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Immunoglobulin A (IgA) nephropathy (IgAN) is the most common primary glomerulonephritis worldwide and it is characterized by mesangial IgA deposition. Asymptomatic hematuria with various degrees of proteinuria is the most common clinical presentation and up to 20%–40% of patients develop end-stage kidney disease within 20 years after disease onset. The pathogenesis of IgAN involves four sequential processes known as the “four-hit hypothesis” which starts with the production of a galactose-deficient IgA1 (gd-IgA1), followed by the formation of anti-gd-IgA1 IgG or IgA1 autoantibodies and immune complexes that ultimately deposit in the glomerular mesangium, leading to inflammation and injury. Although several key questions about the production of gd-IgA1 and the formation of anti-gd-IgA1 antibodies remain unanswered, a growing body of evidence is shedding light on the innate and adaptive immune mechanisms involved in this complex pathogenic process. Herein, we will focus on these mechanisms that, along with genetic and environmental factors, are thought to play a key role in disease pathogenesis.

To assess whether NLRP3 gene promoter methylation was able to discriminate glucocorticoid (GC)-resistant from GC-sensitive idiopathic nephrotic syndrome (INS), patients with minimal change disease (MCD) or focal segmental glomerulosclerosis (FSGS), we measured the methylation level of NLRP3 promoter in DNA from peripheral blood cells of 10 adult patients with GC-resistant FSGS already in hemodialysis and 18 patients with GC-sensitive INS (13 MCD/5 FSGS) and in 21 pediatric patients with INS with MCD/FSGS before starting any treatment. Association of NLRP3 inflammasome with GC resistance was recapitulated in vitro in monocytic cell lines (THP-1 and U937). In both adults and pediatric patients, NLRP3 promoter methylation was significantly reduced in GC-resistant compared with GC-sensitive patients. Indeed, NLRP3 methylation distinguished GC-resistant and GC-sensitive patients (area under the receiver operating characteristic curve [AUROC] 86.7% in adults, p = 0.00019, and 73.5% in children, p = 0.00097). NLRP3 knock-down augmented sensitivity to GCs in THP-1 cells, whereas NLRP3 inflammasome activation lowered GC receptor concentration, increasing GC resistance in U937 cells. Our results uncovered a new biological mechanism by which patients with INS may acquire GC resistance, that could be used in future as a novel noninvasive diagnostic tool. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? ☑ Approximately 80% of patients with idiopathic nephrotic syndrome (INS) respond to glucocorticoids, with the remaining 20% being steroid-resistant. WHAT QUESTION DID THIS STUDY ADDRESS? ☑ Whether NLRP3 gene promoter methylation was able to discriminate glucocorticoid-resistant from glucocorticoid (GC)-sensitive INS. WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? ☑ In both adults and children, NLRP3 promoter methylation was significantly reduced in leukocytes of patients with GC-resistant compared with GC-sensitive INS. NLRP3 inflammasome activation lowered GC receptor concentration and augmented GC resistance, whereas NLRP3 knockdown increased sensitivity to GCs in cell lines representative of monocytes (U937 and THP1). HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? ☑ Our findings uncovered a new biological mechanism whereby patients with INS may develop resistance to GCs that could be used in the future as a novel noninvasive diagnostic tool.

Background: Medullary sponge kidney (MSK) disease is a rare and neglected congenital condition typically associated with nephrocalcinosis/nephrolithiasis, urinary concentration defects, and cystic anomalies in the precalyceal ducts that, although sporadic in the general population, is relatively frequent in renal stone formers. The physiopathologic mechanism associated with this disease is not fully understood, and omics technologies may help address this gap. Summary: The aim of this review was to provide an overview of the current state of the application of proteomics in the study of this rare disease. In particular, we focused on the results of our recent Italian collaborative studies that, analyzing the MSK whole and extracellular vesicle urinary content by mass spectrometry, have displayed the existence of a large and multifactorial MSK-associated biological machinery and identified some main regulatory biological elements able to discriminate patients affected by this rare disorder from those with idiopathic calcium nephrolithiasis and autosomal dominant polycystic kidney disease (including laminin subunit alpha 2, ficolin 1, mannan-binding lectin serine protease 2, complement component 4-binding protein β, sphingomyelin, ephrins). Key Messages: The application of omics technologies has provided new insights into the comprehension of the physiopathology of the MSK disease and identified novel potential diagnostic biomarkers that may replace in future expensive and invasive radiological tests (including CT) and select novel therapeutic targets potentially employable, whether validated in a large cohort of patients, in the daily clinical practice.

IgA nephropathy (IgAN) is a progressive form of kidney disease defined by glomerular deposition of IgA. We performed a genome-wide association study involving 10,146 kidney biopsy-diagnosed IgAN cases and 28,751 matched controls across 17 international cohorts. We defined 30 independent genome-wide significant risk loci jointly explaining 11% of disease risk. A total of 16 loci were novel, including TNFSF4, REL, CD28, CXCL8/PF4V1, LY86, LYN, ANXA3, TNFSF8/15, REEP3, ZMIZ1, RELA, ETS1, IGH, IRF8, TNFRSF13B and FCAR. The SNP-based heritability of IgAN was estimated at 23%. We observed a positive genetic correlation between IgAN and total serum IgA levels, allergy, tonsillectomy, and several infections, and a negative correlation with inflammatory bowel disease. All significant non-HLA loci shared with serum IgA levels had a concordant effect on the risk of IgAN. Moreover, IgAN loci were globally enriched in gene orthologs causing abnormal IgA levels when genetically manipulated in mice. The explained heritability was enriched in the regulatory elements of cells from the immune and hematopoietic systems and intestinal mucosa, providing support for the pathogenic role of extra-renal tissues. The polygenic risk of IgAN was associated with early disease onset, increased lifetime risk of kidney failure, as well as hematuria and several other traits in a phenome-wide association study of 590,515 individuals. In the comprehensive functional annotation analysis of candidate causal genes across genome-wide significant loci, we observed the convergence of biological candidates on a common set of inflammatory signaling pathways and cytokine ligand-receptor pairs, prioritizing potential new drug targets.

Background: Molecular biology has recently added new insights into the comprehension of the physiopathology of the medullary sponge kidney disease (MSK), a rare kidney malformation featuring nephrocalcinosis and recurrent renal stones. Pathogenesis and metabolic alterations associated to this disorder have been only partially elucidated.Methods: Plasma and urine samples were collected from 15 MSK patients and 15 controls affected by idiopathic calcium nephrolithiasis (ICN). Plasma metabolomic profile of 7 MSK and 8 ICN patients was performed by liquid chromatography combined with electrospray ionization tandem mass spectrometry (UHPLC–ESI-MS/MS). Subsequently, we reinterrogated proteomic raw data previously obtained from urinary microvesicles of MSK and ICN focusing on proteins associated with sphingomyelin metabolism. Omics results were validated by ELISA in the entire patients' cohort.Results: Thirteen metabolites were able to discriminate MSK from ICN (7 increased and 6 decreased in MSK vs. ICN). Sphingomyelin reached the top level of discrimination between the two study groups (FC: −1.8, p < 0.001). Ectonucleotide pyrophophatase phosphodiesterase 6 (ENPP6) and osteopontin (SPP1) resulted the most significant deregulated urinary proteins in MSK vs. ICN (p < 0.001). ENPP6 resulted up-regulated also in plasma of MSK by ELISA.Conclusion: Our data revealed a specific high-throughput metabolomics signature of MSK and indicated a pivotal biological role of sphingomyelin in this disease.

Background and Aims Peritoneal dialysis (PD) is the worldwide preferred dialysis treatment for children affected by end stage kidney disease. However, due to the unphysiological composition of PD fluids, the peritoneal mesothelium of these patients may undergo structural and functional alterations, which may cause fibrosis. Several factors may accelerate this process and primary kidney disease may have a causative role. In particular, patients affected by corticosteroid resistant focal segmental glomerulosclerosis (FSGS), a rare glomerular disease leading to nephrotic syndrome, seems more prone to develop peritoneal fibrosis. The mechanism causing this predisposition is still unrecognized. To better define this condition, we carried out, for the first time, a new comprehensive comparative proteomic mass spectrometry analysis of mesothelial exosomes from peritoneal dialysis effluent (PDE). Method We enrolled 12 paediatric patients on PD. Half of them affected by FSGS and the others affected by other disorders (NO FSGS). Exosomes from mesothelial peritoneal cells were isolated by centrifugation and, using a biotinylated antibody and streptavidin magnetic beads. Exosome size was determined by dynamic light scattering, and antigen profile of exosomes was assayed by western blot. Mass spectrometry data were analyzed by unsupervised hierarchical clustering using multidimensional scaling, in order to identify outliers and dissimilarity between samples. The normalized data were used to construct a co-expression network using the weighted gene co-expression network analysis (WGCNA). A weighted adjacency matrix was constructed, and transformed into a topological overlap matrix, which measures the network connectivity of all proteins. To identify the relationship between each module and each clinical trait, we used module eigengenes (MEs) and calculated the correlation between MEs and the clinical traits. To identify the hub proteins of modules that maximize the discrimination between FSGS and NO FSGS samples, we applied T-test, and non-linear support vector machine (SVM) learning. Finally, gene set enrichment analysis was done to build a functional proteins network based on their Gene Ontology (GO) annotations extracted from the Gene Ontology Consortium. Results Our omic study demonstrated that SVM allowed a complete distinction of the whole proteomic exosome mesothelial content of FSGS versus NO FSGS (100% accuracy). Out of the 2490 identified proteins, 40% (995) were involved in endothelial to mesenchymal transition /fibrosis and in the TGF-B pathway. The WGCNA analysis highlighted a total of 40 proteins were that maximize the discrimination between FSGS and NO FSGS patients (Figure 1A). Their expression profile is reported in a heatmap diagram (Figure 1B). Additionally, we performed GO enrichment analysis (Figure 2) and the algorithm identified that some of the discriminative proteins (TIMP1, CTHRC1, SPARC, CHMP4B, COL5A2, ANXA13, FNC2 and CENP-E) were also highly correlated to the peritoneal dialysis vintage, fibrosis, EMT and PM disease. Conclusion Our data demonstrated, for the first time, that mesothelial cells of FSGS are more prone to activate a pro-fibrotic machinery with exosomes having a primary role in this process. Moreover, they indicated that identified FSGS-associated element in mesothelial exosome protein could be employed as potential new biomarkers of mesothelial integrity. Finally, results highlighted that FSGS should be treated with more biocompatible dialysis solution, reduce the length of time on PD and personalize type and regimens of PD to minimize the risk of rapid loss of peritoneal membrane.

Peritoneal dialysis (PD) is the worldwide recognized preferred dialysis treatment for children affected by end-stage kidney disease (ESKD). However, due to the unphysiological composition of PD fluids, the peritoneal membrane (PM) of these patients may undergo structural and functional alterations, which may cause fibrosis. Several factors may accelerate this process and primary kidney disease may have a causative role. In particular, patients affected by steroid resistant primary focal segmental glomerulosclerosis, a rare glomerular disease leading to nephrotic syndrome and ESKD, seem more prone to develop peritoneal fibrosis. The mechanism causing this predisposition is still unrecognized. To better define this condition, we carried out, for the first time, a new comprehensive comparative proteomic mass spectrometry analysis of mesothelial exosomes from peritoneal dialysis effluent (PDE) of 6 pediatric patients with focal segmental glomerular sclerosis (FSGS) versus 6 patients affected by other primary renal diseases (No FSGS). Our omic study demonstrated that, despite the high overlap in the protein milieu between the two study groups, machine learning allowed to identify a core list of 40 proteins, with ANXA13 as most promising potential biomarker, to distinguish, in our patient population, peritoneal dialysis effluent exosomes of FSGS from No FSGS patients (with 100% accuracy). Additionally, the Weight Gene Co-expression Network Analysis algorithm identified 17 proteins, with PTP4A1 as the most statistically significant biomarker associated to PD vintage and decreased PM function. Altogether, our data suggest that mesothelial cells of FSGS patients are more prone to activate a pro-fibrotic machinery. The role of the proposed biomarkers in the PM pathology deserves further investigation. Our results need further investigations in a larger population to corroborate these findings and investigate a possible increased risk of PM loss of function or development of encapsulating peritoneal sclerosis in FSGS patients, thus to eventually carry out changes in PD treatment and management or implement new solutions.

Vogt, Bruno/0000-0002-1548-6387; Dufek, Stephanie/0000-0002-6323-6673; Liu, Lili/0000-0002-2622-9669; Paczek, Leszek/0000-0003-0160-3009; Cui, Zhao/0000-0002-5837-1926; amoroso, antonio/0000-0002-9437-9407; Rosen, Raphael/0000-0003-1025-1965; Loos, Ruth/0000-0002-8532-5087; coenen, marieke/0000-0001-8796-2031; zanoni, francesca/0000-0001-9567-6713; CUCCA, Francesco/0000-0002-7414-1995; Ars, Elisabet/0000-0002-4118-4358; Hamilton, Patrick/0000-0001-6703-3745 WOS:000563559600001 PubMed ID: 32231244 Membranous Nephropathy (MN) is a rare autoimmune cause of kidney failure. Here we report a genome-wide association study (GWAS) for primary MN in 3,782 cases and 9,038 controls of East Asian and European ancestries. We discover two previously unreported loci, NFKB1 (rs230540, OR = 1.25, P = 3.4 x 10(-12)) and IRF4 (rs9405192, OR = 1.29, P = 1.4 x 10(-14)), fine-map the PLA2R1 locus (rs17831251, OR = 2.25, P = 4.7 x 10(-103)) and report ancestry-specific effects of three classical HLA alleles: DRB1*1501 in East Asians (OR = 3.81, P = 2.0 x 10(-49)), DQA1*0501 in Europeans (OR = 2.88, P = 5.7 x 10(-93)), and DRB1*0301 in both ethnicities (OR = 3.50, P = 9.2 x 10(-23) and OR = 3.39, P = 5.2 x 10(-82), respectively). GWAS loci explain 32% of disease risk in East Asians and 25% in Europeans, and correctly re-classify 20-37% of the cases in validation cohorts that are antibody-negative by the serum anti-PLA2R ELISA diagnostic test. Our findings highlight an unusual genetic architecture of MN, with four loci and their interactions accounting for nearly one-third of the disease risk. National Institute for Diabetes and Digestive Kidney Diseases (NIDDK) [RC2-DK116690, R01-DK105124, R01-DK097053, R01-DK108805]; National Institute on Minority Health and Health Disparities (NIMHD)United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute on Minority Health & Health Disparities (NIMHD) [R01-MD009223]; Charles Woodson Clinical Research Fund; Nephrotic Syndrome Study Network Consortium (NEPTUNE) [U54-DK-083912]; Columbia University, Columbia Glomerular Center; Office of Rare Diseases Research, National Center for Advancing Translational Sciences (NCATS); National Institute of Diabetes, Digestive, and Kidney Diseases (NIDDK)United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK); University of MichiganUniversity of Michigan System; NephCure Kidney International; Halpin Foundation; National Key Research and Development Program of China [2016YFC0904100]; Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [81621092]; National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [81870460, 81570598]; Science and Technology Innovation Action Plan of Shanghai Science and Technology Committee [17441902200]; Shanghai Municipal Education Commission, Gaofeng, Clinical Medicine Grant [20152207]; Shanghai Jiao Tong University School of Medicine, Multi-Center Clinical Research Project [DLY201510]; International Cooperation and Exchange Projects of Shanghai Science and Technology Committee [14430721000]; Outstanding Young Scholar Award [81622009]; Shanghai Health and Family Planning Committee Hundred Talents Program [2018BR37]; Seoul National University Hospital Human Biobank, a member of the National Biobank of Korea - Ministry of Health and Welfare, Republic of Korea; MRCMedical Research Council UK (MRC) [MR/J010847/1]; Manchester Academic Health Science Centre [MAHSC 186/200]; Greater Manchester Local Clinical Research Network; Kidneys for Life Charity; David and Elaine Potter Charitable Foundation; St Peter's Trust for Kidney, Bladder and Prostate Research; Kids Kidney Research UKKidney Research UK (KRUK); Kidney Research UKKidney Research UK (KRUK); Italian Ministry of Health grantMinistry of Health, Italy [GR-2011-02350438]; Department of Excellence Grant 2018-2022 - Italian Ministry of Education for the Department of Medical Sciences of the University of Turin; Columbia University; Poznan University of Medical Sciences, Poland; German Ministry of Education and Research (BMBF)Federal Ministry of Education & Research (BMBF) [01ER0804]; KfH Foundation for Preventive Medicine; Bayer Pharma AG; German Research FoundationGerman Research Foundation (DFG) [CRC 1140, KO 3598/3-1, CRC 992]; European Research CouncilEuropean Research Council (ERC) [ERC-2012ADG_2012-0314, 322947]; 7th Framework Programme of the European Community contract [2012-305608]; National Research Agency grant MNaims [ANR-17-CE17-0012-01]; Dutch Kidney Foundation [OW08, KJPB11.021]; National Human Genome Research Institute (NHGRI)United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Human Genome Research Institute (NHGRI); NIMHD We are grateful to all study participants across multiple nephrology centres worldwide for their contributions to this work. This work was supported by the following institutions, grants and funding agencies in the US: Columbia University, Columbia Glomerular Center, National Institute for Diabetes and Digestive Kidney Diseases (NIDDK) grants RC2-DK116690 (K.K., M.K.), R01-DK105124 (K.K.), R01-DK097053 (L.H.B., M.K.), R01-DK108805 (M.G.S.), and National Institute on Minority Health and Health Disparities (NIMHD) grant R01-MD009223 (K.K., A.G.G., A.B.). M.G.S. is additionally supported by the Charles Woodson Clinical Research Fund. The Nephrotic Syndrome Study Network Consortium (NEPTUNE), U54-DK-083912, is a part of the National Institutes of Health (NIH) Rare Disease Clinical Research Network (RDCRN), supported through a collaboration between the Office of Rare Diseases Research, National Center for Advancing Translational Sciences (NCATS) and the National Institute of Diabetes, Digestive, and Kidney Diseases (NIDDK). Additional funding and/or programmatic support for this project has also been provided by the University of Michigan, the NephCure Kidney International and the Halpin Foundation. The recruitment and analysis of the Chinese cohorts were supported by the National Key Research and Development Program of China (2016YFC0904100), Natural Science Foundation of China to the Innovation Research Group (81621092), National Natural Science Foundation of China (No. 81870460, 81570598), Science and Technology Innovation Action Plan of Shanghai Science and Technology Committee (No.17441902200), Shanghai Municipal Education Commission, Gaofeng, Clinical Medicine Grant (No.20152207), Shanghai Jiao Tong University School of Medicine, Multi-Center Clinical Research Project (No.DLY201510), International Cooperation and Exchange Projects of Shanghai Science and Technology Committee (No.14430721000), the Outstanding Young Scholar Award for Zhao Cui (No.81622009), and Shanghai Health and Family Planning Committee Hundred Talents Program for Jingyuan Xie (No.2018BR37). The recruitment of the Korean cohort was supported by the Seoul National University Hospital Human Biobank, a member of the National Biobank of Korea, financed by the Ministry of Health and Welfare, Republic of Korea. P.B. and P.H. acknowledge financial support from MRC project "Autoimmunity in Membranous Nephropathy", grant MR/J010847/1 which funded the sample collection from MN patients across the UK. P.B., P.H. and S.H. acknowledge support from Manchester Academic Health Science Centre (MAHSC 186/200), the Greater Manchester Local Clinical Research Network and Kidneys for Life Charity for supporting research in MN in Manchester. We are grateful to the MENTOR study (clinical trials no. NCT01180036), for contributing blood samples of trial participants. The UK cohort was supported in part by grants from the David and Elaine Potter Charitable Foundation (to S.H.P. and R.K.), St Peter's Trust for Kidney, Bladder and Prostate Research (to D.B., H.C.S., S.H.P. and R.K.), Kids Kidney Research UK and Kidney Research UK (to D.B. and R.K.). The Italian cohorts were supported by the Italian Ministry of Health grant GR-2011-02350438 (G.Z., S.G.) and the Department of Excellence Grant 2018-2022 funded by the Italian Ministry of Education for the Department of Medical Sciences of the University of Turin (A.A.).; The recruitment of Polish cases was sponsored by the Polish Kidney Genetics Network (POLYGENES), a collaborative effort between Columbia University and Poznan University of Medical Sciences, Poland. The full list of POLYGENES collaborators can be found in the Supplementary Materials. The GCKD (German Chronic Kidney Disease) study was funded by grants from the German Ministry of Education and Research (BMBF, No. 01ER0804) and the KfH Foundation for Preventive Medicine, with genotyping supported by Bayer Pharma AG. The list of GCKD investigators can be found in the Supplementary Materials. The work of M.W. and A.K. was funded by the CRC 1140 Initiative and by KO 3598/3-1 and CRC 992 (A.K.) of the German Research Foundation. The work of E.H. and R.A.K.S. was funded by the CRC 1192 from the German Research Foundation (Projects B1 and C1). P.R. is a recipient of European Research Council ERC-2012ADG_2012-0314 grant 322947, 7th Framework Programme of the European Community contract 2012-305608 (European Consortium for High-Throughput Research in Rare Kidney Diseases), and the National Research Agency grant MNaims (ANR-17-CE17-0012-01). The Dutch studies were supported by grants from the Dutch Kidney Foundation to JMH and JFW (Nierstichting Nederland grant OW08 and grant KJPB11.021). We would like to thank the Population Architecture Using Genomics and Epidemiology (PAGE) consortium, funded by the National Human Genome Research Institute (NHGRI) with co-funding from the NIMHD, for providing population controls for this study. For full acknowledgment of the PAGE consortium, please see Supplementary Materials. The funding sources were not involved in the study design, collection, analysis, and interpretation of data, writing of the report, or in the decision to submit the paper for publication.

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 and Aims We have previously shown that the Nod-like receptor protein-3 (NLRP3) gene is over-expressed in leukocytes of many patients with chronic kidney disease, and studies have also shown that hypomethylation and overexpression of NLRP3 are associated with glucocorticoid resistance in acute lymphoblastic leukemia. Therefore, we hypothesized that hypomethylation of the NLRP3 promoter my cause glucocorticoid resistance in patients with Idiopathic Nephrotic Syndrome (INS) associated with minimal change disease (MCD) or focal glomerulosclerosis (FSGS). Method We assessed NLRP3 and Caspase 1 (CASP1) promoter methylation by SNuPE reaction in germline DNA from leukocytes of 14 glucocorticoid-resistant and 18 glucocorticoid-sensitive adult INS patients (discovery cohort) and 7 glucocorticoid-resistant and 14 glucocorticoid-sensitive pediatric INS patients with MCD/FSGS (validation cohort). The effects of NLRP3 inflammasome on glucocorticoid resistance were validated in vitro in human monocytic cell lines (THP-1 and U937). Results Methylation of CpG islands in the CASP1 promoter was undetectable in both groups whereas NLRP3 promoter methylation was significantly lower in glucocorticoid-resistant compared to glucocorticoid-sensitive in both adults and children. The AUROC curve was 81.2% (p=0.0003) in adults and 73.5% (p=0.0002) in children demonstrating the capability of NLRP3 methylation to discriminate glucocorticoid-resistant versus glucocorticoid-sensitive. Activation of the NLRP3 inflammasome by LPS/ATP reduced glucocorticoid receptor expression and increased glucocorticoid resistance in U937. Consistently, knockdown of NLRP3 in THP-1 increased sensitivity to glucocorticoids. Conclusion Our findings demonstrate a novel mechanism whereby INS patients develop resistance to glucocorticoids via leukocyte epigenetic changes of NLRP3 and reveal a new potential clinical biomarker to early detect this condition.

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 Aims: Emerging evidence suggests an association between patatin-like phospholipase domain-containing protein-3 (PNPLA3) rs738409 (I148M protein variant) and risk of chronic kidney disease (CKD), but the mechanisms underpinning this association are poorly understood. Methods: We studied 157 patients with type 2 diabetes (T2DM) who underwent ultrasonography and vibration-controlled transient elastography for diagnosing nonalcoholic fatty liver disease (NAFLD). CKD was defined as estimated glomerular filtration rate (e-GFR) '60 mL/min/1.73 m 2 and/or abnormal albuminuria. We surveyed PNPLA3 mRNA expression in human tissues, using the liver as a positive control, and also measured PNPLA3 mRNA and protein expression levels in human cell lines represented in the kidney and the liver. Results: In all, 112 patients had NAFLD and 43 had CKD. Patients homozygous for the I148M variant (n = 11) had lower e-GFR levels (60.6 ± 11.7 vs 77.8 ± 15.9 vs 83.5 ± 16.5 mL/min/1.73 m 2, P =.0001) and higher prevalence of CKD (63.6% vs 24.2% vs 25.0%, P =.028), compared to those with I/M (n = 66) and I/I (n = 80) PNPLA3 genotype. The association of I148M homozygosity with lower e-GFR levels (P '.0001) and higher risk of CKD (adjusted-odds ratio 6.65; 95% CI 1.65-26.8, P =.008) was independent of liver disease severity (as detected by liver stiffness ≥7kPa) and other risk factors. PNPLA3 mRNA expression was greatest in liver and renal cortex, and podocytes showed high PNPLA3 mRNA and protein levels, comparable to that of hepatocytes and hepatic stellate cells respectively. Conclusions: The PNPLA3 I148M variant was associated with CKD, independently of common renal risk factors and severity of NAFLD PNPLA3 expression levels were particularly high in renal podocytes.

Introduction: Exosomes are nanovesicles that play important functions in a variety of physiological and pathological conditions. They are powerful cell-to-cell communication tool thanks to the protein, mRNA, miRNA, and lipid cargoes they carry. They are also emerging as valuable diagnostic and prognostic biomarker sources. Urinary exosomes carry information from all the cells of the urinary tract, downstream of the podocyte. Rare kidney diseases are a subset of an inherited diseases whose genetic diagnosis can be unclear, and presentation can vary due to genetic, epigenetic, and environmental factors. Areas covered: In this review, we focus on a group of rare and often neglected kidney diseases, for which we have sufficient available literature data on urinary exosomes. The analysis of their content can help to comprehend pathological mechanisms and to identify biomarkers for diagnosis, prognosis, and therapeutic targets. Expert opinion: The foreseeable large-scale application of system biology approach to the profiling of exosomal proteins as a source of renal disease biomarkers will be also useful to stratify patients with rare kidney diseases whose penetrance, phenotypic presentation, and age of onset vary sensibly. This can ameliorate the clinical management.

Medullary sponge kidney (MSK) disease is a rare and neglected kidney condition often associated with nephrocalcinosis/nephrolithiasis and cystic anomalies in the precalyceal ducts. Little is known about the pathogenesis of this disease, so we addressed the knowledge gap using a proteomics approach. The protein content of microvesicles/exosomes isolated from urine of 15 MSK and 15 idiopathic calcium nephrolithiasis (ICN) patients was investigated by mass spectrometry, followed by weighted gene co-expression network analysis, support vector machine (SVM) learning, and partial least squares discriminant analysis (PLS-DA) to select the most discriminative proteins. Proteomic data were verified by ELISA. We identified 2998 proteins in total, 1764 (58.9%) of which were present in both vesicle types in both diseases. Among the MSK samples, only 65 (2.2%) and 137 (4.6%) proteins were exclusively found in the microvesicles and exosomes, respectively. Similarly, among the ICN samples, only 75 (2.5%) and 94 (3.1%) proteins were exclusively found in the microvesicles and exosomes, respectively. SVM learning and PLS-DA revealed a core panel of 20 proteins that distinguished extracellular vesicles representing each clinical condition with an accuracy of 100%. Among them, three exosome proteins involved in the lectin complement pathway maximized the discrimination between MSK and ICN: Ficolin 1, Mannan-binding lectin serine protease 2, and Complement component 4-binding protein &beta, ELISA confirmed the proteomic results. Our data show that the complement pathway is involved in the MSK, revealing a new range of potential therapeutic targets and early diagnostic biomarkers.

Background The epithelial to mesenchymal transition (EMT) is a multi-factorial biological mechanism involved in renal and hepatic fibrosis and the IL-1 beta has been assumed as a mediator of this process although data are not exhaustive. Therefore, the aim of our study was to evaluate the role of this cytokine in the EMT of renal proximal tubular epithelial cells (HK-2) and stellate cells (LX-2) and the protective/anti-fibrotic effect of its inhibition by Canakinumab (a specific human monoclonal antibody targeted against IL-1beta). Methods Both cell types were treated with IL-1 beta (10 ng/ml) for 6 and 24 h with and without Canakinumab (5 μg/ml). As control we used TGF-beta (10 ng/ml). Expression of EMT markers (vimentin, alpha-SMA, fibronectin) were evaluated through western blotting and immunofluorescence. Genes expression for matrix metalloproteinases (MMP)-2 was measured by Real-Time PCR and enzymatic activity by zymography. Cellular motility was assessed by scratch assay. Results IL-1 beta induced a significant up-regulation of EMT markers in both cell types and increased the MMP-2 protein expression and enzymatic activity, similarly to TGF-beta. Moreover, IL-1 beta induced a higher rate of motility in HK-2. Canakinumab prevented all these modifications in both cell types. Conclusions Our results clearly demonstrate the role of IL-1 beta in the EMT of renal/stellate cells and it underlines, for the first time, the therapeutic potential of its specific inhibition on the prevention/minimization of organ fibrosis. Electronic supplementary material The online version of this article (10.1186/s12967-019-1770-1) contains supplementary material, which is available to authorized users.

The administration of Everolimus (EVE), a mTOR inhibitor used in transplantation and cancer, is often associated with adverse effects including pulmonary fibrosis. Although the underlying mechanism is not fully clarified, this condition could be in part caused by epithelial to mesenchymal transition (EMT) of airway cells. To improve our knowledge, primary bronchial epithelial cells (BE63/3) were treated with EVE (5 and 100 nM) for 24 h. EMT markers (α-SMA, vimentin, fibronectin) were measured by RT-PCR. Transepithelial resistance was measured by Millicell-ERS ohmmeter. mRNA and microRNA profiling were performed by Illumina and Agilent kit, respectively. Only high dose EVE increased EMT markers and reduced the transepithelial resistance of BE63/3. Bioinformatics showed 125 de-regulated genes that, according to enrichment analysis, were implicated in collagen synthesis/metabolism. Connective tissue growth factor (CTGF) was one of the higher up-regulated mRNA. Five nM EVE was ineffective on the pro-fibrotic machinery. Additionally, 3 miRNAs resulted hyper-expressed after 100 nM EVE and able to regulate 31 of the genes selected by the transcriptomic analysis (including CTGF). RT-PCR and western blot for MMP12 and CTGF validated high-throughput results. Our results revealed a complex biological network implicated in EVE-related pulmonary fibrosis and underlined new potential disease biomarkers and therapeutic targets.

Il trattamento poli-farmacologico ha determinato, nel corso degli anni, un significativo rallentamento della progressione della malattia renale cronica verso lo stadio di uremia terminale, ma siamo ancora distanti dallo sviluppo di interventi terapeutici in grado di bloccare questo inesorabile e irreversibile processo. Studi clinico-patologici hanno chiaramente dimostrato che il principale elemento coinvolto nel danno renale è la fibrosi tubulo-interstiziale e che il meccanismo patogenetico alla base di questa condizione ha inizio in larga parte nel compartimento tubulare. In particolare, il processo di transizione epitelio-mesenchimale gioca un ruolo importante nella genesi del danno cronico. Durante questo processo, le cellule epiteliali tubulari subiscono un incremento significativo di markers di superficie di natura mesenchimale e, grazie al rimodellamento del citoscheletro e alla degradazione della membrana basale, sono in grado di migrare nell'interstizio dove svolgono un ruolo chiave nel processo patogenetico. In questo contesto, sembra avere un ruolo chiave l'enzima eparanasi, una endo-β-D-glucuronidasi che taglia le catene dell'eparan-solfato a livello di siti specifici intracatena, e partecipa attivamente alla degradazione e al rimodellamento della matrice extracellulare. La degradazione dei vari costituenti dell'ECM, inclusi i proteoglicani eparan-solfato fa-vorisce il rilascio di fattori trofici quali il FGF-2 che induce l'espressione dei marcatori mesenchimali alfa-SMA, VIM e FN, porta alla degradazione della membrana basale mediante la secrezione di metalloproteinasi della matrice ed aumenta la motilità cellulare. L'epressione dell'eparanasi è regolata da fattori di trascrizione, dalla metilazione del DNA e da varie molecole endogene. L'importanza di questo enzima è stata confermata clinicamente dal riscontro di una sua iperespressione in preparati istologici di biopsie effettuate in soggetti affetti da nefropatie croniche (per esempio, nefropatia diabetica). Pertanto visto l'importante ruolo dell'eparanasi sono in fase di standardizzazione numerose strategie per inibire la sua espressione genica e/o la sua attività enzimatica. Infine, è stato proposto il suo possibile utilizzo come biomarker di progressione del danno tubulo-interstiziale da utilizzare routinariamente in ambito nefrologico.

Renal ischemia/reperfusion (I/R) injury occurs in patients undergoing renal transplantation and with acute kidney injury and is responsible for the development of chronic allograft dysfunction as characterized by parenchymal alteration and fibrosis. Heparanase (HPSE), an endoglycosidase that regulates EMT and macrophage polarization, is an active player in the biological response triggered by ischemia/reperfusion (I/R) injury. I/R was induced in vivo by clamping left renal artery for 30 min in wt C57BL/6J mice. Animals were daily treated and untreated with Roneparstat (an inhibitor of HPSE) and sacrificed after 8 weeks. HPSE, fibrosis, EMT-markers, inflammation and oxidative stress were evaluated by biomolecular and histological methodologies together with the evaluation of renal histology and measurement of renal function parameters. 8 weeks after I/R HPSE was upregulated both in renal parenchyma and plasma and tissue specimens showed clear evidence of renal injury and fibrosis. The inhibition of HPSE with Roneparstat-restored histology and fibrosis level comparable with that of control. I/R-injured mice showed a significant increase of EMT, inflammation and oxidative stress markers but they were significantly reduced by treatment with Roneparstat. Finally, the inhibition of HPSE in vivo almost restored renal function as measured by BUN, plasma creatinine and albuminuria. The present study points out that HPSE is actively involved in the mechanisms that regulate the development of renal fibrosis arising in the transplanted organ as a consequence of ischemia/reperfusion damage. HPSE inhibition would therefore constitute a new pharmacological strategy to reduce acute kidney injury and to prevent the chronic pro-fibrotic damage induced by I/R.

Oxidative stress is a well-described imbalance between the production of reactive oxygen species (ROS) and the antioxidant defense system of cells and tissues. The overproduction of free radicals damages all components of the cell (proteins, lipids, nucleic acids) and modifies their physiological functions. As widely described, this condition is a biochemical hallmark of chronic kidney disease (CKD) and may dramatically influence the progression of renal impairment and the onset/development of major systemic comorbidities including cardiovascular diseases. This state is exacerbated by exposure of the body to uremic toxins and dialysis, a treatment that, although necessary to ensure patients’ survival, exposes cells to non-physiological contact with extracorporeal circuits and membranes with consequent mitochondrial and anti-redox cellular system alterations. Therefore, it is undeniable that counteracting oxidative stress machinery is a major pharmacological target in medicine/nephrology. As a consequence, in recent years several new naturally occurring compounds, administered alone or integrated with classical therapies and an appropriate lifestyle, have been proposed as therapeutic tools for CKD patients. In this paper, we reviewed the recent literature regarding the “pioneering” in vivo testing of these agents and their inclusion in small clinical trials performed in patients affected by CKD.

Background Epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells induced by high glucose (HG) levels is a major biological mechanism leading to myofibroblast accumulation in the omentum of patients on peritoneal dialysis (PD). Heparanase (HPSE), an endoglycosidase that cleaves heparan sulfate chains, is involved in the EMT of several cell lines, and may have a major role in this pro-fibrotic process potentially responsible for the failure of dialysis. Its specific inhibition may therefore plausibly minimize this pathological condition. Methods An in vitro study employing several biomolecular strategies was conducted to assess the role of HPSE in the HG-induced mesothelial EMT process, and to measure the effects of its specific inhibition by SST0001, a N-acetylated glycol-split heparin with a strong anti-HPSE activity. Rat mesothelial cells were grown for 6 days in HG (200 mM) culture medium with or without SST0001. Then EMT markers (VIM, α-SMA, TGF-β) and vascular endothelial growth factor (VEGF) (a factor involved in neoangiogenesis) were measured by real-time PCR and immunofluorescence/western blotting. As a functional analysis, trans-epithelial resistance (TER) and permeability to albumin were also measured in our in vitro model using a Millicell-ERS ohmmeter and a spectrophotometer, respectively. Results Our results showed that 200 mM of glucose induced a significant gene and protein up-regulation of VEGF and all EMT markers after 6 days of culture. Intriguingly, adding SST0001 on day 3 reversed these biological and cellular effects. HPSE inhibition also restored the normal TER and permeability lost during the HG treatment. Conclusion Taken together, our data confirm that HG can induce EMT of mesothelial cells, and that HPSE plays a central part in this process. Our findings also suggest that pharmacological HPSE inhibition could prove a valuable therapeutic tool for minimizing fibrosis and avoiding a rapid decline in the efficacy of dialysis in patients on PD, though clinical studies and/or trials would be needed to confirm the clinical utility of this treatment.

A series of monoclonal antibodies (mAbs) are commonly utilized in renal transplantation as induction therapy (a period of intense immunosuppression immediately before and following the implant of the allograft), to treat steroid-resistant acute rejections, to decrease the incidence and mitigate effects of delayed graft function, and to allow immunosuppressive minimization. Additionally, in the last few years, their use has been proposed for the treatment of chronic antibody-mediated rejection, a major cause of late renal allograft loss. Although the exact mechanism of immunosuppression and allograft tolerance with any of the currently used induction agents is not completely defined, the majority of these medications are targeted against specific CD proteins on the T or B cells surface (e.g., CD3, CD25, CD52). Moreover, some of them have different mechanisms of action. In particular, eculizumab, interrupting the complement pathway, is a new promising treatment tool for acute graft complications and for post-transplant hemolytic uremic syndrome. While it is clear their utility in renal transplantation, it is also unquestionable that by using these highly potent immunosuppressive agents, the body loses much of its innate ability to mount an adequate immune response, thereby increasing the risk of severe adverse effects (e.g., infections, malignancies, haematological complications). Therefore, it is extremely important for clinicians involved in renal transplantation to know the potential side effects of monoclonal antibodies in order to plan a correct therapeutic strategy minimizing/avoiding the onset and development of severe clinical complications.