PINK1 Deficiency Ameliorates Cisplatin-Induced Acute Kidney Injury in Rats

Abstract

Mitophagy plays a key role in cleaning damaged and depolarized mitochondria to maintain cellular homeostasis and viability. Although it was originally found in neurodegenerative diseases, mitophagy is reported to play an important role in acute kidney injury. PINK1 and Parkin are key molecules in mitophagy pathway. Here, we used PINK1 knockout rats to examine the role of PINK1/Parkin-mediated mitophagy in cisplatin nephrotoxicity. After cisplatin treatment, PINK1 knockout rats showed lower plasma creatinine and less tubular damage when compared with wild-type rats. Meanwhile, mitophagy indicated by autophagosome formation and LC3B-II accumulation was also attenuated in PINK1 knockout rats. Renal expression of PINK1 and Parkin were down-regulated while BNIP3L was up-regulated by cisplatin treatment, indicating a major role of BNIP3/BNIP3L pathway in cisplatin-induced mitophagy. Transmission electron microscopy showed that PINK1 deficiency inhibited cisplatin-induced mitochondrial fragmentation indicating an involvement of mitochondrial fusion and fission. Renal expression of mitochondrial dynamics related proteins including Fis1, Drp1, Mfn1, Mfn2, and Opa1 were checked by real-time PCR and western blots. The results showed PINK1 deficiency distinctly prevented cisplatin-induced up-regulation of DRP1. Finally, PINK1 deficiency alleviated cisplatin-induced tubular apoptosis indicated by TUNEL assay as well as the expression of caspase3 and cleaved caspase3. Together, these results suggested PINK1 deficiency ameliorated cisplatin-induced acute kidney injury in rats, possibly via inhibiting DRP1-mediated mitochondrial fission and excessive mitophagy.

Keywords: DRP1; PINK1; mitochondrial dynamics; mitophagy; nephrotoxicity.

FIGURE 1

PINK1 deficiency attenuated cisplatin-induced acute kidney injury. PINK1 KO and WT rats were injected with 5 mg/kg cisplatin or saline for 72 h. (A) Plasma level of BUN in PINK1 WT and KO rats after cisplatin treatment. (B) Plasma level of creatinine in PINK1 WT and KO rats after cisplatin treatment. (C) Pathological score of tubular damage. (D) Representative histology of kidney cortex by PAS staining. N = 6 in each group. ^∗P < 0.05, WT + cisplatin vs. WT + saline; ^#P < 0.05, KO + cisplatin vs. WT + cisplatin; ^ΔP < 0.05, KO + cisplatin vs. KO + saline. Data are mean ± SE.

FIGURE 2

PINK1 deficiency prevented cisplatin-induced mitophagy. (A) Representative images of LC3B immunostaining. (B) Representative transmission electron microscope images of renal proximal tubular cells and mitochondria. L indicated the proximal tubular lumen. The arrow indicated a representative mitophagosome. (C) Quantitative analysis of the length/width of mitochondria in proximal tubular cells. (D) Transmission electron microscope images of large vacuoles containing numerous autophagosomes and mitophagosomes in cisplatin treated WT rats. (E) Quantitative analysis of the western blotting of LC3BII. (F) Representative western blot bands of LC3BII in renal cortex. N = 3 in WT + saline group and KO + saline group, N = 4 in WT + cisplatin group and KO + cisplatin group. ^∗P < 0.05, WT + cisplatin vs. WT + saline; ^#P < 0.05, KO + cisplatin vs. WT + cisplatin; ^ΔP < 0.05, KO + cisplatin vs. KO + saline. Data are mean ± SE.

FIGURE 3

Altered expression of PINK1, Parkin and BNIP3L during cisplatin treatment in PINK1 WT and KO rats. (A) Real-time quantitative PCR analysis of Pink1 mRNA expression in renal cortex. (B) Real-time quantitative PCR analysis of Parkin mRNA expression in renal cortex. (C) Quantitative analysis of the western blotting of Parkin. (D) Quantitative analysis of the western blotting of BNIP3L. (E) Representative western blot bands of Parkin and BNIP3L in renal cortex. N = 3 in WT + saline group and KO + saline group, N = 4 in WT + cisplatin group and KO + cisplatin group. ^∗P < 0.05, WT + cisplatin vs. WT + saline; ^#P < 0.05, KO + cisplatin vs. WT + cisplatin; ^ΔP < 0.05, KO + cisplatin vs. KO + saline; ^$P < 0.05, KO + saline vs. WT + saline. Data are mean ± SE.

FIGURE 4

The effect of PINK1 deficiency on mitochondrial dynamics related proteins during cisplatin treatment. (A–E) Real-time quantitative PCR analysis of Fis1, Drp1, Mfn1, Mfn2, and Opa1 mRNA expression in renal cortex. (F) Quantitative analysis of the western blotting of DRP1. (G) Representative western blot bands of DRP1 in renal cortex. N = 3 in WT + saline group and KO + saline group, N = 4 in WT + cisplatin group and KO + cisplatin group. ^∗P < 0.05, WT + cisplatin vs. WT + saline; ^#P < 0.05, KO + cisplatin vs. WT + cisplatin; ^ΔP < 0.05, KO + cisplatin vs. KO + saline; ^$P < 0.05, KO + saline vs. WT + saline. Data are mean ± SE.

FIGURE 5

PINK1 deficiency attenuated cisplatin-induced renal tubular cell apoptosis. (A) Presented are the representative photographs of TUNEL staining of kidney tissues in PINK1 WT and KO rats after cisplatin treatment. (B) Quantitative analysis of TUNEL-positive cells among the various groups. (C,D) Quantitative analysis of the western blotting of cleaved caspase3 and caspase3. (E) Representative western blot bands of cleaved caspase3 and caspase3. N = 3 in WT + saline group and KO + saline group, N = 4 in WT + cisplatin group and KO + cisplatin group. ^∗P < 0.05, WT + cisplatin vs. WT + saline; ^#P < 0.05, KO + cisplatin vs. WT + cisplatin; ^ΔP < 0.05, KO + cisplatin vs. KO + saline; ^$P < 0.05, KO + saline vs. WT + saline. Data are mean ± SE.

FIGURE 6

Schematic representation of possible mechanism related to mitophagy in cisplatin-induced kidney injury in PINK1 KO rats. Cisplatin causes the mitochondrial damage of renal tubular epithelial cells, which induces BNIP3/BNIP3L-mediated mitophagy, DRP1-mediated mitochondrial fission and apoptosis leading to kidney injury. And the excessive mitophagy may have negative feedback inhibition effect on the expression of PINK1 and Parkin and result in the down-regulation of PINK1 and Parkin in WT rats. In PINK1 KO rats, PINK1 deficiency causes a compensatory increase of Parkin and significantly inhibits the up-regulation of DRP1, which regulates mitochondrial fission and apoptosis, and therefore to inhibit the excessive mitophagy and overall kidney damage.