Claudia Curci

Endothelial-to-mesenchymal transition and renal fibrosis in ischaemia/reperfusion injury are mediated by complement anaphylatoxins and Akt pathway

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.

miR-1915 and miR-1225-5p Regulate the Expression of CD133, PAX2 and TLR2 in Adult Renal Progenitor Cells

Adult renal progenitor cells (ARPCs) were recently identified in the cortex of the renal parenchyma and it was demonstrated that they were positive for PAX2, CD133, CD24 and exhibited multipotent differentiation ability. Recent studies on stem cells indicated that microRNAs (miRNAs), a class of noncoding small RNAs that participate in the regulation of gene expression, may play a key role in stem cell self-renewal and differentiation. Distinct sets of miRNAs are specifically expressed in pluripotent stem cells but not in adult tissues, suggesting a role for miRNAs in stem cell self-renewal. We compared miRNA expression profiles of ARPCs with that of mesenchymal stem cells (MSCs) and renal proximal tubular cells (RPTECs) finding distinct sets of miRNAs that were specifically expressed in ARPCs. In particular, miR-1915 and miR-1225-5p regulated the expression of important markers of renal progenitors, such as CD133 and PAX2, and important genes involved in the repair mechanisms of ARPCs, such as TLR2. We demonstrated that the expression of both the renal stem cell markers CD133 and PAX2 depends on lower miR-1915 levels and that the increase of miR-1915 levels improved capacity of ARPCs to differentiate into adipocyte-like and epithelial-like cells. Finally, we found that the low levels of miR-1225-5p were responsible for high TLR2 expression in ARPCs. Therefore, together, miR-1915 and miR-1225-5p seem to regulate important traits of renal progenitors: the stemness and the repair capacity.

Role of let-7b in the regulation of N-acetylgalactosaminyltransferase 2 in IgA nephropathy

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.

Genome-wide scan identifies a copy number variable region at 3p21.1 that influences the TLR9 expression levels in IgA nephropathy patients

Immunoglobulin A nephropathy (IgAN) is a complex multifactorial disease characterized by genetic factors that influence the pathogenesis of the disease. In this context, an intriguing role could be ascribed to copy number variants (CNVs). We performed the whole-genome screening of CNVs in familial IgAN patients, their healthy relatives and healthy subjects (HSs). In the initial screening, we included 217 individuals consisting of 51 biopsy-proven familial IgAN cases and 166 healthy relatives. We identified 148 IgAN-specific aberrations, specifically 105 loss and 43 gain, using a new statistical approach that allowed us to identify aberrations that were concordant across multiple samples. Several CNVs overlapped with regions evidenced by previous genome-wide genetic studies. We focused our attention on a CNV located in chromosome 3, which contains the TLR9 gene and found that IgAN patients characterized by deteriorated renal function carried low copy number of this CNV. Moreover, the TLR9 gene expression was low and significantly correlated with the loss aberration. Conversely, IgAN patients with normal renal function had no aberration and the TLR9 mRNA was expressed at the same level as in HSs. We confirmed our data in another cohort of Greek subjects. In conclusion, here we performed the first genome-wide CNV study in IgAN identifying structural variants that could help the genetic dissection of this complex disease, and pointed out a loss aberration in the chromosome 3, which is responsible for the downregulation of TLR9 expression that, in turn, could contribute to the deterioration of the renal function in IgAN patients.

The Three-Gene Signature in Urinary Extracellular Vesicles from Patients with Clear Cell Renal Cell Carcinoma.

Renal cell carcinoma (RCC) accounts for more than 2% of neoplasias in humans worldwide. Renal biopsy is the gold standard among the diagnostic procedures, but it is invasive and not suitable for all patients. Therefore, new reliable and non-invasive biomarkers for RCC are required. Secretion of extracellular vesicles (EVs), containing RNA molecules that can be transferred between cells, appears to be a common feature of neoplasia. Consistently, cancer-derived EVs are increased in blood and urine. Therefore, urinary samples may be a non-invasive approach for discovering new diagnostic biomarkers. We enrolled 46 patients of whom 33 with clear cell renal cell carcinoma (ccRCC) and 22 healthy subjects (HS). Urinary EVs were isolated by differential centrifugation. Microarray analysis led to the identification of RNA molecules that were validated using RT-qPCR. We found that urinary exosomal shuttle RNA (esRNA) pattern was significantly different in ccRCC patients compared to HS and to non-clear cell RCC (non-ccRCC) and we identified three esRNAs involved in the tumor biology that may be potentially suitable as non-invasive gene signature. GSTA1, CEBPA and PCBD1 esRNA levels were decreased in urine of patients compared with HS. This pattern was specific of the ccRCC and one month after partial or radical nephrectomy the esRNA levels increased to reach the normal level. This study suggests, for the first time, the potential use of the RNA content of urinary EVs to provide a non-invasive first step to diagnose the ccRCC.

Transcriptomics in kidney biopsy is an untapped resource for precision therapy in nephrology: a systematic review

Background The diagnosis of glomerular diseases is based on the evaluation of histological lesions in renal tissue by means of light and electronic microscopy, and immunofluorescence technique. Frozen and archival formalin-fixed paraffin-embedded kidney biopsies represent a stored resource for high-throughput technologies. Transcriptomics makes it possible to study the whole gene-expression profile of cells and tissues in a specific period and/or condition. The results, whether considered alone or integrated with other omics data, could help to improve existing knowledge about the pathogenetic mechanisms of glomerulopathies. Methods This review describes the molecular analysis of histological lesions obtained by transcriptomics in glomerular diseases, such as minimal change disease, focal and segmental glomerular sclerosis, IgA nephropathy, lupus nephritis and diabetic nephropathy. Results Of 716 articles obtained through database searches, 19 relevant articles were considered for the systematic review. Transcriptomics in kidney biopsy from patients with glomerular diseases have generated new insights on a few promising genes, illustrated in each disease section, which may be considered important targets for the care of these diseases. Conclusions Transcriptomics is an untapped resource for precision nephrology. Moreover, the integration of transcriptomics and systems pharmacology could predict the best drug combination to revert a pathological condition by targeting disease-specific molecular networks.

Aberrantly methylated DNA regions lead to low activation of CD4+ T-cells in IgA nephropathy

IgAN (IgA nephropathy) is the most common form of primary glomerulonephritis worldwide and has a strong genetic component. In this setting, DNA methylation could also be an important factor influencing this disease. We performed a genome-wide screening for DNA methylation in CD4(+) T-cells from IgAN patients and found three regions aberrantly methylated influencing genes involved in the response and proliferation of CD4(+) T-cells. Two hypomethylated regions codified genes involved in TCR (T-cell receptor) signalling, TRIM27 (tripartite motif-containing 27) and DUSP3 (dual-specificity phosphatase 3), and an hypermethylated region included the VTRNA2-1 (vault RNA 2-1) non-coding RNA, also known as miR-886 precursor. We showed that the aberrant methylation influences the expression of these genes in IgAN patients. Moreover, we demonstrated that the hypermethylation of the miR-886 precursor led to a decreased CD4(+) T-cell proliferation following TCR stimulation and to the overexpression of TGFβ (transforming growth factor β). Finally, we found a Th1/Th2 imbalance in IgAN patients. The IL (interleukin)-2/IL-5 ratio was notably higher in IgAN patients and clearly indicated a Th1 shift. In conclusion, we identified for the first time some specific DNA regions abnormally methylated in IgAN patients that led to the reduced TCR signal strength of the CD4(+) T-cells and to their anomalous response and activation that could explain the T-helper cell imbalance. The present study reveals new molecular mechanisms underlying the abnormal CD4(+) T-cell response in IgAN patients.

Inhibin-A and Decorin Secreted by Human Adult Renal Stem/Progenitor Cells Through the TLR2 Engagement Induce Renal Tubular Cell Regeneration

Acute kidney injury (AKI) is a public health problem worldwide. Several therapeutic strategies have been made to accelerate recovery and improve renal survival. Recent studies have shown that human adult renal progenitor cells (ARPCs) participate in kidney repair processes, and may be used as a possible treatment to promote regeneration in acute kidney injury. Here, we show that human tubular ARPCs (tARPCs) protect physically injured or chemically damaged renal proximal tubular epithelial cells (RPTECs) by preventing cisplatin-induced apoptosis and enhancing proliferation of survived cells. tARPCs without toll-like receptor 2 (TLR2) expression or TLR2 blocking completely abrogated this regenerative effect. Only tARPCs, and not glomerular ARPCs, were able to induce tubular cell regeneration process and it occurred only after damage detection. Moreover, we have found that ARPCs secreted inhibin-A and decorin following the RPTEC damage and that these secreted factors were directly involved in cell regeneration process. Polysaccharide synthetic vesicles containing these molecules were constructed and co-cultured with cisplatin damaged RPTECs. These synthetic vesicles were not only incorporated into the cells, but they were also able to induce a substantial increase in cell number and viability. The findings of this study increase the knowledge of renal repair processes and may be the first step in the development of new specific therapeutic strategies for renal repair.

A transcriptomics study of hereditary angioedema attacks

Background Hereditary angioedema (HAE) caused by C1‐inhibitor deficiency is a lifelong illness characterized by recurrent acute attacks of localized skin or mucosal edema. Activation of the kallikrein/bradykinin pathway at the endothelial cell level has a relevant pathogenetic role in acute HAE attacks. Moreover, other pathways are involved given the variable clinical expression of the disease in different patients. Objective We sought to explore the involvement of other putative genes in edema formation. Methods We performed a PBMC microarray gene expression analysis on RNA isolated from patients with HAE during an acute attack and compared them with the transcriptomic profile of the same patients in the remission phase. Results Gene expression analysis identified 23 genes significantly modulated during acute attacks that are involved primarily in the natural killer cell signaling and leukocyte extravasation signaling pathways. Gene set enrichment analysis showed a significant activation of relevant biological processes, such as response to external stimuli and protein processing (q < 0.05), suggesting involvement of PBMCs during acute HAE attacks. Upregulation of 2 genes, those encoding adrenomedullin and cellular receptor for urokinase plasminogen activator (uPAR), which occurs during an acute attack, was confirmed in PBMCs of 20 additional patients with HAE by using real‐time PCR. Finally, in vitro studies demonstrated the involvement of uPAR in the generation of bradykinin and endothelial leakage. Conclusions Our study demonstrates the increase in levels of adrenomedullin and uPAR in PBMCs during an acute HAE attack. Activation of these genes usually involved in regulation of vascular tone and in inflammatory response might have a pathogenic role by amplifying bradykinin production and edema formation in patients with HAE.

Role of miR-422a and kallikrein-related peptidase 4 implicated in the development of lupus nephritis. Do we work in this direction?

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of circulating immune complexes in the blood that deposit in the vessel walls of many organs. The involvement of kidney is represented by lupus nephritis (LN) showing different types of histological lesions. SLE is induced by genetic susceptibility due to a deficiency of the first complement components (C2 and C4). However, >28 susceptibility loci contribute to development of the disease [1]. The most prominent defect is represented by low phagocytosis of cell debris. Thus their permanence in the blood induces the attraction of macrophages and dendritic cells that capture fragments of nuclear particles and then interact with Tand B-cells. B-cells are very active in producing large amounts of antinuclear antibodies, whereas T-cells that should regulate B-cell response have abnormal T-helper cell activity and a reduced function of T-suppressor cells. This imbalance causes abnormal production of autoantibodies and circulating immune complexes that are responsible for kidney damage in LN. Clinically, LN manifests a wide range of symptoms, from subclinical microhematuria and/or mild proteinuria to nephrotic syndrome and from normal renal function to progressive deterioration of the kidney. Due to the frequent occurrence of subclinical urinary signs, the early diagnosis of LN is many times misunderstood. Therefore, in the presence of SLE, it is recommended to analyse urine and, in the presence of urinary abnormalities, to perform kidney biopsy, providing information for early therapeutic decisions. Fortunately, not all SLE patients develop LN. A few years ago, Ge et al. [2] demonstrated in mice that the Cgnz1 allele, mapped on the distal region of chromosome 1, conferred kidney resistance to damage, preventing the progression of renal lesions in acute lupus glomerulonephritis. Thus, these mice did not show end-stage kidney disease and early mortality. The region containing the Cgnz1 allele has 45 genes, and the same investigators are generating transgenic lines covering the entire region to investigate whole genes that may be responsible for this kidney resistance to potential immune complex damage. In conclusion, the distal region of mouse chromosome 1 plays an important role for LN susceptibility. It should be noted that the homologous region in human chromosome 1 is located between 160.0 and 160.6 Mb, but there are no studies on the role of human homologous of Cgnz1 on LN development. In this issue, Krasoudaki et al. [3] expand their previous data obtained on microRNA (miRNA) signatures from peripheral blood mononuclear cells to kidney tissue. Elegantly, they first performed miRNA expression profile analysis on human kidney specimens of LN, then validated their results in vitro by human cell culture and in vivo by means of a murine model of LN. Finally, they carried out Western blot and immunohistochemistry in both human and murine kidney tissue to definitely confirm their data. ThemiRNA profiling was performed in 12 formalin-fixed paraffin-embedded (FFPE) kidney biopsy specimens from patients with active LN (8 proliferative and 4 membranous) and 3 normal FFPE kidney specimens collected in the HippoKration Hospital in Thessaloniki, Greece. Sample choice is a very challenging phase to obtain genomic data suitable for RNA analysis. Formalin fixation is widely used to preserve tissue and today represents the greatest stock of archived samples in nephrology. Nevertheless, the RNA obtained from FFPE biopsies may be degraded and fragmented due to the formalin fixation process [4], compelling scientists to increase the number of kidney biopsy samples and to assess refined techniques to overcome this challenge. Many studies have compared the differences in miRNA expression profiles between fresh-frozen and FFPE tissue samples. As shown by Klopfleisch et al. [4], miRNA