Original Report: Laboratory Investigation American
Journal of
Nephrology
Am J Nephrol 2013;38:430–444 DOI: 10.1159/000355970
Received: August 6, 2013 Accepted: September 23, 2013 Published online: November 12, 2013
IQGAP1 Mediates Angiotensin II-Induced Apoptosis of Podocytes via the ERK1/2 MAPK Signaling Pathway Yipeng Liu a Wei Liang a Qian Yang a Zhilong Ren a Xinghua Chen a Dongqing Zha a Pravin C. Singhal b Guohua Ding a a
Division of Nephrology, Renmin Hospital of Wuhan University, Wuhan, China; b Medicine, North Shore-Long Island Jewish Health System, Manhasset, N.Y., USA
Abstract Background/Aims: The mechanism underlying angiotensin II (AngII)-promoted podocyte apoptosis has not been established. IQ domain GTPase-activating protein 1 (IQGAP1) is a scaffolding protein of the mitogen-activated protein kinases (MAPK) signaling pathway, and plays a significant role in apoptosis. The present study evaluates the role of IQGAP1 in AngII-induced podocyte apoptosis. Methods: We randomly assigned 36 male Wistar rats to a normal saline-infused group, an AngII-infused group, or a normal control group, and measured podocyte apoptosis by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and transmission electron microscopic analysis. In addition, we exposed differentiated mouse podocytes to AngII and then assessed apoptosis by flow cytometry and Hoechst-33258 staining. Expression of IQGAP1 was measured by Western blotting, real-time PCR and immunofluorescence assay in vivo and in vitro. IQGAP1 siRNA and MAPK pathway inhibitors were further introduced to investigate the role of IQGAP1 and MAPK signaling in the process. Coimmunoprecipitation was used to evaluate the inter-
© 2013 S. Karger AG, Basel 0250–8095/13/0385–0430$38.00/0 E-Mail [email protected] www.karger.com/ajn
action between ERK1/2 and IQGAP1. Results: AngII promoted podocyte apoptosis in vivo and in vitro. IQGAP1 had a linear distribution along the capillary loops of glomeruli in vivo, and was in the cellular membrane and cytoplasm of cultured podocytes. AngII stimulated IQGAP1 expression and increased phosphorylation of P38, JNK, and ERK1/2. Knockdown of IQGAP1 with siRNA prevented AngII-induced apoptosis of podocytes and reduced AngII-induced phosphorylation of ERK1/2, but not that of P38, JNK. This was accompanied by a reduced interaction between ERK1/2 and IQGAP1. Conclusion: IQGAP1 contributes to AngII-induced apoptosis of podocytes by interacting with the ERK1/2 signaling protein. © 2013 S. Karger AG, Basel
Introduction
Angiotensin II (AngII) is a well-known vasoconstrictor, and elevated levels increase the risk for initiation and progression of chronic kidney disease (CKD) [1, 2]. AngII also has diverse nonhemodynamic effects on renal tissue, and elevated levels can lead to oxidative stress, expansion
Yipeng Liu and Wei Liang contributed equally to this study.
Guohua Ding, MD, PhD Division of Nephrology Renmin Hospital of Wuhan University 238 Jiefang Rd., Wuhan, Hubei 430060 (China) E-Mail ghxding @ gmail.com
Downloaded by: University of British Columbia 142.103.160.110 - 6/18/2016 4:15:42 AM
Key Words Angiotensin II · IQGAP1 protein · ERK1/2 signaling protein · Podocyte · Apoptosis
Cell Culture Conditionally immortalized mouse podocytes were a kind gift from Dr. Peter Mundel (Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Mass., USA) and were cultured under standard conditions [22]. During the proliferation phase, 10 U/ml of recombinant mouse interferon-γ (Sigma-Aldrich) was added to the medium, and the cells were subcultured at 33 ° C. These cells were cultured in a 37 ° C incubator with interferon-γ-free medium for 10–14 days to induce differentiation, and all experiments employed these differentiated podocytes. The differentiated cells were stimulated by AngII (10–8 M) for variable times (0–24 h), or by variable concentrations of AngII (10–12 to 10–6 M) for 6 h. The P38, JNK, and ERK1/2 signaling pathways were inhibited by addition of SB202190 (10 μM, 1 h, Cell Signaling Technology, USA), SP600125 (25 μM, 30 min, Cell Signaling Technology), and U0126 (10 μM, 30 min, Cell Signaling Technology) before addition of AngII, respectively.
Animals All experimental procedures were approved by the ethics committee for animal experiments of Wuhan University. Thirty-six male SPF Wistar rats (140–160 g) were supplied by the Hubei Research Center of Experimental Animals and raised in a temperature- and humidity-controlled laminar flow room under a 12-hour light/12-hour dark cycle, with free access to tap water and standard rat chow. The AngII-infused model employed an osmotic minipump (Alzet, USA) and was established as described previously [9]. Rats were randomly assigned to an AngII-infused (400 ng · kg–1 · min–1, Sigma-Aldrich, USA) group, a normal saline-infused group, or an untreated control group for 14 or 28 days. The 24hour urinary protein was measured on days 7, 14, 21, and 28. Animals were sacrificed on days 14 and 28, and left kidneys were harvested for isolation of glomeruli. The cortex of each right kidney was separated and fixed in 4% phosphate-buffered paraformaldehyde, glutaraldehyde, or a tissue-freezing medium for renal pathological and immunofluorescence analysis.
IQGAP1 and Podocyte Apoptosis
Isolation of Glomeruli Glomeruli were isolated by differential sieving method [23, 24]. The renal cortex was separated and successively passed through three stainless steel sieves with pore sizes of 177 (80 mesh), 125 (120 mesh), and 74 μm (200 mesh). D-Hank’s buffer containing vanadate (phosphatase inhibitor) was used in the continuous rinsing of debris. Glomeruli collected on the last sieve were harvested and used for real-time PCR and Western blotting analysis. Immunofluorescence Assay Frozen kidney sections (5 μm thick) were blocked with 5% BSA for 30 min at 37 ° C; they were incubated with a mixture of guinea pig anti-nephrin polyclonal antibody (1:50, PROGEN Biotechnik, Germany) and rabbit anti-IQGAP1 polyclonal antibody (1: 50, Santa Cruz, USA) overnight at 4 ° C, and then were subjected to a mixture of TRITC-conjugated goat anti-guinea pig IgG (1: 100, Santa Cruz) and FITC-conjugated goat anti-rabbit IgG (1: 100, Thermo scientific, USA) for 90 min at 37 ° C in darkness. The cellclimbing films were fixed with 4% paraformaldehyde for 30 min at 4 ° C, and only stained with IQGAP1 antibody according to the above protocol. All microscopic images were recorded by a 12.8megapixel camera (DP72, Olympus, Japan).
Materials and Methods
Real-Time PCR Total RNA was extracted from podocytes and isolated glomeruli using Trizol reagent (Invitrogen, USA). The concentration of collected RNA was determined by spectrophotography. After the cDNA was synthesized, the study was performed with the real-time fluorescent quantitative PCR machine (Illumina, USA). The specificity of real-time PCR was detected by the melting curve analysis. β-Actin was used as internal standard. The primers used in this study were as follows: IQGAP1, forward 5′-GGGGAAACGTACCAGAGTGA-3′ and reverse 5′-TCTCGAGAAAGCTGCACAGA-3′; β-actin, forward 5′-CACGATGGAGGGGCCGGACTCATC-3′ and reverse 5′-TAAAGACCTCTATGCCAACACAGT-3′. Western Blotting Total protein of glomeruli and podocytes were extracted with lysis buffer (0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 1.0% Triton X-100, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, 20 mM Tris, pH 7.5) that contained a protease inhibitor cocktail (P8340, Sigma-Aldrich,). The sample was centrifuged at 12,000
Am J Nephrol 2013;38:430–444 DOI: 10.1159/000355970
431
Downloaded by: University of British Columbia 142.103.160.110 - 6/18/2016 4:15:42 AM
of the extracellular matrix, production of inflammatory cytokines, disruption of the cytoskeleton, cell cycle abnormalities, and apoptosis [3]. Podocytes are terminally differentiated epithelial cells that are important components of the glomerular filtration barrier and their injury or depletion plays a pivotal role in the onset of proteinuria and progression of glomerular diseases [4–8]. Our previous studies demonstrated that AngII-induced podocyte apoptosis contributed to podocyte injury [9–11]. Numerous molecules are associated with podocyte apoptosis, but the precise mechanism of AngII-induced apoptosis of podocytes is still unknown. IQGAP1 is a scaffolding protein that occurs in the slit diaphragm region of podocytes [12, 13], and contains several functional domains that recruit multiple signaling molecules. This 190-kDa protein provides a platform for diverse cellular functions, including cellular adhesion, migration, polarization [14, 15], and its role in regulating cellular apoptosis gradually attracted attention [16–18]. The mitogen-activated protein kinases (MAPK) signaling family, which consists of P38, JNK, and ERK, is thought to be essential for induction of apoptosis and plays an important role in kidney diseases [19]. Recent reports demonstrated that IQGAP1 functioned as an essential protein scaffold during MAPK signaling [20] and modulated the activities of some signaling proteins in the MAPK signaling pathway [16, 21]. Nevertheless, the role of IQGAP1 in AngII-induced podocyte apoptosis is unclear. Here, we examined the role of IQGAP1 in the podocytes of rats and in podocyte cell cultures in order to determine the role of IQGAP1 in AngII-induced apoptosis of podocytes.
rpm for 20 min at 4 ° C. Equal amounts of boiled protein in loading buffer were separated on an 8–10% sodium dodecyl sulfate-polyacrylamide gel, and then electrophoretically transferred to a nitrocellulose membrane (GE Healthcare, USA). Membranes were incubated with a primary antibody (IQGAP1 rabbit polyclonal antibody, 1:200, Santa Cruz; p-P38 rabbit monoclonal antibody, 1:500, Cell Signaling Technology; P38 rabbit polyclonal antibody, 1:500, Cell Signaling Technology; (p-) JNK rabbit monoclonal antibody, 1:500, Cell Signaling Technology; (p-) ERK1/2 rabbit monoclonal antibody, 1: 500, Cell Signaling Technology, or β-actin mouse monoclonal antibody, control, 1: 1,000, Santa Cruz) overnight at 4 ° C. A horseradish peroxidase-conjugated anti-rabbit or mouse IgG was used as the secondary antibody (1:10,000, Cell Signaling Technology). Bands were visualized by an enhanced chemiluminescence reagent (Santa Cruz) and developed onto film. The integrated optical density of each band was calculated by Gel-Pro Analyzer version 4.5.
Coimmunoprecipitation Analysis Coimmunoprecipitation experiments were conducted according to the manufacturer’s instructions (P2012, Beyotime, China). Briefly, ERK1/2 rabbit monoclonal antibody (1:50, Cell Signaling Technology) was added to the protein sample and rotated overnight at 4 ° C. Then the mixture was loaded with protein A+G agarose and incubated for 3 h at 4 ° C. The centrifuged sediment was saved and mixed with 5× Lane Marker Sample Buffer to make a 1× final solution. After boiling at 95–100 ° C for 5 min, the sample was analyzed by Western blotting for ERK1/2 and IQGAP1.
Transfection of IQGAP1 siRNA Transfection of podocytes with IQGAP1 siRNA was performed according to the HiPerFect Transfection Reagent Handbook (QIAGEN, Germany). Briefly, 3 × 105 cells were seeded in a 6-well plate and incubated with transfection complexes containing 5 nM IQGAP1 siRNA (or a negative control with scrambled siRNA) and 15 μl of Hiperfect transfection reagent under normal growth conditions for 24 h. Measurement of Apoptosis Apoptosis of podocytes in kidney tissue was determined by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay according to the manufacturer’s instructions (C1098, Beyotime). As described previously [25], dewaxed sections (3 μm thick) were incubated with protease K for 30 min at 37 ° C, followed by 3% H2O2 for 10 min at room temperature. Sections were then incubated with a mixture of terminal deoxynucleotidyl transferase (TdT) and biotin-dUTP in a humidified chamber for 1 h at 37 ° C, and were then subjected to streptavidin-horseradish peroxidase for 30 min at room temperature. The slides were stained with DAB, with hematoxylin as a counterstain. The omission of TdT was used as a negative control. Apoptotic podocytes from each glomerular cross-section were counted using the Weibel-Gomez method [26]. The apoptotic podocyte in kidney tissue was also determined by transmission electron microscopic analysis. Briefly, 2.5% glutaraldehyde-fixed 1-mm3 blocks of renal cortices were postfixed with 1% osmic acid, dehydrated in graded ethanols, embedded in EPON, sectioned, stained with uranylacetate and lead citrate, then observed and recorded with a Hitachi H-600 transmission electron microscope (Hitachi, Japan).
432
Statistical Analysis All data are presented as means ± SDs, and statistical analysis was performed using SPSS v17.0. Statistical comparison of groups was conducted using a one-way ANOVA test, and the LSD test was used for multiple comparisons. A p value
Journal of
Nephrology
Am J Nephrol 2013;38:430–444 DOI: 10.1159/000355970
Received: August 6, 2013 Accepted: September 23, 2013 Published online: November 12, 2013
IQGAP1 Mediates Angiotensin II-Induced Apoptosis of Podocytes via the ERK1/2 MAPK Signaling Pathway Yipeng Liu a Wei Liang a Qian Yang a Zhilong Ren a Xinghua Chen a Dongqing Zha a Pravin C. Singhal b Guohua Ding a a
Division of Nephrology, Renmin Hospital of Wuhan University, Wuhan, China; b Medicine, North Shore-Long Island Jewish Health System, Manhasset, N.Y., USA
Abstract Background/Aims: The mechanism underlying angiotensin II (AngII)-promoted podocyte apoptosis has not been established. IQ domain GTPase-activating protein 1 (IQGAP1) is a scaffolding protein of the mitogen-activated protein kinases (MAPK) signaling pathway, and plays a significant role in apoptosis. The present study evaluates the role of IQGAP1 in AngII-induced podocyte apoptosis. Methods: We randomly assigned 36 male Wistar rats to a normal saline-infused group, an AngII-infused group, or a normal control group, and measured podocyte apoptosis by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and transmission electron microscopic analysis. In addition, we exposed differentiated mouse podocytes to AngII and then assessed apoptosis by flow cytometry and Hoechst-33258 staining. Expression of IQGAP1 was measured by Western blotting, real-time PCR and immunofluorescence assay in vivo and in vitro. IQGAP1 siRNA and MAPK pathway inhibitors were further introduced to investigate the role of IQGAP1 and MAPK signaling in the process. Coimmunoprecipitation was used to evaluate the inter-
© 2013 S. Karger AG, Basel 0250–8095/13/0385–0430$38.00/0 E-Mail [email protected] www.karger.com/ajn
action between ERK1/2 and IQGAP1. Results: AngII promoted podocyte apoptosis in vivo and in vitro. IQGAP1 had a linear distribution along the capillary loops of glomeruli in vivo, and was in the cellular membrane and cytoplasm of cultured podocytes. AngII stimulated IQGAP1 expression and increased phosphorylation of P38, JNK, and ERK1/2. Knockdown of IQGAP1 with siRNA prevented AngII-induced apoptosis of podocytes and reduced AngII-induced phosphorylation of ERK1/2, but not that of P38, JNK. This was accompanied by a reduced interaction between ERK1/2 and IQGAP1. Conclusion: IQGAP1 contributes to AngII-induced apoptosis of podocytes by interacting with the ERK1/2 signaling protein. © 2013 S. Karger AG, Basel
Introduction
Angiotensin II (AngII) is a well-known vasoconstrictor, and elevated levels increase the risk for initiation and progression of chronic kidney disease (CKD) [1, 2]. AngII also has diverse nonhemodynamic effects on renal tissue, and elevated levels can lead to oxidative stress, expansion
Yipeng Liu and Wei Liang contributed equally to this study.
Guohua Ding, MD, PhD Division of Nephrology Renmin Hospital of Wuhan University 238 Jiefang Rd., Wuhan, Hubei 430060 (China) E-Mail ghxding @ gmail.com
Downloaded by: University of British Columbia 142.103.160.110 - 6/18/2016 4:15:42 AM
Key Words Angiotensin II · IQGAP1 protein · ERK1/2 signaling protein · Podocyte · Apoptosis
Cell Culture Conditionally immortalized mouse podocytes were a kind gift from Dr. Peter Mundel (Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Mass., USA) and were cultured under standard conditions [22]. During the proliferation phase, 10 U/ml of recombinant mouse interferon-γ (Sigma-Aldrich) was added to the medium, and the cells were subcultured at 33 ° C. These cells were cultured in a 37 ° C incubator with interferon-γ-free medium for 10–14 days to induce differentiation, and all experiments employed these differentiated podocytes. The differentiated cells were stimulated by AngII (10–8 M) for variable times (0–24 h), or by variable concentrations of AngII (10–12 to 10–6 M) for 6 h. The P38, JNK, and ERK1/2 signaling pathways were inhibited by addition of SB202190 (10 μM, 1 h, Cell Signaling Technology, USA), SP600125 (25 μM, 30 min, Cell Signaling Technology), and U0126 (10 μM, 30 min, Cell Signaling Technology) before addition of AngII, respectively.
Animals All experimental procedures were approved by the ethics committee for animal experiments of Wuhan University. Thirty-six male SPF Wistar rats (140–160 g) were supplied by the Hubei Research Center of Experimental Animals and raised in a temperature- and humidity-controlled laminar flow room under a 12-hour light/12-hour dark cycle, with free access to tap water and standard rat chow. The AngII-infused model employed an osmotic minipump (Alzet, USA) and was established as described previously [9]. Rats were randomly assigned to an AngII-infused (400 ng · kg–1 · min–1, Sigma-Aldrich, USA) group, a normal saline-infused group, or an untreated control group for 14 or 28 days. The 24hour urinary protein was measured on days 7, 14, 21, and 28. Animals were sacrificed on days 14 and 28, and left kidneys were harvested for isolation of glomeruli. The cortex of each right kidney was separated and fixed in 4% phosphate-buffered paraformaldehyde, glutaraldehyde, or a tissue-freezing medium for renal pathological and immunofluorescence analysis.
IQGAP1 and Podocyte Apoptosis
Isolation of Glomeruli Glomeruli were isolated by differential sieving method [23, 24]. The renal cortex was separated and successively passed through three stainless steel sieves with pore sizes of 177 (80 mesh), 125 (120 mesh), and 74 μm (200 mesh). D-Hank’s buffer containing vanadate (phosphatase inhibitor) was used in the continuous rinsing of debris. Glomeruli collected on the last sieve were harvested and used for real-time PCR and Western blotting analysis. Immunofluorescence Assay Frozen kidney sections (5 μm thick) were blocked with 5% BSA for 30 min at 37 ° C; they were incubated with a mixture of guinea pig anti-nephrin polyclonal antibody (1:50, PROGEN Biotechnik, Germany) and rabbit anti-IQGAP1 polyclonal antibody (1: 50, Santa Cruz, USA) overnight at 4 ° C, and then were subjected to a mixture of TRITC-conjugated goat anti-guinea pig IgG (1: 100, Santa Cruz) and FITC-conjugated goat anti-rabbit IgG (1: 100, Thermo scientific, USA) for 90 min at 37 ° C in darkness. The cellclimbing films were fixed with 4% paraformaldehyde for 30 min at 4 ° C, and only stained with IQGAP1 antibody according to the above protocol. All microscopic images were recorded by a 12.8megapixel camera (DP72, Olympus, Japan).
Materials and Methods
Real-Time PCR Total RNA was extracted from podocytes and isolated glomeruli using Trizol reagent (Invitrogen, USA). The concentration of collected RNA was determined by spectrophotography. After the cDNA was synthesized, the study was performed with the real-time fluorescent quantitative PCR machine (Illumina, USA). The specificity of real-time PCR was detected by the melting curve analysis. β-Actin was used as internal standard. The primers used in this study were as follows: IQGAP1, forward 5′-GGGGAAACGTACCAGAGTGA-3′ and reverse 5′-TCTCGAGAAAGCTGCACAGA-3′; β-actin, forward 5′-CACGATGGAGGGGCCGGACTCATC-3′ and reverse 5′-TAAAGACCTCTATGCCAACACAGT-3′. Western Blotting Total protein of glomeruli and podocytes were extracted with lysis buffer (0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 1.0% Triton X-100, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, 20 mM Tris, pH 7.5) that contained a protease inhibitor cocktail (P8340, Sigma-Aldrich,). The sample was centrifuged at 12,000
Am J Nephrol 2013;38:430–444 DOI: 10.1159/000355970
431
Downloaded by: University of British Columbia 142.103.160.110 - 6/18/2016 4:15:42 AM
of the extracellular matrix, production of inflammatory cytokines, disruption of the cytoskeleton, cell cycle abnormalities, and apoptosis [3]. Podocytes are terminally differentiated epithelial cells that are important components of the glomerular filtration barrier and their injury or depletion plays a pivotal role in the onset of proteinuria and progression of glomerular diseases [4–8]. Our previous studies demonstrated that AngII-induced podocyte apoptosis contributed to podocyte injury [9–11]. Numerous molecules are associated with podocyte apoptosis, but the precise mechanism of AngII-induced apoptosis of podocytes is still unknown. IQGAP1 is a scaffolding protein that occurs in the slit diaphragm region of podocytes [12, 13], and contains several functional domains that recruit multiple signaling molecules. This 190-kDa protein provides a platform for diverse cellular functions, including cellular adhesion, migration, polarization [14, 15], and its role in regulating cellular apoptosis gradually attracted attention [16–18]. The mitogen-activated protein kinases (MAPK) signaling family, which consists of P38, JNK, and ERK, is thought to be essential for induction of apoptosis and plays an important role in kidney diseases [19]. Recent reports demonstrated that IQGAP1 functioned as an essential protein scaffold during MAPK signaling [20] and modulated the activities of some signaling proteins in the MAPK signaling pathway [16, 21]. Nevertheless, the role of IQGAP1 in AngII-induced podocyte apoptosis is unclear. Here, we examined the role of IQGAP1 in the podocytes of rats and in podocyte cell cultures in order to determine the role of IQGAP1 in AngII-induced apoptosis of podocytes.
rpm for 20 min at 4 ° C. Equal amounts of boiled protein in loading buffer were separated on an 8–10% sodium dodecyl sulfate-polyacrylamide gel, and then electrophoretically transferred to a nitrocellulose membrane (GE Healthcare, USA). Membranes were incubated with a primary antibody (IQGAP1 rabbit polyclonal antibody, 1:200, Santa Cruz; p-P38 rabbit monoclonal antibody, 1:500, Cell Signaling Technology; P38 rabbit polyclonal antibody, 1:500, Cell Signaling Technology; (p-) JNK rabbit monoclonal antibody, 1:500, Cell Signaling Technology; (p-) ERK1/2 rabbit monoclonal antibody, 1: 500, Cell Signaling Technology, or β-actin mouse monoclonal antibody, control, 1: 1,000, Santa Cruz) overnight at 4 ° C. A horseradish peroxidase-conjugated anti-rabbit or mouse IgG was used as the secondary antibody (1:10,000, Cell Signaling Technology). Bands were visualized by an enhanced chemiluminescence reagent (Santa Cruz) and developed onto film. The integrated optical density of each band was calculated by Gel-Pro Analyzer version 4.5.
Coimmunoprecipitation Analysis Coimmunoprecipitation experiments were conducted according to the manufacturer’s instructions (P2012, Beyotime, China). Briefly, ERK1/2 rabbit monoclonal antibody (1:50, Cell Signaling Technology) was added to the protein sample and rotated overnight at 4 ° C. Then the mixture was loaded with protein A+G agarose and incubated for 3 h at 4 ° C. The centrifuged sediment was saved and mixed with 5× Lane Marker Sample Buffer to make a 1× final solution. After boiling at 95–100 ° C for 5 min, the sample was analyzed by Western blotting for ERK1/2 and IQGAP1.
Transfection of IQGAP1 siRNA Transfection of podocytes with IQGAP1 siRNA was performed according to the HiPerFect Transfection Reagent Handbook (QIAGEN, Germany). Briefly, 3 × 105 cells were seeded in a 6-well plate and incubated with transfection complexes containing 5 nM IQGAP1 siRNA (or a negative control with scrambled siRNA) and 15 μl of Hiperfect transfection reagent under normal growth conditions for 24 h. Measurement of Apoptosis Apoptosis of podocytes in kidney tissue was determined by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay according to the manufacturer’s instructions (C1098, Beyotime). As described previously [25], dewaxed sections (3 μm thick) were incubated with protease K for 30 min at 37 ° C, followed by 3% H2O2 for 10 min at room temperature. Sections were then incubated with a mixture of terminal deoxynucleotidyl transferase (TdT) and biotin-dUTP in a humidified chamber for 1 h at 37 ° C, and were then subjected to streptavidin-horseradish peroxidase for 30 min at room temperature. The slides were stained with DAB, with hematoxylin as a counterstain. The omission of TdT was used as a negative control. Apoptotic podocytes from each glomerular cross-section were counted using the Weibel-Gomez method [26]. The apoptotic podocyte in kidney tissue was also determined by transmission electron microscopic analysis. Briefly, 2.5% glutaraldehyde-fixed 1-mm3 blocks of renal cortices were postfixed with 1% osmic acid, dehydrated in graded ethanols, embedded in EPON, sectioned, stained with uranylacetate and lead citrate, then observed and recorded with a Hitachi H-600 transmission electron microscope (Hitachi, Japan).
432
Statistical Analysis All data are presented as means ± SDs, and statistical analysis was performed using SPSS v17.0. Statistical comparison of groups was conducted using a one-way ANOVA test, and the LSD test was used for multiple comparisons. A p value
