Renal NKCC2 Research Articles

Increases of renal sodium transporters before and after the occurrence of the hypertension in spontaneously hypertensive rats

Background: Adult spontaneously hypertensive rats (SHR) have increases of renal sodium transporters associated with hypertension. It remains to be determined whether the increases of renal sodium transporters are precede by occurrence of hypertension and reflected by urine exosomal sodium transporters that may serve as non-invasive biomarkers for the effcacy of anti-hypertensive drugs. Methods: 1. Male WKY rats and SHRs were aged of 4, 7 and 10 weeks respectively (n=5/group). 2. Additional 3 sets of 10-week-old rat (n=5/group) were administered by a 7-day intraperitoneal injections of furosemide (NKCC2 inhibitor, 5mg/kg), hydrochlorothiazide (NCC2 inhibitor, 30mg/kg) and amiloride (ENaC inhibitor, 10mg/kg) respectively. 3. The systolic blood pressure (SBP) was measured with a catheter in carotid artery of rats under anesthesia. Tail-cuff methods were used to monitor the drug responses. 4. All rats were rationally fed 0.4% NaCl agar-gelled diet for 7 days. 5. Exosomes were extracted with ultracentrifuge from the urines. Results: SHRs (4 weeks) exhibited similar SBP. The renal NKCC2, the major apical sodium transporters in thick ascending limb, was more than doubled in SHRs (215±14 % of WKY, student t-test, p<0.05, same as below) while the other sodium transporters were normal. However, SHRs had a higher SBP than WKY at ages of 7 (157±10 vs 125±14, mmHg) and 10 weeks (192±8 vs 128±12). There were no differences for the all age groups in heart rate, body weight, urine volume, urine Na+ and Cl− excretion, and serum Cr, K+, Na+, and Cl− concentration between rat strains. At 10-weeks old, renal (161±24) and urine exosomal (159±21, corrected by UCr, same as below) NKCC2 were higher in SHR than WKY. Frusemide normalized SBP on day 1 (day 0: 190±18 vs 138±13; day 1: 149±13 vs 128±16) in SHR; Renal (129±9) and urine-exosomal (137±12) NCC were higher in SHR than WKY. Hydrochlorothiazide normalized SBP in SHR on day 1 (day 0: 206±4 vs 140±9; day 1: 159±11 vs 144±11). Renal (α:134±16; β:137±25) and urine-exosomal (α:153±13; β:158±12) ENaCs were higher in SHR than WKY. Amiloride diminished the difference in SBP between rat strains on day 1 (day 0: 188±18 vs 138±11; day 1: 164±16 vs 147±7). Conclusions: The increase of renal NKCC2 precedes the elevation of SBP in SHRs. Urine exosomal NKCC2, NCC, ENaCs may reflect their renal levels and be indicative of the corresponding inhibitors on the hypertension model. Natural Science Foundation of China 81970605. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Renal CD81 interacts with sodium potassium 2 chloride cotransporter and sodium chloride cotransporter in rats with lipopolysaccharide-induced preeclampsia.

The kidney regulates blood pressure through salt/water reabsorption affected by tubular sodium transporters. Expanding our prior research on placental cluster of differentiation 81 (CD81), this study explores the interaction of renal CD81 with sodium transporters in preeclampsia (PE). Effects of renal CD81 with sodium transporters were determined in lipopolysaccharide (LPS)-induced PE rats and immortalized mouse renal distal convoluted tubule cells. Urinary exosomal CD81, sodium potassium 2 chloride cotransporter (NKCC2), and sodium chloride cotransporter (NCC) were measured in PE patients. LPS-PE rats had hypertension from gestational days (GD) 6 to 18 and proteinuria from GD9 to GD18. Urinary CD81 in both groups tented to rise during pregnancy. Renal CD81, not sodium transporters, was higher in LPS-PE than controls on GD14. On GD18, LPS-PE rats exhibited higher CD81 in kidneys and urine exosomes, higher renal total and phosphorylated renal NKCC2 and NCC with elevated mRNAs, and lower ubiquitinated NCC than controls. CD81 was co-immunoprecipitated with NKCC2 or NCC in kidney homogenates and co-immunostained with NKCC2 or NCC in apical membranes of renal tubules. In plasma membrane fractions, LPS-PE rats had greater amounts of CD81, NKCC2, and NCC than controls with enhanced co-immunoprecipitations of CD81 with NKCC2 or NCC. In renal distal convoluted tubule cells, silencing CD81 with siRNA inhibited NCC and prevented LPS-induced NCC elevation. Further, PE patients had higher CD81 in original urines, urine exosomes and higher NKCC2 and NCC in urine exosomes than controls. Thus, the upregulation of renal CD81 on NKCC2 and NCC may contribute to the sustained hypertension observed in LPS-PE model. Urine CD81 with NKCC2 and NCC may be used as biomarkers for PE.

Abstract P247: Altered Renal Sodium Transporters Expression Is Associated With Cholinergic-mediated Salt Sensitive Hypertension In Young Spontaneously Hypertensive Rats

High sodium diet is a major contributing factor and has been associated with inflammation in patients suffering from hypertension. Systemic infusion of nicotine induces renal inflammation and the development of premature hypertension in young spontaneously hypertensive rats (SHR) and leads to enhanced expression of the renal Na-K-Cl cotransporter (NKCC2). Sodium sensitivity also plays a role in the development of cholinergic hypertension. We hypothesized that these findings were related to alterations in renal sodium transporters expression. We continuously infused SHR and Wistar Kyoto (WKY, normotensive control) rats with saline or nicotine (15mg/kg/day) and fed either a low-salt (LSD, 0.01% NaCl) or high-salt (HSD, 4% NaCl) diet for 2 weeks. Renal homogenates were assessed by western blot for the presence of the epithelial sodium channel (ENaC), NKCC2, Na-Cl co-transporter (NCC) and Serum and glucocorticoid regulated kinase 1 (SGK-1). Nicotine infusion in WKY induced 1.9-fold increase (p=0.003) in renal expression of SGK-1 under LSD, compared to saline-infused controls. The sharp increase in SGK-1 in nicotine-infused WKY on LSD was accompanied by a 1.9-fold increase in NCC (p=0.004), a 1.3-fold increase in the cleaved alpha ENaC, a 1.2-fold increase in the uncleaved alpha ENaC, and a 1.5-fold increase in the beta-ENaC (p=0.02). Both saline and nicotine infused WKY showed very little expression of renal NKCC2 in either LSD or HSD. There was no effect of either diet or nicotine infusion on expression of SGK-1, NCC, or ENaC subunits in SHR. Nicotine had no effect on NKCC2 expression in SHR on LSD, but HSD alone induced a 2.25-fold increase in renal NKCC2 expression compared to LSD in saline infused SHR (p=0.006). Nicotine infusion further induced a 58% increase in renal NKCC2 expression for SHR fed HSD (p=0.004). Here we show an effect of nicotine to appropriately increase the expression of NCC and ENaC in WKY under LSD and a trend towards decreased expression under HSD. In contrast, nicotine not only has no effect on NCC and ENaC under LSD in SHR but leads to a paradoxical increase in NKCC2 under HSD. We conclude that inappropriate regulation of renal sodium transporters likely plays a role in cholinergic-mediated renal inflammation and hypertension.

Abstract 042: A Novel (pro)renin Receptor Variant Prr24 As A Specific Renin Receptor And Key Regulator Of Nkcc2 And Urine Concentrating Capability

(Pro)renin receptor (PRR) has emerged as a key regulator of renal handling of Na + and water, and blood pressure (BP). Renal action of PRR is at least in part mediated through its ability to activate tissue prorenin and renin. In preliminary studies, investigation of a “non-specific” protein band recognized by an anti-PRR antibody against the extracellular domain led to identification of a novel 24-kDa isoform of PRR, termed as PRR24. PRR24 was encoded by a novel cDNA with part of 5’UTR arising from retention of intron 3 and an ATG in exon 5 as an alternative start codon so that this isoform overlaps completely with PRR except that N-terminal 136 amino acids are missing. PRR24 selectively bound and activated renin but not prorenin whereas PRR non-selectively interacted both prorenin and renin. PRR24 protein and mRNA expression was exclusively detected in the renal medulla but not renal cortex or extrarenal organs. Within the kidney, PRR24 mRNA was restricted to medullary thick ascending limb (mTAL) as assessed by RT-PCR on microdissected nephron segments. Using CRISP/Cas9 strategy, we generated a mouse model with mutagenesis of the start codon in exon 5 (termed PRR24 mut ) by replacing methionine with glutamine in PRR. Renal medullary protein expression of PRR24 was barely detectable contrasting unchanged PRR abundance in the null mice. PRR24 mut mice was born at Mendelian ratio and didn’t exhibit developmental abnormalities or mortality as seen in systemic PRR KO mice. PRR24 mut mice developed polyuria, hypoosmotic urine, polydipsia, hypotension (radiotelemetry MAP: WT 120 + 0.8 vs. Mutant 112 + 1.4 mmHg; p<0.05). Plasma volume assessed indirectly by hematocrit and directly assessed by using a fluorescent tracer was consistently reduced by 5-10%. Renal expression of NKCC2 was downregulated by 70% in parallel with reduced diuretic response to furosemide. Following 24-h water deprivation, the upregulation of NKCC2 expression was blunted in PRR24 mut mice associated with impaired urine concentrating capability as assessed by urine volume and osmolality. Overall, PRR24 is a novel PRR variant and serves as a specific renin receptor to regulate renal NKCC2 expression to ultimately determine urine concentrating capability.

The Hypertension Induced by Clozapine is Related with Increased Renal Sodium Transporters in Mice

IntroductionHypertension is caused by multi‐factors, and the pathogenesis is linked to, generally, the increases in the protein expressions and functions of renal sodium transport proteins along the nephron. One of the major side effects of clozapine (common antipsychotic drug) is hypertension. The mechanism is unknown.Hypothesisthe pathogenesis of the clozapine‐induced hypertension is associated with increases of renal sodium transport proteins.Methodswe examined the blood pressures (BP) and protein expressions of major sodium transport proteins profiled along the renal tubules in the C57Bl6/J male mice (9 weeks old, n=4‐5/group) treated with clozapine at three doses of 5,10 and 20 mg/ kg for 1 week via intraperitoneal injection.Resultblood pressures (tail‐cuff, BP98A, softron, Japan) were higher in clozapine at 20mg/kg/day than vehicle (SBP: 130±1.64 vs 109±1.39, DBP: 87 ±4.12 vs 69±2.10 mmHg, n=5/group) and not altered at 5 & 10 mg/kg/day. There were no differences in BW, serum/urine creatinine and electrolytes except for a slight increase in serum sodium at dose of 20 mg/kg (152.8±0.71 vs 150.6±0.40, mmol/L). In renal homogenates, total protein abundance of NaPi2 (western blotting,199±26% of vehicle) NKCC2(184±20), NCC(213±14),and ENaC‐α(217±47),were higher at 20 mg/kg than vehicle. No increases were found in protein expressions of NHE3, αNKA, ENaC‐β and ENaC‐γ. The expressions of renin, AT1R and ACE at 20mg/kg were similar between groups while ACE2 was decreased (42±10). No increases were found in protein expressions of pNKCC2 in 5&10mg/kg(100±50,45±15); NCC was increased at 5 mg/kg (175±9); α NKA was increased in 5mg/kg (216±5) and 10mg/kg (163±2) groups.In plasma membrane enriched fraction (17k), NKCC2(204±43) and ENaC‐α(381±28)were higher at 20 mg/kg than vehicle. In intracellular membrane enriched fraction (200k), NKCC2(521±127) was higher than vehicle. Phosphorylated NKCC2 was increased in renal homogenates(331±35),17k(427±87) 200k(521±127) but WNK4 was not changed.Additional mice were treated with clozapine at 20mg/kg/day via osmotic mini‐pump for 1 week (mini‐pump) or administrated by intraperitoneal injection for 2 weeks (2wks). Increases of renal NKCC2 (mini‐pump:180±21, 2wks:153±18) and NCC (mini‐pump:171±22, 2 wks:160±23) were also found in those models.Conclusionsincreases of renal sodium transport proteins, mainly pNKCC2 and NKCC2, contribute to the dose‐related increase of BP in mice treated with clozapine and may play an important role in the pathogenesis of the clozapine‐induced hypertension.

Abstract 11991: Effects of Clozapine at Different Doses on Blood Pressure and Renal Sodium Transporters in Mice

Introduction: one of major side effects of clozapine (common antipsychotic drug) is hypertension. Kidney regulates blood pressure by initiating and responding to renin-angiotensin-aldosterone system (RAAS) physiologically and pathophysiologically;the pathogenesis of hypertension is linked to, generally, the increases in the protein expressions and functions of renal sodium transport proteins along the nephron with or without RAAS activation. Methods: we examined the blood pressures (BP) and protein expressions of major sodium transport proteins profiled along the renal tubules in the C57Bl6/J male mice (9 weeks old, n=4-5/group) treated with clozapine at three doses of 5,10 and 20 mg/ kg for 1 week via intraperitoneal injection. Result: blood pressures (tail-cuff, BP98A, softron, Japan) were higher in clozapine at 20mg/kg/day than vehicle (SBP: 130±1.64 vs 109±1.39, DBP: 87 ±4.12 vs 69±2.10 mmHg, n=5/group) and not altered at 5 & 10 mg/kg/day. BW was similar between groups. There were no differences in serum/urine creatinine and electrolytes except for a slight increase in serum sodium at dose of 20 mg/kg (152.8±0.71 vs 150.6±0.40, mmol/L).The renal protein expressions of NaPi2 (western blotting, 199±26 , % of vehicle), NKCC2(184±20), NCC(213±14), and ENaC-α(217±47)were higher at 20 mg/kg than vehicle. No increases were found in protein expressions of NHE3, αNKA, ENaC-β and ENaC-γ. The expressions of renin, AT1R and ACE at 20mg/kg were similar between groups while ACE2 was decreased (42±10). Renal protein expression of NKCC2 was increased in 5mg/kg (360±80, % of vehicle) and 10mg/kg (247±50) groups; NCC was increased at 5 mg/kg (175±9); α NKA was increased in 5mg/kg (216±5) and 10mg/kg (163±2) groups. Additional mice were treated with clozapine at 20mg/kg/day via osmotic mini-pump for 1 week (mini-pump) or administrated by intraperitoneal injection for 2 weeks (2wks). Increases of renal NKCC2 (mini-pump:180±21, 2wks:153±18) and NCC (mini-pump:171±22, 2 wks:160±23) were also found in those models. Conclusions: increases of renal sodium transport proteins, mainly NCC and NKCC2, contribute to the dose-related increase of BP in mice treated with clozapine and may play an important role in the pathogenesis of theclozapine-induced hypertension.

Abstract P213: Furosemide-mediated Suppression Of P 38 MAPK Signaling Abrogates Cholinergic-mediated Salt Sensitive Hypertension In Young Spontaneously Hypertensive Rats

Hypertension is a major cause of premature death worldwide. Inflammatory macrophages play a key role in the pathogenesis of hypertension. Our lab has observed that nicotinic acetylcholine receptors (nAChR) activation potentiates salt-sensitive hypertension in Spontaneously Hypertensive Rats (SHR). Cholinergic receptor activation causes infiltration of CD68 + macrophages into the renal cortex/medulla junction and results in enhanced expression of renal sodium-potassium chloride cotransporter (NKCC2). To determine whether or not NKCC2 plays a role in the activation of inflammatory pathway furosemide, a selective pharmacologic blocker of NKCC2, was used to block NKCC2 expression in SHR in current study. Young pre-hypertensive SHR, receiving nicotine infusion (15mg/kg/day), were fed HSD (4% NaCl) and treated with either saline or furosemide (20mg/kg/day) intraperitoneal injections. There was a 16% increase (158.5±7.3 mmHg to 183.6±7.0 mmHg) in the systolic blood pressure in SHR that received saline injections, compared to no increase in SHR that received furosemide (155±6.0 mmHg to 148±3.6 mmHg) (p<0.0016). There was over a two-fold increase in fractional excretion of sodium (FENa, p=0.03) in the furosemide group, compared to saline controls. Although furosemide injections did not alter renal NKCC2 expression, there was nearly 50% reduction in phosphorylated p38 MAPK (p=0.0002) and a 25% decrease in renal infiltration of inflammatory CD68 + macrophages in SHR receiving furosemide injections, compared to saline controls. Interestingly, there was also a significant reduction in urinary albumin in response to furosemide injections (14.7±5.7 control vs 3.3±0.9 furosemide, p=0.027). Based on these findings, we conclude that furosemide results in cessation of the development of hypertension via interruption of a p38-MAPK dependent inflammatory pathway.

Abstract P216: Cholinergic-mediated Salt-Sensitive Hypertension In A Genetic Model Of Essential Hypertension

Inflammation plays a key role in the pathogenesis of hypertension. High dietary sodium intake has been associated with inflammation in patients suffering from high blood pressure & atherosclerosis. Our lab has shown that development of hypertension in a genetic model of human essential hypertension, the Spontaneously Hypertensive Rat (SHR), is dependent on nicotinic acetylcholine receptor (nAChR) mediated renal inflammation, which leads to enhanced expression of the renal sodium-potassium chloride cotransporter (NKCC2). Based on these findings we hypothesized that sodium sensitivity may play a role in the development of cholinergic hypertension. To investigate this hypothesis, we infused young pre-hypertensive SHR and Wistar Kyoto (WKY, normotensive control) rats with either saline or nicotine (15mg/kg/day) and placed them on either low-salt (LSD, 0.01% NaCl) or high-salt (HSD, 4% NaCl) diet for 2 wks. There was no change in systolic blood pressure (SBP) in saline or nicotine infused WKY with either LSD or HSD. Similarly, there was no difference between the SBP of saline infused SHR with either LSD or HSD. Interestingly, nicotine infusion did not change SBP in LSD fed SHR, but did result in ~20% increase in SBP in HSD fed SHR (122.0 ± 0.7 vs. 146.2 ± 0.2mmHg, p= 0.01). HSD alone induced a 2.25-fold increase in renal NKCC2 expression compared to LSD in saline infused SHR (p=0.006). In comparison to saline, nicotine infusion further induced a 58% increase in renal NKCC2 expression in SHR fed HSD (p=0.004). Renal NKCC2 expression did not change in WKY with either diet or nicotine infusion. Nicotine infused SHR on a HSD also manifested a 16% sodium retention compared to saline infused SHR (2.1 ± 0.1 mM/gBW vs. 2.5 ± 0.1 mM/gBW, p = 0.017). Nicotine infused SHR had 1.7 times more renal IL-6 than saline-infused SHR (1442. ± 212 vs 826 ± 103 pg/25mg total protein, p=0.04) and 9 times more renal IL-6 than nicotine-infused WKY controls (1442. ± 212 vs 148. ± 71 pg/25mg total protein, p=0.0041) under HSD conditions. Taken together with our previously published data, we conclude that NKCC2 likely plays a role in cholinergic-mediated renal inflammation and hypertension.

Renal NKCC2 Is Dual Regulated by the Synergy of 20-HETE and High-Fat Diet in CYP4F2 Transgenic Mice

Introduction: 20-Hydroxyeicosatetraenoic acid (20-HETE) is the metabolite of cytochrome P450, which modulates blood pressure by inhibiting renal sodium transport. However, the molecular mechanisms underlying the role of 20-HETE in the development of obesity-related hypertension remain unclear, necessitating this study. Methods: Cytochrome P450 4F2 (CYP4F2) transgenic mice fed high-fat diet (HFD) were used as research animal models. The expression of renal ion transport molecules targeted by 20-HETE was evaluated by real-time PCR and Western blot (WB). The regulatory effect of 20-HETE and HFD on renal Na⁺-K⁺-2Cl⁻ cotransporter, isoform 2 (NKCC2) was explored by immunoprecipitation, WB, and luciferase assay. Results: A 2-week HFD feeding dramatically decreased protein abundance but increased renal NKCC2 mRNA expression in CYP4F2 transgenic mice. The decrease in NKCC2 protein was demonstrated to be due to ubiquitination induced by the synergy between 20-HETE and HFD. The increased PPAR-γ protein in CYP4F2 transgenic mice fed HFD and the activation of rosiglitazone on the luciferase reporter construct of the NKCC2 promoter demonstrated that the increase in NKCC2 mRNA in CYP4F2 transgenic mice fed HFD was a consequence of elevated PPAR-γ protein induced by the synergy between 20-HETE and HFD. Conclusions: Our data demonstrated that the synergy between 20-HETE and HFD could decrease NKCC2 protein via posttranslational ubiquitination, which was thought to be the main mechanism underlying the short-term effect in response to HFD and might be responsible for the adaptive modulation of renal NKCC2 to resist sodium retention. Moreover, the increased NKCC2 mRNA expression via PPAR-γ-induced transcriptional regulation was thought to be the main mechanism underlying the long-term effect in response to HFD and plays a pivotal role in the development of obesity-related hypertension.

Abstract 16216: Increases in Renal Nkcc2 and Ncc Preceded the Elevation of Blood Pressure in Mice Treated With Clozapine

One of major side effects of clozapine, a common antipsychotic drug, is hypertension. In order to explore the pathogenesis of the drug-induced hypertension, we examined the renal sodium transport proteins and BP in the C57Bl6/J male mice treated with clozapine at different doses for 1 week. Clozapine at 20mg/kg/day via osmotic mini-pump increased SBP ( 110±0.6,mmHg, femoral artery, under anesthesia) and DBP (76.7±1) relative to vehicle (97.6±1.2/71.2±2.7) while the renal protein expression of NKCC2 (180±21, % of vehicle) and NCC (171±22), and ENaC-α (140±7), β(166±21) and γ(130±7) was increased without changes in NHE3 and α1-NKA (n=5/group). Clozapine at lower doses (5&10mg/kg/day,IP) did not change the conscious SBP and DBP monitored daily by tail-cuff, even on day 7 in 5 mg/kg group (115.61±1.6/90.1±1.3) and in 10mg/kg group (116.8±2/89.0±1.6) relative to vehicle (111.9±1.5/86.9±1.4) (n=4/group). Urinary excretion of Na + & K + and serum concentrations of Na + , K + , Cl - , creatinine were similar in all groups. However, renal protein expression of NKCC2 was increased in 5mg/kg (360±80, % of vehicle) and 10mg/kg (247±50) groups; NCC was increased at 5 mg/kg (175±9); α1-NKA was increased in 5mg/kg (216±5) and 10mg/kg (163±2) groups. No increases were found in the protein expression of NHE3 and ENaCs. Those increases in renal NKCC2 &NCC at 5 & 10 mg/kg were consistent with those at 20 mg/kg. AT1R was increased at 5 (221±20) &10mg/kg (255±21) while renin (186±15) and ACE1 (186±5), not ACE2, were increased at 5 mg/kg suggesting an activation of RAAS in kidney. NOX4, not NOX2, was increased at 5 mg/kg (453±69) while NOS3, not NOS1&2, was decreased at 5 (60±21) and 10mg/kg (64±5) groups. TNFα, not IL-6 was increased at 5 (222±18) &10 mg/kg (321±26). In cultured mouse distal convoluted tubule cells, clozapine also increased NCC and α1-NKA at 1nM (169±4;156±7) and 10nm(175±23;183±15) respectively ( 24h,n=4/group). Those changes in kidney, including increases in sodium transport proteins, RAAS components, ROS generation enzymes, inflammation factors and decrease in NO synthase, preceded the elevation of BP suggesting that direct or indirect regulations of clozapine on those proteins may play an important role in the pathogenesis of the drug-induced hypertension.

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.

Female mice retain more sodium than male mice but maintain lower blood pressure during 30 days on a high salt diet (1135.1)

High salt consumption contributes to hypertension. The kidneys must excrete ingested sodium in order to maintain normal blood pressure. Our previous study showed that female mice had lower renal excretion of sodium (Nae) than male mice for a 15‐day period of high salt consumption. The goal of this study was to determine if sex differences in Nae and blood pressure (BP) occur from a 30‐day period of high salt consumption. Intact female and male 12‐week old mice (n=6/group) consumed a 4% salt diet in metabolic cages for 30 days. Nae was determined from daily measurements of urine sodium concentration and urine volume. Sodium intake (Nai) was determined from daily food intake. BP was measured via the tail‐cuff method. Real‐time quantitative PCR was used to measure expression of renal sodium transporters. For the 30‐day period, average daily Nae/Nai (%) was significantly lower in female mice compared to male mice (53.3±2.7 vs 68.1±1.8, respectively, p<0.0001) in addition to the mean BP (mmHg) (78.4 ±1.0 vs 84.9 ±1.2 respectively, <0.0005). Expression of the renal NKCC2 mRNA was over 5‐fold higher in the female kidney. We conclude that female mice retain more sodium and maintain lower BP during high salt consumption compared to male mice. This study indicates that female mice exposed to a high salt diet are protected from hypertension for a period of 30 days despite higher sodium retention.Grant Funding Source: Supported by OK INBRE 8P20GM103447 Summer Research Program

Adenylyl Cyclase 6 Enhances NKCC2 Expression and Mediates Vasopressin-Induced Phosphorylation of NKCC2 and NCC

Arginine vasopressin (AVP) affects kidney function via vasopressin V2 receptors that are linked to activation of adenylyl cyclase (AC) and an increase in cyclic adenosine monophosphate formation. AVP/cyclic adenosine monophosphate enhance the phosphorylation of the Na-K-2Cl cotransporter (NKCC2) at serine residue 126 (pS126 NKCC2) and of the Na-Cl cotransporter (NCC) at threonine 58 (pT58 NCC). The isoform(s) of AC involved in these responses, however, were unknown. Phosphorylation of S126 NKCC2 and T58 NCC, induced by the V2 receptor agonist (1-desamino-8-D-arginine vasopressin) in wild-type mice, is lacking in knockout mice for AC isoform 6 (AC6). With regard to NKCC2 phosphorylation, the stimulatory effect of 1-desamino-8-D-AVP and the defect in AC6(-/-) mice seem to be restricted to the medullary portion of the thick ascending limb. AC6 is also a stimulator of total renal NKCC2 protein abundance in medullary and cortical thick ascending limb. Consequently, mice lacking AC6 have lower NKCC2 expression and a mild Bartter syndrome-like phenotype, including lower plasma concentrations of K+ and H+ and compensatory upregulation of NCC. Increased AC6-independent phosphorylation of NKCC2 at S126 might help to stabilize NKCC2 activity in the absence of AC6. Renal AC6 determines total NKCC2 expression and mediates vasopressin-induced NKCC2/NCC phosphorylation. These regulatory mechanisms, which are defective in AC knockout mice, are likely responsible for the observed mild Bartter syndrome.

Synergistical effect of 20-HETE and high salt on NKCC2 protein and blood pressure via ubiquitin–proteasome pathway

We previously generated a cytochrome P450 4F2 (CYP4F2) transgenic mouse model and demonstrated that overexpressed CYP4F2 and overproduced 20-HETE in the kidneys contribute to the increase of blood pressure in the CYP4F2 transgenic mice with normal salt intake. We currently expect to elucidate a potential mechanism of salt-related hypertension whereby diverse levels of 20-HETE interact with dietary salt on Na(+)-K(+)-2Cl(-) cotransporter, isoform 2 (NKCC2) in the kidneys of the transgenic and wild-type mice with high salt intake. High salt intake reduced about 85% abundance of renal NKCC2 protein in the transgenic mice and about 24% in the wild-type mice by Western blot. Furthermore, we first found that NKCC2 was ubiquitinated and interacted with Nedd4-2 by immunoprecipitation in the transgenic mice with high salt intake. In addition, inhibition of 20-HETE synthesis or proteasome activity reversed the reduction of NKCC2 expression induced by 20-HETE and high salt intake. These results suggest that 20-HETE and high salt intake synergistically decrease the expression of NKCC2 protein via Nedd4-2-mediated ubiquitin-proteasome pathway, and thereby modulate natriuresis and blood pressure. We propose that diverse levels of 20-HETE have diverse effects on blood pressure in different salt concentrations.

EFFECT OF HIGH SALT ON RENAL NKCC2 IN CYP4F2 transgenic mice

Cytochrome P450 4F2 (CYP4F2) catalyzes the ω-hydroxylation of arachidonic acid (AA) to 20-HETE. We previously generated a CYP4F2 transgenic (TG) mouse model, and demonstrated that overexpressed CYP4F2 elevates 20-HETE production and blood pressure in the TG mice, indicating 20-HETE plays a prohypertensive role via vasoconstriction in CYP4F2 TG mice. To investigate antihypertensive action of 20-HETE via natriuresis in CYP4F2 TG mice , we fed TG mice a high salt (4% NaCl) diet for 2 weeks, and measured their systolic blood pressure (SBP), urine sodium concentration, urine volume, and urinary 20-HETE excretion. The expression of renal Na + -K + -2Cl - cotransporter, isoform 2 (NKCC2) was detected by Real-time PCR and Western blot. The results showed that SBP was not changed, but the urinary sodium excretion and urinary 20-HETE excretion were promoted by high salt intake in TG mice. NKCC2 protein was reduced by high salt intake, but its mRNA was not. These data suggest that 20-HETE of CYP4F2 TG mice exert natriuresis in renal adaptation to elevated Na + intake, in which reduction of renal NKCC2 protein was involved through high salt-induced posttranscriptional regulation.

A minor role of WNK3 in regulating phosphorylation of renal NKCC2 and NCC co-transporters in vivo

SummaryMutations in WNK1 and WNK4 kinase genes have been shown to cause a human hereditary hypertensive disease, pseudohypoaldosteronism type II (PHAII). We previously discovered that WNK kinases phosphorylate and activate OSR1/SPAK kinases that regulate renal SLC12A family transporters such as NKCC2 and NCC, and clarified that the constitutive activation of this cascade causes PHAII. WNK3, another member of the WNK kinase family, was reported to be a strong activator of NCC/NKCC2 when assayed in Xenopus oocytes, suggesting that WNK3 also plays a major role in regulating blood pressure and sodium reabsorption in the kidney. However, it remains to be determined whether WNK3 is in fact involved in the regulation of these transporters in vivo. To clarify this issue, we generated and analyzed WNK3 knockout mice. Surprisingly, phosphorylation and expression of OSR1, SPAK, NKCC2 and NCC did not decrease in knockout mouse kidney under normal and low-salt diets. Similarly, expression of epithelial Na channel and Na/H exchanger 3 were not affected in knockout mice. Na+ and K+ excretion in urine in WNK3 knockout mice was not affected under different salt diets. Blood pressure in WNK3 knockout mice was not lower under normal diet. However, lower blood pressure was observed in WNK3 knockout mice fed low-salt diet. WNK4 and WNK1 expression was slightly elevated in the knockout mice under low-salt diet, suggesting compensation for WNK3 knockout by these WNKs. Thus, WNK3 may have some role in the WNK-OSR1/SPAK-NCC/NKCC2 signal cascade in the kidney, but its contribution to total WNK kinase activity may be minimal.

Prenatal programming of renal salt wasting resets postnatal salt appetite, which drives food intake in the rat

Sodium retention has been proposed as the cause of hypertension in the LP rat (offspring exposed to a maternal low-protein diet in utero) model of developmental programming because of increased renal NKCC2 (Na+/K+/2Cl- co-transporter 2) expression. However, we have shown that LP rats excrete more rather than less sodium than controls, leading us to hypothesize that LP rats ingest more salt in order to maintain sodium balance. Rats were fed on either a 9% (low) or 18% (control) protein diet during pregnancy; male and female offspring were studied at 4 weeks of age. LP rats of both sexes held in metabolism cages excreted more sodium and urine than controls. When given water to drink, LP rats drank more and ate more food than controls, hence sodium intake matched excretion. However, when given a choice between saline and water to drink, the total volume of fluid ingested by LP rats fell to control levels, but the volume of saline taken was significantly larger [3.8±0.1 compared with 8.8±1.3 ml/24 h per 100 g of body weight in control and LP rats respectively; P<0.001]. Interestingly food intake also fell to control levels. Total body sodium content and ECF (extracellular fluid) volumes were greater in LP rats. These results show that prenatal programming of renal sodium wasting leads to a compensatory increase in salt appetite in LP rats. We speculate that the need to maintain salt homoeostasis following malnutrition in utero stimulates greater food intake, leading to accelerated growth and raised BP (blood pressure).

K Influx Parameters in Rat Aortic Vascular Smooth Muscle Cells (RASMCs)

Background RASMCs serve as major experimental tool in cardiovascular research. Here we studied further the effect of cell culture passage on K influxes with Rb as congener. Methods Rb uptake was measured by atomic absorption spectrophotometry in RASMCs ± inhibitors of the Na/K pump (ouabain, O), Na-K-2Cl cotransport (NKCC, bumetanide, B), and K-Cl cotransport (KCC, Cl replacement by sulfamate). Rb influx was calculated in nmol/mg protein × time, with O inhibition maximized for NKCC and KCC measurements. RASMC identity was verified immunologically. Results Positive IF anti-αactin staining. Rb uptake nonlinear with time in the absence (total, T) and presence of 1 mM O, and both O and 10 μM B. Rb flux by NKCC {O-insensitive (OIS) – [O+B-insensitive, OBIS]) = > 30% than that by the Na/K pump (T- OIS). Presence of O during the flux alone or flux + preincubation inhibited the Na/K pump, IC50 = 0.14 or 0.17 mM and 45% and 60% of T at 2 mM O, respectively. At 2 mM O, B inhibited 80% of OIS Rb influx with an IC50 = 0.1 μM. OBIS KCC activity (53%) saturated with increasing Cl mol fractions, remainder in 100% sulfamate due to channel Rb influx. KCC Vmax = ~ 1 and 2 nmol/(mg protein × min), and Km = 89 and 59 mM [Cl]o, with Hill n=1, in old vs young RASMCs passages, respectively. Conclusions Na/K pump, NKCC and KCC measurement required inhibitor presence in both preincubation + flux. Apparent Na/K pump O affinity was consistent with the low rodent α1Na/K ATPase isoform O affinity and NKCC B affinity s close to that for renal NKCC2. KCC parameters, except Hill n, were age-dependent, consistent with KCC1, 3 & 4 isoforms present in RASMCs.

The Na+–K+–2Cl− cotransporter and the osmotic stress response in a model salt transport epithelium

Epithelia are physiologically exposed to osmotic stress resulting in alteration of cell volume in several aspects of their functioning; therefore, the activation of 'emergency' systems of rapid cell volume regulation is fundamental in their physiology. In this review, the physiological response to osmotic stress, particularly hypertonic stress, was described in a salt-transporting epithelium, the intestine of the euryhaline teleost European eel. This epithelium is physiologically exposed to changes in extracellular osmolarity and represents a good physiological model for functional studies on cellular volume regulation, permitting the study of volume regulated ion transport mechanisms in a native tissue. An absorptive form of the cotransporter, homologue of the renal NKCC2, localized on the apical membrane, was found in the intestine of the euryhaline teleost European eel. This cotransporter accounts for the luminal uptake of Cl-; it operates in series with a basolateral Cl- conductance and presumably a basolateral electroneutral KCl cotransport and in parallel with a luminal K+ conductance. The ion transport model described for eel intestine, based on the operation of an absorptive luminal Na+-K+-2Cl-, is basically the same as the model that has been proposed for the thick ascending limb (cTAL) of the mammalian renal cortex. This paper focuses on the role of Na+-K+-2Cl- cotransport in the responses to hypertonic stress in the eel intestine and the role of cytoskeleton (either actin-based or tubulin based) is discussed.

Renal Na-K-Cl cotransporter NKCC2 in Dahl salt-sensitive rats.

Dahl salt-sensitive (DS) rats are characterized by enhanced NaCl reabsorption in the loop of Henle, but the responsible ion transport protein is unknown. To investigate renal Na-K-Cl cotransporter NKCC2 function and expression in DS rats under a low-salt diet. NKCC2 functioning was assessed in vitro by measuring bumetanide-sensitive rubidium uptake and cytosolic chloride concentration in isolated medullary thick ascending limb (mTAL) tubules, and in vivo by measuring the salidiuretic action of orally given bumetanide. NKCC2 expression was assessed by Western blot analysis of outer medullary proteins using T4 monoclonal antibody. mTAL tubules from DS rats exhibited significantly higher bumetanide-sensitive rubidium uptake (85.1 +/- 4.8 versus 66.2 +/- 4.4 nmol/min per mg protein in DS and DR, (Dahl salt-resistant) rats, respectively; P = 0.011) and significantly higher cytosolic chloride (32.8 +/- 1.7 versus 25.0 +/- 1.5 mmol/l in DS and DR rats, respectively). Moreover, DS rats showed a significantly higher (P < 0.001) natriuretic response to bumetanide (1.13 +/- 0.05 versus 0.64 +/- 0.09 mmole/3 h in DS and DR rats, respectively). Finally, Western blot analysis revealed less NKCC2 expression in DS rats. We conclude that DS rats have increased renal NKCC2 activity, thus explaining, at least in part, their genetic renal inability to excrete sodium. Moreover, DS rats have a decreased renal NKCC2 expression, which can be a compensatory phenomenon against NKCC2 hyperactivity.