Na-Cl Cotransporter Research Articles

Roles of WNK4 and SPAK in K+-mediated dephosphorylation of the NaCl cotransporter.

Phosphorylation of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) is altered rapidly in response to changes in extracellular K+ concentration ([K+]). High extracellular [K+] is believed to activate specific phosphatases to dephosphorylate NCC, thereby reducing its activity. This process is defective in the human disease familial hyperkalemic hypertension, in which extracellular [K+] fails to dephosphorylate NCC, suggesting an interplay between NCC-activating and NCC-inactivating switches. Here, we explored the role of STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and intracellular Cl- concentration in the rapid effects of extracellular K+ on NCC phosphorylation. SPAK was found to be rapidly dephosphorylated in vitro in human embryonic kidney cells and ex vivo in kidney slices by high [K+]. Acute high-K+ challenge resulted in DCT1-specific SPAK dephosphorylation in vivo and dissolution of SPAK puncta. In line with the postulate of interplay between activating and inactivating switches, we found that the "on" switch, represented by with no lysine kinase 4 (WNK4)-SPAK, must be turned off for rapid NCC dephosphorylation by high [K+]. Longer-term WNK-SPAK-mediated stimulation, however, altered the sensitivity of the system, as it attenuated rapid NCC dephosphorylation due to acute K+ loading. Although blockade of protein phosphatase (PP)1 increased NCC phosphorylation at baseline, neither PP1 nor PP3, singly or in combination, was essential for NCC dephosphorylation. Overall, our data suggest that NCC phosphorylation is regulated by a dynamic equilibrium between activating kinases and inactivating phosphatases, with kinase inactivation playing a key role in the rapid NCC dephosphorylation by high extracellular K+.NEW & NOTEWORTHY Although a great deal is known about mechanisms by which thiazide-sensitive NaCl cotransporter is phosphorylated and activated, much less is known about dephosphorylation. Here, we show that rapid dephosphorylation by high K+ depends on the Cl- sensitivity of with no lysine kinase 4 and the rapid dephosphorylation of STE20/SPS1-related proline-alanine-rich protein kinase, primarily along the early distal convoluted tubule.

Zinc Deficiency Drives Renal NFkB Activation

Background Zn2+ deficiency (ZnD) is a worldwide problem. In the United States, 14% of Americans are Zn2+ deficient, which represents 1 out of 7 people. In ZnD populations, the prevalence of hypertension is higher. In our recent studies, we demonstrated that ZnD induces hypertension by promoting renal Na+ reabsorption by the sodium chloride co-transporter (NCC). However, the exact molecular mechanisms involved in NCC upregulation were undefined. Nuclear factor-κB (NFκB) is a transcription factor found to play a role in ZnD-mediated detrimental effects throughout the body. Hypothesis As such, we hypothesized that ZnD drives renal NFκB activation. Experimental Design To examine the effects of ZnD on renal NFκB activation, adult, male C57Bl/6 mice were fed a ZnA- or ZnD-diet for 6 weeks. NFκB expression and nuclear translocation were examined by immunohistochemistry. To confirm the role of Zn2+ in NFκB regulation, mouse distal convoluted tubular (mDCT) cells were treated with the Zn2+ chelator - N,N,Nʹ,Nʹ-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) or vehicle ± Zn2+ supplementation. Results In ZnD mice, NFκB protein expression and nuclear localization were increased compared to ZnA mice. Consistently, in mDCT cells, TPEN-induced ZnD stimulated NFκB expression and nuclear translocation. However, Zn2+ supplementation reversed TPEN-induced NFκB upregulation. Conclusion These results indicate that 1) NFκB is a Zn2+-regulated nuclear transcription factor, and 2) ZnD drives renal NFkB activation. Significance NFκB represents a potential mediator that drives ZnD-induced NCC upregulation and consequently renal Na+ reabsorption and hypertension.

Phosphorylation of NCC and its Association with The Actin Cytoskeleton Linker Protein Ezrin in the Diabetic Kidney is Reduced by Dapagliflozin Treatment

Ezrin, an actin cytoskeleton linker protein is known to regulate various membrane proteins in the kidney. The Sodium Chloride Cotransporter (NCC) is responsible for 5-10% of salt reabsorption from the filtrate in the distal convoluted tubules. Dapagliflozin, an SGLT2 inhibitor, is a novel drug in the treatment of type 2 diabetes mellitus. We hypothesize that dapagliflozin decreases sodium reabsorption and blood pressure by attenuating the association between NCC and ezrin in the distal convoluted tubules in the hypertensive diabetic db/db mice. Twelve week-old male db/db mice with a blood glucose of more than 300 mg/dl were salt-loaded (4% NaCl) for 10 days to induce hypertension. The mice were subjected to metabolic cage studies for 24-hour urine collections in order to measure urinary electrolytes, creatinine, and albumin. Blood pressure was measured by the tail-cuff method to evaluate the effect of dapagliflozin or vehicle given by oral gavage (10 mg/kg of body weight per day for 2 weeks). At the end of the study the mice were euthanized, and the left kidney was formalin-fixed and paraffin-embedded for 3,3′-Diaminobenzidine (DAB) double staining for phospho-NCC and ezrin proteins while the right kidney was homogenized for Western blotting. Western blot analysis showed attenuation of total NCC and ezrin proteins in the diabetic db/db mice given an oral gavage of dapagliflozin compared to vehicle. DAB analysis showed reduced expression of the actin cytoskeleton linker protein ezrin and phosphorylated NCC in the dapagliflozin treated group compared to the control group. Taken together, we show dapagliflozin decreases sodium retention and blood pressure by reducing the density of renal NCC at the luminal membrane and the association between NCC and the actin cytoskeleton.

NFkB Mediates Renal Sodium Retention via the Sodium Chloride Cotransporter in Zinc Deficiency‐Induced Hypertension

Background : Zinc deficiency (ZnD) is comorbid with diseases such as kidney disease and diabetes. Individuals in these populations have a higher prevalence of hypertension. Recently, we reported that ZnD promotes hypertension in mice. The blood pressure elevations were accompanied with increased Na+ retention via the renal Sodium Chloride Cotransporter (NCC). Although our published results indicate that zinc plays a critical role in blood pressure and NCC regulation, the mechanisms involved were not investigated. Hypothesis : Since nuclear factor-kB (NFkB) mediates ZnD-induced detrimental effects, we tested the hypothesis that NFκB drives ZnD-induced NCC upregulation and subsequently Na+ retention and hypertension. Experimental Design : To determine the role of NFκB in ZnD-induced renal Na+ retention and hypertension, adult male C57Bl/6 mice were fed a ZnD diet (5 weeks) followed by administration of the NFκB inhibitor - caffeic acid phenethyl ester (CAPE; 1 week). Systolic blood pressure and urinary Na+ concentrations were examined. To examine if NFkB mediates ZnD-induced NCC upregulation, mouse distal convoluted tubule (mDCT) cells were treated with the zinc chelator N, N, N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) ± CAPE, a NFκB inhibitor. After 24 hours, NCC mRNA and protein expressions, as well as activation, were assessed by qRT-PCR, Western blot and immunofluorescence, respectively. Results : In mice, ZnD promoted hypertension and renal Na+ retention. Further, increased NCC expression was accompanied with enhanced NFkB expression in ZnD mice. However, CAPE administration lowered blood pressure and elevated urinary Na+ concentrations. In mDCT cells, TPEN-induced NCC upregulation was prevented by CAPE treatment. Conclusions : Together, these results demonstrate that 1) NFkB is a novel NCC regulator and 2) NFkB mediates ZnD-induced renal Na+ retention and hypertension. These novel findings indicate that ZnD drives renal NFkB activation thereby stimulating NCC-mediated Na+ retention and promoting hypertension. Significance : This study identifies NFkB as a possible pharmacological target to mitigate hypertension.

Zinc Deficiency‐Induced NFκB Activation Promotes Hypertension by Driving Renal Na + Reabsorption

Background : Zinc deficiency (ZnD) is a common comorbidity with chronic disorders including diabetes and kidney disease. Patients with these diseases also have a higher incidence of hypertension. Recently, we reported that a zinc deficient diet is sufficient to induce hypertension in mice. Changes in blood pressure were accompanied by increased Na+ reabsorption via the renal sodium chloride cotransporter (NCC). Although our novel results indicate that zinc plays a critical role in NCC regulation and its downstream effects on blood pressure, the specific mechanisms involved are unknown. Hypothesis : Since nuclear factor-κB (NFκB) is a key transcription factor implicated in ZnD-mediated effects, we tested the hypothesis that ZnD stimulates renal NFκB activation which drives NCC upregulation and thereby promotes hypertension. Experimental Design : Adult, male C57BL/6 mice were fed a zinc adequate (ZnA; 50ppm) or a ZnD (1ppm) diet for 6 weeks. To examine the role of NFκB in ZnD-induced hypertension, mice were switched from a ZnA-diet to a ZnD-diet for 5 weeks. After, ZnD mice were administered caffeic acid phenethyl ester (CAPE), an NFκB inhibitor, for 1 week. In mice, NCC expression (qRT-PCR) and blood pressure (tail-cuff) were measured. To further investigate the role of NFκB in NCC expression (qRT-PCR and Western blot), mouse distal convoluted tubule (mDCT) cells were treated with TPEN (a zinc chelator) or vehicle (DMSO) ± CAPE. Results : Compared to ZnA mice, NCC mRNA abundance was increased in ZnD mice (7.625 ± 1.747 vs 1.0 ± 0.127) and was accompanied by enhanced renal NFκB nuclear localization. In mDCT cells, TPEN-induced ZnD stimulated NCC mRNA expression (3.152 ± 0.966 vs 1.0 ± 0.174) and NFκB upregulation (1.573 ± 0.117 vs 1.0 ± 0). However, NFκB inhibition by CAPE prevented TPEN-induced NCC mRNA (0.636 ± 0.178 vs 3.152 ± 0.966) and protein (1.039 ± 0.018 vs 2.335 ± 0.062) expression in mDCT cells. Finally, CAPE administration lowered blood pressure (154.5 ± 5.42 vs 138.1 ± 1.97 mm Hg) in ZnD mice. Summary : These results demonstrate that 1) renal NFκB is a Zn2+-regulated transcription factor, 2) NFκB is involved in NCC regulation and 3) NFκB mediates ZnD-induced blood pressure increases. Significance : Taken together, these data indicate that ZnD stimulates NFκB activation thereby driving NCC upregulation and hypertension.

Update on Treatment of Hypertension After Renal Transplantation.

To incorporate novel findings on pathophysiology and treatment of posttransplant hypertension. (1) The sodium retaining effects of CNIs are mediated by stimulation of the thiazide-sensitive sodium chloride co-transporter in the distal convoluted tubule and in this regard chlorthalidone was proven to be an effective antihypertensive drug in renal transplantation. (2) Local and not systemic activation of the renin-angiotensin-aldosterone system plays a crucial role in the pathogenesis of posttransplant hypertension. (3) Recent randomized controlled trials failed to prove the presumed superiority of renin-angiotensin blockers in kidney transplantation. (4) Steroid-free and mammalian target of rapamycin-based immunosuppressive drug combinations did not show favorable effects on blood pressure control. (5) In a recent report the risk of non-melanoma skin cancer was higher with thiazide diuretics. But the increased cancer risk in transplant recipients is mainly attributed to comorbidities, such as diabetes and hypertension and of course to the transplantation condition itself or the obligatory application of immunosuppression, and has little to do with the antihypertensive medication Actual recommendations about BP targets in adult renal transplant recipients are coming from a post hoc analysis of a large randomized trial with another primary endpoint. Unless convincing studies on treatment of hypertension after renal transplantation are available, the ESC/ESH Guidelines 2018 should apply for these patients.

Effects of Chronic Potassium Supplementation and the Accompanying Anion on Blood Pressure

Increased dietary NaCl is associated with higher blood pressure (BP), but dietary potassium supplementation can reduce BP and reduce ‘salt sensitivity’ of BP, improving cardiovascular (CV) outcomes. Rodents fed a high K+ diet often exhibit reduced activity of the sodium-chloride cotransporter NCC, promoting natiuresis, which is a proposed major mechanism for mediating the effects of K+ on BP. However, the role of the accompanying anion during K+ supplementation is a matter of debate, especially considering that low plasma chloride levels are associated with better CV outcomes. Recently, the effect of extreme K+ diets on rodent BP seemed inconsistent with patterns of BP modulation seen in humans. Therefore, the aim of this study was to establish how BP is altered during chronic alteration of dietary potassium intake alongside alternative anions and/or NaCl supplementation. Male mice implanted with telemetric BP monitors were initially normalized to a control diet of NaCl (0.3% Na+) and KCl (1.05% K+), and subsequently maintained on this control diet or a high NaCl (1.57% Na+) (HS) diet for 7 weeks. Subsequently, mice were stratified to receive either a high KCl (5.25% K+) or a high K-citrate (KCit, 5.25% K+) diet during control or high NaCl intake for a subsequent 3 weeks. Finally, a subset of mice were switched to a zero K+ diet with either control or high NaCl level. Throughout the experiment BP, heart rate, activity, urinary ion excretion and plasma Na+/K+/Cl- levels were monitored. 2 weeks 0 K+ diet significantly increased BP, an effect augmented under HS diet. A trend to reduce BP was apparent under control and HS diet during chronic KCl feeding (> effect during resting period), whereas chronic KCit feeding only tended to reduce BP during HS diet. Plasma K+ levels inversely correlated with BP, with chronic KCl inducing a significant increase of 0.5mM, whereas chronic KCit feeding had no significant effect. Plasma aldosterone was also significantly increased 3.5 fold by chronic KCl feeding but not significantly by KCit diet. Total and phosphorylated NCC levels were significantly increased on 0 K+ diet. KCl and KCit feeding during control NaCl or HS intake reduced total NCC levels, whereas phosphorylated NCC was only reduced during chronic KCl intake. All dietary conditions had minimal effect on levels of WNK/SPAK pathway proteins, but αENaC (cleaved/uncleaved) levels were increased, substantially more during KCit feeding. Pendrin abundance was also significantly elevated by KCit feeding. In summary, we show a negative correlation of BP with plasma K+, consistent with human interventional studies. Chronic KCl has a greater ability to lower BP than KCit feeding. This may be related to the ability of KCl to lower NCC phosphorylation, or that large increases in αENaC and pendrin levels during KCit feeding oppose the BP lowering effects of reduced total NCC. As aldosterone levels were increased more with KCl, a differential role of aldosterone also needs to be considered.

Deletion of renal Nedd4-2 abolishes the effect of high sodium intake (HS) on Kir4.1, ENaC, and NCC and causes hypokalemia during high HS.

Neural precursor cell expressed developmentally downregulated protein 4-2 (Nedd4-2) regulates the expression of Kir4.1, thiazide-sensitive NaCl cotransporter (NCC), and epithelial Na+ channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN), and Nedd4-2 deletion causes salt-sensitive hypertension. We now examined whether Nedd4-2 deletion compromises the effect of high-salt (HS) diet on Kir4.1, NCC, ENaC, and renal K+ excretion. Immunoblot analysis showed that HS diet decreased the expression of Kir4.1, Ca2+-activated large-conductance K+ channel subunit-α (BKα), ENaCβ, ENaCγ, total NCC, and phospho-NCC (at Thr53) in floxed neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4lfl/fl) mice, whereas these effects were absent in kidney-specific Nedd4-2 knockout (Ks-Nedd4-2 KO) mice. Renal clearance experiments also demonstrated that Nedd4-2 deletion abolished the inhibitory effect of HS diet on hydrochlorothiazide-induced natriuresis. Patch-clamp experiments showed that neither HS diet nor low-salt diet had an effect on Kir4.1/Kir5.1 currents of the distal convoluted tubule in Nedd4-2-deficient mice, whereas we confirmed that HS diet inhibited and low-salt diet increased Kir4.1/Kir5.1 activity in Nedd4lflox/flox mice. Nedd4-2 deletion increased ENaC currents in the ASDN, and this increase was more robust in the cortical collecting duct than in the distal convoluted tubule. Also, HS-induced inhibition of ENaC currents in the ASDN was absent in Nedd4-2-deficient mice. Renal clearance experiments showed that HS intake for 2 wk increased the basal level of renal K+ excretion and caused hypokalemia in Ks-Nedd4-2-KO mice but not in Nedd4lflox/flox mice. In contrast, plasma Na+ concentrations were similar in Nedd4lflox/flox and Ks-Nedd4-2 KO mice on HS diet. We conclude that Nedd4-2 plays an important role in mediating the inhibitory effect of HS diet on Kir4.1, ENaC, and NCC and is essential for maintaining normal renal K+ excretion and plasma K+ ranges during long-term HS diet.NEW & NOTEWORTHY The present study suggests that Nedd4-2 is involved in mediating the inhibitory effect of high salt (HS) diet on Kir4.1/kir5.1 in the distal convoluted tubule, NaCl cotransporter function, and epithelial Na+ channel activity and that Nedd4-2 plays an essential role in maintaining K+ homeostasis in response to a long-term HS diet. This suggests the possibility that HS intake could lead to hypokalemia in subjects lacking proper Nedd4-2 E3 ubiquitin ligase activity in aldosterone-sensitive distal nephron.

Activation of the kidney sodium chloride cotransporter by the β2-adrenergic receptor agonist salbutamol increases blood pressure

The thiazide-sensitive sodium-chloride-cotransporter (NCC) in the kidney distal convoluted tubule (DCT) plays an essential role in sodium and potassium homeostasis. Here, we demonstrate that NCC activity is increased by the β2-adrenoceptor agonist salbutamol, a drug prevalently used to treat asthma. Relative to β1-adrenergic receptors, the β2-adrenergic receptors were greatly enriched in mouse DCT cells. In mice, administration of salbutamol increased NCC phosphorylation (indicating increased activity) within 30 minutes but also caused hypokalemia, which also increases NCC phosphorylation. In exvivo kidney slices and isolated tubules, salbutamol increased NCC phosphorylation in the pharmacologically relevant range of 0.01-10 μM, an effect observed after 15 minutes and maintained at 60 minutes. Inhibition of the inwardly rectifying potassium channel (Kir) 4.1 or the downstream with-no-lysine kinases (WNKs) and STE20/SPS1-related proline alanine-rich kinase (SPAK) pathway greatly attenuated, but did not prevent, salbutamol-induced NCC phosphorylation. Salbutamol increased cAMP in tubules, kidney slices and mpkDCT cells (model of DCT). Phosphoproteomics indicated that protein phosphatase 1 (PP1) was a key upstream regulator of salbutamol effects. A role for PP1 and the PP1 inhibitor 1 (I1) was confirmed in tubules using inhibitors of PP1 or kidney slices from I1 knockout mice. On normal and high salt diets, salbutamol infusion increased systolic blood pressure, but this increase was normalized by thiazide suggesting a role for NCC. Thus, β2-adrenergic receptor signaling modulates NCC activity via I1/PP1 and WNK-dependent pathways, and chronic salbutamol administration may be a risk factor for hypertension.

Downregulation of Cullin 3 Ligase Signaling Pathways Contributes to Hypertension in Preeclampsia.

Background: Preeclampsia (PE) is a leading cause of maternal and perinatal morbidity and mortality; however, its etiology and pathophysiology remain obscure. PE is initiated by inadequate spiral artery remodeling and subsequent placental ischemia/hypoxia, which stimulates release of bioactive factors into maternal circulation, leading to hypertension and renal damage.Methods and Results: Abundance of key components of cullin 3-ring ubiquitin ligase (CRL3), including cullin 3 (CUL3) and its neddylated modification, and adaptors including Kelch-like 2 (KLHL2) and Rho-related BTB domain containing protein 1 was all decreased in spiral arteries and placentas of PE patients. Similar changes were found in aortic tissues and renal distal tubules of pregnant mice treated with Nω-nitro-l-arginine methyl ester hydrochloride. The downregulation of CRL3 function led to accumulation of with-no-lysine kinases, phosphodiesterase 5, and RhoA in vessels and renal distal tubules, which promoted vasoconstriction and Na–Cl cotransporter activation in the distal convoluted tubule (DCT), as well as vascular and DCT structure remodeling. Proton pump inhibitor esomeprazole partially restored CRL3 function. In vitro studies have shown that increased abundance of JAB1, a component of the COP9 signalosome, inhibited CUL3 neddylation and promoted the expression of hypoxia-inducible factor 1α, which downregulated peroxisome proliferator–activated receptor γ and further promoted CUL3 inactivation. KLHL3/2 was degraded by increased autophagy.Conclusion: These findings support that the downregulation of CRL3 function disrupts the balance of vasoconstriction and vasodilation and aggravates excess reabsorption of sodium in PE.

Dietary Magnesium Insufficiency Induces Salt-Sensitive Hypertension in Mice Associated With Reduced Kidney Catechol-O-Methyl Transferase Activity.

[Figure: see text].

DIVERSIFIED RENAL TUBULAR EPITHELIAL REMODELING AND WATER BALANCE IN GITELMAN'S SYNDROME AND THIAZIDE-TREATED HYPERTENSIVE PATIENTS

Objective: Mutations leading to loss of function of NaCl cotransporter (NCC) in the distal convoluted tubule is the basis defect of Gitelman's Syndrome (GS), which is clinically characterized by hypokaliemic metabolic alkalosis, hypomagnesemia, hypocalciuria and normal or low blood pressure with increased renin activity and high aldosterone levels. NCC is the target of thiazide diuretics (TZ) and clinical and laboratory findings of GS patients resemble those on chronic thiazide diuretics treatment. From a physiopathological perspective, this similarity in term of site of defect or site of action (NCC in distal convoluted tubule) for GS or TZ-treated patients respectively, should not alter their urine-concentrating or diluting ability. We aimed to study water balance pathways in these 2 clinical settings. Design and method: We investigated two groups of patients: 1) GS (n = 5, age range 21–43, 2 females and 3 males, mean concentration of blood potassium 2.9 mmol/L, bicarbonate 34 mmol/L and magnesium 1.2 mg/dl, normotensive) and 2) TZ hypertensive patients (n = 11, age range 26–48, 4 female and 7 males, blood pressure normalized with chronic thiazide type or like diuretics including hydrochlorothiazide, chlortalidone and indapamide maintained for at least 2 months) to evaluate their water balance pathway by determining plasma and urine osmolarity and to assess through urine aquaporin 2 (AQP2)/creatinine ratio their sensitivity to antidiuretic hormone. These measurements were repeated three times in the same patients. Results: Four out of five GS patients showed a urinary AQP2/creatinine ratio lower than TZ-treated patients (mean value of AQP2/creatinine of GS vs TZ-treated p < .05), associated with lower urine osmolarity (p = 0.03). Conclusions: Our study shows that water balance in these two groups may be differently handled: A diversified renal tubular epithelial remodeling of medullary collecting duct cells related to TZ treatment could be suggested, leading to an increased prevalence of principal cells on intercalated cells, thus increasing free water reabsorption.

PENDRIN REDUCTION IS ASSOCIATED WITH ACUTE INTRAVENOUS SODIUM CHLORIDE LOADING IN PATIENTS WITH PRIMARY ALDOSTERONISM

Objective: Primary aldosteronism (PA) is a common, potentially curable form of hypertension characterised by excessive, autonomous aldosterone production. In the distal renal tubule, aldosterone is involved in volume homeostasis by modulation of NaCl reabsorption via the epithelial Na+ channel (ENaC), the NaCl cotransporter (NCC) and the Cl-/HCO3- exchanger (pendrin). Na+ loading suppresses plasma aldosterone but its effects on these proteins in humans remain unclear. Design and method: Urine samples were collected before and 1 h after a 4 h 2L 0.9% saline infusion from 13 (F9/M5) subjects including 10 confirmed with PA and 3 in which PA was excluded [herein designated low renin hypertension (LRH)]. Urinary exosomes were harvested by ultracentrifugation and analysed by LC-MS/MS. Results: Plasma aldosterone decreased (P < 0.05) from 623 [312, 2210] (median [range]) to 338 [197, 2330] pmol/L in confirmed PA and from 223 [180, 256] to 117 [110, 133] pmol/L in LRH. There were no changes in plasma K+ (pre 3.4 [2.6, 4.3] vs post 3.6 [2.6, 3.9] mmol/L in PA; pre 4.1 [3.6, 4.1] vs post 4.0 [3.9, 4.0] mmol/L in LRH). LC-MS/MS demonstrated decreases in abundance of pendrin (21.5%, P < 0.01) and aquaporin-2 (AQP2) (42.9%, P < 0.01) in urinary exosomes and a trend towards a decrease in NCC (20%, P = 0.07) in patients with PA. No changes were seen in those with LRH. PA patients had higher baseline pendrin abundance (1.2 [1.0, 2.1]) than LRH patients (0.7 [0.5, 1.0]) (P < 0.05), but there was no difference in baseline AQP2. In all 13 subjects combined, baseline pendrin correlated positively with plasma aldosterone (P = 0.02, r2 = 0.32) and negatively with plasma K+ (P = 0.02, r2 = 0.35), whilst no correlations were observed between AQP2 and aldosterone or K+. Delta Pendrin (P = 0.04, r2 = 0.3) and delta AQP2 (P = 0.01, r2 = 0.58) positively correlated with delta plasma aldosterone. Conclusions: In PA, reduction in pendrin during saline infusion may be due to falling plasma aldosterone in response to NaCl loading. Although also correlating with reduction in aldosterone, a more likely explanation for the reduction in AQP2 may be a fall in plasma vasopressin induced by water loading. Different observations in LRH require validation with larger numbers.

RNA-SEQ REVEALS HSP90 AS A REGULATOR FOR INTERLEUKIN 6-MEDIATED ACTIVATION OF NCC VIA THE MINERALOCORTICOID RECEPTOR

Objective: Hypertension (HTN) is characterized by increased Na+ reabsorption and systemic inflammation including increased cytokines such as IL6. Clinically, IL6 is increased in hypertensive patients. Previous data from our laboratory has revealed a mechanism for IL6-mediated activation of the mineralocorticoid receptor (MR), leading to increased Na+ transporter activation and HTN via reactive oxygen species (ROS) and Rac1. We hypothesized that IL6 induces a specific transcriptome in the late distal nephron (DCT2) and used RNA-seq to investigate. Design and method: Mouse DCT2 (mDCT15) cells were treated with IL6 (100ng/mL, 24hrs) or vehicle (n = 4, each), then total RNA isolated. Data from the Illumina-sequenced transcriptome underwent comparative analysis to identify significant differentially expressed transcripts between vehicle and IL6 treated cells. Results: Differential gene expressed revealed multiple targets, including HSP90 (3.03x10-6, p-adj) encoding for heat shock protein (HSP) 90. HSP90 has been previously shown as crucial for ligand binding, MR nuclear translocation and activation. To investigate whether inhibition of HSP90 would affect IL6-mediated MR activation of distal Na+ transporters, such as the sodium chloride cotransporter (NCC), a thiazide-sensitive Na+ uptake was performed on transwells in vehicle, IL6 (100ng/mL) or IL6-Gel (geldanamycin, HSP90 inhibitor; 5uM) in mDCT15 cells (n = 3). IL6 treatment increased Na+ uptake compared (1666 ± 12 nmol/mg/20 min vs.1449 ± 7; p < 0.001). With HSP90 inhibition, Na+ uptake was reduced (1499 ± 12; p < 0.001). Conclusions: Using RNA-seq, we have uncovered specific transcripts activated within the late distal nephron (DCT2) following IL6, including HSP90, which we confirmed to be an important and novel player in IL6-mediated activation of NCC.

Sex differences in prenatal programming of hypertension by dexamethasone.

Prenatal dexamethasone has been shown to increase blood pressure in male offspring but the mechanism for the increase in blood pressure is unclear. The present study examined if prenatal programming by maternal injection of dexamethasone on days 15 and 16 of gestation affected the blood pressure comparably in female and male offspring. Our hypothesis was that males would be affected by prenatal dexamethasone to a greater extent than females and that either an increase in renal tubular transporter abundance or an increase in renin or aldosterone system would be associated with hypertension with prenatal programming. Prenatal dexamethasone increased blood pressure at two months and six months of age and resulted in proteinuria and albuminuria at six months in male but not female rat offspring. There was no effect of prenatal dexamethasone on blood pressure and proteinuria at one month in male and in female offspring. While prenatal dexamethasone increased male renal thick ascending limb sodium potassium two chloride cotransporter protein abundance at two months, prenatal dexamethasone on days 15 and 16 of gestation did not affect transporter abundance in males at other ages, nor did it affect proximal tubule sodium/hydrogen exchanger or distal convoluted tubule sodium chloride cotransporter protein abundance at any age. There was no difference in systemic renin or aldosterone in the prenatal dexamethasone group compared to same sex controls. In conclusion, male but not female offspring have an increase in blood pressure and urinary protein excretion with prenatal dexamethasone. The increase in blood pressure with prenatal programming was not associated with a consistent increase in renal tubular transporter protein abundance, nor plasma renin activity and serum aldosterone.

POS-098 GITELMAN SYNDROM, HYPOMAGNESEMIA AND VENOUS THROMBOSIS: AN INTRIGUING ASSOSIATION

Gietelman syndrome is an autosomal recessive condition that results from defects in the sodium chloride cotransporter encoded by SLC12A3 , resulting in primary salt wasting tubulopathy with hypokalemia, hypomagnesemia, hypocalciuria and failure to thrive. Cardiac arrhythmia and seizures are the most dangerous complications due to these metabolic disorders. We report a rare complication which is deep venous thrombosis.

DIETARY SODIUM INTAKE DOES NOT ALTER RENAL POTASSIUM HANDLING AND BLOOD PRESSURE IN HEALTHY YOUNG MALES

Objective: High salt intake and potassium deficiency are involved in the pathogenesis of hypertension and cardiovascular diseases. The effects of sodium intakes on renal handling of potassium are scarcely studied. The objective of this study is to assess the renal handling of potassium in condition of low, normal and high sodium intake. Design and method: In a cross-over study, we assessed the effect of sodium on renal potassium handling in 16 healthy males assigned to three 7-day periods on low (LSD, 3 g NaCl/d), normal (NSD, 6 g NaCl/d) and high (HSD, 15 g NaCl/d) sodium diet with constant potassium intake. Contributions of distal NaCl co-transporter and epithelial sodium channel in the collecting system on potassium and sodium handling were assessed at steady-state by acute response to 100 mg oral hydrochlorothiazide and with addition of 10 mg of amiloride to hydrochlorothiazide, respectively. Results: Diurnal blood pressure slightly increased from 119.30 ± 7.95 mmHg under LSD to 123.00 ± 7.50 mmHg, (P = 0.02) under HSD, while estimated glomerular filtration rate increased from 133.20 ± 34.68 ml/min under LSD to 187.00 ± 49.10 under HSD, (P = 0.005). Twenty-four hours potassium excretion remained stable on all sodium intakes (66.28 ± 19.12 mmol/24 h under LSD; 55.91 ± 21.17 mmol/24 h under NSD and 66.81 ± 20.72 under HSD, P = 0.9). The hydrochlorothiazide-induced natriuresis was the highest under HSD (30.22 ± 12.53 mmol/h), and the lowest under LSD (15.38 ± 8.94 mmol/h, P = 0.02). Hydrochlorothiazide increased kaliuresis and amiloride decreased kaliuresis similarly on all 3 diets. Conclusions: Neither spontaneous nor diuretic-induced potassium excretion were influenced by sodium intake in healthy male subjects. However, the respective contribution of the distal convoluted tubule and the collecting duct to renal sodium handling was dependent on dietary sodium intake.

Genetic analysis of the Gitelman syndrome coexisting with Osteogenesis imperfecta

Gitelman syndrome (GS) is an autosomal recessive disorder caused by loss-of-function mutations in SLC12A3, which encodes the Na-Cl cotransporter (NCC). Osteogenesis imperfecta (OI) is an autosomal dominant disorder caused by the inheritance of mutations mainly in the COL1A1 gene, resulting in bone fragility and deformity. In this study, we aimed to investigate the clinical and genetic manifestations in a 7-year-old boy with OI, who had electrolyte abnormalities and his four family members.Complete sequence analysis of COL1A1 revealed a novel mutation, c.268G>T, p.Glu90del. The gene mutation of OI in the patient's older brother was inherited from his mother, and the younger brother had no mutation. Two pathogenic mutations (c.179C>T, p.Thr60Met and c.1763C>T, p.Ala588Val) in SLC12A3 resulting in GS were also identified in the patient. The OI-related genetic mutation in the patient was consistent with that in the patient's mother. The GS-related genetic mutations were inherited from each parent.This study is the first to identify compound heterozygous variants in the SLC12A3 gene and a novel mutation in the COL1A1 gene in patients with OI and GS. Our findings indicate that genetic analysis is recommended to differentiate GS from BS, as clinical manifestations do not provide an accurate diagnosis.

Role of KLHL3 and dietary K+ in regulating KS-WNK1 expression.

The physiological role of the shorter isoform of with no lysine kinase (WNK)1 that is exclusively expressed in the kidney (KS-WNK1), with particular abundance in the distal convoluted tubule, remains elusive. KS-WNK1, despite lacking the kinase domain, is nevertheless capable of stimulating the NaCl cotransporter, apparently through activation of WNK4. It has recently been shown that a less severe form of familial hyperkalemic hypertension featuring only hyperkalemia is caused by missense mutations in the WNK1 acidic domain that preferentially affect cullin 3 (CUL3)-Kelch-like protein 3 (KLHL3) E3-induced degradation of KS-WNK1 rather than that of full-length WNK1. Here, we show that full-length WNK1 is indeed less impacted by the CUL3-KLHL3 E3 ligase complex compared with KS-WNK1. We demonstrated that the unique 30-amino acid NH2-terminal fragment of KS-WNK1 is essential for its activating effect on the NaCl cotransporter and recognition by KLHL3. We identified specific amino acid residues in this region critical for the functional effect of KS-WNK1 and KLHL3 sensitivity. To further explore this, we generated KLHL3-R528H knockin mice that mimic human mutations causing familial hyperkalemic hypertension. These mice revealed that the KLHL3 mutation specifically increased expression of KS-WNK1 in the kidney. We also observed that in wild-type mice, the expression of KS-WNK1 was only detectable after exposure to a low-K+ diet. These findings provide new insights into the regulation and function of KS-WNK1 by the CUL3-KLHL3 complex in the distal convoluted tubule and indicate that this pathway is regulated by dietary K+ levels.NEW & NOTEWORTHY In this work, we demonstrated that the kidney-specific isoform of with no lysine kinase 1 (KS-WNK1) in the kidney is modulated by dietary K+ and activity of the ubiquitin ligase protein Kelch-like protein 3. We analyzed the role of different amino acid residues of KS-WNK1 in its activity against the NaCl cotransporter and sensitivity to Kelch-like protein 3.

WNK4 kinase: from structure to physiology.

With no lysine kinase-4 (WNK4) belongs to a serine-threonine kinase family characterized by the atypical positioning of its catalytic lysine. Despite the fact that WNK4 has been found in many tissues, the majority of its study has revolved around its function in the kidney, specifically as a positive regulator of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule of the nephron. This is explained by the description of gain-of-function mutations in the gene encoding WNK4 that causes familial hyperkalemic hypertension. This disease is mainly driven by increased downstream activation of the Ste20/SPS1-related proline-alanine-rich kinase/oxidative stress responsive kinase-1-NCC pathway, which increases salt reabsorption in the distal convoluted tubule and indirectly impairs renal K+ secretion. Here, we review the large volume of information that has accumulated about different aspects of WNK4 function. We first review the knowledge on WNK4 structure and enumerate the functional domains and motifs that have been characterized. Then, we discuss WNK4 physiological functions based on the information obtained from in vitro studies and from a diverse set of genetically modified mouse models with altered WNK4 function. We then review in vitro and in vivo evidence on the different levels of regulation of WNK4. Finally, we go through the evidence that has suggested how different physiological conditions act through WNK4 to modulate NCC activity.