Potassium Effects on NCC Are Attenuated during Inhibition of Cullin E3–Ubiquitin Ligases

The thiazide-sensitive sodium chloride cotransporter (NCC) plays a vital role in maintaining sodium (Na+) and potassium (K+) homeostasis. NCC activity is modulated by with-no-lysine kinases 1 and 4 (WNK1 and WNK4), the abundance of which is controlled by the RING-type E3 ligase Cullin 3 (Cul3) and its substrate adapter Kelch-like protein 3. Dietary K+ intake has an inverse correlation with NCC activity, but the mechanism underlying this phenomenon remains to be fully elucidated. Here, we investigated the involvement of other members of the cullin family in mediating K+ effects on NCC phosphorylation (active form) and abundance. In kidneys from mice fed diets varying in K+ content, there were negative correlations between NCC (phosphorylated and total) and active (neddylated) forms of cullins (Cul1, 3, 4, and 5). High dietary K+ effects on phosphorylated NCC were attenuated in Cul3 mutant mice (CUL3-Het/Δ9). Short-term (30 min) and long-term (24 h) alterations in the extracellular K+ concentration did not affect cullin neddylation levels in ex vivo renal tubules. In the short term, the ability of high extracellular K+ to decrease NCC phosphorylation was preserved in the presence of MLN4924 (pan-cullin inhibitor), but the response to low extracellular K+ was absent. In the long term, MLN4924 attenuated the effects of high extracellular K+ on NCC phosphorylation, and responses to low extracellular K+ were absent. Our data suggest that in addition to Cul3, other cullins are involved in mediating the effects of K+ on NCC phosphorylation and abundance.

The Pharmacological Inhibition of CaMKII Regulates Sodium Chloride Cotransporter Activity in mDCT15 Cells

The thiazide-sensitive sodium chloride cotransporter (NCC) in the distal convoluted tubule is responsible for reabsorbing up to one-tenth of the total filtered load of sodium in the kidney. The actin cytoskeleton is thought to regulate various transport proteins in the kidney but the regulation of the NCC by the actin cytoskeleton is largely unknown. Here, we identify a direct interaction between the NCC and the cytoskeletal protein filamin A in mouse distal convoluted tubule (mDCT15) cells and in the native kidney. We show that the disruption of the actin cytoskeleton by two different mechanisms downregulates NCC activity. As filamin A is a substrate of the Ca2+/calmodulin-dependent protein kinase II (CaMKII), we investigate the physiological significance of CaMKII inhibition on NCC luminal membrane protein expression and NCC activity in mDCT15 cells. The pharmacological inhibition of CaMKII with the compound KN93 increases the active form of the NCC (phospho-NCC) at the luminal membrane and also increases NCC activity in mDCT15 cells. These data suggest that the interaction between the NCC and filamin A is dependent on CaMKII activity, which may serve as a feedback mechanism to maintain basal levels of NCC activity in the distal nephron.

Activation of the Thiazide-Sensitive Sodium-Chloride Cotransporter by Beta3-Adrenoreceptor in the Distal Convoluted Tubule

We previously showed that the beta-3 adrenergic receptor (BAR3) is expressed in most segments of the nephron where its agonism promotes a potent antidiuretic effect. We localized BAR3 in distal convoluted tubule (DCT) cells expressing the thiazide-sensitive sodium-chloride cotransporter (NCC). Aim of this study is to investigate the possible functional role of BAR3 on NCC modulation in DCT cells. Here, we found that, in mice, the knockout of BAR3 was paralleled by a significant attenuation of NCC phosphorylation, paralleled by reduced expression and activation of STE-20/SPS1-related proline-alanine-rich kinase (SPAK) and WNKs the main kinases involved in NCC activation. Conversely, in BAR1/2 knockout mice, we found reduced NCC abundance with no changes in the phosphorylation state of NCC. Moreover, selective BAR3 agonism promotes both SPAK and NCC activation in wild-type mouse kidney slices. In conclusion, our findings suggest a novel role for BAR3 in the regulation of NCC in DCT.

Gitelman Syndrome in a Pregnant Woman

Background: Gitelman syndrome (GS) is an autosomal recessive inherited salt-losing tubulopathy resulted from a loss-of-function mutation in the gene SLC12A3 encoding the thiazide-sensitive sodium-chloride cotransporter (NCCT) protein located in the distal renal tubules. Investigations revealed hypokalemia, metabolic alkalosis, hypomagnesemia, hypocalciuria and increased activity of renin-angiotensin-aldosterone system. There have been very few case reports on Gitelman syndrome in pregnancy, and some cases showed adverse consequences of the fetus.Case presentation: We presented a case report of a pregnant female with hypokalemia, a large amount of intravenous potassium was required to maintain a relatively normal level of serum potassium.Therefore, further laboratory examinations and whole blood DNA sequencing were carried out. The patient was eventually diagnosed with Gitelman syndrome. In terms of treatment, the amount of potassium supplementation was gradually reduced, and magnesium supplementation was intermittently provided at the same time to maintain the patient's serum potassium at about 3.0mmol/L and serum magnesium at about 0.8mmol/L. Obstetric ultrasound during hospitalization indicated normal fetal development, and the patient was discharged from hospital after her condition improved.Conclusions: The clinical manifestations of GS are non-specific, and there is a lack of evidence-based treatment guidelines for pregnant GS patients, so multidisciplinary management of pregnant GS women is essential. Treatment should be cautious and individual, and the electrolytes should be closely monitored to avoid complications caused by electrolyte disturbance and strive to obtain a good maternal and fetal outcome.

Gitelman Syndrome: A Rare Case of Hypokalaemia and a Novel Mutation

Gitelman syndrome (GS) is a hereditary renal tubulopathy caused by mutations in the SLC12A3 gene which encodes the thiazide-sensitive apical sodium-chloride cotransporter. GS is characterized by hypokalaemia, hypomagnesaemia and metabolic alkalosis. Treatment is based on potassium and magnesium replacement ad eternum. We present the case of a young man with palpitations and persistent hypokalaemia, who was diagnosed with GS. Genetic testing revealed 2 mutations in the gene SLC12A3 of combined heterozygosity, both considered pathological. Interestingly, 1 of these mutations was not yet described in the literature or in the reviewed databases. We also discuss the clinical approach and the specificities of managing this rare hereditary renal tubulopathy.

Thiazide-Sensitive NCC (Sodium-Chloride Cotransporter) in Human Metabolic Syndrome

The thiazide-sensitive sodium-chloride cotransporter (NCC;SLC12A3) is central to sodium and blood pressure regulation. Metabolic syndrome induces NCC upregulation generating sodium-sensitive hypertension in experimental animal models. We tested the role of NCC in sodium sensitivity in hypertensive humans with metabolic syndrome. Conversely, oral potassium induces NCC downregulation producing potassium-induced natriuresis. We determined the time course and magnitude of potassium-induced natriuresis compared with the natriuresis following hydrochlorothiazide (HCTZ) as a reference standard. We studied 19 obese hypertensive humans with metabolic syndrome during 13-day inpatient confinement. We determined sodium sensitivity by change in 24-hour mean systolic pressure by automated monitor from days 5 (low sodium) to 10 (high sodium). We determined NCC activity by standard 50 mg HCTZ sensitivity test (day 11). We determined potassium-induced natriuresis following 35 mmol KCl (day 13). We determined (1) whether NCC activity was greater in sodium-sensitive versus sodium-resistant participants and correlated with sodium sensitivity and (2) time course and magnitude of potassium-induced natriuresis following 35 mmol KCl directly compared with 50 mg HCTZ. NCC activity was not greater in sodium-sensitive versus sodium-resistant humans and did not correlate with sodium sensitivity. Thirty-five-millimoles KCl produced a rapid natriuresis approximately half that of 50 mg HCTZ with a greater kaliuresis. Our investigation tested a key hypothesis regarding NCC activity in human hypertension and characterized potassium-induced natriuresis following 35 mmol KCl compared with 50 mg HCTZ. In obese hypertensive adults with metabolic syndrome ingesting a high-sodium diet, 35 mmol KCl had a net natriuretic effect approximately half that of 50 mg HCTZ.

The Role of Serum Chloride in Acute and Chronic Heart Failure: A Narrative Review

Clinical guidelines include diuretics for the treatment of heart failure (HF), not to decrease mortality but to decrease symptoms and hospitalizations. More attention has been paid to the worse outcomes, including mortality, associated with continual diuretic therapy due to hypochloremia. Studies have revealed a pivotal role for serum chloride in the pathophysiology of HF and is now a target of treatment to decrease mortality. The prognostic value of serum chloride in HF has been the subject of much attention. Mechanistically, the macula densa, a region in the renal juxtaglomerular apparatus, relies on chloride levels to sense salt and volume status. The recent discovery of with-no-lysine (K) (WNK) protein kinase as an intracellular chloride sensor sheds light on the possible reason of diuretic resistance in HF. The action of chloride on WNKs results in the upregulation of the sodium-potassium-chloride cotransporter and sodium-chloride cotransporter receptors, which could lead to increased electrolyte and fluid reabsorption. Genetic studies have revealed that a variant of a voltage-sensitive chloride channel (CLCNKA) gene leads to almost a 50% decrease in current amplitude and function of the renal chloride channel. This variant increases the risk of HF. Several trials exploring the prognostic value of chloride in both acute and chronic HF have shown mostly positive results, some even suggesting a stronger role than sodium. However, so far, interventional trials exploring serum chloride as a therapeutic target have been largely inconclusive. This study is a review of the pathophysiologic effects of hypochloremia in HF, the genetics of chloride channels, and clinical trials that are underway to investigate novel approaches to HF management.