PACSIN2 and septin 7 in diabetic kidney disease : Roles in intracellular trafficking and actin cytoskeleton organisation in podocytes

Background. Diabetes mellitus is a metabolic disease characterised by the inability of the body to maintain stable glycaemia. Diabetic kidney disease (DKD), the renal complication of diabetes, is the leading cause of end-stage renal disease in Europe and in the USA. Approximately 40% of individuals with diabetes develop this microvascular complication that may eventually require dialysis or kidney transplant. DKD is characterised by progressive loss of the permselectivity of the glomerular filtration barrier and declining glomerular filtration rate. The pathological mechanisms of DKD are not fully elucidated but podocyte injury is involved. In particular, alterations in the expression or localisation of nephrin, changes in the regulation of the actin cytoskeleton organisation and defects in the insulin signalling pathway are involved. This work characterises the renal phenotype of a novel transgenic mouse model of hyperglycaemia triggered by hypoinsulinaemia due to the defective β-cell mass growth in the pancreas. It also aims to evaluate the expression, the role and the phosphorylation status of protein kinase C and casein kinase 2 substrate in neurons (PACSIN2) in the trafficking of nephrin and the regulation of the actin cytoskeleton organisation in the context of DKD in podocytes. Further, the thesis refines how septin 7 hinders the trafficking of glucose transporter 4 (GLUT4) storage vesicles, as septin 7 was recently found to inhibit glucose uptake in podocytes. Results. The characterisation of hyperglycaemic E1-DN mice revealed in homozygous mice an increased urine volume as well as albuminuria, the severity of which correlated with the hyperglycaemia. The kidneys developed changes typical of human DKD, such as tubular proliferation and atrophy, mesangial expansion and glomerular basement membrane thickening, lower expression of nephrin, foot process effacement and podocyte apoptosis. Next, we found that PACSIN2 expression was elevated in the glomeruli of obese Zucker Diabetic Fatty (ZDF) rats, a model of advanced DKD. Interestingly, the obese ZDF rats had albuminuria and altered localisation of nephrin. We found that overexpression of PACSIN2 increased both endocytosis and recycling of nephrin. This may rely on the newly identified PACSIN2-nephrin-rabenosyn-5 protein complex. Interestingly, the interaction of PACSIN2 with nephrin was stimulated by treating podocytes with palmitate, the most abundant free fatty acid (FFA) in the circulation and elevated in diabetes. Our data using isolated glomeruli from ZDF rats and human, as well as using cultured podocytes treated with sera from diabetic patients with normal kidney function or microalbuminuria suggested that PACSIN2 phosphorylation at serine 313 is increased in the context of DKD but not diabetes alone. We identified that palmitate induced the phosphorylation of PACSIN2 at S313 in a protein kinase C-dependent manner. Finally, we found that overexpression of PACSIN2 altered the actin cytoskeleton organisation and morphology of podocytes in culture, and that constitutively phosphorylated PACSIN2 at S313 showed milder effect. In the work concentrating on the trafficking of GLUT4 in podocytes, we found that septin 7 forms a complex with nephrin and nonmuscle myosin IIA (NMIIA) at the plasma membrane in the docking and fusion site of GLUT4 storage vesicles (GSVs). We showed that removal of septin 7 and activation of NMIIA in the docking and fusion site are essential for insulin-stimulated glucose uptake. Also, the presence of septin 7 and the activity of NMIIA at the docking and fusion site appeared to be regulated by circulating factors in the serum of individuals with type 1 diabetes and macroalbuminuria. Conclusion. In this thesis, we identified that PACSIN2, the expression of which is increased in podocytes in DKD, regulates the localisation of nephrin in cultured podocytes. In vitro, PACSIN2 also regulates the actin cytoskeleton organisation depending on its phosphorylation at S313, also elevated in DKD. We found that septin 7, together with NMIIA, regulates the docking and fusion of GSVs with the plasma membrane in podocytes. Thus, we propose that PACSIN2, septin 7 and NMIIA are central molecules in podocytes, and participate in the progression towards DKD by regulating the organisation of the actin cytoskeleton and the trafficking of nephrin and GLUT4.Diabeettinen munuaistauti on diabeteksen vakava komplikaatio, jonka kehittymiseen vaikuttavat korkea verensokeri, rasva-aineenvaihdunnan muutokset ja korkea verenpaine. Taudille on tyypillistä etenevä munuaisten toiminnan heikentyminen, mikä voi johtaa loppuvaiheen munuaistautiin vaatien dialyysihoitoa tai munuaissiirteen. Väitöskirjan ensimmäinen osatyö keskittyy tutkimaan uuden diabeettisen hiirimallin munuaisvauriota. Kyseisen mallin hiirten haiman betasolut tuottavat normaalia vähemmän insuliinia, jonka seurauksena hiirille kehittyy diabeettista munuaistautia vastaavia muutoksia, kuten valkuaisvirtsaisuutta, glomeruluksen tyvikalvon paksuuntumista, nefriini-valkuaisaineen ilmentymisen vähentymistä ja podosyyttien (glomeruluksen epiteelisolujen) solukuolemaa. Väitöskirjan seuraavissa osatöissä tutkitaan munuaisen toiminnalle välttämättömien podosyyttien vauriomekanismeja molekyylitasolla. Havaitsimme, että PACSIN2-valkuaisaineen ilmentyminen ja fosforylaatio ovat kohonneet kokeellisen diabetesmallin glomeruluksissa. In vitro-kokeet viljellyillä soluilla osoittivat, että muutokset PACSIN2:n ilmentymisessä vaikuttivat nefriinin solunsisäiseen kuljetukseen ja muutokset fosforylaatiossa puolestaan vaikuttivat aktiinitukirangan järjestäytymiseen. Näin ollen PACSIN2 saattaa liittyä diabeettisen munuaistaudin syntyyn vaikuttamalla podosyyttien vesikkelikuljetuksen ja aktiinitukirangan säätelyyn. Väitöskirjan viimeinen osatyö keskittyy tutkimaan septiini 7:n merkitystä podosyyttien glukoosin sisäänoton säätelyssä. Havaitsimme, että sekä septiini 7 että myosiini IIA ilmentyvät solukalvolla alueella, johon glukoosinkuljettaja 4-valkuaisainetta kuljettavat vesikkelit ohjautuvat ja kiinnittyvät. Työn päähavainto oli, että septiini 7 ja myosiini IIA säätelevät näiden vesikkeleiden kuljetusta, ja siten glukoosin sisäänottoa podosyytteihin, vastakkaisin tavoin. Tämän väitöskirjan keskeisiä tuloksia olivat uuden diabeettisen eläinmallin munuaistaudin analysointi ja podosyyteissä havaittavien munuaisvaurioon johtavien vauriomekanismien tunnistaminen ja karakterisointi. Tuloksia voidaan hyödyntää kehitettäessä uusia diabeettisen munuaistaudin etenemistä hidastavia hoitokeinoja

Development of new processes to protect zinc against corrosion, suitable for on-site use

Protection against corrosion of metals is well known as an important issue in numerous fields. In all cases, the improvement of durability of these metals has to be connected to the development of environmentally friendly processes. Sol–gel protective coatings have shown excellent chemical stability and enhanced corrosion resistance for zinc substrates. Further, the sol–gel method, used as technique of surface protection, showed the potential for the replacement of toxic pre-treatments. This paper highlights the recent developments and applications of silane based sol–gel coatings on zinc substrates. Then, the challenges for industrial transfer of the developed process are also discussed because this process presents a disadvantage for on-site use, which is the too time-consuming thermal treatment. So, the goal of this study was to determine the convenient experimental conditions to reduce the duration of heat treatment of the hybrid sol–gel layer, compatible with the severe industrial requirements, without reducing the protection against corrosion. To reach this objective, a correlation between the results of chemical analyses and the protection against corrosion efficiency was established

PACSIN proteins in vivo : Roles in development and physiology

Protein kinase C and casein kinase substrate in neurons (PACSINs), or syndapins (synaptic dynamin-associated proteins), are a family of proteins involved in the regulation of cell cytoskeleton, intracellular trafficking and signalling. Over the last twenty years, PACSINs have been mostly studied in the in vitro and ex vivo settings, and only in the last decade reports on their function in vivo have emerged. We first summarize the identification, structure and cellular functions of PACSINs, and then focus on the relevance of PACSINs in vivo. During development in various model organisms, PACSINs participate in diverse processes, such as neural crest cell development, gastrulation, laterality development and neuromuscular junction formation. In mouse, PACSIN2 regulates angiogenesis during retinal development and in human, PACSIN2 associates with monosomy and embryonic implantation. In adulthood, PACSIN1 has been extensively studied in the brain and shown to regulate neuromorphogenesis, receptor trafficking and synaptic plasticity. Several genetic studies suggest a role for PACSIN1 in the development of schizophrenia, which is also supported by the phenotype of mice depleted of PACSIN1. PACSIN2 plays an essential role in the maintenance of intestinal homeostasis and participates in kidney repair processes after injury. PACSIN3 is abundant in muscle tissue and necessary for caveolar biogenesis to create membrane reservoirs, thus controlling muscle function, and has been linked to certain genetic muscular disorders. The above examples illustrate the importance of PACSINs in diverse physiological or tissue repair processes in various organs, and associations to diseases when their functions are disturbed.Peer reviewe

Importance of the reconciliation method to handle experimental data in refrigeration and power cycle: application to a reversible heat pump/organic Rankine cycle unit integrated in a positive energy building

Experimental data is often the result of long and costly experimentations. Many times, measurements are used directly without (or with few) analysis and treatment. This paper therefore presents a detailed methodology to use steady-state measurements efficiently in the analysis of a thermodynamic cycle. The reconciliation method allows to correct each measurement as little as possible, taking its accuracy into account, in order to satisfy all constraints and to evaluate the most probable physical state. The reconciliation method should be used for multiple reasons. First, this method allows to close energy and mass balances exactly, which is needed for predictive models. Also, it allows determining some unknowns that are not or that cannot be measured precisely. Furthermore, it fully exploits the collected measurements with redundancy and it allows to know which sensor should be checked or replaced if necessary. An application of this method is presented in the case of a reversible HP/ORC unit. This unit is a modified heat pump which is able to work as an organic Rankine cycle by reversing its cycle. Combined with a passive house comprising a solar roof and a ground heat exchanger, it allows to get a plus energy house. In this study case, the oil mass fraction is not measured despite of its strong influence on the results. The reconciliation method allows to evaluate it. The efficiency of this method is proven by comparing the error on the outputs of steady-state models of compressor and exchangers. An example is given with the prediction of the pinch-point of an evaporator. In this case, the normalized root mean square deviation (NRMSD) is decreased from 14.3 % to 4.1 % when using the reconciliation method. This paper proves the efficiency of the method and also that the method should be considered more often when dealing with experimentation

Experimental investigation of a Scroll unit used as a compressor and as an expander in a Heat Pump/ORC reversible unit

This paper presents an innovative system comprising a heat pump connected to a solar roof and a geothermal heat exchanger. This heat pump is able to invert its cycle and operate as an ORC. By using the whole rooftop (hereunder called “absorber”) as a heat source, a large amount of heat is generated throughout the year. This heat is used in priority to cover the building annual heating needs, while the surplus heat (mainly during summer months) is utilized to generate electricity in the so-called HP/ORC module. The same module can be used in winter time as an efficient heat pump which radically simplifies the complexity of the total system compared to competitive products. The main advantage of the proposed technology is related to the reversibility of scroll machines, which have proven to operate efficiently both as a compressor and as an expander. This paper focuses on three aspects: design of the prototype, results from experiments and validation of models based on the experimental data. To size the unit, the expander and the different exchangers are modeled thanks to semi-empirical correlations calibrated on experimental data. The sizing of the prototype is based on yearly simulations with a simulation step of one hour. The algorithm optimizes the operating mode based on the domestic heat demand, the meteorological conditions and the available heat in the storage. This paper demonstrates the feasibility of such a prototype with promizing experimentally-achieved performance (4.1% overall efficiency in ORC mode and a COP of 4.2 in HP mode

Experimental Investigation Of A Thermally Integrated Carnot Battery Using A Reversible Heat Pump/Organic Rankine Cycle

The growth of renewable energy requires flexible, low-cost and efficient electrical storage systems to balance the mismatch between energy supply and demand. The Carnot battery (or Pumped Thermal Energy Storage) converts electric energy to thermal energy with a heat pump (HP) when electricity production is greater than demand; when electricity demand outstrips production, the Carnot battery generates power from two thermal storage reservoirs (Rankine mode). Classical Carnot batteries architectures do not achieve more than 60% roundtrip electric efficiency. However, innovative architectures, using waste heat recovery (thermally integrated Carnot batteries) are able to reach electrical power production of the power cycle larger than the electrical power consumption of the heat pump (power-to-power-ratio), increasing the value of the technology. It can be shown that the optimization of such a technology is a trade-off between the maximization of the power and the power-to-power ratio (depending on electricity prices among others). In this paper, the full development of a prototype of thermally integrated Carnot battery using a reversible heat pump/organic Rankine cycle (HP/ORC) is described. It includes the selection of the nominal design point, the architecture, the components and the sizing. This first experimental campaign showed a roundtrip electrical energy ratio of 72.5% with ORC efficiency of 5% (temperature lift is equal to 49 K) and COP of HP of 14.4 (temperature lift is equal to 8 K). These results are very encouraging because the performance can easily be improved (probably up to 100% roundtrip electrical energy ratio) by optimizing the volumetric machine, working at larger scale, optimizing the control and thermal insulation. Also, the performance of the main components (volumetric machine and heat exchangers) is analyzed

Performance characteristics of a new structured packing

A new structured packing using carbon fibres, called Sepcarb® 4D, is presented. This packing has several attractive properties, such as high voidage (ε=94%) and high effective area (a=420 m2 m−3). These properties are advantageous for packing used as a gas–liquid contactor for separation units. To determine the internal characteristics of this packing, we performed several experiments using a 150-mm-internal-diameter column. Firstly, hydrodynamics experiments were conducted using an air–water counter current flow to determine the pressure drop (for both dry and wet packing) and flooding point. Secondly, the mass transfer efficiency was determined in terms of HETP (height equivalent to theoretical plate) by total reflux experiments with an n-heptane/cyclohexane mixture at atmospheric pressure. Hydrodynamic performance and mass transfer efficiency were compared with those of packings generally used in distillation and absorption