Cellular, molecular, genomic, transcriptomic and proteomic approaches for studying renal pathology
Our laboratory of Oncology/Molecular Pathology at Dept of Experimental Medicine has acquired competence in the field of renal pathology. Combining cellular, molecular, genomic, transcriptomic and proteomic approaches we have produced interesting results studying renal cell carcinoma (RCC) that represents 3% of all adult malignant tumors and whose diagnosis is difficult and often late because of lack of validated RCC markers. We established primary cultures of normal and RCC tissue that retain the phenotype of the original tissue, providing a more homogeneous and enhanced cytological material. In these primary cultures has been evidenced, by a proteomic approach, the differential expression of proteins of various cell compartments, and the AnxA3 protein has been well characterized. On these cultures is ongoing the genomic, transcriptomic and microRNA profiling. To enhance chances of finding gene products related to RCC pathogenesis and progression, normal and tumor primary cultures are now treated with RNA interference (RNAi) for silencing genes involved in metabolic pathways altered in RCC. The genes silenced are chosen among those we previously found differentially expressed. The RNAi treatment hopefully will increase the information about the role of studied genes in RCC pathogenesis. The data obtained will be used to determine a cluster of signals able to distinguish between healthy and RCC individuals, to build diagnostic reagents and as a base to study new drugs.
The identification of a resident renal stem cell (SC) may have clinical relevance in the field of cellular therapy and the definition of a cancer stem cell (CSC) may have a role for the proposal of new pathogenetic hypothesis, for detecting new markers and for developing new therapies targeted to CSC. Data concerning SC in kidney or CSC in RCC are till now incomplete and contradictory and the identity of any renal SC or CSC has not been readily forthcoming. Since the markers used in literature for the identification of a stem cell population of the kidney and RCC are promiscuous and are not able to identify and isolate a purified stem cell population, we use for the isolation a functional approach based on “sphere forming assay”. The disaggregated cells of normal kidney and RCC tissues cultured in suspension at low density and with specific medium form “nephrospheres”. We characterize the cells that compose the nephrospheres by immunofluorescence, FACS and Real Time PCR, evaluating typical stem cell markers and renal markers. We found that some genes of epithelial differentiation have a lower expression and genes related to stemness are over expressed in the cell that compose normal and cancer nephrospheres if compared to already differentiated cells. The cells that compose normal nephrospheres form three-dimensional structures similar to tubules and glomeruli if cultivated into semisolid substrates, and can differentiate into epithelial and podocytic lineage with specific culture media. To identify the stem cell population inside the nephrospheres we use the lipophilic fluorescent dye, PKH26. The more fluorescent cells (PKHhigh), retain the dye and likely are the relative quiescent stem cells, some less fluorescent, active replicating cells (PKHlow/neg), likely are the progenitor cells already committed. These population can be sorted by FACS and only PKHhigh population can form filial nephrospheres of clonal origin as demonstrating by limiting dilution assay. We are going to better characterize the PKHhigh stem cell population by high-throughput transcriptomic approach to find out the phenotype of the adult renal stem cell. The complete characterization of the PKHhigh renal stem cell will be useful for attempts of cellular therapy in renal pathologies. The identification of the renal stem cell phenotype could also give more information about the CSC compartment in RCC to better comprehend the cellular and molecular basis of RCC.
Diabetic nephropathy (DN) is characterized by tubulointerstitial fibrosis that arises not only from “activation” of renal fibroblasts, but also from tubular epithelial cells, that become collagen producing cells, throughout the epithelial-mesenchymal transition (EMT) process. To study the molecular mechanisms of EMT in DN we use an in vitro model of diabetic tubular EMT, establishing well-characterized primary cultures of human tubular epithelial cells (HUTECs) cultured with high glucose concentration. In these cultures the EMT development can be proved by i) morphological changes, evaluated by contrast phase microscopy; ii) immunocytofluorescence and FACS analysis, evaluating the downexpression of epithelial and the overexpression of mesenchymal markers; iii) molecular analysis by Real-Time PCR and 1D-Western blot. In this well-characterized in vitro model of tubular EMT we evidenced an increase of stress fibres and focal adhesions, typical of EMT process. This increment is correlated in the treated cells with the modulation of metabolic pathways in which are involved the Rho and Rac GTPase proteins whose activity play a role in cytoskeleton remodelling. These data induce us to further use this EMT model in order to better investigate the modulation of tubular EMT by silencing genes of interest with siRNA technology. Moreover, in this in vitro model of human primary cultures treated with high glucose has been performed the first overall proteomic approach to study EMT. The identification of the proteins differentially expressed will help to clarify the molecular pathways involved in the early steps of tubular EMT and will help the detection of predictive markers of diabetic nephropathy development.
Arg (Abl related gene) tyrosine kinase inhibits cellular migration by attenuating actomyosin contractility and regulating focal adhesion dynamics through its kinase domain and C-terminal region containing two F-actin and one microtubule binding domains. Alternative splicing of 5’- and 3’- regions of the human Arg mRNA gives rise to eight isoforms (1BLCTL, 1BSCTL, 1BLCTS, 1BSCTS, 1ALCTL, 1ASCTL, 1ALCTS, 1ASCTS) differing at N- termini and in the first C-terminal F-actin binding domain, as described by our laboratory. We analyse the role of the eight different Arg isoforms in regulating cell morphology, adhesion and cytoskeletal organization by transfecting COS-7 cells with the Arg isoforms, cloned into the pFlag CMV-2 expression vector. Immunofluorescence analysis, by confocal microscopy and ImageJ software, evidenced for the different Arg isoforms similar cytoplasmatic distribution and colocalization with F-actin and tubulin, and a different capacity to inhibit stress fibers and cellular spreading and to induce shorter filopodia-like cytoplasmatic extroflections. Studies on the exact role of Arg isoforms on morphology, spreading, and focal adhesions of cells stimulated by fibronectin are ongoing. Based on the different expression of Arg isoforms in normal and neoplastic cells, their different role in cell dynamic regulation might be important for metastatic diffusion. Furthermore, Arg expression, at transcript and protein level, has been studied also in our in vitro EMT model evidencing a downregulation of Arg that correlates with cytoskeleton rearrangements occurring during EMT development.
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