Potassium Activates mTORC2-dependent SGK1 Phosphorylation to Stimulate Epithelial Sodium Channel: Role in Rapid Renal Responses to Dietary Potassium.

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 .

WNK1 is a chloride-stimulated scaffold that regulates mTORC2 activity and ion transport.

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.

Nephrin and Podocin mRNA Detection in Urine Sediment of Dogs with Chronic Kidney Disease: Preliminary Observations.

Introduction: Dogs with chronic kidney disease (CKD) may have alterations in the glomerular filtration barrier, including podocyte loss. Detection of podocyte mRNA in urine could be useful for assessing podocyturia in dogs with kidney disease. The objective of this study was to evaluate the presence of nephrin mRNA (NPHS1) and podocin mRNA (NPHS2) in urine sediments of dogs with naturally occurring CKD and healthy dogs. Material and Methods: Twenty-four dogs, 14 with CKD and 10 as healthy controls, underwent clinical evaluation. The dogs with CKD were divided into two groups, according to the International Renal Interest Society criteria: stage 1 or 2 CKD (n = 5) and stage 3 or 4 CKD (n = 9). Urine was collected by catheterisation or free catch and RNA isolation from the urine sediments was optimised using glycogen as a co-precipitant. Detection of NPHS1 and NPHS2 in the sediment samples was performed using quantitative real-time PCR. Results: Both types of mRNA were detected in samples from all groups, but the percentages of detection were higher in the group of dogs with stage 1 or 2 CKD and lower in the group of dogs with stage 3 or 4 disease. Conclusion: Physiological podocyturia was observed in healthy dogs, and the results suggest differential podocyturia in dogs with CKD, according to the stage of the disease, i.e. an increase in podocyturia in dogs at stage 1 or 2 and a reduction in podocyturia in dogs at stage 3 or 4.

Imbalance in the ratio between mineralocorticoid and glucocorticoid receptors and neurodegeneration in the dentate gyrus of aged dogs.

Background and Aim: Cortisol binds to mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) found in the hippocampus. The balanced expression of these receptors is essential to neuronal survival as MR and GR activations have antiapoptotic and proapoptotic effects, respectively. Given the aging changes in dogs' dentate gyrus (DG) and the possible involvement of cortisol receptors in this process, this study aimed to evaluate the expression of MR and GR and neuronal degeneration in this hippocampal region of aged dogs. Materials and Methods: This study included cadaveric histologic hippocampus sections from six dogs aged 10 years and older (AG group) and 12 young/adult dogs aged up to 8 years (YAd group). Nissl staining and immunohistochemistry were performed to identify cells and investigate MR and GR expression, respectively. Furthermore, fluorescent labeling (fluoro-Jade B) was used to detect degenerating neurons. Results: The AG group's polymorphic layer of the DG had a lower cell count (16%) and more degenerating neurons than the YAd group. In addition to these cellular changes, the AG group had lower MR immunoreactivity and MR-to-GR ratio. Furthermore, the lowest MR expression was associated with neuronal degeneration in the polymorphic layer of the DG of dogs. Conclusion: An imbalance in the MR-to-GR ratio was observed in the polymorphic layer of the DG of aged dogs, along with lower MR expression and a greater number of degenerating neurons. These findings have clinical implications for understanding the decline in hippocampal memory formation associated with cognitive changes in aged dogs.

Signaling pathways involved in the rapid biphasic effect of aldosterone on Na+/H+ exchanger in rat proximal tubule cells.

The receptors and signaling pathways for nongenomic effects of aldosterone (Aldo) on the proximal Na+/H+ exchanger are still unknown; therefore, the aim of this study was to investigate the mineralocorticoid receptor (MR) and/or glucocorticoid receptor (GR) participation in rapid Aldo effects on NHE1 (basolateral Na+/H+ exchanger isoform) and cytosolic calcium concentration ([Ca2+]i). In addition, phospholipase C (PLC), protein kinase C (PKC), and mitogen-activated protein kinase kinase (MEK) involvement in signaling pathways of such effects was evaluated, using immortalized proximal tubule cells of rat (IRPTC) as an experimental model. MR and GR expression was investigated using reverse transcription polymerase chain reaction and immunoblotting. The intracellular pH recovery rate (after acid loading) and [Ca2+]i were determined by the probes BCECF-AM and FURA 2-AM, respectively. Aldo (10-12 M) promoted a moderate increase in [Ca2+]i and stimulation of NHE1, whereas Aldo (10-6 M) greatly increased the [Ca2+]i, but inhibited the NHE1. BAPTA-AM (a calcium chelator), GR antagonism and inhibition of PLC, PKC and MEK pathway abolished the biphasic and dose-dependent effect of Aldo on NHE1 and decreased the [Ca2+]i; whereas MR do not appear to participate in this rapid signaling in IRPTC cells. The reduction of GR content, by gene silencing, abolished the Aldo effect on NHE1, in low concentration, confirming the importance of this receptor in the rapid modulation of proximal sodium and hydrogen transports.

The effects of angiotensin-(1-7) on the exchanger NHE3 and on [Ca2+]i in the proximal tubules of spontaneously hypertensive rats.

The acute effects of angiotensin-1-7 [ANG-(1-7)] on the reabsorptive bicarbonate flow (J[Formula: see text]) were evaluated using stationary microperfusion in vivo in the proximal tubules of spontaneously hypertensive rats (SHR) and their normotensive controls, Wistar-Kyoto (WKY) rats, using a microelectrode sensitive to H+ In WKY rats, the control J[Formula: see text] was 2.40 ± 0.10 nmol·cm-2·s-1 (n = 120); losartan (10-7 M) or A779 (10-6 M, a specific Mas antagonist), alone or in combination with losartan, decreased the J[Formula: see text] ANG-(1-7) had biphasic effects on J[Formula: see text]: at 10-9 M, it inhibited, and at 10-6, it stimulated the flow. S3226 [10-6 M, a specific Na+-H+ exchanger 3 (NHE3) antagonist] decreased J[Formula: see text] and changed the stimulatory effect of ANG-(1-7) to an inhibitory one but did not alter the inhibitory action of ANG-(1-7). In SHR, the control J[Formula: see text] was 2.04 ± 0.13 nmol·cm-2·s-1 (n = 56), and A779 and/or losartan reduced the flow. ANG-(1-7) at 10-9 M increased J[Formula: see text], and ANG-(1-7) at 10-6 M reduced it. The effects of A779, losartan, and S3226 on the J[Formula: see text] were similar to those found in WKY rats, which indicated that in SHR, the ANG-(1-7) action on the NHE3 was via Mas and ANG II type 1. The cytosolic calcium in the WKY or SHR rats was ~100 nM and was increased by ANG-(1-7) at 10-9 or 10-6 M. In hypertensive animals, a high plasma level of ANG-(1-7) inhibited NHE3 in the proximal tubule, which mitigated the hypertension caused by the high plasma level of ANG II.

Direct action of aldosterone on bicarbonate reabsorption in in vivo cortical proximal tubule.

The direct action of aldosterone (10(-12) M) on net bicarbonate reabsorption (J(HCO(3)(-))) was evaluated by stationary microperfusion of an in vivo middle proximal tubule (S2) of rat kidney, using H ion-sensitive microelectrodes. Aldosterone in luminally perfused tubules caused a significant increase in J(HCO(3)(-)) from a mean control value of 2.84 +/- 0.08 [49/19 (n degrees of measurements/n degrees of tubules)] to 4.20 +/- 0.15 nmol.cm(-2).s(-1) (58/10). Aldosterone perfused into peritubular capillaries also increased J(HCO(3)(-)), compared with basal levels during intact capillary perfusion with blood. In addition, in isolated perfused tubules aldosterone causes a transient increase of cytosolic free calcium ([Ca(2+)](i)), monitored fluorometrically. In the presence of ethanol (in similar concentration used to prepare the hormonal solution), spironolactone (10(-6) M, a mineralocorticoid receptor antagonist), actinomycin D (10(-6) M, an inhibitor of gene transcription), or cycloheximide (40 mM, an inhibitor of protein synthesis), the J(HCO(3)(-)) and the [Ca(2+)](i) were not different from the control value; these drugs also did not prevent the stimulatory effect of aldosterone on J(HCO(3)(-)) and on [Ca(2+)](i). However, in the presence of RU 486 alone [10(-6) M, a classic glucocorticoid receptor (GR) antagonist], a significant decrease on J(HCO(3)(-)) and on [Ca(2+)](i) was observed; this antagonist also inhibited the stimulatory effect of aldosterone on J(HCO(3)(-)) and on [Ca(2+)](i). These studies indicate that luminal or peritubular aldosterone (10(-12) M) has a direct nongenomic stimulatory effect on J(HCO(3)(-)) and on [Ca(2+)](i) in proximal tubule and that probably GR participates in this process. The data also indicate that endogenous aldosterone stimulates J(HCO(3)(-)) in middle proximal tubule.