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

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http://urn.fi/URN:ISBN:978-951-51-5721-8
Title: PACSIN2 and septin 7 in diabetic kidney disease: Roles in intracellular trafficking and actin cytoskeleton organisation in podocytes
Author: Dumont, Vincent
Contributor: University of Helsinki, Faculty of Medicine
Doctoral Programme in Biomedicin
Publisher: Helsingin yliopisto
Date: 2020-01-18
URI: http://urn.fi/URN:ISBN:978-951-51-5721-8
http://hdl.handle.net/10138/308781
Thesis level: Doctoral dissertation (article-based)
Abstract: 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.
Subject: Biomedicine
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