Introduction Precise regulation of kidney potassium excretion is essential for maintaining plasma potassium concentration within narrow limits in the face of wide variations in intake. Aldosterone has long been considered the key regulator of tubule potassium secretion. However, increasing evidence suggests that it plays a relatively limited role over a wide range of potassium ingestion. Here we investigated the roles of WNK1 kinase and the mTORC2 regulatory complex in potassium secretion in the distal nephron of mice. Methods We generated knockout (KO) mice where alternatively mTORC2 or the potassium-sensitive kinase, WNK1, were selectively disrupted in the late distal convoluted tubule and early connecting tubule (DCT2/CNTe), an important site of potassium secretion. KO mice were subjected to an acute increase in dietary potassium, after which fluid and electrolyte parameters were measured, and subcellular localization and activity of key ion channels were assessed using immunofluorescence and patch clamp, respectively. Phosphorylation state of mTORC2 targets was determined using Western blot. Results In mice lacking mTORC2 in the DCT2/CNTe, urinary potassium excretion failed to increase adequately in response to high potassium intake, while sodium, chloride and net urine output were excessive, resulting in hyperkalemia and fluid volume wasting. Immunofluorescence and patch clamp revealed that active apical membrane sodium channels (αENaC) in DCT2/CNe failed to increase. Furthermore, KO mice lacking WNK1 in the DCT2/CNTe similarly have diminished potassium excretion and diminished mTORC2-dependent phosphorylation of key targets involved in potassium secretion. Conclusions These findings strongly support the idea that a WNK1-mTORC2-dependent signaling pathway acts selectively in the DCT2/CNTe to regulate potassium secretion in an aldosterone-independent manner.

Disclosure: R.J. Weber: None. E. Takagi: None. J. Demko: None. B. Saha: None. J. Oses-Prieto: None. A. Burlingame: None. D. Pearce: None. Background: Aldosterone is the primary mineralocorticoid in humans and its dysregulation is an important driver of hypertension. Aldosterone has two primary roles – augmenting potassium (K+) excretion and maintenance of blood pressure (BP). Much of this activity occurs in the distal nephron by ENaC translocation to the plasma membrane in response to mineralocorticoid receptor (MR) activation. However, this signaling pathway is modulated by mTORC2-dependent post-translational modifications. mTORC2 is a kinase complex that responds to both local electrolytes and circulating hormones, including angiotensin II (AngII) and insulin, to augment kaliuresis and sodium resorption by unleashing a kinase cascade that ultimately enhances translocation of ENaC and ROMK to the plasma membrane. Our lab has previously demonstrated that mice lacking mTORC2 are aldosterone resistant and has elucidated downstream pathways. However, there has not yet been a global evaluation of the interaction between mTORC2 and aldosterone. The majority of the known signaling changes are mediated by kinases; thus, we chose to perform phosphoproteomics. We hypothesized that by taking an unbiased approach, we may uncover novel mTORC2-dependent phosphorylation events secondary to stimulation by K+ and AngII. Methods: We utilized a transgenic mouse line with an inducible, nephron-specific mTORC2 knockout (TRKO) via a Pax-8 driven Cre and floxed Rictor, a necessary subunit of mTORC2. We used tail cuff blood pressure (BP) measurements and scanned a series of diets with varied K+ content. Wild type (WT) and TRKO mice were exposed to high or low K+ diets or injected subcutaneously with AngII. Subsequently, bilateral nephrectomy was performed, and slices of renal cortex were collected. We performed high throughput phosphoproteomic analysis of these slices using tandem mass tagged labeling. Results and Conclusions: We found that a 3% K+ diet decreased systolic BP in WT mice but paradoxically increased systolic BP in TRKOs. This correlated with a marked increase in serum aldosterone in TRKOs. We used this diet for phosphoproteomic analysis compared to a low K+ diet (0.5%) and compared to stimulation with AngII in both WT and TRKO mice. Phosphorylation of several well-established proteins directly downstream of mTORC2 was reduced in TRKO mice - AKT2, PKC, NEDD4-2, and Rictor itself. However, we also identified several ion channels and GPCRs not previously known to be downstream of mTORC2. Additionally, we used published kinase prediction tools to predict downstream kinases disrupted by mTORC2 deletion. Known downstream kinases were again confirmed and, in addition, we predict a family of CAMK kinases is linked to aldosterone signaling in the distal nephron which we are currently further exploring. Funding: Grants from NIH (T32- DK007418, R01-DK56695) and The James Hilton Manning and Emma Austin Manning Foundation. Presentation: Saturday, July 12, 2025

Background:The kidney proximal tubule is uniquely responsible for reabsorption of filtered glucose and gluconeogenesis (GNG). Insulin stimulates glucose transport and suppresses GNG in the proximal tubule, however, the signaling mechanisms and coordinated regulation of these processes remain poorly understood. The kinase complex mTORC2 is critical for regulation of growth, metabolism, solute transport, and electrolyte homeostasis in response to a wide array of inputs. Here we examined its role in the regulation of renal glucose reabsorption and GNG. Methods:Rictor, an essential component of mTORC2, was knocked out using the Pax8-LC1 system to generate inducible tubule specific Rictor knockout (TRKO) mice. These animals were subjected to fasting, refeeding, and variation in dietary K + . Metabolic parameters including glucose homeostasis and renal function were assessed in balance cages. Kidneys and livers were also harvested for molecular analysis of gluconeogenic enzymes, mTORC2-regulated targets, and plasma membrane glucose transporters. Results:On a normal chow diet, TRKO mice had marked glycosuria despite indistinguishable blood glucose relative to WT controls. Kidney plasma membrane showed lower SGLT2 and SGLT1 in the fed state, supporting reduced renal glucose reabsorption. Additional metabolic testing provided evidence for renal insulin resistance with elevated fasting insulin, impaired pyruvate tolerance, elevated hemoglobin A1c, and increased renal gluconeogenic enzymes in the fasted and fed states. These effects were correlated with reduced downstream phosphorylation of Akt and the transcription factor FOXO4, identifying a novel role of FOXO4 in the kidney. Interestingly, high dietary K + prevented glycosuria and excessive GNG in TRKO mice, despite persistent reduction in mTORC2 substrate phosphorylation. Conclusion:Renal tubule mTORC2 is critical for coordinated regulation of sodium-glucose cotransport by SGLT2 and SGLT1 as well as renal GNG. Dietary K + promotes glucose reabsorption and suppresses GNG independently of insulin signaling and mTORC2, potentially providing an alternative signaling mechanism in states of insulin resistance. SIGNIFICANCE STATEMENT:The kidney contributes to regulation of blood glucose through reabsorption of filtered glucose and gluconeogenesis. This study shows that mTORC2 and dietary potassium coordinate the regulation of sodium-glucose cotransport and glucose production in the kidney via independent mechanisms. New insights into the regulation of these processes in the kidney offer promising implications for diabetes mellitus management and treatment.

Background: The activity of the epithelial sodium ion channel, ENaC, and ENaC-dependent potassium (K+) secretion through ROMK are influenced by both aldosterone-dependent and independent mechanisms in response to dietary potassium (K+). While ENaC activity in the late connecting tubule (CNT) and cortical collecting duct (CCD) depends on aldosterone, it is aldosterone-independent in the late distal convoluted tubule (DCT2) and early CNT (eCNT). Aldosterone-stimulated ENaC activity relies on SGK1, transcriptionally upregulated by aldosterone and subsequently phosphorylated and activated by mTOR complex-2 (mTORC2). Recent evidence suggests that high potassium can rapidly activate mTORC2/SGK1 signaling to stimulate ENaC. In this study, we explore mTORC2’s role in K+ secretion and aldosterone-independent ENaC regulation. Methods: Rictor, a core component of mTORC2, was selectively knocked out (KO'd) in DCT2, CNT and CD by utilizing Calbindin as Cre-driver (CRKO mice) or late CNT and CD, by employing AQP2 as the Cre-driver (ARKO mice). Both wild-type (WT) and KO mice were subjected to a high potassium (HK) diet for short-term (4 hours) or medium-term (48 hours) periods, enabling the monitoring of both the early response and prolonged adaptation to the HK diet. Parameters assessed include urinary and blood electrolyte levels, renal transporter expression, activity and localization, and mTORC2 target phosphorylation. Results: On a normal K+ diet, both KO mice maintained Na+ and K+ balance but CRKO mice had elevated aldosterone levels. After short-term (4-hour) HK intake, CRKO mice had disrupted Na+ and K+ balance, reduced K+ excretion and hyperkalemia. In contrast, ARKO mice maintained Na+ and K+ balance with elevated aldosterone. On a prolonged HK diet (48 hours), CRKO mice had severe hyperkalemia, reduced GFR and significantly elevated aldosterone level. Immunofluorescence studies indicated markedly reduced apical localization of active ENaC in DCT2/eCNT of CRKO mice. Conversely, ENaC apical localization in the late CNT and CCD was mostly unaffected. Phosphorylation of mTORC2 targets involved in ENaC regulation, such as SGK1, and SGK1’s target, Nedd4-2, was also reduced in CRKO mice. ARKO mice maintained ion balance with normal K+ levels but significantly increased aldosterone levels and maintained ENaC apical localization in the late CNT and CCD. Conclusions: The data suggest that high aldosterone can compensate mTORC2 deficiency and maintain ENaC activity in the late CNT and CCD. However, mTORC2 activity is crucial for maintaining ENaC activity in the late DCT and early CNT (aldosterone-independent) and preserving potassium balance during high dietary potassium intake. National Institutes of Health (R01- DK56695), James Hilton Manning and Emma Austin Manning Foundation. 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.

PURPOSE OF REVIEW:Maintenance of plasma K + concentration within a narrow range is critical to all cellular functions. The kidneys are the central organ for K + excretion, and robust renal excretory responses to dietary K + loads are essential for survival. Recent advances in the field have challenged the view that aldosterone is at the center of K + regulation. This review will examine recent findings and propose a new mechanism for regulating K + secretion. RECENT FINDINGS:Local aldosterone-independent response systems in the distal nephron are increasingly recognized as key components of the rapid response to an acute K + load, as well as playing an essential role in sustained responses to increased dietary K + . The master kinase mTOR, best known for its role in mediating the effects of growth factors and insulin on growth and cellular metabolism, is central to these aldosterone-independent responses. Recent studies have shown that mTOR, particularly in the context of the "type 2" complex (mTORC2), is regulated by K + in a cell-autonomous fashion. SUMMARY:New concepts related to cell-autonomous K + signaling and how it interfaces with aldosterone-dependent regulation are emerging. The underlying signaling pathways and effectors of regulated K + secretion, as well as implications for the aldosterone paradox and disease pathogenesis are discussed.

Background: Insulin promotes renal proximal tubule glucose reabsorption and suppresses gluconeogenesis (GNG). The kinase mTORC2 is critical for insulin signaling in multiple cell types. In the kidney tubules, mTORC2 knockout has recently been shown to cause glycosuria via reduced plasma membrane SGLT2 and SGLT1 as well as inappropriately increased renal GNG. Potassium (K+) also plays an important role in systemic glucose homeostasis. However, the overall importance of K+ for the regulation of renal glucose reabsorption and GNG is poorly understood. Methods: Rictor, an essential component of mTORC2, was knocked out using the Pax8-LC1 system to generate inducible tubule specific Rictor knockout (TRKO) mice. To examine the role of dietary K+ on renal glucose homeostasis, TRKO mice and wild-type (WT) littermates were fed with either a normal 0.5% K+ diet or high 3% K+ diet while in balance cages. Kidney tissue was harvested after feeding and processed to study plasma membrane glucose transporters, gluconeogenic enzymes, and mTORC2-regulated targets by western blot and qPCR. Results: On a normal K+ diet, TRKO mice had marked glycosuria despite indistinguishable blood glucose relative to WT controls. Kidney plasma membrane fractions showed decreased SGLT2 and SGLT1, supporting reduced renal glucose reabsorption. Additional metabolic testing provided evidence for renal insulin resistance with elevated fasting insulin, impaired pyruvate tolerance, elevated hemoglobin A1c, and increased renal gluconeogenic enzymes. On a high K+ diet, glycosuria resolved and plasma membrane SGLT2 and SGLT1 were restored in TRKO mice. In addition, TRKO animals fed with a high K+ diet had suppressed gluconeogenic enzymes compared to WT mice. Regardless of dietary K+ content, TRKO animals had similar reductions in phosphorylation of mTORC2 substrates compared to WT controls. Conclusion: Renal tubule mTORC2 is critical for coordinated glucose reabsorption by SGLT2 and SGLT1 as well as suppression of renal GNG. High dietary K+ promotes sodium-glucose cotransport by restoring plasma membrane SGLT2 and SGLT1 in TRKO mice. High dietary K+ also prevents excessive GNG in TRKO mice. High K+ appears to regulate these processes independently of mTORC2 and its downstream substrates, such as Akt. High K+ could potentially provide an alternative signaling mechanism to regulate renal glucose reabsorption and GNG in states of insulin resistance. Grants from the NIH (T32, R01) and The James Hilton Manning and Emma Austin Manning Foundation. 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.

Journal of the American Society of Nephrology 34(11S):p 542, November 2023. | DOI: 10.1681/ASN.20233411S1542b

Significance Statement Rapid renal responses to ingested potassium are essential to prevent hyperkalemia and also play a central role in blood pressure regulation. Although local extracellular K + concentration in kidney tissue is increasingly recognized as an important regulator of K + secretion, the underlying mechanisms that are relevant in vivo remain controversial. To assess the role of the signaling kinase mTOR complex-2 (mTORC2), the authors compared the effects of K + administered by gavage in wild-type mice and knockout mice with kidney tubule-specific inactivation of mTORC2. They found that mTORC2 is rapidly activated to trigger K + secretion and maintain electrolyte homeostasis. Downstream targets of mTORC2 implicated in epithelial sodium channel regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated in wild-type, but not knockout, mice. These findings offer insight into electrolyte physiologic and regulatory mechanisms. Background Increasing evidence implicates the signaling kinase mTOR complex-2 (mTORC2) in rapid renal responses to changes in plasma potassium concentration [K + ]. However, the underlying cellular and molecular mechanisms that are relevant in vivo for these responses remain controversial. Methods We used Cre-Lox–mediated knockout of rapamycin-insensitive companion of TOR (Rictor) to inactivate mTORC2 in kidney tubule cells of mice. In a series of time-course experiments in wild-type and knockout mice, we assessed urinary and blood parameters and renal expression and activity of signaling molecules and transport proteins after a K + load by gavage. Results A K + load rapidly stimulated epithelial sodium channel (ENaC) processing, plasma membrane localization, and activity in wild-type, but not in knockout, mice. Downstream targets of mTORC2 implicated in ENaC regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated in wild-type, but not knockout, mice. We observed differences in urine electrolytes within 60 minutes, and plasma [K + ] was greater in knockout mice within 3 hours of gavage. Renal outer medullary potassium (ROMK) channels were not acutely stimulated in wild-type or knockout mice, nor were phosphorylation of other mTORC2 substrates (PKC and Akt). Conclusions The mTORC2-SGK1-Nedd4-2-ENaC signaling axis is a key mediator of rapid tubule cell responses to increased plasma [K + ] in vivo . The effects of K + on this signaling module are specific, in that other downstream mTORC2 targets, such as PKC and Akt, are not acutely affected, and ROMK and Large-conductance K + (BK) channels are not activated. These findings provide new insight into the signaling network and ion transport systems that underlie renal responses to K + in vivo .

Background: The kinase, mTOR complex-2 (mTORC2), is implicated in the regulation of renal K+ secretion and maintenance of renal sodium and potassium homeostasis. Recent evidences suggest differential regulation of epithelial sodium channel, ENaC, and ENaC-dependent K+ secretion along the distal nephron under various levels of dietary K perturbations. However, the role of mTORC2 in this nephron segment-specific control of K+ secretion is not fully understood yet. Here we have generated multiple mouse models with renal tubular segment specific mTORC2 KO, and studied its physiological consequences as well as the ability of these mice to respond to dietary potassium load to assess the role of mTORC2 in K+ secretion. Methods: mTORC2 was inactivated in either the entire nephron or selectively in different segments of the distal nephron using segment-specific Cre-Lox-mediated knockout (KO) of Rictor (a core component of mTORC2). WT and KO mice were subjected to a high K diet for 2 days. Urinary and blood parameters, and renal expression and activity of signaling molecules and transport proteins were assessed. Results: At 48 h on a high K+ diet, WT mice demonstrated increased activated ENaC and ROMK in the plasma membrane with normal plasma [K+] and moderately elevated aldosterone level. Downstream targets of mTORC2 implicated in ENaC regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated. At 24 h on HK diet, the entire tubule-specific Rictor knockout (TRKO) mice had significantly higher urinary Na/K ratio, greater UNa*V (net urine Na excretion) and reduced UK*V than WT, consistent with a functional defect in ENaC. By 48h, TRKO mice displayed severe pathophysiologic changes, including marked weight loss, severe hyperkalemia and volume depletion along with significantly higher aldosterone level and BUN than WT. Activation of ENaC and ROMK and phosphorylation of both SGK1 and Nedd4-2 were also severely impaired in TRKO mice. Knocking out of Rictor at the distal part of the DCT and at CNT, resulted in nearly similar pathophysiologic responses to the HK load, which included weight loss, reduced food intake, and urine output, significantly higher urinary Na/K ratio, BUN and plasma aldosterone level, hyperkalemia and abnormal urinary K+ excretion, as compared to the WT mice. Interestingly, deletion of Rictor in more distal part of the CNT and CCD, resulted in only milder abnormal phenotype than the entire nephron- or DCT2/CNT-specific Rictor KO mice on a high K diet. Conclusions: Overall, the data suggest that mTORC2 activity in the late DCT and CNT plays a major role in adjusting renal K+ secretion in response to an increased dietary K+ load. National Institutes of Health (R01- DK56695), James Hilton Manning and Emma Austin Manning Foundation 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.

Journal of the American Society of Nephrology 34(11S):p 35, November 2023. | DOI: 10.1681/ASN.20233411S135a

Methods: Rictor is a critical component of the mTORC2 complex. Tubule-specific Rictor knockout (TRKO) mice were made using doxycycline inducible Pax8-Cre Rictor-flox. Male and female TRKO mice and their control littermates were used for all experiments. Tolerance tests were performed with intraperitoneal glucose (1g/kg), insulin (0.75U/kg), and pyruvate (2g/kg) after overnight fasts. Refeeding at the time of sacrifice to induced insulin signaling; mice were fasted for 18 hours then refed 4 hours. Whole kidney relative mRNA was measured via RT-PCR. Kidney plasma membrane and cytosolic proteins were separated using the BioVision Plasma Membrane Protein Extraction Kit, and protein abundance was measured with western blotting. Results: There were no differences in serum glucose during refeeding experiments, glucose tolerance tests, or insulin tolerance tests between TRKO and control mice at any timepoints (n=9 per group). However, the mean ± standard error of mean (SEM) urine glucose concentration was 472.5±181.2mg/dL in TRKO mice compared to 30.8±5.0mg/dL in control animals during refeeding (n=9 per group; p<0.01). Serum glucose was higher in TRKO mice compared to controls (n=7 per group) after giving the gluconeogenic substrate pyruvate at 60 (234.4±15.0 vs 189.4±9.1mg/dL; p<0.01) and 90 minutes (194.3±7.4 vs 148.9±5.9mg/dL; p<0.01). TRKO mice (n=8) also had elevated hemoglobin A1c (HbA1c) compared to control mice (n=6 per group) after 3 months on a 0.5% K+ diet (6.00±0.21% vs 5.23±0.11%; p<0.01). Refed TRKO mice kidneys compared to controls had significantly higher relative mRNA of PEPCK (3.74±0.76 vs 1.18±0.15AU; p<0.01) and G6Pase (4.24±1.02 vs 1.37±0.29AU; p<0.01) (n=15 per group). Refed TRKO mice kidneys compared to controls also had elevated protein abundance of PEPCK (0.54±0.05 vs 0.28±0.03AU; p<0.001) but no difference in G6Pase (0.62±0.03 vs 0.49±0.05AU; ns). Kidneys from refed TRKO and control mice showed no differences in relative mRNA of SGLT2, SGLT1, or GLUT2 (n=15 per group). Kidneys from TRKO and control mice also showed no difference in plasma membrane protein abundance of SGLT2 and GLUT2 (n=8 per group). Conclusion: This study demonstrates that insulin signaling through mTORC2 is critical for suppression of renal GNG and complete reabsorption of glucose. Increased serum glucose during pyruvate tolerance testing, increased HbA1c, increased gluconeogenic gene transcription (i.e., PEPCK, G6Pase), and increased PEPCK protein abundance all support increased renal GNG in TRKO mice. Glycosuria was present in TRKO mice despite no difference in serum glucose between TRKO and control mice, suggesting that mTORC2 is important for both renal GNG and glucose reabsorption. Future studies will use TRKO mice to further evaluate glucose transporters and elucidate the mechanism of glycosuria. NIDDK Support, T32DK007219; Diacomp, 5U24DK115255-040 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.

mTORC2 is a multi-subunit kinase complex that is central to multiple essential signaling pathways. Two core subunits, Rictor and mSin1 distinguish it from its relative, mTORC1 and support context-dependent phosphorylation of its substrates. mTORC2 structures have been determined previously, however, important questions remain, particularly regarding structural determinants of substrate specificity and context dependent activities. We used cryo-EM to obtain high resolution structures of the human mTORC2 apo-complex, as well as structures in the presence of substrates, Akt and SGK1. Specific predictions suggested by substrate-induced structural changes were tested in functional assays. First, side chain interactions between Rictor and mTOR that prevent recruitment of mTORC1 substrates and confer resistance to the mTORC1 inhibitor rapamycin were visualized for the first time in the apo-state, demonstrating the steric occlusion that prevents mTORC2 interaction with mTORC1 substrates and rapamycin. Also in the apo-state, mSin1 was seen to form extensive contacts with Rictor, including a pair of short α-helices nestled between two Rictor helical repeat clusters, followed by an extended strand, which makes multiple weak contacts with Rictor helical cluster 1. In co-complex structures, SGK1, but not Akt, markedly altered the conformation of the mSin1 N-terminal extended strand, disrupting multiple weak interactions while inducing a large rotation of mSin1/Arg-83, which comes to interact with a negative patch within Rictor. Mutation of Arg-83 to Ala selectively disrupted mTORC2 dependent phosphorylation of SGK1 but not of Akt, supporting context-dependent substrate selection. These findings provide new structural and functional insights into mTORC2 specificity and context-dependent activities.

ABSTRACT Mammalian (or mechanistic) target of rapamycin complex 2 (mTORC2) is a kinase complex that targets predominantly Akt family proteins, SGK1 and protein kinase C (PKC), and has well-characterized roles in mediating hormone and growth factor effects on a wide array of cellular processes. Recent evidence suggests that mTORC2 is also directly stimulated in renal tubule cells by increased extracellular K+ concentration, leading to activation of the Na+ channel, ENaC, and increasing the electrical driving force for K+ secretion. We identify here a signaling mechanism for this local effect of K+. We show that an increase in extracellular [K+] leads to a rise in intracellular chloride (Cl−), which stimulates a previously unknown scaffolding activity of the protein ‘with no lysine-1’ (WNK1) kinase. WNK1 interacts selectively with SGK1 and recruits it to mTORC2, resulting in enhanced SGK1 phosphorylation and SGK1-dependent activation of ENaC. This scaffolding effect of WNK1 is independent of its own kinase activity and does not cause a generalized stimulation of mTORC2 kinase activity. These findings establish a novel WNK1-dependent regulatory mechanism that harnesses mTORC2 kinase activity selectively toward SGK1 to control epithelial ion transport and electrolyte homeostasis.

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.