Dopamine acutely decreases apical membrane Na/H exchanger NHE3 protein in mouse renal proximal tubule

Abstract

Background: Dopamine is a principal natriuretic hormone in mammalian Na+ homeostasis. Dopamine acutely alters glomerular filtration rate (GFR) and decreases Na+ absorption in both the proximal and distal nephron. Proximal tubule natriuresis is effected through inhibition of the apical membrane Na/H exchanger NHE3.

Methods: We examined whether dopamine directly and acutely decreases apical membrane NHE3 protein using renal tissue in two in vitro systems: renal cortical slices and in vitro perfused single tubules. After incubation with dopamine, NHE3 activity was measured by 22Na flux and NHE3 antigen was measured by immunoblot in apical membrane and total cellular membranes.

Results: Direct application of dopamine to either cortical slices or microperfused tubules acutely decreases NHE3 activity and antigen at the apical membrane of the proximal tubule. No change in total cellular NHE3 was detected.

Conclusion: One mechanism by which dopamine causes natriuresis is via direct and acute reduction of NHE3 protein at the apical membrane via changes in NHE3 protein trafficking.

Fig. 1. Effect of dopamine on NHE3 antigen: Time response

(A) Representative experiments. Mouse renal cortical slices were incubated with 10⁻⁵ mol/L dopamine (DA) and apical and total cortical membranes were prepared and NHE3 and β-actin antigen were measured by immunoblot. Con is control. (B) Summary of the effect of dopamine on NHE3 antigen. Y-axis shows the abundance of NHE3 antigen in the apical membrane vesicles as a percentage of the vehicle-treated controls. Symbols denote means ± SE. The number of independent experiments for each time point is N = 8, 5 minutes; N = 4, 10 minutes; N = 4, 15 minutes; N = 8, 30 minutes; N = 3, 60 minutes. *P < 0.05 (unpaired t test); ^#P = 0.08.

Fig. 2. Effect of dopamine on NHE3 activity and NHE3 antigen on the same apical membrane vesicles

Mouse renal cortical slices were incubated with 10⁻⁵ mol/L dopamine (DA) for either 5 or 30 minutes and apical and cortical membranes were prepared for NHE3 activity (pH gradient driven ²²Na flux) and NHE3 antigen (immunoblot) assays. The left panel shows NHE3 activity normalized to membrane protein and NHE3 activity from dopamine-treated tissue is expressed as percentage of the vehicle-treated ones. The middle panel shows the quantity of NHE3 antigen expressed also as a percentage of vehicle. The right panel shows NHE3 activity divided by NHE3 antigen and expressed as dopamine as percentage of control. The number of independent experiments = 4 pairs for either 5 or 30 minutes. *P < 0.05 (unpaired t test).

Fig. 3. Effect of dopamine on NHE3 antigen: Dose response

(A) Representative experiment. Mouse renal cortical slices were incubated with various concentration of dopamine (DA) for 30 minutes and apical and cortical membranes were prepared and NHE3 and β-actin antigen were measured by immunoblot. (B) Summary of all experiments. Y-axis shows the abundance of NHE3 antigen in the apical membrane vesicles as a percentage of the vehicle-treated controls. Each symbol represents the mean ± SE of three to four independent experiments. The number of independent experiments for each dose were N = 3, 10⁻⁷ mol/L; N = 4, 10⁻⁶ mol/L; N = 8, 10⁻⁵ mol/L; N = 4, 10⁻⁴ mol/L. *P < 0.05 (unpaired t test).

Fig. 4. Immunohistochemical staining of NHE3 antigen in mouse kidney

Mouse kidneys were perfusion fixed and stained with anti-NHE3 as described in the Methods section. (A) Whole kidney overview. (B and C) Proximal tubule S1, S2, and S3 segment and thick ascending limb (TAL) are labeled.

Fig. 4. Immunohistochemical staining of NHE3 antigen in mouse kidney

Mouse kidneys were perfusion fixed and stained with anti-NHE3 as described in the Methods section. (A) Whole kidney overview. (B and C) Proximal tubule S1, S2, and S3 segment and thick ascending limb (TAL) are labeled.

Fig. 5. Single-tubule immunohistochemistry: Effect of dopamine (DA) on proximal convoluted tubule

Microdissected proximal convoluted tubules were perfused with 10⁻⁵ mol/L dopamine in the lumen or the bath for 30 minutes and fixed and stained for NHE3 immunohistochemistry as described in the Methods section. Three independent experiments showed similar results.

Fig. 6. Single-tubule immunohistochemistry: Effect of dopamine (DA) on proximal straight tubule

Microdissected proximal convoluted tubules were perfused with 10⁻⁵ mol/L dopamine in the lumen or the bath for 30 minutes and fixed and stained for NHE3 immunohistochemistry as described in the Methods section. Three independent experiments showed similar results.