Arachidonic acid reverses cholesterol and zinc inhibition of human voltage-gated proton channels.

Unlike other members of the voltage-gated ion channel superfamily, voltage-gated proton (Hv) channels are solely composed of voltage sensor domains without separate ion-conducting pores. Due to their unique dependence on both voltage and transmembrane pH gradients, Hv channels normally open to mediate proton efflux. Multiple cellular ligands were also found to regulate the function of Hv channels, including Zn2+, cholesterol, polyunsaturated arachidonic acid, and albumin. Our previous work showed that Zn2+ and cholesterol inhibit the human voltage-gated proton channel (hHv1) by stabilizing its S4 segment at resting state conformations. Released from phospholipids by phospholipase A2 in cells upon infection or injury, arachidonic acid regulates the function of many ion channels, including hHv1. In the present work, we examined the effects of arachidonic acid on purified hHv1 channels using liposome flux assays and revealed underlying structural mechanisms using single-molecule FRET. Our data indicated that arachidonic acid strongly activates hHv1 channels by promoting transitions of the S4 segment toward opening or "preopening" conformations. Moreover, we found that arachidonic acid even activates hHv1 channels inhibited by Zn2+ and cholesterol, providing a biophysical mechanism to activate hHv1 channels in nonexcitable cells upon infection or injury.

Heparanase inhibition as a systemic approach to protect the endothelial glycocalyx and prevent microvascular complications in diabetes.

BACKGROUND: Diabetes mellitus is a chronic disease which is detrimental to cardiovascular health, often leading to secondary microvascular complications, with huge global health implications. Therapeutic interventions that can be applied to multiple vascular beds are urgently needed. Diabetic retinopathy (DR) and diabetic kidney disease (DKD) are characterised by early microvascular permeability changes which, if left untreated, lead to visual impairment and renal failure, respectively. The heparan sulphate cleaving enzyme, heparanase, has previously been shown to contribute to diabetic microvascular complications, but the common underlying mechanism which results in microvascular dysfunction in conditions such as DR and DKD has not been determined. METHODS: In this study, two mouse models of heparan sulphate depletion (enzymatic removal and genetic ablation by endothelial specific Exotosin-1 knock down) were utilized to investigate the impact of endothelial cell surface (i.e., endothelial glycocalyx) heparan sulphate loss on microvascular barrier function. Endothelial glycocalyx changes were measured using fluorescence microscopy or transmission electron microscopy. To measure the impact on barrier function, we used sodium fluorescein angiography in the eye and a glomerular albumin permeability assay in the kidney. A type 2 diabetic (T2D, db/db) mouse model was used to determine the therapeutic potential of preventing heparan sulphate damage using treatment with a novel heparanase inhibitor, OVZ/HS-1638. Endothelial glycocalyx changes were measured as above, and microvascular barrier function assessed by albumin extravasation in the eye and a glomerular permeability assay in the kidney. RESULTS: In both models of heparan sulphate depletion, endothelial glycocalyx depth was reduced and retinal solute flux and glomerular albumin permeability was increased. T2D mice treated with OVZ/HS-1638 had improved endothelial glycocalyx measurements compared to vehicle treated T2D mice and were simultaneously protected from microvascular permeability changes associated with DR and DKD. CONCLUSION: We demonstrate that endothelial glycocalyx heparan sulphate plays a common mechanistic role in microvascular barrier function in the eye and kidney. Protecting the endothelial glycocalyx damage in diabetes, using the novel heparanase inhibitor OVZ/HS-1638, effectively prevents microvascular permeability changes associated with DR and DKD, demonstrating a novel systemic approach to address diabetic microvascular complications.

Effects of topical application of resveratrol on tight junction barrier and antimicrobial compound production in lactating goat mammary glands.

In mammary glands, the formation of less-permeable tight junctions (TJs) and the production of antimicrobial compounds like lactoferrin and defensins are important for preventing mastitis. Resveratrol, a polyphenol contained in red grapes, is known to protect mammary epithelial cells (MECs) from oxidative stress; however, oral administration of resveratrol causes a decrease in certain biological processes through conjugation and metabolic conversion. In this study, we determined the beneficial effects of resveratrol on TJs and antimicrobial compounds in cultured goat MECs by adding it to the medium, and in lactating goat mammary glands by topical application for percutaneous absorption. TJ barrier function was evaluated by transepithelial resistance and expression or localization pattern of claudins for culture model in vitro and by somatic cell count, Na+, albumin, and IgG in milk for topical application in vivo. Concentrations of antimicrobial compounds and cytokines were measured using ELISA. Activation of STAT3 was evaluated by Western blotting. Resveratrol strengthened TJ barrier function by upregulating claudin-3 in cultured MECs and topical application to udders reduced somatic cell count, Na+, albumin, and IgG in milk. Resveratrol increased ß-defensin and S100A7 levels in cultured MECs and milk. In addition, resveratrol down-regulated cytokine production and STAT3 pathway. These findings suggest that the topical application of resveratrol to udders may be effective in preventing mastitis.

Effect of SGLT2 Inhibitors on Discontinuation of Renin-angiotensin System Blockade: A Joint Analysis of the CREDENCE and DAPA-CKD Trials.

SIGNIFICANCE STATEMENT: Angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) are foundational therapy for CKD but are underused, in part because they are frequently withheld and not restarted due to hyperkalemia, AKI, or hospitalization. Consequently, ensuring persistent use of ACE inhibitors and ARBs in CKD has long been a major clinical priority. In this joint analysis of the CREDENCE and DAPA-CKD trials, the relative risk of discontinuation of ACE inhibitors and ARBs was reduced by 15% in patients randomized to sodium-glucose cotransporter 2 (SGLT2) inhibitors. This effect was more pronounced in patients with urine albumin:creatinine ratio ≥1000 mg/g, for whom the absolute benefits of these medications are the greatest. These findings indicate that SGLT2 inhibitors may enable better use of ACE inhibitors and ARBs in patients with CKD. BACKGROUND: Strategies to enable persistent use of renin-angiotensin system (RAS) blockade to improve outcomes in CKD have long been sought. The effect of SGLT2 inhibitors on discontinuation of RAS blockade has yet to be evaluated. METHODS: We conducted a joint analysis of canagliflozin and renal events in diabetes with established nephropathy clinical evaluation (CREDENCE) and dapagliflozin and prevention of adverse outcomes in CKD (DAPA-CKD), two randomized, double-blind, placebo-controlled, event-driven trials of SGLT2 inhibitors in patients with albuminuric CKD. The main outcome was time to incident temporary or permanent discontinuation of RAS blockade, defined as interruption of an ACE inhibitor or ARB for at least 4 weeks or complete cessation during the double-blind on-treatment period. Cox regression analyses were used to estimate the treatment effects from each trial. Hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) were pooled with fixed effects meta-analysis to obtain summary treatment effects, overall and across key subgroups. RESULTS: During median follow-up of 2.2 years across both trials, 740 of 8483 (8.7%) patients discontinued RAS blockade. The relative risk for discontinuation of RAS blockade was 15% lower in patients randomized to receiving SGLT2 inhibitors (HR, 0.85; 95% CI, 0.74 to 0.99), with consistent effects across trials ( P -heterogeneity = 0.92). The relative effect on RAS blockade discontinuation was more pronounced among patients with baseline urinary albumin:creatinine ratio ≥1000 mg/g (pooled HR, 0.77; 95% CI, 0.63 to 0.94; P -heterogeneity = 0.009). CONCLUSIONS: In patients with albuminuric CKD with and without type 2 diabetes, SGLT2 inhibitors facilitate the use of RAS blockade. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: ClinicalTrials.gov, NCT02065791 and NCT03036150 . PODCAST: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2023_11_21_JASN0000000000000248.mp3.

Hepatoprotective effect of methanol fruit extract of Punica granatum L in highly active antiretroviral therapy-induced toxicity in Wistar rats.

Prolonged use of Highly Active Antiretroviral Therapy (HAART) has been linked to toxicity, particularly hepatotoxicity. There are few effective drugs for HAART patients that promote hepatic cell regeneration and prevent liver injury. Therefore, the purpose of this study was to investigate the hepato-protective activity of Methanol fruit extract of Punica granatum (MFEPG) in HAART-administered rats. Thirty rats weighing between 150-200 g were randomly divided into six groups and each group comprised of five rats. Distilled water was given to the rats in group one. Only HAART was given to the rats in group two. MFEPG at doses of 100 and 400 mg/kg was given to the rats in groups three and four. MFEPG dosages of 100 and 400 mg/kg along with HAART were given to the rats in groups five and six, respectively. All treatments were via oral gavage daily for 40 days. Under halothane anesthesia, all rats were sacrificed on day 41. Liver tissues were utilized for lipid peroxidation marker; Malondialdehyde (MDA), antioxidant enzymes; Superoxide dismutase (SOD) and Catalase (CAT) and histological evaluation, while blood samples were examined for biochemical parameters (AST, ALT, ALP, Total cholesterol, Total protein, and Albumin). The HAART-treated group exhibited a significantly higher amount of the lipid peroxidation end product; MDA, and significantly lower levels of antioxidant enzymes; SOD, and CAT. Liver enzymes and total cholesterol were significantly increased with a significant reduction in Total protein and Albumin levels in the HAART-treated group. Conversely, the liver function biomarkers were returned to normal levels in the HAART and MFEPG-treated groups. Histopathological studies revealed that when HAART-exposed rats were treated with MFEPG, both the biochemical and histological results significantly improved. Thus, the antioxidant activity of MFEPG provides protection against HAART-induced liver oxidative damage. More research is needed to determine the safety of using MFEPG in humans.

Microalbuminuria in Rats Treated with D-Nitroarginine Methyl Ether.

Microalbuminuria is an early symptom and prognostic marker of the progression of renal pathology. The analysis of the role of anionic components of the renal glomeruli in the albumin retention and the development of a model of minimal changes in the glomerular filter leading to the appearance of microalbuminuria are relevant. The effect of organic cations D-arginine methyl esters (D-AME) and D-nitroarginine (D-NAME) on the excretion of albumin by the kidneys in rats was studied. D-AME had no effect on urinary albumin excretion in rats. D-NAME caused microalbuminuria, which persisted for more than a day and sharply increased after injection of vasopressin. The number of anionic sites labeled with polyethyleneimine decreased in the structures of the glomerular filter. D-NAME-induced microalbuminuria can later serve as a model for studying nephroprotective or damaging factors.

Glycolytic enzyme PFKFB3 regulates sphingosine 1-phosphate receptor 1 in proangiogenic glomerular endothelial cells under diabetic condition.

Glomerular angiogenesis is a characteristic feature of diabetic nephropathy (DN). Enhanced glycolysis plays a crucial role in angiogenesis. The present study was designed to investigate the role of glycolysis in glomerular endothelial cells (GECs) in a mouse model of DN. Mouse renal cortex and isolated glomerular cells were collected for single-cell and RNA sequencing. Cultured GECs were exposed to high glucose in the presence (proangiogenic) and absence of a vascular sprouting regimen. MicroRNA-590-3p was delivered by lipofectamine in vivo and in vitro. In the present study, a subgroup of GECs with proangiogenic features was identified in diabetic kidneys by using sequencing analyses. In cultured proangiogenic GECs, high glucose increased glycolysis and phosphofructokinase/fructose bisphosphatase 3 (PFKFB3) protein expression, which were inhibited by overexpressing miRNA-590-3p. Mimics of miRNA-590-3p also increased receptor for sphingosine 1-phosphate (S1pR1) expression, an angiogenesis regulator, in proangiogenic GECs challenged with high glucose. Inhibition of PFKFB3 by pharmacological and genetic approaches upregulated S1pR1 protein in vitro. Mimics of miRNA-590-3p significantly reduced migration and angiogenic potential in proangiogenic GECs challenged with high glucose. Ten-week-old type 2 diabetic mice had elevated urinary albumin levels, reduced renal cortex miRNA-590-3p expression, and disarrangement of glomerular endothelial cell fenestration. Overexpressing miRNA-590-3p via perirenal adipose tissue injection restored endothelial cell fenestration and reduced urinary albumin levels in diabetic mice. Therefore, the present study identifies a subgroup of GECs with proangiogenic features in mice with DN. Local administration of miRNA-590-3p mimics reduces glycolytic rate and upregulates S1pR1 protein expression in proangiogenic GECs. The protective effects of miRNA-590-3p provide therapeutic potential in DN treatment.NEW & NOTEWORTHY Proangiogenetic glomerular endothelial cells (GECs) are activated in diabetic nephropathy. High glucose upregulates glycolytic enzyme phosphofructokinase/fructose bisphosphatase 3 (PFKFB3) in proangiogenetic cells. PFKFB3 protects the glomerular filtration barrier by targeting endothelial S1pR1. MiRNA-590-3p restores endothelial cell function and mitigates diabetic nephropathy.

Structural Studies Reveal that Endosomal Cations Promote Formation of Infectious Coxsackievirus A9 A-Particles, Facilitating RNA and VP4 Release.

Coxsackievirus A9 (CVA9), an enterovirus, is a common cause of pediatric aseptic meningitis and neonatal sepsis. During cell entry, enterovirus capsids undergo conformational changes leading to expansion, formation of large pores, externalization of VP1 N termini, and loss of the lipid factor from VP1. Factors such as receptor binding, heat, and acidic pH can trigger capsid expansion in some enteroviruses. Here, we show that fatty acid-free bovine serum albumin or neutral endosomal ionic conditions can independently prime CVA9 for expansion and genome release. Our results showed that CVA9 treatment with albumin or endosomal ions generated a heterogeneous population of virions, which could be physically separated by asymmetric flow field flow fractionation and computationally by cryo-electron microscopy (cryo-EM) and image processing. We report cryo-EM structures of CVA9 A-particles obtained by albumin or endosomal ion treatment and a control nonexpanded virion to 3.5, 3.3, and 2.9 Å resolution, respectively. Whereas albumin promoted stable expanded virions, the endosomal ionic concentrations induced unstable CVA9 virions which easily disintegrated, losing their genome. Loss of most of the VP4 molecules and exposure of negatively charged amino acid residues in the capsid's interior after expansion created a repulsive viral RNA-capsid interface, aiding genome release. IMPORTANCE Coxsackievirus A9 (CVA9) is a common cause of meningitis and neonatal sepsis. The triggers and mode of action of RNA release into the cell unusually do not require receptor interaction. Rather, a slow process in the endosome, independent of low pH, is required. Here, we show by biophysical separation, cryogenic electron microscopy, and image reconstruction that albumin and buffers mimicking the endosomal ion composition can separately and together expand and prime CVA9 for uncoating. Furthermore, we show in these expanded particles that VP4 is present at only ~10% of the occupancy found in the virion, VP1 is externalized, and the genome is repelled by the negatively charged, repulsive inner surface of the capsid that occurs due to the expansion. Thus, we can now link observations from cell biology of infection with the physical processes that occur in the capsid to promote genome uncoating.

Cerebrolysin Attenuates Exacerbation of Neuropathic Pain, Blood-spinal Cord Barrier Breakdown and Cord Pathology Following Chronic Intoxication of Engineered Ag, Cu or Al (50-60 nm) Nanoparticles.

Neuropathic pain is associated with abnormal sensations and/or pain induced by non-painful stimuli, i.e., allodynia causing burning or cold sensation, pinching of pins and needles like feeling, numbness, aching or itching. However, no suitable therapy exists to treat these pain syndromes. Our laboratory explored novel potential therapeutic strategies using a suitable composition of neurotrophic factors and active peptide fragments-Cerebrolysin (Ever Neuro Pharma, Austria) in alleviating neuropathic pain induced spinal cord pathology in a rat model. Neuropathic pain was produced by constrictions of L-5 spinal sensory nerves for 2-10 weeks period. In one group of rats cerebrolysin (2.5 or 5 ml/kg, i.v.) was administered once daily after 2 weeks until sacrifice (4, 8 and 10 weeks). Ag, Cu and Al NPs (50 mg/kg, i.p.) were delivered once daily for 1 week. Pain assessment using mechanical (Von Frey) or thermal (Hot-Plate) nociceptive showed hyperalgesia from 2 weeks until 10 weeks progressively that was exacerbated following Ag, Cu and Al NPs intoxication in nerve lesioned groups. Leakage of Evans blue and radioiodine across the blood-spinal cord barrier (BSCB) is seen from 4 to 10 weeks in the rostral and caudal cord segments associated with edema formation and cell injury. Immunohistochemistry of albumin and GFAP exhibited a close parallelism with BSCB leakage that was aggravated by NPs following nerve lesion. Light microscopy using Nissl stain exhibited profound neuronal damages in the cord. Transmission electron microcopy (TEM) show myelin vesiculation and synaptic damages in the cord that were exacerbated following NPs intoxication. Using ELISA spinal tissue exhibited increased albumin, glial fibrillary acidic protein (GFAP), myelin basic protein (MBP) and heat shock protein (HSP 72kD) upregulation together with cytokines TNF-α, IL-4, IL-6, IL-10 levels in nerve lesion that was exacerbated following NPs intoxication. Cerebrolysin treatment significantly reduced hyperalgesia and attenuated BSCB disruption, edema formation and cellular changes in nerve lesioned group. The levels of cytokines were also restored near normal levels with cerebrolysin treatment. Albumin, GFAP, MABP and HSP were also reduced in cerebrolysin treated group and thwarted neuronal damages, myelin vesiculation and cell injuries. These neuroprotective effects of cerebrolysin with higher doses were also effective in nerve lesioned rats with NPs intoxication. These observations suggest that cerebrolysin actively protects spinal cord pathology and hyperalgesia following nerve lesion and its exacerbation with metal NPs, not reported earlier.

Lack of EGFR catalytic activity in hepatocytes improves liver regeneration following DDC-induced cholestatic injury by promoting a pro-restorative inflammatory response.

Despite the well-known hepatoprotective role of the epidermal growth factor receptor (EGFR) pathway upon acute damage, its specific actions during chronic liver disease, particularly cholestatic injury, remain ambiguous and unresolved. Here, we analyzed the consequences of inactivating EGFR signaling in the liver on the regenerative response following cholestatic injury. For that, transgenic mice overexpressing a dominant negative mutant human EGFR lacking tyrosine kinase activity (ΔEGFR) in albumin-positive cells were submitted to liver damage induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), an experimental model resembling human primary sclerosing cholangitis. Our results show an early activation of EGFR after 1-2 days of a DDC-supplemented diet, followed by a signaling switch-off. Furthermore, ΔEGFR mice showed less liver damage and a more efficient regeneration following DDC injury. Analysis of the mechanisms driving this effect revealed an enhanced activation of mitogenic/survival signals, AKT and ERK1/2-MAPKs, and changes in cell turnover consistent with a quicker resolution of damage in response to DDC. These changes were concomitant with profound differences in the profile of intrahepatic immune cells, consisting of a shift in the M1/M2 balance towards M2 polarity, and the Cd4/Cd8 ratio in favor of Cd4 lymphocytes, overall supporting an immune cell switch into a pro-restorative phenotype. Interestingly, ΔEGFR livers also displayed an amplified ductular reaction, with increased expression of EPCAM and an increased number of CK19-positive ductular structures in portal areas, demonstrating an overexpansion of ductular progenitor cells. In summary, our work supports the notion that hepatocyte-specific EGFR activity acts as a key player in the crosstalk between parenchymal and non-parenchymal hepatic cells, promoting the pro-inflammatory response activated during cholestatic injury and therefore contributing to the pathogenesis of cholestatic liver disease. © 2022 The Pathological Society of Great Britain and Ireland.

Albumin alleviated esketamine-induced neuronal apoptosis of rat retina through downregulation of Zn2+-dependent matrix metalloproteinase 9 during the early development.

AIMS: Esketamine upregulates Zn2+-dependent matrix metalloproteinase 9 (MMP9) and increases the neuronal apoptosis in retinal ganglion cell layer during the early development. We aimed to test whether albumin can alleviate esketamine-induced apoptosis through downregulating Zn2+-dependent MMP9. METHODS: We investigate the role of Zn2+ in esketamine-induced neuronal apoptosis by immunofluorescence. MMP9 protein expression and enzyme activity were investigated by zymography in situ., western blot and immunofluorescence. Whole-mount retinas from P7 Sprague-Dawley rats were used. RESULTS: We demonstrated that esketamine exposure increased Zn2+ in the retinal GCL during the early development. Zn2+-dependent MMP9 expression and enzyme activity up-regulated, which eventually aggravated apoptosis. Albumin effectively down-regulated MMP9 expression and activity via binding of free zinc, ultimately protected neurons from apoptosis. Meanwhile albumin treatment promoted activated microglia into multi-nucleated macrophagocytes and decreased the inflammation. CONCLUSION: Albumin alleviates esketamine-induced neuronal apoptosis through decreasing Zn2+ accumulation in GCL and downregulating Zn2+-dependent MMP9.

Inhibition of Hepatic Stellate Cell Activation Suppresses Tumorigenicity of Hepatocellular Carcinoma in Mice.

Transdifferentiation (or activation) of hepatic stellate cells (HSCs) to myofibroblasts is a key event in liver fibrosis. Activated HSCs in the tumor microenvironment reportedly promote tumor progression. This study analyzed the effect of an inhibitor of HSC activation, retinol-binding protein-albumin domain III fusion protein (R-III), on protumorigenic functions of HSCs. Although conditioned medium collected from activated HSCs enhanced the migration, invasion, and proliferation of the hepatocellular carcinoma cell line Hepa-1c1c7, this effect was not observed in Hepa-1c1c7 cells treated with conditioned medium from R-III-exposed HSCs. In a subcutaneous tumor model, larger tumors with increased vascular density were formed in mice transplanted with Hepa-1c1c7+HSC than in mice transplanted with Hepa-1c1c7 cells alone. Intriguingly, when Hepa-1c1c7+HSC-transplanted mice were injected intravenously with R-III, a reduction in vascular density and extended tumor necrosis were observed. In an orthotopic tumor model, co-transplantation of HSCs enhanced tumor growth, angiogenesis, and regional metastasis accompanied by increased peritumoral lymphatic vessel density, which was abolished by R-III. In vitro study showed that R-III treatment affected the synthesis of pro-angiogenic and anti-angiogenic factors in activated HSCs, which might be the potential mechanism underlying the R-III effect. These findings suggest that the inhibition of HSC activation abrogates HSC-induced tumor angiogenesis and growth, which represents an attractive therapeutic strategy.

Initiation of the SGLT2 inhibitor canagliflozin to prevent kidney and heart failure outcomes guided by HbA1c, albuminuria, and predicted risk of kidney failure.

BACKGROUND: Sodium glucose co-transporter-2 (SGLT2) inhibitors reduce the risk of kidney and heart failure events independent of glycemic effects. We assessed whether initiation of the SGLT2 inhibitor canagliflozin guided by multivariable predicted risk based on clinical characteristics and novel biomarkers is more efficient to prevent clinical outcomes compared to a strategy guided by HbA1c or urinary-albumin-creatinine ratio (UACR) alone. METHODS: We performed a post-hoc analysis of the CANVAS trial including 3713 patients with available biomarker measurements. We compared the number of composite kidney (defined as a sustained 40% decline in eGFR, chronic dialysis, kidney transplantation, or kidney death) and composite heart failure outcomes (defined as heart failure hospitalization or cardiovascular (CV) death) prevented per 1000 patients treated for 5 years when canagliflozin was initiated in patients according to HbA1c ≥ 7.5%, UACR, or multivariable risk models consisting of: (1) clinical characteristics, or (2) clinical characteristics and novel biomarkers. Differences in the rates of events prevented between strategies were tested by Chi2-statistic. RESULTS: After a median follow-up of 6.1 years, 144 kidney events were recorded. The final clinical model included age, previous history of CV disease, systolic blood pressure, UACR, hemoglobin, body weight, albumin, estimated glomerular filtration rate, and randomized treatment assignment. The combined biomarkers model included all clinical characteristics, tumor necrosis factor receptor-1, kidney injury molecule-1, matrix metallopeptidase-7 and interleukin-6. Treating all patients with HbA1c ≥ 7.5% (n = 2809) would prevent 33.0 (95% CI 18.8 to 43.3 ) kidney events at a rate of 9.6 (95% CI 5.5 to 12.6) events prevented per 1000 patients treated for 5 years. The corresponding rates were 5.8 (95% CI 3.4 to 7.9), 16.6 (95% CI 9.5 to 22.0) (P < 0.001 versus HbA1c or UACR approach), and 17.5 (95% CI 10.0 to 23.0) (P < 0.001 versus HbA1c or UACR approach; P = 0.54 versus clinical model). Findings were similar for the heart failure outcome. CONCLUSION: Initiation of canagliflozin based on an estimated risk-based approach prevented more kidney and heart failure outcomes compared to a strategy based on HbA1c or UACR alone. There was no apparent gain from adding novel biomarkers to the clinical risk model. These findings support the use of risk-based assessment using clinical markers to guide initiation of SGLT2 inhibitors in patients with type 2 diabetes.

Albumin protects the liver from tumor necrosis factor α-induced immunopathology.

Besides its oncotic power, albumin exerts pleiotropic actions, including binding, transport, and detoxification of endogenous and exogenous molecules, antioxidant activity, and modulation of immune and inflammatory responses. In particular, recent studies have demonstrated that albumin reduces leukocyte cytokine production. Here, we investigated whether albumin also has the ability to protect tissues from the damaging actions of these inflammatory mediators. We circumscribed our investigation to tumor necrosis factor (TNF) α, which exemplifies the connection between immunity and tissue injury. In vivo experiments in analbuminemic mice showed that these mice exhibit a more pronounced response to a model of TNFα-mediated liver injury induced by the administration of lipopolysaccharide (LPS) and D-galactosamine (D-gal). A tissue protective action against LPS/D-gal liver injury was also observed during the administration of human albumin to humanized mice expressing the human genes for albumin and neonatal Fc receptor (hAlb+/+ /hFcRn+/+ ) with preestablished carbon tetrachloride (CCl4 )-induced early cirrhosis. The cytoprotective actions of albumin against TNFα-induced injury were confirmed ex vivo, in precision-cut liver slices, and in vitro, in primary hepatocytes in culture. Albumin protective actions were independent of its scavenging properties and were reproduced by recombinant human albumin expressed in Oryza sativa. Albumin cytoprotection against TNFα injury was related to inhibition of lysosomal cathepsin B leakage accompanied by reductions in mitochondrial cytochrome c release and caspase-3 activity. These data provide evidence that in addition to reducing cytokines, the albumin molecule also has the ability to protect tissues against inflammatory injury.

Prenatal glucocorticoid administration accelerates the maturation of fetal rat hepatocytes.

BACKGROUND: Prenatal glucocorticoid (GC) is clinically administered to pregnant women who are at risk of preterm birth for the maturation of cardiopulmonary function. Preterm and low-birth-weight infants often experience liver dysfunction after birth because their livers are immature. However, the effects of prenatal GC administration on the liver remain unclear. We aimed to investigate the effects of prenatal GC administration on the maturation of liver hepatocytes in preterm rats. METHODS AND RESULTS: Dexamethasone (DEX) was administered to pregnant Wistar rats on gestational days 17 and 19 before cesarean section. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the mRNA levels of albumin, hepatocyte nuclear factor-4 alpha (HNF4α), hepatocyte growth factor (HGF), thymus cell antigen 1 (Thy-1), cyclin B, and Cyclin-dependent kinase 1 (CDK1) in the liver samples. Immunohistochemical staining and enzyme-linked immunosorbent assay were performed to examine protein production. The hepatocytes enlarged because of growth and prenatal DEX administration. Albumin, HNF4α, and HGF levels increased secondary to growth and prenatal DEX administration. The levels of the cell cycle markers cyclin B and CDK1 gradually decreased during growth and with DEX administration. CONCLUSIONS: The results suggest that prenatal GC administration leads to hepatocyte maturation via expression of HNF4α and HGF in preterm fetuses.

Ticagrelor and Dapagliflozin Have Additive Effects in Ameliorating Diabetic Nephropathy in Mice with Type-2 Diabetes Mellitus.

PURPOSE: Ticagrelor and dapagliflozin can suppress the activation of the NOD-like receptor 3 (NLRP3)-inflammasome and activate AMP-activated protein kinase (AMPK). The anti-inflammatory effects of dapagliflozin has been shown to depend on AMPK activation. Dapagliflozin and ticagrelor have been shown to have additive effects on the progression of diabetic cardiomyopathy in BTBR ob/ob mice with type-2 diabetes. We assessed whether dapagliflozin and ticagrelor have additive effects on the activation of the NLRP3-inflammasome and the progression of diabetic nephropathy in mice with type-2 diabetes. METHODS: Eight-week-old BTBR received either no-drug, dapagliflozin (1.5 mg/kg/d), ticagrelor (100 mg/kg/d), or their combination for 12 weeks. Blood was assessed weekly for glucose and urine for glucose and albumin. After 12 weeks, blood creatinine, cystatin C, inflammasome activation, and insulin were assessed by ELISA. Renal cortex samples were assessed by hematoxylin and eosin and periodic acid-Schiff staining. RT-PCR and immunoblotting were used to evaluate fibrosis and the activation of Akt, AMPK and the inflammasome. RESULTS: Both ticagrelor and dapagliflozin reduced serum creatinine and cystatin C levels and urinary albumin. Both drugs attenuated the increase in glomerular area and mesangial matrix index. Both drugs decreased collagen-1 and collagen-3 expression and the activation of the NLRP3-inflammasome. Both drugs increased P-AMPK levels, but only dapagliflozin increased P-Akt levels. Overall, the protective effects of dapagliflozin and ticagrelor were additive. CONCLUSIONS: Dapagliflozin and ticagrelor attenuated the progression of diabetic nephropathy in BTBR ob/ob mice with additive effects of the combination. This was associated with AMPK activation and reduced activation of the NLRP3 inflammasome, whereas only dapagliflozin increased Akt activation.

Lack of renoprotective effects of targeting the endothelin A receptor and (or) sodium glucose transporter 2 in a mouse model of Type 2 diabetic kidney disease.

Two recent clinical trials, using sodium glucose cotransporter (SGLT2) or endothelin-A receptor (ET-A) blocker, reported the first efficacious treatments in 18 years to slow progression of diabetic kidney disease (DKD). We hypothesized that combined inhibition of SGLT2 and ET-A receptor may confer greater protection against renal injury than either agent alone. Uninephrectomized male db/db mice were randomized to four groups: vehicle, SGLT2 inhibitor (dapagliflozin (dapa), 1 mg/kg/day), ET-A blocker (atrasentan (atra), 5 mg/kg/day), or dual treatment from 10 weeks until 22 weeks of age. At 10 weeks of age, no differences were observed in body weight, blood glucose or urinary albumin excretion among the four groups. At 16 and 22 weeks of age, body weight was lower and blood glucose levels higher in the vehicle and atra groups compared with dapa- and dual-treated groups. No notable differences were observed among the four groups in urinary albumin excretion at weeks 16 and 22. Histological analysis showed mild glomerulosclerosis and tubular injury (<5%) in all four groups with reduced glomerulosclerosis in the dual treatment group compared with vehicle. Individual or combined treatment with an SGLT2 inhibitor and (or) an ET-A antagonist did not confer renoprotective effects in this model.

Albumin-induced premature senescence in human renal proximal tubular cells and its relationship with intercellular fibrosis.

The presence of senescent cells is associated with renal fibrosis. This study aims to investigate the effect of albumin-induced premature senescence on tubulointerstitial fibrosis and its possible mechanism in vitro. Different concentrations of bovine serum albumim (BSA) with or without si-p21 are used to stimulate HK-2 cells for 72 h, and SA-ß-gal activity, senescence-associated secretory phenotypes (SASPs), LaminB1 are used as markers of senescence. Immunofluorescence staining is performed to characterize the G2/M phase arrest between the control and BSA groups. Alterations in the DNA damage marker γ-H2AX, fibrogenesis, and associated proteins at the G2/M phase, such as p21, p-CDC25C and p-CDK1, are evaluated. Compared with those in the control group, the SA-ß-gal activity, SASP, and γ-H2AX levels are increased in the BSA group, while the level of LaminB1 is decreased. Meanwhile, HK-2 cells blocked at the G2/M phase are significantly increased under the stimulation of BSA, and the levels of p21, p-CDC25C and p-CDK1, as well as fibrogenesis are also increased. When p21 expression is inhibited, the levels of p-CDC25C and p-CDK1 are decreased and the G2/M phase arrest is improved, which decreases the production of fibrogenesis. In conclusion, BSA induces renal tubular epithelial cell premature senescence, which regulates the G2/M phase through the CDC25C/CDK1 pathway, leading to tubulointerstitial fibrosis.

Oral administration of potato peel extract affects serum blood metabolites, liver function and ameliorating oxidative stress induced in rabbits exposed to cold stress.

This study investigated the effect of potato peel extract (PPE), orally administrated to rabbits, on serum blood metabolites and ameliorating oxidative stress induced by cold stress under Egyptian winter conditions. Twenty-four bucks grouped into three treatments (8 animals per group) were used for the experiment. The animals received 1.5 ml of water orally, containing 0 (PPE0), 25 (PPE25) or 50 (PPE50) mg PPE/kg live weight. Bucks were randomly assigned into three homogenous equal groups according to the level of PPE. Treatments were applied to each animal every two days over a period of three months including one month as an adaptation period. At the 8th week of the experiment, blood samples were collected from each buck and at the end of the experiment, bucks were slaughtered, and some organs were collected and weighed. The PPE improved (p < 0.05) blood total protein, albumin, globulin and glucose. The blood concentration of total lipid, cholesterol, triglyceride, low density lipoprotein and very low-density lipoprotein (were increased (p < 0.02) in PPE rabbits. Furthermore, PPE extract doses decreased (p < 0.001) oxidant thiobarbituric reactive substance (TBARS) in both blood and liver. Other liver and blood antioxidant system enzymes such as catalase, glutathione peroxidase, and superoxide dismutase were improved (p < 0.005) by PPE supplementation. Overall, oral administration of PPE up to 50 mg/kg live weight can have positive effects on rabbit health under cold stress.

Hemodynamic Response to Fluid Boluses in Patients with Single-Ventricle Parallel Circulation.

The purpose of this study is to assess the effect of fluid bolus in response to a hypotensive episode by evaluating high-fidelity hemodynamic data obtained from children with single-ventricle anatomy and parallel circulation. Single center, retrospective analysis of hemodynamic and oximetric data after fluid bolus administrations within the first 2 weeks post-surgery. A baseline (- 60 to - 10 min), hypotensive episode (- 10 to 0 min), and response interval (0 to 60 min) were defined to quantify the dynamics of vital signs. The responses assessed include heart rate, blood pressure, oxygen saturation, oxygen extraction ratios, and pulmonary-to-systemic flow ratios. Mixed effects models were used to account for the repeated measures over the response interval. The analysis included 67 fluid boluses. There is a decrease in heart rate and an increase in blood pressure during the response in comparison to the hypotensive time. These vitals rapidly return to the baseline values. The boluses induced a significant decrease in renal and cerebral oxygen extraction ratios, with no significant change in arterial oxygen saturation or pulmonary-to-systemic flow ratio. The type of bolus (normal saline versus albumin) did not affect the response in blood pressure. However, in comparison with albumin, normal saline had a more favorable effect on the renal and cerebral oxygen extraction ratios. This study demonstrates that fluid boluses are an effective rescue medication for hypotensive episodes in children with parallel circulation by improving hemodynamics, as well as markers of oxygen delivery. The type of bolus (normal saline versus albumin) did not affect the blood pressure response. However, normal saline had a more pronounced effect on the renal and cerebral oxygen extraction ratios than albumin.