Human Serum Albumin Is an Essential Component of the Host Defense Mechanism Against Clostridium difficile Intoxication.

Background: The pathogenic effects of Clostridium difficile are primarily attributable to the production of the large protein toxins (C difficile toxins [Tcd]) A (TcdA) and B (TcdB). These toxins monoglucosylate Rho GTPases in the cytosol of host cells, causing destruction of the actin cytoskeleton with cytotoxic effects. Low human serum albumin (HSA) levels indicate a higher risk of acquiring and developing a severe C difficile infection (CDI) and are associated with recurrent and fatal disease. Methods: We used a combined approach based on docking simulation and biochemical analyses that were performed in vitro on purified proteins and in human epithelial colorectal adenocarcinoma cells (Caco-2), and in vivo on stem cell-derived human intestinal organoids and zebrafish embryos. Results: Our results show that HSA specifically binds via its domain II to TcdA and TcdB and thereby induces their autoproteolytic cleavage at physiological concentrations. This process impairs toxin internalization into the host cells and reduces the toxin-dependent glucosylation of Rho proteins. Conclusions: Our data provide evidence for a specific HSA-dependent self-defense mechanism against C difficile toxins and provide an explanation for the clinical correlation between CDI severity and hypoalbuminemia.

Hemicentin 1 influences podocyte dynamic changes in glomerular diseases.

Different complex mechanisms control the morphology of podocyte foot processes and their interactions with the underlying basement membrane. Injuries to this system often cause glomerular dysfunction and albuminuria. The present study aimed at identifying early markers of glomerular damage in diabetic nephropathy. For this purpose, we performed a microarray analysis on kidneys of 3-wk-old peroxisome proliferator-activated receptor-γ (PPARγ)-null and AZIP/F1 mice, which are two models of diabetic nephropathy due to lipodystrophy. This was followed by functional annotation of the enriched clusters of genes. One of the significant changes in the early stages of glomerular damage was the increase of hemicentin 1 (HMCN1). Its expression and distribution were then studied by real-time PCR and immunofluorescence in various models of glomerular damage and on podocyte cell cultures. HMCN1 progressively increased in the glomeruli of diabetic mice, according to disease severity, as well as in puromycin aminonucleoside (PA)-treated rats. Studies on murine and human podocytes showed an increased HMCN1 deposition upon different pathological stimuli, such as hyperglycemia, transforming growth factor-ß (TGF-ß), and PA. In vitro silencing studies showed that HMCN1 mediated the rearrangements of podocyte cytoskeleton induced by TGF-ß. Finally, we demonstrated an increased expression of HMCN1 in the kidneys of patients with proteinuric nephropathies. In summary, our studies identified HMCN1 as a new molecule involved in the dynamic changes of podocyte foot processes. Its increased expression associated with podocyte dysfunction points to HMCN1 as a possible marker for the early glomerular damage occurring in different proteinuric nephropathies.

BDNF repairs podocyte damage by microRNA-mediated increase of actin polymerization.

Idiopathic focal segmental glomerulosclerosis (FSGS) is a progressive and proteinuric kidney disease that starts with podocyte injury. Podocytes cover the external side of the glomerular capillary by a complex web of primary and secondary ramifications. Similar to dendritic spines of neuronal cells, podocyte processes rely on a dynamic actin-based cytoskeletal architecture to maintain shape and function. Brain-derived neurotrophic factor (BDNF) is a pleiotropic neurotrophin that binds to the tropomyosin-related kinase B receptor (TrkB) and has crucial roles in neuron maturation, survival, and activity. In neuronal cultures, exogenously added BDNF increases the number and size of dendritic spines. In animal models, BDNF administration is beneficial in both central and peripheral nervous system disorders. Here we show that BDNF has a TrkB-dependent trophic activity on podocyte cell processes; by affecting microRNA-134 and microRNA-132 signalling, BDNF up-regulates Limk1 translation and phosphorylation, and increases cofilin phosphorylation, which results in actin polymerization. Importantly, BDNF effectively repairs podocyte damage in vitro, and contrasts proteinuria and glomerular lesions in in vivo models of FSGS, opening a potential new perspective to the treatment of podocyte disorders.

Small molecule membrane transporters in the mammalian podocyte: a pathogenic and therapeutic target.

The intriguingly complex glomerular podocyte has been a recent object of intense study. Researchers have sought to understand its role in the pathogenesis of common proteinuric diseases such as minimal change disease and focal segmental glomerular sclerosis. In particular, considerable effort has been directed towards the anatomic and functional barrier to macromolecular filtration provided by the secondary foot processes, but little attention has been paid to the potential of podocytes to handle plasma proteins beyond the specialization of the slit diaphragm. Renal membrane transporters in the proximal tubule have been extensively studied for decades, particularly in relation to drug metabolism and elimination. Recently, uptake and efflux transporters for small organic molecules have also been found in the glomerular podocyte, and we and others have found that these transporters can engage not only common pharmaceuticals but also injurious endogenous and exogenous agents. We have also found that the activity of podocyte transporters can be manipulated to inhibit pathogen uptake and efflux. It is conceivable that podocyte transporters may play a role in disease pathogenesis and may be a target for future drug development.

Brain-Derived Neurotrophic Factor-Loaded Low-Temperature-Sensitive liposomes as a drug delivery system for repairing podocyte damage.

Podocytes, cells of the glomerular filtration barrier, play a crucial role in kidney diseases and are gaining attention as potential targets for new therapies. Brain-Derived Neurotrophic Factor (BDNF) has shown promising results in repairing podocyte damage, but its efficacy via parenteral administration is limited by a short half-life. Low temperature sensitive liposomes (LTSL) are a promising tool for targeted BDNF delivery, preserving its activity after encapsulation. This study aimed to improve LTSL design for efficient BDNF encapsulation and targeted release to podocytes, while maintaining stability and biological activity, and exploiting the conjugation of targeting peptides. While cyclic RGD (cRGD) was used for targeting endothelial cells in vitro, a homing peptide (HITSLLS) was conjugated for more specific uptake by glomerular endothelial cells in vivo. BDNF-loaded LTSL successfully repaired cytoskeleton damage in podocytes and reduced albumin permeability in a glomerular co-culture model. cRGD conjugation enhanced endothelial cell targeting and uptake, highlighting an improved therapeutic effect when BDNF release was induced by thermoresponsive liposomal degradation. In vivo, targeted LTSL showed evidence of accumulation in the kidneys, and their BDNF delivery decreased proteinuria and ameliorated kidney histology. These findings highlight the potential of BDNF-LTSL formulations in restoring podocyte function and treating glomerular diseases.

Albuminuria and glomerular damage in mice lacking the metabotropic glutamate receptor 1.

The metabotropic glutamate (mGlu) receptor 1 (GRM1) has been shown to play an important role in neuronal cells by triggering, through calcium release from intracellular stores, various signaling pathways that finally modulate neuron excitability, synaptic plasticity, and mechanisms of feedback regulation of neurotransmitter release. Herein, we show that Grm1 is expressed in glomerular podocytes and that a glomerular phenotype is exhibited by Grm1(crv4) mice carrying a spontaneous recessive inactivating mutation of the gene. Homozygous Grm1(crv4/crv4) and, to a lesser extent, heterozygous mice show albuminuria, podocyte foot process effacement, and reduced levels of nephrin and other proteins known to contribute to the maintenance of podocyte cell structure. Overall, the present data extend the role of mGlu1 receptor to the glomerular filtration barrier. The regulatory action of mGlu1 receptor in dendritic spine morphology and in the control of glutamate release is well acknowledged in neuronal cells. Analogously, we speculate that mGlu1 receptor may regulate foot process morphology and intercellular signaling in the podocyte.

High-salt diet increases glomerular ACE/ACE2 ratio leading to oxidative stress and kidney damage.

BACKGROUND: Angiotensin II (AngII) contributes to salt-driven kidney damage. In this study, we aimed at investigating whether and how the renal damage associated with a high-salt diet could result from changes in the ratio between angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2). METHODS: Forty-eight rats randomly allocated to three different dietary contents of salt were studied for 4 weeks after undergoing a left uninephrectomy. We focussed on kidney functional, structural and molecular changes. At the same time, we studied kidney molecular changes in 20 weeks old Ace2-knockout mice (Ace2KO), with and without ACE inhibition. RESULTS: A high salt content diet significantly increased the glomerular ACE/ACE2 ratio. This was associated with increased oxidative stress. To assess whether these events were related, we measured renal oxidative stress in Ace2KO, and found that the absence of ACE2 promoted oxidative stress, which could be prevented by ACE inhibition. CONCLUSION: One of the mechanisms by which a high-salt diet leads to renal damage seems to be the modulation of the ACE/ACE2 ratio which in turn is critical for the cause of oxidative stress, through AngII.

5-Azacytidine Downregulates the Proliferation and Migration of Hepatocellular Carcinoma Cells In Vitro and In Vivo by Targeting miR-139-5p/ROCK2 Pathway.

BACKGROUND: For hepatocellular carcinoma (HCC), effective therapeutic approaches are lacking. As aberrant gene methylation is a major contributor to HCC development, demethylating drugs such as 5-azacytidine (5-Aza) have been proposed. As most 5-Aza mechanisms of action are unknown, we investigated its phenotypic/molecular effects. METHODS: 5-Aza effects were examined in the human HCC cell lines JHH-6/HuH-7 and in the rat cell-line N1-S1. We also employed a xenograft mouse model (HuH-7), a zebrafish model (JHH-6), and an orthotopic syngeneic rat model (N1-S1) of HCC. RESULTS: 5-Aza downregulated cell viability/growth/migration/adhesion by upregulating miR-139-5p, which in turn downregulated ROCK2/cyclin D1/E2F1 and increased p27kip1, resulting in G1/G0 cell accumulation. Moreover, a decrease in cyclin B1 and an increase in p27kip1 led to G2/M accumulation. Finally, we observed a decrease in MMP-2 levels, a stimulator of HCC cell migration. Aza effects were confirmed in the mouse model; in the zebrafish model, we also demonstrated the downregulation of tumor neo-angiogenesis, and in the orthotopic rat model, we observed impaired N1-S1 grafting in a healthy liver. CONCLUSION: We demonstrate for the first time that 5-Aza can impair HCC development via upregulation of miR-139-5p, which in turn impairs the ROCK2/cyclin D1/E2F1/cyclin B1 pro-proliferative pathway and the ROCK2/MMP-2 pro-migratory pathway. Thus, we provide novel information about 5-Aza mechanisms of action and deepen the knowledge about the crosstalk among ROCK2/cyclin D1/E2F1/cyclin B1/p27kip1/MMP-2 in HCC.

Xenograft Zebrafish Models for the Development of Novel Anti-Hepatocellular Carcinoma Molecules.

Hepatocellular carcinoma (HCC) is the sixth most common type of tumor and the second leading cause of tumor-related death worldwide. Liver cirrhosis is the most important predisposing factor for HCC. Available therapeutic approaches are not very effective, especially for advanced HCC, which is the most common form of the disease at diagnosis. New therapeutic strategies are therefore urgently needed. The use of animal models represents a relevant tool for preclinical screening of new molecules/strategies against HCC. However, several issues, including animal husbandry, limit the use of current models (rodent/pig). One animal model that has attracted the attention of the scientific community in the last 15 years is the zebrafish. This freshwater fish has several attractive features, such as short reproductive time, limited space and cost requirements for husbandry, body transparency and the fact that embryos do not show immune response to transplanted cells. To date, two different types of zebrafish models for HCC have been developed: the transgenic zebrafish and the zebrafish xenograft models. Since transgenic zebrafish models for HCC have been described elsewhere, in this review, we focus on the description of zebrafish xenograft models that have been used in the last five years to test new molecules/strategies against HCC.

Podocyte glutamatergic signaling contributes to the function of the glomerular filtration barrier.

Podocytes possess the complete machinery for glutamatergic signaling, raising the possibility that neuron-like signaling contributes to glomerular function. To test this, we studied mice and cells lacking Rab3A, a small GTPase that regulates glutamate exocytosis. In addition, we blocked the glutamate ionotropic N-methyl-d-aspartate receptor (NMDAR) with specific antagonists. In mice, the absence of Rab3A and blockade of NMDAR both associated with an increased urinary albumin/creatinine ratio. In humans, NMDAR blockade, obtained by addition of ketamine to general anesthesia, also had an albuminuric effect. In vitro, Rab3A-null podocytes displayed a dysregulated release of glutamate with higher rates of spontaneous exocytosis, explained by a reduction in Rab3A effectors resulting in freedom of vesicles from the actin cytoskeleton. In addition, NMDAR antagonism led to profound cytoskeletal remodeling and redistribution of nephrin in cultured podocytes; the addition of the agonist NMDA reversed these changes. In summary, these results suggest that glutamatergic signaling driven by podocytes contributes to the integrity of the glomerular filtration barrier and that derangements in this signaling may lead to proteinuric renal diseases.

Extra-Intestinal Effects of C.difficile Toxin A and B: An In Vivo Study Using the Zebrafish Embryo Model.

C.difficile infection (CDI) is not a merely "gut-confined" disease as toxemia could drive the development of CDI-related extra-intestinal effects. These effects could explain the high CDI-associated mortality, not just justified by diarrhea and dehydration. Here, the extra-intestinal effects of toxin A (TcdA) and B (TcdB) produced by C. difficile have been studied in vivo using the zebrafish embryo model. Noteworthy, protective properties of human serum albumin (HSA) towards toxins-induced extra-intestinal effects were also addressed. Zebrafish embryos were treated with TcdA, TcdB and/or HSA at 24 h post-fertilization. Embryos were analyzed for 48 h after treatment to check vital signs and morphological changes. Markers related to cardio-vascular damage and inflammation were evaluated by Real-Time quantitative PCR and/or western blotting. Both toxins induced cardiovascular damage in zebrafish embryos by different mechanisms: (i) direct toxicity (i.e., pericardial edema, cardiac chambers enlargement, endothelial alteration); (ii) increased hormonal production and release (i.e., atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP)), (iii) alteration of the vascular system through the increase of the vascular endothelial growth factor (VEGF-A) levels, as well as of its receptors, (iv) pro-inflammatory response through high cytokines production (i.e., CXCL8, IL1B, IL6 and TNFα) and (v) cell-mediated damage due to the increase in neutrophils number. In addition to cardiovascular damage, we observe skin alteration and inflammation. Finally, our data indicate a protective effect of HSA toward the toxins induced extra-intestinal effects. Together, our findings can serve as a starting point for humans' studies to substantiate and understand the extra-intestinal effects observed in CDI patients.

HMGA1 promotes breast cancer angiogenesis supporting the stability, nuclear localization and transcriptional activity of FOXM1.

BACKGROUND: Breast cancer is the most common malignancy in women worldwide. Among the breast cancer subtypes, triple-negative breast cancer (TNBC) is the most aggressive and the most difficult to treat. One of the master regulators in TNBC progression is the architectural transcription factor HMGA1. This study aimed to further explore the HMGA1 molecular network to identify molecular mechanisms involved in TNBC progression. METHODS: RNA from the MDA-MB-231 cell line, silenced for HMGA1 expression, was sequenced and, with a bioinformatic analysis, molecular partners HMGA1 could cooperate with in regulating common downstream gene networks were identified. Among the putative partners, the FOXM1 transcription factor was selected. The relationship occurring between HMGA1 and FOXM1 was explored by qRT-PCR, co-immunoprecipitation and protein stability assays. Subsequently, the transcriptional activity of HMGA1 and FOXM1 was analysed by luciferase assay on the VEGFA promoter. The impact on angiogenesis was assessed in vitro, evaluating the tube formation ability of endothelial cells exposed to the conditioned medium of MDA-MB-231 cells silenced for HMGA1 and FOXM1 and in vivo injecting MDA-MB-231 cells, silenced for the two factors, in zebrafish larvae. RESULTS: Here, we discover FOXM1 as a novel molecular partner of HMGA1 in regulating a gene network implicated in several breast cancer hallmarks. HMGA1 forms a complex with FOXM1 and stabilizes it in the nucleus, increasing its transcriptional activity on common target genes, among them, VEGFA, the main inducer of angiogenesis. Furthermore, we demonstrate that HMGA1 and FOXM1 synergistically drive breast cancer cells to promote tumor angiogenesis both in vitro in endothelial cells and in vivo in a zebrafish xenograft model. Moreover, using a dataset of breast cancer patients we show that the co-expression of HMGA1, FOXM1 and VEGFA is a negative prognostic factor of distant metastasis-free survival and relapse-free survival. CONCLUSIONS: This study reveals FOXM1 as a crucial interactor of HMGA1 and proves that their cooperative action supports breast cancer aggressiveness, by promoting tumor angiogenesis. Therefore, the possibility to target HMGA1/FOXM1 in combination should represent an attractive therapeutic option to counteract breast cancer angiogenesis.

Gadolinium tissue deposition in the periodontal ligament of mice with reduced renal function exposed to Gd-based contrast agents.

Gadolinium deposition in tissue is linked to nephrogenic systemic fibrosis (NSF): a rare disorder occurring in patients with severe chronic kidney disease and associated with administration of Gd-based contrast agents (GBCAs) for Magnetic Resonance Imaging (MRI). It is suggested that the GBCAs prolonged permanence in blood in these patients may result in a Gd precipitation in peripheral or central organs, where it initiates a fibrotic process. In this study we investigated new sites of retention/precipitation of Gd in a mouse model of renal disease (5/6 nephrectomy) receiving two doses (closely after each other) of a linear GBCA. Two commercial GBCAs (Omniscan® and Magnevist®) were administered at doses slightly higher than those used in clinical practice (0.7 mmol/kg body weight, each). The animals were sacrificed one month after the last administration and the explanted organs (kidney, liver, femur, dorsal skin, teeth) were analysed by X-ray fluorescence (XRF) at two synchrotron facilities. The XRF analysis with a millimetre-sized beam at the SYRMEP beamline (Elettra, Italy) produced no detectable levels of Gd in the examined tissues, with the notable exception of the incisors of the nephrectomised mice. The XRF analyses at sub-micron resolution performed at ID21 (ESRF, France) allowed to clearly localize Gd in the periodontal ligaments of teeth both from Omniscan® and Magnevist® treated nephrectomised mice. The latter results were further confirmed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The study prompts that prolonged permanence of GBCAs in blood may result in Gd retention in this particular muscular tissue, opening possibilities for diagnostic applications at this level when investigating Gd-related toxicities.

Corrigendum: Treponema pallidum (syphilis) antigen TpF1 induces angiogenesis through the activation of the IL-8 pathway.

This corrects the article DOI: 10.1038/srep18785.

Cell-autonomous and cell non-autonomous downregulation of tumor suppressor DAB2IP by microRNA-149-3p promotes aggressiveness of cancer cells.

The tumor suppressor DAB2IP contributes to modulate the network of information established between cancer cells and tumor microenvironment. Epigenetic and post-transcriptional inactivation of this protein is commonly observed in multiple human malignancies, and can potentially favor progression of tumors driven by a variety of genetic mutations. Performing a high-throughput screening of a large collection of human microRNA mimics, we identified miR-149-3p as a negative post-transcriptional modulator of DAB2IP. By efficiently downregulating DAB2IP, this miRNA enhances cancer cell motility and invasiveness, facilitating activation of NF-kB signaling and promoting expression of pro-inflammatory and pro-angiogenic factors. In addition, we found that miR-149-3p secreted by prostate cancer cells induces DAB2IP downregulation in recipient vascular endothelial cells, stimulating their proliferation and motility, thus potentially remodeling the tumor microenvironment. Finally, we found that inhibition of endogenous miR-149-3p restores DAB2IP activity and efficiently reduces tumor growth and dissemination of malignant cells. These observations suggest that miR-149-3p can promote cancer progression via coordinated inhibition of DAB2IP in tumor cells and in stromal cells.

HIV-1 Tat reduces nephrin in human podocytes: a potential mechanism for enhanced glomerular permeability in HIV-associated nephropathy.

OBJECTIVE: To determine whether HIV-1 Tat may directly alter glomerular permeability in HIV-associated nephropathy (HIVAN). DESIGN: Heavy proteinuria is a hallmark of HIVAN. The slit diaphragm is the ultimate glomerular filtration barrier critical for maintaining the efficiency of the ultrafiltration unit of the kidney. In this study, we evaluated the direct effect of Tat protein on the permeability of isolated glomeruli and on the expression of nephrin, the main slit diaphragm component, by human cultured podocytes. METHODS: Permeability was studied by measuring the permeability to albumin in isolated rat glomeruli. We also evaluated the expression of nephrin in human cultured podocytes by using immunofluorescence and Western blot. RESULTS: We found that Tat increased albumin permeability in isolated glomeruli, and rapidly induced the redistribution and loss of nephrin in cultured podocytes. Pretreatment of glomeruli and podocytes with blocking antibodies showed that Tat reduced nephrin expression by engaging vascular endothelial growth factor receptors types 2 and 3 and the integrin alphavbeta3. Pre-incubation of podocytes with two platelet-activating factor (PAF) receptor antagonists prevented the loss and redistribution of nephrin induced by Tat, suggesting that PAF is an intracellular mediator of Tat action. Tat induced a rapid PAF synthesis by podocytes. When podocytes transfected to overexpress PAF-acetylhydrolase, the main catabolic enzyme of PAF, were stimulated with Tat, the redistribution and loss of nephrin was abrogated. CONCLUSION: The present results define a mechanism by which Tat may reduce nephrin expression in podocytes, thus increasing glomerular permeability. This provides new insights in the understanding of HIVAN pathogenesis.

Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS.

CD40/CD40 ligand (CD40L) dyad, a co-stimulatory bi-molecular complex involved in the adaptive immune response, has also potent pro-inflammatory actions in haematopoietic and non-haematopoietic cells. We describe here a novel role for soluble CD40L (sCD40L) as modifier of glomerular permselectivity directly acting on glomerular epithelial cells (GECs). We found that stimulation of CD40, constitutively expressed on GEC cell membrane, by the sCD40L rapidly induced redistribution and loss of nephrin in GECs, and increased albumin permeability in isolated rat glomeruli. Pre-treatment with inhibitors of CD40-CD40L interaction completely prevented these effects. Furthermore, in vivo injection of sCD40L induced a significant reduction of nephrin and podocin expression in mouse glomeruli, although no significant increase of urine protein/creatinine ratio was observed after in vivo injection. The same effects were induced by plasma factors partially purified from post-transplant plasma exchange eluates of patients with focal segmental glomerulosclerosis (FSGS), and were blocked by CD40-CD40L inhibitors. Moreover, 17 and 34 kDa sCD40L isoforms were detected in the same plasmapheresis eluates by Western blotting. Finally, the levels of sCD40Lwere significantly increased in serum of children both with steroid-sensitive and steroid-resistant nephrotic syndrome (NS), and in adult patients with biopsy-proven FSGS, compared to healthy subjects, but neither in children with congenital NS nor in patients with membranous nephropathy. Our results demonstrate that sCD40L directly modifies nephrin and podocin distribution in GECs. Moreover, they suggest that sCD40L contained in plasmapheresis eluates from FSGS patients with post-transplant recurrence may contribute, presumably cooperating with other mediators, to FSGS pathogenesis by modulating glomerular permeability.

The renal phenotype of allopurinol-treated HPRT-deficient mouse.

Excess of uric acid is mainly treated with xanthine oxidase (XO) inhibitors, also called uricostatics because they block the conversion of hypoxanthine and xanthine into urate. Normally, accumulation of upstream metabolites is prevented by the hypoxanthine-guanine phosphoribosyltransferase (HPRT) enzyme. The recycling pathway, however, is impaired in the presence of HPRT deficiency, as observed in Lesch-Nyhan disease. To gain insights into the consequences of purine accumulation with HPRT deficiency, we investigated the effects of the XO inhibitor allopurinol in Hprt-lacking (HPRT-/-) mice. Allopurinol was administered in the drinking water of E12-E14 pregnant mothers at dosages of 150 or 75 µg/ml, and mice sacrificed after weaning. The drug was well tolerated by wild-type animals and heterozygous HPRT+/- mice. Instead, a profound alteration of the renal function was observed in the HPRT-/- model. Increased hypoxanthine and xanthine concentrations were found in the blood. The kidneys showed a yellowish appearance, diffuse interstitial nephritis, with dilated tubules, inflammatory and fibrotic changes of the interstitium. There were numerous xanthine tubular crystals, as determined by HPLC analysis. Oil red O staining demonstrated lipid accumulation in the same location of xanthine deposits. mRNA analysis showed increased expression of adipogenesis-related molecules as well as profibrotic and proinflammatory pathways. Immunostaining showed numerous monocyte-macrophages and overexpression of alpha-smooth muscle actin in the tubulointerstitium. In vitro, addition of xanthine to tubular cells caused diffuse oil red O positivity and modification of the cell phenotype, with loss of epithelial features and appearance of mesenchymal characteristics, similarly to what was observed in vivo. Our results indicate that in the absence of HPRT, blockade of XO by allopurinol causes rapidly developing renal failure due to xanthine deposition within the mouse kidney. Xanthine seems to be directly involved in promoting lipid accumulation and subsequent phenotype changes of tubular cells, with activation of inflammation and fibrosis.

Rapid and cost-effective xenograft hepatocellular carcinoma model in Zebrafish for drug testing.

We developed a novel, rapid and cost-effective Zebrafish xenograft model of hepatocellular carcinoma (HCC) for drug screening in the disease. Following injection into the yolk sack of Zebrafish larvae of the human HCC cell line JHH6 stained by a vital dye, tumor mass growth was followed by fluorescence microscopy and by human Ki67 quantification. Tumor induced neo-angiogenesis was evaluated by alkaline phosphatase staining of the vessels, by using the Tg(fli1:EGFP)y1 strain of Zebrafish and by the quantification of the zebrafish vascular endothelial growth factor and of its receptor. We show that it is feasible to micro-inject JHH6 in Zebrafish larvae, that injected cells can grow for different days and that this induces a marked neo-angiogenesis. Finally, we show that our model allows testing the effects of anti-HCC drugs such as Bortezomib. Compared to more complex HCC mouse models, our model is far less expensive, faster to set up and does not need immunosuppressant treatment. Finally, the model makes use of JHH6, an aggressive form of HCC cell line never tested before in Zebrafish. In conclusion, the possibility to test anti HCC/neo-angiogenesis drugs makes our JHH6 model useful to select therapeutic molecules for a highly vascularized tumor such as HCC.