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The small GTPase protein RhoA has two effectors, ROCK (Rho-associated protein kinase 1) and mDIA1 (protein diaphanous homolog 1), which cooperate reciprocally. However, temporal regulation of RhoA and its effectors in obesity-induced kidney damage remains unclear. Here, we investigated the role of RhoA activation in the proximal tubules at the early and late stages of obesity-induced kidney damage. In mice, a three-week high-fat-diet induced proximal tubule hypertrophy and damage without increased albuminuria, and RhoA/mDIA1 activation without ROCK activation. Conversely, a 12-week high-fat diet induced proximal tubule hypertrophy, proximal tubule damage, increased albuminuria, and RhoA/ROCK activation without mDIA1 elevation. Proximal tubule hypertrophy resulting from cell cycle arrest accompanied by downregulation of the multifunctional cyclin-dependent kinase inhibitor p27Kip1 was elicited by RhoA activation. Mice overexpressing proximal tubule-specific and dominant-negative RHOA display amelioration of high-fat diet-induced kidney hypertrophy, cell cycle abnormalities, inflammation, and renal impairment. In human proximal tubule cells, mechanical stretch mimicking hypertrophy activated ROCK, which triggered inflammation. In human kidney samples from normal individuals with a body mass index of about 25, proximal tubule cell size correlated with body mass index, proximal tubule cell damages, and mDIA1 expression. Thus, RhoA activation in proximal tubules is critical for the initiation and progression of obesity-induced kidney damage. Hence, the switch in the downstream RhoA effector in proximal tubule represents a transition from normal to pathogenic kidney adaptation and to body weight gain, leading to obesity-induced kidney damage.

Sleep exerts modulatory effects on the cerebral cortex. Whether sleep modulates local connectivity in the cortex or only individual neural activity, however, is poorly understood. Here we investigated functional connectivity, that is, covarying activity between neurons, during spontaneous sleep–wake states and during and after sleep deprivation using calcium imaging of identified excitatory/inhibitory neurons in the motor cortex. Functional connectivity was estimated with a statistical learning approach glasso and quantified by “the probability of establishing connectivity (sparse/dense)” and “the strength of the established connectivity (weak/strong).” Local cortical connectivity was sparse in non-rapid eye movement (NREM) sleep and dense in REM sleep, which was similar in both excitatory and inhibitory neurons. The overall mean strength of the connectivity did not differ largely across spontaneous sleep–wake states. Sleep deprivation induced strong excitatory/inhibitory and dense inhibitory, but not excitatory, connectivity. Subsequent NREM sleep after sleep deprivation exhibited weak excitatory/inhibitory, sparse excitatory, and dense inhibitory connectivity. These findings indicate that sleep–wake states modulate local cortical connectivity, and the modulation is large and compensatory for stability of local circuits during the homeostatic control of sleep, which contributes to plastic changes in neural information flow.

Dysregulation of nicotinamide adenine dinucleotide (NAD +) metabolism contributes to the initiation and progression of age-associated diseases, including chronic kidney disease (CKD). Nicotinamide N-methyltransferase (NNMT), a nicotinamide (NAM) metabolizing enzyme, regulates both NAD + and methionine metabolism. Although NNMT is expressed abundantly in the kidney, its role in CKD and renal fibrosis remains unclear. We generated NNMT-deficient mice and a unilateral ureter obstruction (UUO) model and conducted two clinical studies on human CKD to investigate the role of NNMT in CKD and fibrosis. In UUO, renal NNMT expression and the degraded metabolites of NAM increased, while NAD + and NAD + precursors decreased. NNMT deficiency ameliorated renal fibrosis; mechanistically, it (1) increased the DNA methylation of connective tissue growth factor (CTGF), and (2) improved renal inflammation by increasing renal NAD + and Sirt1 and decreasing NF-κB acetylation. In humans, along with CKD progression, a trend toward a decrease in serum NAD + precursors was observed, while the final NAD + metabolites were accumulated, and the level of eGFR was an independent variable for serum NAM. In addition, NNMT was highly expressed in fibrotic areas of human kidney tissues. In conclusion, increased renal NNMT expression induces NAD + and methionine metabolism perturbation and contributes to renal fibrosis.

Ketone bodies including β-hydroxybutyrate (β-OHB) have been shown to protect against ischemic tissue injury when present at low concentrations. We evaluated the impact of β-OHB on renal ischemia/reperfusion injury (IRI). Mice were treated with a continuous infusion of β-OHB using an osmotic mini-pump before and after IRI. We also tested the effects of increasing endogenous serum β-OHB levels by fasting. Renal IRI was attenuated by β-OHB treatment compared to saline control, with similar results in the fasting condition. β-OHB treatment reduced the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells and increased expression of forkhead transcription factor O3 (FOXO3), an upstream regulator of pyroptosis. Although β-OHB treatment did not impact markers of apoptosis, it decreased the expression of caspase-1 and proinflammatory cytokines, indicating that β-OHB blocked pyroptosis. In a human proximal tubular cell line exposed to hypoxia and reoxygenation, β-OHB reduced cell death in a FOXO3-dependent fashion. Histone acetylation was decreased in kidneys exposed to IRI and in proximal tubular cells exposed to hypoxia and reoxygenation, and this effect was ameliorated by β-OHB through the inactivation of histone deacetylases. In vitro, β-OHB treatment restored histone acetylation at the FOXO3 promoter. Consistent with epigenetic molecular effects, the renoprotective effects of β-OHB were still observed when the continuous infusion was stopped at the time of IRI. Thus, β-OHB attenuates renal IRI through anti-pyroptotic effects, likely mediated by an epigenetic effect on FOXO3 expression.

A large number of neurons form cell assemblies that process information in the brain. Recent developments in measurement technology, one of which is calcium imaging, have made it possible to study cell assemblies. In this study, we aim to extract cell assemblies from calcium imaging data. We propose a clustering approach based on non-negative matrix factorization (NMF). The proposed approach first obtains a similarity matrix between neurons by NMF and then performs spectral clustering on it. The application of NMF entails the problem of model selection. The number of bases in NMF affects the result considerably, and a suitable selection method is yet to be established. We attempt to resolve this problem by model averaging with a newly defined estimator based on NMF. Experiments on simulated data suggest that the proposed approach is superior to conventional correlation-based clustering methods over a wide range of sampling rates. We also analyzed calcium imaging data of sleeping/waking mice and the results suggest that the size of the cell assembly depends on the degree and spatial extent of slow wave generation in the cerebral cortex.

Sleep is generally viewed as a period of recovery, but how the supply of cerebral blood flow (CBF) changes across sleep/wake states has remained unclear. Here, we directly observe red blood cells (RBCs) within capillaries, where the actual substance exchange between the blood and neurons/glia occurs, by two-photon microscopy. Across multiple cortical areas, average capillary CBF is largely increased during rapid eye movement (REM) sleep, whereas it does not differ between periods of active wakefulness and non-REM sleep. Capillary RBC flow during REM sleep is further elevated following REM sleep deprivation, suggesting that capillary CBF reflects REM sleep pressure. At the molecular level, signaling via adenosine A2a receptors is crucial; in A2a-KO mice, capillary CBF upsurge during REM sleep is dampened, and effects of REM sleep pressure are abolished. These results provide evidence regarding the dynamics of capillary CBF across sleep/wake states and insights to the underlying mechanisms., 睡眠中の脳のリフレッシュ機構を解明. 京都大学プレスリリース. 2021-08-27.

Background The activation of NAD+-dependent deacetylase, Sirt1, by the administration of nicotinamide mononucleotide (NMN) ameliorates various aging-related diseases. Methods Diabetic db/db mice were treated with NMN transiently for 2 weeks and observed for effects on diabetic nephropathy (DN). Results At 14 weeks after the treatment period, NMN attenuated the increases in urinary albumin excretion in db/db mice without ameliorating hemoglobin A1c levels. Short-term NMN treatment mitigated mesangium expansion and foot process effacement, while ameliorating decreased Sirt1 expression and increased claudin-1 expression in the kidneys of db/db mice. This treatment also improved the decrease in the expression of H3K9me2 and DNMT1. Short-term NMN treatment also increased kidney concentrations of NAD+ and the expression of Sirt1 and nicotinamide phosphoribosyltransferase (Nampt), and it maintained nicotinamide mononucleotide adenyltransferase1 (Nmnat1) expression in the kidneys. In addition, survival rates improved after NMN treatment. Conclusions Short-term NMN treatment in early-stage DN has remote renal protective effects through the upregulation of Sirt1 and activation of the NAD+ salvage pathway, both of which indicate NMN legacy effects on DN.

Under diabetic conditions, sodium–glucose cotransporter 2 (SGLT2) for glucose uptake in proximal tubules (PTs) increases, whereas NAD+-dependent protein deacetylase silent mating type information regulation 2 homolog 1 (Sirtuin-1; SIRT1) for PT survival decreases. Therefore, we hypothesized that increased glucose influx by SGLT2 reduces SIRT1 expression. To test this hypothesis, db/db mice with diabetes and high-glucose (HG)-cultured porcine PT LLC-PK1 cells in a two-chamber system were treated with the SGLT2 inhibitor canagliflozin. We also examined SIRT1 and SGLT2 expression in human kidney biopsies. In db/db mice, SGLT2 expression increased with concomitant decreases in SIRT1, but was inhibited by canagliflozin. For determination of the polarity of SGLT2 and SIRT1 expression, LLC-PK1 cells were seeded into Transwell chambers (pore size, 0.4 µm; Becton Dickinson, Oxford, UK). HG medium was added to either or to both of the upper and lower chambers, which corresponded to the apical and basolateral sides of the cells, respectively. In this system, the lower chamber with HG showed increased SGLT2 and decreased SIRT1 expression. Canagliflozin reversed HG-induced SIRT1 downregulation. Gene silencing and inhibitors for glucose transporter 2 (GLUT2) blocked HG-induced SGLT2 expression upregulation. Gene silencing for the hepatic nuclear factor-1α (HNF-1α), whose nuclear translocation was enhanced by HG, blocked HG-induced SGLT2 expression upregulation. Similarly, gene silencing for importin-α1, a chaperone protein bound to GLUT2, blocked HG-induced HNF-1α nuclear translocation and SGLT2 expression upregulation. In human kidney, SIRT1 immunostaining was negatively correlated with SGLT2 immunostaining. Thus, under diabetic conditions, SIRT1 expression in PTs was downregulated by an increase in SGLT2 expression, which was stimulated by basolateral HG through activation of the GLUT2/importin-α1/HNF-1α pathway.

The amount, quality, and diurnal pattern of sleep change greatly during development. Developmental changes of sleep/wake architecture are in a close relationship to brain development. The fragmentation of wake episodes is one of the salient features in the neonatal period, which is also observed in mature animals and human individuals lacking neuropeptide orexin/hypocretin signaling. This raises the possibility that developmental changes of lateral hypothalamic orexin neurons are relevant to the development of sleep/wake architecture. However, little information is available on morphological and physiological features of developing orexin neurons. To address the cellular basis for maturation of the sleep/wake regulatory system, we investigated the functional development of orexin neurons in the lateral hypothalamus. The anatomical development as well as the changes in the electrophysiological characteristics of orexin neurons was examined from embryonic to postnatal stages in orexin-EGFP mice. Prepro-orexin promoter activity was detectable at embryonic day (E) 12.0, followed by expression of orexin A after E14.0. The number of orexin neurons and their membrane capacitance reached similar levels to adults by postnatal day (P) 7, while their membrane potentials, firing rates, and action potential waveforms were developed by P21. The hyperpolarizing effect of serotonin, which is a major inhibitory signal for adult orexin neurons, was detected after E18.0 and matured at P1. These results suggest that the expression of orexin peptides precedes the maturation of electrophysiological activity of orexin neurons. The function of orexin neurons gradually matures by 3 weeks after birth, coinciding with maturation of sleep/wake architecture.

Sleep, a common event in daily life, has clear benefits for brain function, but what goes on in the brain when we sleep remains unclear. Sleep was long regarded as a silent state of the brain because the brain seemingly lacks interaction with the surroundings during sleep. Since the discovery of electrical activities in the brain at rest, electrophysiological methods have revealed novel concepts in sleep research. During sleep, the brain generates oscillatory activities that represent characteristic states of sleep. In addition to electrophysiology, opto/chemogenetics and two-photon Ca2+ imaging methods have clarified that the sleep/wake states organized by neuronal and glial ensembles in the cerebral cortex are transitioned by neuromodulators. Even with these methods, however, it is extremely difficult to elucidate how and when neuromodulators spread, accumulate, and disappear in the extracellular space of the cortex. Thus, real-time monitoring of neuromodulator dynamics at high spatiotemporal resolution is required for further understanding of sleep. Toward direct detection of neuromodulator behavior during sleep and wakefulness, in this review, we discuss developing imaging techniques based on the activation of G-protein-coupled receptors that allow for visualization of neuromodulator dynamics.

Cortical neurons fire intermittently and synchronously during non-rapid eye movement sleep (NREMS), in which active and silent periods are referred to as ON and OFF periods, respectively. Neuronal firing rates during ON periods (NREMS-ON-activity) are similar to those of wakefulness (W-activity), raising the possibility that NREMS-ON neuronal-activity is fragmented W-activity. To test this, we investigated the patterning and organization of cortical spike trains and of spike ensembles in neuronal networks using extracellular recordings in mice. Firing rates of neurons during NREMS-ON and W were similar, but showed enhanced bursting in NREMS with no apparent preference in occurrence, relative to the beginning or end of the on-state. Additionally, there was an overall increase in the randomness of occurrence of sequences comprised of multi-neuron ensembles in NREMS recorded from tetrodes. In association with increased burst firing, somatic calcium transients were increased in NREMS. The increased calcium transients associated with bursting during NREM may activate calcium-dependent, cell-signaling pathways for sleep related cellular processes.

Summary: Nicotinamide adenine dinucleotide (NAD+) metabolism plays a critical role in kidneys. We previously reported that decreased secretion of a NAD+ precursor, nicotinamide mononucleotide (NMN), from proximal tubules (PTs) can trigger diabetic albuminuria. In the present study, we investigated the role of NMN-producing enzyme nicotinamide phosphoribosyltransferase (Nampt) in diabetic nephropathy. The expression of Nampt in PTs was downregulated in streptozotocin (STZ)-treated diabetic mice when they exhibited albuminuria. This albuminuria was ameliorated in PT-specific Nampt-overexpressing transgenic (TG) mice. PT-specific Nampt-conditional knockout (Nampt CKO) mice exhibited TBM thickening and collagen deposition, which were associated with the upregulation of the profibrogenic gene TIMP-1. Nampt CKO mice also exhibited the downregulation of sirtuins, particularly in Sirt6. PT-specific Sirt6-knockout mice exhibited enhanced fibrotic phenotype resembling that of Nampt CKO mice with increased Timp1 expression. In conclusion, the Nampt-Sirt6 axis in PTs serves as a key player in fibrogenic extracellular matrix remodeling in diabetic nephropathy. : Muraoka et al. reveal that the Nampt-Sirt6 axis in proximal tubules serves as a key player in fibrogenic extracellular matrix remodeling in diabetic nephropathy. Keywords: NAD, Nampt, Sirt6, metabo-fibrosis, NMN, TBM thickening, diabetic nephropathy

Sleep is a physiological process not only for the rest of the body but also for several brain functions such as mood, memory, and consciousness. Nevertheless, the nature and functions of sleep remain largely unknown due to its extremely complicated nature and lack of optimized technology for the experiments. Here we review the recent progress in the biology of the mammalian sleep, which covers a wide range of research areas: the basic knowledge about sleep, the physiology of cerebral cortex in sleeping animals, the detailed morphological features of thalamocortical networks, the mechanisms underlying fluctuating activity of autonomic nervous systems during rapid eye movement sleep, the cutting-edge technology of tissue clearing for visualization of the whole brain, the ketogenesis-mediated homeostatic regulation of sleep, and the forward genetic approach for identification of novel genes involved in sleep. We hope this multifaceted review will be helpful for researchers who are interested in the biology of sleep.

Background The lipodystrophy-like phenotype has been suggested in early chronic kidney disease (CKD). It includes adipose tissue atrophy, systemic insulin resistance (IR), dyslipidemia and ectopic lipid accumulation. To elucidate its pathogenesis, we investigated the role of two uremic toxins that affect insulin sensitivity: an endogenous nitric oxide synthase inhibitor, and asymmetric dimethylarginine (ADMA) and indoxyl sulfate (IS). Methods Six-week-old Sprague-Dawley rats were rendered CKD by subtotal nephrectomy (Nx) and compared with sham-operated rats. Cultured 3T3-L1 fibroblasts were differentiated into mature adipocytes with or without ADMA. Transgenic (Tg) mice overexpressing each isoform of ADMA degrading enzyme, dimethylarginine dimethylaminohydrolase 1 (DDAH1) and DDAH2 were subject to Nx and their phenotypes were investigated. Results In Nx rats, IR was evident and insulin stimulation failed to activate insulin signaling in adipose tissues. Adipose tissue weight, adipocyte size and adipocyte differentiation marker expressions decreased as a consequence of IR in Nx. Tissue lipid content in the liver and muscle increased in Nx rats. Tissue levels of ADMA, IS and oxidative stress increased in the adipose tissue of Nx rats. Both DDAH1 and DDAH2 expressions decreased, and a putative IS receptor, aryl hydrocarbon receptor, expression increased in the adipose tissue of Nx rats. ADMA inhibited adipocyte differentiation, triglyceride accumulation and insulin signaling, which were reversed by pretreatment with cGMP. In each type of Tg mice overexpressing DDAH1 or DDAH2, all lipodystrophy-like phenotypes induced by Nx were reversed. Conclusions In mild CKD, dysregulation of the ADMA/DDAH pathway in adipose tissue triggers lipodystrophy-like phenotype changes, including ectopic fat depositions.

Background The role of gut microbiota in the progression of chronic kidney disease (CKD) has not been fully elucidated. Methods Renal failure was induced in 6-week-old spontaneously hypertensive rats by 5/6 nephrectomy (Nx). We analyzed the gut microbiota population to identify the relevant species potentially involved in inducing renal damage. Human colon Caco-2 cells were used to delineate the mechanism involved in the molecular changes in the gut of Nx rats. Results Nx rats showed an increase in Bacteroides (Bact) and a decrease in Lactobacillus (Lact) species compared with sham-operated rats. Lact, but not Bact, populations were significantly associated with urinary protein excretion. Treatment of Nx rats with 1 × 10(10) CFU/kg/day Lact ameliorated increased urinary protein excretion and higher serum levels of the uremic toxins, indoxyl sulfate and p-cresyl sulfate, and serum urea nitrogen levels. Lact also attenuated systemic inflammation in Nx rats, as evaluated by serum lipopolysaccharide, interleukin-6 and C-reactive protein levels. Histologically, renal sclerosis in Nx rats was restored by Lact treatment. A reduction in the expression of tight junction proteins and the Toll-like receptor 2 (TLR2), a putative Lact receptor, in the colons of Nx rats were mitigated by Lact. Treatment of Caco-2 cells with indole downregulated tight junction protein expression, which was abolished by exposure to Lact. The effects of Lact were reversed by treatment with OxPAPC, a TLR inhibitor. Similarly, the increase in the permeability of the Caco-2 cell monolayer was reversed by the administration of Lact. Lact upregulated TLR2 expression in Caco-2 cells. Lact also attenuated the increase in serum indoxyl sulfate and urea levels and urinary protein excretion in Nx rats even in the pseudogerm-free environment. Conclusions Lact supplementation mitigated the systemic inflammation and proteinuria associated with renal failure, suggesting that in the gut microbiota, Lact plays a protective role against the progression of CKD.

In this study, we examined the association between chronic kidney disease (CKD) and insulin resistance. In a patient cohort with nondiabetic stages 2-5 CKD, estimated glomerular filtration rate (eGFR) was negatively correlated and the plasma aldosterone concentration was independently associated with the homeostasis model assessment of insulin resistance. Treatment with the mineralocorticoid receptor blocker spironolactone ameliorated insulin resistance in patients, and impaired glucose tolerance was partially reversed in fifth/sixth nephrectomized rats. In these rats, insulin-induced signal transduction was attenuated, especially in the adipose tissue. In the adipose tissue of nephrectomized rats, nuclear mineralocorticoid receptor expression, expression of the mineralocorticoid receptor target molecule SGK-1, tissue aldosterone content, and expression of the aldosterone-producing enzyme CYP11B2 increased. Mineralocorticoid receptor activation in the adipose tissue was reversed by spironolactone. In the adipose tissue of nephrectomized rats, asymmetric dimethylarginine (ADMA; an uremic substance linking uremia and insulin resistance) increased, the expression of the ADMA-degrading enzymes DDAH1 and DDAH2 decreased, and the oxidative stress increased. All of these changes were reversed by spironolactone. In mature adipocytes, aldosterone downregulated both DDAH1 and DDAH2 expression, and ADMA inhibited the insulin-induced cellular signaling. Thus, activation of mineralocorticoid receptor and resultant ADMA accumulation in adipose tissue has, in part, a relevant role in the development of insulin resistance in CKD.

Narcolepsy-cataplexy is a debilitating disorder of sleep/wakefulness caused by a loss of orexin-producing neurons in the lateroposterior hypothalamus. Genetic or pharmacologic orexin replacement ameliorates symptoms in mouse models of narcolepsy-cataplexy. We have recently discovered a potent, nonpeptide OX2R-selective agonist, YNT-185. This study validates the pharmacological activity of this compound in OX2R-transfected cells and in OX2R-expressing neurons in brain slice preparations. Intraperitoneal, and intracerebroventricular, administration of YNT-185 suppressed cataplexy-like episodes in orexin knockout and orexin neuron-ablated mice, but not in orexin receptor-deficient mice. Peripherally administered YNT-185 also promotes wakefulness without affecting body temperature in wild-type mice. Further, there was no immediate rebound sleep after YNT-185 administration in active phase in wild-type and orexin-deficient mice. No desensitization was observed after repeated administration of YNT-185 with respect to the suppression of cataplexy-like episodes. These results provide a proof-of-concept for a mechanistic therapy of narcolepsy-cataplexy by OX2R agonists.

Nicotinamide N-methyltransferase (NNMT) catalyses the reaction between nicotinamide (NAM) and S-adenosylmethionine to produce 1-methylnicotinamide and S-adenosylhomocysteine. Recently, this enzyme has also been reported to modulate hepatic nutrient metabolism, but its role in the liver has not been fully elucidated. We developed transgenic mice overexpressing NNMT to elucidate its role in hepatic nutrient metabolism. When fed a high fat diet containing NAM, a precursor for nicotinamide adenine dinucleotide (NAD)

Summary: Altered DNA methylation plays an important role in the onset and progression of kidney disease. However, little is known about how the changes arise in disease states. Here, we report that KAT5-mediated DNA damage repair is essential for the maintenance of kidney podocytes and is associated with DNA methylation status. Podocyte-specific KAT5-knockout mice develop severe albuminuria with increased DNA double-strand breaks (DSBs), increased DNA methylation of the nephrin promoter region, and decreased nephrin expression. Podocyte KAT5 expression is decreased, whereas DNA DSBs and DNA methylation are increased in diabetic nephropathy; moreover, KAT5 restoration by gene transfer attenuates albuminuria. Furthermore, KAT5 decreases DNA DSBs and DNA methylation at the same nephrin promoter region, which indicates that KAT5-mediated DNA repair may be related to DNA methylation status. These results suggest a concept in which an environment of DNA damage repair, which occurs with decreased KAT5, may affect DNA methylation status. : Hishikawa et al. reveal that KAT5-mediated DNA repair is essential for podocyte maintenance and is related to changes in DNA methylation status. Decreased podocyte KAT5 expression may contribute to the pathophysiology of diabetic nephropathy, suggesting a therapeutic target. Keywords: podocyte, DNA damage repair, DNA methylation, diabetic nephropathy

The involvement of tissue ischemia in obesity-induced kidney injury remains to be elucidated. Compared with low fat diet (LFD)-mice, high fat diet (HFD)-fed mice became obese with tubular enlargement, glomerulomegaly and peritubular capillary rarefaction, and exhibited both tubular and glomerular damages. In HFD-fed mice, despite the increase in renal pimonidazole-positive areas, the expressions of the hypoxia-responsive genes such as Prolyl-hydroxylase PHD2, a dominant oxygen sensor, and VEGFA were unchanged indicating impaired hypoxic response. Tamoxifen inducible proximal tubules (PT)-specific Phd2 knockout (Phd2-cKO) mice and their littermate control mice (Control) were created and fed HFD or LFD. Control mice on HFD (Control HFD) exhibited renal damages and renal ischemia with impaired hypoxic response compared with those on LFD. After tamoxifen treatment, HFD-fed knockout mice (Phd2-cKO HFD) had increased peritubular capillaries and the increased expressions of hypoxia responsive genes compared to Control HFD mice. Phd2-cKO HFD also exhibited the mitigation of tubular damages, albuminuria and glomerulomegaly. In human PT cells, the increased expressions of hypoxia-inducible genes in hypoxic condition were attenuated by free fatty acids. Thus, aberrant hypoxic responses due to dysfunction of PHD2 caused both glomerular and tubular damages in HFD-induced obese mice. Phd2-inactivation provides a novel strategy against obesity-induced kidney injury.

Sleep is a behavior conserved from invertebrates to vertebrates, and tightly regulated in a homeostatic manner. The molecular and cellular mechanism determining the amount of rapid eye movement sleep (REMS) and non-REMS (NREMS) remains unknown. Here we identified two dominant mutations affecting sleep/wakefulness through an electroencephalogram/electromyogram-based screening of randomly mutagenized mice. A splicing mutation of the Sik3 protein kinase gene causes a profound decrease in total wake time, due to an increase in inherent sleep need. Sleep deprivation affects regulatory-site phosphorylation of the kinase. Sik3 orthologues regulate sleep also in fruit flies and roundworms. A missense mutation of the leak cation channel NALCN reduces the total amount and episode duration of REMS, apparently by increasing the excitability of REMS-inhibiting neurons. Our results substantiate the utility of forward genetic approach for sleep behaviors in mice, demonstrating the role of SIK3 and NALCN in regulating the amount of NREMS and REMS, respectively.

A growing body of evidence suggests that dopamine plays a role in sleep-wake regulation, but the dopamine-producing brain areas that control sleep-wake states are unclear. In this study, we chemogenetically activated dopamine neurons in the ventral midbrain of mice to examine the role of these neurons in sleep-wake regulation. We found that activation of dopamine neurons in the ventral tegmental area (VTA), but not in the substantia nigra, strongly induced wakefulness, although both cell populations expressed the neuronal activity marker c-Fos after chemogenetic stimulation. Analysis of the pattern of behavioral states revealed that VTA activation increased the duration of wakefulness and decreased the number of wakefulness episodes, indicating that wakefulness was consolidated by VTA activation. The increased wakefulness evoked by VTA activation was completely abolished by pretreatment with the dopamine D

Objective: Peroxisome proliferator–activated receptor (PPAR)-γ ligand, pioglitazone (PIO), is reported to induce edema especially in postmenopausal women. The aim of this study was to elucidate the mechanism for PIO-induced sodium retention and to discover the therapeutic strategy for the PIO-induced changes in renal sodium handling. Methods and Procedures: Zucker obese rats were ovariectomized and were given PIO for 8 weeks. Renal sodium excretion and renal expressions of several enzymes that generate natriuretic substances were examined. Results: Sodium excretion was decreased in ovariectomized (OVX) rats that were given PIO when compared with OVX rats that were not. Urinary nitrites/nitrates excretion was decreased in OVX rats, but was restored by PIO. The expressions of nitric oxide synthases (NOSs) and cyclooxygenases-1/2 (COX-1/2) were unaltered. Similarly, the expression of epithelial sodium channels (ENaC), identified as a PPAR-γ-regulated gene product, was unaffected. In contrast, the expression of cytochrome P450 4A (CYP4A) was increased in OVX rats, and was downregulated by PIO. Co-treatment of OVX rats with PIO and PPAR-α ligand, fenofibrate, a putative inducer of CYP4A, restored not only the impaired sodium excretion but also the downregulated CYP4A expression. Discussion: PIO-induced sodium retention is specific in female OVX rats. Ovariectomy decreases renal NO production, but upregulates renal CYP4A expression to compensate for renal sodium balance. In this setting, PIO downregulates CYP4A, leading to sodium retention. Furthermore, PPAR-α ligands can provide a novel strategy for preventing the PIO-induced sodium retention.

Cardiovascular events are accelerated in chronic kidney disease (CKD). Although deranged nitric oxide (NO) pathways and asymmetric dimethylarginine (ADMA) cause endothelial dysfunction, no direct evidence for coronary artery endothelial dysfunction in CKD has been documented. CKD was induced in male dogs by heminephrectomy (1/2Nx) or five-sixths nephrectomy (5/6Nx). After 4 wk, renal ablation reduced GFR (control 76 [54 to 85]; 1/2Nx 38 [29 to 47]; 5/6Nx 15 [12 to 46] ml/min) and elevated plasma ADMA (control 1.88 [1.68 to 2.54]; 1/2Nx 2.51 [2.11 to 3.55]; 5/6Nx 3.84 [2.16 to 3.95] micromol/L). Coronary circulatory responses to acetylcholine revealed marked increases in coronary blood flow in control group (83 +/- 17% increment) but blunted responses in 1/2Nx (34 +/- 8% increment) and 5/6Nx (20 +/- 4% increment). The acetylcholine-induced changes in epicardial arteriolar diameter, using needle-lens probe charge-coupled device videomicroscopy, showed similar results. The responsiveness to sodium nitroprusside did not differ among three groups. Plasma nitrite/nitrate levels decreased in 1/2Nx and 5/6Nx, and the mRNA expressions of dimethylarginine dimethylaminohydrolase-II (DDAH-II), ADMA-degrading enzyme, and endothelial NO synthase (eNOS) in coronary arteries were downregulated in 1/2Nx and 5/6Nx. Finally, 4-wk treatment with all-trans retinoic acid restored the impaired endothelium-dependent vasodilation and reversed the expression of eNOS but not DDAH-II. Coronary endothelial function is impaired in the early stage of CKD. The dysfunction is attributed to the downregulation of eNOS and/or DDAH-II in coronary arteries. Furthermore, the manipulation of NO pathways may constitute a therapeutic strategy for the prevention of coronary dysfunction in CKD.

Pontine omnipause neurons (OPNs) are inhibitory neurons projecting to saccade-related premotor burst neurons. OPNs exhibit sustained discharge during fixations and cease firing before and during saccades. The pause in OPN discharge releases the burst neurons from tonic inhibition, resulting in generation of saccadic eye movements. OPNs are thought to receive two major inhibitory inputs during saccades: an early component that determines the pause onset and a late component that controls the pause duration. Although there is evidence that numerous glycinergic and GABAergic terminals contact OPNs, their physiological roles remain unclear. To reveal functions of glycinergic and GABAergic inputs, we investigated effects of iontophoretic application of strychnine, a glycine receptor antagonist, and bicuculline, a GABAA receptor antagonist, on discharge patterns of OPNs in alert cats. Application of strychnine reduced the ratio of pause duration to saccade duration. Analysis of the timing of pause relative to saccades showed that pause onset was delayed and pause end was advanced. These effects were observed for saccades in all directions. Application of bicuculline, in contrast, had no effect on the OPN pause duration or timing. Both strychnine and bicuculline increased tonic firing rate during intersaccadic intervals. These results suggest that glycinergic, but not GABAergic, afferents convey inhibitory signals that determine the onset as well as duration of pause in OPN activity during saccades.

Objectives— Dimethylarginie dimethylaminohydrolase (DDAH) is a degrading enzyme for asymmetrical dimethylarginine, an endogenous NO synthase inhibitor. The molecular mechanism for DDAH-induced vascular endothelial growth factor (VEGF) expression was examined. Methods and Results— Although the transfection of expression vectors for 2 isoforms of DDAH, DDAH1, or DDAH2 increased DDAH activity in bovine aortic endothelial cells and human umbilical vein endothelial cells, expression and secretion of VEGF were increased only in DDAH2-transfected cells. Knocking down the DDAH2 gene reduced VEGF production, and DDAH2 overexpression enhanced both proliferation and migration of endothelial cells. The VEGF promoter activity was increased by DDAH2 transfection, which was not blocked by an NO synthase (NOS) inhibitor but required the Sp1 sites. DDAH2 overexpression increased nuclear protein levels bound to Sp1 oligonucleotides in endothelial cells. Sp1 small interfering RNA blocked DDAH2-induced upregulation of VEGF. DDAH2 transfection increased nuclear and threonine-phosphorylation levels of Sp1 in a protein kinase A (PKA)–dependent manner. Protein–protein interaction between DDAH2 and PKA was enhanced in DDAH2-transfected cells. Conclusions— DDAH2 upregulated the expression of VEGF through Sp1-dependent and NO/NOS system-independent promoter activation. DDAH2-increased Sp1 DNA binding activity was PKA dependent. These mechanisms may provide a novel therapeutic strategy for VEGF-related vasculopathies such as atherosclerosis.

Rho/Rho-kinase plays an important role not only in the vasoconstrictor mechanism but also in cellular morphology, motility, adhesion, and proliferation. This pathway also serves to modulate the structure and function of various kidney cells including tubular epithelial cells, mesangial cells, and podocytes. The inhibition of the Rho/Rho-kinase pathway elicits marked increases in renal blood flow in vivo and dilates both afferent and efferent arterioles preconstricted by angiotensin II in vitro. In renal injury, intrarenal angiotensin II is reported to be activated, which subsequently would upregulate the Rho-kinase pathway. A selective Rho-kinase inhibitor, fasudil, has recently been shown to improve renal damage resulting from hypertensive glomerulosclerosis, unilateral ureteral obstruction (for interstitial renal fibrosis) and subtotal nephrectomy. Of interest, fasudil upregulated the expression of p27kip1, a cyclin-dependent kinase inhibitor, and increased the p27kip1 immuno-positive cells in both glomeruli and tubulointerstitium with the use of immunohistochemistry. Collectively, the Rho-kinase pathway is involved in the pathogenesis of renal injury. Clinical application of this type of therapy however awaits further investigations. Keywords:: Rho-kinase, renal hemodynamics, fasudil, renal injury, p27kip1

Background and aims We previously demonstrated that acetylcholine elicited nitric oxide-dependent sustained and endothelium-derived hyperpolarizing factor (EDHF)-dependent transient dilation of afferent arterioles. The present study examined whether free radicals interacted with nitric oxide-dependent and EDHF-dependent vasodilator mechanisms in renal microvessels of salt-sensitive hypertension, using the isolated perfused hydronephrotic kidney. Methods and results Following the pretreatment with indomethacin (100 micromol/L) with or without nitro- l-arginine methylester (100 micromol/L), the effect of acetylcholine on noradrenaline (0.3 micromol/L)-induced constriction was evaluated in kidneys from Dahl salt-sensitive and salt-resistant rats. Although acetylcholine (0.01-10 micromol/L) caused dose-dependent and sustained vasodilation of afferent arterioles, attenuated dilation was observed in Dahl salt-sensitive rats, compared with that in salt-resistant rats (58 +/- 4 vs 101 +/- 11% reversal at 10 micromol/L acetylcholine). In the presence of nitro- l-arginine methylester, acetylcholine elicited only transient dilation, with vasodilator response blunted in Dahl salt-sensitive rats (64 +/- 4 vs 100 +/- 9% reversal at 10 micromol/L acetylcholine). Furthermore, chronic (8-10 weeks) treatment with tempol caused partial restoration of acetylcholine (10 micromol/L)-induced sustained arteriolar dilation (71 +/- 3% reversal), but complete reversal of transient dilation (92 +/- 4% reversal). Finally, acute treatment with tempol not only improved the sustained component of the acetylcholine-induced dilation but also restored the impaired responsiveness of transient dilation in Dahl salt-sensitive rats. Conclusion Both sustained (nitric oxide-mediated) and transient (EDHF-mediated) components of acetylcholine-induced afferent arteriolar dilation were attenuated in Dahl salt-sensitive rats, which was attributed, in part, to enhanced free radical activity. A reversal of the sustained and transient vasodilation by the acute tempol treatment suggests possible interaction between free radicals and EDHF as well as increased bioavailability of nitric oxide.

We examined the role of free radicals in renal microvascular tone induced by various vasoactive stimuli. Isolated perfused rat hydronephrotic kidneys were used for direct visualization of renal microcirculation. The effect of tempol on angiotensin II-, norepinephrine-, KCl-, and pressure-induced afferent arteriolar constriction was evaluated. Under angiotensin II-induced constriction, tempol (3 mmol/L) caused 57 +/- 8% dilation of afferent arterioles. In contrast, tempol elicited only 38 +/- 8% and 26 +/- 9% dilation of norepinephrine- and KCl-induced constriction. Similarly, myogenic response induced by elevating renal arterial pressure from 80 to 180 mmHg was resistant to the vasodilator action of tempol (22 +/- 7% inhibition). Furthermore, tempol failed to reverse nitro-L-arginine methylester-induced afferent constriction, nor had vasodilator effect on the angiotensin II-induced constriction in the presence of nitro-L-arginine methylester. In contrast, nitroprusside elicited marked vasodilation of angiotensin II- (97 +/- 5% reversal) and norepinephrine-induced afferent constriction (89 +/- 6% reversal), but had less effect on KCl- (46 +/- 8% reversal) and pressure-induced constriction (26 +/- 9% reversal). These different actions were also observed when polyethylene-glycolated superoxide dismutase was used as an antioxidant. In conclusion, the role of free radicals in afferent arteriolar tone varies, depending on the underlying vasoconstrictor stimuli, with greater contribution of free radicals to angiotensin II-induced constriction. The heterogeneity in the responsiveness to free radical scavengers is attributed to both magnitude of free radicals produced and sensitivity of the underlying vasoconstrictors to nitric oxide.

Although nifedipine and other conventional calcium antagonists elicit preferential vasodilation of renal afferent arterioles, we demonstrate that mibefradil and nickel, T-type calcium channel blockers, reverse the angiotensin II-induced constriction of both afferent and efferent arterioles. Since the angiotensin II-induced vasoconstriction involves inositol trisphosphate (IP3)-induced calcium release from the sarcoplasmic reticulum in the afferent arteriole, and both IP3- and protein kinase C (PKC)-mediated pathways in the efferent arteriole, we investigated the cellular mechanism for the mibefradil-induced dilation of angiotensin II-constricted renal arterioles, using the isolated perfused hydronephrotic rat kidney. Mibefradil caused a dose-dependent dilation of angiotensin II-constricted afferent and efferent arterioles, with 88 ± 9% and 74 ± 10% reversal observed at 1 μmol/L, respectively. The blockade of PKC by staurosporine did not alter the mibefradil-induced vasodilator responses of either arterioles (P > 0.5). In contrast, the pretreatment with thapsigargin, which predominantly blocked the IP3-mediated intracellular calcium release, prevented the afferent arteriolar constrictor response to angiotensin II, but caused a significant constriction of efferent arterioles. The subsequent addition of mibefradil had no effect on the efferent arteriolar diameter. Furthermore, the efferent arteriolar constriction induced by direct PKC activation by phorbol myristate acetate was refractory to mibefradil, but completely reversed by LOE908, a nonselective cation channel blocker. In summary, mibefradil markedly dilates the angiotensin II-induced renal arteriolar constriction; the action of mibefradil is most likely mediated by the inhibition of the IP3-mediated pathway, but the inhibitory action on the PKC pathway appears modest.

We examined the role of Rho/Rho-kinase in renal afferent and efferent arteriolar tone induced by angiotensin (Ang) II, KCl and elevated renal arterial pressure (from 80 to 180 mm Hg), using isolated perfused rat hydronephrotic kidney. In the condition with no vasoconstrictor stimuli, Y-27632, a Rho-kinase inhibitor, dilated only afferent (from 11.6 ± 0.4 to 14.1 ± 0.5 µm) but not efferent arterioles (from 11.6 ± 0.2 to 12.6 ± 0.7 µm) at 10–5 mol/l. During renal vasoconstriction by Ang II, Y-27632 restored the afferent arteriolar constriction (141 ± 10% reversal at 10–5 mol/l), whereas the ability of Y-27632 to inhibit the Ang II-induced efferent arteriolar constriction was diminished (73 ± 7% reversal). A similar action was observed with fasudil, another Rho-kinase inhibitor. Furthermore, Y-27632 impaired myogenic afferent arteriolar constriction, with 117 ± 17% inhibition at 10–5 mol/l. The inhibition by Y-27632 of the myogenic vasoconstriction was almost the same as that of the Ang II-induced tone of this vessel type. However, Y-27632 had a modest effect on KCl-induced vasoconstriction of afferent arterioles. In conclusion, the present study demonstrates a predominant role of Rho/Rho-kinase in mediating the basal and Ang II-induced tone of afferent, but not efferent, arterioles. Furthermore, the role of Rho/Rho-kinase in afferent arteriolar constriction differs, with a substantial contribution to Ang II-induced and myogenic constriction but a minimal role in depolarization-induced constriction. Since Ang II-induced, KCl-induced and myogenic constriction of afferent arterioles require calcium entry through voltage-dependent calcium channels, the interaction between Rho/Rho-kinase and the calcium entry pathway may determine the afferent arteriolar tone induced by these stimuli.

Renal afferent arterioles (AFF) regulate glomerular capillary pressure through two main mechanisms: the myogenic response (MYO) and tubuloglomerular feedback (TGF). Because Rho-kinase and nitric oxide synthase (NOS) are established factors that modulate vascular tone, we examined the role of these factors in pressure-induced AFF tone in Wistar-Kyoto rats and in spontaneously hypertensive rats (SHR) using an intravital CCD camera. Elevated renal perfusion pressure elicited marked AFF constriction that was partially inhibited by gadolinium, furosemide and fasudil, which inhibit MYO, TGF and Rho-kinase, respectively; however, this AFF constriction was completely blocked by combined treatment with fasudil+gadolinium or fasudil+furosemide. S-methyl-L-thiocitrulline (SMTC) partially reversed the fasudil-induced inhibition of TGF-mediated, but not that of MYO-mediated, AFF constriction. In SHR, the pressure-induced AFF response was enhanced, and MYO- and TGF-induced constriction were exaggerated. In the presence of gadolinium, SMTC partially mitigated the fasudil-induced inhibition of TGF-mediated AFF constriction. Immunoblot analyses demonstrated that both Rho-kinase activity and neuronal NOS were augmented in SHR kidneys. In conclusion, Rho-kinase contributes to MYO- and TGF-mediated AFF responses, and these responses are enhanced in SHR. Furthermore, neuronal NOS-induced nitric oxide modulates the TGF mechanism. This mechanism constitutes a target for Rho-kinase in TGF-mediated AFF constriction.

Rho-kinase plays an important role in hypertension and is reported to interfere with insulin signaling through serine phosphorylation of insulin receptor substrate-1 (IRS-1) in cultured vascular smooth muscle cells. We therefore examined the role of Rho-kinase in the development of insulin resistance in Zucker obese rats. In skeletal muscles and aortic tissues of Zucker obese rats, activation of RhoA/Rho-kinase was observed. Long-term Rho-kinase inhibition by 4 wk treatment with fasudil (a Rho-kinase inhibitor) not only reduced blood pressure but corrected glucose and lipid metabolism, with improvement in serine phosphorylation of IRS-1 and insulin signaling in skeletal muscles. Direct visualization of skeletal muscle arterioles with an intravital CCD videomicroscope demonstrated that both acetylcholine- and sodium nitroprusside-induced vasodilations were blunted, which were restored by the fasudil treatment. Furthermore, both fasudil and Y-27632 prevented the serine phosphorylation of IRS-1 induced by insulin and/or tumor necrosis factor-alpha in skeletal muscle cells. Collectively, Rho-kinase is responsible for the impairment of insulin signaling and may constitute a critical mediator linking between metabolic and hemodynamic abnormalities in insulin resistance.

Sirtuin 1 (Sirt1), a NAD[superscript +]-regulated deacetylase with numerous known positive effects on cellular and whole-body metabolism, is expressed in the renal cortex and medulla. It is known to have protective effects against age-related disease, including diabetes. Here we investigated the protective role of Sirt1 in diabetic renal damage. We found that Sirt1 in proximal tubules (PTs) was downregulated before albuminuria occurred in streptozotocin-induced or obese (db/db) diabetic mice. PT-specific SIRT1 transgenic and Sirt1 knockout mice showed prevention and aggravation of the glomerular changes that occur in diabetes, respectively, and nondiabetic knockout mice exhibited albuminuria, suggesting that Sirt1 in PTs affects glomerular function. Downregulation of Sirt1 and upregulation of the tight junction protein Claudin-1 by SIRT1-mediated epigenetic regulation in podocytes contributed to albuminuria. We did not observe these phenomena in 5/6 nephrectomized mice. We also demonstrated retrograde interplay from PTs to glomeruli using nicotinamide mononucleotide (NMN) from conditioned medium, measurement of the autofluorescence of photoactivatable NMN and injection of fluorescence-labeled NMN. In human subjects with diabetes, the levels of SIRT1 and Claudin-1 were correlated with proteinuria levels. These results suggest that Sirt1 in PTs protects against albuminuria in diabetes by maintaining NMN concentrations around glomeruli, thus influencing podocyte function., Japan. Ministry of Education, Culture, Sports, Science and Technology (Grant 22790800)

The role of dimethylarginine dimethylaminohydrolase 2 (DDAH2) in glucose metabolism is unknown. Here, we generated DDAH2 transgenic (Tg) mice. These mice had lower plasma glucose levels (60 min: 298±32 vs. 418±35 mg/dl; 120 min: 205±15 vs. 284±20 mg/dl) and higher insulin levels (15 min: 2.1±0.2 vs. 1.5±0.1 ng/ml; 30 min: 1.8±0.1 vs. 1.5±0.1 ng/ml) during intraperitoneal glucose tolerance tests when fed a high-fat diet (HFD) compared with HFD-fed wild-type (WT) mice. Glucose-stimulated insulin secretion (GSIS) was increased in Tg islets by 33%. Pancreatic asymmetrical dimethylarginine, nitric oxide, and oxidative stress levels were not correlated with improvements in insulin secretion in Tg mice. Secretagogin, an insulin vesicle docking protein, was up-regulated by 2.7-fold in Tg mice and in pancreatic MIN-6 cells overexpressing DDAH2. GSIS in MIN-6 cells was dependent on DDAH2-induced secretagogin expression. Pancreatic Sirt1, DDAH2, and secretagogin were down-regulated in HFD-fed WT mice by 70, 75, and 85%, respectively. Overexpression of Sirt1 overexpression by 3.9-fold increased DDAH2 and secretagogin expression in MIN-6 cells by 3.2- and 2.5-fold, respectively. DDAH2 overexpression improved GSIS in pancreas-specific Sirt1-deficient mice. In summary, the Sirt1/DDAH2/secretagogin pathway is a novel regulator of GSIS.

The renin-angiotensin-aldosterone system(RAAS) plays a crucial role in the regulation of physiological homeostasis and diseases such as hypertension, coronary artery disease and chronic renal failure. In this cascade, the ACE/Ang II/AT1 receptor axis induces pathological effects, such as vasoconstriction, cell proliferation and fibrosis. Recently the ACE2/Ang(1-7)/Mas receptor axis has been recognized as a negative regulator of the RAAS. ACE2 metabolizes Ang II into Ang(1-7), which has opposite properties of Ang II through Mas receptor activation. Both animal and human studies provide strong evidence that the ACE2/Ang(1-7)/Mas receptor axis is protective for end-organ damage. Therefore, the ACE2/Ang(1-7)/Mas receptor axis could be a therapeutic target for coronary artery disease and chronic renal failure.

The allometric relationships between resting metabolism (VO 2 ) and body mass ( M ), VO 2 = a i M b , are considered a fundamental law of nature. A distinction though needs to be made between the ontogeny (within a species) and phylogeny (among species) of metabolism. However, the nature and significance of the intraspecific allometry (ontogeny of metabolism) have not been established in fishes. In this study, we present experimental evidence that a puffer fish ranging 0.0008–3 g in wet body mass has four distinct allometric phases in which three stepwise increases in scaling constants ( a i , i = 1–4), i.e. ontogenetic phase shifts in metabolism, occur with growth during its early life stages at around 0.002, 0.01 and 0.1 g, keeping each scaling exponent constant in each phase ( b = 0.795). Three stepwise increases in a i accompanied behavioural and morphological changes and three peaks of severe cannibalism, in which the majority of predation occurred on smaller fish that had a lower value of a i . Though fishes are generally highly fecund, producing a large number of small eggs, their survivability is very low. These results suggest that individuals with the ability to rapidly grow and step up ‘ a i ’ develop more anti-predator adaptation as a result of the decreased predatory risk.

Although endothelial dysfunction, defined as abnormal vasoreactivity, is a common early finding in individuals with type 2 diabetes, the endothelium has not been known to regulate metabolism. As PPARgamma, a transcriptional regulator of energy balance, is expressed in endothelial cells, we set out to investigate the role of endothelial cell PPARgamma in metabolism using mice that lack PPARgamma in the endothelium and BM (gammaEC/BM-KO). When gammaEC/BM-KO mice were fed a high-fat diet, they had decreased adiposity and increased insulin sensitivity compared with control mice, despite increased serum FFA and triglyceride (TG) levels. After fasting or olive oil gavage, gammaEC/BM-KO mice exhibited significant dyslipidemia and failed to respond to the FFA and TG lowering effects of the PPARgamma agonist rosiglitazone. BM transplantation studies, which reconstituted hematopoietic PPARgamma, established that these metabolic phenotypes were due to endothelial PPARgamma deficiency. We further found that the impairment in TG-rich lipoprotein metabolism in gammaEC/BM-KO mice was associated with fatty acid-mediated lipoprotein lipase inhibition and changes in a PPARgamma-regulated endothelial cell transcriptional program. Despite their metabolic improvements, high-fat diet-fed gammaEC/BM-KO mice had impaired vasoreactivity. Taken together, these data suggest that PPARgamma in the endothelium integrates metabolic and vascular responses and may contribute to the effects of PPARgamma agonists, thus expanding what endothelial function and dysfunction may entail.

Within the nervous system, estradiol was originally characterized through its critical role in sexual maturation and reproduction. However, it is now firmly established that estradiol affects a diverse array of brain functions including learning and memory, fine motor control, pain perception and mood. In parallel with this increased scope, new mechanisms of estradiol action are being elucidated. Traditionally, estradiol was known to work through intracellular estrogen receptors (ERalpha and ERbeta, which regulate transcription by binding to estrogen response elements (EREs). These functions, critical for sexual behavior, are termed "genomic" and are distinct from "nongenomic" effects whereby estradiol produces rapid changes in neuronal function by processes initiated at the membrane surface. Recent advances have demonstrated that these nongenomic effects are also mediated by classical estrogen receptors that are localized to the plasma membrane. Further, stimulation of estrogen receptors on the membrane surface by estradiol activates G-protein-coupled receptor (GPCR) signaling, which can lead to both short- and long-term changes in neuronal excitability. Related, the brain itself synthesizes estrogens, allowing for localized signaling by the steroid that is less dependent on gonadal secretion. This review outlines highlights from both established and recent findings of estrogen action, how these diverse mechanisms act in concert to regulate nervous system function, and outlines several questions that still remain regarding the effects of estrogen in brain.

Peroxisome proliferator activated receptor-gamma (PPARgamma) ligands increase nitric oxide (NO) production and reduce systemic blood pressure. Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor degraded by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which has two isoforms, DDAH-I and -II. In order to elucidate the mechanism whereby PPARgamma ligands affect NO metabolism, their effects on the DDAH-ADMA pathway were investigated. Six-week-old male Wister-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were maintained with or without pioglitazone (PIO), a PPARgamma ligand. After 4 weeks, serum ADMA levels and urinary daily NO excretion were analyzed. Tissue DDAH expression was examined by real-time polymerase chain reaction (PCR), immunoblotting, and immunohistochemistry. The results showed that PIO decreased serum ADMA and increased urinary NO excretion in both WKY and SHR. Also in both strains, the expression level of DDAH-II in the kidney was increased at transcriptional levels, although the DDAH-I level was unaffected. PIO lowered blood pressure in SHR, but not in WKY. We also demonstrated that PIO induced DDAH-II protein expression in Marbin-Dubin Canine Kidney (MDCK) cells, a renal tubular cell line. In conclusion, a PPARgamma ligand was here found to increase NO production partly by upregulating tissue DDAH-II expression and decreasing systemic ADMA levels. This mechanism constitutes a direct action on renal tubular cells, but is less likely to be responsible for the blood pressure-lowering effects of PPARgamma ligands. Since ADMA is one of the risk factors for cardiovascular events, this study provides compelling evidence that PPARgamma ligands have the potential for reducing cardiovascular risks.

The present study examined the role of L-/T-type Ca channels and the interaction between these channels and protein kinase C (PKC) in hypertension. The isolated perfused hydronephrotic rat kidney model was used to visualize directly the renal microvascular effects of L-/T-type Ca channel blockers (nifedipine and mibefradil, respectively). Nifedipine reversed the angiotensin II-induced constriction of afferent, but not efferent, arterioles in kidneys from Wistar-Kyoto rats (WKY), and similar magnitude in dilation was observed in spontaneously hypertensive rats (SHR). Although mibefradil elicited dilation of both arterioles, the afferent arteriolar dilation was less in SHR than in WKY (57+/-5% vs. 80+/-4% reversal at 1 micrommol/L). The pretreatment with staurosporine did not alter the angiotensin II-induced afferent arteriolar constriction in WKY, but attenuated this response in SHR. Furthermore, staurosporine enhanced the nifedipine-induced afferent arteriolar dilation (62+/-3% vs. 50+/-3% reversal at 10 nmol/L), and restored the attenuated afferent arteriolar response to mibefradil in SHR. The pretreatment with thapsigargin (a blocker of IP3-mediated intracellular calcium release) prevented the angiotensin II-induced afferent arteriolar constriction in WKY, but caused a significant constriction of afferent arterioles in SHR and efferent arterioles in WKY and SHR; in this setting, mibefradil did not alter efferent arteriolar tone. In conclusion, although both L-type (nifedipine) and T-type Ca channel blockers (mibefradil) exerted potent vasodilation of rat renal microvessels, these actions were modified by PKC, which determined the afferent arteriolar sensitivity to these blockers in SHR. Furthermore, the enhancement in nifedipine-induced afferent arteriolar dilation by staurosporine in SHR suggests that L-type Ca channel activity is augmented in hypertensive animals.

Angiotensin II enhances the development of atherosclerotic lesion in which cellular proliferation and/or migration are critical steps. Although cyclin-dependent kinase inhibitor, p27, and Rho/Rho-kinase pathway have recently been implicated as factors regulating these events cooperatively, their role in vivo has not been fully elucidated. We evaluated the contribution of p27 and Rho-kinase to angiotensin II-induced vascular injury using p27-deficient mice. Two-week angiotensin II (1500 ng/kg per minute SC) infusion elicited similar degrees of elevation in systolic blood pressure in wild-type mice (159±5 mm Hg) and p27-deficient mice (157±5 mm Hg; P >0.05). Angiotensin II infusion to wild-type mice resulted in increases in the medial thickness of aorta, proliferating cell number, and monocyte/macrophage infiltration within the vasculature. In p27-deficient mice, however, these changes were more prominent than those in wild-type mice. Treatment of wild-type mice with fasudil, a selective Rho-kinase inhibitor, did not alter blood pressure but significantly upregulated p27 expression, decreased medial thickness of aorta, reduced proliferating cell number, and prevented monocyte/macrophage infiltration. These protective effects of fasudil were attenuated in p27-deficient mice. In conclusion, p27 constitutes an important modulator of angiotensin II–induced monocyte/macrophage infiltration and vascular remodeling, which is mediated in part by Rho-kinase stimulation. Inhibition of Rho-kinase activity improves angiotensin II–induced vascular injury through p27-dependent and p27-independent mechanisms.

Although peroxisome proliferator-activated receptor γ (PPARγ) ligands have an antihypertensive effect in vivo, the precise mechanism has not been fully elucidated. We examined their effects on Rho/Rho kinase pathway, a key regulator of vascular tone. In cultured rat aortic smooth muscle cells (RASMC), Rho kinase stimulated by angiotensin II was suppressed by the pretreatment with pioglitazone and troglitazone, and these effects were explained by the inhibition of the Rho translocation to the cell membrane. We evaluated the role of Vav, a GTP/GDP exchange factor upregulating Rho kinase activity, and Src homology region 2–containing protein tyrosine phosphatase-2 (SHP-2), a protein tyrosine phosphatase that dephosphorylated Vav and subsequently inactivated Rho kinase. Both pioglitazone and troglitazone upregulated SHP-2, particularly in the cytosolic fraction, and the SHP-2-bound Vav, and reduced the phosphorylation of Vav. Furthermore, 4-week treatment with pioglitazone lowered systolic blood pressure in spontaneously hypertensive rats (SHR) and suppressed the Rho/Rho kinase activity in aortic tissues isolated from SHR. Consistently, the expression of SHP-2 was upregulated in vascular tissues from pioglitazone-treated SHR. The phosphorylated Vav was increased in SHR, compared with that in normotensive Wistar–Kyoto rats (WKY), which was mitigated by pioglitazone. Finally, both basal and angiotensin II–stimulated levels of Rho kinase activity were greater in RASMC from SHR than those from WKY, and the enhanced Rho kinase activity was blocked by pioglitazone or troglitazone in both strains. Collectively, PPARγ ligands inhibit the Rho/Rho kinase pathway through upregulation of cytosolic SHP-2 expression and inactivation of Vav, and may contribute to the hemodynamic, in addition to metabolic, action in hypertensive metabolic syndrome. The full text of this article is available online at http://circres.ahajournals.org.

Although available evidence demonstrates that obesity manifests insulin resistance and causes glomerular sclerosis, it has not been determined whether insulin resistance alters the renal microvascular reactivity. This study examined whether insulin- and acetylcholine (ACH)-induced vasodilation was impaired in Zucker obese rats, and attempted to clarify the change in myogenic afferent arteriolar constriction, a determinant of glomerular pressure. Isolated perfused hydronephrotic rat kidneys were used to visualize the renal microcirculation. In Zucker lean rats, insulin (10 to 300 [mu ]U/mL) inhibited norepinephrine (NE)-induced afferent and efferent arteriolar constriction in a dose-dependent manner, with 112 % [plusmn] 8% and 98% [plusmn] 8% reversal at 300 [mu ]U/mL Similarly, ACH elicited dose-dependent dilation of these vessels. In Zucker obese rats, by contrast, afferent and efferent arterioles failed to dilate in response to insulin, and manifested diminished vasodilator responses to acetylcholine In the presence of nitro- L -arginine methylester (LNAME; 100 [mu ]mol/L), ACH (10 [mu ]mol/L) induced transient afferent arteriolar dilation (121% [plusmn] 9% reversal) in Zucker lean rats, whereas this response was blunted in obese rats (72% [plusmn] 8% reversal) Furthermore, myogenic afferent arteriolar constriction by elevating renal arterial pressure to 180 mm Hg was diminished in Zucker obese rats ([minus ]14% [plusmn] 3% decrement in diameter), compared with that in lean rats ([minus ]23% [plusmn] 2% decrement) Finally, the impairment in these vasodilator and vasoconstrictor responses was partially prevented by troglitazone, an insulin-sensitizing agent. Collectively, in insulin resistance, renal microvessels are refractory to the vasodilator action of insulin. Furthermore, [ldquo ]renal insulin resistance[rdquo ] is associated with the impaired vasodilator responses to ACH-induced nitric oxide (NO) and the diminished vasoconstrictor responses to pressure. The blunted myogenic afferent arteriolar constriction would allow glomerular hypertension, and in concert with the impaired endothelium-dependent vasodilation, could be responsible for the development of glomerular injury in obesity.

Both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) are established as important factors determining the vascular tone. The relative contribution of these factors to the renal microvascular tone, however, has not been delineated.Isolated perfused hydronephrotic rat kidneys were used to characterize the relative role of NO and EDHF in mediating the tone of interlobular arteries (ILA) and afferent arterioles (AFF).During the norepinephrine constriction, acetylcholine (ACH, 1 micromol/l) induced a sustained vasodilation of ILA (90 +/- 9% reversal) and AFF (117 +/- 13% reversal). In the presence of nitro-L-arginine methylester (LNAME), the ACH-induced vasodilation of ILA and AFF was converted to transient dilation, with only 53 +/- 7 and 32 +/- 7% reversal observed 10 min after 1 micromol/l ACH (i.e sustained phase). In contrast, LNAME had no effect on the initial phase of ACH-induced dilation. In the presence of apamin + charybdotoxin, the initial vasodilator response to ACH (1 micromol/l) was diminished (ILA, from 108 +/- 8 to 46 +/- 9%; AFF, from 108 +/- 14 to 58 +/- 8%), whereas no impairment was observed in sustained phases. Furthermore, the magnitude of the vasoconstriction caused by LNAME was greater at smaller vessel segments. Finally, the LNAME-induced inhibition of the sustained phase of ACH-induced vasodilation was greater as the vessel diameter decreased.That the relative contribution of NO and EDHF differs, with a greater role of NO in the basal tone and ACH-induced vasodilation at smaller vascular segments of ILA and AFF.

Effect of fasudil on Rho-kinase and nephropathy in subtotally nephrectomized spontaneously hypertensive rats. Background Although Rho-kinase is reported to play an important role in vascular injury, the contribution of Rho-kinase to the progression of renal injury remains unestablished. Methods We examined the effect of fasudil, a Rho-kinase inhibitor, on the progression of renal injury in subtotally nephrectomized spontaneously hypertensive rats (SHR). Rats were randomly assigned to three groups: sham-operated SHR; salt-loaded subtotally nephrectomized rats (SHR-subtotal nephrectomy); SHR-subtotal nephrectomy given fasudil for 6weeks (SHR-subtotal nephrectomy + fasudil; 3mg/kg/day). Renal morphologic and molecular analysis as well as urinary protein excretion was evaluated. Results In SHR-subtotal nephrectomy treated with fasudil, systolic blood pressure was not significantly different from that in SHR-subtotal nephrectomy without fasudil (208 ± 8mm Hg vs. 217 ± 14mm Hg). Urinary protein excretion was markedly increased in SHR-subtotal nephrectomy (124 ± 16mg/day), but this increase was significantly suppressed by fasudil (79 ± 12mg/day). Renal histologic examination revealed that fasudil improved glomerular and tubulointerstitial injury scores with parallel amelioration of proliferating cell nuclear antigen-positive and ED-1–positive cell infiltration. Furthermore, Western blot analyses showed that both expression and activity of Rho-kinase were enhanced in SHR-subtotal nephrectomy, compared with those in SHR without nephrectomy, and fasudil suppressed Rho-kinase activity. Finally, fasudil up-regulated the expression of p27 kip1 , a cyclin-dependent kinase inhibitor, and increased the p27 kip1 immunopositive cells in both glomeruli and tubulointerstitium with the use of immunohistochemistry. Conclusion Rho-kinase pathway is involved in the pathogenesis of renal injury. Furthermore, the inhibition of Rho-kinase may constitute a therapeutic strategy for the treatment of renal injury in part through the p27 kip1 up-regulation and the subsequent inhibition of cell proliferation and macrophage recruitment.