Contributed equally
Diabetic nephropathy (DN) is one of the most common microvascular complications associated with diabetes mellitus (DM); the incidence has been predicted to reach 7.7% by 2030 on a global scale. Krüppel-like factor 5 (KLF5) is involved in numerous important biological processes; however, the potential effects of KLF5 on podocytes in patients with diabetic nephrotic (DN) have not yet been investigated. In the present study, synaptopodin expression in podocytes was investigated using an immunofluorescence assay. Following this, the proliferation of podocytes was investigated using an MTT assay. In addition, KLF5 was overexpressed in podocytes, and cell cycle arrest and apoptosis was subsequently investigated using flow cytometry. Western blotting and reverse transcription-quantitative polymerase chain reaction assays were performed to detect the expression levels of genes involved in the cell cycle and apoptosis, and the extracellular signal-regulated protein kinase (ERK)/p38 mitogen-activated protein (MAP) kinase pathway. The results demonstrated that treatment with puromycin aminonucleoside (PAN) suppressed the proliferation of podocytes in a dose- and time-dependent manner, and overexpression of KLF5 induced cell cycle arrest of podocytes regulated by PAN. Furthermore, overexpression of KLF5 was revealed to have inhibited PAN-induced apoptosis of podocytes, and that overexpression of KLF5 suppressed the ERK/p38 MAP kinase pathway in podocytes induced by PAN. Therefore, the results of the present study suggested that KLF5 may represent a potential therapeutic target for treatment of patients with DN.
Diabetic mellitus (DM) is a progressive disease and is usually associated with numerous complications (
Krüppel-like factors (KLFs) are a type of transcriptional regulatory factor. KLFs are associated with cell proliferation, migration, apoptosis and tissue remodeling (
Puromycin aminonucleoside (PAN) may disrupt the morphology of podocytes, trigger the overproduction of reactive oxygen species and induce nephrosis. Therefore, PAN is frequently used to establish a nephropathy model (
Podocytes (MPC-5 cells) were acquired from the Bena Culture Collection (Beijing, China). Podocytes were cultured in Ham Nutrient Mix F12-Dulbecco modified Eagle medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 1% penicillin-streptomycin G (Biochrom, Ltd., Cambridge, UK) and 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). Podocytes were seeded in 6-well plates at a concentration of 3×105 cells/ml and subsequently treated with PBS (control), or with PAN (0, 5, 10, 20, 40, 60, 80 and 100 µg/ml) for 0, 6, 12, 24 and 48 h time intervals at 37°C.
Mouse KLF5 complementary (c)DNA clone was purchased from GeneCopoeia Inc. (Rockville, MD, USA). KLF5 was amplified with PrimSTAR DNA polymerase (Takara Bio, Inc., Otsu, Japan) using primers with methylation-sensitive
Treated podocytes (2×103 cells/well) were seeded into a 96-well plate. The cells were treated with PAN at different concentrations (0, 5, 10, 20, 40, 60, 80 and 100 µg/ml) and maintained at 37°C for 0, 6, 12, 24 and 48 h time intervals. Following this, 20 µl of MTT solution was added into each well and incubated for 30 min. A total of 150 µl dimethylsulfoxide was subsequently was added into each well. Finally, a microplate reader was used to determine the absorbance at 490 nm. Treatment with 60 µg/ml PAN for 6 h was selected for subsequent analysis as this resulted in decreased cell viability (described below).
Podocytes were plated in 6-well plates at a density of 1×106 cells per well. Cells were treated with PBS or 60 µg/ml PAN for 6 h at 37°C. KLF5 overexpression (1 ug) or negative control plasmids (pcDNA 3.1; 1 µg) were transfected into podocytes using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 12 h following transfection, the cells were used for subsequent experimentation.
Total RNA was obtained using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). RNA was reversely transcribed to cDNA using a miScript II RT Kit (Qiagen GmbH, Hilden, Germany). The temperature protocol or RT was 25°C for 5 min, 37°C for 60 min, 85°C for 5 min and then held at 4°C. The mRNA expression levels were subsequently determined using the SYBR-Green PCR Master Mix kit (Takara Bio, Inc.) and the ABI 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were set as: 5 min pretreatment at 95°C, followed by 28 cycles of 95°C for 15 sec and 60°C for 30 sec, a final extension at 72°C for 10 min. The specific primers used were as follows: KLF5 forward, 5′-TTTCTGTCCCTACCCAGCAG-3′ and reverse, 5′-AGTAAGTGGCCTGTTGTGGA-3′; Bax forward, 5′-GAGCTGCAGAGGATGATTGC-3′ and reverse, 5′-CCAATGTCCAGCCCATGATG-3′; Bcl-2 forward, 5′-GCCTTCTTTGAGTTCGGTGG-3′ and reverse, 5′-GAAATCAAACAGAGGCCGCA-3′; Caspase-3 forward, 5′-TTGCCACCTGTCCAGTTTTG-3′ and reverse, 5′-AGGAGTGAGTGGTCTTGCTC-3′; caspase-8 forward, 5′-TTTCTGTCCCTACCCAGCAG-3′ and reverse, 5′-AGTAAGTGGCCTGTTGTGGA-3′; Caspase-9 forward, 5′-GCCCCATATGATCGAGGACA-3′ and reverse, 5′-CAGAAACGAAGCCAGCATGT-3′; cyclin D1 forward, 5′-GCTGCTCCTGGTGAACAAGC-3′ and reverse, 5′-TTGCGTCTCAGCTCAGGGAC-3′; and c-myc forward, 5′-CCACAGCAAACCTCCTCACA-3′ and reverse, 5′-TCCAACTTGACCCTCTTGGC-3′. GAPDH forward, 5′-GGGTCCCAGCTTAGGTTCAT-3′; GAPDH reverse, 5′-CATTCTCGGCCTTGACTGTG-3′. Data were quantified using the 2−ΔΔCq method (
Total proteins were prepared using a radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing a protease inhibitor cocktail (P8340; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Protein concentrations were determined using a Bradford Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins (30 µg) were separated via 10% SDS-PAGE gels and then transferred onto polyvinyl difluoride (PVDF) membranes (PerkinElmer, Inc., Waltham, MA, USA). Following this, the membranes were blocked with 5% skimmed milk (BD Biosciences, Franklin Lakes, NJ, USA) at room temperature for 2 h. The membranes were subsequently incubated with the following primary antibodies overnight at 4°C: Anti-GAPDH (1:2,000; cat. no. ab8245; Abcam, Cambridge, UK), anti-KLF5 (1:1,000; cat. no. ab24331; Abcam), anti-B cell lymphoma 2 (Bcl-2) associated X (Bax; 1:1,000; cat. no. ab32503; Abcam), Bcl-2 (1:1,000; cat. no. ab32124; Abcam), anti-caspase-3 (1:1,500; cat. no. ab13586; Abcam), anti-caspase-8 (1:1,500; cat. no. ab25901; Abcam), anti-caspase-9 (1:1,500; cat. no. ab25758; Abcam), anti-cyclin D1 (1:1,000; cat. no. ab134175; Abcam), anti-c-myc (1:1,000; cat. no. ab39688; Abcam), anti-phosphorylated (p)-extracellular signal-regulated protein kinase (ERK)1/2 (1:1,200; cat. no. 4370; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-ERK1/2 (1:1,000; cat. no. ab184699; Abcam), anti-p-p38 (1:1,000; cat. no. ab47363; Abcam), anti-p38 (1:1,000; cat. no. ab170099; Abcam). PVDF membranes were subsequently incubated with a horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. sc-2004, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 1 h. Finally, the proteins were visualized using ECL Western Blotting Substrate (Pierce; Thermo Fisher Scientific, Inc.) in an enhanced chemiluminescence detection system (GE Healthcare, Chicago, IL, USA). The gray value was determined by Quantity One 4.6.2 software (Bio-Rad Laboratories, Inc.).
Podocytes were fixed with 4% paraformaldehyde at 4°C for 20 min, and then permeabilized with 0.2% Triton X-100 for 3 min. Following this, cells were washed with PBS and then blocked using 10% goat serum (Thermo Fisher Scientific, Inc.) for 30 min at room temperature. Cells were subsequently incubated with 4 µg/ml anti-synaptopodin antibodies (cat. no. ab220345; Abcam) overnight at 4°C. Following this, cells were washed using PBS and then incubated with Alexa-Fluor® 633-conjugated secondary antibodies (1:500; A-21070, Thermo Fisher Scientific, Inc.) at 37°C for 1 h. Cells were subsequently stained using 4′-6-diamidino-2-phenylindole (Thermo Fisher Scientific, Inc.) for 5 min at room temperature. A fluorescence microscope (magnification, ×200; Olympus Corporation, Tokyo, Japan) was used to visualize the results.
Podocytes were treated as described above. Cells (2×105 cells/well) were seeded into 24-well plates and incubated overnight at 37°C. To investigate the cell cycle, cells were centrifuged at 1,000 × g for 5 min at 4°C and subsequently fixed using 70% (v/v) ethanol for 1 h at 4°C. Following this, cells were washed with PBS and then incubated with 500 µl propidium iodide (PI)/RNase buffer (BD Biosciences, Franklin Lakes, NJ, USA) at room temperature for 30 min. Finally, a FACS-Calibur flow cytometer (BD Biosciences) was used to investigate cell cycle distribution, and ModFit LT 2.0 software (Verity Software House, Inc., Topsham, ME, USA) was used to analyze the results. To investigate apoptosis, cells were washed with PBS and then re-suspended with 0.5 ml binding buffer containing 5 µl Annexin V-fluorescein isothiocyanate and PI double stain (BD Biosciences) for 20 min at room temperature in the dark. Following this, the apoptotic rate was determined using a FACS-Calibur flow cytometer (BD Biosciences) and ModFit LT 2.0 software.
All data were analyzed by SPSS 13.0 (SPSS, Inc., Chicago, IL, USA) using one-way analysis of variance with Tukey's test. Data are presented as the mean ± standard deviation. All experiments were performed in triplicate. P<0.05 was considered to indicate a statistically significant difference.
Initially, synaptopodin expression in podocytes was investigated using immunofluorescence. The results revealed that the expression of synaptopodin was positive in podocytes (
To further investigate the potential role of KLF5 in podocytes, KLF5 was overexpressed in podocytes via transfection with pCDNA3.1-KLF5 plasmids. RT-qPCR and western blot assays were performed to determine the expression levels of KLF5. The results demonstrated that KLF5 expression was significantly increased in podocytes transfected with pCDNA3.1-KLF5 compared with the NC group (
The results revealed that the proportion of cells in the G1 phase was increased in the PAN group compared with the control group (
Flow cytometry was performed to determine whether KLF5 has an important role in the apoptosis of podocytes. The results demonstrated that the apoptosis of podocytes was significantly increased in the PAN group compared with the NC group (P<0.001;
MAP kinases have important roles in numerous cellular functions and are regulated via independent upstream activation cascades (
DN is one of the most common complications associated with diabetes and has become the most frequent causative factor resulting in the development of end-stage renal disease (
Podocytes and the slit diaphragm within foot processes are critical components of the glomerular filtration barrier (
KLF-5 is an important member of the KLF protein family, and is located at chromosome 13q21 and encodes a 55 kDa protein that contains 457 amino acids (
MAP kinases are serine/threonine-specific protein kinases (
Limitations of the present study included that all experiments were performed
Not applicable.
The present study was supported by the Natural Science Fund project in Shandong Province (grant no. 2017GSF21116).
All data generated and/or analyzed during this study are included in this published article.
YL wrote the main manuscript. YL, XH and XS performed the experiments. YL and ZH designed the study. XS and XH performed data analysis. YL, XS and ZH contributed to manuscript revisions. All authors reviewed the manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
PAN inhibited the proliferative ability of podocytes in a dose- and time-dependent manner. (A) Synaptopodin expression in podocytes was detected via immunofluorescence assay using a fluorescence microscope (magnification, ×200). (B) Cell viability in podocytes was analyzed using an MTT assay following g treatment with PBS (control), or PAN (5, 10, 20, 40, 60, 80 and 100 µg/ml) for 0, 6, 12, 24 and 48 h time intervals. PAN, puromycin aminonucleoside. *P<0.05 vs. 0 h.
KLF5 was overexpressed in podocytes. Podocytes were transfected with either empty vectors (control) or pCDNA3.1-KLF5. (A) Relative mRNA expression levels of KLF5 were determined by reverse transcription-quantitative polymerase chain reaction assays. (B) Protein expression levels of KLF5 were analyzed via western blot analysis, and quantitative values were determined based on the gray values. ***P<0.001. KLF5, Krüppel-like factor 5.
Overexpression of KLF5 induced cell cycle arrest of podocytes mediated by PAN. Podocytes were treated with either PBS (control), 60 µg/ml PAN, NC + PAN, KLF5 + PAN. (A) Cell cycle distribution in podocytes was investigated using flow cytometry, and the number of cells was quantitatively analyzed. (B) Reverse transcription-quantitative polymerase chain reaction assays were used to determine cyclin D1 and c-myc expression levels in treated podocytes. (C) Western blot assays were performed to determine cyclin D1 and c-myc expression levels in treated podocytes. (D) Cyclin D1 and c-myc protein expression levels were determined according to the gray values. *P<0.05, **P<0.01, ***P<0.001. PAN, puromycin aminonucleoside; KLF5, Krüppel-like factor 5; NC, negative control.
Overexpression of KLF5 inhibited PAN-induced apoptosis of podocytes. Podocytes were treated with PBS (control), PAN (60 µg/ml), NC + PAN or KLF5 + PAN. (A) Cell apoptosis was investigated using flow cytometry in treated podocytes, and the number of apoptotic cells were quantitatively analyzed. (B) mRNA expression levels of Bax, Bcl-2, caspase-3, caspase-8 and caspase-9 were determined via by reverse transcription-quantitative polymerase chain reaction assays. (C) Protein expression levels of Bax, Bcl-2, caspase-3, caspase-8 and caspase-9 were investigated using western blot analyses, and quantitative analysis was performed using base gray values and GAPDH. *P<0.05, **P<0.01 and ***P<0.001. PAN, puromycin aminonucleoside; KLF5, Krüppel-like factor 5; NC, negative control; Bcl-2, B cell lymphoma 2; Bax, Bcl-2 associated X; PI, propidium iodide; FITC, fluorescein isothiocyanate.
Overexpression of KLF5 suppressed the ERK/p38 MAP kinase pathway in podocytes induced by PAN. Podocytes were treated with PBS (control), 60 µg/ml PAN, NC + PAN, KLF5 + PAN. (A) Western blot assays were performed to determine p-ERK1/2 and ERK1/2 protein levels. (B) Western blot assays were performed to determine p-p38 and p38 protein levels. ***P<0.001. PAN, puromycin aminonucleoside; p-, phosphorylated; ERK, extracellular signal-regulated protein kinase; KLF5, Krüppel-like factor 5; NC, negative control.