Geniposide ameliorated sepsis-induced acute kidney injury by activating PPARγ

Effects of GE on sepsis-induced kidney injury and dysfunction

The severity of CLP surgery-caused kidney injury was determined by HE and PAS staining. The result showed that the structure of glomerulus was clear and complete, and the kidney tubules were tightly packed in control and sham group. However, in CLP group, glomerulus was shrunken and the kidney tubules exhibited diffuse expansion. Then, GE treatment weakened these histological changes with a dose-dependent way, in agreement with the quantified result (Figure 1A, 1C). Besides, PAS staining showed a severe histological kidney injury as glomerular basement membranes markedly thickened companied with glomerular hypertrophy in CLP group, which was attenuated by GE treatment (Figure 1B).

Figure 1. Effect of GE on sepsis-induced kidney injury. Sepsis-induced kidney injury model in BALB/c mice was conducted using cecal ligation and puncture (CLP) surgery, and the mice were received GE 1 h after the CLP surgery at different doses (10 mg/kg, 20 mg/kg and 40 mg/kg) three times (once every 6 h). 24 h after the surgery, H&E staining and PAS staining were performed to observe the histopathological changes (A, B). The histological injury was quantified (C). ***p<0.001 vs="" the="" sham="" group.="" ###p<0.001="" CLP="" group.<="" p=""/>

NGAL, KIM1, Scr, BUN, ScysC, IL-18, α-GST and albumin are identified to be markers in acute kidney [15, 16]. As shown in Figure 2A–2G, the levels of KIM1, Scr, BUN, ScysC, IL-18, α-GST and albumin were similar in each group before CLP surgery, respectively. 24 h after surgery, levels of these markers were increased significantly, and an obvious decrease of them were observed in GE group, especially at 20 mg/kg and 40 mg/kg. Besides, protein expressions of NGAL and KIM1 were both upregulated in CLP group, but downregulated after the treatment of GE with a dose-dependent manner (Figure 2H–2I).

Figure 2. Effect of GE on sepsis-induced kidney dysfunction. Levels of serum creatinine (Scr) (A), blood urea nitrogen (BUN) (B) and serum cystain C (ScysC) (C) in serum and α glutathione S transferase (α-GST) (F) and albumin (G) in urine were measured by an automatic biochemical analyzer. The levels of kidney injury molecule-1 (KIM1) (D) and IL-18 (E) were determined using their corresponding ELISA kits. The mRNA level and protein expression of NGAL were measured using qRT-PCR and western blot, respectively (H, I). ***p<0.001 vs="" the="" sham="" group.="" #,="" ##,="" ###p<0.05,="" 0.01,="" 0.001="" CLP="" group.<="" p=""/>

Effect of GE on sepsis-induced inflammatory response and oxidative injury

Inflammatory cytokines in blood were detected by ELISA assay. The results in Figure 3A–3E displayed that CLP surgery markedly increased the levels of pro-inflammatory mediators such as TNF-α, IL-6, IL-1β, and MCP-1, and decreased the level of anti-inflammatory mediator such as IL-10, while these changes were significantly reversed by introduction of GE. Additionally, the oxidative stress-associated factors were also assessed by corresponding test kits. The results in Figure 3F–3H showed that the levels of ROS, SOD and MDA were higher, and the level of GSH-px was lower in CLP group than those in control group, hinting the severe oxidative stress after CLP surgery. As expected, while GE treatment reversed the effects caused by CLP surgery. These results together suggested that GE could effectively alleviated sepsis-induced inflammatory response and oxidative injury.

Figure 3. Effect of GE on sepsis-induced inflammatory response and oxidative injury. The concentrations of inflammatory cytokines in blood including tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, IL-1β, and monocyte chemotactic protein 1 (MCP-1) in blood were measured using ELISA kit (A–E). Superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-px) and reactive oxygen species (ROS) were measured using their corresponding test kits (F–I). ***p<0.001 vs="" the="" sham="" group.="" #,="" ##,="" ###p<0.05,="" 0.01,="" 0.001="" CLP="" group.<="" p=""/>

Effect of GE on sepsis-induced vascular permeability

Changes in vascular permeability are important in the pathogenesis of sepsis-induced organ injury [17]. Therefore, we investigated the changes of kidney vascular permeability using Evans blue dye. The kidney tissues isolated from mice showed that the kidney in CLP group exhibited black, compared with the control tissues, and after the treatment of GE (20 mg/kg and 40 mg/kg), the kidney turned to be nearly normal (Figure 4A). Then the amount of Evans blue dye leakage reflected the degree of kidney vascular permeability. As shown in Figure 4B, CLP significantly increased kidney vascular permeability, which was alleviated by GE. These results suggested that GE could effectively ameliorate sepsis-induced kidney vascular permeability.

Figure 4. Effect of GE on sepsis-induced vascular permeability. Evans blue dye was injected intraperitoneally into the mice, and kidney tissues were collected and observed after 24 h (A). The amount of Evans blue dye in the supernatant of kidney tissue was analyzed by detecting absorbance at 620 nm (B). ***p<0.001 vs="" the="" sham="" group.="" ##,="" ###p<0.01,="" 0.001="" CLP="" group.<="" p=""/>

Effect of GE on sepsis-induced kidney tissue apoptosis

As an important reason for sepsis-induced kidney injury, apoptosis of cells in kidney could result in damage to the septic kidney [3]. The apoptotic cells were detected using TUNEL assay. Compared to the control group, significant promotion of the number of TUNEL-positive cells was observed in CLP group. However, GE obviously declined the number of TUNEL-positive cells at a dose-dependent manner (Figure 5A). Besides, the positive cells were quantified to indicate cellular apoptosis of the kidney tissue (Figure 5B). Consistent with the results of TUNEL assay, the CLP surgery also provoked reduction of Bcl-2 and elevation of Bax and cleaved caspase 3, and the treatment of GE remarkably increased the expression of Bcl-2 and decreased the expression of Bax and cleaved caspase 3(Figure 5C–5F). These results displayed that GE could dramatically reduce apoptotic activity in the cells of the kidney during sepsis.

Figure 5. Effect of GE on sepsis-induced kidney tissue apoptosis. The apoptosis of cells in kidney was detected using TUNEL assay (A). The TUNEL-positive cells were quantified (B). The expression levels of apoptosis-related proteins such as Bcl-2, Bax, cleaved caspase 3 and caspase 3 were measured with western blot (C–F). ***p<0.001 vs="" the="" sham="" group.="" #,="" ##,="" ###p<0.05,="" 0.01,="" 0.001="" CLP="" group.<="" p=""/>

Effect of GE on PPARγ expression

To further understand the mechanism of GE in sepsis-induced kidney injury, we measured the effect of GE on PPARγ signaling using western blot and immune histochemical analysis. In immunohistochemical analysis, the expression of PPARγ was markedly reduced in CLP group in contrast to the control group, which was increased by the GE treatment (Figure 6A). Besides, the western blot assay also exhibited a similar change as the protein expression of PPARγ was distinctly lowered after CLP surgery, and the decrease was alleviated by the treatment of GE (Figure 6B).

Figure 6. Effect of GE on PPARγ expression. The expression of PPARγ in kidney tissue of each group was determined by immunohistochemical analysis (A). Besides, western blot was performed to determine the protein expression of PPARγ (B, C). ***p<0.001 vs="" the="" sham="" group.="" ###p<0.001="" CLP="" group.<="" p=""/>

Inhibition of PPARγ abolished the effect of GE

To investigate whether the activation of PPARγ plays an indispensable role in the protective function of GE in sepsis-induced kidney injury, GW9662, a PPARγ antagonist, was introduced into HK-2 cells. Here, LPS-induced HK-2 cells were used to simulate sepsis-induced kidney injury. In LPS-induced HK-2 cells, the mRNA level of PPARγ was decreased, which was improved under GE treatment in a dose-dependent manner (Figure 7A). The detection of inflammatory mediators of each group showed that LPS significantly increased the levels of TNF-α, IL-6, IL-1β, and MCP-1, and decreased the level of IL-10, consistent with those results discovered in mice with sepsis-induced kidney injury. The treatment of GE significantly reversed the expression changes of TNF-α, IL-6, IL-1β, and MCP-1, and IL-10 (Figure 7B–7F). In addition, GE alleviated the elevated cell apoptotic rate induced by LPS (Figure 7G, 7H). However, the suppressive effects of GE on inflammatory response and cell apoptosis were significantly diminished by GW9662, suggesting that the activation of PPARγ was necessary for GE to exert its function, and GE might exert the protective role in kidney injury by activating PPARγ.

Figure 7. Inhibition of PPARγ abolished the effect of GE. LPS-induced HK-2 cells was utilized to simulate sepsis-induced kidney injury. Cells were treated with GE (100 μg/ml, 200 μg/ml, 300 μg/ml), and the mRNA level of PPARγ was detected by qRT-PCR (A). GW9662, a PPARγ antagonist, was employed to treat HK-2 cells, and then the concentrations of inflammatory cytokines including TNF-α, IL-6, IL-10, IL-1β, and MCP-1 in each group were measured using ELISA kit (B–F). Cell apoptotic rate in each group was determined via usage of flow cytometry analysis (G, H). **, ***p<0.01, 0.001="" vs="" the="" control="" group.="" ##,="" ###p<0.01,="" LPS="" $$,="" $$$="" p<0.01,="" LPS+GE.<="" p=""/>

Experimental animals and experimental design

All BALB/c mice (male; 25-30 g) were obtained from Vital River Laboratories Co., Ltd. (Beijing, China) and kept in a pathogen-free room with standard laboratory diet and water ad libitum, with stable room temperature (22-24 °C) and 12 h light/dark cycle. All animal experiments were performed following Nursing and Use Guidance for Animal Experiment Operation of National Institutes Health.

Mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Sepsis was induced in mice undergoing cecal ligation and puncture (CLP) surgery as previously described [39]. Briefly, the abdominal area was shaved and disinfected, and a midline vertical incision of 2 to 3 cm was made in the abdomen. The cecum was then ligated from the top and punctured twice by piercing the cecum with an 18-gauge needle. After a small amount of stool was squeezed from the puncture hole, the cecum was reinserted afterwards into the abdominal cavity and the incision was sutured. Pre-warmed saline was then administrated intraperitoneally for resuscitation. The sham-operated mice underwent CLP surgery except cecal ligation and perforation. To explore the protective effect of GE, GE groups received an intraperitoneal injection of GE (NO. #SML0153; Sigma-Aldrich, St. Louis, MO, USA) 1 h after the CLP surgery at different doses (10 mg/kg, 20 mg/kg and 40 mg/kg) three times (once every 6 h). 24 h after the surgery, mice were euthanized, and blood samples and kidney tissues were gathered for further analysis.

Analysis of inflammatory cytokines and oxidative stress-associated factors

The concentrations of inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, IL-1β, and monocyte chemotactic protein 1 (MCP-1) in blood were measured using corresponding enzyme-linked immunosorbent assay (ELISA) kits (R&D System, Minneapolis, MN, USA). Superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-px) and reactive oxygen species (ROS) were measured using their corresponding test kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Real-time quantitative PCR analysis

Total RNA was extracted from kidney tissues of different groups using TRIzol reagent, and then reversely transcribed into cDNA using PrimeScript Reagent Kit (Takara, Dalian, China). Subsequently, Real-time PCR was performed using the SYBR Mixture (Takara) to determine the gene levels. GAPDH was utilized as an internal control, and the quantification of relative transcript levels was assessed with the comparative threshold (Ct) method.

Western blot

Total protein was extracted from kidney tissues of different groups using RIPA cell lysis buffer, and then quantified using a BCA kit. The same amount of protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. Subsequently, the membranes were blocked with 5% nonfat milk and next incubated with primary antibodies at 4 °C overnight, followed by rinse with PBS for three times and incubation with anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibodies. The bands were developed using enhanced chemiluminescence reagents (Pierce, Rockford, IL, USA).

Determination of kidney function

Blood samples were made by centrifugation at 3000 rpm for 10 min, and then levels of blood urea nitrogen (BUN), serum creatinine (Scr) and serum cystain C (ScysC) in serum and α glutathione S transferase (α-GST) and albumin in urine were measured by an automatic biochemical analyzer (AU400, Olympus, Japan). The levels of kidney injury molecule 1 (KIM1) and IL-18 were determined using their corresponding ELISA kits (R&D System). The expression of neutrophil gelatinase-associated lipocalin (NGAL) and KIM1 was determined using western blot.

Histopathological examination

Kidney tissue samples were collected 24 h after surgery, fixed in 10% paraformaldehyde, embedded in paraffin, and cut into sections with 4 μm-thickness. The sections were then stained with hematoxylin and eosin (H&E). Additionally, the sections were stained with periodic acid-schiff (PAS). The histological changes of kidney were observed under a light microscope (Olympus IX71, Tokyo, Japan) at 200X magnification.

TUNEL analysis

The apoptosis of kidney tissues was measured using TUNEL Assay Kit (KeyGEN BioTECH, Jiangsu, China) as the manufacturer instructed. After deparaffinization and hydration, the sections were washed with PBS, and treated with proteinase K working solution. After that, sections were cultivated with TUNEL reaction mixture at 37 °C for 60 min, followed by incubation with DAPI for 10 min. The results were observed through the fluorescence microscope (Olympus, Japan).

Evans blue staining

To study vascular leakage from kidney tissues, Evans blue dye (Sigma-Aldrich, St. Louis, MO, USA) was injected intraperitoneally into the mice 30 minutes before sacrifice, and kidney tissues were collected and viewed. Then, Kidney tissues were weighed and homogenized in 1 ml formamide and incubated at 55 °C for 18 h. After centrifugation, the supernatant was collected, and the amount of Evans blue dye in the supernatant was analyzed by detecting the absorbance at 620 nm and calculated according to a standard curve of Evans blue dye.

Immunohistochemical analysis

Kidney tissues were fixed with paraformaldehyde, embedded in paraffin, and cut into sections for immunostaining with antibody against PPARγ. Subsequently, sections were deparaffinized using a graded series of ethanol, and stained using primary antibody against PPARγ. After washing with PBS, sections were incubated with EnVision+/HRP/Rb (DAKO, Glostrup, Denmark) for 30 min at room temperature. The staining was visualized using 3, 3’-diaminobenzidine (DAB) substrate and then counterstained with hematoxylin for 30 s. All sections were photographed using an Olympus BH2 microscope (Olympus).

Cell culture and treatment

HK-2 cells were obtained from China Center for Type Culture Collection (CCTCC, Wuhan, China) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere with 5% CO₂.

For experimental study, HK-2 cells were pretreated with GE (300 μg/ml) for 1 h, or 10 μM GW9662 (a PPARγ antagonist) for 30 min before GE incubation (100, 200 and 300 μg/ml) and stimulated with LPS (1 μg/ml) for 24 h.

Flow cytometry

For apoptotic rate analysis of LPS-induced HK-2 cells, flow cytometry was performed using an Annexin-V-FITC Apoptotic Detection kit (KeyGEN) as guided by the manufacturer. Briefly, after treatment for 24 h, cells were rinsed with PBS and stained with PI/FITC-Annexin V in the presence of RNase A (Sigma-Aldrich). Next, cells were grown for 30 min at room temperature in the dark. A FACScan flow cytometer (BD Biosciences, San Jose, CA, USA) was employed to detect apoptosis within 1 h.

Statistical analysis

All data were analyzed using Graphpad 4.0 software (Graphpad software, Inc., CA, USA) and SPSS 18.0 software (SPSS Inc., Chicago, IL, USA) and expressed as mean ± standard deviation (SD) from at least three independent experiments. Differences between groups were examined for statistical significance by variance analysis (ANOVA). A P value<0.05 was="" accepted="" as="" statistically="" significant.<="" p="">