High glucose induces an activated state of partial epithelial-mesenchymal transition in human primary tubular cell cultures.

Tubulointerstitial fibrosis is observed in diabetic nephropathy. It is still debated whether tubular cells, undergoing epithelial-mesenchymal transition (EMT) in high glucose (HG) conditions, may contribute to interstitial fibrosis development. In this study, we investigated the phenotypic and molecular EMT-like changes and the alteration of inflammatory and fibrogenic secretome induced by HG in human primary tubular cell cultures. Taking advantage of this in vitro cell model composed of proximal and distal tubular cells, we showed that HG-treated tubular cells acquired a fibroblast-like morphology with increased cytoplasmic stress fibers, maintaining the expression of the epithelial markers specific of proximal and distal tubular cells. HG increased Snail1, miRNA210 and Vimentin mesenchymal markers, decreased N-cadherin expression and migration ability of primary tubular cells, while E-cadherin expression and focal adhesion distribution were not affected. Furthermore, HG treatment of tubular cells altered the inflammatory cytokine secretion creating a secretome able to enhance the proliferation and migration of fibroblasts. Our findings show that HG promotes an activated state of partial EMT in human tubular primary cells and induces a pro-inflammatory and pro-fibrogenic microenvironment, supporting the active role of tubular cells in diabetic nephropathy onset.

The cross-talk between Abl2 tyrosine kinase and TGFß1 signalling modulates the invasion of clear cell Renal Cell Carcinoma cells.

Clear cell Renal Cell Carcinoma (ccRCC) is the most common and metastatic urological cancer. Molecular players of ccRCC progression and metastasis are not completely known. Here, using primary cell cultures from patients' specimens, we found that TGFß1/Smad signalling is more activated in high versus low grade ccRCC and inversely correlates with Abl2 tyrosine kinase protein expression. TGFß1 treatment increased ubiquitination and degradation of Abl2 protein in ccRCC cell lines by TGFß1/Smad pathway activation and reactive oxygen species production. 3D invasion and matrix degradation assays showed that Abl2 promoted TGFß1-induced ccRCC cell invasion and maturation of invadopodia, a hallmark of tumour invasion and metastasis. Our findings define Abl2 as a new downstream molecule of TGFß1 signalling and putative target to counteract advanced ccRCC.

DNA Damage in Circulating Hematopoietic Progenitor Stem Cells as Promising Biological Sensor of Frailty.

Frailty is an age-related syndrome that exposes individuals to increased vulnerability. Although it is potentially reversible, in most cases it leads to negative outcomes, including mortality. The different methods proposed identify frailty after the onset of clinical manifestations. An early diagnosis might make it possible to manage the frailty progression better. The frailty pathophysiology is still unclear although mechanisms, in particular, those linked to inflammation and immunosenescence, have been investigated. A common feature of several clinical aspects involved in senescent organisms is the increase of oxidative stress, described as one of the major causes of deoxyribonucleic acid (DNA) damage accumulation in aged cells including the adult stem cell compartment. Likely, this accumulation is implicated in frailty status. The oxidative status of our frail, pre-frail, and non-frail population was characterized. In addition, the DNA damage in hematopoietic cells was evidenced by analyzing the peripheral blood mononuclear cell and their T lymphocyte, monocyte, circulating hematopoietic progenitor stem cell (cHPSC) subpopulations. The phosphorylation of C-terminal of histone H2AX at amino acid Ser 139 (γ-H2AX), which occurs at the DNA double-strand break focus, was evaluated. In our frail population, increased oxidative stress and a high level of DNA damage in cHPSC were found. This study may have potential implications because the increment of DNA damage in cHPSC could be suggestive of an organism impairment preceding the evident frailty. In addition, it may open the possibility for attenuation of frailty progression throughout specific drugs acting on preventing DNA damage or removing damaged cells.

Integration of Lipidomics and Transcriptomics Reveals Reprogramming of the Lipid Metabolism and Composition in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is fundamentally a metabolic disease. Given the importance of lipids in many cellular processes, in this study we delineated a lipidomic profile of human ccRCC and integrated it with transcriptomic data to connect the variations in cancer lipid metabolism with gene expression changes. Untargeted lipidomic analysis was performed on 20 ccRCC and 20 paired normal tissues, using LC-MS and GC-MS. Different lipid classes were altered in cancer compared to normal tissue. Among the long chain fatty acids (LCFAs), significant accumulations of polyunsaturated fatty acids (PUFAs) were found. Integrated lipidomic and transcriptomic analysis showed that fatty acid desaturation and elongation pathways were enriched in neoplastic tissue. Consistent with these findings, we observed increased expression of stearoyl-CoA desaturase(SCD1) and FA elongase 2 and 5 in ccRCC. Primary renal cancer cells treated with a small molecule SCD1 inhibitor (A939572) proliferated at a slower rate than untreated cancer cells. In addition, after cisplatin treatment, the death rate of tumor cells treated with A939572 was significantly greater than that of untreated cancer cells. In conclusion, our findings delineate a ccRCC lipidomic signature and showed that SCD1 inhibition significantly reduced cancer cell proliferation and increased cisplatin sensitivity, suggesting that this pathway can be involved in ccRCC chemotherapy resistance.

Integrated Genomic, Functional, and Prognostic Characterization of Atypical Chronic Myeloid Leukemia.

Atypical chronic myeloid leukemia (aCML) is a BCR-ABL1-negative clonal disorder, which belongs to the myelodysplastic/myeloproliferative group. This disease is characterized by recurrent somatic mutations in SETBP1, ASXL1 and ETNK1 genes, as well as high genetic heterogeneity, thus posing a great therapeutic challenge. To provide a comprehensive genomic characterization of aCML we applied a high-throughput sequencing strategy to 43 aCML samples, including both whole-exome and RNA-sequencing data. Our dataset identifies ASXL1, SETBP1, and ETNK1 as the most frequently mutated genes with a total of 43.2%, 29.7 and 16.2%, respectively. We characterized the clonal architecture of 7 aCML patients by means of colony assays and targeted resequencing. The results indicate that ETNK1 variants occur early in the clonal evolution history of aCML, while SETBP1 mutations often represent a late event. The presence of actionable mutations conferred both ex vivo and in vivo sensitivity to specific inhibitors with evidence of strong in vitro synergism in case of multiple targeting. In one patient, a clinical response was obtained. Stratification based on RNA-sequencing identified two different populations in terms of overall survival, and differential gene expression analysis identified 38 significantly overexpressed genes in the worse outcome group. Three genes correctly classified patients for overall survival.

PKHhigh/CD133+/CD24- Renal Stem-Like Cells Isolated from Human Nephrospheres Exhibit In Vitro Multipotency.

The mechanism upon which human kidneys undergo regeneration is debated, though different lineage-tracing mouse models have tried to explain the cellular types and the mechanisms involved. Different sources of human renal progenitors have been proposed, but it is difficult to argue whether these populations have the same capacities that have been described in mice. Using the nephrosphere (NS) model, we isolated the quiescent population of adult human renal stem-like PKHhigh/CD133+/CD24- cells (RSC). The aim of this study was to deepen the RSC in vitro multipotency capacity. RSC, not expressing endothelial markers, generated secondary nephrospheres containing CD31+/vWf+ cells and cytokeratin positive cells, indicating the coexistence of endothelial and epithelial commitment. RSC cultured on decellularized human renal scaffolds generated endothelial structures together with the proximal and distal tubular structures. CD31+ endothelial committed progenitors sorted from nephrospheres generated spheroids with endothelial-like sprouts in Matrigel. We also demonstrated the double commitment toward endothelial and epithelial lineages of single RSC. The ability of the plastic RSC population to recapitulate the development of tubular epithelial and endothelial renal lineages makes these cells a good tool for the creation of organoids with translational relevance for studying the parenchymal and endothelial cell interactions and developing new therapeutic strategies.

36-kDa Annexin A3 Isoform Negatively Modulates Lipid Storage in Clear Cell Renal Cell Carcinoma Cells.

The adipocyte-like morphology of clear cell renal cell carcinoma (ccRCC) cells results from a grade-dependent neutral lipid accumulation; however, the molecular mechanism and role in renal cancer progression have yet to be clarified. ccRCC shows a gene expression signature consistent with adipogenesis, and the phospholipid-binding protein annexin A3 (AnxA3), a negative regulator of adipocyte differentiation, is down-regulated in RCC and shows a differential expression pattern for two isoforms of 36 and 33 kDa. Using primary cell cultures and cell lines, we investigated the involvement of AnxA3 isoforms in lipid storage modulation of ccRCC cells. We found that the increased accumulation of lipids into ccRCC cells correlated with a decrease of the 36/33 isoform ratio. Treatment with adipogenic medium induced a significant increment of lipid storage in ccRCC cells that had a low 36-kDa AnxA3 expression and 36/33 ratio. The 36-kDa AnxA3 silencing in ccRCC cells increased lipid storage induced by adipogenic medium. These data suggest that 36-kDa AnxA3 negatively modulates the response to adipogenic treatment and may act as negative regulator of lipid storage in ccRCC cells. The subcellular distribution of AnxA3 in the cellular endocytic compartment suggests its involvement in modulation of vesicular trafficking, and it might serve as a putative mechanism of lipid storage regulation in ccRCC cells, opening novel translational outcomes.

The 1ALCTL and 1BLCTL isoforms of Arg/Abl2 induce fibroblast activation and extra cellular matrix remodelling differently.

The fibrotic tissue and the stroma adjacent to cancer cells are characterised by the presence of activated fibroblasts (myofibroblasts) which play a role in creating a supportive tissue characterised by abundant extracellular matrix (ECM) secretion. The myofibroblasts remodel this tissue through secreted molecules and modulation of their cytoskeleton and specialized contractile structures. The non-receptor protein tyrosine kinase Arg (also called Abl2) has the unique ability to bind directly to the actin cytoskeleton, transducing diverse extracellular signals into cytoskeletal rearrangements. In this study we analysed the 1ALCTL and 1BLCTL Arg isoforms in Arg-/- murine embryonal fibroblasts (MEF) cell line, focusing on their capacity to activate fibroblasts and to remodel ECM. The results obtained showed that Arg isoform 1BLCTL has a major role in proliferation, migration/invasion of MEF and in inducing a milieu able to modulate tumour cell morphology, while 1ALCTL isoform has a role in MEF adhesion maintaining active focal adhesions. On the whole, the presence of Arg in MEF supports the proliferation, activation, adhesion, ECM contraction and stiffness, while the absence of Arg affected these myofibroblast features.This article has an associated First Person interview with the first author of the paper.

Integrated multi-omics characterization reveals a distinctive metabolic signature and the role of NDUFA4L2 in promoting angiogenesis, chemoresistance, and mitochondrial dysfunction in clear cell renal cell carcinoma.

An altered metabolism is involved in the development of clear cell - renal cell carcinoma (ccRCC), and in this tumor many altered genes play a fundamental role in controlling cell metabolic activities. We delineated a large-scale metabolomic profile of human ccRCC, and integrated it with transcriptomic data to connect the variations in cancer metabolism with gene expression changes. Moreover, to better analyze the specific contribution of metabolic gene alterations potentially associated with tumorigenesis and tumor progression, we evaluated the transcription profile of primary renal tumor cells. Untargeted metabolomic analysis revealed a signature of an increased glucose uptake and utilization in ccRCC. In addition, metabolites related to pentose phosphate pathway were also altered in the tumor samples in association with changes in Krebs cycle intermediates and related metabolites. We identified NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4-like 2 (NDUFA4L2) as the most highly expressed gene in renal cancer cells and evaluated its role in sustaining angiogenesis, chemoresistance, and mitochondrial dysfunction. Finally, we showed that silencing of NDUFA4L2 affects cell viability, increases mitochondrial mass, and induces ROS generation in hypoxia.

Nephrosphere-Derived Cells Are Induced to Multilineage Differentiation when Cultured on Human Decellularized Kidney Scaffolds.

In end-stage chronic kidney disease, the option of organ transplantation is limited because of the scarce availability of kidneys. The combination of stem cell research, regenerative medicine, and tissue engineering seems a promising approach to produce new transplantable kidneys. Currently, the possibility to repopulate naturally obtained scaffolds with cells of different sources is advancing. Our aim was to test, for the first time, whether the nephrosphere (NS) cells, composed by renal stem/progenitor-like cells, were able to repopulate different nephron portions of renal extracellular matrix scaffolds obtained after decellularization of human renal tissue slices. Our decellularization protocol enabled us to obtain a completely acellular renal scaffold while maintaining the extracellular matrix structure and composition in terms of collagen IV, laminin, and fibronectin. NS cells, cultured on decellularized renal scaffolds with basal medium, differentiated into proximal and distal tubules as well as endothelium, as highlighted by histology and by the specific expression of epithelial cytokeratin 8.18, proximal tubular CD10, distal tubular cytokeratin 7, and endothelial von Willebrand factor markers. Endothelial medium promoted the differentiation toward the endothelium, whereas epithelial medium promoted the differentiation toward the epithelium. NS cells seem to be a good tool for scaffold repopulation, paving the way for experimental investigations focused on whole-kidney reconstruction.

The glucose and lipid metabolism reprogramming is grade-dependent in clear cell renal cell carcinoma primary cultures and is targetable to modulate cell viability and proliferation.

Clear cell renal cell carcinoma (ccRCC) has a poor prognosis despite novel biological targeted therapies. Tumor aggressiveness and poor survival may correlate with tumor grade at diagnosis and with complex metabolic alterations, also involving glucose and lipid metabolism. However, currently no grade-specific metabolic therapy addresses these alterations. Here we used primary cell cultures from ccRCC of low- and high-grade to investigate the effect on energy state and reduced pyridine nucleotide level, and on viability and proliferation, of specific inhibition of glycolysis with 2-deoxy-D-glucose (2DG), or fatty acid oxidation with Etomoxir. Our primary cultures retained the tissue grade-dependent modulation of lipid and glycogen storage and aerobic glycolysis (Warburg effect). 2DG affected lactate production, energy state and reduced pyridine nucleotide level in high-grade ccRCC cultures, but the energy state only in low-grade. Rather, Etomoxir affected energy state in high-grade and reduced pyridine nucleotide level in low-grade cultures. Energy state and reduced pyridine nucleotide level were evaluated by ATP and reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) dye quantification, respectively. 2DG treatment impaired cell proliferation and viability of low-grade ccRCC and normal cortex cultures, whereas Etomoxir showed a cytostatic and cytotoxic effect only in high-grade ccRCC cultures. Our data indicate that in ccRCC the Warburg effect is a grade-dependent feature, and fatty acid oxidation can be activated for different grade-dependent metabolic needs. A possible grade-dependent metabolic therapeutic approach in ccRCC is also highlighted.

Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells.

Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy.

Major Action of Endogenous Lysyl Oxidase in Clear Cell Renal Cell Carcinoma Progression and Collagen Stiffness Revealed by Primary Cell Cultures.

Human clear cell renal cell carcinoma (ccRCC) is therapy resistant; therefore, it is worthwhile studying in depth the molecular aspects of its progression. In ccRCC the biallelic inactivation of the VHL gene leads to stabilization of hypoxia-inducible factors (HIFs). Among the targets of HIF-1α transcriptional activity is the LOX gene, which codes for the inactive proenzyme (Pro-Lox) from which, after extracellular secretion and proteolysis, derives the active enzyme (Lox) and the propeptide (Lox-PP). By increasing stiffness of extracellular matrix by collagen crosslinking, Lox promotes tumor progression and metastasis. Lox and Lox-PP can reenter the cells where Lox promotes cell proliferation and invasion, whereas Lox-PP acts as tumor suppressor because of its Ras recision and apoptotic activity. Few data are available concerning LOX in ccRCC. Using an in vitro model of ccRCC primary cell cultures, we performed, for the first time in ccRCC, a detailed study of endogenous LOX and also investigated their transcriptomic profile. We found that endogenous LOX is overexpressed in ccRCC, is involved in a positive-regulative loop with HIF-1α, and has a major action on ccRCC progression through cellular adhesion, migration, and collagen matrix stiffness increment; however, the oncosuppressive action of Lox-PP was not found to prevail. These findings may suggest translational approaches for new therapeutic strategies in ccRCC.

Arg tyrosine kinase modulates TGF-ß1 production in human renal tubular cells under high-glucose conditions.

Renal tubular cells are involved in the tubular interstitial fibrosis observed in diabetic nephropathy. It is debated whether epithelial-mesenchymal transition (EMT) affects tubular cells, which under high-glucose conditions overproduce transforming growth factor-ß (TGF-ß), a fibrogenic cytokine involved in interstitial fibrosis development. Our study investigated the involvement of non-receptor tyrosine kinase Arg (also called Abl2) in TGF-ß production. Human primary tubular cell cultures exposed to high-glucose conditions were used. These cells showed an elongated morphology, stress fibers and vimentin increment but maintained most of the epithelial marker expression and distribution. In these cells exposed to high glucose, which overexpressed and secreted active TGF-ß1, Arg protein and activity was downregulated. A further TGF-ß1 increase was induced by Arg silencing with siRNA, as with the Arg tyrosine kinase inhibitor Imatinib. In the cells exposed to high glucose, reactive oxygen species (ROS)-dependent Arg kinase downregulation induced both RhoA activation, through p190RhoGAPA (also known as ARHGAP35) modulation, and proteasome activity inhibition. These data evidence a new specific involvement of Arg kinase into the regulation of TGF-ß1 expression in tubular cells under high-glucose conditions and provide cues for new translational approaches in diabetic nephropathy.

PKH(high) cells within clonal human nephrospheres provide a purified adult renal stem cell population.

The existence and identification of adult renal stem cells is a controversial issue. In this study, renal stem cells were identified from cultures of clonal human nephrospheres. The cultured nephrospheres exhibited the activation of stem cell pathways and contained cells at different levels of maturation. In each nephrosphere the presence of 1.12-1.25 cells mirroring stem cell properties was calculated. The nephrosphere cells were able to generate three-dimensional tubular structures in 3D cultures and in vivo. In clonal human nephrospheres a PKH(high) phenotype was isolated using PKH26 epifluorescence, which can identify quiescent cells within the nephrospheres. The PKH(high) cells, capable of self-renewal and of generating a differentiated epithelial, endothelial and podocytic progeny, can also survive in vivo maintaining the undifferentiated status. The PKH(high) status, together with a CD133(+)/CD24(-) phenotype, identified a homogeneous cell population displaying in vitro self-renewal and multipotency capacity. The resident adult renal stem cell population isolated from nephrospheres can be used for the study of mechanisms that regulate self-renewal and differentiation in adult renal tissue as well as in renal pathological conditions.

One isoform of Arg/Abl2 tyrosine kinase is nuclear and the other seven cytosolic isoforms differently modulate cell morphology, motility and the cytoskeleton.

The non-receptor tyrosine kinase Abelson related gene (Arg/Abl2) regulates cell migration and morphogenesis by modulating the cytoskeleton. Arg promotes actin-based cell protrusions and spreading, and inhibits cell migration by attenuating stress fiber formation and contractility via activation of the RhoA inhibitor, p190RhoGAP, and by regulating focal adhesion dynamics also via CrkII phosphorylation. Eight full-length Arg isoforms with different N- and C-termini are endogenously expressed in human cells. In this paper, the eight Arg isoforms, subcloned in the pFLAG-CMV2 vector, were transfected in COS-7 cells in order to study their subcellular distribution and role in cell morphology, migration and cytoskeletal modulation. The transfected 1BSCTS Arg isoform has a nuclear distribution and phosphorylates CrkII in the nucleus, whilst the other isoforms are detected in the cytoplasm. The 1BLCTL, 1BSCTL, 1ASCTS isoforms were able to significantly decrease stress fibers, induce cell shrinkage and filopodia-like protrusions with a significant increase in p190RhoGAP phosphorylation. In contrast, 1ALCTL, 1ALCTS, 1ASCTL and 1BLCTS isoforms do not significantly decrease stress fibers and induce the formation of retraction tail-like protrusions. The 1BLCTL and 1ALCTL isoforms have different effects on cell migration and focal adhesions. All these data may open new perspectives to study the mechanisms of cell invasiveness.