Introduction: AHSG, a serum glycoprotein with recognized anti-calcification activity, has also been suggested to modulate both bone formation and resorption. Though the bulk of AHSG is mostly synthesized in the liver, it has been claimed that also bone cells might produce it. However, the extent of the bone AHSG production and the potential controlling factors remain to be definitively proven.A relevant number of studies support the notion that FGF23, a bone-derived hormone, not only regulates the most important mineral metabolism (MM) related factors (phosphate, parathyroid hormone, vitamin D, etc.), but might be also involved in cardiovascular (CV) outcome, both in chronic kidney disease (CKD) patients and in the general population. Furthermore, in addition to some direct autocrine and paracrine effects in bone, FGF23 has been suggested to interact with AHSG.In this study we investigated if AHSG is really produced by bone cells, and if its bone production is related and/or controlled by FGF23, using cultured bone cells, according to a new method recently published by our group.Results: Our data show that AHSG is consistently produced in osteocytes and to a far lesser extent in osteoblasts. Both FGF23 addition to the culture medium and its over-expression in osteocytes were associated with a consistent increase of both AHSG mRNA and protein, while FGF23 silencing was followed by opposite effects. Though most of these results were largely affected by the blockage of FGF23 receptors, the role of these receptors in the different experimental sets is still not completely clarified. In addition, we found that FGF23 and AHSG proteins co-localized both in cytoplasm and nucleus, which suggests a possible reciprocal interactivity.Conclusions: Our data not only confirm that AHSG is produced in bone, mainly in osteocytes, but show for the first time that its production is modulated by FGF23. Since both proteins play important roles in the bone and cardiovascular pathology, these results add new pieces to the puzzling relationship between bone and vascular pathology, in particular in CKD patients, prompting future investigations in this field. (c) 2015 Elsevier Inc. All rights reserved.

Refers to: D. Mattinzoli, M.P. Rastaldi, M. Ikehata, S. Armelloni, C. Pignatari, L.A. Giardino, M. Li, C.M. Alfieri, A. Regalia, D. Riccardi, P. Messa FGF23-regulated production of Fetuin-A (AHSG) in osteocytes Bone, Volume 83, February 2016, Pages 35-47

Proteinuria is a common symptom of glomerular diseases and is due to leakage of proteins from the glomerular filtration barrier, a three-layer structure composed by two post-mitotic highly specialized and interdependent cell populations, i.e. glomerular endothelial cells and podocytes, and the basement membrane in between. Despite enormous progresses made in the last years, pathogenesis of proteinuria remains to be completely uncovered. Studies in the field could largely benefit from an in vitro model of the glomerular filter, but such a system has proved difficult to realize. Here we describe a method to obtain and utilize a three-dimensional podocyte-endothelial co-culture which can be largely adopted by the scientific community because it does not rely on special instruments nor on the synthesis of devoted biomaterials. The device is composed by a porous membrane coated on both sides with type IV collagen. Adhesion of podocytes on the upper side of the membrane has to be preceded by VEGF-induced maturation of endothelial cells on the lower side. The co-culture can be assembled with podocyte cell lines as well as with primary podocytes, extending the use to cells derived from transgenic mice. An albumin permeability assay has been extensively validated and applied as functional readout, enabling rapid drug testing. Additionally, the bottom of the well can be populated with a third cell type, which multiplies the possibilities of analyzing more complex glomerular intercellular signaling events. In conclusion, the ease of assembly and versatility of use are the major advantages of this three-dimensional model of the glomerular filtration barrier over existing methods. The possibility to run a functional test that reliably measures albumin permeability makes the device a valid companion in several research applications ranging from drug screening to intercellular signaling studies.

Progresses in podocyte biology have been strictly connected with genetic advances; the identification of genes mutated in familial and sporadic forms of nephrotic syndrome has been followed by functional studies of the encoded proteins, revealing numerous properties of the cell. The molecules uncovered so far belong to three main categories: a) proteins located at the slit diaphragm, the intercellular junction which laterally connects podocyte processes and is responsible for selectivity of the glomerular filter, b) molecules involved in regulation of actin dynamics, which are essential for the maintenance of podocyte structure and function, and c) molecules belonging to intracellular organelles, such as mitochondria and lysosomes, which are central players in podocyte metabolism. Considering the key role of the podocyte in health and disease of the glomerular filter, better knowledge of this cell is a pre-requisite for developing targeted therapies of glomerular diseases.

Podocytes are postmitotic renal glomerular cells with multiple ramifications that extend from the cell body. Processes departing from a podocyte interdigitate with corresponding projections from neighboring cells and form an intricate web that enwraps the glomerular capillary completely. Podocyte processes are interconnected by the slit diaphragm, an adhesion junction mostly formed by Ig-like molecules, cadherins/protocadherins, ephrin/eph, and neurexin molecules organized in an assembly that resembles synaptic junctions. Podocyte failure is primarily or secondarily implicated in all forms of proteinuric glomerular diseases, as confirmed by the morphological changes of their elaborate cell architecture detectable by electron microscopy. Importantly, mutations of podocyte proteins are responsible for the most severe forms of congenital nephrotic syndrome. In the last 15 years, progressive technological advances have aided the study of podocyte biology and pathology, confirming the relevance of podocyte molecules and signaling pathways for the function of the glomerular filter. This review will examine the most important and newest discoveries in the field, which is rapidly evolving, hopefully leading to a detailed knowledge of this fascinating cell and to the development of specific therapeutic options for proteinuric diseases.

The analysis of cellular and molecular profiles represents a powerful tool in many biomedical applications to identify the mechanisms underlying the pathological changes. The improvement of cellular starting material and the maintenance of the physiological status in the sample preparation are very useful. Human umbilical vein endothelial cells (HUVEC) are a model for prediction of endothelial dysfunction. HUVEC are enzymatically removed from the umbilical vein by collagenase. This method provides obtaining a good sample yield. However, the obtained cells are often contaminated with blood cells and fibroblasts. Methods based on negative selection by in vitro passages or on the use of defined marker are currently employed to isolate target cells. However, these approaches cannot reproduce physiological status and they require expensive instrumentation. Here we proposed a new method for an easy, tag-less and direct isolation of HUVEC from raw umbilical cord sample based on the gravitational field-flow fractionation (GrFFF). This is a low-cost, fully biocompatible method with low instrumental and training investments for flow-assisted cell fractionation. The method allows obtaining pure cells without cell culture procedures as starting material for further analysis; for example, a proper amount of RNA can be extracted. The approach can be easily integrated into clinical and biomedical procedures.

BACKGROUND:Donovanosis is a chronic bacterial illness, progressive and indolent, which normally attacks the skin and mucous membranes in the genital and perigenital regions.CASE:An 18-year-old pregnant female presented with large, hypertrophic lesions in the ano-genital region. HIV serology was negative. Pap smear revealed a CIN 1 associated with HPV infection. Biopsy yielded macrophages laden with Gram-negative Donovan bodies.SUMMARY AND CONCLUSION:A diagnosis of vulvar and perianal donovanosis was reached; the patient decided to terminate the pregnancy and was treated with azithromycin, which led to clinical resolution.

Gli sviluppi della biologia e della genetica del podocita sono stati finora strettamente associati perché l’analisi genetica di soggetti affetti da sindrome nefrosica ha permesso l’individuazione e lo studio di molecole fondamentali per il mantenimento della struttura e della funzione podocitaria. Tali molecole possono essere raggruppate in tre categorie principali: a) proteine appartenenti allo slit diaphragm, la giunzione che connette lateralmente i processi podocitari ed è responsabile della selettività del filtro glomerulare, b) molecole coinvolte nelle dinamiche del citoscheletro di actina, quindi nel mantenimento della complessa struttura ramificata del podocita, e c) molecole appartenenti ad organuli intracellulari, quali mitocondri e lisosomi, che presiedono al metabolismo cellulare. Considerando la centralità del podocita nel mantenimento della barriera di filtrazione glomerulare, la conoscenza completa dei meccanismi che regolano la biologia e la patologia podocitaria è un requisito fondamentale per lo sviluppo di terapie mirate delle malattie glomerulari. Podocytes: genetics and biology Progresses in podocyte biology have been strictly connected with genetic advances; the identification of genes mutated in familial and sporadic forms of nephrotic syndrome has been followed by functional studies of the encoded proteins, revealing numerous properties of the cell. The molecules uncovered so far belong to three main categories: a) proteins located at the slit diaphragm, the intercellular junction which laterally connects podocyte processes and is responsible for selectivity of the glomerular filter, b) molecules involved in regulation of actin dynamics, which are essential for the maintenance of podocyte structure and function, and c) molecules belonging to intracellular organelles, such as mitochondria and lysosomes, which are central players in podocyte metabolism. Considering the key role of the podocyte in health and disease of the glomerular filter, better knowledge of this cell is a pre-requisite for developing targeted therapies of glomerular diseases.