Acetylbritannilactone attenuates contrast-induced acute kidney injury through its anti-pyroptosis effects

Abstract

Contrast-induced acute kidney injury (CI-AKI) is a severe complication caused by intravascular applied radial contrast media (CM). Pyroptosis is a lytic type of cell death inherently associated with inflammation response and the secretion of pro-inflammatory cytokines following caspase-1 activation. The aim of the present study was to investigate the protective effects of acetylbritannilactone (ABL) on iopromide (IOP)-induced acute renal failure and reveal the underlying mechanism. In vivo and in vitro, IOP treatment caused renal damage and elevated the caspase-1 (+) propidium iodide (PI) (+) cell count, interleukin (IL)-1β and IL-18 levels, lactate dehydrogenase (LDH) release, and the relative expression of nucleotide-binding domain, leucine-rich repeat containing protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), and gasdermin D (GSDMD), suggesting that IOP induces AKI via the activation of pyroptosis. Furthermore, the pretreatment of ABL partly mitigated the CI-AKI, development of pyroptosis, and subsequent kidney inflammation. These data revealed that ABL partially prevents renal dysfunction and reduces pyroptosis in CI-AKI, which may provide a therapeutic target for the treatment of CM-induced AKI.

Keywords: Acetylbritannilactone; Contrast media; acute kidney injury; pyroptosis.

Figure 1. IOP affected the percentage of caspase-1(+) PI (+) cells, cell viability, IL-1β levels, IL-18 levels, and LDH release activity in HK-2 cells in dosage- and time-dependent manner

HK-2 cells were incubated with IOP at the concentration of 0–160 mg I/ml for 2 h. In addition, HK-2 cells were cultured with IOP at 80 mg I/ml for 0–120 min, respectively. Cells were collected for follow-up experiments. (A–D) The percentage of caspase-1(+) PI (+) cells was determined by flow cytometry. (E,F) Exhibited the effect of IOP on cell viability. (G,H) The IL-1β levels were examined by ELISA. (I,J) IL-18 levels were detected by ELISA. (K,L) Showed the effect of IOP on LDH release activity. *P<0.05, **P<0.01.

Figure 2. IOP blocked the expression of proteins related to pyroptosis in dosage- and time-dependent ways

(A) HK-2 cells were incubated with IOP at a concentration of 0–160 mg I/ml for 2 h, respectively. Proteins were extracted for Western blot. The relative expression of NLRP3 (B), ASC (C), cleaved caspase-1 (D), mature GSDMD (E), and IL-1β (F) was normalized to GAPDH. (G) HK-2 cells were incubated with IOP (80 mg I/ml) for 0–120 min, respectively. Proteins were isolated for Western blot. The relative expression of NLRP3 (H), ASC (I), cleaved caspase-1 (J), mature GSDMD (K), and IL-1β (L) was calculated by the normalization to GAPDH. *P<0.05, **P<0.01.

Figure 3. ABL partly mitigated the effect of IOP on HK-2 cells

HK-2 cells were randomly divided into four groups: control, ABL, IOP, and ABL + IOP groups. Cells in the ABL + IOP group were pretreated with ABL (100 μM) for 2 h and then incubated with IOP (80 mg I/ml) for 1 h. Cells in ABL or IOP groups were given ABL for 2 h or IOP for 1 h, respectively. The control group was administered with the same amount of vehicle. (A,B) The percentage of caspase-1 (+) PI (+) cells of four groups was determined by flow cytometry. (C) Cell viability was examined by CCK-8 kits. (D,E) IL-1β and IL-18 levels were detected by ELISA kits. (F) Shows the LDH release activity of the four groups. (G–Q) Protein or total RNA was extracted for Western blot or RT-PCR. The protein levels of NLRP3, ASC, caspase-1, GSDMD, and IL-1β were normalized to GAPDH. The mRNA levels of NLRP3, ASC, caspase-1, GSDMD, and IL-1β were normalized to GAPDH. Compared with the control group, ^aP<0.01 and ^bP<0.01 in the ABL+IOP group vs IOP group.

Figure 4. ABL partly mitigated IOP-induced AKI via suppressing pyroptosis

Mice were assigned randomly into five groups: control group (Cont, n=6), vehicle group (Veh, n=6), ABL group (ABL, n=6), IOP group (n=6), and ABL + IOP group (n=6). Mice in the Cont or ABL group were given saline or ABL (25 mg/kg) via gavage administration for 6 days, respectively. Mice in the IOP + ABL group were first gavage administered with ABL for 6 days. On the sixth day, mice in IOP and IOP + ABL groups were injected intraperitoneally with indomethacin, NG-nitro-l-arginine methyl ester, and IOP. Mice in the Veh group were administered with the same amount of vehicle (methanol). (A,B) Serum creatinine and urea levels were determined by ELISA. (C) Kidney tissues were extracted for HE stain. Scale bar = 100 μm. (D,E) show the kidney weight/body weight (%) and tubular injury score. (F,G) Serum IL-18 and IL-1β levels were determined by ELISA. (H) exhibits the LDH release activity. (I) Proteins were extracted for Western blot. (J–N) The relative protein levels of NLRP3, ASC, cleaved caspase-1, mature GSDMD, and IL-1β were normalized to GAPDH. (O) Schema depicting the mechanisms for renoprotection by ABL against IOP-induced AKI. In vivo and in vitro, the application of IOP significantly up-regulated the expression of NLRP3, caspase-1, ASC, and mature GSDMD, in turn promoting the release of pro-inflammatory cytokines IL-1β and IL-18 to induce pyroptosis and AKI. However, the pretreatment of ABL partly reversed the pyroptosis gene alterations in IOP-injured kidney and ameliorated AKI.