Data di Pubblicazione:
2013
Citazione:
A METABOLOMIC, STRUCTURAL AND FUNCTIONAL STUDY OF POLYCYSTIN1 AND NEPHROCYSTIN1 / V. Mannella ; docente guida: M. Duranti ; correlatrice: G. Musco ; coordinatore: F. Bonomi. UNIVERSITA' DEGLI STUDI DI MILANO, 2013 Feb 19. 25. ciclo, Anno Accademico 2012. [10.13130/mannella-valeria_phd2013-02-19].
Abstract:
Abstract Nephronophthisis (NPHP) and Autosomal Dominant Polycystic Kidney Disease (ADPKD) are two genetic renal cystic diseases that lead to End Stage Renal Disease (ESRD) in childhood or adolescence and at the late-middle age, respectively1. At present the mechanisms at the basis of cystogenesis in both diseases are poorly understood and pharmacological therapies are still lacking.
On the one hand NPHP is characterized by kidney tubular atrophy and cysts formation occurring primarily at the cortico-medullary border, that leads to a reduction of kidneys volume. It is caused by mutations in 11 different genes. The most common form of NPHP is the juvenile one (NPHP type 1), which is caused, in most of the cases, by homozygous deletion of NPHP1 gene (2q13 chromosome). NPHP1 gene encodes for nephrocystin1 (NPHP1), a 732 amino acids long cytoplasmatic protein. It presents a widespread expression, but the pathogenic consequences due to loss-of-function are mainly confined to the kidney. In particular, NPHP1 is localized to cell-cell junctions, cilia and cell-matrix adhesion sites and it is involved in signaling transduction, cell-cell adhesion and cell polarity2-4.
On the other hand the hallmark of ADPKD is the formation of bilateral cysts that leads to an increase of kidneys volume weight5,6. The disease is caused by mutations in two genes: Pkd1 gene (16p13.3 chromosome) which is mutated in 85% of cases and Pkd2 (4q21 chromosome) which is mutated in the remaining part of cases. These genes encode respectively for polycystin1 (PC1), a large plasma membrane receptor 4302 amino acids long and polycystin2 (PC2), a 968 amino acids long calcium channel. The two proteins interact through their C-terminal coiled coil domains and are involved in common signaling pathways, thus explaining the same phenotype5,7,8.
In this thesis we focused our attention on two new possible roles of PC1: the regulation of metabolism and its interaction with NPHP1. To disentangle PC1 role as “metabolic regulator” and its possible interaction with NPHP1 through its coiled coil domain, we have selected nuclear magnetic resonance (NMR) spectroscopy as primary investigation technique.
PC1 as metabolic regulator. During a routine culture of mouse embryonic fibroblasts (MEFs) our collaborators (Dr. Boletta’s group in San Raffaele Hospital, Milan, Italy) have observed that the knock out (Pkd1 KO) MEFs for PC1 acidify the medium faster than the wild type (WT) ones. This finding rises the hypothesis that PC1 is involved in the regulation of cellular metabolism. Hence, exploiting nuclear magnetic resonance (NMR) spectroscopy, we have applied a metabolomic approach in order to investigate the metabolic pathways and the signaling cascade deregulated by the loss of PC1. Importantly, the metabolomic analysis was performed both in cells and in mouse kidneys (WT and Pkd1 KO) in order to compare the results both in vitro and in vivo. The analysis of the 1D 1H NMR spectra of the media conditioned by the Pkd1 KO and by the WT MEF cells (exometabolome) has highlighted some metabolic differences: the exometabolome of the Pkd1 KO MEF cells displays an increased content of lactate, glutamate and alanine, presenting features similar to tumor cell metabolism9-11whereas the exometabolome of the WT MEF cells has an increased content of choline, formate, glucose, glycine and pyruvate. The two metabolites that vary mostly between the two cell lines are glucose and lactate, as highlighted by PCA analysis. In particular Pkd1 KO MEF cells display a major consumption of glucose, which is in turn related to a major production of lactate, showing the aerobic glycolysis, also called Warburg effect, a metabolic hallmark usually observed in cancer cells.
In order to pro
Tipologia IRIS:
Tesi di dottorato
Keywords:
ADPKD ; NPHP ; PC1 ; NPHP1 ; metabolomics ; protein structure ; NMR
Elenco autori:
V. Mannella
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