High baseline ROMK activity in mouse late distal convoluted and early connecting tubule (DCT2/CNT) probably contributes to aldosterone-independent K+ secretion

The renal outer medullary K+ channel (ROMK) is co-localized with the epithelial Na+ channel (ENaC) in late distal convoluted tubule (DCT2), connecting tubule (CNT) and cortical collecting duct (CCD). ENaC-mediated Na+ absorption generates the electrical driving force for ROMK-mediated tubular K+ secretion which is critically important for maintaining renal K+ homeostasis. ENaC activity is aldosterone-dependent in late CNT and early CCD (CNT/CCD) but aldosterone-independent in DCT2 and early CNT (DCT2/CNT). This suggests that under baseline conditions with low plasma aldosterone ROMK-mediated K+ secretion mainly occurs in DCT2/CNT. Therefore, we hypothesized that baseline ROMK activity is higher in DCT2/CNT than in CNT/CCD. To test this hypothesis, patch-clamp experiments were performed in DCT2/CNT and CNT/CCD microdissected from mice maintained on standard diet. In single-channel recordings from outside-out patches we detected typical ROMK channel activity in both DCT2/CNT and CNT/CCD and confirmed that ROMK is the predominant K+ channel in the apical membrane. Amiloride-sensitive (ΔIami) and tertiapin-sensitive (ΔITPNQ) whole-cell currents were determined to assess ENaC and ROMK activity, respectively. As expected, baseline ΔIami was high in DCT2/CNT (~370 pA) but low in CNT/CCD (~60 pA). Importantly, ΔITPNQ was significantly higher in DCT2/CNT than in CNT/CCD (~810 pA versus ~350 pA). We conclude that high ROMK activity in DCT2/CNT is critical for aldosterone-independent renal K+ secretion under baseline conditions. A low potassium diet significantly reduced ENaC but not ROMK activity in DCT2/CNT. This suggests that modifying ENaC activity in DCT2/CNT plays a key regulatory role in adjusting renal K+ excretion to dietary K+ intake.

ROMK channels are inhibited in the aldosterone-sensitive distal nephron (ASDN) of renal-tubule Nedd4-2 deficient mice.

We used whole-cell-recording to examine renal-outer-medullary-K+ channel (ROMK or Kir1.1) and epithelial-Na+-Channel (ENaC) in late-distal-convoluted-tubule (DCT2)/initial-connecting-tubule (iCNT) and in the cortical-collecting-duct (CCD) of kidney-tubule-specific Nedd4-2 knockout mice (Ks-Nedd4-2-KO) and floxed-Nedd4l mice (control). TPNQ-sensitive K+ currents (ROMK) were smaller in both DCT2/iCNT and CCD of Ks-Nedd4-2-KO mice on normal diet than control mice. Neither high-dietary-salt-intake (HS) nor low-dietary-salt-intake (LS) had a significant effect on ROMK activity in the DCT2/iCNT and CCD of control and Ks-Nedd4-2-KO mice. In contrast, high-dietary-K+-intake (HK) increased while low-dietary-K+-intake (LK) decreased TPNQ-sensitive K+ currents in floxed-Nedd4l mice. However, effects of dietary-K+ intake on ROMK channel activity were absent in Ks-Nedd4-2-KO mice since neither HK nor LK significantly affected TPNQ-sensitive K+ currents in DCT2/iCNT and CCD. Moreover. TPNQ-sensitive K+ currents in DCT2/iCNT and the CCD of Ks-Nedd4-2-KO mice on HK were similar to the control mice on LK. Amiloride-sensitive Na+ currents in DCT2/iCNT and CCD were significantly higher in Ks-Nedd4-2-KO mice than floxed-Nedd4l mice on normal-K+-diet. HK increased ENaC activity of DCT2/iCNT only in the control mice but HK stimulated ENaC of the CCD in both control and Ks-Nedd4-2-KO mice. Moreover, HK-induced increase in amiloride-sensitive Na+-currents was larger in Ks-Nedd4-2-KO mice than the control mice. Deletion of Nedd4-2 increased WNK1 expression and abolished the HK-induced inhibition of WNK1. We conclude that deletion of Nedd4-2 increases ENaC activity but decreases ROMK activity in aldosterone-sensitive distal nephron (ASDN) and that HK fails to stimulate ROMK but robustly increases ENaC activity in the CCD of Nedd4-2-deficeint mice.

Critical role of the mineralocorticoid receptor in aldosterone-dependent and aldosterone-independent regulation of ENaC in the distal nephron

The epithelial sodium channel (ENaC) constitutes the rate-limiting step for sodium absorption in the aldosterone-sensitive distal nephron (ASDN) comprising the late distal convoluted tubule (DCT2), the connecting tubule (CNT) and the collecting duct. Previously, we demonstrated that ENaC activity in the DCT2/CNT transition zone is constitutively high and independent of aldosterone, in contrast to its aldosterone dependence in the late CNT and initial cortical collecting duct (CNT/CCD). The mineralocorticoid receptor (MR) is expressed in the entire ASDN. Its activation by glucocorticoids is prevented through 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) abundantly expressed in the late but probably not the early part of ASDN. We hypothesized that ENaC function in the early part of the ASDN is aldosterone-independent but may depend on MR activated by glucocorticoids due to low 11β-HSD2 abundance. To test this hypothesis, we used doxycycline-inducible nephron-specific MR-deficient mice (MR KO). Whole-cell ENaC currents were investigated in isolated nephron fragments from DCT2/CNT or CNT/CCD transition zones using the patch-clamp technique. ENaC activity was detectable in CNT/CCD of control mice but absent or barely detectable in the majority of CNT/CCD preparations from MR KO mice. Importantly, ENaC currents in DCT2/CNT were greatly reduced in MR KO mice compared to ENaC currents in DCT2/CNT of control mice. Immunofluorescence for 11β-HSD2 was abundant in CCD, less prominent in CNT and very low in DCT2. We conclude that MR is critically important for maintaining aldosterone-independent ENaC activity in DCT2/CNT. Aldosterone-independent MR activation is probably mediated by glucocorticoids due to low expression of 11β-HSD2.

The Angiotensin II Type 1 Receptor-Associated Protein Attenuates Angiotensin II-Mediated Inhibition of the Renal Outer Medullary Potassium Channel in Collecting Duct Cells

Adjustments in renal K+ excretion constitute a central mechanism for K+ homeostasis. The renal outer medullary potassium (ROMK) channel accounts for the major K+ secretory route in collecting ducts during basal conditions. Activation of the angiotensin II (Ang II) type 1 receptor (AT1R) by Ang II is known to inhibit ROMK activity under the setting of K+ dietary restriction, underscoring the role of the AT1R in K+ conservation. The present study aimed to investigate whether an AT1R binding partner, the AT1R-associated protein (ATRAP), impacts Ang II-mediated ROMK regulation in collecting duct cells and, if so, to gain insight into the potential underlying mechanisms. To this end, we overexpressed either ATRAP or β-galactosidase (LacZ; used as a control), in M-1 cells, a model line of cortical collecting duct cells. We then assessed ROMK channel activity by employing a novel fluorescence-based microplate assay. Experiments were performed in the presence of 10−10 M Ang II or vehicle for 40 min. We observed that Ang II-induced a significant inhibition of ROMK in LacZ, but not in ATRAP-overexpressed M-1 cells. Inhibition of ROMK-mediated K+ secretion by Ang II was accompanied by lower ROMK cell surface expression. Conversely, Ang II did not affect the ROMK-cell surface abundance in M-1 cells transfected with ATRAP. Additionally, diminished response to Ang II in M-1 cells overexpressing ATRAP was accompanied by decreased c-Src phosphorylation at the tyrosine 416. Unexpectedly, reduced phospho-c-Src levels were also found in M-1 cells, overexpressing ATRAP treated with vehicle, suggesting that ATRAP can also downregulate this kinase independently of Ang II-AT1R activation. Collectively, our data support that ATRAP attenuates inhibition of ROMK by Ang II in collecting duct cells, presumably by reducing c-Src activation and blocking ROMK internalization. The potential role of ATRAP in K+ homeostasis and/or disorders awaits further investigation.

Loss of Adam10 disrupts ion transport in immortalised kidney collecting duct cells

The kidney cortical collecting duct (CCD) comprises of principal cells (PC), intercalated cells (IC) and the recently discovered intermediate cell type. Kidney pathology in a mouse model of the syndrome of apparent aldosterone excess (SAME) revealed plasticity of the cortical collecting duct (CCD), with altered principal cell (PC): intermediate cell: intercalated cell (IC) ratio. The self-immortalized mouse CCD cell line, mCCDcl1, shows functional characteristics of PCs but displays a range of cell types, including intermediate cells, making it ideal to study plasticity. We knocked out Adam10, a key component of the Notch pathway, in mCCDcl1 cells, using CRISPR-Cas9 technology, and isolated independent clones, which exhibited severely affected sodium transport capacity and loss of aldosterone response. Single-cell RNA sequencing revealed significantly reduced expression of major PC-specific markers, such as Scnn1g (γ-ENaC) and Hsd11b2 (11ßHSD2), but no significant changes in transcription of components of the Notch pathway were observed. Immunostaining in the knockout clone confirmed the decrease in expression of γ-ENaC and importantly, showed an altered, diffuse distribution of PC and IC markers, suggesting altered trafficking in the Adam10 knockout clone as an explanation for the loss of polarisation.

Decreased 11β-Hydroxysteroid Dehydrogenase Type 2 Expression in the Kidney May Contribute to Nicotine/Smoking-Induced Blood Pressure Elevation in Mice

Chronic nicotine exposure significantly increases hypertensive risk in smokers, but the underlying mechanisms are poorly understood. In the kidneys, 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) catalyzes the conversion from active into inactive glucocorticoids and plays a pivotal role in the regulation of blood pressure. We hypothesized that nicotine-induced blood pressure elevation is in part mediated by change in renal 11β-HSD2 leading to higher MR (mineralocorticoid receptor) occupancy. Here, we show that nicotine exposure markedly decreased the expression and activity of renal 11β-HSD2 and increased the mean systolic arterial pressure in C57BL/6J mice. Reduction of renal 11β-HSD2 expression by nicotine was correlated with the suppression of C/EBPβ (CCAAT/enhancer-binding protein-β) and activation of Akt protein kinase phosphorylation (pThr 308 Akt/PKB) within the kidney. Conversely, nicotine-treated mice had elevated renal MR and epithelial sodium channel-α abundance. Treatment with the MR antagonist spironolactone significantly decreased the elevated mean systolic blood pressure and corrected ENaC along with inhibition of pThr 308 Akt/PKB within the kidney in nicotine-treated mice. Suppression of Akt/PKB activation by spironolactone was accompanied by upregulation of renal C/EBPβ and amelioration of nicotine-mediated reduction of 11β-HSD2. Addition of nicotine to mouse renal cortical collecting duct M1 cells downregulated 11β-HSD2 and stimulated MR expression, and these effects are likely mediated by activation of Akt coupled inhibition of C/EBPβ. These findings suggest that nicotine-mediated suppression of 11β-HSD2 in the kidney may contribute to the development of nicotine/smoking-induced hypertension through decreasing the intrarenal deactivation of glucocorticoids. Spironolactone may prove useful in protecting against the hypertensive risks of nicotine/smoking.

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

Nedd4-2 regulates the expression of Kir4.1, thiazide-sensitive Na-Cl-cotransporter (NCC) and epithelial Na+ channel (ENaC) in aldosterone-sensitive-distal-nephron (ASDN) and Nedd4-2-deletion causes salt-sensitive hypertension. We now examine whether Nedd4-2-deletion compromises the effect of high salt (HS) on Kir4.1, NCC, ENaC and renal K+ excretion. Immunoblotting showed that HS decreased the expression of Kir4.1, Ca2+-activated-big-conductance K+-channel subunit-a (BKa), ENaCb, ENaCg, total NCC (tNCC) and phosphor-NCC (pNCC at Thr53) in Nedd4lflox/flox mice while these effects were absent in kidney-specific Nedd4-2-knockout (Ks-Nedd4-2 KO) mice. Patch-clamp experiments showed that neither HS nor low salt (LS) had effect on Kir4.1/Kir5.1 currents of the distal convoluted tubule (DCT) in Nedd4-2-deficient mice while we confirmed that HS inhibited and LS increased Kir4.1/Kir5.1 activity in Nedd4lflox/flox mice. Nedd4-2-deletion increased ENaC currents in the ASDN and this increase was more robust in the cortical-collecting-duct (CCD) than in the DCT. Also, HS-induced inhibition of ENaC currents in the ASDN was absent in Nedd4-2-deficient mice. Renal clearance experiments showed that HS intake for two-weeks increased the basal level of renal K+ excretion and caused hypokalemia in Ks-Nedd4-2-KO mice but not in Nedd4lflox/flox mice. In contrast, plasma Na+ concentrations were similar in Nedd4lflox/flox and Ks-Nedd4-2 KO mice on HS. We conclude that Nedd4-2 plays an important role in mediating inhibitory effect of HS on the Kir4.1, ENaC and NCC; and is essential for maintaining a normal renal K+ excretion and plasma K+ ranges during long-term HS.

Unilateral Nephrectomy Stimulates ERK and Is Associated With Enhanced Na Transport

Nephron loss initiates compensatory hemodynamic and cellular effects on the remaining nephrons. Increases in single nephron glomerular filtration rate and tubular flow rate exert higher fluid shear stress (FSS) on tubules. In principal cell (PC) culture models FSS induces ERK, and ERK is implicated in the regulation of transepithelial sodium (Na) transport, as well as, proliferation. Thus, we hypothesize that high tubular flow and FSS mediate ERK activation in the cortical collecting duct (CCD) of solitary kidney which regulates amiloride sensitive Na transport and affects CCD cell number. Immunoblotting of whole kidney protein lysate was performed to determine phospho-ERK (pERK) expression. Next, sham and unilateral nephrectomized mice were stained with anti-pERK antibodies, and dolichos biflorus agglutinin (DBA) to identify PCs with pERK. Murine PCs (mpkCCD) were grown on semi-permeable supports under static, FSS, and FSS with U0126 (a MEK1/2 inhibitor) conditions to measure the effects of FSS and ERK inhibition on amiloride sensitive Na short circuit current (Isc). pERK abundance was greater in kidney lysate of unilateral vs. sham nephrectomies. The total number of cells in CCD and pERK positive PCs increased in nephrectomized mice (9.3 ± 0.4 vs. 6.1 ± 0.2 and 5.1 ± 0.5 vs. 3.6 ± 0.3 cell per CCD nephrectomy vs. sham, respectively, n > 6 per group, p < 0.05). However, Ki67, a marker of proliferation, did not differ by immunoblot or immunohistochemistry in nephrectomy samples at 1 month compared to sham. Next, amiloride sensitive Isc in static mpkCCD cells was 25.3 ± 1.7 μA/cm2 (n = 21), but after exposure to 24 h of FSS the Isc increased to 41.4 ± 2.8 μA/cm2 (n = 22; p < 0.01) and returned to 19.1 ± 2.1 μA/cm2 (n = 18, p < 0.01) upon treatment with U0126. Though FSS did not alter α- or γ-ENaC expression in mpkCCD cells, γ-ENaC was reduced in U0126 treated cells. In conclusion, pERK increases in whole kidney and, specifically, CCD cells after nephrectomy, but pERK was not associated with active proliferation at 1-month post-nephrectomy. In vitro studies suggest high tubular flow induces ERK dependent ENaC Na absorption and may play a critical role in Na balance post-nephrectomy.