Blocking Periostin Prevented Development of Inflammation in Rhabdomyolysis-Induced Acute Kidney Injury Mice Model

Figures

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  Figure 1. (In vivo experiments) The methods of the in vivo experiment schedule. 1. The day before the injection, the mice were abstained from drinking. 2. On the day of the injection, the mice were anesthetized by inhalation or intravenous injection, and 50% glycerol (diluted with normal saline) was injected into the thigh muscle. 3. The water deprivation was cancelled. 4. Acute renal failure due to rhabdomyolysis developed on day 3. (In vitro experiments) The methods of the in vitro experiment schedule. Cell: NIH3T3 fibroblast, medium: DMEM + 10% FBS, stimulus: Hemin (100 μM), stimulation time: 6 h, evaluation item: inflammation markers.

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  Figure 2. Periostin expression in the kidney of the rhabdomyolysis model. The expression of periostin in the kidney was highly increased, peaking at approximately day 3 after 50% glycerol injection. The relative gene expression was quantified at day 0–42 after operation by a real-time polymerase chain reaction using specific primers for periostin. * p < 0.05 vs. day 0. n = 3–4.

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  Figure 3. The levels of serum creatinine kinase (A) and creatinine (B) in wildtype and Pn-null rhabdomyolysis mouse model. (A) Serum creatinine kinase was quantified at 6 h later after glycerol injection. ** p < 0.05 vs. Non-treatment. N = 3–5. The levels of serum creatinine (B) was quantified at day 0 and 3 after operation. ** p < 0.05 vs. day 0, and day 3 (PnKO). N = 3–5.

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  Figure 4. (A) The tubular damage in Hematoxylin-Eosin (H-E) staining (×100). Tubular damage in wildtype and Pn-null rhabdomyolysis mouse model kidney. Periostin-null mice exhibited reduced tubular damage in the kidney. (B) The evaluation of the degree of tubular damage in wildtype and Pn-null rhabdomyolysis mouse model kidney. The tubular damage was defined by tubular epithelial necrosis, intratubular debris, and loss of brush border. The tubular damage score was 0: normal, 1: 1–25% damaged, 2: 26–50% damaged, 3: 51–75% damaged, and 4: 76–100% damaged. We examined 40 fields at random. Injury score (%) = (numbers of injured tubules/number of whole tubules) × 100. ** p < 0.01 vs. day 0 (WT) and day 3 (PnKO) n = 6.

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  Figure 5. The expression of periostin and inflammation markers in the kidney of the rhabdomyolysis model. All markers of inflammation declined in the Pn-null rhabdomyolysis mouse model compared with the wildtype rhabdomyolysis mouse model. ** p < 0.01 vs. day 0 (WT) and day 3 (PnKO). n = 6.

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  Figure 6. The expression of periostin and inflammation markers in the NIH3T3 fibroblasts after Hemin stimulation. All markers of inflammation declined in the periostin-knockdown NIH3T3 cells with hemin compared with the wildtype NIH3T3 fibroblasts with hemin. ** p < 0.05 vs. day 0 (WT) and day 3 (PnKO). n = 4.