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Identifier 000385226
Title Μηχανισμοί νεφρικής βλάβης στην οικογενή αμυλοειδική πολυνευροπάθεια
Alternative Title Mechanisms of renal insult in familial amyloid polyneuropathy
Author Πετράκης, Ιωάννης
Thesis advisor Αμοιρίδης, Γεώργιος
Πλαίτάκης, Ανδρέας
Δαφνής, Ευγένιος
Abstract Introduction: Familial Amyloid Polyneuropathy (FAP) has been first described 60 years ago. Nowadays consists one of the commonest forms of hereditary amyloidosis. It is inherited through the autosomal dominant way of transmittance. Clinical features vary, but the dominant one is polyneuropathy due to small nerve fibers insult. Till present all FAP patients carry spot mutations of transthyretin gene and present amyloid deposition in various tissues. Renal amyloid deposition and renal injury has been a not so common feature in FAP patients. As far as FAP pathogenesis is concerned oligomeric non-fibrilar transthyretin species induce apoptosis, increase pro-inflammatory cytokines and induce oxidative stress within target tissues. Scope: The present study had the intention to examine mechanisms of renal insult in FAP animal models. It specifically examined urinary albumin excretion (UAE) in FAP animal models. Furthermore it examined the way with which various components of the slit diaphragm (nephrin and podocin), podocyte number, mRNA levels of WT1, glomerular basement membrane (GBM) thickness, foot process width (FPW), change due to partial shortage of Hsf-1. In addition it examined, the effect of different environmental conditions in TTRV30M glomerular deposition, TTRV30M renal gene expression, TTRV30M podocytic localization, as well as caspase 3 activation. Furthermore sex impact under the administration of rapamycin in tissue TTRV30M deposition was examined. Glomerular levels of bax/bcl-2 were examined in relation to transgenic mice gender. Materials and Methods: Transgenic animals (C57Bl/6-Tg(6.0 TTRMet30)15Imeg –hTTRV30M, CARD, Kumamoto Japan) were used for experimentation. Oviduct transfer of cryopreserved embryos was performed. Young animals (10-16 months old), and Old animals (16-21) with or without TTRVal30Met gene were used for 24 hrs urinary collection. UAE was normalized over body weight (BW) of laboratory animals. UAE was examined with the use of ELISA (Bethyl laboratories). Logarithmic transformation of data was performed and test Chi square was used for statistical analysis. Statistical analysis was performed with SPSS 19 (IBM) software. Pearson’s R was used for correlation analysis. Statistical significance was set at .05. In addition in an animal group 8 months old rapamycin (0.4 mg/kg BW) was 9 administered i.p for 2 months. In this group urinary collections were performed. The kidneys were acutely frozen in liquid nitrogen. Five μm thick cryosections were fixed with paraformaldehyde solution 4% and were incubated with rabbit anti-human transthyretin antibody (DAKO), bax anti mouse antibody (Abcam), bcl-2 anti mouse antibody (Abcam). Renal tissue was further stained for PAS, Silver Stain, Masson, Congo Red (CR) and H&E. Confocal microscopy was utilized for analyzing immunofluorescence sections (Leica, SP). Leica confocal software was used for image analysis. Data were examined for normality. In case of non normality logarithmic transformation was performed. Statistical significance was set at .05 and was examined with the use of test Chi square, and independent samples T test. SPSS 19 software was used for statistical analysis. In addition it was examined whether TTRV30M deposition is affected by different environmental conditions (SPF vs non-SPF housing conditions). Glomeruli from transgenic animals were examined under direct and indirect confocal microscopy fluorescence analysis for the presence of TTRV30M deposition, serum amyloid P (SAP), activation of caspase 3 and nephrin. Co-localization rate and amount of co-localization was estimated with the use of Image J software (NIH). TTRV30M localization was achieved with the use of immunoelectron microscopy. Levels of TTRV30M, and nephrin gene expression were estimated with the use reverse transcription real time pcr (RT-PCR). In parallel renal tissue genes nephrin, podocin, WT-1 from Hsf1 hemizygous TTRV30M mice were studied with the use of Western Blot (WB), RT-PCR, electron microscopy (GBM thickness, FPW), and immunofluorescence. Results: 78.6% of Young TTRV30M animals had UAE above median, while 21.4% of non transgenic animals had UAE above median (Χ2=0.583, p=0.445). 100% of old TTRV30M animals had UAE above median, while 0% of non transgenic animals, had UAE above median (X2 =8.6, p=0.003,R=0.496, p=0.002). As far as the rapamycin treated mice are concerned mean UAE = 778.9 +/- 6.0, while control mice had ean UAE =815.3 +/- 3.3 p=0.97. In all studied transgenic mice renal tissue was CR negative. Glomeruli from male transgenic rapamycin treated mice had less mesangial expansion when compared to DMSO treated control mice (X2 =26.56; p΄&λτ0.001). Glomeruli from female transgenic rapamycin treated mice had more mesangial expansion when compared to DMSO treated control mice (X2 =16.6; p=0.001). Male transgenic rapamycin treated mice had 17.6% of glomeruli with fluorescence intensity above median, while female transgenic rapamycin treated mice had 89,8% of 10 glomeruli with fluorescence intensity above median(X 2 =72.4; p΄&λτ0.001). No difference was observed for fluorescence intensity of male and female control mice (DMSO treated X2 =2.12; p=0.18), while fluorescence intensity was intermediate male and female mice treated with rapamycin. Male transgenic rapamycin treated mice had 28.6% of glomeruli with a bax/bcl2 ratio above median, while female transgenic rapamycin treated mice had 88.9% of glomeruli with a bax/bcl2 ratio above median(X2 =27.71; p΄&λτ0.001). Non SPF housed TTRV30M mice had increased transthyretin glomerular deposition when compared to SPF housed animals. In addition increased glomerular caspase3 activation was observed in non SPF transgenic mice in relation to SPF transgenic animals. Podocytic caspase3 activation was elevated irrespectively to housing conditions. Hsf1 hemizygous mice had lower nephrin and podocin content but higher podocyte number than Hsf1 homozygous mice. Transthyretin deposition was increased in glomeruli of Hsf1 hemizygous mice while no influence was observed in WT1, nephrin and podocin expression levels. Increased GBM thickness and FPW was observed in Hsf1 hemizygous mice. Conclusions: Human TTRV30M transgene presence increases UAE, and this is accompanied by glomerular transthyretin deposition. Male transgenic rapamycin treated mice had less mesangial expansion to femal transgenic rapamycin treated mice. In addition more glomeruli of male transgenic rapamycin treated mice had increased TTRV30M deposition when compared to female counterparts. Increased bax/bcl2 ratio was observed in the glomeruli of female transgenic rapamycin treated mice in relation to male counterparts. Therefore rapamycin induces differential effects under the influence of sex either detrimental or beneficial. This interaction is not accompanied by UAE increase. Different environmental conditions (SPF, non SPF) affect glomerular transthyretin deposition, intrapodocytic transthyretin localization. Under these influences TTRV30M deposition causes an increase in caspase 3 activation. Under the effect of Hsf1 hemizygosity, TTRV30M deposition has catastrophic effects in GBM thickness, PFP width and slit diaphragm constitution without affecting podocin and nephrin gene expression. Therefore mechanisms of renal insult in FAP could be strongly be affected by the combination of environmental and genetic factors.
Language English
Subject Amyloidosis
Enviromental effects on renal disease
Podocytes
Transgenic mouse models of renal disease
Transthyretin
mechanisms of renal insult
Αμυλοείδωση
Διαγονιδιακά μοντέλα νεφρικής νόσου
Μηχανισμοί νεφρικής βλάβης
Περιβαλλοντική επίδραση και νεφρική νόσος
Ποδοκύτταρα
Τρανσθυρετίνη
Issue date 2014-07-24
Collection   School/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
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