Urea transporters are a family of urea-selective channel proteins expressed in multiple tissues that play an important role in the urine-concentrating mechanism of the mammalian kidney. Previous studies have shown that knockout of urea transporter (UT)-B, UT-A1/A3, or all UTs leads to urea-selective diuresis, indicating that urea transporters have important roles in urine concentration. Here, we sought to determine the role of UT-A1 in the urine-concentrating mechanism in a newly developed UTA1–knockout mouse model. Phenotypically, daily urine output in UT-A1–knockout mice was nearly 3-fold that of WT mice and 82% of all-UT–knockout mice, and the UT-A1–knockout mice had significantly lower urine osmolality than WT mice. After 24-h water restriction, acute urea loading, or high-protein (40%) intake, UT-A1–knockout mice were unable to increase urine-concentrating ability. Compared with all-UT–knockout mice, the UT-A1–knockout mice exhibited similarly elevated daily urine output and decreased urine osmolality, indicating impaired urea-selective urine concentration. Our experimental findings reveal that UT-A1 has a predominant role in urea-dependent urine-concentrating mechanisms, suggesting that UTA1 represents a promising diuretic target.
Cycoloxygenase-2 (COX-2) induction is prevalent in a variety of (brain and peripheral) injury models where COX-2 levels correlate with disease progression. Thus, COX-2 has been widely explored for anti-inflammatory therapy with COX-2 inhibitors, which proved to be effective in reducing the pain and inflammation in patients with arthritis and menstrual cramps, but they have not provided any benefit to patients with chronic inflammatory neurodegenerative disease. Recently, two COX-2 drugs, rofecoxib and valdecoxib, were withdrawn from the United States market due to cardiovascular side effects. Thus, future anti-inflammatory therapy could be targeted through a specific prostanoid receptor downstream of COX-2. The PGE2 receptor EP2 is emerging as a pro-inflammatory target in a variety of CNS and peripheral diseases. Here we highlight the latest developments on the role of EP2 in diseases, mechanism of activation, and small molecule discovery targeted either to enhance or to block the function of this receptor.
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Roman Alpatov;
Bluma J. Lesch;
Mika Nakamoto-Kinoshita;
Andres Blanco;
Shuzhen Chen;
Alexandra Stuetzer;
Karim J. Armache;
Matthew D. Simon;
Chao Xu;
Muzaffar Ali;
Jernej Murn;
Slandjana Prisic;
Tatiana G. Kutateladze;
Christopher R. Vakoc;
Jinrong Min;
Robert E. Kingston;
Wolfgang Fischle;
Stephen Warren;
David C. Page;
Yang Shi
Fragile X syndrome, a common form of inherited intellectual disability, is caused by loss of the fragile X mental retardation protein FMRP. FMRP is present predominantly in the cytoplasm, where it regulates translation of proteins that are important for synaptic function. We identify FMRP as a chromatin-binding protein that functions in the DNA damage response (DDR). Specifically, we show that FMRP binds chromatin through its tandem Tudor (Agenet) domain in vitro and associates with chromatin in vivo. We also demonstrate that FMRP participates in the DDR in a chromatin-binding-dependent manner. The DDR machinery is known to play important roles in developmental processes such as gametogenesis. We show that FMRP occupies meiotic chromosomes and regulates the dynamics of the DDR machinery during mouse spermatogenesis. These findings suggest that nuclear FMRP regulates genomic stability at the chromatin interface and may impact gametogenesis and some developmental aspects of fragile X syndrome.
The EP2 receptor has emerged as a therapeutic target with exacerbating role in disease pathology for a variety of peripheral and central nervous system disorders. We and others have recently demonstrated beneficial effects of EP2 antagonists in preclinical models of neuroinflammation and peripheral inflammation. However, it was earlier reported that mice with global EP2 knockout (KO) display adverse phenotypes on fertility and blood pressure. Other studies indicated that EP2 activation with an agonist has a beneficial effect of healing fractured bone in animal models. These results impeded the development of EP2 antagonists, and EP2 antagonism as therapeutic strategy. To determine whether treatment with EP2 antagonist mimics the adverse phenotypes of the EP2 global KO mouse, we tested two EP2 antagonists TG11–77. HCl and TG6–10–1 in mice and rats while they are on normal or high-salt diet, and by two different administration protocols (acute and chronic). There were no adverse effects of the antagonists on systolic and diastolic blood pressure, heart rate, respiratory function in mice and rats regardless of rodents being on a regular or high salt diet. Furthermore, chronic exposure to TG11–77. HCl produced no adverse effects on blood cell counts, bone-volume and bone-mineral density in mice. Our findings argue against adverse effects on cardiovascular and respiratory systems, blood counts and bone structure in healthy rodents from the use of small molecule reversible antagonists for EP2, in contrast to the genetic ablation model. This study paves the way for advancing therapeutic applications of EP2 antagonists against diseases involving EP2 dysfunction.
Astrocytes are relatively resistant to injury compared to neurons and oligodendrocytes. Here, we report transient region-specific loss of astrocytes in mice early after pilocarpine-induced status epilepticus (SE). In the dentate hilus, immunoreactivity for glial acidic fibrillary protein (GFAP) was decreased, and the number of healthy appearing GFAP- or S100β-positive cells was significantly reduced (≥ 65%) 1 and 3 days after pilocarpine-induced SE. Many remaining GFAP-positive cells were shrunken, and 1 day after SE electron microscopy revealed numerous electron-dense degenerating astrocyte processes and degenerating glial somata in the hilus. Degeneration of GFAP-expressing cells may be linked to hilar neuronal death, because we did not observe loss of astrocytes after kainate-induced SE, after which hilar neurons remained intact. Ten days after SE, hilar GFAP immunoreactivity had returned, partially from GFAP-positive cells in the hilus. Unlike control mice, many GFAP-positive hilar processes originated from cell bodies located in the subgranular zone (SGZ). To investigate whether proliferation contributes to hilar repopulation, we injected 5-bromo-2′-deoxyuridine (BrdU) 3 days after SE. Five hours later and up to 31 days after SE, many BrdU/GFAP colabeled cells were found in the hilus and the SGZ, some with hilar processes, indicating that proliferation in both areas contributes to generation of hilar astrocytes and astrocyte processes. In contrast to pilocarpine-induced SE in mice, astrocyte degeneration was not found after pilocarpine-induced SE in rats. These findings demonstrate astrocyte degeneration in the mouse dentate hilus specifically in the mouse pilocarpine epilepsy model, followed by astrogenesis leading to hilar repopulation.
Background: Recently, two enteric neuronal cell lines, one fetal and the other post-natal (IM-PEN), have been developed from the H-2Kb-tsA58 transgenic mouse (immortomouse). However, their electrophysiological properties are not known. The goal of this study was to determine the electrical excitability and ionic conductance of the immortalized postnatal enteric neuronal (IM-PEN) cell line.
Methods: Whole cell patch clamp studies, immunohistochemistry and RT-PCR were performed on differentiated IM-PEN cells following propagation at 33 C and differentiation at 37 C.
Results: Differentiated IM-PEN cells stained positively for the neuron specific markers βIII-tubulin and PGP9.5. The mRNA for several ion channels expressed in enteric neurons were detected by RT-PCR. In current clamp, the resting membrane potential was -24.6 ± 2.1 mV (n = 6) for IM-FEN and -29.8 ± 0.9 mV (n = 30) for IM-PEN. Current injections from Vh -80 mV resulted in passive responses but not action potentials. Depolarizing pulses in the whole cell voltage clamp configuration from Vh -80 mV elicited small nifedipine-sensitive inward currents. Additionally, outward currents with slow deactivating tail currents were blocked by niflumic acid and low chloride solution. A volume-regulated anion current was elicited by hypo-osmotic solution and inhibited by 10 μM DCPIB. Growth with rabbit gastrointestinal smooth muscle did not yield significant differences in the active properties of the IM-PEN cell line. Transient expression of L-type Ca2+ channels produced large inward currents demonstrating a working mechanism for protein folding and transport.
Conclusion: The electrophysiological characteristics of IM-PEN cells suggest that chloride channels in IM-PEN cells play an important role in their resting state, and membrane trafficking of some of the ion channels may preclude their electrical excitability.
Rift Valley fever virus (RVFV) causes outbreaks of severe disease in people and livestock throughout Africa and the Arabian Peninsula. Human RVFV infections generally manifest as a self-limiting febrile illness, but in some individuals, the disease can progress to a fatal encephalitis or hemorrhagic syndrome. Little is known about the host characteristics that predispose development of more severe disease. Early in infection, interferon-mediated antiviral responses are critical for controlling RVFV replication, but the roles of downstream adaptive immune responses in determining clinical outcome have not been examined. Here, using a C57BL/6 mouse disease model, we evaluated the roles of B cells and T cells in RVFV pathogenesis. Given the profound inhibition of the innate response by the viral NSs protein and rapid course of wild-type infection, we utilized an attenuated RVFV lacking NSs to examine host responses following primary infection. Experiments utilizing B-cell-deficient mice or targeted T cell depletions of wild-type mice demonstrated that B cells and CD4 + T cells, but not CD8 + T cells, were critical for mediating viral clearance, even in the presence of a functional innate response. One-third of CD4 + -depleted mice developed severe neurologic disease following infection, in contrast to virus-infected mock-depleted mice that showed no clinical signs. CD4 + T cells were required for robust IgG and neutralizing antibody responses that correlated with RVFV clearance from peripheral tissues. Furthermore, CD4 + -depleted mice demonstrated significantly stronger proinflammatory responses relative to controls, suggesting CD4 + T cells regulate immune responses to RVFV infection. Together, these results indicate CD4 + T cells are critical determinants of RVFV pathogenesis and play an important role in preventing onset of neurologic disease.
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Michihiro Kobayashi;
Yang Lin;
Akansha Mishra;
Chris Shelly;
Rui Gao;
Colton W Reeh;
Paul Zhiping Wang;
Rongwen Xi;
Yunlong Liu;
Pamela Wenzel;
Eliver Ghosn;
Yan Liu;
Momoko Yoshimoto
The self-renewal ability is a unique property of fetal-derived innate-like B-1a lymphocytes, which survive and function without being replenished by bone marrow (BM) progenitors. However, the mechanism by which IgM-secreting mature B-1a lymphocytes self-renew is poorly understood. In this study, we showed that Bmi1 was critically involved in this process. Although Bmi1 is considered essential for lymphopoiesis, the number of mature conventional B cells was not altered when Bmi1 was deleted in the B cell lineage. In contrast, the number of peritoneal B-1a cells was significantly reduced. Peritoneal cell transfer assays revealed diminished self-renewal ability of Bmi1-deleted B-1a cells, which was restored by additional deletion of Ink4-Arf, the well-known target of Bmi1. Fetal liver cells with B cell-specific Bmi1 deletion failed to repopulate peritoneal B-1a cells, but not other B- 2 lymphocytes after transplantation assays, suggesting that Bmi1 may be involved in the developmental process of B-1 progenitors to mature B-1a cells. Although Bmi1 deletion has also been shown to alter the microenvironment for hematopoietic stem cells, fatassociated lymphoid clusters, the reported niche for B-1a cells, were not impaired in Bmi1-/- mice. RNA expression profiling suggested lysine demethylase 5B (Kdm5b) as another possible target of Bmi1, which was elevated in Bmi1-/- B-1a cells in a stress setting and might repress B-1a cell proliferation. Our work has indicated that Bmi1 plays pivotal roles in self-renewal and maintenance of fetal-derived B-1a cells.
Urea transporters (UT) play a vital role in the mechanism of urine concentration and are recognized as novel targets for the development of salt-sparing diuretics. Thus, UT inhibitors are promising for development as novel diuretics. In the present study, a novel UT inhibitor with a diarylamide scaffold was discovered by high-throughput screening. Optimization of the inhibitor led to the identification of a promising preclinical candidate, N-[4-(acetylamino)phenyl]-5-nitrofuran-2-carboxamide (1H), with excellent in vitro UT inhibitory activity at the submicromolar level. The half maximal inhibitory concentrations of 1H against UT-B in mouse, rat, and human erythrocyte were 1.60, 0.64, and 0.13 μmol/L, respectively. Further investigation suggested that 8 μmol/L 1H more powerfully inhibited UT-A1 at a rate of 86.8% than UT-B at a rate of 73.9% in MDCK cell models. Most interestingly, we found for the first time that oral administration of 1H at a dose of 100 mg/kg showed superior diuretic effect in vivo without causing electrolyte imbalance in rats. Additionally, 1H did not exhibit apparent toxicity in vivo and in vitro, and possessed favorable pharmacokinetic characteristics. 1H shows promise as a novel diuretic to treat hyponatremia accompanied with volume expansion and may cause few side effects.
Human respiratory syncytial virus (RSV) is an important pathogen causing acute lower respiratory tract disease in children. The RSV attachment glycoprotein (G) is not required for infection, as G-null RSV replicates efficiently in several cell lines. Our laboratory previously reported that the viral fusion (F) protein is a determinant of strain-dependent pathogenesis. Here, we hypothesized that virus dependence on G is determined by the strain specificity of F. We generated recombinant viruses expressing G and F, or null for G, from the laboratory A2 strain (Katushka RSV-A2GA2F [kRSV-A2GA2F] and kRSV-GstopA2F) or the clinical isolate A2001/2-20 (kRSV-2-20G2-20F and kRSV-Gstop2-20F). We quantified the virus cell binding, entry kinetics, infectivity, and growth kinetics of these four recombinant viruses in vitro. RSV expressing the 2-20 G protein exhibited the greatest binding activity. Compared to the parental viruses expressing G and F, removal of 2-20 G had more deleterious effects on binding, entry, infectivity, and growth than removal of A2 G. Overall, RSV expressing 2-20 F had a high dependence on G for binding, entry, and infection.