Cell-cycle mechanisms involved in podocyte proliferation in cellular lesion of focal segmental glomerulosclerosis

Cell-Cycle Mechanisms Involved in Podocyte Proliferation in Cellular Lesion of Focal Segmental Glomerulosclerosis Suxia Wang, MD, Ji Hoon Kim, MD, Kyung Chul Moon, MD, Hye Kyoung Hong, MSc, and Hyun Soon Lee, MD ● Background: Podocyte injury may induce podocyte proliferation, which results in glomerular scarring. The cellular lesion, seen in some patients with primary focal segmental glomerulosclerosis (FSGS), is characterized by proliferation of cells covering the sclerotic or collapsed glomerular tufts. Cell-cycle mechanisms by which podocyte proliferation occurs in the cellular lesion of FSGS are unclear. Methods: We examined expression patterns of cyclin D1; cyclin E; cyclin A; cyclin B1; cyclin-dependent kinase (CDK)2; CDK4; such CDK inhibitors as p21WAF1/CIP1 (p21), p27kip1 (p27), and p57kip2 (p57); and Wilms’ tumor protein-1 (WT-1) in 12 renal biopsy specimens with the cellular lesion of FSGS and 6 renal biopsy specimens with no detectable abnormalities by immunohistochemistry and immunoelectron microscopy. Messenger RNA (mRNA) expression patterns of cyclin D1, cyclin E, p21, p27, and p57 were evaluated further by in situ hybridization. Results: In controls, immunostaining for cyclin A, cyclin B1, CDK2, CDK4, and p21 was almost negligible, but positive signals for cyclin D1, cyclin E, p27, and p57 were observed in glomerular epithelial cells (GECs). In the cellular lesion of FSGS, positive signals for cyclin E, cyclin A, cyclin B1, CDK2, and p21 were present in GEC nuclei, in which WT-1, p27, p57, and cyclin D1 were undetected. Immunoelectron microscopy showed that cyclin E–, CDK2-, and p21-specific gold particles were increased significantly in GEC nuclei in the cellular lesion in which cyclin D- and p57-specific particles were absent compared with controls. An in situ hybridization study showed specific signals of cyclin D1, cyclin E, p21, p27, and p57 mRNA in GECs forming the cellular lesion of FSGS. Conclusion: Our results suggest that damaged podocytes may inhibit p27 and p57 protein expression, but activate a cyclin D1–independent cell-cycle mechanism and mitotic cell cyclins to promote GEC proliferation in the cellular lesion of FSGS. Am J Kidney Dis 43:19-27. © 2004 by the National Kidney Foundation, Inc. INDEX WORDS: Glomerular epithelial cells (GECs); cyclins; cyclin-dependent kinase (CDK); cyclin-dependent kinase (CDK) inhibitors; glomerulosclerosis.

M

ATURE PODOCYTES or visceral glomerular epithelial cells (GECs) are growtharrested terminally differentiated cells. The inability of podocytes to proliferate after glomerular injury may underlie the development of glomerulosclerosis.1 Conversely, podocyte proliferation superimposed on sclerotic or collapsed glomerular tufts characterizes the cellular lesion of focal segmental glomerulosclerosis (FSGS)2,3 or its subgroup collapsing glomerulopathy.4 This cellular lesion is clinically important because it is considered an initial lesion that leads to glomerular scarring.2-5 Furthermore, patients with compared with those without the cellular lesion or collapsing glomerulopathy have a greater prevalence of end-stage renal disease.5 Cell proliferation is controlled at the cell-cycle level by cell-cycle regulatory proteins. Activation of specific cyclin-dependent kinases (CDKs) by a partner cyclin in each phase of the cell cycle leads to cell proliferation. Cyclin D and cyclin E are responsible for progression of the G1/S phase, whereas the S/G2/M phase is promoted by cyclin A and cyclin B (reviewed in6). Cyclin D activates CDK4 during the G1 phase.7 Cyclin E and cyclin A activate CDK2,8 which is essential for DNA

synthesis.9 Cyclin-CDK complexes are regulated negatively by CDK inhibitors,10 which include p21WAF1/CIP1 (p21), p27kip1 (p27), and p57kip2 (p57).11 The reduced proliferative capacity of mature podocytes in vivo may be the consequence of CDK inhibitor activity.11,12 p27 and p57 are constitutively expressed in mature podocytes.12-15 In terms of p21 expression in the normal glomerulus, 1 group found it absent in healthy adults,12 whereas others claimed it is expressed in healthy children.16 In the cellular lesion of FSGS, GECs were negative for p27 and p57,12,15 but positive for p21.12 The positive regulators of cell cycle, cyclin A

From the Department of Pathology, Seoul National University College of Medicine, Seoul, Korea. Received July 7, 2003; accepted in revised form September 15, 2003. Supported in part by the Korea-China Young Scientists Exchange Program of the Korean Science and Engineering Foundation (S.W.). Address reprint requests to Hyun Soon Lee, MD, Department of Pathology, Seoul National University College of Medicine, Chongno-gu, Yongon-dong 28, Seoul 110-799, Korea. E-mail: [email protected] © 2004 by the National Kidney Foundation, Inc. 0272-6386/04/4301-0002$30.00/0 doi:10.1053/j.ajkd.2003.09.010

American Journal of Kidney Diseases, Vol 43, No 1 (January), 2004: pp 19-27

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WANG ET AL

and cyclin B1, are absent in mature podocytes,14,15 although cyclin D1 is present.14,17 In the cellular lesion of FSGS or collapsing glomerulopathy, cyclin A is expressed segmentally,15,17 but cyclin D1 is absent.17 The role of cyclin-CDK complexes and CDK inhibitors in the regulation of podocyte proliferation in the cellular lesion of FSGS is still unclear. Thus, we undertook to examine expression patterns of cyclin D1, cyclin E, cyclin A, cyclin B1, CDK2, CDK4, p21, p27, and p57 protein and messenger RNA (mRNA) collectively in the cellular lesion of FSGS. MATERIALS AND METHODS

Patients Twelve patients with a diagnosis of the cellular lesion of FSGS were selected for this study. No patient had a history of diabetes mellitus, cyanotic heart disease, reflux nephropathy, obesity, autoimmune disease, human immunodeficiency virus infection, or drug abuse. Control specimens were obtained during kidney donation for transplantation from a 35-year-old man and 5 patients with microscopic hematuria, but no detectable abnormalities. Renal biopsy specimens with more than 10 glomeruli were processed for light, electron, and immunofluorescence microscopy. For light microscopy, kidney tissues were fixed overnight in 4% paraformaldehyde at 4°C. Patient age ranged from 21 to 72 years, with a mean age of 39 ⫾ 17 years. The man:woman ratio was 7:5. Two patients (17%) showed hypertension, defined as blood pressure greater than 140/90 mm Hg. Seven patients (58%) had nephroticrange proteinuria, with protein excretion of 3.5 g/d or greater at the time of biopsy. Renal insufficiency, defined as a serum creatinine level greater than 1.5 mg/dL (⬎133 ␮mol/L), was observed in 2 cases.

Immunohistochemistry An avidin-biotin-peroxidase procedure (Dakopatts, Glostrup, Denmark) was used for antibody localization. All primary antibodies were obtained from Santa Cruz Biotechnology Inc (Santa Cruz, CA), except for anti-p27 (Dakopatts). Paraffin-embedded kidney sections were deparaffinized serially. Subsequently, these sections were baked in a microwave oven for 10 minutes, then incubated overnight at 4°C with rabbit anticyclin A, anticyclin B1, anti-CDK4, and anti-p57, and antihuman Wilms’ tumor protein-1 (WT-1); mouse anti-p27, at a dilution of 1 in 50 to 1 in 200. To show the presence of CDK2, sections were treated with proteinase K for 15 minutes, then incubated overnight with rabbit anti-CDK2 at a dilution of 1 in 200. To show the presence of p21, sections were baked at 100°C under high pressure for 10 minutes, then incubated overnight with rabbit anti-p21 at a dilution of 1 in 100. The other sections were baked in a microwave oven for 10 minutes, then incubated with rabbit anticyclin E and anticyclin D1 at a dilution of 1 in 2,000 and 1 in 100 for 2 hours at room temperature, respectively. Biotinylated goat antirabbit immunoglobulin or antimouse

immunoglobulin (Dakopatts) was used as a secondary antibody. Endogenous peroxidase activity was quenched with methanol–hydrogen peroxide solution. Streptavidin-conjugated horseradish peroxidase complex incubation was performed, followed by the addition of diaminobenzidine. Sections were counterstained with hematoxylin and periodic acid–Schiff reagent. As positive controls for CDK2 and CDK4, tissue sections from normal tonsil and breast carcinoma were used, respectively. Control experiments were performed by omitting the primary antibody or replacing it with the corresponding nonimmune serum. Expression and distribution patterns of cyclin D1, cyclin E, cyclin A, cyclin B1, CDK2, CDK4, p21, p27, and p57 in glomeruli were assessed by 2 pathologists (S.W. and H.S.L.) independently in a blinded manner. For each glomerulus showing the cellular lesion of FSGS, cell-cycle regulatory protein expression was analyzed quantitatively. First, numbers of GECs in the cellular lesion of FSGS and adjacent segments or glomeruli were counted separately. Next, the number of GECs that stained positive for each antibody was counted in both areas to obtain percentages of podocytes that express cell-cycle regulatory proteins in the cellular lesion of FSGS and the nonsclerotic area.

Immunogold Electron Microscopy Immunoelectron microscopy was performed in 3 cases of FSGS and 3 controls. Briefly, samples were immersed in a periodate-lysine-paraformaldehyde solution for 2 hours at 4°C and dehydrated in a graded ethanol series at progressively lower temperatures to ⫺35°C. Tissue was transferred to a 1:1 mixture of 100% ethanol and Lowicryl K4M (Chemische Werke Lowi, Waldkraiburg, Germany) for 2 hours at ⫺35°C, then transferred to 100% Lowicryl at ⫺35°C for 2 hours. Ultrathin sections were mounted on Formvar (Ernest F. Fullam Inc, Latham, NY) coated 100mesh nickel grids, which were incubated overnight at 4°C with primary antibodies, exposed to 10 nm of goldconjugated secondary antibodies for 2 hours at room temperature, and stained with uranyl acetate and then with lead citrate. Control experiments were performed by omitting or replacing the primary antibodies with the corresponding nonimmune serum.

Generation of Digoxigenin-Labeled Riboprobes and In Situ Hybridization Histochemistry Total cellular RNA was isolated from cultured human mesangial cells and used to synthesize complementary DNAs (cDNAs) for cyclin D and cyclin E with Superscript II (Gibco BRL, Paisley, UK) according to the manufacturer’s protocol. Using the cDNA mixture together with the sense and antisense primers of cyclin D118 and cyclin E,19 polymerase chain reaction was performed. In addition, a 2.1-kb cDNA for p21,20 a 0.7-kb cDNA for p27,21 and a 1.5-kb cDNA for p5722 were obtained from American Type Culture Collection (Rockville, MD). For amplification of cDNA templates, polymerase chain reaction was performed using T7 and T3 promoters as primers. Two oppositely oriented promoters served to provide 2 transcripts of the same template; 1 was complementary (antisense) and the other was identical (sense) to the target mRNA. Digoxigenin (DIG)-

CELL CYCLE AND CELLULAR LESION OF FSGS

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Fig 1. Immunoperoxidase staining for WT-1, cyclin D1 (Cyc D1), CDK4, cyclin E (Cyc E), cyclin A (Cyc A), cyclin B1 (Cyc B1), CDK2, p21, p27, and p57 in controls and the cellular lesion of FSGS. (Cyc D1, CDK4, Cyc E, Cyc A, Cyc B1, CDK2, p21, p27, and p57 in columns of FSGS.) Serial sections. (Original magnification ⴛ200.)

labeled riboprobes were generated by using an RNA labeling in vitro transcription kit (DIG RNA labeling kit; Boehringer Mannheim, Mannheim, Germany). In situ hybridization was performed in 4 cases of FSGS and 4 controls by using paraffin-embedded renal tissues.

RESULTS

Immunohistochemistry WT-1. Control biopsy specimens stained with anti–WT-1 showed nuclear staining in all podocytes. In FSGS specimens, there was a loss of

WT-1 in GECs covering the sclerotic segments (Fig 1). Cyclin D1. In controls, some GECs expressed nuclear staining for cyclin D1. In cases in which the cellular lesion of FSGS was present, cyclin D1 was almost negligible in the cellular lesion and adjacent segments, except for a few isolated GECs (Table 1; Fig 1). CDK4. No immunostaining for CDK4 was observed in control biopsy specimens. In cases in

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WANG ET AL Table 1.

Cell-Cycle Regulatory Protein Expression in the Cellular Lesion of FSGS

No. of Glomeruli Examined Cellular lesion of FSGS (n ⫽ 12) Sclerotic area Nonsclerotic area Controls (n ⫽ 6)

No. of Podocytes Examined

Cyclin D1

CDK4*

Cyclin E

Cyclin A

Cyclin B1

CDK2

p21

p27

p57

47 ⫾ 21 (20-80) 15 ⫾ 2 (13-19) 15 ⫾ 6 (3-29)

2⫾3 4⫾4 40 ⫾ 14‡

8 ⫾ 12 1⫾2 0

91 ⫾ 8 49 ⫾ 17‡ 40 ⫾ 24‡

34 ⫾ 15 16 ⫾ 25‡ 11 ⫾ 27‡

15 ⫾ 3 0 0

40 ⫾ 27 3 ⫾ 7‡ 0

16 ⫾ 11 0 0

8⫾7 92 ⫾ 8‡ 91 ⫾ 4‡

1⫾2 58 ⫾ 23‡ 88 ⫾ 10‡

Cell-Cycle Regulatory Proteins (% positive)

3.6 ⫾ 1.5† (1-6)

11 ⫾ 3.6 (8-15)

NOTE. Data expressed as mean ⫾ SD (range). *Mostly perinuclear staining. †Glomeruli showing the cellular lesion of FSGS. ‡P ⬍ 0.05 versus sclerotic area in the cellular lesion of FSGS by Wilcoxon’s rank-sum test.

which the cellular lesion of FSGS was present, GECs occasionally showed nuclear membrane or perinuclear staining for CDK4 in the cellular lesion (Table 1; Fig 1). In nonsclerotic glomeruli, CDK4 staining was almost negligible. Cyclin E. In controls, GECs and parietal epithelial cells frequently expressed nuclear staining for cyclin E. In cases with the cellular lesion of FSGS, some GECs in nonsclerotic glomeruli also showed positive cyclin E staining. In the cellular lesion of FSGS, diffuse nuclear staining for cyclin E was apparent in hyperplastic GECs (Table 1; Fig 1). Cyclin A. Cyclin A rarely was positive in glomeruli of control biopsy specimens. In patients with the cellular lesion of FSGS, several GECs showed cyclin A positivity in nonsclerotic glomeruli. In the cellular lesion, cyclin A staining was observed in nuclei of some hyperplastic GECs (Table 1; Fig 1). Cyclin B1. In controls, glomerular cells were negative for cyclin B1. In FSGS specimens, no cyclin B1 staining was present in the nonsclerotic area. In the cellular lesion of FSGS, GECs occasionally showed nuclear and/or cytoplasmic staining for cyclin B1 (Table 1; Fig 1). CDK2. In controls, glomerular cells were negative for CDK2. In specimens of the cellular lesion of FSGS, only a few isolated GECs showed CDK2 immunostaining in nonsclerotic glomeruli. In GECs of the cellular lesion of FSGS, segmental positive nuclear staining was observed (Table 1; Fig 1). p21. In controls, glomerular cells were negative for p21. In FSGS specimens, no immunostaining for p21 was observed in the nonsclerotic area. In the cellular lesion of FSGS, GECs occa-

sionally showed nuclear staining for p21 (Table 1; Fig 1). p27. In controls, immunostaining for p27 was observed in nuclei of most GECs. In patients with cellular FSGS, p27 was positive on GECs in nonsclerotic glomeruli. In the cellular lesion, GECs showed no staining except for a few positive foci (Table 1; Fig 1). p57. In controls, most GECs expressed nuclear staining for p57. In FSGS specimens, p57 frequently was positive on GECs in nonsclerotic glomeruli. In the cellular lesion, loss of podocyte immunostaining for p57 was observed (Table 1; Fig 1). Immunoelectron Microscopy Cyclin D1. In controls, several gold particles were observed in nuclei of GECs (Fig 2A). In the cellular lesion of FSGS, no particle density labeling for cyclin D1 in GEC nuclei was present (Table 2; Fig 2B). Cyclin E. In controls, several gold particles were observed in GEC nuclei (Fig 2C). In the cellular lesion of FSGS, particle density labeling for cyclin E was increased markedly in GEC nuclei (Table 2; Fig 2D). CDK2. In controls, immunogold density for CDK2 was absent in GECs (Fig 2E). However, in the cellular lesion of FSGS, several gold particles were present, scattered in GEC nuclei (Table 2; Fig 2F). p21. In controls, immunogold density for p21 was largely absent in GECs (Fig 2G). In the cellular lesion of FSGS, GEC nuclei frequently were labeled with this antibody (Table 2; Fig 2H). p57. In normal adult human glomeruli, p57 antibody mainly labeled GEC nuclei (Fig 2I). In

CELL CYCLE AND CELLULAR LESION OF FSGS

Fig 2. Electron micrographs of gold-labeled antibodies to (A, B) cyclin D (arrows), (C, D) cyclin E (arrows), (E, F) CDK2, (G, H) p21, and (I, J) p57 in GEC nuclei in (A, C, E, G, I) controls and (B, D, F, H, J) the cellular lesion of FSGS. Abbreviations: scl, sclerotic lesion; p, podocytes. (Original magnification [A] ⴛ31,170, [B] ⴛ3,300, [C] ⴛ29,500, [D] ⴛ31,800, [E] ⴛ10,800, [F] ⴛ23,900, [G] ⴛ9,500, [H] ⴛ25,600, [I] ⴛ25,500, and [J] ⴛ5,780.)

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Table 2. Occurrence of Immunogold Particles for Cell-Cycle Regulatory Proteins in Podocytes

0

ally expressed p57 mRNA. In the cellular lesion of FSGS, GECs showed a positive signal for p57 mRNA (Fig 3J). Tubular epithelial cells occasionally showed the characteristic message for the p57 gene. No hybridization was detected in sections probed by sense riboprobes for cyclin D1, cyclin E, p21, p27, and p57 (Fig 3K and L).

27 ⫾ 10

DISCUSSION

No. of Particles/Nucleus Cyclin D1 Cyclin E

Cellular lesion of FSGS (N ⫽ 3) Controls (N ⫽ 3)

0 9⫾2

CDK2

p21

34 ⫾ 10 12 ⫾ 6 14 ⫾ 5 9

0

0

p57

NOTE. Data expressed as mean ⫾ SD.

the cellular lesion of FSGS, no particle density labeling for p57 was observed in GEC nuclei (Table 2; Fig 2J). In Situ Hybridization Cyclin D1 mRNA. In normal adult human glomeruli, a weak signal for cyclin D1 mRNA was observed in several GECs (Fig 3A). In the cellular lesion of FSGS, GECs expressed a specific message for cyclin D1 mRNA (Fig 3B). Cyclin E mRNA. A relatively weak signal for cyclin E mRNA was shown in GECs and mesangial cells in control renal biopsy specimens (Fig 3C). All biopsy specimens containing the cellular lesion of FSGS showed intense signals for cyclin E mRNA in GECs covering sclerotic segments (Fig 3D). In nonsclerotic glomeruli, glomerular cells showed an enhanced signal for cyclin E transcripts. Tubular epithelial cells frequently showed the characteristic message for the cyclin E gene. p21 mRNA. In controls, several GECs showed a weak signal for p21 mRNA (Fig 3E). Parietal epithelial cells rarely expressed p21 mRNA. In the cellular lesion of FSGS, hyperplastic GECs showed a distinctive signal for p21 mRNA (Fig 3F). Tubular epithelial cells showed a strong message for the p21 gene. p27 mRNA. In normal adult human glomeruli, GECs frequently showed a distinctive signal for p27 mRNA (Fig 3G). Endothelial and parietal epithelial cells also expressed p27 mRNA. In the cellular lesion of FSGS, a specific message for p27 mRNA was observed in GECs (Fig 3H). p57 mRNA. In control renal biopsy specimens, GECs showed a distinctive signal for p57 mRNA (Fig 3I). Parietal epithelial cells occasion-

These studies confirm and extend previous analyses of cell-cycle regulatory proteins in the cellular lesion of FSGS. A new finding of this study is that GECs in the cellular lesion of FSGS showed increased staining for cyclin E, cyclin B1, and CDK2 despite the lack of active cyclin D1-CDK4 complex. In agreement with previous reports, we observed decreased staining for p27 and p57, but increased staining for p21 and cyclin A in the cellular lesion. Podocyte injury, the first event to occur in the natural progression of FSGS,2,3 results in the loss of podocyte differentiation markers,23-25 as we observed in this study. In the present study, most GECs in the cellular lesion of FSGS were negative for cyclin D1, whereas GECs in control biopsy specimens expressed nuclear staining for cyclin D1. In agreement with our results, Barisoni et al17 observed the loss of cyclin D1 in collapsing glomerulopathy. Furthermore, CDK4 was expressed mainly in perinuclear areas of GECs in the cellular lesion, suggesting that CDK4 is not activated in this lesion despite increased biosynthesis of the molecule. Cyclin D activates CDK4 and CDK6 during the mid-G1 phase,7,10 and these active CDK complexes then enter the nuclear compartment. Thus, the lack of nuclear staining for cyclin D1 and CDK4 in GECs in the cellular lesion suggests that damaged GECs do not form an active cyclin D1-CDK4 complex to trigger aberrant cell-cycle progression. We first show that most hyperplastic GECs overlying sclerotic glomerular tufts showed positive staining for cyclin E or markedly increased immunogold density for cyclin E in nuclei. In addition, several GECs showed positive immunostaining for CDK2 in the cellular lesion. Cyclin E levels increase in late G1, when it associates with and activates CDK2, and peak suddenly at the G1/S boundary.8 G1/S transition results in

Fig 3. Detection of (A, B, K, L) cyclin D, (C, D) cyclin E, (E, F) p21, (G, H) p27, and (I, J) p57 mRNA in renal biopsy specimens of (A, C, E, G, I, K) healthy controls and (B, D, F, H, J, L) patients with FSGS. In FSGS, renal biopsy sections hybridized with probes specific for (B) cyclin D, (D) cyclin E, (F) p21 (arrows), (H) p27, and (J) p57 mRNA show specific signals in GECs of sclerotic segments. (K, L) Control in situ hybridization with sense cyclin D ribroprobe. (Original magnification ⴛ200.)

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DNA synthesis and cell proliferation.26 Cyclin E is controlled internally, whereas cyclin D is controlled by extracellular signals. In addition, cyclin E can functionally replace cyclin D1, thereby obviating the need for cyclin D1 in cell-cycle progression.27 Thus, our immunostaining results for cyclin E and cyclin D suggest cyclin E is a major G1 cyclin that supplements cyclin D1 deficiency to promote podocyte proliferation in the cellular lesion. We also show that several GECs in the cellular lesion expressed cyclin B1. Increased cyclin B1 podocyte expression has never been described in the cellular lesion of human FSGS, although it was shown in human immunodeficiency virus transgenic mice, a model characterized by podocyte proliferation.28 Furthermore, we observed that some GECs show nuclear staining for cyclin A in the cellular lesion, in agreement with previous reports.15,17 Cyclin A levels peak in late G1 and persist through G2, whereas cyclin B levels increase at the end of the S phase and continue throughout the G2 phase.29 Cyclin B is required for mitosis.30 Cyclin B1-cdc2 (formerly CDK1) complexes are imported into the nucleus shortly before breakdown of the nuclear membrane, at the end of the prophase.31 Thus, enhanced expression of cyclin B1 and cyclin A in GECs, together with activated CDK2 in the present study, suggest that mitotic cyclins could contribute to increased podocyte numbers in the cellular lesion of FSGS. In the present study, most GECs in the cellular lesion of FSGS were negative for p27 and p57, although several GECs showed strong nuclear staining for p21. These findings are similar to those of previous reports.12,15 Loss of p27 and p57 in the cellular lesion may explain the mechanism by which podocytes replicate. However, the reason for de novo expression of p21 in some GECs in the cellular lesion is unclear. Similar to proliferating cell nuclear antigen, p21 expression increases during proliferation and remains bound to CDK complexes in proliferating cells, possibly facilitating DNA synthesis.32 Nonetheless, p21 is a universal inhibitor of CDKs and inhibits G1 CDK33,34 and cyclin B-cdc2 at the G2/M boundary.35 In experimental glomerular diseases, p21 limits the kinase activity of CDK211 and GEC proliferation.36 Whether increased expression of p21 in the cellular lesion of FSGS serves

WANG ET AL

a protective role to limit further podocyte proliferation or contributes to cell proliferation remains to be clarified. Our in situ hybridization study shows that cyclin D1, cyclin E, p21, p27, and p57 mRNA are expressed in GECs. Furthermore, we show that specific signals of cyclin E and p21 mRNA are overexpressed consistently in GECs within the cellular lesion of FSGS, suggesting the increased expression of cyclin E and p21 is caused by enhanced transcription. Despite the lack of cyclin D1, p27, and p57 protein expression in the cellular lesion of FSGS, specific signals for their mRNAs were shown in GECs forming this lesion. The different expression patterns of these proteins and mRNAs are difficult to explain. We only conjecture that increased proteolysis of cyclin D1, p27, and p57 proteins may contribute to the decreased expression of these proteins in the cellular lesion. In summary, our results suggest that damaged podocyes may inhibit p27 and p57 protein expression, bypass a requirement for G1 phase cyclin D1, and activate cyclin E, cyclin A, CDK2, and cyclin B1, thus contributing to podocyte proliferation in the cellular lesion of FSGS. REFERENCES 1. Kriz W: Progressive renal failure—Inability of podocytes to replicate and the consequences for development of glomerulosclerosis. Nephrol Dial Transplant 11:1738-1742, 1996 2. Schwartz MM, Lewis EJ: Focal segmental glomerulosclerosis: The cellular lesion. Kidney Int 28:968-974, 1985 3. Schwartz MM, Korbet SM: Primary focal segmental glomerulosclerosis: Pathology, histological variants, and pathogenesis. Am J Kidney Dis 22:874-883, 1993 4. Meyrier AY: Collapsing glomerulopathy: Expanding interest in a shrinking tuft. Am J Kidney Dis 33:801-803, 1999 5. Schwartz MM, Evans J, Bain R, Korbet SM: Focal segmental glomerulosclerosis: Prognostic implications of the cellular lesions. J Am Soc Nephrol 10:1900-1907, 1999 6. Shankland SJ, Wolf G: Cell cycle regulatory proteins in renal disease: Role in hypertrophy, proliferation, and apoptosis. Am J Physiol Renal Physiol 278:F515-F529, 2000 7. Kato J, Matsushime H, Hiebert SW, Ewen ME, Sherr CJ: Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin Ddependent kinase CDK4. Genes Dev 7:331-342, 1993 8. Ohtsubo M, Theodoras AM, Schumacher J, Roberts JM, Pagano M: Human cyclin E, a nuclear protein essential for the G1-to-S phase transition. Mol Cell Biol 15:26122624, 1995

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9. Girard F, Strausfeld U, Fernandez A, Lamb N: Cyclin A is required for the onset of DNA replication in mammalian fibroblasts. Cell 67:1169-1179, 1991 10. Sherr CJ, Roberts JM: Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 9:1149-1163, 1995 11. Shankland SJ, Floege J, Thomas SE, et al: Cyclin kinase inhibitors are increased during experimental membranous nephropathy: Potential role in limiting glomerular epithelial cell proliferation in vivo. Kidney Int 52:404-413, 1997 12. Shankland SJ, Eitner F, Hudkins KL, Goodpaster T, D’Agati V, Alpers CE: Differential expression of cyclindependent kinase inhibitors in human glomerular disease: Role in podocyte proliferation and maturation. Kidney Int 58:674-683, 2000 13. Combs HL, Shankland SJ, Setzer SV, Hudkins KL, Alpers CE: Expression of the cyclin kinase inhibitor, p27kip1, in developing and mature human kidney. Kidney Int 53:892896, 1998 14. Nagata M, Nakayama K, Terada Y, Hoshi S, Watanabe T: Cell cycle regulation and differentiation in the human podocyte lineage. Am J Pathol 153:1511-1520, 1998 15. Nagata M, Horita S, Shu Y, et al: Phenotypic characteristics and cyclin-dependent kinase inhibitors repression in hyperplastic epithelial pathology in idiopathic focal segmental glomerulosclerosis. Lab Invest 80:869-880, 2000 16. Srivastava T, Garola RE, Whiting JM, Alon US: Cell-cycle regulatory proteins in podocyte cell in idiopathic nephrotic syndrome of childhood. Kidney Int 63:1374-1381, 2003 17. Barisoni L, Mokrzycki M, Sablay L, Nagata M, Yamase H, Mundel P: Podocyte cell cycle regulation and proliferation in collapsing glomerulopathies. Kidney Int 58:137-143, 2000 18. Oya M, Schmidt B, Schmitz-Dra¨ ger BJ, Schulz WA: Expression of G1/S transition regulatory molecules in human urothelial cancer. Jpn J Cancer Res 89:719-726, 1998 19. Schmidt BA, Rose A, Steinhoff C, Strohmeyer T, Hartmann M, Ackermann R: Up-regulation of cyclindependent kinase 4/cyclin D2 expression but down-regulation of cyclin-dependent kinase 2/cyclin E in testicular germ cell tumors. Cancer Res 61:4214-4221, 2001 20. Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ: The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75:805-816, 1993 21. Rasmussen UB, Wolf C, Mattei MG, et al: Identification of a new interferon-alpha-inducible gene (p27) on human chromosome 14q32 and its expression in breast carcinoma. Cancer Res 53:4096-4101, 1993 22. Matsuoka S, Edwards MC, Bai C, et al: p57KIP2, a

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structurally distinct member of the p21cip1 Cdk inhibitor family, is a candidate tumor suppressor gene. Genes Dev 9:650-662, 1995 23. Bariety J, Bruneval P, Hill G, Irinopoulou T, Mandet C, Meyrier A: Posttransplantation relapse of FSGS is characterized by glomerular epithelial cell transdifferentiation. J Am Soc Nephrol 12:261-274, 2001 24. Barisoni L, Kriz W, Mundel P, D’Agati V: The dysregulated podocyte phenotype: A novel concept in the pathogenesis of collapsing idiopathic focal segmental glomerulosclerosis and HIV-associated nephropathy. J Am Soc Nephrol 10:51-61, 1999 25. Kim BK, Hong HK, Kim JH, Lee HS: Differential expression of nephrin in acquired human proteinuric diseases. Am J Kidney Dis 40:964-973, 2002 26. Sherr CJ: G1 phase progression: Cycling on cue. Cell 79:551-555, 1994 27. Geng Y, Whoriskey W, Park MY, et al: Rescue of cyclin D1 deficiency by knocking cyclin E. Cell 97:767-777, 1999 28. Petermann AT, Pippin J, Hiromura K, et al: Mitotic cell cycle proteins increase in podocytes despite lack of proliferation. Kidney Int 63:113-122, 2003 29. Nilsson I, Hoffmann I: Cell cycle regulation by the Cdc25 phosphatase family. Prog Cell Cycle Res 4:107-114, 2000 30. Draetta B: cdc2 activation: The interplay of cyclin binding and Thr 161 phosphorylation. Trends Cell Biol 3:287-289, 1993 31. Hagting A, Jackman M, Simpson K, Pines J: Translocation of cyclin B1 to the nucleus at prophase requires a phosphorylation-dependent nuclear import signal. Curr Biol 9:680-689, 1999 32. Zhang H, Xiong Y, Beach D: Proliferating cell nuclear antigen and p21 are components of multiple cell cycle kinase complexes. Mol Biol Cell 4:897-906, 1993 33. Harper JW, Elledge SJ, Keyomarsi K, et al: Inhibition of cyclin-dependent kinases by p21. Mol Biol Cell 6:387400, 1995 34. Nagata M, Tomari S, Kanemoto K, Usui J, Lemley KV: Podocytes, parietal cells, and glomerular pathology: The role of cell cycle proteins. Pediatr Nephrol 18:3-8, 2003 35. Barboule N, Lafon C, Chadebech P, Vidal S, Valette A: Involvement of p21 in the PKC-induced regulation of the G2/M cell cycle transition. FEBS Lett 444:32-37, 1999 36. Kim Y-G, Alpers CE, Brugarolas J, Johnson RJ, Couser WG, Shankland SJ: The cyclin kinase inhibitor p21CIP1/WAF1 limits glomerular epithelial cell proliferation in experimental glomerulonephritis. Kidney Int 55:2349-2361, 1999