LPS removal reduces CD80-mediated albuminuria in critically ill patients with Gram-negative sepsis.

LPS-induced sepsis is a leading cause of acute kidney injury (AKI) in critically ill patients. LPS may induce CD80 expression in podocytes with subsequent onset of proteinuria, a risk factor for progressive chronic kidney disease (CKD) frequently observed after AKI. This study aimed to investigate the therapeutic efficacy of LPS removal in decreasing albuminuria through the reduction of podocyte CD80 expression. Between January 2015 and December 2017, 70 consecutive patients with Gram-negative sepsis-induced AKI were randomized to either have coupled plasma filtration and adsorption (CPFA) added to the standard care ( n = 35) or not ( n = 35). To elucidate the possible relationship between LPS-induced renal damage, proteinuria, and CD80 expression in Gram sepsis, a swine model of LPS-induced AKI was set up. Three hours after LPS infusion, animals were treated or not with CPFA for 6 h. Treatment with CPFA significantly reduced serum cytokines, C-reactive protein, procalcitonin, and endotoxin levels in patients with Gram-negative sepsis-induced AKI. CPFA significantly lowered also proteinuria and CD80 urinary excretion. In the swine model of LPS-induced AKI, CD80 glomerular expression, which was undetectable in control pigs, was markedly increased at the podocyte level in LPS-exposed animals. CPFA significantly reduced LPS-induced proteinuria and podocyte CD80 expression in septic pigs. Our data indicate that LPS induces albuminuria via podocyte expression of CD80 and suggest a possible role of timely LPS removal in preventing the maladaptive repair of the podocytes and the consequent increased risk of CKD in sepsis-induced AKI.

Peroxisome proliferator-activated receptor γ-mediated induction of microRNA-145 opposes tumor phenotype in colorectal cancer.

UNLABELLED: MicroRNAs (miRNAs) regulate diverse biological processes by inhibiting translation or inducing degradation of target mRNAs. miR-145 is a candidate tumor suppressor in colorectal carcinoma (CRC). Colorectal carcinogenesis involves deregulation of cellular processes controlled by a number of intertwined chief transcription factors, such as PPARγ and SOX9. Since PPAR family members are able to modulate complex miRNAs networks, we hypothesized a role of miRNA-145 in the interaction between PPARγ and SOX9 in colorectal carcinogenesis. To address this issue, we evaluated gene expression in tissue specimens of CRC patients and we took advantage of invitro models represented by CRC derived cell lines (CaCo2, SW480, HCT116, and HT-29), employing PPARγ activation and/or miRNA-145 ectopic overexpression to analyze how their interplay impact the expression of SOX9 and the development of a malignant phenotype. RESULTS: PPARγ regulates the expression of miR-145 by directly binding to a PPAR response element (PPRE) in its promoter at -1207/-1194bp from the transcription start site. The binding is essential for miR-145 upregulation by PPARγ upon rosiglitazone treatment. Ectopic expression of miR-145, in turn, regulates SOX9 expression through the binding to specific seed motifs. The PPARγ-miR-145-SOX9 axis overarches cell cycle progression, invasiveness and differentiation of CRC derived cell lines. Together, these results suggest that miR-145 is a novel target of PPARγ, acts as a tumor suppressor in CRC cell lines and is a key regulator of intestinal cell differentiation by directly targeting SOX9, a marker of undifferentiated progenitors in the colonic crypts.

Interplay between SOX9, ß-catenin and PPARγ activation in colorectal cancer.

Colorectal carcinogenesis relies on loss of homeostasic mechanisms regulating cell proliferation, differentiation and survival. These cell processes have been reported to be influenced independently by transcription factors activated downstream of the Wnt pathway, such as SOX9 and ß-catenin, and by the nuclear receptor PPARγ. The purpose of this study was to explore the expression levels and functional link between SOX9, ß-catenin and PPARγ in the pathogenesis of colorectal cancer (CRC). We evaluated SOX9, ß-catenin and PPARγ expression levels on human CRC specimens by qPCR and immunoblot detection. We tested the hypothesis that PPARγ activation might affect SOX9 and ß-catenin expression using four colon cancer cell lines (CaCo2, SW480, HCT116, and HT29 cells). In CRC tissues SOX9 resulted up-regulated at both mRNA and protein levels when compared to matched normal mucosa, ß-catenin resulted up-regulated at protein levels, while PPARG mRNA and PPARγ protein levels were down-regulated. A significant relationship was observed between high PPARG and SOX9 expression levels in the tumor tissue and female gender (p=0.005 and p=0.04, respectively), and between high SOX9 expression in the tumor tissue and age (p=0.04) and microsatellite instability (MSI), in particular with MSI-H (p=0.0002). Moreover, treatment with the synthetic PPARγ ligand rosiglitazone induced different changes of SOX9 and ß-catenin expression and subcellular localization in the colon cancer cell lines examined. In conclusion, SOX9, ß-catenin and PPARγ expression levels are deregulated in the CRC tissue, and in colon cancer cell lines ligand-dependent PPARγ activation unevenly influences SOX9 and ß-catenin expression and subcellular localization, suggesting a variable mechanistic role in colon carcinogenesis.

Endothelial dysfunction and renal fibrosis in endotoxemia-induced oliguric kidney injury: possible role of LPS-binding protein.

INTRODUCTION: The pathophysiology of endotoxemia-induced acute kidney injury (AKI) is characterized by an intense activation of the host immune system and renal resident cells by lipopolysaccharide (LPS) and derived proinflammatory products. However, the occurrence of renal fibrosis in this setting has been poorly investigated. The aim of the present study was to investigate the possible association between endothelial dysfunction and acute development of tissue fibrosis in a swine model of LPS-induced AKI. Moreover, we studied the possible effects of coupled plasma filtration adsorption (CPFA) in this setting. METHODS: After 9 hours from LPS infusion and 6 hours of CPFA treatment, histologic and biochemical changes were analyzed in pigs. Apoptosis and endothelial dysfunction were assessed on renal biopsies. The levels of LPS-binding protein (LBP) were quantified with enzyme-linked immunosorbent assay (ELISA). Endothelial cells (ECs) were stimulated in vitro with LPS and cultured in the presence of swine sera and were analyzed with FACS and real-time RT-PCR. RESULTS: In a swine model of LPS-induced AKI, we observed that acute tubulointerstitial fibrosis occurred within 9 hours from LPS injection. Acute fibrosis was associated with dysfunctional alpha-smooth muscle actin (α-SMA)+ ECs characterized by active proliferation (Ki-67+) without apoptosis (caspase-3-). LPS led to EC dysfunction in vitro with significant vimentin and N-cadherin expression and increased collagen I mRNA synthesis. Therapeutic intervention by citrate-based CPFA significantly prevented acute fibrosis in endotoxemic animals, by preserving the EC phenotype in both peritubular capillaries and renal arteries. We found that the removal of LBP from plasma was crucial to eliminate the effects of LPS on EC dysfunction, by blocking LPS-induced collagen I production. CONCLUSIONS: Our data indicate that EC dysfunction might be pivotal in the acute development of tubulointerstitial fibrosis in LPS-induced AKI. Selective removal of the LPS adaptor protein LBP might represent a future therapeutic option to prevent EC dysfunction and tissue fibrosis in endotoxemia-induced AKI.

Functional characterization of the OFD1 protein reveals a nuclear localization and physical interaction with subunits of a chromatin remodeling complex.

Oral-facial-digital (OFD) type I syndrome is an X-linked dominant disease (MIM311200) characterized by malformations of oral cavity, face, and digits and by cystic kidneys. We previously identified OFD1, the gene responsible for this disorder, which encodes for a centrosomal protein with an unknown function. We now report that OFD1 localizes both to the primary cilium and to the nucleus. Moreover, we demonstrate that the OFD1 protein is able to self-associate and that this interaction is mediated by its coiled-coil rich region. Interestingly, we identify an OFD1-interacting protein RuvBl1, a protein belonging to the AAA(+)-family of ATPases, which has been recently associated to cystic kidney in zebrafish and to ciliary assembly and function in Chlamydomonas reinhardtii. We also provide experimental evidence that OFD1, together with RuvBl1, is able to coimmunoprecipitate with subunits of the human TIP60 histone acetyltransferase (HAT) multisubunit complex. On the basis of these results, we hypothesize that OFD1 may be part of a multi-protein complex and could play different biological functions in the centrosome-primary cilium organelles as well as in the nuclear compartment.

Mutational spectrum of the oral-facial-digital type I syndrome: a study on a large collection of patients.

Oral-facial-digital type I (OFDI) syndrome is a male-lethal X-linked dominant developmental disorder belonging to the heterogeneous group of oral-facial-digital syndromes (OFDS). OFDI is characterized by malformations of the face, oral cavity, and digits. Central nervous system (CNS) abnormalities and cystic kidney disease can also be part of this condition. This rare genetic disorder is due to mutations in the OFD1 gene that encodes a centrosome/basal body protein necessary for primary cilium assembly and for left-right axis determination, thus ascribing OFDI to the growing number of disorders associated to ciliary dysfunction. We now report a mutation analysis study in a cohort of 100 unrelated affected individuals collected worldwide. Putative disease-causing mutations were identified in 81 patients (81%). We describe 67 different mutations, 64 of which represent novel mutations, including 36 frameshift, nine missense, 11 splice-site, and 11 nonsense mutations. Most of them concentrate in exons 3, 8, 9, 12, 13, and 16, suggesting that these exons may represent mutational hotspots. Phenotypic characterization of the patients provided a better definition of the clinical features of OFDI syndrome. Our results indicate that renal cystic disease is present in 60% of cases >18 years of age. Genotype-phenotype correlation did not reveal significant associations apart for the high-arched/cleft palate most frequently associated to missense and splice-site mutations. Our results contribute to further expand our knowledge on the molecular basis of OFDI syndrome.

Identification of GLA gene deletions in Fabry patients by Multiplex Ligation-dependent Probe Amplification (MLPA).

Fabry disease is an under-recognized X-linked lysosomal disorder, due to alpha-galactosidase A deficiency. Most of the mutations in the GLA gene are detectable using genomic sequencing analysis. However, deletions of one or more exons or deletion encompassing the entire gene are undetectable, especially in heterozygous females. The Multiplex Ligation-dependent Probe Amplification (MLPA) is an efficient tool for discovering these rearrangements. In this study two novel different deletions were detected using MLPA assay on two Fabry patients, both resulted mutation negative by sequencing analysis. These data suggest that this screening should be systematically included in genetic testing surveys of patients with Fabry disease.

Nano-LC-MS/MS for the identification of proteins trapped in sorbent cartridges used for coupled plasma filtration-adsorption treatments of healthy pigs.

A dedicated proteomic approach based on nano-Liquid Chromatography coupled with tandem mass spectrometry in ion trap is proposed for the analysis of proteins trapped in sorbent resin cartridges used to remove inflammatory mediators from blood by coupled plasma filtration adsorption (CPFA). The final purpose of the proposed proteomic approach was to obtain a reference map of plasma proteins trapped in CPFA sorbents used for the extracorporeal blood purification of healthy pigs, with the potential impact to design new bio-filters able to control the inflammatory imbalance under pathological conditions, such as severe sepsis. The five main steps of the proteomics analysis, (i) protein extraction from resin cartridges, (ii) two-dimensional gel electrophoresis (2D-PAGE) for protein separation and profiling, (iii) in-gel proteolytic digestion, (iv) tandem mass analysis of peptides resulting from enzymatic cleavage and (v) bioinformatics, for protein identification and post-processing validation of MS/MS data sets, have been carefully evaluated. Prior to electrophoresis, the efficiency of different extraction solutions and procedures to recovery plasma proteins trapped into the sorbents were tested. Then, a rapid one-step procedure for protein extraction was optimized. Protein bands corresponding to the main plasma proteins, namely porcine serum albumin, serotransferrin and immunoglobulins, were identified. In addition, the presence of haptoglobin, hemopexin, α-1 acid glycoprotein and fetuin-A, that are known as acute-phase reaction proteins, was observed, suggesting that CPFA resins led to a non-specifically protein depletion from plasma, rather than targeting specific molecules.

Semaphorin 3F expression is reduced in pregnancy complicated by preeclampsia. An observational clinical study.

BACKGROUND AND OBJECTIVE: Preeclampsia is a systemic disorder, affecting 2-10% of pregnancies, characterized by a deregulated pro- and anti-angiogenic balance. Semaphorin 3F is an angiogenesis inhibitor. We aimed to investigate whether semaphorin 3F expression is modulated in preeclampsia. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS: We performed two observational single center cohort studies between March 2013 and August 2014. In the first we enrolled 110 consecutive women, undergoing an elective cesarean section; in the second we included 150 consecutive women undergoing amniocentesis for routine clinical indications at 16-18 week of gestation. Semaphorin 3F concentration was evaluated in maternal peripheral blood, venous umbilical blood and amniotic fluid, along with its placenta protein expression at the time of delivery in the first study group and in the amniotic fluid at 16-18 weeks of gestation in the second study group. RESULTS: In the first study 19 patients presented at delivery with preeclampsia. Semaphorin 3F placenta tissue expression was significantly reduced in preeclampsia. In addition, semaphorin 3F level at delivery was significantly lower in serum, amniotic fluid and venous umbilical blood of preeclamptic patients compared with normal pregnant women. In the prospective cohort study 14 women developed preeclampsia. In this setting, semaphorin 3F amniotic level at 16-18 weeks of gestation was reduced in women who subsequently developed preeclampsia compared to women with a normal pregnancy. ROC curve analysis showed that semaphorin 3F amniotic levels could identify women at higher risk of preeclampsia. CONCLUSIONS: Semaphorin 3F might represent a predictive biomarker of preeclampsia.

Establishment and characterization of a highly immunogenic human renal carcinoma cell line.

Renal cell carcinoma (RCC) is the most common kidney cancer, and accounts for ~3% of all adult malignancies. RCC has proven refractory to conventional treatment modalities but appears to be the only histological form that shows any consistent response to immunotherapeutic approaches. The development of a clinically effective vaccine remains a major strategic target for devising active specific immunotherapy in RCC. We aimed to identify a highly immunogenic antigenic format for immunotherapeutic approaches, so as to boost immune responses in RCC patients. We established and cloned an immunogenic cell line, RCC85#21 named Elthem, which was derived from a non-aggressive and non-metastatic clear cell carcinoma. The cell line characterization was performed by genomics (real-time PCR, genome instability), proteomics (two dimensional electrophoresis, mass spectro-metry) and immunological analysis (mixed lymphocytes tumor cell cultures). Real-time PCR confirmed the RCC85#21 cell expression of tumor antigens and cytokine genes. No difference in microsatellite instability (MSI) in RCC85#21 cell line was found as compared to control, loss of heterozygosity was observed in the RCC85#21 clone, but not in the renal cancer cell lines from which it was generated. The image analysis of RCC85#21 by two-dimensional gels showed 700±26 spots and 119 spots were identified by mass spectrometry analysis. RCC85#21 promoted a significant RCC-specific T cells activation by exhibiting a cytotoxic phenotype after mixed lymphocyte and tumor cell cultures. CD8+ T cells isolated from RCC patients displayed an elevated reactivity against RCC85#21 and efficiently lysed the RCC85#21 clone. The RCC85#21 immunogenic cell line will be suitable for immune stimulation. The identification of novel tumor associated antigens will allow the evaluation of the immune response in vitro and, subsequently, in vivo paving the way for new immunotherapeutic strategies in the RCC setting.

Exposure to low- vs iso-osmolar contrast agents reduces NADPH-dependent reactive oxygen species generation in a cellular model of renal injury.

Contrast-induced nephropathy represents the third cause of hospital-acquired acute renal failure. This study investigated the effects of low- vs iso-osmolar contrast medium (CM) exposure on NADPH-dependent reactive oxygen species (ROS) generation by tubular cells. X-ray attenuation of iohexol, iopamidol, and iodixanol was assessed at equimolar iodine concentrations and their effects on human renal proximal tubular cells (PTCs) were evaluated with equally attenuating solutions of each CM. Cytotoxicity, apoptosis, and necrosis were investigated by trypan blue exclusion, MTT assay, and annexin V/propidium iodide assay, respectively. ROS production was assessed by DCF assay, NADPH oxidase activity by the lucigenin-enhanced chemiluminescence method, and Nox4 expression by immunoblot. Yielding the same X-ray attenuation, CM cytotoxicity was assessed in PTCs at equimolar iodine concentrations. More necrosis was present after incubation with iohexol and iopamidol than after incubation with equal concentrations of iodixanol. Iohexol and iodixanol at low iodine concentrations induced less cytotoxicity than iopamidol. Moreover, both iohexol and iopamidol induced more apoptosis than iodixanol, with a dose-dependent effect. ROS generation was significantly higher with iopamidol and iohexol compared to iodixanol. NADPH oxidase activity and Nox4 protein expression significantly increased after exposure to iopamidol and iohexol, with a dose-dependent effect, compared with iodixanol. CM-induced Nox4 expression and activity depended upon Src activation. In conclusion, at angiographic concentrations, iodixanol induces fewer cytotoxic effects on cultured tubular cells than iohexol and iopamidol along with a lower induction of Nox4-dependent ROS generation. This enzyme may, thus, represent a potential therapeutic target to prevent iodinated CM-related oxidative stress.

Pentraxin 3: a novel biomarker for predicting progression from prostatic inflammation to prostate cancer.

Pentraxin-3 (PTX3) is a member of the pentraxin family of innate immune regulators, which includes C-reactive protein (CRP). PTX3 has been implicated in angiogenesis, proliferation, and immune escape in cancer. In the present study, we evaluated PTX3 tissue expression and serum concentration as a biomarker to discriminate prostatic inflammation and benign prostatic hyperplasia (BPH) from prostate cancer, and to determine whether PTX3 status may predict progression from BPH to prostate cancer. We analyzed 40 patients with biopsy-proven BPH who underwent a second prostate biopsy 12 to 36 months later when they were diagnosed with prostate cancer or inflammation/BPH (n = 20 patients each group). Furthermore, we evaluated PTX3 serum concentrations in an independent set of patients with biopsy-proven inflammation/BPH (n = 61) and prostate cancer (n = 56). We found reduced PTX3 tissue expression in patients with prostatic inflammation/BPH compared with patients who developed prostate cancer. In the latter group, there was an increase in PTX3 tissue expression between the first and second prostate biopsy. PTX3 serum levels were also higher in patients with prostate cancer than in patients with inflammation/BPH. In contrast, there was no difference in serum PSA or CRP levels in these two groups. ROC curve analysis confirmed the reliability of PTX3 serum levels in predicting prostate cancer development, identifying a cutoff value of 3.25 ng/mL with a sensitivity and a specificity of 89.3% and 88.5%, respectively. In summary, our results encourage further evaluation of PTX3 as a tissue biopsy and blood-borne biomarker to discriminate BPH from prostate cancer.

JAK3 in clear cell renal cell carcinoma: mutational screening and clinical implications.

OBJECTIVES: Janus Kinase 3 (JAK3) mediates cytokine signaling and T-cell activation. We hypothesized that JAK3 mutations may contribute to the development and progression of clear cell renal cell carcinoma (ccRCC). To test this hypothesis, we performed mutational screening and functional studies. PATIENTS AND METHODS: This hospital-based case-control study included 50 patients with ccRCC and 100 age- and gender-matched controls. Both genomic and tumor DNA were extracted. All 23 JAK3 exons were amplified by PCR and analyzed by denaturing high-performance liquid chromatography and automatic sequencing. Effects of JAK3 mutations on interleukin-2-stimulated peripheral T-cells were analyzed by confocal laser-scanning microscopy and immunoprecipitation. RESULTS: Four different JAK3 germline missense mutations (p.Gln13Lys; p.Arg925Ser; p.Ala677Thr, p.Val722Ile) were found in a total of 7 ccRCC patients (14%), but in none of the controls (P = 0.0006). All germline mutations were similarly detected in the tumors. An additional somatic missense mutation (p.Tyr238Cys) was found in a patient who had a germline mutation. Four of the mutations have not been previously described (p.Gln13Lys; p.Arg925Ser; p.Ala677Thr, p.Tyr238Cys). Patients with JAK3 mutations more frequently presented with metastases (3 out of 4 [75%] vs. 4 out of 46 [9%]; P = 0.004) and had poorer survival (P = 0.049). In p.Arg925Ser and p.Ala677Thr/p.Val722Ile, functional analyses showed abnormal JAK3 and STAT5 tyrosine phosphorylation and a reduction of JAK3/STAT5 interaction. CONCLUSIONS: JAK3 mutations are found in a subset of ccRCC patients and may be associated with ccRCC development and a greater risk of metastases. JAK3 function is compromised in p.Arg925Ser and p.Ala677Thr/p.Val722Ile. Future studies with a larger number of patients need to confirm these findings.