Fabrication of gradient hydrogels using a thermophoretic approach in microfluidics.

The extracellular matrix presents spatially varying physical cues that can influence cell behavior in many processes. Physical gradients within hydrogels that mimic the heterogenous mechanical microenvironment are useful to study the impact of these cues on cellular responses. Therefore, simple and reliable techniques to create such gradient hydrogels are highly desirable. This work demonstrates the fabrication of stiffness gradient Gellan gum (GG) hydrogels by applying a temperature gradient across a microchannel containing hydrogel precursor solution. Thermophoretic migration of components within the precursor solution generates a concentration gradient that mirrors the temperature gradient profile, which translates into mechanical gradients upon crosslinking. Using this technique, GG hydrogels with stiffness gradients ranging from 20 to 90 kPa over 600µm are created, covering the elastic moduli typical of moderately hard to hard tissues. MC3T3 osteoblast cells are then cultured on these gradient substrates, which exhibit preferential migration and enhanced osteogenic potential toward the stiffest region on the gradient. Overall, the thermophoretic approach provides a non-toxic and effective method to create hydrogels with defined mechanical gradients at the micron scale suitable forin vitrobiological studies and potentially tissue engineering applications.

Quality of life in people with multiple sclerosis receiving glatiramer acetate or interferon in Greek clinical practice.

Aim: To evaluate glatiramer acetate (GA) or IFN-ß effects on quality of life (QoL) in people with relapsing/remitting multiple sclerosis (PwRRMS) in Greece. Methods: A prospective, practice-based study. QoL/function/symptoms were assessed by seven questionnaires/scales. Results: Significant increases in Short Form-36 (SF-36) health survey scores occurred with GA in four of the eight domains and three of the eight domains at 6 and 12 months, respectively, versus baseline. Similar and significant SF-36 score improvements occurred with GA in treatment-naive PwRRMS. SF-36 scores were unaffected in GA-treated, IFN-ß treatment-experienced PwRRMS, or with IFN-ß versus baseline. Slight improvements in fatigue and sexual satisfaction were evident (6 months). No deteriorations were seen in the other four instruments. Conclusion: The findings show that 12-month treatment with GA, but not IFN-ß, improved certain QoL parameters in treatment-naive PwRRMS.

People with relapsing/remitting multiple sclerosis (PwRRMS) are treated with drugs, for example, glatiramer acetate (GA) or IFN-ß. We checked if these drugs improved quality of life (QoL) in PwRRMS in Greece. QoL was measured by seven questionnaires, asking many questions on aspects of life. One survey showed significant improvements with GA treatment in almost half of the question groups. Similar improvements in this survey were seen with GA in patients who had no other previous treatments. No changes were seen in GA-treated PwRRMS who previously received IFN-ß, or treated with IFN-ß alone. Slight improvements in fatigue and sexual satisfaction were seen. No QoL deteriorations were seen in the other four questionnaires. Twelve months of GA treatment, but not IFN-ß, improved certain QoL parameters in treatment-naive PwRRMS.

Distinct profiles of LRRK2 activation and Rab GTPase phosphorylation in clinical samples from different PD cohorts.

Despite several advances in the field, pharmacodynamic outcome measures reflective of LRRK2 kinase activity in clinical biofluids remain urgently needed. A variety of targets and approaches have been utilized including assessments of LRRK2 itself (levels, phosphorylation), or its substrates (e.g. Rab10 or other Rab GTPases). We have previously shown that intrinsic kinase activity of LRRK2 isolated from PBMCs of G2019S carriers is elevated, irrespective of disease status. In the present study we find that phosphorylation of Rab10 is also elevated in G2019S carriers, but only those with PD. Additionally, phosphorylation of this substrate is also elevated in two separate idiopathic PD cohorts, but not in carriers of the A53T mutation in α-synuclein. In contrast, Rab29 phosphorylation was specifically reduced in urinary exosomes from A53T and idiopathic PD patients. Taken together, our findings highlight the need for the assessment of multiple complimentary targets for a more comprehensive picture of the disease.

Characteristics of respiratory microdroplet nuclei on common substrates.

To evaluate the role of common substrates in the transmission of respiratory viruses, in particular SARS-CoV-2, uniformly distributed microdroplets (approx. 10 µm diameter) of artificial saliva were generated using an advanced inkjet printing technology to replicate the aerosol droplets and subsequently deposited on five substrates, including glass, polytetrafluoroethylene, stainless steel, acrylonitrile butadiene styrene and melamine. The droplets were found to evaporate within a short timeframe (less than 3 s), which is consistent with previous reports concerning the drying kinetics of picolitre droplets. Using fluorescence microscopy and atomic force microscopy, we found that the surface deposited microdroplet nuclei present two distinctive morphological features as the result of their drying mode, which is controlled by both interfacial energy and surface roughness. Nanomechanical measurements confirm that the nuclei deposited on all substrates possess similar surface adhesion (approx. 20 nN) and Young's modulus (approx. 4 MPa), supporting the proposed core-shell structure of the nuclei. We suggest that appropriate antiviral surface strategies, e.g. functionalization, chemical deposition, could be developed to modulate the evaporation process of microdroplet nuclei and subsequently mitigate the possible surface viability and transmissibility of respiratory virus.

Val66Met polymorphism is associated with decreased likelihood for pediatric headache and migraine.

Background: Migraine is a complex multifactorial disorder and its pathogenesis still remains unclear. Evidence suggests the involvement of the activated trigeminovascular pathway, in which BDNF seems to play an important role. Therefore, BDNF polymorphisms are promising candidate susceptibility factors.Aim: BDNF rs6265 functional polymorphism was analyzed in order to determine its possible association with pediatric headache and migraine risk.Methods: The research included 120 consecutive pediatric patients who were diagnosed with headache and 120 healthy controls. The diagnosis was in compliance with the International Classification of Headache Disorders. Blood samples were collected from all participants and genotyped for rs6265.Results: BDNF rs6265 was significantly associated with decreased headache risk, particularly in the dominant model [Odds Ratio, OR (95% confidence interval, C.I.): 0.47 (0.26-0.85), p = 0.011] and the log-additive model [OR (95% C.I.): 0.48 (0.28-0.82), p = 0.0053]. During the sensitivity analysis, the associations were also maintained among patients with migraine.Conclusions: This is the first study to reveal a significant association of this BDNF variant with headache risk. Additionally, Val66Met was also for the first time related to decreased childhood migraine risk.

Collecting duct cells show differential retinoic acid responses to acute versus chronic kidney injury stimuli.

Retinoic acid (RA) activates RA receptors (RAR), resulting in RA response element (RARE)-dependent gene expression in renal collecting duct (CD). Emerging evidence supports a protective role for this activity in acute kidney injury (AKI) and chronic kidney disease (CKD). Herein, we examined this activity in RARE-LacZ transgenic mice and by RARE-Luciferase reporter assays in CD cells, and investigated how this activity responds to neurotransmitters and mediators of kidney injury. In RARE-LacZ mice, Adriamycin-induced heavy albuminuria was associated with reduced RA/RAR activity in CD cells. In cultured CD cells, RA/RAR activity was repressed by acetylcholine, albumin, aldosterone, angiotensin II, high glucose, cisplatin and lipopolysaccharide, but was induced by aristolochic acid I, calcitonin gene-related peptide, endothelin-1, gentamicin, norepinephrine and vasopressin. Compared with age-matched normal human CD cells, CD-derived renal cystic epithelial cells from patients with autosomal recessive polycystic kidney disease (ARPKD) had significantly lower RA/RAR activity. Synthetic RAR agonist RA-568 was more potent than RA in rescuing RA/RAR activity repressed by albumin, high glucose, angiotensin II, aldosterone, cisplatin and lipopolysaccharide. Hence, RA/RAR  in CD cells is a convergence point of regulation by neurotransmitters and mediators of kidney injury, and may be a novel therapeutic target.

Whole exome sequencing establishes diagnosis of Charcot-Marie-Tooth 4J, 1C, and X1 subtypes.

BACKGROUND: Charcot-Marie-Tooth (CMT) hereditary polyneuropathies pose a diagnostic challenge. Our aim here is to describe CMT patients diagnosed by whole exome sequencing (WES) following years of fruitless testing. METHODS/RESULTS: Three patients with polyneuropathy suspected to be genetic in origin, but not harboring PMP22 gene deletion/duplication, were offered WES. The first patient, a 66-year-old man, had been suffering from progressive weakness and atrophies in the lower and upper extremities for 20 years. Due to ambiguous electrophysiological findings, immune therapies were administered to no avail. Twelve years after PMP22 deletion/duplication testing, WES revealed two pathogenic variants in the FIG4 gene (p.Ile41Thr and p.Phe598fs, respectively), as a cause of CMT 4J. The second patient, a 19-year-old man, had been suffering from hearing and gait impairment since at least his infancy, and recently presented with weakness and dystonia of the lower extremities. In this patient, WES identified the p.Leu122Val LITAF gene variant in heterozygous state, suggesting the diagnosis of CMT 1C, several years after initial genetic analyses. The third patient, a 44-year-old man, presented with progressive weakness and atrophies of the lower and upper extremities since the age of 17 years old. In this patient, WES identified the hemizygous p.Arg164Gln pathogenic variant in the GJB1 gene, establishing the diagnosis of CMT X1, 8 years after testing for PMP22 deletion/duplication. CONCLUSION: Novel diagnostic techniques, such as WES, offer the possibility to decipher the cause of CMT subtypes, ending the diagnostic Odyssey of the patients and sparing them from unnecessary and potentially harmful treatments.

Linkage between endosomal escape of LNP-mRNA and loading into EVs for transport to other cells.

RNA-based therapeutics hold great promise for treating diseases and lipid nanoparticles (LNPs) represent the most advanced platform for RNA delivery. However, the fate of the LNP-mRNA after endosome-engulfing and escape from the autophagy-lysosomal pathway remains unclear. To investigate this, mRNA (encoding human erythropoietin) was delivered to cells using LNPs, which shows, for the first time, a link between LNP-mRNA endocytosis and its packaging into extracellular vesicles (endo-EVs: secreted after the endocytosis of LNP-mRNA). Endosomal escape of LNP-mRNA is dependent on the molar ratio between ionizable lipids and mRNA nucleotides. Our results show that fractions of ionizable lipids and mRNA (1:1 molar ratio of hEPO mRNA nucleotides:ionizable lipids) of endocytosed LNPs were detected in endo-EVs. Importantly, these EVs can protect the exogenous mRNA during in vivo delivery to produce human protein in mice, detected in plasma and organs. Compared to LNPs, endo-EVs cause lower expression of inflammatory cytokines.

The cortical excitability profile of patients with the G209A SNCA mutation versus patients with sporadic Parkinson's disease: A transcranial magnetic stimulation study.

Mutations in the α-synuclein gene are a rare cause of Parkinson's disease. We investigated, by single-pulse TMS, the cortical excitability profile of nine α-synuclein patients in comparison with 24 idiopathic PD patients, subdivided into "akinetic" (n=17) and "tremor-dominant" (n=7) subgroups. The comparative assessment of rest motor threshold, active MEP and Silent Period Input/Output curves indicated that the cortical excitability of α-Synuclein patients is similar to patients with the "akinetic" form of PD. Both groups of patients exhibited differences in excitatory and inhibitory brain circuits from "tremor-dominant" patients indicating that varying clinical phenotypes are associated with differential profiles of corticospinal excitability.

Identification of RNA-binding proteins in exosomes capable of interacting with different types of RNA: RBP-facilitated transport of RNAs into exosomes.

The RNA that is packaged into exosomes is termed as exosomal-shuttle RNA (esRNA); however, the players, which take this subset of RNA (esRNA) into exosomes, remain largely unknown. We hypothesized that RNA binding proteins (RBPs) could serve as key players in this mechanism, by making complexes with RNAs and transporting them into exosomes during the biosynthesis of exosomes. Here, we demonstrate the presence of 30 RBPs in exosomes that were shown to form RNA-RBP complexes with both cellular RNA and exosomal-RNA species. To assess the involvement of these RBPs in RNA-transfer into exosomes, the gene transcripts encoding six of the proteins identified in exosomes (HSP90AB1, XPO5, hnRNPH1, hnRNPM, hnRNPA2B1, and MVP) were silenced by siRNA and subsequent effect on esRNA was assessed. A significant reduction of total esRNA was observed by post-transcriptional silencing of MVP, compared to other RBPs. Furthermore, to confirm the binding of MVP with esRNA, a biotinylated-MVP was transiently expressed in HEK293F cells. Higher levels of esRNA were recovered from MVP that was eluted from exosomes of transfected cells, as compared to those of non-transfected cells. Our data indicate that these RBPs could end up in exosomes together with RNA molecules in the form of RNA-ribonucleoprotein complexes, which could be important for the transport of RNAs into exosomes and the maintenance of RNAs inside exosomes. This type of maintenance may favor the shuttling of RNAs from exosomes to recipient cells in the form of stable complexes.

Blink reflex habituation in migraine and chronic tension-type headache.

ΑBSTRACT: R1 and R2 blink reflex latencies were investigated blind in 55 patients with chronic tension-type headache, 55 patients with migraine, and 55 headache-free controls. Standard electrical stimulation of the supraorbital nerve was applied and the response was recorded from the ipsilateral orbicularis oculi muscles. There were no R1 or R2 latency differences between the three groups. During migraine attacks we observed a statistically significant reduction of R2 amplitude and area. The main finding of our study was the elicitation of the late R2" response at different interstimulus intervals in migraine patients compared to the tension-type headache and control groups. This could be considered an indication of habituation mechanism hyperexcitability, although further investigation is needed to confirm these findings and establish the neurophysiologic basis. This study suggests that blink reflex studies can be used routinely as a non-evasive and inexpensive method for the evaluation of headache patients.

Defective Autophagy in Diabetic Retinopathy.

PURPOSE: Müller cells (MCs) are a major source of VEGF in diabetic retinopathy (DR). Vascular endothelial growth factor is the main therapeutic target for treating DR. This study aimed to investigate whether autophagy is involved in MC response under high glucose (HG). METHODS: Rat retinal Müller cells (rMCs) were exposed to normal or high glucose in and out of presence of pharmacologic inhibitors and activators and small interfering RNA (siRNA) for p62/SQTSM1 for 24 hours. RESULTS: High glucose induces increase of early and late autophagic markers, accumulation of p62/SQTSM1 and endoplasmic reticulum (ER) stress response associated with apoptosis augmentation (P < 0.01). The inhibition of autophagy in HG leads to higher rMC apoptotic rate (P < 0.001). By silencing the p62/SQTSM1, ER stress is ameliorated (p<0.0001), preventing apoptosis. Retinal MCs in HG treated with rapamycin (mTOR inhibitor) show autophagy machinery activation and reestablishment of cargo degradation, protecting cells from apoptosis (P < 0.0001). Rapamycin improves lysosomal proteolytic activity by improving cathepsin L activity restoring autophagic cargo degradation, and preventing increased VEGF release (P < 0.0001). In experimental model of diabetes, Beclin-1 and p62/SQTSM-1 were found to be marked increased in retinas from diabetic Wystar Kyoto rats compared with control group (P < 0.003) with reduction of cathepsin L activity. CONCLUSIONS: High glucose upregulates autophagy but accumulates p62/SQTSM1 cargo due to lysosomal dysfunction, leading to massive VEGF release and cell death of rMCs. Lysosomal impairment and autophagic dysfunction are early events present in the pathogenesis of diabetic retinopathy (DR). This might be valuable for developing a novel therapeutic strategy to treat DR.

The use of green tea polyphenols for treating residual albuminuria in diabetic nephropathy: A double-blind randomised clinical trial.

Prior research has shown that in experimental diabetes mellitus, green tea reduces albuminuria by decreasing podocyte apoptosis through activation of the WNT pathway. We investigated the effect of green tea polyphenols (GTP) on residual albuminuria of diabetic subjects with nephropathy. We conducted a randomised, double-blind study in 42 diabetic subjects with a urinary albumin-creatinine ratio (UACR) >30 mg/g, despite administration of the maximum recommended dose of renin-angiotensin (RAS) inhibition. Patients were randomly assigned to two equal groups to receive either GTP (containing 800 mg of epigallocatechin gallate, 17 with type 2 diabetes and 4 with type 1 diabetes) or placebo (21 with type 2 diabetes) for 12 weeks. Treatment with GTP reduced UACR by 41%, while the placebo group saw a 2% increase in UACR (p = 0.019). Podocyte apoptosis (p = 0.001) and in vitro albumin permeability (p < 0.001) were higher in immortalized human podocytes exposed to plasma from diabetic subjects compared to podocytes treated with plasma from normal individuals. In conclusion, GTP administration reduces albuminuria in diabetic patients receiving the maximum recommended dose of RAS. Reduction in podocyte apoptosis by activation of the WNT pathway may have contributed to this effect.

Motor and Nonmotor Features of Carriers of the p.A53T Alpha-Synuclein Mutation: A Longitudinal Study.

BACKGROUND: G209A SNCA mutation carriers represent an important group of genetic PD. We describe motor and nonmotor features of G209A SNCA mutation carriers. METHODS: Longitudinal clinical assessments over 2 years were collected in 22 symptomatic and 8 asymptomatic G209A SNCA mutation carriers. Motor and nonmotor rating scales were administered. Correlations were performed between clinical variables and disease duration or age. Penetrance was calculated using Kaplan-Meier survival curves. RESULTS: Asymptomatic carriers did not manifest clear premotor symptoms, but symptomatic carriers often reported that olfactory dysfunction and rapid eye movement sleep behavior disorder preceded motor symptoms. Prominent motor decline and deterioration of autonomic and cognitive function occurred at follow-up; such nonmotor features correlated with disease duration, but not age. Disease penetrance was estimated at around 90%. CONCLUSIONS: This study may help to inform clinical trials and provide the basis for studies of disease modifiers in genetic synucleinopathy cohorts. © 2016 International Parkinson and Movement Disorder Society.

Conditioned Medium from Early-Outgrowth Bone Marrow Cells Is Retinal Protective in Experimental Model of Diabetes.

Bone marrow-derived cells were demonstrated to improve organ function, but the lack of cell retention within injured organs suggests that the protective effects are due to factors released by the cells. Herein, we tested cell therapy using early outgrowth cells (EOCs) or their conditioned media (CM) to protect the retina of diabetic animal models (type 1 and type 2) and assessed the mechanisms by in vitro study. Control and diabetic (db/db) mice (8 weeks of age) were randomized to receive a unique intravenous injection of 5×105EOCs or 0.25 ml thrice weekly tail-vein injections of 10x concentrated CM and Wystar Kyoto rats rendered diabetic were randomized to receive 0.50 ml thrice weekly tail-vein injections of 10x concentrated CM. Four weeks later, the animals were euthanized and the eyes were enucleated. Rat retinal Müller cells (rMCs) were exposed for 24 h to high glucose (HG), combined or not with EOC-conditioned medium (EOC-CM) from db/m EOC cultures. Diabetic animals showed increase in diabetic retinopathy (DR) and oxidative damage markers; the treatment with EOCs or CM infusions significantly reduced this damage and re-established the retinal function. In rMCs exposed to diabetic milieu conditions (HG), the presence of EOC-CM reduced reactive oxygen species production by modulating the NADPH-oxidase 4 system, thus upregulating SIRT1 activity and deacetylating Lys-310-p65-NFκB, decreasing GFAP and VEGF expressions. The antioxidant capacity of EOC-CM led to the prevention of carbonylation and nitrosylation posttranslational modifications on the SIRT1 molecule, preserving its activity. The pivotal role of SIRT1 on the mode of action of EOCs or their CM was also demonstrated on diabetic retina. These findings suggest that EOCs are effective as a form of systemic delivery for preventing the early molecular markers of DR and its conditioned medium is equally protective revealing a novel possibility for cell-free therapy for the treatment of DR.

Conjugate eye deviation as predictor of acute cortical and subcortical ischemic brain lesions.

OBJECTIVES: Non-enhanced computed tomography (NECT) of the brain is used to exclude intracranial hemorrhage in patients who are considered for treatment with tissue plasminogen activator due to stroke symptoms. However, early infarct signs on NECT have low sensitivity for ischemic stroke. It was hypothesized that horizontal conjugate eye deviation (average ocular gaze deviation-OGD >14°) on NECT predicts ischemic brain injury on a second detailed examination. PATIENTS AND METHODS: Patients who underwent brain NECT within three hours after the onset of stroke symptoms and subsequently had brain CT scan with intravenous contrast or MRI were potential participants. OGD was measured from the cross-sectional image including both globes at their maximum diameter. RESULTS: 73 subjects were studied (mean age 64.2±20.8 years) with a median interval (interquartile range) of 56 h (22-109.3 h) between NECT and the second examination. On NECT, 24 of 73 (32.9%) subjects had OGD >14°. Of 32 individuals with acute ischemic injury on the second examination, 19 (59.4%) had OGD >14° on NECT. OGD >14° was associated with increased risk of ischemic injury: OR=10.5 (95% confidence interval 3.33-33.9); P=0.002. OGD >14° had significantly higher sensitivity and negative predictive value than early infarct signs on NECT (59.4% vs. 21.9% and 73.5% vs. 59.7%, respectively; P<0.05), and similar specificity and positive predictive value (87.8% vs. 90.2% and 79.2% vs. 63.6%; P>0.05). CONCLUSION: In the presence of stroke symptoms, average OGD >14° on the initial brain NECT is early predictor of ischemic brain injury.

Polyphenol-enriched cocoa protects the diabetic retina from glial reaction through the sirtuin pathway.

Cocoa is rich in flavonoids, which are potent antioxidants with established benefits for cardiovascular health but unproven effects on neurodegeneration. Sirtuins (SIRTs), which make up a family of deacetylases, are thought to be sensitive to oxidation. In this study, the possible protective effects of cocoa in the diabetic retina were assessed. Rat Müller cells (rMCs) exposed to normal or high glucose (HG) or H2O2 were submitted to cocoa treatment in the presence or absence of SIRT-1 inhibitor and small interfering RNA The experimental animal study was conducted in streptozotocin-induced diabetic rats randomized to receive low-, intermediate-, or high-polyphenol cocoa treatments via daily gavage for 16 weeks (i.e., 0.12, 2.9 or 22.9 mg/kg/day of polyphenols). The rMCs exposed to HG or H2O2 exhibited increased glial fibrillary acidic protein (GFAP) and acetyl-RelA/p65 and decreased SIRT1 activity/expression. These effects were cancelled out by cocoa, which decreased reactive oxygen species production and PARP-1 activity, augmented the intracellular pool of NAD(+), and improved SIRT1 activity. The rat diabetic retinas displayed the early markers of retinopathy accompanied by markedly impaired electroretinogram. The presence of diabetes activated PARP-1 and lowered NAD(+) levels, resulting in SIRT1 impairment. This augmented acetyl RelA/p65 had the effect of up-regulated GFAP. Oral administration of polyphenol cocoa restored the above alterations in a dose-dependent manner. This study reveals that cocoa enriched with polyphenol improves the retinal SIRT-1 pathway, thereby protecting the retina from diabetic milieu insult.

Theobromine increases NAD⁺/Sirt-1 activity and protects the kidney under diabetic conditions.

Reduction in sirtuin 1 (Sirt-1) is associated with extracellular matrix (ECM) accumulation in the diabetic kidney. Theobromine may reduce kidney ECM accumulation in diabetic rats. In the current study, we aimed to unravel, under diabetic conditions, the mechanism of kidney ECM accumulation induced by a reduction in Sirt-1 and the effect of theobromine in these events. In vitro, we used immortalized human mesangial cells (iHMCs) exposed to high glucose (HG; 30 mM), with or without small interfering RNA for NOX4 and Sirt-1. In vivo, spontaneously hypertensive rats (SHR) were rendered diabetic by means of streptozotocin and studied after 12 wk. The effects of treatment with theobromine were investigated under both conditions. HG leads to a decrease in Sirt-1 activity and NAD(+) levels in iHMCs. Sirt-1 activity could be reestablished by treatment with NAD(+), silencing NOX4, and poly (ADP-ribose) polymerase-1 (PARP-1) blockade, or with theobromine. HG also leads to a low AMP/ATP ratio, acetylation of SMAD3, and increased collagen IV, which is prevented by theobromine. Sirt-1 or AMPK blockade abolished these effects of theobromine. In diabetic SHR, theobromine prevented increases in albuminuria and kidney collagen IV, reduced AMPK, elevated NADPH oxidase activity and PARP-1, and reduced NAD(+) levels and Sirt-1 activity. These results suggest that in diabetes mellitus, Sirt-1 activity is reduced by PARP-1 activation and NAD(+) depletion due to low AMPK, which increases NOX4 expression, leading to ECM accumulation mediated by transforming growth factor (TGF)-ß1 signaling. It is suggested that Sirt-1 activation by theobromine may have therapeutic potential for diabetic nephropathy.

Inactivation of AMPK mediates high phosphate-induced extracellular matrix accumulation via NOX4/TGFß-1 signaling in human mesangial cells.

BACKGROUND/AIMS: High phosphate (Pi) levels and extracellular matrix (ECM) accumulation are associated with chronic kidney disease progression. However, how high Pi levels contribute to ECM accumulation in mesangial cells is unknown. The present study investigated the role and mechanism of high Pi levels in ECM accumulation in immortalized human mesangial cells (iHMCs). METHODS: iHMCs were exposed to normal (0.9 mM) or increasing Pi concentrations (2.5 and 5 mM) with or without diferent blockers or activators. NOX4, phosphorylated AMPK (p-AMPK), phosphorylated SMAD3 (p-SMAD3), fibronectin (F/N), collagen IV (C-IV) and alpha-smooth muscle actin (α-SMA) expression was assessed via western blot and immunofluorescence. Lucigenin-enhanced chemiluminescence, and dihydroethidium (DHE) assessed NADPH oxidase activity and superoxide (SO), respectively. RESULTS: In iHMCs, a Pi transporter blocker (PFA) abrogated high Pi-induced AMPK inactivation, increase in NADPH oxidase-induced reactive oxygen species (ROS) levels, NOX4, p-SMAD3, α-SMA and C-IV expression. AMPK activation by AICAR, NOX4 silencing or NADPH oxidase blocker prevented high Pi-induced DHE levels, p-SMAD3, F/N, C-IV and α-SMA expression. CONCLUSION: AMPK inactivation with NOX4-induced ROS formation and transforming growth factor ß-1 (TGFß-1) signaling activation mediates high Pi-induced ECM accumulation in iHMCs. Maneuvers increasing AMPK or reducing NOX4 activity may contribute to renal protection under hyperphosphatemia.

Increase in AMPK brought about by cocoa is renoprotective in experimental diabetes mellitus by reducing NOX4/TGFß-1 signaling.

The aims of the present study were to investigate, in diabetes mellitus (DM), the mechanism of NOX4 up-regulation, its link with 5' adenosine monophosphate-activated protein kinase (AMPK) inactivation and transforming growth factor (TGF) ß-1 signaling in determining the accumulation of kidney extracellular matrix (ECM), and the possible action of cocoa enriched with polyphenols (CH) in these events. After 16 weeks of DM, spontaneously hypertensive rats showed increased kidney TGFß-1 levels and expression of phosphorylated smad2, collagen IV and fibronectin in parallel with elevated NOX4 expression and reduced phosphorylated AMPK. CH treatment in diabetic rats prevented all of these abnormalities. In immortalized human mesangial cells exposed to high glucose (HG), or TGFß-1, CH, nicotinamide adenine dinucleotide phosphate blocker, or silencing NOX4 ameliorated enhanced phosphorylated smad2 and collagen IV. Reduction in phosphorylated AMPK induced by HG or TGFß-1 was ameliorated by CH or activation of AMPK, which reduced phosphorylation of smad2 and collagen IV via reduction in NOX4 expression. The effects of CH were abolished by AMPK blockade. These results suggest that inactivation in AMPK leads to NOX4 up-regulation, activation of TGFß-1 signaling and increased ECM accumulation. Additionally, increased TGF-ß1 per se leads to the amplification of ECM production by reducing AMPK and promoting the activation of NOX4. It is suggested that the activation of AMPK by CH followed by reduction in NOX4/TGFß-1 signaling may have a therapeutic potential in diabetic nephropathy.