Constitutive endocytosis and recycling of NKCC2 in rat thick ascending limbs

Abstract

The Na-K-2Cl cotransporter (NKCC2) mediates NaCl absorption by the thick ascending limb of Henle's loop (THAL). Exocytosis and endocytosis regulates surface expression of most transporters. However, little is known about the mechanism of NKCC2 trafficking in the absence of stimulating hormones and whether this mechanism contributes to regulation of steady-state surface expression of apical NKCC2 in the THAL. We tested whether NKCC2 undergoes constitutive endocytosis that regulates steady-state surface NKCC2 and NaCl reabsorption in THALs. We measured steady-state surface NKCC2 levels and the rate of NKCC2 endocytosis by surface biotinylation and Western blot and confocal microscopy of isolated perfused rat THALs. We observed constitutive NKCC2 endocytosis over 30 min that averaged 21.5 ± 2.7% of the surface pool. We then tested whether methyl-β-cyclodextrin (MβCD), a compound that inhibits endocytosis by chelating membrane cholesterol, blocked NKCC2 endocytic retrieval. We found that 30-min treatment with MβCD (5 mM) blocked NKCC2 endocytosis by 81% (P < 0.01). Blockade of endocytosis by MβCD induced accumulation of NKCC2 at the apical membrane as demonstrated by a 60 ± 16% (P < 0.05) increase in steady-state surface expression and enhanced apical surface NKCC2 immunostaining in isolated, perfused THALs. Acute treatment with MβCD did not change the total pool of NKCC2. MβCD did not affect NKCC2 trafficking when it was complexed with cholesterol before treatment. Inhibition endocytosis with MβCD enhanced NKCC2-dependent NaCl entry by 57 ± 16% (P < 0.05). Finally, we observed that a fraction of retrieved NKCC2 recycles back to the plasma membrane (36 ± 7%) over 30 min. We concluded that constitutive NKCC2 trafficking maintains steady-state surface NKCC2 and regulates NaCl reabsorption in THALs. These are the first data showing an increase in apical membrane NKCC2 in THALs by altering the rates of constitutive NKCC2 trafficking, rather than by stimulation of hormone-dependent signaling.

Fig. 1.

Na-K-2Cl cotransporter (NKCC2) undergoes constitutive endocytosis in thick ascending limbs of Henle's loop (THALs). A: representative Western blot of the endocytic protocol for NKCC2 in THALs. Expression of steady-state surface NKCC2 before (lane 2; basal surface NKCC2) and after (lanes 3–6) treatment with sodium 2-mercaptoethanesulfonate (MesNa) to strip surface biotinylated proteins. After stripping, THALs were incubated for 0 (lane 3), 7.5 (lane 4), 15 (lane 5), and 30 min (lane 6) at 37°C to allow protein trafficking. THALs were cooled, and endocytosed NKCC2 (protected from MesNa) was precipitated, detected by Western blotting, and measured by densitometry. As a control, intracellular GAPDH was not detected in the surface fraction, indicating the absence of intracellular protein biotinylation with NHS-SS-biotin as described in materials and methods. B: cumulative data for constitutive NKCC2 endocytosis in THAL suspensions. Constitutive NKCC2 endocytosis over time averaged 8.9 ± 1.5% at 7.5 min, 12.9 ± 2.2% at 15 min, and 21.7 ± 3.3% at 30 min (n = 6). The remaining signal from the stripped sample (lane 3) was always subtracted from the experimental samples, and thus data were expressed as percentage of the MesNa-stripped fraction (see results). Error bars represent SE.

Fig. 2.

Effect of methyl-β-cyclodextrin (MβCD) on apical endocytosis of a fluid-phase marker in isolated. perfused THALs. A: representative confocal images of isolated and perfused THALs. THALs were incubated with apical FITC-dextran (10-kDa marker, 5 mg/ml) for 10 min, after treatment with vehicle (top) or MβCD (5 mM, bottom) for 20 min (z-section shows only cells at the bottom of the tubule, closer to the coverslip). B: cumulative data for fluid-phase endocytic marker FITC-dextran (10 kDa) in THALs. The number of vesicles (punctae) per cell were decreased from 13 ± 2 to 3 ± 2 (*P < 0.05) in MβCD-treated THALs (see results). Error bars represent SE.

Fig. 3.

Effect of MβCD on constitutive NKCC2 endocytosis in THALs. A: representative Western blot for a single experiment showing constitutive NKCC2 endocytosis in basal condition or in the presence of MβCD. B: THALs were pretreated with vehicle or MβCD for 20 min before biotinylation. After biotinylation, samples were incubated at 37°C for 0, 7.5, 15, and 30 min in the presence of vehicle (●) or MβCD (■). Treatment of THALs with MβCD (5 mM) blocked constitutive NKCC2 endocytosis (basal = 8.95 ± 1.58% at 7.5 min, 11.0 ± 1.9% at 15 min, 21.5 ± 2.7% at 30 min; MβCD = 2.4 ± 0.4% at 7.5 min, 2.7 ± 0.6% at 15 min, 2.9 ± 0.9% at 30 min, n = 6, *P < 0.05). Error bars represent SE. Note: the vehicle experiments at time point 7.5 min, were performed in a separate group of animals.

Fig. 4.

Effect of cholesterol-saturated MβCD on NKCC2 endocytosis in THALs. Effect of MβCD (5 mM), MβCD/cholesterol (Chol), or Chol alone on constitutive NKCC2 endocytosis measured at 30 min. MβCD/Chol does not significantly affect NKCC2 endocytosis. Basal = 17.3 ± 1.8%, MβCD = 2.1 ± 0.3%, MβCD/Chol = 13.5 ± 1.4%, and Chol (2 μg/ml) = 12.3 ± 2.3%. Data are expressed as percentage of the MesNa-stripped fraction after 30 min of endocytosis (n = 6, *P < 0.05).

Fig. 5.

Effect of Chol-saturated MβCD on steady-state surface NKCC2 in THALs. A: cumulative data showing the effect of MβCD (5 mM), MβCD/Chol, or Chol alone on steady-state surface NKCC2 levels in THALs. Samples were treated for 20 min at 37°C, then biotinylated as described in materials and methods, and NKCC2 was detected by Western blotting and quantified by densitometry. Basal = 100%, MβCD = 184 ± 20%, MβCD/Chol = 110 ± 13%, and Chol = 106 ± 16% (n = 5, *P < 0.05). Data are expressed as a percent of baseline surface NKCC2. Error bars represent SE. B: representative blot showing total NKCC2 expression in THALs treated with vehicle (lanes 1 and 2), MβCD (lanes 3 and 4), MβCD/Chol (lanes 5 and 6), or Chol (lanes 7 and 8).

Fig. 6.

MβCD increases apical surface NKCC2 staining in isolated, perfused medullary THALs. A: representative confocal micrograph showing apical surface NKCC2 staining in isolated, perfused medullary THALs under control (unstimulated) conditions (left) and in a THAL treated with MβCD (5 mM) for 20 min (right). B: cumulative data for apical NKCC2 immunofluorescence staining under basal conditions and in THALs treated with MβCD (5 mM). The mean fluorescent intensity in the apical membrane was measured in 15–20 THAL cells in each tubule and then averaged (basal: 91.5 + 6.0%, MβCD: 203.5 ± 12.5%, P < 0.05, n = 4). Error bars represent SE.

Fig. 7.

Effect of MβCD on net Cl⁻ reabsorption. Cumulative data showing the effect of MβCD (5 mM) on net Cl⁻ reabsorption. THALs were isolated and perfused as described in materials and methods. THALs were treated with either vehicle or MβCD (5 mM) in the luminal and basolateral side for 20 min at 37°C. Net Cl⁻ reabsorption was measured as described in materials and methods. MβCD (5 mM) enhanced net Cl⁻ reabsorption from 80 ± 9 to 126 ± 20 pmol·min⁻¹·mm⁻¹ of THAL (n = 5, *P < 0.04). Error bars represent SE.

Fig. 8.

Retrieved NKCC2 recycles back to the plasma membrane. A: representative Western blot showing recycling of NKCC2 in THALs. Lane 1, baseline steady-state surface NKCC2; lane 2, surface NKCC2 after stripping of surface biotin with MesNa; lane 3, 30-min retrieval with one and two rounds of MesNa stripping; lanes 4–6, 30-min retrieval plus incubation at 37°C for 7.5, 15, or 30 min and second round of MesNa stripping (the decrease in the NKCC2 retrieved for 30 min is due to second round stripping of proteins recycling back to the surface). The total pool of NKCC2 did not change over time (bottom). B: cumulative data for NKCC2 recycling in THAL suspensions. THAL surface proteins were biotinylated, incubated at 37°C for 30 min to allow endocytosis, and then the remaining biotin in surface proteins was stripped (first-round stripping). Then, THALs were warmed to 37°C to allow recycling of retrieved biotinylated proteins back to the cell surface, and surface biotin was stripped for a second time. Biotinylated proteins were isolated, and NKCC2 was measured by Western blotting. The fraction of NKCC2 recycled was expressed as a percentage of endocytosed NKCC2 at 30 min (n = 5). C: cumulative data for stability of biotinylated surface NKCC2 in THALs. THAL surface proteins were biotinylated as described in materials and methods and incubated at 37°C for 60 or 120 min. Biotinylated proteins were isolated, and NKCC2 was detected by Western blotting and quantified by densitometry (0 min: 100%, 60 min: 63.9 ± 6.4%, 120 min: 51.9 ± 3.7%, n = 4, *P < 0.05). Data are expressed as a percentage of baseline surface NKCC2. Error bars represent SE.