Role of Thioredoxin 1 in Impaired Renal Sodium Excretion of hD 5 R F173L Transgenic Mice

Figures

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  Figure 1. Blood pressure and renal function in hD 5 R F173L ‐TG mice. A, SBP, DBP, and MAP measured from the carotid artery in anesthetized 4‐month‐old hD 5 R WT‐TG and hD 5 R F173L‐TG mice (*P<0.05 compared with hD 5 R WT‐TG mice; n=5). B, SBP measured by the tail‐cuff method in conscious, nonanesthetized 4‐ and 8‐month‐old hD 5 R WT‐TG and hD 5 R F173L‐TG mice (*P<0.05 compared with hD 5 R WT‐TG mice; # P<0.05 compared with others; n=5). C and D, Urine volume (C) and UNa (D) in 4‐ and 8‐month‐old hD 5 R WT‐TG and hD 5 R F173L‐TG mice (*P<0.05 compared with hD 5 R WT‐TG mice; # P<0.05 compared with others; n=5). BW indicates body weight; DBP, diastolic blood pressure; hD 5 R F173L ‐TG, human dopamine D5 receptor mutant F173L transgenic; hD 5 R WT‐TG, human dopamine D5 receptor wild‐type transgenic; MAP, mean arterial blood pressure; SBP, systolic blood pressure; V, urine volume; UNa, urine sodium excretion.

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  Figure 2. Renal function and blood pressure in fenoldopam‐treated hD 5 R WT ‐TG and hD 5 R F173L‐TG mice. A and B, Urine volume (A) and UNa (B) in hD 5 R WT‐TG and hD 5 R F173L‐TG mice treated with fenoldopam (*P<0.05 compared with hD 5 R F173L‐TG mice; # P<0.05 compared with control; n=5). C, MAP measured from the carotid artery in anesthetized fenoldopam‐infused hD 5 R WT‐TG and hD 5 R F173L‐TG mice (*P<0.05 compared with hD 5 R WT‐TG mice; n=5). hD 5 R F173L ‐TG indicates human dopamine D5 receptor mutant F173L transgenic; hD 5 R WT‐TG, human dopamine D5 receptor wild‐type transgenic; MAP, mean arterial blood pressure; V, urine volume; UNa, urine sodium excretion.

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  Figure 3. Increased oxidative stress in hD 5 R F173L ‐TG mice. A, Serum MDA levels in hD 5 R WT‐TG and hD 5 R F173L‐TG mice (*P<0.05 compared with hD 5 R WT‐TG mice; n=5). B and C, Fluorescence microscopy images (B) and quantification (C) of renal ROS production in hD 5 R WT‐TG and hD 5 R F173L‐TG mice (*P<0.05 compared with hD 5 R WT‐TG mice; n=10). AOD indicates average optical density; hD 5 R F173L ‐TG indicates human dopamine D5 receptor mutant F173L transgenic; hD 5 R WT‐TG, human dopamine D5 receptor wild‐type transgenic; MDA, malondialdehyde; ROS, reactive oxygen species.

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  Figure 4. Trx1 expression and function in hD 5 R WT‐TG and hD 5 R F173L‐TG mice and hD 5 R WT‐ and hD 5 R F173L‐transfected cells. A and B, Trx1 mRNA (A) and protein expression (B) in hD 5 R WT‐TG and hD 5 R F173L‐TG mice (*P<0.05 compared with hD 5 R WT‐TG mice; n=8). C and D, Trx1 mRNA (C) and protein expression (D) in hD 5 R WT‐ and hD 5 R F173L‐transfected cells (*P<0.05 compared with control; n=6). E, Effect of exogenous recombinant human Trx1 protein on Na+‐K+‐ATPase activity in hD 5 R WT‐transfected and hD 5 R F173L‐transfected RPT cells. RPT cells were incubated with the recombinant human Trx1 protein (20 μg/mL) for 24 hours (*P<0.05 compared with other treatments; n=11). F, Trx1 protein expression in primary cultures of RPT cells from hD 5 R WT‐TG and hD 5 R F173L‐TG mice (*P<0.05 compared with hD 5 R WT‐TG mice; n=5). G, Na+‐K+‐ATPase activity in primary cultures of RPT cells from hD 5 R WT‐TG and hD 5 R F173L‐TG mice (*P<0.05 compared with hD 5 R WT‐TG mice; n=11). GAPDH indicates glyceraldehyde‐3‐phosphate dehydrogenase; hD 5 R F173L –TG, human dopamine D5 receptor mutant F173L transgenic; hD 5 R WT‐TG, human dopamine D5 receptor wild‐type transgenic; RPT, renal proximal…

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  Figure 5. Mechanism of renal D5R regulation of Trx1 expression. A, Role of PLC in regulation of Trx1 expression by D5R. U73122, a PLC inhibitor, blocked the stimulatory effect of fenoldopam on Trx1 expression in primary cultures of RPT cells from D1dr knockout mice (*P<0.05 compared with other treatments; n=6; Fen: 10−6 mol/L, 24 hours; U73122: 10−6 mol/L). B, Role of PKC in regulation of Trx1 expression by D5R. Staurosporine, a PKC inhibitor, blocked the stimulatory effect of Fen on Trx1 expression in primary cultures of RPT cells from D1dr knockout mice. (*P<0.05 compared with others; n=6; Fen: 10−6 mol/L, 24 hours; Stau: 10−8 mol/L). C, Trx1 expression in primary cultures of RPT cells from hD 5 R WT‐TG and hD 5 R F173L‐TG mice treated with Fen (*P<0.05 compared with control; # P<0.05 compared with hD 5 R WT‐TG; n=6; Fen: 10−6 mol/L, 24 hours). D, PKC activity in primary cultures of RPT cells from hD 5 R WT‐TG and hD 5 R F173L‐TG mice treated with Fen (*P<0.05 compared with other treatments; n=3; Fen: 10−6 mol/L, 30 minutes). D5R indicates dopamine D5 receptor; D1dr, dopamine D1 receptor; Fen, fenoldopam; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; hD 5 R F173L, human dopamine D5…

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  Figure 6. Phosphorylation of renal D5R and its association with Gαs/Gαq in hD 5 R WT‐TG and hD 5 R F173L‐TG mice. A and B, Serine (A) and threonine (B) phosphorylation of D5R in hD 5 R WT‐TG and hD 5 R F173L‐TG mice. Samples were immunoprecipitated with anti‐D5R antibody and immunoblotted with anti‐phosphoserine or ‐phosphothreonine antibody (*P<0.05 compared with hD 5 R WT‐TG mice; n=6). C and D, Coimmunoprecipitation of D5R and Gαq (C) or Gαs (D) in kidney homogenates from hD 5 R WT‐TG and hD 5 R F173L‐TG mice. Homogenates were immunoprecipitated with anti‐D5R antibody and immunoblotted with anti‐Gαq or anti‐Gαs antibody. For positive control, anti‐Gαq or ‐Gαs antibody was used instead of anti‐D5R antibody for immunoprecipitation, and for negative control (NC), IgG was used instead of anti‐D5R antibody for immunoprecipitation (*P<0.05 compared with hD 5 R WT‐TG mice; n=5 or 6). D5R indicates dopamine D5 receptor; Gαs, G protein alpha s subunit; Gαq, G protein alpha q subunit; hD 5 R F173L ‐TG, human dopamine D5 receptor mutant F173L transgenic; hD 5 R WT‐TG, human dopamine D5 receptor wild‐type transgenic; IB, immunoblotting; IgG, immunoglobulin G; IP, immunoprecipitation; NC, negative…