The complex cyto-architecture of the podocyte is critical for glomerular permselectivity. The present study characterizes the expression of nestin, an intermediate filament protein, in human kidneys. In normal kidneys, nestin was detected at the periphery of glomerular capillary loops. Colabeling showed nestin was expressed in WT1-positive cells. Within the podocyte, nestin immunoreactivity was present in the cell body and primary process. This was supported by immunoelectron microscopy. Nestin also colocalized with vimentin in the periphery of capillary loops but not in the mesangium. Nestin was not detected in other structures of the adult human kidney. To determine the potential role of nestin in proteinuria, nestin was examined in kidney biopsies from patients with or without proteinuria. These patients were diagnosed with IgA nephropathy with mild mesangial expansion but without proteinuria, IgA nephropathy with proteinuria, membranous nephropathy (MN), and focal segmental glomerular sclerosis (FSGS). The distribution of nestin in these biopsies was similar to that in the normal kidney. Semiquantitative analysis of immunostaining showed that glomerular nestin expression in IgA nephropathy without proteinuria was not different from normal kidney; however, nestin expression in kidneys of patients with IgA nephropathy and proteinuria, or MN and FSGS with proteinuria was significantly reduced compared with normal kidney (P < 0.01). Reduced nestin mRNA expression in the patients with IgA nephropathy with proteinuria and FSGN was also observed by quantitative real-time PCR. These studies suggest that nestin may play an important role in maintaining normal podocyte function in the human kidney.
- IgA nephropathy
- cytoskeletal protein
podocytes are highly specialized cells with a complex cyto-architecture. Accumulating evidence indicates that a well-developed cytoskeleton is required for the formation of podocyte process and plays an important role in establishing selective permeability of the glomerular filtration barrier (4, 19). The podocyte cytoskeleton has also been suggested to be critical in counterbalancing mechanical stress, preventing outward ballooning of the vessel and preserving the normal architecture of the glomerular tuft (9, 10). Cytoskeletal proteins can be classified into three major groups: microtubules, intermediate filaments, and microfilaments. Studies have shown that actin microfilaments (such as α-actinin4) are localized mainly in foot processes and its expression is important for the formation of the glomerular filtration barrier (19). Microtubules, which are localized mainly in the cell body and primary processes, are reported to participate in podocyte process formation (9). Intermediate filament proteins, such as vimentin and desmin, are also detected in the cell body and primary processes of the podocyte (18, 22), but their roles in the podocyte have not been elucidated. The precise mechanisms by which podocyte cytoskeletal proteins are organized and their potential significance in the pathogenesis of podocyte injury in kidney diseases remain to be fully explored.
Nestin is a cytoskeleton-associated class VI intermediate filament protein (15) that was originally identified in stem cells and progenitor cells in the central nervous system, as well as in peripheral organs (2, 6, 8, 11, 12, 14). Recently, nestin has also been detected in the glomerular podocytes of rodents (1, 23, 24). In contrast to its expression in progenitor cells, where it disappears once cells become differentiated, nestin is expressed in fully differentiated podocytes (1). Since nestin has been reported to interact with all three classes of cytoskeletal proteins, it may be involved in the organization of the cellular cytoskeleton (15) and may therefore also play an important role in the maintenance of normal podocyte function. To determine the potential significance of nestin expression in human podocytes, we examined nestin expression in normal human kidney and diseased kidneys with or without podocyte injury.
Diseased kidney tissues were obtained from needle biopsies of 28 patients with proteinuria and four patients diagnosed with IgA nephropathy without proteinuria but with mild mesangial expansion (IgA-np). The kidney tissues were obtained with informed consent to be used for research purposes after the diagnostic workup was completed. The study's protocol was approved by the Fudan University Huashan Hospital Ethics Committee. Of the 28 patients with proteinuria, the pathological diagnosis included IgA nephropathy (IgA-p, n = 17, urinary protein >1 g/24 h), membranous nephropathy (MN, n = 8), and focal segmental glomerular sclerosis (FSGS, n = 3). Twenty-four-hour urinary protein excretion and renal function of these patients at the time of biopsy are shown in Table 1. Normal kidney tissues were obtained from surgical nephrectomy because of renal tumors (n = 6). Podocyte foot process effacement was seen in kidney tissues from all patients with proteinuria, as demonstrated by electron microscopy. No foot process effacement was observed in renal biopsies from patients with IgA without proteinuria. No pathological findings were observed in normal kidney tissues.
The renal specimens were obtained according to standard diagnostic renal biopsy procedure and immediately fixed with 4% paraformaldehyde. The tissues were then processed using a Leica semienclosed tissue processor (model TP1020; Leica, Wetzler, Germany). Sections (4 μm) were cut and stained with mouse anti-nestin antibody (1:1,000, catalog no. 611658; BD Transduction Lab, Sanprego, CA) and visualized with diaminiobenzidine (Vectastain; Vector Laboratories). For semiquantitative nestin immunohistochemistry, after optimization of conditions, nestin immunostaining of all the samples was completed in two separate experiments. The same control kidney samples were included in both experiments. Similar nestin staining pattern and intensity in two separate experiments were observed for each individual tissue specimen, indicating that the results from two experiments were comparable. Nestin expression levels were assessed semiquantitatively using morphometric analysis (Motic Images Advanced 3.2) by experienced personnel who were blinded to the patient's information. Glomeruli (10–15/slide) were randomly chosen for morphometric analysis. Nestin expression level was calculated as positive area × optical density/glomerular area.
Cryostat sections (5 μm) were blocked with 10% normal donkey serum for 20 min. Sections were then incubated with primary antibodies for 60 min. After washing, the sections were incubated in Cy2- or Cy3-conjugated anti-IgG secondary antibody (Jackson Immunoresearch Laboratories) for 30 min.
Nestin immunohistochemistry and immunofluorescence presented in this paper were performed by using a mouse anti-nestin antibody from BD Transduction Lab (catalog no. 611658; Sanprego, CA). The expression of nestin in the podocyte of human kidney was further confirmed by a goat anti-nestin antibody from Santa Cruz (catalog no. SC21249, 1:100) for immunofluorescence and a mouse anti-nestin antibody from Boster (catalog no. BA1289; Wuhan, China) for both immunofluorescence and immunohistochemistry. These antibodies produced a similar nestin distribution in the human kidney (data not shown). Other antibodies used for immunofluorescent studies included: a rabbit anti-laminin antibody (1:40, catalog no. AB2034, Chemicon International, Temecula, CA); a goat anti-Vimentin antibody (1:100, Sigma) and a rabbit anti-WT1 antibody (1:20, Santa Cruz).
A preembedding immunoperoxidase labeling approach was used. Cryostat sections of kidney tissue were cut at 35 μm and processed as free-floating sections. After blocking, nestin antibody (1:400) was applied for 72 h at 4°C, followed by biotinylated donkey anti-mouse IgG overnight at 4°C. Peroxidase activity was detected using 3,3-diaminobenzidine as the chromogen. The sections were stained en bloc with uranylacetate in maleate buffer, osmicated in 1% OsO4 for 2 h, dehydrated, and embedded in epon. Areas of interest were selected under a light microscope, excised with a razor blade, and mounted onto preformed epon blocks. Thin sections were cut with an ultramicrotome E (Leica) and examined under an immunoelectron microscope.
Total RNA was extracted from renal biopsy specimens containing ∼10 glomeruli using Qiagen RNeasy Micro kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Real-time PCR was conducted using Bio-Rad Icycler5 with Master Mix from Toyobo Biotech (Osaka, Japan). The primers and probes were as follows: human GAPDH: forward, 5′-ATGCTGGCGCTGAGTACGT-3′, reverse: 5′-AGCCCCAGCCTTCTCCAT-3′; human GAPDH probe: FAM-5′-TGGAGTCCACTGGCGTCTTCA-3′-TAMRA; human nestin: forward, 5′-CGCCACCGAGGTTCTGAA-3′, reverse, 5′-CTGACCCTCGTTTCTAGGTTCTG-3′; and human nestin probe: FAM-5′-CGACCTCGACGTTAAGGGATCCTGG-3′-TAMRA. Gene expression values were calculated based on the comparative threshold cycle (Ct) method detailed in Applied Biosystems User Bulletin Number 2. Nestin mRNA expression levels were normalized to the GAPDH mRNA, and displayed relative to normal kidney nestin mRNA levels.
Nestin is expressed in podocytes of adult human kidney.
In normal human kidneys, anti-nestin antibody detected strong immunoreactivity in the periphery of glomerular capillary loops (Fig. 1). Costaining with anti-laminin antibody, which labels glomerular basement membrane, showed that nestin immunoreactivity was localized outside the glomerular basement membrane (Fig. 2, A–C). Costaining with the podocyte marker WT1 showed that nestin immunoreactivity localized to the cytosol of WT1-positive cells (Fig. 2, D–F). These studies are consistent with nestin expression in the podocytes of human kidney. Colabeling with vimentin, which is expressed in both the podocyte and the mesangial cells, showed that nestin colocalized with vimentin only in a subset of cells at the periphery of the capillary loops (Fig. 2, G–I), also supporting predominant nestin expression in the podocyte. In some areas, nestin immunoreactivity was linear or coarsely granular along the glomerular basement membrane (Figs. 2–4), consistent with podocyte process expression of nestin. Immunoelectron microscopy showed that nestin immunoreactivity was predominantly localized to the primary processes above the foot processes (Fig. 3). Nestin was also detected in the cell body, but it was mainly at the origin of the primary processes (Fig. 3). No nestin was observed in the foot processes by immunoelectron microscopy (Fig. 3). No nestin immunoreactivity was detected in tubular structures or in the vasculature of normal adult kidney (Fig. 1).
Glomerular nestin expression is reduced in kidney tissues from patients with proteinuria.
To examine the potential association of nestin expression with podocyte injury and proteinuria, kidney nestin expression in patients with IgA nephropathy with or without proteinuria, and nephrotic patients with MN and FSGS was examined (see Table 1). The distribution of nestin immunoreactive protein in diseased kidney tissues was similar to that seen in normal kidney, i.e., predominantly in the periphery of glomerular loops, consistent with podocyte expression (Fig. 4). Semiquantitative morphometric analysis showed that glomerular nestin expression levels in IgA nephropathy with mild mesangial expansion and without proteinuria were not different from normal kidney (Fig. 5A). In contrast, glomerular nestin expression in IgA nephropathy with proteinuria was significantly reduced compared with normal kidney and kidney tissues from IgA nephropathy without proteinuria (Fig. 5A). Reduced glomerular nestin expression was also observed in MN and FSGS (Fig. 5A) in which proteinuria was typically present.
Reduced nestin expression in the kidney samples from patients with proteinuria was further supported by mRNA expression levels measured by quantitative RT-PCR. As shown in Fig. 5B, renal nestin mRNA expression in IgA nephropathy with proteinuria and FSGS were significantly lower than in normal kidney. Nestin mRNA expression in IgA nephropathy without proteinuria was not different from normal control. Nestin mRNA expression in MN was lower than that in normal kidney, but this difference did not achieve statistical significance (P = 0.065). These studies support an association of reduced nestin expression and proteinuria that reflects abnormal podocyte function.
To determine whether glomerular nestin expression levels were associated with treatment response, 17 IgA patients were treated and followed for 12 mo. According to response of proteinuria to treatment, the patients were divided into three groups: no remission (urinary protein was decreased by <50%), partial remission (urinary protein was decreased by >50%, but remained ≥300 mg/24 h), and complete remission (urinary protein was <300 mg/24 h). The initial urinary protein levels and treatments in these 17 patients are listed in Table 2. As shown in Fig. 6, glomerular nestin levels were not significantly different among patients with differing treatment responses, although there appeared to be a trend toward increased nestin in patients with a better treatment response.
The present studies demonstrate that as in mice, nestin is selectively expressed in the glomerular podocytes of adult human kidney. Furthermore, podocyte nestin expression is reduced in patients with proteinuria.
Nestin has been reported to be expressed in progenitor cells in a number of tissues, including smooth muscle cells, vascular endothelial cells, and pancreatic islets (2, 6, 8, 11, 12, 14). Nestin expression disappears when these organs or tissues are fully developed. Therefore, nestin has been proposed to be a marker of stem cells (11, 20). In the developing murine kidney, nestin immunoreactivity has been demonstrated in precursor cells that contribute to metanephric mesenchyme but not ureteric bud-derived structures (1). Nestin has also been detected in podocytes of rodents (1, 23, 24). In contrast to its expression in progenitor cells, nestin expression in the podocyte is only detected once the glomerulus becomes mature (1). The present studies further demonstrate that in adult human kidney, nestin is selectively expressed in podocytes, suggesting a potential role for nestin in this specialized epithelial cell.
Podocytes are specialized epithelial cells that envelop the portion of the glomerular capillary loop facing the urinary space and comprise a major permeability barrier between capillary lumen and urinary space (16, 19). Dysfunction of the podocyte is associated with proteinuria and may also contribute to sclerotic damage of the glomeruli in chronic kidney disease (7, 10, 16). Normal podocyte function is dependent on maintenance of their unique shape including elongated primary processes, secondary processes, and foot processes that affix to the glomerular capillary endothelium (19). Formation and maintenance of the podocyte processes depend on a well-developed and complex cytoskeleton (19). In the cell body and primary process, microtubules and intermediate filaments predominate. The intermediate filament proteins identified in podocytes include vimentin and desmin (19). Microfilaments are mainly localized in foot process (19). Studies show that the microfilaments form loop-shaped bundles along the longitudinal axis of the foot processes. The ends of the actin bundles appear to be anchored in the dense cytoplasm at the “soles” of the foot processes that constitute the major filtration barrier (10). The bends of the actin loops are located at the transition of the foot process and primary processes and may be connected to the microtubules by the microtubule-associated protein (19). These studies not only demonstrate a distinct localization of different cytoskeletal proteins within the podocytes but are also consistent with interaction among cytoskeletal proteins within the podocyte to maintain and regulate podocyte function. However, the precise mechanism by which these cytoskeletal proteins are organized and regulated has not been fully elucidated.
The present studies identify nestin as another intermediate filament protein selectively expressed in podocytes of adult human kidney. Expression of nestin in the podocyte is supported by several lines of evidence: 1) nestin immunoreactive protein was detected only in cells residing outside laminin-positive glomerular basement membrane, 2) nestin expressing cells were labeled by WT1 antibody, and 3) nestin colocalized with vimentin only in a subpopulation of cells that localized at the periphery of the glomeruli. Vimentin has been reported to be expressed in both podocytes and mesangial cells (18, 22), and it appears that nestin colocalizes with vimentin in podocyte but not mesangial cells, consistent with restriction of nestin expression to the podocytes. Within the podocytes, immunoelectron microscopy shows that nestin is primarily localized to the primary processes that cover the foot processes. Recent studies described a subpodocyte space that is a restricted region located between the filtration barrier and the outer boundary formed by primary process, cell body, and anchoring process. The subpodocyte space covers 58% to 65% of the glomerular filtration barrier and drains into Bowman's space through exit pores (17). Interestingly, Neal et al. (17) show that the subpodocyte space was altered during increased renal perfusion pressure, suggesting a dynamic reaction to the increase in filtration. Whether nestin in the primary process is involved in this dynamic regulation of subpodocyte space modulating filtration remains to be explored.
As is typical for intermediate filament proteins, nestin is characterized by an α-helical central “rod” domain that contains repeated hydrophobic heptad motifs (11). Unlike other intermediate filament proteins, nestin contains a short NH2 terminus and an unusually long COOH terminus (11). Nestin is unable to self-assemble (3), most likely because of its very short NH2 terminus (a domain necessary for intermediate filament assembly). The characteristic long COOH terminus of nestin contains binding sites for microtubules and microfilament (11, 13, 21), consistent with the possibility that nestin plays an important role in coordinating or bridging cytoskeletal proteins.
To further determine the potential significance of nestin expression in kidney diseases with podocyte dysfunction, renal biopsies from patients with proteinuria were examined. The results show that podocyte nestin protein expression is significantly reduced in kidneys with podocyte effacement including MN, FSGS, and IgA nephropathy. This result is supported by quantitative real-time PCR, which shows significantly reduced nestin mRNA expression in kidney samples from patients with FSGS and IgA nephropathy with proteinuria. The mRNA levels of nestin in the kidney of patients with MN are also lower compared with normal kidney, but the difference did not reach statistical significance (P = 0.065), probably due to the limited number of samples. Most strikingly, glomerular nestin expression in IgA nephropathy without foot process effacement and proteinuria remains unchanged. In patients with IgA nephropathy, there appeared to be a trend toward increased glomerular nestin in patients with a better treatment response, but the difference is not statistically significant. This result is inconclusive because of the limited patient number. These observations suggest that reduction of renal nestin expression is associated with podocyte dysfunction in patients with proteinuria. This result is supported by a recently published animal study by Wagner et al. (23), showing reduced podocyte nestin expression in an inducible podocyte injury mouse model with proteinuria. The study by Wagner et al. (23) shows that the transcription factor WT1 is required for nestin expression in the podocytes. Reduced WT1 expression in injured podocytes is responsible for reduced nestin expression (23). The mechanism by which podocyte nestin expression is reduced in patients with proteinuria remains to be explored. In contrast, in the puromycin aminonucleoside-induced nephrotic model, glomerular nestin expression appeared to be significantly increased (24). The reason for this difference is not clear. The different insults may be responsible for the varied response of nestin expression seen.
In summary, the present studies characterize nestin expression in adult human kidney, demonstrating that nestin expression is restricted to glomerular podocytes. Podocyte nestin expression was reduced in human kidney samples with podocyte foot process effacement and proteinuria. The present studies suggest that nestin may play an important role in maintaining normal function of the podocyte in human kidney.
This work is supported by the Natural Science Foundation of China (NSFC) Grant 30570861 and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-071876 (to C.-M. Hao), NSFC 30670978 (to J. Chen), the 211 Project of Administration of Education, China (to Y. Gu) and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-37097 and a Veterans Affairs Merit Award (to M. D. Breyer).
The authors thank Dr. Hui Cai from the Center for Health Service Research at Vanderbilt University, Nashville, TN, for his assistance in data analysis and Weiyu Zhufor technical assistance.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 the American Physiological Society