application-retinoic-acid-to-obtain-osteocytes-cultures-from-primary

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico

Although patients with chronic renal failure are increasing worldwide, many aspects of kidney biology remain to be elucidated. Recent research has uncovered several molecular properties of the glomerular filtration barrier, in which podocytes, highly differentiated, ramified cells that enwrap the glomerular basement membrane, have been reported to be mainly responsible for filter's selectivity. We previously described that podocytes express Rab3A, a GTPase restricted to cell types that are capable of highly regulated exocytosis, such as neuronal cells. Here, we first demonstrate by a proteomic study that Rab3A in podocytes coimmmunoprecipitates with molecules once thought to be synapse specific. We then show that podocytes possess structures resembling synaptic vesicles, which contain glutamate, coexpress Rab3A and synaptotagmin 1, and undergo spontaneous and stimulated exocytosis and recycling, with glutamate release. Finally, from the results of a cDNA microarray study, we describe the presence of a series of neuron- and synapse-specific molecules in normal human glomeruli and confirm the glomerular protein expression of both metabotropic and ionotropic glutamate receptors. These data point toward a synaptic-like mechanism of communication among glomerular cells, which perfectly fits with the molecular composition of the glomerular filter and puts in perspective several previous observations, proposing a different working hypothesis for understanding glomerular signaling dynamics.

Glomerular podocytes contain neuron-like functional synaptic vesicles.

Human tumors constitutively release endosome-derived microvesicles, transporting a broad array of biologically active molecules with potential modulatory effects on different immune cells. Here, we report the first evidence that tumor-released microvesicles alter myeloid cell function by impairing monocyte differentiation into dendritic cells and promoting the generation of a myeloid immunosuppressive cell subset. CD14+ monocytes isolated from healthy donors and differentiated with interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor in the presence of tumor-derived microvesicles turned into HLA-DR(-/low) cells, retaining CD14 expression and failing to up-regulate costimulatory molecules, such as CD80 and CD86. These phenotypic changes were paralleled by a significant release of different cytokines, including IL-6, tumor necrosis factor-alpha, and transforming growth factor-beta (TGF-beta), and a dose-dependent suppressive activity on activated T-cell-proliferation and cytolytic functions, which could be reversed by anti-TGF-beta-neutralizing antibodies. Microvesicles isolated from plasma of advanced melanoma patients, but not from healthy donors, mediated comparable effects on CD14+ monocytes, skewing their differentiation toward CD14+HLA-DR-/low cells with TGF-beta-mediated suppressive activity on T-cell-functions. Interestingly, a subset of TGF-beta-secreting CD14+HLA-DR- cells mediating suppressive activity on T lymphocytes was found to be significantly expanded in peripheral blood of melanoma patients compared with healthy donors. These data suggest the development in cancer patients of an immunosuppressive circuit by which tumors promote the generation of suppressive myeloid cells through the release of circulating microvesicles and without the need for cell-to-cell contact. Therapeutic interventions on the crucial steps of this pathway may contribute to restore tumor/immune system interactions favoring T-cell-mediated control of tumor growth in cancer patients.

Human tumor-released microvesicles promote the differentiation of myeloid cells with transforming growth factor-beta-mediated suppressive activity on T lymphocytes.

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.

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

Vacuolar-H(+)-ATPase plays a critical role in the cellular balance of protons, thus regulating intracellular pH and contributing to apoptosis resistance, drug resistance, and invasive and metastatic behavior of cells. NiK-12192, a vacuolar-H(+)-ATPase inhibitor, caused a reduction in the volume and/or acidity of lysosomes, a polarization of alphavbeta5 integrin distribution, and a number of floating live cells, whereas signs of apoptosis appeared only after 72 h of treatment. In conclusion, NiK-12192, by affecting vacuolar- H(+)-ATPase activity (and intracellular pH), causes a modification of structures crucial for cell adhesion and induces cell death, likely by a modality involving an anoikis-mediated apoptosis.

Biological effects of a new vacuolar-H,-ATPase inhibitor in colon carcinoma cell lines.

Cannabinoid receptor 1 (CB1) is localized in the central nervous system and in peripheral tissues involved in energy metabolism control. However, CB1 receptors are also expressed at low level within the glomeruli, and the aim of this study was to investigate their potential relevance in the pathogenesis of proteinuria in experimental type 1 diabetes.

Cannabinoid receptor 1 blockade ameliorates albuminuria in experimental diabetic nephropathy.

Uromodulin-associated kidney diseases (UAKD) are autosomal-dominant disorders characterized by alteration of urinary concentrating ability, tubulo-interstitial fibrosis, hyperuricaemia and renal cysts at the cortico-medullary junction. UAKD are caused by mutations in UMOD, the gene encoding uromodulin. Although uromodulin is the most abundant protein secreted in urine, its physiological role remains elusive. Several in vitro studies demonstrated that mutations in uromodulin lead to endoplasmic reticulum (ER) retention of mutant protein, but their relevance in vivo has not been studied. We here report on the generation and characterization of the first transgenic mouse model for UAKD. Transgenic mice that express the C147W mutant uromodulin (Tg(Umod)(C147W)), corresponding to the well-established patient mutation C148W, were compared with expression-matched transgenic mice expressing the wild-type protein (Tg(Umod)(wt)). Tg(Umod)(C147W) mice recapitulate most of the UAKD features, with urinary concentrating defect of renal origin and progressive renal injury, i.e. tubulo-interstitial fibrosis with inflammatory cell infiltration, tubule dilation and specific damage of the thick ascending limb of Henle's loop, leading to mild renal failure. As observed in patients, Tg(Umod)(C147W) mice show a marked reduction of urinary uromodulin excretion. Mutant uromodulin trafficking to the plasma membrane is indeed impaired as it is retained in the ER of expressing cells leading to ER hyperplasia. The Tg(Umod)(C147W) mice represent a unique model that recapitulates most of the features associated with UAKD. Our data clearly demonstrate a gain-of-toxic function of uromodulin mutations providing insights into the pathogenetic mechanism of the disease. These findings may also be relevant for other tubulo-interstitial or ER-storage disorders.

A transgenic mouse model for uromodulin-associated kidney diseases shows specific tubulo-interstitial damage, urinary concentrating defect and renal failure.

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.

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

Nephrin is an immunoglobulin-like adhesion molecule first discovered as a major component of the podocyte slit diaphragm, where its integrity is essential to the function of the glomerular filtration barrier. Outside the kidney, nephrin has been shown in other restricted locations, most notably in the central nervous system (CNS) of embryonic and newborn rodents. With the aim of better characterizing nephrin expression and its role in the CNS of adult rodents, we studied its expression pattern and possible binding partners in CNS tissues and cultured neuronal cells and compared these data to those obtained in control renal tissues and podocyte cell cultures. Our results show that, besides a number of locations already found in embryos and newborns, endogenous nephrin in adult rodent CNS extends to the pons and corpus callosum and is expressed by granule cells and Purkinje cells of the cerebellum, with a characteristic alternating expression pattern. In primary neuronal cells we find nephrin expression close to synaptic proteins and demonstrate that nephrin co-immunoprecipitates with Fyn kinase, glutamate receptors and the scaffolding molecule PSD95, an assembly that is reminiscent of those made by synaptic adhesion molecules. This role seems to be confirmed by our findings of impaired maturation and reduced glutamate exocytosis occurring in Neuro2A cells upon nephrin silencing. Of note, we disclose that the very same nephrin interactions occur in renal glomeruli and cultured podocytes, supporting our hypothesis that podocytes organize and use similar molecular intercellular signalling modules to those used by neuronal cells.

Nephrin expression in adult rodent central nervous system and its interaction with glutamate receptors.

[Synaptic-like signals at the filtration barrier: the role of nephrin].

The relative contribution of genetic factors and dietary patterns to glomerular damage in healthy individuals and prediabetic conditions is currently unclear. All Rab3A knockout (KO) mice spontaneously develop macroalbuminuria, but only male mice exhibit a glucose-intolerant phenotype, thus making the model suitable to examine the impact of a diet on preexisting podocyte damage.

Proteinuria and glomerular damage in Rab3A knockout mice chronically fed a high-glucose diet.

BAMBI is a type I TGFβ receptor antagonist, whose in vivo function remains unclear, as BAMBI(-/-) mice lack an obvious phenotype.

BAMBI regulates angiogenesis and endothelial homeostasis through modulation of alternative TGFβ signaling.

MicroRNAs (miRNAs), a class of small non-protein-encoding RNAs, regulate gene expression via suppression of target mRNAs. MiRNAs are present in body fluids in a remarkable stable form as packaged in microvesicles of endocytic origin, named exosomes. In the present study, we have assessed miRNA expression in urinary exosomes from type 1 diabetic patients with and without incipient diabetic nephropathy. Results showed that miR-130a and miR-145 were enriched, while miR-155 and miR-424 reduced in urinary exosomes from patients with microalbuminuria. Similarly, in an animal model of early experimental diabetic nephropathy, urinary exosomal miR-145 levels were increased and this was paralleled by miR-145 overexpression within the glomeruli. Exposure of cultured mesangial cells to high glucose increased miR-145 content in both mesangial cells and mesangial cells-derived exosomes, providing a potential mechanism for diabetes-induced miR-145 overexpression. In conclusion, urinary exosomal miRNA content is altered in type 1 diabetic patients with incipient diabetic nephropathy and miR-145 may represent a novel candidate biomarker/player in the complication.

Urinary exosomal microRNAs in incipient diabetic nephropathy.

Poor prognosis and limited therapeutic options characterize immunoglobulin light chain (AL) amyloidosis with major heart involvement. Reliable experimental models are needed to study light chains (LC)-heart interactions and to explore strategies for prevention of cardiac damage. We have exploited the nematode C. elegans as a novel tool, since its pharynx is evolutionarily related to the vertebrate heart. Our data demonstrate that the pharyngeal pumping of C. elegans is significantly and selectively reduced by LC from AL patients suffering from cardiomyopathy, but not by amyloid LC with different organ tropism or non-amyloidogenic LC from multiple myeloma. This functional alteration is dependent on the LC concentration and results in persistent pharyngeal dysfunction and in a significant reduction of the worms' lifespan. These manifestations are paralleled by an increase of mitochondrial reactive oxygen species and can be prevented by treatment with antioxidant agents. In conclusion, these data indicate that this nematode-based assay is a promising surrogate model for investigating the heart-specific toxicity of amyloidogenic LC and for a rapid screening of new therapeutic strategies.

Article	Year
Glomerular podocytes contain neuron-like functional synaptic vesicles. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 16585060	2006
Human tumor-released microvesicles promote the differentiation of myeloid cells with transforming growth factor-beta-mediated suppressive activity on T lymphocytes. Cancer research\| PubMed ID: 16982774	2006
Podocyte glutamatergic signaling contributes to the function of the glomerular filtration barrier. Journal of the American Society of Nephrology : JASN\| PubMed ID: 19578006	2009
Biological effects of a new vacuolar-H,-ATPase inhibitor in colon carcinoma cell lines. Annals of the New York Academy of Sciences\| PubMed ID: 19723111	2009
Cannabinoid receptor 1 blockade ameliorates albuminuria in experimental diabetic nephropathy. Diabetes\| PubMed ID: 20068137	2010
A transgenic mouse model for uromodulin-associated kidney diseases shows specific tubulo-interstitial damage, urinary concentrating defect and renal failure. Human molecular genetics\| PubMed ID: 20472742	2010
Albuminuria and glomerular damage in mice lacking the metabotropic glutamate receptor 1. The American journal of pathology\| PubMed ID: 21356376	2011
Nephrin expression in adult rodent central nervous system and its interaction with glutamate receptors. The Journal of pathology\| PubMed ID: 21630272	2011
[Synaptic-like signals at the filtration barrier: the role of nephrin]. Giornale italiano di nefrologia : organo ufficiale della Società italiana di nefrologia\| PubMed ID: 22028253	2011
Proteinuria and glomerular damage in Rab3A knockout mice chronically fed a high-glucose diet. Nephron. Experimental nephrology\| PubMed ID: 22472623	2012
BAMBI regulates angiogenesis and endothelial homeostasis through modulation of alternative TGFβ signaling. PloS one\| PubMed ID: 22761782	2012
Urinary exosomal microRNAs in incipient diabetic nephropathy. PloS one\| PubMed ID: 24223694	2013
A Caenorhabditis elegans-based assay recognizes immunoglobulin light chains causing heart amyloidosis. Blood\| PubMed ID: 24665135	2014

Alessandro Corbelli

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