Human CD133 + Renal Progenitor Cells Induce Erythropoietin Production and Limit Fibrosis after Acute Tubular Injury

Persistent alterations of the renal tissue due to maladaptive repair characterize the outcome of acute kidney injury (AKI), despite a clinical recovery. Acute damage may also limit the renal production of erythropoietin, with impairment of the hemopoietic response to ischemia and possible lack of its reno-protective action. We aimed to evaluate the effect of a cell therapy using human CD133(+) renal progenitor cells on maladaptive repair and fibrosis following AKI in a model of glycerol-induced rhabdomyolysis. In parallel, we evaluated the effect of CD133(+) cells on erythropoietin production. Administration of CD133(+) cells promoted the restoration of the renal tissue, limiting the presence of markers of injury and pro-inflammatory molecules. In addition, it promoted angiogenesis and protected against fibrosis up to day 60. No effect of dermal fibroblasts was observed. Treatment with CD133(+) cells, but not with PBS or fibroblasts, limited anemia and increased erythropoietin levels both in renal tissue and in circulation. Finally, CD133(+) cells contributed to the local production of erythropoietin, as observed by detection of circulating human erythropoietin. CD133(+) cells appear therefore an effective source for cell repair, able to restore renal functions, including erythropoietin release, and to limit long term maldifferentiation and fibrosis

Quinagolide Treatment Reduces Invasive and Angiogenic Properties of Endometrial Mesenchymal Stromal Cells

Endometrial mesenchymal stromal cells (E-MSCs) extensively contribute to the establishment and progression of endometrial ectopic lesions through formation of the stromal vascular tissue, and support to its growth and vascularization. As E-MSCs lack oestrogen receptors, endometriosis eradication cannot be achieved by hormone-based pharmacological approaches. Quinagolide is a non-ergot-derived dopamine receptor 2 agonist reported to display therapeutic effects in in vivo models of endometriosis. In the present study, we isolated E-MSCs from eutopic endometrial tissue and from ovarian and peritoneal endometriotic lesions, and we tested the effect of quinagolide on their proliferation and matrix invasion ability. Moreover, the effect of quinagolide on E-MSC endothelial differentiation was assessed in an endothelial co-culture model of angiogenesis. E-MSC lines expressed dopamine receptor 2, with higher expression in ectopic than eutopic ones. Quinagolide inhibited the invasive properties of E-MSCs, but not their proliferation, and limited their endothelial differentiation. The abrogation of the observed effects by spiperone, a dopamine receptor antagonist, confirmed specific dopamine receptor activation. At variance, no involvement of VEGFR2 inhibition was observed. Moreover, dopamine receptor 2 activation led to downregulation of AKT and its phosphorylation. Of interest, several effects were more prominent on ectopic E-MSCs with respect to eutopic lines. Together with the reported effects on endometrial and endothelial cells, the observed inhibition of E-MSCs may increase the rationale for quinagolide in endometriosis treatment

The DCR protein TTC3 affects differentiation and Golgi compactness in neurons through specific actin-regulating pathways

In neuronal cells, actin remodeling plays a well known role in neurite extension but is also deeply involved in the organization of intracellular structures, such as the Golgi apparatus. However, it is still not very clear which mechanisms may regulate actin dynamics at the different sites. In this report we show that high levels of the TTC3 protein, encoded by one of the genes of the Down Syndrome Critical Region (DCR), prevent neurite extension and disrupt Golgi compactness in differentiating primary neurons. These effects largely depend on the capability of TTC3 to promote actin polymerization through signaling pathways involving RhoA, ROCK, CIT-N and PIIa. However, the functional relationships between these molecules differ significantly if considering the TTC3 activity on neurite extension or on Golgi organization. Finally, our results reveal an unexpected stage-dependent requirement for F-actin in Golgi organization at different stages of neuronal differentiation.status: publishe

Molecular and functional characterization of urine-derived podocytes from patients with Alport syndrome

Alport syndrome (AS) is a genetic disorder involving mutations in the genes encoding collagen IV α3, α4 or α5 chains, resulting in the impairment of glomerular basement membrane. Podocytes are responsible for production and correct assembly of collagen IV isoforms; however, data on the phenotypic characteristics of human AS podocytes and their functional alterations are currently limited. The evident loss of viable podocytes into the urine of patients with active glomerular disease enables their isolation in a non-invasive way. Here we isolated, immortalized, and subcloned podocytes from the urine of three different AS patients for molecular and functional characterization. AS podocytes expressed a typical podocyte signature and showed a collagen IV profile reflecting each patient's mutation. Furthermore, RNA-sequencing analysis revealed 348 genes differentially expressed in AS podocytes compared with control podocytes. Gene Ontology analysis underlined the enrichment in genes involved in cell motility, adhesion, survival, and angiogenesis. In parallel, AS podocytes displayed reduced motility. Finally, a functional permeability assay, using a podocyte-glomerular endothelial cell co-culture system, was established and AS podocyte co-cultures showed a significantly higher permeability of albumin compared to control podocyte co-cultures, in both static and dynamic conditions under continuous perfusion. In conclusion, our data provide a molecular characterization of immortalized AS podocytes, highlighting alterations in several biological processes related to extracellular matrix remodelling. Moreover, we have established an in vitro model to reproduce the altered podocyte permeability observed in patients with AS. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland..status: publishe

Opportunities and progress in open AWE hardware

<p><b>A.</b> 1 DIV primary hippocampal neurons plated on biochemistry dishes were treated for 24h with vehicle or with the ROCK inhibitor Y27632. Cells were then lysed and the expression of the indicated proteins was analyzed by western blotting with the indicated antibodies. In the left panel the ratio between phopshorylated and non-phosporylated forms of myosin light chain (MLC, positive control) and of Cofilin was quantified. pMLC and pCofilin indicate the phosphorylated forms of the two proteins. The result represents the average of three independent experiments. <b>B–D.</b> Primary hippocampal neurons were co-transfected with the CMV expression plasmids encoding GFP-TTC3 and with HA empty plasmid (Ctrl), HA tagged wild type LIMK (wt) or with the indicated HA-tagged LIMK mutants. Cells were then processed for IF to reveal the expression of the encoded proteins and with anti-GM130 antibodies. The percentage of differentiated cells (C) and of cells with intact Golgi (D) was then quantified. Scale bars = 10 μm; error bars = SEM; **P<0.01, ***P<0.001, ns = non significant, two tails Student T-test.</p

Effects of altered TTC3 levels on neuronal differentiation.

<p><b>A–B.</b> Hippocampal neurons extracted from E18.5 rats were electroporated by nucleofection with the indicated plasmids. 24 hours (A) or 48 hours (B) after plating, cells were processed for immunofluorescence and the morphology of GFP-positive cells was assessed to analyze their distribution across the indicated differentiation stages. <b>C.</b> Selected frames from time-lapse series of neurons transfected as in panel A, undergoing differentiation in culture. For full movies, see supporting material. <b>D–E.</b> Hippocampal neurons were nucleofected with control or TTC3-specific sh-RNA-expressing plasmids, plated and allowed to differentiate 72 hours in culture. Cells were then processed for IF with anti-Tau antibodies and the axonal length was then quantified with ImageJ. <b>F.</b> Overexpression of GFP-TTC3 (TTC3) is able to rescue the phenotype induced by TTC3 downregulation. Cells were co-electroporated with sh-RNA-expressing plasmids together with GFP-empty (empty) or GFP-TTC3 (TTC3). After 72h, cells were than analyzed as in panel E. Scale bars = 10 μm; error bars = Standard Error of the Mean (SEM); *P<0.05, **P<0.01, two tails Student T-test.</p

Effects of actin-affecting drugs on the neurite-extension phenotypes induced by modulating TTC3 levels.

<p><b>A.</b> Hippocampal neurons were electroporated by nucleofection with the indicated plasmids and treated after 6 hours with vehicle (DMSO) or with 1 μM Cytochalasin-D (CytoD). 18 hours later cells were then processed for IF. Neurites were revealed by the GFP signal. <b>B.</b> Quantification of the percentage of differentiated cells in hippocampal neurons treated as in panel A. Differentiated cells were defined as those bearing at least one neurite longer than twice the cell body. <b>C.</b> Quantification of the average number of neurites in hippocampal neurons treated as in panel A. <b>D.</b> Hippocampal neurons were nucleofected with control or with TTC3-specific sh-RNA-expressing plasmids, plated and allowed to differentiate 54 hours in culture. Cells were then treated with 5 nM Jasplakinolide (Jaspla) or with vehicle for additional 18 hours and processed for IF to reveal GFP and Tau. The length of the main neurite (Tau-positive axon) was then quantified. Scale bars = 10 μm; error bars = SEM; *P<0.05, ***P<0.001, two tails Student T-test.</p

Effects of altered TTC3 levels on Golgi compactness.

<p><b>A–B.</b> Hippocampal neurons were electroporated by nucleofection with the indicated plasmids. Cells were processed for immunofluorescence with anti-GM130 antibodies 24 or 48 hours after plating. The percentage of cells with compact (normal) or fragmented Golgi morphology was then quantified (B). Note the compact morphology of the Golgi in the cell transfected with the GFP control plasmid (first cell from left in panel A). The other three cells in panel A are different examples of TTC3-overexpressing cells with disrupted Golgi, analyzed 24 hours after transfection. <b>C–D.</b> Confocal images of 7 DIV primary hippocampal neurons co-stained for TTC3 and GM130 after standard PFA fixation (C) or after pre-extraction with saponin (D). The right panels show a false color imange in which the white pixels represent the areas of colocalization (CL) of TTC3 and GM130 (cyan and magenta, respectively). <b>E.</b> Pixel intensity plots of the TTC3 and GM130 immunoreactivities in exemplar cells treated as in panels C and D. AU = arbitrary units. <b>F.</b> Hippocampal neurons were nucleofected with control or TTC3-specific sh-RNA-expressing plasmids (RNAi-TTC3), plated and allowed to differentiate 72 hours in culture. Cells were then processed for IF with GM130 antibodies and the Golgi compactness was measured (see Material and methods). Scale bars = 5 μm; error bars = SEM; **P<0.01, ***P<0.001, two tails Student T-test.</p