The Expression Of Nestin In Human Kidney And Involved Mechanisms

PART I the expression of Nest in in the normal adult kidneyBackground podocytes are highly specialized cells with a voluminous cell body. It's location, It's architecture, and it's relevance are unique. Their most prominent features are interdigitated foot processes. These foot processes are bridged by the slit diaphragm, which plays a major role in establishing selective permeability of the glomerular filtration barrier .They have adapted themselves to facilitating the bulk flow of the glomerular filtrate through the intercellular spaces or filtration slits. Nestin is a cytoskeleton associated class VI intermediate filament first identified in mitotically active central and peripheral nervous system progenitor cells that give rise to both neurons and glia during early neurogenesis. Subsequently, nestin is detected in progenitor cells of non-neuronal tissues, including muscle, the heart, pancreas and the skin follicle. Recently nestin expression has also been reported in adult tissues, such as skin and central nervous system, but is mainly restricted to areas of regeneration. Owing to its characteristic expression pattern, nestin is generally considered a marker of stem cells or progenitor cell. The present study show in the adult human kidney, Nestin expression is restricted to differentiated podocytes.However, wether Nestin can be expressed in human kidney remains explored.The present studies showed the expression of Nestin in the human kidney and identified the localization in the podocyte.Methods The expression of Nestin was detected by immunofluoresence; Normal kidneys from surgical nephrectomy were obtained during resection of kidneys for renal tumor. The expression of Nestin was examined by the immunohistochemistry and immunofluoresence; With the immunoelectron microscope, the location of Nestin in the podocyte was detected.Results Nestin was selectively expressed in the glomerular podocytes. Co-staining with anti-laminin antibody,which labels glomerular basement membrane, demonstrated that nestinimmunoreactivity was localized outside the glomerular basement membraneCo-staining with WT1 antibody showed that nestin immunoreactive protein waslocalized in the cytosol of the cells that were positive for wt1, a marker of podocyte.Immunoelectron microscopy showed nestin immunoreactivity in the cell body as wellas in primary and secondary processConclusions (1) Nestin was selectively expressed in the adult human glomerularpodocytes.(2) Nestin was localized in the cell body as well as in primary and secondary processPART II The effect of ncstin expression in podocyte1, The interacting proteins with NestinBackground Nestin is a large protein (>1600 amino acids).As is typical for IF proteins, nestin is characterized by an α -helical 'rod' domain that contains repeated hydrophobic heptad motif. The 240KD protein nestin contains a short N-terminus and an unusually long C-terminus. Nestin is unable to self-assemble, most likely because of its very short N-terminus(a domain necessary for IF assembly);therefore, nestin requires the presence of other IF proteins,such as vimentin or desmin,to assemble into heterodimers and mixed polymers. Since Nestin is reported to interact with all three major classes of cytoskeletal proteins, Nestin may be involved in the organization of cellular cytoskeletons,and may therefore play an important role in the maintenance normal podocyte function.To determine the portential significance of Nestin in podocyte,we examine the interaction of Nestin with other IF. Methods Immortalized murine podocytes were cultured .Co-expression of nestin with vimentin and α -internexin in the cultured podocye with immunofluoresence double stains. Podocyte protein extract was immunoprecipitated by anti nestin antibody. The immunoprecipitated proteins were separated on SDS-PAGE and blotted for vimentin α -internexin and nestin. The total cell lysate and normal mouse IgG were used as the positive and negative controlsResults. Immortalized mouse podocyte clones(MPC)grown under nonpermissive conditions display characteristics of differentiated arborized-type. Nestin is organized into fibroblast-like stress fibers extending into the processes of the differentiated cells and it forms a cage-like structure surrounding the nuclear. Immunofluoscence showed that nestin and vimentin ,nestin and α -internexin co-localized in the adult human podocyte. In cultured differentiated podocytes, nestin vimentin and α -internexin immunofluorescence demonstrated filamentous staining in cytosol and processes, and nestin co-localized with vimentin and α -internexin. Immunoprecipitation using anti-nestin antibody not only precipitated nestin, but also precipitated vimentin and α-interexin detected by immunoblot via an anti-vimentin and α -internexin antibody. With the same way, α -actinin4 could not be detected . Conclusion Nestin could physically interact with vimentin and α -internexin, but notwith α -actinin42, The role of Nestin in the maintance the structure of podocyteBackground Two decades ago, Nestin was first identified in a population ofproliferating neuronal cells, which was recognized as neuronal stem cell. Later, moreand more functions were identified in non-neuronal cells. such as the important role incoordinating or bridging cytoskeletal proteins; in regulation of the assembly anddisassembly of IF, participating in remodeling of the cell. But the nestin expressionand function in the differentiated cells have no reports. The present studies show thatnestin is not only a special protein in the stem cell and rapidly dividing progenitor cell,but also can express in differentiated podocyte. It is suggested that nestin may play animportant role in maintaining normal structure of podocyte .But the precise role ofNestin in the processes structure remains to be fully elucidated.Methods The immortalized murine podocytes were cultured. In differentiatedpodocytes,the expression of nestin was knock-down by RNAi. The effect of nestinknock-down was examined by western blotting and immunofluoresence. The cellswith the projections longer than maximal length of the cell body in siRNA or controlvector transfected cells were counted respectively.Results Nestin siRNA markedly reduced or abolished nestin expression .Moreimportantly, siRNA nestin knock-down was associated with significantly reducedprocess formation. In cells transfected with nestin siRNA, the percentage of cells withprocesses was significantly lower than cells transfected with control vector(77% ±6.3% vs 16%±4.6%,n=3,p<0.01).Conclusion nestin may play an important role in maintaining normal function ofpodocyte .part IIIThe expression of nestin in the human disease kidneyBackground The visceral glomerular epitheliar cell,also known as the podocyte, plays an important role in the maintenance of renal glomerular function .This cell type is highly specialized and its foot processes together with the interposed slit diaphragm(SD) form the final barrier to urinary protein loss. Effacement of foot processes is the morphological basement of proteinuria .To maintain an intact glomerular filter barrier ,podocyte-podocyte interactions and podocyte interactions with the glomerular basement membrane are essential. Formation and maintenance of these podocyte processes depend on a well-developed and complex cytoskeleton. The podocyte cytoskeleton is also suggested to play a critical role in counterbalancing the mechanical stretch stress,preventing outward ballooning of the vessel and preserving the normal architecture of the glomerular tuft.Our cellular experiments showed that Nestin may be involved in the intermediate filament arrangement and the processes structure maintanace .The present studies demonstrated the relationship between the podocyte Nestin expression and proteinuria.It is suggested the reduction of Nestin expression is associated with podocyte injury. The mechanism by which Nestin is involved in maintaining normal podocyte function and the role of Nestin in diseased podocyte remains to be explored.Methods Kidney tissues were obtained from needle biopsies of 25 patients with nephritic syndrome and four patients diagnosed with IgA nephropathy with mild mesangial expansion (IgA GN).The pathological diagnosis of these patients with proteinuria was minimal change disease (MCD, n=10),membranous nephropathy(MN, n=8,) focal segmental glomerular sclerosis(FSGS, n=3), diabetic nephropathy (DN, n=4). The expression of nestin in these diseases was detected by immunohistrochemistry .Series sections of renal biopsies were stained with nestin and WT1 respectively. Nestin expression levels were assessed semi-quantitatively using morphometric analysis.Results Semi-quantitative morphometric analysis , which examined both area and signal intensity of staining, showed that glomerular nestin expression levels in IgAGN did not differ from normal control. However, glomerular nestin expression levels in MCD,MN were significantly lower compared to normal kidney and IgAGN. In FSGS ,nestin expression in sclerotic area was substantially reduced or absent.this appears to be due to loss of podocytes in these areas, since WT1was absent in the same sclerotic area in a continuous series of sections. Glomerular nestin expressionlevel was inversely correlated with 24 hour proteinuria.Conclusions Podocyte nestin expression reduced in kidneys with podocyte foot processes effacement and proteinuria. The present studies suggest that nestin may play an important role in maintaining normal function of podocyte in human kidney.