Ubiquitination of NKCC2 by the cullin-RING E3 ubiquitin ligase family in the thick ascending limb of the loop of Henle.

The Na+/K+/2Cl- cotransporter (NKCC2) in the thick ascending limb of the loop of Henle (TAL) mediates NaCl reabsorption. cGMP, the second messenger of nitric oxide and atrial natriuretic peptide, inhibits NKCC2 activity by stimulating NKCC2 ubiquitination and decreasing surface NKCC2 levels. Among the E3 ubiquitin ligase families, the cullin-RING E3 ubiquitin ligase (CRL) family is the largest. Cullins are molecular scaffold proteins that recruit multiple subunits to form the CRL complex. We hypothesized that a CRL complex mediates the cGMP-dependent increase in NKCC2 ubiquitination in TALs. Cullin-1, cullin-2, cullin-3, cullin-4A, and cullin-5 were expressed at the protein level, whereas the other members of the cullin family were expressed at the mRNA level, in rat TALs. CRL complex activity is regulated by neuronal precursor cell-expressed developmentally downregulated protein 8 (Nedd8) to cullins, a process called neddylation. Inhibition of cullin neddylation blunted the cGMP-dependent increase in ubiquitinated NKCC2 while increasing the expression of cullin-1 by threefold, but this effect was not seen with other cullins. CRL complex activity is also regulated by cullin-associated Nedd8-dissociated 1 (CAND1). CAND1 binds to cullins and promotes the exchange of substrate-recognition proteins to target different proteins for ubiquitination. CAND1 inhibition exacerbated the cGMP-dependent increase in NKCC2 ubiquitination and decreased surface NKCC2 expression. Finally, cGMP increased neddylation of cullins. We conclude that the cGMP-dependent increase in NKCC2 ubiquitination is mediated by a CRL complex. To the best of our knowledge, this is the first evidence that a CRL complex mediates NKCC2 ubiquitination in native TALs.NEW & NOTEWORTHY The Na+/K+/2Cl- cotransporter (NKCC2) reabsorbs NaCl by the thick ascending limb. Nitric oxide and atrial natriuretic peptide decrease NaCl reabsorption in thick ascending limbs by increasing the second messenger cGMP. The present findings indicate that cGMP increases NKCC2 ubiquitination via a cullin-RING ligase complex and regulates in part surface NKCC2 levels. Identifying the E3 ubiquitin ligases that regulate NKCC2 expression and activity may provide new targets for the development of specific loop diuretics.

cGMP induces degradation of NKCC2 in the thick ascending limb via the ubiquitin-proteasomal system.

The thick ascending limb of Henle's loop (TAL) reabsorbs NaCl via the apical Na+-K+-2Cl- cotransporter (NKCC2). NKCC2 activity is regulated by surface NKCC2 levels. The second messenger cGMP decreases NKCC2 activity by decreasing surface NKCC2 levels. We found that surface NKCC2 undergoes constitutive degradation. Therefore, we hypothesized that cGMP decreases NKCC2 levels by increasing NKCC2 ubiquitination and proteasomal degradation. We measured surface NKCC2 levels by biotinylation of surface proteins, immunoprecipitation of NKCC2, and ubiquitin in TALs. First, we found that inhibition of proteasomal degradation blunts the cGMP-dependent decrease in surface NKCC2 levels [vehicle: 100%, db-cGMP (500 µM): 70.3 ± 9.8%, MG132 (20 µM): 97.7 ± 5.0%, and db-cGMP + MG132: 103.3 ± 3.4%, n = 5, P < 0.05]. We then found that cGMP decreased the internalized NKCC2 pool and that this effect was prevented by inhibition of the proteasome but not the lysosome. Finally, we found that NKCC2 is constitutively ubiquitinated in TALs and that cGMP enhances the rate of NKCC2 ubiquitination [vehicle: 59 ± 14% and db-cGMP (500 µM): 111 ± 25%, n = 5, P < 0.05]. We conclude that NKCC2 is constitutively ubiquitinated and that cGMP stimulates NKCC2 ubiquitination and proteasomal degradation. Our data suggest that the cGMP-induced NKCC2 ubiquitination and degradation may contribute to the cGMP-induced decrease of the NKCC2-dependent NaCl reabsorption in TALs.

Fructose acutely stimulates NKCC2 activity in rat thick ascending limbs by increasing surface NKCC2 expression.

The thick ascending limb (TAL) reabsorbs 25% of the filtered NaCl through the Na+-K+-2Cl- cotransporter (NKCC2). NKCC2 activity is directly related to surface NKCC2 expression and phosphorylation. Higher NaCl reabsorption by TALs is linked to salt-sensitive hypertension, which is linked to consumption of fructose in the diet. However, little is known about the effects of fructose on renal NaCl reabsorption. We hypothesized that fructose, but not glucose, acutely enhances TAL-dependent NaCl reabsorption by increasing NKCC2 activity via stimulation of surface NKCC2 levels and phosphorylation at Thr96/101. We found that fructose (5 mM) increased transport-related O2 consumption in TALs by 11.1 ± 3.2% ( P < 0.05). The effect of fructose on O2 consumption was blocked by furosemide. To study the effect of fructose on NKCC2 activity, we measured the initial rate of NKCC2-dependent thallium influx. We found that 20 min of treatment with fructose (5 mM) increased NKCC2 activity by 58.5 ± 16.9% ( P < 0.05). We then used surface biotinylation to measure surface NKCC2 levels in rat TALs. Fructose increased surface NKCC2 expression in a concentration-dependent manner (22 ± 5, 49 ± 10, and 101 ± 59% of baseline with 1, 5, and 10 mM fructose, respectively, P < 0.05), whereas glucose or a glucose metabolite did not. Fructose did not change NKCC2 phosphorylation at Thre96/101 or total NKCC2 expression. We concluded that acute fructose treatment increases NKCC2 activity by enhancing surface NKCC2 expression, rather than NKCC2 phosphorylation. Our data suggest that fructose consumption could contribute to salt-sensitive hypertension by stimulating NKCC2-dependent NaCl reabsorption in TALs.

Direct renal effects of a fructose-enriched diet: interaction with high salt intake.

Consumption of fructose has increased during the last 50 years. Excessive fructose consumption has a detrimental effect on mammalian health but the mechanisms remain unclear. In humans, a direct relationship exists between dietary intake of added sugars and increased risk for cardiovascular disease mortality (52). While the causes for this are unclear, we recently showed that fructose provided in the drinking water induces a salt-dependent increase in blood pressure in Sprague-Dawley rats in a matter of days (6). However, little is known about the effects of fructose in renal salt handling and whether combined intake of high fructose and salt can lead to salt-sensitive hypertension before the development of metabolic abnormalities. The long-term (more than 4 wk) adverse effects of fructose intake on renal function are not just due to fructose but are also secondary to alterations in metabolism which may have an impact on renal function. This minireview focuses on the acute effect of fructose intake and its effect on salt regulation, as they affect blood pressure.

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 Thr(96)/Thr(101) 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 Thr(96) and Thr(101) 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 Thr(96) and Thr(101) 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 Thr(96) and Thr(101) 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.

Dynamin2, clathrin, and lipid rafts mediate endocytosis of the apical Na/K/2Cl cotransporter NKCC2 in thick ascending limbs.

Steady-state surface levels of the apical Na/K/2Cl cotransporter NKCC2 regulate NaCl reabsorption by epithelial cells of the renal thick ascending limb (THAL). We reported that constitutive endocytosis of NKCC2 controls NaCl absorption in native THALs; however, the pathways involved in NKCC2 endocytosis are unknown. We hypothesized that NKCC2 endocytosis at the apical surface depends on dynamin-2 and clathrin. Measurements of steady-state surface NKCC2 and the rate of NKCC2 endocytosis in freshly isolated rat THALs showed that inhibition of endogenous dynamin-2 with dynasore blunted NKCC2 endocytosis by 56 ± 11% and increased steady-state surface NKCC2 by 67 ± 27% (p < 0.05). Expression of the dominant negative Dyn2K44A in THALs slowed the rate of NKCC2 endocytosis by 38 ± 8% and increased steady-state surface NKCC2 by 37 ± 8%, without changing total NKCC2 expression. Inhibition of clathrin-mediated endocytosis with chlorpromazine blunted NKCC2 endocytosis by 54 ± 6%, while preventing clathrin from interacting with synaptojanin also blunted NKCC2 endocytosis by 52 ± 5%. Disruption of lipid rafts blunted NKCC2 endocytosis by 39 ± 4% and silencing caveolin-1 by 29 ± 4%. Simultaneous inhibition of clathrin- and lipid raft-mediated endocytosis completely blocked NKCC2 internalization. We concluded that dynamin-2, clathrin, and lipid rafts mediate NKCC2 endocytosis and maintain steady-state apical surface NKCC2 in native THALs. These are the first data identifying the endocytic pathway for apical NKCC2 endocytosis.

Molecular regulation of NKCC2 in the thick ascending limb.

The kidney plays an essential role in blood pressure regulation by controlling short-term and long-term NaCl and water balance. The thick ascending limb of the loop of Henle (TAL) reabsorbs 25-30% of the NaCl filtered by the glomeruli in a process mediated by the apical Na(+)-K(+)-2Cl(-) cotransporter NKCC2, which allows Na(+) and Cl(-) entry from the tubule lumen into TAL cells. In humans, mutations in the gene coding for NKCC2 result in decreased or absent activity characterized by severe salt and volume loss and decreased blood pressure (Bartter syndrome type 1). Opposite to Bartter's syndrome, enhanced NaCl absorption by the TAL is associated with human hypertension and animal models of salt-sensitive hypertension. TAL NaCl reabsorption is subject to exquisite control by hormones like vasopressin, parathyroid, glucagon, and adrenergic agonists (epinephrine and norepinephrine) that stimulate NaCl reabsorption. Atrial natriuretic peptides or autacoids like nitric oxide and prostaglandins inhibit NaCl reabsorption, promoting salt excretion. In general, the mechanism by which hormones control NaCl reabsorption is mediated directly or indirectly by altering the activity of NKCC2 in the TAL. Despite the importance of NKCC2 in renal physiology, the molecular mechanisms by which hormones, autacoids, physical factors, and intracellular ions regulate NKCC2 activity are largely unknown. During the last 5 years, it has become apparent that at least three molecular mechanisms determine NKCC2 activity. As such, membrane trafficking, phosphorylation, and protein-protein interactions have recently been described in TALs and heterologous expression systems as mechanisms that modulate NKCC2 activity. The focus of this review is to summarize recent data regarding NKCC2 regulation and discuss their potential implications in physiological control of TAL function, renal physiology, and blood pressure regulation.

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 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-ß-cyclodextrin (MßCD), a compound that inhibits endocytosis by chelating membrane cholesterol, blocked NKCC2 endocytic retrieval. We found that 30-min treatment with MßCD (5 mM) blocked NKCC2 endocytosis by 81% (P < 0.01). Blockade of endocytosis by MßCD 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 MßCD did not change the total pool of NKCC2. MßCD did not affect NKCC2 trafficking when it was complexed with cholesterol before treatment. Inhibition endocytosis with MßCD 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.

cAMP stimulates apical exocytosis of the renal Na(+)-K(+)-2Cl(-) cotransporter NKCC2 in the thick ascending limb: role of protein kinase A.

The apical renal Na(+)-K(+)-2Cl(-) cotransporter NKCC2 mediates NaCl absorption by the thick ascending limb (TAL) of Henle's loop. cAMP stimulates NKCC2 by enhancing steady-state apical membrane levels of this protein; however, the trafficking and signaling mechanisms by which this occurs have not been studied. Here, we report that stimulation of endogenous cAMP levels with either forskolin/3-isobutyl-1-methylxanthine (IBMX) or the V2 receptor agonist [deamino-Cys(1),d-Arg(8)]vasopressin increases steady-state surface NKCC2 and that the protein kinase A (PKA) inhibitor H-89 blocks this effect. Confocal imaging of apical surface NKCC2 in isolated perfused TALs confirmed a stimulatory effect of cAMP on apical trafficking that was blocked by PKA inhibition. Selective stimulation of PKA with the agonist N(6)-benzoyl-cAMP (500 microm) stimulated steady-state surface NKCC2, whereas the Epac-selective agonist 8-p-chlorophenylthio-2'-O-methyl-cAMP (100 and 250 microm) had no effect. To explore the trafficking mechanism by which cAMP increases apical NKCC2, we measured cumulative apical membrane exocytosis and NKCC2 exocytic insertion in TALs. By monitoring apical FM1-43 fluorescence, we observed rapid stimulation of apical exocytosis (2 min) by forskolin/IBMX. We also found constitutive exocytic insertion of NKCC2 in TALs over time, which was increased by 3-fold in the presence of forskolin/IBMX. PKA inhibition blunted cAMP-stimulated exocytic insertion but did not affect the rate of constitutive exocytosis. We conclude that cAMP stimulates steady-state apical surface NKCC2 by stimulating exocytic insertion and that this process is highly dependent on PKA but not Epac.

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.