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.

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.

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.

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.

Using Copy Number Alterations to Identify New Therapeutic Targets for Bladder Carcinoma.

Bladder cancer represents the ninth most widespread malignancy throughout the world. It is characterized by the presence of two different clinical and prognostic subtypes: non-muscle-invasive bladder cancers (NMIBCs) and muscle-invasive bladder cancers (MIBCs). MIBCs have a poor outcome with a common progression to metastasis. Despite improvements in knowledge, treatment has not advanced significantly in recent years, with the absence of new therapeutic targets. Because of the limitations of current therapeutic options, the greater challenge will be to identify biomarkers for clinical application. For this reason, we compared our array comparative genomic hybridization (array-CGH) results with those reported in literature for invasive bladder tumors and, in particular, we focused on the evaluation of copy number alterations (CNAs) present in biopsies and retained in the corresponding cancer stem cell (CSC) subpopulations that should be the main target of therapy. According to our data, CCNE1, MYC, MDM2 and PPARG genes could be interesting therapeutic targets for bladder CSC subpopulations. Surprisingly, HER2 copy number gains are not retained in bladder CSCs, making the gene-targeted therapy less interesting than the others. These results provide precious advice for further study on bladder therapy; however, the clinical importance of these results should be explored.

Chromosomal imbalances in human bladder urothelial carcinoma: similarities and differences between biopsy samples and cancer stem-like cells.

BACKGROUND: The existence of two distinct groups of tumors with different clinical characteristic is a remarkable feature of transitional cell carcinomas (TCCs) of the bladder. More than 70% are low-grade (LG) non-infiltrating (NI) cancers at diagnosis, but 60-80% of them recur at least one time and 10-20% progress in stage and grade. On the other hand, about 20% of tumors show muscle invasion (IN) and have a poor prognosis with <50% survival after 5 years. This study focuses on the complexity of the bladder cancer genome, and for the first time to our knowledge, on the possibility to compare genomic alterations of in vitro selected cancer stem-like cells (CSCs), and their original biopsy in order to identify different genomic signature already present in the early stages of tumorigenesis of LG and HG tumors. METHODS: We initially used conventional chromosome analysis on TCC biopsies with different histotypes (LG vs HG) in order to detect rough differences between them. Then, we performed array comparative genomic hybridization (aCGH) on 10 HG and 10 LG tumors providing an overview of copy number alterations (CNAs). Finally, we made a comparison of the overall CNAs in 16 biopsies and their respective CSCs isolated from them. RESULTS: Our findings indicate that LG and HG bladder cancer differ with regard to their genomic profile even in the early stage of tumorigenesis; moreover, we identified a subgroup of LG samples with a higher tendency to lose genomic regions which could represent a more aggressive phenotype. CONCLUSIONS: The outcomes not only provide valuable information to deeper studying TCC carcinogenesis, but also could help in the clinic for diagnosis and prognosis of patients who will benefit from a more aggressive therapy.

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.

Biological heterogeneity of putative bladder cancer stem-like cell populations from human bladder transitional cell carcinoma samples.

Transitional cell carcinoma (TCC) is the most common type of bladder cancer. Emerging evidence has suggested that the capability of a tumor to grow and propagate is dependent on a small subset of cells, the cancer stem-like cells (CSCs) or tumor initiating cells. We report on the isolation and biological characterization of putative bladder CSC populations from primary TCCs. Isolated cells were induced to proliferate in stem cell culture conditions (serum-free medium containing mitogenic growth factors). The proliferating cells formed spheroids (urospheres) and their abilities for extensive proliferation and self-renewal were assayed. Their positivity for several stem cell markers (CD133, Oct-3/4, nestin, and cytokeratins) was also assessed by immunofluorescence tests and they could have the potential to differentiate in the presence of serum. In stem cell culture conditions they gradually showed loss of proliferation, adherence to the substrate, and morphological changes, which might reflect their progressive acquisition of differentiative capacity and loss of self-renewal ability. To evaluate if effective cell selection occurred after isolation, conventional cytogenetic studies on fresh chromosome spreads immediately after isolation and after culture were carried out. In addition, a molecular cytogenetic study by UroVysion assay was carried out on paraffin-embedded tissue sections and on fresh and after culture nuclei preparations. The data collected indicated important karyotype changes and a positive selection for hypo- or near-diploid cells, losing the complexity present in fresh tumors.

Complete genital prosthetization in patients treated with bilateral orchiectomy for metachronous testicular cancer.

UNLABELLED: Testicular malignant tumours represent 1% of neoplasms. The probability of developing a contralateral tumour is between 3% and 7%. We report two cases of complete genital prosthetization in two patients treated with bilateral orchiectomy for metachronous testicular cancer. In our knowledge no previous case is reported in literature. CASE REPORTS: A 46-year-old patient and a 49-year-old patient underwent bilateral radical orchiectomy for metachronous cancers of the testis. The first patient complained the complete loss of libido and complete impotence. For this reason he began a substitutive hormonal therapy with testosterone esters obtaining re-establishment of the masculine phenotype. With the reappearance of the libido and physical efficiency, the patient showed the intention to recover sexual activity and he was treated first by phosphodiesterase type 5 (PDE-5) inhibitors and then he began a cycle of prostaglandin El1(PGE1) but unfortunately without a satisfactory sexual performance. The second man maintained libido, lost sexual efficiency and showed hypogonadism. Substitutive therapy with testosterone enanthate obtained normalization of testosterone values. A psychosexual assessment highlighted a depressive state related to the absence of sexual performance due to an erectile deficit, that was treated first by PDE-5 inhibitors and then by intracavernous injections of PGEI without benefits. After individual and couple counselling both of the patients submitted both semirigid mono component penile prosthesis and bilateral testicular prostheses implantation by means of a single penile-scrotal incision. Both the patients regained sexual activity and were satisfied with the cosmetic appearance. CONCLUSION: We underline the importance of the collaboration between the andrologist and the psycho-sexologist and the on-going collaboration with the patient. This allows the clinician to appraise the situation completely and to work out a common solution built around the patient to obtain a full recovery of his identity.

Giant renal cyst complicated with acquired crossed renal ectopia, hypertension and renal cell carcinoma: case report and review of the literature.

Simple renal cysts are acquired kidney lesion that are described as spherical, smoothed surfaced, with a serous, sub-yellow content. They are generally considered as a harmless anomaly, however cases of complicated renal cysts have been reported. We present a case of an enormous renal cyst (the biggest ever described) containing more than 25 L of fluid mimicking ascites, complicated with controlateral displacement of ipsilateral functional kidney and intra-abdominal organs, renal cell carcinoma and hypertension. Particular attention is carried in the analysis of the literature about different aspects of giant renal cysts like the ability to grow to a very giant size, the association with hypertension and renal cell carcinoma, the sensibility of the most important examinations to reveals malignancy and management.

Numb chin syndrome: the presenting symptom of a metastatic prostate carcinoma.

Numb chin syndrome is a sensory neuropathy characterized by numbness involving the distribution of the mental nerve that could be an uncommon manifestation of metastatic malignancy. Bony metastases are common in patients with advanced prostate carcinoma and involving preferentially vertebrae, sternum, pelvic bones, ribs and femurs. We report a case in an 82-year-old man presenting a history of mental neuropathy as the isolated presenting symptom of a widespread metastatic prostate cancer Numb chin syndrome was describe in some reviews as a late component of a previously diagnosed disease but this report underline the importance of this neuropathy as the isolated presenting symptom of a widespread metastatic prostate cancer. This event is very rare and enumerates four cases in the world literature.

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.

Testosterone supplementation in hypogonadal men: our personal experience.

INTRODUCTION AND OBJECTIVES: Around thirty years of age, we assist to a physiological, progressive and slow involution of the testicular function accompanied by a fall of the plasmatic levels of testosterone. Rarely hypogonadism is manifest in so young age with signs and symptoms, unless it is subsequent to severe events. After 50 years of age, it can present with a series of aspects that negatively influence the physical and sexual efficiency. The aim of this study is to prove if the replacement therapy with testosterone is able to restore a physiological gonadal function, without incurring in unpleasant side effects to prostate, liver and cardiac muscle. MATERIAL AND METHODS: We report our experience on 123 patients over 60 years of age (mean age 71) and 12 patients over 30 (mean age 37). These last ones were submitted to andrologic assessment after genital trauma. Both groups of patients have reached our observation from December 2000 to June 2003 for important asthenia and decrease of libido associated with impairment of secondary sexual characters and erectile deficit of various degrees. Respectively 76 and 12 patients did not show contraindications for testosterone therapy and they began injections with testosterone for at least 6 months. RESULTS AND CONCLUSIONS: Although compliance to testosterone treatment has not been equal in the time in all the subjects, undesirable effects are not highlighted. All the patients have reported complete or partial recovery of physical efficiency and improvement of sexual life, evaluated by means of IIEF scale and ADAM-AMS questionnaires. The replacement pharmacological treatment with testosterone improves the physical and sexual performance of the adult patient affected by symptomatic hypogonadism.

Leiomyoma of the seminal vesicles.

We report the case of a 37-year-old man suffering from dysuria, frequency, acute renal failure followed by bowel distress with rectal tenesmus. PSA was 6.19 ng/ml. CT of abdomen and pelvis revealed a probable prostatic-related pelvis mass. TRUS was not carried out due to intolerance. The patient underwent laparotomy through a lower midline incision that confirmed a firm, even if not involving the adjacent organs, considerable pelvic mass adhering to the left seminal vesicle and involving the right one. Therefore the right seminal vesicle and the mass originating from it were removed. Three months follow-up showed a clinically recovered patient, free of voiding and intestinal symptoms, back to normal working activity.

[Extracorporeal shock wave lithotripsy as surgical therapy in the kidney-ureter calculosis]. / Extracorporeal shock wave lithotripsy as surgical therapy in the kidney-ureter calculosis.

INTRODUCTION: The coming of the extra-corporeal shock waves lithotripsy (ESWL) represented one of the main progress in medicine of all times. MATERIALS AND METHODS: From January '84 up to March '99, 7.508 patients underwent extracorporeal shock wave lithotripsy (ESWL) for renal, ureteral and bladder stones with a total of 13.032 treatments. 6.329 kidney stones, 2.165 ureteral-calculosis and 52 bladder stones, for a total amount of 8.546 stones were treated. Seven different lithotripters have been used: radiologic-based Dornier HM3 with electrohydraulic shock waves production, radiologic-based modified-Dornier HM3 with electrohydraulic shock waves production, ultrasonography-based Dornier MPL 9000 with electrohydraulic shock waves production, ultrasonography-based EDAP LT 01 with piezoelectric shock waves production, ultrasonography and radiologic-based EDAP LT 02 with piezoelectric shock waves production, ultrasonography-based Piezolith 2200 and the Piezolith 2300 only with piezoelectric shock waves production. In 1,451 patients an auxiliary action was necessary. RESULTS: Only a treatment was sufficient in 5.337 patients (77.7%), two in 1.497, three in 507 patients, four in 248 patients, five in 109, six in 55, seven in 34, eight in 9, nine in 12, ten in 5, eleven in a patient, twelve in a patient, thirteen in a patient, fourteen in two patients and fifteen in a patient. The patients who had a negative abdomen radiograph or ultrasound after two months were considered "stone-free". We have considered urolithies smaller than 3 mm as fragments, with the possibility of natural expulsion, non conditioning lying behind. Totally the results was: 5.950 patients "stone-free", 753 patients with fragments, 257 patients with non-broken calculi and 547 patients with dust only. The "stainstrasse" with spontaneous resolution and the one with instrumental resolution, the hyperpyrexia, the serious colics, the symptomatic renal haematomas and the intolerance to the treatment (vomiting and nausea) have been considered as complications. CONCLUSIONS: Nowadays, except for the cases in which are necessary admission to hospital and urgency treatment, since imminent colics are present, the ESWL may be performed in day-hospital. Since the first years of employment the ESWL has solved almost all the cases of urolithiasis. Nevertheless the experience has proved the extreme aggressiveness and inappropriate characteristics of a number sometimes too high of re-treatments. The ESWL should maintain the feature of non-spread methodic when it is possible to use it in cases solving with two--maximum three--treatments.

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.

DNA copy number alterations and PPARG amplification in a patient with multifocal bladder urothelial carcinoma.

BACKGROUND: Bladder cancer is the seventh most common cancer worldwide and over 90% are transitional cell carcinoma (TCC). At the first time of diagnosis at least 70% of TCC present as superficial bladder cancer. Because the clinical outcome of superficial bladder tumors is relatively unpredictable, there is a pressing need to identify markers that may predict tumor recurrence and progression and new treatment strategies. CASE PRESENTATION: We present a unique case of a 67-year old male who underwent total cystectomy after repeated trans-urethral resections of the bladder for multifocal non-muscle invasive bladder cancer. The first and the third tumor were diagnosed as high grade non-infiltrating (HGNI), while the second as carcinoma in situ (CIS). We performed both array comparative genomic hybridization and a targeted chromosomal profile by UroVysion in order to detect copy number variations (CNVs) that may be involved with tumor recurrence and progression. The overall data from this study provide new evidence for the monoclonal origin of urothelial tumor multifocality as several genetic changes were found in different tumors of the same patient. From the analysis of shared CNVs two gained regions emerged at 3p25.2 and 12q23.2, including PPARG and ASCL1 genes, respectively. The copy number level of these genes would seem inversely mutually correlated and highly dependent on histological grade, because the highest level of amplification at 3p25.2 was evidenced in the two HGNI samples, while the highest level of copy number gain at 12q23.2 was reported in the CIS. CONCLUSION: We provide new evidence on the role of PPARG in initiation and maintenance of bladder cancer. For the first time we also suggest a possible explanation for the elevated expression of PPARG in this type of tumor through a focal high level amplification at 3p25.2. Furthermore, a new gene, ASCL1, emerged as a potential candidate to assist PPARG in bladder carcinogenesis.