The mineralocorticoid hormone aldosterone plays a crucial role in the control of Na+ and K+ balance, blood volume, and arterial blood pressure, by acting in the aldosterone-sensitive distal nephron (ASDN) and stimulating a complex transcriptional, translational, and cellular program. Because the complexity of the aldosterone response is still not fully appreciated, we aimed at identifying new elements in this pathway. Here, we demonstrate that the expression of the proto-oncogene PIM3 (Proviral Integration Site of Moloney Murine Leukemia Virus 3), a serine/threonine kinase belonging to the calcium/calmodulin-regulated group of kinases, is stimulated by aldosterone in vitro (mCCDcl1 cells), ex vivo (mouse kidney slices), and in vivo in mice. Characterizing a germline Pim3-/- mouse model, we found that these mice have an upregulated Renin-Angiotensin-Aldosterone System (RAAS), with high circulating aldosterone and plasma renin activity levels on both standard or Na+ -deficient diet. Surprisingly, we did not observe any obvious salt-losing phenotype in Pim3 KO mice as shown by normal blood pressure, plasma and urinary electrolytes, as well as unchanged expression levels of the major Na+ transport proteins. These observations suggest that the potential effects of the loss of the Pim3 gene are physiologically compensated. Indeed, the 2 other family members of the PIM kinase family, PIM1 and PIM2 are upregulated in the kidney of Pim3-/- mice, and may therefore be involved in such compensation. In conclusion, our data demonstrate that the PIM3 kinase is a novel aldosterone-induced protein, but its precise role in aldosterone-dependent renal homeostasis remains to be determined.

Since many years now, scientists believe that a great credit for the blood pressure regulation goes to the kidneys, as their inability in maintaining salt balance and water homeostasis can result in the development of hypertension, the most common disease in the human population. Aldosterone is the key hormone in the control of sodium balance, blood volume and blood pressure, acting in the aldosterone‐sensitive distal nephron (ASDN) and stimulating a complex transcriptional, translational and cellular program.We have carried out a gene expression profiling in a mouse cortical collecting duct cell model (mpkCCD), stimulated or not by aldosterone and identified the PIM‐3 Ser/Thr kinase as a novel aldosterone‐induced protein. PIM kinases (PIM‐1, ‐2 and ‐3), and in particular PIM‐3, are overexpressed in different tumors and are known to be regulators of apoptosis, cell proliferation and cell metabolism. PIM‐3, the PIM kinase family member most expressed in the kidney, has similar substrate specificities to other Ser/Thr kinases (e.g. SGK1, PKB), and despite its circadian expression in several organs including the kidney, its renal role is largely unknown.Here we studied a possible new role of PIM‐3 in the regulation of renal salt and water handling, both in vitro (mCCDcl1 cells) and in vivo (PIM‐3 KO mice). In mCCD cells, we confirmed that PIM‐3 expression is stimulated by aldosterone in a dose dependent manner and observed that shRNA‐based suppression of PIM‐3 reduces the aldosterone‐mediated sodium currents as well as the activation of the aldosterone‐mineralocorticoid receptor pathway. In PIM‐3 KO mice, we found decreased body weight, increased circulating renin activity and elevated plasma aldosterone levels compared to control littermates. Moreover, challenging WT and KO mice with standard, low Na+ or high‐Na+ diet, we observed a tendency of impaired expression of the most important electrolytes transporters in case of PIM‐3 deficiency, leading to a different sodium and water handling between WT and KO mice.In conclusion, our data suggest a possible novel function of the PIM‐3 kinase and its potentially important role in the control of sodium and water homeostasis to finely regulate blood pressure.

The thiazide-sensitive NaCl cotransporter (NCC) is important for renal salt handling and blood-pressure homeostasis. The canonical NCC-activating pathway consists of With-No-Lysine (WNK) kinases and their downstream effector kinases SPAK and OSR1, which phosphorylate NCC directly. The upstream mechanisms that connect physiological stimuli to this system remain obscure. Here, we have shown that aldosterone activates SPAK/OSR1 via WNK1. We identified 2 alternatively spliced exons embedded within a proline-rich region of WNK1 that contain PY motifs, which bind the E3 ubiquitin ligase NEDD4-2. PY motif-containing WNK1 isoforms were expressed in human kidney, and these isoforms were efficiently degraded by the ubiquitin proteasome system, an effect reversed by the aldosterone-induced kinase SGK1. In gene-edited cells, WNK1 deficiency negated regulatory effects of NEDD4-2 and SGK1 on NCC, suggesting that WNK1 mediates aldosterone-dependent activity of the WNK/SPAK/OSR1 pathway. Aldosterone infusion increased proline-rich WNK1 isoform abundance in WT mice but did not alter WNK1 abundance in hypertensive Nedd4-2 KO mice, which exhibit high baseline WNK1 and SPAK/OSR1 activity toward NCC. Conversely, hypotensive Sgk1 KO mice exhibited low WNK1 expression and activity. Together, our findings indicate that the proline-rich exons are modular cassettes that convert WNK1 into a NEDD4-2 substrate, thereby linking aldosterone and other NEDD4-2-suppressing antinatriuretic hormones to NCC phosphorylation status.

Background/Aims: Thiazolidinediones (TZDs, like rosiglitazone (RGZ)) are peroxisome proliferator-activated receptor γ (PPARγ) agonists used to treat type 2 diabetes. Clinical limitations include TZD-induced fluid retention and body weight (BW) increase, which are inhibited by amiloride, an epithelial-sodium channel (ENaC) blocker. RGZ-induced fluid retention is maintained in mice with αENaC knockdown in the collecting duct (CD). Since ENaC in the connecting tubule (CNT) rather than in CD appears to be critical for normal NaCl retention, we aimed to further explore the role of ENaC in CNT in RGZ-induced fluid retention. Methods: Mice with conditional inactivation of αENaC in both CNT and CD were used (αENaC lox/lox AQP2-Cre; ‘αENaC-CNT/CD-KO') and compared with littermate controls (αENaC lox/lox mice; ‘WT'). BW was monitored and total body water (TBW) and extracellular fluid volume (ECF) were determined by bioelectrical impedance spectroscopy (BIS) before and after RGZ (320 mg/kg diet for 10 days). Results: On regular NaCl diet, αENaC-CNT/CD-KO had normal BW, TBW, ECF, hematocrit, and plasma Na+, K+, and creatinine, associated with an increase in plasma aldosterone compared with WT. Challenging αENaC-CNT/CD-KO with a low NaCl diet unmasked impaired NaCl and K homeostasis, consistent with effective knockdown of αENaC. In WT, RGZ increased BW (+6.1%), TBW (+8.4%) and ECF (+10%), consistent with fluid retention. These changes were significantly attenuated in αENaC-CNT/CD-KO (+3.4, 1.3, and 4.3%). Conclusion: Together with the previous studies, the current results are consistent with a role of αENaC in CNT in RGZ-induced fluid retention, which dovetails with the physiological relevance of ENaC in this segment.

Ubiquitylation is crucial for regulating numerous cellular functions. In the kidney, ubiquitylation regulates the epithelial Na+channel ENaC. The importance of this process is highlighted in Liddle's syndrome, where mutations interfere with ENaC ubiquitylation, resulting in constitutive Na+reabsorption and hypertension. There is emerging evidence that NCC, involved in hypertensive diseases, is also regulated by ubiquitylation. Here, we discuss the current knowledge and recent findings in this field.

The E3 ubiquitin ligase NEDD4-2 (encoded by the Nedd4L gene) regulates the amiloride-sensitive epithelial Na+ channel (ENaC/SCNN1) to mediate Na+ homeostasis. Mutations in the human β/γENaC subunits that block NEDD4-2 binding or constitutive ablation of exons 6-8 of Nedd4L in mice both result in salt-sensitive hypertension and elevated ENaC activity (Liddle syndrome). To determine the role of renal tubular NEDD4-2 in adult mice, we generated tetracycline-inducible, nephron-specific Nedd4L KO mice. Under standard and high-Na+ diets, conditional KO mice displayed decreased plasma aldosterone but normal Na+/K+ balance. Under a high-Na+ diet, KO mice exhibited hypercalciuria and increased blood pressure, which were reversed by thiazide treatment. Protein expression of βENaC, γENaC, the renal outer medullary K+ channel (ROMK), and total and phosphorylated thiazide-sensitive Na+Cl- cotransporter (NCC) levels were increased in KO kidneys. Unexpectedly, Scnn1a mRNA, which encodes the αENaC subunit, was reduced and proteolytic cleavage of αENaC decreased. Taken together, these results demonstrate that loss of NEDD4-2 in adult renal tubules causes a new form of mild, salt-sensitive hypertension without hyperkalemia that is characterized by upregulation of NCC, elevation of β/γENaC, but not αENaC, and a normal Na+/K+ balance maintained by downregulation of ENaC activity and upregulation of ROMK.

Constitutive Nedd4‐2 knockout (KO) mice show Na+ retention and salt‐sensitive hypertension. To determine the role of renal Nedd4‐2 in adult mice, we generated inducible renal tubule‐specific Nedd4‐2 KO mice (Nedd4‐2Pax8/LC1) using a combination of the Tet‐On and Cre‐loxP systems. Under standard and high‐Na+ diets, KO displayed decreased plasma aldosterone but normal Na+/K+ balance. Under high Na+ diet, KO mice showed hypercalciuria and hypertension, both treated by thiazides. NCC expression and phosphorylation were increased in the KO. Interestingly, KO showed increased intracellular β‐ and γENaC abundance, but decreased α‐ and γENaC proteolytic cleavage and decreased αENaC mRNA and protein expression, suggesting down‐regulated ENaC. In contrast, ROMK protein expression and cell surface localization were increased in the distal convoluted tubules and connecting tubules of KO, in keeping with the absence of hyperkalemia. Thus, in adult mice, loss of renal Nedd4‐2 causes a pseudohypoaldosteronism type II‐like syndrome with NCC up‐regulation leading to salt‐sensitive hypertension and hypercalciuria, suggesting that Nedd4‐2 primarily targets NCC. The accompanying hypoaldosteronism with down‐regulation of ENaC‐mediated Na+ reabsorption together with an up‐regulation of K+ secretion via ROMK likely contributes to the compensated phenotype of the KO mice and explains the maintained Na+/K+ balance.

NCC and ENaC are inhibited by the E3 ubiquitin ligase Nedd4‐2 (JASN, 2011) and elimination of two canonical SGK1 phosphorylation sites on Nedd4‐2 (S222A and S328A), renders the Nedd4‐2 mediated inhibition of NCC and ENaC irrecoverable by SGK1. In search of new regulatory proteins, we found that the NH2 terminal domain of WNK3 interacts with Nedd4‐2. In Xenopus laevis oocytes inhibition of NCC by Nedd4‐2 is completely reversed in the presence of WNK3. In oocytes injected with NCC alone, WNK3 increases NCC activity by 264 ± 22 %, while in the presence of NCC and Nedd4‐2, the WNK3‐induced increase was of 353 ± 44 %. Additionally, in contrast to SGK1, WNK3 further activates NCC in the presence of double mutant Nedd4‐2 S222A/S328A to 501 ± 48 %. Thus, in the presence of wild type or mutant Nedd4‐2, WNK3 is a more potent stimulator of NCC. In the absence or presence of Nedd4‐2, NCC activation by WNK3 is prevented by elimination of WNK3 histidine 1497 and glycine 1508 that have been shown to be critical for interaction between WNKs (TBiochem J, 2011). In order to test for non‐specific WNK interactions we tested WNK4 and saw no difference in the Nedd4‐2 mediated inhibition of NCC. Thus, we propose that WNK3 is a regulator of the Nedd4‐2‐NCC pathway.

Aldosterone enhances renal K + excretion via ROMK channels and stimulates apical epithelial Na + channels (ENaC) to provide the electrochemical gradient for K + secretion. Here we studied MR flox /AQP2 cre knockout mice (KO) with targeted inactivation of the mineralocorticoid receptor (MR) in the renal collecting system (CS). The mice tolerate K + loading (5% K + , 2d) without major illness, but show slightly reduced food intake indicating K + avoidance. Immunoblotting and immunohistochemistry reveal a K + diet‐induced increase in ROMK protein abundance and apical localization that is similar for the CS of control and KO mice. However, KO mice show less K + diet‐induced α‐ and γENaC proteolytic activation and the apical translocation of ENaC is restricted to the remaining MR‐positive late DCT. KO mice have a reduced abundance and phosphorylation of the NaCl‐cotransporter (NCC) in the DCT. This NCC down‐regulation is observed already under basal conditions, but becomes even more pronounced on 5% K + diet. Thus, the K + diet‐induced activation of ENaC, but not of ROMK, appears to depend on MR. In KO mice, the loss of the MR‐dependent activation of ENaC in the CS might be compensated, at least in part, by down‐regulation of NCC, which increases Na + delivery to cells with remaining ENaC activity, and hence improves the electrochemical gradient for K + secretion.

The mechanisms through which aldosterone promotes apparently opposite effects like salt reabsorption and K+secretion remain poorly understood. The identification, localization, and physiological analysis of ion transport systems in distal nephron have revealed an intricate network of interactions between several players, revealing the complex mechanism behind the aldosterone paradox. We review the mechanisms involved in differential regulation of ion transport that allow the fine tuning of salt and K+balance.

Regulation of renal Na(+) transport is essential for controlling blood pressure, as well as Na(+) and K(+) homeostasis. Aldosterone stimulates Na(+) reabsorption by the Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) and by the epithelial Na(+) channel (ENaC) in the late DCT, connecting tubule, and collecting duct. Aldosterone increases ENaC expression by inhibiting the channel's ubiquitylation and degradation; aldosterone promotes serum-glucocorticoid-regulated kinase SGK1-mediated phosphorylation of the ubiquitin-protein ligase Nedd4-2 on serine 328, which prevents the Nedd4-2/ENaC interaction. It is important to note that aldosterone increases NCC protein expression by an unknown post-translational mechanism. Here, we present evidence that Nedd4-2 coimmunoprecipitated with NCC and stimulated NCC ubiquitylation at the surface of transfected HEK293 cells. In Xenopus laevis oocytes, coexpression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreased NCC activity and surface expression. SGK1 prevented this inhibition in a kinase-dependent manner. Furthermore, deficiency of Nedd4-2 in the renal tubules of mice and in cultured mDCT(15) cells upregulated NCC. In contrast to ENaC, Nedd4-2-mediated inhibition of NCC did not require the PY-like motif of NCC. Moreover, the mutation of Nedd4-2 at either serine 328 or 222 did not affect SGK1 action, and mutation at both sites enhanced Nedd4-2 activity and abolished SGK1-dependent inhibition. Taken together, these results suggest that aldosterone modulates NCC protein expression via a pathway involving SGK1 and Nedd4-2 and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.

To investigate the role of the mineralocorticoid receptor (MR) in renal ENaC-mediated sodium reabsorption, we have previously used the Cre-loxP system to generate mice with principal-cell specific MR ablation (MRAQP2Cremice). To restrict Cre expression to principal cells, we have used the regulatory elements of the mouse aquaporin-2 (AQP2) gene to drive Cre expression. Since AQP2 is already expressed during renal development, MR ablation took place long before the analysis performed at the adult stage. To investigate whether the early onset of MR ablation affected the adult renal sodium handling, we developed a transgene expressing the CreERT2fusion protein under control of the regulatory elements of the AQP2 gene (AQP2CreERT2). Immunofluorescence revealed MR loss in the collecting duct (CD) and late connecting tubule after induction of MR ablation by tamoxifen in MRAQP2CreERT2mice that equals the MR loss in MRAQP2Cremice. Surprisingly, tamoxifen-independent MR loss is observed in CDs of noninduced mutants without affecting circulating aldosterone levels. Under a low-salt diet, the induced ablation of MR at the adult stage recapitulates the renal sodium wasting observed in mice with constitutive early-onset MR ablation. The AQP2CreERT2transgene is a new tool for investigating in vivo the function of genes downstream of MR in renal ENaC-mediated sodium reabsorption by inducible somatic gene inactivation.

Mutations in α, β, or γ subunits of the epithelial sodium channel (ENaC) can downregulate ENaC activity and cause a severe salt-losing syndrome with hyperkalemia and metabolic acidosis, designated pseudohypoaldosteronism type 1 in humans. In contrast, mice with selective inactivation of αENaC in the collecting duct (CD) maintain sodium and potassium balance, suggesting that the late distal convoluted tubule (DCT2) and/or the connecting tubule (CNT) participates in sodium homeostasis. To investigate the relative importance of ENaC-mediated sodium absorption in the CNT, we used Cre-lox technology to generate mice lacking αENaC in the aquaporin 2-expressing CNT and CD. Western blot analysis of microdissected cortical CD (CCD) and CNT revealed absence of αENaC in the CCD and weak αENaC expression in the CNT. These mice exhibited a significantly higher urinary sodium excretion, a lower urine osmolality, and an increased urine volume compared with control mice. Furthermore, serum sodium was lower and potassium levels were higher in the genetically modified mice. With dietary sodium restriction, these mice experienced significant weight loss, increased urinary sodium excretion, and hyperkalemia. Plasma aldosterone levels were significantly elevated under both standard and sodium-restricted diets. In summary, αENaC expression within the CNT/CD is crucial for sodium and potassium homeostasis and causes signs and symptoms of pseudohypoaldosteronism type 1 if missing.

Germline inactivation of the mineralocorticoid receptor (MR) gene in mice results in postnatal lethality as a result of massive loss of sodium and water. The knockout mice show impaired epithelial sodium channel (ENaC) activity in kidney and colon. For determination of the role of renal MR in aldosterone-driven ENaC-mediated sodium reabsorption, mice with principal cell MR deficiency were generated using the Cre-loxP system. For driving Cre recombinase expression in principal cells, the regulatory elements of the mouse aquaporin 2 (AQP2) gene were used. Mutant mice (MR(AQP2Cre)) were obtained by crossing AQP2Cre mice with mice that carried a conditional MR allele. Under standard diet, MR(AQP2Cre) mice develop normally and exhibit unaltered renal sodium excretion but show strongly elevated aldosterone levels. Increased renal sodium and water excretion, resulting in continuous loss of body weight, occur under low-sodium diet. Immunofluorescence revealed that the loss of MR and apical ENaC staining is restricted to principal cells of the collecting duct (CD) and late connecting tubule (CNT) and that MR is crucial for ENaC trafficking to the apical membrane. These results demonstrate that inactivation of MR in CD and late CNT can be compensated under standard diet but no longer when sodium supply is limited. Because the mutant mice show preserved renal ENaC activity, this study provides evidence that the late distal convoluted tubule and early CNT can compensate to a large extent deficient ENaC-mediated sodium reabsorption in late CNT and CD.