RIP‐3 and HMGB‐1 are upregulated in adult hypertensive male spontaneously hypertensive rats (SHR).

Abstract

BackgroundNecrotic cell death is a hallmark of hypertension. We previously published that male SHR have greater renal necrosis compared to age matched female SHR. Consistent with this, inhibition of necrosis attenuated maturation induced increases in blood pressure (BP) in male, but not female SHR. However, the mechanisms underlying sex differences in renal necrotic cell death in SHR are unknown. The goal of the current study is to test the hypothesis that maturation in male SHR drives increases in necrosis and upregulation of key proteins mediating necrotic cell death, receptor‐interacting protein kinase 3 (RIP‐3) and high mobility group box 1 (HMGB‐1).MethodsMale and female SHR were euthanized at 6 (pre‐hypertensive, sexually immature; n=6) or 15 weeks of age (hypertensive, sexually mature; n=6). The right kidney was isolated and processed for flow cytometric analysis of cell death using 7AAD. The left kidney was isolated for Western blot analysis of total RIP‐3, phosphorylated RIP‐3 and HMGB‐1. Protein expression was normalized to β‐actin. All data are expressed as mean ± standard error.ResultsRenal necrosis was comparable between 6 week old male and female SHR (expressed as % total kidney cells: 2.4±0.2 vs. 1.8±0.1, respectively) and increased with age only in males (5.4±0.2 vs. 1.7±0.3, respectively; Psex<0.0001; Page<0.0001; Pinteraction<0.0001). To gain insight into what mediates sex differences in necrosis in 15 week old SHR, protein expression of RIP‐3 and HMGB‐1 were determined in 6 vs. 15 week old male SHR and in 15 week old male and female SHR. Protein expression of RIP‐3 (1±0.1 vs. 0.7±0.1; P=0.008; n=5–6), p‐RIP‐3 (1±0.1 vs. 0.7±0.03; P=0.008; n=5–6) and HMGB‐1 (1±0.06 vs. 0.7±0.1; P=0.005; n=5–6) were all greater in 15 week old vs 6 week old male SHR. 15 week old male SHR also had greater protein expression of RIP‐3 (1±0.1 vs. 0.4±0.1; P=0.003; n=6), p‐RIP‐3 (1±0.2 vs. 0.4±0.1; P=0.02; n=6) and HMGB‐1 (1±0.2 vs. 0.6±0.1; P=0.02; n=6) compared to age matched females.ConclusionIn contrast to adult, hypertensive SHR, sex differences in renal necrosis were absent in young, sexually immature pre‐hypertensive SHR. Consistent with greater renal necrosis, key proteins of necrotic cell death machinery were also upregulated in hypertensive male SHR compared to either age matched hypertensive females or young, pre‐hypertensive male SHR. Future studies are needed to test the hypothesis that RIP‐3 activation and the subsequent HMGB1 release potentially underlie greater renal necrosis in hypertensive male SHR compared to age matched females.