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, 9 (4), 1694-1707
eCollection

Melatonin Promoted Renal Regeneration in Folic Acid-Induced Acute Kidney Injury via Inhibiting Nucleocytoplasmic Translocation of HMGB1 in Tubular Epithelial Cells

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Melatonin Promoted Renal Regeneration in Folic Acid-Induced Acute Kidney Injury via Inhibiting Nucleocytoplasmic Translocation of HMGB1 in Tubular Epithelial Cells

Fengming Zhu et al. Am J Transl Res.

Abstract

Melatonin (N-acetyl-5-methoxytryptamine), a circadian-regulating hormone, has been reported to exert a protective role during acute kidney injury (AKI) induced by renal ischemia-reperfusion injury (I/R). High-mobility group box 1 (HMGB1) is a novel member of the damage-associated molecular pattern (DAMP) family, and has been verified to be an inflammatory cytokine mediating AKI induced by I/R and cisplatin. However, the effect of melatonin on HMGB1, as well as the relationship of these two with folic acid induced AKI are elusive. In this study, we sought to identify the role of melatonin on folic acid induced AKI and its association with HMGB1. Pretreatment with melatonin significantly attenuated folic acid-induced increase in serum creatinine and BUN levels, renal tubular epithelial cell (TEC) apoptosis, and the infiltration of inflammatory cells and secretion of cytokines. Moreover, melatonin pretreatment promoted renal tubular proliferation and improved cell cycle arrest of TECs after folic acid-induced renal damage. This protective role of melatonin was closely related to the inhibition of nucleocytoplasmic translocation of HMGB1 in TECs. These data provide a strong proof that administering melatonin prior to folic acid insult may shed light on a potential treatment for AKI.

Keywords: Acute kidney injury; HMGB1; melatonin; nucleocytoplasmic translocation; regeneration.

Conflict of interest statement

None.

Figures

Figure 1
Figure 1
Melatonin pretreatment improved renal function in folic acid-induced AKI in mice. (A) Scheme of the experimental plan, which showed that mice were treated with folic acid or pretreated with melatonin. (B) Gross appearance of representative kidneys from mice in each group. (C) Statistical analysis of the ratio of the left kidney to body weight. Serum creatinine (D) and BUN (E) levels among different groups. Group Normal, mice without treatment; Group NaHCO3, mice treated with vehicle; Group Mel, mice treated with melatonin alone; Group Folic Acid, mice treated with folic acid alone; Group FA+Mel, folic acid treated mice pretreated with melatonin for 1 week. N=5/group. Data were presented as means ± SEM. *P<0.05, **P<0.01, ***P<0.001.
Figure 2
Figure 2
Melatonin pretreatment attenuated renal pathological injury induced by folic acid. Representative photomicrographs of hematoxylin and eosin (HE) (A) and periodic acid-Schiff (PAS) (B) stained kidney sections. Nucleated cells in the black rectangule in (A) represented inflammatory cells. Black arrows in (B): represented the proximal tubule brush border; Triangles in (B): represented the expansion of tubules; Pentagon in (B): represented casts. Kidney sections immunolabeled with primary antibodies against LTL (C) and Kim-1 (D) were shown. LTL: lotus tetraglonolobus lectin; Kim-1: kidney injury molecule-1. (E) The general infiltration of inflammatory cells were quantified in 8 visual fields from HE stained sections. (F) Tubular damage was scored semiquantitatively using PAS-stained kidney sections. The remaining tubules were analysed by LTL staining (G). Injured tubules were assessed by Kim-1 staining (H). The gene expression level of intrarenal Kim-1 was assessed by RT-PCR (I). N=5/group. Scale bars =50 μm. Data were presented as the means ± SEM. *P<0.05, **P<0.01, ***P<0.001.
Figure 3
Figure 3
Melatonin pretreatment reduced infiltration of inflammatory cells and secretion of inflammatory cytokines. Kidney sections were immunolabeled with primary antibodies against Ly6G (A), CD3 (B) and F4/80 (C). The number of neutrophils (D), lymphocytes (E) and macrophages (F) infiltrated into kidneys were analysed in 8 visual fields using IF staining. (G-I) Real-time PCR of IL-6, IL-1β and TNF-α was performed to evaluate the expression levels of inflammatory cytokines in the kidney. N=5/group. Scale bars =50 μm. Data were presented as the means ± SEM. *P<0.05, **P<0.01, ***P<0.001.
Figure 4
Figure 4
Melatonin pretreatment mitigated the apoptosis of TECs. Kidney sections were immunolabeled with primary antibodies against TUNEL (A). The number of apoptotic tubules was analysed in 8 visual fields using TUNEL staining (D). The expression of Bax (B) and Bcl-2 (C) was assessed using immunoblotting. Quantitation of Bax (E) and Bcl-2 (F) was normalized to GAPDH. Statistics analyzed using unpaired t test (Mann-Whitney U test). N=5/group. Scale bars =50 μm. *P<0.05, **P<0.01, ***P<0.001. Values were means ± SEM.
Figure 5
Figure 5
Melatonin pretreatment promoted the proliferation and improved cell cycle arrest of TECs. Representative photographs of kidney sections immunolabeled with primary antibodies against Ki67, and double-stained with Ki67 and pH3 were shown (A). The proliferation of epithelial cells was assessed in 8 visual fields using Ki67 staining (B). Cells arrested in G2/M phase were analysed in 8 visual fields using double-staining with Ki67 and PH3 (C). Statistics analyzed using unpaired t test (Mann-Whitney U test). N=5/group. Scale bars =50 μm. *P<0.05, **P<0.01, ***P<0.001. Values were means ± SEM.
Figure 6
Figure 6
Melatonin pretreatment inhibited nucleocytoplasmic shuttling of HMGB1 in TECs. Representative photographs of kidney sections immunolabeled with primary antibodies against HMGB1 through IHC (A) and IF (B). Numbers of cytoplasmic HMGB1-positive tubules were calculated in (C) and (D). Total HMGB1 (E) in total protein and cytoplasmic HMGB1 (F) in cytoplastic protein were immunoblotted. The expression levels were normalized to GAPDH (G). (H) Statistics analyzed using unpaired t test (Mann-Whitney U test). N=5/group. Scale bars =50 μm. *P<0.05, **P<0.01, ***P<0.001. Values were means ± SEM.

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