Fluorofenidone Alleviates Renal Fibrosis by Inhibiting Necroptosis Through RIPK3/MLKL Pathway

Abstract

Cell death and sterile inflammation are major mechanisms of renal fibrosis, which eventually develop into end-stage renal disease. "Necroptosis" is a type of caspase-independent regulated cell death, and sterile inflammatory response caused by tissue injury is strongly related to necrosis. Fluorofenidone (AKF-PD) is a novel compound shown to ameliorate renal fibrosis and associated inflammation. We investigated whether AKF-PD could alleviate renal fibrosis by inhibiting necroptosis. Unilateral ureteral obstruction (UUO) was used to induce renal tubulointerstitial fibrosis in C57BL/6J mice. AKF-PD (500 mg/kg) or necrostatin-1 (Nec-1; 1.65 mg/kg) was administered simultaneously for 3 and 7 days. Obstructed kidneys and serum were harvested after euthanasia. AKF-PD and Nec-1 ameliorated renal tubular damage, inflammatory-cell infiltration, and collagen deposition, and the expression of proinflammatory factors (interlukin-1β, tumor necrosis factor [TNF]-α) and chemokines (monocyte chemoattractant protein-1) decreased. AKF-PD or Nec-1 treatment protected renal tubular epithelial cells from necrosis and reduced the release of lactate dehydrogenase in serum. Simultaneously, production of receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like protein (MLKL) was also reduced 3 and 7 days after UUO. AKF-PD and Nec-1 significantly decreased the percentage of cell necrosis, inhibiting the phosphorylation of MLKL and RIPK3 in TNF-α- and Z-VAD-stimulated human proximal tubular epithelial (HK-2) cells. In conclusion, AKF-PD and Nec-1 have effective anti-inflammatory and antifibrotic activity in UUO-induced renal tubulointerstitial fibrosis, potentially mediated by the RIPK3/MLKL pathway.

Keywords: fluorofenidone; inflammation; necroptosis; receptor-interacting protein kinase 3-mixed lineage kinase domain-like protein pathway; renal fibrosis.

FIGURE 1

AKF-PD and Nec-1 attenuated the histological changes in the obstructed kidney after UUO on days 3 and 7 . (A), (B) AKF-PD and Nec-1 attenuated tubulointerstitial injury in UUO on days 3 and 7 (×200) (H&E staining of kidney sections, ×200). The red arrow points to the dilated area of the renal tubule; the yellow arrow points to the infiltration area of inflammatory cells; the black arrow points to the renal interstitial fibrosis area. (C), (D) AKF-PD and Nec-1 alleviated extracellular matrix deposition in UUO on days 3 and 7 (Masson’s trichrome staining of kidney sections, ×200). All data are presented as means ± SD, n ≥ 5; *p < 0.05 versus sham, #p < 0.05 versus UUO. Data were analyzed by one-way ANOVA. AKF-PD, fluorofenidone; Nec-1, necrostatin-1; UUO, unilateral ureteral obstruction.

FIGURE 2

AKF-PD and Nec-1 reduced the infiltration of macrophage and the expression of inflammatory factors and chemokines in UUO. (A), (B) AKF-PD and Nec-1 affected F4/80 protein expression in the obstructed kidneys stained by immunohistochemistry (×200). (C) IL-1β, (D) TNF-α and MCP-1, (E) mRNA expression in the kidneys, (F) IL-1β protein expression in UUO kidneys on days 3. (G) TNF-α protein expression in UUO kidneys on day 3. All data are presented as means ± SD, n ≥ 5. *p < 0.05 versus sham, #p < 0.05 versus UUO. Data were analyzed by one-way ANOVA. AKF-PD, fluorofenidone; Nec-1, necrostatin-1; UUO, unilateral ureteral obstruction.

FIGURE 3

AKF-PD and Nec-1 alleviate cell necrosis in UUO. (A) Cell morphology of renal tissue observed by transmission electron microscopy (×5,000). ①: Cell membrane perforation or deformation; ②: nuclear membrane prolapse or rupture; ③: cytoplasmic transparency; ④: chromatin condenses into irregular plaque; ⑤: organelle swelling. (B) The level of the released LDH in the serum. All data are presented as means ± SD, n ≥ 5. *p < 0.05 versus sham, #p < 0.05 versus UUO. Data were analyzed by one-way ANOVA. AKF-PD, fluorofenidone; Nec-1, necrostatin-1; UUO, unilateral ureteral obstruction; LDH, lactate dehydrogenase.

FIGURE 4

AKF-PD and Nec-1 inhibited the expression of RIPK3 and MLKL in UUO. (A)–(D) The expression of RIPK3 and MLKL in UUO kidneys on days 3 and 7 detected by Western blots. All data are presented as means ± SD, n ≥ 5. *p < 0.05 versus sham, #p < 0.05 versus UUO. Data were analyzed by one-way ANOVA. AKF-PD, fluorofenidone; Nec-1, necrostatin-1; UUO, unilateral ureteral obstruction; RIPK, receptor-interacting protein kinase; MLKL, mixed lineage kinase domain-like protein.

FIGURE 5

AKF-PD and Nec-1 inhibited RIPK3- and p-MLKL–related necroptosis. (A) Percentages of necrotic cells determined by FACS analysis using annexin V-FITC and PI. (B)–(D) The expression of RIPK3, MLKL, and p-MLKL stimulated with TNF-α and Z-VAD in HK-2 cells detected by Western blots. All data are presented as means ± SD, n = 3. *p < 0.05 versus control, #p < 0.05 versus TNF-α+Z-VAD group. Data were analyzed by one-way ANOVA. AKF-PD, fluorofenidone; Nec-1, necrostatin-1; RIPK, receptor-interacting protein kinase; MLKL, mixed lineage kinase domain-like protein.