Leonel Malacrida

Delayed mTOR Inhibition with Low Dose of Everolimus Reduces TGFβ Expression, Attenuates Proteinuria and Renal Damage in the Renal Mass Reduction Model

Background The immunosuppressive mammalian target of rapamycin (mTOR) inhibitors are widely used in solid organ transplantation, but their effect on kidney disease progression is controversial. mTOR has emerged as one of the main pathways regulating cell growth, proliferation, differentiation, migration, and survival. The aim of this study was to analyze the effects of delayed inhibition of mTOR pathway with low dose of everolimus on progression of renal disease and TGFβ expression in the 5/6 nephrectomy model in Wistar rats. Methods This study evaluated the effects of everolimus (0.3 mg/k/day) introduced 15 days after surgical procedure on renal function, proteinuria, renal histology and mechanisms of fibrosis and proliferation. Results Everolimus treated group (EveG) showed significantly less proteinuria and albuminuria, less glomerular and tubulointerstitial damage and fibrosis, fibroblast activation cell proliferation, when compared with control group (CG), even though the EveG remained with high blood pressure. Treatment with everolimus also diminished glomerular hypertrophy. Everolimus effectively inhibited the increase of mTOR developed in 5/6 nephrectomy animals, without changes in AKT mRNA or protein abundance, but with an increase in the pAKT/AKT ratio. Associated with this inhibition, everolimus blunted the increased expression of TGFβ observed in the remnant kidney model. Conclusion Delayed mTOR inhibition with low dose of everolimus significantly prevented progressive renal damage and protected the remnant kidney. mTOR and TGFβ mRNA reduction can partially explain this anti fibrotic effect. mTOR can be a new target to attenuate the progression of chronic kidney disease even in those nephropathies of non-immunologic origin.

Measurements of absolute concentrations of NADH in cells using the phasor FLIM method.

We propose a graphical method using the phasor representation of the fluorescence decay to derive the absolute concentration of NADH in cells. The method requires the measurement of a solution of NADH at a known concentration. The phasor representation of the fluorescence decay accounts for the differences in quantum yield of the free and bound form of NADH, pixel by pixel of an image. The concentration of NADH in every pixel in a cell is obtained after adding to each pixel in the phasor plot a given amount of unmodulated light which causes a shift of the phasor towards the origin by an amount that depends on the intensity at the pixel and the fluorescence lifetime at the pixel. The absolute concentration of NADH is obtained by comparison of the shift obtained at each pixel of an image with the shift of the calibrated solution.

Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures

Using LAURDAN spectral imaging and spectral phasor analysis we concurrently studied the growth and hydration state of subcellular organelles (lamellar body-like, LB-like) from live A549 lung cancer cells at different post-confluence days. Our results reveal a time dependent two-step process governing the size and hydration of these intracellular LB-like structures. Specifically, a first step (days 1 to 7) is characterized by an increase in their size, followed by a second one (days 7 to 14) where the organelles display a decrease in their global hydration properties. Interestingly, our results also show that their hydration properties significantly differ from those observed in well-characterized artificial lamellar model membranes, challenging the notion that a pure lamellar membrane organization is present in these organelles at intracellular conditions. Finally, these LB-like structures show a significant increase in their hydration state upon secretion, suggesting a relevant role of entropy during this process.

Model-free methods to study membrane environmental probes: a comparison of the spectral phasor and generalized polarization approaches.

In this note, we present a discussion of the advantages and scope of model-free analysis methods applied to the popular solvatochromic probe LAURDAN, which is widely used as an environmental probe to study dynamics and structure in membranes. In particular, we compare and contrast the generalized polarization approach with the spectral phasor approach. To illustrate our points we utilize several model membrane systems containing pure lipid phases and, in some cases, cholesterol or surfactants. We demonstrate that the spectral phasor method offers definitive advantages in the case of complex systems.

PTEN deficiency and AMPK activation promote nutrient scavenging and anabolism in prostate cancer cells

We report that PTEN-deficient prostate cancer cells use macropinocytosis to survive and proliferate under nutrient stress. PTEN loss increased macropinocytosis only in the context of AMPK activation, revealing a general requirement for AMPK in macropinocytosis and a novel mechanism by which AMPK promotes survival under stress. In prostate cancer cells, albumin uptake did not require macropinocytosis, but necrotic cell debris proved a specific macropinocytic cargo. Isotopic labeling confirmed that macropinocytosed necrotic cell proteins fueled new protein synthesis in prostate cancer cells. Supplementation with necrotic debris, but not albumin, also maintained lipid stores, suggesting that macropinocytosis can supply nutrients other than amino acids. Nontransformed prostatic epithelial cells were not macropinocytic, but patient-derived prostate cancer organoids and xenografts and autochthonous prostate tumors all exhibited constitutive macropinocytosis, and blocking macropinocytosis limited prostate tumor growth. Macropinocytosis of extracellular material by prostate cancer cells is a previously unappreciated tumor-microenvironment interaction that could be targeted therapeutically.Significance: As PTEN-deficient prostate cancer cells proliferate in low-nutrient environments by scavenging necrotic debris and extracellular protein via macropinocytosis, blocking macropinocytosis by inhibiting AMPK, RAC1, or PI3K may have therapeutic value, particularly in necrotic tumors and in combination with therapies that cause nutrient stress. Cancer Discov; 8(7); 866-83. ©2018 AACR.See related commentary by Commisso and Debnath, p. 800This article is highlighted in the In This Issue feature, p. 781.

Adenosine triphosphate–dependent calcium signaling during ventilator-induced lung injury is amplified by hypercapnia

ABSTRACT Adenosine triphosphate (ATP) is released by alveolar epithelial cells during ventilator-induced lung injury (VILI) and regulates fluid transport across epithelia. High CO2 levels are observed in patients with “permissive hypercapnia,” which inhibits alveolar fluid reabsorption (AFR) in alveolar epithelial cells. The authors set out to determine whether VILI affects AFR and whether the purinergic pathway is modulated in cells exposed to hypercapnia. Control group was compared against VILI (tidal volume [Vt] = 35 mL/kg, zero positive end-expiratory pressure [PEEP]) and protective ventilation (Vt = 6 mL/kg, PEEP = 10 cm H2O) groups. Lung mechanics, histology, and AFR were evaluated. Alveolar epithelial cells (AECs) were loaded with Fura 2-AM to measure intracellular calcium in the presence ATP (10 μM) at 5% or 10% CO2 as compared with baseline. High tidal volume ventilation impairs lung mechanics and AFR. Hypercapnia (HC) increases intracellular calcium levels in response to ATP stimulation. HC + ATP is the most detrimental combination decreasing AFR. Purinergic signaling in AECs is modulated by high CO2 levels via increased cytosolic calcium. The authors reason that this modulation may play a role in the impairment of alveolar epithelial functions induced by hypercapnia.

A multidimensional phasor approach reveals LAURDAN photophysics in NIH-3T3 cell membranes

Mammalian cell membranes have different phospholipid composition and cholesterol content, displaying a profile of fluidity that depends on their intracellular location. Among the dyes used in membrane studies, LAURDAN has the advantage to be sensitive to the lipid composition as well as to membrane fluidity. The LAURDAN spectrum is sensitive to the lipid composition and dipolar relaxation arising from water penetration, but disentangling lipid composition from membrane fluidity can be obtained if time resolved spectra could be measured at each cell location. Here we describe a method in which spectral and lifetime information obtained in different measurements at the same plane in a cell are used in the phasor plot providing a solution to analyze multiple lifetime or spectral data through a common visualization approach. We exploit a property of phasor plots based on the reciprocal role of the phasor plot and the image. In the phasor analysis each pixel of the image is associated with a phasor and each phasor maps to pixels and features in the image. In this paper the lifetime and spectral fluorescence data are used simultaneously to determine the contribution of polarity and dipolar relaxations of LAURDAN in each pixel of an image.

Spectral phasor analysis reveals altered membrane order and function of root hair cells in Arabidopsis dry2/sqe1-5 drought hypersensitive mutant

Biological membranes allow the regulation of numerous cellular processes, which are affected when unfavorable environmental factors are perceived. Lipids and proteins are the principal components of biological membranes. Each lipid has unique biophysical properties, and, therefore the lipid composition of the membrane is critical to maintaining the bilayer structure and functionality. Membrane composition and integrity are becoming the focus of studies aiming to understand how plants adapt to its environment. In this study, using a combination of di-4-ANEPPDHQ fluorescence and spectral phasor analysis, we report that the drought hypersensitive/squalene epoxidase (dry2/sqe1-5) mutant with reduced major sterols such as sitosterol and stigmasterol in roots presented higher membrane fluidity than the wild type. Moreover, analysis of endomembrane dynamics showed that vesicle formation was affected in dry2/sqe1-5. Further analysis of proteins associated with sterol rich micro domains showed that dry2/sqe1-5 presented micro domains function altered.

El salbutamol mejora la contractilidad diafragmática en la obstrucción crónica de la vía aérea

INTRODUCTION Chronic airflow obstruction in conditions such as chronic obstructive pulmonary disease is associated with respiratory muscle dysfunction. Our aim was to study the effects of salbutamol-a beta-adrenergic agonist known to improve muscle strength in physiologic and pathologic conditions-on diaphragm contractility in an animal model of chronic airway obstruction achieved by tracheal banding. MATERIALS AND METHODS Twenty-four Sprague-Dawley rats were randomized into a control group and 3 tracheal banding groups, 1 that received acute salbutamol treatment, 1 that received chronic salbutamol treatment, and 1 that received nothing. Arterial blood gases, acid-base balance, and in vitro diaphragmatic contractility were evaluated by measuring peak twitch tension, contraction time, contraction velocity, half-relaxation time, relaxation velocity, and force-frequency curves. RESULTS The 3 study groups had significantly reduced arterial pH and increased PaCO2 and bicarbonate levels compared to the control group (P<.05). The untreated tracheal banding group had significantly reduced peak twitch tension and contraction velocity, and a significantly lower force-frequency curve in comparison with the other groups (P<.05). The chronic treatment group had a higher relaxation velocity than the untreated study group (P<.05). The mean (SE) peak twitch tension values were 6.46 (0.90)N/cm(2) for the control group, 3.28 (0.55)N/cm(2) for the untreated tracheal banding group, 6.18 (0.71)N/cm(2) for the acute treatment group, and 7.09 (0.59)N/cm(2) for the chronic treatment group. CONCLUSIONS Diaphragmatic dysfunction associated with chronic airflow obstruction improves with both the acute and chronic administration of salbutamol. The mechanisms involved in respiratory muscle dysfunction warrant further study.

Salbutamol Improves Diaphragmatic Contractility in Chronic Airway Obstruction

Introduction: Chronic airfl ow obstruction in conditions such as chronic obstructive pulmonary disease is associated with respiratory muscle dysfunction. Our aim was to study the effects of salbutamol—a β2adrenergic agonist known to improve muscle strength in physiologic and pathologic conditions—on diaphragm contractility in an animal model of chronic airway obstruction achieved by tracheal banding. Materials and Methods: Twenty-four Sprague-Dawley rats were randomized into a control group and 3 tracheal banding groups, 1 that received acute salbutamol treatment, 1 that received chronic salbutamol treatment, and 1 that received nothing. Arterial blood gases, acid-base balance, and in vitro diaphragmatic contractility were evaluated by measuring peak twitch tension, contraction time, contraction velocity, halfrelaxation time, relaxation velocity, and force-frequency curves. Results: The 3 study groups had signifi cantly reduced arterial pH and increased PaCO2 and bicarbonate levels compared to the control group (P<.05). The untreated tracheal banding group had signifi cantly reduced peak twitch tension and contraction velocity, and a signifi cantly lower force-frequency curve in comparison with the other groups (P<.05). The chronic treatment group had a higher relaxation velocity than the untreated study group (P<.05). The mean (SE) peak twitch tension values were 6.46 (0.90) N/cm 2