TORCing up the importance of calcium signaling.

The glomerular filtration barrier is made up of fenestrated endothelium, the endothelial surface layer, glomerular basement membrane, and podocytes forming a slit diaphragm between interdigitating foot processes and the subpodocyte space.1 Disruption of the filtration barrier results in loss of permselectivity and macromolecules such as albumin in urine. There is evidence to support the relative importance of each component of the filtration barrier in maintaining its integrity.2–5 Identification of mutations in a number of genes causing familial FSGS has resulted in significant advances in the understanding of proteinuric kidney disease.3,6 –10 In vitro studies focused on podocyte signaling11,12 combined with evidence of increased calcium transients in TRPC6 mutations seen in familial FSGS9,10 support a role for calcium signaling in podocyte dysfunction and disruption of the glomerular filtration barrier. In this issue of JASN, Vassiliadis et al.13 treated ex vivo rat glomeruli with protamine sulfate (PS) to recapitulate minimal change disease in an animal model. They confirmed podocyte foot process effacement and albumin leak from glomeruli occurring as early as 45 minutes after administration of PS. Importantly, using Fluo-4 fluorescence, they showed that intracellular calcium increased in isolated glomeruli almost immediately after treatment with PS. Pretreatment with SKF96365 (a selective inhibitor of receptor-mediated calcium entry), gadolinium (a general ion channel blocker), or EGTA (a Ca chelator) prevented the release of albumin from glomeruli. This is significant because it suggests that aberrant increased calcium signaling is an early and, moreover, a necessary event in the development of albumin leakage. NFAT-dependent transcription is induced by TRPC6 through calcineurin in both podocytes12 and cardiomyocytes,14 with TRPC6 and NFAT fulfilling a pathogenic positive-feedback loop in cardiac hypertrophy.15 Calcineurin also directly dephosphorylates the actin-stabilizing protein synaptopodin, leaving it prone to Cathepsin L (CATL)-mediated degradation. This leads to NFAT-independent alterations in actin polymerization and proteinuria.16 Vassiliadis et al.13 tested the hypothesis that calcium-mediated proteinuria and foot process effacement in the PS-treated glomeruli are mediated through the calcineurin/synaptopodin axis. This was confirmed by the observation of decreased expression of fulllength (110 kD) synaptopodin, which was prevented by pretreatment with E64, a CATL inhibitor. Inhibition of synaptopodin cleavage by CATL directly with E64 or indirectly with calcineurin inhibitor FK506 or cyclosporine reduced albumin leak from the pretreated glomeruli. The mammalian target of rapamycin (mTOR) signaling in the glomerulus has been the focus of intense investigation recently.17,18 mTOR is a widely expressed serine/threonine kinase of 289 kDa that exists as part of two multimeric complexes: TORC1 and TORC2. TORC1 includes the key scaffolding protein Raptor (regulatory associated protein of mTOR), whereas TORC2 includes Rictor (rapamycin-insensitive companion of mTOR).19 As the nomenclature suggests, rapamycin binding to FKBP12 (FK-binding protein of 12 kDa) acutely inhibits mTORC1, whereas mTORC2 is relatively insensitive to acute effects of rapamycin.20,21 mTORC1 regulates proliferation and inhibits autophagy, thereby controlling cell size,21 whereas mTORC2 controls cell survival with phosphorylation of the AKT and protein kinase C (PKC).20 The balance of activity of the TOR signaling, which is described in a number of reviews, and the context in which it happens will determine the net effect on the cell.20,22 Vassiliadis et al.13 investigated whether mTOR signaling was involved in the calcium-dependent albumin leakage in the PS injury model. Indeed, rapamycin ameliorates the effects of PS administration in a dosage-dependent manner. They carefully investigated the downstream components of the mTORC1 and mTORC2 pathways and observed that phosphorylation of the mTORC1 targets 4E-BP1 and 70S6K was not enhanced by PS treatment, indicating the PS-induced changes did not require mTORC1 activation. In a similar manner, PKC and serine 473 of Akt, the downstream targets of mTORC2, were also investigated. Phosphorylation of Akt at serine 473 is activated by PS treatment, whereas PKC is unchanged. Accordingly, rapamycin treatment dramatically inhibits Akt Ser473 phosphorylation, whereas it only partially attenuates phosphorylation of mTORC1 targets 4E-BP1 and 70S6K. Acute inhibition of the mTORC2 complex by rapamycin is discordant with previous reports20; however, the authors cite the high dosage used in this study as a possible explanation. It is known that Akt is also activated by an alternate pathPublished online ahead of print. Publication date available at www.jasn.org.