Interleukin-1beta, but not interleukin-6, enhances renal and systemic endothelin production in vivo

Abstract

The inflammatory cytokines IL-1beta and IL-6 have been shown to stimulate production of endothelin-1 (ET-1) by several cell types in vitro, but their effects on renal ET-1 production in vivo are not known. To test whether IL-1beta and IL-6 stimulate renal ET-1 production and release in vivo, urine was collected from male C57BL/6 mice over 24-h periods at baseline and on days 7 and 14 of a 14-day subcutaneous infusion of IL-1beta (10 ng/h), IL-6 (16 ng/h), or vehicle. By day 14, plasma ET-1 was significantly increased by IL-1beta infusion (1.7 +/- 0.1 vs. 0.8 +/- 0.1 pg/ml for vehicle, P < 0.001). Compared with vehicle infusion, IL-1beta infusion induced significant increases in urinary ET-1 excretion rate and urine flow but did not affect conscious mean arterial pressure (telemetry). IL-1beta infusion significantly increased renal cortical and medullary IL-1beta content (ELISA) and prepro-ET-1 mRNA expression (quantitative real-time PCR). In contrast, 14 days of IL-6 infusion had no significant effect on plasma ET-1 or urinary ET-1 excretion rate. To determine whether IL-1beta stimulates ET-1 release via activation of NF-kappaB, inner medullary collecting duct (IMCD-3) cells were incubated for 24 h with IL-1beta, and ET-1 release and NF-kappaB activation were measured (ELISA). IL-1beta activated NF-kappaB and increased ET-1 release in a concentration-dependent manner. The effect of IL-1beta on ET-1 release could be partially inhibited by pretreatment of IMCD-3 cells with an inhibitor of NF-kappaB activation (BAY 11-7082). These results indicate that IL-1beta stimulates renal and systemic ET-1 production in vivo, providing further evidence that ET-1 participates in inflammatory responses.

Fig. 1.

Effects of 14-day subcutaneous infusion of IL-6 on renal function. Mice were placed in metabolic cages for 24 h at weekly intervals before (baseline) and during IL-6 or vehicle infusion. A: urinary endothelin-1 (ET-1) excretion rate (U_ET-1V). B: urine flow (V). C: urinary Na⁺ excretion rate (U V). D: food intake. E: water intake. F: body weight. Values are means ± SE; n = 6 and 7 mice for vehicle and IL-6 groups, respectively, except for food intake, where n = 5 and 6, respectively. Repeated-measures ANOVA was used to test whether responses were affected by treatment group (P_group) or time (P_time) and whether responses differed between groups in a time-dependent manner (P_{g * t}). NS, not statistically significant.

Fig. 2.

Effect of 14-day subcutaneous infusion of IL-1β on renal tissue. A: renal cortical and medullary IL-1β concentration, expressed per milligram of total protein. Values are means ± SE for 8 and 7 vehicle- and IL-1β-infused mice, respectively. B: relative expression of prepro-ET-1 mRNA in renal cortex (n = 7 and 6 in vehicle- and IL-1β-infused mice) and renal medulla (n = 5–7). Values are means ± SE. *P < 0.05 vs. vehicle (by unpaired Student's t-test).

Fig. 3.

Effects of 14-day subcutaneous infusion of IL-1β on renal function. Mice were placed in metabolic cages at weekly intervals before (baseline) and during IL-1β (n = 7) or vehicle (n = 8) infusion. A: urinary ET-1 excretion rate. B: urine flow. C: urinary Na⁺ excretion rate. D: food intake. E: water intake. F: body weight. Repeated-measures ANOVA was used to test whether responses were affected by treatment group (P_group) or time (P_time) and whether responses differed between groups in a time-dependent manner (P_{g * t}). Post hoc contrasts are as follows: *P < 0.05 vs. baseline for the same group; †P < 0.05 vs. vehicle at specified time point; ‡P < 0.05 vs. week 1 for the same group.

Fig. 4.

Telemetry recordings before and during 14-day IL-1β or vehicle infusion. Values are group means ± SE of 24-h average core body temperature (A), mean arterial pressure (MAP, B), and heart rate (beats/min, C). Data were collected from 5 animals in all groups, with core body temperature measured in 1 set of mice and hemodynamic data measured in a separate set of mice. Vertical dashed line indicates start of subcutaneous infusion. Repeated-measures ANOVA was used to test whether core body temperature was affected by treatment group (P_group) or time (P_time) and whether responses differed between groups in a time-dependent manner (P_{g * t}). Post hoc contrasts are as follows: *P < 0.05 vs. vehicle at the same time point.

Fig. 5.

Concentration-dependent effects of IL-1β on inner medullary collecting duct (IMCD-3) cell ET-1 release (A) and activation of the p65 subunit of NF-κB (B). Cells were incubated for 24 h with IL-1β at the doses shown. Values are means ± SE; n = 3–4. *P < 0.05 vs. 0 pg/ml (by Dunnett's post hoc test).