distal colon from DSS-treated mice, spontaneous contractions decreased markedly, and nicotine caused contraction without relaxation in half of the preparations tested. Nicotine-induced relaxation in the presence of bethanechol was significantly decreased in the DSS-treated distal colon without changing bethanechol-induced contractions. These data suggest that 1) responses to nicotine differ dependent on colon regions, 2) DSS treatment predominantly caused nicotine-sensitive neurogenic changes in distal colon, and 3) DSS treatment may reverse the direction of nicotine-evoked responses in the colon, in mice.
We previously demonstrated that the growth hormone (GH)-releaser diet ameliorated β-amyloid (Aβ) (1-42)–induced memory impairment, but the underlying mechanism remained to be characterized. We show here that the GH-releaser diet significantly attenuated Aβ(1-42)-induced impairment in context-dependent conditioned fear, with a reduction in GH levels and changes in hippocampal acetylcholine, acetylcholinesterase, choline acetyltransferase, insulin-like growth factor (IGF)-1, and IGF-1–receptor activity in mice. JB-1, an IGF-1–receptor antagonist, significantly blocked GH-releaser diet–mediated pharmacological actions. Our results suggest that the GH-releaser diet prevents Aβ(1-42)-induced cognitive deficits via stimulation of the hippocampal IGF-1 receptor.
Gabapentin was developed as an anticonvulsant, but has also been used to alleviate hyperalgesia in neuropathic pain. In this study, the protective effect of gabapentin against N-methyl-D-aspartate (NMDA)-induced excitotoxicity in rat hippocampal CA1 neurons was investigated. Pre-treatment with gabapentin reduced the degree of neuronal damage induced by NMDA exposure in cultured hippocampal slices. Patch-clamp studies revealed that gabapentin significantly inhibited the NMDA receptor–activated ion current in dissociated hippocampal CA1 neurons, resulting in suppression of glutamate-induced neuronal injury. These results show that gabapentin may exert protective effects against glutamate-induced neuronal injury at least in part by inhibiting the NMDA receptor–activated ion current.
K+ channels are key molecules in the progression of several cancer types and considered to be potential targets for cancer therapy. We examined the gene expressions of voltage-gated (Kv), Ca2+-activated (KCa), and two-pore domain (K2P) K+-channel subtypes in needle-biopsy samples of human prostate cancer (PCa) by real-time PCR and compared them with those in PCa epithelial cell lines. The expression of Kv1.3, KCa1.1, KCa3.1, and K2P1 markedly increased in the PCa group with Gleason score of 5 – 6 (GS5–6) but significantly decreased in the GS8–9 group. This malignancy grade–dependent K+-channel expression pattern may provide a convenient marker to understand PCa progression level.
We recently reported that physiological concentrations of 17β-estradiol partially down-regulate cardiac rapidly-activating delayed rectifier K+ currents (hERG currents) independently of estrogen-receptor signaling. To determine if other estrogens have the same effect as that of 17β-estradiol, we investigated receptor-independent effects of estrone, estrone 3-sulfate, and estriol on hERG currents in patch-clamped estrogen-negative HEK293 cells. Only estrone 3-sulfate partially suppressed hERG currents in a receptor-independent manner by modifying the gating. The concentration-dependence of estrone 3-sulfate revealed that physiological levels of circulating estrone 3-sulfate can modulate hERG currents to the maximal extent in both women and men at any age.