199 research outputs found
A nanoporous surface is essential for glomerular podocyte differentiation in three-dimensional culture.
Although it is well recognized that cell-matrix interactions are based on both molecular and geometrical characteristics, the relationship between specific cell types and the three-dimensional morphology of the surface to which they are attached is poorly understood. This is particularly true for glomerular podocytes - the gatekeepers of glomerular filtration - which completely enwrap the glomerular basement membrane with their primary and secondary ramifications. Nanotechnologies produce biocompatible materials which offer the possibility to build substrates which differ only by topology in order to mimic the spatial organization of diverse basement membranes. With this in mind, we produced and utilized rough and porous surfaces obtained from silicon to analyze the behavior of two diverse ramified cells: glomerular podocytes and a neuronal cell line used as a control. Proper differentiation and development of ramifications of both cell types was largely influenced by topographical characteristics. Confirming previous data, the neuronal cell line acquired features of maturation on rough nanosurfaces. In contrast, podocytes developed and matured preferentially on nanoporous surfaces provided with grooves, as shown by the organization of the actin cytoskeleton stress fibers and the proper development of vinculin-positive focal adhesions. On the basis of these findings, we suggest that in vitro studies regarding podocyte attachment to the glomerular basement membrane should take into account the geometrical properties of the surface on which the tests are conducted because physiological cellular activity depends on the three-dimensional microenvironment
Positive effects of a novel non-peptidyl low molecular weight radical scavenger in renal ischemia/reperfusion: a preliminary report
Ischemia/reperfusion (I/R) is one of the most common causes of acute kidney injury. Reactive oxygen species have been recognized to be an important contributor to the pathogenesis of I/R injury. We hypothesize that a non-peptidyl low molecular weight radical scavenger (IAC) therapy may counteract this factor, ultimately providing some protection after acute phase renal I/R injury. The aim of this preliminary study was to assess the ability of IAC to reduce acute kidney injury in C57BL/6 mice after 30-minute of bilateral ischemia followed by reperfusion. The rise in serum creatinine level was higher in C57BL/6 control mice after I/R when compared to IAC (1 mg)-treated mice. Control mice showed greater body weight loss compared to IAC-treated mice, and at pathology, reduced signs of tubular necrosis were also evident in IAC-treated mice. These preliminary evidences lay the basis for more comprehensive studies on the positive effects of IAC as a complementary therapeutic approach for acute phase renal I/R injury
NeuroD Expression in Podocytes and Interrelationships with Nephrin at Both Nuclear and Cytoplasmic Sites
Background/Aims The research of genes implicated in kidney glomerular function, eliciting cell fate program, is always at the forefront in nephrological studies. Several neurological molecules have been recently the object of study not only for their involvement in the central nervous system differentiation but also for their importance in the functionality of other organs and for mature phenotype, as in kidney. NeuroD, in CNS, is related to two functional roles, the early survival and the differentiation. The aim of our study was to ascertain the presence of NeuroD transcription factor in glomeruli and to understand which targets and mechanisms NeuroD controls. Methods: We used immunofluorescence (IF) studies on both human and mice renal tissues and on cultured podocytes to describe NeuroD distribution; then we investigated NeuroD binding to the nephrin promoter region in cultured podocytes by chromatin-immuno-precipitation (ChIP) assay. The overexpression of NeuroD in podocytes was used to establish first its role in nephrin synthesis, evaluated by real-Time quantitative (RTq) PCR and western-blot (WB) and successively to determine the recovery of cell morphology after adriamycin injury, measuring foot processes length. Results: We identified NeuroD transcription factor in glomeruli, in the same cells positive for WT1 and synaptopodin, namely podocytes; subsequently we observed a differentiation dependent NeuroD distribution in cultured podocytes, and a consistent link of NeuroD with the Nephrin promoter leading to the regulation of Nephrin translation and transcription. Our data also describes NeuroD expression in cytoplasm as phosphoprotein linked to nephrin and actinin4. Preliminary experiments seem to indicate NeuroD involved in dynamics of cell shape regulation after adriamycin injury. Conclusion: we propose that NeuroD possess in podocytes a dual ability acting in the nucleus as a transcription factor and in cytoplasm stabilizing cell shape
Role of Sema4C in TGF-β1-induced mitogen-activated protein kinase activation and epithelialâmesenchymal transition in renal tubular epithelial cells
Background. The p38 mitogen-activated protein kinase (p38 MAPK) is an important intracellular signal transduction pathway involved in TGF-β1-induced epithelialâmesenchymal transition (EMT). Sema4C, a member of the semaphorin family, was found to be essential for the activation of p38 MAPK. However, the role of Sema4C in promoting TGF-β1-induced EMT is unclear
Early involvement of cellular stress and inflammatory signals in the pathogenesis of tubulointerstitial kidney disease due to UMOD mutations
Autosomal dominant tubulointerstitial kidney disease (ADTKD) is an inherited disorder that causes progressive kidney damage and renal failure. Mutations in the UMOD gene, encoding uromodulin, lead to ADTKD-UMOD related. Uromodulin is a GPI-anchored protein exclusively produced by epithelial cells of the thick ascending limb of Henle's loop. It is released in the tubular lumen after proteolytic cleavage and represents the most abundant protein in human urine in physiological condition. We previously generated and characterized a transgenic mouse model expressing mutant uromodulin (TgUmodC147W) that recapitulates the main features of ATDKD-UMOD. While several studies clearly demonstrated that mutated uromodulin accumulates in endoplasmic reticulum, the mechanisms that lead to renal damage are not fully understood. In our work, we used kidney transcriptional profiling to identify early events of pathogenesis in the kidneys of TgUmodC147Wmice. Our results demonstrate up-regulation of inflammation and fibrosis and down-regulation of lipid metabolism in young TgUmodC147Wmice, before any functional or histological evidence of kidney damage. We also show that pro-inflammatory signals precede fibrosis onset and are already present in the first week after birth. Early induction of inflammation is likely relevant for ADTKD-UMOD pathogenesis and related pathways can be envisaged as possible novel targets for therapeutic intervention
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 numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models.
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications
Discoidin domain receptor-1 and periostin: New players in chronic kidney disease
The incidence and prevalence of chronic kidney disease represents an important problem for public health. In renal diseases, the main histologic alterations derive from the development of renal fibrosis which results from the loss of the balance between proand anti-fibrotic factors. Tyrosine kinase receptors (RTKs) and matricellular proteins (MPs) are nowadays studied as potential modulators of renal injury. RTKs regulate cell cycle, migration, metabolism and cellular differentiation. Discoidin domain receptor-1 (DDR-1) is an RTK that has been extensively studied in cancer, and lung and renal diseases. It modulates inflammatory recruitment, extracellular matrix deposition and fibrosis; in renal diseases, it appears to act independently of the underlying disease. MPs regulate cell-matrix interactions and matrix accumulation, cellular adhesion and migration, and expression of inflammatory cells. Periostin is an MP, mainly studied in bone, heart, lung and cancer. Several studies demonstrated that it mediates cellmatrix interactions, migration of inflammatory cells and development of fibrosis. Recently, it has been reported in several nephropathies. In this review, we discuss the potential pathological roles of DDR-1 and periostin focussing on the kidney in both experimental models and human diseases
Podocyte developmental defects caused by adriamycin in zebrafish embryos and larvae: A novel model of glomerular damage
The zebrafish pronephros is gaining popularity in the nephrology community, because embryos are easy to cultivate in multiwell plates, allowing large number of experiments to be conducted in an in vivo model. In a few days, glomeruli reach complete development, with a structure that is similar to that of the mammalian counterpart, showing a fenestrated endothelium and a basement membrane covered by the multiple ramifications of mature podocytes. As a further advantage, zebrafish embryos are permeable to low molecular compounds, and this explains their extensive use in drug efficacy and toxicity experiments. Here we show that low concentrations of adriamycin (i.e. 10 and 20 \u3bcM), when dissolved in the medium of zebrafish embryos at 9 hours post-fertilization and removed after 48 hours (57 hpf), alter the development of podocytes with subsequent functional impairment, demonstrated by onset of pericardial edema and reduction of expression of the podocyte proteins nephrin and wt1. Podocyte damage is morphologically confirmed by electron microscopy and functionally supported by increased clearance of microinjected 70 kDa fluorescent dextran. Importantly, besides pericardial edema and glomerular damage, which persist and worsen after adriamycin removal from the medium, larvae exposed to adriamycin 10 and 20 \u3bcM do not show any myocardiocyte alterations nor vascular changes. The only extra-renal effect is a transient delay of cartilage formation that rapidly recovers once adriamycin is removed. In summary, this low dose adriamycin model can be applied to analyze podocyte developmental defects, such as those observed in congenital nephrotic syndrome, and can be taken in consideration for pharmacological studies of severe early podocyte injury
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