Regulation of renal brush-border glucose transport in response to metabolic dysregulation

Diabetic nephropathy is a consequence of hyperglycaemia-induced renal cell damage and a major contributor to end-stage renal disease in later stages of diabetes. Previous studies, in type I diabetes, observed PKC-βI-dependent GLUT2 recruitment to the proximal tubule brush-border membrane (BBM) as a direct result of elevated plasma glucose. The up-regulation of glucose transporters at the proximal tubule BBM causes a rise in intracellular glucose concentration; tubular injury in diabetic nephropathy is postulated to be a result of increased GLUT-mediated glucose entry into the proximal tubule cell. Augmented glucose transport across the proximal tubule BBM is likely to elevate glucose reabsorption and exacerbate hyperglycaemia during diabetes. Thus identification of regulators of this process may offer novel therapeutic targets to reduce renal glucose handling during hyperglycaemia. Studies described in this thesis were designed to elucidate the modulation of glucose transporters in models of metabolic syndrome associated with diabetes. Type II diabetes, the prevalent form of the disease, was found to elevate both SGLT- and GLUT-mediated glucose transport across the proximal tubule BBM, with an accompanying increase in glucose transporter expression. In rodent models of metabolic syndrome, induced by feeding studies, an elevation in PKC-βI-dependent GLUT2 recruitment to the proximal tubule BBM was also observed. However SGLT1 expression at the proximal tubule BBM did not consistently mirror that of GLUT2. In studies presented in this thesis, the sweet taste receptor heterodimer (T1R2/3) was identified at the proximal tubule BBM and this sweet taste sensor exhibited a tight correlation with SGLT1 in all models of metabolic dyregulation studied. Furthermore, stimulation of T1R2/3, by the artificial sweetener saccharin, resulted in an increase in SGLT-mediated glucose transport across the proximal tubule BBM. The data reported in this thesis provide evidence that GLUT2 expression at the proximal tubule BBM is not solely a response to hyperglycaemia but also a renal response to whole-body metabolic dysregulation. From these studies it is hypothesised that PKC-βI-mediated GLUT2 recruitment to the proximal tubule BBM occurs via a SGLT-T1R2/3-dependent and –independent pathway

Diet-induced iron deficiency in rats impacts small intestinal calcium and phosphate absorption

Aims: Recent reports suggest that iron deficiency impacts both intestinal calcium and phosphate absorption, although the exact transport pathways and intestinal segment responsible have not been determined. Therefore, we aimed to systematically investigate the impact of iron deficiency on the cellular mechanisms of transcellular and paracellular calcium and phosphate transport in different regions of the rat small intestine. // Methods: Adult, male Sprague-Dawley rats were maintained on a control or iron-deficient diet for 2 weeks and changes in intestinal calcium and phosphate uptake were determined using the in situ intestinal loop technique. The circulating levels of the hormonal regulators of calcium and phosphate were determined by ELISA, while the expression of transcellular calcium and phosphate transporters, and intestinal claudins were determined using qPCR and western blotting. Results: Diet-induced iron deficiency significantly increased calcium absorption in the duodenum but had no impact in the jejunum and ileum. In contrast, phosphate absorption was significantly inhibited in the duodenum and to a lesser extent the jejunum, but remained unchanged in the ileum. The changes in duodenal calcium and phosphate absorption in the iron-deficient animals were associated with increased claudin 2 and 3 mRNA and protein levels, while levels of parathyroid hormone, fibroblast growth factor-23 and 1,25-dihydroxy vitamin D3 were unchanged. // Conclusion: We propose that iron deficiency alters calcium and phosphate transport in the duodenum. This occurs via changes to the paracellular pathway, whereby upregulation of claudin 2 increases calcium absorption and upregulation of claudin 3 inhibits phosphate absorption

Extracellular vesicles released from p18 overexpressing pulmonary endothelial cells are barrier protective – potential implications for acute respiratory distress syndrome

The novel endosome protein, p18, and the early endosome GTPase, Rab4, play a significant role in protecting the pulmonary vasculature against permeability associated with acute respiratory distress syndrome. Recently, endothelial-derived extracellular vesicles have been identified to play a key role in the endothelial permeability associated with acute respiratory distress syndrome. Therefore, we investigated the effect of these microparticles, released from endothelial cells overexpressing p18 and Rab4, on pulmonary endothelial barrier function. Endothelial-derived extracellular vesicles isolated from lung microvascular endothelial cells which overexpressed cDNA for wild-type p18 protected a naïve monolayer against lipopolysaccharide-induced permeability. In contrast, endothelial-derived extracellular vesicles from cells overexpressing the non-endosomal binding p18 mutant (p18N39) exerted no protective effect on the endothelial monolayer. Cells overexpressing either dominant active or inactive Rab4 released endothelial-derived extracellular vesicles which had no effect on lipopolysaccharide-induced permeability. miRNA analysis and permeability studies of endothelial-derived extracellular vesicle isolated from wild-type p18-overexpressing cells demonstrates that let-7i-5p, miR-96-5p, and miR-137-3p are endothelial-derived extracellular vesicle cargo which exert protective effects on the pulmonary endothelium. Finally, we observed down-regulation of p18 protein expression in both the lung and endothelium in an in vivo and in vitro model of acute respiratory distress syndrome. These results demonstrate that endothelial-derived extracellular vesicle released from cells overexpressing p18, but not Rab4, contain miRNA cargo which likely promote a barrier-protective effect on the pulmonary endothelium in settings of acute respiratory distress syndrome. Findings indicate the importance of p18 in the pulmonary vasculature and demonstrate that targeting this protein may provide a novel therapeutic strategy to reduce endothelial permeability associated with acute respiratory distress syndrome

Artificial sweeteners negatively regulate pathogenic characteristics of two model gut bacteria, E. coli and E. faecalis

Artificial sweeteners (AS) are synthetic sugar substitutes that are commonly consumed in the diet. Recent studies have indicated considerable health risks which links the consumption of AS with metabolic derangements and gut microbiota perturbations. Despite these studies, there is still limited data on how AS impacts the commensal microbiota to cause pathogenicity. The present study sought to investigate the role of commonly consumed AS on gut bacterial pathogenicity and gut epithelium-microbiota interactions, using models of microbiota (Escherichia coli NCTC10418 and Enterococcus faecalis ATCC19433) and the intestinal epithelium (Caco-2 cells). Model gut bacteria were exposed to different concentrations of the AS saccharin, sucralose, and aspartame, and their pathogenicity and changes in interactions with Caco-2 cells were measured using in vitro studies. Findings show that sweeteners differentially increase the ability of bacteria to form a biofilm. Co-culture with human intestinal epithelial cells shows an increase in the ability of model gut bacteria to adhere to, invade and kill the host epithelium. The pan-sweet taste inhibitor, zinc sulphate, effectively blocked these negative impacts. Since AS consumption in the diet continues to increase, understanding how this food additive affects gut microbiota and how these damaging effects can be ameliorated is vital

Artificial Sweeteners Disrupt Tight Junctions and Barrier Function in the Intestinal Epithelium through Activation of the Sweet Taste Receptor, T1R3

The breakdown of the intestinal epithelial barrier and subsequent increase in intestinal permeability can lead to systemic inflammatory diseases and multiple-organ failure. Nutrition impacts the intestinal barrier, with dietary components such as gluten increasing permeability. Artificial sweeteners are increasingly consumed by the general public in a range of foods and drinks. The sweet taste receptor (T1R3) is activated by artificial sweeteners and has been identified in the intestine to play a role in incretin release and glucose transport; however, T1R3 has not been previously linked to intestinal permeability. Here, the intestinal epithelial cell line, Caco-2, was used to study the effect of commonly-consumed artificial sweeteners, sucralose, aspartame and saccharin, on permeability. At high concentrations, aspartame and saccharin were found to induce apoptosis and cell death in intestinal epithelial cells, while at low concentrations, sucralose and aspartame increased epithelial barrier permeability and down-regulated claudin 3 at the cell surface. T1R3 knockdown was found to attenuate these effects of artificial sweeteners. Aspartame induced reactive oxygen species (ROS) production to cause permeability and claudin 3 internalization, while sweetener-induced permeability and oxidative stress was rescued by the overexpression of claudin 3. Taken together, our findings demonstrate that the artificial sweeteners sucralose, aspartame, and saccharin exert a range of negative effects on the intestinal epithelium through the sweet taste receptor T1R3

Saccharin and Sucralose Protect the Glomerular Microvasculature In Vitro against VEGF-Induced Permeability

Diabetic kidney disease (DKD) has become a global health concern, with about 40% of people living with type 1 and type 2 diabetes mellitus developing DKD. Upregulation of vascular endothelial growth factor (VEGF) in the kidney is a significant pathology of DKD associated with increased glomerular vascular permeability. To date, however, current anti-VEGF therapies have demonstrated limited success in treating DKD. Recent studies have shown that artificial sweeteners exhibit anti-VEGF potential. The aim of this study was therefore to assess the effects of aspartame, saccharin, and sucralose on VEGF-induced leak using an in vitro model of the glomerular endothelium. Saccharin and sucralose but not aspartame protected against VEGF-induced permeability. Whilst the sweeteners had no effect on traditional VEGF signalling, GC-MS analysis demonstrated that the sweetener sucralose was not able to enter the glomerular endothelial cell to exert the protective effect. Chemical and molecular inhibition studies demonstrated that sweetener-mediated protection of the glomerular endothelium against VEGF is dependent on the sweet taste receptor, T1R3. These studies demonstrate the potential for sweeteners to exert a protective effect against VEGF-induced increased permeability to maintain a healthy endothelium and protect against vascular leak in the glomerulus in settings of DKD

Agonists for bitter taste receptors T2R10 and T2R38 attenuate LPS-induced permeability of the pulmonary endothelium in vitro

One of the hallmarks of acute respiratory distress syndrome (ARDS) is an excessive increase in pulmonary vascular permeability. In settings of ARDS, the loss of barrier integrity is mediated by cell–cell contact disassembly and actin remodelling. Studies into molecular mechanisms responsible for improving microvascular barrier function are therefore vital in the development of therapeutic targets for reducing vascular permeability seen in ARDS. Bitter taste receptors (T2Rs) belong to the superfamily of G-protein-coupled receptors found in several extraoral systems, including lung epithelial and smooth muscle cells. In the present study, we show for the first time that several T2Rs are expressed in human pulmonary arterial endothelial cells (HPAECs). Our results focus on those which are highly expressed as: T2R10, T2R14 and T2R38. Agonists for T2R10 (denatonium) and T2R38 (phenylthiourea), but not T2R14 (noscapine), significantly attenuated lipopolysaccharide (LPS)-induced permeability and VE-cadherin internalisation in HPAECs. In T2R10- or T2R38-siRNA knockdown cells, these endothelial-protective effects were abolished, indicating a direct effect of agonists in regulating barrier integrity. Our further findings indicate that T2R10 and T2R38 exert their barrier-protective function through cAMP but via Rac1-dependent and independent pathways, respectively. However, using an in vivo model of ARDS, the T2R38 agonist, phenylthiourea, was not able to protect against pulmonary edema formation. Taken together, these studies identify bitter taste sensing in the pulmonary endothelium to regulate barrier integrity in vitro through cAMP-Rac1 signalling

Sore eyes as the most significant symptom experienced by people with COVID-19. A comparison between pre- and during-COVID-19 states

Objective: Conjunctivitis has been reported in people suffering from COVID-19. However, many ocular symptoms are associated with the term ‘conjunctivitis’ which may be misleading. It is also unknown whether ocular symptoms were different in chronic sufferers of anterior eye diseases, when they were experienced or how long they lasted for compared with other COVID-19 symptoms. Methods: An online structured questionnaire obtained self-report data from people who had a confirmed diagnosis of COVID-19. Data for the type, frequency and duration of different COVID-19 symptoms were ascertained. Anterior eye symptoms experienced by participants in the pre-COVID-19 state were compared with during the COVID-19 state. Results: Data from 83 participants showed that the most reported COVID-19 symptoms were dry cough (66%), fever (76%), fatigue (90%) and loss of smell/taste (70%). The three most common ocular symptoms experienced by participants were photophobia (18%), sore eyes (16%) and itchy eyes (17%). The frequency of sore eyes was significantly higher (p=0.002) during COVID-19 state (16%) compared with pre-COVID-19 state (5%). There were no differences between males and females (p>0.05). 81% of participants reported to have experienced ocular symptoms within 2 weeks of other COVID-19 symptoms, and 80% reported they lasted for less than 2 weeks. Conclusion: The most significant ocular symptom experienced by people suffering from COVID-19 was sore eyes. Other symptoms associated with other types of conjunctivitis, such as mucous discharge and gritty eyes linked to bacterial infection, did not reach significance. The term ‘conjunctivitis’ is too broad and should be used with caution

Effect of α7 nicotinic acetylcholine receptor activation on cardiac fibroblasts: A mechanism underlying RV fibrosis associated with cigarette smoke exposure

INTRODUCTION: Right ventricular dysfunction is associated with numerous smoking-related illnesses including chronic obstructive pulmonary disease (COPD) where it is present even in absence of pulmonary hypertension. It is unknown if exposure to cigarette smoke has direct effects on RV function and cardiac fibroblast proliferation or collagen synthesis. In this study, we evaluated cardiac function and fibrosis in mice exposed to cigarette smoke (CS) and determined mechanisms of smoke-induced changes in cardiac fibroblast signaling and fibrosis. METHODS: AKR mice were exposed to cigarette smoke for six weeks followed by echocardiography and evaluation of cardiac hypertrophy, collagen content, and pulmonary muscularization. Proliferation and collagen content were evaluated in primary isolated rat cardiac fibroblasts (CF) exposed to cigarette smoke extract (CSE) or nicotine. Markers of cell proliferation, fibrosis, and proliferative signaling were determined by immunoblot or Sircol collagen assay. RESULTS: Mice exposed to CS had significantly decreased RV function as determined by TAPSE. There were no changes in LV parameters. RV collagen content was significantly elevated but there was no change in RV hypertrophy or pulmonary vascular muscularization. CSE directly increased cardiac fibroblast proliferation and collagen content in CF. Nicotine alone reproduced these effects. CSE and nicotine-induced fibroblast proliferation and collagen content were mediated through α7 nicotinic acetylcholine receptors and were dependent on PKC-α, PKC-δ, and reduced p38-MAPK phosphorylation. CONCLUSION: CS and nicotine have direct effects on cardiac fibroblasts to induce proliferation and fibrosis which may negatively affect right heart function