Protective effect of photodegradation product of nifedipine against tumor necrosis factor alpha-induced oxidative stress in human glomerular endothelial cells

Recently, increasing evidence suggests that the antihypertensive drug nifedipine acts as a protective agent for endothelial cells, and that the activity is unrelated to its calcium channel blocking. Nitrosonifedipine (NO-NIF) is metabolically and photochemically produced from nifedipine, and NO-NIF has been recognized as a contaminant of nifedipine because it has no antihypertensive effect. Treatment of tumor necrosis factor-α (TNF-α) suppressed the cell viability and facilitated the expression of Inter-Cellular Adhesion Molecule 1(ICAM-1) in human glomerular endothelial cells (HGECs) though, pretreatment of NO-NIF significantly recovered the TNF-α-induced cell damage to the same extent as Trolox-C did, and suppressed the ICAM-1 expression in a concentration dependent manner. In addition, NO-NIF inhibited the cell toxicity induced by cumene hydroperoxide, which hampers the integrity of cell membrane through oxidative stress, as effective as Trolox-c. These data suggest that NO-NIF is a candidate for a new class of antioxidative drug that protect cells against oxidative stress in glomerular endothelial cells

Development of a novel automatic ascites filtration and concentration equipment with multi‐ring‐type roller pump units for cell‐free and concentrated ascites reinfusion therapy

Cell‐free and concentrated ascites reinfusion therapy (CART) is an effective therapy for refractory ascites. However, CART is difficult to perform as ascites filtration and concentration is a complicated procedure. Moreover, the procedure requires the constant assistance of a clinical engineer or/and the use of an expensive equipment for the multi‐purpose blood processing. Therefore, we developed a CART specialized equipment (mobility CART [M‐CART]) that could be used safely with various safety measures and automatic functions such as automatic washing of clogged filtration filter and self‐regulation of the concentration ratio. Downsizing, lightning of the weight, and automatic processing in M‐CART required the use of newly developed multi‐ring‐type roller pump units. This equipment was approved under Japanese regulations in 2018. In performing 41 sessions of CART (for malignant ascites, 22 sessions; and hepatic ascites, 19 sessions) using this equipment in 17 patients, no serious adverse event occurred. An average of 4494 g of ascites was collected and the total amount of ascites was processed in all the sessions without any trouble. The mean weight of the processed ascites was 560 g and the mean concentration ratio was 8.0. The ascites were processed at a flow rate of 50 mL/min. The mean ascites processing time was 112.5 minutes and a 106.5‐minutes (95.2%) ascites processing was performed automatically. The operator responded to alarms or support information 3.2 times on average (3.1 minutes, 2.1% of ascites processing time). Human errors related to ascites processing were detected by M‐CART at 0.4 times per session on average and were appropriately addressed by the operator. The frequencies of automatic washing of clogged filtration filter and self‐regulation of the concentration ratio were 31.7% and 53.7%, respectively. The mean recovery rates (recovery dose) of protein, albumin, and immunoglobulin G were 72.9%, 72.9%, and 71.2% (65.9 g, 34.9 g, and 13.2 g), respectively. Steroids were administered in 92.7% of the sessions to prevent fever and the mean increase in body temperature was 0.53°C. M‐CART is a compact and lightweight automatic CART specialized equipment that can safely and easily process a large quantity of ascites without the constant assistance of an operator

GFAP-immunoreactive astrocytes in the ventral anterior-lateral complex of the thalamus (VAL) of WT, homozygous <i>Slc19a3</i> KO, and <i>Slc19a3</i> KI mice.

<p>A, C, E, G. Low-magnification images of reactive astrocytes in the thalamus of a WT mouse fed with a conventional diet at postnatal day 35 (A); a homozygous KO mouse fed with thiamine 0.60 mg/100 g food for 5 days (C) and for 12 days (E); and a KI mouse fed with thiamine 0.60 mg/100 g for 14 days (G). B, D, F, and H are high-magnification images of the VAL area (insets) shown in panels of A, C, E, and G, respectively. Scale bars, 500 μm (upper panel) and 100 μm (lower panel).</p

Thiamine concentrations in the blood and brain of WT, homozygous <i>Slc19a3</i> KO, and <i>Slc19a3</i> KI mice.

<p>Whole blood and cerebrum homogenates of KO and KI mice were obtained at 5 and 14 days of thiamine restriction, respectively. A. Thiamine concentration in whole blood (nmol/mL). B. Thiamine concentration in cerebrum homogenates (nmol/g wet weight). Bullets, individual thiamine concentrations; and bars, mean values.</p

Temporal profile of NeuN-immunostaining in the hippocampus of WT, homozygous <i>Slc19a3</i> KO, and <i>Slc19a3</i> KI mice.

<p>NeuN-immunoreactive neurons in the submedial nucleus of the thalamus (SMT) and ventral anterior-lateral complex of the thalamus (VAL). A. Low magnification of a Nissl-stained brain slice at the level of the hippocampus (HIP). B. High-magnification overview of the specific regions of the thalamic area of a WT mouse. C. Closer view of a region contained in the inset of panel B. D–E. Brain slices of a homozygous KO mouse fed with thiamine 1.71 mg/100 g food for 12 days (D), thiamine 0.6 mg/100 g food for 5 days (E) and for 12 days (F). G–I. Brain slices of a homozygous KI mouse fed with thiamine 1.71 mg/100 g food for 14 days (G), thiamine 0.6 mg/100 g food for 14 days (H) and thiamine 0.27 mg/100 g food for 14 days (I). Scale bar, 200 μm. VPM: ventral posteromedial nucleus of the thalamus, CM: central medial nucleus of the thalamus, VM: ventral medial nucleus of the thalamus, mtt: mammillothalamic tract.</p

High-dose thiamine prevents brain lesions and prolongs survival of <i>Slc19a3</i>-deficient mice

<div><p>SLC19A3 deficiency, also called thiamine metabolism dysfunction syndrome-2 (THMD2; OMIM 607483), is an autosomal recessive neurodegenerative disorder caused by mutations in <i>SLC19A3</i>, the gene encoding thiamine transporter 2. To investigate the molecular mechanisms of neurodegeneration in SLC19A3 deficiency and whether administration of high-dose thiamine prevents neurodegeneration, we generated homozygous Slc19a3 E314Q knock-in (KI) mice harboring the mutation corresponding to the human SLC19A3 E320Q, which is associated with the severe form of THMD2. Homozygous KI mice and previously reported homozygous <i>Slc19a3</i> knock-out (KO) mice fed a thiamine-restricted diet (thiamine: 0.60 mg/100 g food) died within 30 and 12 days, respectively, with dramatically decreased thiamine concentration in the blood and brain, acute neurodegeneration, and astrogliosis in the submedial nucleus of the thalamus and ventral anterior-lateral complex of the thalamus. These findings may bear some features of thiamine-deficient mice generated by pyrithiamine injection and a thiamine-deficient diet, suggesting that the primary cause of THMD2 could be thiamine pyrophosphate (TPP) deficiency. Next, we analyzed the therapeutic effects of high-dose thiamine treatment. When the diet was reverted to a conventional diet (thiamine: 1.71 mg/100 g food) after thiamine restriction, all homozygous KO mice died. In contrast, when the diet was changed to a high-thiamine diet (thiamine: 8.50 mg/100 g food) after thiamine restriction, more than half of homozygous KO mice survived, without progression of brain lesions. Unexpectedly, when the high-thiamine diet of recovered mice was reverted to a conventional diet, some homozygous KO mice died. These results showed that acute neurodegeneration caused by thiamine deficiency is preventable in most parts, and prompt high-dose thiamine administration is critical for the treatment of THMD2. However, reduction of thiamine should be performed carefully to prevent recurrence after recovery of the disease.</p></div

Recovery of homozygous <i>Slc19a3</i> KO mice with a high-thiamine diet after thiamine restriction.

<p>Homozygous KO mice were fed a thiamine-restricted diet (thiamine 0.60 mg/100 g food) for 2 days and then changed to a high-thiamine diet (thiamine 8.50 mg/100 g food) for 28 days. A. Low magnification of the NeuN-immunoreactive neurons in the thalamic area in a recovered homozygous KO mouse. B. High magnification of the inset of panel A. The submedial nucleus of the thalamus (SMT) and ventral anterior-lateral complex of the thalamus (VAL) are shown. C, GFAP immunostaining of the serial sections of B, respectively. Scale bar, 500 μm. The mean numbers of NeuN-immunoreactive neurons in the thalamic area and cortex of 3 homozygous KO mice are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180279#pone.0180279.g004" target="_blank">Fig 4A–4C</a>.</p

Fluoro-Jade C staining of WT, homozygous KO mice fed with a thiamine-restricted diet, and homozygous KO mice fed with a thiamine-restricted diet for 2 days and reverted to a high-thiamine diet.

<p>Brain slices of a WT and homozygous KO mouse fed with thiamine 1.71 mg/100 g food for 8 days, and surviving homozygous KO mice with high-thiamine diet showing the SMT (A, B, C), VAL (D, E, F), and cortex (G, H, I). Overview of the specific regions of the thalamic area of a WT mouse (J), with red boxes indicating the region of each part. Scale bar, 100 μm.</p

Survival rates of <i>Slc19a3</i> KO and KI mice fed with a conventional (thiamine: 1.71 mg/100 g food) and thiamine-deficient (thiamine: 0.60 or 0.27 mg/100 g food) diet.

<p>A. Survival rates of <i>Slc19a3</i> KO mice. B. Survival rates of <i>Slc19a3</i> KI mice. Thick lines, wild type (+/+); broken lines, heterozygous (+/-); and dotted lines, homozygous mice (-/-).</p

Numbers of NeuN-immunoreactive neurons in the thalamic area and cortex of WT, homozygous <i>Slc19a3</i> KO and KI mice.

<p>The numbers of NeuN-immunoreactive neurons of homozygous KO (A) and KI (D) mice in the submedial nucleus of the thalamus (SMT) (0.13 mm²); homozygous KO (B) and KI (E) mice in the ventral anterior-lateral complex of the thalamus (VAL) (0.2 mm²); and homozygous KO (C) and KI (F) mice in the cortex (0.2 mm²). Mean numbers of three experiments are shown. One-way ANOVA, two-way ANOVA, and the Steel–Dwass test were used for statistical comparisons. Asterisks (*) indicate that the numbers of NeuN-immunoreactive neurons of KO and KI mice fed with thiamine-restricted diets were significantly different from those of WT mice (first bar of each graph) at p < 0.05.</p