Irene Londono

Control of glycogen synthase through ADIPOR1-AMPK pathway in renal distal tubules of normal and diabetic rats

Diabetic nephropathies are characterized by glycogen accumulation in distal tubular cells, which eventually leads to their apoptosis. The present study aims to determine whether adiponectin and AMPK are involved in the regulation of glycogen synthase (GS) in these structures. Western blots of isolated distal tubules revealed the presence of adiponectin receptor ADIPOR1, catalytic AMPK subunits alpha(1) and alpha(2), their phosphorylated active forms, and the glycogen-binding AMPK subunit beta(2). ADIPOR2 was not detected. Expression levels of ADIPOR1, AMPKalpha(1), AMPKalpha(2), and AMPKbeta(2) were increased in streptozotocin-treated diabetic rats, whereas phosphorylated active AMPK levels were strongly decreased. Immunohistochemistry revealed the presence of ADIPOR1 on the luminal portion of distal tubules and thick ascending limb cells. Catalytic subunits alpha(1) and alpha(2), their phosphorylated active forms, and the glycogen-binding subunit beta(2) were also found in the same cells, confirming immunoblot results. In vitro, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR; 2 mM) and globular adiponectin (10 mug/ml) activated catalytic AMPK in distal tubules isolated from kidneys of normal rats but much more weakly in those from diabetic rats. GS inhibition paralleled AMPK activation in both groups of animals: active GS levels were low in control animals and elevated in diabetic ones. Finally, glucose-6-phosphate, an allosteric activator of GS, was also increased in diabetic rats. These results demonstrate that in distal tubular cells, adiponectin through luminal ADIPOR1 activates AMPK, leading to the inhibition of GS. During hyperglycemia, this regulation is altered, which may explain, at least in part, the accumulation of large glycogen deposits.

Caspase activation regulates the extracellular export of autophagic vacuoles.

The endothelium plays a central role in the regulation of vascular wall cellularity and tone by secreting an array of mediators of importance in intercellular communication. Nutrient deprivation of human endothelial cells (EC) evokes unconventional forms of secretion leading to the release of nanovesicles distinct from apoptotic bodies and bearing markers of multivesicular bodies (MVB). Nutrient deficiency is also a potent inducer of autophagy and vesicular transport pathways can be assisted by autophagy. Nutrient deficiency induced a significant and rapid increase in autophagic features, as imaged by electron microscopy and immunoblotting analysis of LC3-II/LC3-I ratios. Increased autophagic flux was confirmed by exposing serum-starved cells to bafilomycin A1. Induction of autophagy was followed by indices of an apoptotic response, as assessed by microscopy and poly (ADP-ribose) polymerase cleavage in absence of cell membrane permeabilization indicative of necrosis. Pan-caspase inhibition with ZVAD-FMK did not prevent the development of autophagy but negatively impacted autophagic vacuole (AV) maturation. Adopting a multidimensional proteomics approach with validation by immunoblotting, we determined that nutrient-deprived EC released AV components (LC3I, LC3-II, ATG16L1 and LAMP2) whereas pan-caspase inhibition with ZVAD-FMK blocked AV release. Similarly, nutrient deprivation in aortic murine EC isolated from CASP3/caspase 3-deficient mice induced an autophagic response in absence of apoptosis and failed to prompt LC3 release. Collectively, the present results demonstrate the release of autophagic components by nutrient-deprived apoptotic human cells in absence of cell membrane permeabilization. These results also identify caspase-3 as a novel regulator of AV release.

Apoptosis of Tubular Epithelial Cells in Glycogen Nephrosis During Diabetes

The important problem of the fate of glycogen-accumulating clear cells in glycogen nephrosis is still unsettled. In this study, we examine whether apoptosis plays a relevant role in the development of diabetic glycogen nephrosis and explore the involvement of the Fas/Fas-L system and the activation of the caspase cascade. Diabetes was induced in rats by streptozotocin injection. Glycogen-accumulating clear cells were identified in renal tissues of hyperglycemic rats. They were found to be concentrated in the thick ascending limbs and distal tubules. Large cellular glycogen accumulations were confirmed by biochemical assays and enzyme-gold cytochemistry. Clear cells displayed apoptotic features such as Annexin V binding, nuclear TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling), and the simultaneous occurrence of Fas, Annexin V, and TUNEL positivity. Western blot analysis demonstrated enhanced expression of Fas receptor/ligand and the activation of the caspase cascade in these cells because cleaved forms of the caspase-3, -8, and -9 were detected. Furthermore, active caspase-3 was located in nuclei by immunoelectron microscopy. Our results indicate that epithelial cells in thick ascending limbs and distal tubules that develop glycogen nephrosis in response to hyperglycemia undergo Fas/Fas-L mediated cell death. Thus, apoptosis could be playing a significant role in renal epithelial cell deletion during diabetes.

Association of AMP-activated protein kinase subunits with glycogen particles as revealed in situ by immunoelectron microscopy.

Immunogold cytochemistry was applied to reveal the intracellular location of AMP-activated protein kinase (AMPK) subunits in liver tissue of normal rats fed ad libitum. AMPK α and β subunits were located both in the cytosol and in close association with rosettes of glycogen particles (α particles). To reveal their true in situ association with glycogen, particular tissue processing conditions that retain glycogen in the cells were required. These included fixation with a combination of glutaraldehyde and paraformaldehyde, followed by postfixation with osmium tetroxide and lead citrate and embedding in Epon. Processing by less-stringent fixation conditions and embedding in Lowicryl led to the extraction of the glycogen deposits, which in turn resulted in the absence of any labeling. This indicates that the loss of glycogen deposits leads to the loss of closely associated proteins. Labeling for the α1 and α2 subunits of AMPK was found to be about 2-fold greater over glycogen than over cytosol, whereas labeling for β1 was 8-fold higher over the glycogen particles than over the cytosol. Immunogold combined with morphometric analysis demonstrated that the β1 subunits are located at the periphery of the glycogen rosettes, consistent with a recent hypothesis developed via biochemical approaches.

New means to assess neonatal inflammatory brain injury

Preterm infants are especially vulnerable to infection-induced white matter injury, associated with cerebral palsy, cognitive and psychomotor impairment, and other adverse neurological outcomes. The etiology of such lesions is complex and multifactorial. Furthermore, timing and length of exposure to infection also influence neurodevelopmental outcomes. Different mechanisms have been posited to mediate the observed brain injury including microglial activation followed by subsequent release of pro-inflammatory species, glutamate-induced excitotoxicity, and vulnerability of developing oligodendrocytes to cerebral insults. The prevalence of such neurological impairments requires an urgent need for early detection and effective neuroprotective strategies. Accordingly, noninvasive methods of monitoring disease progression and therapy effectiveness are essential. While diagnostic tools using biomarkers from bodily fluids may provide useful information regarding potential risks of developing neurological diseases, the use of magnetic resonance imaging/spectroscopy has emerged as a promising candidate for such purpose. Various pharmacological agents have demonstrated protective effects in the immature brain in animal models; however, few studies have progressed to clinical trials with promising results.

Neonatal microglia: The cornerstone of brain fate

Microglia, mainly known for their role in innate immunity and modulation of neuroinflammation, play an active role in central nervous system development and homeostasis. Depending on the context and environmental stimuli, microglia adopt a broad spectrum of activation status from pro-inflammatory, associated with neurotoxicity, to anti-inflammatory linked to neuroprotection. Pro-inflammatory microglial activation is a key hallmark of white matter injury in preterm infants and is involved in developmental origin of adult neurological diseases. Characterization of neonatal microglia function in brain development and inflammation has allowed the investigation of promising therapeutic targets with potential long-lasting neuroprotective effects. True prevention of neuro-degenerative diseases might eventually occur as early as the perinatal period.

Imaging of an Inflammatory Injury in the Newborn Rat Brain with Photoacoustic Tomography

Background The precise assessment of cerebral saturation changes during an inflammatory injury in the developing brain, such as seen in periventricular leukomalacia, is not well defined. This study investigated the impact of inflammation on locoregional cerebral oxygen saturation in a newborn rodent model using photoacoustic imaging. Methods 1 mg/kg of lipopolysaccharide(LPS) diluted in saline or saline alone was injected under ultrasound guidance directly in the corpus callosum of P3 rat pups. Coronal photoacoustic images were carried out 24 h after LPS exposure. Locoregional oxygen saturation (SO2) and resting state connectivity were assessed in the cortex and the corpus callosum. Microvasculature was then evaluated on cryosection slices by lectin histochemistry. Results Significant reduction of SO2 was found in the corpus callosum; reduced SO2 was also found in the cortex ipsilateral to the injection site. Seed-based functional connectivity analysis showed that bilateral connectivity was not affected by LPS exposure. Changes in locoregional oxygen saturation were accompanied by a significant reduction in the average length of microvessels in the left cortex but no differences were observed in the corpus callosum. Conclusion Inflammation in the developing brain induces marked reduction of locoregional oxygen saturation, predominantly in the white matter not explained by microvascular degeneration. The ability to examine regional saturation offers a new way to monitor injury and understand physiological disturbance non-invasively.

Glomerular Basement Membrane Selective Permeability in Short-term Streptozotocin-induced Diabetic Rats

In diabetes, the glomerular basement membrane undergoes thickening and structural alterations with loss of glomerular permselectivity properties. However, the onset of the alterations at early phases of diabetes is unclear. Aiming to determine the functional and structural alterations of the glomerular wall in the early stages of diabetes, we have studied the distribution of endogenous circulating albumin and type IV collagen in the glomerular basement membrane, using the immunocytochemical approach. The streptozotocin-injected hyperglycemic rat was our animal model. Renal tissues were examined after 10 days, 2, 4 and 6 months of hyperglycemia. Upon immunogold labelings, changes in the glomerular permeability to endogenous albumin were found altered as early as upon ten days of hyperglycemia. In contrast, no structural modifications were detected at this time point. Indeed, glomerular basement membrane thickening and an altered type IV collagen labeling distribution were only observed after four months of hyperglycemia, suggesting that functional alterations take place early in diabetes prior to any structural modification. In order to evaluate the reversibility of the glomerular alterations, two-month-old diabetic animals were treated with insulin. These animals showed a significant restoring of their glomerular permselectivity. Our results suggest a link between glycemic levels and alteration of glomerular permeability in early stages of diabetes, probably through high levels of glycated serum proteins.

Expression differences in mitochondrial and secretory chaperonin 60 (Cpn60) in pancreatic acinar cells

Abstract In pancreatic acinar cells, chaperonin Cpn60 is present in all the cellular compartments involved in protein secretion as well as in mitochondria. To better understand the role Cpn60 plays in pancreatic secretion, we have evaluated its changes under experimental conditions known to alter pancreatic secretion. Quantitative protein A–gold immunocytochemistry was used to reveal Cpn60 in pancreatic acinar cells. Cpn60 immunolabelings in cellular compartments involved in secretion were found to decrease in acute pancreatitis as well as upon stimulation of secretion and in starvation conditions. A major increase in Cpn60 was recorded in diabetic condition. This was normalized by insulin treatment. Although in certain situations changes in secretory enzymes and in Cpn60 correlate well, in others, nonparallel secretion seemed to take place. In contrast, expression of mitochondrial Cpn60 in acinar cells appeared to remain stable in all conditions except starvation, where its levels decreased. Expression of Cpn60 in the secretory pathway and in mitochondria thus appears to behave differently, and Cpn60 in the secretory pathway must be important for quality control and integrity of secretion.

Psammomys obesus, a particularly important animal model for the study of the human diabetic nephropathy

The Psammomys obesus lives in natural desert habitat on low energy (LE) diet, however when maintained in laboratory conditions with high energy (HE) diet it exhibits pathological metabolic changes resembling those of type 2 diabetes. We have evaluated and correlated the histopathology, metabolic and functional renal alterations occurring in the diabetic Psammomys. Renal function determined by measuring glomerular filtration rate (GFR), protein excretion, protein/creatinine ratio and morpho-immunocytochemical evaluations were performed on HE diet diabetic animals and compared to LE diet control animals. The diabetic animals present a 54% increase in GFR after one month of hyperglycemic condition and a decrease of 47% from baseline values after 4 months. Protein excretion in diabetic animals was 5 folds increased after 4 months. Light microscopy showed an increase in glomeruli size in the diabetic Psammomys, and electron microscopy and immunocytochemical quantitative evaluations revealed accumulation of basement membrane material as well as frequent splitting of the glomerular basement membrane. In addition, glycogen-filled Armanni-Ebstein clear cells were found in the distal tubules including the thick ascending limbs of the diabetic animals. These renal complications in the Psammomys, including changes in GFR with massive proteinuria sustained by physiological and histopathological changes, are very similar to the diabetic nephropathy in human. The Psamommys obesus represents therefore a reliable animal model of diabetic nephropathy.