Podocytes in culture: past, present, and future

Human genetic and in vivo studies have helped to define the critical importance of podocytes for kidney function in health and disease. However, as in any other research area, by default these approaches do not allow for mechanistic studies. Such mechanistic studies require the availability of cells grown ex vivo (i.e., in culture) with the ability to directly study mechanistic events and control the environment such that specific hypotheses can be tested. A seminal breakthrough came about a decade ago with the documentation of differentiation in culture of primary rat and human podocytes and the subsequent development of conditionally immortalized differentiated podocyte cell lines that allow deciphering the decisive steps of differentiation and function of ‘in vivo’ podocytes. Although this paper is not intended to provide a comprehensive review of podocyte biology, nor their role in proteinuric renal diseases or progressive glomerulosclerosis, it will focus specifically on several aspects of podocytes in culture. In particular, we will discuss the scientific and research rationale and need for cultured podocytes, how podocyte cell-culture evolved, and how cultured podocytes are currently being used to uncover novel functions of podocytes that can then be validated in vivo in animal or human studies. In addition, we provide a detailed description of how to properly culture and characterize podocytes to avoid potential pitfalls

Real estate lease-backed securities

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Urban Studies and Planning, 1997."September 1997."Includes bibliographical references (leaves 83-84).by John P. McMurray and Samuel M. Mundel.S.M

Effect of cyclosporin A on proteinuria in the course of glomerulopathy associated with WT1 mutations

Denys–Drash syndrome (DDS) is characterized by progressive glomerulopathy caused by diffuse mesangial sclerosis (DMS), genitourinary defects, and a higher risk of developing Wilms’ tumor. It is commonly assumed that the DMS is unresponsive to any medications. In this report, we present a patient with Denys–Drash syndrome, in whom the cyclosporine A (CsA) was found to induce total remission. This observation and observations of other authors confirm that in genetic forms of nephrotic syndrome, the proteinuric effect of CsA may be due to a non-immunologic mechanism. We confirm the beneficial effect of CsA treatment in DDS; however, the potential nephrotoxicity of this drug will probably not allow long-term use

Rewiring neuronal microcircuits of the brain via spine head protrusions--a role for synaptopodin and intracellular calcium stores.

Neurological diseases associated with neuronal death are also accompanied by axonal denervation of connected brain regions. In these areas, denervation leads to a decrease in afferent drive, which may in turn trigger active central nervous system (CNS) circuitry rearrangement. This rewiring process is important therapeutically, since it can partially recover functions and can be further enhanced using modern rehabilitation strategies. Nevertheless, the cellular mechanisms of brain rewiring are not fully understood. We recently reported a mechanism by which neurons remodel their local connectivity under conditions of network-perturbance: hippocampal pyramidal cells can extend spine head protrusions (SHPs), which reach out toward neighboring terminals and form new synapses. Since this form of activity-dependent rewiring is observed only on some spines, we investigated the required conditions. We speculated, that the actin-associated protein synaptopodin, which is involved in several synaptic plasticity mechanisms, could play a role in the formation and/or stabilization of SHPs. Using hippocampal slice cultures, we found that ~70 % of spines with protrusions in CA1 pyramidal neurons contained synaptopodin. Analysis of synaptopodin-deficient neurons revealed that synaptopodin is required for the stability but not the formation of SHPs. The effects of synaptopodin could be linked to its role in Ca(2+) homeostasis, since spines with protrusions often contained ryanodine receptors and synaptopodin. Furthermore, disrupting Ca(2+) signaling shortened protrusion lifetime. By transgenically reintroducing synaptopodin on a synaptopodin-deficient background, SHP stability could be rescued. Overall, we show that synaptopodin increases the stability of SHPs, and could potentially modulate the rewiring of microcircuitries by making synaptic reorganization more efficient

Precisely tracking childhood death

Little is known about the specific causes of neonatal and under-five childhood death in high-mortality geographic regions due to a lack of primary data and dependence on inaccurate tools, such as verbal autopsy. To meet the ambitious new Sustainable Development Goal 3.2 to eliminate preventable child mortality in every country, better approaches are needed to precisely determine specific causes of death so that prevention and treatment interventions can be strengthened and focused. Minimally invasive tissue sampling (MITS) is a technique that uses needle-based postmortem sampling, followed by advanced histopathology and microbiology to definitely determine cause of death. The Bill & Melinda Gates Foundation is supporting a new surveillance system called the Child Health and Mortality Prevention Surveillance network, which will determine cause of death using MITS in combination with other information, and yield cause-specific population-based mortality rates, eventually in up to 12-15 sites in sub-Saharan Africa and south Asia. However, the Gates Foundation funding alone is not enough. We call on governments, other funders, and international stakeholders to expand the use of pathology-based cause of death determination to provide the information needed to end preventable childhood mortality

Isolating and Culturing Mouse Podocyte Cells to Study Diabetic Nephropathy

Diabetic nephropathy is associated with injury and loss of podocytes, specialized epithelial cells that are critical for glomerular filtration. This chapter describes a method of isolating and culturing podocyte cells from mouse adult kidneys. In this way, podocytes with genetic modifications can be obtained from transgenic animals and they can be used to study the effects of the diabetic environment in vitro

Establishment of Protein Delivery Systems Targeting Podocytes

Podocytes are uniquely structured cells that are critical to the kidney filtration barrier. Their anatomic location on the outer side of the glomerular capillaries expose podocytes to large quantities of both plasma and urinary components and thus are reachable for drug delivery. Recent years have made clear that interference with podocyte-specific disease pathways can modulate glomerular function and influence severity and progression of glomerular disease.Here, we describe studies that show efficient transport of proteins into the mammalian cells mouse 3T3 fibroblasts and podocytes, utilizing an approach termed profection. We are using synthetic lipid structures that allow the safe packing of proteins or antibodies resulting in the subsequent delivery of protein into the cell. The uptake of lipid coated protein is facilitated by the intrinsic characteristic of cells such as podocytes to engulf particles that are physiologically retained in the extracellular matrix. Profection of the restriction enzyme MunI in 3T3 mouse fibroblasts caused an increase in DNA degradation. Moreover, purified proteins such as beta-galactosidase and the large GTPase dynamin could be profected into podocytes using two different profection reagents with the success rate of 95-100%. The delivered beta-galactosidase enzyme was properly folded and able to cleave its substrate X-gal in podocytes. Diseased podocytes are also potential recipients of protein cargo as we also delivered fluorophore labeled IgG into puromycin treated podocytes. We are currently optimizing our protocol for in vivo profection.Protein transfer is developing as an exciting tool to study and target highly differentiated cells such as podocytes

Nephrin Is Expressed on the Surface of Insulin Vesicles and Facilitates Glucose-Stimulated Insulin Release

Nephrin, an immunoglobulin-like protein essential for the function of the glomerular podocyte and regulated in diabetic nephropathy, is also expressed in pancreatic beta-cells, where its function remains unknown. The aim of this study was to investigate whether diabetes modulates nephrin expression in human pancreatic islets and to explore the role of nephrin in beta-cell function. Nephrin expression in human pancreas and in MIN6 insulinoma cells was studied by Western blot, PCR, confocal microscopy, subcellular fractionation, and immunogold labeling. Islets from diabetic (n = 5) and nondiabetic (n = 7) patients were compared. Stable transfection and siRNA knockdown in MIN-6 cells/human islets were used to study nephrin function in vitro and in vivo after transplantation in diabetic immunodeficient mice. Live imaging of green fluorescent protein (GFP)-nephrin-transfected cells was used to study nephrin endocytosis. Nephrin was found at the plasma membrane and on insulin vesicles. Nephrin expression was decreased in islets from diabetic patients when compared with nondiabetic control subjects. Nephrin transfection in MIN-6 cells/pseudoislets resulted in higher glucose-stimulated insulin release in vitro and in vivo after transplantation into immunodeficient diabetic mice. Nephrin gene silencing abolished stimulated insulin release. Confocal imaging of GFP-nephrin-transfected cells revealed nephrin endocytosis upon glucose stimulation. Actin stabilization prevented nephrin trafficking as well as nephrin-positive effect on insulin release. Our data suggest that nephrin is an active component of insulin vesicle machinery that may affect vesicle-actin interaction and mobilization to the plasma membrane. Development of drugs targeting nephrin may represent a novel approach to treat diabetes

A novel fragment derived from the β chain of human fibrinogen, β43–63, is a potent inhibitor of activated endothelial cells in vitro and in vivo

Background: Angiogenesis and haemostasis are closely linked within tumours with many haemostatic proteins regulating tumour angiogenesis. Indeed we previously identified a fragment of human fibrinogen, fibrinogen E-fragment (FgnE) with potent anti-angiogenic properties in vitro and cytotoxic effects on tumour vessels in vivo. We therefore investigated which region of FgnE was mediating vessel cytotoxicity. Methods: Human dermal microvascular endothelial cells (ECs) were used to test the efficacy of peptides derived from FgnE on proliferation, migration, differentiation, apoptosis and adhesion before testing the efficacy of an active peptide on tumour vasculature in vivo. Results: We identified a 20-amino-acid peptide derived from the β chain of FgnE, β43–63, which had no effect on EC proliferation or migration but markedly inhibited the ability of activated ECs to form tubules or to adhere to various constituents of the extracellular matrix – collagen IV, fibronectin and vitronectin. Furthermore, our data show that β43–63 interacts with ECs, in part, by binding to αvβ3, so soluble αvβ3 abrogated β43–63 inhibition of tubule formation by activated ECs. Finally, when injected into mice bearing tumour xenografts, β43–63 inhibited tumour vascularisation and induced formation of significant tumour necrosis. Conclusions: Taken together, these data suggest that β43–63 is a novel anti-tumour peptide whose anti-angiogenic effects are mediated by αvβ3

Primary cilia disappear in rat podocytes during glomerular development

Most tubular epithelial cell types express primary cilia, and mutations of primary-cilium-associated proteins are well known to cause several kinds of cystic renal disease. However, until now, it has been unclear whether mammalian podocytes express primary cilia in vivo. In this study, we determined whether primary cilia are present in the podocytes of rat immature and mature glomeruli by means of transmission electron microscopy of serial ultrathin sections. In immature glomeruli of fetal rats, podocytes express the primary cilia with high percentages at the S-shaped body (88 ± 5%, n = 3), capillary loop (95 ± 4%, n =  4), and maturing glomerulus (76 ± 13%, n = 5) stages. The percentage of ciliated podocytes was significantly lower at the maturing glomerulus stage than at the former two stages. In mature glomeruli of adult rats, ciliated podocytes were not found at all (0 ± 0%, n = 11). These findings indicate that the primary cilia gradually disappear in rat podocytes during glomerular development. Since glomerular filtration rate increases during development, the primary cilia on the podocytes are subjected to a stronger bending force. Thus, the disappearance of the primary cilia presumably prevents the entry of excessive calcium-ions via the cilium-associated polycystin complexes and the disturbance of intracellular signaling cascades in mature podocytes