Syndecan-1 Shedding Inhibition to Protect Against Ischemic Acute Kidney Injury Through HGF Target Signaling Pathway

Abstract

Background: The hepatocyte growth factor (HGF) target pathway plays pivotal renoprotective roles after acute kidney injury. Syndecan-1 (SDC-1) serves as the coreceptor for HGF. Shedding of SDC-1 is involved in various pathological processes. Thus, we hypothesized that ischemia/reperfusion injury induced SDC-1 shedding, and inhibiting SDC-1 shedding would protect against kidney injury by potentiating activation of the HGF receptor mesenchymal epithelial transition factor (c-Met).

Methods: Expression of SDC-1 and its sheddases were observed in kidneys of sham and ischemia/reperfusion (I/R) mice. To inhibit SDC-1 shedding, mice were injected with the sheddase inhibitor GM6001 before I/R surgery, and then, renal inflammation, tubular apoptosis, and activation of the c-Met/AKT/glycogen synthase kinase-3β (GSK-3β) pathway were analyzed. In vitro, human proximal tubular cell lines were pretreated with GM6001 under hypoxia/reperfusion conditions. The apoptosis and viability of cells and expression of c-Met/AKT/GSK-3β pathway components were evaluated. The relationship was further confirmed by treatment with SU11274, a specific inhibitor of phospho-c-Met.

Results: Shedding of SDC-1 was induced after ischemia/reperfusion injury both in vivo and in vitro. GM6001 pretreatment suppressed SDC-1 shedding, alleviated renal inflammation and tubular apoptosis, and upregulated phosphorylation of the c-Met/AKT/GSK-3β pathway. In vitro, pretreatment with GM6001 also decreased hypoxia/reperfusion-induced cell apoptosis and promoted activation of the c-Met pathway. In addition, the cytoprotective role of GM6001 was attenuated by suppressing c-Met phosphorylation with SU11274.

Conclusions: Our findings suggest that inhibiting I/R-induced SDC-1 shedding protected against ischemic acute kidney injury by potentiating the c-Met/AKT/GSK-3β pathway.

FIGURE 1.

The distribution of SDC-1 in the kidney and shedding of SDC-1 was increased after IRI. A-B, Representative photomicrographs of immunohistochemical staining for SDC-1 of renal sections from sham and I/R-operated mice. A, Syndecan-1-positive immunohistochemical staining was mainly observed in the cortico-medullary zone of the kidney, with little expression in the medulla. Syndecan-1 was localized at both the basolateral and luminal sides of tubular cells. According to PAS staining, SDC-1 was primarily expressed in the glycogen structure on the luminal sides of tubular cells; B, compared with sham-operated kidney, SDC-1–positive immunohistochemical staining appeared in the medulla of the kidney. C, The ELISA results of serum soluble SDC-1 levels in sham and I/R groups. D, The relative mRNA expression levels of MMP7, MMP9, and ADAM17 in kidneys from the 2 groups. The expression levels of MMP7, MMP9, and ADAM17 were all increased, and the elevation of MMP7 mRNA level was the most evident; E, Western blot analysis of SDC-1 and MMP7 in I/R challenged kidneys. F-G, Representative photomicrographs and quantitative analysis of MMP7 immunohistochemical staining of renal sections from mice in the sham and I/R groups. The data are shown as the mean ± SEM, **P < 0.01; ***P < 0.001; n = 6.

FIGURE 2.

Inhibiting SDC-1 shedding by pretreatment with GM6001 protected against IR-induced renal injury. Mice in the GM6001 + I/R group were pretreated with GM6001 (100 mg/kg) 18 hours before I/R surgery. A, The serum soluble SDC-1 levels in I/R and I/R + GM6001 groups determined by ELISA. B, Serum creatinine levels in the 3 groups. C, Representative images of kidney sections stained with PAS in sham, I/R and I/R + GM6001 groups. D, Assessment of morphologic injury quantified with histologic scores in each group. The data are presented as the mean ± SEM; ***P < 0.001 vs sham group; ^#P < 0.05 and ^##P < 0.01 vs I/R group; n = 6.

FIGURE 3.

Pretreatment with GM6001 attenuated I/R-induced inflammatory cell infiltration and cytokine production. A, Representative images of LY6G (marker for neutrophils) immunohistochemical staining in renal sections from sham, I/R and I/R + GM6001 groups. B, Number of Ly6G⁺ cells in renal sections. C, Representative images of F4/80 (marker for macrophages) immunohistochemical staining in renal sections from sham, I/R and I/R + GM6001 groups. D, Number of F4/80⁺ cells in renal sections. E, The relative mRNA expression levels of IL-6, TNF-α, and IL-10 in kidneys from the 3 groups. F, Protein concentration of inflammatory and anti-inflammatory cytokines in kidneys from each group. The data are presented as the mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001 vs sham group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs I/R group; n = 6.

FIGURE 4.

Pretreatment with GM6001 inhibited I/R-induced apoptosis of tubular epithelial cells. A, Representative immunofluorescence images of TUNEL staining of kidneys from mice in the sham, I/R and I/R + GM6001 groups. Nuclei were stained with DAPI (blue), and green fluorescence represents TUNEL-positive nuclei. B, Quantitative analysis of TUNEL-positive cells in renal sections from the 3 groups. C, Western blot analysis of Bax in samples from sham, I/R and I/R + GM6001 groups. D, Western blot analysis of Bcl2 from these groups; E, Quantitative analysis of the Bax/Bcl2 expression ratio in the 3 groups. F, Representative immunoblots of the cleaved and total caspase-3 expression in these groups and the ratio of cleaved and total caspase-3. The data are presented as the mean ± SEM; **P < 0.01 and ***P < 0.001 vs sham group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs I/R group (n = 6).

FIGURE 5.

H/R-induced shedding of SDC-1 and inhibition of SDC-1 shedding by GM6001 alleviated apoptosis of HK-2 cells. A, The concentration of soluble SDC-1 in HK-2 cell culture medium determined by ELISA under H/R conditions; B-C, Western blot analysis of sydencan-1 and MMP7 expression in H/R-treated HK-2 cells; D, HK-2 cells in the GM6001 + H/R groups were administered different doses of GM6001 (5 μM, 10 μM, 20 μM, 50 μM, or 100 μM) before H/R treatment. GM6001 inhibited SDC-1 shedding in a dose-dependent manner. The data are presented as the mean ± SEM. *P < 0.05; **P < 0.01 (n = 6) E, Cell viability measured by CCK8. The cell viability was increased when cells were pretreated with 10 μM GM6001 under hypoxia but decreased with an increase of the GM6001 dose. The data are presented as the mean ± SEM; *P < 0.05; **P < 0.01 (n = 6). F, Typical images of Annexin-V/PI staining in cells pretreated with GM6001 (10 μM) under hypoxia, evaluated by flow cytometry. G, Quantitative analysis of the apoptosis ratio using FlowJo Version X software. H, Representative immunoblots of Bax and Bcl2 in samples from control, H/R, and H/R + GM6001 (10 μM) groups. The data are presented as the mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001 vs control group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs H/R group; n = 4 for flow cytometry analysis; n = 6 for the other studies.

FIGURE 6.

Inhibition of syndecan-1 shedding improved activation of the c-Met/AKT/GSK-3β pathway in vivo and in vitro. A-C, Representative immunoblots of the renal protein expression levels of p-Met, c-Met, p-AKT, AKT, p-GSK-3β and GSK-3β, and quantitative analysis of the phosphorylation level of c-Met, AKT, and GSK-3β in sham, I/R and I/R + GM6001 groups. The data are presented as the mean ± SEM; **P < 0.01 vs sham group; ^##P < 0.01 and ^###P < 0.001 vs IR group; n = 6. D-F, Representative immunoblots of p-Met, c-Met, p-AKT, AKT, p-GSK-3β and GSK-3β and quantitative analysis of the phosphorylation level of c-Met, AKT, and GSK-3β in samples from control, H/R and H/R + GM6001 groups. The data are presented as the mean ± SEM; **P < 0.01 and ***P < 0.001 vs control group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs H/R group; n = 6.

FIGURE 7.

Inhibiting phosphorylation of c-Met with SU11274 blunted the protective role of GM6001. A, HK-2 cells were administered SU11274 (1 μM, 5 μM, or 10 μM) 18 hours before the use of GM6001 (10 μM) under H/R conditions. Representative immunoblots and quantitative analysis of p-Met and c-Met levels in these groups. Phosphorylation of c-Met was blocked by treating HK-2 cells with 1, 5, or 10 μM SU11274. B-C, Representative immunoblots of p-AKT, AKT, p-GSK-3β, and GSK-3β expression and statistical analysis of the p-AKT and p-GSK-3β protein levels in samples from control, H/R, H/R + GM6001, and H/R + GM6001 + SU11274 (1 μM) groups. D, Representative immunoblots of Bax and Bcl2 expression and quantitative analysis of Bax and Bcl2 levels and the Bax/Bcl2 expression ratio in control, H/R, H/R + GM6001 and H/R + GM6001 + SU11274 (1 μM) groups. E, Measurement of cell viability using CCK8 in HK-2 cells pretreated with 1 μM SU11274 18 hours before the use of GM6001 under hypoxia. F, Apoptosis of HK-2 cells pretreated with GM6001 (10 μM) and/or SU11274 (1 μM) under hypoxia evaluated by flow cytometry. The data are presented as the mean ± SEM; **P < 0.01 and ***P < 0.001 vs control group; ^#P < 0.05, ^###P < 0.01, and ^####P < 0.001 vs H/R group; ^$$P < 0.01 and ^$$$P < 0.001 vs H/R + GM6001 group; n = 6.

FIGURE 8.

Mechanism of the renoprotective function of SDC-1 shedding inhibition in I/R-induced kidney injury. A. the main significant results in this study; B, the diagram depicting the pathways involved in SDC-1. As the coreceptor of HGF, SDC-1 facilitates activation of c-Met and its downstream signal transduction. ischemia/reperfusion improves the activity of SDC-1 shedding mediated by MMPs. However, as a self-protection mechanism of tubular epithelial cells, the production of HGF was upregulated evidently at the same time, resulting in the activation of the downstream pathway AKT/GSK-3β in I/R challenged kidneys. GM6001 pretreatment suppresses shedding of SDC-1, resulting in a more evident increase in the phosphorylation of c-Met/AKT/GSK-3β, an elevation in Bcl2 expression, a reduction in Bax and cleaved caspase-3 activation, and downregulation of inflammatory cytokine production, consequently inhibiting apoptosis and inflammation.