Fig. 5.

Necrostatin-1 reduces MLKL expression on the apical membrane of proximal tubules and ameliorates IRI. Male WT and Rgmb cKO mice at 2 mo of age were injected with the vehicle (DMSO) or necrostatin (Nec)-1 (1.65 mg/kg body weight) 30 min before they underwent 40 min of bilateral renal pedicle clipping. Mice were killed 24 h after reperfusion. (A) Serum creatinine levels were measured in WT and Rgmb cKO mice. (B) Tubular injury scores were presented. (C) Expression of RIP3 and MLKL protein expression. Kidney lysates were used for Western blotting analysis for RIP3 and MLKL. RIP3 and MLKL levels relative to GAPDH levels were quantified by densitometry. (D) Immunofluorescence for MLKL and megalin in the outer medulla of kidneys from WT and Rgmb cKO mice injected with DMSO or Nec-1 before I/R. (E) MLKL levels in membranous fractions. Membranous proteins were isolated from kidneys of WT and Rgmb cKO mice with IRI, treated with and without Nec-1, and were subjected to Western blotting for MLKL. α1-subunit of Na⁺/K⁺ ATPase was used as the control for membranous proteins. n = 6–10 for A and B; n = 5 for D. *P < 0.05; **P < 0.01.