Inflammation compromises renal dopamine D1 receptor function in rats

Abstract

We tested the effects of inflammation on renal dopamine D1 receptor signaling cascade, a key pathway that maintains sodium homeostasis and blood pressure during increased salt intake. Inflammation was produced by administering lipopolysaccharide (LPS; 4 mg/kg ip) to rats provided without (normal salt) and with 1% NaCl in drinking water for 2 wk (high salt). Control rats had saline injection and received tap water. We found that LPS increased the levels of inflammatory cytokines, interleukin-6, and tumor necrosis factor-alpha in the rats given either normal- or high-salt intake. Also, these rats had higher levels of oxidative stress markers, malondialdehyde and nitrotyrosine, and lower levels of antioxidant enzyme superoxide dismutase in the renal proximal tubules (RPTs). The nuclear levels of transcription factors NF-kappaB increased and Nrf2 decreased in the RPTs in response to LPS in rats given normal and high salt. Furthermore, D1 receptor numbers, D1 receptor proteins, and D1 receptor agonist (SKF38393)-mediated (35)S-GTPgammaS binding decreased in the RPTs in these rats. The basal activities of Na-K-ATPase in the RPTs were similar in control and LPS-treated rats given normal and high salt. SKF38393 caused inhibition of Na-K-ATPase activity in the primary cultures of RPTs treated with vehicle but not in the cultures treated with LPS. Furthermore, LPS caused an increase in blood pressure in the rats given high salt but not in the rats given normal salt. These results suggest that LPS differentially regulates NF-kappaB and Nrf2, produces inflammation, decreases antioxidant enzyme, increases oxidative stress, and causes D1 receptor dysfunction in the RPTs. The LPS-induced dysfunction of renal D1 receptors alters salt handling and causes hypertension in rats during salt overload.

Fig. 1.

LPS increases inflammatory markers TNF-α and IL-6 in rats given normal (LPS) and high salt (HS+LPS). TNF-α (A) in the renal proximal tubules (RPTs) and IL-6 (B) in the plasma were measured by ELISA. Results are means ± SE from 4–5 experiments (n = 4–5 animals). *Significantly different from control rats.

Fig. 2.

LPS increases oxidative and nitrative stress markers and decreases anti-oxidant superoxide dismutase (SOD) enzyme in RPTs of rats. A: malondialdehyde (MDA) levels in rats given normal (LPS) and high salt (HS+LPS). B, left: representative immunoblot of protein nitrotyrosine; right: quantification of protein nitrotyrosine bands A, B, and C in control (lane 1) and LPS-treated rats given normal (lane 2) and high salt (lane 3). C, top: representative immunoblot of SOD and GAPDH; bottom: bars are ratios between the densities of SOD and GAPDH. Results are means ± SE from 4–5 experiments (n = 4–5 animals). *Significantly different from control. #Significantly different from LPS in A and control band B in B. $Significantly different from control band C in C.

Fig. 3.

Nuclear levels of NF-κB increase and of Nrf2 decrease in RPTs in response to LPS. A, top: representative immunoblot of NF-κB and protein loading control histone-4 in control (lane 1) and LPS-treated rats given normal (lane 2) and high salt (lane 3). Bottom: bars represent the ratios of the densities of NF-κB and histone-4. B, top: representative immunoblot of Nrf2 and protein loading control histone-4 in control (lane 1) and LPS-treated rats given normal (lane 2) and high salt (lane 3). Bottom: bars represent the ratios between the densities of Nrf2 and histone-4. Results are means ± SE from 4–5 experiments (n = 4–5 animals). *Significantly different from control. #Significantly different from LPS.

Fig. 4.

LPS decreases D1 receptor numbers and the levels of D1 receptor proteins in the membranes of RPTs. A: radio D1 receptor antagonist ³H-SCH23390 binding in the membranes of RPTs. B, top: representative immunoblot of D1 receptor proteins in the membranes of RPTs from control (lane 1) and LPS-treated rats given normal (lane 2) and high salt (lane 3). Bottom: bars represent the densities of D1 receptor protein. C, top: representative immunoblot blot of D1 receptors and protein loading control GAPDH in the homogenates of RPTs from control (lane 1) and LPS-treated rats given normal (lane 2) and high salt (lane 3). Bottom: bars represent the ratios of the densities of D1 receptors and GAPDH. Results are means ± SE from 4–5 experiments (n = 4–5 animals). *Significantly different from control. #Significantly different from LPS.

Fig. 5.

LPS decreases D1 receptor agonist-mediated receptor G protein coupling and attenuates inhibitory response of the agonist on Na-K-ATPase. A: ³⁵S-GTPγS binding in response to D1 receptor agonist SKF38393 (10⁻⁶ M), an index of D1 receptor G protein coupling, in RPT membranes of control (open bars) and LPS-treated rats given normal (hatched bars) and high salt (filled bars). B: ⁸⁶Rubidium uptake, an index of Na-K-ATPase activity, in primary cultures of RPTs treated with (hatched bars) and without (open bars) LPS (1 μg/ml, overnight). The ability of D1 receptor agonist (1 μM, 10 min) to inhibit Na-K-ATPase activity in cultures treated with vehicle (open bars) attenuates in cultures prior treated with LPS (hatched bars). Results are means ± SE from 4–5 experiments (n = 4–5 animals). *Significantly different from vehicle in controls in A and B.

Fig. 6.

LPS increases systolic but not diastolic blood pressure (BP) in LPS-treated rats on high-salt diet. Rats given high salt are identified by filled bars. Rats given normal salt are identified by open and hatched bars. Results are means ± SE from 4–5 experiments (n = 4–5 animals). *Significantly different from control (open bars). #Significantly different from LPS-treated rats given normal salt (hatched bars).