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BACKGROUND AND AIMS Several models have been proposed to describe the pathogenesis of Immunoglobulin A nephropathy (IgAN) and, among them, the multihit model where the gut-microbiota may play an important role. These models explain the pathogenesis of IgAN caused by the production of aberrant IgA, but it is believed that further predisposing factors are present, including immunological, genetic, environmental or nutritional factors. Recently, the role of IL-6 in IgAN pathogenesis is becoming increasingly important. It is essential for the deposition of glomerular immunoglobulin A and the development of renal disease in Cd37-deficient mice, although the pathogenetic mechanisms that determine its increase are not well known. A possible hypothesis emerges from our recent work on genome-wide DNA methylation screening in patients with IgAN, which identified, among other findings, a hypermethylated region comprising Vault 2–1 RNA (VTRNA2-1), a non-RNA coding also known as a precursor of miR-886 (pre-mi-RNA). Consistently, VTRNA2-1 expression was found downregulated in IgAN patients. Here we studied the involvement of the VTRNA2-1/PKR/CREB/IL-6 pathway in IgAN. METHOD Total RNA were isolated from PBMCs of IgAN patients, transplanted IgAN patients (TP-IgAN), non-IgAN transplanted patients (TP) and healthy subjects (HS). VTRNA2-1, CREB and PKR transcripts were evaluated by RT-PCR. Total and phosphorylated PKR, CREB and Il-6 proteins were evaluated by ELISA. Poly (I: C), a synthetic analogue of dsRNA and Pfizer-BioNTech COVID-19 COMIRNATY vaccine were used to transfect patient PBMCs. PKR inhibitor imoxin (C16) 1 µM was used to stimulate patient PBMCs. RESULTS Here we confirm that VTRNA2-1 transcript was down-regulated in native and transplanted IgAN subjects compared to HS and non IgAN transplanted patients, with a decrease of 30- and 100-folds, respectively (P Since PKR is normally activated by bacterial and viral RNA, we hypothesized that these microorganisms can further activate the PKR/CREB/IL-6 pathway leading to an excess of IL-6 production. This may explain both the high levels of IL-6, and infection involvement in the disease, and cases of IgAN associated with COVID-19 infection or with COVID-19 RNA-vaccination, and recent data showing microbiota involvement in IgAN. Effectively, we found that IgAN PMBCs stimulated with RNA poly(I: C) or the COVID-19 RNA-vaccine showed a significant increase in IL-6 levels compared to not-stimulated PBMCs (P Finally, we showed that the IL-6 secretion can be reduced by the PKR inhibitor imoxin (fold decrease of 5-folds in IgAN and TP-IgAN patients; P CONCLUSION In conclusion, the discovery of the upregulated VTRNA2-1/PKR/CREB/IL-6 pathway in IgAN patients may provide a new pathogenic mechanism in IgAN and may be useful for the development of novel therapeutic approaches, likely by modulating the VTRNA2-1 methylation level in IgAN patients.

BACKGROUND AND AIMS The complex system of human adult renal stem/progenitor cells (ARPCs) and their crucial role in the renewal of epithelial renal cells and regenerative processes in the adult kidneys have been recently discovered. In the human kidney, CD133+/CD24+ ARPCs are present within different segments of the nephron as a population with epithelial expansion, self-renewal and differentiation capabilities. Understanding the factors that regulate the ARPCs behaviour is fundamental to completely take advantage of their potential and to develop treatments for renal injury. Recent studies showed that long no-coding RNA (lncRNAs) are essential to establish developmental patterning and maintain the stem cell pluripotency network, further underscoring their important role in stem cell biology/technology and cellular reprogramming. lncRNAs can be dysregulated in different types of diseases and can also modulate the cellular senescence processes. For the first time, we studied their function in ARPCs showing that they express high levels of a particular lncRNA, HOTAIR, influencing cell senescence. METHOD Whole-genome lncRNA expression was performed by Agilent microarray. lncRNA expression was validated by real-time PCR. CRISPR/Cas9 system has been used to knock-down HOTAIR lncRNA. SA-β-Gal experiments were used to evaluate cellular senescence in normal ARPCs and ARPCs knock-out for HOTAIR. By ELISA, it was evaluated the expression of secreted anti-ageing protein Klotho. Fluorescence-activated cell sorting (FACS) was applied to measure CD133 and protein p15 expression in normal and transfected cells. Chromatin immunoprecipitation assay (ChIP) was used to evaluate H3K27me3 in the promoter of p15. RESULTS We studied the lncRNA profile of renal proximal tubular cells (RPTEC) and tubular ARPCs. We found 611 lncRNAs specifically expressed in ARPCs compared with RPTECs (Fold Change > 2; FDR We discovered that ARPCs produce α-Klotho at much higher levels compared to RPTEC, at a mean concentration of 200 pg/mL versus 33 pg/mL (Fold increase = 6.6; P CONCLUSION These data demonstrated that HOTAIR regulates the self-renewal capacity of ARPCs and prevents them from becoming senescent in the short term. Moreover, HOTAIR influences ARPC ability to secrete high levels of α-Klotho, and those renal progenitors, through α-Klotho secretion, are able to influence its levels in surrounding tissues and to modulate, therefore, kidney ageing.

Background and Aims EVs (Extracellular vesicles) are circulating microparticles able to mediate cell-to cell communication by carrying proteins, DNA, RNA or antibodies on their surface. EV are emerging as pivotal in renal Antibody-mediated rejection (AMR), one of the major causes of transplant failure associated to a massive complement activation. AMR is characterized by endothelial to mesenchymal transition (EndMT) and the occurrence of tubular accelerated senescence process, known as graft “Inflammaging”. However, the potential role of EVs in accelerating the renal aging and graft fibrosis after AMR is not well understood. Method Plasma EVs were isolated from 10 Acute AMR (AAMR), 10 Chronic AMR (CAMR) patients, 5 stable graft transplanted patients and 5 healthy volunteers. EVs were isolated by ultracentrifugation at 100.000 g for 1h at 4°C, quantified by Nanoparticle Tracking Analysis (Nanosight, NTA EV/ml) and characterized by FACS (Attune Nxt). RPTEC and endothelial cell culture were incubated with EVs (5e+4 EVs/cells target for 24h. MTT test was performed to assess cell viability. Cellular senescence was investigated by qPCR for p21, p53, Klotho and CYP1B1 and SA-β-gal staining were performed. To assess EndMT analysis for CD31, VE-cadherin, Vimentin, Collagen I was performed by FACS. mRNA level of C3 and CFH were also measured by qPCR and C4d deposits were evaluated in endothelial cell colture by IF. Renal biopsies were then analyzed for inflammaging (p16INK4a) and EndMT markers (CD31/αSMA) by IHC and IF. Results The Nanosight analysis showed significant differences in the amount of-EVs count per ml of plasma in AMR patients compared to healthy subjects. EVs appeared to be significantly augmented in acute AMR, in a higher manner than chronic AMR (NTA, p=0.0154). By cytofluorimetric analysis, the endothelial markers CD31 and VE- cadherin appeared to be significantly increased compared of healthy control, indicating a predominant endothelial EV origin (p Conclusion These results suggest a putative role for circulating AMR derived-EVs in inducing the tubular inflammaging by local complement activation and early fibrosis by EndMT. The EVs cargo characterization that is ongoing might highlight novel targets for therapeutic intervention.

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 Sepsis-induced acute kidney injury (AKI) is a growing health care problem, refractory to conventional treatments. This disease is characterized by an overwhelmed immune response against a primary insult that become responsible for renal dysfunction and poor outcome. Therapeutic strategies based on blood purification have been developed for the treatment of this disease. The use of polymethyl methacrylate (PMMA) membrane hemofilter in continuous hemodiafiltration (CHDF) modality showed better hemodynamic stability and efficient renal support in chronic dialysis maintenance. Here we investigated the efficacy of Hemofeel PMMA membrane (TORAY, Japan) in interfering with Complement activation and renal damage in a swine model of sepsis-induced AKI. Method After 3 hours from LPS infusion, 7 hours of PMMA-CVVH treatment or 7 hours of polysulfone (PSF)-CVVH were performed. Animals were sacrificed after 24h from LPS infusion. Histologic and renal function parameters were analyzed in all pigs. Pentraxin-3 (PTX3) and C5b-9 deposits were assessed on renal biopsies. Systemic Complement activation was evaluated by Wieslab kit. Gene expression profile was obtained from isolated PBMCs by Agilent SurePrint G3 Porcine Gene Expression Microarrays. Genespring and R software were used for the analysis. Results were validated by Real-time PCR. Results Analysis of renal biopsies from septic pigs presented increased interstitial leucocyte infiltrate, extensive collagen deposition and diffuse glomerular thrombi compared to healthy pigs (p2 ; false discovery rate Conclusion Our data suggest that LPS induced AKI is characterized by activation of Classical and alternative Complement pathways resulting in significant renal tissue damage. By interfering with complement activation and inflammatory response, PMMA membrane might prevent dysfunctional activation of resident renal cells with prevention of sepsis-induced AKI.

Background and Aims Cisplatin, is a nonspecific cytotoxic agent that primarily interferes with cellular DNA replication and the cell cycle, nevertheless it lacks tumor selectivity and acts also in normal cells. The most serious adverse reaction of cisplatin is Acute Kidney Injury (AKI), limiting its use and efficacy in chemotherapy. Cisplatin nephrotoxicity is observed in more than 30% of older patients, however the mechanism of nephrotoxicity remains unclear and specific preventive measures are not available. Today, there is an urgent need for specific nephroprotective strategies to be used during cisplatin chemotherapy. Recently, we found that tubular stem/progenitor cells (tARPC) are able to protect the tubular epithelial (RPTEC) from cisplatin induced injury, preserving their proliferation and inhibiting apoptosis. The aim of this study was to identify the molecular mechanisms involved in tARPC-mediated resistance to cisplatin. Method Co-cultures of RPTEC cells and tARPCs were exposed to cisplatin (2.5 µM) for 6 h and then kept in culture for 96 h. Gene expression profile was obtained from tARPCs and RPTECs by Agilent SurePrint G3 Human Gene Expression Microarrays. Genespring and R software were used for the analysis. Gene expression data were validated by Real-time PCR. Extracellular vesicles were isolated from cell culture supernatant by miRCURY Exosome Cell/Urine/CSF Kit (Qiagen) and RNA contained in extracellular vesicles was purified, analyzed in quality by Bioanalyzer (RNA nano) and evaluated by qPCR. The BrdU assay and caspase 3 were used to measure proliferation and apoptosis levels. Immunohistochemical expression of activated caspase-3 was used as a marker of apoptosis in RPTECs. Results By a whole-genome gene expression analysis, we found 107 genes specifically modulated by RPTECs in response to cisplatin and, among these, 30 genes induced by ARPCs following the cisplatin damage. In particular, we found a strong upregulation of the CYP1B1 gene (false discovery rate corrected p value These data support the hypothesis that ARPCs are sensor of cisplatin damaged-RPTEC and confers cisplatin resistance by transferring CYP1B1 gene in extracellular vesicles. Conclusion This is the first evidence of a cisplatin-induced overexpression of CYP1b1 in renal epithelial cells as a defense mechanism against cisplatin toxicity. This is consistent with our previous data showing that renal progenitors are resistant to cisplatin. The findings may have biological and clinical significance in terms of their implications in cellular communications and potential use of CYP1B1 as biomarkers for AKI induced by cisplatin or as protective agent.

Background and Aims The IgA nephropathy (IgAN) is the most frequent primitive glomerulonephritis. In the last years, the role of mucosal immunity in IgAN, together with that of the gut microbiota in the activation of innate and adaptive immune cells, has gained importance. Particularly interesting is the role of the microbiota and intestinal immunity in IgAN. BAFF and APRIL can be produced by the intestinal epithelium, in response to signals triggered by TLRs once activated by the commensal bacteria present in the intestinal lumen, representing the link between microbiota and intestinal immunity. To date, even if hypothesized, this relationship in IgAN patients has not been investigated. Here, we studied the intestinal-renal axis connections analyzing levels of BAFF, April and intestinal-activated B cells in IgAN patients. Method Serum and fecal samples were collected from 44 IgAN patients, 22 non-IgA glomerulonephritides (controls) and 22 healthy subjects (HS) with similar clinical features. BAFF and APRIL serum levels were measured by ELISA assay. Metabolomic analysis of fecal microbiome was performed using Biochrom 30 series amino acid analyzer and gas-chromatography mass spectrometry/solid-phase microextraction (GC-MS/SPME) analysis. B cell subsets were investigated by FACS. Results IgAN patients had increased serum levels of BAFF cytokine compared to the control group of patients with non-IgA glomerulonephritis and compared with HS (p We correlated serum BAFF levels with fecal concentration of 5 different metabolites of 30 IgAN patients, which were previously investigated for the fecal microbiota. These organic compounds had been found at significantly higher levels in the feces of IgAN patients compared to HS. Serum BAFF levels positively correlated with the levels of fecal metabolites: 4-(1,1,3,3-tetramethylbutyl) phenol (r2 = 0.2882, p = 0.0027), p-tert-butyl-phenol (r2 = 0.386, p = 0.0003), methyl neopentyl phthalic acid (r2 = 0.3491, p =0.0007), hexadecyl ester benzoic acid (r2 = 0.2832, p =0.003) and furanone A (r2 = 0.1743, p = 0.024). Serum levels of APRIL were significantly increased in IgAN patients respect to control groups (4.49 ± 0.54 vs 2.27 ± 1 ng/ml, p=0.0014). We found a correlation between APRIL and serum creatinine (r2 = 0.159, p =0.04) and eGFR (r2 = 0.2395, p =0.0082), while no correlation was found between APRIL and fecal metabolite levels in IgAN patients. In addition, we found that subjects with IgAN have a significantly higher proportion of circulating Bregs, Memory B cells and IgA secreting-plasmablasts activated at the intestinal level (CCR9+INTB7+) compared to HS. Conclusion The results of our study showed for the first time an important correlation of serum levels of BAFF with intestinal microbiota in patients with IgAN, confirming the hypothesis of the pathogenic role of intestinal mucosal hyperresponsiveness in the IgAN patients. The intestinal-renal axis plays a crucial role in Berger's glomerulonephritis, whose complex pathogenesis may contribute several factors as genetics, pathogens and food.

BACKGROUND Chronic T cell-mediated rejection (TCMR) in kidney graft is characterized by reduction of the vessel lumen with marked intimal thickening, fibrous hyperplasia of the small renal arteries and leukocyte infiltrates. The aim of this study was to find specific gene expression profiles in chronic TCMR kidney biopsies. METHODS RNA extracted from archival formalin-fixed, paraffin-embedded renal biopsies was used for gene expression profiling. Our study included 14 patients with chronic TCMR and 10 with acute TCMR. Fifty-two cadaveric donors were used as controls. The results were validated in an independent set of kidney biopsies. RESULTS We identified 616 and 243 differentially expressed genes with a fold change ≥1.5 and a false discovery rate

Background IgA nephropathy (IgAN) is characterized by aberrant O-glycosylation in the hinge region of IgA1. The early step in O-glycan formation is the attachment of N-acetylgalactosamine (GalNAc) to the serine/threonine of the hinge region; the process is catalysed by UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2). In our previous work, the microarray analysis on peripheral blood mononuclear cells (PBMCs) identified an upregulated miRNA called let-7b. Methods To study the molecular mechanisms in which let-7b was involved, we performed a bioinformatic analysis to predict their target genes. To validate biologically let-7b targets, we performed transient transfection experiments ex vivo using PBMCs from an independent group of IgAN patients and healthy blood donors (HBDs). Results Bioinformatic analysis revealed that GALNT2 is the potential target of let-7b. We found this miRNA significantly upregulated in PBMCs of IgAN patients compared with HBDs. Then, we demonstrated in ex-vivo experiments that let-7b decreased GALNT2 levels in PBMCs of IgAN patients, whereas the loss of let-7b function in PBMCs of HBDs led to an increase of GALNT2 mRNA and its protein level. Finally, we found that upregulation of let-7b occurred also in B-lymphocytes from IgAN patients. Conclusions Our results give novel additional information on the abnormal O-glycosylation process of IgA1 in IgAN patients. This study provides evidence for another important miRNA-based regulatory mechanism of the O-glycosylation process in which the deregulated expression of let-7b is associated with altered expression of GALNT2. This finding could be taken into consideration for new therapeutic approaches in IgAN because other serum glycosylated proteins do not display abnormal glycosylation.

Background Increasing evidence demonstrates a phenotypic plasticity of endothelial cells (ECs). Endothelial-to-mesenchymal transition (EndMT) contributes to the development of tissue fibrosis. However, the pathogenic factors and signalling pathways regulating this process in ischaemia/reperfusion (I/R) injury are still poorly understood. Methods We investigated the possible role of complement in the induction of this endothelial dysfunction in a swine model of renal I/R injury by using recombinant C1 inhibitor in vivo. Results Here, we showed that I/R injury reduced the density of renal peritubular capillaries and induced tissue fibrosis with generation of CD31(+)/α-SMA(+) and CD31(+)/FPS-1(+) cells indicating EndMT. When we inhibited complement, the process of EndMT became rare, with preserved density of peritubular capillaries and significant reduction in renal fibrosis. When we activated ECs by anaphylatoxins in vitro, C3a and C5a led to altered endothelial phenotype with increased expression of fibroblast markers and decrease expression of specific endothelial markers. The activation of Akt pathway was pivotal for the C3a and C5a-induced EndMT in vitro. In accordance, inhibition of complement in vivo led to the abrogation of Akt signalling, with hampered EndMT and tissue fibrosis. Conclusions Our data demonstrate a critical role for complement in the acute induction of EndMT via the Akt pathway. Therapeutic inhibition of these systems may be essential to prevent vascular damage and tissue fibrosis in transplanted kidney.