GJB5 association with BRAF mutation and survival in cutaneous malignant melanoma.

BACKGROUND: Gap-junctional intercellular communication is crucial for epidermal cellular homeostasis. Inability to establish melanocyte-keratinocyte contact and loss of the intercellular junction's integrity may contribute to melanoma development. Connexins, laminins and desmocollins have been implicated in the control of melanoma growth, where their reduced expression has been reported in metastatic lesions. OBJECTIVES: The aim of this study was to investigate connexin 31·1 (GJB5) expression and identify any association with BRAF mutational status, prognosis of patients with melanoma and mitogen-activated protein kinase (MAPK) inhibitor (MAPKi) treatment. METHODS: GJB5 expression was measured at RNA and protein level in melanoma clinical samples and established cell lines treated (or not) with BRAF and MEK inhibitors (MEKi), as well as in cell lines which developed MAPKi resistance. Findings were further validated and confirmed by analysis of independent datasets. RESULTS: Our analysis reveals significant downregulation of GJB5 expression in metastatic melanoma lesions compared with primary ones and in BRAF-mutated vs. BRAF-wildtype (BRAFWT ) melanomas. Likewise, GJB5 expression is significantly lower in BRAFV600E compared with BRAFWT cell lines and increases on MAPKi treatment. MAPKi-resistant melanoma cells display a similar expression pattern compared with BRAFWT cells, with increased GJB5 expression associated with morphological changes. Enhancement of BRAFV600E expression in BRAFWT melanoma cells significantly upregulates miR-335-5p expression with consequent downregulation of GJB5, one of its targets. Furthermore, overexpression of miR-335-5p in two BRAFWT cell lines confirms specific GJB5 protein downregulation. Reverse transcriptase quantitative polymerase chain reaction analysis also revealed upregulation of miR-335 in BRAFV600E melanoma cells, which is significantly downregulated in cells resistant to MEKi. Our data were further validated using the TCGA_SKCM dataset, where BRAF mutations associate with increased miR-335 expression and inversely correlate with GJB5 expression. In clinical samples, GJB5 underexpression is also associated with patient overall worse survival, especially at early stages. CONCLUSIONS: We identified a significant association between metastases/BRAF mutation and low GJB5 expression in melanoma. Our results identify a novel mechanism of gap-junctional protein regulation, suggesting a prognostic role for GJB5 in cutaneous melanoma.

The new Oxford Clinico-Pathological Classification of IgA nephropathy.

The new Oxford Classification of the Pathology of IgA nephropathy originated from an international collaborative effort of the Working Group of the International IgA Nephropathy Network and Renal Pathology Society. It provides a new common language to categorizing glomerular and tubulo-interstitial lesions in IgA nephropathy having a proved effect on progression. Although retrospective in design and requiring future prospective validation, the Oxford collaboration defined and scored four pathologic parameters having an influence on outcome (independently of clinical information at renal biopsy or during follow-up); including mesangial (M) and endocapillary (E) proliferation (hypercellularity), glomerulosclerosis (S) and tubular atrophy and interstitial fibrosis (T). The scheme will likely become known as the OXFORD-MEST scoring system.

Oxidative stress in IgA nephropathy.

IgA nephropathy (IgAN) is characterized by mesangial deposits of IgA1, likely due to accumulation of IgA immune complexes. The activation of intracellular signaling mostly results in oxidative stress, as detected in mesangial cells cultured with aberrantly glycosylated IgA or IgA aggregates and in renal biopsies of patients with IgAN. Signs of altered oxidation/antioxidation balance have been detected in sera and/or in erythrocytes of patients with IgAN, including increased levels of lipoperoxide or malondialdehyde and reduced activity of superoxide dismutase, catalase and glutathione peroxidase. Moreover, increased levels of a marker of oxidative stress, advanced oxidation protein products (AOPPs), have been reported to be significantly associated with proteinuria and disease progression in patients with IgAN. AOPPs are often carried by albumin and can in turn enhance the oxidative stress in the circulation. Recent research suggests that the nephrotoxicity of aberrantly glycosylated IgA1 in IgAN is enhanced in the presence of systemic signs of oxidative stress, and it is tempting to hypothesize that the level of the oxidative milieu conditions the different expression and progression of IgAN.

Toll-like receptor 4 expression is increased in circulating mononuclear cells of patients with immunoglobulin A nephropathy.

We investigated Toll-like receptors (TLR-3, -4 and -7) expression in circulating mononuclear cells of patients with immunoglobulin A nephropathy (IgAN), a disease with debated relationships with mucosal immunity. TLR-4 expression (detected by fluorescence activated cell sorter) and mRNA transcriptional levels (Taqman) were significantly higher in patients with IgAN than in healthy controls (P = 0.00200 and P = 0.0200). TLR-3 and TLR-7 were not modified significantly. In IgAN patients proteinuria was correlated significantly with TLR-4 expression (P = 0.0312). In a group of nephrotic syndromes, TLR-3, -4 and -7 expression was similar to healthy controls. A significant difference in TLR-4 expression and mRNA levels was found between very active IgAN patients (proteinuria > 1 g/1.73 m(2)/day in association with severe microscopic haematuria) and inactive patients (proteinuria < 0.5 g/1.73 m(2)/day, with absent or minimal haematuria). No correlation with levels of aberrantly glycosylated IgA1, age, renal biopsy features or therapy was found. This study shows for the first time an up-regulation of TLR-4 in circulating mononuclear cells of patients with IgAN, particularly in association with proteinuria and heavy microscopic haematuria.

[Non-immunological therapy in nephropathies leading to chronic renal failure and in post-transplant chronic renal dysfunction]. / La terapia non immunologica delle nefropatie con insufficienza renale cronica e della disfunzione cronica da trapianto.

With its aging population, the Western world is experiencing a significant increase in the prevalence of chronic kidney disease, which has actually been defined as ''pandemic''. The high mortality and comorbidity, especially in terms of cardiovascular disease, have led to a significant increase in hospitalizations and rising public health expenditure, setting a trend that will become unsustainable in the next decades, even in the most developed countries. These epidemiological data have underlined the need for prevention campaigns and urged researchers and clinicians to develop new and more specific treatments able to slow down the progression of chronic kidney disease towards dialysis. To obtain such results, a deeper understanding of the pathogenetic mechanisms of nephropathy progression is mandatory. Once sclerosis is established and the initial pathogenetic noxa, whether or not involving the glomeruli, has been extinguished, the sclerotic progression of different nephropathies follows a standard pathway, irrespective of the initial cause. The achievement of an effective therapy for this condition, in native kidneys as well as transplanted organs, is the third-millennium challenge for nephrologists. In this review we will first describe the main risk factors and pathogenetic mechanisms involved in nephropathy progression and then discuss the results obtained with drugs that basic research has identified as potentially useful in experimental animal models as well as rigorous clinical trials.

[Audit on quality of life of patients with chronic kidney disease on dialysis and after transplant]. / Audit sulla qualità di vita nei pazienti in insufficianza renale cronica, dialisi e trapianto.

Since the improvement of quality of life (QOL) in patients with chronic disease on dialysis (D-CKD) has been demonstrated to have significant effects on clinical outcome, QOL has been included among the principal targets for these patients. QOL is among the objectives of the Healthy People 2010 program of the United Nations and CKD is one of the 18 focus areas of the program. The process of improving clinical outcome for D-CKD patients is obviously correlated with the continuing attempts to improve the adequacy of dialysis, and this remains a milestone. Some recent studies, including the HEMO study, have demonstrated that the present standards for dialytic efficiency are adequate for morbidity and mortality outcomes. However, these concepts are rapidly evolving and there are emerging factors which should be monitored, such as a) frequency of hospitalization, b) QOL, c) patient's satisfaction, and d) transplantation rate. Each aspect should be taken into consideration when the general well-being of D-CKD patients is at stake. However, there is a lack of validated standards, and there are confounding effects related to different geographical areas, different degrees of morbidity, and different therapies. Health is defined by the WHO as the complete feeling of well-being, and transplantation obviously is a way to overcome the many difficulties encountered by D-CKD patients. It is true that QOL after transplant is affected by uncertainty about the final result, fear of having to go back on dialysis, or anger about unexpected complications. For these reasons special questionnaires have been designed for transplanted patients (e.g., SF-36). These indicate an improvement of QOL for transplanted compared with D-CKD patients, although QOL remains below the level of that of the healthy population. Post-transplant QOL tends to improve over time and will become superimposable to that of the healthy population in the long run. It is of paramount importance that each dialysis and transplantation center provide psychological support not only to patients but also to doctors and nurses.

Children with chronic organ failure possibly ending in organ transplantation: a survey in an Italian region of 5,000,000 inhabitants.

AIM: The Italian Piedmont region sponsored in 2005 a population-based registry to assess the epidemiology of childhood chronic organ failure involving kidneys, liver, heart or lungs. METHODS: Patients in chronic organ failure who were younger than 18 years were selected, and entered the registry when accomplishing the standard failure criteria for each organ. The cases were reported by the general paediatricians of the region and integrated with the data gathered by the Children University Hospital, a tertiary care centre. RESULTS: In Piedmont (647,727 inhabitants < 18 years), a total of 146 children (217 cases per million of paediatric population) were found to be affected by chronic organ failure (mean age 10 years; range 0-17). The organ failure involved kidneys in 68 subjects (48%), liver in 24 (17%), heart in 21 (15%) and lungs in 28 (20%), and was severe in 32 subjects (6 on transplantation waiting list). The most represented disease leading to chronic renal failure was renal hypodysplasia (79%). Chronic liver failure was mostly caused by biliary atresia (30%), autoimmune hepatitis (25%) and Wilson's disease (21%). Dilated cardiomyopathy (62%) and surgically treated congenital cardiopathy were the two leading causes of chronic heart failure. The most represented disease leading to chronic lung failure was cystic fibrosis (89%). CONCLUSION: This is the first report of the literature focusing on the epidemiology of chronic organ failure in children encompassing a region of 4,000,000 inhabitants. This clinical condition is rare, but medically and socially very demanding not only in childhood but the life along, as most of these patients will need solid organ transplantation decades later.

Angiotensin antagonists and fish oil for treating IgA nephropathy.

In IgA nephropathy (IgAN), ACE inhibitors (ACE-I) and angiotensin receptor blockers (ARB) are beneficial against hypertension, and their anti-proteinuric effect has been clearly demonstrated. However, sub-analyses of IgAN patients enrolled in large studies failed to prove a benefit against progression to renal failure. The European Community Biomed Concerted Action - a placebo-controlled randomized controlled trial begun in 1995 - in children and adults (9-35 years old) with proteinuria > 1 < 3.5 g/day/1.73 m(2) and normal or moderately reduced renal function proved the significant benefit of ACE-I on progression of kidney disease. The combination of ACE-I and ARB in proteinuric normotensive IgAN patients showed greater antiproteinuric effect and the COOPERATE trial also reported a superior effect of combination therapy in protecting against renal function deterioration. Treating IgAN with fish oil has a good rationale for renal inflammation as well as for prevention of cardiovascular morbidity. However, the published reports gave conflicting conclusions and also very recent data did not show significant benefits. In conclusion, ACE-I and ARB have a definite role in treating IgAN, particularly the hypertensive and proteinuric forms. These patients should be treated to target BP to <130/70 mm Hg and proteinuria <0.5 g/day.

[Immunoglobulin A (IgA) and its cellular receptors: Recent advances and new pathogenetical hypothesis]. / L' immunoglobulina A (IgA) ed i suoi recettori cellulari nella nefropatia a depositi IgA: recenti conoscenze e nuove ipotesi patogenetiche.

Researchers have always been focusing their interest on IgA nephropathy, a nephropathy named after a class of immunoglobulin, and on circulating IgA in these patients. However, serum level studies conducted on IgA and IgA containing immune complexes have not fulfilled the expectations of providing useful tools for diagnosis and prognosis of this renal disease. Over recent years interest has grown on qualitative properties of IgA, particularly IgA1, the mostly represented subclass in renal deposits. The sugar composition of the short carbohydrate chains characteristic of the IgA1 hinge region has been thoroughly investigated, and a defective glycosylation of serum IgA1 in IgAN patients was detected with increase in desialylated and degalactosylated carbohydrate chains, and exposure of internal N-acetylgalactosamine residues. These aberrantly glycosylated IgA1 have the properties of self-aggregation, auto-antibody enhancement with IgA1/IgG immunocomplexes formation, and activation of complement and nuclear transcription factors such as NF-kB. Among others, aberrantly glycosylated IgA1 react with FC RI receptor on peripheral lymphomonocytes and, after shedding, the complex is released in form of macromolecular IgA. At mesangial level the nephrotoxicity of aberrantly glycosylated IgA1 seems to be mediated by the transferrin receptor (TfR) binding, selectively expressed on mesangial cells. This report aims at reviewing the most recent knowledge on IgA synthesis alterations and the subsequent biological effects on the pathogenesis of this nephritis so widespread in Europe.

[Kidney transplantation in children]. / Il trapianto di rene nel bambino.

Indications, procedures, complications, pharmacokinetics and outcomes of renal transplantation are different in children and in adults. Subjects <18 yrs old, are often included in a unique list as in Italy, benefiting from donors <15 yrs old, and the waiting time is reduced to <12 months in 71% of cases. The risk of thrombosis limits the use of donors <2 yrs and trans-plantation in infants <1 yr. The age at kidney transplantation is <5 yrs in 20-30% of children. In Italy living-related trans-plantation (LRT) is performed in 7% of cases, while in the USA it is more common (57%) and is often pre-emptive before entering dialysis (24%). Current therapy tends to reduce steroid treatment doses and, optimizing induction therapy with IL-2R inhibitors, using tacrolimus or mycophenolate or sirolimus. Transplanted patient survival is better in children than in adults (94-98% at 5 yrs). Infections, cardiovascular diseases and neoplasia induce 34, 15 and 12% of deaths, respectively, at 10 yrs; morbidity for infections and lymphoproliferative disease is increasing. Acute rejections declined from 70% in 1987 to 31% in 2002 in cadaveric transplantation (CT) and renal survival at 3 yrs increased from 50% in 1985 to 82% for CT and up to 92% in LRT. In adolescents (11-17 yrs old) renal survival is lower than in infants and in adults <65 yrs old. Renal losses are due to chronic transplant nephropathy (32%), vascular thrombosis (13%) and the recurrence of the original nephropathy (focal glomerulosclerosis up to 50%, membrano-proliferative glomerulonephritis up to 30%, and primary hyperoxaluria up to 90% if combined kidney-liver transplantation is not performed). Growth improves after transplantation particularly in children <5 yrs, while it is not completely satisfactory in adolescents. Overall, results indicate that kidney transplantation in children has very much improved and will offer in the near future even more favorable outcomes.

The role of nasal tolerance in a model of IgA nephropathy induced in mice by Sendai virus.

Mucosal antigenic exposure is implicated in pathogenesis of IgA nephropathy. Although IgG and/or IgM codeposits may promote disease, protracted mucosal antigenic exposure reduces IgG and IgM antibody, a process termed mucosal tolerance. We immunized mice intranasally with infectious or inactivated Sendai virus for 6 or 14 weeks. Anti-virus IgG remained high in mice given infectious virus for 14 weeks, but decreased after 6 weeks in mice given inactivated virus; IgA antibody remained high in both groups. Upon viral challenge, glomerular IgG and complement deposits and the frequency of hematuria, all equal after 6 weeks of immunization, were lower in mice immunized with inactivated virus for 14 weeks but remained high in mice given infectious virus; glomerular IgA increased over time in both immunized groups. Viremia in a non-tolerized immune host can promote glomerulonephritis with IgG and complement codeposits and glomerular dysfunction. These preliminary experiments may guide future, more mechanistic, investigation.

[Glomerulonephritis and renal sclerosis: new therapeutic proposals (review)]. / Glomerulonefriti e sclerosi renale: nuove proposte terapeutiche.

The treatment of immuno-mediated glomerulonephritides is presently based upon a limited series of drugs. Albeit evidence-based medicine relies solely upon controlled trials, there is a need to follow new perspectives with an open mind, since they may lead to tomorrow's therapy. Several original and innovative approaches to treat inflammatory glomerular diseases have been recently reported, including drugs designed to limit the effect of pro-inflammatory and pro-sclerotic cytokines (recombinant monoclonal antibodies, receptor antagonists, gene therapy providing viral transfection of genes, antisense oligonucleotides, aptamers, inhibition of transcription factors, active immunization). Moreover, newer options are being proposed, as in the case of enhancing natural anti-inflammatory cytokines or intracellular signalling limiting inflammation. Some of these proposals, which are briefly reviewed in this article, are likely to enter soon clinical investigation and to become in the next future standard treatment for glomerular diseases.

[Biocompatibility of the acetate in the dialysis fluid]. / Biocompatibilità dell'acetato contenuto nel bagno di dialisi.

PURPOSE: Dialytic vasculopathy is a major morbidity and mortality risk factor in patients undergoing chronic dialysis treatment. Among the pathogenetic factors some are related to the uremic condition, others are due to biocompatible reactions to dialytic materials. Endothelial cells (EC) are the target of the mediators released during bioincompatible reactions, and the related effects could be considered the initial event eliciting the vasculopathy pathogenesis. Among the others, we focused our attention on the role played in this process by the inducible isoform of nitric oxide (NO) synthase (iNOS). In previous studies we demonstrated that bioincompatible membranes, as well as acetate-containing dialysis buffers stimulate iNOS gene expression and activity in endothelial cells in culture. In this study, we planned to evaluate the potential role of a new dialysis buffer in which acetate has been substituted with HCl as a stabilizer. METHODS: ECs were incubated for 12 h at 37 degrees C with different dialysis buffers: acetate (Acet), standard bicarbonate (Bic), acetate-free buffer (AF) and HCl-bicarbonate (BicHCl). We evaluated in reverse transcriptase polymerase chain reaction (RT-PCR) the gene transcription for iNOS, the NOS activity (as the production of H3 citrulline from H3 arginine by ionic exchange chromatography), EC proliferative (H3 thymidine incorporation) and pro-apoptotic rate (TUNEL analysis) and the nuclear translocation of the transcriptional factor NF-kappaB (EMSA). RESULTS: Acetate, even in the low concentration present in Bic was able to induce a significant iNOS gene transcription (results expressed as relative units and referred to basal values: Acet 1.9 +/- 0.01 fold increase, p<0.01; Bic 1.45 +/- 0.03 p<0.05; BicHCl 1.24 +/- 0.01; AF 1.17 +/- 0.02) and translation. Acetate at concentrations both of 3 mmoL and 38 mmoL (present in the bicarbonate buffer) significantly increased the enzymatic NOS activity vs unconditioned ECs: Acet 3.46 +/- 0.3, p<0.0005; Bic 1.69 +/- 0.2, p<0.005; BicHCl 1.24 +/- 0.15; AF 1.17 +/- 0.05. The EC proliferative index was significantly depressed by acetate containing dialysis buffers (unconditioned ECs 100%, Acet 38 +/- 15%, p<0.01; Bic 65 +/- 6%, p<0.05; AF 87 +/- 8%; BicHCl 75 +/- 6%). The percentage of apoptotic ECs was significantly increased by buffers contain-ing Acet vs BicHCl and AF. Finally, acetate at the concentrations present in Acet and Bic activated and promoted the nuclear translocation of the transcriptional factor NF-kappaB in ECs (p<0.01 vs unconditioned cells). CONCLUSIONS: The acetate-free dialysis buffers have better biocompatibility and potentially down-modulate the flogistic and sclerotic processes responsible for dialytic vasculopathy.

[A severe case of nephrotic syndrome]. / Un caso difficile di sindrome nefrosica.

A 14-year-old girl presented at the Renal Unit with clinical and laboratory pictures of a severe nephrotic syndrome. It was done a kidney biopsy which detected a picture of "minimal change". As the patient had no benefit from the prednisone therapy, she underwent to another biopsy which detected, this time, a Focal Glomerulosclerosis. Despite the immunosuppressive therapy, the renal function deteriorated and, therefore, the patient began dialysis. After a while, the girl received a cadaveric kidney transplantation but the nephrotic syndrome recurred again and the patient went back to chronic dialysis.

[Neonatal chronic kidney failure associated with cyclo-oxygenase-2 inhibitors administered during pregnancy]. / Insufficienza renale cronica neonatale associata ad assunzione di un inibitore della ciclo-ossigenasi-2 durante le gravidanza.

Non-steroidal anti-inflammatory drugs (NSAID) are used since years as tocolytic due to their capacity to inhibit cyclo-oxygenase (COX) expressed in uterus and fetal membranes, fundamental for labour initiation and maintenance. The use of nimesulide, a COX-2 selective NSAID, has been recently proposed due to its capacity to selectively inhibit the enzyme expressed in the myometrium and endometrium. A case of neonatal irreversible end stage renal failure after maternal assumption of nimesulide as tocolytic for 6 week is reported. Cesarean section at the 32nd week due to oligohydramnios gave birth to a baby girl of 2090 g, in good general conditions, without signs of respiratory distress and of visible abnormalities. From birth she displayed oligo-anuria which required dialytic substitutive therapy from the second day of life. At US scan both kidneys had normal diameters for gestational age slightly increased echogenicity and a reduced cortico-medullary differentiation. On the 20th day of life she had a surgical renal biopsy for the persistence of oligo-anuria, showing fetal glomeruli, without lymphocytic interstitial infiltrate, and normal tubuli without evidence of necrosis. She is now 16 months old and under automated peritoneal dialysis on a home dialysis program. The occurrence of chronic renal failure in strict relationship with maternal nimesulide assumption in this case is strongly suggestive for a pharmacological damage, either direct or mediated by renin angiotensin inhibition, and possibly modulated by genetic factors, likely to account for the different outcome of similarly treated patients. A cautious use of this drug as long term tocolytic should be recommended while waiting for ad hoc experimental and clinical evidences of safeness.