Gustavo R. Ares

cGMP decreases surface NKCC2 levels in the thick ascending limb: role of phosphodiesterase 2 (PDE2)

NaCl absorption in the medullary thick ascending limb of the loop of Henle (THAL) is mediated by the apical Na/K/2Cl cotransporter (NKCC2). Hormones that increase cGMP, such as nitric oxide (NO) and natriuretic peptides, decrease NaCl absorption by the THAL. However, the mechanism by which cGMP decreases NaCl absorption in THALs is not known. We hypothesized that cGMP decreases surface NKCC2 levels in the THAL. We used surface biotinylation to measure surface NKCC2 levels in rat THAL suspensions. We tested the effect of the membrane-permeant cGMP analog dibutyryl-cGMP (db-cGMP) on surface NKCC2 levels. Incubating THALs with db-cGMP for 20 min decreased surface NKCC2 levels in a concentration-dependent manner (basal=100%; db-cGMP 100 microM=77+/-7%; 500 microM=54+/-10% and 1,000 microM=61+/-8%). A different cGMP analog 8-bromo-cGMP (8-Br-cGMP) also decreased surface NKCC2 levels by 25%, (basal=100%; 8-Br-cGMP=75+/-5%). Incubation of isolated, perfused THALs with db-cGMP decreased apical surface NKCC2 labeling levels as measured by immunofluorescence and confocal microscopy. cGMP-stimulated phosphodiesterase 2 (PDE2) mediates the inhibitory effect of NO on NaCl absorption by THALs. Thus we examined the role of PDE2 and found that PDE2 inhibitors blocked the effect of db-cGMP on surface NKCC2. Also, a nonstimulatory concentration of db-cAMP blocked the cGMP-induced decrease in surface NKCC2. Finally, db-cGMP inhibited THAL net Cl absorption by 48+/-4%, and this effect was completely blocked by PDE2 inhibition. We conclude that cGMP decreases NKCC2 levels in the apical membrane of THALs and that this effect is mediated by PDE2. This is an important mechanism by which cGMP inhibits NaCl absorption by the THAL.

Constitutive endocytosis and recycling of NKCC2 in rat thick ascending limbs

The Na-K-2Cl cotransporter (NKCC2) mediates NaCl absorption by the thick ascending limb of Henles 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.

Hyperphosphorylation of Na-K-2Cl Cotransporter in Thick Ascending Limbs of Dahl Salt-Sensitive Rats

Salt-sensitive hypertension involves a renal defect preventing the kidney from eliminating excess NaCl. The thick ascending limb of Henle loop reabsorbs ≈30% of filtered NaCl via the apical Na-K-2Cl cotransporter (NKCC2). Higher NKCC2 activity and Cl reabsorption have been reported in the thick ascending limbs from Dahl salt-sensitive rats (DSS) fed normal salt. NKCC2 activity is primarily regulated by protein trafficking and phosphorylation at Thr96/Thr101 via STE20- and SPS1-related proline and alanine-rich kinases and oxidative stress-responsive kinase 1. However, the mechanism for enhanced NKCC2 activity in DSS is unclear. We hypothesized that DSS exhibit enhanced NKCC2 trafficking and higher NKCC2 phosphorylation compared with Dahl salt-resistant rats on normal salt diet. We measured steady state surface NKCC2 expression and phosphorylation at Thr96 and Thr101 by surface biotinylation and Western blot. In DSS, the surface:total NKCC2 ratio was enhanced by 25% compared with Dahl salt-resistant rats (P<0.05) despite lower NKCC2 expression. Total NKCC2 phosphorylation at Thr96 and Thr101 was enhanced ≈5-fold in DSS thick ascending limbs. Moreover, total STE20- and SPS1-related proline and alanine-rich kinases expression, kidney-specific STE20- and SPS1-related proline and alanine-rich kinases, and oxidative stress-responsive kinase 1 were not different between strains, although STE20- and SPS1-related proline and alanine-rich kinases/oxidative stress-responsive kinase 1 phosphorylation was enhanced by 60% (P<0.05) in DSS rats, suggesting increased activity. We concluded that phosphorylation of NKCC2 Thr96 and Thr101 and surface:total NKCC2 ratio are enhanced in DSS rats. These differences in NKCC2 may be, in part, responsible for higher NKCC2 activity and abnormally enhanced thick ascending limb NaCl reabsorption in DSS rats.