Intestinal Na+, K+, 2Cl- cotransporter 2 plays a crucial role in hyperosmotic transitions of a euryhaline teleost.

Euryhaline fishes, such as the red drum (Sciaenops ocellatus), must quickly transition between hyperosmotic and hypoosmotic physiological strategies. When freshwater individuals transition to seawater they are exposed to increased diffusive water loss and ion gain. To maintain osmoregulatory balance these animals must drink and absorb seawater through the intestine, followed by ion excretion at the gills. The ability of fishes to transition between strategies can limit the magnitude of osmotic shock that can be tolerated. Here, we demonstrate that red drum can tolerate direct transfer from freshwater to full-strength seawater with marginal impacts on osmotic balance, as indicated by plasma and muscle ion concentration, as well as muscle water. Seawater transition is concurrent with a significant increase in intestinal fluid volume. Typical patterns of osmoregulatory plasticity were observed in the gill with increased expression of nkcc1 and cftr Expression changes in the anterior intestine were observed after 24 h for nkcc2 with smaller and later responses observed for slc26a3, slc26a6, and nbc Immunofluorescence staining demonstrated similar patterns of NKCC localization in freshwater and seawater intestines; however, reduced basolateral staining of V-type ATPase was observed in seawater. Electrophysiological preparations demonstrated that seawater fish had increased absorptive current in the anterior intestine, which was significantly reduced in the presence of 10 µmol/L bumetanide. Overall, these results suggest that nkcc2 plays a crucial role during hyperosmotic transitions, and may be a more important complement to the well-known bicarbonate secretion pathway than generally considered.

Effect of vasopressin on Na(+)-K(+)-2Cl(-) cotransporter (NKCC) and the signaling mechanisms on the murine late distal colon.

It has been demonstrated that the antidiuretic hormone vasopressin is able to regulate the expression of Na-K-Cl cotransporters (NKCC1 and NKCC2) in the kidney. The present study investigated the effects of long- and short-term administration of vasopressin on NKCC and the possible signaling mechanism of vasopressin in the mouse distal colon using the siRNA, real-time PCR, western blotting and Ussing chambers method. The results showed the presence of NKCC2 expression in the colon, which was verified with a siRNA technique. The mRNA and protein expression level of NKCC2 significantly increased by about 40% and 90% respectively in response to restricting water intake to 1ml/day/20g for 7 days. In contrast, the NKCC1 expression level was unchanged in the colon. To determine the short-term activation of NKCC2 by vasopressin in vitro, we found that the administration of vasopressin caused a 3-fold increase in mouse colon NKCC2 phosphorylation, which was detected with phosphospecific antibody R5. In addition, the Ussing chamber results showed that NKCC2, cAMP and Ca(2+) signaling pathway may be involved in the vasopressin-induced response. Further, adenylate cyclase inhibitor MDL-12330A and PKA inhibitor H89 and Ca(2+) chelator BAPTA-AM reversed the vasopressin induced NKCC2 phosphorylation level increase by about 35%, 28% and 42% respectively suggesting vasopressin stimulate NKCC2 phosphorylation increase mediated by cAMP-PKA and Ca(2+) signaling in the colon. Collectively, these data suggest that the expression and phosphorylation of NKCC2 are increased in the colon by vasopressin stimulation, in association with enhanced activity of the vasopressin/cAMP and Ca(2+) pathways.

Renal sodium transporters are increased in urinary exosomes of cyclosporine-treated kidney transplant patients.

BACKGROUND/AIMS: Cyclosporine (CsA) is a calcineurin inhibitor widely used as an immunosuppressant in organ transplantation. Previous studies demonstrated the relationship between CsA and renal sodium transporters such as the Na-K-2Cl cotransporter in the loop of Henle (NKCC2). Experimental models of CsA-induced hypertension have shown an increase in renal NKCC2. METHODS: Using immunoblotting of urinary exosomes, we investigated in CsA-treated kidney transplant patients (n = 39) the excretion of NKCC2 and Na-Cl cotransporter (NCC) and its association with blood pressure (BP) level. We included 8 non-CsA-treated kidney transplant patients as a control group. Clinical data, immunosuppression and hypertension treatments, blood and 24-hour urine tests, and 24-hour ambulatory BP monitoring were recorded. RESULTS: CsA-treated patients tended to excrete a higher amount of NKCC2 than non-CsA-treated patients (mean ± SD, 175 ± 98 DU and 90 ± 70.3 DU, respectively; p = 0.05) and showed higher BP values (24-hour systolic BP 138 ± 17 mm Hg and 112 ± 12 mm Hg, p = 0.003; 24-hour diastolic BP, 83.8 ± 9.8 mm Hg and 72.4 ± 5.2 mm Hg, p = 0.015, respectively). Within the CsA-treated group, there was no correlation between either NKCC2 or NCC excretion and BP levels. This was confirmed by a further analysis including potential confounding factors. On the other hand, a significant positive correlation was observed between CsA blood levels and the excretion of NKCC2 and NCC. CONCLUSION: Overall, these results support the hypothesis that CsA induces an increase in NKCC2 and NCC in urinary exosomes of renal transplant patients. The fact that the increase in sodium transporters in urine did not correlate with the BP level suggests that in kidney transplant patients, other mechanisms could be implicated in CsA-induced hypertension.

Localization and vasopressin regulation of the Na⁺-K⁺-2Cl⁻ cotransporter in the distal colonic epithelium.

AIM: To investigate whether Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) is expressed in the mouse distal colonic epithelia and whether it is regulated by vasopressin in the colon. METHODS: The mRNA expression of NKCC2 in the mouse colonic mucosa was examined by reverse transcription-polymerase chain reaction. NKCC trafficking in the colon stimulated by 1-D-amino(8-D-arginine)-vasopressin (dDAVP) infusion (10 ng/mouse, intraperitoneal injection ) within 15 min, 30 min and 1h was investigated by laser confocal scanning microscopy. Total and membrane NKCC2 expression in the colonic mucosa from control and dDAVP-treated mice was detected by Western blotting. Short circuit current method was performed to determine regulation of NKCC2 by vasopressin in the colon. RESULTS: NKCC2 was predominantly located in the apical region of the surface of the distal colonic epithelia; by comparison, a large amount of NKCC1 was distributed in the basolateral membrane of the lower crypt epithelia of the mouse distal colon. Short-term treatment with dDAVP, a V2-type receptor-specific vasopressin analog, induced NKCC2 re-distribution, i.e., NKCC2 traffics to the apical membrane after dDAVP stimulation. In contrast, no obvious NKCC1 membrane translocation was observed. Western blotting results confirmed that membrane NKCC2 had significantly higher abundance in the dDAVP-treated mouse colonic mucosa relative to that in the untreated control, which is consistent with our immunostaining data. Moreover, the short-circuit current method combined with a NKCC2 inhibitor demonstrated that NKCC2 was also activated by serosal vasopressin in isolated distal colonic mucosa. CONCLUSION: Our results provide direct evidence that vasopressin also plays an important role in the colonic epithelia by stimulating NKCC2 trafficking to the apical membrane and inducing NKCC2-mediated ion transport.