Association of a reactive intermediate derived from 1′,6‐dihydroxy metabolite with benzbromarone‐induced hepatotoxicity

Treatment with benzbromarone can be associated with liver injury, but the detailed mechanism remains unknown. Our recent studies demonstrated that benzbromarone was metabolized to 1′,6‐dihydroxybenzbromarone and followed by formation of reactive intermediates that were trapped by glutathione, suggesting that the reactive intermediates may be responsible for the liver injury. The aim of this study was to clarify whether the reactive intermediates derived from 1′,6‐dihydroxybenzbromarone is a risk factor of liver injury in mice. An incubation study using mouse liver microsomes showed that the rates of formation of 1′,6‐dihydroxybenzbromarone from benzbromarone were increased by pretreatment with dexamethasone. Levels of a hepatic glutathione adduct derived from 1′,6‐dihydroxybenzbromarone were increased by pretreatment with dexamethasone. Furthermore, plasma alanine amino transferase activities were increased in mice treated with benzbromarone after pretreatment with dexamethasone. The results suggest that the reactive intermediate derived from 1′,6‐dihydroxybenzbromarone may be associated with liver injury.     

A multi-body dynamics study on a weight-drop test of rat brain injury

Traumatic brain injury (TBI), induced by impact of an object with the head, is a major health problem worldwide. Rats are a well-established animal analogue for study of TBI and the weight-drop impact-acceleration (WDIA) method is a well-established model in rats for creating diffuse TBI, the most common form of TBI seen in humans. However, little is known of the biomechanics of the WDIA method and, to address this, we have developed a four-degrees-of-freedom multi-body mass-spring-damper model for the WDIA test in rats. An analytical expression of the maximum skull acceleration, one of the important head injury predictor, was derived and it shows that the maximum skull acceleration is proportional to the impact velocity but independent of the impactor mass. Furthermore, a dimensional analysis disclosed that the maximum force on the brain and maximum relative displacement between brain and skull are also linearly proportional to impact velocity. Additionally, the effects of the impactor mass were examined through a parametric study from the developed multi-body dynamics model. It was found that increasing impactor mass increased these two brain injury predictors.     

Involvement of Nox2 and Nox4 NADPH oxidases in early brain injury after subarachnoid hemorrhage

Oxidative stress is responsible for a poor prognosis of subarachnoid hemorrhage (SAH) patients. Nox2 has been shown to participate in SAH-induced early brain injury (EBI). Nox4 is another major subtype of Nox family widely expressed in central nervous system (CNS). Here, we investigated the role of Nox4 and whether there was a synergistic effect of Nox2 and Nox4 in SAH-induced EBI. Clinical brain biopsies of four patients with traumatic brain injury (TBI) and perihematomal brain tissue from six subjects with SAH were examined. Gp91ds-tat (a specific inhibitor of Nox2), GKT137831 (a specific inhibitor of Nox4), and apocynin (a non-specific Nox inhibitor) were used to test the role of Nox2 and Nox4. The protein levels of Nox2 and Nox4 were elevated in rat neurons and astrocytes at 12?h after SAH, and in cultured brain microvascular endothelial cells at 24?h after exposure to OxyHb. Similarly, there were higher Nox2 and Nox4 protein levels in perihematomal neurons and astrocytes in SAH patients than that in brain tissue from subjects with TBI. In SAH rat model, gp91ds-tat and GKT137831 could reduce SAH-induced neuronal death and degeneration, whereas apocynin did not induce a more intense neuroprotection. Consistently, in in vitro SAH model, siRNA-mediated silencing of Nox2 and Nox4 suppressed the OxyHb-induced neuronal apoptosis, whereas Nox2 and Nox4 co-knockdown also did not show a remarkable overlay effect. In conclusion, Nox4 should contribute to the pathological processes in SAH-induced EBI, and there was not an overlay effect of Nox2 inhibition and Nox4 inhibition on preventing SAH-induced EBI.     

The neuronal differentiation microenvironment is essential for spinal cord injury repair

Spinal cord injury (SCI) often leads to substantial disability due to loss of motor function and sensation below the lesion. Neural stem cells (NSCs) are a promising strategy for SCI repair. However, NSCs rarely differentiate into neurons; they mostly differentiate into astrocytes because of the adverse microenvironment present after SCI. We have shown that myelin-associated inhibitors (MAIs) inhibited neuronal differentiation of NSCs. Given that MAIs activate epidermal growth factor receptor (EGFR) signaling, we used a collagen scaffold-tethered anti-EGFR antibody to attenuate the inhibitory effects of MAIs and create a neuronal differentiation microenvironment for SCI repair. The collagen scaffold modified with anti-EGFR antibody prevented the inhibition of NSC neuronal differentiation by myelin. After transplantation into completely transected SCI animals, the scaffold-linked antibodies induced production of nascent neurons from endogenous and transplanted NSCs, which rebuilt the neuronal relay by forming connections with each other or host neurons to transmit electrophysiological signals and promote functional recovery. Thus, a scaffold-based strategy for rebuilding the neuronal differentiation microenvironment could be useful for SCI repair.     

Improvement in Double Staining With Fluoro-Jade C and Fluorescent Immunostaining: FJC Staining Is Not Specific to Degenerating Mature Neurons

Fluoro-Jade C (FJC) staining has been used to detect degenerating neurons in tissue sections. It is a simple and easy staining procedure and does not depend on the manner of cell death. In some experiments, double staining with FJC and fluorescent immunostaining (FI) is required to identify cell types. However, pretreatment for FJC staining contains some processes that are harsh to fluorophores, and the FI signal is greatly reduced. To overcome this issue, we improved the double staining protocol to acquire clear double-stained images by introducing the labeled streptavidin–biotin system. In addition, several studies indicate that FJC can label non-degenerating glial cells, including resting/reactive astrocytes and activated microglia. Moreover, our previous study indicated that degenerating mesenchymal cells were also labeled by FJC, but it is still unclear whether FJC can label degenerating glial cells. Acute encephalopathy model mice contained damaged astrocytes with clasmatodendrosis, and 6-aminonicotinamide-injected mice contained necrotic astrocytes and oligodendrocytes. Using our improved double staining protocol with FJC and FI, we detected FJC-labeled degenerating astrocytes and oligodendrocytes with pyknotic nuclei. These results indicate that FJC is not specific to degenerating neurons in some experimental conditions:     

CGRP is essential for protection against alveolar epithelial cell necroptosis by activating the AMPK/L-OPA1 signaling pathway during acute lung injury

Alveolar epithelial cell (AEC) necroptosis is critical to disrupt the alveolar barrier and provoke acute lung injury (ALI). Here, we define calcitonin gene-related peptide (CGRP), the most abundant endogenous neuropeptide in the lung, as a novel modulator of AEC necroptosis in lipopolysaccharide (LPS)-induced ALI. Upon LPS-induced ALI, overexpression of Cgrp significantly mitigates the inflammatory response, alleviates lung tissue damage, and decreases AEC necroptosis. Similarly, CGRP alleviated AEC necroptosis under the LPS challenge in vitro. Previously, we identified that long optic atrophy 1 (L-OPA1) deficiency mediates mitochondrial fragmentation, leading to AEC necroptosis. In this study, we discovered that CGRP positively regulated mitochondrial fusion through stabilizing L-OPA1. Mechanistically, we elucidate that CGRP activates AMP-activated protein kinase (AMPK). Furthermore, the blockade of AMPK compromised the protective effect of CGRP against AEC necroptosis following the LPS challenge. Our study suggests that CRGP-mediated activation of the AMPK/L-OPA1 axis may have potent therapeutic benefits for patients with ALI or other diseases with necroptosis.     

Coconut products alleviate hyperglycaemic,hyperlipidimic and nephropathy indices in streptozotocin-induced diabetic wistar rats

Type 2 diabetes mellitus (T2DM) is a chronic and one of the most common metabolic diseases affecting large proportion of world population. Diabetes-induced changes in lipid and renal parameters are major risk factors contributing to diabetic complications such as diabetic nephropathy and cardiovascular diseases. Due to adverse effects associated with pharmacological intervention in the T2DM treatment, there is an increased interest in the research focussing on identifying novel plant based therapeutic agents. Here we report the effects of various coconut products on diabetic, lipid and renal parameters in streptozotocin (STZ)-induced diabetic rat model. Diabetic rats demonstrated a significant increase in serum glucose, and glycated haemoglobin levels (HbA1c). Lipid parameters including triglycerides, total cholesterol, low density lipoprotein cholesterol (LDL-cholesterol) and very low density lipoprotein cholesterol (VLDL-cholesterol) were found to be significantly increased, while high density lipoprotein cholesterol (HDL-cholesterol) was significantly declined in diabetic rats. Diabetic rats also displayed increased serum and kidney creatinine, urea, and total protein levels and increased urine glucose, urea, albumin and creatinine levels. Contrastingly, treatment with virgin and filtered coconut oils, coconut water and coconut milk resulted in a significant reversal in the levels of above studied parameters in diabetic rats. Further, these coconut products markedly prevented diabetes induced histopathological changes in kidney tissue. Collectively, the data demonstrate the antidiabetic, hypolipidemic and renal protective properties of various coconut products and underscore the importance of regular consumption of plant based medicinal products in the treatment of T2DM and its complications.     

Human kidney anion exchanger 1 interacts with kinesin family member 3B (KIF3B)

Impaired trafficking of human kidney anion exchanger 1 (kAE1) to the basolateral membrane of α-intercalated cells of the kidney collecting duct leads to the defect of the Cl⁻/ exchange and the failure of proton (H⁺) secretion at the apical membrane of these cells, causing distal renal tubular acidosis (dRTA). In the sorting process, kAE1 interacts with AP-1 mu1A, a subunit of AP-1A adaptor complex. However, it is not known whether kAE1 interacts with motor proteins in its trafficking process to the plasma membrane or not. We report here that kAE1 interacts with kinesin family member 3B (KIF3B) in kidney cells and a dileucine motif at the carboxyl terminus of kAE1 contributes to this interaction. We have also demonstrated that kAE1 co-localizes with KIF3B in human kidney tissues and the suppression of endogenous KIF3B in HEK293T cells by small interfering RNA (siRNA) decreases membrane localization of kAE1 but increases its intracellular accumulation. All results suggest that KIF3B is involved in the trafficking of kAE1 to the plasma membrane of human kidney α-intercalated cells.     

Vesicle-associated membrane protein-2 (VAMP2) mediates cAMP-stimulated renin release in mouse juxtaglomerular cells

Renin is essential for blood pressure control. Renin is stored in granules in juxtaglomerular (JG) cells, located in the pole of the renal afferent arterioles. The second messenger cAMP stimulates renin release. However, it is unclear whether fusion and exocytosis of renin-containing granules is involved. In addition, the role of the fusion proteins, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment proteins), in renin release from JG cells has not been studied. The vesicle SNARE proteins VAMP2 (vesicle associated membrane protein 2) and VAMP3 mediate cAMP-stimulated exocytosis in other endocrine cells. Thus, we hypothesized that VAMP2 and/or -3 mediate cAMP-stimulated renin release from JG cells. By fluorescence-activated cell sorting, we isolated JG cells expressing green fluorescent protein and compared the relative abundance of VAMP2/3 in JG cells versus total mouse kidney mRNA by quantitative PCR. We found that VAMP2 and VAMP3 mRNA are expressed and enriched in JG cells. Confocal imaging of primary cultures of JG cells showed that VAMP2 (but not VAMP3) co-localized with renin-containing granules. Cleavage of VAMP2 and VAMP3 with tetanus toxin blocked cAMP-stimulated renin release from JG cells by ~50% and impaired cAMP-stimulated exocytosis by ~50%, as monitored with FM1-43. Then we specifically knocked down VAMP2 or VAMP3 by adenoviral-mediated delivery of short hairpin silencing RNA. We found that silencing VAMP2 blocked cAMP-induced renin release by ~50%. In contrast, silencing VAMP3 had no effect on basal or cAMP-stimulated renin release. We conclude that VAMP2 and VAMP3 are expressed in JG cells, but only VAMP2 is targeted to renin-containing granules and mediates the stimulatory effect of cAMP on renin exocytosis.