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Address correspondence to Masanori Hara, M.D., Ph.D., Iwamuro Health Promotion Center, Niigatashi-Nishikanku Iwamuro-onsen 772-1, 953-0104, Niigata, Japan
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MissouriRenal Pathology, Arkana Laboratories, Little Rock, Arkansas
Mitotic catastrophe (MC) is a major cause of podocyte loss in vitro and in vivo. We evaluated urine samples (n = 184 urine samples from diabetic patients; n = 41 patients) from diabetic patients and determined the presence of podocytes in the urine and studied their characteristics, specifically asking whether apoptosis versus MC is present. We also evaluated diabetic glomeruli in renal biopsy specimens by electron microscopy (n = 54). A battery of stains including the antibody to podocalyxin (PCX) were used. PCX and podocytes (PCX+podo) showed nuclear morphologies such as a i) mononucleated normal shape (8.7%), ii) large and abnormal shape (3.8%), iii) multinucleated with or without micronucleoli (31.2%), iv) mitotic spindles (8.2%), v) single nucleus and denucleation combined (10.3%), and vi) denucleation only (37.0%). Large size/abnormal shape, multinucleation, mitotic spindles, and a combination of single nucleus and denucleation were considered features of MC (53.5%). Dual staining of PCX+podo was positive for Glepp 1 (50%), whereas none of PCX+podo were positive for nephrin, podocin, leukocyte, or parietal epithelial cell markers (cytokeratin 8), annexin V, cleaved caspase-3, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Ten percent of PCX+podo were positive for phosphorylated vimentin. Electron microscopy identified cellular and nuclear podocyte changes characteristic of MC. The majority of urine podocytes in diabetic patients showed MC, not apoptosis. This noninvasive approach may be clinically useful in determining progressive diabetic nephropathy or response to therapeutic intervention.
Glomerulosclerosis in human and experimental glomerular diseases is associated with podocytopenia, which is defined as a decrease in the number of visceral podocytes lining the glomerular capillaries.
Apoptosis was thought to be a major mechanism by which glomerular podocytes are lost, characterized by complex pathways of initiation and inhibition of an eventually irreversible process (programed cell death). Numerous in vivo and in vitro studies have provided biochemical evidence of apoptosis in experimental glomerular disease (caspase, DNA fragmentation assays, and so forth), but morphologic characteristics of apoptosis such as cell membrane blebbing, membrane protrusions, nuclear condensation, and cell fragmentation (apoptotic bodies) is lacking.
Typical findings of advanced diabetes are mesangial sclerosis and glomerular basement membrane thickening. Podocytes invariably show cytoplasmic hypertrophy and foot process effacement, detachment of the cell body, but no condensed nuclei or cell fragmentation to suggest apoptosis.
Apoptosis is a complex process mediated by cell-cycle signaling pathways, including p53 signaling, which is inhibited by the mouse double-minute 2 homolog (MDM2). However, MDM2 inhibition with nutlin-3 in the Adriamycin mouse model did not trigger apoptotic podocyte death but induced G2/M podocyte arrest, preventing aberrant nuclear division, resulting in glomerular basement membrane detachment of aneuploid podocytes, a feature of MC both in vitro and in vivo.
Numerous studies found viable podocytes shedding in the urine, in part thought to be caused by physiologic aging and also increased shedding in active glomerular disease.
Effects of angiotensin-converting enzyme inhibitor, angiotensin II receptor antagonist and calcium antagonist on urinary podocytes in patients with IgA nephropathy.
MC is molecularly heterogeneous, at least in carcinogenesis. For example, aberrant cyclin B1–dependent kinase cyclin dependent kinase 1, pololike kinases and aurora kinases, cell-cycle checkpoint proteins, survivin, tumor protein 53, caspases, and members of the B-cell lymphoma 2 family were shown.
Podocytes in mitosis have not been assessed systematically in human glomerulonephritides, but most experienced renal pathologists have seen occasional mitotic or binucleated podocytes.
Here, we examined the extent of podocyturia in diabetic patients by analyzing the podocytes shed in their urine, looking specifically for morphologic evidence of cell death, such as MC or apoptosis.
Materials and Methods
Patients, Urine Samples, and Kidney Specimens
Urine samples of patients with type 2 diabetes (n = 41), and absent clinical history of another glomerular disease or cancer, were obtained from urine collected at a hospital visit at the Nephrology Clinic of Yoshida Hospital (Japan). The study was approved by the ethics committee of Yoshida Hospital. Informed consent was obtained from all patients. The clinical profiles of the patients are shown in Table 1. Urine samples were examined by immunofluorescence (IF) using podocyte markers as follows.
Table 1Patient Characteristics
Clinical characteristic
Microalbuminuria
Macroalbuminuria
Total patients, n
8
33
Age, years
63.5 ± 2.7
68.0 ± 3.0
Sex, male/female, n
4/4
17/16
SBP, mmHg
135.4 ± 4.5
136.1 ± 4.4
DBP, mmHg
76.6 ± 3.3
74 ± 2.3
eGFR, mL per minute/1.73m2
72.5 ± 5.4
44.1 ± 4.4
Albuminuria, mg/L
201.6 ± 24.2
1480.1 ± 440.2
Urinary podocytes, cells/mL
0.3 ± 0.1
0.8 ± 0.2
Data are expressed as means ± SEM, unless otherwise indicated.
Ten milliliters of urine was centrifuged at 1710 × g for 5 minutes and the sediments were air-dried on glass slides, fixed with 95% alcohol, and treated with skim milk, followed by conventional IF using the primary and secondary antibodies. A total of 184 urinary podocyte samples were prepared for various stains from the 41 patients. The reproducibility of sample preparation was tested previously,
and confirmed for this study using two urine podocyte samples examined by seven observers (data not shown). Factors that might influence the assay, such as the temperature and duration of storage, were evaluated and were found to have minimal effect on the assay. Urine podocyte numbers were counted using an in-house–produced antibody podocalyxin PCX (see below). Individual PCX-positive cells with whole-cell shape were counted and expressed as cells/10 mL. A separate score was generated for urine casts with PCX-positive cells. A scale was generated as follows: 0, 1+, 2+, and 3+, based on the number of casts per high-power field, where 0 = none, 1+ = fewer than 0.5 casts, 2+ = 0.5 to 2 casts, and 3+ = 3 or more casts.
The morphologic appearance of the nuclear shape in podocytes was evaluated with hematoxylin staining applied at the end of the IF procedure.
Dual IF staining was performed on PCX+ cells; antibodies were labeled appropriately for primary and secondary antibodies.
PCX Antibody Generation
A monoclonal antibody against human native PCX to detect urinary podocytes was generated. The immunogen was native PCX prepared from isolated normal glomeruli from a nephrectomy.
Isolated glomeruli were extracted in 0.2% (vol/vol) Triton X-100 (Sigma-Aldrich K.K., Tokyo, Japan) in phosphate-buffered saline containing protease inhibitors. The extract was incubated with wheat germ agglutinin–Sepharosel (Sigma-Aldrich K.K.); after washing, the sialic acid–rich material that bound to the wheat germ agglutinin column was removed with N-acetyl-β–glucopyranoside. Balb/c mice were immunized with 50 μg wheat germ agglutinin–bound PCX. Spleen cells were fused according to standard procedures. Clones producing anti-PCX antibody were screened by indirect immunofluorescence on cryostat sections of human kidneys and characterized further by Western blot analysis and immunoprecipitation. A number of positive clones were identified. Finally, three clones (22A4, 3H11, and 4D5) were obtained and confirmed as monoclonal antibodies against human native PCX. Among the three antibodies, 22A4 was chosen for detecting urinary podocytes. IF 22A4 antibody on frozen human kidney sections from nephrectomy and Western blot findings are shown in Figure 1, A and B. Representative findings of urinary podocytes are shown in Figure 1, C and D.
Figure 1Characterization of the anti-podocalyxin (PCX) antibody (22A4). A: Normal kidney immunofluorescence staining with 22A4: glomerular capillary loop staining (mainly on the podocyte aspect) is strongly positive; small vessels around the glomerulus also stain weakly. There is no staining along the Bowman's capsule. B: Western blot of 22A4 using extracts from isolated human glomeruli as a positive control. The band with 160 to 170 kDa is strongly stained; this is the appropriate molecular weight of human podocalyxin. C: Representative urinary podocytes stained with 22A4 in urine from patient with IgA nephropathy. D: Representative electron microscopy of urinary podocytes from patient with Henoch–Schönlein purpura (pre-embedding immuno-EM with 22A4). Original magnification: ×40 (A); ×10 (C); ×5000 (D). EM, electron microscopy.
For dual-immunofluorescent staining, protein A–bound fraction was labeled with Alexa Fluor 555 according to the instruction's from Thermo Fisher Scientific (Waltham, MA). Glepp1 antibody, a gift from Roger Wiggins (University of Michigan Medical Center, Ann Arbor, MI), is a mouse monoclonal antibody to Glepp1 (clone 4C3).
Nephrin antibody, a gift from Dr. Kunimasa Yan (Kyorin University School of Medicine, Mikata, Tokyo, Japan), a rabbit polyclonal antibody to human nephrin.
Podocin rabbit polyclonal antibody was from Immuno-Biological Laboratories (Minneapolis, MN). Cytokeratin 8 mouse monoclonal antibody, which stains parietal epithelial cells and injured tubular epithelial cells, panleukocyte (human CD45) rabbit polyclonal antibody, and caspase 3 rabbit polyclonal antibody were from Abcam (Cambridge, MA). Macrophage mouse monoclonal antibody to human CD68 (clone KP1) was from Dako (Kyoto, Japan). Phosphorylated vimentin antibody (4A4), a marker of mitosis, was a gift from Dr. Masaki Inagaki (Aichi Cancer Institute, Nagoya, Japan).
Annexin V staining was performed with the Annexin V assay kit (Medical and Biological Laboratories Co, Ltd, Nagoya, Japan) using fresh urine sediment on the glass slide without air drying, and Alexa-conjugated 22A4.
Apoptosis also was examined by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method using the ApopTag Fluorescein In Situ Apoptosis Detection Kit (Merck Millipore, Merck KGaA, Darmstadt, Germany).
Jurkat cells activated by sodium valproate were used as a positive control. Cells showed apoptosis with Annexin V and caspase 3 immunofluorescence and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay.
Renal Biopsy Specimens
Consecutive renal biopsy specimens (n = 54) were reviewed retrospectively. Renal biopsy specimens with a diagnosis of diabetes archived at Arkana Laboratories were retrieved (n = 54) (institutional review board exempt). All patients presented with significant proteinuria; 36 of 54 presented with nephrotic range proteinuria. The demographic data of the patients whose renal biopsy specimens were used are shown in Table 2. Electron microscopic examination was performed on routinely obtained micrographs (5 to 10 images) of consecutive biopsies performed for diagnostic purposes.
Table 2Demographic Data of Patients with Diabetic Glomerulosclerosis (n = 54)
Characteristic
Values
Age, years
40.0 ± 15.6
Sex, male/female
48%/52%
Ethnicity, n (%)
Caucasian
26 (48)
African American
20 (37)
Hispanic
5 (9)
Others
3 (6)
Hematuria, n (%)
18 (33.3)
Proteinuria, n (%)
54 (100)
Nephrotic range, n (%)
36 (66.7)
Serum creatinine, mg/dL
3.0 ± 2.2
Age and serum creatinine are expressed as means ± SEM.
: i) enlarged podocytes with irregular nucleus, ii) multinucleation, iii) presence of micronucleus (round DNA aggregates close to the nucleus) or mitotic spindle, and iv) denucleation (invisible nucleus).
Statistics
Continuous variables were presented as means ± SD and were compared using the two-tailed t-test or one-way analysis of variance. Categoric variables were presented as a percentage and were compared using the χ2 or the Fisher exact test, where appropriate.
Results
Podocytes Detached in the Urine Show Morphologic Characteristics of MC
A total of 184 urinary podocyte samples were prepared for various stains from 41 patients. The urinary podocytes in the sediments had various nuclear shapes classified into the following six groups: A, B, D, J, and K (Table 3); representative features are shown in Figure 2. A total of 3.8% of urinary podocytes in MC had a micronucleus (Figure 2, H and M). The urinary podocytes in groups C, D, I, and J had definitive MC findings according to the criteria described above in Materials and Methods. Podocytes with normal nuclear appearance were consistent with physiologic shedding (Figure 2, A and E). In Figure 2, B and F show enlarged and irregular podocyte nucleus; C and G show binucleated podocytes; D shows podocyte binucleation and micronucleolus; I shows trinucleated podocyte; J and N show podocyte in mitosis. Another lesion seen in group K was denucleation (seemingly absent nucleus), a presumed feature of MC. Taken together, 54.4% of urinary podocytes had morphologic characteristics of MC (Table 3). Moreover, approximately one third of the podocytes with presumptive denudation showed faint multinucleation when carefully re-evaluated (Figure 2, K, L, O, and P). This may have been owing to poor hematoxylin staining of dying or poorly preserved urinary podocytes (degenerating cells stain poorly). If these podocytes were included in the definitive MC groups, approximately 66% to 67% of urinary podocytes in diabetic patients showed MC.
Table 3Nuclear Characteristics of Urinary Podocytes (n = 184)
Figure 2Nuclear staining of urinary podocalyxin (PCX)+ podocytes with various morphologic appearances. A set of immunofluorescence and hematoxylin staining (A–D and I–L show anti-PCX staining, and E–H and M–P show hematoxylin). A and E: Normal nuclear morphology. B and F: Enlarged and irregular nucleus. C and G: Binucleated. D and H: Binucleated and micronucleus (arrow). I and M: Trinucleated podocyte and micronucleus (arrow). J and N: Mitotic figure. K and O: Single nucleus. L, and P: Denucleation. E–H and M–P: Hematoxylin stain. Original magnification, ×40 (A–P).
Sixty-two urinary podocyte samples from patients with diabetes were examined for apoptosis by IF using annexin, caspase 3, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (Figure 3). Apoptosis was not detected by any of the three different apoptosis assays.
On the contrary, the mitosis marker phosphorylated vimentin was positive for 5 of 25 urinary podocytes (20%). A representative image of phosphorylated vimentin staining is shown in Figure 3.
Figure 3Apoptosis assays. Annexin V, caspase 3, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) are entirely negative. Twenty percent of urinary podocytes show positive with immunofluorescence staining with phosphorylated vimentin. Original magnification, ×20 (all images). LM, light microscopy; PCX, podocalyxin.
Urinary Podocytes Do Not Express Leukocyte Antigen or Renal Tubular or Parietal Epithelial Cell Markers
Forty urinary podocyte samples from patients with diabetes were examined for leukocyte markers such as macrophage and panleukocyte stains and dual immunohistochemistry (with PCX). None of the urinary podocytes were positive for leukocyte markers.
Ninety-five urinary podocytes were examined for podocyte markers such as nephrin, podocin, Glepp1, and renal tubular marker cytokeratin 8. None of the urinary podocytes were positive for nephrin and podocin; 50.0% of urinary podocytes were positive for Glepp1. No urinary podocytes were positive for cytokeratin 8. Representative images are shown in Figure 4.
Figure 4Podocyte markers in kidney sections and urine from diabetic patients. Top row: Immunofluorescence staining on normal kidney sections (control). Bottom row: Podocyte markers (nephrin, podocin, and Glepp1) and renal tubular and parietal epithelial cell marker (cytokeratin 8) on urinary podocytes; dual staining with antipodocalyxin antibody. Original magnification, ×40 (all images). PCX, podocalyxin.
Urine Sediments Contain Podocytes and Podocyte Cell Debris
The shape of urinary podocytes was examined carefully and various morphologic staining patterns were present, such as diffuse PCX staining on the whole cell (Figure 5A ), spotty PCX staining (Figure 5B), granular staining (Figure 5, E and F), and combined spotty and granular staining (Figure 5, C and D). Cellular destruction is shown diagrammatically in Figure 5G. Because no other epithelial cells are potentially PCX positive, we propose that cell debris with a granular PCX pattern is released from destroyed PCX+ cells and finally entrapped in casts.
Figure 5Destruction process of urinary podocytes. A–F: PCX immunofluorescence shows various features of diabetic urine podocytes in vivo. A: Intact podocyte. B: Enlarged cell body with large granular debris. C and D: Enlarged cell body containing variable-size granules. E and F: Cast containing granular cell debris. G: Proposed concept of destruction of urine podocytes with mitotic catastrophe. Original magnification, ×40 (all images).
A positive correlation was found between the number of urinary podocytes and cast scores (P < 0.001) (Figure 6B). These findings may suggest a destructive process of urinary podocytes starting as an intact cell and ending trapped in casts through various phases of cell disintegration (Figure 6A).
Figure 6Urinary podocytes and cast scores. A: Urinary podocytes with the whole cell stained with podocalyxin (PCX). B: Urinary podocytes correlate with casts containing PCX-positive granular structures (cells/10 mL urine). n = 14 urinary podocytes (B). P < 0.001 podocytes versus casts. Original magnification, ×40 (all images).
Renal Biopsy Specimens Show No Evidence for Apoptosis but Occasional MC Electron Microscopy
Multinucleated podocytes were observed in 8 of 54 specimens (Table 4 and Figure 7). Other findings included podocyte detachment and podocyte hypertrophy (a known feature of diabetes).
Because podocyte multinucleation is recognized as a specific feature of aberrant mitosis, we hypothesized that mitotic podocytes may be associated with proteinuria. However, in this cohort, no statistical correlation with proteinuria was found (Table 5) (P = 0.1).
Figure 7Electron microscopy of diabetic glomeruli with mitotic catastrophe (MC). A–D: Podocyte binucleation (arrows), a typical MC feature (A and B), diffuse foot process effacement (C; arrowhead), and podocyte hypertrophy (D; star) are found in human diabetic glomeruli as described in Table 4. Scale bars = 10 μm.
The present study is the first detailed morphologic evaluation of urinary podocytes from diabetic patients. By using podocyte-specific molecular markers and dual staining on synaptopodin-positive cells, morphologic characteristics of MC were observed in approximately half of urinary podocytes. No apoptotic podocytes were found by morphology or immunohistochemistry in these urine samples. Podocytes with MC also were found in diabetic renal biopsy specimens, although not so frequently. There were no apoptotic podocytes seen by electron microscopy in these biopsy specimens. The results from urine and biopsy examinations clearly show that MC instead of apoptosis may be involved in the detachment of podocytes from the glomerular basement membrane in the diabetic kidney. The present study is the first to describe the involvement of MC in human diabetic podocytes in situ or captured in the urine, suggesting that a major cause for podocyte loss in diabetic kidney disease is MC.
MC is caused by incomplete assembly of the chromosomes with the mitotic spindle in prometaphase, which leads to aberrant chromosome segregation.
or irregularly shaped nuclei, indicating that podocytes indeed can enter the cell cycle and increase DNA synthesis, although they are terminally differentiated cells unable to complete mitotic division; cytokinesis would require complete reorganization of the actin cytoskeleton. Multinucleation, micronuclei, irregular-shaped nuclei, and abnormal mitosis clearly showed that MC occurred in podocytes from diabetic patients. Phosphorylated vimentin is a specific intermediate filament type III, expressed predominantly during mitosis in malignant cancer cells and sarcomas resulting in multinucleation, therefore it is considered a cell mitosis marker.
In this study, phosphorylated vimentin was detected in urinary podocytes, supporting our hypothesis that urinary podocytes from diabetic patients enter the cell cycle. Notably, none of the patients in this study had cancer.
Tubular epithelial cells were excluded by selection of cells with dual staining (podocyte marker+/cytokeratin 8-). These results are significant because they point to a previously poorly recognized mechanism of diabetic glomerular injury progression.
Independent recent studies have shown that hyperglycemia induces increased MDM2 in diabetic podocytes and is associated with MC.
MDM2 is a cell-cycle regulator and its action is mediated at least in part via downstream Notch 1 signaling. MDM2 knock-down decreases Notch signaling.
Podocyte apoptosis is thought to happen very fast and it is possible that apoptotic cells disintegrate beyond morphologic detection or by apoptosis assays. The present study showed no detection of apoptotic podocytes in the urine, or in situ by electron microscopy. Taking into consideration the findings both in in situ glomeruli and podocytes in urine samples, it seems highly unlikely that apoptosis is a cause of podocyte loss in human diabetes.
This comprehensive morphologic study shows for the first time that urinary podocytes have varied and heterogeneous morphology and range from degenerative to viable podocytes. The podocytes with MC or apoptosis are considered dying cells. However, viable podocytes reportedly were recovered from urine and grown in culture.
In addition to these dead or viable podocytes, it is possible that podocyte progenitors are present in urine. Recently, it was shown that a subpopulation of progenitor cells in the parietal epithelium of the Bowman's capsule act as a source of progenitors for visceral podocytes.
These cells show a mixed phenotype between parietal epithelial cells and podocytes. The present study showed no urinary podocytes with parietal cell markers, such as cytokeratin 8.
Anti-PCX monoclonal antibody was used as a podocyte marker based on previous studies that showed stable staining not altered in various glomerular diseases.
In addition, the anti-PCX antibody showed excellent specificity for visceral podocytes, reacting with visceral glomerular epithelium exclusively, and not with parietal epithelial cells along the Bowman's capsule (Figure 1A). We propose that this anti-PCX antibody is most reliable to detect urinary podocytes. Nephrin and podocin also are used as podocyte markers, but these are not suitable for detecting urinary podocytes, perhaps because the expression of these molecules is down-regulated in various glomerular diseases including diabetes,
or because fallen podocytes have no slit diaphragm where nephrin is expressed. Glepp1 also seems to be a good urinary podocyte marker, but PCX is better because the Glepp1 antibody only labeled a fraction of PCX+ urinary podocytes. On the other hand, macrophages may express podocyte markers including podocalyxin under pathologic conditions.
This possibility was less likely in this study because urinary podocytes did not express macrophage or leukocyte markers.
Dying podocytes with MC have a variable appearance just before glomerular basement membrane detachment. When dying podocytes in the sediments were examined carefully, various stages of cell destruction were seen (Figure 2). Larger fragments of cell debris appear to become gradually smaller, before turning granular. The majority of these granular structures seem to be entrapped in casts, as seen in other glomerular diseases.
The number of urinary podocytes correlated well with the number of PCX-positive casts, indicating a destruction process (Figure 6). The PCX-positive granular structures originated from podocytes, so the actual podocyte numbers in the urine may in fact be higher, although the countable urinary podocytes are not so numerous (approximately 0 to 10 cells/10 mL
A correlation between the progression of glomerular sclerosis and PCX+ cells in urinary sediments of patients with a variety of kidney diseases has been shown.
In addition, changes in semiquantitative measures of podocyturia seem to correlate directly with disease activity as assessed by renal biopsy and to decrease with treatment, suggesting that urinary shedding of podocytes may represent a real-time measure of podocyte loss from the glomerulus.
Shedding of podocytes in urine is found in other diseases such as lupus and IgA nephropathy, and it is possible that in such diseases podocyturia may be higher compared with diabetes, characterized by relatively longer progression times.
The present study was executed meticulously to address the question of how podocytes may die in human diabetic kidney disease. MC, and not apoptosis, appears to be the major type of cell death. Further studies may show the clinical relevance of detecting podocytes in diabetic urine and whether this noninvasive approach may determine the effect of conventional or new therapies on MC-induced podocyte loss in diabetic kidney disease.
Acknowledgments
We thank Hidaki Muraki for technical assistance with immunohistochemistry; Roger Wiggins (University of Michigan Medical Center, Ann Arbor, MI) for kindly providing Glepp1, nephrin, and 4A4 antibodies; Dr. Kunimasa Yan (Tokyo); and Dr. Masaki Inagaki (Nagoya, Japan), respectively.
K.O. and D.-F.D. performed experiments and collected data; and M.H. and H.L. designed the study and wrote the manuscript.
Effects of angiotensin-converting enzyme inhibitor, angiotensin II receptor antagonist and calcium antagonist on urinary podocytes in patients with IgA nephropathy.
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