By 4 weeks of age, glomerular scarring and vacuolization of podocytes are evident. Similarly, human podocytes treated with the MTOR inhibitor rapamycin accumulated autophagosomes and autophagolysosomes. Taken together, these results suggest that disruption of the autophagic pathway may play a role in the pathogenesis of proteinuria in patients treated with MTOR inhibitors. The mammalian target of rapamycin (MTOR) is an evolutionarily conserved serine-threonine kinase that interacts with regulatory associated protein of MTOR (Rptor) or Rptor impartial companion of MTOR (Rictor) to form mTORC1 and mTORC2 complexes, respectively. In turn, mTORC1 and mTORC2 regulate different aspects of MTOR function. mTORC1 is a key regulator of cellular metabolism, including protein translation, ribosomal biogenesis, cell growth and proliferation, and suppression of autophagy in response to amino acids, growth factors, and elevated cellular ATP levels.1mTORC2 is regulated primarily by growth factors to promote actin cytoskeletal rearrangement, cell survival, and cell cycle progression.2In mammalian cells, rapamycin and other MTOR inhibitors associate with the FKBP12 protein, and together they directly bind MTOR to prevent the RPTOR-MTOR interaction and thus inhibit mTORC1 function.3In certain cell types, including the podocyte, chronic inhibition of MTOR by rapamycin also results in downregulation of mTORC2 functions. 46Although this mechanism of action has not been completely elucidated, data in podocytes suggest that prolonged rapamycin treatment directly downregulates MTOR and Rictor, both of which are required for mTORC2 function.6 Sirolimus (rapamycin) was originally proposed as an immunosuppressant to prevent rejection of solid organ transplants. There were expectations that MTOR inhibitors would replace nephrotoxic calcineurin inhibitors (CNIs). In one prospective trial, patients treated with sirolimus or switched to sirolimus from CNIs had comparable rates of biopsy-confirmed acute allograft rejection and 2-year graft survival to those treated with CNIs.7In addition, sirolimus-treated patients had fewer malignancies and a better estimated GFR (eGFR) at 24 months if their baseline eGFR was >40 ml/min. Because of their antiproliferative and antiangiogenic effects, indications for MTOR inhibitors have expanded to include treatment of various cancers such as renal cell carcinoma, nonmalignant conditions such as autosomal dominant polycystic kidney disease (AD-PKD), and primary glomerulopathies.812 Despite its potential advantages in the transplant Tezosentan setting, evidence that sirolimus causesde novoor worsening proteinuria is unequivocal. In one randomized clinical trial in which patients with AD-PKD received sirolimus or placebo, the group receiving an MTOR inhibitor had a significantly higher median urine protein/creatinine ratio.9Similarly, in a recent open-label randomized clinical trial in which 503 renal transplant patients were randomized to an everolimus-based CNI-free regime or standard CNI therapy, those taking everolimus had a significantly higher 24-hour urine protein excretion. 13Although subnephrotic increases in proteinuria may result from glomerular or tubular injury, the small incidence of reported cases of patients developing full-blown nephrotic syndrome after treatment with rapamycin14suggests that this glomerular filtration barrier is usually affected, at least in this subset of patients. Severalin vitroand patient biopsy studies have addressed a role for MTOR in the glomerulus. One group described thrombotic microangiopathic glomerular lesions in Tezosentan renal biopsies from five patients who developed proteinuria when treated with sirolimus.15These Tezosentan lesions were associated with downregulation of vascular endothelial growth factor A (VEGFA) expression in podocytes, a molecular mechanism that has been associated with thrombotic microangiopathy in patients with pre-eclampsia16and in those treated with anti-VEGFA agents.17Another small case series describes three instances ofde novoFSGS in patients treated with sirolimus, characterized by focal loss of PAX2, synaptopodin, and VEGFA.14Although not all patients with proteinuria who take sirolimus have a distinct glomerular lesion, Stalloneet al.performed a biopsy study showing that sirolimus treatment was associated with decreased expression of synaptopodin, DKFZp781B0869 podocin, CD2AP, and nephrin in podocytes.18Cell culture studies support the results of these biopsy studies and further suggest a role for MTOR in regulating actin and slit-diaphragmassociated proteins in the podocyte.6Finally, genetic deletion ofRptoralone or bothRictorandRptorfrom podocytes results in glomerular injury in mice by an unknown mechanism.19These data suggest that inhibition of MTOR signaling within the podocyte may play a complex role to promote proteinuria in patients. Given the well recognized proteinuric effect of MTOR inhibitors, we were interested in understanding its mechanism. To explore the role of MTORin vivo, we developed a mouse model with a podocyte-selective deletion of theMtorgene (Mtorpod-KO). A conditionalMtorallele was generated using BAC recombineering to place loxP sites around the first three exons of theMtorgene (Physique 1A). The successful generation of a floxedMtorallele was confirmed by Southern blot (Physique 1B). In this mouse, Cre-mediated excision results in a nullMtorallele, abolishing function of both mTORC1 and mTORC2 complexes. Because chronic use of mTOR inhibitors downregulates both mTORC1 and mTORC2 functions, we chose to delete theMtorgeneas opposed to its partnersRptororRictorto obtain the most complete knockdown and simulate the clinical effects of MTOR inhibitors. == Physique 1. == Generation of podocyte-selective knockout of theMtorgene. (A) BAC recombineering construct. LoxP sites (triangles) are inserted around the first three exons.