Sodium Thiosulfate Ameliorates Renovascular Hypertension-Induced Renal Dysfunction and Injury in Rats.

BACKGROUND/AIMS: Arterial stenosis activates the renin-angiotensin-aldosterone system subsequently resulting in renovascular hypertension (RVHT) and renal oxidative injury. We explored the effect of sodium thiosulfate (STS, Na2S2O3), a developed antioxidant in clinical trial, on RVHT-induced hypertension and renal oxidative injury in rats. METHODS: We induced RVHT in male Wistar rats with bilaterally partial ligation of renal arteries in the 2-kidney 2-clip model. We evaluated the STS effect on RVHT-induced oxidative injury and apoptosis by a chemiluminescence amplification method, Western blot, and immunohistochemistry. RESULTS: We found STS displayed a dose-dependent antioxidant H2O2 activity and adapted the maximal scavenging H2O2 activity of STS at the dosage of 0.1 g/kg intraperitoneally 3 times/week for 4 weeks in RVHT rats. RVHT induced a significant elevation of arterial blood pressure, blood reactive oxygen species amount, neutrophil infiltration, 4-HNE and NADPH oxidase gp91 expression, Bax/Bcl-2/poly(ADP-ribose) polymerase (PARP)-mediated apoptosis formation, blue Masson-stained fibrosis, and urinary protein level. STS treatment significantly reduced hypertension, oxidative stress, neutrophil infiltration, fibrosis, and Bax/Bcl-2/PARP-mediated apoptosis formation and depressed the urinary protein level in the RVHT models. CONCLUSION: Our results suggest that STS treatment could ameliorate RVHT hypertension and renal oxidative injury through antioxidant, antifibrotic, and antiapoptotic mechanisms.

Adipose-Derived Stem Cells and Their Derived Microvesicles Ameliorate Detrusor Overactivity Secondary to Bilateral Partial Iliac Arterial Occlusion-Induced Bladder Ischemia.

(1) Background: We established a new bladder ischemia rat model through bilateral partial iliac arterial occlusion (BPAO) and investigated the therapeutic effect of adipose-derived stem cells (ADSCs) and ADSC-derived microvesicles (MVs); (2) Methods: The study included four groups: (1) sham, (2) BPAO, (3) BPAO + ADSCs, and (4) BPAO + ADSC-derived MVs. Female Wistar rats with BPAO were injected with ADSCs or ADSC-derived MVs through the femoral artery. Doppler flowmetry and real-time laser speckle contrast imaging were performed to quantify blood flow in the common iliac arteries and bladder microcirculation. A 24-h behavior study and transcystometrogram were conducted after 2 weeks. Bladder histology, immunostaining, and lipid peroxidation assay were performed. The expressions of P2X2, P2X3, M2, and M3 receptors and nerve growth factor (NGF) were evaluated; (3) Results: BPAO significantly reduced bladder microcirculation, intercontraction interval (ICI), and bladder volume and increased the amplitude of nonvoiding contraction, neutrophil infiltration, and malondialdehyde and NGF levels. ADSCs and ADSC-derived MVs significantly ameliorated these effects. The results of Western blot showed that the BPAO group exhibited the highest expression of M3 and P2X2 receptors. ADSCs significantly attenuated the expressions of M2 and P2X2 receptors. ADSC-derived MVs significantly attenuated the expressions of M3 and P2X2 receptors; (4) Conclusions: ADSCs and ADSC-derived MVs ameliorated the adverse effects of BPAO including bladder overactivity, bladder ischemia, and oxidative stress. Inflammation, muscarinic signaling, purinergic signaling, and NGF might be involved in the therapeutic mechanism.

Sulforaphane improves voiding function via the preserving mitochondrial function in diabetic rats.

BACKGROUND: Hyperglycemia evoked oxidative stress contributing to diabetes (DM)-induced voiding dysfunction. We explored whether antioxidant sulforaphane,a NF-E2-related nuclear factor erythroid-2 (Nrf-2) activator, may ameliorate DM-induced bladder dysfunction. METHODS: DM was induced by streptozotocin and sulforaphanewas administered before DM induction.Bladder reactive oxygen species (ROS) were determined by an ultrasensitive chemiluminescence analyzer. Mitochondrial function index mitochondrial Bax and cytosolic cytochrome c, antioxidant defense Nrf-2/HO-1, endoplasmic reticulum stress marker ATF-6/CHOP, and caspase 3/PARP were evaluated by Western blot. RESULTS: DM increased Keap1 and reduced Nrf-2 expression, associated with increase of bladder ROS, mitochondrial Bax translocation, cytosolic cytochrome c release, ATF-6/CHOP, caspase-3/PARP in bladders which resulted in voiding dysfunction by increased intercontraction intervals and micturition duration. However, sulforaphanesignificantly increased nuclear Nrf-2/HO-1axis expression, decreased bladder ROS amount, mitochondrial Bax translocation, cytochrome c release, ATF-6/CHOP and caspase 3/PARP/apoptosis, thereby improved the voiding function by the shortened intercontraction intervals and micturition duration. CONCLUSION: We suggest that sulforaphanevia activating Nrf-2/HO-1 signaling preserved mitochondrial function and suppressed DM-induced ROS, endoplasmic reticulum stress, apoptosis and voiding dysfunction.

Repetitively hypoxic preconditioning attenuates ischemia/reperfusion-induced liver dysfunction through upregulation of hypoxia-induced factor-1 alpha-dependent mitochondrial Bcl-xl in rat.

Repetitive hypoxic preconditioning (HP) enforces protective effects to subsequently severe hypoxic/ischemic stress. We hypothesized that HP may provide protection against ischemia/reperfusion (I/R) injury in rat livers via hypoxia-induced factor-1 alpha (HIF-1α)/reactive oxygen species (ROS)-dependent defensive mechanisms. Female Wistar rats were exposed to hypoxia (15 h/day) in a hypobaric hypoxic chamber (5500 m) for HP induction, whereas the others were kept in sea level. These rats were subjected to 45 min of hepatic ischemia by portal vein occlusion followed by 6 h of reperfusion. We evaluated HIF-1α in nuclear extracts, MnSOD, CuZnSOD, catalase, Bad/Bcl-xL/caspase 3/poly-(ADP-ribose)-polymerase (PARP), mitochondrial Bcl-xL, and cytosolic cytochrome C expression with Western blot and nitroblue tetrazolium/3-nitrotyrosine stain. Kupffer cell infiltration and terminal deoxynucleotidyl transferase-mediated nick-end labeling method apoptosis were determined by immunocytochemistry. The ROS value from liver surface and bile was detected by an ultrasensitive chemiluminescence-amplification method. Hepatic function was assessed with plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. HP increased nuclear translocation of HIF-1α and enhanced Bcl-xL, MnSOD, CuZnSOD, and catalase protein expression in a time-dependent manner. The response of HP enhanced hepatic HIF-1α, and Bcl-xL expression was abrogated by a HIF-1α inhibitor YC-1. Hepatic I/R increased ROS levels, myeloperoxidase activity, Kupffer cell infiltration, ALT and AST levels associated with the enhancement of cytosolic Bad translocation to mitochondria, release of cytochrome C to cytosol, and activation of caspase 3/PARP-mediated apoptosis. HP significantly ameliorated hepatic I/R-enhanced oxidative stress, apoptosis, and mitochondrial and hepatic dysfunction. In summary, HP enhances HIF-1α/ROS-dependent cascades to upregulate mitochondrial Bcl-xL protein expression and to confer protection against I/R injury in the livers.

Glycine tomentella hayata extract and its ingredient daidzin ameliorate cyclophosphamide-induced hemorrhagic cystitis and oxidative stress through the action of antioxidation, anti-fibrosis, and anti-inflammation.

We explored the therapeutic potential of intragastric administration of traditional Chinese medicine Glycine tomentella Hayata (I-Tiao-Gung [ITG]) extract and its major component Daidzin on cyclophosphamide (CYP)-induced cystitis, oxidative stress, fibrosis, inflammation, and bladder hyperactivity in rats. Female Wistar rats were divided into control, CYP (200 mg/kg), CYP+ITG (1.17 g/kg/day), and CYP+Daidzin (12.5 mg/kg/day) groups. We measured the voiding function by the transcystometrogram and evaluated the pathology with the hematoxylin and eosin and Masson stain. We determined the bladder reactive oxygen species (ROS) amount by an ultrasensitive chemiluminescence analyzer, the expression of 3-nitrotyrosine (3-NT) and NADPH oxidase 4 (NOX4) by Western blot and the expression of multiple cytokine profiles, including matrix metalloproteinase (MMP)-8 and tissue inhibitor of metalloproteinase (TIMP)-1 through a cytokine array. ITG extract contains 1.07% of Daidzin through high-performance liquid chromatography. The effect of ITG extract and Daidzin in scavenging hydrogen peroxide activity was more efficient than distilled water. CYP-induced higher urination frequency, shorter intercontraction interval, and lower maximal voiding pressure in the bladders and these symptoms were significantly ameliorated in CYP+ITG and CYP+Daidzin groups. The amount of in vivo bladder ROS and the expression of 3-NT and NOX4 expressions were significantly increased in CYP group but were efficiently decreased in the CYP+ITG and CYP+Daidzin groups. CYP-induced fibrosis, hemorrhage, leukocyte infiltration, and edema in the bladders were significantly attenuated in the CYP+ITG and CYP+Daidzin groups. These results suggested that ITG extract and its active component Daidzin effectively improved CYP-induced oxidative stress, inflammation, and fibrosis through inhibiting the MMP-8, TIMP-1, and oxidative stress.

I-Tiao-Gung extract through its active component daidzin improves cyclophosphamide-induced bladder dysfunction in rat model.

AIMS: We explored the therapeutic potential of intragastric administration traditional Chinese medicine Glycine tomentella Hayata (I-Tiao-Gung, ITG) extract and its active component Daidzin on cyclophosphamide (CYP)-induced cystitis and bladder hyperactivity in rats. METHODS: Female Wistar rats were divided into control, CYP (200 mg/kg), CYP + ITG (1.17 g/kg/day), CYP + Daidzin (12.5 mg/kg/day), and 1 week of ITG preconditioning with CYP (ITG + CYP) groups. We determined the trans cystometrogram associated with external urethral sphincter electromyogram, and the expression of M2 and M3 muscarinic and P2 × 2 and P2 × 3 purinergic receptors by Western blot in these animals. RESULTS: ITG extract contains 1.07% of Daidzin and 0.77% of Daidzein by high-performance liquid chromatography. Daidzin was more efficient than Daidzein in scavenging H2 O2 activity by a chemiluminescence analyzer. CYP induced higher frequency, shorter intercontraction interval, lower maximal voiding pressure, lower threshold pressure, and Phase-2 emptying contraction with a depressed external urethral sphincter electromyogram activity, and hemorrhagic cystitis in the bladders. The altered parameters by CYP were significantly improved in CYP + ITG, CYP + Daidzin, and ITG + CYP groups. The P2 × 2 and P2 × 3 expressions were significantly upregulated in CYP group, but were depressed in CYP + ITG, CYP + Daidzin, and ITG + CYP groups. The M2 expression was not significantly different among these five groups. The M3 expression was significantly upregulated in CYP group, but was significantly depressed in CYP + ITG, CYP + Daidzin, and ITG + CYP groups. CONCLUSIONS: These data suggest that ITG extract through its active component Daidzin effectively improved CYP-induced cystitis by the action of restoring Phase 2 activity and inhibiting the expressions of P2 × 2, P2 × 3, and M3 receptors.

l-Theanine inhibits proinflammatory PKC/ERK/ICAM-1/IL-33 signaling, apoptosis, and autophagy formation in substance P-induced hyperactive bladder in rats.

AIMS: Upregulation of substance P (SP) and neurokinin-1 receptor (NK1R) activation induces pro-inflammatory bladder hyperactivity through the PKC/ERK/NF-κB/ICAM-1/IL-33 signaling pathways to increase the leukocyte infiltration and adhesion leading to reactive oxygen species (ROS) production, autophagy, and apoptosis. l-Theanine is a unique non-protein-forming amino acid present in tea (Camellia sinensis [L.] O. Kuntze) with its antioxidant, anti-inflammatory, and relaxation effects to improve cognition, mood, gastric ulcer injury, and cerebral ischemia/reperfusion injury, and posttraumatic stress disorder. We explored the protective effect of l-theanine on SP-induced bladder hyperactivity. METHODS: In urethane-anesthetized female Wistar rats, we explored the transcystometrogram, pelvic nerve activity, proinflammatory PKC/ERK/NF-κB/ICAM-1/IL-33 signaling, apoptosis-related Caspase 3/poly-(ADP-ribose)-polymerase (PARP), and autophagy-mediated LC3 II expression by Western blot, electrophoretic-mobility shift assay and immunohistochemistry, bladder ROS amount by a ultrasensitive chemiluminescence method, and possible ROS sources from the different leukocytes by specific stains in SP-evoked hyperactive bladder. RESULTS: l-Theanine dose-dependently depressed H2 O2 and HOCl activity in vitro. In urethane-anesthetized female Wistar rats, intra-arterial SP through NK1R activation increased voiding frequency (shortened intercontraction intervals) associated with the increase in bladder nerve activity, proinflammatory PKC/ERK/NF-κB/ICAM-1/IL-33 signaling, Caspase 3/PARP-mediated apoptosis, LC3 II-mediated autophagy, ROS amount, neutrophils adhesion, CD68 (monocyte/macrophage) infiltration, and mast cells degranulation in the hyperactive bladder. Intragastrical l-theanine (15 mg/kg) twice daily for 2 weeks efficiently ameliorated all the enhanced parameters in the SP-treated hyperactive bladder. CONCLUSIONS: In conclusion, l-theanine through antioxidant and anti-inflammatory actions ameliorates SP-induced bladder hyperactivity via the inhibition of proinflammatory PKC/ERK/NF-κB/ICAM-1/IL-33 signaling, oxidative stress, bladder nerve hyperactivity, apoptosis, and autophagy. Neurourol. Urodynam. 36:297-307, 2017. © 2016 Wiley Periodicals, Inc.

Catechins Blunt the Effects of oxLDL and its Primary Metabolite Phosphatidylcholine Hydroperoxide on Endothelial Dysfunction Through Inhibition of Oxidative Stress and Restoration of eNOS in Rats.

BACKGROUND/AIMS: We explored the effects of catechins (decaffeinated green tea extracts containing (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate and (-)-epigallocatechin gallate) on atherosclerosis risk factors, oxidized low-density lipoprotein (oxLDL) and its primary metabolite, phosphatidylcholine hydroperoxide (PCOOH) induced oxidative injury in cultured endothelial cell line and rats. METHODS: We used endothelial cell line and male Wistar rats to determine the effect of catechins on oxLDL or PCOOH induced oxidative injury including apoptosis, H2O2 level, vascular responses and urinary 8-isoprostane and nitrite/nitrate concentration. Plasma catechins concentration was determined by a CoulArray HPLC. Responses of aortic and renal vasoconstriction were evaluated by a transonic meter and a full-field laser perfusion imager. RESULTS: PCOOH administration significantly increased H2O2 amounts and cell apoptosis and decreased endothelial nitric oxide synthase (eNOS) expression in the cultured endothelial cells. Catechins pretreatment significantly reduced PCOOH-elevated H2O2 amounts, endothelial cell apoptosis and partly recovered eNOS expression. Intravenous administration of oxLDL, PCOOH or H2O2, not native LDL, significantly decreased renal and aortic blood flow associated with enhanced ICAM-1 expression and 4-hydroxynoneal (4-HNE) accumulation, and decreased eNOS expression in the male Wistrar rats. One hour after oral intake of green tea extracts, 4 peaks of catechins were found in the rat plasma. The increased plasma catechins significantly inhibited oxLDL-, PCOOH- or H2O2-induced renal and aortic vasoconstriction, decreased urinary 8-isoprostane levels, renal ICAM-1 expression and 4-HNE accumulation, and restored nitrite/nitrate amounts and eNOS activity. CONCLUSIONS: Our data suggests that catechins pretreatment decrease PCOOH-induced endothelial apoptosis and arterial vasoconstriction through the action of H2O2 inhibition and eNOS restoration.

Ba-Wei-Di-Huang-Wan through its active ingredient loganin counteracts substance P-enhanced NF-κB/ICAM-1 signaling in rats with bladder hyperactivity.

Overt bladder afferent activation may exacerbate endogenous substance P (SP) release to induce intercellular adhesion molecule-1 (ICAM-1)-mediated inflammation and reactive oxygen species (ROS) production leading to hyperactive bladder. Ba-Wei-Die-Huang-Wan (BWDHW), a traditional Chinese medicine, has been used to treat lower urinary tract symptoms in patients by undefined mechanisms. We explored the possible mechanisms and the active components of BWDHW on exogenous SP-induced bladder hyperactivity. BWDHW contained six major components: loganin, paeoniflorin, 5-hydroxymethylfurfural, cinnamic acid, cinnamaldehyde, and paeonol by high-performance liquid chromatography. In urethane-anesthetized female Wistar rats, we evaluated transcystometrogram, pelvic afferent nerve activity by electrophysiologic recording techniques, ICAM-1 expression by Western blot and immunohistochemistry, ROS amount by an ultrasensitive chemiluminescence method and possible ROS sources from the different leukocytes by specific stains in SP-treated bladder. BWDHW and its major component loganin dose-dependently inhibited H2 O2 and HOCl activity in vitro. Intragastrical BWDHW (250 mg/kg) and loganin (5 mg/kg) twice daily for 2 weeks did not affect the baseline micturition parameters. Intra-arterial SP (20 µg/rat) through neurokinin-1 receptor activation increased voiding frequency (shortened intercontraction intervals), pelvic afferent nerve activity, bladder NF-κB/ICAM-1 expression, bladder ROS amount, neutrophils adhesion to venous endothelium, CD68 (monocyte/macrophage), and mast cell infiltration in the inflamed bladder. BWDHW and loganin pretreatment significantly depressed SP-enhanced pelvic afferent nerve activity, bladder NF-κB/ICAM-1 expression, leukocyte infiltration, and ROS amount, and subsequently improved bladder hyperactivity. In conclusion, our results suggest that BWDHW and its active component loganin improves bladder hyperactivity via inhibiting SP/neurokinin-1 receptor signaling and depressing NF-κB/ICAM-1 mediated oxidative injury and inflammation. Neurourol. Urodynam. 35:771-779, 2016. © 2015 Wiley Periodicals, Inc.

Pretreatment of Sialic Acid Efficiently Prevents Lipopolysaccharide-Induced Acute Renal Failure and Suppresses TLR4/gp91-Mediated Apoptotic Signaling.

BACKGROUND/AIMS: Lipopolysaccharides (LPS) binding to Toll-like receptor 4 (TLR4) activate NADPH oxidase gp91 subunit-mediated inflammation and oxidative damage. Recognizing the high binding affinity of sialic acid (SA) with LPS, we further explored the preventive potential of SA pretreatment on LPS-evoked acute renal failure (ARF). METHODS: We determined the effect of intravenous SA 30 min before LPS-induced injury in urethane-anesthetized female Wistar rats by evaluating kidney reactive oxygen species (ROS) responses, renal and systemic hemodynamics, renal function, histopathology, and molecular mechanisms. RESULTS: LPS time-dependently reduced arterial blood pressure, renal microcirculation, and increased blood urea nitrogen and creatinine in the rats. LPS enhanced monocyte/macrophage infiltration and ROS production, and subsequently impaired kidneys with the enhancement of TLR4/NADPH oxidase gp91/Caspase 3/poly-(ADP-ribose)-polymerase (PARP)-mediated apoptosis in the kidneys. SA pretreatment effectively alleviated LPS-induced ARF. The levels of LPS-increased ED-1 infiltration and ROS production in the kidney were significantly depressed by SA pretreatment. Furthermore, SA pretreatment significantly depressed TLR4 activation, gp91 expression, and Caspase 3/PARP induced apoptosis in the kidneys. CONCLUSION: We suggest that pretreatment of SA significantly and preventively attenuated LPS-induced detrimental effects on systemic and renal hemodynamics, renal ROS production and renal function, as well as, LPS-activated TLR4/gp91/Caspase3 mediated apoptosis signaling.

Monascus Adlay and Monacolin K Attenuates Arterial Thrombosis in Rats through the Inhibition of ICAM-1 and Oxidative Stress.

BACKGROUND/AIMS: Monascus Adlay (MA) prepared from fungal fermentation of Monascus purpureus inoculating with cooked adlay contains high content of monakolin K (MK) and phenolic compounds. We explored whether MA and MK improve FeCl3-induced arterial thrombosis in rats. METHODS: The rats were divided into control, FeCl3-treated rat carotid artery occlusion (TTO), TTO determined with one-week MA, and TTO determined with one-week MK. We compared MA or MK effects on oxidative stress by chemiluminescence amplification and immunohistochemistry, TTO by a transonic system, NFκB, ICAM-1, endoplasmic reticulum stress CHOP and Nrf2 signaling by western blotting. RESULTS: MA or MK efficiently depressed O2-, H2O2 and HOCl levels, platelet activation and aggregation and H2O2-enhanced ICAM-1 and VCAM-1 expression in the endothelial cells. FeCl3 significantly increased NFκB p65, 3-nitrotyrosine, CHOP and ICAM-1 expression, and decreased nuclear Nrf2 translocation and induces arterial thrombus formation. MA or MK pretreatment significantly elongated the level of FeCl3-induced TTO compared to TTO group, significantly decreased proinflammatory NF-κB/ICAM-1 signaling, endoplasmic reticulum stress CHOP expression and decreased thrombotic area. MA or MK significantly preserved nuclear Nrf2 translocation. MA and MK exerted a similar protective effect in attenuating thrombus formation. CONCLUSIONS: We suggest MA is better than MK to improve FeCl3-induced arterial thrombosis.

Predicting stroke volume and arterial pressure fluid responsiveness in liver cirrhosis patients using dynamic preload variables: A prospective study of diagnostic accuracy.

BACKGROUND: Predicting whether a fluid challenge will elicit 'fluid responsiveness' in stroke volume (SV) and arterial pressure is crucial for managing hypovolaemia and hypotension. Pulse pressure variation (PPV), SV variation (SVV) and the plethysmographic variability index (PVI) have been shown to predict SV fluid responsiveness, and the PPV/SVV ratio has been shown to predict arterial pressure fluid responsiveness under various conditions. However, these variables have not been investigated in liver cirrhosis patients. OBJECTIVE: The objective was to evaluate SV and arterial pressure fluid responsiveness in liver cirrhosis patients by using dynamic preload and vascular tone variables. DESIGN: A prospective study of diagnostic accuracy. SETTINGS: A single-centre trial conducted from November 2013 to April 2015. PATIENTS: Thirty-one adult patients, recipients of a living donor liver transplantat. INTERVENTION: An intraoperative fluid challenge with 10 ml kg of 0.9% normal saline. MAIN OUTCOME MEASURES: PPV, SVV, cardiac index and systemic vascular resistance index were measured using the Pulse index Continuous cardiac system. The PVI and perfusion index were measured using the Masimo Radical 7 co-oximeter. The PPV, SVV and PVI were measured to investigate SV fluid responsiveness, and the PPV/SVV ratio, perfusion index and systemic vascular resistance index were measured to investigate arterial pressure fluid responsiveness. RESULTS: The areas under the receiver operating characteristic curves for PPV, SVV and PVI were 0.794, 0.754 and 0.800, respectively (all P < 0.001). The cut-off values for PPV, SVV and PVI were 10% (sensitivity 78.3%, specificity 79.5%), 12% (sensitivity 69.6%, specificity 71.8%) and 11% (sensitivity 95.7%, specificity 59.0%), respectively. However, all investigated vascular tone variables failed to predict arterial pressure and fluid responsiveness. CONCLUSION: Dynamic preload variables predicted SV fluid responsiveness. Therefore, these variables can be used for fluid management in liver cirrhosis patients receiving mechanical ventilation. In contrast, vascular tone variables did not predict arterial pressure fluid responsiveness in liver cirrhosis patients. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT01971333.

Quercetin-Rich Guava (Psidium guajava) Juice in Combination with Trehalose Reduces Autophagy, Apoptosis and Pyroptosis Formation in the Kidney and Pancreas of Type II Diabetic Rats.

We explored whether the combination of anti-oxidant and anti-inflammatory guava (Psidium guajava) and trehalose treatment protects the kidney and pancreas against Type II diabetes (T2DM)-induced injury in rats. We measured the active component of guava juice by HPLC analysis. T2DM was induced in Wistar rats by intraperitoneal administration of nicotinamide and streptozotocin and combination with high fructose diets for 8 weeks. The rats fed with different dosages of guava juice in combination with or without trehalose for 4 weeks were evaluated the parameters including OGTT, plasma insulin, HbA1c, HOMA-IR (insulin resistance) and HOMA-ß (ß cell function and insulin secretion). We measured oxidative and inflammatory degrees by immunohistochemistry stain, fluorescent stain, and western blot and serum and kidney reactive oxygen species (ROS) by a chemiluminescence analyzer. High content of quercetin in the guava juice scavenged H2O2 and HOCl, whereas trehalose selectively reduced H2O2, not HOCl. T2DM affected the levels in OGTT, plasma insulin, HbA1c, HOMA-IR and HOMA-ß, whereas these T2DM-altered parameters, except HbA1c, were significantly improved by guava and trehalose treatment. The levels of T2DM-enhanced renal ROS, 4-hydroxynonenal, caspase-3/apoptosis, LC3-B/autophagy and IL-1ß/pyroptosis were significantly decreased by guava juice and trehalose. The combination with trehalose and guava juice protects the pancreas and kidney against T2DM-induced injury.

Laser speckle contrast imaging for assessing microcirculatory changes in multiple splanchnic organs and the gracilis muscle during hemorrhagic shock and fluid resuscitation.

OBJECTIVE: Hemorrhagic shock induces both macrocirculatory and microcirculatory impairment. Persistent microcirculatory dysfunction is associated with the dysfunction of multiple organs, especially in the splanchnic organs. However, few studies have simultaneously investigated microcirculation in multiple organs. In the present study, we used laser speckle contrast imaging to simultaneously investigate microcirculatory changes secondary to hemorrhagic shock and after fluid resuscitation among multiple splanchnic organs and the gracilis muscle. MATERIALS AND METHODS: 72 male Wistar rats were subjected to sham operation, hemorrhagic shock (total blood loss of 30mL/kg) and saline resuscitation. Macrocirculatory parameters, including the mean arterial pressure (MAP) and heart rate, and microcirculatory parameters, including microcirculatory blood flow intensity and tissue oxygen saturation in the liver, kidney, intestine (mucosa, serosal muscular layer, and Peyer's patch), and gracilis muscle were compared in a period of 3h. RESULTS: Hemorrhagic shock induced a significant reduction of microcirculatory blood flow intensity in the kidney and intestine (especially the mucosa). Tissue oxygen saturation reduction secondary to hemorrhagic shock was comparable among the various splanchnic organs but lower than the gracilis muscle. Fluid resuscitation restored the MAP but not the microcirculatory blood flow in the intestine and the tissue oxygen saturation in each splanchnic organ. CONCLUSION: Hemorrhagic shock induced the largest reduction in microcirculatory blood flow intensity in the intestinal mucosa. By comparison, the reduction of tissue oxygen saturation was not significantly different among the various splanchnic organs. Although fluid resuscitation restored the MAP, the intestinal microcirculation remained damaged.

Effects of different types of fluid resuscitation for hemorrhagic shock on splanchnic organ microcirculation and renal reactive oxygen species formation.

INTRODUCTION: Fluid resuscitation is an indispensable procedure in the acute management of hemorrhagic shock for restoring tissue perfusion, particularly microcirculation in splanchnic organs. Resuscitation fluids include crystalloids, hypertonic saline (HTS), and synthetic colloids, and their selection affects the recovery of microcirculatory blood flow and reactive oxygen species (ROS) formation, which is often evident in the kidney, following reperfusion. In this study, the effects of acute resuscitation with 0.9% saline (NS), 3% HTS, 4% succinylated gelatin (GEL), and 6% hydroxyethyl starch (HES) 130/0.4 were compared in a hemorrhagic shock rat model to analyze restoration of microcirculation among various splanchnic organs and the gracilis muscle and reperfusion-induced renal ROS formation. METHODS: A total of 96 male Wistar rats were subjected to sham operation (sham group), hemorrhagic shock (control group), and resuscitation with NS, HTS, GEL and HES. Two hours after resuscitation, changes in the mean arterial pressure (MAP), serum lactate level and the microcirculatory blood flow among various splanchnic organs, namely the liver, kidney, and intestine (mucosa, serosal muscular layer, and Peyer's patch), and the gracilis muscle, were compared using laser speckle contrast imaging. Renal ROS formation after reperfusion was investigated using an enhanced in vivo chemiluminescence (CL) method. RESULTS: Microcirculatory blood flow was less severely affected by hemorrhaging in the liver and gracilis muscle. Impairment of microcirculation in the kidney was restored in all resuscitation groups. Resuscitation in the NS group failed to restore intestinal microcirculation. Resuscitation in the HTS, GEL, and HES groups restored intestinal microcirculatory blood flow. By comparison, fluid resuscitation restored hemorrhagic shock-induced hypotension and decreased lactatemia in all resuscitation groups. Reperfusion-induced in vivo renal ROS formation was significantly higher in the GEL and HES groups than in the other groups. CONCLUSION: Although fluid resuscitation with NS restored the MAP and decreased lactatemia following hemorrhagic shock, intestinal microcirculation was restored only by other volume expanders, namely 3% HTS, GEL, and HES. However, reperfusion-induced renal ROS formation was significantly higher when synthetic colloids were used.

Effect of Adenoviral Catalase Gene Transfer on Renal Ischemia/Reperfusion Injury in Rats.

Ischemia/reperfusion (I/R) may through overt H2O2-induced pathophysiologic mechanisms lead to renal dysfunction. We explore whether catalase (CAT) protein overexpression by adenoviral CAT gene (Adv-CAT) transfection may improve ischemia/reperfusion-induced renal dysfunction. We augmented renal CAT expression by intrarenal arterial Adv-CAT administration with renal venous clamping in avertin-anesthetized female Wistar rats. After Adv-CAT transfection, we examined the CAT expression, location and effects on blood urea nitrogen (BUN) and urinary tubular injury biomarkers by biochemical assays, microcirculation by a laser perfusion imager, renal H2O2 amount by a chemiluminescent analyzer and molecular mechanisms including cytosolic cytochrome C leakage, apoptosis, autophagy and phospho- Akt (p-Akt)/phospho-endothelial nitric oxide (p-eNOS)/nitric oxide (NO) signaling by western blotting, immunohistochemistry and immunofluorescence. Adv-CAT enhanced 2.6-fold renal CAT protein expression primarily located in the proximal and distal tubules and renal vessels. Ischemia/reperfusion increased cytosolic cytochrome C leakage, renal H2O2-dependent level, autophagic Beclin-1/Atg5-Atg12/LC3 II expression, apoptotic Bax/Bcl-2/caspase 3/poly-(ADP-ribose)-polymerase fragments (PARP) expression and terminal deoxynucleotidyl transferasemediated nick-end labeling (TUNEL) stains and BUN and urinary glutathione S-transferase (GST) levels leading to proximal tubular injury. Ischemia/reperfusion also decreased renal microvascular blood flow associated with the inhibited renal expression of p-Akt and p-eNOS and NO production. Adv-CAT significantly improved the reduction in renal microvascular blood flow, reduced ischemia/reperfusion-enhanced oxidative stress, Beclin-1/Atg5-Atg12/LC3 II-meidated autophagy, Bax/Bcl-2/caspase 3/PARP-mediated apoptotic signaling, TUNEL stains, urinary GST level, and restored the p-Akt/p-eNOS/NO signaling in the kidney. Treatment of phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002, deleted Adv-CAT-induced p-Akt/p-eNOS/NO protective signaling. In conclusion, our results suggest Adv-CAT gene transfer counteracts H2O2-induced ischemia/reperfusion injury through preserving p-Akt/p-eNOS/NO pathway in the rat kidney.

Alterations in peripheral purinergic and muscarinic signaling of rat bladder after long-term fructose-induced metabolic syndrome.

PURPOSE: We explored the pathophysiologic mechanisms of long-term fructose-induced lower urinary tract symptoms (LUTS) in rats. METHODS: Male Wistar rats were fed with fructose for 3 or 6 months. Biochemical and transcystometric parameters were compared between fructose-fed and age-matched normal-diet rats. Pelvic nerve and external urethral sphincter-electromyogram activity recordings were performed to investigate fructose effects on neural control of bladders. Mitochondrial structure, ATP and acetylcholine content and purinergic and muscarinic cholinergic receptors were examined. Cytosolic cytochrome C staining by Western blot and immunocytochemistry for mitochondrial injury and PGP 9.5 stain for nerve density were also determined. RESULTS: The fructose-fed rats with higher plasma triglyceride, LDL and fasting glucose levels displayed LUTS with increased frequency and suppressed voiding contractile amplitude in phase 1 and phase 2 duration versus normal-diet control. Fructose feeding altered the firing types in pelvic afferent and efferent nerves and external urethral sphincter-electromyogram activity. Increased mast cell number, disrupted and swollen mitochondria, increased cytosolic cytochrome C stain and expression and decreased nerve density in bladder smooth muscle layers appeared in the fructose-fed rats. Fructose feeding also significantly reduced ATP and acetylcholine content and enhanced protein expression of postsynaptic P(2)X(1), P(2)X(2) and P(2)X(3) purinergic receptors and M(2) and M(3) muscarinic cholinergic receptors expression in the smooth muscles of urinary bladder. CONCLUSION: Long-term fructose feeding induced neuropathy and myopathy in the urinary bladders. Impaired mitochondrial integrity, reduced nerve density, ATP and acetylcholine content and upregulation of purinergic and muscarinic cholinergic receptors expression may contribute to the bladder dysfunction of fructose-fed animals.

Sodium thiosulfate through preserving mitochondrial dynamics ameliorates oxidative stress induced renal apoptosis and ferroptosis in 5/6 nephrectomized rats with chronic kidney diseases.

Chronic kidney disease (CKD) progression may be evoked through dysregulated mitochondrial dynamics enhanced oxidative stress and inflammation contributing to high cardiovascular morbidity and mortality. Previous study has demonstrated sodium thiosulfate (STS, Na2S2O3) could effectively attenuate renal oxidative injury in the animal model of renovascular hypertension. We explored whether the potentially therapeutic effect of STS is available on the attenuating CKD injury in thirty-six male Wistar rats with 5/6 nephrectomy. We determined the STS effect on reactive oxygen species (ROS) amount in vitro and in vivo by an ultrasensitive chemiluminescence-amplification method, ED-1 mediated inflammation, Masson's trichrome stained fibrosis, mitochondrial dynamics (fission and fusion) and two types of programmed cell death, apoptosis and ferroptosis by western blot and immunohistochemistry. Our in vitro data showed STS displayed the strongest scavenging ROS activity at the dosage of 0.1 g. We applied STS at 0.1 g/kg intraperitoneally 5 times/week for 4 weeks to these CKD rats. CKD significantly enhanced the degree in arterial blood pressure, urinary protein, BUN, creatinine, blood and kidney ROS amount, leukocytes infiltration, renal 4-HNE expression, fibrosis, dynamin-related protein 1 (Drp1) mediated mitochondrial fission, Bax/c-caspase 9/c-caspase 3/poly (ADP-ribose) polymerase (PARP) mediated apoptosis, iron overload/ferroptosis and the decreased xCT/GPX4 expression and OPA-1 mediated mitochondrial fusion. STS treatment significantly ameliorated oxidative stress, leukocyte infiltration, fibrosis, apoptosis and ferroptosis and improved mitochondrial dynamics and renal dysfunction in CKD rats. Our results suggest that STS as drug repurposing strategy could attenuate CKD injury through the action of anti-mitochondrial fission, anti-inflammation, anti-fibrosis, anti-apoptotic, and anti-ferroptotic mechanisms.