Postembryonic development and aging of the appendicular skeleton in Ambystoma mexicanum

Background: The axolotl is a key model to study appendicular regeneration. The limb complexity resembles that of humans in structure and tissue components; however, axolotl limbs develop postembryonically. In this work, we evaluated the postembryonic development of the appendicular skeleton and its changes with aging. Results: The juvenile limb skeleton is formed mostly by Sox9/Col1a2 cartilage cells. Ossification of the appendicular skeleton starts when animals reach a length of 10 cm, and cartilage cells are replaced by a primary ossification center, consisting of cortical bone and an adipocyte-filled marrow cavity. Vascularization is associated with the ossification center and the marrow cavity formation. We identified the contribution of Col1a2-descendants to bone and adipocytes. Moreover, ossification progresses with age toward the epiphyses of long bones. Axolotls are neotenic salamanders, and still ossification remains responsive to l-thyroxine, increasing the rate of bone formation. Conclusions: In axolotls, bone maturation is a continuous process that extends throughout their life. Ossification of the appendicular bones is slow and continues until the complete element is ossified. The cellular components of the appendicular skeleton change accordingly during ossification, creating a heterogenous landscape in each element. The continuous maturation of the bone is accompanied by a continuous body growth.Fil: Riquelme Guzmán, Camilo. Technische Universität Dresden; AlemaniaFil: Schuez, Maritta. Technische Universität Dresden; AlemaniaFil: Böhm, Alexander. Technische Universität Dresden; AlemaniaFil: Knapp, Dunja. Technische Universität Dresden; AlemaniaFil: Edwards Jorquera, Sandra. Technische Universität Dresden; AlemaniaFil: Ceccarelli, Alberto Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; ArgentinaFil: Chara, Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Argentina de la Empresa; Argentina. Technische Universität Dresden; AlemaniaFil: Rauner, Martina. Universitätsklinikum Carl Gustav Carus; AlemaniaFil: Sandoval Guzmán, Tatiana. Universitätsklinikum Carl Gustav Carus; Alemania. Technische Universität Dresden; Alemani

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

TRPML regula la migración y fagocitosis en hemocitos de Drosophila melanogaster mediante la liberación localizada de calcio desde los lisosomas

Tesis entregada a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al grado de Doctor en Ciencias Biológicas con mención en Biología Molecular, Celular y Neurociencias.Lysosomal physiology has been shown to be important for the correct functioning of immune cells, including their migratory and their phagocytic properties. In this work, we aimed to explore the migratory process of immune cells and understand the connection between cytoskeletal dynamics and lysosomal degradative capacity. Specifically, we characterized the role of the calcium permeable channel Trpml in the process of cell migration, as well as that of the immune response and its connection with lysosomal physiology, using as model of study the Drosophila melanogaster macrophages, called hemocytes. Hemocytes are the cellular component of the immune response in Drosophila, performing key functions during development, as well as in immune defense in response to bacterial or parasitic infection. These cells are highly migratory in vivo and, importantly, present a substantial degree of functional and structural conservation with mammalian innate immune cells. Conveniently, the Drosophila genome contains only one gene coding for the Trpml channel, and its mutant phenotype resembles that of Mucolipidosis type IV patients, which bear a loss of function mutation in MCOLN1, the human homolog of the Drosophila trpml gene. Results presented in this thesis demonstrate that Trpml plays a fundamental role in phagolysosomal degradation and in vivo hemocyte migration, and that it participates in these processes through partially independent mechanisms. The most likely scenario is that Trpml is important for the degradative process by favoring phagolysosomal fusion and further phagocytic degradation. This is supported by the fact that calcium ions are needed for the fusion of phagosomal and lysosomal membranes. On the other hand, for the migratory process, localized Trpml activation from the lysosomes in the rear of polarized hemocytes, would promote the local activation of non-muscle myosin-II, which is necessary event to trigger cell body translocation during movement.La fisiología lisosomal ha mostrado ser importante para el correcto funcionamiento de células inmunes, abarcando tanto sus propiedades migratorias como fagocíticas. En este trabajo, se quiso ahondar en el proceso migratorio de células del sistema inmune para comprender el nexo existente entre la dinámica del citoesqueleto y la capacidad degradativa de los lisosomas. En particular, se caracterizó el rol del canal lisosomal permeable a calcio, Trpml, en el proceso de migración celular, al igual que la respuesta inmune y su vínculo con la fisiología lisosomal, usando como modelo de estudio, los macrófagos de Drosophila melanogaster denominados hemocitos. Los hemocitos, son el componente celular de la respuesta inmune de Drosophila, cumpliendo funciones claves durante el desarrollo, así como durante la defensa ante infección por microorganismos o parásitos. Estas células son altamente migratorias in vivo, e importantemente, presentan un sustancial grado de conservación funcional y estructural con las células del sistema inmune innato de mamíferos. Convenientemente, Drosophila posee sólo un gen que codifica para el canal Trpml, cuyo fenotipo mutante semeja al de pacientes con Mucolipidosis tipo IV, quienes presentan pérdida de función del gen homólogo que codifica Trpml en humanos, MCOLN1. Los resultados presentados en esta tesis demuestran que Trpml cumple un rol fundamental en la degradación fagolisosomal, y la migración de hemocitos in vivo, y que estaría participando en estos dos procesos mediante mecanismos parcialmente independientes. A partir de los resultados obtenidos podemos proponer que Trpml es fundamental para el procesamiento degradativo, mediante el favorecimiento de la fusión fagolisosomal, y consecuente degradación fagocítica. Esto posiblemente se sustenta en la necesidad de iones Ca2+ para la fusión de las membranas del fagosoma con el lisosoma. Por su parte, en el proceso migratorio, la activación localizada de Trpml desde los lisosomas en la región posterior de células polarizadas, promovería la activación de Miosina-II no muscular en esta región, evento necesario para la translocación del cuerpo celular.Conicyt 21140289, Proyectos Redes 140004, Fondecyt 1140522, Anillo ACT 1401 y Fondap 15090007

Epithelial tricellular junctions act as interphase cell shape sensors to orient mitosis.

International audienceThe orientation of cell division along the long axis of the interphase cell--the century-old Hertwig's rule--has profound roles in tissue proliferation, morphogenesis, architecture and mechanics. In epithelial tissues, the shape of the interphase cell is influenced by cell adhesion, mechanical stress, neighbour topology, and planar polarity pathways. At mitosis, epithelial cells usually adopt a rounded shape to ensure faithful chromosome segregation and to promote morphogenesis. The mechanisms underlying interphase cell shape sensing in tissues are therefore unknown. Here we show that in Drosophila epithelia, tricellular junctions (TCJs) localize force generators, pulling on astral microtubules and orienting cell division via the Dynein-associated protein Mud independently of the classical Pins/Gαi pathway. Moreover, as cells round up during mitosis, TCJs serve as spatial landmarks, encoding information about interphase cell shape anisotropy to orient division in the rounded mitotic cell. Finally, experimental and simulation data show that shape and mechanical strain sensing by the TCJs emerge from a general geometric property of TCJ distributions in epithelial tissues. Thus, in addition to their function as epithelial barrier structures, TCJs serve as polarity cues promoting geometry and mechanical sensing in epithelial tissues

Control of lysosomal-mediated cell death by the pH-dependent calcium channel RECS1

Programmed cell death is regulated by the balance between activating and inhibitory signals. Here, we have identified RECS1 (responsive to centrifugal force and shear stress 1) [also known as TMBIM1 (transmembrane BAX inhibitor motif containing 1)] as a proapoptotic member of the TMBIM family. In contrast to other proteins of the TMBIM family, RECS1 expression induces cell death through the canonical mitochondrial apoptosis pathway. Unbiased screening indicated that RECS1 sensitizes cells to lysosomal perturbations. RECS1 localizes to lysosomes, where it regulates their acidification and calcium content, triggering lysosomal membrane permeabilization. Structural modeling and electrophysiological studies indicated that RECS1 is a pH-regulated calcium channel, an activity that is essential to trigger cell death. RECS1 also sensitizes whole animals to stress in vivo in Drosophila melanogaster and zebrafish models. Our results unveil an unanticipated function for RECS1 as a proapoptotic component of the TMBIM family that ignites cell death programs at lysosomes.Peer reviewe

Trpml controls actomyosin contractility and couples migration to phagocytosis in fly macrophages.

International audiencePhagocytes use their actomyosin cytoskeleton to migrate as well as to probe their environment by phagocytosis or macropinocytosis. Although migration and extracellular material uptake have been shown to be coupled in some immune cells, the mechanisms involved in such coupling are largely unknown. By combining time-lapse imaging with genetics, we here identify the lysosomal Ca2+ channel Trpml as an essential player in the coupling of cell locomotion and phagocytosis in hemocytes, the Drosophila macrophage-like immune cells. Trpml is needed for both hemocyte migration and phagocytic processing at distinct subcellular localizations: Trpml regulates hemocyte migration by controlling actomyosin contractility at the cell rear, whereas its role in phagocytic processing lies near the phagocytic cup in a myosin-independent fashion. We further highlight that Vamp7 also regulates phagocytic processing and locomotion but uses pathways distinct from those of Trpml. Our results suggest that multiple mechanisms may have emerged during evolution to couple phagocytic processing to cell migration and facilitate space exploration by immune cells