|
|
|
|
 |
|||
|
|||
doi:10.1369/jhc.6A7058.2007
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Volume 55 (4): 411-421, 2007
Copyright ©The Histochemical Society, Inc.
Nestin Expression in Adult and Developing Human Kidney
Department of Pharmacology "Giorgio Segre" (EB,MR,LF), Department of Human Pathology and Oncology, Policlinico Le Scotte (RO,SM,PT), and Department of Evolutionary Biology (LE), University of Siena, Siena, Italy
Correspondence to: Eugenio Bertelli, Dept. of Pharmacology "Giorgio Segre", Section of Anatomy, University of Siena, Via Aldo Moro 4, I-53100 Siena, Italy. E-mail: bertelli5{at}unisi.it
 |
Summary |
|---|
 Top Summary IntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
Nestin is considered a marker of neurogenic and myogenic precursor cells. Its arrangement is regulated by cyclin-dependent kinase 5 (CDK5), which is expressed in murine podocytes. We investigated nestin expression in human adult and fetal kidney as well as CDK5 presence in adult human podocytes. Confocal microscopy demonstrated that adult glomeruli display nestin immunoreactivity in vimentin-expressing cells with the podocyte morphology and not in cells bearing the endothelial marker CD31. Glomerular nestin-positive cells were CDK5 immunoreactive as well. Western blotting of the intermediate filament-enriched cytoskeletal fraction and coimmunoprecipitation of nestin with anti-CDK5 antibodies confirmed these results. Nestin was also detected in developing glomeruli within immature podocytes and a few other cells. Confocal microscopy of experiments conducted with antibodies against nestin and endothelial markers demonstrated that endothelial cells belonging to capillaries invading the lower cleft of S-shaped bodies and the immature glomeruli were nestin immunoreactive. Similar experiments carried out with antibodies raised against nestin and
-smooth muscle actin showed that the first mesangial cells that populate the developing glomeruli expressed nestin. In conclusion, nestin is expressed in the human kidney from the first steps of glomerulogenesis within podocytes, mesangial, and endothelial cells. This expression, restricted to podocytes in mature glomeruli, appears associated with CDK5. (J Histochem Cytochem 55:411–421, 2007)
Key Words: nestin • podocytes • cyclin-dependent kinase 5 • CD31 • -smooth muscle actin • immunohistochemistry • confocal microscopy
|
Introduction |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
THE RENAL CORPUSCLE consists of a tuft of blood capillaries surrounded by Bowman's capsule, a bowl-shaped double-layered epithelial structure. The two epithelial layers of the capsule, though continuous at the vascular pole of the corpuscle, are separated by an interval, the urinary space, into which the primary urine is ultrafiltrated. The inner layer of the capsule, tightly applied to the blood capillaries, is also referred to as the visceral layer and is formed of highly specialized epithelial cells called podocytes because of their peculiar shape. Along with the basement membrane and the fenestrated endothelial cells, these cells constitute the glomerular filtration barrier, whose integrity is essential for the filtration and proper composition of the primary urine. Several functions have been assigned to podocytes, including key roles in determining the permeability properties of the glomerular filter, in counteracting intraglomerular hydrostatic pressure, and in the synthesis of basement membrane components (Pavenstädt et al. 2003
). Even though some of these functions are more supposed than definitely proven (Pavenstädt et al. 2003), the importance of podocytes in maintaining glomerular homeostasis and proper renal function is testified to by the development of proteinuria and progressive glomerulosclerosis upon their injury (Kriz et al. 1994; Ruggenenti et al. 2001; Gubler 2003). Podocytes display a very complex morphology that must be preserved to accomplish their delicate functions (Bariety et al. 1998; Nakahama et al. 1999; Hir et al. 2001; Kerajaschki 2001). The shape of podocytes is sui generis, with a cell body that protrudes into the capsule lumen and long primary processes anchored to the basement membrane by extending secondary processes referred to as foot processes. The foot processes of adjacent podocytes interdigitate on the surface of the basement membrane, leaving a space between them, the filtration slit, which is occupied by a thin diaphragm. The complex and ramified shape of podocytes has led some investigators to compare them with neurons. Indeed, an increasing number of features are described as being shared by the two cell types. First, podocytes and neurons are postmitotic terminally differentiated cells (Nagata et al. 1998; Gubler 2003); second, they express common sets of proteins including synaptopodin (Mundel et al. 1997; Czarnecki et al. 2005), nephrin (Putaala et al. 2001), cyclin-dependent kinase 5 (CDK5) (Griffin et al. 2004), olfactomedin-related glycoprotein (Kondo et al. 2000), a type III receptor protein tyrosine phosphatase (Beltran et al. 2003), and Rab3A and Rabphilin-3a (Rastaldi et al. 2003). Third, some transcription factors such as N-myc (Hirvonen et al. 1989) and Wilms' tumor suppressor protein (Armstrong et al. 1992) are at least transiently expressed either in neurons or podocytes. Finally, if on one hand the mechanism for the formation of podocyte processes is somehow similar to the development of neuronal dendrites (Kobayashi et al. 2004), on the other hand it has recently been shown that podocytes possess neuronal-like functional synaptic vesicles (Rastaldi et al. 2006). In this respect, the fact that CDK5 has been shown to regulate podocyte morphology (Griffin et al. 2004), coupled with the finding that CDK5 in neuronal progenitor ST15A cells and in C2C12 myoblasts is associated with nestin and governs its arrangement (Sahlgren et al. 2003), prompted us to investigate if nestin was expressed by human podocytes as well. Here we report that nestin is present in human podocytes, is associated with CDK5 and vimentin, and represents an early marker of developing glomeruli where it can be detected in the vesicles (the first epithelial structure consisting of polarized cells) and in the podocyte layer of the S-shaped bodies, in endothelial cells of blood capillaries invading the lower cleft of the S-shaped bodies and, finally, in mesangial precursor cells. |
Materials and Methods |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
Antibodies
Primary antibodies were purchased as follows: mouse anti-nestin monoclonal antibody (MAb5326) and rabbit anti-nestin polyclonal antibody (Ab5922; Chemicon, Temecula, CA); mouse anti-SMA MAb (clone 1A4; Neomarkers, Fremont, CA); mouse anti-CD31 (clone JC70A) and anti-CD34 MAbs (clone QBEnd-10) from DakoCytomation (Glostrup, Denmark); mouse anti-vimentin MAb (clone V9; Sigma, St Louis, MO); and rabbit anti-CDK5 PAb C-8 and rabbit anti-vimentin (H-84) (Santa Cruz Biotechnology; Santa Cruz, CA).
Secondary antibodies were obtained from the following sources: double-labeling-grade tetramethylrhodamine isothiocyanate (TRITC)-conjugated donkey anti-mouse IgG and TRITC-conjugated donkey anti-rabbit IgG from Chemicon; fluorescein isothiocyanate-conjugated (FITC) goat anti-mouse IgG antibody from Sigma; double-labeling-grade horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG from Zymed (South San Francisco, CA); and rabbit anti-mouse IgG from DakoCytomation.
Tissues
Cases and controls were taken consecutively and retrospectively from the surgical and autoptic files of the department of Human Pathology and Oncology. Adult kidney samples were obtained from surgical nephrectomy for carcinoma (n=10, age range 24–78 years, median age 60 years). Kidneys were fixed in 4% buffered formalin for 24–48 hr, and normal tissue, far from the neoplasia, was sampled and paraffin embedded.
Fetal kidneys were obtained from autopsies for spontaneous abortion or induced termination of pregnancy (TP) (n=23; gestational age range, 16–37 weeks; median gestational age 22 weeks). Macerated and malformed stillbirths were not included in the study. The cause of spontaneous fetal death was placental in origin and included abruptio placentae, true cord knot, acute massive placenta infarcts, acute chorioamnionitis; and conditions associated with prolonged stress such as fetal growth restriction and other morphological signs of chronic suffering, as well as maternal preeclampsia and eclampsia, were excluded from this study. TP stemmed from either minor malformations unrelated to alterations in the urinary system or maternal (personal) reasons. A complete autopsy was carried out between the second and twelfth hour after fetal expulsion.
Selected samples for biochemical analysis were immediately frozen in liquid nitrogen and stored at –80C. Other fresh tissues (only from adult kidneys) were sampled for immunofluorescence histochemistry, cryoprotected with Killik Frozen Medium (Bio Optica; Milan, Italy), and promptly frozen in liquid nitrogen. Ten-µm-thick sections were cut at –25C, air-dried, fixed in acetone at –20C for 10 min, and stored at –80C.
Immunohistochemistry
Immunohistochemistry to detect nestin was performed as previously described (Toti et al. 2005). Three- to 5-µm sections were cut from each specimen, mounted on electrostatically charged slides, and dried. Sections were dewaxed, rehydrated, and washed in TBS. TBS was used for all subsequent washes and for antibody dilutions. Nestin antigenic sites were unmasked by heating sections in 0.01 M citrate buffer (pH 6.0) with a microwave oven (two 5-min cycles at 750 W) and were subsequently rinsed for 10 min in 3% hydrogen peroxide to block endogenous peroxidase. Slides were then incubated for 1 hr at room temperature with anti-nestin MAb (working dilution 1:200). The ABC system (Vector Laboratories; Burlingame, CA) was applied on sections of fetal samples, and the reaction was developed with 1 mg/ml 3,3'-diaminobenzidine tetrahydrochloride (Sigma) dissolved in TBS containing 0.3% H2O2. Sections of adult kidneys were incubated with a bridge antibody anti-mouse IgG for 30 min at room temperature, and the reactions were developed by the alkaline phosphatase–anti-alkaline phosphatase technique (Dakopatts; DakoCytomation) with new fuchsin. Harris hematoxylin was used for nuclear counterstaining. Negative controls were prepared by substituting the primary antibody with the corresponding preimmune serum. Cognate sections were stained with hematoxylin and eosin to check for renal pathologies or malformations.
Confocal Microscopy
A TCS 4D Leica (Wetzlar, Germany) confocal microscope was used to evaluate double-labeling experiments carried out on frozen sections of adult kidney and on formalin-fixed, paraffin-embedded sections of fetal kidney.
Frozen sections were incubated 2 hr at room temperature with anti-vimentin MAb (working dilution 1:50) or anti-CD31 MAb (working dilution 1:100) followed by FITC-conjugated anti-mouse IgG for 1 hr at room temperature; the second primary antibody, anti-nestin PAb (working dilution 1:200), was applied overnight at 4C followed for 1 hr at room temperature by the appropriate TRITC-conjugated secondary antibody (working dilution 1:50). Other experiments were carried out incubating sections with anti-nestin MAb (working dilution 1:100) or anti-CD31 MAb (working dilution 1:100) for 2 hr at room temperature, followed for 1 hr by TRITC-conjugated anti-mouse IgG (working dilution 1:50). Rabbit anti-CDK5 was then applied overnight at 4C, followed by HRP-conjugated anti-rabbit IgG for 1 hr at room temperature. Application of fluorescein-labeled tyramide amplification reagent (Perkin-Elmer; Boston, MA) catalyzed a HRP-driven reaction that resulted in the deposition of a fluorescent precipitate.
Sections of embryonic kidney were dewaxed, rehydrated, and processed as follows: they were incubated with rabbit anti-nestin PAb for 1 hr at room temperature followed by the ABC system according to manufacturer's instructions (Vector Laboratories); the second primary antibody, anti-CD34 MAb (working dilution 1:100) or anti-SMA MAb (working dilution 1:200), was applied overnight at 4C followed for 1 hr at room temperature by TRITC-conjugated anti-mouse IgG (working dilution 1:50). Final application of fluorescein-labeled tyramide amplification reagent (Perkin-Elmer) catalyzed a HRP-driven reaction that resulted in the deposition of a fluorescent precipitate on nestin antigenic sites.
SDS/PAGE Electrophoresis, Coimmunoprecipitation, and Western Blotting
To confirm our immunohistochemical results, Western blotting analysis with anti-nestin antibody was carried out on proteins of the intermediate filament-enriched cytoskeletal fraction (IFCF) of adult and fetal kidney, separated by SDS-PAGE. IFCFs were prepared as previously reported (Achtstaetter et al. 1986; Carapelli et al. 2004; Toti et al. 2005) and stored at –80C until use. Just before use, aliquots of IFCF were thawed and mixed with Laemmli sample buffer.
Immunoprecipitation experiments were carried out on total homogenates of adult renal cortex. Pieces of renal cortex were placed in homogenization buffer (50 mM Tris/HCl, 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, 1% NP-40, 1 mM PMSF, 2 µg/ml aprotinin, pH 7.4) and homogenized with a Dounce homogenizer. The suspension was centrifuged at 5000 x g for 10 min at 4C, and the protein content was measured with the microBCA protein assay reagent kit (Pierce; Rockford, IL). Samples were stored at –80C until use. Five hundred µl of homogenate was precleared with protein A–Sepharose (Sigma), centrifuged, and the pellet of Sepharose beads was placed in Laemmli sample buffer. Two µl of anti-CDK5 IgG or 3 µl of anti-vimentin PAb was added to the supernatant and incubated overnight at 4C, followed by 2 hr with the protein A–Sepharose. Immunocomplexes were washed three times with buffer, and the final pellet was resuspended in Laemmli sample buffer.
IFCFs, immunoprecipitates, and proteins released from the Protein A–Sepharose complexes used for the preclearings were separated by electrophoresis through a 6% polyacrylamide gel and transferred to nitrocellulose in a Bio-Rad Transblot apparatus (BioRad; Segrate, Italy). A 5% (w/v) solution of skim milk in TBS was used to quench nonspecific protein binding. Membranes were incubated overnight at room temperature with 1:2000 anti-nestin MAb in 5% (w/v) solution of skim milk in TBS. Specific bands were detected using an electrochemiluminescense kit (Roche Diagnostics; Milan, Italy).
|
Results |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
Adult Kidney
In adult kidney, nestin staining was restricted to glomeruli, smooth muscle cells of the arterioles, and some endothelial cells (Figures 1A –1C). The highest intensity of staining was referred to podocytes where nestin immunoreactivity was consistent (Figures 1A–1C) and to the tunica media of some arterioles. Even though with a variable degree of intensity, endothelial cells of arterioles and of some blood capillaries were nestin labeled as well (Figure 1A).
|
Glomerular nestin-expressing cells resembled podocytes as to their location and morphology. However, to confirm immunohistochemical data obtained on formalin-fixed, paraffin-embedded material, a series of double-immunofluorescence experiments were performed on frozen sections and analyzed with the confocal microscope. A well-known marker of podocytes, vimentin, was used along with nestin in double-labeling experiments. In the glomerulus, vimentin is expressed at high levels only within podocytes, mesangial, and endothelial cells being reported as showing possibly only a much weaker labeling (Bachmann et al. 1983
; Holthöfer et al. 1984; Stamenkovic et al. 1986). Other podocyte markers like podocin, synaptopodin, nephrin, ZO-1, and podocalixyn could not be used for our purpose in vivo because they were all concentrated prevalently in the foot processes where intermediate filaments are not present, and their staining hardly overlapped with nestin. These experiences showed that all nestin-immunoreactive cells were, indeed, highly vimentin-expressing cells as well (Figures 1D–1F). Moreover, nestin/vimentin double-labeled cells clearly resembled podocytes, displaying long immunoreactive primary processes (Figures 1G–1I). Double-labeling experiments carried out using nestin and the endothelial marker CD31 (Naruse et al. 2000) showed that the relationships occurring between nestin-positive cells and endothelial cells were those expected between podocytes and the glomerular capillaries (Figures 1J–1L).
Because it has been reported that CDK5 is expressed in murine podocytes (Griffin et al. 2004), and that it plays a key role in the regulation of nestin organization (Sahlgren et al. 2003), we performed CDK5/nestin double-labeling experiments that showed a certain degree of colocalization of the two proteins (Figures 2A
–2F). Moreover, because CDK5 has been previously reported in endothelial cells (Sharma et al. 2004), additional experiments have been carried out to rule out the possibility that CDK5 staining had to be referred in part to the glomerular endothelium. Indeed, confocal microscopy of CDK5/CD31 double-labeled sections showed two different staining patterns with no detectable colocalizations of the two antigens in the renal corpuscles (Figures 2G–2I). Remarkably, CDK5 fluorescence appeared frequently arranged parallel to the glomerular endothelium (Figure 2I).
|
To confirm that the antibodies used in this study recognized actual nestin, we carried out Western blot analysis with anti-nestin MAb on renal IFCF of adult individuals from total homogenates of adult kidneys. Depending on samples, one or two nestin-immunoreactive bands could be unveiled in the IFCF migrating at 240 and 280 kDa (Figure 3A ), the lower band being constant. Moreover, to confirm that CDK5/nestin and nestin/vimentin colocalizations were due to their actual association, additional Western blotting experiments with anti-nestin MAb were carried out on CDK5 and vimentin immunoprecipitates. The presence of a double-nestin-immunoreactive band of the appropriate molecular mass (240 and 280 kDa) in the immunoprecipitates demonstrated that nestin coimmunoprecipitated with vimentin and CDK5, confirming on the one hand that nestin and vimentin coassemble together even in podocytes and suggesting on the other hand that the association of nestin and CDK5 observed in C2C12 myoblasts and ST15A neuronal precursors (Sahlgren et al. 2003
) occurs in human podocytes as well (Figure 3B).
|
Fetal Kidney
Development of human metanephric kidney begins at gestational week 4–5 and is said to terminate after the 35th week of gestation (Nagata et al. 1993
). In this period a nephrogenic zone located under the renal capsule continues to add new developing glomeruli, the older being located deeper in the kidney and the newer just under the capsule. For this reason, in a single section of fetal kidney it is possible to appreciate all the stages of glomerular development in a sort of gradient of maturation going from the surface to the depth of the organ. The steps of glomerular maturation are morphologically divided into the following stages: vesicle (V), S-shaped body (S), capillary loop (C), and maturing glomerulus (M) (Naruse et al. 2000; Pavenstädt et al. 2003). At low magnification, nestin staining on fetal kidney sections clearly appeared located in glomeruli at all the maturation stages (Figure 4A
). Although less constantly, a certain degree of staining was apparent even at the V stage (Figure 4B). Then, at the S stage, nestin labeling became consistent and intense within the cell layer that eventually would differentiate into podocytes (Figure 4C). At the same stage, nestin was also clearly located within cells that populated the cleft of the S-shaped body, from which both mesangial and endothelial glomerular cells are known to differentiate. At the C stage, podocytes were the only glomerular cell elements incontrovertibly nestin immunoreactive (Figure 4D). However, a certain degree of nestin staining in presumptive mesangial cells was also seen at this stage. At the M stage, some cells of the parietal layer of the Bowman's capsule were nestin immunoreactive (Figure 4E), whereas all podocytes were morphologically well developed and maintained their intense nestin staining (Figures 4E and 4F). Outside the glomeruli, nestin was expressed by capillary endothelial cells (Figures 4B and 4D) and smooth muscle cells of the arterial wall (data not shown). To corroborate nestin expression in developing kidneys, the IFCFs of fetal kidneys were immunoblotted with a MAb against nestin. A double-immunoreactive band comigrating with nestin from adult kidneys was observed in several preparations (Figure 3C). When bands of the proper molecular mass were not detectable, they were always substituted by immunoreactive bands of lower molecular mass (data not shown), likely representing degradation products as previously reported (Sahlgren et al. 2001).
|
Confocal microscopy analysis of double-labeling experiments was carried out to identify nestin+ cells in the early stages of nephrogenesis. As a mesangial marker, we chose
-smooth muscle actin (-SMA), which has been reported to be expressed in activated and fetal mesangial cells (Alpers et al. 1992; Naruse et al. 2000), whereas we used CD34 as a marker for the endothelium (Fina et al. 1990; Ito et al. 1995; Naruse et al. 2000). -SMA was constantly expressed in the S (Figure 5B
), C (Figure 5E), and M (Figures 5H and 5K) stages. Nestin/SMA double-labeled cells were visible at the S (Figures 5A–5C) and C (Figures 5D–5F) stages and within the smaller glomeruli (Figures 5G–5I) at the M stage. Mid-sized glomeruli did not show any overlapping between SMA and nestin stainings, whereas smooth muscle cells of the afferent and efferent arterioles expressed both antigens (Figures 5J–5L).
|
CD34 appeared early in the developing glomeruli at the S stage. It is believed that as soon as the lower cleft of the S-shaped body is formed, blood capillaries invade it or differentiate in loco from mesenchymal precursors (Naruse et al. 2000
). In our preparations, CD34-positive blood capillaries were already visible at this stage (Figure 5N). Glomeruli at later stages of maturation displayed an increasing complex tuft of capillary loops (Figures 5Q and 5T). Double-labeled CD34/nestin cells were detected in the S-shaped bodies where all, or almost all, endothelial cells appeared nestin immunoreactive (Figures 5M–5O). At the C stage, only a few CD34-positive endothelial cells coexpressed nestin (Figures 5P–5U), and the few nestin-expressing cells that could not be referred to the layer of podocytes and did stain with the anti-CD34 antibody were most likely mesangial cells. Finally, at the M stage no double-labeled cells were detectable (Figures 5V–5X). |
Discussion |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
Nestin, a high molecular mass intermediate filament (IF), was first described as expressed in central nervous system stem cells (Lendahl et al. 1990
). Since then, several other tissues and cells, including skeletal and cardiac myogenic cells (Sejersen and Lendahl 1993; Kachinsky et al. 1995), Leydig cells (Lobo et al. 2004), adrenocortical cells (Bertelli et al. 2002; Toti et al. 2005), adult and embryonic pancreas (Lardon et al. 2002; Klein et al. 2003; Delacour et al. 2004; Street et al. 2004), and many other cell types (for review, see Wiese et al. 2004) have been reported to contain nestin. On the whole, however, stable expression of nestin in cells from adult individuals is considered a rare phenomenon and a marker of stem or progenitor cells (Lendahl et al. 1990; Wiese et al. 2004). The present investigation, demonstrating that nestin is constitutively expressed in adult human podocytes, should change this view. Moreover, as nestin has been recently shown in mouse and rat podocytes as well (Chen et al. 2006; Wagner et al 2006; Zou et al. 2006), its presence in human kidney confirms that it should play a fundamental role in the biology of the renal corpuscle. Because of its frequent species-specific variability, it is important to evaluate the pattern of expression of IF proteins in human samples. Even considering the same animal species, cells can change IF content once they are grown in culture (Franke et al. 1979; Virtanen et al. 1981). To mention a few examples, we recall membranous-epithelial (M-) cells that, in addition to cytokeratins, display vimentin in rabbits but not in other species (Jepson and Clark 1998; Carapelli et al. 2004) and pancreatic duct cells that express cytokeratin 20 in rats and pigs (Bouwens 1998; Bertelli et al. 2001; Bertelli and Bendayan 2005) but not in humans and mice (Bouwens 1998). However, many other cases could be listed and even podocytes differ in IF content from one animal species to another (Yaoita et al. 1999; Zou et al. 2006) showing, accordingly, that the molecular arrangement of the IF network can be quite diverse, depending on the animals. Nestin is expressed in undifferentiated and dividing cells in the central nervous system and in myogenic tissues (Lendahl et al. 1990; Sejersen and Lendahl 1993). Upon differentiation, nestin is downregulated and replaced by other IFs. Nevertheless, it is re-expressed upon injury and in regenerating phenomena (Frisen et al. 1995; Vaittinen et al. 2001). These features have led some investigators to propose nestin as a marker of multilineage progenitor cells (Wiese et al. 2004). Nestin expression has also been correlated to increased motility and tumor invasiveness (Dahlstrand et al. 1992; Thomas et al. 2004). It is evident that podocytes cannot be placed into such a framework. Podocytes are non-motile, postmitotic terminally differentiated cells that re-enter the cell cycle only in a limited set of diseases, namely, HIV glomerulopathy, collapsing glomerulopathy, and the cellular variant of focal segmental glomerulosclerosis (Pavenstädt et al. 2003).
Nestin has been functionally linked to CDK5. CDK5 seems to play a fundamental role in triggering the exit of neurons from the cell cycle and in inducing differentiation (Cicero and Herrup 2005). In addition, CDK5 is important in regulating the differentiation of muscle cells and the rearrangement of the nestin network (Lazaro et al. 1997; Sahlgren et al. 2003). To carry out its kinase activity, CDK5 requires an activator, p35, which has been reported to associate with nestin as well (Sahlgren et al. 2003). When activated, CDK5 phosphorylates nestin and, in this way, probably regulates nestin disassembling because phosphorylated nestin is detected exclusively in the Triton X-100 soluble fraction (Sahlgren et al. 2003). Our results demonstrate for the first time that in vivo nestin and CDK5 are coexpressed and associated in adult human podocytes. Moreover, our data show that, whereas CDK5 is diffuse throughout the entire cytoplasm, pedicels included, the nestin network is located in the cell body and in the primary processes, but not in the foot processes. This is in accordance with the notion that the IF network does not extend into foot processes (Drenckhahn and Franke 1988; Pavenstädt et al. 2003). CDK5 in podocytes likely plays multiple roles because it is probably responsible for the differentiation and exit from the cell cycle (Griffin et al. 2004). However, an additional and pivotal role could be the regulation of IF network extension, preventing it from invading foot processes. In support of this view, nestin disassembling has been reported as regulated by the active complex CDK5/p35 (Sahlgren et al. 2003), which has been spotted mainly gathered at the podocyte cell processes (Griffin et al. 2004).
Nestin, however, is not capable of forming IFs on its own, as it must copolymerize with other type III IF proteins (Sjoberg et al. 1994; Steinert et al. 1999). In accordance with the well-known expression of vimentin in podocytes (Holthöfer et al 1984; Stamenkovic et al. 1986; Moll et al. 1991), our immunocytochemical and biochemical results confirm that the nestin partner in human podocytes is vimentin. The persistence of nestin expression in adult postmitotic terminally differentiated cells, in contrast to the more general rule that would restrict nestin to undifferentiated and dividing cells, implies an involvement of nestin in peculiar roles specific for podocytes. The possibility of a structural strengthening of the podocyte IF network is not convincing because nestin, far from reinforcing vimentin IFs, has been shown to make them less stable (Steinert et al. 1999). Nestin/vimentin heterodimers, however, have been proposed to be located at the periphery of a core of vimentin homodimers with the long nestin carboxyl terminal that sticks out of the filament. The importance of this arrangement would reside in the ability of the carboxyl domain to interact with microfilaments and microtubules cross-linking the three distinct components of the cytoskeleton (Herrmann and Aebi 2000). These interconnections could result, therefore, in a net advantage for the mechanical stability of podocytes. Another possibility is that nestin may serve a regulatory function for the assembly state of vimentin because it has been demonstrated in mitotic cells where vimentin IFs, in spite of their phosphorylated state, disassemble only when nestin is coexpressed (Chou et al. 2003). In this respect, based also on our results, a functional chain linking CDK5, nestin, and vimentin could be envisioned in podocytes: active CDK5/p35 complex within foot processes would phosphorylate nestin which, in turn, could regulate vimentin disassembly, preventing the IF network from extending into the pedicels. This view is supported by the observation that, in puromomycin-induced nephrosis, nestin and vimentin are both overexpressed (Zou et al. 2006) and that foot process effacement is characterized by the presence of IFs within the cytoplasmic sheet that, replacing pedicels, covers the basement membrane (Kubosawa and Kondo 1994). At any rate, the proper architecture of the IF network seems essential to maintain foot process integrity. This is confirmed by experiments of nestin silencing with siRNA where the effect of nestin knock-down resulted in a marked decrease of podocyte processes (Chen et al. 2006).
The present investigation shows that nestin IF is expressed in human podocytes starting from the very beginning of their differentiation. The pattern of nestin expression during human glomerulogenesis shares some resemblances with that which has been observed in rat and mouse (Chen et al. 2006; Wagner et al. 2006; Zou et al. 2006) but also shows significant differences. In contrast to mouse developing kidney where nestin has been detected within podocytes only in mature glomeruli (Chen et al. 2006), in humans nestin appears to be expressed from the first stage of glomerulogenesis (V stage). Throughout the following stages of development, nestin staining becomes more intense, localizing preferentially in the epithelial cells of the visceral layer. This behavior seems closer to what has been reported in rat developing kidney (Zou et al. 2006). On the other hand, whereas nestin expression in human differentiating glomerular endothelial cells is in accordance with mouse glomerulogenesis (Chen et al. 2006), our finding of a further expression in mesangial precursors seems to be unique to human immature glomeruli.
In Western blotting, nestin is usually reported as migrating as a double-immunoreactive band at 220 and 240 kDa (Sultana et al. 1998; Sahlgren et al. 2001; Lobo et al. 2004; Wiese et al. 2004). However, our evaluation gives considerably higher molecular masses, 240 and 280 kDa. Our values, in accordance with other studies (Messam et al. 2000; Chou et al. 2003; Toti et al. 2005; Zou et al. 2006), point toward nestin heavy posttranslational modifications as possibly serving particular functions in podocytes and in a few other particular cell types. Otherwise, considering that the molecular mass predicted by the amino acid sequence should be only 177 kDa and that posttranslational modifications should add 100 kDa to the molecule, the existence of alternative splicings of nestin gene is a possibility that should also be considered. However, more studies are required to clarify this point.
In conclusion, the present investigation has shown, for the first time, that human podocytes express nestin and that this is associated in vivo with CDK5, which is also located within the foot processes where nestin is not detectable. Moreover, we have demonstrated that, during glomerulogenesis, nestin is expressed in differentiating and mature podocytes, in some endothelial cells and, for the first time, in mesangial precursors.
|
Acknowledgments |
|---|
This work was supported by PAR2004 (quota servizi) (to EB) and PRIN2004 (to PT).
|
Footnotes |
|---|
Received for publication July 13, 2006; accepted December 20, 2006
|
Literature Cited |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
Achtstaetter T, Hatzfeld M, Quinlan RA, Parmelee DC, Franke WW (1986) Separation of cytokeratin polypeptides by gel electrophoretic and chromatographic techniques and their identification by immunoblotting. Methods Enzymol 134:355–371[Medline]
Alpers CE, Seifert RA, Hudkins KL, Johnson RJ, Bowen-Pope DF (1992) Developmental patterns of PDGF B-chain, PDGF-receptor, and -actin expression in human glomerulogenesis. Kidney Int 42:390–399[Medline]
Armstrong JF, Pritchard-Jones K, Bickmore WA, Hastie ND, Bard JBL (1992) The expression of the Wilms' tumour gene, WT1, in the developing mammalian embryo. Mech Dev 40:85–97
Bachmann S, Kriz W, Kuhn C, Franke WW (1983) Differentiation of cell types in the mammalian kidney by immunofluorescence microscopy using antibodies to intermediate filament proteins and desmoplakins. Histochemistry 77:365–394[CrossRef][Medline]
Bariety J, Nochy D, Mandet C, Jacquot C, Glotz D, Meyrier A (1998) Podocytes undergo phenotypic changes and express macrophagic-associated markers in idiopathic collapsing glomerulopathy. Kidney Int 53:918–925[CrossRef][Medline]
Beltran PJ, Bixby JL, Masters BA (2003) Expression of PTPRO during mouse development suggests involvement in axono-genesis and differentiation of NT-3 and NGF-dependent neurons. J Comp Neurol 465:384–395
Bertelli E, Bendayan M (2005) Association between endocrine pancreas and ductal system. More than an epiphenomenon of endocrine differentiation and development? J Histochem Cytochem 53:1071–1086
Bertelli E, Regoli M, Lucattelli M, Bastianini A, Fonzi L (2002) Nestin expression in rat adrenal gland. Histochem Cell Biol 117:371–377[CrossRef][Medline]
Bertelli E, Regoli M, Orazioli D, Bendayan M (2001) Association between islets of Langerhans and pancreatic ductal system in adult rat. Where endocrine and exocrine meet together? Diabetologia 44:575–584[CrossRef][Medline]
Bouwens L (1998) Cytokeratins and cell differentiation in the pancreas. J Pathol 184:234–239[CrossRef][Medline]
Carapelli A, Regoli M, Nicoletti C, Ermini L, Fonzi L, Bertelli E (2004) Rabbit tonsil-associated M cells express cytokeratin 20 and take up particulate antigen. J Histochem Cytochem 52:1323–1331
Chen J, Boyle S, Zhao M, Su M, Takahashi K, Davis L, DeCaestecker M, et al. (2006) Differential expression of the intermediate filament protein nestin during renal development and its localization in adult podocytes. J Am Soc Nephrol 17:1283–1291
Chou Y-H, Khuon S, Herrmann H, Goldman RD (2003) Nestin promotes the phosphorylation-dependent disassembly of vimentin intermediate filaments during mitosis. Mol Biol Cell 14:1468–1478
Cicero S, Herrup K (2005) Cyclin-dependent kinase 5 is essential for neuronal cell cycle arrest and differentiation. J Neurosci 25:9658–9668
Czarnecki K, Haas CA, Bas Orth C, Deller T, Frotscher M (2005) Postnatal development of synaptopodin expression in the rodent hippocampus. J Comp Neurol 490:133–144[CrossRef][Medline]
Dahlstrand J, Collins VP, Lendahl U (1992) Expression of the class VI intermediate filament nestin in human central nervous system tumors. Cancer Res 52:5334–5341
Delacour A, Nepote V, Trump A, Herrera PL (2004) Nestin expression in pancreatic exocrine cell lineages. Mech Dev 121:3–14[CrossRef][Medline]
Drenckhahn D, Franke R-P (1988) Ultrastructural organization of contractile and cytoskeletal proteins in glomerular podocytes of chicken, rat and man. Lab Invest 59:673–682
Fina L, Molgaard HV, Robertson D, Bradley NJ, Monaghan P, Delia D, Sutherland DR, et al. (1990) Expression of the CD34 gene in vascular endothelial cells. Blood 75:2417–2426
Franke WW, Schid E, Winter S, Osborn M, Weber K (1979) Widespread occurrence of intermediate-sized filaments of the vimentin-type in cultured cells from diverse vertebrates. Exp Cell Res 123:25–46[CrossRef][Medline]
Frisen J, Johansson CB, Török C, Risling M, Lendahl U (1995) Rapid, widespread and long-lasting induction of nestin contributes to the generation of glial scar tissue after CNS injury. J Cell Biol 131:453–464
Griffin SV, Hiromura K, Pippin J, Petermann AT, Blonski MJ, Krofft R, Takahashi S, et al. (2004) Cyclin-dependent dinase 5 is a regulator of podocyte differentiation, proliferation, and morphology. Am J Pathol 165:1175–1185
Gubler M-C (2003) Podocyte differentiation and hereditary proteinuria/nephrotic syndromes. J Am Soc Nephrol 14:S22–S26
Herrmann H, Aebi U (2000) Intermediate filaments and their associates: multi-talented structural elements specifying cytoarchitecture and cytodynamics. Curr Opin Cell Biol 12:79–90[CrossRef][Medline]
Hir ML, Keller C, Eschmann V, Hahnel B, Hosser H, Kriz W (2001) Podocytes bridges between the tuft and Bowman's capsule: an early event in experimental crescentic glomerulonephritis. J Am Soc Nephrol 12:2060–2071
Hirvonen H, Sandberg M, Kalimo H, Hukkanen V, Vuorio E, Salmi TT, Alitalo K (1989) The N-myc proto-oncogene and IGF-II growth factor mRNAs are expressed by distinct cells in human fetal kidney and brain. J Cell Biol 108:1093–1104
Holthöfer H, Miettinen A, Letho VP, Lehtonen E, Virtanen I (1984) Expression of vimentin and cytokeratin types of intermediate filament proteins in developing and adult human kidneys. Lab Invest 50:552–559[Medline]
Ito A, Nomura S, Hirota S, Suda J, Suda T, Kitamura Y (1995) Enhanced expression of CD34 messenger RNA by developing endothelial cells of mice. Lab Invest 72:532–538
Jepson MA, Clark MA (1998) Studying M cells and their role in infection. Trends Microbiol 6:359–365[CrossRef][Medline]
Kachinsky AM, Dominov JA, Miller JB (1995) Intermediate filaments in cardiac myogenesis: nestin in the developing mouse heart. J Histochem Cytochem 43:843–847[Abstract]
Kerajaschki D (2001) Caught flat-footed: podocyte damage and the molecular bases of focal glomerulosclerosis. J Clin Invest 108:1583–1587[CrossRef][Medline]
Klein T, Ling Z, Heimberg H, Madsen OD, Heller RS, Serup P (2003) Nestin is expressed in vascular endothelial cells in the adult human pancreas. J Histochem Cytochem 51:697–706
Kobayashi N, Gao S, Chen J, Saito K, Miyawaki K, Li C, Pan L, et al. (2004) Process formation of the renal glomerular podocyte: is there common molecular machinery for processes of podocytes and neurons? Anat Sci Int 79:1–10[CrossRef][Medline]
Kondo D, Yamamoto T, Yaoita E, Danielson PE, Kobayashi H, Ohshiro K, Funaki H, et al. (2000) Localization of olfactomedin-related glycoprotein isoform (BMZ) in the golgi apparatus of glomerular podocytes in rat kidneys. J Am Soc Nephrol 11:803–813
Kriz W, Hackenthal E, Nobiling R, Sakai T, Elger M (1994) A role for podocytes to counteract capillary wall distension. Kidney Int 45:369–376[Medline]
Kubosawa H, Kondo Y (1994) Modulation of cytoskeletal organization of podocytes during the course of aminonucleoside nephrosis in rats. Pathol Int 44:578–586[Medline]
Lardon J, Rooman I, Bouwens L (2002) Nestin expression in pancreatic stellate cells and angiogenic endothelial cells. Histochem Cell Biol 117:535–540[CrossRef][Medline]
Lazaro JB, Kitzmann M, Poul MA, Vandromme M, Lamb NJ, Fernandez A (1997) Cyclin dependent kinase 5, cdk5, is a positive regulator of myogenesis in mouse C2 cells. J Cell Sci 110:1251–1260[Abstract]
Lendahl U, Zimmerman LB, McKay RD (1990) CNS stem cells express a new class of intermediate filament protein. Cell 60:585–595[CrossRef][Medline]
Lobo MV, Arenas MI, Alonso FJ, Gomez G, Bazan E, Paino CL, Fernandez E, et al. (2004) Nestin, a neuroectodermal stem cell marker molecule, is expressed in Leydig cells of the human testis and in some specific cell types from human testicular tumours. Cell Tissue Res 316:369–376[CrossRef][Medline]
Messam CA, Hou J, Major EO (2000) Coexpression of nestin in neural and glial cells in the developing human CNS defined by a human-specific anti-nestin antibody. Exp Neurol 161:585–596[CrossRef][Medline]
Moll R, Hage C, Thoenes W (1991) Expression of intermediate filament proteins in fetal and adult human kidney: modulations of intermediate filament patterns during development and in damaged tissue. Lab Invest 65:74–86
Mundel P, Heid HW, Mundel TM, Krüger M, Reiser J, Kriz W (1997) Synaptopodin: an actin-associated protein in telencephalic dendrites and renal podocytes. J Cell Biol 139:193–204
Nagata M, Kakayama K, Terada Y, Hoshi S, Watanabe T (1998) Cell cycle regulation and differentiation in the human podocyte lineage. Am J Pathol 153:1511–1520
Nagata M, Yamaguchi Y, Ito K (1993) Loss of mitotic activity and the expression of vimentin in glomerular epithelial cells of developing human kidneys. Anat Embryol (Berl) 187:175–179
Nakahama H, Obata K, Matsuyama T, Sugita M, Horio M, Oka K, Moriyama T (1999) Effect of a novel immunosuppressant, FK506, on autoimmune glomerulonephritis in Brown Norway rats. Nephron 81:215–220[CrossRef][Medline]
Naruse K, Fujieda M, Miyazaki E, Hayashi Y, Toi M, Fukui T, Kuroda N, et al. (2000) An immunohistochemical study of developing glomeruli in human fetal kidneys. Kidney Int 57:1836–1846[CrossRef][Medline]
Pavenstädt H, Kriz W, Kretzler M (2003) Cell biology of the glomerular podocyte. Physiol Rev 83:253–307
Putaala H, Soininen R, Kilpeläinen P, Wartiovaara J, Tryggvason K (2001) The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death. Hum Mol Genet 10:1–8
Rastaldi MP, Armelloni S, Berra S, Calvaresi N, Corbelli A, Giardino LA, Li M, et al. (2006) Glomerular podocytes contain neuron-like functional synaptic vesicles. FASEB J 20:976–978
Rastaldi MP, Armelloni S, Berra S, Li M, Pesaresi M, Poczewski H, Langer B, et al. (2003) Glomerular podocytes possess the synaptic vesicle molecule Rab3A and its specific effector Rabphilin-3a. Am J Pathol 163:889–899
Ruggenenti P, Schieppati A, Remuzzi G (2001) Progression, remission, regression of chronic renal diseases. Lancet 357:1601–1608[CrossRef][Medline]
Sahlgren CM, Mikhailov A, Hellman J, Chou Y-H, Lendahl U, Goldman RD, Eriksson JE (2001) Mitotic reorganization of the intermediate filament protein nestin involves phosphorylation by cdc2 kinase. J Biol Chem 276:16456–16463
Sahlgren CM, Mikhailov A, Vaittinen S, Pallari H-M, Kalimo H, Pant HC, Eriksson JE (2003) Cdk5 regulates the organization of nestin and its association with p35. Mol Cell Biol 23:5090–5106
Sejersen T, Lendahl U (1993) Transient expression of the intermediate filament nestin during skeletal muscle development. J Cell Sci 106:1292–1300
Sharma MR, Tuszynski GP, Sharma MC (2004) Angiostatin-induced inhibition of endothelial cell proliferation/apoptosis is associated with the down-regulation of cell cycle regulatory protein cdk5. J Cell Biochem 91:398–409[CrossRef][Medline]
Sjoberg G, Jiang WQ, Ringertz NR, Lendahl U, Sejersen T (1994) Colocalization of nestin and vimentin/desmin in skeletal muscle cells demonstrated by three-dimensional fluorescence digital imaging microscopy. Exp Cell Res 214:447–458[CrossRef][Medline]
Stamenkovic I, Skalli O, Gabbiani G (1986) Distribution of intermediate filament proteins in normal and diseased human kidney. Am J Pathol 125:465–475[Abstract]
Steinert PM, Chou Y-H, Prahlad V, Parry DAD, Marekov LN, Wu KC, Jang S-I, et al. (1999) A high molecular weight intermediate filament-associated protein in BHK-21 cells is nestin, a type VI intermediate filament protein. J Biol Chem 274:9881–9890
Street CN, Lakey JR, Seeberger K, Helms L, Rajotte RV, Shapiro AM, Korbutt GS (2004) Heterogenous expression of nestin in human pancreatic tissue precludes its use as an islet precursor marker. J Endocrinol 180:213–225[Abstract]
Sultana S, Zhou R, Sadagopan MS, Skalli O (1998) Effects of growth factors and basement membrane proteins on the phenotype of U-373 MG glioblastoma cells as determined by the expression of intermediate filament proteins. Am J Pathol 153:1157–1168
Thomas SK, Messam CA, Spengler BA, Biedler JL, Ross RA (2004) Nestin is a potential mediator of malignancy in human neuroblastoma cells. J Biol Chem 279:27994–27999
Toti P, Regoli M, Nesi G, Occhini R, Bartolommei S, Fonzi L, Bertelli E (2005) Nestin expression in normal adrenal gland and adrenocortical tumors. Histol Histopathol 20:1115–1120[Medline]
Vaittinen S, Lukka R, Sahlgren CM, Hurme T, Rantanen J, Lendahl U, Eriksson JE, et al. (2001) The expression of the intermediate filament nestin as related to vimentin and desmin in regenerating skeletal muscle. J Neuropathol Exp Neurol 60:588–597[Medline]
Virtanen I, Letho V-P, Lethonen E, Varto T, Stenman S, Kurki P, Wager O, et al. (1981) Expression of intermediate filaments in cultured cells. J Cell Sci 50:45–63[Medline]
Wagner N, Wagner KD, Scholz H, Kirschner KM, Schedl A (2006) The intermediate filament protein nestin is expressed in the developing kidney and heart and might be regulated by the Wilms' tumor suppressor Wt1. Am J Physiol Regul Integr Comp Physiol 291:R779–787
Wiese C, Rolletschek A, Kania G, Blyszczuk P, Tarasov KV, Tarasova Y, Wersto RP, et al. (2004) Nestin expression—a property of multi-lineage progenitor cells? Cell Mol Life Sci 61:2510–2522[CrossRef][Medline]
Yaoita E, Franke WW, Yamamoto T, Kawasaki K, Kihara I (1999) Identification of renal podocytes in multiple species: higher vertebrates are vimentin positive/lower vertebrates are desmin positive. Histochem Cell Biol 111:107–115[CrossRef][Medline]
Zou J, Yaoita E, Watanabe Y, Yoshida Y, Nameta M, Li H, Qu Z, et al. (2006) Upregulation of nestin, vimentin, desmin in rat podocytes in response to injury. Virchows Arch 448:485–492[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  P. S. Thorner, M. Ho, V. Eremina, Y. Sado, and S. Quaggin Podocytes Contribute to the Formation of Glomerular Crescents J. Am. Soc. Nephrol., March 1, 2008; 19(3): 495 - 502. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||