ILT3(high)ILT4(high) dendritic cells (DCs) may cause anergy in CD4(+)CD45RO(+)CD25(+) T cells transforming them into regulatory T cells (Tregs). Here, we tested whether chronic exposure to rapamycin may modulate this immunoregulatory pathway in renal transplant recipients. Forty renal transplant patients with biopsy-proven chronic allograft nephropathy and receiving calcineurin inhibitors were randomly assigned to either calcineurin inhibitor dose reduction or withdrawal with rapamycin introduction. At conversion and 2 years thereafter, we measured the rapamycin effects on circulating DCs (BDCA1/BDCA2 and ILT3/ILT4 expression), CD4(+)/CD25(high)/Foxp3(+) Tregs, CD8(+)/CD28(-) T cells, and the Th1/Th2 balance in graft biopsies. In rapamycin-treated patients, peripheral BDCA2(+) cells were significantly increased along with ILT3/ILT4(+) DCs. The number of circulating CD4(+)/CD25(high)/Foxp3(+)/CTLA4(+) Tregs, CD8(+)CD28(-) T cells, and HLA-G serum levels were higher in the rapamycin-treated group. The number of ILT3/ILT4(+)BDCA2(+) DC was directly and significantly correlated with circulating Tregs and CD8(+)CD28(-) T cells. ILT3/ILT4 expression was increased in kidney biopsies at the end of the study period along with a significant bias toward a Th2 response within the graft only in the rapamycin-treated patients. Thus, rapamycin induces the upregulation of ILT3 and ILT4 on the DC surface, and this effect is associated with an increase in the number of Tregs and expansion of the CD8(+)CD28(-) T cell population. This suggests that mTOR inhibition may promote a novel immunoregulatory pathway.

In some tumours, despite a wild-type p53 gene, the p53 pathway is inactivated by alterations in its regulators or by unknown mechanisms, leading to resistance to cytotoxic therapies. Understanding the mechanisms of functional inactivation of wild-type p53 in these tumours may help to define prospective targets for treating cancer by restoring p53 activity. Recently, we identified TRIM8 as a new p53 modulator, which stabilizes p53 impairing its association with MDM2 and inducing the reduction of cell proliferation. In this paper we demonstrated that TRIM8 deficit dramatically impairs p53-mediated cellular responses to chemotherapeutic drugs and that TRIM8 is down regulated in patients affected by clear cell Renal Cell Carcinoma (ccRCC), an aggressive drug-resistant cancer showing wild-type p53. These results suggest that down regulation of TRIM8 might be an alternative way to suppress p53 activity in RCC. Interestingly, we show that TRIM8 expression recovery in RCC cell lines renders these cells sensitive to chemotherapeutic treatments following p53 pathway re-activation. These findings provide the first mechanistic link between TRIM8 and the drug resistance of ccRCC and suggest more generally that TRIM8 could be used as enhancer of the chemotherapy efficacy in cancers where p53 is wild-type and its pathway is defective.

Renal cell carcinoma (RCC) accounts for approximately 3% of adult malignancies, and clear cell RCC (ccRCC), that has a high metastatic index and high relapse rate, is the most common histological subtype. The identification of new biomarkers in ccRCC is fundamental for stratifying patients into prognostic risk groups and to guide therapy. The renoprotective antiaging gene, αKlotho, has recently been found to work as a tumor suppressor in different human cancers. Here, we evaluated αKlotho expression in tissue and serum of ccRCC patients and correlated it with disease progression. Tissue αKlotho expression was studied by quantitative RT-PCR and immunohistochemistry. In addition, soluble serum αKlotho levels were preoperatively measured in 160 patients who underwent nephrectomy for RCC with ELISA. Estimates of cancer-specific (CSS) and progression-free survival (PFS) were calculated according to the Kaplan-Meier method. Multivariate analysis was performed to identify the most significant variables for predicting CSS and PFS. αKlotho protein levels were significantly decreased in RCC tissues compared with normal tissues (P < 0.01) and the more advanced the disease, the more evident the down-regulation. This trend was also observed in serum samples. Statistically significant differences resulted between serum αKlotho levels and tumor size (P = 0.003), Fuhrman grade (P = 0.007), and clinical stage (P = 0.0004). CSS and PFS were significantly shorter in patients with lower levels of αKlotho (P < 0.0001 and P = 0.0004, respectively). At multivariate analysis low serum levels of αKlotho were independent adverse prognostic factors for CSS (HR = 2.11; P = 0.03) and PFS (HR = 2.18; P = 0.03).These results indicate that a decreased αKlotho expression is correlated with RCC progression, and suggest a key role of declining αKlotho in the onset of cancer metastasis.

Protein phosphorylation is considered a key event in signal transduction. Peripheral blood mononuclear cells (PBMCs) are a critical component of the immune system. The analysis of PBMCs phosphoproteome might help elucidate the signaling pathways essential to their biological role in health, immunological diseases and cancer. Enrichment of phosphoproteins becomes a prerequisite for phosphoproteome analysis and conventionally requires a multi-step procedure and sophisticated equipments. In this study, we standardized 2D-PAGE phosphoproteome analysis of PBMCs and compared two phosphoprotein enrichment methods, lanthanum chloride precipitation and affinity micro-column. Further, the different specificity for PBMCs phosphorylated proteins of each method was investigated.

Glucose-6-phosphate isomerase (GPI), also known as phosphoglucose isomerase, was initially identified as the second glycolytic enzyme that catalyzes the interconversion of glucose-6-phosphate to fructose-6-phosphate. Later studies demonstrated that GPI was the same as the autocrine motility factor (AMF), and that it mediates its biological effects through the interaction with its surface receptor (AMFR/gp78). In this study, we assessed the role of GPI/AMF as a prognostic factor for clear cell renal cell carcinoma (ccRCC) cancer-specific (CSS) and progression-free survival (PFS). In addition, we evaluated the expression and localization of GPI/AMF and AMFR, using tissue microarray-based immunohistochemistry (TMA-IHC), indirect immunofluorescence (IF), and confocal microscopy analysis.Primary renal tumor and nonneoplastic tissues were collected from 180 patients who underwent nephrectomy for ccRCC. TMA-IHC and IF staining showed an increased signal for both GPI and AMFR in cancer cells, and their colocalization on plasma membrane. Kaplan-Meier curves showed significant differences in CSS and PFS among groups of patients with high versus low GPI expression. In particular, patients with high tissue levels of GPI had a 5-year survival rate of 58.8%, as compared to 92.1% for subjects with low levels (P < 0.0001). Similar findings were observed for PFS (56.8% vs 93.3% at 5 years). At multivariate analysis, GPI was an independent adverse prognostic factor for CSS (HR = 1.26; P = 0.001), and PFS (HR = 1.16; P = 0.01).In conclusion, our data suggest that GPI could serve as a marker of ccRCC aggressiveness and a prognostic factor for CSS and PFS.

Primary membranous nephropathy (PMN) is a renal-specific autoimmune disease caused by circulating autoantibodies that target glomerular podocyte antigens (PLA2R/THSD7A). However, very little is known on the molecular mechanisms controlling B cell response in this nephropathy. The present study was aimed at correlating the serum levels of B cell activators BAFF/BLyS and APRIL with the presence of anti-PLA2R antibodies in PMN patients and with long-term clinical outcome. To this aim, 51 patients with anti-PLA2R-positive biopsy-proven PMN and nephrotic range proteinuria (>3.5 g/24 hours) were enrolled between January 2009 and December 2015 and treated with conventional 6-month immunosuppressive therapy. After 6 months, 29 patients (56.9%) cleared circulating anti-PLA2R, while in remaining 22 (43.1%), they persisted. Intriguingly, in the first group, baseline serum levels of BAFF/BLyS and APRIL were significantly lower than those in the second one. Moreover, after 6 months of immunosuppressive therapy, an overall reduction in both cytokine serum levels was observed. However, in PMN patients with anti-PLA2R clearance, this reduction was more prominent, as compared with those with anti-PLA2R persistence. When related to clinical outcome, lower baseline BAFF/BLyS (<6.05 ng/mL) and APRIL (<4.20 ng/mL) serum levels were associated with significantly higher probability to achieve complete or partial remission after 24-month follow-up. After dividing the entire study cohort into three groups depending on both cytokine baseline serum levels, patients with both BAFF/BLyS and APRIL below the cut-off showed a significantly higher rate of complete or partial remission as compared with patients with only one cytokine above the cut-off, while the composite endpoint was achieved in a very low rate of patients with both cytokines above the cut-off. Taken together, these results provide new insights into the role of BAFF/BLyS and APRIL in both the pathogenesis of anti-PLA2R-positive PMN and the response to immunosuppressive therapy.

Kidney transplant recipients (KTRs) have been considered as patients at higher risk of SARS-CoV-2-related disease severity, thus COVID-19 vaccination was highly recommended. However, possible interferences of different immunosuppression with development of both humoral and T cell-mediated immune response to COVID-19 vaccination have not been determined. Here we evaluated the association between mTOR-inhibitors (mTOR-I) and immune response to mRNA BNT162b2 (Pfizer-BioNTech) vaccine in KTR. To this aim 132 consecutive KTR vaccinated against COVID-19 in the early 2021 were enrolled, and humoral and T cell-mediated immune response were assessed after 4-5 weeks. Patients treated with mTOR-I showed significantly higher anti-SARS-CoV-2 IgG titer (p = .003) and higher percentages of anti-SARS-CoV-2 S1/RBD Ig (p = .024), than those without. Moreover, SARS-CoV-2-specific T cell-derived IFNγ release was significantly increased in patients treated with mTOR-I (p < .001), than in those without. Multivariate analysis confirmed that therapy with mTOR-I gained better humoral (p = .005) and T cell-mediated immune response (p = .005) in KTR. The presence of mTOR-I is associated with a better immune response to COVID-19 vaccine in KTR compared to therapy without mTOR-I, not only by increasing vaccine-induced antibodies but also by stimulating anti-SARS-CoV-2 T cell response. These finding are consistent with a potential beneficial role of mTOR-I as modulators of immune response to COVID-19 vaccine in KTR.

Clear cell renal cell carcinoma (ccRCC) is the most common malignant renal epithelial tumor and also the most deadly. To identify molecular changes occurring in ccRCC, in the present study we performed a genome wide analysis of its entire complement of mRNAs. Gene and exon-level analyses were carried out by means of the Affymetrix Exon Array platform. To achieve a reliable detection of differentially expressed cassette exons we implemented a novel methodology that considered contiguous combinations of exon triplets and candidate differentially expressed cassette exons were identified when the expression level was significantly different only in the central exon of the triplet. More detailed analyses were performed for selected genes using quantitative RT-PCR and confocal laser scanning microscopy. Our analysis detected over 2,000 differentially expressed genes, and about 250 genes alternatively spliced and showed differential inclusion of specific cassette exons comparing tumor and non-tumoral tissues. We demonstrated the presence in ccRCC of an altered expression of the PTP4A3, LAMA4, KCNJ1 and TCF21 genes (at both transcript and protein level). Furthermore, we confirmed, at the mRNA level, the involvement of CAV2 and SFRP genes that have previously been identified. At exon level, among potential candidates we validated a differentially included cassette exon in DAB2 gene with a significant increase of DAB2 p96 splice variant as compared to the p67 isoform. Based on the results obtained, and their robustness according to both statistical analysis and literature surveys, we believe that a combination of gene/isoform expression signature may remarkably contribute, after suitable validation, to a more effective and reliable definition of molecular biomarkers for ccRCC early diagnosis, prognosis and prediction of therapeutic response.

Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial, ear, and renal anomalies. The most common gene mutated in BOR patients is EYA1, the human homolog of the Drosophila eyes absent gene, while mutations in SIX1 gene, the human homolog of sine oculis, encoding a DNA binding protein interacting with EYA1, have been reported less frequently. Recently, mutations in another SIX family member, SIX5, have been described in BOR patients, however, this association has not been confirmed by other groups.

Fabry disease is an under-recognized X-linked lysosomal disorder, due to alpha-galactosidase A deficiency. Most of the mutations in the GLA gene are detectable using genomic sequencing analysis. However, deletions of one or more exons or deletion encompassing the entire gene are undetectable, especially in heterozygous females. The Multiplex Ligation-dependent Probe Amplification (MLPA) is an efficient tool for discovering these rearrangements. In this study two novel different deletions were detected using MLPA assay on two Fabry patients, both resulted mutation negative by sequencing analysis. These data suggest that this screening should be systematically included in genetic testing surveys of patients with Fabry disease.

CD40 crosslinking plays an important role in regulating cell migration, adhesion and proliferation in renal cell carcinoma (RCC). CD40/CD40L interaction on RCC cells activates different intracellular pathways but the molecular mechanisms leading to cell scattering are not yet clearly defined. Aim of our study was to investigate the main intracellular pathways activated by CD40 ligation and their specific involvement in RCC cell migration. CD40 ligation increased the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH (2)-terminal kinase (JNK) and p38 MAPK. Furthermore, CD40 crosslinking activated different transcriptional factors on RCC cell lines: AP-1, NFkB and some members of the Nuclear Factor of Activated T cells (NFAT) family. Interestingly, the specific inhibition of NFAT factors by cyclosporine A, completely blocked RCC cell motility induced by CD40 ligation. In tumor tissue, we observed a higher expression of NFAT factors and in particular an increased activation and nuclear migration of NFATc4 on RCC tumor tissues belonging to patients that developed metastases when compared to those who did not. Moreover, CD40-CD40L interaction induced a cytoskeleton reorganization and increased the expression of integrin β1 on RCC cell lines, and this effect was reversed by cyclosporine A and NFAT inhibition. These data suggest that CD40 ligation induces the activation of different intracellular signaling pathways, in particular the NFATs factors, that could represent a potential therapeutic target in the setting of patients with metastatic RCC.

Chronic antibody-mediated rejection (CAMR) is the major cause of kidney transplant failure. The molecular mechanisms underlying this event are still poorly defined and this lack of knowledge deeply influences the potential therapeutic strategies. The aim of our study was to analyze the phosphoproteome of peripheral blood mononuclear cells (PBMCs), to identify cellular signaling networks differentially activated in CAMR. Phosphoproteins isolated from PBMCs of biopsy proven CAMR, kidney transplant recipients with normal graft function and histology and healthy immunocompetent individuals, have been investigated by proteomic analysis. Phosphoproteomic results were confirmed by Western blot and PBMCs' confocal microscopy analyses. Overall, 38 PBMCs samples were analyzed. A differential analysis of PBMCs' phosphoproteomes revealed an increase of lactotransferrin, actin-related protein 2 (ARPC2) and calgranulin-B in antibody-mediated rejection patients, compared to controls. Increased expression of phosphorylated ARPC2 and its correlation to F-actin filaments were confirmed in CAMR patients. Our results are the first evidence of altered cytoskeleton organization in circulating immune cells of CAMR patients. The increased expression of phosphorylated ARPC2 found in the PBMCs of our patients, and its association with derangement of F-actin filaments, might suggest that proteins regulating actin dynamics in immune cells could be involved in the mechanism of CAMR of kidney grafts.

Background: The correlation between the severity of hemolytic uremic syndrome related to Shiga toxin-producing Escherichia coli (STEC-HUS) and involvement of the complement system has been examined in a small number of studies, with conflicting results. In the present study, we investigated whether serum C3 levels on admission are associated with neurologic involvement. Methods: To this purpose, 68 consecutive STEC-HUS patients were recruited and main clinical and laboratory variables ad hospital admission were compared between those with or without neurologic involvement. Results: STEC-HUS patients who developed neurologic involvement (NI) showed significant higher leukocyte count, C-reactive protein and hemoglobin, and lower sodium levels as compared with those without. Interestingly, baseline serum levels of C3 were significantly lower in patients with NI as compared with those without (p < 0.001). Moreover, when stratified according to need of Eculizumab rescue therapy due to severe NI, patients treated with this drug showed baseline C3 serum levels significantly lower than those who were not (p < 0.001). Low C3 was independent risk factor for NI in our patients' population when entered as covariate in a multivariate logistic regression analysis including other major variables previously proposed as possible predictors of poor prognosis in STEC-HUS (for instance, leukocyte count, c-reactive protein, sodium levels) (HR 6.401, 95%CI 1.617-25.334, p = 0.008 for C3). To underline the role of complement in the worsening of STEC-HUS patients' clinical conditions and outcomes, all patients were divided into two groups according to the baseline lower vs. normal serum levels of C3 and the main data on care needs were assessed. Interestingly more patients with lower C3 serum levels required renal replacement therapy (p = 0.024), anti-hypertensive therapy (p = 0.011), Intensive Care Unit admission (p = 0.009), and longer hospitalization (p = 0.003), thus displaying significantly more severe disease features as compared with those with normal C3 serum levels. Conclusions: Our data suggests that children with STEC-HUS with decreased C3 concentrations at admission are more likely to develop neurologic involvement and are at increased risk of having severe clinical complications.

TRIM8 plays a key role in controlling the p53 molecular switch that sustains the transcriptional activation of cell cycle arrest genes and response to chemotherapeutic drugs. The mechanisms that regulate TRIM8, especially in cancers like clear cell Renal Cell Carcinoma (ccRCC) and colorectal cancer (CRC) where it is low expressed, are still unknown. However, recent studies suggest the potential involvement of some microRNAs belonging to miR-17-92 and its paralogous clusters, which could include TRIM8 in a more complex pathway.

Renal cell carcinoma (RCC) is the most common kidney cancer, and accounts for ~3% of all adult malignancies. RCC has proven refractory to conventional treatment modalities but appears to be the only histological form that shows any consistent response to immunotherapeutic approaches. The development of a clinically effective vaccine remains a major strategic target for devising active specific immunotherapy in RCC. We aimed to identify a highly immunogenic antigenic format for immunotherapeutic approaches, so as to boost immune responses in RCC patients. We established and cloned an immunogenic cell line, RCC85#21 named Elthem, which was derived from a non-aggressive and non-metastatic clear cell carcinoma. The cell line characterization was performed by genomics (real-time PCR, genome instability), proteomics (two dimensional electrophoresis, mass spectro-metry) and immunological analysis (mixed lymphocytes tumor cell cultures). Real-time PCR confirmed the RCC85#21 cell expression of tumor antigens and cytokine genes. No difference in microsatellite instability (MSI) in RCC85#21 cell line was found as compared to control, loss of heterozygosity was observed in the RCC85#21 clone, but not in the renal cancer cell lines from which it was generated. The image analysis of RCC85#21 by two-dimensional gels showed 700±26 spots and 119 spots were identified by mass spectrometry analysis. RCC85#21 promoted a significant RCC-specific T cells activation by exhibiting a cytotoxic phenotype after mixed lymphocyte and tumor cell cultures. CD8+ T cells isolated from RCC patients displayed an elevated reactivity against RCC85#21 and efficiently lysed the RCC85#21 clone. The RCC85#21 immunogenic cell line will be suitable for immune stimulation. The identification of novel tumor associated antigens will allow the evaluation of the immune response in vitro and, subsequently, in vivo paving the way for new immunotherapeutic strategies in the RCC setting.

Malignancies are one of the main causes of mortality in diabetic patients; however, to date, very limited data have been reported on the specific influence of type 2 diabetes mellitus (T2DM) on the survival of patients with renal cell carcinoma (RCC). In the present long-term retrospective study, we investigated whether T2DM may influence the overall survival (OS), cancer-specific survival (CSS), and progression-free survival (PFS) in patients with surgically treated RCC. Medical records of 924 patients treated by radical or partial nephrectomy for sporadic, unilateral RCC were reviewed. Patients with type-1 DM and with T2 DM receiving insulin treatment were excluded. Survival estimates were calculated according to the Kaplan-Meier method and compared with the log-rank test. Univariate and multivariate analyses were performed using the Cox regression model.Of the 924 RCC patients, 152 (16.5%) had T2DM. Mean follow-up was 68.5 months. Mean OS was 41.3 and 96.3 months in T2DM and non-T2DM patients, respectively (P < 0.0001).The estimated CSS rates at 1, 3, and 5 years in T2DM versus non-T2DM patients were 63.4% versus 76.7%, 30.4% versus 56.6%, and 16.3% versus 48.6%, respectively (P = 0.001). Mean PFS was significantly lower (31.5 vs 96.3 months; P < 0.0001) in the T2DM group. At multivariate analysis, T2DM was an independent adverse prognostic factor for OS (hazard ratio [HR]  = 3.44; 95% confidence interval [CI]:2.40-4.92), CSS (HR = 6.39; 95% CI: 3.78-10.79), and PFS (HR = 4.71; 95% CI: 3.11-7.15). In conclusion, our findings suggest that patients with RCC and pre-existing T2DM have a shorter OS, increased risk of recurrence, and higher risk for kidney cancer mortality than those without diabetes.

Pentraxin-3 (PTX3) belongs to the pentraxine family, innate immune regulators involved in angiogenesis, proliferation and immune escape in cancer. Here, we evaluated PTX3 tissue expression and serum levels as biomarkers of clear cell renal cell carcinoma (ccRCC) and analyzed the possible role of complement system activation on tumor site. A 10-year retrospective cohort study including patients undergoing nephrectomy for ccRCC was also performed. PTX3 expression was elevated in both neoplastic renal cell lines and tissues, while it was absent in both normal renal proximal tubular cells (HK2) and normal renal tissues. Analysis of complement system activation on tumor tissues showed the co-expression of PTX3 with C1q, C3aR, C5R1 and CD59, but not with C5b-9 terminal complex. RCC patients showed higher serum PTX3 levels as compared to non-neoplastic patients (p<0.0001). Higher PTX3 serum levels were observed in patients with higher Fuhrman grade (p<0.01), lymph node (p<0.0001), and visceral metastases (p<0.001). Patients with higher PTX3 levels also showed significantly lower survival rates (p=0.002). Our results suggest that expression of PTX3 can affect the immunoflogosis in the ccRCC microenvironment, by activating the classical pathway of CS (C1q) and releasing pro-angiogenic factors (C3a, C5a). The up-regulation of CD59 also inhibits the complement-mediated cellular lysis.

Pentraxins are a family of evolutionarily conserved pattern recognition molecules with pivotal roles in innate immunity and inflammation, such as opsonization of pathogens during bacterial and viral infections. In particular, the long Pentraxin 3 (PTX3) has been shown to regulate several aspects of vascular and tissue inflammation during solid organ transplantation. Our study investigated the role of PTX3 as possible modulator of Complement activation in a swine model of renal ischemia/reperfusion (I/R) injury. We demonstrated that I/R injury induced early PTX3 deposits at peritubular and glomerular capillary levels. Confocal laser scanning microscopy revealed PTX3 deposits co-localizing with CD31+ endothelial cells. In addition, PTX3 was associated with infiltrating macrophages (CD163), dendritic cells (SWC3a) and myofibroblasts (FSP1). In particular, we demonstrated a significant PTX3-mediated activation of classical (C1q-mediated) and lectin (MBL-mediated) pathways of Complement. Interestingly, PTX3 deposits co-localized with activation of the terminal Complement complex (C5b-9) on endothelial cells, indicating that PTX3-mediated Complement activation occurred mainly at the renal vascular level. In conclusion, these data indicate that PTX3 might be a potential therapeutic target to prevent Complement-induced I/R injury.

Mammalian microRNAs (miR) regulate the expression of genes relevant for the development of adaptive and innate immunity against cancer. Since T cell dysfunction has previously been reported in patients with renal cell carcinoma (RCC; clear cell type), we aimed to analyze these immune cells for genetic and protein differences when compared to normal donor T cells freshly after isolation and 35 days after in vitro stimulation (IVS) with HLA-matched RCC tumor cells.

To investigate the molecular mechanisms underlying altered T cell response in renal cell carcinoma (RCC) patients, we compared autologous and allogeneic CD8(+) T cell responses against RCC line from RCC patients and their HLA-matched donors, using mixed lymphocyte/tumor cell cultures (MLTCs). In addition, we analyzed the expression of molecules associated with cell cycle regulation. Autologous MLTC responder CD8(+) T cells showed cytotoxic activity against RCC cell lines; however the analysis of the distribution of CD8(+) T-cell subsets revealed that allogenic counterparts mediate superior antitumor efficacy. In RCC patients, a decreased proliferative response to tumor, associated with defects in JAK3/STAT5/6 expression that led to increased p27KIP1 expression and alterations in the cell cycle, was observed. These data define a molecular pathway involved in cell cycle regulation that is associated with the dysfunction of tumor-specific CD8(+) effector cells. If validated, this may define a therapeutic target in the setting of patients with RCC.