Background
Diabetic nephropathy (DN) is the leading cause of chronic kidney disease (CKD), which has been recognized as a public health disease worldwide because it creates a high risk of cardiovascular disease and other complications [
1‐
4]. Accumulating evidence has shown that glomerular podocytes, which are glomerular visceral epithelial cells residing on the urinary side of the glomerular basement membrane (GBM), play a critical role in the pathogenesis of DN by maintaining the glomerular structure and filtration barrier [
5‐
8].
Klotho was initially identified as an anti-ageing gene [
9‐
12]. There are three members in this family: α-, β-, and γ-klotho [
13]. In this paper, the term klotho in the subsequent text refers to α-klotho. Klotho is a single transmembrane protein that consists of a small intracellular domain in the C terminus and a large extracellular domain in the N terminus [
12,
14]. Membrane-bound klotho is suggested to be a coreceptor mainly for FGF23 signalling, regulating kidney phosphate excretion [
15‐
17]. The extracellular domain can be cleaved by a disintegrin and metalloproteinase (ADAM)-10 and ADAM-17 at the α cut site to become soluble klotho, which contains two functional repeats named Kl1 and Kl2 and are involved in the circulation [
12,
18,
19]. Generally, soluble klotho, which refers to this degradation fragment of membrane-bound klotho, is the functional form and is widely present in the blood, urine and cerebrospinal fluid [
20‐
22]. Soluble klotho has been suggested to be an ideal biomarker for CKD based on findings from clinical and animal studies that klotho deficiency seems to be significantly associated with podocyte injury and kidney fibrosis in human kidneys [
23]. Moreover, extracellular supplementation is considered a novel therapeutic strategy for CKD to restore klotho levels and/or promote endogenous expression [
24,
25]. Previous studies indicated that soluble klotho could inhibit transforming growth factor β1 (TGF-β1) activity to reduce kidney fibrosis by directly binding to the TGF-β1 receptor and preventing TGF-β1 signalling [
24,
26].
The normal function of podocytes depends on their actin cytoskeleton. Previous studies revealed that Rho family GTPases regulate podocyte actin dynamics, and abnormalities in Rho GTPase activity may result in podocyte mobility and induce proteinuria [
7,
27‐
30]. Slit-Robo GTP activating protein 2a (SRGAP2a), a member of the Rho GTPase large family, has been shown to be primarily enriched in podocytes and is tightly correlated with the estimated glomerular filtration rate (eGFR) and proteinuria of DN patients [
7]. The expression of SRGAP2a was shown to be decreased in DN patients and
db/db mice, while increasing SRGAP2a levels in podocytes in
db/db mice efficiently rescued the DN state [
7]. Recent studies revealed that the expression of SRGAP2a decreased significantly in the presence of high glucose (HG) or TGF-β1, and exogenous SRGAP2a largely rescued podocytes from HG- and TGF-β1-induced damage [
7,
31]. SRGAP2a has been shown to play protective roles against podocyte injury and proteinuria in DN [
7,
31]. In addition, ROS production was a key player progressing diabetic kidney diseases and was closely associated with TGF-β1 signaling [
32,
33].
Therefore, there is a close relationship between the levels of klotho, SRGAP2a, TGF-β1 and ROS signaling in CKD, including DN. However, whether klotho attenuates kidney injury by regulating SRGAP2a in podocytes remains unclear. In this study, we cultured differentiated rat podocytes and measured the expression of SRGAP2a, the reactive oxygen species (ROS) levels, and key factors in TGF-β1 and ROS signalling with or without exogenous klotho under HG conditions and analysed the relationship between klotho and SRGAP2a. And we confirmed the in vitro findings with diabetic rat models. The results provide theoretical support for klotho protein as a novel therapeutic strategy for treating DN patients.
Materials and methods
Primary podocyte culture
We isolated two rat kidneys under aseptic conditions and dissected and gently ground the kidney cortex. The tissues were rinsed by sequential passage through 100 μm and 200 μm sieves at 4 °C. Glomeruli were collected on a 200 μm sieves, and 2 g/L type IV collagenase (Sigma, china) was properly digested at 37 °C for about 15 min. Under the observation with the inverted microscope, when a few cells were dissociated in the medium, added completed Dulbecco's modified Eagle's medium (DMEM) (Hyclone, USA) to terminate the digestion. Centrifuge and wash twice at 1000 r/min for 10 min, and resuspended in DMEM, inoculated into three 75 cm2 culture flasks lined with rat tail collagen and incubated at 37 °C and 5% CO2 for 7–8 days. The majority of cells observed with an electron microscope (Leica TCS SP8 STED, Germany) were primary podocytes.
Immunofluorescence staining
Primary podocytes were cultured for another 7 days after being trypsinized and subsequently passaged. Podocyte morphology was observed with an inverted phase contrast microscope (Leica TCS SP8 STED, Germany) when the cells covered the bottom of the dish. The cells were fixed with cold acetone and processed for immunofluorescence staining for the podocyte markers nephrin and synaptopodin.
Cell transfection
All siRNAs used in this study were synthesized by GenePharma (Suzhou). The siRNA-TGF-β1 (TGF-β1 siRNA-2) sequence consisted of a 21-nucleotide sense strand (5'-GACAAGUUCAAGCAGAGUACA-3') and an antisense strand (5'-UACUCUGCUUGAACU UGUCAU-3'). Podocytes were inoculated in 24-well plates and transfected with the designed siRNAs to mediate TGF-β1 silencing with Lipofectamine™ 2000 transfection reagent according to the manufacturer’s instructions (Thermo Fisher, US). Twenty-four hours after transfection, the podocytes were treated with HG and harvested for subsequent analysis such as SRGAP2a immunofluorescence staining, qRT-PCR and western blotting.
Quantitative real-time PCR (qRT-PCR)
Podocyte samples were treated with mannitol (100 mM), glucose (100 mM), glucose (100 mM) plus acetylcysteine (10 μM) and glucose (100 mM) plus klotho (2000 pM) for 24 h, and samples without additional sugar treatment were used as negative controls. Then, the medium was removed from the 6-well cell culture plate, and 1 × PBS solution (Sangon Shanghai) was added to wash the cells gently. Next, the 6-well plate was placed on ice, and 800 μl of TRIzol reagent (Sigma, US) was added to each sample, which were repeatedly pipetted to dislodge all adherent cells, and the cells were transferred to 1.5 ml EP tubes. The total RNA of each sample was extracted according to the manufacturer’s instructions and reverse transcribed with the PrimeScript™ RT reagent kit (TaKaRa, Japan). qRT-PCR was performed with SYBR Green detection mix (TaKaRa, Japan). The relative expression levels of genes in this study were normalized to actin expression, analysed by the 2
−ΔΔCt method, and summarized from three separately harvested podocyte samples. The primers used for qRT-PCR were showed in Supplemental Table
1.
Western blot analysis
Podocyte samples were treated as described above for qRT-PCR. Total protein was extracted from each sample to prepare cell lysates using RIPA Lysis Buffer (Sangon, Shanghai, China) and stored at -20 °C. The bicinchoninic acid (BCA) protein quantification method was used to ensure that the concentration of each sample was basically equal. Protein samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes with electrophoresis systems (Tanon VE180 and Tanon VE186, Shanghai, China). The PVDF membranes were blocked with 5% (w/v) skimmed milk powder for 2 h and incubated at 4 °C overnight with the following primary antibodies: rabbit anti-small mother against decapentaplegic (Smad)2/3 (Abcam, ab202445, diluted to 1:1000), phosphorylated (p)-Smad2/3 (Abcam, ab280888, diluted to 1:500), Smad7 (Abcam, ab216428, diluted to 1:500) and NAD(P)H oxidase 4 (NOX4) (Abcam, ab133303, diluted to 1:2000). After being washed with 1 × PBS solution (Sangon, Shanghai, China) three times, the membranes were incubated with HRP-labelled goat anti-rabbit IgG secondary antibodies (Abcam, ab205718, diluted to 1:10,000). Immunoreactivity was determined with enhanced chemiluminescence (ECL) reagent (Thermo Fisher, US). A gel imaging system (BIO-RAD Gel Doc XR + , US) and software (BIO-RAD Image Lab Software, Version 5.1 and SPSS 20.0) were used for imaging and statistical analysis. GAPDH was used as an internal control to ensure equal protein loading.
Fluorescein-conjugated phalloidin staining
The podocyte cytoskeletal remodelling with different treatments in this study were detected by fluorescein-conjugated phalloidin staining using test kits (APExBIO, Beijing, China) according to the manufacturer's instructions.
Animal experiment
The Wistar rats purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. were divided into two groups randomly. The control group rats were with free access to water and normal diet. The modelled DN rats were supplied with high-fat-sugar diet (10% lard, 10% egg yolk powder, 5% sucrose), free access to water, and intraperitoneal injected with streptozotocin (70 mg/kg) six week later to induced DN. The DN rats were divided into two groups randomly. The klotho intervention rats were intraperitoneal injected with klotho proteins (0.02 mg/kg) for 7 d consecutively. The control and DN group were intraperitoneal injected with normal saline. The health status was monitored by a specific veterinarian. All animals were euthanized and sacrificed by cervical dislocation after treatment, and the kidney tissues were removed. All experiments with animals were approved by the Institutional Animal Care and Use Committee of Soochow University.
Immunohistochemistry
The kidney tissues were embedded in paraffin and cut into 4-μm in thickness. After dewaxed and hydrated, the sections were washed with distilled water containing 3% hydrogen peroxidase to reduce endogenous oxidase activity. Then the tissue sections were incubated with anti-SRGAP2a antibody (Abcam, ab121977), anti-nephrin (Abcam, ab216692), anti-synaptopodin (Abcam, ab224491) for 2 h at room temperature, and subsequently, incubated with goat-anti-rabbit antibody at room temperature for 40 min. The degree of staining was determined by developing with diaminobenzidine (DAB) chromogen (Bio-Rad, Inc., CA, USA).
Intracellular ROS generation analysis
We measured intracellular ROS generation with the fluorescent probe DCFH-DA, which is hydrolysed and generates nonfluorescent DCFH after passing through the cell membrane. The intracellular ROS oxidizes DCFH to produce fluorescent DCF. The level of DCF fluorescence intensity indicates the level of intracellular ROS.
Podocyte samples were treated as described above for qRT-PCR or western blotting, washed with cold 1 × PBS solution once, resuspended in serum-free DMEM and incubated with DCFH-DA (10 μM)) for 30 min at 37 °C. The samples were mixed every 3–5 min to ensure good contact between the probes and podocytes. After that, the podocytes were washed with serum-free DMEM three times and resuspended in 1 × PBS solution. Finally, the podocyte samples were analysed by flow cytometry (Life Attune NxT, US). FlowJo 10 software was used for data analysis.
Statistical analysis
Sigma Plot 12.0 and SPSS 20.0 were used for statistical analysis. All data are presented as the means ± SD. Independent group comparisons were performed using Student’s t-test or one-way ANOVA with Bonferroni’s post hoc test. A value of P < 0.05 was considered statistically significant.
Discussion
DN is a common complication of diabetes mellitus, the major cause of kidney failure and the leading cause of CKD [
8,
35]. Podocyte injury, which is clinically indicated by progressive decline in the eGFR and increased proteinuria, plays a critical role in the pathogenesis of DN [
5‐
8]. The specific function of podocytes is strongly dependent on the actin cytoskeletons of the interdigitating foot process between neighbouring podocytes [
5,
7]. SRGAP2a was strongly suggested to protect podocyte function and structure [
7]. In this study, we found that the expression of SRGAP2a was significantly decreased in cultured podocytes under HG conditions. And the DN rats also revealed downregulation of SRGAP2a expression. These results were consistent with the investigation showing that the SRGAP2a level was significantly decreased in DN patients [
7]. These findings indicated that the expression of SRGAP2a might be essential for proper podocyte activity.
TGF-β signalling regulates a series of biological processes and is indispensable in animal development [
36]. Smad transcription factors occupy the core of this pathway [
37]. According to this study, TGF-β1 silencing stimulated SRGAP2a expression under HG conditions in podocytes. In addition, previous studies revealed that TGF-β1 significantly reduced the podocyte level of SRGAP2a [
7]. In summary, the molecular functions of SRGAP2a seemed to be tightly associated with TGF-β1/Smad signalling. ROS inhibition resulted in the upregulation of podocyte SRGAP2a expression which indicated that SRGAP2a might be involved in ROS signalling. Previous research indicated that HG-induced ROS production increased TGF-β1 level in DN [
32]. Overall, we suggest that SRGAP2a plays protective roles against kidney diseases via the TGF-β1/Smad/ROS signalling axis. Increased ROS generation (oxidative stress) is consequently associated with the hyperglycaemia underlying DN [
38‐
40]. Thus, we hypothesised that reducing oxidative stress might facilitate SRGAP2a expression to prevent podocyte injury in DN, since in diabetic mice, reducing oxidative stress efficiently improved kidney structure and function [
41].
On the one hand, the serum klotho protein level is one of the early markers of kidney diseases, as the level declines at the early stage of CKD and continuously decreases with the progression of CKD [
24]. On the other hand, klotho protein is considered to be a novel therapeutic target [
22,
24,
42]. In this study, we found that the expression of SRGAP2a significantly increased after the addition of klotho to HG solutions compared with the expression of SRGAP2a in HG-treated cultured podocytes. This might indicate that klotho protein actually stimulated SRGAP2a expression in podocytes to protect against injury. In addition, the expression of key factors in the TGF-β1/Smad/ROS signalling pathway changed after klotho administration. The expression of NOX4 and Smad2/3 was greatly enhanced with HG treatment but significantly downregulated when klotho was added to the HG treatment. However, the expression of Smad7, an inhibitory Smad serving as a competitor for receptor-regulated Smad proteins such as Smad2/3, was contrary to the results of Smad2/3 and NOX4. The protein level of p-Smad2/3 showed the same trend as that of Smad2/3 and NOX4 in the presence of HG and exogenous klotho. These results suggested that klotho could regulate the expression of key factors in TGF-β1/Smad/ROS signalling that are strongly associated with SRGAP2a in podocytes. NOX4 is the major source of ROS in the kidney [
38‐
40]. NOX4-derived ROS mediate TGF-β1/Smad signalling in many disease processes. In this study, ROS generation was analysed by flow cytometry and was enhanced under HG conditions but reduced significantly after klotho administration. In summary, exogenous klotho reduced ROS generation, which might benefit the expression of SRGAP2a to prevent podocyte injury in DN. The TGF-β1/Smad/ROS signalling pathway may regulate actin activity in kidney myofibroblast activation, human pulmonary artery smooth muscle cells and kidney interstitial fibrosis [
34,
43,
44]. In this study, klotho remodelled the actin fibers in DN rats. Thus, klotho may prevent podocyte dysfunction by regulating the expression of SRGAP2a and key factors of the associated regulatory pathway.
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