Fig 9 - uploaded by Natalie Homer
Content may be subject to copyright.
Source publication
Na(+) reabsorption from the distal renal tubule involves electroneutral and electrogenic pathways, the latter promoting K(+) excretion. The relative activities of these two pathways are tightly controlled, participating in the minute-to-minute regulation of systemic K(+) balance. The pathways are interdependent: the activity of the NaCl co-transpor...
Contexts in source publication
Context 1
... -treated groups). The combined natriuretic effect of BZM and HCTZ in Na ϩ -deprived mice was subadditive ( Fig. 9 D ). However, in contrast to the results obtained on the control diet, HCTZ induced an acute kaliuresis in Na ϩ -deprived mice (Fig. 9 E ) and a leftward shift in the flow-kaliuresis relationship (Fig. 9 F ). The urinary Na ϩ -to-K ϩ concentration ratio after HCTZ was similar in Na ϩ -replete and Na ϩ -deprived mice (Fig. 9 G ). We tested the hypothesis that acute inhibition of NCC transport activity would enhance ENaC-mediated Na ϩ reabsorption and thereby induce kaliuresis. Our results disprove this: the natriuretic effect of HCTZ was not enhanced during ENaC blockade and a bolus of HCTZ did not enhance urinary K ϩ excretion in mice maintained on a control diet. clearance. Thiazides are commonly used to study NCC function in mice, but there is little agreement on the optimal dose or the best sampling strategy. In renal clearance studies, bolus doses of HCTZ have ranged from 0.05 to 30 mg/kg and urine samples have been obtained immediately after drug administration for a period of up to 120 min in anesthetized mice or up to 6 h in conscious mice (4, 5, 25). The validity of such a diuretic pharmacotyping approach is critically dependent on the experimental design: the diuretics and their doses should be selected so that they exert ...
Context 2
... -treated groups). The combined natriuretic effect of BZM and HCTZ in Na ϩ -deprived mice was subadditive ( Fig. 9 D ). However, in contrast to the results obtained on the control diet, HCTZ induced an acute kaliuresis in Na ϩ -deprived mice (Fig. 9 E ) and a leftward shift in the flow-kaliuresis relationship (Fig. 9 F ). The urinary Na ϩ -to-K ϩ concentration ratio after HCTZ was similar in Na ϩ -replete and Na ϩ -deprived mice (Fig. 9 G ). We tested the hypothesis that acute inhibition of NCC transport activity would enhance ENaC-mediated Na ϩ reabsorption and thereby induce kaliuresis. Our results disprove this: the natriuretic effect of HCTZ was not enhanced during ENaC blockade and a bolus of HCTZ did not enhance urinary K ϩ excretion in mice maintained on a control diet. clearance. Thiazides are commonly used to study NCC function in mice, but there is little agreement on the optimal dose or the best sampling strategy. In renal clearance studies, bolus doses of HCTZ have ranged from 0.05 to 30 mg/kg and urine samples have been obtained immediately after drug administration for a period of up to 120 min in anesthetized mice or up to 6 h in conscious mice (4, 5, 25). The validity of such a diuretic pharmacotyping approach is critically dependent on the experimental design: the diuretics and their doses should be selected so that they exert ...
Context 3
... -treated groups). The combined natriuretic effect of BZM and HCTZ in Na ϩ -deprived mice was subadditive ( Fig. 9 D ). However, in contrast to the results obtained on the control diet, HCTZ induced an acute kaliuresis in Na ϩ -deprived mice (Fig. 9 E ) and a leftward shift in the flow-kaliuresis relationship (Fig. 9 F ). The urinary Na ϩ -to-K ϩ concentration ratio after HCTZ was similar in Na ϩ -replete and Na ϩ -deprived mice (Fig. 9 G ). We tested the hypothesis that acute inhibition of NCC transport activity would enhance ENaC-mediated Na ϩ reabsorption and thereby induce kaliuresis. Our results disprove this: the natriuretic effect of HCTZ was not enhanced during ENaC blockade and a bolus of HCTZ did not enhance urinary K ϩ excretion in mice maintained on a control diet. clearance. Thiazides are commonly used to study NCC function in mice, but there is little agreement on the optimal dose or the best sampling strategy. In renal clearance studies, bolus doses of HCTZ have ranged from 0.05 to 30 mg/kg and urine samples have been obtained immediately after drug administration for a period of up to 120 min in anesthetized mice or up to 6 h in conscious mice (4, 5, 25). The validity of such a diuretic pharmacotyping approach is critically dependent on the experimental design: the diuretics and their doses should be selected so that they exert ...
Context 4
... -treated groups). The combined natriuretic effect of BZM and HCTZ in Na ϩ -deprived mice was subadditive ( Fig. 9 D ). However, in contrast to the results obtained on the control diet, HCTZ induced an acute kaliuresis in Na ϩ -deprived mice (Fig. 9 E ) and a leftward shift in the flow-kaliuresis relationship (Fig. 9 F ). The urinary Na ϩ -to-K ϩ concentration ratio after HCTZ was similar in Na ϩ -replete and Na ϩ -deprived mice (Fig. 9 G ). We tested the hypothesis that acute inhibition of NCC transport activity would enhance ENaC-mediated Na ϩ reabsorption and thereby induce kaliuresis. Our results disprove this: the natriuretic effect of HCTZ was not enhanced during ENaC blockade and a bolus of HCTZ did not enhance urinary K ϩ excretion in mice maintained on a control diet. clearance. Thiazides are commonly used to study NCC function in mice, but there is little agreement on the optimal dose or the best sampling strategy. In renal clearance studies, bolus doses of HCTZ have ranged from 0.05 to 30 mg/kg and urine samples have been obtained immediately after drug administration for a period of up to 120 min in anesthetized mice or up to 6 h in conscious mice (4, 5, 25). The validity of such a diuretic pharmacotyping approach is critically dependent on the experimental design: the diuretics and their doses should be selected so that they exert ...
Context 5
... with the inhibition of Na reabsorption in the diluting segment (32, 50). As a consequence, HCTZ caused a reduction in plasma osmolality and plasma Na ϩ concentration ( Table 2). Conversely, HCTZ caused a relative increase in hematocrit (Table 2). Natriuretic response to BZM. We used BZM to inhibit ENaC, as this compound exhibits less off-target inhibition of Na ϩ /H ϩ exchanger 2 than amiloride (22, 59). A bolus of BZM elicited a natriuresis that was sustained for at least 110 min. The magnitude of this response was larger in mice that received 2 mg/kg than in those that received 0.2 mg/kg, but no further increases were observed in mice that received 8 mg/kg (Fig. 6 A ). There was no significant hemodynamic effect of any of the three doses except for an apparent increase in GFR after 60 min with the highest dose (Fig. 6, B–D ). Therefore, for our definitive experiment ( clearance protocol D ), we chose a bolus dose of 2 mg/kg BZM followed by a continuous infusion of 1 mg·kg Ϫ 1 ·h Ϫ 1 . This regime induced maximal ENaC blockade that remained stable over the study period (Fig. 7 A , vehicle- treated group), without affecting blood pressure, RBF, or GFR (Fig. 7, B–D ). Interaction between HCTZ and BZM. In light of the results described above, we arrived at a renal clearance protocol capable of examining the acute response to NCC inhibition superimposed on constant ENaC blockade ( clearance protocol D ; Fig. 1). We used this to test our central hypothesis: that the natriuresis induced by HCTZ would be augmented by concomitant ENaC blockade. However, during BZM infusion, HCTZ elicited a natriuresis of a magnitude similar to that in diuretic- naïve mice (cf. Figs. 7 A and 3 A ). An analysis of the HCTZ- induced increment in FE Na ( FE Na ) in individual BZM-treated and BZM-naïve mice confirmed that the response did not differ between these groups and, therefore, that the natriuretic effects of HCTZ and BZM were additive (see Fig. 9 C ). (The results were similar if expressed as change in net urinary Na ϩ excretion; data not shown.) Kaliuretic response to HCTZ. HCTZ induced a sustained reduction in TTKG, indicative of suppressed K ϩ secretion in the cortical CD (Fig. 8 A ). Although there was a transient increase in net urinary K ϩ excretion immediately after HCTZ administration, we attribute this to a dead-space artifact (Fig. 8 B ); HCTZ did not affect fractional K ϩ excretion (Fig. 8 C ). Any effect of HCTZ on renal K ϩ excretion was dominated by its effects on the urine flow rate (Fig. 8 D ). In contrast, BZM induced a sharp reduction in TTKG and a dissociation between urinary flow rate and K ϩ excretion (Figs. 8 E and 9 F ). HCTZ had no effect on plasma K ϩ concentration (Table 2). Effect of dietary Na ϩ restriction. Our failure to detect either a supra-additive interaction between HCTZ and BZM or an acute kaliuretic response to HCTZ alone might be due to low ENaC activity in mice maintained on a control (0.25% Na ϩ ) diet. Therefore, we repeated the experiments in mice adapted to dietary Na ϩ restriction (3 days of 0.01% Na ϩ ), a maneuver that suppressed baseline FE Na (Fig. 9, A and B ). There were trends toward increased natriuretic responses to HCTZ alone and BZM alone (Fig. 9, A and B ), but these did not reach statistical significance ( P Ͼ 0.05 by t -test to compare HCTZ-induced or BFZ-induced changes in FE Na between 0.25% Na ϩ - and ...
Context 6
... with the inhibition of Na reabsorption in the diluting segment (32, 50). As a consequence, HCTZ caused a reduction in plasma osmolality and plasma Na ϩ concentration ( Table 2). Conversely, HCTZ caused a relative increase in hematocrit (Table 2). Natriuretic response to BZM. We used BZM to inhibit ENaC, as this compound exhibits less off-target inhibition of Na ϩ /H ϩ exchanger 2 than amiloride (22, 59). A bolus of BZM elicited a natriuresis that was sustained for at least 110 min. The magnitude of this response was larger in mice that received 2 mg/kg than in those that received 0.2 mg/kg, but no further increases were observed in mice that received 8 mg/kg (Fig. 6 A ). There was no significant hemodynamic effect of any of the three doses except for an apparent increase in GFR after 60 min with the highest dose (Fig. 6, B–D ). Therefore, for our definitive experiment ( clearance protocol D ), we chose a bolus dose of 2 mg/kg BZM followed by a continuous infusion of 1 mg·kg Ϫ 1 ·h Ϫ 1 . This regime induced maximal ENaC blockade that remained stable over the study period (Fig. 7 A , vehicle- treated group), without affecting blood pressure, RBF, or GFR (Fig. 7, B–D ). Interaction between HCTZ and BZM. In light of the results described above, we arrived at a renal clearance protocol capable of examining the acute response to NCC inhibition superimposed on constant ENaC blockade ( clearance protocol D ; Fig. 1). We used this to test our central hypothesis: that the natriuresis induced by HCTZ would be augmented by concomitant ENaC blockade. However, during BZM infusion, HCTZ elicited a natriuresis of a magnitude similar to that in diuretic- naïve mice (cf. Figs. 7 A and 3 A ). An analysis of the HCTZ- induced increment in FE Na ( FE Na ) in individual BZM-treated and BZM-naïve mice confirmed that the response did not differ between these groups and, therefore, that the natriuretic effects of HCTZ and BZM were additive (see Fig. 9 C ). (The results were similar if expressed as change in net urinary Na ϩ excretion; data not shown.) Kaliuretic response to HCTZ. HCTZ induced a sustained reduction in TTKG, indicative of suppressed K ϩ secretion in the cortical CD (Fig. 8 A ). Although there was a transient increase in net urinary K ϩ excretion immediately after HCTZ administration, we attribute this to a dead-space artifact (Fig. 8 B ); HCTZ did not affect fractional K ϩ excretion (Fig. 8 C ). Any effect of HCTZ on renal K ϩ excretion was dominated by its effects on the urine flow rate (Fig. 8 D ). In contrast, BZM induced a sharp reduction in TTKG and a dissociation between urinary flow rate and K ϩ excretion (Figs. 8 E and 9 F ). HCTZ had no effect on plasma K ϩ concentration (Table 2). Effect of dietary Na ϩ restriction. Our failure to detect either a supra-additive interaction between HCTZ and BZM or an acute kaliuretic response to HCTZ alone might be due to low ENaC activity in mice maintained on a control (0.25% Na ϩ ) diet. Therefore, we repeated the experiments in mice adapted to dietary Na ϩ restriction (3 days of 0.01% Na ϩ ), a maneuver that suppressed baseline FE Na (Fig. 9, A and B ). There were trends toward increased natriuretic responses to HCTZ alone and BZM alone (Fig. 9, A and B ), but these did not reach statistical significance ( P Ͼ 0.05 by t -test to compare HCTZ-induced or BFZ-induced changes in FE Na between 0.25% Na ϩ - and ...
Context 7
... with the inhibition of Na reabsorption in the diluting segment (32, 50). As a consequence, HCTZ caused a reduction in plasma osmolality and plasma Na ϩ concentration ( Table 2). Conversely, HCTZ caused a relative increase in hematocrit (Table 2). Natriuretic response to BZM. We used BZM to inhibit ENaC, as this compound exhibits less off-target inhibition of Na ϩ /H ϩ exchanger 2 than amiloride (22, 59). A bolus of BZM elicited a natriuresis that was sustained for at least 110 min. The magnitude of this response was larger in mice that received 2 mg/kg than in those that received 0.2 mg/kg, but no further increases were observed in mice that received 8 mg/kg (Fig. 6 A ). There was no significant hemodynamic effect of any of the three doses except for an apparent increase in GFR after 60 min with the highest dose (Fig. 6, B–D ). Therefore, for our definitive experiment ( clearance protocol D ), we chose a bolus dose of 2 mg/kg BZM followed by a continuous infusion of 1 mg·kg Ϫ 1 ·h Ϫ 1 . This regime induced maximal ENaC blockade that remained stable over the study period (Fig. 7 A , vehicle- treated group), without affecting blood pressure, RBF, or GFR (Fig. 7, B–D ). Interaction between HCTZ and BZM. In light of the results described above, we arrived at a renal clearance protocol capable of examining the acute response to NCC inhibition superimposed on constant ENaC blockade ( clearance protocol D ; Fig. 1). We used this to test our central hypothesis: that the natriuresis induced by HCTZ would be augmented by concomitant ENaC blockade. However, during BZM infusion, HCTZ elicited a natriuresis of a magnitude similar to that in diuretic- naïve mice (cf. Figs. 7 A and 3 A ). An analysis of the HCTZ- induced increment in FE Na ( FE Na ) in individual BZM-treated and BZM-naïve mice confirmed that the response did not differ between these groups and, therefore, that the natriuretic effects of HCTZ and BZM were additive (see Fig. 9 C ). (The results were similar if expressed as change in net urinary Na ϩ excretion; data not shown.) Kaliuretic response to HCTZ. HCTZ induced a sustained reduction in TTKG, indicative of suppressed K ϩ secretion in the cortical CD (Fig. 8 A ). Although there was a transient increase in net urinary K ϩ excretion immediately after HCTZ administration, we attribute this to a dead-space artifact (Fig. 8 B ); HCTZ did not affect fractional K ϩ excretion (Fig. 8 C ). Any effect of HCTZ on renal K ϩ excretion was dominated by its effects on the urine flow rate (Fig. 8 D ). In contrast, BZM induced a sharp reduction in TTKG and a dissociation between urinary flow rate and K ϩ excretion (Figs. 8 E and 9 F ). HCTZ had no effect on plasma K ϩ concentration (Table 2). Effect of dietary Na ϩ restriction. Our failure to detect either a supra-additive interaction between HCTZ and BZM or an acute kaliuretic response to HCTZ alone might be due to low ENaC activity in mice maintained on a control (0.25% Na ϩ ) diet. Therefore, we repeated the experiments in mice adapted to dietary Na ϩ restriction (3 days of 0.01% Na ϩ ), a maneuver that suppressed baseline FE Na (Fig. 9, A and B ). There were trends toward increased natriuretic responses to HCTZ alone and BZM alone (Fig. 9, A and B ), but these did not reach statistical significance ( P Ͼ 0.05 by t -test to compare HCTZ-induced or BFZ-induced changes in FE Na between 0.25% Na ϩ - and ...
Citations
... However, it may be that the reduction in NCC in the current study was not sufficient to cause natriuresis, and concurrent inhibition of Na þ transport in the proximal tubule and loop of Henle is required. [33][34][35] Furthermore, in mice there is a biphasic response to high K þ loading which is characterized by an initial natriuresis and kaliuresis over an initial 3-hour period, followed by a sustained kaliuresis. These functional changes were accompanied by a rapid and sustained dephosphorylation of NCC and a late up-regulation of proteolytically activated epithelial sodium channel. ...
Introduction:
The putative "renal-K switch" mechanism links dietary potassium intake with sodium retention and involves activation of the sodium chloride (NaCl) cotransporter (NCC) in the distal convoluted tubule in response to low potassium intake, and suppression in response to high potassium intake. This study examined NCC abundance and phosphorylation (phosphorylated NCC [pNCC]) in urinary extracellular vesicles (uEVs) isolated from healthy adults on a high sodium diet to determine tubular responses to alteration in potassium chloride (KCl) intake.
Methods:
Healthy adults maintained on a high sodium (∼4.5 g [200 mmol]/d) low potassium (∼2.3 g [60 mmol]/d) diet underwent a 5-day run-in period followed by a crossover study, with 5-day supplementary KCl (active phase, Span-K 3 tablets (potassium 24 mmol) thrice daily) or 5-day placebo administrated in random order and separated by 2-day washout. Ambulatory blood pressure (BP) and biochemistries were assessed, and uEVs were analyzed by western blotting.
Results:
Among the 18 participants who met analysis criteria, supplementary KCl administration (vs. placebo) was associated with markedly higher levels of plasma potassium and 24-hour urine excretion of potassium, chloride, and aldosterone. KCl supplementation was associated with lower uEV levels of NCC (median fold change (KCl/Placebo) = 0.74 [0.30-1.69], P < 0.01) and pNCC (fold change (KCl/Placebo) = 0.81 [0.19-1.75], P < 0.05). Plasma potassium inversely correlated with uEV NCC (R2 = 0.11, P = 0.05).
Conclusions:
The lower NCC and pNCC in uEVs in response to oral KCl supplementation provide evidence to support the hypothesis of a functional "renal-K switch" in healthy human subjects.
... This presumes that K 1 secretion is limited by Na 1 delivery, so that the shift of Na 1 absorption from the early DCT to these downstream segments enhances electrogenic Na 1 absorption and K 1 secretion. However, the studies of Good and Wright (46) would suggest that flow rate, and not luminal Na 1 concentration, is the critical factor that affects K 1 secretion, and studies by Hunter et al. (80) also suggest that inhibiting NCC with hydrochlorothiazide does not necessarily increase electrogenic Na 1 reabsorption or produce a kaliuresis. Thus, alternative explanations merit consideration. ...
... This is thought to underlie, at least in part, urinary K + wasting and hypokalemia resulting from diuretic treatment. However, acute pharmacological inhibition of NCC-mediated transport using thiazide diuretics does not enhance K + secretion to levels seen with dietary K + loads (Yang et al., 2021) and in some cases does not acutely increase K + excretion at all (Hunter et al., 2014). However, proximal inhibition of Na + transport will ensure that adequate Na + is delivered to the distal segments to participate in K + secretion. ...
The kidneys regulate levels of Na⁺ and K⁺ in the body by varying urinary excretion of the electrolytes. Since transport of each of the two ions can affect the other, controlling both at the same time is a complex task. The kidneys meet this challenge in two ways. Some tubular segments change the coupling between Na⁺ and K⁺ transport. In addition, transport of Na⁺ can shift between segments where it is coupled to K⁺ reabsorption and segments where it is coupled to K⁺ secretion. This permits the kidney to maintain electrolyte balance with large variations in dietary intake.
... Importantly, however, increased Na + delivery due to NCC inhibition in-and-ofitself does not stimulate K + secretion. In particular, Hunter et al. showed that acute NCC inhibition using hydrocholorthiazide does not trigger a kaliuresis [70], and Ayasse et al. found that the effect of furosemide to induce a kaliuresis depends on ENaC expression [11]. In contrast, K + administration rapidly stimulates ENaC activity concomitantly with NCC inhibition, even prior to a significant rise in aldosterone, an effect which is only partially inhibited by MR blockade with eplerenone [161]. ...
... Several-non mutually exclusive-theories have been proposed for this "aldosterone paradox," which remains not fully understood [7,136]. Regulation of upstream electroneutral Na + reabsorption may contribute to the regulation of electrogenic Na + reabsorption [97]; however, it is not sufficient [70], and other factors must obtain. Numerous hormonal and local factors that regulate ENaC and ROMK may be implicated [54,84,137,180]. ...
Regulated Na⁺ transport in the distal nephron is of fundamental importance to fluid and electrolyte homeostasis. Further upstream, Na⁺ is the principal driver of secondary active transport of numerous organic and inorganic solutes. In the distal nephron, Na⁺ continues to play a central role in controlling the body levels and concentrations of a more select group of ions, including K⁺, Ca⁺⁺, Mg⁺⁺, Cl⁻, and HCO3⁻, as well as water. Also, of paramount importance are transport mechanisms aimed at controlling the total level of Na⁺ itself in the body, as well as its concentrations in intracellular and extracellular compartments. Over the last several decades, the transporters involved in moving Na⁺ in the distal nephron, and directly or indirectly coupling its movement to that of other ions have been identified, and their interrelationships brought into focus. Just as importantly, the signaling systems and their components—kinases, ubiquitin ligases, phosphatases, transcription factors, and others—have also been identified and many of their actions elucidated. This review will touch on selected aspects of ion transport regulation, and its impact on fluid and electrolyte homeostasis. A particular focus will be on emerging evidence for site-specific regulation of the epithelial sodium channel (ENaC) and its role in both Na⁺ and K⁺ homeostasis. In this context, the critical regulatory roles of aldosterone, the mineralocorticoid receptor (MR), and the kinases SGK1 and mTORC2 will be highlighted. This includes a discussion of the newly established concept that local K⁺ concentrations are involved in the reciprocal regulation of Na⁺-Cl⁻ cotransporter (NCC) and ENaC activity to adjust renal K⁺ secretion to dietary intake.
... Results are expressed as fractional excretion of Na + as mean ± SEM. Following equation is used: FE Na+ = [(Urine Na+ × Plasma creatinine )/(Plasma Na+ × Urine creatine )] [52]. ...
The serine protease prostasin (CAP1/Prss8, channel-activating protease-1) is a confirmed in vitro and in vivo activator of the epithelial sodium channel ENaC. To test whether proteolytic activity or CAP1/Prss8 abundance itself are required for ENaC activation in the kidney, we studied animals either hetero- or homozygous mutant at serine 238 (S238A; Prss8cat/+ and Prss8cat/cat), and renal tubule-specific CAP1/Prss8 knockout (Prss8PaxLC1) mice. When exposed to varying Na+-containing diets, no changes in Na+ and K+ handling and only minor changes in the expression of Na+ and K+ transporting protein were found in both models. Similarly, the α- or γENaC subunit cleavage pattern did not differ from control mice. On standard and low Na+ diet, Prss8cat/+ and Prss8cat/cat mice exhibited standard plasma aldosterone levels and unchanged amiloride-sensitive rectal potential difference indicating adapted ENaC activity. Upon Na+ deprivation, mice lacking the renal CAP1/Prss8 expression (Prss8PaxLC1) exhibit significantly decreased plasma aldosterone and lower K+ levels but compensate by showing significantly higher plasma renin activity. Our data clearly demonstrated that the catalytic activity of CAP1/Prss8 is dispensable for proteolytic ENaC activation. CAP1/Prss8-deficiency uncoupled ENaC activation from its aldosterone dependence, but Na+ homeostasis is maintained through alternative pathways.
... In humans, robust diuretic effects are observed with HCT doses of 1-2 mg/kg body weight (18,19). Significantly higher thiazide doses are needed in mice to stimulate natriuresis, in case of HCT typically doses of 20 -50 mg/kg body weight are applied (20)(21)(22)(23)(24)(25)(26)(27)(28). The reason for this discrepancy is not entirely clear, but likely due to differences in the pharmacokinetics of thiazides between mice and humans. ...
Thiazides are associated with glucose intolerance and new onset diabetes mellitus, but the molecular mechanisms remain elusive. The aim of this study was to decipher the molecular basis of thiazide-induced glucose intolerance. In mice, hydrochlorothiazide induced a pathological glucose tolerance, characterized by reduced first phase insulin secretion but normal insulin sensitivity. In vitro , thiazides inhibited glucose-and sulfonylurea-stimulated insulin secretion in islets and the murine β-cell line Min6 at pharmacologically relevant concentrations. Inhibition of insulin secretion by thiazides was CO 2 /HCO 3 ⁻ -dependent, not additive to unselective carbonic anhydrase (CA) inhibition with acetazolamide and independent of extracellular potassium. In contrast, insulin secretion was unaltered in islets of mice lacking the known molecular thiazide targets NCC (SLC12A3) or NDCBE (SLC4A8). CA expression profiling with subsequent knock-down of individual CA isoforms suggested mitochondrial CA5b as molecular target. In support of these findings, thiazides significantly attenuated Krebs cycle anaplerosis through reduction of mitochondrial oxalacetate synthesis. CA5b KO mice were resistant to thiazide-induced glucose intolerance, and insulin secretion of islets isolated from CA5b KO mice was unaffected by thiazides.
In summary, our study reveals attenuated insulin secretion due to inhibition of the mitochondrial CA5b isoform in β-cells as molecular mechanism of thiazide-induced glucose intolerance.
... Pharmacological inhibition of the channels could result in different outcomes compared with knockout animal studies. For example, it was reported that inhibition of NCC transport activity by pharmacological means does not increase Na + reabsorption through ENaC (Hunter et al., 2014). Here, we used the available pharmacological tools to exclude the discrepancy of the different genetic models and establish the primary role of basolateral K ir 4.1/K ir 5.1 channels (Wu, Su, et al., 2020) in regulating ENaC in CCD. ...
Background and Purpose
Inwardly rectifying K⁺ (Kir) channels located on the basolateral membrane of epithelial cells of the distal nephron play a crucial role in K⁺ handling and BP control, making these channels an attractive target for the treatment of hypertension. The purpose of the present study was to determine how the inhibition of basolateral Kir4.1/Kir5.1 heteromeric K⁺ channel affects epithelial sodium channel (ENaC)‐mediated Na⁺ transport in the principal cells of cortical collecting duct (CCD).
Experimental Approach
The effect of fluoxetine, amitriptyline and recently developed Kir inhibitor, VU0134992, on the activity of Kir4.1, Kir4.1/Kir5.1 and ENaC were tested using electrophysiological approaches in CHO cells transfected with respective channel subunits, cultured polarized epithelial mCCDcl1 cells and freshly isolated rat and human CCD tubules. To test the effect of pharmacological Kir4.1/Kir5.1 inhibition on electrolyte homeostasis in vivo and corresponding changes in distal tubule transport, Dahl salt‐sensitive rats were injected with amitriptyline (15 mg·kg⁻¹·day⁻¹) for 3 days.
Key Results
We found that inhibition of Kir4.1/Kir5.1, but not the Kir4.1 channel, depolarizes the cell membrane, induces the elevation of intracellular Ca²⁺ concentration and suppresses ENaC activity. Furthermore, we demonstrate that amitriptyline administration leads to a significant drop in plasma K⁺ level, triggering sodium excretion and diuresis.
Conclusion and Implications
The present data uncover a specific role of the Kir4.1/Kir5.1 channel in the modulation of ENaC activity and emphasize the potential for using Kir4.1/Kir5.1 inhibitors to regulate electrolyte homeostasis and BP.
... Consistent with the idea that NCC-dependent sodium delivery does not always influence K + secretion through electrogenic Na/K exchange. For example, Hunter et al. (2014) found that the kaliuretic response to HCTZ in mice is delayed compared to natriuresis. Our data reveal a new autocrine regulatory pathway (PGE2-EP1) that offers one mechanism to explain how sodium delivery from the DCT influences potassium secretory machinery in the ASDN, beyond electrogenic sodium potassium exchange. ...
Aberrant activation of with-no-lysine kinase (WNK)-STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) kinase signaling in the distal convoluted tubule (DCT) causes unbridled activation of the thiazide-sensitive sodium chloride cotransporter (NCC), leading to familial hyperkalemic hypertension (FHHt) in humans. Studies in FHHt mice engineered to constitutively activate SPAK specifically in the DCT (CA-SPAK mice) revealed maladaptive remodeling of the aldosterone sensitive distal nephron (ASDN), characterized by decrease in the potassium excretory channel, renal outer medullary potassium (ROMK), and epithelial sodium channel (ENaC), that contributes to the hyperkalemia. The mechanisms by which NCC activation in DCT promotes remodeling of connecting tubule (CNT) are unknown, but paracrine communication and reduced salt delivery to the ASDN have been suspected. Here, we explore the involvement of prostaglandin E2 (PGE2). We found that PGE2 and the terminal PGE2 synthase, mPGES1, are increased in kidney cortex of CA-SPAK mice, compared to control or SPAK KO mice. Hydrochlorothiazide (HCTZ) reduced PGE2 to control levels, indicating increased PGE2 synthesis is dependent on increased NCC activity. Immunolocalization studies revealed mPGES1 is selectively increased in the CNT of CA-SPAK mice, implicating low salt-delivery to ASDN as the trigger. Salt titration studies in an in vitro ASDN cell model, mouse CCD cell (mCCD-CL1), confirmed PGE2 synthesis is activated by low salt, and revealed that response is paralleled by induction of mPGES1 gene expression. Finally, inhibition of the PGE2 receptor, EP1, in CA-SPAK mice partially restored potassium homeostasis as it partially rescued ROMK protein abundance, but not ENaC. Together, these data indicate low sodium delivery to the ASDN activates PGE2 synthesis and this inhibits ROMK through autocrine activation of the EP1 receptor. These findings provide new insights into the mechanism by which activation of sodium transport in the DCT causes remodeling of the ASDN.
... There are several methods (direct and indirect) to evaluate RBF in mice [30][31][32], including the use of an ultrasound device [17][18][19]; however, their results are generally difficult to evaluate. In the present study, the data of RBF at 0 h were comparable between all groups; therefore, we could evaluate each group relative to each other. ...
Background
Galectin-9 (Gal-9) is a multifunctional lectin that moderates inflammation and organ damage. In this study, we tested whether Gal-9 has a protective role in the pathogenesis of endotoxemic acute kidney injury.Methods
We examined the levels of Gal-9 in control mice after lipopolysaccharide (LPS) administration. We developed Gal-9 knockout (KO) mice that lack Gal-9 systemically and evaluated the role of Gal-9 in LPS-induced proinflammatory cytokines, vascular permeability, and renal injury.ResultsGal-9 levels were increased in the plasma, kidney, and spleen within 4 h after LPS administration to wild-type mice. Gal-9 deficiency did not affect the LPS-induced increase in plasma tumor necrosis factor-α levels at 1 h or vascular permeability at 6 h. Lower urine volume and reduced creatinine clearance were observed in Gal-9-KO mice compared with wild-type mice after LPS administration. Gal-9-KO mice had limited improvement in urine volume after fluid resuscitation compared with wild-type mice. LPS reduced the body temperature 12 h after its administration. Hypothermia had disappeared in wild-type mice by 24 h, whereas it was sustained until 24 h in Gal-9-KO mice. Importantly, maintaining body temperature in Gal-9-KO mice improved the response of urine flow to fluid resuscitation.Conclusion
Deficiency in Gal-9 worsened LPS-induced hypothermia and kidney injury in mice. The accelerated hypothermia induced by Gal-9 deficiency contributed to the blunted response to fluid resuscitation.
... This supports the concept of a saturable ENaC-dependent K + secretion. In alignment with this concept, a more recent in vivo study addressed the electrolyte excretion after acute pharmacologic NCC inhibition with thiazides (Hunter et al., 2014). This maneuver is comparable to furosemide administration as it increases Na + delivery to the ENaC-expressing tubular segments without direct acute effects on ENaC. ...
... In control mice, with assumable low basal ENaC activity, no acute thiazide-induced kaliuresis was detected. Interestingly, in mice kept a few days on a low Na + intake, a thiazide-induced kaliuresis could be detected (Ayasse et al., 2017;Hunter et al., 2014). In contrast, Li et al. (2017) reported a significant thiazide-induced kaliuresis in mice kept on standard rodent chow. ...
Background:
In the aldosterone-sensitive distal nephron (ASDN), epithelial sodium channel (ENaC)-mediated Na+ absorption drives K+ excretion. K+ excretion depends on the delivery of Na+ to the ASDN and molecularly activated ENaC. Furosemide is known as a K+ wasting diuretic as it greatly enhances Na+ delivery to the ASDN. Here, we studied the magnitude of acute furosemide-induced kaliuresis under various states of basal molecular ENaC activity.
Methods:
C57/Bl6J mice were subjected to different dietary regimens that regulate molecular ENaC expression and activity levels. The animals were anesthetized and bladder-catheterized. Diuresis was continuously measured before and after administration of furosemide (2 µg/g BW) or benzamil (0.2 µg/g BW). Flame photometry was used to measure urinary [Na+ ] and [K+ ]. The kidneys were harvested and, subsequently, ENaC expression and cleavage activation were determined by semiquantitative western blotting.
Results:
A low K+ and a high Na+ diet markedly suppressed ENaC protein expression, cleavage activation, and furosemide-induced kaliuresis. In contrast, furosemide-induced kaliuresis was greatly enhanced in animals fed a high K+ or low Na+ diet, conditions with increased ENaC expression. The furosemide-induced diuresis was similar in all dietary groups.
Conclusion:
Acute furosemide-induced kaliuresis differs greatly and depends on the a priori molecular expression level of ENaC. Remarkably, it can be even absent in animals fed a high Na+ diet, despite a marked increase of tubular flow and urinary Na+ excretion. This study provides auxiliary evidence that acute ENaC-dependent K+ excretion requires both Na+ as substrate and molecular activation of ENaC.
