Apoptosis DOI 10.1007/s10495-015-1134-0

ORIGINAL PAPER

Podocyte hypertrophy precedes apoptosis under experimental diabetic conditions Sun Ha Lee1 • Sung Jin Moon2 • Jisun Paeng1 • Hye-Young Kang1 • Bo Young Nam1 • Seonghun Kim1 • Chan Ho Kim1 • Mi Jung Lee1 • Hyung Jung Oh1 • Jung Tak Park1 • Seung Hyeok Han1 • Tae-Hyun Yoo1 Shin-Wook Kang1

•

Ó Springer Science+Business Media New York 2015

Abstract Podocyte hypertrophy and apoptosis are two hallmarks of diabetic glomeruli, but the sequence in which these processes occur remains a matter of debate. Here we investigated the effects of inhibiting hypertrophy on apoptosis, and vice versa, in both podocytes and glomeruli, under diabetic conditions. Hypertrophy and apoptosis were inhibited using an epidermal growth factor receptor inhibitor (PKI 166) and a pan-caspase inhibitor (zAsp-DCB), respectively. We observed significant increases in the protein expression of p27, p21, phospho-eukaryotic elongation factor 4E-binding protein 1, and phospho-p70 S6 ribosomal protein kinase, in both cultured podocytes exposed to high-glucose (HG) medium, and streptozotocininduced diabetes mellitus (DM) rat glomeruli. These increases were significantly inhibited by PKI 166, but not by zAsp-DCB. In addition, the amount of protein per cell, the relative cell size, and the glomerular volume were all significantly increased under diabetic conditions, and these changes were also blocked by treatment with PKI 166, but not zAsp-DCB. Increased protein expression of cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase, together with increased Bax/Bcl-2 ratios, were also observed in HG-stimulated podocytes and DM glomeruli. Treatment

Sun Ha Lee and Sung Jin Moon contributed equally to this work. & Shin-Wook Kang [email protected] 1

Department of Internal Medicine, College of Medicine, Brain Korea 21 for Medical Science, Severance Biomedical Science Institute, Yonsei University, 134 Shinchon-Dong Seodaemoon-Gu, Seoul 120-752, Korea

2

Department of Internal Medicine, College of Medicine, International St. Mary’s Hospital, Catholic Kwandong University, Incheon, Korea

with either zAsp-DCB or PKI 166 resulted in a significant attenuation of these effects. Both PKI 166 and zAsp-DCB also inhibited the increase in number of apoptotic cells, as assessed by Hoechst 33342 staining and TUNEL assay. Under diabetic conditions, inhibition of podocyte hypertrophy results in attenuated apoptosis, whereas blocking apoptosis has no effect on podocyte hypertrophy, suggesting that podocyte hypertrophy precedes apoptosis. Keywords Apoptosis  Caspase  Diabetic nephropathy  Epidermal growth factor  Hypertrophy  Podocyte

Introduction Diabetic nephropathy, the leading cause of end-stage renal disease worldwide, is characterized pathologically by glomerular hypertrophy, and clinically by proteinuria [1]. Even though accumulating evidence indicates that the diabetic milieu per se, hemodynamic changes, and local growth factors, such as angiotensin II and transforming growth factor-b (TGF-b), are all involved in the pathogenesis of diabetic nephropathy [2–4], the underlying pathways mediating these processes are not well understood. Kidney size is typically increased in diabetes, even at the time of diagnosis [5]. This is primarily due to glomerular and tubular hypertrophy, but inflammatory cell infiltration, extracellular matrix (ECM) accumulation, and hemodynamic factors also contribute [6–9]. In addition, some low-grade proliferation of glomerular cells is present in the early stage of diabetic nephropathy [10, 11]. Cell culture experiments using mesangial cells under high-glucose conditions, and in vivo studies using various models of diabetes, have indicated a biphasic growth response

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[12]. Self-limited proliferation occurs initially, and is followed by cell cycle arrest and hypertrophy [13]. In contrast to mesangial cells, mature podocytes do not actively synthesize DNA nor proliferate under normal conditions [14]. However, proliferation of podocytes is observed in certain glomerular diseases, such as HIV nephropathy [15], collapsing glomerulopathy [16], and the cellular variant of focal segmental glomerulosclerosis [17]. Furthermore, podocytes are demonstrated to become hypertrophic under diabetic conditions, resulting in increased cell size [18, 19]. Apoptosis, which is defined as programmed cell death, removes damaged or unwanted cells, and has been implicated in the pathogenesis of numerous diseases, such as malignancy, systemic lupus erythematosus, and Alzheimer’s disease [20]. Various renal diseases, including diabetic nephropathy, have been shown to feature apoptotic cell death [21–24]. Cell death by apoptosis is believed to be involved in the process of mesangial cell loss in the late stage of diabetic nephropathy, suggesting that apoptosis may be a homeostatic mechanism regulating mesangial cell numbers [25]. Apoptosis is also considered to be one of the underlying causes of podocyte loss in diabetic nephropathy, contributing to the development of albuminuria [23, 24]. Podocyte hypertrophy and apoptosis are two hallmarks of diabetic glomeruli, but the sequence in which these processes occur remains a matter of debate. It has been suggested that podocyte hypertrophy in diabetic nephropathy is simply a compensatory phenomenon, triggered to cover the denuded glomerular basement membrane that is a result of apoptosis-mediated podocyte loss [26, 27]. Several studies, however, have found that the density of podocytes, rather than their absolute number, is reduced in the early stage of diabetic nephropathy in humans and in experimental diabetic models [28]. A recent study by Jung et al. showed that the expression of apoptosis-related molecules and cyclin-dependent kinase inhibitors (CKIs) differed according to glomerular size in diabetic nephropathy, indicating that hypertrophy alone occurred in less hypertrophic glomeruli, and it was only once they became sufficiently large that apoptosis began to predominate [29]. Moreover, a recent morphometric analysis of diabetic nephropathy revealed that podocyte hypertrophy preceded glomerular hypertrophy and glomerulosclerosis [30, 31]. Based on these findings, some researchers have inferred that podocyte hypertrophy is a prominent early feature of diabetic nephropathy prior to other pathologic findings, including podocyte loss [32]. To resolve this controversy, it necessary to clarify the order in which hypertrophy and apoptosis occur in the process of diabetic nephropathy. The aim of this study was to investigate the effects of inhibiting hypertrophy on apoptosis, and vice versa, under diabetic conditions. For this purpose, we treated high-glucose (HG)-stimulated

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podocytes and glomeruli from diabetic (DM) rats with a selective epidermal growth factor receptor tyrosine kinase (EGFR) inhibitor, which was reported to abrogate glomerular hypertrophy in experimental diabetic rats, or with a pan-caspase inhibitor. We then assessed the podocytes and glomeruli for changes in cell/glomerular size and changes in expression of apoptosis-related molecules and CKIs.

Materials and methods Podocyte culture Conditionally immortalized mouse podocytes were provided by Dr. Peter Mundel (Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA) and were cultured as described previously [33]. Briefly, frozen podocytes were first grown under permissive conditions at 33 °C in RPMI-1640 media containing 10 % fetal bovine serum (FBS), 50 U/mL interferon-c (INF-c), and 100 U/mL of penicillin/streptomycin in collagen-coated flasks, and the INF-c tapered down to 10 U/ mL in successive passages. Cells were then trypsinized and subcultured without INF-c (nonpermissive conditions) and allowed to differentiate at 37 °C with media changed on alternate days. Differentiation of podocytes grown for 14 days at 37 °C was confirmed by the identification of synaptopodin, a podocyte differentiation marker, by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Immunocytochemical staining for proliferating cell nuclear antigen (PCNA) was also performed before and after thermoshifting to verify that the cells were growth arrested. After confirming differentiation of podocytes, the medium was changed to serum-free RPMI medium containing 5.6 mM glucose (normal glucose; NG), 5.6 mM glucose and 24.4 mM mannitol (NG ? M), or 30 mM glucose (high-glucose; HG). Mannitol was used as a control to compare the osmotic effect of HG. Cultures were stimulated with either 10 lM PKI 166 (Novartis, Basel, Switzerland), an EGFR inhibitor, or 10-7 M zAsp-DCB (N-aspartyl-2,6-dichlorobenzoyloxy-methylketone; Alexis Biochemicals, San Diego, CA, USA), a pan-caspase inhibitor. Control cultures received no treatment. At 48 h after the media change, cells were harvested for protein analyses. In another set of study, siRNA for mouse eukaryotic elongation factor 4E binding protein 1 (4EBP1) was used to inhibit podocyte hypertrophy. The concentrations of 4EBP1 siRNA (Dhamacon, Lafayette, CO, USA, catalog no. M-058681-01) were determined based on the preliminary experiments with three different concentrations of 10, 50, and 100 nM siRNA. 4EBP1 siRNA were

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transiently transfected into differentiated podocytes with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for 24 h. Transfected and nontransfected podocytes were serum restricted for 24 h, after which the medium was changed to RPMI medium containing NG, NG ? 100 nM 4EBP1 siRNA, HG, or HG ? 100 nM 4EBP1 siRNA. After 48 h, cells were harvested. Assessment of podocyte hypertrophy Hypertrophy of cultured podocytes was assessed by calculating the amount of protein per cell (cellular protein/cell number) and by flow cytometry. After seeding podocytes on 100-mm dishes, the medium was changed as described above. After 48 h the cells were harvested with 0.05 % trypsin and 0.25 mmol/L ethylenediaminetetraacetic acid, pelleted at 1,5009g for 5 min, and resuspended in PBS. Podocytes were enumerated using a hemocytometer, and then lysed in 0.5 M NaOH for measurement of total protein content using a modified Lowry method. To determine the cell size directly, cells were harvested by trypsinization after 48 h of treatment as described above, fixed with 75 % methanol, washed, and incubated with 100 lg/mL RNAse and 10 lg/mL propidium iodide in PBS for 1 h at 37 °C. Samples were analyzed by forward light scattering on a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and data were processed with Cell Quest Pro software (BD Biosciences). Animals All animal studies were conducted using a protocol approved by the Committee for the Care and Use of Laboratory Animals of Yonsei University College of Medicine. Forty-eight 9 to 10-week old male Sprague– Dawley rats, weighing 250–280 g, were used. Twenty-four rats were injected intraperitoneally with diluent (control), and the other 24 rats were injected once with 65 mg/kg streptozotocin (STZ) [diabetes mellitus (DM)]. Tail vein blood glucose levels above 300 mg/dL on the third day post-injection were considered indicative of successful induction of diabetes. Eight rats from each group were used to evaluate the effect of the EGFR inhibitor PKI 166. After confirming diabetes, eight rats each from the control and DM groups were treated with 100 mg/kg/day of PKI 166 by gavage for three months. The dose of PKI 166 was determined based on the previous study by Wassef et al. [34]. To investigate the effect of the pan-caspase inhibitor zAsp-DCB, another 16 rats were used. After confirmation of diabetes, eight rats from each group were treated for 3 months with zAsp-DCB (2 mg/day) via subcutaneously implanted osmotic minipumps (Durect Corp., Cupertino, CA, USA). A bolus dose of 2-mg zAsp-DCB or vehicle

was injected immediately before mini-pump implantation surgery. This dose was obtained from a previous study [35]. Administration of drugs was initiated on the 3rd day after STZ injection, at the time when diabetes was confirmed. The rats were given free access to water and standard laboratory chow during the study period. Body weights were assessed monthly, and kidney weights were measured at the time of sacrifice. Blood glucose concentrations and 24-h urinary albumin, measured using a glucometer and enzyme-linked immunosorbent assay (Nephrat II, Exocell, INC., Philadelphia, PA, USA), respectively, were measured at the time of sacrifice.

Isolation of glomeruli Glomeruli were isolated by sieving. Purity of the glomerular preparation was greater than 98 %, as determined by light microscopy. Morphometric measurement of glomerular volume Glomerular volumes (VG) of isolated glomeruli were calculated as described previously [34, 36]. Briefly, tissue sections were subjected to periodic acid-Schiff-staining. The surface areas of 50 glomeruli cut at the vascular pole traced along the outline of the capillary loops were determined using a computer-assisted color image analyzer (Image-Pro Ver. 2.0, Media Cybernetics, Silver Spring, MD, USA). VG was calculated using the equation: VG = b/ k 9 (Area)3/2, where b = 1.38 (the shape coefficient for spheres), and k = 1.1 (the size distribution coefficient). Total RNA extraction Total ribonucleic acid (RNA) was extracted from cultured mouse podocytes as previously described [37]. Briefly, addition of 700 lL of RNA STAT-60 reagent (Tel-Test, Inc., Friendswood, TX, USA) was added, the mixture was vortexed and stored for 5 min at room temperature, then, 160 lL of chloroform was added, and the mixture was shaken vigorously for 30 s. After 3 min, the mixture was centrifuged at 12,0009g for 15 min at 4 °C and the upper aqueous phase containing the extracted RNA was transferred to a new tube. RNA was precipitated from the aqueous phase by 400 lL of isopropanol and was pelleted with centrifugation at 12,0009g for 30 min at 4 °C. The RNA precipitate was washed with 70 % ice-cold ethanol, dried using Speed Vac, and dissolved in diethyl pyrocarbonate (DEPC)treated distilled water. Podocyte RNA yield and quality were assessed based on spectrophotometric measures at the wavelengths of 260 and 280 nm.

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Reverse transcription (RT) First-strand complementary deoxyribonucleic acid (cDNA) was made by using a Boehringer Mannheim cDNA synthesis kit (Boehringer Mannheim GmbH, Mannheim, Germany). Two micrograms of total RNA extracted from cultured cells was reverse transcribed using 10 lM random hexanucleotide primer, 1 mM, deoxynucleoside triphosphate (dNTP), 8 mM MgCl2, 30 mM KCl, 50 mM Tris– HCl, pH 8.5, 0.2 mM dithiothreitol (DTT), 25 U RNAse inhibitor, and 40 U avian myeloblastoma virus (AMV) reverse transcriptase. The mixture was incubated at 30 °C for 10 min and 42 °C for 1 h, followed by inactivation of enzyme at 99 °C for 5 min. Cellular RNA from each plate was similarly reverse transcribed. Polymerase chain reaction (PCR) The primers used for synaptopodin and 18 s amplification were as follows: synaptopodin, sense 50 -TATCAACCAGAACCCGTC-30 , antisense 50 -AATCAAGTGTGC0 0 CATTCG-3 ; and 18 s, sense 5 -AGTCCCTGCCCHTTTG TACACA-30 , antisense 50 -GATCCGAGGGCCTCACTAAAC-30 . cDNAs from 25 ng RNA of the cultured cells per reaction tube were used for amplification. PCR was performed with 1 U TaKaRa Ex TaqÒ DNA Polymerase (Takara Bio Inc, Otsu, Japan), 20 lM dNTP, and 20 pM sense and antisense primers in a volume of 20 lL containing 1 9 PCR buffer. The PCR conditions were as follows: 35 cycles of denaturation at 94.5 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for one minute. Initial heating at 95 °C for nine minutes and final extension at 72 °C for seven minutes were performed for all PCRs. After PCR, the products were visualized on 2 % agarose gels. Western blot (WB) analysis Isolated glomeruli and cultured cells harvested from plates were lysed in sodium dodecyl sulfate (SDS) sample buffer (2 % SDS, 10 mM Tris–HCl, pH 6.8, 10 % (vol/vol) glycerol). Samples of 50-lg protein were treated with Laemmli sample buffer, heated at 100 °C for 5 min, and electrophoresed at 50 lg/lane in an 8-12 % acrylamide denaturing SDS–polyacrylamide gel. Proteins were then transferred to a Hybond-ECL membrane using a Hoeffer semidry blotting apparatus (Hoeffer Instruments, San Francisco, CA, USA). Membranes were incubated in blocking buffer (1 9 phosphate-buffered saline (PBS), 0.1 % Tween-20, and 5 % non-fat milk) for 1 h at room temperature, followed by an overnight incubation at 4 °C in the presence of polyclonal antibodies at 1:1,000 dilutions. Antibodies used were as follows: EGFR (catalog no.

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2646), phospho-EGFR (catalog no. 2231) (Cell Signaling, Inc., Beverly, MA, USA), p27 (catalog no. sc-528), p21 (catalog no. sc-6246) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), phospho-4EBP1 (p4EBP1, catalog no. 2855), phospho-p70 S6 ribosomal protein kinase (pp70S6K, catalog no. 9202), cleaved poly (ADP-ribose) polymerase (PARP, catalog no. 9544) (Cell Signaling, Inc.), Bax (catalog no. sc-493), Bcl-2 (catalog no. sc-492) (Santa Cruz Biotechnology, Inc.), cleaved caspase-3 (catalog no. 9661, Cell Signaling, Inc.), and b-actin (catalog no. A5441, Sigma Chemical Co., St Louis, MO, USA). Membranes were then washed once for 15 min and twice for 5 min in 1 9 PBS with 0.1 % Tween-20, before incubation in blocking buffer containing a 1:1,000 dilution of horseradish peroxidase-linked goat anti-rabbit IgG (catalog no. sc-2004) or goat anti-mouse IgG (catalog no. sc-2005) (Santa Cruz Biotechnology, Inc.). The washes were repeated, and membranes were developed with a chemiluminescent reagent (ECL; Amersham Life Science, Inc.). Band densities were determined using the TINA image analysis software (Raytest, Straubenhardt, Germany). Hoechst 33342 staining and FACS aasay with Annexin V/PI staining We assessed apoptosis using Hoechst 33342 (Molecular Probes, Eugene, OR, USA) staining and FACS analysis. In Hoechst 33342-stained cultured podocytes, cells with condensed nuclei were considered apoptotic. The percentage of apoptotic cells in cultured podocytes was determined by examining at least 300 cells per condition, at 9400 magnification. The Annexin-V-FITC Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA, catalog no. 556547) was also used to detect apoptosis by flow cytometry. Cells were exposed to various conditions of treatment and after 48 h, they were harvested and processed according to the manufacturer’s instructions. Cells were considered viable if FITC Annexin V and PI staining were all negative; early apoptotic if FITC Annexin V staining was positive with negative PI staining; and late apoptotic or already dead if both FITC Annexin V and PI staining were positive. TUNEL assay and double immunofluorescence (IF) staining with WT-1 and cleaved caspase-3 We also assessed a terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling (TUNEL) assay (Chemicon International, Temecula, CA, USA). TUNELpositive cells were considered apoptotic in the glomeruli of formalin-fixed renal tissue. The percentage of apoptotic cells in tissue samples was determined by examining at least 300 cells per condition or 30 glomeruli, respectively,

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at 9400 magnification. Double IF staining with WT-1 and cleaved caspase-3 was also performed to identify which cells within the glomeruli were apoptotic. Slices of kidney for IF staining were snap-frozen in optimal cutting temperature (OCT) solution and 4 lm sections of tissues were utilized. Slides were fixed in acetone for 10 min at 4 °C, air dried for 10 min at room temperature, and blocked with 10 % donkey serum for 20 min at room temperature. For cleaved caspase-3 staining, the monoclonal antibody to cleaved caspase-3 was diluted in 1:200 with antibody diluent (DAKO, Glostrup, Denmark) and was applied for 3 h at room temperature. After washing, FITC-conjugated anti-mouse IgG antibody (Research Diagnostics, Inc., Flanders, NJ, USA, catalog no. 43R-1313) was added for 60 min. For WT-1 double staining, a 1:200 dilution of polyclonal anti-WT-1 antibody (Santa Cruz Biotechnology, Inc.) was applied, followed by Cy3 (red)-conjugated antigoat IgG antibody (Research Diagnostics, catalog no. 43RID040CY).

significance, the expression of phospho-EGFR was significantly higher in HG-stimulated podocytes compared to NG cells (p \ 0.01) (Fig. 2), suggesting EGFR was activated under high glucose conditions. p27, p21, p4EBP1, and pp70S6K protein levels To examine the effect of the EGFR inhibitor and the pancaspase inhibitor on HG-induced podocyte hypertrophy, we investigated inhibitor-induced changes in p27, p21, p4EBP1, and pp70S6K protein levels in cultured podocytes. Compared to cells cultured under NG conditions, we observed 1.9-, 1.7-, 3.1-, and 2.5-fold increases in p27, p21, p4EBP1, and pp70S6K protein levels in HG-cultured podocytes, respectively. These changes were significantly diminished following treatment with PKI 166 (Fig. 3a), but not with zAsp-DCB (Fig. 3b). Mannitol had no effect on the levels of these proteins. Podocyte hypertrophy

Statistical analysis All values are expressed as the mean ± SEM. Statistical analysis was performed using the statistical package SPSS for Windows Ver. 15.0 (SPSS, Inc., Chicago, IL, USA). Results were analyzed using the Kruskal–Wallis nonparametric test for multiple comparisons. Significant differences by the Kruskal–Wallis test were confirmed by the Mann–Whitney U test. p values less than 0.05 were considered to be statistically significant.

To examine the effect of the EGFR inhibitor and the pancaspase inhibitor on HG-induced podocyte hypertrophy, we assessed cellular hypertrophy by establishing the ratio of total cellular protein to cell number and the relative cell size using flow cytometry. Both the amount of protein per cell and the relative cell size were significantly greater in podocytes cultured under HG conditions compared to NG cells (p \ 0.05), suggesting that HG treatment induces podocyte hypertrophy. These effects were significantly blocked by PKI 166 treatment (Fig. 4a, b), whereas zAspDCB had no effect (Fig. 4c, d).

Results Cultured podocyte studies

Bax, Bcl-2, cleaved caspase-3, and cleaved poly (ADPribose) polymerase protein levels

Identification of differentiated podocytes Differentiated podocytes were verified by RT-PCR and WB for synaptopodin. Synapotopodin mRNA and protein were demonstrated in cultured podocytes at 37 °C but not in cells cultured at 33 °C (Fig. 1a). In contrast, the expression of PCNA was detectable in nearly all cells cultured at 33 °C, but completely disappeared in cultured podocytes at 37 °C (Fig. 1b). Activation of EGFR pathway in podocytes under HG conditions WB analyses for phospho-EGFR and total EGFR were performed to clarify whether HG per se activated the EGFEGFR pathway in cultured podocytes. Even though the increase in total EGFR expression did not reach statistical

To examine the effect of the EGFR inhibitor and the pan-caspase inhibitor on HG-induced podocyte apoptosis, we assessed the Bax, Bcl-2, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) protein levels in cultured podocytes. Compared to NG-cultured podocytes, podocytes exposed to HG medium showed significantly increased levels of Bax, cleaved caspase-3, and cleaved PARP, but a significantly decreased level of Bcl-2 (p \ 0.01). The increase in the ratio of Bax to Bcl2, and the cleaved caspase-3 and cleaved PARP protein levels in HG-stimulated podocytes were significantly attenuated by treatment with either PKI 166 or zAsp-DCB (p \ 0.05 to p \ 0.01), indicating that both drugs inhibit podocyte apoptosis under HG conditions (Fig. 5). Mannitol had no effect on the expression of apoptosis-related molecules.

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Fig. 1 Synaptopodin and proliferating cell nuclear antigen (PCNA) expression in podocytes cultured at 33 °C (undifferentiated state) or 37 °C (differentiated state) (number of experiments = 4). a Synaptopodin mRNA and protein were demonstrated in differentiated

podocytes but not in undifferentiated cells. b In contrast, the expression of PCNA was detectable in nearly all undifferentiated cells, but completely disappeared in differentiated podocytes

Fig. 2 EGFR expression in podocytes exposed to 5.6 mM glucose (NG) or 30 mM glucose (HG) (representative of four blots). Even though the increase in total EGFR expression did not reach statistical significance, the expression of phosphoEGFR was significantly higher in HG-stimulated podocytes compared to NG cells, suggesting EGFR was activated under high glucose conditions. *p \ 0.01 versus NG group

Hoechst 33342 staining and FACS assay with Annexin V/PI staining To examine the effect of the EGFR inhibitor and the pancaspase inhibitor on HG-induced podocyte apoptosis,

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Hoechst 33342 staining was used to identify apoptotic cells in podocytes cultures. The number of apoptotic cells was significantly increased in HG-stimulated podocytes (p \ 0.01), and this increase was significantly blocked by both PKI 166 and zAsp-DCB treatment (p \ 0.05)

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Fig. 3 A representative Western blot of p27, p21, phospho-eukaryotic elongation factor 4E-binding protein 1 (p4EBP1), and phosphop70 S6 ribosomal protein kinase (pp70S6K) in cultured podocytes exposed to serum-free RPMI medium containing 5.6 mM glucose (NG), NG ? 24.4 mM mannitol (NG ? M), or 30 mM glucose (HG) with or without 10 lM PKI 166 or 10-7 M zAsp-DCB (representative of five blots). a Compared to NG cells, there were 1.9-, 1.7-, 3.1-, and 2.5-fold increases in p27, p21, p4EBP1, and pp70S6K protein

expression in cultured HG-stimulated podocytes, and these changes were significantly diminished by PKI 166 treatment. b In contrast, the increases in p27, p21, p4EBP1, and pp70S6K protein levels in cultured podocytes under HG conditions were not altered by zAspDCB. *p \ 0.05 versus NG and NG ? M groups;  p \ 0.05 versus HG group; #p \ 0.01 versus NG and NG ? M groups; àp \ 0.01 versus HG group

(Fig. 6a). FACS assay with Annexin V/PI staining also revealed that that the increase in apoptotic podocytes under HG conditions was significantly abrogated by PKI 166 as well as zAsp-DCB treatment (Fig. 6b).

Animal studies

The impact of 4EBP1 siRNA on podocytes hypertrophy and apoptosis To examine the consequence of inhibiting another hypertrophic factor besides EGF, we used siRNA of 4EBP1 to inhibit 4EBP1, which was known to play a critical role in podocyte hypertrophy. 4EBP1 siRNA treatment significantly inhibited podocyte hypertrophy, assessed by the amount of protein per cell (p \ 0.05) (Fig. 7a). The increases in phospho- and total 4EBP1, the ratio of Bax to Bcl-2, cleaved caspase-3, and cleaved PARP protein expression in HG-stimulated podocytes were also significantly ameliorated by 4EBP1 siRNA (p \ 0.001 to p \ 0.01) (Fig. 7b). Moreover, FACS assay showed that the increase in apoptotic podocytes exposed to HG medium was significantly attenuated by 4EBP1 siRNA (Fig. 7c). These findings indicated that 4EBP1 siRNA abrogated not only podocyte hypertrophy but also apoptosis under HG conditions.

Animal data After the 3-month experimental period, body weight was significantly greater in control rats compared to the DM group (620 ± 9 vs. 324 ± 9 g, p \ 0.001). The ratios of kidney weight to body weight in the DM group were significantly higher than those in control rats (1.19 ± 0.12 vs. 0.52 ± 0.06 %, p \ 0.01), and administration of PKI 166, but not zAsp-DCB, significantly diminished this effect (0.98 ± 0.09 %, p \ 0.05; 1.13 ± 0.08 %, p = 0.78). Furthermore, the increase in albuminuria in the DM group (2.51 ± 0.27 mg/day) was significantly reduced by treatment with either PKI 166 or zAsp-DCB (0.89 ± 0.12 and 1.07 ± 0.11 mg/day, respectively, p \ 0.05). Conversely, neither PKI 166 nor zAsp-DCB affected body weight and blood glucose levels in control and DM rats (Table 1). Glomerular p27, p21, p4EBP1, and pp70S6K protein levels To examine the effect of the EGFR inhibitor and the pancaspase inhibitor on glomerular hypertrophy in DM rats, we assessed p27, p21, p4EBP1, and pp70S6K protein

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Apoptosis b Fig. 4 Assessment of cellular hypertrophy in cultured podocytes

exposed to serum-free RPMI medium containing 5.6 mM glucose (NG) or 30 mM glucose (HG) with or without 10 lM PKI 166 or 10-7 M zAsp-DCB (number of experiments = 4). a,b Compared to NG cells, both the amount of protein per cell and the relative cell size were significantly greater in podocytes cultured under HG conditions, and these changes were significantly inhibited by PKI 166 treatment. c,d In contrast, zAsp-DCB had no effect on cellular hypertrophy in HG-stimulated podocytes. *p \ 0.05 versus NG group;  p \ 0.05 versus HG group

levels in isolated glomeruli. Figure 8 shows a representative Western blot of the lysates of sieved glomeruli from each group. Compared to control rats, we observed 1.6-, 1.4-, 2.5-, and 2.8-fold increases in glomerular p27, p21, p4EBP1, and pp70S6K protein levels, respectively, in the DM group (p \ 0.05 to p \ 0.01). These increases were significantly attenuated in by PKI 166 treatment (p \ 0.05 to p \ 0.01), but not zAsp-DCB treatment. Glomerular hypertrophy To examine the effect of the EGFR inhibitor and the pancaspase inhibitor on glomerular hypertrophy in DM rats, we used morphometric measurements to determine glomerular volume. The mean glomerular volume at three months was significantly larger in the DM group compared to the control group (1.24 ± 0.07 9 106 vs. 0.92 ± 0.04 9 106 lm3, p \ 0.05). PKI 166 treatment significantly diminished the

Fig. 5 A representative Western blot of Bax, Bcl-2, cleaved caspase3 (c-caspase-3), and cleaved poly (ADP-ribose) polymerase (c-PARP) in cultured podocytes exposed to serum-free RPMI medium containing 5.6 mM glucose (NG), NG ? 24.4 mM mannitol (NG ? M), or 30 mM glucose (HG) with or without 10 lM PKI 166 or 10-7 M zAsp-DCB (representative of five blots). a Compared to NG cells, HG-treated podocytes showed significantly increased levels of Bax,

increase in glomerular volume in DM rats (0.98 ± 0.05 9 106 lm3, p \ 0.05), whereas administration of zAsp-DCB had no effect (1.25 ± 0.05 9 106 lm3). Glomerular Bax, Bcl-2, cleaved caspase-3, and cleaved PARP protein levels To examine the effect of the EGFR inhibitor and the pancaspase inhibitor on podocyte apoptosis under diabetic conditions, Bax, Bcl-2, cleaved caspase-3, and cleaved PARP protein levels were determined in isolated glomeruli. Compared to the control group, DM rats showed significantly increased glomerular Bax, cleaved caspase-3, and cleaved PARP levels, and a significantly decreased Bcl-2 level (p \ 0.01). The increases in the ratio of Bax to Bcl-2, and the levels of glomerular cleaved caspase-3 and cleaved PARP protein in the DM group were significantly attenuated by treatment with either PKI or zAsp-DCB (p \ 0.05 to p \ 0.01), indicating that both drugs inhibit apoptosis in DM glomeruli (Fig. 9). TUNEL assay and double IF staining with WT-1 and cleaved caspase-3 To examine the effect of the EGFR and pan-caspase inhibitors on podocyte apoptosis under diabetic conditions,

cleaved caspase-3, and cleaved PARP, but a significantly decreased level of Bcl-2, and these changes were significantly attenuated by PKI 166 treatment. b zAsp-DCB also significantly ameliorated HGinduced changes in apoptosis-related molecules. * p \ 0.01 versus NG and NG ? M groups;  p \ 0.05 versus HG group; #p \ 0.01 versus HG group

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Fig. 6 Apoptosis in cultured podocytes exposed to serum-free RPMI medium containing 5.6 mM glucose (NG), NG ? 24.4 mM mannitol (NG ? M), or 30 mM glucose (HG) with or without 10 lM PKI 166 or 10-7 M zAsp-DCB (number of experiments = 4). a The number of apoptotic cells assessed by Hoechst 33342 staining was significantly increased in HG-stimulated podocytes, and this increase was

significantly attenuated by both PKI 166 and zAsp-DCB treatment. b FACS assay with Annexin V/PI staining also revealed that that the increase in apoptotic podocytes under HG conditions was significantly abrogated by PKI 166 as well as zAsp-DCB treatment. *p \ 0.01 versus NG and NG ? M groups,  p \ 0.05 versus HG group; # p \ 0.01 versus HG group

we assessed apoptosis in glomeruli using a TUNEL assay. The number of apoptotic cells in glomeruli was significantly increased in the DM group compared to the control group (p \ 0.001), and this increase was significantly inhibited by either PKI 166 or zAsp-DCB treatment (p \ 0.01; Fig. 10a). Meanwhile, double IF staining with WT-1 and cleaved caspase-3 revealed that apoptotic cells within DM glomeruli were mainly podocytes (Fig. 10b).

in vitro and in vivo, suggesting that podocyte hypertrophy precedes apoptosis under diabetic conditions. Glomerular hypertrophy is a characteristic feature of diabetic nephropathy, and is attributed mainly to glomerular cell hypertrophy and extracellular matrix (ECM) accumulation [38]. Unlike glomerular mesangial cells, which are known to proliferate in the early stage of diabetic nephropathy [39], mature podocytes do not actively undergo proliferation under diabetic conditions [40, 41]. Instead, the size of individual podocytes increases in diabetic nephropathy. Some investigators have suggested that podocyte hypertrophy is merely a compensatory phenomenon, triggered to cover the denuded glomerular basement membrane that is a result of diabetes-induced podocyte loss [26]. However, accumulating evidence indicates that renal enlargement is a very early feature of experimental diabetic nephropathy. SeyerHansen found that kidney weight was increased by 15 % at 3 days after diabetes induction and by 90 % at

Discussion Podocyte hypertrophy and apoptosis play an important role in the pathogenesis of diabetic nephropathy; however, little is known about the order in which these events occur. In this study, we demonstrate that inhibition of hypertrophy results in diminished podocyte apoptosis, whereas blocking apoptosis has no effect on podocyte hypertrophy, both

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Fig. 7 Cell hypertrophy and apoptosis in cultured podocytes exposed to exposed to serum-free RPMI medium containing 5.6 mM glucose (NG) or 30 mM glucose (HG) with or without 100 nM 4EBP1 siRNA (number of experiments = 5). a The increase in the amount of protein per cell in HG-stimulated podocytes was significantly ameliorated by 4EBP1 siRNA treatment. *p \ 0.05 versus NG group;  p \ 0.05 versus HG group. b The increases in phospho-4EBP1 (p-4EBP1) and

total 4EBP1, the ratio of Bax to Bcl-2, cleaved caspase-3 (c-caspase3) and cleaved PARP (c-PARP) protein levels in podocytes cultured under HG conditions were significantly attenuated by 4EBP1 siRNA treatment. c FACS assay also showed that the increase of apoptotic podocytes exposed to HG medium was significantly abrogated by 4EBP1 siRNA. *p \ 0.01 versus NG group;  p \ 0.001 versus HG group; #p \ 0.01 versus HG group

Table 1 Physiological and renal parameters of control (C) and diabetic rats (DM) treated with PKI 166 and zAsp-DCB C (n = 8)

C ? PKI 166 (n = 8)

C ? zAsp-DCB (n = 8)

DM (n = 8)

DM ? PKI 166 (n = 8)

DM ? zAsp-DCB (n = 8)

Body weight (Bwt, g)

620 ± 9

612 ± 10

622 ± 10

324 ± 9*

335 ± 7*

329 ± 9*

Kidney Wt (g)

3.22 ± 0.10

3.06 ± 0.11

3.11 ± 0.14

3.86 ± 0.11#

3.28 ± 0.14 

3.72 ± 0.13

#

Kidney Wt/Bwt (%)

0.52 ± 0.06

0.50 ± 0.08

0.50 ± 0.08

1.19 ± 0.12

0.98 ± 0.09 

1.13 ± 0.08

Glucose (mg/dL)

108 ± 4

113 ± 6

115 ± 6

486 ± 13*

495 ± 11*

478 ± 11*

24 h UAE (mg/day)

0.40 ± 0.03

0.36 ± 0.05

0.45 ± 0.04

*

2.51 ± 0.27

0.89 ± 0.12

 

1.07 ± 0.11 

* p \ 0.001 versus C, C ? PKI 166, and C ? zAsp-DCB groups #

p \ 0.01 versus C, C ? PKI 166, and C ? zAsp-DCB groups

 

p \ 0.05 versus DM group

42 days in female diabetic Winstar rats induced by a single injection of STZ (40 mg/kg) [42]. Another study reported a 62 % increase in kidney weight at six days after streptozotocin (STZ)-induced diabetes [43]. They also showed that hyperplasia and hypertrophy contributed equally to renal enlargement. Recently, Jung et al. found

that the Bax and cleaved caspase-3 protein levels were significantly increased and the number of podocytes was significantly decreased in more hypertrophied diabetic glomeruli, but not in less hypertrophied glomeruli, suggesting that podocyte loss follows glomerular hypertrophy [29].

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Apoptosis

Fig. 8 A representative Western blot of glomerular p27, p21, phospho-eukaryotic elongation factor 4E binding protein 1 (p4EBP1), and phospho-p70 S6 ribosomal protein kinase (pp70S6K) in control (C) and diabetic rats (DM) treated with or without 100 mg/ kg/day of PKI 166 or 2 mg/day of zAsp-DCB for 3 months (representative of five blots). a Compared to C rats, there were 1.6, 1.4-, 2.5-, and 2.8-fold increases in glomerular p27, p21, p4EBP1,

and pp70S6K protein levels, respectively, in the DM group, and these increases were significantly attenuated by PKI 166 treatment. b In contrast, the increases in the p27, p21, p4EBP1, and pp70S6K protein levels in DM glomeruli were not altered by zAsp-DCB. *p \ 0.05 versus C group;  p \ 0.05 versus DM group; #p \ 0.01 versus C group; àp \ 0.01 versus DM group

Fig. 9 A representative Western blot of glomerular Bax, Bcl-2, cleaved caspase-3 (c-caspase-3), and cleaved poly (ADP-ribose) polymerase (c-PARP) in control (C) and diabetic rats (DM) treated with or without 100 mg/kg/day of PKI 166 or 2 mg/day of zAsp-DCB for 3 months (representative of five blots). a The significant increases

in the ratio of Bax to Bcl-2, and c-caspase-3 and c-PARP protein expression in DM glomeruli were significantly abrogated by PKI 166 treatment. b zAsp-DCB also significantly ameliorated the changes in apoptosis-related molecules in DM glomeruli. *p \ 0.01 versus C group;  p \ 0.01 versus DM group; #p \ 0.05 versus DM group

Cell hypertrophy under diabetic conditions requires the combined effect of mitogen-induced entry into the cell cycle and subsequent arrest at the G1/S interphase [41].

This cell cycle arrest has been demonstrated to be principally mediated by cyclin-dependent kinases (CDKs) and their inhibitors (CKIs), such as p27 and p21 [18, 44, 45].

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Apoptosis

Fig. 10 Apoptosis assessed by TUNEL assay and double immunofluorescence (IF) staining with WT-1 and cleaved caspase-3 in control (C) and diabetic rats (DM) treated with or without 100 mg/ kg/day of PKI 166 or 2 mg/day of zAsp-DCB for 3 months. a The number of apoptotic cells in glomeruli was significantly increased in

DM rats compared to control C rats, and this increase was significantly attenuated by treatment with either PKI 166 or zAspDCB. b Double IF staining revealed that apoptotic cells within DM glomeruli were mainly podocytes. *p \ 0.001 versus C groups;   p \ 0.01 versus DM group

Numerous previous studies have found that down-regulation of CDKs and enhanced expression of CKIs are closely associated with cell hypertrophy under diabetic conditions. The mammalian target of rapamycin pathway along with its downstream signal molecules such as 4EBP1 and p70S6 K, final effectors of mRNA translation, has also been shown to be activated in diabetic nephropathy and to be associated with diabetes-induced cell hypertrophy [46, 47]. Therefore, we regarded the increases in P27, P21, phospho-4EBP1, and phospho-p70S6 K as indicators of cellular hypertrophy in addition to the amount of protein per cell and relative cell size assessed by flow cytometry. In the present study, PKI 166, an EGFR blocker, was used to examine the consequence of inhibiting podocyte hypertrophy on apoptosis. TGF-b and angiotensin II are considered key molecules in the pathogenesis of diabetic nephropathy [3, 4, 48]. However, these two molecules are involved in both podocyte hypertrophy and apoptosis under diabetic conditions, making it impossible to investigate the impact of blocking one event on the other. Therefore, we used PKI 166 due to its anti-hypertrophic, but not antiapoptotic, effect on podocytes. EGFR was found to be

present in podocytes and to be activated by angiotensin II, an end product of the renin-angiotensin system, which was activated within podocytes under diabetic conditions [49]. Moreover, previous studies suggested that the EGF-EGFR system played a role in diabetes-related renal and glomerular hypertrophy in experimental rats [50]. There was a threefold increase in urinary EGF excretion and a three- to six-fold increase in renal EGF mRNA levels in STZ-induced diabetic rats, compared to control rats. In contrast, serum EGF levels were comparable between the two groups. Furthermore, administration of PKI 166 significantly inhibited the increase in kidney weight observed in experimental diabetic rats by 30 % at week 3 of diabetes, in addition to reducing albuminuria [34]. Glomerular hypertrophy was also significantly reduced in STZ-induced diabetic TGR(mRen-2)27 rats treated with PKI 166 for 16 weeks. This treatment was also associated with podocyte preservation and reduction of albuminuria [51]. The results of this study show that PKI 166 treatment significantly inhibits the increase in albuminuria and glomerular volume and the decrease in podocyte number as well as podocyte hypertrophy in vitro, which is consistent

123

Apoptosis

with most previous reports. Since PKI 166 was reported exert a pro-apoptotic effect on some cells, we performed additional in vitro experiments using siRNA of 4EBP1, which was known to play a critical role in podocyte hypertrophy [52] and had never been demonstrated to be involved in apoptosis of non-malignant cells. The results of additional experiments revealed that treatment of HG-stimulated podocytes with 4EBP1 siRNA not only abrogated podocyte hypertrophy but also ameliorated podocyte apoptosis. Considering our findings that podocyte apoptosis under diabetic conditions is significantly attenuated by PKI 166 and 4EBP1 siRNA, it is possible to conclude that preservation of podocyte number can be attributed to restricted diabetes-induced podocyte hypertrophy. Wassef et al. demonstrated that apoptosis in the kidney, assessed by TUNEL staining and active caspase-3 immunohistochemical staining, was significantly increased in diabetic rats treated with PKI 166, compared to vehicletreated diabetic rats [34]. These data are not consistent with the results of the current study. It is important to note, however, that in the aforementioned study, diabetes was induced by tail vein injection of 50 mg/kg STZ and PKI was administered for only 3 weeks and that apoptosis was almost restricted to the tubulointerstitial area. Moreover, the expression of PCNA and 5-bromo-20 -deoxyuridine were significantly increased in renal tubules in diabetic rats. Based on these findings and the results of a number of previous reports of an apoptotic effect of PKI 166 on various cancer cell lines, including epidermoid carcinoma [53], renal cell cancer [54], and pancreatic cancer [55], we conclude that PKI 166 has an anti-proliferative effect on actively proliferating cells by inducing apoptosis. In contrast, in the presence of TGF-b, EGF is known to exert a hypertrophic rather than a hyperplastic effect [56]. Since TGF-b expression is significantly increased in diabetic glomeruli and TGF-b is regarded as a principal mediator of the pathogenesis of diabetic glomerulopathy, we hypothesized that the EGF-EGFR axis might be involved in podocyte hypertrophy under diabetic conditions. This was verified in the present study by demonstrating that EGFR was activated in podocytes under HG conditions and diabetes-induced podocyte hypertrophy both in vitro and in vivo was significantly inhibited by PKI 166. Furthermore, similar to the report by Wassef et al., an increase in apoptosis in glomeruli was not found in diabetic rats treated with PKI 166 [34]. This may be due to the presence of minimal or no actively proliferating cells in diabetic glomeruli. The number of podocytes is decreased in the glomeruli of human diabetic patients and experimental diabetic animals [57, 58]. Podocyte number usually reflects the balance between podocyte loss and proliferation, and apoptosis has been considered an important cause underlying podocyte loss in diabetic nephropathy. In addition, administration of an angiotensin II receptor blocker, an aldosterone antagonist,

123

or anti-oxidants reduced albuminuria and podocyte apoptosis in experimental diabetic animals [59–61], suggesting that podocyte apoptosis is closely associated with the development of albuminuria in diabetes. In this study, the increase in albuminuria in diabetic rats was significantly diminished by both PKI 166 and a pan-caspase inhibitor. The pan-caspase inhibitor also significantly inhibited podocyte apoptosis, but not podocyte hypertrophy. Taken together, these findings suggest that in diabetic nephropathy, albuminuria is associated more closely with podocyte apoptosis than hypertrophy. In conclusion, under diabetic conditions, inhibition of podocyte hypertrophy reduced apoptosis, whereas blocking apoptosis had no effect on podocyte hypertrophy, suggesting that podocyte hypertrophy precedes apoptosis. Based on these findings, targeting podocyte hypertrophy may be effective in the treatment of diabetic nephropathy. Acknowledgments This work was supported by the Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, the National Research Foundation of Korea (NRF) Grant funded by the Korea government (MSIP) (No. NRF-2011-0030086), and a Grant of the Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (HI12C0646). Confilicts of interest interests.

All the authors declared no competing

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