Molecular regulation of NKCC2 in blood pressure control and hypertension.

PURPOSE OF REVIEW: The apical Na/K/2Cl cotransporter (NKCC2) mediates NaCl reabsorption by the thick ascending limb, contributing to maintenance of blood pressure (BP). Despite effective NKCC2 inhibition by loop diuretics, these agents are not viable for long-term management of BP due to side effects. Novel molecular mechanisms that control NKCC2 activity reveal an increasingly complex picture with interacting layers of NKCC2 regulation. Here, we review the latest developments that shine new light on NKCC2-mediated control of BP and potential new long-term therapies to treat hypertension. RECENT FINDINGS: Emerging molecular NKCC2 regulators, often binding partners, reveal a complex overlay of interacting mechanisms aimed at fine tuning NKCC2 activity. Different factors achieve this by shifting the balance between trafficking steps like exocytosis, endocytosis, recycling and protein turnover, or by balancing phosphorylation vs. dephosphorylation. Further molecular details are also emerging on previously known pathways of NKCC2 regulation, and recent in-vivo data continues to place NKCC2 regulation at the center of BP control. SUMMARY: Several layers of emerging molecular mechanisms that control NKCC2 activity may operate simultaneously, but they can also be controlled independently. This provides an opportunity to identify new pharmacological targets to fine-tune NKCC2 activity for BP management.

Adenine acts in the kidney as a signaling factor and causes salt- and water-losing nephropathy: early mechanism of adenine-induced renal injury.

Chronic adenine feeding is extensively used to develop animal models of chronic renal failure with metabolic features resembling those observed in humans. However, the mechanism by which adenine induces renal failure is poorly understood. In this study, we examined the early effects of adenine on water metabolism and salt balance in rats placed in metabolic cages and fed control or adenine-containing diets for 7 days. Molecular and functional studies demonstrated that adenine-fed rats exhibited a significant reduction in food intake, polyuria, polydipsia, decreased urine osmolality, and increased salt wasting. These effects are independent of changes in food intake and result from a coordinated downregulation of water channel aquaporin-2 (AQP2) and salt transporter (Na+-K+-Cl- cotransporter 2; NKCC2) in the collecting duct and medullary thick ascending limb, respectively. As a result, adenine-fed rats exhibited massive volume depletion, as indicated by a significant body weight loss, increased blood urea nitrogen, and increased hematocrit and hemoglobin levels, all of which were significantly corrected with NaCl replacement. Adenine-induced urinary concentrating defect was not corrected by exogenous arginine vasopressin (AVP), and it correlated with reduced cAMP production in vivo and in vitro. In conclusion, adenine acts on renal tubules as a signaling molecule and causes nephrogenic diabetes insipidus with salt wasting, at least, by directly interfering with AVP V2 receptor signaling with subsequent downregulation of NKCC2 and AQP2 in the kidney. The combination of renal fluid loss and decreased food intake with subsequent massive volume depletion likely plays an important role in the development of early prerenal failure that progresses to chronic kidney disease in long-term adenine feeding.

Treatment with an SLC12A1 antagonist inhibits tumorigenesis in a subset of hepatocellular carcinomas.

A central aim in cancer research is to identify genes with altered expression patterns in tumor specimens and their potential role in tumorigenesis. Most types of tumors, including hepatocellular carcinoma (HCC), are heterogeneous in terms of genotype and phenotype. Thus, traditional analytical methods like the t-test fail to identify all oncogenes from expression profiles. In this study, we performed a meta-Cancer Outlier Profile Analysis (meta-COPA) across six microarray datasets for HCC from the GEO database. We found that gene SLC12A1 was overexpressed in the Hep3B cell line, compared with five other HCC cell lines and L02 cells. We also found that the upregulation of SLC12A1 was mediated by histone methylation within its promoter region, and that SLC12A1 is a positive regulator of the WNK1/ERK5 pathway. Consistent with in vitro results, treatment with the SLC12A1 antagonist Bumetanide delayed tumor formation and reduced Hep3B cell tumor size in mouse xenografts. In summary, our research reveals a novel subset of HCCs that are sensitive to SLC12A1 antagonist treatment, thereby offering a new strategy for precision HCC treatment.

OS9 Protein Interacts with Na-K-2Cl Co-transporter (NKCC2) and Targets Its Immature Form for the Endoplasmic Reticulum-associated Degradation Pathway.

Mutations in the renal specific Na-K-2Cl co-transporter (NKCC2) lead to type I Bartter syndrome, a life-threatening kidney disease featuring arterial hypotension along with electrolyte abnormalities. We have previously shown that NKCC2 and its disease-causing mutants are subject to regulation by endoplasmic reticulum-associated degradation (ERAD). The aim of the present study was to identify the protein partners specifically involved in ERAD of NKCC2. To this end, we screened a kidney cDNA library through a yeast two-hybrid assay using NKCC2 C terminus as bait. We identified OS9 (amplified in osteosarcomas) as a novel and specific binding partner of NKCC2. Co-immunoprecipitation assays in renal cells revealed that OS9 association involves mainly the immature form of NKCC2. Accordingly, immunocytochemistry analysis showed that NKCC2 and OS9 co-localize at the endoplasmic reticulum. In cells overexpressing OS9, total cellular NKCC2 protein levels were markedly decreased, an effect blocked by the proteasome inhibitor MG132. Pulse-chase and cycloheximide-chase assays demonstrated that the marked reduction in the co-transporter protein levels was essentially due to increased protein degradation of the immature form of NKCC2. Conversely, knockdown of OS9 by small interfering RNA increased NKCC2 expression by increasing the co-transporter stability. Inactivation of the mannose 6-phosphate receptor homology domain of OS9 had no effect on its action on NKCC2. In contrast, mutations of NKCC2 N-glycosylation sites abolished the effects of OS9, indicating that OS9-induced protein degradation is N-glycan-dependent. In summary, our results demonstrate the presence of an OS9-mediated ERAD pathway in renal cells that degrades immature NKCC2 proteins. The identification and selective modulation of ERAD components specific to NKCC2 and its disease-causing mutants might provide novel therapeutic strategies for the treatment of type I Bartter syndrome.

Inhibition of Mitochondrial Complex-1 Prevents the Downregulation of NKCC2 and ENaCα in Obstructive Kidney Disease.

Ureteral obstruction with subsequent hydronephrosis is a common clinical complication. Downregulation of renal sodium transporters in obstructed kidneys could contribute to impaired urinary concentrating capability and salt waste following the release of a ureteral obstruction. The current study was undertaken to investigate the role of mitochondrial complex-1 inhibition in modulating sodium transporters in obstructive kidney disease. Following unilateral ureteral obstruction (UUO) for 7 days, a global reduction of sodium transporters, including NHE3, α-Na-K-ATPase, NCC, NKCC2, p-NKCC2, ENaCα, and ENaCγ, was observed, as determined via qRT-PCR and/or Western blotting. Interestingly, inhibition of mitochondrial complex-1 by rotenone markedly reversed the downregulation of NKCC2, p-NKCC2, and ENaCα. In contrast, other sodium transporters were not affected by rotenone. To study the potential mechanisms involved in mediating the effects of rotenone on sodium transporters, we examined a number of known sodium modulators, including PGE2, ET1, Ang II, natriuretic peptides (ANP, BNP, and CNP), and nitric oxide synthases (iNOS, nNOS, and eNOS). Importantly, among these modulators, only BNP and iNOS were significantly reduced by rotenone treatment. Collectively, these findings demonstrated a substantial role of mitochondrial dysfunction in mediating the downregulation of NKCC2 and ENaCα in obstructive kidney disease, possibly via iNOS-derived nitric oxide and BNP.

Mechanisms of guanylin action on water and ion absorption at different regions of seawater eel intestine.

Guanylin (GN) inhibited water absorption and short-circuit current (Isc) in seawater eel intestine. Similar inhibition was observed after bumetanide, and the effect of bumetanide was abolished by GN or vice versa, suggesting that both act on the same target, Na(+)-K(+)-2Cl(-) cotransporter (NKCC), which is a key player for the Na(+)-K(+)-Cl(-) transport system responsible for water absorption in marine teleost intestine. However, effect of GN was always greater than that of bumetanide: 10% greater in middle intestine (MI) and 40% in posterior intestine (PI) for Isc, and 25% greater in MI and 34% in PI for water absorption. After treatment with GN, Isc decreased to zero, but 20-30% water absorption still remained. The remainder may be due to the Cl(-)/HCO3 (-) exchanger and Na(+)-Cl(-) cotransporter (NCC), since inhibitors for these transporters almost nullified the remaining water absorption. Quantitative PCR analysis revealed the presence of major proteins involved in water absorption; the NKCC2ß and AQP1 genes whose expression was markedly upregulated after seawater acclimation. The SLC26A6 (anion exchanger) and NCCß genes were also expressed in small amounts. Consistent with the inhibitors' effect, expression of NKCC2ß was MI > PI, and that of NCCß was MI << PI. The present study showed that GN not only inhibits the bumetanide-sensitive Na(+)-K(+)-Cl(-) transport system governed by NKCC2ß, but also regulates unknown ion transporters different from GN-insensitive SLC26A6 and NCC. A candidate is cystic fibrosis transmembrane conductance regulator Cl(-) channel, as demonstrated in mammals, but its expression is low in eel intestine, and its role may be minor, as indicated by the small effect of its inhibitors.