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Extracellular vesicles (EV) are emerging mediators in several diseases. However, their role in the pathophysiology of antibody-mediated allograft rejection (AMR) has been poorly investigated. Here, we investigated the role of EV isolated from AMR patients in inducing tubular senescence and endothelial to mesenchymal transition (EndMT) and analyzed their miRNA expression profile. By multiplex bead flow cytometry, we characterized the immunophenotype of plasma AMR-derived EV and found a prevalent platelet and endothelial cell origin. In vitro, AMR-derived EV induced tubular senescence by upregulating SA-β Gal and CDKN1A mRNA. Furthermore, AMR-derived EV induced EndMT. The occurrence of tubular senescence and EndMT was confirmed by analysis of renal biopsies from the same AMR patients. Moreover, AMR-derived EV induced C3 gene upregulation and CFH downregulation in tubular epithelial cells, with C4d deposition on endothelial cells. Interestingly, RNase-mediated digestion of EV cargo completely abrogated tubular senescence and EndMT. By microarray analysis, miR-604, miR-515-3p, miR-let-7d-5p, and miR-590-3p were significantly upregulated in EV from AMR group compared with transplant controls, whereas miR-24-3p and miR-29a-3p were downregulated. Therefore, EV-associated miRNA could act as active player in AMR pathogenesis, unraveling potential mechanisms of accelerated graft senescence, complement activation and early fibrosis that might lead to new therapeutic intervention.

Cisplatin is one of the most effective chemotherapeutic agents strongly associated with nephrotoxicity. Tubular adult renal progenitor cells (tARPC) can regenerate functional tubules and participate in the repair processes after cisplatin exposition. This study investigated the molecular mechanisms underlying the protective effect of tARPC on renal epithelium during cisplatin nephrotoxicity. By performing a whole-genome transcriptomic analysis, we found that tARPC, in presence of cisplatin, can strongly influence the gene expression of renal proximal tubular cell [RPTEC] by inducing overexpression of CYP1B1, a member of the cytochrome P450 superfamily capable of metabolizing cisplatin and of hypoxia/cancer-related lncRNAs as MIR210HG and LINC00511. Particularly, tARPC exerted renoprotection and regeneration effects via extracellular vesicles (EV) enriched with CYP1B1 and miR-27b-3p, a well-known CYP1B1 regulatory miRNA. The expression of CYP1B1 by tARPC was confirmed by analyzing biopsies of cisplatin-treated renal carcinoma patients that showed the colocalization of CYP1B1 with the tARPC marker CD133. CYP1B1 was also overexpressed in urinary EV purified from oncologic patients that presented nephrotoxicity episodes after cisplatin treatment. Interestingly CYP1B1 expression significantly correlated with creatinine and eGFR levels. Taken together, our results show that tARPC are able to counteract cisplatin-induced nephrotoxicity via CYP1B1 release through EV. These findings provide a promising therapeutic strategy for nephrotoxicity risk assessment that could be related to abundance of renal progenitors.

Chronic kidney disease (CKD) is actually considered a public health priority according to the increasing number of patients affected by this condition: this casuistry is not only related to specific glomerular, tubular or autoimmune diseases or a consequence of acute kidney injury (AKI) episodes leading to organ failure, but it is tied to the progression of life expectancy and the impact of comorbidities such as cardiovascular disease, diabetes and cancer. CKD and its comorbidities promote low grade inflammatory status with an impact on patients’ clinical conditions, reducing their possibility for kidney transplantation or graft survival and their survival when receiving renal replacement therapies such as hemodialysis (HD) or peritoneal dialysis (PD). CKD is often referred to as an ageing accelerator: innate immune system dysregulation, in the uraemic proinflammatory milieu, is involved in this accelerated senescence phenomena and pentraxins, particularly Pentraxin-3 (PTX-3), are of particular interest in the development of kidney disease. A complete understanding of the mechanism of CKD progression, innate immune system involvement and a proper definition of PTX-3 role in kidney disease, could redefine the approach for diagnosis and a more centered patients’ management to slow down CKD progression over time and reduce its clinical and social impact.

Hepatitis C virus (HCV) adopts several immune evasion mechanisms such as interfering with innate immunity or promoting T-cell exhaustion. However, the recent direct-antiviral agents (DAAs) rapidly eliminate the virus, and the repercussions in terms of immune system balance are unknown. Here we compared the PBMCs transcriptomic profile of patients with HCV chronic infection at baseline (T0) and 12 weeks after the end of the therapy (SVR12) with DAAs. 3862 genes were differently modulated, identifying oxidative phosphorylation as the top canonical pathway differentially activated. Therefore, we dissected PBMCs bioenergetic profile by analyzing mitochondrial respiration and glycolysis at 4 timepoints: T0, 4 weeks of therapy, end of therapy (EoT), and SVR12. Maximal and reserve respiratory capacity considerably increased at EoT, persisting until SVR12. Notably, over time a significant increase was observed in respiratory chain (RC) complexes protein levels and the enzymatic activity of complexes I, II, and IV. Mitochondrial-DNA integrity improved over time, and the expression of mitochondrial biogenesis key regulators such as TFAM, Nrf-1, and PPARGC1A significantly increased at SVR12; hence, RC complexes synthesis and mitochondrial respiration were supported after treatment. HCV clearance with DAAS profoundly changed PBMCs bioenergetic profile, suggesting the immunometabolism study as a new approach to the understanding of viral immune evasion mechanisms and host adaptations during infections and therapies.

Monoclonal gammopathy of undetermined significance (MGUS) represents the pre-clinical stage of Multiple Myeloma (MM) with the 5% of MGUS progresses to MM. Although the progression from MGUS to MM has not been completely characterized, it is possible to monitor the DNA modifications of patients diagnosed with MGUS to detect early specific genomic abnormalities, including copy number variations (CNV). The CNVs of chromosome 1q and chromosome 13q are associated with a worse prognosis in MM. In the present study, we showed that it is possible to monitor the 1q21 gain and 13q deletion frequencies in gDNA using digital PCR. The CNV analysis of three cell lines with a well-characterized cytogenetic profile were compared with measures performed by a real-time PCR approach and with a digital PCR approach. Then, we analyzed CNVs in CD138(+) plasma cells isolated from bone marrow of MGUS and MM patients. Our results show that digital PCR and targeted DNA monitoring represent a specific and accurate technique for the early detection of specific genomic abnormalities both in MM and in MGUS patients. Our results could represent a remarkable advancement in MM and MGUS diagnosis and in CNV analysis for the evaluation of the risk of progression from MGUS to MM.

Background Hemodialysis patients present a dramatic increase in cardiovascular morbidity/mortality. Circulating immune cells, activated by both uremic milieu and dialysis, play a key role in the pathogenesis of dialysis-related vascular disease. The aim of our study was to identify, through a high-throughput approach, differences in gene expression profiles in the peripheral blood mononuclear cells (PBMCs) of patients treated with on-line hemodiafiltration and bicarbonate hemodialysis. Methods The transcriptomic profile was investigated in PBMCs isolated from eight patients on on-line hemodiafiltration and eight patients on bicarbonate hemodialysis by microarray analysis. The results were evaluated by statistical and functional pathway analysis and validated by real time PCR (qPCR) in an independent cohort of patients (on-line hemodiafiltration N = 20, bicarbonate hemodialysis n = 20). Results Eight hundred and forty-seven genes were differentially expressed in patients treated with on-line hemodiafiltration and bicarbonate hemodialysis. Thirty-seven functional gene networks were identified and atherosclerosis signaling was the top canonical pathway regulated by on-line hemodiafiltration. Among the genes of this pathway, on-line hemodiafiltration was associated with a reduced expression of Platelet-derived growth factor A chain (PDGF A), Clusterin, Monoamine Oxidase A, Interleukin-6 (IL-6) and Vascular Endothelial Growth Factor C (VEGF-)C and with an increase of Apolipoprotein E. qPCR confirmed the microarray results. Platelet derived growth factor AA (PDGF-AA), IL-6 and VEGF-C serum levels were significantly lower in the on-line hemodiafiltration group. Finally, 10 patients previously on bicarbonate hemodialysis were switched to on-line hemodiafiltration and PBMCs were harvested after 6 months. The qPCR results from this perspective group confirmed the modulation of atherosclerotic genes observed in the cross-sectional analysis. Conclusions Our data suggest that type of dialysis (on-line hemodiafiltration versus bicarbonate hemodialysis) may modulate the expression of several genes involved in the pathogenesis of atherosclerotic disease.

BACKGROUND AND AIMS Diabetes mellitus remains the most frequent cause of end-stage renal disease (ESRD) in developing countries. The main determinant of the onset of nephropathy in diabetes is hyperglycemia which brings to glycation of albumin and haemoglobin with higher concentration of AGEs (advanced glycation end products), finally leading to fibrosis, inflammation and albuminuria. Glycated albumin (GA) was introduced as an alternative to hemoglobin A1c (HbA1c) for glycemic control in the CKD population, with some advantages for diabetic patients with diabetic kidney disease (DKD), since it is not influenced by anemia and associated treatments and it is not affected by serum albumin levels. Although some evidences in literature demonstrated that GA is superior to HbA1c in assessing blood glucose control in DKD (Gan T et al. J Diabetes Complications. 2018); however, KDIGO Guidelines still recommend HbA1c, although underlying some limitations in the context of DKD, meaning that new markers are needed in order to monitor this class of diabetic patients. Kidney disease in diabetic patients can include different kind of renal damage ranging from real diabetic nephropathy (DN, with Kimmelstiel–Wilson lesions, glomerular basal membrane thickening, proliferation of mesangial matrix) to nondiabetic renal disease (NDRD), in the presence or absence of real DN, thus influencing prognosis and treatment. The purpose of the study was to evaluate the clinical utility of the GA dosage, alone or with other molecular and clinical markers as noninvasive biomarkers for the prediction of the progression and type of kidney damage in the diabetic patient. METHOD We enrolled 108 diabetic patients in follow-up at our center, 90 with a biopsy-proven diagnosis of kidney disease and 18 with no kidney dysfunction. Among the 90 patients, 51 had real diabetic nephropathy (DN) or mixed forms (DN + NDRD) and 39 had non-diabetic renal disease (NDRD). GA dosage was obtained by QuantILab® Glycated Albumin assay (Instrumentation Laboratory SpA, Milan, Italy). qPCR was used to evaluate urinary levels of miR27b-3p, a biomarker that predicts the kind of renal damage in diabetic patients and is related to kidney fibrosis (Conserva F et al. Sci Rep. 2019). Statistical analysis was performed using ‘SPSS Statistical Software.’ RESULTS We observed that GA values had a significantly different distribution among DN, NDRD and diabetic patients without renal dysfunction (P = 0.003); conversely, there was no statistical difference in HbA1c distribution although a trend was visible. In the entire cohort, only GA levels, but not HbA1c levels, were significantly correlated with fasting glucose (P = 0.001) and the duration of diabetes (P = .001). In the single classes, only in DN patients there was a significant correlation between GA and diabetes duration (P = .02) and GA and HbA1c (P = 0.01) but not in the NDRD class (P = 0.43). We then applied a multivariate logistic regression model to discriminate real DN and NDRD. We demonstrated that GA is the only significant predictor of real DN in a model corrected for sex, age and serum creatinine {P = 0.009; OR = 0.757–0.962; [95% confidence interval (CI)]}, whereas the same logistic regression model with HbA1c demonstrated no significance (P = 0.082). In the same cohort, we also evaluated miR-27b-3p expression levels, whose levels are correlated with renal fibrosis in DN, and we observed that there was a significant and direct correlation also with GA levels (P = 0.001), thus suggesting the possibility to integrate these biomarkers to predict the kind of renal damage in diabetic patients. CONCLUSION Our study described for the first time the role played by GA as a diagnostic marker in the identification of the different kind of renal damage in diabetic patients. All these evidences support the need to introduce GA as an index of glycemic control to be associated with other molecular markers, such as miRNAs, in order to noninvasively diagnose the kind of renal damage in diabetic patients.

BACKGROUND AND AIMS Diabetic Nephropathy (DN) is the major causes of end-stage renal failure. DN diagnosis is based on typical histological features (Kimmelstiel–Wilson lesions, glomerular basal membrane thickening and proliferation of mesangial matrix). Not all diabetic patients with kidney disease develop true DN: kidney impairment can be due to a nondiabetic renal disease (NDRD) in the presence or absence of real DN. Thus, there is the need for noninvasive biomarkers, related to specific pathogenic processes, to discriminate DN and NDRD, and/or to predict DN onset. In the case of DN, there is extensive evidence in literature of genetic contribution to disease susceptibility and lots of efforts aim at the identification of specific loci. The research group demonstrated that a characteristic feature of DN is an increase in Lys63-ubiquitinated proteins at tubular level that leads to epithelial-to-mesenchymal transition (EMT) and finally to the progression of the tubular-interstitial fibrosis and the renal damage in DN patients (Pontrelli P et al. FASEB J 2017). Ube2V1 is the unique E2 enzyme known for producing Lys63-linked ubiquitin chains. Moreover, two miRNAs (miR27b-3p e miR1228-3p), which interact specifically with Ube2V1 transcript, predict the type of renal damage in diabetic patients and are related with kidney fibrosis (Conserva F et al. Sci Rep 2019). The goal of this project was to identify single nucleotide polymorphisms (SNPs) able to predict the different kind of renal damage and the progression of kidney disease in diabetic patients. METHOD We selected by UCSC genome, 10 HapMap SNPs of patients within coding and regulatory sequences both of miR27b-3p and miR1228-3p and Ube2V1 gene in order to evaluate their diagnostic and prognostic potential. The patients enrolled in this study were diabetic patients with a biopsy-proven DN diagnosis (DN), diabetic patients with a biopsy-proven diagnosis of other nephropathy than DN (NDRD), diabetic patients without clinical signs of impaired renal function (T2D), diabetic patients with a biopsy-proven coexistence of both conditions (ND + NDRD), non-diabetic patients with glomerulonephritis (CKD), non-diabetic patients without renal damage (CTRL). The DNA of 201 subjects (patients and controls) was isolated from blood samples, and 10 HapMap SNPs (rs3802456, rs4744422, rs10761364, rs7853195 in the control region of miR27b-3p; rs2306692, rs4759277, rs4759044, rs17547610, rs4759275 in the control region of miR1228-3p; rs761214 in the UBE2v1 gene) for each patient were analyzed using TaqMan real-time PCR. Glomerular and tubulointerstitial fibrosis in kidney biopsies was quantified on Sirius Red staining using the Aperio Imagescope software. RESULTS The analyzed SNPs showed a different genotype frequency among all the patients’ classes. Interestingly, SNPs rs4759275, rs4759277, rs4744422 and rs3802456 showed a statistically significant difference in genotype frequency comparing DN patients with CEU Population (Northern and Western European Ancestry in Utah) (P CONCLUSION Our data demonstrated that the allelic forms of the analyzed SNPs are related to the different kind of renal damage in diabetic patients. Their prognostic and diagnostic potential could represent the starting point to create a new noninvasive diagnosis system based on clinical and genotyping data.

BACKGROUND AND AIMS Replication of the enveloped SARS-COV2 virus can alter lipidomic composition and metabolism of infected cells [1]. These alterations commonly result in a decline in HDL, total cholesterol and LDL, and an increase in triglyceride levels in COVID-19 patients. Furthermore, the ‘cytokine storm’ subsequent to release of inflammatory cytokines can severely impair lipid homeostasis. Importantly, decreased HDL-cholesterol correlates with severity of COVID-19 infection and represents a significant prognostic factor in predicting poor clinical outcomes [2]. Similarly, it has been observed that COVID-19 patients’ recovery is accompanied by a rise in serum HDL levels. Pharmacological intervention that aims to restore ApoA-1 or functional HDL particles may have beneficial roles for clinical outcome of COVID-19 patients and has recently been approved for compassionate use [3]. SARS-CoV 2 spike proteins S1 and S2 can bind free cholesterol and HDL-bound cholesterol, facilitating virus entry by binding the ACE2 co-receptor Scavenger Receptor-BI (SR-BI) [4]. When activated at the trans-membrane level, SR-BI signalling culminates in Ser1173-eNOS phosphorylation with both anti-inflammatory and anti-apoptotic effect. We hypothesized that SARS-COV2 binding promoted SR-BI internalization, so that it could not exert its essential protective function. Therefore, the aim of this study is to evaluate the effects of CER-001, a mimetic HDL, in antagonizing this process. METHOD Endothelial and tubular (RPTEC) cells were exposed to S1, S2 and S1 + S2 (50–250 nM) with or without CER-001 (CER-001 50–500 ug/mL) and cholesterol (10–50 uM). Apoptosis tests (MTT and AnnV/PI) were performed. Internalization of SR-BI, ACE2 with S1 and activation of eNOS was evaluated by FACS analysis. SR-BI and ACE2 expression were evaluated on kidney biopsies from COVID-19 patients. RESULTS At concentrations used, the exposition of S1, S2 and S1 + S2 in the presence of CER-001 and cholesterol did not induce apoptosis of endothelial cells and RPTEC. Endothelial and tubular cells stimulated by S1, in presence of cholesterol, showed an increased intracellular level of SR-BI and ACE-2, with significantly reduced eNOS phosphorylation compared to baseline (P < 0.05). The treatment with CER-001 reversed trans-membrane SR-BI levels and eNOS phosphorylation to baseline values. The detection of S1 spike protein by endothelial cells immunohistochemistry revealed an increased level in S1-exposed cells with cholesterol and reduced S1 intracellular positive staining in CER-001-exposed cells (P < 0.05). Interestingly, S1-exposed cells without cholesterol appeared not to be capable of mediating S1 spike protein internalization. Consistent with in vitro results, analysis of renal biopsies from COVID-19 patients with proteinuria showed increased SR-BI and ACE-2 cytoplasmic signals and reduced expression at the apical domain of injured tubules. CONCLUSION Our data confirmed the key role of lipid profile in SARS-COV2 infection, evaluating the molecular signalling involved in HDL metabolism and inflammatory processes, and could offer new therapeutic strategies for COVID-19 patients.

BackgroundDelayed graft function (DGF) leads to a reduced graft survival. Donors’ features have been always considered as key pathogenic factors in this setting. The aim of our study was to evaluate the recipients’ characteristics in the development of DGF.MethodsWe enrolled 932 kidney graft recipients from 466 donors; 226 recipients experienced DGF. In 290 donors, both recipients presented with early graft function (EGF, group A), in 50 both recipients experienced DGF (group B), and in 126 one recipient presented with DGF and the other with EGF (group C). In group C, we selected 7 couples of DGF/EGF recipients and we evaluated the transcriptomic profile by microarray on circulating mononuclear cells harvested before transplantation. Results were validated by qPCR in an independent group of 25 EGF/DGF couples.FindingsIn the whole study group, DGF was associated with clinical characteristics related to both donors and recipient. In group C, DGF was significantly associated with body mass index, hemodialysis, and number of mismatches. In the same group, we identified 411 genes differently expressed before transplantation between recipients discordant for the transplant outcome. Those genes were involved in immune dysfunction and inflammation. In particular, we observed a significant increase in DGF patients in the expression of C–C chemokine receptor type 2 (CCR2), the monocyte chemoattractant protein-1 (MCP-1) receptor. CCR-2 upregulation was confirmed in an independent cohort of patients.ConclusionsOur results suggest that recipients’ clinical/immunological features, potentially modulated by dialysis, are associated with the development of DGF independently of donors’ features.

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.

Involvement of T lymphocytes in kidney transplantation is a well-developed topic; however, most of the scientific interest focused on the different type of CD4+ lymphocyte subpopulations. Few authors, instead, investigated the role of CD8+ T cells in renal transplantation and how deleterious they can be to long-term allograft survival. Recently, there has been a renewed interest in the CD8+ T cells involvement in the transplantation field with the aim to investigate the immunological mechanisms underlying the infiltration of CD8+ T cells and their biological functions in human kidney allografts. The purpose of the present review is to highlight the role of allo-reactive cytotoxic T lymphocytes (CTLs) CD8

miRNAs are short, single-stranded RNA molecules that function as posttranscriptional regulators of gene expression. miRNAs represent ideal biomarkers since they can also circulate in the bloodstream as well as in other biological fluids such as urine, saliva, and cerebrospinal fluid.miRNAs play an important role in the regulation of immune cells including cytotoxic T-lymphocytes. Circulating miRNAs can be analyzed by Real-Time PCR or microarray profiling; however data normalization represents still an unsolved problem due to the lack of widely validated house-keeping miRNAs candidates.Digital PCR (dPCR) is an end-point PCR method that is used for absolute quantification. In this chapter we will describe the applications of Digital PCR for the analysis of miRNAs that can influence immune response in serum samples and we will report a specific protocol that can be used to analyze miRNAs using the QuantStudio™ 3D Digital PCR System. The advantage of this method consists in the possibility to highlight weaker differences in miRNA circulating molecules that can be useful to monitor CTLs behavior in pathological conditions or after therapeutic intervention.

The glomerular endothelial glycocalyx (GEnGlx) forms the first part of the glomerular filtration barrier (GFB). We have previously shown that excess mineralocorticoid receptor (MR) activation causes GEnGlx damage and albuminuria. Damage to the GEnGlx occurs early in the pathogenesis of diabetic nephropathy (DN). Here we sought to determine whether MR antagonism with spironolactone could prevent the development of albuminuria in diabetes, by preserving the GEnGlx to maintain the GFB. Streptozotocin-induced diabetic Wistar rats developed increased glomerular albumin permeability (Ps’alb) and albuminuria, with associated GEnGlx loss and increases in plasma and urine active matrix metalloproteinase 2 (MMP2). MR antagonism reduced urine active MMP2, preserved the GEnGlx, restored Ps’alb to control values and prevented diabetes-induced albuminuria progression. Enzymatic degradation of the GEnGlx, with hyaluronidase, reversed the effect of MR antagonism in diabetic rats, confirming the importance of GEnGlx preservation in this model. Using this model we validated a novel fluorescent profile peak-to-peak confocal imaging technique and applied it to assess GEnGlx damage on renal biopsies from patients with DN and compared them to healthy controls. We confirmed that GEnGlx loss occurs in human DN and may contribute to the disease phenotype. Taken together our work suggests GEnGlx preservation as an important novel mechanism for reno-protection by MR inhibitors in diabetes.Translational StatementMineralocorticoid receptor (MR) antagonists reduce albuminuria in diabetic nephropathy (DN), but side effects limit their clinical utility, and the mechanism is unknown. This paper demonstrates that MR antagonism prevented the development of glomerular endothelial glycocalyx (GEnGlx) dysfunction and albuminuria. Highly specific, enzyme mediated, removal of the GEnGlx abolished this effect, confirming the importance of EnGlx preservation in this model. Our data also confirm that GEnGlx damage occurs in human DN and may contribute to the clinical phenotype. This work suggests directly targeting glycocalyx preservation in DN could reproduce the protective effects seen with MR inhibition, whilst avoiding side effects.

The mammalian target of rapamycin inhibitor (mTOR-I) Rapamycin, a drug widely used in kidney transplantation, exerts important anti-cancer effects, particularly in Kaposi's Sarcoma (KS), through several biological interactions. In this in vivo and in vitro study, we explored whether the activation of the autophagic pathway through the low-affinity receptor for nerve growth factor, p75NTR , may have a pivotal role in the anti-cancer effect exerted by Rapamycin in S. Our Kimmunohistochemistry results revealed a significant hyper-activation of the autophagic pathway in KS lesions. In vitro experiments on KS cell lines showed that Rapamycin exposure reduced cell viability by increasing the autophagic process, in the absence of apoptosis, through the transcriptional activation of p75NTR via EGR1. Interestingly, p75NTR gene silencing prevented the increase of the autophagic process and the reduction of cell viability. Moreover, p75NTR activation promoted the upregulation of phosphatase and tensin homolog (PTEN), a tumour suppressor that modulates the PI3K/Akt/mTOR pathway. In conclusion, our in vitro data demonstrated, for the first time, that in Kaposi's sarcoma, autophagy triggered by Rapamycin through p75NTR represented a major mechanism by which mTOR inhibitors may induce tumour regression. Additionally, it suggested that p75NTR protein analysis could be proposed as a new potential biomarker to predict response to Rapamycin in kidney transplant recipients affected by Kaposi's sarcoma.

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.

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19), resulting in acute respiratory disease, is a worldwide emergency. Because recently it has been found that SARS-CoV is dependent on host transcription factors (TF) to express the viral genes, efforts are required to understand the molecular interplay between virus and host response. By bioinformatic analysis, we investigated human TF that can bind the SARS-CoV-2 sequence and can be involved in viral transcription. In particular, we analysed the key role of TF involved in interferon (IFN) response. We found that several TF could be induced by the IFN antiviral response, specifically some induced by IFN-stimulated gene factor 3 (ISGF3) and by unphosphorylated ISGF3, which were found to promote the transcription of several viral open reading frame. Moreover, we found 22 TF binding sites present only in the sequence of virus infecting humans but not bat coronavirus RaTG13. The 22 TF are involved in IFN, retinoic acid signalling and regulation of transcription by RNA polymerase II, thus facilitating its own replication cycle. This mechanism, by competition, may steal the human TF involved in these processes, explaining SARS-CoV-2's disruption of IFN-I signalling in host cells and the mechanism of the SARS retinoic acid depletion syndrome leading to the cytokine storm. We identified three TF binding sites present exclusively in the Brazilian SARS-CoV-2 P.1 variant that may explain the higher severity of the respiratory syndrome. These data shed light on SARS-CoV-2 dependence from the host transcription machinery associated with IFN response and strengthen our knowledge of the virus's transcription and replicative activity, thus paving the way for new targets for drug design and therapeutic approaches.

CVD remains the major cause of mortality with graft functioning in Kidney transplant recipients (KTRs), with an estimated risk of CV events about 50-fold higher than in the general population. Many strategies have been considered to reduce the CV risk such as the use of mTOR inhibitors. We evaluate whether chronic mTOR inhibition might influence CV aging in KTRs studying the molecular mechanisms involved in this effect. We retrospectively analyzed 210 KTRs with stable graft function on therapy with CNI and mycophenolic acid (Group A, 105 pts.), or with CNI and mTORi (Everolimus, Group B, 105 pts.). The presence of mTOR inhibitor in immunosuppressive therapy was associated to increase serum levels of Klotho with concomitant reduction in FGF-23, with a significant decrease in left ventricular mass. In addition, KTRs with mTORi improved mitochondrial function/biogenesis in PBMC with more efficient oxidative phosphorylation, antioxidant capacity and glutathione peroxidase activity. Finally, group B KTRs presented reduced levels of inflammaging markers such as reduced serum pentraxin-3 and p21ink expression in PBMC. In conclusion, we demonstrated that mTOR inhibition in immunosuppressive protocols prevents the occurrence and signs of CV aging in KTRs.

Diabetic nephropathy (DN) is the most frequent cause of end-stage renal disease. Tubulointerstitial accumulation of lysine 63 (K63)-ubiquitinated (Ub) proteins is involved in the progression of DN fibrosis and correlates with urinary miR-27b-3p downregulation. We explored the renoprotective effect of an inhibitor of K63-Ub (NSC697923), alone or in combination with the ACE-inhibitor ramipril, in vitro and in vivo. Proximal tubular epithelial cells and diabetic DBA/2J mice were treated with NSC697923 and/or ramipril. K63-Ub protein accumulation along with α-SMA, collagen I and III, FSP-1, vimentin, p16INK4A expression, SA-α Gal staining, Sirius Red, and PAS staining were measured. Finally, we measured the urinary albumin to creatinine ratio (uACR), and urinary miR-27b-3p expression in mice. NSC697923, both alone and in association with ramipril, in vitro and in vivo inhibited hyperglycemia-induced epithelial to mesenchymal transition by significantly reducing K63-Ub proteins, α-SMA, collagen I, vimentin, FSP-1 expression, and collagen III along with tubulointerstitial and glomerular fibrosis. Treated mice also showed recovery of urinary miR-27b-3p and restored expression of p16INK4A. Moreover, NSC697923 in combination with ramipril demonstrated a trend in the reduction of uACR. In conclusion, we suggest that selective inhibition of K63-Ub, when combined with the conventional treatment with ACE inhibitors, might represent a novel treatment strategy to prevent the progression of fibrosis and proteinuria in diabetic nephropathy and we propose miR-27b-3p as a biomarker of treatment efficacy.

miRNAs are short, single-stranded RNA molecules that function as posttranscriptional regulators of gene expression. miRNAs represent ideal biomarkers since they can also circulate in the bloodstream as well as in other biological fluids such as urine, saliva, and cerebrospinal fluid.miRNAs play an important role in the regulation of immune cells including cytotoxic T-lymphocytes. Circulating miRNAs can be analyzed by Real-Time PCR or microarray profiling; however data normalization represents still an unsolved problem due to the lack of widely validated house-keeping miRNAs candidates.Digital PCR (dPCR) is an end-point PCR method that is used for absolute quantification. In this chapter we will describe the applications of Digital PCR for the analysis of miRNAs that can influence immune response in serum samples and we will report a specific protocol that can be used to analyze miRNAs using the QuantStudio™ 3D Digital PCR System. The advantage of this method consists in the possibility to highlight weaker differences in miRNA circulating molecules that can be useful to monitor CTLs behavior in pathological conditions or after therapeutic intervention.

Involvement of T lymphocytes in kidney transplantation is a well-developed topic; however, most of the scientific interest focused on the different type of CD4+ lymphocyte subpopulations. Few authors, instead, investigated the role of CD8+ T cells in renal transplantation and how deleterious they can be to long-term allograft survival. Recently, there has been a renewed interest in the CD8+ T cells involvement in the transplantation field with the aim to investigate the immunological mechanisms underlying the infiltration of CD8+ T cells and their biological functions in human kidney allografts. The purpose of the present review is to highlight the role of allo-reactive cytotoxic T lymphocytes (CTLs) CD8+ subset in allograft kidney recipients and their related clinical complications.

Pentraxin 3 (PTX3) is an essential component of the innate immune system and a recognized modulator of Complement cascade. The role of Complement system in the pathogenesis of prostate cancer has been largely underestimated. The aim of our study was to investigate the role of PTX3 as possible modulator of Complement activation in the development of this neoplasia. We performed a single center cohort study; from January 2017 through December 2018, serum and prostate tissue samples were obtained from 620 patients undergoing prostate biopsy. A group of patients with benign prostatic hyperplasia (BPH) underwent a second biopsy within 12–36 months demonstrating the presence of a prostate cancer (Group A, n = 40) or confirming the diagnosis of BPH (Group B, N = 40). We measured tissue PTX3 protein expression together with complement activation by confocal microscopy in the first and second biopsy in group A and B patients. We confirmed that that PTX3 tissue expression in the first biopsy was increased in group A compared to group B patients. C1q deposits were extensively present in group A patients co-localizing and significantly correlating with PTX3 deposits; on the contrary, C1q/PTX3 deposits were negative in group B. Moreover, we found a significantly increased expression of C3a and C5a receptors within resident cells in group A patient. Interestingly, C1q/PTX3 deposits were not associated with activation of the terminal Complement complex C5b-9; moreover, we found a significant increase of Complement inhibitor CD59 in cancer tissue. Our data indicate that PTX3 might play a significant pathogenic role in the development of this neoplasia through recruitment of the early components of Complement cascade with hampered activation of terminal Complement pathway associated with the upregulation of CD59. This alteration might lead to the PTX3-mediated promotion of cellular proliferation, angiogenesis and insensitivity to apoptosis possible leading to cancer cell invasion and migration.

Background: Lung transplantation is still the best therapeutic option for cystic fibrosis (CF) patients, but, unfortunately, immunosuppressive therapies, often employed at high dosages to avoid acute rejection, may induce severe complications. In particular, patients treated with high dose of mammalian target of rapamycin inhibitors (mTOR-Is) may experience lung fibrosis (including bronchiolitis obliterans-organizing pneumonia). Although epithelial to mesenchymal transition (EMT) of airway cells has a central role in this process, the complete biological machinery is not completely clarified.Methods: In order to improve our knowledge on this process, primary bronchial epithelial cells carrying F508del mutation were treated with 5 and 100 nM everolimus (EVE) for 24 hours. Subsequently, RNA was hybridized to the Human HT-12 v3 Expression BeadChip (Illumina). Real-Time PCR was, then, used to validate microarray results and to measure major EMT biomarkers. Trans-epithelial resistance was measured by Millicell-ERS ohmmeter.Results: High dosage EVE induced a significant up-regulation of 42 genes and a down-regulation of 12 genes. After pathway analysis by Gene Set Enrichment Analysis and Ingenuity Pathway Analysis, we found that most of them were implicated in the pro-inflammatory pathway. Real-Time PCR validated these results and revealed that, in addition to pro-inflammatory genes (IL-1α, IL-8, Pim-1 Oncogene), EVE at high dosage was able to up-regulate major EMT biomarkers (such as: alpha-smooth muscle actin, connective tissue growth factor and metalloproteinase 12). In lung, EMT is the convergence point between inflammation and the progression of fibrotic damage. Additionally, EVE at this dosage reduced the trans-epithelial resistance (altering tight junction strength). In contrast, lower EVE did not trigger similar effects.Conclusions: We demonstrated that high dose EVE may trigger a pro-inflammatory/fibrotic biological machinery in bronchial epithelial cells from CF patients. Our results, although obtained in vitro, suggest that solid organ transplant recipients affected by CF should be treated with the lowest possible dose of mTOR inhibitors to minimize/avoid the onset and development of lung complications. In vivo studies might be useful to confirm our hypothesis.

Background The novel SARS-CoV-2 causing the pandemic acute respiratory disease COVID-19 is considered a worldwide emergency since no vaccines or effective antiviral are still available. As virus are dependent on the host transcriptional factors (TFs) to express the viral genes, efforts are required to understand the molecular interplay between virus and host response. Methods By bioinformatic analysis, we investigated human TFs involved in viral transcription. In particular, we analyzed the key role of the TF induced by Interferon regulatory factors (IRFs) exploiting the way of antiviral response related to interferon antiviral cytokines released during an infection. We compared SARS-CoV-2 consensus sequence from Italian patients with sequences in Coronaviridae ViPR database, to identify elements and TF binding sites that were present uniquely in the Italian viral sequence.Results Several TFs were induced by IRFs, others were induced by IFN-stimulated gene factor 3 (ISGF3) and by un-phosphorylated ISGF3 that was found to promote the transcription of several viral open reading frame. These data shed light on SARS-CoV2 dependence from the host transcription machinery associated to interferon-related antiviral response. We found binding sites for 11 TF present only in sequence of virus infecting humans and among these IRF8, IRF4, GLIS2, POU4F2 that were expressed in response to the viral infection and induced by interferon. POU4F2 binds in ORF1ab promoter; IRF8, and IRF4 both bind within the ORF1ab gene.Conclusions Our data suggest that SARS-CoV-2 can exploit the interferon-related host response, inducing the expression of genes by host TF involved in the regulation of transcription by RNA polymerase II, thus facilitating its own replication cycle.These data strengthen our knowledge about the transcription and replicative activity of the virus and could pave the way for new targets drug design.

Background Diabetic kidney disease (DKD) remains one of the leading causes of premature death in diabetes. DKD is classified on albuminuria and reduced kidney function (estimated glomerular filtration rate (eGFR)) but these have modest value for predicting future renal status. There is an unmet need for biomarkers that can be used in clinical settings which also improve prediction of renal decline on top of routinely available data, particularly in the early stages. The iBEAt study of the BEAt-DKD project aims to determine whether renal imaging biomarkers (magnetic resonance imaging (MRI) and ultrasound (US)) provide insight into the pathogenesis and heterogeneity of DKD (primary aim) and whether they have potential as prognostic biomarkers in DKD (secondary aim). Methods iBEAt is a prospective multi-centre observational cohort study recruiting 500 patients with type 2 diabetes (T2D) and eGFR ≥30 ml/min/1.73m2. At baseline, blood and urine will be collected, clinical examinations will be performed, and medical history will be obtained. These assessments will be repeated annually for 3 years. At baseline each participant will also undergo quantitative renal MRI and US with central processing of MRI images. Biological samples will be stored in a central laboratory for biomarker and validation studies, and data in a central data depository. Data analysis will explore the potential associations between imaging biomarkers and renal function, and whether the imaging biomarkers improve the prediction of DKD progression. Ancillary substudies will: (1) validate imaging biomarkers against renal histopathology; (2) validate MRI based renal blood flow measurements against H2O15 positron-emission tomography (PET); (3) validate methods for (semi-)automated processing of renal MRI; (4) examine longitudinal changes in imaging biomarkers; (5) examine whether glycocalyx and microvascular measures are associated with imaging biomarkers and eGFR decline; (6) explore whether the findings in T2D can be extrapolated to type 1 diabetes. Discussion iBEAt is the largest DKD imaging study to date and will provide valuable insights into the progression and heterogeneity of DKD. The results may contribute to a more personalised approach to DKD management in patients with T2D. Trial registration Clinicaltrials.gov (NCT03716401).

Background and Aims Diabetic Nephropathy (DN) is the primary cause of end stage renal disease (ESRD). Our group demonstrated that in DN an accumulation of lysine63 (K63)-ubiquitinated proteins at tubular level is involved in the progression of renal damage, in particular renal fibrosis. Current treatments do not provide complete renoprotection and targeted therapies that prevent fibrosis or delay its progression are still lacking. Aim of the present study was to evaluate the renoprotective effect of specific drug and their combinations, including an inhibitor of K63 ubiquitination (K63Ub) and/or an anti-hypertensive agent, in vitro and in vivo in a murine model of DN. Method Renal Proximal Tubule Epithelial Cells (HK2) were pre-incubated with a specific inhibitor of K63Ub and/or with the ACE-inhibitor Ramipril. Accumulation of K63 ubiquitinated proteins along with α-sma expression, indicator of epithelial-to-mesenchymal transition (EMT), were analyzed through immunofluorescence and western blotting. The same drug combination was also tested in streptozotocin (STZ)-treated DBA/2J mice, a model of human DN. In mice, K63Ub was evaluated by IHC, while renal fibrosis was evaluated by Sirius red and Collagen III expression. Urinary albuminuria was measured by ELISA. Results We observed that the association of the specific K63Ub inhibitor with Ramipril was able to block hyperglycemia-induced EMT in HK2 cells by significantly reducing α-sma expression, when compared to single drugs alone (p Conclusion Our data demonstrated and confirmed the importance of K63Ub in the progression of renal fibrosis in vitro and in vivo. We proposed and patented a novel combination of drugs that ameliorates both fibrosis and proteinuria in DN. Novel treatment regimens could represent an important goal for reducing the incidence of ESRD related to diabetes complication.

Background and Aims The recent discovery of a complex system of human Adult renal stem/progenitor cells (ARPCs) dedicated to the renewal of epithelial renal cells has allowed scientists to better understand how the regenerative process can occur in the adult kidney. ARPCs have a great potential in view of developing future treatments for both acute and chronic renal injury. However, to completely take advantage of their capability it is essential to study the factors regulating the stem cell behavior. Recently, long noncoding RNAs (lncRNAs) have been recognized as a crucial class of gene expression regulators. lncRNAs have several distinguishing characteristics that provide particular regulatory functions, including cell- and tissue-specific expression and the ability to transduce higher-order spatial information. Several lncRNAs modulate some somatic stem cell renewal or differentiation, while others promote a differentiation program. Their functions are often helped by proteic co-factor that convey the ability to activate or repress gene expression or to post-transcriptionally regulate other RNAs. Furthermore, numerous lncRNAs have been shown to act as competing endogenous RNAs, where the lncRNAs are proposed to bind to and compete miRNAs away from cognate mRNA targets. The aim of the study was to evaluate the basal expression profile of lncRNA expressed specifically in ARPCs. Method lncRNA expression profile was obtained from ARPCs and renal proximal tubular cells (RPTECs) alone or following cisplatin damage induction. We used Agilent SurePrint G3 Human Gene Expression Microarrays providing comprehensive coverage of genes and transcripts using the latest annotation databases. Genespring and R software were used for the analysis. lncRNA expression was validated by Real-time PCR. CrispR/Cas9 system has been used to knock-down specific lncRNA. Results We compared lncRNA expression between ARPCs and RPTECs: 45 lncRNA were differently modulated in ARPCs vs RPTECs (Fold change 1.5; FDR Conclusion ARPCs express specific lncRNAs that could explain some of the ARPC stemness properties and their capacity to repair damage induced by cisplatin. Our findings suggest that lncRNA may represent a novel therapeutic target in acute and chronic renal injury.

Background and Aims Chronic antibody-mediated rejection (CAMR) is a multifactorial pathological condition that can affect more that 40-50% of kidney allografts. Its clinical evolution is often silent, and this can delay its diagnosis. Although many advances in understanding the biological mechanisms underlying its onset/development, at the moment, no reliable non-invasive diagnostic biomarkers are available in clinic. Method We enrolled 29 patients with biopsy-proven CAMR, 29 stable transplant recipients (controls). Subsequently, the microarray profile of mRNA isolated from peripheral blood mononuclear cells (PBMCs) from 10 randomly selected CAMR patients and 10 controls was evaluated by Agilent technology. For the bioinformatics analysis we employed several statistical algorithms (including ANOVA and Kruskal-Wallis tests adjusted for multiple-tests) and a Weighted Gene Correlation Network Analysis (WGCNA) to select genes biologically associated in co-expressed networks. Results 935 genes resulted differentially expressed between the two study groups, demonstrating the large biological impact of this pathological condition on circulating immune-inflammatory cells. After WGCNA co-expression analysis, a group of genes (enclosed in a single co-expression module, 13 genes) resulted highly discriminating CAMR versus controls (p Conclusion Our study confirmed the role of the type 1 interferon pathway in CAMR and underlined its capability to promote C1QB expression. These effects could contribute to better define the role of innate immunity in CAMR. Finally, C1QB, if validated in a large cohort of patients, could represent a new valuable molecular diagnostic biomarker for this complication.

Background and objectives Active antibody-mediated rejection is the main cause of kidney transplant loss, sharing with SLE the alloimmune response and the systemic activation of the IFN-α pathway. IgE-mediated immune response plays a key role in the development of SLE nephritis and is associated with IFN-α secretion. The aim of our study was to investigate IgE-mediated immune response in antibody-mediated rejection. Design, setting, participants, & measurements This was a cross-sectional study of 56 biopsy-proven antibody-mediated rejection study participants, 80 recipients with normal graft function/histology (control), 16 study participants with interstitial fibrosis/tubular atrophy, and six participants with SLE. We evaluated graft IgE deposition, tryptase (a mast cell marker), and CD203 (a specific marker of activated basophils) by immunofluorescence/confocal microscopy. In addition, we measured serum concentration of human myxovirus resistance protein 1, an IFN-α–induced protein, and anti-HLA IgE. Results We observed a significantly higher IgE deposition in tubules and glomeruli in antibody-mediated rejection (1766±79 pixels) and SLE (1495±43 pixels) compared with interstitial fibrosis/tubular atrophy (582±122 pixels) and control (253±50 pixels). Patients with antibody-mediated rejection, but not control patients and patients with interstitial fibrosis/tubular atrophy, presented circulating anti-HLA IgE antibodies, although with a low mean fluorescence intensity. In addition, immunofluorescence revealed the presence of both mast cells and activated basophils in antibody-mediated rejection but not in control and interstitial fibrosis/tubular atrophy. The concentration of circulating basophils was significantly higher in antibody-mediated rejection compared with control and interstitial fibrosis/tubular atrophy. MxA serum levels were significantly higher in antibody-mediated rejection compared with control and correlated with the extent of IgE deposition. Conclusions Our data suggest that IgE deposition and the subsequent recruitment of basophils and mast cells within the kidney transplant might play a role in antibody-mediated rejection.