Tubulointerstitial injury and proximal tubule albumin transport in early diabetic nephropathy induced by type 1 diabetes mellitus

A decrease in the tubular expression of albumin endocytic transporters megalin and cubilin has been associated with diabetic nephropathy (DN), but there are no comprehensive studies to date relating early tubulointerstitial injury and the effect of the disease on both transporters in type 1 diabetes mellitus (T1DM). We used eight-week-old male C57BL/6 mice divided into two groups; one of them received the vehicle (control group), while the other received the vehicle + 200 mg/kg streptozotocin (T1DM). Ten weeks after the injection, we evaluated plasma insulin, enzymuria, urinary vitamin D-binding protein (VDBP), tubulointerstitial fibrosis and proximal tubule histology, markers of autophagy, and megalin and cubilin levels. We found a reduction in tubular protein reabsorption (albumin and VDBP as specific substances carried by both transporters) with increased tubulointerstitial injury, development of fibrosis, thickening of tubular basement membrane, and an increase in tubular cell metalloproteases. This was associated with a decrease in the renal expression of megalin and cubilin. We also observed an increase in the amount of cellular vesicles of the phagocytic system in the tubules, which could be linked to an alteration of normal intracellular trafficking of both receptors, thus affecting the normal function of transporters in early stages of DN. In diabetic animals, the added effects of tubulointerstitial injury, the decreases in megalin and cubilin expression, and an altered intracellular trafficking of these receptors, seriously affect protein reabsorptionFil: Giraud Billoud, Maximiliano German. Universidad del Desarrollo. Facultad de Medicina Clínica Alemana; Chile. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Ezquer, Fernando. Universidad del Desarrollo. Facultad de Medicina Clínica Alemana; ChileFil: Bahamonde, Javiera. Universidad del Desarrollo. Facultad de Medicina Clínica Alemana; ChileFil: Ezquer, Marcelo. Universidad del Desarrollo. Facultad de Medicina Clínica Alemana; Chil

Mesenchymal Stem Cell Therapy in Type 1 Diabetes Mellitus and Its Main Complications: From Experimental Findings to Clinical Practice

Type 1 diabetes mellitus (T1DM) is a complex multifactorial disorder which involves a loss of self-tolerance leading to the autoimmune destruction of pancreatic β−cells. Exogenous insulin administration cannot mimic precise pancreatic β-cell regulation of glucose homeostasis, thereby leading to severe long-term complications. Pancreas or islet transplant only provides partial exogenous insulin independence and induces several adverse effects, including increased morbidity and mortality. The scientific community and diabetic patients are thus, still waiting for an effective therapy which could preserve the remaining β-cells, replenish islet mass and protect newly-generated β-cells from autoimmune destruction. Mesenchymal stem cells (MSCs) have been envisioned as a promising tool for T1DM treatment over the past few years, since they could differentiate into glucose-responsive insulin-producing cells. Their immunomodulatory and proangiogenic roles can be used to help arrest β-cell destruction, preserve residual β-cell mass, facilitate endogenous β-cell regeneration and prevent disease recurrence, thereby making them ideal candidates for the comprehensive treatment of diabetic patients. This review focuses on recent pre-clinical data supporting MSC use in regenerating β-cell mass and also in treating several T1DM-associated complications. Clinical trial results and the ongoing obstacles which must be addressed regarding the widespread use of such therapy are also discussed.Fil: Ezquer, Marcelo. Universidad del Desarrollo. Facultad de Medicina Clínica Alemana; ChileFil: Arango Rodriguez, Martha. Universidad del Desarrollo. Facultad de Medicina Clínica Alemana; ChileFil: Giraud Billoud, Maximiliano German. Universidad del Desarrollo. Facultad de Medicina Clínica Alemana; Chile. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Ezquer, Fernando. Universidad del Desarrollo. Facultad de Medicina Clínica Alemana; Chil

Diabetic nephropathy, autophagy and proximal tubule protein endocytic transport: A potentially harmful relationship

Diabetic nephropathy (DN) is the most frequent cause of chronic renal failure. Until now, the pathophysiological mechanisms that determine its development and progression have not yet been elucidated. In the present study, we evaluate the role of autophagy at early stages of DN, induced in type 2 diabetes mellitus (T2DM) mouse, and its association with proximal tubule membrane endocytic receptors, megalin and cubilin. In T2DM animals we observed a tubule-interstitial injury with significantly increased levels of urinary GGT and ALP, but an absence of tubulointerstitial fibrosis. Kidney proximal tubule cells of T2DM animals showed autophagic vesicles larger than those observed in the control group, and an increase in the number of these vesicles marked with LBPA by immunofluorescence. Furthermore, a significant decrease in the ratio of LC3II/LC3I isoforms and in p62 protein expression in DN affected animals is shown. Finally, we observed a marked increase in urinary albumin and vitamin D binding-protein levels in T2DM animals as well as a significant decrease in expression of megalin in the renal cortex. These results indicate an alteration of the tubular endocytic transporters in DN, which could be related to autophagic dysfunction, which would in turn result in impaired organelle recycling, thus contributing to the progression of this disease.Fil: Giraud Billoud, Maximiliano German. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Fader Kaiser, Claudio Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Agüero, Rocio. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Ezquer, Fernando. Universidad del Desarrollo; ChileFil: Ezquer, Marcelo. Universidad del Desarrollo; Chil

Spatial maps and oscillations in the healthy hippocampus of Octodon degus, a natural model of sporadic Alzheimer’s disease

The Octodon degus is a South American rodent that is receiving increased attention as a potential model of aging and sporadic late-onset Alzheimer’s disease (AD). Impairments in spatial memory tasks in Octodon degus have been reported in relation to either advanced AD-like disease or hippocampal lesion, opening the way to investigate how the function of hippocampal networks affects behavior across AD stages. However, no characterization of hippocampal electrophysiology exists in this species. Here we describe in young, healthy specimens the activity of neurons and local field potential rhythms during spatial navigation tasks with and without objects. Our findings show similarities between the Octodon degus and laboratory rodents. First, place cells with characteristics similar to those found in rats and mice exist in the CA1 subfield of the Octodon degus. Second, the introduction of objects elicits novelty-related exploration and an increase in activity of CA1 cells, with location specific and unspecific components. Third, oscillations of the local field potential are organized according to their spectral content into bands similar to those found in laboratory rodents. These results suggest a common framework of underlying mechanisms, opening the way to future studies of hippocampal dysfunction in this species associated to aging and disease.Fil: Mugnaini, Matías. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Polania, Diana. Universidad de Chile; ChileFil: Díaz, Yannina Constanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Ezquer, Marcelo. Universidad del Desarrollo; ChileFil: Ezquer, Fernando. Universidad del Desarrollo; ChileFil: Deacon, Robert M. J.. Universidad de Chile; ChileFil: Cogram, Patricia. Universidad de Chile; Chile. University of California at Irvine; Estados UnidosFil: Kropff, Emilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Propagation of gravity waves and spread F in the low-latitude ionosphere over Tucumán, Argentina, by continuous Doppler sounding: first results

Results of systematic analysis of propagation directions and horizontal velocities of gravity waves (GWs) and spread F structures in low-latitude ionosphere (magnetic inclination ~27°) in Tucumán region, Argentina, are presented. Measurements were carried out by multipoint continuous Doppler system during 1 year from December 2012 to November 2013. It was found that meridian propagation of GWs dominated and that southward propagation prevailed in the local summer. Oblique spread structures observed in Doppler shift spectrograms and associated with spread F propagated roughly eastward at velocities from ~70 to ~180 m/s and were observed at night from ~ September to ~ March. The velocities were computed for 182 events and the azimuths for 64 events. Continuous Doppler sounding makes it possible to analyze more events compared to optical observations often used for propagation studies since the measurements do not depend on weather.Fil: Chum, J.. Institute of Atmospheric Physics; República ChecaFil: Miranda Bonomi, Fernando Alberto. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnología. Departamento de Electricidad, Electrónica y Computación. Laboratorio de Telecomunicaciones; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fišer, J.. Institute of Atmospheric Physics; República ChecaFil: Cabrera, M. A.. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnología. Departamento de Electricidad, Electrónica y Computación. Laboratorio de Telecomunicaciones; Argentina. Universidad Tecnológica Nacional. Facultad Regional Tucuman; ArgentinaFil: Ezquer, Rodolfo Gerardo. Universidad Tecnológica Nacional. Facultad Regional Tucuman; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnología. Departamento de Física. Laboratorio de Ionosfera; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Burešová, D.. Institute of Atmospheric Physics; República ChecaFil: Laštovička, J.. Institute of Atmospheric Physics; República ChecaFil: Baše, J.. Institute of Atmospheric Physics; República ChecaFil: Hruška, F.. Institute of Atmospheric Physics; República ChecaFil: Molina, Maria Graciela. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnología. Departamento de Ciencias de la Computación; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ise, Juan Eduardo. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnología. Departamento de Electricidad, Electrónica y Computación. Laboratorio de Telecomunicaciones; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cangemi, José Ignacio. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnología. Departamento de Electricidad, Electrónica y Computación. Laboratorio de Telecomunicaciones; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Šindelářová, T.. Institute of Atmospheric Physics; República Chec

Human adipose-derived mesenchymal stem cell-conditioned medium ameliorates polyneuropathy and foot ulceration in diabetic BKS db/db mice

Background: Diabetic polyneuropathy (DPN) is the most common and early developing complication of diabetes mellitus, and the key contributor for foot ulcers development, with no specific therapies available. Different studies have shown that mesenchymal stem cell (MSC) administration is able to ameliorate DPN; however, limited cell survival and safety reasons hinder its transfer from bench to bedside. MSCs secrete a broad range of antioxidant, neuroprotective, angiogenic, and immunomodulatory factors (known as conditioned medium), which are all decreased in the peripheral nerves of diabetic patients. Furthermore, the abundance of these factors can be boosted in vitro by incubating MSCs with a preconditioning stimulus, enhancing their therapeutic efficacy. We hypothesize that systemic administration of conditioned medium derived from preconditioned MSCs could reverse DPN and prevent foot ulcer formation in a mouse model of type II diabetes mellitus. Methods: Diabetic BKS db/db mice were treated with systemic administration of conditioned medium derived from preconditioned human MSCs; conditioned medium derived from non-preconditioned MSCs or vehicle after behavioral signs of DPN was already present. Conditioned medium or vehicle administration was repeated every 2 weeks for a total of four administrations, and several functional and structural parameters characteristic of DPN were evaluated. Finally, a wound was made in the dorsal surface of both feet, and the kinetics of wound closure, re-epithelialization, angiogenesis, and cell proliferation were evaluated. Results: Our molecular, electrophysiological, and histological analysis demonstrated that the administration of conditioned medium derived from non-preconditioned MSCs or from preconditioned MSCs to diabetic BKS db/db mice strongly reverts the established DPN, improving thermal and mechanical sensitivity, restoring intraepidermal nerve fiber density, reducing neuron and Schwann cell apoptosis, improving angiogenesis, and reducing chronic inflammation of peripheral nerves. Furthermore, DPN reversion induced by conditioned medium administration enhances the wound healing process by accelerating wound closure, improving the re-epithelialization of the injured skin and increasing blood vessels in the wound bed in a skin injury model that mimics a foot ulcer. Conclusions: Studies conducted indicate that MSC-conditioned medium administration could be a novel cell-free therapeutic approach to reverse the initial stages of DPN, avoiding the risk of lower limb amputation triggered by foot ulcer formation and accelerating the wound healing process in case it occurs.Fil: De Gregorio, Cristian. Universidad del Desarrollo; ChileFil: Contador, David. Universidad del Desarrollo; ChileFil: Díaz, Diego. Universidad del Desarrollo; ChileFil: Cárcamo, Constanza. Universidad del Desarrollo; ChileFil: Santapau, Daniela. Universidad del Desarrollo; ChileFil: Lobos Gonzalez, Lorena. Universidad del Desarrollo; ChileFil: Acosta, Cristian Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Campero, Mario. Universidad de Chile; ChileFil: Carpio, Daniel. Universidad Austral de Chile; ChileFil: Gabriele, Caterina. University Of Catanzaro; ItaliaFil: Gaspari, Marco. University Of Catanzaro; ItaliaFil: Aliaga Tobar, Victor. Universidad de Chile; ChileFil: Maracaja Coutinho, Vinicius. Universidad de Chile; ChileFil: Ezquer, Marcelo. Universidad del Desarrollo; ChileFil: Ezquer, Fernando. Universidad del Desarrollo; Chil

The Antidiabetic Effect of MSCs Is Not Impaired by Insulin Prophylaxis and Is Not Improved by a Second Dose of Cells

Type 1 diabetes mellitus (T1D) is due to autoimmune destruction of pancreatic beta-cells. Previously, we have shown that intravenously administered bone marrow-derived multipotent mesenchymal stromal cells (MSCs) allows pancreatic islet recovery, improves insulin secretion and reverts hyperglycemia in low doses streptozotocin (STZ)-induced diabetic mice. Here we evaluate whether insulin prophylaxis and the administration of a second dose of cells affect the antidiabetic therapeutic effect of MSC transplantation. Insulitis and subsequent elimination of pancreatic beta-cells was promoted in C57BL/6 mice by the injection of 40 mg/kg/day STZ for five days. Twenty-four days later, diabetic mice were distributed into experimental groups according to if they received or not insulin and/or one or two doses of healthy donor-derived MSCs. Three and half months later: glycemia, pancreatic islets number, insulinemia, glycated hemoglobin level and glucose tolerance were determined in animals that did not received exogenous insulin for the last 1.5 months. Also, we characterized MSCs isolated from mice healthy or diabetic. The therapeutic effect of MSC transplantation was observed in diabetic mice that received or not insulin prophylaxis. Improvements were similar irrespective if they received one or two doses of cells. Compared to MSCs from healthy mice, MSCs from diabetic mice had the same proliferation and adipogenic potentials, but were less abundant, with altered immunophenotype and no osteogenic potential

Improving Cell Recovery: Freezing and Thawing Optimization of Induced Pluripotent Stem Cells

Achieving good cell recovery after cryopreservation is an essential process when working with induced pluripotent stem cells (iPSC). Optimized freezing and thawing methods are required for good cell attachment and survival. In this review, we concentrate on these two aspects, freezing and thawing, but also discuss further factors influencing cell recovery such as cell storage and transport. Whenever a problem occurs during the thawing process of iPSC, it is initially not clear what it is caused by, because there are many factors involved that can contribute to insufficient cell recovery. Thawing problems can usually be solved more quickly when a certain order of steps to be taken is followed. Under optimized conditions, iPSC should be ready for further experiments approximately 4–7 days after thawing and seeding. However, if the freezing and thawing protocols are not optimized, this time can increase up to 2–3 weeks, complicating any further experiments. Here, we suggest optimization steps and troubleshooting options for the freezing, thawing, and seeding of iPSC on feeder-free, Matrigel™-coated, cell culture plates whenever iPSC cannot be recovered in sufficient quality. This review applies to two-dimensional (2D) monolayer cell culture and to iPSC, passaged, frozen, and thawed as cell aggregates (clumps). Furthermore, we discuss usually less well-described factors such as the cell growth phase before freezing and the prevention of osmotic shock during thawing

Characterization of diabetic neuropathy progression in a mouse model of type 2 diabetes mellitus

Diabetes mellitus (DM) is one of most common chronic diseases with an increasing incidence in most countries. Diabetic neuropathy (DN) is one of the earliest and main complications of diabetic patients, which is characterized by progressive, distal-to-proximal degeneration of peripheral nerves. The cellular and molecular mechanisms that trigger DN are highly complex, heterogeneous and not completely known. Animal models have constituted a valuable tool for understanding diabetes pathophysiology; however, the temporal course of DN progression in animal models of type 2 diabetes (T2DM) is not completely understood. In this work, we characterized the onset and progression of DN in BKS diabetic (db/db) mice, including the main functional and histological features observed in the human disease. We demonstrated that diabetic animals display progressive sensory loss and electrophysiological impairments in the early-to-mid phases of the disease. Furthermore, we detected an early decrease in intraepidermal nerve fiber (IENF) density in 18-week-old diabetic mice, which is highly associated with sensory loss and constitutes a reliable marker of DN. Other common histological parameters of DN – like Schwann cells apoptosis and infiltration of CD3+ cells in the sciatic nerve – were altered in mid-to-late phases of the disease. Our results support the general consensus that DN evolves from initial functional to late structural changes. This work aimed to characterize the progression of DN in a reliable animal model sharing the main human disease features, which is necessary to assess new therapies for this complex disease. Finally, we also aimed to identify an effective temporal window where these potential treatments could be successfully applied