Aldosterone-sensitive Distal Nephron Research Articles

Sex- and Cell-Specific Changes in BKα Protein Expression in the Aldosterone-Sensitive Distal Nephron (ASDN) in Response to Dietary K+

Sex- and Cell-Specific Changes in BKα Protein Expression in the Aldosterone-Sensitive Distal Nephron (ASDN) in Response to Dietary K+

Abstract 60: Atlas of Distal Nephron Mineralocorticoid Receptor-Dependent Transcriptome Reveals Novel Aldosterone Actions

Aldosterone regulation of Na+ and K+ balance and blood pressure are mediated by the mineralocorticoid receptor (MR, Nr3c2). Although several aldosterone/MR-dependent genes have been identified, their regulation cannot fully explain how aldosterone activates electrogenic Na+- K+ exchange in the aldosterone-sensitive distal nephron (ASDN). Here, we apply RNA-Seq and comprehensive downstream analysis to define the complete inventory of MR-regulated genes. MRfl/fl/Pax8,rtTA/LC1 mice were used to knock out the Nr3c2 gene in an inducible and kidney-specific manner. RNA-Seq analysis was conducted in the micro-dissected connecting tubule and cortical collecting duct tubule segments, followed by multimodal bioinformatic analyses. Direct MR-dependent gene regulation was validated in vitro on immortalized collecting duct cells, ex vivo with isolated distal nephron segments and gene reporter assays. All known aldosterone-response genes, including Sgk1, Scnn1a, Ndrg2, Per3, Tsc22d3, Zbtb16, MLPH, and Atp1a1 were significantly decreased in MR KO mice. 1193 additional genes were down-regulated in the MR KO, and 526 of these have a Mineralocorticoid Responsive Element (MRE), revealing the MR-dependent transcriptome is much larger than previously imagined. MREs were found in most genes with closed chromatin regions, suggesting MR binding might pioneer transcription for most of its regulated genes. The architecture of some MR-regulated genes, highlighted by the co-regulation of 7 Kallikrein genes, points to a novel mechanism of MR-regulation involving epigenetic remodeling in topology-associated domains. Pathway enrichment analysis revealed that MR-regulated genes are highly enriched in mitochondria-associated metabolic processes, and direct MR regulation of Klf15 was identified as a key transcriptional driver. In summary, the inventory of MR-regulated genes in the ASDN is much larger than previously imagined, involving new mechanisms of gene regulation. In addition to pathways that directly up-regulate transport, the data suggest that aldosterone-MR may directly influence metabolism to make energy-consuming transport highly efficient.

Abnormal activation of the mineralocorticoid receptor in the aldosterone-sensitive distal nephron contributes to fructose-induced salt-sensitive hypertension.

Fructose high-salt (FHS) diets increase blood pressure (BP) in an angiotensin II (Ang II)-dependent manner. Ang II stimulates aldosterone release, which, by acting on the mineralocorticoid receptor (MR), regulates Na + reabsorption by the aldosterone-sensitive distal nephron (ASDN). The MR can be transactivated by glucocorticoids, including those locally produced by 11β-HSD1. The epithelial sodium channel (ENaC) is a key transporter regulated by MRs. We hypothesized that fructose-induced salt-sensitive hypertension depends in part on abnormal activation of MRs in the ASDN with consequent increases in ENaC expression. We found that aldosterone-upregulated genes in mice ASDN, significantly overlapped with 74 genes upregulated by FHS in the rat kidney cortex (13/74; p≤1x10 -8 ), and that these 74 genes are prominently expressed in rat ASDN cells. Additionally, the average z-score expression of mice-aldosterone-upregulated genes is highly correlated with FHS compared to glucose high-salt (GHS) in the rat kidney cortex (Pearson correlation; r=0.66; p≤0.005). There were no significant differences in plasma aldosterone concentrations between the FHS and GHS. However, 11β-HSD1 transcripts were upregulated by FHS (log 2 FC=0.26, p≤0.02). FHS increased BP by 23±6 mmHg compared to GHS, and blocking MRs with eplerenone prevented this increase. Additionally, inhibiting ENaC with amiloride significantly reduced BP in FHS from 148±6 to 134±5 mmHg (p≤0.019). Compared to GHS, FHS increased total and cleaved αENaC protein by 89±14 % (p≤0.03) and 47±16 % (p≤0.01) respectively. FHS did not change β- or γ-subunit expression. These results suggest that fructose-induced salt-sensitive hypertension depends, in part, on abnormal Na + retention by ENaC, resulting from the activation of MRs by glucocorticoids.

Epithelial Na + Channel Activation after Bile Duct Ligation with Mineralocorticoid Receptor Blockade.

Sodium and fluid retention in liver disease are classically thought to result from reduced effective circulating volume and stimulation of the renin-angiotensin-aldosterone system (RAAS). However, evidence of fluid retention in patients without RAAS activation suggests the involvement of additional mechanisms. In vitro, bile acids activate the epithelial Na+ channel (ENaC) found in the aldosterone-sensitive distal nephron. If this occurs in vivo, ENaC may become activated in liver disease even with antagonism of aldosterone signaling. To test this, we performed bile duct ligation to induce liver disease and increase circulating bile acids in mice given spironolactone to antagonize aldosterone signaling. We analyzed effects on blood, urine and body composition. We also determined the effects of taurocholic acid, a primary conjugated bile acid elevated in liver disease, on ion fluxes in microperfused rabbit collecting ducts. Bile duct ligation increased benzamil-sensitive natriuresis compared to sham, indicating ENaC activation. These effects were not explained by effects on ENaC expression, cleavage, or localization. Bile duct ligated mice also gained significantly more fluid than sham-operated animals. Blocking ENaC reversed fluid gains in bile duct ligated mice but had no effect in shams. In dissected collecting ducts from rabbits, which express ENaC, taurocholic acid stimulated net Na+ absorption. Our results provide experimental evidence for a novel aldosterone-independent mechanism for sodium and fluid retention in liver disease.

MTORc2 in the distal convoluted tubule (DCT) stimulates renal K+ excretion by inhibiting basolateral Kir4.1/Kir5.1 and Na-Cl−cotransporter (NCC) during increasing dietary K+ intake

Background: The mTORc2 is a multiprotein complex and the core components of mTORc2 contain mTOR (a serine/threonine protein kinase) and RICTOR (rapamycin insensitive companion of mTOR). Previous studies have strongly suggested the role of mTORc2 in the regulation of renal K+ excretion by stimulating epithelial Na+ channels (ENaC) and ROMK (Kir1.1) in the aldosterone-sensitive distal nephron (ASDN). The mice with deletion of RICTOR in the distal nephron or in the renal tubules are hyperkalemic during increasing dietary K+ intake. A large body of evidence has demonstrated that HK-induced inhibition of NCC is essential for stimulating renal K+ excretion and this is achieved by inhibiting the basolateral Kir4.1/Kir5.1 heterotetramer in the DCT. However, the role of mTORc2 in the regulation of basolateral Kir4.1/Kir5.1 heterotetramer in the DCT and NCC is not explored. Thus, the aim of the present study is to explore whether mTORc2 in the DCT regulates the basolateral Kir4.1/Kir5.1 of the DCT, NCC activity and renal K+ excretion. Method: We have generated DCT-specific RICTOR KO mice (DCT-RICTOR-KO) with C57BL6/J background by crossing the mice expressing Slc12a3-Cre-ERT2 with Rictorflox/flox mice. We used patch-clamp-technique to examine basolateral Kir4.1/Kir5.1 in early-DCT, immunoblotting and immunofluorescence to examine NCC expression and in vivo measurement of urinary K+-excretion to determine baseline renal K+-excretion (EK). We also used metabolic cage to examine the body weight (BW), food/water intakes, urinary Na+ (UNa) /K+ (UK) excretion rate, and plasma Na+ and K+ concentrations in male(m)/female(f) DCT-RICTOR KO mice and Slc12a3-Cre-ERT2 -Rictorflox/flox mice (control). Results: Overnight-HK intake stimulates RICTOR expression in the kidney and increases Rictor mRNA in the DCT. The deletion of RICTOR in the DCT was confirmed by immunofluorescence staining showing that RICTOR is expressed in parvalbumin-positive tubule (DCT) of control mice but is almost absent in DCT-RICTOR-KO mice. Deletion of RICTOR in the DCT increased the Kir4.1/Kir5.1-mediated K+-currents, hyperpolarized DCT membrane and increased the expression of pWNK4 and pNCC. While the two genotypes did not have significant difference regarding BW, food/water intakes, UNa and plasma Na+ concentrations, m/f DCT-RICTOR KO mice had a significantly lower UK and higher plasma K+ concentrations than their corresponding control. In vivo measurement of baseline renal K+ excretion (EK) also shows that renal-EK was lower and plasma K+ was higher in DCT-RICTOR-KO mice than corresponding control mice. Also, overnight-HK did not inhibit Kir4.1/Kir5.1 activity in the DCT and failed to inhibit the expression of pNCC in DCT-RICTOR-KO mice. Overnight-HK stimulated renal-EK in control mice, but this effect was attenuated in DCT-RICTOR-KO mice. Thus, overnight-HK induced hyperkalemia in DCT-RICTOR-KO mice but not in control mice. Conclusion: The mTORc2 of the DCT plays a significant role in mediating the effect of HK intake on Kir4.1/Kir5.1 activity and NCC expression. Since mTORC2 also stimulates ENaC and ROMK in the ASDN, an increase in Na+-delivery is expected to stimulate ENaC-ROMK-dependent K+-excretion and flow-stimulated renal K+-excretion. The work is supported by NIH Grants DK136491 (LDH) and DK133220 (WHW). 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.

A conserved interaction between the GRIP and finger domains in ENaC subunit is critical for proteolytic activation

The epithelial sodium channel (ENaC) mediates Na+ transport in several epithelia, including the lung and the aldosterone-sensitive distal nephron. ENaC is a heterotrimer comprising ⍺βγ or δβγ subunits. Though sharing similar structural folds as other ENaC/Degenerin family members, ENaC subunits have a unique extracellular GRIP (Gating Release of Inhibition by Proteolysis) domain, and the a and g subunits have polybasic sites that can be cleaved by proteases. Aldosterone promotes double cleavage of the α and γ subunits at these sites, leading to release of the first two b-strands of the GRIP domain (P1 and P2) and channel activation. Structural and sequence conservation suggest that the interaction between P1 and the channel are mediated by a Pro-Leu motif in P1 and a conserved Trp residue in helix α2 of the finger domain. We hypothesized that this interaction is critical for the inhibitory properties of the imbedded tract, defined by P1, and thus channel activation upon release. To test our hypothesis, we substituted the conserved Trp with Ala or Phe in each of the human ENaC subunits and performed two-electrode voltage clamp experiments in Xenopus oocytes to measure the effects on function. Compared to wild type channels, we found that ⍺W251F, βW218A, βW218F, γW229F, and δW396F had no effect on amiloride-sensitive currents. Interestingly, δW396A decreased amiloride-sensitive current by 20% (p=0.01). However, amiloride-sensitive currents were greatly enhanced for Ala mutations in subunits subject to cleavage: ⍺W251A (p=0.02) and γW229A (p=0.01). In line with channel activation, ⍺W251A weakened sensitivity to extracellular Na+ (Na+ self-inhibition), and γW229A completely lost its sensitivity to both extracellular Na+ and chymotrypsin. We also tested the effect of inhibitory peptides (α8 and γ11) corresponding to the imbedded inhibitory tracts (i.e., P1 of the GRIP domain) of the ⍺ and γ subunits. ⍺W251A was insensitive to 10 μM ⍺8 compared to wild type ENaC (85% inhibition), and γW229A was insensitive to 100 μM γ11 compared to wild type ENaC (40% inhibition). Collectively, our results provide evidence for the idea that the interaction between the Pro-Leu motif in GRIP domain’s P1 β-strand and the conserved Trp in the finger domain’s α2 helix is critical for the autoinhibitory properties of P1, and therefore activation upon P1 release after proteolysis of the ⍺ and γ subunits. R01 DK125439 Kashlan (PI) ENaC Regulation by Biliary Factors. 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.

Role of paraoxonase 3 in regulating ENaC-mediated Na+ transport in the distal nephron.

Paraoxonase 3 (PON3) is expressed in the aldosterone-sensitive distal nephron, where filtered Na+ is reabsorbed mainly via the epithelial Na+ channel (ENaC) and Na+ -coupled co-transporters. We previously showed that PON3 negatively regulates ENaC through a chaperone mechanism. The present study aimed to determine the physiological role of PON3 in renal Na+ and K+ homeostasis. Pon3 knockout (KO) mice had higher amiloride-induced natriuresis and lower plasma [K+ ] at baseline. Single channel recordings in split-open tubules showed that the number of active channels per patch was significantly higher in KO mice, resulting in a higher channel activity in the absence of PON3. Although whole kidney abundance of ENaC subunits was not altered in Pon3 KOs, ENaC gamma subunit was more apically distributed within the connecting tubules and cortical collecting ducts of Pon3 KO kidneys. Additionally, small interfering RNA-mediated knockdown of PON3 in cultured mouse cortical collecting duct cells led to an increased surface abundance of ENaC gamma subunit. As a result of lower plasma [K+ ], sodium chloride co-transporter phosphorylation was enhanced in the KO kidneys, a phenotype that was corrected by a high K+ diet. Finally, PON3 expression was upregulated in mouse kidneys under dietary K+ restriction, potentially providing a mechanism to dampen ENaC activity and associated K+ secretion. Taken together, our results show that PON3has a role in renal Na+ and K+ homeostasis through regulating ENaC functional expression in the distal nephron. KEY POINTS: Paraoxonase 3 (PON3) is expressed in the distal nephron of mouse kidneys and functions as a molecular chaperone to reduce epithelial Na+ channel (ENaC) expression and activity in heterologous expression systems. We examined the physiological role of PON3 in renal Na+ and K+ handling using a Pon3 knockout (KO) mouse model. At baseline, Pon3 KO mice had lower blood [K+ ], more functional ENaC in connecting tubules/cortical collecting ducts, higher amiloride-induced natriuresis, and enhanced sodium chloride co-transporter (NCC) phosphorylation. Upon challenge with a high K+ diet, Pon3 KO mice had normalized blood [K+ ] and -NCC phosphorylation but lower circulating aldosterone levels compared to their littermate controls. Kidney PON3 abundance was altered in mice under dietary K+ loading or K+ restriction, providing a potential mechanism for regulating ENaC functional expression and renal Na+ and K+ homeostasis in the distal nephron.

Kir7.1 knockdown and inhibition alter renal electrolyte handling but not the development of hypertension in Dahl salt-sensitive rats.

High K+ supplementation is correlated with a lower risk of the composite of death, major cardiovascular events, and ameliorated blood pressure, but the exact mechanisms have not been established. Inwardly rectifying K+ (Kir) channels expressed in the basolateral membrane of the distal nephron play an essential role in maintaining electrolyte homeostasis. Mutations in this channel family have been shown to result in strong disturbances in electrolyte homeostasis, among other symptoms. Kir7.1 is a member of the ATP-regulated subfamily of Kir channels. However, its role in renal ion transport and its effect on blood pressure have yet to be established. Our results indicate the localization of Kir7.1 to the basolateral membrane of aldosterone-sensitive distal nephron cells. To examine the physiological implications of Kir7.1, we generated a knockout of Kir7.1 (Kcnj13) in Dahl salt-sensitive (SS) rats and deployed chronic infusion of a specific Kir7.1 inhibitor, ML418, in the wild-type Dahl SS strain. Knockout of Kcnj13 (Kcnj13-/-) resulted in embryonic lethality. Heterozygous Kcnj13+/- rats revealed an increase in K+ excretion on a normal-salt diet but did not exhibit a difference in blood pressure development or plasma electrolytes after 3 wk of a high-salt diet. Wild-type Dahl SS rats exhibited increased renal Kir7.1 expression when dietary K+ was increased. K+ supplementation also demonstrated that Kcnj13+/- rats excreted more K+ on normal salt. The development of hypertension was not different when rats were challenged with high salt for 3 wk, although Kcnj13+/- rats excrete less Na+. Interestingly, chronic infusion of ML418 significantly increased Na+ and Cl- excretion after 14 days of high salt but did not alter salt-induced hypertension development. Here, we found that reduction of Kir7.1 function, either through genetic ablation or pharmacological inhibition, can influence renal electrolyte excretion but not to a sufficient degree to impact the development of SS hypertension.NEW & NOTEWORTHY To investigate the role of the Kir7.1 channel in salt-sensitive hypertension, its function was examined using complementary genetic and pharmacological approaches. The results revealed that although reducing Kir7.1 expression had some impact on maintaining K+ and Na+ balance, it did not lead to a significant change in the development or magnitude of salt-induced hypertension. Hence, it is probable that Kir7.1 works in conjunction with other basolateral K+ channels to fine-tune membrane potential.

Defining corticosteroid-regulated sodium transport within the distal nephron

The aldosterone (ALDO)-sensitive distal nephron is typically defined as: the late distal convoluted tubule (DCT2), connecting tubule (CNT) and cortical collecting duct (CCD). ALDO-sensitivity is defined by 11βHSD2 (11β) activity (which inactivates corticosterone, CORT); mineralocorticoid (MR) rather than glucocorticoid receptor (GR) expression; and the target epithelial sodium channel (ENaC). Recent studies have reported ALDO-insensitive, but MR-dependent ENaC activity in DCT2/CNT and that CORT-stimulated ENaC may be relevant in MR antagonism as 4th line anti-hypertensive therapy. Whilst functional evidence of ALDO-insensitive ENaC activity is compelling, corticosteroid-regulated Na + transport along the distal nephron is not fully defined. AIM: to determine 11βHSD2, MR and GR expression alongside Na + transporter localisation in murine distal nephron and assess the endogenous receptor(s) underpinning steroid-stimulated ENaC activity.Male C57BL/6 mouse kidney sections were subject to multiplex immunofluorescence (11β, MR and GR) and immunohistochemical labelling (NCC, TRPV5, γ-ENaC and AQP2). Images were co-registered, annotated and analysed using Halo® software. Steroid-induced ENaC-mediated currents were measured in mCCD cl1 and primary CD cells. Carbenoxolone (CBX), mifepristone (MF) or PF-03882845 (PF) were used to inhibit 11β, GR and MR, respectively.Distal nephron segments were defined as: DCT - NCC; DCT/CNT - NCC and TRPV5; CNT - TRPV5; CNT/CCD - TRPV5 and γ-ENaC/AQP2; CCD - γ-ENaC/AQP2. We did not detect NCC and γ-ENaC co-localisation. Average cells/section, DCT: 874±39; DCT/CNT: 238±31; CNT: 198±63; CNT/CCD: 831±282; CCD: 2262±231 ( n=3). Negligible numbers of cells expressed both MR and GR.DCT was predominantly 11β- (~90%) and GR dominant (GR:MR ratio ~7:1). In 11β+ cells (~10%), GR was also dominant (GR:MR ratio ~4:1). DCT/CNT and CNT were mainly 11β- (~85 and ~75%, respectively), where MR and GR were similar. In 11β+ cells, MR and GR were similar in DCT/CNT but MR was greater in CNT (MR:GR ratio ~4:1). CNT/CCD contained a near equal split of 11β- (~52%) and 11β+ (~48%) cells. MR and GR were similar in 11β- cells, but 11β+ cells were almost exclusively MR (MR:GR ratio ~37:1). In CCD, ~35% of cells were 11β-, MR and GR were similar, and ~65% cells were 11β+, these were predominantly MR (MR:GR ratio ~30:1).In mCCD cl1 ( n=8-11) and primary CD cells ( n=6), ALDO-induced ENaC activity was inhibited by MR ( p<0.001) but not GR antagonists. DEX-induced ENaC activity was inhibited by GR ( p<0.001) but not MR antagonists. CORT-induced ENaC activity was modestly inhibited by MR ( p<0.05) but not by GR antagonists.Our data suggest that the DCT is a glucocorticoid-responsive segment, with 11β+ cells becoming progressively more dominant from CNT to CCD. The appearance of ENaC in the late CNT and particularly CCD, alongside 11β and MR, suggest these segments reflect the mineralocorticoid-responsive, and thus aldosterone-sensitive distal nephron. Kidney Research UK, The Royal Society This is the full abstract presented at the American Physiology Summit 2023 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.

Behavior of the renal kallikrein in spontaneously hypertensive rats: Influence of sexual hormones and aldosterone-sensitive distal nephron ion channels

The renal kallikrein-kinin system (RKKS) has been related to blood pressure control and sodium and water balance. We have previously shown that female spontaneously hypertensive rats (SHR) have high urinary kallikrein activity (UKa) and lower blood pressure (BP) than males whereas ovariectomy stimulates UKa and diminishes BP. We also showed that high K+ intake and prepuberal gonadectomy (Gx) diminish BP with a concomitant increase in UKa and plasma aldosterone levels. Since kallikrein co-localize in the same distal nephron segments of aldosterone effectors, we explored the effect of pharmacological blockage of aldosterone receptor, epithelial Na+ (ENaC) and the rectifying outer medulla K+ (ROMK) channels in different gonad contexts on the gene expression, renal tissue content and urine release of kallikrein. Klk1 gene expression was determined by real-time PCR and enzymatic activity of kallikrein by the amidolytic method. We found that the inhibition of the aldosterone receptor by spironolactone increases kallikrein renal tissue storage and decreases its urinary activity, especially in Gx rats. Moreover, ENaC blockade by benzamil increases the renal content of kallikrein without affecting synthesis or excretion, especially in females and Gx animals, while the inhibition of ROMK by glibenclamide increases the synthesis and renal content of kallikrein only in intact male animals. We concluded that RKKS regulation showed sexual dimorphism and seemed to be modulated by sex hormones throughout a process involving aldosterone and the aldosterone-sensitive ion channels..

Functional characterization of ion channels expressed in kidney organoids derived from human induced pluripotent stem cells.

Kidney organoids derived from human or rodent pluripotent stem cells have glomerular structures and differentiated/polarized nephron segments. Although there is an increasing understanding of the patterns of expression of transcripts and proteins within kidney organoids, there is a paucity of data regarding functional protein expression, in particular on transporters that mediate the vectorial transport of solutes. Using cells derived from kidney organoids, we examined the functional expression of key ion channels that are expressed in distal nephron segments: the large-conductance Ca2+-activated K+ (BKCa) channel, the renal outer medullary K+ (ROMK, Kir1.1) channel, and the epithelial Na+ channel (ENaC). RNA-sequencing analyses showed that genes encoding the pore-forming subunits of these transporters, and for BKCa channels, key accessory subunits, are expressed in kidney organoids. Expression and localization of selected ion channels was confirmed by immunofluorescence microscopy and immunoblot analysis. Electrophysiological analysis showed that BKCa and ROMK channels are expressed in different cell populations. These two cell populations also expressed other unidentified Ba2+-sensitive K+ channels. BKCa expression was confirmed at a single channel level, based on its high conductance and voltage dependence of activation. We also found a population of cells expressing amiloride-sensitive ENaC currents. In summary, our results show that human kidney organoids functionally produce key distal nephron K+ and Na+ channels.NEW & NOTEWORTHY Our results show that human kidney organoids express key K+ and Na+ channels that are expressed on the apical membranes of cells in the aldosterone-sensitive distal nephron, including the large-conductance Ca2+-activated K+ channel, renal outer medullary K+ channel, and epithelial Na+ channel.

Interleukin 6 mediated activation of the mineralocorticoid receptor in the aldosterone-sensitive distal nephron.

Hypertension is characterized by increased sodium (Na+) reabsorption along the aldosterone-sensitive distal nephron (ASDN) as well as chronic systemic inflammation. Interleukin-6 (IL-6) is thought to be a mediator of this inflammatory process. Interestingly, increased Na+ reabsorption within the ASDN does not always correlate with increases in aldosterone (Aldo), the primary hormone that modulates Na+ reabsorption via the mineralocorticoid receptor (MR). Thus, understanding how increased ASDN Na+ reabsorption may occur independent of Aldo stimulation is critical. Here, we show that IL-6 can activate the MR by activating Rac1 and stimulating the generation of reactive oxygen species (ROS) with a consequent increase in thiazide-sensitive Na+ uptake. Using an in vitro model of the distal convoluted tubule (DCT2), mDCT15 cells, we observed nuclear translocation of eGFP-tagged MR after IL-6 treatment. To confirm the activation of downstream transcription factors, mDCT15 cells were transfected with mineralocorticoid response element (MRE)-luciferase reporter constructs; then treated with vehicle, Aldo, or IL-6. Aldosterone or IL-6 treatment increased luciferase activity that was reversed with MR antagonist cotreatment, but IL-6 treatment was reversed by Rac1 inhibition or ROS reduction. In both mDCT15 and mpkCCD cells, IL-6 increased amiloride-sensitive transepithelial Na+ current. ROS and IL-6 increased 22Na+ uptake via the thiazide-sensitive sodium chloride cotransporter (NCC). These results are the first to demonstrate that IL-6 can activate the MR resulting in MRE activation and that IL-6 increases NCC-mediated Na+ reabsorption, providing evidence for an alternative mechanism for stimulating ASDN Na+ uptake during conditions where Aldo-mediated MR stimulation may not occur.

Role of inwardly rectifying K+ channel 5.1 (Kir5.1) in the regulation of renal membrane transport.

Kir5.1 interacts with Kir4.2 in proximal tubule and with Kir4.1 in distal convoluted tubule (DCT), connecting tubule (CNT) and cortical collecting duct (CCD) to form basolateral-K+-channels. Kir4.2/Kir5.1 and Kir4.1/Kir5.1 play an important role in regulating Na+/HCO3--transport of the proximal tubule and Na+/K+ -transport in the DCT/CNT/CCD. The main focus of this review is to provide an overview of the recent development in the field regarding the role of Kir5.1 regulating renal electrolyte transport in the proximal tubule and DCT. Loss-of-function-mutations of KCNJ16 cause a new form of tubulopathy, characterized by hypokalaemia, Na+-wasting, acid-base-imbalance and metabolic-acidosis. Abnormal bicarbonate transport induced by loss-of-function of KCNJ16-mutants is recapitulated in Kir4.2-knockout-(Kir4.2 KO) mice. Deletion of Kir5.1 also abolishes the effect of dietary Na+ and K+-intakes on the basolateral membrane voltage and NCC expression/activity. Long-term high-salt intake or high-K+-intake causes hyperkalaemic in Kir5.1-deficient mice. Kir4.2/Kir5.1 activity in the proximal tubule plays a key role in regulating Na+, K+ and bicarbonate-transport through regulating electrogenic-Na+-bicarbonate-cotransporter-(NBCe1) and type 3-Na+/H+-exchanger-(NHE3). Kir4.1/Kir5.1 activity of the DCT plays a critical role in mediating the effect of dietary-K+ and Na+-intakes on NCC activity/expression. As NCC determines the Na+ delivery rate to the aldosterone-sensitive distal nephron (ASDN), defective regulation of NCC during high-salt and high-K+ compromises renal K+ excretion and K+ homeostasis.

Analysis of the Hypoxic Response in a Mouse Cortical Collecting Duct-Derived Cell Line Suggests That Esrra Is Partially Involved in Hif1α-Mediated Hypoxia-Inducible Gene Expression in mCCDcl1 Cells.

The kidney is strongly dependent on a continuous oxygen supply, and is conversely highly sensitive to hypoxia. Controlled oxygen gradients are essential for renal control of solutes and urine-concentrating mechanisms, which also depend on various hormones including aldosterone. The cortical collecting duct (CCD) is part of the aldosterone-sensitive distal nephron and possesses a key function in fine-tuned distal salt handling. It is well known that aldosterone is consistently decreased upon hypoxia. Furthermore, a recent study reported a hypoxia-dependent down-regulation of sodium currents within CCD cells. We thus investigated the possibility that cells from the cortical collecting duct are responsive to hypoxia, using the mouse cortical collecting duct cell line mCCDcl1 as a model. By analyzing the hypoxia-dependent transcriptome of mCCDcl1 cells, we found a large number of differentially-expressed genes (3086 in total logFC< −1 or >1) following 24 h of hypoxic conditions (0.2% O2). A gene ontology analysis of the differentially-regulated pathways revealed a strong decrease in oxygen-linked processes such as ATP metabolic functions, oxidative phosphorylation, and cellular and aerobic respiration, while pathways associated with hypoxic responses were robustly increased. The most pronounced regulated genes were confirmed by RT-qPCR. The low expression levels of Epas1 under both normoxic and hypoxic conditions suggest that Hif-1α, rather than Hif-2α, mediates the hypoxic response in mCCDcl1 cells. Accordingly, we generated shRNA-mediated Hif-1α knockdown cells and found Hif-1α to be responsible for the hypoxic induction of established hypoxically-induced genes. Interestingly, we could show that following shRNA-mediated knockdown of Esrra, Hif-1α protein levels were unaffected, but the gene expression levels of Egln3 and Serpine1 were significantly reduced, indicating that Esrra might contribute to the hypoxia-mediated expression of these and possibly other genes. Collectively, mCCDcl1 cells display a broad response to hypoxia and represent an adequate cellular model to study additional factors regulating the response to hypoxia.

Inwardly rectifying K+ channels 4.1 and 5.1 (Kir4.1/Kir5.1) in the renal distal nephron.

The inwardly rectifying potassium channel (Kir) 4.1 (encoded by KCNJ10) interacts with Kir5.1 (encoded by KCNJ16) to form a major basolateral K+ channel in the renal distal convoluted tubule (DCT), connecting tubule (CNT), and the cortical collecting duct (CCD). Kir4.1/Kir5.1 heterotetramer plays an important role in regulating Na+ and K+ transport in the DCT, CNT, and CCD. A recent development in the field has firmly established the role of Kir4.1/Kir5.1 heterotetramer of the DCT in the regulation of thiazide-sensitive Na-Cl cotransporter (NCC). Changes in Kir4.1/Kir5.1 activity of the DCT are an essential step for the regulation of NCC expression/activity induced by dietary K+ and Na+ intakes and play a role in modulating NCC by type 2 angiotensin II receptor (AT2R), bradykinin type II receptor (BK2R), and β-adrenergic receptor. Since NCC activity determines the Na+ delivery rate to the aldosterone-sensitive distal nephron (ASDN), a distal nephron segment from late DCT to CCD, Kir4.1/Kir5.1 activity plays a critical role not only in the regulation of renal Na+ absorption but also in modulating renal K+ excretion and maintaining K+ homeostasis. Thus, Kir4.1/Kir5.1 activity serves as an important component of renal K+ sensing mechanism. The main focus of this review is to provide an overview regarding the role of Kir4.1 and Kir5.1 of the DCT and CCD in the regulation of renal K+ excretion and Na+ absorption.

MO068: Paraoxonase 2 Protects the Kidney Tissue from Aldosterone-Promoted Oxidative Stress in Milan Normotensive Rats Strain

Abstract BACKGROUND AND AIMS Kidney plays a pivotal role on the maintenance of blood pressure by the regulation of sodium reabsorption. Aldosterone sensitive distal nephron finely tunes salt reabsorption through several transporters, including the epithelial sodium channel (ENaC). Alteration in ENaC function leads to clinical relevant conditions associated with high or low blood pressure when gain or loss of function mutations occur, respectively. Recently, accumulating evidence suggests the putative role of renal Paraoxonase 2 (PON2) as regulator of ENaC function. PON2 is a membrane-associated enzyme showing hydrolytic activity against 3-oxo-C12 homoserine lactone, a signaling molecule produced by bacteria and mainly Pseudomonas aeruginosa. PON2 also exhibits antioxidant property. In the distal nephron, PON2 is expressed in principal cells, and in vitro it physically interacts with ENaC subunits and inhibits ENaC channel activity in Xenopus oocytes likely by modulating channel assembly in the endoplasmic reticulum. However, the role of PON2 in the kidney is still not known. In the present study, we investigate whether PON2 is regulated by changes in aldosterone state and its role in aldosterone-induced oxidative stress in the kidneys. METHOD We addressed these issues utilizing both in vivo and in vitro approaches, such as RESULTS We first demonstrated that in Milan normotensive rats strain, PON2 is upregulated by low-salt state according with α-ENaC activation. This regulation is aldosterone sensitive since pretreatment with mineralocorticoid receptor antagonist spironolactone prevented the stimulatory effects of sodium restricted diet. Furthermore, low-sodium diet inducing PON2 upregulation was associated with reduced ROS levels, more specifically superoxide levels, but treatment with spironolactone prevented this beneficial effect, by increasing ROS level in kidney cortex. Since antihypertensive drugs spironolactone and canrenone contain a lactone residue in their molecular structure, we tested whether they could directly interact with PON2. We demonstrated in vitro that spironolactone and canrenone were able to inhibit PON2 lactonase activity and so to directly interfere with PON2 function. Finally, Milan hypertensive rats, carrying a mutation of α-adducin as determinant of their hypertensive phenotype, presented with higher level of ROS while PON2 was upregulated. CONCLUSION These results demonstrate PON2 as a novel target of steroid hormone aldosterone. PON2 may serve as a potential ROS scavenger mechanism protecting the kidneys from aldosterone-promoted oxidative stress.

Localization and Functional Characterization of the Cannabinoid Receptor Type 1 (CB1R) in the Kidney

IntroductionCannabis and synthetic cannabinoid consumption is increasing worldwide. The impact of their consumption on human health including the risk of kidney disease and its associated complications is uncertain. The kidney, among other organs, produces endogenous cannabinoids (endocannabinoids, ECs) that act on the G‐protein‐coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R). ECs and CB1R agonists acutely act as diuretics with variable effects on Na+, K+, and HCO3– excretion. As the aldosterone sensitive distal nephron (ASDN) is responsible for the final renal regulation of total body electrolyte, acid/base, and water balance that is mediated by principal cells (PCs) and intercalated cells (ICs), we sought to examine the cell specific expression of components of the EC system in the mouse collecting duct and the acute changes in urine composition after administration of the cannabinoid receptor agonist WIN55,212‐2 (WIN).MethodsCB1R and AQP2 localization were examined by immunofluorescence microscopy in cryosections of kidneys harvested from non‐manipulated male C57BL/6 mice (7‐12 weeks of age) using protein‐specific Abs. A subset of whole animal metabolic studies were performed in mice that were acid‐loaded by provision of 20 mM NH4Cl and 1% sucrose added to the drinking water x 7d. On the day of experimentation, mice were allowed to spontaneously void, then injected SC with either 1.5 mg/kg WIN or vehicle control (10% Tween‐80 and 10% DMSO in 1x PBS) in a volume &lt;1% BW. Urine was collected for up to 4hr for measurement of volume, pH, osmolality, Na+ and K+.ResultsIn the ASDN, CB1R was localized to AQP2(‐) cells, and thus presumably ICs. Acid‐loaded mice treated with WIN (n=5) had similar UpH, UNaV, and UKV to untreated mice (n= 4) but had a higher urine output (UOP) (24.1 ± 6.5 vs. 13.6 ±1.2 µL/gBW/4hr; (P=0.02) and a lower UOsm (822 ± 335 vs. 1912 ± 293 mmol/kg H2O; P=0.001). WIN‐treated non‐acid loaded mice (n=7) urinated more frequently (3.6 ± 2.2 vs. 0.4 ± 0.5 micturitions/2 hr; P=0.05), had a lower UOsm (315 ± 82 vs. 1449 ± 553 mmol/kg H2O; P&lt;0.0001), and lower UKV (0.4 ± 0.2 vs. 0.9 ± 0.3 µmol/gBW/2hrs; P=0.002) compared to controls (n=8), whereas UNaV and UpH were similar. Furthermore, WIN reduced the apical/cytoplasmic ratio of AQP2 expression in PCs of the inner medulla by 43% (n=209 cells in 4 mice) compared to controls (n=183 cells in 4 mice) (P&lt;0.0001).ConclusionICs express CB1R. Although UpH and Na+ excretion were unaffected in either acid‐loaded or non‐acid loaded mice by WIN, the cannabinoid receptor agonist induced diabetes insipidus, a disorder characterized by the excretion of large volumes of dilute urine, and reduced K+ excretion in non‐acid loaded mice. Efforts are underway to determine whether this effect is central or nephrogenic. These results promise to reveal how cannabis affects kidney function and to identify potential targets for novel therapeutics to treat water and electrolyte disorders.

Phosphorylation‐Dependent Endocytosis of ROMK Limits Urinary Potassium Loss

ROMK channels in the aldosterone‐sensitive distal nephron are tightly regulated to maintain potassium balance. In response to potassium restriction, ROMK channels are internalized from the apical membrane, safeguarding against extensive potassium loss. Previously, we found the clathrin adaptor protein, ARH, directly binds to ROMK to stimulate endocytosis (Fang et al, JCI ‘09). WNK signaling pathway has been implicated in activating ROMK endocytosis, but the mechanism is not clear, and physiological significance of ARH‐mediated ROMK regulation remains uncertain. To tackle these questions, we explored how WNK kinases activate ARH and compared the homeostatic response to dietary potassium restriction in wild type and ARH KO mice.In vitro phosphorylation studies revealed ARH is phosphorylated by ERK, which in turn is activated by L‐WNK1 or WNK4. Examination of the phosphorylation cascade revealed that dietary potassium restriction increases phosphorylation of ERK and ARH‐S14 in the kidney, coincident with phospho‐activation of the WNKs, and reduction of ROMK apical surface expression. We found phosphorylation of ARH at S14 prevents its degradation, stimulates its membrane association, and this enhances ROMK endocytosis. ARH KO mice exhibit an increase in glycosylated ROMK in the renal cortex without changes in the abundance of the core‐glycosylated or unglycosylated forms, consistent with inhibition of post‐endocytic degradation. In dietary potassium deficiency, ARH KO mice are unable to reduce ROMK protein to the same extent as WT mice, and thus exhibit excessive urinary potassium loss compared to WT mice.These studies identify ARH as a downstream effector of the WNK/ERK pathway that stimulates ROMK endocytosis in potassium deficiency.

Regulation of BK channel expression by Paraoxonase 3

Paraoxonase 3 (PON3) is a molecular chaperone that is primarily expressed in the aldosterone‐sensitive distal nephron (ASDN) where the fine tuning of Na+ and K+ homeostasis occurs. The flow‐induced K+ secretion in the distal nephron is mediated by large conductance Ca2+‐activated K+ (BK) channels in intercalated cells (ICs). As PON3 co‐localizes with BK in the ICs and Pon3 KO mice have altered blood [K+] at baseline, we hypothesize that PON3 has a role in regulating BK expression and functional activity.To determine whether the functional expression of BK channels is regulated by PON3, the pore‐forming α subunits of mouse BK (αBK) was expressed in HEK293 cells with or without mouse PON3. We performed co‐immunoprecipitation (co‐IP) to examine whether PON3 interacts with αBK. We also examined the effects of PON3 on αBK expression and BK channel activity using biochemical and electrophysiological approaches.When expressed in HEK293 cells, PON3 was pulled down by the αBK‐IP while αBK was detected in the PON3‐pulldown, only in cells expressing both proteins. Our data suggest that there are direct or indirect interactions between PON3 and αBK, the pore‐forming subunit of BK channels. We performed whole cell patch clamp to measure BK channel activity in HEK293 cells. we found that IBTX‐sensitive whole cell K+ currents were reduced approximately 50% by PON3. Consistently, both whole cell and surface abundance of αBK was significantly lower in cells co‐expressing PON3. Together, our data suggest that PON3 negatively regulates the expression and activity of BK channels.In conclusion, we identified the BK channel as a novel target of the chaperone PON3. Future studies will determine mechanisms by which PON3 regulates BK channel functional expression as well as physiological consequences of this regulatory interaction in mouse kidney.

Low Salt Delivery Triggers Autocrine Release of Prostaglandin E2 From the Aldosterone-Sensitive Distal Nephron in Familial Hyperkalemic Hypertension Mice.

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.