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1.  Karyopherins: potential biological elements involved in the delayed graft function in renal transplant recipients 
BMC Medical Genomics  2014;7:14.
Immediately after renal transplantation, patients experience rapid and significant improvement of their clinical conditions and undergo considerable systemic and cellular modifications. However, some patients present a slow recovery of the renal function commonly defined as delayed graft function (DGF). Although clinically well characterized, the molecular mechanisms underlying this condition are not totally defined, thus, we are currently missing specific clinical markers to predict and to make early diagnosis of this event.
We investigated, using a pathway analysis approach, the transcriptomic profile of peripheral blood mononuclear cells (PBMC) from renal transplant recipients with DGF and with early graft function (EGF), before (T0) and 24 hours (T24) after transplantation.
Bioinformatics/statistical analysis showed that 15 pathways (8 up-regulated and 7 down-regulated) and 11 pathways (5 up-regulated and 6 down-regulated) were able to identify DGF patients at T0 and T24, respectively. Interestingly, the most up-regulated pathway at both time points was NLS-bearing substrate import into nucleus, which includes genes encoding for several subtypes of karyopherins, a group of proteins involved in nucleocytoplasmic transport. Signal transducers and activators of transcription (STAT) utilize karyopherins-alpha (KPNA) for their passage from cytoplasm into the nucleus. In vitro functional analysis demonstrated that in PBMCs of DGF patients, there was a significant KPNA-mediated nuclear translocation of the phosphorylated form of STAT3 (pSTAT3) after short-time stimulation (2 and 5 minutes) with interleukin-6.
Our study suggests the involvement, immediately before transplantation, of karyopherin-mediated nuclear transport in the onset and development of DGF. Additionally, it reveals that karyopherins could be good candidates as potential DGF predictive clinical biomarkers and targets for pharmacological interventions in renal transplantation. However, because of the low number of patients analyzed and some methodological limitations, additional studies are needed to validate and to better address these points.
PMCID: PMC3975142  PMID: 24625024
Delayed graft function; Renal transplantation; Microarray
2.  Monoclonal Antibody Therapy and Renal Transplantation: Focus on Adverse Effects 
Toxins  2014;6(3):869-891.
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.
PMCID: PMC3968366  PMID: 24590384
renal transplantation; adverse effects; toxicity; Basiliximab; Rituximab; Eculizumab; malignancy; infection; toxicity
3.  Everolimus-induced epithelial to mesenchymal transition in immortalized human renal proximal tubular epithelial cells: key role of heparanase 
Everolimus (EVE) is a drug widely used in several renal transplant protocols. Although characterized by a relatively low nephrotoxicity, it may induce several adverse effects including severe fibro-interstitial pneumonitis. The exact molecular/biological mechanism associated to these pro-fibrotic effects is unknown, but epithelial to mesenchymal transition (EMT) may have a central role. Additionally, heparanase, an enzyme recently associated with the progression of chronic allograft nephropathy, could contribute to activate this machinery in renal cells.
Several biomolecular strategies (RT-PCR, immunofluorescence, zymography and migration assay) have been used to assess the capability of EVE (10, 100, 200 and 500 nM) to induce an in vitro heparanase-mediated EMT in wild-type (WT) and Heparanase (HPSE)-silenced immortalized human renal epithelial proximal tubular cells (HK-2). Additionally, microarray technology was used to find additional biological elements involved in EVE-induced EMT.
Biomolecular experiments demonstrated a significant up-regulation (more than 1.5 fold increase) of several genes encoding for well known EMT markers [(alpha-smooth muscle actin (α-SMA), Vimentin (VIM), Fibronectin (FN) and matrix metalloproteinase-9 (MMP9)], enhancement of MMP9 protein level and increment of cells motility in WT HK2 cells treated with high concentrations of EVE (higher than 100 nM). Similarly, immunofluorescence analysis showed that 100 nM of EVE increased α-SMA, VIM and FN protein expression in WT HK2 cells. All these effects were absent in both HPSE- and AKT-silenced cell lines. AKT is a protein having a central role in EMT. Additionally, microarray analysis identified other 2 genes significantly up-regulated in 100 nM EVE-treated cells (p < 0.005 and FDR < 5%): transforming growth factor beta-2 (TGFβ2) and epidermal growth factor receptor (EGFR). Real-time PCR analysis validated microarray.
Our in vitro study reveals new biological/cellular aspects of the pro-fibrotic activity of EVE and it demonstrates, for the first time, that an heparanase-mediated EMT of renal tubular cells may be activated by high doses of this drug. Additionally, our results suggest that clinicians should administer the adequate dosage of EVE in order to increase efficacy and reduce adverse effects. Finally heparanase could be a new potential therapeutic target useful to prevent/minimize drug-related systemic fibrotic adverse effects.
PMCID: PMC4222256  PMID: 24256696
Epithelial-mesenchymal transition; Everolimus; Heparanase; mTOR; Tubular cells
4.  Downregulation of Nuclear-Encoded Genes of Oxidative Metabolism in Dialyzed Chronic Kidney Disease Patients 
PLoS ONE  2013;8(10):e77847.
Mitochondria, essential eukaryotic cells organelles defined as the “powerhouse of the cell” because of their ability to produce the vast majority of energy necessary for cellular metabolism, may have a primary role in the oxidative stress-related intracellular machinery associated to chronic kidney disease (CKD).
To better assess this research assumption, we decided to study the key factors regulating mitochondrial oxidative metabolism in CKD patients in peritoneal dialysis (PD, n = 15) using several bio-molecular methodologies.
RT-PCR experiments demonstrate that the expression level of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) and nuclear respiratory factor-1 (NRF-1), two genes primarily involved in mitochondrial biogenesis and functions, were significantly hypo-expressed in peripheral blood mononuclear cells of PD patients compared to healthy subjects (HS, n = 15). Additionally, mRNA levels of several PGC1-α downstream target genes (TFAM, COX6C,COX7C, UQCRH and MCAD) were profoundly down-regulated in PD cells. TFAM protein analysis confirmed gene-expression results. High plasmatic concentration of Malondialdehyde found in PD patients, confirmed the contribution of the oxidative stress to these biological effects. Finally, Nuclear factor erythroid-derived 2-like 2 (NRF2 or NFE2L2), a transcription factor for numerous antioxidant/detoxifying enzymes and one of its target genes, superoxide dismutase-2 mitochondrial (SOD2) were up-regulated in PD compared to HS.
Our results revealed, for the first time, that CKD-PD patients’ PBMC, through a complex intracellular biochemical machinery, are able to modulate their mitochondrial functions probably in the attempt to reduce oxidative metabolic damage and to turn on a valuable defense cellular strategy against oxidative stress.
PMCID: PMC3810143  PMID: 24204994
5.  Systemic and Nonrenal Adverse Effects Occurring in Renal Transplant Patients Treated with mTOR Inhibitors  
The mammalian target of rapamycin inhibitors (mTOR-I), sirolimus and everolimus, are immunosuppressive drugs largely used in renal transplantation. The main mechanism of action of these drugs is the inhibition of the mammalian target of rapamycin (mTOR), a regulatory protein kinase involved in lymphocyte proliferation. Additionally, the inhibition of the crosstalk among mTORC1, mTORC2, and PI3K confers the antineoplastic activities of these drugs. Because of their specific pharmacological characteristics and their relative lack of nephrotoxicity, these inhibitors are valid option to calcineurine inhibitors (CNIs) for maintenance immunosuppression in renal transplant recipients with chronic allograft nephropathy. However, as other immunosuppressive drugs, mTOR-I may induce the development of several adverse effects that need to be early recognized and treated to avoid severe illness in renal transplant patients. In particular, mTOR-I may induce systemic nonnephrological side effects including pulmonary toxicity, hematological disorders, dysmetabolism, lymphedema, stomatitis, cutaneous adverse effects, and fertility/gonadic toxicity. Although most of the adverse effects are dose related, it is extremely important for clinicians to early recognize them in order to reduce dosage or discontinue mTOR-I treatment avoiding the onset and development of severe clinical complications.
PMCID: PMC3789319  PMID: 24151517
6.  Hairy cell leukemia in kidney transplantation: lesson from a rare disorder 
We report here on the diagnosis and successful treatment of a case of hairy cell leukemia (HCL) that arose 15 years after kidney transplantation in a 51-year-old patient. As soon as the diagnosis was made, HCL was treated with 2-CDA, obtaining complete hematological remission. Immunosuppression with the calcineurin inhibitor cyclosporin was maintained, and the graft was preserved. In kidney transplant recipients supported with immunosuppressive drugs, post-transplant lymphoproliferative diseases (PTLDs) are frequent and typically related to immunosuppression via a loss of control of infectious/EBV-related proliferative stimuli. To date, HCL has not been considered among PTLDs. Recently, however, the oncogenic mutation V600E of the BRAF protein kinase has been found to be a hallmark of HCL, and calcineurin inhibitors have been shown to interfere with signaling downstream of V600E BRAF early on by counteracting senescence-associated mechanisms that protect against the oncogenic potential of the mutated kinase. Such a biochemical link between the oncogene-dependent signaling and calcineurin inhibitor activities suggests that HCL in transplanted patients might be a peculiar type of PTLD based on the presence of a specific mutation. This mechanism might also be involved in other neoplasias bearing the same or similar mutations, such as melanoma and non-melanoma skin cancer.
PMCID: PMC3750510  PMID: 23927433
Hairy cell leukemia; Kidney transplantation; BRAF; V600E BRAF; Immunosuppression; Calcineurin inhibitors; Post-transplant lymphoproliferative disease; Post-transplant cancer; Treatment
7.  Genome-wide association analyses identify 18 new loci associated with serum urate concentrations 
Köttgen, Anna | Albrecht, Eva | Teumer, Alexander | Vitart, Veronique | Krumsiek, Jan | Hundertmark, Claudia | Pistis, Giorgio | Ruggiero, Daniela | O’Seaghdha, Conall M | Haller, Toomas | Yang, Qiong | Tanaka, Toshiko | Johnson, Andrew D | Kutalik, Zoltán | Smith, Albert V | Shi, Julia | Struchalin, Maksim | Middelberg, Rita P S | Brown, Morris J | Gaffo, Angelo L | Pirastu, Nicola | Li, Guo | Hayward, Caroline | Zemunik, Tatijana | Huffman, Jennifer | Yengo, Loic | Zhao, Jing Hua | Demirkan, Ayse | Feitosa, Mary F | Liu, Xuan | Malerba, Giovanni | Lopez, Lorna M | van der Harst, Pim | Li, Xinzhong | Kleber, Marcus E | Hicks, Andrew A | Nolte, Ilja M | Johansson, Asa | Murgia, Federico | Wild, Sarah H | Bakker, Stephan J L | Peden, John F | Dehghan, Abbas | Steri, Maristella | Tenesa, Albert | Lagou, Vasiliki | Salo, Perttu | Mangino, Massimo | Rose, Lynda M | Lehtimäki, Terho | Woodward, Owen M | Okada, Yukinori | Tin, Adrienne | Müller, Christian | Oldmeadow, Christopher | Putku, Margus | Czamara, Darina | Kraft, Peter | Frogheri, Laura | Thun, Gian Andri | Grotevendt, Anne | Gislason, Gauti Kjartan | Harris, Tamara B | Launer, Lenore J | McArdle, Patrick | Shuldiner, Alan R | Boerwinkle, Eric | Coresh, Josef | Schmidt, Helena | Schallert, Michael | Martin, Nicholas G | Montgomery, Grant W | Kubo, Michiaki | Nakamura, Yusuke | Tanaka, Toshihiro | Munroe, Patricia B | Samani, Nilesh J | Jacobs, David R | Liu, Kiang | D’Adamo, Pio | Ulivi, Sheila | Rotter, Jerome I | Psaty, Bruce M | Vollenweider, Peter | Waeber, Gerard | Campbell, Susan | Devuyst, Olivier | Navarro, Pau | Kolcic, Ivana | Hastie, Nicholas | Balkau, Beverley | Froguel, Philippe | Esko, Tõnu | Salumets, Andres | Khaw, Kay Tee | Langenberg, Claudia | Wareham, Nicholas J | Isaacs, Aaron | Kraja, Aldi | Zhang, Qunyuan | Wild, Philipp S | Scott, Rodney J | Holliday, Elizabeth G | Org, Elin | Viigimaa, Margus | Bandinelli, Stefania | Metter, Jeffrey E | Lupo, Antonio | Trabetti, Elisabetta | Sorice, Rossella | Döring, Angela | Lattka, Eva | Strauch, Konstantin | Theis, Fabian | Waldenberger, Melanie | Wichmann, H-Erich | Davies, Gail | Gow, Alan J | Bruinenberg, Marcel | Study, LifeLines Cohort | Stolk, Ronald P | Kooner, Jaspal S | Zhang, Weihua | Winkelmann, Bernhard R | Boehm, Bernhard O | Lucae, Susanne | Penninx, Brenda W | Smit, Johannes H | Curhan, Gary | Mudgal, Poorva | Plenge, Robert M | Portas, Laura | Persico, Ivana | Kirin, Mirna | Wilson, James F | Leach, Irene Mateo | van Gilst, Wiek H | Goel, Anuj | Ongen, Halit | Hofman, Albert | Rivadeneira, Fernando | Uitterlinden, Andre G | Imboden, Medea | von Eckardstein, Arnold | Cucca, Francesco | Nagaraja, Ramaiah | Piras, Maria Grazia | Nauck, Matthias | Schurmann, Claudia | Budde, Kathrin | Ernst, Florian | Farrington, Susan M | Theodoratou, Evropi | Prokopenko, Inga | Stumvoll, Michael | Jula, Antti | Perola, Markus | Salomaa, Veikko | Shin, So-Youn | Spector, Tim D | Sala, Cinzia | Ridker, Paul M | Kähönen, Mika | Viikari, Jorma | Hengstenberg, Christian | Nelson, Christopher P | Consortium, CARDIoGRAM | Consortium, DIAGRAM | Consortium, ICBP | Consortium, MAGIC | Meschia, James F | Nalls, Michael A | Sharma, Pankaj | Singleton, Andrew B | Kamatani, Naoyuki | Zeller, Tanja | Burnier, Michel | Attia, John | Laan, Maris | Klopp, Norman | Hillege, Hans L | Kloiber, Stefan | Choi, Hyon | Pirastu, Mario | Tore, Silvia | Probst-Hensch, Nicole M | Völzke, Henry | Gudnason, Vilmundur | Parsa, Afshin | Schmidt, Reinhold | Whitfield, John B | Fornage, Myriam | Gasparini, Paolo | Siscovick, David S | Polašek, Ozren | Campbell, Harry | Rudan, Igor | Bouatia-Naji, Nabila | Metspalu, Andres | Loos, Ruth J F | van Duijn, Cornelia M | Borecki, Ingrid B | Ferrucci, Luigi | Gambaro, Giovanni | Deary, Ian J | Wolffenbuttel, Bruce H R | Chambers, John C | März, Winfried | Pramstaller, Peter P | Snieder, Harold | Gyllensten, Ulf | Wright, Alan F | Navis, Gerjan | Watkins, Hugh | Witteman, Jacqueline C M | Sanna, Serena | Schipf, Sabine | Dunlop, Malcolm G | Tönjes, Anke | Ripatti, Samuli | Soranzo, Nicole | Toniolo, Daniela | Chasman, Daniel I | Raitakari, Olli | Kao, W H Linda | Ciullo, Marina | Fox, Caroline S | Caulfield, Mark | Bochud, Murielle | Gieger, Christian
Nature genetics  2012;45(2):145-154.
Elevated serum urate concentrations can cause gout, a prevalent and painful inflammatory arthritis. By combining data from >140,000 individuals of European ancestry within the Global Urate Genetics Consortium (GUGC), we identified and replicated 28 genome-wide significant loci in association with serum urate concentrations (18 new regions in or near TRIM46, INHBB, SFMBT1, TMEM171, VEGFA, BAZ1B, PRKAG2, STC1, HNF4G, A1CF, ATXN2, UBE2Q2, IGF1R, NFAT5, MAF, HLF, ACVR1B-ACVRL1 and B3GNT4). Associations for many of the loci were of similar magnitude in individuals of non-European ancestry. We further characterized these loci for associations with gout, transcript expression and the fractional excretion of urate. Network analyses implicate the inhibins-activins signaling pathways and glucose metabolism in systemic urate control. New candidate genes for serum urate concentration highlight the importance of metabolic control of urate production and excretion, which may have implications for the treatment and prevention of gout.
PMCID: PMC3663712  PMID: 23263486
8.  A specific immune transcriptomic profile discriminates chronic kidney disease patients in predialysis from hemodialyzed patients 
BMC Medical Genomics  2013;6:17.
Chronic kidney disease (CKD) patients present a complex interaction between the innate and adaptive immune systems, in which immune activation (hypercytokinemia and acute-phase response) and immune suppression (impairment of response to infections and poor development of adaptive immunity) coexist. In this setting, circulating uremic toxins and microinflammation play a critical role. This condition, already present in the last stages of renal damage, seems to be enhanced by the contact of blood with bioincompatible extracorporeal hemodialysis (HD) devices. However, although largely described, the cellular machinery associated to the CKD- and HD-related immune-dysfunction is still poorly defined. Understanding the mechanisms behind this important complication may generate a perspective for improving patients outcome.
To better recognize the biological bases of the CKD-related immune dysfunction and to identify differences between CKD patients in conservative (CKD) from those in HD treatment, we used an high-throughput strategy (microarray) combined with classical bio-molecular approaches.
Immune transcriptomic screening of peripheral blood mononuclear cells (1030 gene probe sets selected by Gene-Ontology) showed that 275 gene probe sets (corresponding to 213 genes) discriminated 9 CKD patients stage III-IV (mean ± SD of eGFR: 32.27±14.7 ml/min) from 17 HD patients (p < 0.0001, FDR = 5%). Seventy-one genes were up- and 142 down-regulated in HD patients. Functional analysis revealed, then, close biological links among the selected genes with a pivotal role of PTX3, IL-15 (up-regulated in HD) and HLA-G (down-regulated in HD). ELISA, performed on an independent testing-group [11 CKD stage III-IV (mean ± SD of eGFR: 30.26±14.89 ml/min) and 13 HD] confirmed that HLA-G, a protein with inhibition effects on several immunological cell lines including natural killers (NK), was down-expressed in HD (p = 0.04). Additionally, in the testing-group, protein levels of CX3CR1, an highly selective chemokine receptor and surface marker for cytotoxic effector lymphocytes, resulted higher expressed in HD compared to CKD (p < 0.01).
Taken together our results show, for the first time, that HD patients present a different immune-pattern compared to the un-dialyzed CKD patients. Among the selected genes, some of them encode for important biological elements involved in proliferation/activation of cytotoxic effector lymphocytes and in the immune-inflammatory cellular machinery. Additionally, this study reveals new potential diagnostic bio-markers and therapeutic targets.
PMCID: PMC3655909  PMID: 23663527
9.  A new mechanism of action of sulodexide in diabetic nephropathy: inhibits heparanase-1 and prevents FGF-2-induced renal epithelial-mesenchymal transition 
Epithelial-mesenchymal transition of tubular cells is a widely recognized mechanism that sustains interstitial fibrosis in diabetic nephropathy (DN). The signaling of FGF-2, a growth factor involved in this mechanism, is regulated by glycosaminoglycans. Heparanase-1, an endoglycosidase that cleaves heparan sulfate, is implicated in the pathogenesis of diabetic nephropathy and is necessary to FGF-2 for the induction of tubular cells transition. Well known Heparanase-1 inhibitors are heparin(s) and sulodexide, a low-molecular weight heparin – dermatan sulphate blend, which is effective in the treatment of DN.
We have investigated the inhibition by sulodexide and its components of Heparanase-1 by an ELISA assay. We have analyzed its effect on the epithelial-mesenchymal transition of tubular cells by real time gene expression analysis, zymography and migration assay.
Results show that sulodexide is an effective heparanase-1 inhibitor, exclusively in virtue to the heparin component, with an IC50 of 5 μg/ml. In FGF-2 treated tubular cells, sulodexide also prevents the over-expression of the mesenchymal markers αSMA, vimentin and fibronectin and the motility increase, i.e. the epithelial-mesenchymal transition of tubular cells. Moreover, sulodexide prevents FGF-2 induced heparanase-1 and MMP9 increase switching off the autocrine loop that FGF-2 activates to support its signal.
The findings highlight the capacity of sulodexide to inhibit heparanase-1 and to control tubular fibrosis triggered by epithelial-mesenchymal transition. In conclusion, these sulodexide activities support the value of this agent in controlling the progression of nephropathy to renal failure.
PMCID: PMC3562257  PMID: 23095131
Diabetic nephropathy; Epithelial-mesenchymal transition; Fibrosis; Heparanase-1; Sulodexide; Tubular cells
10.  Evaluation of Hepcidin Isoforms in Hemodialysis Patients by a Proteomic Approach Based on SELDI-TOF MS 
The hepatic iron regulator hormone hepcidin consists, in its mature form, of 25 amino acids, but two other isoforms, hepcidin-20 and hepcidin-22, have been reported, whose biological meaning remains poorly understood. We evaluated hepcidin isoforms in sera from 57 control and 54 chronic haemodialysis patients using a quantitative proteomic approach based on SELDI-TOF-MS. Patients had elevated serum levels of both hepcidin-25 and hepcidin-20 as compared to controls (geometric means: 7.52 versus 4.69 nM, and 4.06 versus 1.76 nM, resp., P < .05 for both). The clearance effects of a single dialysis session by different dialysis techniques and membranes were also investigated, showing an average reduction by 51.3% ± 29.2% for hepcidin-25 and 34.2% ± 28.4% for hepcidin-20 but only minor differences among the different dialysis modalities. Measurement of hepcidin isoforms through MS-based techniques can be a useful tool for better understanding of their biological role in hemodialysis patients and other clinical conditions.
PMCID: PMC2857619  PMID: 20414466

Results 1-10 (10)