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. 2016 Jul 12;17(7):1051.
doi: 10.3390/ijms17071051.

Stanniocalcin-1 Protects a Mouse Model from Renal Ischemia-Reperfusion Injury by Affecting ROS-Mediated Multiple Signaling Pathways

Dajun Liu  1 , Huiping Shang  2 , Ying Liu  3
Affiliations

Stanniocalcin-1 Protects a Mouse Model from Renal Ischemia-Reperfusion Injury by Affecting ROS-Mediated Multiple Signaling Pathways

Dajun Liu et al. Int J Mol Sci. .

Abstract

Stanniocalcin-1 (STC-1) protects against renal ischemia-reperfusion injury (RIRI). However, the molecular mechanisms remain widely unknown. STC-1 inhibits reactive oxygen species (ROS), whereas most ROS-mediated pathways are associated with ischemic injury. Therefore, to explore the mechanism, the effects of STC-1 on ROS-medicated pathways were studied. Non-traumatic vascular clamps were used to establish RIRI mouse models. The serum levels of STC-1, interleukin-6 (IL-6), interferon (IFN) γ, P53, and capase-3 were measured by ELISA kits. Superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by fluorescence spectrofluorometer. All these molecules changed significantly in a RIRI model mouse when compared with those in a sham control. Kidney cells were isolated from sham and model mice. STC-1 was overexpressed or knockout in these kidney cells. The molecules in ROS-medicated pathways were measured by real-time quantitative PCR and Western blot. The results showed that STC-1 is an effective ROS scavenger. The serum levels of STC-1, MDA and SOD activity were increased while the serum levels of IL-6, iIFN-γ, P53, and capase-3 were decreased in a model group when compared with a sham control (p < 0.05). Furthermore, the levels of STC-1,p53, phosphorylated mitogen-activated protein kinase kinase (p-MEKK-1), c-Jun N-terminal kinase (p-JNK), extracellular signal-regulated kinase (p-ERK), IkB kinase (p-IKK), nuclear factor (NF) κB, apoptosis signal-regulating kinase 1 (ASK-1) and caspase-3 changed significantly in kidney cells isolated from a RIRI model when compared to those isolated from a sham control (p < 0.05). Meanwhile, STC-1 overexpression or silence caused significant changes of the levels of these ROS-mediated molecules. Therefore, STC-1 maybe improve anti-inflammation, anti-oxidant and anti-apoptosis activities by affecting ROS-mediated pathways, especially the phospho-modifications of the respective proteins, resulting in the increase of SOD and reduce of capase-3, p53, IL-6 and IFN-γ.

Keywords: apoptosis signal-regulating kinase 1; caspase-3; extracellular signal-regulated kinase; p-IkB kinase; p-NF-κB; phosphorylated mitogen-activated protein kinase kinase; protein kinase C; reactive oxygen species-mediated pathways; renal ischemia-reperfusion injury; stanniocalcin-1.

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Figures

Figure 1
Figure 1
Creatinine clearance rate in mice. RIRI, Renal ischemia-reperfusion injury. All data were presented as mean values ± S.D. n = eight in each group. * p < 0.05 compared to the sham group.
Figure 2
Figure 2
The serum biochemical and immunological parameters in different treated mice: (A) the serum protein levels of IL-6; (B) the serum protein levels of IFN-γ; (C) the serum protein levels of p53; (D) the serum protein levels of caspase-3; (E) the serum activity of SOD; (F) the serum protein levels of MDA; and (G) the serum protein levels of STC-1. Among 16 mice, eight mice were used to create RIRI models and another eight mice were used as a sham group. All data were presented as mean values ± S.D. * p < 0.05 compared to the model group.
Figure 3
Figure 3
ROS production in different cells. Among 16 mice, eight mice were used to create RIRI models as a model group and eight mice were used as a sham group. Renal progenitor cells were isolated from the kidney cortex of RIRI model and sham mice after 16-h surgery. For the kidney cells isolated from eight model mice, they were further subdivided into three groups based on different treatment options (model group (MG); STC-1 expression group (MSG), the mice were transfected with STC-1 gene to overexpress STC-1; and STC-1 shRNA group (MSShG), the mice were transfected with STC-1 shRNA to knockdown STC-1). In the same way, the kidney cells from eight sham mice were subdivided into another three groups based on different treatment options sham groups (sham group (SG); STC-1 expression group (SSG), the mice were transfected with STC-1 gene to overexpress STC-1; and STC-1 shRNA group (SSShG), the mice were transfected with STC-1 shRNA to knockdown STC-1). The ROS levels were measured from all the cells cultured for 24 h. All data were presented as the mean values ± S.D. * p < 0.05 via a model group and # p < 0.05 via a sham group.
Figure 4
Figure 4
The effects of STC-1 on the mRNA levels of ROS-mediated molecules. Among 16 mice, eight mice were used to create RIRI models as a model group and eight mice were used as a sham group. Renal progenitor cells were isolated from the kidney cortex of RIRI model and sham mice after 16-h surgery. For the kidney cells isolated from eight model mice, they were further subdivided into three groups based on different treatment options (model group (MG); STC-1 expression group (MSG), the mice were transfected with STC-1 gene to overexpress STC-1; and STC-1 shRNA group (MSShG), the mice were transfected with STC-1 shRNA to knockdown STC-1). In the same way, the kidney cells from 8 sham mice were subdivided into another three groups based on different treatment options (sham group (SG); STC-1 expression group (SSG), the mice were transfected with STC-1 gene to overexpress STC-1; and STC-1 shRNA group (SSShG), the mice were transfected with STC-1 shRNA to knockdown STC-1). The mRNA levels were measured from the same part of renal tissues obtained after 4-h surgery: (A) the changes of mRNA levels of STC-1 in different groups; (B) the changes of mRNA levels of PKC-α in different groups; (C) the changes of mRNA levels of ERK in different groups; (D) the changes of mRNA levels p53 in different groups; (E) the changes of mRNA levels of IKK in different groups; (F) the changes of mRNA levels of MEKK-1 in different groups; (G) the changes of mRNA levels of JNK in different groups; (H) the changes of mRNA levels of ASK-1 in different groups; (I) the changes of mRNA levels of caspase-3 in different groups; and (J) the changes of mRNA levels of NF-κB in different groups. All data were normalized to actin, and presented as the mean values ± S.D. * p < 0.05 compared to the model group and # p < 0.05 compared to the sham group.
Figure 5
Figure 5
The effects of STC-1 on the protein levels of PKC-α, p-IKK, p-MEKK-1, p-NF-κB and caspase-3. Among 16 mice, eight mice were used to create RIRI models as a model group and eight mice were used as a sham group. Renal progenitor cells were isolated from the kidney cortex of RIRI model and sham mice after 16-h surgery. For the kidney cells isolated from eight model mice, they were further subdivided into three groups based on different treatment options (model group (MG); STC-1 expression group (MSG), the mice were transfected with STC-1 gene to overexpress STC-1; and STC-1 shRNA group (MSShG), the mice were transfected with STC-1 shRNA to knockdown STC-1). In the same way, the kidney cells from 32 sham mice were subdivided into another three groups based on different treatment options (sham group (SG); STC-1 expression group (SSG), the mice were transfected with STC-1 gene to overexpress STC-1; and STC-1 shRNA group (SSShG), the mice were transfected with STC-1 shRNA to knockdown STC-1). The mRNA levels were measured from the same part of renal tissues obtained after 4-h surgery: (A) the changes of protein levels of STC-1 in different groups; (B) the changes of protein levels of PKC-α in different groups; (C) the changes of protein levels of p-ERK in different groups; (D) the changes of protein levels p53 in different groups; (E) the changes of protein levels of p-IKK in different groups; (F) the changes of protein levels of p-MEKK-1 in different groups; (G) the changes of protein levels of p-JNK in different groups; (H) the changes of protein levels of ASK-1 in different groups; (I) the changes of protein levels of caspase-3 in different groups; (J) the changes of protein levels of NF-κB in different groups; and (K) the ratio of non-phosphate proteins and phosphate proteins. All data were normalized to actin, and presented as the mean values ± S.D. * p < 0.05 via model group and # p < 0.05 via sham group.
Figure 5
Figure 5
The effects of STC-1 on the protein levels of PKC-α, p-IKK, p-MEKK-1, p-NF-κB and caspase-3. Among 16 mice, eight mice were used to create RIRI models as a model group and eight mice were used as a sham group. Renal progenitor cells were isolated from the kidney cortex of RIRI model and sham mice after 16-h surgery. For the kidney cells isolated from eight model mice, they were further subdivided into three groups based on different treatment options (model group (MG); STC-1 expression group (MSG), the mice were transfected with STC-1 gene to overexpress STC-1; and STC-1 shRNA group (MSShG), the mice were transfected with STC-1 shRNA to knockdown STC-1). In the same way, the kidney cells from 32 sham mice were subdivided into another three groups based on different treatment options (sham group (SG); STC-1 expression group (SSG), the mice were transfected with STC-1 gene to overexpress STC-1; and STC-1 shRNA group (SSShG), the mice were transfected with STC-1 shRNA to knockdown STC-1). The mRNA levels were measured from the same part of renal tissues obtained after 4-h surgery: (A) the changes of protein levels of STC-1 in different groups; (B) the changes of protein levels of PKC-α in different groups; (C) the changes of protein levels of p-ERK in different groups; (D) the changes of protein levels p53 in different groups; (E) the changes of protein levels of p-IKK in different groups; (F) the changes of protein levels of p-MEKK-1 in different groups; (G) the changes of protein levels of p-JNK in different groups; (H) the changes of protein levels of ASK-1 in different groups; (I) the changes of protein levels of caspase-3 in different groups; (J) the changes of protein levels of NF-κB in different groups; and (K) the ratio of non-phosphate proteins and phosphate proteins. All data were normalized to actin, and presented as the mean values ± S.D. * p < 0.05 via model group and # p < 0.05 via sham group.
Figure 5
Figure 5
The effects of STC-1 on the protein levels of PKC-α, p-IKK, p-MEKK-1, p-NF-κB and caspase-3. Among 16 mice, eight mice were used to create RIRI models as a model group and eight mice were used as a sham group. Renal progenitor cells were isolated from the kidney cortex of RIRI model and sham mice after 16-h surgery. For the kidney cells isolated from eight model mice, they were further subdivided into three groups based on different treatment options (model group (MG); STC-1 expression group (MSG), the mice were transfected with STC-1 gene to overexpress STC-1; and STC-1 shRNA group (MSShG), the mice were transfected with STC-1 shRNA to knockdown STC-1). In the same way, the kidney cells from 32 sham mice were subdivided into another three groups based on different treatment options (sham group (SG); STC-1 expression group (SSG), the mice were transfected with STC-1 gene to overexpress STC-1; and STC-1 shRNA group (SSShG), the mice were transfected with STC-1 shRNA to knockdown STC-1). The mRNA levels were measured from the same part of renal tissues obtained after 4-h surgery: (A) the changes of protein levels of STC-1 in different groups; (B) the changes of protein levels of PKC-α in different groups; (C) the changes of protein levels of p-ERK in different groups; (D) the changes of protein levels p53 in different groups; (E) the changes of protein levels of p-IKK in different groups; (F) the changes of protein levels of p-MEKK-1 in different groups; (G) the changes of protein levels of p-JNK in different groups; (H) the changes of protein levels of ASK-1 in different groups; (I) the changes of protein levels of caspase-3 in different groups; (J) the changes of protein levels of NF-κB in different groups; and (K) the ratio of non-phosphate proteins and phosphate proteins. All data were normalized to actin, and presented as the mean values ± S.D. * p < 0.05 via model group and # p < 0.05 via sham group.
Figure 6
Figure 6
STC-1 acts a modulator for renal injury via affecting ROS-mediated multiple pathways. STC-1 is an effective ROS scavenger, which affects the levels of phosphorylated mitogen-activated protein kinase kinase kinase (p-MEKK-1), c-Jun N-terminal kinase (p-JNK), nuclear factor (NF) κB, extracellular signal-regulated kinase (p-ERK), IkB kinase (p-IKK), apoptosis signal-regulating kinase 1 (ASK-1), p53 and caspase-3. STC-1 affects ROS-mediated IKK-NF-κB, PKC-ERK-NF-κB, ASK-1-NF-κB, p53 and MEKK-JNK pathways, and improves anti-inflammation, anti-oxidant and anti-apoptosis activities by increasing level of superoxide dismutase (SOD) and reducing the level of capase-3, p53, interleukin-6 (IL-6) and IFN-γ.
Figure 7
Figure 7
The flowchart of the study. Among 16 mice, eight mice were used to create RIRI models as a model group and another eight mice were used as a sham group. The RIRI model was evaluated by comparing the creatine clearance rate between RIRI models and sham controls. The serum levels of interleukin-6 (IL-6), interferon (IFN) γ, P53, capase-3, superoxide dismutase (SOD) and malondialdehyde (MDA) were measured in these mice. Renal progenitor cells were isolated from the kidney cortex of RIRI model and sham mice after 16-h surgery. For the kidney cells isolated from eight model mice, they were further subdivided into three groups based on different treatment options (model group (MG); STC-1 expression group (MSG), the mice were transfected with STC-1 gene to overexpress STC-1; and STC-1 shRNA group (MSShG), the mice were transfected with STC-1 shRNA to knockdown STC-1). In the same way, the kidney cells from 32 sham mice were subdivided into another three groups based on different treatment options (sham group (SG); STC-1 expression group (SSG), the mice were transfected with STC-1 gene to overexpress STC-1; and STC-1 shRNA group (SSShG), the mice were transfected with STC-1 shRNA to knockdown STC-1). Real-time quantitative PCR and Western blot analysis analyzed the levels of Stanniocalcin-1 (STC-1), apoptosis signaling kinase (ASK-1), protein kinase C (PKC-α), phosphate mitogen-activated protein kinase kinase kinase (p-MEKK-1), MEKK-1, phosphate c-Jun N-terminal kinase (p-JNK), JNK, nuclear factor (NF) κB, phosphate extracellular signal-regulated kinase (p-ERK), ERK, phosphate IkB kinase (p-IKK), IKK, and caspase-3.
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