Lysosomal dysfunction induced by changes in albumin's tertiary structure: Potential key factor in protein toxicity during diabetic nephropathy.

AIMS: Increased amounts of protein, in particular albumin within renal tubular cells (TBCs), induce the expression of inflammatory and fibrogenic mediators, which are adverse prognostic factors in tubulointerstitial fibrosis and diabetic nephropathy (DN). We sought to assess the participation of the thiol-linked tertiary structure of albumin in the mechanism of protein toxicity in a model of TBCs. MATERIALS AND METHODS: Cultured human renal proximal tubular cells, HK-2, were exposed to isolated albumin from patients with and without DN (Stages 0, 1 and 4). The magnitude of change of the albumin tertiary structure, cell viability (LDH leakage), apoptosis (Annexin V), transdifferentiation and reticulum endoplasmic stress (Western blot and flow cytometry) and lysosomal enzyme activity were assessed. KEY FINDINGS: We found that albumin from Stage 4 patients presented >50% higher thiol-dependent changes of tertiary structure compared to Stages 0 and 1. Cells incubated with Stage 4 albumin displayed 5 times less viability, accompanied by an increased number of apoptotic cells; evidence of profibrogenic markers E-cadherin and vimentin and higher expression of epithelial-to-mesenchymal transition markers α-SMA and E-cadherin and of endoplasmic reticulum stress protein GRP78 were likewise observed. Moreover, we found that cathepsin B activity in isolated lysosomes showed a significant inhibitory effect on albumin from patients in advanced stages of DN and on albumin that was intentionally modified. SIGNIFICANCE: Overall, this study showed that thiol-dependent changes in albumin's tertiary structure interfere with the lysosomal proteolysis of renal TBCs, inducing molecular changes associated with interstitial fibrosis and DN progression.

Is gestational diabetes mellitus in obese women predicted by oxidative damage in red blood cells?

Obesity in pregnant women has been associated with an increased risk of maternal complications, including gestational diabetes mellitus (GDM), a process that is related to oxidative stress (OS). To evaluate the biomarkers of OS in red blood cells (RBCs), we assigned 80 pregnant women to one of three groups: control (n = 28), overweight (n = 26) and obese (n = 26). Then, we measured in plasma, the levels of glucose, triacylglycerol (TAG), insulin, free fatty acids (FFAs), leptin and cytokines (e.g. interleukin-6 [IL-6] and tumor necrosis factor-alpha [TNF-alpha]) and OS biomarkers, such as lipohydroperoxides (LHP), malondialdehyde (MDA) and protein carbonylation (PC) in RBCs. We found significant positive correlations between OS biomarkers, body mass index (BMI) and pregnancy progression. Seven (26.9%) obese women who were diagnosed with GDM at 24-28 weeks of pregnancy showed significantly increased concentrations of FFAs, insulin, leptin, TNF-alpha and biomarkers of OS measured at 12-13 weeks of gestation. We propose to quantify LHP, MDA and PC in membranes of erythrocytes as possible markers to diagnose GDM from weeks 12-14.

RBC membrane damage and decreased band 3 phospho-tyrosine phosphatase activity are markers of COPD progression.

Injury to red blood cell (RBC) membrane by oxidative stress is of clinical importance in chronic obstructive pulmonary disease (COPD) which leads to oxidative stress (OE) during disease progression. Here, we studied the impact of this stress on injury to RBC membrane. Blood samples from both healthy volunteers (HV, n = 11) and controlled COPD patients (n=43) were divided according to their GOLD disease stage (I=7, II=21, III=10, IV=5). Plasma levels of paraoxonase (PON) activity, protein carbonyls (PC), conjugate dienes, lipohydroperoxides (LPH) and malondialdehyde (MDA) were determined and the PTPase, and the oxidative parameters were measured in RBC ghosts. Plasma from patients with COPD showed an increased oxidation of lipids and proteins, that correlated with the disease progression. PON activity decreased from GOLD stages II to IV and correlated with an increase in LPH (p less than 0.0001, r = -0.8115). There was evidence of an increase in the oxidative biomarkers in RBCs, while the PTPase activity was diminished in stage III and IV of COPD. In conclusion, OE-induced injury associated with COPD is associated with an oxidative damage to the RBC membrane, with a concomitant decrease in the PTPase activity and altered function of anionic exchanger (AE1).

Airborne particulate matter PM2.5 from Mexico City affects the generation of reactive oxygen species by blood neutrophils from asthmatics: an in vitro approach.

BACKGROUND: The Mexico City Metropolitan Area is densely populated, and toxic air pollutants are generated and concentrated at a higher rate because of its geographic characteristics. It is well known that exposure to particulate matter, especially to fine and ultra-fine particles, enhances the risk of cardio-respiratory diseases, especially in populations susceptible to oxidative stress. The aim of this study was to evaluate the effect of fine particles on the respiratory burst of circulating neutrophils from asthmatic patients living in Mexico City. METHODS: In total, 6 subjects diagnosed with mild asthma and 11 healthy volunteers were asked to participate. Neutrophils were isolated from peripheral venous blood and incubated with fine particles, and the generation of reactive oxygen species was recorded by chemiluminescence. We also measured plasma lipoperoxidation susceptibility and plasma myeloperoxidase and paraoxonase activities by spectrophotometry. RESULTS: Asthmatic patients showed significantly lower plasma paraoxonase activity, higher susceptibility to plasma lipoperoxidation and an increase in myeloperoxidase activity that differed significantly from the control group. In the presence of fine particles, neutrophils from asthmatic patients showed an increased tendency to generate reactive oxygen species after stimulation with fine particles (PM2.5). CONCLUSION: These findings suggest that asthmatic patients have higher oxidation of plasmatic lipids due to reduced antioxidant defense. Furthermore, fine particles tended to increase the respiratory burst of blood human neutrophils from the asthmatic group.On the whole, increased myeloperoxidase activity and susceptibility to lipoperoxidation with a concomitant decrease in paraoxonase activity in asthmatic patients could favor lung infection and hence disrupt the control of asthmatic crises.

Perspectivas del uso de antioxidantes como coadyuvantes en el tratamiento del asma / Perspectives in the use of antioxidants for the treatment of asthma

Al tratarse de un proceso inflamatorio, en el asma hay participación e incremento en la generación de especies reactivas del oxígeno, dando lugar a un desequilibrio oxidante/antioxidante, fenómeno que se ha descrito como estrés oxidante, que causa daño a diferentes biomoléculas. La utilización de agentes antioxidantes exógenos o activadores de antioxidantes endógenos como coadyuvantes de la terapéutica del paciente asmático, es una posibilidad a discutir.

Oxidative stress seen in bronchial asthma can damage different kinds of biomolecules; this oxidant/antioxidant imbalance results from an increment in the production of oxygen reactive species. The utilization of exogenous antioxidant agents or promoters of endogenous antioxidants can be seen as an alternative therapy for asthma that is worth discussing.

Estrés oxidante en asma / Oxidative stress in asthma

El asma es una enfermedad inflamatoria crónica del tracto respiratorio de etiología aún desconocida; sin embargo, nuevas evidencias han involucrado al estrés oxidante, en el que la participación e incremento en la generación de especies reactivas del oxígeno por diferentes sistemas bioquímicos, superan a los mecanismos antioxidantes en el ambiente de las vías respiratorias del asmático, lo cual es acompañado de alteraciones inducidas por radicales libres que involucran daño estructural y modificaciones metabólicas presentes, a nivel sistémico y en el tracto respiratorio.

Asthma is a chronic inflammatory disease of the airways: its precise etiology is still unknown. New evidence points to oxydative stress, in which the participation and increment of reactive species of oxygen by several biochemical systems overwhelms the anti oxidant mechanisms of the airways; this, in conjunction with changes induced by free radicals involving systemic and local respiratory structural damage and metabolic changes.

Participación d las especies reactivas del oxígeno en las enfermedades pulmonares

Las especies reactivas de oxígeno son moléculas (O2•-, HO • , NO • ), muy reactivas debido a que en el último orbital tienen un electrón no pareado (radical libre), lo cual confiere inestabilidad física. Se incluyen en las especies reactivas de oxígeno a moléculas precursoras de los radicales libres (H2O2, HONO2-). Estas especies participan en procesos fisiológicos en el organismo. Cuando la generación de especies reactivas de oxígeno supera a los mecanismos de inactivación, se presenta el estado metabólico de estrés oxidante que se caracteriza por daños moleculares y celulares que conducen a predisposición o modificación de diversos padecimientos crónico-degenerativos. Entre las enfermedades pulmonares en que se ha demostrado la participación de las especies reactivas de oxígeno, destacan el síndrome de insuficiencia respiratoria progresiva, la enfermedad pulmonar obstructiva crónica y el asma. Se analizan las características del estrés oxidante en estos padecimientos.

Reactive Oxygen Species (ROS) are very reactive molecules (O2•-, HO•, NO• ) since they have a single and unpaired electron in the last orbital (free radical) which confers them physical instability. Free radical precursors such as H2O2 , HONO2- are considered ROS. These species are important in the physiological processes. When ROS production exceeds the inactivation mechanisms, oxidative stress takes place. This stress is characterized by molecular and cellular damage which predisposes to or modifies chronic-degenerative diseases. Among pulmonary diseases in which ROS participation has been proved are ARDS, COPD and asthma. The aim of this paper was to analyze the mechanisms of oxidative stress that lead to those illnesses.

Lack of voltage-dependent calcium channel opening during the calcium influx induced by progesterone in human sperm. Effect of calcium channel deactivation and inactivation.

Progesterone induces calcium influx and acrosomal exocytosis in human sperm. Pharmacologic evidence suggests that voltage-dependent calcium channels (VDCCs) are involved. In this study, membrane potential (Vm) and intracellular calcium concentration ([Ca(2+)](i)) were monitored simultaneously to assess the effect of VDCC gating on the calcium influx triggered by progesterone. Holding the Vm to values that maintained VDCCs in a deactivated (-71 mV) closed state inhibited the calcium influx induced by progesterone by approximately 40%. At this Vm, the acrosomal reaction induced by progesterone, but not by A23187, was inhibited. However, when the Vm was held at -15 mV (which maintains VDCCs in an inactivated closed state), the progesterone-induced calcium influx was stimulated. Furthermore, the progesterone and voltage-dependent calcium influxes were additive. These findings indicate that progesterone does not produce VDCC gating in human sperm.

Stimulation of voltage-dependent calcium channels during capacitation and by progesterone in human sperm.

To fertilize, mammalian sperm must undergo two sequential steps that require activation of calcium entry mechanisms, capacitation and acrosomal exocytosis, induced in the latter case by the egg zona pellucida glycoprotein ZP3 or by progesterone. Voltage-dependent calcium channels (VDCC) could participate in these processes. Since patch clamp recordings are extremely difficult in mature sperm, the activity of VDCC has been alternatively analyzed with optical detectors of membrane potential and intracellular calcium in sperm populations. Using this approach, we previously reported that in human sperm there is a voltage-dependent calcium influx system that strongly indicates that human sperm are endowed with functional VDCC. In this study we developed evidence indicating that calcium influx through VDCC is significantly stimulated during sperm in vitro capacitation and by progesterone action, which is present in the follicular fluid that surrounds the egg. The observed effects of capacitation and progesterone on VDCC may be physiologically significant for sperm-egg interaction.

Comunicación de gametos a distancia. Quimiotaxis y quimiocinesis del espermatozoide en mamíferos / At distance gamet communication. chemotaxis and chemokinesis of spermatozoo in mammals

La comunicación a distancia mediada químicamente entre el esperamatozoide o el óvulo como parte del proceso de fecundación a sido intensamente estudiado en especies de invertebrados con fecundación externa, sin embargo, su estudio en vertebrados, y especialmente mamíferos, abre un campo de gran interés. Los principales quimioatractantes en mamíferos que han sido encontrados incluyen a: péptidos de 1 a 20 KDa, proteínas, hormonas como progesterona, oxitocina, adrenalina y recientemente se han encontrado receptortes presentes en la cabeza del espermatozoide que están asociados a la enzima Guanilil ciclasa, vía proteína G, así como receptores de tipo olfatorio, rodopsina cinasa y arrestina. El estudio de la quimiotaxis permite además de entender algunos procesos de fecundación en gametos humanos y aplicarlos en los programas de reproducción asistida, puede ser de gran importancia en los programas de control prenatal

Especies reactivas de oxígeno en el espermatozoide / Oxygen reactive species in the spermatozoa

Los radicales libres de oxígeno son especies químicas de este elemento formadas mediante reacciones enzimáticas y no enzimáticas y han sido implicados en muchos procesos patológicos y fisiológicos. Las especies químicas de oxígeno más reactivas son el peróxido de hidrógeno y los radicales libres anión superóxido e hidróxilo, siendo este último el más reactivo. El espermatozoide de mamíferos, incluyendo el del humano producen radicales libres de oxígeno y peróxido de hidrógeno; sin embargo esta última célula normalmente posee mecanismos enzimáticos para protegerse del posible daño que pudiera causarle estos agentes tóxicos. A pesar de ello, el espermatozoide es bastante suceptible al "estrés oxidativo", probablemente debido a su alto contenido de ácidos grasos insaturados. Este hecho puede tener importancia en la patología de ciertos tipos de infertilidad masculina como lo es la oligozoospermia, ya que se ha demostrado que en estos casos, se presenta una producción incontrolada de especies reactivas de oxígeno por parte del espermatozoide

Mecanismo de acción de hormonas esteroides I: estrógenos / Mechanism of action of steroid hormones I: estrogens

Las hormonas esteroides son moléculares muy versátiles: a pesar de estar relacionada entre sí por su estructura química, cumplen funciones muy diversas, e incluso antagónicas. Su mecanismo de acción no esta aclarado totalmente. Los estrógenos participan en la regulación de prácticamente todos los eventos reproductivos y sexuales de la hembra, sin embargo, las acciones intracelulares por las que se llevan a cabo no se conocen adecuadamente las interrogantes. Se acepta en la actualidad la existencia de un receptor citoplásmico y/o nuclear, sin aclarar satisfactoriamente cómo las hormonas llegan hasta el núcleo. Los eventos endócrinos que se expresan rápidamente (segundos) se deben a una posible interacción con la membrana celular. El propósito de esta revisión es analizar y conciliar los datos reportados sobre el mecanismo de acción de estrogenos.