Long term high glucose exposure induces premature senescence in retinal endothelial cells.

Purpose: Features of cellular senescence have been described in diabetic retinal vasculature. The aim of this study was to investigate how the high glucose microenvironment impacts on the senescence program of retinal endothelial cells. Methods: Human retinal microvascular endothelial cells were cultured under control and high glucose conditions of 5 mM and 25 mM D-glucose, respectively. Isomeric l-glucose was used as the osmotic control. Cells were counted using CASY technology until they reached their Hayflick limit. Senescence-associated ß-Galactosidase was used to identify senescent cells. Endothelial cell functionality was evaluated by the clonogenic, 3D tube formation, and barrier formation assays. Cell metabolism was characterized using the Seahorse Bioanalyzer. Gene expression analysis was performed by bulk RNA sequencing. Retinal tissues from db/db and db/+ mice were evaluated for the presence of senescent cells. Publicly available scRNA-sequencing data for retinas from Akimba and control mice was used for gene set enrichment analysis. Results: Long term exposure to 25 mM D-Glucose accelerated the establishment of cellular senescence in human retinal endothelial cells when compared to 5 mM D-glucose and osmotic controls. This was shown from 4 weeks, by a significant slower growth, higher percentages of cells positive for senescence-associated ß-galactosidase, an increase in cell size, and lower expression of pRb and HMGB2. These senescence features were associated with decreased clonogenic capacity, diminished tubulogenicity, and impaired barrier function. Long term high glucose-cultured cells exhibited diminished glycolysis, with lower protein expression of GLUT1, GLUT3, and PFKFB3. Transcriptomic analysis, after 4 weeks of culture, identified downregulation of ALDOC, PFKL, and TPI1, in cells cultured with 25 mM D-glucose when compared to controls. The retina from db/db mice showed a significant increase in acellular capillaries associated with a significant decrease in vascular density in the intermediate and deep retinal plexuses, when compared to db/+ mice. Senescent endothelial cells within the db/db retinal vasculature were identified by senescence-associated ß-galactosidase staining. Analysis of single cell transcriptomics data for the Akimba mouse retina highlighted an enrichment of senescence and senescence-associated secretory phenotype gene signatures when compared to control mice. Conclusion: A diabetic-like microenvironment of 25 mM D-glucose was sufficient to accelerate the establishment of cellular senescence in human retinal microvascular endothelial cells.

Author Correction: Controlling parameters and characteristics of electrochemical biosensors for enhanced detection of 8-hydroxy-2'-deoxyguanosine.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Vascular Regeneration for Ischemic Retinopathies: Hope from Cell Therapies.

Retinal vascular diseases, such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, ocular ischemic syndrome and ischemic optic neuropathy, are leading causes of vision impairment and blindness. Whilst drug, laser or surgery-based treatments for the late stage complications of many of these diseases are available, interventions that target the early vasodegenerative stages are lacking. Progressive vasculopathy and ensuing ischemia is an underpinning pathology in many of these diseases, leading to hypoperfusion, hypoxia, and ultimately pathological neovascularization and/or edema in the retina and other ocular tissues, such as the optic nerve and iris. Therefore, repairing the retinal vasculature may prevent progression of ischemic retinopathies into late stage vascular complications. Various cell types have been explored for their vascular repair potential. Endothelial progenitor cells, mesenchymal stem cells and induced pluripotent stem cells are studied for their potential to integrate with the damaged retinal vasculature and limit ischemic injury. Clinical trials for some of these cell types have confirmed safety and feasibility in the treatment of ischemic diseases, including some retinopathies. Another promising avenue is mobilization of endogenous endothelial progenitors, whereby reparative cells are moved from their niche to circulating blood to target and home into ischemic tissues. Several aspects and properties of these cell types have yet to be elucidated. Nevertheless, we foresee that cell therapy, whether through delivery of exogenous or enhancement of endogenous reparative cells, will become a valuable and beneficial treatment for ischemic retinopathies.

Controlling parameters and characteristics of electrochemical biosensors for enhanced detection of 8-hydroxy-2'-deoxyguanosine.

This work discusses the parameters and characteristics required on the development of a scalable and reliable electrochemical sensor board for detecting 8-hydroxy-2'-deoxyguanosine (8-OHdG), an oxidative stress biomarker for diabetic nephropathy, cancer and Parkinson's disease. We used Printed Circuit Board (PCB) technology to make a precise, low-cost bare sensor board. ZnO nanorods (NRs) and ZnO NRs: reduced graphene oxide (RGO) composites were used as a pathway for antibody immobilization on the working electrode (WE). The parameters and characteristics of the WE were controlled for enhancing the quality of the electrochemical sensor board. Thickness of the gold and the presence of ZnO NRs or their composite on the WE have influence on charge transference process and reproducibility of the sensor board. The amount of the antibody, and its incubation period are crucial to avoid saturation of the sites during immobilization step and reduce the cost of the sensor. Our ZnO NRs-based electrochemical sensor board showed high sensitivity and selectivity to 8-OHdG with detection capacity in the range of 0.001-5.00 ng.mL-1. The successful application of our immunosensor to detect 8-OHdG in urine was evidenced.

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.

Dual effect of advanced glycation end products in pancreatic islet apoptosis.

BACKGROUND: Loss of ß-cell function hastens deterioration of metabolic control in type 2 diabetes patients. Besides amyloid deposit and glucolipotoxicity, advanced glycation end products (AGEs) acting through their receptors (RAGE) seem to contribute to this process by promoting islet apoptosis. In order to investigate the role of AGEs in ß-cell deterioration, we evaluated the temporal and dose effects of AGE compounds on apoptosis rate, reactive oxygen species generation and expression of pro-apoptotic and anti-apoptotic genes in cultured islets. METHODS: Rat pancreatic islets were exposed or not for 24, 48, 72 and 96 h to albumin modified by glycoaldehyde. Apoptosis, reactive oxygen species and superoxide content and NADPH oxidase activity were evaluated as well as RNA expression of the genes Ager (codes for RAGE), Bax, Bcl2 and Nfkb1. RESULTS: In 24 and 48 h, glycoaldehyde elicited a decrease in apoptosis rate in comparison with the control condition concomitantly with a reduction in Bax/Bcl2 RNA ratio and in Nfkb1 RNA expression. In contrast, after 72 and 96 h, glycoaldehyde promoted an increase in apoptosis rate concomitantly with an increase in Bax/Bcl2 RNA ratio and in Nfkb1 RNA expression. In 24 h, glycoaldehyde elicited a decrease in the islet content of reactive oxygen species, whereas after 48 and 72 h, it promoted an opposite effect, increasing superoxide generation. The NADPH oxidase inhibitor VAS2870 attenuated superoxide production, implicating NADPH oxidase as an important source of reactive oxygen species in islets exposed to AGEs. CONCLUSIONS: Albumin modified by glycoaldehyde exerted a dual effect in cultured pancreatic islets, being protective against apoptosis after short exposure but pro-apoptotic after prolonged exposure.

Tempol reduces podocyte apoptosis via PARP signaling pathway in experimental diabetes mellitus.

BACKGROUND/AIMS: In diabetic hypertensive rats, tempol reduces albuminuria by restoring the redox imbalance. Increased formation of reactive oxygen species leading to activation of poly(ADP-ribose) polymerase (PARP)-1 and podocyte loss by apoptosis contribute to albuminuria in diabetes mellitus (DM). In the present study, we investigated the hypothesis that in DM tempol reduces albuminuria by inhibition of PARP-induced podocyte apoptosis. METHODS: DM was induced in 4-week-old spontaneously hypertensive rats by streptozotocin. Mouse and human podocyte cell lines were cultured in normal or high-glucose conditions, with or without tempol and/or a PARP-1 inhibitor, PJ34. RESULTS: In diabetic rats, tempol treatment did not affect plasma glucose levels or systolic blood pressure. Albuminuria was higher in diabetic rats, and it was reduced by tempol. DM leads to an elevation of glomerular apoptotic cells and to podocyte loss; both were prevented by tempol treatment. DM increases the expression of poly(ADP-ribose)-modified proteins in isolated glomeruli, and it was reduced by tempol. In vitro, high glucose increased caspase-3 activity and led to a higher number of apoptotic cells that were prevented by tempol and the PARP-1 inhibitor. CONCLUSION: In DM, tempol reduces albuminuria associated with reduction of podocyte apoptosis and decreasing oxidative stress via PARP signaling.

Spironolactone improves nephropathy by enhancing glucose-6-phosphate dehydrogenase activity and reducing oxidative stress in diabetic hypertensive rat.

Spironolactone (SPR), a mineralocorticoid receptor blocker, diminishes hyperglycemia-induced reduction in glucose-6-phosphate dehydrogenase (G6PD) activity, improving oxidative stress damage. This study investigated whether SPR ameliorates nephropathy by increasing G6PD activity and reducing oxidative stress in spontaneously hypertensive diabetic rats (SHRs). The streptozotocin-induced diabetic rats received or not SPR 50 mg/kg per day, for eight weeks. A human mesangial cell line was cultured in normal or high glucose conditions, with or without SPR, for 24 h. Plasma glucose levels and systolic blood pressure were unaltered by diabetes or by SPR treatment. Albuminuria, fibronectin expression, 8-OHdG urinary levels, lipid peroxidation and p47phox expression were higher in the diabetic rats compared with the control and were reduced by SPR. The antioxidant GSH/GSSG ratio was reduced in the diabetic rats and the treatment reestablished it. Diabetes-induced SGK1 up-regulation was inhibited by SPR. Reactive oxygen species (ROS) and superoxide production induced by NADPH oxidase were increased by hyperglycemia and high glucose, in vivo and in vitro, respectively, and were reduced with SPR. Hyperglycemia and high glucose decreased G6PD activity, which was restored with SPR. These results suggest that SPR ameliorates nephropathy in diabetic SHRs by restoring G6PD activity and diminishes oxidative stress without affecting glycaemia and blood pressure.

p53-Mediated oxidative stress and tubular injury in rats with glycerol-induced acute kidney injury.

BACKGROUND/AIMS: p53 is a transcriptional factor that responds to severe cell damage promoting the transcription of proapoptotic and prooxidant genes. In this study, we evaluated the role of p53 activation in glycerol-induced acute kidney injury (Gly-AKI). METHODS: Rats were treated with p53 inhibitor (pifithrin-α) in the moment we injected glycerol. Renal function, renal histology (HE), TUNEL labeling, cleaved caspase-3 staining, renal p53, Bax, PUMA, Bcl-2, p21 and survivin expressions, renal lipid and DNA oxidative markers, and the expression of antioxidant enzymes (Mn-SOD, HO-1, and NAD(P)H:quinone-oxidoreductase-1) were evaluated. RESULTS: Gly-AKI rats showed an increased renal expression of phosphorylated-p53. The p53 inhibitor attenuated renal impairment and significantly reduced tubular injury. The expression of the oxidative markers was significantly reduced in treated rats. Proapoptotic and prooxidant proteins Bax and PUMA were overexpressed in Gly-AKI rats and reduced in treated rats. On the contrary, antiapoptotic Bcl-2, p21, and survivin showed a tendency to increase in treated rats. The antioxidant enzymes' expression remained elevated or increased in treated rats. CONCLUSION: On the whole, p53 inhibition was protective in the short term. The oxidative stress subsided and the transcription tipped toward prosurvival genes; consequently tubular injury was attenuated in treated rats.

Antioxidant SOD mimetic prevents NADPH oxidase-induced oxidative stress and renal damage in the early stage of experimental diabetes and hypertension.

AIMS: The presence of hypertension increases renal oxidative stress by increasing NADPH oxidase-dependent superoxide production and by decreasing antioxidant defense in the early stage of experimental diabetes mellitus (DM). In the present study, we investigated whether the administration of an antioxidant mimetic of the superoxide dismutase (SOD) (tempol) corrects the oxidative imbalance and oxidative stress-induced renal injury in the presence of DM and hypertension. METHODS: DM was induced in spontaneously hypertensive rats (SHR) by streptozotocin at 4 weeks of age. The diabetic rats either did or did not receive tempol for 20 days. Oxidative-stress parameters and indices of renal injury were evaluated. RESULTS: Tempol reestablished the imbalance in redox status induced by DM. It elevated the expression of renal antioxidant extracellular SOD, p < 0.0001; decreased (p = 0.049) the production of renal NADPH-dependent superoxide production, and diminished (p = 0.016) a marker of oxidative stress-induced DNA damage, 8-hydroxy-2'-deoxyguanosine. Reduction of oxidative stress markers was associated with reduction in renal damage parameters associated with DN. DM-induced albuminuria and elevation in renal expression of collagen IV were reduced to the level observed in control rats. CONCLUSION: We conclude that an imbalance in renal redox status is associated with markers of renal injury in the early stage of DM and hypertension. Antioxidant treatment reestablished the redox status and prevented oxidative stress-induced renal damage.

Hypertension increases pro-oxidant generation and decreases antioxidant defense in the kidney in early diabetes.

AIMS: The combination of hypertension and diabetes exacerbates renal oxidative stress. The aim of the present study was therefore to evaluate the pro-oxidant and antioxidant mechanisms responsible for the induction of renal oxidative stress in the presence of hypertension and diabetes mellitus. METHODS: Diabetes was induced in spontaneously hypertensive rats (SHR) and their genetically normotensive control Wistar-Kyoto (WKY) rats by streptozotocin at 12 weeks of age. After 10 days, pro-oxidant, antioxidant and oxidative stress parameters were evaluated in the renal tissue. RESULTS: NADPH oxidase-dependent superoxide generation in the renal cortex was significantly elevated in WKY and SHR diabetic (D) groups compared to the respective control (C) groups (p < 0.005, n = 5). However, the highest level of superoxide generation was observed in the SHR-D group compared to all other groups. The expression of the gp91phox subunit of NADPH oxidase was significantly elevated in the SHR-D (p < 0.05, n = 5), but not in the WKY-D group, compared to the respective control groups. The renal cortical extracellular-superoxide dismutase level was found to be markedly decreased in the SHR groups compared to the WKY groups (p < 0.05, n = 5). The antioxidant glutathione level was found to be lower in the SHR-D (p = 0.03, n = 15), but not in the WKY-D group, compared to the respective control groups. Finally, nitrotyrosine and 8-hydroxy-2'-deoxyguanosine, markers of oxidative stress, were found to be similar in the kidneys of WKY-C and WKY-D, but were elevated in the SHR-D compared to the SHR-C group. CONCLUSION: We therefore conclude that hypertension increases pro-oxidant generation and decreases antioxidant defense, and thereby induces renal oxidative stress in early diabetes.

Protamine-induced epithelial barrier disruption involves rearrangement of cytoskeleton and decreased tight junction-associated protein expression in cultured MDCK strains.

Natural and synthetic polycationic proteins, such as protamine, have been used to reproduce the tissue injury and changes in epithelial permeability caused by positively charged substances released by polymorphonuclear cells during inflammation. Protamine has diverse and often conflicting effects on epithelial permeability. The effects of this polycation on the distribution and expression of tight junction (TJ)-associated proteins have not yet been investigated. In this work, we examined the influence of protamine on paracellular barrier function and TJ structure using two strains of the epithelial Madin-Darby canine kidney (MDCK) cell line that differed in their TJ properties ("tight" TJ-strain I and "leaky" TJ-strain II). Protamine induced concentration-, time- and strain-dependent alterations in transepithelial electrical resistance (Rt) only when applied to apical or apical+basolateral monolayer surfaces, indicating a polarity of action. In MDCK II cells, protamine (50 microg/ml) caused a significant increase in Rt that returned to control values after 2 h. However, the treatment of this MDCK strain with a higher concentration of protamine (250 microg/ml) significantly decreased the Rt after 30 min. In contrast, treated MDCK I monolayers showed a significant decrease in Rt after apical treatment with protamine at both concentrations. The protamine-induced decrease in Rt was paralleled by an increase in the phenol red basal-to-apical flux in both MDCK strains, suggesting disruption of the paracellular barrier. Marked changes in cytoskeletal F-actin distribution/polymerization and a significant reduction in the junctional expression of the tight junctional proteins occludin and claudin-1 but subtle alterations in ZO-1 were observed following protamine-elicited paracellular barrier disruption. In conclusion, protamine induces alterations in the epithelial barrier function of MDCK monolayers that may involve the cytoskeleton and TJ-associated proteins. The various actions of protamine on epithelial function may reflect different degrees of interaction of protamine with the plasma membrane and different intracellular processes triggered by this polycation.