QiShenYiQi Inhibits Tissue Plasminogen Activator–Induced Brain Edema and Hemorrhage after Ischemic Stroke in Mice

Background: Thrombolysis with tissue plasminogen activator (tPA) remains the only approved drug therapy for acute ischemic stroke. However, delayed tPA treatment is associated with an increased risk of brain hemorrhage. In this study, we assessed whether QiShenYiQi (QSYQ), a compound Chinese medicine, can attenuate tPA-induced brain edema and hemorrhage in an experimental stroke model.Methods: Male mice were subjected to ferric chloride-induced carotid artery thrombosis followed by mechanical detachment of thrombi. Then mice were treated with QSYQ at 2.5 h followed by administration of tPA (10 mg/kg) at 4.5 h. Hemorrhage, infarct size, neurological score, cerebral blood flow, Evans blue extravasation, FITC-labeled albumin leakage, tight and adherens junction proteins expression, basement membrane proteins expression, matrix metalloproteinases (MMPs) expression, leukocyte adhesion, and leukocyte infiltration were assessed 24 h after tPA administration.Results: Compared with tPA alone treatments, the combination therapy of QSYQ and tPA significantly reduced hemorrhage, infarction, brain edema, Evans blue extravasation, albumin leakage, leukocyte adhesion, MMP-9 expression, and leukocyte infiltration at 28.5 h after stroke. The combination also significantly improved the survival rate, cerebral blood flow, tight and adherens junction proteins (occludin, claudin-5, junctional adhesion molecule-1, zonula occludens-1, VE-cadherin, α-catenin, β-catenin) expression, and basement membrane proteins (collagen IV, laminin) expression. Addition of QSYQ protected the downregulated ATP 5D and upregulated p-Src and Caveolin-1 after tPA treatment.Conclusion: Our results show that QSYQ inhibits tPA-induced brain edema and hemorrhage by protecting the blood-brain barrier integrity, which was partly attributable to restoration of energy metabolism, protection of inflammation and Src/Caveolin signaling activation. The present study supports QSYQ as an effective adjunctive therapy to increase the safety of delayed tPA thrombolysis for ischemic stroke.

QiShenYiQi Pills Attenuates Ischemia/Reperfusion-Induced Cardiac Microvascular Hyperpermeability Implicating Src/Caveolin-1 and RhoA/ROCK/MLC Signaling

Aims: Coronary microvascular hyperpermeability is an important contributor to ischemia or reperfusion (I/R) injury. However, the effective strategy for this insult remains limited. This study aimed to explore the protective effect of the compound Chinese medicine QiShenYiQi Pills (QSYQ) against coronary microvascular hyperpermeability after cardiac I/R with focusing on the underlying mechanism.Methods and Results: Male Sprague-Dawley rats under anesthesia were subjected to occlusion of left coronary anterior descending artery followed by reperfusion. QSYQ was administrated 90 min before ischemia initiation. Human cardiac microvascular endothelial cells (HCMECs) underwent hypoxia or reoxygenation (H/R) challenge with QSYQ administrated 1 h prior to hypoxia. QSYQ exhibited effects on attenuating microvascular damage and albumin leakage after I/R injury, showing a role in maintaining endothelial junctions, caveolae, and collagen in basement membrane (BM) of microvessels. Study using HCMECs disclosed that QSYQ protected endothelial barrier from impairment by H/R, attenuating the decline of respiratory chain complex I and ATP synthase, activation of Src/caveolin-1 and increase of RhoA/ROCK/p-MLC, MMP-9, and CTSS. PP2, a Src inhibitor, partially imitated the effect of QSYQ.Conclusions: The QSYQ was able to prevent I/R-induced cardiac microvascular hyperpermeability via a mechanism involving Src/caveolin-1 and RhoA/ROCK/MLC signaling.

The Bradykinin B2 Receptor Agonist (NG291) Causes Rapid Onset of Transient Blood–Brain Barrier Disruption Without Evidence of Early Brain Injury

Background: The blood–brain barrier (BBB) describes the brain’s highly specialized capillaries, which form a dynamic interface that maintains central nervous system (CNS) homeostasis. The BBB supports the CNS, in part, by preventing the entry of potentially harmful circulating molecules into the brain. However, this specialized function is challenging for the development of CNS therapeutics. Several strategies to facilitate drug delivery into the brain parenchyma via disruption of the BBB have been proposed. Bradykinin has proven effective in disrupting mechanisms across the blood–tumor barrier. Unfortunately, bradykinin has limited therapeutic value because of its short half-life and the undesirable biological activity elicited by its active metabolites.Objective: To evaluate NG291, a stable bradykinin analog, with selective agonist activity on the bradykinin-B2 receptor and its ability to disrupt the BBB transiently.Methods: Sprague Dawley rats and CD-1 mice were subjected to NG291 treatment (either 50 or 100 μg/kg, intravenously). Time and dose-dependent BBB disruption were evaluated by histological analysis of Evans blue (EB) extravasation. Transcellular and paracellular BBB leakage were assessed by infiltration of 99mTc-albumin (66.5 KDa) and 14C-sucrose (340 Da) radiolabeled probes into the brains of CD-1 mice treated with NG291. NG291 influence on P-glycoprotein (P-gp) efflux pump activity was evaluated by quantifying the brain accumulation of 3H-verapamil, a known P-gp substrate, in CD-1 mice.Results: NG291-mediated BBB disruption was localized, dose-dependent, and reversible as measured by EB extravasation. 99mTc-albumin leakage was significantly increased by 50 μg/kg of NG291, whereas 100 μg/kg of NG291 significantly augmented both 14C-sucrose and 99mTc-albumin leakage. NG291 enhanced P-gp efflux transporter activity and was unable to increase brain uptake of the P-gp substrate pralidoxime. NG291 did not evoke significant short-term neurotoxicity, as it did not increase brain water content, the number of Fluoro-Jade C positive cells, or astrocyte activation.Conclusion: Our findings strongly suggest that NG291 increases BBB permeability by two different mechanisms in a dose-dependent manner and increases P-gp efflux transport. This increased permeability may facilitate the penetration into the brain of therapeutic candidates that are not P-gp substrates.

Insulin-activated store-operated Ca2+ entry via Orai1 induces podocyte actin remodeling and causes proteinuria

Podocyte, the gatekeeper of the glomerular filtration barrier, is a primary target for growth factor and Ca2+ signaling whose perturbation leads to proteinuria. However, the effects of insulin action on store-operated Ca2+ entry (SOCE) in podocytes remain unknown. Here, we demonstrated that insulin stimulates SOCE by VAMP2-dependent Orai1 trafficking to the plasma membrane. Insulin-activated SOCE triggers actin remodeling and transepithelial albumin leakage via the Ca2+-calcineurin pathway in podocytes. Transgenic Orai1 overexpression in mice causes podocyte fusion and impaired glomerular filtration barrier. Conversely, podocyte-specific Orai1 deletion prevents insulin-stimulated SOCE, synaptopodin depletion, and proteinuria. Podocyte injury and albuminuria coincide with Orai1 upregulation at the hyperinsulinemic stage in diabetic (db/db) mice, which can be ameliorated by the suppression of Orai1-calcineurin signaling. Our results suggest that tightly balanced insulin action targeting podocyte Orai1 is critical for maintaining filter integrity, which provides novel perspectives on therapeutic strategies for proteinuric diseases, including diabetic nephropathy.

Tofacitinib Ameliorates Retinal Vascular Leakage in a Murine Model of Diabetic Retinopathy with Type 2 Diabetes

We have previously reported that inhibition of the Janus kinase 1 (JAK1) signaling ameliorates IL-17A-mediated blood-retinal barrier (BRB) dysfunction. Higher levels of IL-17A have been observed in the blood and intraocular fluids in patients with diabetic retinopathy (DR), in particular those with diabetic macular oedema. This study aimed to understand whether JAK1 inhibition could prevent BRB dysfunction in db/db mice, a model of type 2 diabetes (T2D). An in vitro study showed that high glucose treatment disrupted the junctional distribution of claudin-5 in bEnd3 cells and ZO-1 in ARPE19 cells and that tofacitinib citrate treatment prevented high glucose-mediated tight junction disruption. Albumin leakage, accompanied by increased levels of the phosphorylated form of JAK1 (pJAK1), was observed in three-month-old db/db mice. Treatment of two-and-a-half-month-old db/db mice with tofacitinib citrate for two weeks significantly reduced retinal albumin leakage and reduced pJAK1 expression. pJAK1 expression was also detected in human DR retina. Our results suggest that JAK1 inhibition can ameliorate BRB dysfunction in T2D, and JAK1 inhibitors such as tofacitinib citrate may be re-purposed for the management of diabetic macular oedema.

IL-17A Damages the Blood–Retinal Barrier through Activating the Janus Kinase 1 Pathway

Blood–retinal barrier (BRB) dysfunction underlies macular oedema in many sight-threatening conditions, including diabetic macular oedema, neovascular age-related macular degeneration and uveoretinitis. Inflammation plays an important role in BRB dysfunction. This study aimed to understand the role of the inflammatory cytokine IL-17A in BRB dysfunction and the mechanism involved. Human retinal pigment epithelial (RPE) cell line ARPE19 and murine brain endothelial line bEnd.3 were cultured on transwell membranes to model the outer BRB and inner BRB, respectively. IL-17A treatment (3 days in bEnd.3 cells and 6 days in ARPE19 cells) disrupted the distribution of claudin-5 in bEnd.3 cells and ZO-1 in ARPE19 cells, reduced the transepithelial/transendothelial electrical resistance (TEER) and increased permeability to FITC-tracers in vitro. Intravitreal (20 ng/1 μL/eye) or intravenous (20 ng/g) injection of recombinant IL-17A induced retinal albumin leakage within 48 h in C57BL/6J mice. Mechanistically, IL-17A induced Janus kinase 1 (JAK1) phosphorylation in bEnd.3 but not ARPE19 cells. Blocking JAK1 with Tofacitinib prevented IL-17A-mediated claudin-5 dysmorphia in bEnd.3 cells and reduced albumin leakage in IL-17A-treated mice. Our results suggest that IL-17A can damage the BRB through the activating the JAK1 signaling pathway, and targeting this pathway may be a novel approach to treat inflammation-induced macular oedema.

Canagliflozin protects against sepsis capillary leak syndrome by activating endothelial α1AMPK

Sepsis capillary leak syndrome (SCLS) is an independent prognostic factor for poor sepsis outcome. We previously demonstrated that α1AMP-activated protein kinase (α1AMPK) prevents sepsis-induced vascular hyperpermeability by mechanisms involving VE-cadherin (VE-Cad) stabilization and activation of p38 mitogen activated protein kinase/heat shock protein of 27 kDa (p38MAPK/HSP27) pathway. Canagliflozin, a sodium-glucose co-transporter 2 inhibitor, has recently been proven to activate AMPK in endothelial cells. Therefore, we hypothesized that canagliflozin could be of therapeutic potential in patients suffering from SCLS. We herein report that canagliflozin, used at clinically relevant concentrations, counteracts lipopolysaccharide-induced vascular hyperpermeability and albumin leakage in wild-type, but not in endothelial-specific α1AMPK-knockout mice. In vitro, canagliflozin was demonstrated to activate α1AMPK/p38MAPK/HSP27 pathway and to preserve VE-Cad’s integrity in human endothelial cells exposed to human septic plasma. In conclusion, our data demonstrate that canagliflozin protects against SCLS via an α1AMPK-dependent pathway, and lead us to consider novel therapeutic perspectives for this drug in SCLS.

Effects of on-line hemodiafiltration regimens and dialysate composition on serum concentrations of magnesium and calcium ions

Background Low-ionized magnesium and high-ionized calcium levels are associated with increased cardiovascular mortality in patients undergoing dialysis. We examined the effects of the dilution method, substitution volume, and dialysate of on-line hemodiafiltration on the total and ionized magnesium and calcium levels. Methods Eighteen patients were randomly assigned to three dialysate groups: two acetic acid dialysate groups and one citrate dialysate group. Five treatment conditions were applied: pre-diluted on-line hemodiafiltration, post-diluted on-line hemodiafiltration, and hemodialysis. Results The total and ionized serum levels of magnesium and calcium were evaluated and found to be unaffected by the dilution methods and substitution volumes. The albumin leakage was approximately 3 g/session under the pre-dilution and hemodiafiltration conditions, and approximately 4–5 g/session under the post-dilution condition. The ionized magnesium concentration decreased in the citrate dialysate group. Conclusion The on-line hemodiafiltration parameters had a negligible effect on ionized magnesium and calcium; however, the use of citrate dialysate decreased the ionized magnesium levels, probably because of chelation. Trial registration 000028172. The study was registered on July 11 2017.

Suitability of α1-microglobulin reduction rate as a biomarker of removal efficiency of online hemodiafiltration: a retrospective cohort study

Background Online hemodiafiltration (OL-HDF), whether in pre-dilution OL-HDF (pre-HDF) or post-dilution OL-HDF (post-HDF), is conducted to efficiently remove low molecular weight proteins from the blood of patients requiring dialysis. β2-microglobulin (β2-MG) and α1-microglobulin (α1-MG) are used as biomarkers to evaluate removal efficiency of OL-HDF. We aimed to evaluate the relationship between β2-MG and α1-MG reduction rates and the amount of albumin leakage. Furthermore, we statistically analyzed the relationship between the α1-MG reduction rate and α1-MG removal amount, and its suitability as a biomarker for evaluating the removal efficiency of OL-HDF. Methods We collected the results of regularly conducted routine evaluations to assess the efficiency of OL-HDF from cases of patients undergoing maintenance dialysis at our clinic from 2018 to 2019. Data on was collected on both pre-HDF and post-HDF sessions. β2-MG and α1-MG reduction rates were analyzed. Regression analysis on reduction rates showed a significant correlation between the α1-MG reduction rate and the α1-MG removal amount. Results We conducted 435 tests on OL-HDF efficiency in 87 cases undergoing maintenance dialysis at our clinic in 2018 and 2019. There were 80.7 ± 4.5% for the β2-MG reduction rate, 33.8 ± 9.4% for the α1-MG reduction rate, and 3.9 ± 1.8 g/s for the amount of albumin leakage. There was no correlation between the β2-MG reduction rate and the α1-MG reduction rate, or between the amount of albumin leakage and β2-MG reduction rate. Conclusion α1-MG reduction rate was found to correlate with its removal amount, demonstrating its suitability as a biomarker for evaluating the removal efficiency of OL-HDF. Trial registration Retrospectively registered.

Despite initial recovery of GFR, long-term renal functions deteriorate following short periods of unilateral ureteral obstruction

Following the release of short periods of unilateral ureteral obstruction (UUO), glomerular filtration rate (GFR) recovers by time. However, research in experimental animal models has demonstrated the presence of an ongoing element of renal interstitial fibrosis a few weeks following UUO reversal. Interstitial fibrosis can cause deterioration in GFR, and it is not known whether it leads to an ongoing slow deterioration in other renal functions despite the apparent initial recovery postreversal. To investigate this, rats underwent a 72-h reversible UUO. Renal functions of nonobstructed and previously obstructed kidneys were measured 1, 4, and 18 mo postreversal. GFR in nonobstructed and previously obstructed kidneys was similar up to 18 mo postreversal. However, there was ongoing tubulointerstitial fibrosis, and the degree of tubular atrophy and dilatation deteriorated by time. This was associated with an increase in urinary albumin leakage and alterations in renal injury markers, proinflammatory and profibrotic cytokines, and p53 from 4 mo onward despite the recovery in GFR. In conclusion, several aspects of renal functions continue to deteriorate following reversal of relatively short periods of UUO despite the initial recovery in GFR. This might stimulate further research in this area and might have clinical implications in terms of determining the best time for intervention following acute ureteral obstruction and long-term monitoring of these individuals.