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Titolo/Abstract/Parole chiave


Durante, Chiara (2009) CALCIUM HOMEOSTASIS AND POLYCYSTIN-2 EXPRESSION IN T-LYMPHOBLASTS OF PKD SUBJECTS. Tesi di Dottorato , Università degli studi di Ferrara.

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    Mutations of polycystin-1 (PC1) and polycystin-2 (PC2), coded by PKD1 and PKD2 genes, account for approximately 85 and 15% of Autosomal Dominant Polycystic Kidney Disease (ADPKD) cases, a common and important inherited kidney disorder. PC2 is a calcium-permeable and -regulated channel, interacting with the membrane receptor PC1, both colocalizing in the ‘primary cilium’ of epithelial cells as a flow-sensitive mechanosensor involved in signal-transduction. Diagnostic tools to decrease ADPKD are little in progress. Renal ultrasound, the principal diagnostic procedure, not allows an early diagnosis. The molecular characterization of the mutations can have a prognostic value, the progression to ESRD occurring more rapidly in PKD1 patients. The genetic test is, however, very expensive, time consuming, many missense variants remaining without a known function. This study is addressed to define whether PC1 and PC2 are expressed and play some functional role in T-lymphoblasts (HTL). These cells are easily obtainable from peripheral blood, differently from kidney cells and transgenic mice that represent canonical models for the study of PKD gene expression and function. In particular, attention has been focused on the possible relation between abnormal PCs levels and Ca2+ homeostasis in HTL obtained from ADPKD patients and thus carrying a mutation of either PKD1 or PKD2 gene. Expression studies have also considered fibrocystin/polyductin (FC1), mutated in the recessive form of the disease, known to be functionally related to PCs in vivo. Reduced cytoplasmic Ca2+ levels or other cellular abnormalities in ADPKD HTL will provide further information on the extrarenal role of PCs. Moreover, as HTL are easily and quickly obtainable from peripheral blood cells, positive results may lead to an ADPKD diagnosis based on quantitative and functional evaluation of PC1/PC2 channel complex. Peripheral blood samples from 31 (13 F) controls and 34 PKD patients (21 F) including some genetically characterized ADPKD1 subjects, have been considered in the present study and compared to B-lymphoblastoid cell lines (LCLs) and human adult and embryonal kidney cell lines. Results obtained demonstrate that PC1 and PC2 are expressed in HTL, although at a lower level than in kidney cells. Since PC1 is not correctly quantifiable in HTL, no comparison between PC1 levels in ADPKD HTL and controls is reported. On the contrary, the degree of PC2 expression in HTL, which appear comparable to that of B-LCLs, does not differ in ADPKD and non-ADPKD HTL (PC2 relative abitrary units, 0.572 ± 0.228 SD vs 0.553 ± 0.176 SD). Few cases with very low PC2 levels (lower than 2 fold the SD of ADPKD1 average values) and showing a less severe disease because entered in dialysis very late (over 70 y. o.), may carry a PKD2 mutation, which should be expected in 15% of ADPKD. Also the investigation of FC1, mutated in the recessive form of PKD, and of FCL, its homologue highly expressed in activated T-cells, does not reveal quantitative alteration in the level of combined FC1/FCL proteins in ADPKD HTL. As far as the function of PC1/PC2 channel complex is concerning, in HTL average values of PAF-evoked [Ca2+] are lower in ADPKD than in non-ADPKD (78.96 ± 9.67 SE nM vs 124.51 ± 16.87 nM, p<0.05), and a same reduction is present in ADPKD PBL (112.03 ± 13.87 nM SE vs 183.32 ± 20.54 nM, p<0.01). These results show that PCs have a functional role in T-cells. However, the large and overlapping distribution of the data in both PBL and HTL does not allow the use of [Ca2+] differences to distinguish individual ADPKD and non-ADPKD cases. A further marked cellular abnormality observed in ADPKD HTL is consistent with the anti-apoptotic action of PC1: ADPKD HTL death is higher compared to normal HTL (after 2 days of culture the % of trypan blue positve cells is 7.6 ± 2.8 SD vs 24.1 ± 9.7, p<0.05). Moreover, the observation of a larger size of cell aggregates in ADPKD compared to control HTL, is consistent with the PCs-dependent positive control of cell scattering/migration of renal epithelial cells. Alterations, presumably located at membrane level, are also present in ADPKD neutrophils which are basically more activated, but less responsive to stimuli, than control cells. Overall these findings indicate that aploinsufficiency of PKD1, the more probable involved gene in these patients, is detectable in HTL as an impairment of the PC1/PC2 channel, reduced cell survival and altered aggregation, thus playing PC1/PC2 a still undefined role in HTL. Aploinsufficiency in these cells seems, however, not sufficient to discriminate between unique ADPKD and not ADPKD subjects.

    Tipologia del documento:Tesi di Dottorato (Tesi di Dottorato)
    Data:27 Febbraio 2009
    Relatore:Del Senno, Laura
    Coordinatore ciclo:Bernardi, Francesco
    Istituzione:Università degli studi di Ferrara
    Struttura:Dipartimento > Biochimica e biologia molecolare
    Soggetti:Area 05 - Scienze biologiche > BIO/11 Biologia molecolare
    Parole chiave:calcium, adpkd, polycystins
    Depositato il:22 Lug 2009 15:08


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