Novel effects of ouabain in autosomal dominant polycystic kidney disease cystogenesis

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in Pkd1 or Pkd2, genes encoding for polycystin-1 (PC-1) and polycystin-2 (PC-2), respectively. ADPKD is characterized by the progressive growth of numerous fluid-filled renal cysts. Cyst formation and growth depends on proliferation of the cyst-lining epithelial cells and fluid secretion into the cyst lumen. ADPKD cystogenesis is highly influenced by non-genomic factors, many of which elicit their effects via cAMP-dependent pathways. Understanding mechanisms mediating the effects of cystogenic agents is crucial for the future development of ADPKD therapy. Previous work has shown that cells derived from the epithelial-lining of renal cysts from patients with ADPKD (ADPKD cells) have an increased affinity for the hormone ouabain. ADPKD cells respond to ouabain by an increased rate of cell proliferation. This effect depends on binding of ouabain to the Na,K-ATPase which induces activation of Src kinase, epidermal growth factor receptor (EGFR), and the extracellular regulated kinase (ERK1/2) pathway. The objective of the current study was to determine the role of ouabain in mechanisms of fluid secretion and cyst growth in ADPKD. Studies were carried out in human ADPKD cells, embryonic kidneys from the Pkd1m1Bei mouse model, and M-1 mouse cortical collecting duct cells. Results of this study show that physiologic concentrations of ouabain enhance cAMP-dependent fluid secretion and cyst growth of ADPKD cells grown in culture as monolayers or in three-dimensional structures resembling cysts. Additionally, ouabain potentiated the cAMP-dependent growth of cyst-like dilations in metanephric kidneys from the Pkd1m1Bei mouse model. These effects were mediated via activation of the Na,K-ATPase signaling apparatus, located at the basolateral domain of ADPKD cells. Intracellular mediators of ouabain's response included the EGFR-Src-ERK pathway. Ouabain alone did not increase fluid secretion and cyst growth. Rather, ouabain treatment altered the phenotype of ADPKD cells to allow enhanced responses to cAMP agonists. The potentiating effect of ouabain on cAMP-induced fluid secretion was associated with the capacity of ouabain to stimulate anion secretion via the apically located cystic fibrosis transmembrane conductance regulator (CFTR). Moreover, ouabain increased membrane expression of the CFTR. Finally, ouabain decreased Na,K-ATPase membrane expression and ion transport at the basolateral membrane of ADPKD cells. The increased ouabain sensitivity of ADPKD cells depends on an abnormally high affinity of the Na,K-ATPase for ouabain. Increased ouabain affinity of the Na,K-ATPase was associated with abnormal expression of the C-terminus of PC-1 in M-1 cells. Altogether, the study of ouabain's effects in ADPKD have uncovered a novel role for ouabain as a physiologic agent that influences renal cyst growth in ADPKD. In addition, it has identified a new mechanism in ADPKD cystogenesis, important for the progression of the disease

Mechanistic target of rapamycin complex 1 signaling links hypoxia to increased igfbp-1 phosphorylation in primary human decidualized endometrial stromal cells

Insulin-like growth factor-1 (IGF-1) bioavailability in pregnancy is governed by IGF binding protein (IGFBP-1) and its phosphorylation, which enhances the affinity of IGFBP-1 for the growth factor. The decidua is the predominant source of maternal IGFBP-1; however, the mechanisms regulating decidual IGFBP-1 secretion/phosphorylation are poorly understood. Using decidualized primary human endometrial stromal cells (HESCs) from first-trimester placenta, we tested the hypothesis that mTORC1 signaling mechanistically links hypoxia to decidual IGFBP-1 secretion/phosphorylation. Hypoxia inhibited mechanistic target of rapamycin (mTORC1) (p-P70-S6K/Thr389, −47%, p = 0.038; p-4E-BP1/Thr70, −55%, p = 0.012) and increased IGFBP-1 (total, +35%, p = 0.005; phosphorylated, Ser101/+82%, p = 0.018; Ser119/+88%, p = 0.039; Ser 169/+157%, p = 0.019). Targeted parallel reaction monitoring-mass spectrometry (PRM-MS) additionally demonstrated markedly increased dual IGFBP-1 phosphorylation (pSer98+Ser101; pSer169+Ser174) in hypoxia. IGFBP-1 hyperphosphorylation inhibited IGF-1 receptor autophosphorylation/ Tyr1135 (−29%, p = 0.002). Furthermore, silencing of tuberous sclerosis complex 2 (TSC2) activated mTORC1 (p-P70-S6K/Thr389, +68%, p = 0.038; p-4E-BP1/Thr70, +30%, p = 0.002) and reduced total/site-specific IGFBP-1 phosphorylation. Importantly, TSC2 siRNA prevented inhibition of mTORC1 and the increase in secretion/site-specific IGFBP-1 phosphorylation in hypoxia. PRM-MS indicated concomitant changes in protein kinase autophosphorylation (CK2/Tyr182; PKC/Thr497; PKC/Ser657). Overall, mTORC1 signaling mechanistically links hypoxia to IGFBP-1 secretion/phosphorylation in primary HESC, implicating decidual mTORC1 inhibition as a novel mechanism linking uteroplacental hypoxia to fetal growth restriction

Increased IGFBP-1 phosphorylation in response to leucine deprivation is mediated by CK2 and PKC

Insulin-like growth factor binding protein-1 (IGFBP-1), secreted by fetal liver, is a key regulator of IGF-I bioavailability and fetal growth. IGFBP-1 phosphorylation decreases IGF-I bioavailability and diminishes its growth-promoting effects. Growth-restricted fetuses have decreased levels of circulating essential amino acids. We recently showed that IGFBP-1 hyperphosphorylation (pSer101/119/169) in response to leucine deprivation is regulated via activation of the amino acid response (AAR) in HepG2 cells. Here we investigated nutrient-sensitive protein kinases CK2/PKC/PKA in mediating IGFBP-1 phosphorylation in leucine deprivation. We demonstrated that leucine deprivation stimulated CK2 activity (enzymatic assay) and induced IGFBP-1 phosphorylation (immunoblotting/MRM-MS). Inhibition (pharmacological/siRNA) of CK2/PKC, but not PKA, prevented IGFBP-1 hyperphosphorylation in leucine deprivation. PKC inhibition also prevented leucine deprivation-stimulated CK2 activity. Functionally, leucine deprivation decreased IGF-I-induced-IGF-1R autophosphorylation when CK2/PKC were not inhibited. Our data strongly support that PKC promotes leucine deprivation-induced IGFBP-1 hyperphosphorylation via CK2 activation, mechanistically linking decreased amino acid availability and reduced fetal growth

Anxiety and depression mediate the relationship between insomnia symptoms and the personality traits of conscientiousness and emotional stability

This study examined the relationship between the Big Five personality traits and insomnia symptoms in the general population. Additionally, the mediating role of anxiety and depression was examined. Participants (N=625) completed online measures of the big five personality traits and insomnia severity. Insomnia symptoms were independently related to extroversion, agreeableness, conscientiousness, emotional stability, anxiety and depression in univariate analysis. Linear regression determined conscientiousness and emotional stability to be the only traits predicting insomnia symptoms. However, these relationships were at least partially mediated by anxiety and depression. Whilst reduced levels of conscientiousness and emotional stability has previously associated with poor sleep and insomnia, the current outcomes shed light on the mechanisms which serve to mediate this relationship

Preferential attention towards the eye-region amongst individuals with insomnia

People with insomnia often perceive their own facial appearance as more tired compared with the appearance of others. Evidence also highlights the eye-region in projecting tiredness cues to perceivers, and tiredness judgements often rely on preferential attention towards this region. Using a novel eye-tracking paradigm, this study examined: (i) whether individuals with insomnia display preferential attention towards the eye-region, relative to nose and mouth regions, whilst observing faces compared with normal-sleepers; and (ii) whether an attentional bias towards the eye-region amongst individuals with insomnia is self-specific or general in nature. Twenty individuals with DSM-5 Insomnia Disorder and 20 normal-sleepers viewed 48 neutral facial photographs (24 of themselves, 24 of other people) for periods of 4000 ms. Eye movements were recorded using eye-tracking, and first fixation onset, first fixation duration and total gaze duration were examined for three interest-regions (eyes, nose, mouth). Significant group × interest-region interactions indicated that, regardless of the face presented, participants with insomnia were quicker to attend to, and spent more time observing, the eye-region relative to the nose and mouth regions compared with normal-sleepers. However, no group × face × interest-region interactions were established. Thus, whilst individuals with insomnia displayed preferential attention towards the eye-region in general, this effect was not accentuated during self-perception. Insomnia appears to be characterized by a general, rather than self-specific, attentional bias towards the eye-region. These findings contribute to our understanding of face perception in insomnia, and provide tentative support for cognitive models of insomnia demonstrating that individuals with insomnia monitor faces in general, with a specific focus around the eye-region, for cues associated with tiredness

Improving the behavioral realism of global integrated assessment models:An application to consumers’ vehicle choices

A large body of transport sector-focused research recognizes the complexity of human behavior in relation to mobility. Yet, global integrated assessment models (IAMs), which are widely used to evaluate the costs, potentials, and consequences of different greenhouse gas emission trajectories over the medium-to-long term, typically represent behavior and the end use of energy as a simple rational choice between available alternatives, even though abundant empirical evidence shows that real-world decision making is more complex and less routinely rational. This paper demonstrates the value of incorporating certain features of consumer behavior in IAMs, focusing on light-duty vehicle (LDV) purchase decisions. An innovative model formulation is developed to represent heterogeneous consumer groups with varying preferences for vehicle novelty, range, refueling/recharging availability, and variety. The formulation is then implemented in the transport module of MESSAGE-Transport, a global IAM, although it also has the generic flexibility to be applied in energy-economy models with varying set-ups. Comparison of conventional and ‘behaviorally-realistic’ model runs with respect to vehicle purchase decisions shows that consumer preferences may slow down the transition to alternative fuel (low-carbon) vehicles. Consequently, stronger price-based incentives and/or non-price based measures may be needed to transform the global fleet of passenger vehicles, at least in the initial market phases of novel alternatives. Otherwise, the mitigation burden borne by other transport sub-sectors and other energy sectors could be higher than previously estimated. More generally, capturing behavioral features of energy consumers in global IAMs increases their usefulness to policy makers by allowing a more realistic assessment of a more diverse suite of policies

Joint effect of phosphorus limitation and temperature on alkaline phosphatase activity and somatic growth in Daphnia magna

Alkaline phosphatase (AP) is a potential biomarker for phosphorus (P) limitation in zooplankton. However, knowledge about regulation of AP in this group is limited. In a laboratory acclimation experiment, we investigated changes in body AP concentration for Daphnia magna kept for 6 days at 10, 15, 20 and 25°C and fed algae with 10 different molar C:P ratios (95–660). In the same experiment, we also assessed somatic growth of the animals since phosphorus acquisition is linked to growth processes. Overall, non-linear but significant relationships of AP activity with C:P ratio were observed, but there was a stronger impact of temperature on AP activity than of P limitation. Animals from the lowest temperature treatment had higher normalized AP activity, which suggests the operation of biochemical temperature compensation mechanisms. Body AP activity increased by a factor of 1.67 for every 10°C decrease in temperature. These results demonstrate that temperature strongly influences AP expression. Therefore, using AP as a P limitation marker in zooplankton needs to consider possible confounding effects of temperature. Both temperature and diet affected somatic growth. The temperature effect on somatic growth, expressed as the Q10 value, responded non-linearly with C:P, with Q10 ranging between 1.9 for lowest food C:P ratio and 1.4 for the most P-deficient food. The significant interaction between those two variables highlights the importance of studying temperature-dependent changes of growth responses to food quality