Small GTP-binding protein GDP dissociation stimulator influences cisplatin-induced acute kidney injury via PERK-dependent ER stress

Abstract

Cisplatin is a common anticancer drug, but its frequent nephrotoxicity limits its clinical use. Small GTP-binding protein GDP dissociation stimulator (smgGDS), a small GTPase chaperone protein, was considerably downregulated during cisplatin-induced acute kidney injury (CDDP-AKI), especially in renal tubular epithelial cells. SmgGDS-knockdown mice was established and found that smgGDS knockdown promoted CDDP-AKI, as demonstrated by an increase in serum creatine, blood urea nitrogen levels and the appearance of tubular patterns. RNA sequencing suggested that protein kinase RNA-like ER kinase (PERK), which bridges mitochondria-associated ER membranes, was involved in smgGDS knockdown following CDDP-AKI, and then identified that smgGDS knockdown increased phosphorylated-PERK in vivo and in vitro. Furthermore, we confirmed that smgGDS deficiency aggravated apoptosis and ER stress in vivo and in vitro. And the ER stress inhibitor 4-Phenylbutyric acid and the inhibition of PERK phosphorylation mitigated smgGDS deficiency-induced ER stress related apoptosis following cisplatin treatment, while the eIF2α phosphorylation inhibitor could not reverse the smgGDS deficiency accelerated cell death. Furthermore, the over-expression of smgGDS could reverse the ER stress and apoptosis caused by CDDP. Overall, smgGDS regulated PERK-dependent ER stress and apoptosis, thereby influencing renal damage. This study identified a target for diagnosing and treating cisplatin-induced acute kidney injury.

Fig. 1. SmgGDS decreased in CDDP-AKI.

a The HE and PAS staining of C57BL/6 J mice kidney treated with saline or cisplatin (scale bar: 50 μm); (b) the kidney injury score of C57BL/6 J mice kidney treated with saline or cisplatin; (c, d) serum creatinine and blood urea nitrogen determined at 72 h after CDDP or saline injection in C57BL6/J mice; (e) immunofluorescence staining of smgGDS (red) and cadherin-16 (green) in the kidney cortex of C57BL/6 J mice treated with saline or CDDP for 72 h (magnification: ×400, scale bar: 50 μm); (f) immunofluorescence staining of smgGDS (red) and cadherin-16 (green) in TCMK-1 treated with CDDP or saline (control group) (magnification: ×1000, scale bar: 50 μm); (g, h) western blot of smgGDS in kidney (n = 6) and TCMK-1 (n = 3) protein expression treated with saline or CDDP and quantified by ImageJ. CDDP, cisplatin; TCMK-1, mouse renal tubular epithelial cells; DAPI, nuclei; smgGDS, small GTP-binding protein GDP dissociation stimulator. All data presented are the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2. SmgGDS insufficiency promoted the cisplatin-induced AKI.

a Coronal sections of the kidney from WT and KD mice injected with CDDP or saline intraperitoneally for 72 h; (b) the ratio of kidney and body weight (n = 6); (c) representative images of HE, PAS, and immunohistochemistry staining of NGAL of kidneys collected from WT and KD mice injected with CDDP or saline intraperitoneally for 72 h (magnification: ×200 HE and PAS upper panel, scale bar: 50 μm; ×400 HE, PAS and immunohistochemistry staining of NGAL lower panel, scale bar: 50 μm); (d) the injury score was evaluated as described in “Methods” (n = 6); (e, f) serum creatinine and blood urea nitrogen determined at 72 h after CDDP or saline injection (n = 6); (g) western blot KIM-1 and NGAL protein expression in the kidney collected from WT and KD mice injected with CDDP or saline and quantified by ImageJ (n = 6). WT wildtype, KD smgGDS^+/−, CDDP cisplatin, HE hematoxylin–eosin staining, PAS periodic acid–Schiff staining, DAPI nuclei, smgGDS small GTP-binding protein GDP dissociation stimulator, NGAL neutrophil gelatinase-associated lipocalin, KIM-1 kidney injury molecule 1. All data presented are the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3. PERK involved in smgGDS-mediated CDDP-AKI process.

Transcriptome sequencing analysis of vector and smgGDS-knockdown TCMK-1 treated with CDDP (NC + CDDP vs. KD + CDDP). a Scheme of RNA-seq and bioinformatics analysis to screen the effector molecule of smgGDS knockdown; a volcano plot (b) and heat map (c) of different genes compared with NC + CDDP with KD + CDDP (n = 4, Log₂^{(fold change)} ≥ 1.5, P < 0.05); (d) TOP10 genes related with the cell death and biological process of smgGDS knockdown affected in the CDDP-AKI; (e) KEGG analysis of top 20 pathways; (f) protein expression of PERK and p-PERK in vivo quantified by ImageJ (n = 6); (g) protein expression of PERK and p-PERK in vitro quantified by ImageJ (n = 3). WT wildtype, KD smgGDS^+/−, CDDP cisplatin, smgGDS small GTP-binding protein GDP dissociation stimulator, KEGG Kyoto Encyclopedia of Genes and Genomes. All data presented are the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4. SmgGDS insufficiency enhanced mitochondria-dependent apoptosis in CDDP-AKI.

a Western blot to detect the expression and activation of Caspase-3, PARP, Bcl-2, and BAX in the kidneys of WT and KD mice injected with CDDP or saline intraperitoneally for 72 h was quantified by ImageJ (n = 6); (b) western blot to detect the protein expression and activation of Caspase-3, Bcl-2, and BAX in TCMK-1 treated with CDDP or saline was quantified by ImageJ (n = 3); (c) representative images of apoptotic cells labeled by TUNEL in the kidney cortex of WT and KD mice injected with CDDP or saline intraperitoneally for 72 h, and the TUNEL-positive cells were counted as described in the “Methods” Section (scale bar: 50 μm). d Apoptosis detection by flow cytometry labeled with PI and Annexin V FITC in NC and KD TCMK-1 treated with CDDP or saline; CDDP cisplatin, NC control group of TCMK-1, KD knockdown of smgGDS in TCMK-1, WT wildtype, KD smgGDS^+/−, TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; smgGDS, small GTP-binding protein GDP dissociation stimulator. All data presented are the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5. SmgGDS insufficiency aggravated endoplasmic reticulum stress in CDDP-AKI.

a Western blot to detect the protein expression of eIF2α, p- eIF2α, GRP78, ATF4 and CHOP in the kidneys of WT and KD mice injected with CDDP or saline intraperitoneally for 72 h; (b) western blot to detect the protein expression of eIF2α, p- eIF2α, GRP78, ATF4 and CHOP in TCMK-1 treated with CDDP or saline; (c) transmission electron microscopy to observe the morphology of subcellular organelles in the kidneys of WT and KD mice injected with CDDP or saline (magnification: ×25.0 k, scale bar: 500 nm); WT wildtype, KD smgGDS^+/−, DAPI nuclei, CDDP cisplatin, smgGDS, small GTP-binding protein GDP dissociation stimulator. All data presented are the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 6. Suppressed of endoplasmic reticulum stress reduced apoptosis in CDDP-induced AKI.

a Apoptosis detected by flow cytometry labeled with PI and Annexin V FITC in NC and KD TCMK-1 treated with CDDP or saline and/or 4-PBA; (b) western blot to detect the influence of 4-PBA to the protein expression of smgGDS, p-PERK, PERK, GRP78, ATF4, CHOP, Cleaved-Caspase3, Pro-Caspase3, Bcl-2, and BAX in NC and KD TCMK-1 treated with CDDP or saline; 4-PBA 4-phenylbutyric acid, CDDP cisplatin, smgGDS small GTP-binding protein GDP dissociation stimulator. All data presented are the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 7. SmgGDS insufficiency increased apoptosis via PERK-dependent ER stress.

a The protein expression and phosphorylation of PERK and eIF2α in CDDP or saline-treated TCMK-1 treated with PERKi (5 nM) or ISRIB (25 nM); (b) the protein expression and activation related to apoptosis including Caspase-3, Bcl-2, BAX, GRP78, ATF4, and CHOP normalized by β-actin in CDDP or saline-treated TCMK-1 treated with PERKi (5 nM); (c) apoptosis detected by flow cytometry labeled by PI and Annexin V FITC in CDDP or saline-treated NC and KD TCMK-1 treated with PERKi (5 nM); (d) the mitochondria membrane potential detection in CDDP or saline-treated NC and KD TCMK-1 treated with PERKi (5 nM, scale bar: 20 μm). CDDP cisplatin, PERKi selective PERK phosphorylation inhibitor, smgGDS small GTP-binding protein GDP dissociation stimulator, JC-1 mitochondrial membrane potential probe. All data presented are the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 8. SmgGDS overexpression alleviated CDDP-induced apoptosis via PERK-dependent ER stress.

a Apoptosis detected by flow cytometry labeled by 7-AAD and Annexin V PE in CDDP or saline-treated OE-NC and OE TCMK-1 treated with CCT020312 (10 μM); (b) the protein expression and activation related to apoptosis including smgGDS, Caspase-3, Bcl-2, and BAX normalized by β-actin in CDDP or saline-treated OE-NC and OE TCMK-1 treated with CCT020312 (10 μM); (c) the protein expression including smgGDS, GRP78, ATF4, and CHOP normalized by β-actin in CDDP or saline-treated OE-NC and OE TCMK-1 treated with CCT020312 (10 μM). CDDP, cisplatin; CCT020312, PERK phosphorylation agonist; OE overexpression of smgGDS in TCMK-1, smgGDS small GTP-binding protein GDP dissociation stimulator. All data presented are the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.