mTOR signaling in renal ion transport

The mammalian target of rapamycin (mTOR) signaling pathway is crucial in maintaining cell growth and metabolism. The mTOR protein kinase constitutes the catalytic subunit of two multimeric protein complexes called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). As such, this pathway is indispensable for many organs, including the kidney. Since its discovery, mTOR has been associated with major renal disorders such as acute kidney injury, chronic kidney disease, and polycystic kidney disease. On top of that, emerging studies using pharmacological interventions and genetic disease models have unveiled mTOR role in renal tubular ion handling. Along the tubule, mTORC1 and mTORC2 subunits are ubiquitously expressed at mRNA level. Nevertheless, at the protein level, current studies suggest that a tubular segment‐specific balance between mTORC1 and mTORC2 exists. In the proximal tubule, mTORC1 regulates nutrients transports through various transporters located in this segment. On the other hand, in the thick ascending limb of the loop of Henle, both complexes play a role in regulating NKCC2 expression and activity. Lastly, in the principal cells of the collecting duct, mTORC2 determines Na+ reabsorption and K+ excretion by regulating of SGK1 activation. Altogether, these studies establish the relevance of the mTOR signaling pathway in the pathophysiology of tubular solute transport. Despite extensive studies on the effectors of mTOR, the upstream activators of mTOR signaling remain elusive in most nephron segments. Further understanding of the role of growth factor signaling and nutrient sensing is essential to establish the exact role of mTOR in kidney physiology.


| INTRODUCTION
The mechanistic target of rapamycin (mTOR) pathway is a central regulator of cell metabolism and proliferation. Upon activation by nutrients and/or growth factors, mTOR coordinates the synthesis and recycling of essential biomolecules such as proteins, lipids, and nucleotides sustaining cell growth ( Figure 1). 1 Consequently, aberrant mTOR signaling has been associated with several diseases including cancers, neurodegenerative diseases, and metabolic diseases. 1 In the kidney, mTOR has been identified as a central player in several kidney diseases such as acute kidney injury (AKI) and chronic kidney disease (CKD) (reviewed in 2,3 ). In AKI, mTOR activity supports renal tissue maintenance and prevents cell death after acute injury. After kidney transplantation, usage of mTOR inhibitors delays graft renal function in both pre-and clinical studies but does not prevent recovery. [4][5][6][7] In CKD, a consensus of how mTOR plays a role in disease progression is less defined compared to AKI. Studies using nephrectomy rat models treated with everolimus, mTORC1 inhibitor, have shown both ameliorative and deleterious effects of inhibiting mTOR in association with CKD. [8][9][10] In the last decade, the identification of novel Mendelian disorders and the development of animal knockout models have uncovered a role of mTOR signaling beyond cell metabolism and proliferation. The mTOR complex has been implicated in regulating ion transport along the kidney tubule. For instance, renal transplant patients prescribed with sirolimus as an immunosuppressive agent often experience side effects such as hypomagnesemia and hypermagnesuria. Sirolimus, also known as rapamycin, forms a complex with the 12-kilo Dalton FK506-binding protein (FKBP12) to inhibit mTORC1. [11][12][13] The mechanisms by which mTOR activity is important for the handling of renal ion transport are debatable. Therefore, this review aims to provide an overview of mTOR signaling along the kidney tubule and to identify the current knowledge gaps and perspectives. F I G U R E 1 mTOR signaling in the cell. mTOR constitutes the kinetic subunit of two multimeric protein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to stimulation by various nutrients and stress while mTORC2 is largely activated through insulin/PI3K signaling. mTORC1 activation sustains cell growth, proliferation, and metabolism, and mTORC2 activation plays a role in cytoskeleton dynamics, sodium and potassium homeostasis, and apoptosis. 12-kDa FK506-binding protein (FKBP12), 5′ adenosine monophosphateactivated protein kinase (AMPK), DEP domain containing mTOR interacting protein (DEPTOR), extracellular signal-regulated kinase (Erk), insulin receptor substrate (IRS), mammalian lethal with SEC13 protein 8 (mLST8), mechanistic target of rapamycin (mTOR), mTOR complex 1 (mTORC1), mTOR complex 2 (mTORC2), phosphatidylinositol-3,4,5-triphosphate (PIP 3 ), phosphatidylinositol-4, 5-bisphosphate (PIP 2 ), phosphoinositide-3-kinase (PI3K), protein kinase B (Akt), pyruvate dehydrogenase kinase 1 (PDK1), rapamycin-insensitive companion of mTOR (Rictor), RAS homologue enriched in brain (Rheb), Ras-related GTP binding proteins (Rag) A-D, regulatory-associated protein of mTOR (Raptor), ribosomal s6 kinase (RSK), stress, activated protein kinase-interacting protein 1 (Sin1), tuberous sclerosis complex 1 (TSC1), tuberous sclerosis complex 1 (TSC2).

| mTOR SIGNALING
mTOR is an evolutionary conserved serine-threonine kinase, which forms the catalytic subunit of two functionally distinct multiprotein complexes: (1) mTOR complex 1 (mTORC1) is the rapamycin-sensitive protein complex characterized by the expression of an adapter protein called RAPTOR and (2) mTORC2 is largely insensitive to rapamycin and contains the adapter protein RICTOR (Figure 1). [11][12][13][14][15][16][17][18][19] The two protein complexes are activated by different upstream signals; mTORC1 is sensitive to nutrients, growth factors, and stress, while mTORC2 is activated by phosphoinositide 3 kinases (PI3Ks) and protein kinase B (Akt) signaling in response to insulin and growth factor signaling ( Figure 1). A more in-depth evaluation of the mTOR complexes subunits, including components upstream and downstream of mTOR, has been reviewed elsewhere. 1,20,21 Due to its ability to integrate upstream metabolic stimuli, mTORC1 is often called as a nutrient sensor ( Figure 1). Nutrients in the forms of glucose, lipids, and amino acids trigger mTORC1 activation while energy depletion and oxidative stress inhibit this process. Moreover, growth factors can induce nutrients uptake and regulate mTORC1 activity this way. Furthermore, growth factors such as insulin and IGF-1 could directly stimulate mTORC1 via the PI3K that enhances Akt signaling. This in turn dampens the inhibition of the guanosine triphosphate (GTP)-ase RAS homologue enriched in brain (Rheb) by the tuberous sclerosis complex (TSC) 1 and 2. 22,23 Rheb is a wellknown allosteric activator of the mTORC1 that resides on the lysosomal surface. 24,25 Amino acid sensing plays an important part in mTORC1 recruitment to the lysosomal surface via the Ras-related GTP binding proteins (Rag GTPases). 26,27 In mammals, four members of the Rag GTPases are known: RagA-D. These Rag GTPases are tethered to the lysosomal surface through a pentameric protein complex called Ragulator ( Figure 2). 28,29 In the presence of amino acids, RagA or RagB binds GTP and forms a heterodimer with RagC or RagD that binds GDP. [30][31][32] In this conformation and nucleotide-binding state, RagA/B and RagC/D heterodimeric complexes recruit mTORC1 to the lysosomal surface. 33,34 The nucleotide binding of the Rag GTPases depends on the activity of the vacuolar H + -ATPase (V-ATPase), which promotes the guanosine-nucleotide exchange factor (GEF) property of the Ragulator on RagA/B ( Figure 2). 28,35 Recently, a study by Amemiya et al. revealed that amino acid also stimulates Ca 2+ intracellular uptake and calmodulin activity, and through the TSC2/Rheb axis, this process activates mTORC1 activity independent of Rag GTPases. 36 Ultimately, mTORC1 integrates these stimuli to exert its main role in controlling cell growth and metabolism.
To sustain cell growth and metabolism, mTORC1 promotes anabolic activities such as nucleotide, protein, and lipid synthesis via S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and inhibits catabolic activities such as autophagy via transcription factor EB (TFEB), Unc-51-like autophagy activating kinase 1 (ULK1), and autophagy-related 13 (Atg13). [37][38][39][40][41][42][43] TFEB is also known as the master regulator of the lysosomal biogenesis via transcription regulation of the Coordinated Lysosomal Expression and Regulation (CLEAR) network. [44][45][46] Extensive recent studies into mTORC1 have changed the view on the way this protein complex behaves; instead of an unselective manner of switching on and off its downstream targets, different cues (e.g., specific Rag GTPases dimerization) can determine the substrate specificity of mTORC1 in a selective manner, 47,48 indicating that delicate balance of upstream signals is needed to drive mTORC1 activity.
Unlike vast knowledge of mTORC1, far less is known about the regulation and effectors of mTORC2 owing to the lack of mTORC2-specific inhibitors. Based on most recent studies, mTORC2 localizes to different subcellular compartments and is differentially regulated based on its localization (discussed in more detail elsewhere 49 ). The most well-characterized activator of mTORC2 at the plasma membrane is insulin through the PI3K pathway ( Figure 1). Upon stimulation by growth factors, PI3K phosphorylates phosphatidylinositol-4, 5-bisphosphate (PIP 2 ) at the plasma membrane, pro-F I G U R E 2 Regulation of Rag GTPases at the lysosome. RagA-D sense the intra-and extracellular amino acids via a number of upstream regulators described in detail elsewhere. 150 This multimeric protein complex is tethered to the lysosomal surface through interaction with the Ragulator complex, which also serves as the guanosine-nucleotide exchange factor (GEF) of RagA/B. Ragulator activation is regulated by the V-ATPase. Ras-related GTP binding proteins (Rag) A-D, vacuolar H + -ATPase (V-ATPase).

Ragulator
RagA/B RagC/D GTP GDP Lysosome Amino acids V-ATPase ducing phosphatidylinositol-3,4,5-triphosphate (PIP 3 ), which co-recruits Akt and mTORC2 to the plasma membrane and directly releases autoinhibition of mTORC2 to stimulate Akt phosphorylation. 50,51 mTORC2 is known to phosphorylate Akt at the Ser473 site, and together with PDK1-dependent phosphorylation of Akt at Thr308, strengthen Akt activation. 52 53 On the other hand, Angiotensin II drives mTORC2 activity to specifically phosphorylate the Ser442 site of serum and glucocorticoid-induced protein kinase 1 (SGK1) at a perinuclear compartment and not of Akt. 54 Other than Akt and SGK1, a few other mTORC2 targets have been identified; these are mainly members of the A, G, and C (AGC) kinases such as protein kinase Cα (PKCα) and SGK3. 16,17,55,56 Through these substrates, the role of mTORC2 in regulating cell survival, cytoskeleton dynamics, and sodium and potassium homeostasis has been established. In the following sections, we will highlight recent findings on the roles of mTOR pathway in regulating ion transport along the kidney tubule and thus, potential involvement in tubular disorders ( Figure 3).

| mTOR IN THE PROXIMAL TUBULE
The proximal tubule (PT) is considered the workhorse of the kidney, accounting for 65% of the tubular transport of Na + , K + , H 2 O, and Ca 2+ . Moreover, the segment is solely responsible for the reabsorption of glucose, amino acids, and small proteins. As these transport processes are highly energy demanding, the PT is rich in mitochondria. PT cells depend on beta-oxidation for energy metabolism and are contributing to body glucose production (±25% of the total) by gluconeogenesis. As all these metabolic and transport processes are taking place simultaneously, the PT cells are highly dynamic. PT cells are required to respond rapidly to nutrient-stress or low oxygen conditions, to ensure sufficient reabsorption of ions and solutes. Consequently, nutrient sensors mTOR and AMPK have been implicated in many proximal tubular reabsorption pathways.
Extensive characterization of transgenic mouse models in which mTOR complexes subunits were deleted has been instrumental in our understanding of mTOR in the PT. Whole tubular-specific mTORC1-mTORC2 KO mice, generated using the Pax8-Cre and Raptor fl/fl /Rictor fl/fl system, demonstrate a Fanconi-like syndrome of proteinuria, hyperphosphaturia, and glucosuria, suggesting a PT involvement. In particular, mTORC1 was demonstrated to be essential for endocytosis of proteins. In mTORC1 KO mice the formation of clathrin-coated vesicles was reduced, despite preserved Megalin-Cubulin expression.
In recent years, several large clinical trials have demonstrated the protective role of SGLT2 inhibitors for renal function decline in patients with and without T2DM. 58,59 Suppression of mTORC1 signaling has an important role on the renal protective effects of SGLT2 inhibitors. As inhibition of SGLT2 increases glucose excretion in urine and activates AMPK, mTORC1 is suppressed, and autophagy is restored. 60 Additionally, SGLT2 inhibitor treatment induced elevated ketone body levels, and mTORC1 suppression may be involved in the SGLT2 inhibitor-mediated renoprotective effect. 60 Sirolimus-induced phosphaturia is common in renal transplant patients, and therefore, several groups have examined the role of mTORC1 signaling in regulation PT phosphate reabsorption. Indeed, sirolimus-treated mice display phosphaturia, which is in line with clinical observations. 61,62 Initial studies demonstrating that mTORC1 activated NaPi-IIa and NaPi-IIb in Xenopus laevis oocytes 62,63 could not be reproduced in mice. The expression and activity of major phosphate transporters (NaPi-IIa, NaPi-IIc, and Pit-2) were unaltered in all reported studies. 57,61,62 The pathophysiological mechanism explaining sirolimusinduced phosphaturia is therefore still not understood.
Although mTORC1 has been the prime focus of study in the PT, the insulin-sensitive mTORC2 may also regulate the expression of ion transporters. Insulin-stimulated sodium-bicarbonate co-transporter 1 (NBCe1) activity is dependent on the Akt2/mTORC2, independent of mTORC1 function. 64 However, the expression levels of mTORC2-specific proteins in the PT are much lower than mTORC1 subunits, and mTORC2 KO mice have a relatively mild phenotype. 57,65 Consequently, the physiological relevance of mTORC2 in the PT remains unclear, because most studies have been executed in cell lines or isolated tubules.
Altogether these studies demonstrate that mTORC1 acts as the main nutrient sensor and mTORC2 responds primarily to insulin signaling. Although these pathways may simultaneously be activated, e.g., in diabetic nephropathy, mTORC1 and mTORC2 have independent downstream targets and physiological function in the PT.

ASCENDING LIMB
The thick ascending limb of Henle's loop (TAL) section of the nephron is important in the countercurrent multiplication, in which energy is used to generate an osmotic gradient along the loop of Henle. The activity of the Na + -K + -2Cl − co-transporter 2 (NKCC2) generates a voltage gradient that is important to drive the passive transport of divalent cations such as Ca 2+ and Mg 2+ from the pro-urine into the blood compartment through Claudin16/19-positive tight junctions. Due to the large energy demand in this segment, the mTOR pathway plays an important role in regulating electrolyte transport. Our current understanding on the role of mTOR in the TAL is driven by a large number of recent studies on genetic diseases, genetic animal models, and pharmacological interventions. [66][67][68][69][70] Mice with kidney-specific Raptor deletion (Raptor fl/fl *K-spCre) experienced polyuria and polydipsia accompanied with elevated urinary excretion of Ca 2+ and Mg 2+ . 66 F I G U R E 3 Role of mTOR complexes along the nephron. In the different nephron sections, mTOR signaling has been shown to regulate nutrients and ion transport machineries. In the PT, mTORC1 modulates clathrin-coated vesicles formation, glucose, and amino acid transport via SGLT2, B 0 AT1, y + LAT1-4F2hc, and NBC1. Additionally, mTORC1 affects phosphate resorption in the PT although the molecular mechanisms are not yet clear. In the TAL, both mTORC1 and 2 play a role in regulating Na + transport via NKCC2. mTORC1 affects NKCC2 expression through an unknown mechanism. Upstream of mTORC1, RagD might regulate Mg 2+ and Ca 2+ transport through mTORC1-mediated intervention on Claudin16/19 tight junction proteins or via NKCC2. Furthermore, mTORC2 alters NKCC2 phosphorylation by direct intervention on OSR1 phosphorylation independent of the WNKs. Upstream of mTORC2, oxidative stress in form of H 2 O 2 increases mTORC2 activity although the mechanisms are still elusive. In the principal cells of the CD, mTORC2 plays a role in dictating Na + and K + transport. mTORC2 regulates SGK1 upon stimulation via the insulin/PI3K pathway and therefore, ENaC and ROMK activity. Moreover, K + and Cl − levels might affect mTORC2 regulation via WNK1. 4F2 cell-surface antigen heavy chain (4F2hc), 5′ adenosine monophosphate-activated protein kinase (AMPK), chloride channel Kb (ClC-Kb), collecting duct (CD), electrogenic sodium/ bicarbonate co-transporter 1 (NBCe1), epithelial sodium channel (ENaC), hydrogen peroxide (H 2 O 2 ), inwardly rectifying potassium channel 4.1 (Kir4.1), inwardly rectifying potassium channel 5.1 (Kir5.1), mineralocorticoid receptor (MR), mTOR complex 1 (mTORC1), mTOR complex 2 (mTORC2), Na + -K + -2Cl − co-transporter 2 (NKCC2), oxidative stress-responsive kinase (OSR1), phosphoinositide-3-kinase (PI3K), principal cell (PC), proximal tubule (PT), Ras-related GTP binding proteins (Rag) A-D, renal outer medullary K + channel (ROMK), serum and glucocorticoid-induced protein kinase 1 (SGK1), sodium/glucose co-transporter 2 (SGLT2), system B 0 neutral amino acid transporter 1 (B 0 AT1), thick ascending limb (TAL), with-no-lysine kinase 1 (WNK1), with-no-lysine kinases (WNKs), Y + L amino acid transporter 1 (y + LAT1). Response to furosemide was blunted in these animals accompanied by loss of mTORC1 activity and reduced expressions of NKCC2 and NCC, indicating that expression of Raptor gene affects the countercurrent multiplication in the TAL. 66 Interestingly, Raptor and Rictor double knockout (Raptor fl/fl *Rictor fl/fl *KspCre) leads to lethality in the mice compared to Raptor deletion, suggesting that mTORC1 and 2 work together to maintain normal tubular homeostasis. 66 In another animal study, sirolimus treatment in Wistar rats induced lower K + and Mg 2+ serum levels and greater urinary excretion of K + and Mg 2+ . 67 Similar to the Raptor fl/fl *KspCre mice, the sirolimus-treated rats experienced polyuria and polydipsia. Importantly, sirolimus treatment resulted in lower NKCC2 protein expression while cortical TRPM6 protein expression was increased, suggesting a potential compensatory attempt for the decreased passive Mg 2+ transport in the TAL. 67 Of note: the Ksp-Cre promoter only targets the TAL, distal convoluted tubule (DCT), connecting tubule (CNT), and collecting duct (CD) and demonstrates varied efficacies in these segments. 71 The role of mTORC2 in TAL is less comprehended compared to mTORC1; however, several studies have hinted at the potential involvement of mTORC2 in regulating NKCC2 via kinase activities. A number of kinases are involved in NKCC2 phosphorylation including the Ste20-and SPS1-related proline and alanine-rich kinase (SPAK) and its homolog oxidative stress-responsive kinase (OSR1), 72,73 protein kinase A (PKA), 74 and AMP-activated kinase (AMPK). 75 In response to tonicity changes, NKCC2 phosphorylation is activated via SPAK and OSR1 and withno-lysine kinases (WNKs). 76 A previous study has demonstrated that OSR1 is also directly activated by mTORC2 and not via WNK1 using HeLa cells. Importantly, knockdown of an important component of mTORC2, Sin1, resulted in lower endogenous NKCC1 activity in these HeLa cells. It has not been studied yet if NKCC2 activity would also be disrupted in Sin1 knockdown cells. 77 In addition to kinase activity, mTORC2 might regulate NKCC2 activity under oxidative stress. Oxidative stress in the form of hydrogen peroxide (H 2 O 2 ) infusion in SD rats resulted in higher mTORC2 activation (phosphorylation of AKT) followed by increased intracellular Na + concentration and phosphorylation of NKCC2 and Na-K-ATPase. 68 These effects were partially blocked by the addition of PP242, an inhibitor of both mTORC1 and 2. 68 This natriuretic property of PP242 has been previously observed in Dahl salt-sensitive (SS) rats. 78 Of note, as PP242 does not only inhibit mTORC2 but also inhibits mTORC1, 79 it cannot be concluded that mTORC2 activity alone is responsible for mediating H 2 O 2 anti-natriuretic effects. Therefore, it would be important to further investigate if mTORC2-specific inhibition or downregulation would result in the same outcome. Nevertheless, whether H 2 O 2 /mTORC2-induced NKCC2 activation is through the kinase activity described above or whether it is via a different pathway is still unexplored.

PT
Upstream regulators of the mTOR pathway have also been implicated in ion transport regulation. We have recently described a novel disease called Autosomal Dominant Kidney Hypomagnesemia RRAGD (ADKH-RRAGD) in which the patients suffered from three key symptoms: hypomagnesemia, nephrocalcinosis, and dilated cardiomyopathy. The kidney phenotypes in these patients are reminiscent of the symptoms seen in Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC) patients where Claudin 16/19 are mutated. [80][81][82][83] Using in vitro model, it was discovered that the patient RRAGD mutations led to hyperactivation of mTORC1 as reflected by increased S6K phosphorylation. Therefore, it is possible that mTOR also regulates the passive transport of Ca 2+ and Mg 2+ in the TAL. Whether the TAL segment is the main culprit of this kidney tubulopathy and whether RagD regulates this process indirectly through mTORC1 or NKCC2 or directly on the Claudins are still unexplored.
Taken together, the abovementioned studies demonstrated that mTORC1 and mTORC2 maintain tubular function in the TAL by the regulation of NKCC2 activity. Nevertheless, the molecular mechanisms of how mTOR regulates NKCC2 expression are still elusive. Future research should aim to untangle the direct regulatory mechanism of NKCC2 transcription/translation and metabolic effects.

| mTOR IN THE DISTAL CONVOLUTED TUBULE
The DCT is an important segment in the regulation of Na + , K + , and Mg 2+ excretion. [84][85][86] Transcellular reabsorption of Na + and Mg 2+ via the thiazide-sensitive Na + -Cl −co-transporter (NCC) and the transient receptor potential melastatin type 6 (TRPM6) cation channel, respectively, is tightly regulated by paracrine and endocrine action. Using the previously described mouse models, the role of mTORC1 and mTORC2 in the DCT was characterized. 66,71 Raptor fl/fl *KspCre mice display a reduced expression of NCC. 66 Although this suggests an activating role of mTORC1 on NCC function, mice treated with mTOR inhibitor sirolimus demonstrated normal NCC expression. 87 Although the cause for this discrepancy remains unclear, it may be dependent on the background of the mouse strains or duration of mTOR inhibition. The effects of mTORC1 on TRPM6 expression are conflicting. Whereas Da Silva et al. reported increased TRPM6 protein expression in sirolimus-treated rats, Ikari et al. demonstrated reduced TRPM6 mRNA expression in rapamycintreated NRK-52e cells. 67,88 Consequently, whether the cause of rapamycin-induced hypomagnesemia can be attributed to reduced Mg 2+ reabsorption in the TAL or in the DCT remains unclear.
Insulin signaling has been demonstrated to activate NCC and TRPM6 activity in the DCT. 89,90 Upon insulin receptor activation, an intracellular signaling cascade including PI3K, Akt, and mTORC2 results in the phosphorylation of NCC and TRPM6. Indeed, mTOR was identified to phosphorylate NCC at positions T58 and S124. 89,91 Nevertheless, Rictor fl/fl *KspCre mice display a normal thiazide-response, suggesting normal NCC function. 71

| mTOR IN THE COLLECTING DUCT
The CD determines the final urinary composition, by fine-tuning Na + , K + , and H 2 O reabsorption. In the CD, the principal cells (PCs) express the epithelial sodium channel (ENaC) and renal outer medullary K + channel (ROMK), which exchange Na + and K + under the control of aldosterone. In recent years, high K + -induced inhibition of NCC in the DCT was demonstrated to indirectly increase K + secretion in the CD. In this mechanism termed "the Potassium Switch," the Na + delivery to the CD is a key determinant of K + secretion, in addition to RAAS activation. 86 A seminal work by the groups of David Pearce and Tobias Huber has established mTORC2 signaling as an essential regulator of Na + reabsorption and K + secretion in the CD. Initially described in mpkCCD cells, mTORC2 was demonstrated to regulate SGK1, as its phosphorylation could be inhibited by mTOR inhibitor PP242 while mTORC1-inhibitor rapamycin could not inhibit insulininduced SGK1 phosphorylation. 92 As SGK1 is an essential regulator of ENaC and ROMK activity, subsequent studies examined the effects of mTORC2 on these ion channels in the CD. In line with the in vitro findings, mTOR inhibition using PP242 and AZD8055 caused reduced SGK1 phosphorylation and substantial natriuresis in mice. 93 Indeed, Grahammer et al. demonstrated impaired SGK1 phosphorylation in Rictor fl/fl *KspCre mice resulting in hyperkalemia and impaired kaliuresis in response to high K + diets. 71 Whether ENaC or ROMK is the primary target of mTORC2-SGK1 signaling is under debate. 71,93 However, as the activity of ENaC and ROMK is closely linked, both channels will probably be activated by the signaling pathway. In agreement with these studies, Chen et al. showed that deletion of mTOR from the PCs in mTOR fl/fl *Aqp2Cre mice CD resulted in reduction of all three ENaC subunits at the protein level and attenuated the ability to maintain Na + homeostasis in response to low NaCl diet and benzamil injection. 94 Although deletion of mTOR would influence both mTORC1 and mTORC2 activity, and the current study did not investigate which protein complex mainly played a role in their mice model, SGK1 phosphorylation was decreased in the cortex of KO mice along with increased ubiquitination of α-ENaC, in line with the current mTORC2-SGK1 signaling pathway consensus. 94 Aside from SGK1, SGK3 also modulates Na + transport in the CD through ENaC activity indirectly in a similar manner as SGK1. 95 It is currently not known if mTOR signaling is responsible for SGK3-mediated activation of these transporters. However, similar to SGK1, as SGK3 is also activated through the PDK1-mTORC2 phosphorylation pathway, 96,97 it would be interesting to investigate if mTORC2 is responsible to stimulate SGK3 actions on the ion transporters.
Several physiological stimuli may initiate mTORC2 signaling in the CD. Whereas classic insulin stimulation was used to study PI3K-mTORC2 activation in the CD in initial studies, recent findings demonstrate that the K + and Cl − may directly activate mTORC2 as well. In mpk-CCD cells and native mouse CCD cells, K + itself activates mTORC2-SGK1 signaling to stimulate ENaC and enhance K + excretion. 98 This process seems to be dependent on the modulation of mTORC2 activity by WNK1, which acts as a K + and Cl − sensor. 98 Indeed, an extracellular K + leads to a rise in intracellular chloride Cl − , which allows WNK1 to selectively bind SGK1 and recruits mTORC2. 99 Extracellular K + levels thereby directly regulate K + secretion, independent of RAAS activation and Na + delivery to the CD. 98 Thus, mTORC2 signaling provides an alternative mechanism to induce K + secretion, in addition to the potassium switch.
Although mTORC2 is considered the most relevant mTOR complex for CD ion transport, several groups have examined mTORC1 function as well. An increased renal perfusion pressure (RPP) was demonstrated to activate mTORC1 signaling and infiltration of immune cells into the kidney. Interestingly, this was associated with increased AQP2 and unchanged α-ENaC expression. 100 In contrast, CD-specific TSC1 KO mice, which are considered as a model for mTORC1 hyperactivation, have dedifferentiated CD cells and reduced AQP2, α-ENaC, and ROMK1 expression. 101 In both models, the effects were reversible by rapamycin, including a rose in α-ENaC expressions. 100,101 Altogether, these studies suggest that the role of mTORC1 in the CD may be dependent on the activation of additional pathways, which may include downstream targets and/or interaction with immune cells.

| EXPRESSION OF mTOR COMPLEX COMPONENTS ALONG THE TUBULE
Based on our analysis of current studies in the sections above, we identified that mTORC1 and mTORC2 seem to function in a tubular segment-specific manner. To visualize if this correlates with their expression patterns, using the RNA sequencing data by Chen et al. 65 we have generated a heat map of mRNA expression of the mTOR complex subunits along the mouse renal tubule (Figure 4). While Mtor is relatively enriched in the proximal tubule (PT) segment 2 and 3, the rest of the subunits (i.e., Deptor, Mapkap1, Mlst8, Rictor, and Rptor) are ubiquitously expressed along all segments with a relatively high expression in the descending thin limb of the loop of Henle. Therefore, we observed no immediate correlation between the transcriptomes of mTOR subunits and the functional assessment at protein levels in the renal tubule.

| mTOR IN KIDNEY DISEASE
Given the evident roles of mTOR in maintaining renal tubular ion transport, dysregulation of this signaling pathway gives rise to tubular disorders. In the previous parts of this review, segement-specific kidney disease have been discussed. In this last section, we will discuss how mTOR is involved in common kidney diseases that affect multiple nephron segments.

| Autosomal dominant polycystic kidney disease
Autosomal dominant polycystic kidney disease (ADPKD) is the most common nephropathy characterized by enlarged kidneys due to the progressive development of fluid-filled renal cysts that deforms the urine-concentrating ability of the kidneys. This results in polyuria early in the disease development and could eventually lead to loss of renal function and end-stage renal disease. Around 85% of ADPKD is caused by mutations in the PKD1 gene, which encodes for polycystin-1 (PC1), and 15% by the PKD2 gene encoding for polycystin-2 (PC2). 102 Interestingly, other than cyst formation, ciliary dysfunction is a common hallmark seen in different ADPKD forms; defects in primary cilia result in F I G U R E 4 mRNA expression levels of mTOR complex subunits along the renal tubule. mRNA expression levels of the subunits of mTORC1 (i.e., Deptor, Mlst8, Mtor, and Rptor) and of mTORC2 (i.e., Deptor, Mapkap1, Mlst8, Mtor, and Rictor) obtained from RNA sequencing data of microdissected mouse kidney tubule segments (https://esbl.nhlbi.nih.gov/MRECA/ Nephr on/). 65   The primary cilia are non-motile cilia that play a key role in developmental signaling functions, chemosensing, and mechanosensing. As both PC1 and PC2 are localized to the primary cilium, the polycystins are crucial to maintaining tubular epithelium integrity through the function of primary cilia. 106,107 Loss of polycystins results in enhanced cell proliferation, cell size, apoptosis, and remodeling of the epithelial membrane. 108 Given the important role of the polycystins in maintaining primary ciliary functions, and that primary cilia are crucial for epithelial membrane integrity, loss of PKD1 and PKD2 could lead to aberrant signaling pathways that contribute to the pathogenesis of ADPKD, including mTOR. This hypothesis was further corroborated by cases of individuals with infantile PKD in which a large deletion often occurs on chromosome 16p where PKD1 and TSC2 genes lie adjacent to one another. 109 This suggests that PC1 and Tuberin, the product of TSC2, work together to modulate cytogenesis. Indeed, the cytoplasmic tail of PC1 has been shown to interact with tuberin. 110 Tuberin can form a complex with Rheb to suppress mTORC1 activity. Thus, loss of PKD1 resulted in the overactivation of mTOR pathway in the cyst-lining epithelial cells in patient and mouse models, promoting cyst formation. 110 Moreover, increased ERK activity has been observed upon loss of PKD1, resulting in increased mTORC1 activity. 108 Loss of cilia also leads to inappropriate mTORC1 activation via the inhibition of the basal body compartment that reduces AMPK-mediated mTORC1 inhibition, resulting in increased cell size. 111 Due to clear evidence of mTORC1 involvement in ADPKD pathogenesis, the usage of mTORC1 inhibitors for ADPKD treatment has been tested in many preclinical and clinical trials. In Han:SPRD rat model of ADPKD, usage of sirolimus and rapamycin has been shown to slow down disease progression. 112,113 Follow-up rodent studies in Pkd1 and Pkd2 mouse models treated with these inhibitors also demonstrated success in decreasing cyst growth and improving the loss of kidney function ultimately. 114,115 Shillingford et al. have reported that rapamycin treatment in ADPKD transplant-recipient patients resulted in shrinkage of enlarged polycystic kidney size. 110 However, the results of clinical trials have been less successful compared to animal models. Despite reports of small changes in kidney size with everolimus compared to placebo, no improvement in renal functions was noted. 116 In another smaller clinical trial, sirolimus treatments managed to arrest kidney enlargement; however, renal function was not ameliorated. 117 These discrepant clinical trial results may be explained by the short period of treatment as well as the heterogeneous patient population with diverse adverse effects. Of note, the disparity between preclinical and clinical trials might be owed to the difference in life span, genetic background, and metabolism in the context of ADPKD development in rodents and in patients. 118 This further complicates the translation of treatment from animal models to clinical trials and therefore, warrants more in-depth future research to address this gap.

| Diabetic kidney disease
Diabetic kidney disease (DKD) frequently appears as a complication of diabetes type 1 and 2; about one-third of type 1 diabetes and 40% of type 2 diabetes patients develop DKD. DKD is a progressive disease that is characterized by a decline in kidney function and structural changes in the kidney. Risk factors of DKD include both susceptibility factors such as race, age, and sex and progression factors such as diet, hypertension, hyperglycaemia, and obesity. 119 More recent studies have unveiled more factors contributing to DKD progression, including alterations of the classical signaling pathways in the renal tubule. In the PT, nutrient overload in the context of DKD affects mTORC1 activity. mTORC1 is activated potentially due to reduced AMPK activity and increased insulin action. 120 mTORC1 hyperactivation is associated with a metabolic switch that impairs lipolysis and gluconeogenesis. 60,121 Consequently, autophagosome formation is inhibited and lipid deposition in lysosomes is increased. 122 Continuous overactivation of mTORC1 and inhibition of autophagy, therefore, make the PT incapable of dealing with cellular stress and breakdown of waste products. DKD-induced mTORC1 activation is therefore associated with tubulo-interstitial fibrosis and loss of renal function. Rapamycin treatment has been demonstrated to restore autophagy and reduce DKD-associated fibrosis. 123,124 Additionally, Forsythiaside targets oxidative stress and mTOR signaling pathway and may thereby protect against DKD nephropathy. 125

| Nephropathic cystinosis
Nephropathic cystinosis is a rare autosomal recessive disease belonging to the family of lysosomal storage disorders. It is caused by mutations in the lysosomal cysteine transporter, cystinosin, encoded by the CTNS gene. [126][127][128] Thus, this disease is characterized by the accumulation of cysteine in all organs, and treatment mainly involves oral supplementation of cysteamine, a cysteine-depleting agent. 129,130 Cystinosis occurs in three clinical forms: infantile, which is the most severe, and frequent, juvenile, and adult cystinosis. In infantile cystinosis (OMIM 219800), renal Fanconi syndrome (RFS) is often the first manifestation presenting in the first year of life. RFS refers to a generalized PT dysfunction that leads to polyuria and urinary loss of low-molecular-weight (LMW) protein, glucose, amino acids, and phosphate. RFS in infantile cystinosis is not resolved by cysteamine supplementation, suggesting that cysteine accumulation is not the main cause of RFS in this disease.
Interestingly, cystinosin has recently been demonstrated to play a role in regulating mTORC1 activity as the absence of cystinosin in cells leads to downregulation of mTORC1 activity. 131,132 Moreover, recent in vitro studies showed that cystinosin interacts with various components of the mTORC1 pathway such as the V-ATPases, Ragulator, and Rag GTPases, and loss of cystinosin resulted in TFEB downregulation. 132,133 Similar to the clinical findings, cysteamine treatment failed to rescue these defects, suggesting that cystinosin might have an additional function than an amino acids transporter and potentially, as an upstream regulator of the mTORC1 signaling pathway. 132,133 The molecular mechanisms that lead to the clinical manifestations are currently still elusive. Nonetheless, as deletion of mTORC1 components in mice led to RFS-like symptoms as we have described in the PT section above, 57 it would be interesting to study if RFS in infantile cystinosis is caused by mTORC1 defects due to lack of cystinosin.

| Salt-induced hypertension
Large blood pressure changes in response to a highsalt diet (defined by >5 g per day by World Health Organization 134 ) and an impaired ability of individuals to excrete sodium and water through urine are characteristics of salt-sensitive hypertension. 135 In these individuals, the difference in blood pressure between salt-loaded and salt-depleted states is >10 mm Hg. 136 This condition has been reported to be determined by many factors including genetic background, race and ethnicity, age, body mass, diet, and other underlying diseases. 135 Other than hypertension, renal inflammation due to the infiltration of immune cells that are causing renal injury constitutes a clinical manifestation of salt-induced hypertension. 137,138 In SS rats, renal mTORC1 activity was upregulated after being given a high-salt (4.0% NaCl) diet for 3 days. Moreover, treatment with rapamycin decreased saltinduced hypertension and protected SS rats against renal injury. Additionally, chronic infusion of H 2 O 2 into the renal interstitium of salt-resistant SD rats results in increased mTORC1 activity. 139 High-salt diet in SS rats is known to elevate the production of ROS such as O 2 − and H 2 O 2 , a process mediated by NOX4. 68,[140][141][142] Taken together, these findings suggest that a high-salt diet-induced production of ROS might mediate mTORC1 activation in SS rats. Indeed, deletion of Nox4 in SS rats decreased renal mTORC1 activity, attenuated hypertension, and protected against renal injury; rapamycin treatment did not further augment these outcomes, suggesting that NOX4/H 2 O 2 / mTORC1 pathway drives the pathogenesis of salt-induced hypertension. 143 Another study from the same group further corroborated the involvement of mTOR in saltinduced hypertension. Inhibition of both mTORC1 and 2 with PP242 resulted in the prevention of salt-induced hypertension and renal injury in SS rats upon a high-salt diet, more potently than rapamycin treatment alone. 78,139 These observations are accompanied by a reduction of mTORC1 activity. 78 Altogether, these studies suggest that mTORC1 and 2 might work together in the pathogenesis of salt-induced hypertension.

| PERSPECTIVES
The development of tubule-specific knockout mouse models and studying human genetic and acquired disease have significantly improved our understanding of mTOR signaling in the kidney tubular segments. Interestingly, most studies have focused on the PT and the CD, leaving other segments such as the DCT largely unexplored. Interestingly, the thin descending limb has relatively high expression levels of many mTOR subunits, 65 but the role of mTOR in this segment remain unclear.
As most pharmacological and transgenic models for mTOR signaling represent the pathophysiological state, the physiological cues that determine mTOR activity remain to be determined. For example, mTORC1 activity is essential for the activation of many transporters in the PT as we have described above. However, which growth factors or nutrient sensing mechanisms are the upstream activators is unknown. Along the same lines, amino acid sensing via RagD has been associated with impaired ion transport in the TAL. 69 Why amino acid sensing in these cells is of importance has never been examined. mTOR signaling is coupled to other nutrient-sensing pathways, e.g., AMPK and insulin signaling. 22,23,50,144 Nevertheless, the interaction of mTOR activity and energy metabolism is largely unexplored. As mitochondrial dysfunction has been demonstrated to cause Fanconi and Gitelman-like syndromes, 145,146 it would be interesting to study mTOR signaling in the larger context of energy metabolism in the kidney. The recent advances in singlecell transcriptomics, proteomics, and metabolomics techniques will be instrumental to study metabolism at a nephron-segment specific level. [147][148][149] ACKNOWLEDGMENTS Jeroen de Baaij was financially supported by the Netherlands Organization of Scientific Research (NWO