mTOR: Pumping Nutrients into Tubules

Recent studies have revealed that the mammalian target of rapamycin (mTOR) plays an important role in both physiologic and pathophysiologic cellular processes in kidney cells.¹ mTOR is an evolutionarily conserved protein kinase that forms two distinct multiprotein kinase complexes termed mTORC1 and mTORC2. Rapamycin is a specific and preponderant allosteric inhibitor for mTORC1. Rapamycin forms a complex with the endogenous FKBP12 protein to interact with the FKBP12-rapamycin binding (FRB) domain of mTOR kinase in mTORC1. Binding this drug-protein complex to the FRB domain blocks the accessibility of substrates to the active site of mTOR kinase and ultimately, disrupts the formation of mTORC1. A component of mTORC2, Rictor, structurally hinders the FRB domain from the rapamycin-FKBP12 complex, which confers mTORC2 resistant to rapamycin.² However, prolonged rapamycin treatment often inhibits cellular mTORC2 activity, likely due to a blockade of mTORC2 formation through its interaction with newly translated mTOR kinase.³ Although a series of potent ATP–competitive mTOR kinase inhibitors, which inhibit both mTORC1 and mTORC2, has recently been developed and is widely used in research, specific inhibitors for mTORC2 remain underdeveloped.

Rapamycin-sensitive mTORC1 plays a key role in stimulating cellular anabolic processes, such as protein and lipid biosynthesis to support cell growth, differentiation, and proliferation. In contrast, it inhibits autophagy, a major catabolic process that maintains cellular energy and nutrient homeostasis by recycling unnecessary and damaged molecules and/or organelles through their degradation.

mTORC1 activation requires both growth factor and amino acids, especially leucine, arginine, and glutamine.⁴ Amino acids, such as leucine, stimulate mTORC1 translocalization to the lysosomal membrane through the activation of Rag small GTPases. On the lysosomal membrane, mTORC1 is directly activated by another small GTPase, Rheb, activity of which is enhanced by the growth factor-PI3K-Akt pathway.

Although aberrant activation of mTORC1 in glomerular podocytes leads to their injury/loss and causes glomerular dysfunction,⁵ in renal proximal and distal tubular cells, aberrant activation of mTORC1 causes their proliferation and cystic formation.⁶ In contrast, loss of functional mTORC1 in podocytes by ablating an essential mTORC1 component, Raptor, also causes podocyte dysfunction.⁷ These genetic observations indicate that both aberrant activation and loss of mTORC1 activity in podocytes cause vulnerability for highly differentiated podocytes, which lack a capability of proliferation.

Clinical data showed that reduction of serum potassium and phosphorus and induction of tubular LMW proteinuria can be seen in patients treated with sirolimus (rapamycin) after renal transplantation,^1,8 suggesting that prolonged sirolimus treatment causes defects of tubular reabsorption.

However, physiologic roles of mTORC1 and/or mTORC2 in the regulation of renal tubular absorption system have remained elusive. To investigate roles of each mTORC function in renal tubules, Grahammer et al.,⁹ as described in this issue of the Journal of the American Society of Nephrology (JASN), generate inducible conditional mTORC1 anord/mTORC2 knockout mouse models. Importantly, mice lacking functional mTORC1 but not mTORC2 in renal tubular cells displayed glucosuria, phosphaturia, aminoaciduria, low molecular weight proteinuria, and albuminuria, which are all pathologic phenotypes seen in Fanconi syndrome.

Renal Fanconi syndrome is characterized by renal tubular dysfunction and vitamin D–resistant metabolic bone disease.¹⁰ Fanconi syndrome is often due to hereditary autosomal recessive Mendelian disorders, such as cystinosis, type 1 glycogen storage disease, and Wilson disease. Fanconi syndrome can also be acquired, and it is then most often reversible and has adult onset. Many metabolic toxins, such as heavy metals, and therapeutic drugs, including tetracycline, gentamycin, and ifosfamide, cause tubular cell damage and induce the reabsorptive dysfunctions characteristic of the Fanconi syndrome.

In fact, in mTORC1 knockout mice, uptake of horseradish peroxidase and fluorescence-labeled lactoglobulin into the proximal tubules was significantly reduced compared with those in wild-type mice, indicating that the activity of mTORC1 is required for both fluid face and receptor-mediated endocytosis.⁹

Proteomics analyses revealed that the expression of certain amino acid transporters, such as B0AT1 (Slc6a19), Y+LAT-1 (Slc7a7), and 4F2HC (Slc3a2), was reduced in mTORC1 knockout tubules.

In addition, the expression of proteins, such as DOCK8, RAB10, and sorting nexin 8, proteins important for maintaining cell polarity and endocytosis, was also mitigated in mTORC1 knockout tubules. However, the expression of major scavenger receptors, megalin and cubilin, was maintained in mTORC1 knockout tubules.⁹ Consistent with the in vivo observations, cultured proximal tubular cells treated with rapamycin or ATP–competitive mTOR kinase inhibitors (Torin1 and PP242) had significantly reduced endocytosis. Interestingly, PF-4708671, a specific S6K1 inhibitor, or knockdown of S6K with the shRNA also blocked endocytosis in proximal tubular cells, suggesting that loss of S6K1 activity in mTORC1 knockout mice may cause a reduction of endocytosis in tubular cells. These observations may also imply that defects of the reabsorption system in tubular cells in mTORC1 knockout mice are caused by not only a reduction of functional proteins involved in endocytosis but also, an inhibition of enzymatic activity of these proteins, which is likely through their post-translational modifications. Indeed, the study also showed that phosphorylation levels of certain amino acid transporters (e.g., B0AT1) and a glucose transporter (SGLT2) were decreased in mTORC1 knockout tubules. However, it remains unclear whether S6K1 and/or mTORC1 directly phosphorylate these proteins and stimulate their functions under physiologic conditions. It will be interesting to determine the activity of endocytosis in the proximal tubules of S6K1 knockout mice.

Although the data presented in this study clearly indicated that the physiologic levels of mTORC1 activity are essential for renal tubular cell functions, the important question from this study is whether loss of mTORC1 activity is involved in the mechanisms underlying any inherited or acquired renal Fanconi syndrome.

Intriguingly, recent studies have shown that the activity of mTORC1 is diminished in cystinosis,^11,12 which is the most frequent cause of renal Fanconi syndrome in children. Cystinosis is an autosomal recessive lysosomal storage disorder caused by mutations in the CTNS gene encoding the lysosomal cysteine-proton symporter, cystinosin. Loss of functional cystinosin leads to the accumulation of cysteine within the lysosome and lysosomal dysfunction, which causes damage in many tissues and organs, including renal tubular cells. Antignac and colleagues¹¹ recently showed that cystinosin interacts with vATPases, Ragulator (RagA/B guanine nucleotide exchange factor), and Rag small GTPases, which are essential compartments for amino acid–induced mTORC1 recruitment on the lysosomal membrane for its activation.

Loss of functional cystinosin significantly blocks amino acid–induced mTORC1 lysosomal localization and its activation in mouse proximal tubular cells. Levtchenko and colleagues¹² also showed that amino acid–induced mTORC1 activation is delayed in human proximal tubular cells that lack functional cystinosin, although they proposed that mTORC1 is rather retained on the lysosomal membrane under amino acid starvation conditions. Although it remains elusive whether forced mTORC1 activation restores function, including endocytosis, in dysfunctional cystinosin null proximal tubular cells, these studies and the data described by Grahammer et al.⁹ in this issue of the JASN suggest that loss of physiologic mTORC1 activation may be an important mechanism underlying renal Fanconi syndrome due to lysosomal perturbation.

Additionally, it has been shown that many acquired and several genetic cases of renal Fanconi syndrome were associated with mitochondrial dysfunction in renal tubular cells.^13,14 The renal tubular reabsorption system requires robust energy, and an impairment of ATP production inhibits Na/K ATPase activity, which is required for maintaining electrochemical sodium gradient across the luminal membrane. Failure of establishing the sodium gradient decreases the net reabsorption of various solutes through Na/H exchange, Na/glucose, Na/PO₄, and Na/amino acid cotransporters. Importantly, it has been postulated that intact mitochondrial function and high concentrations of ATP are required for the mTOR kinase to phosphorylate its substrates. Furthermore, the activity of mTORC1 also plays an important role in mitochondrial biogenesis.¹⁵ Thus, the reduction of intact mitochondrial function may further mitigate the generation of fresh mitochondria through mTORC1 inhibition. Consistent with this idea, Grahammer et al.⁹ observed that numbers of mitochondria were reduced in mTORC1 knockout renal tubular cells. Thus, it is conceivable that an impairment in mitochondrial function seen in certain renal Fanconi syndromes likely contributes to the reduction of cellular mTORC1 activity in proximal tubular cells, which may further disturb the capacity of reabsorption system in the cells. Thus, growing evidence, including the results in the work by Grahammer et al.,⁹ will set the stage for additional studies to explore the roles of mTORC1 as a signaling nexus in the maintenance of renal proximal tubular cells and the development of the devastating pathologic consequences of Fanconi syndrome.

Disclosures

None.