BACKGROUND. Semaphorin3a (Sema3a), a chemorepellant guidance protein, plays crucial roles in neural, cardiac and peripheral vascular patterning. Sema3a is expressed, within the kidney, in the developing nephron, mature podocytes and collecting tubules. Administration of recombinant Sema3a to wild-type mice induces foot process effacement and fusion and reversible albuminuria. Nephrotic-range proteinuria was reported in 64% of renal transplant recipients converted from a calcineurin inhibitor-based to a rapamycin-based immunosuppressive regimen. We previously demonstrated that rapamycin inhibits in vivo and in vitro, in a dose-dependent manner, the expression of the main components of podocyte-cytoskeleton and slit-diaphragm. Thus, the aim of the present study was to investigate whether rapamycin may modulate Sema3a expression, in the attempt to evaluate the role of Sema3a in mTOR inhibition-induced proteinuria.

METHODS. Rapamycin effect on Sema3a protein expression was evaluated in vitro, by confocal microscopy and western blotting, using an immortalized human podocyte cell line. Specifically, the cells were incubated 24 hours in serum-free medium and, then, exposed to rapamycin for 48 hours at the final concentration of 5, 10, 20 or 50 ng/ml. We, then, investigated whether in cultured podocytes 48 hours incubation at different Sema3a concentrations (20,50,100 ng/ml) modulated slit diaphragm-associated proteins-expression by western blotting. RESULTS and CONCLUSIONS. Rapamycin increased Sema3a protein expression in a dose dependent manner with a peak at 20 ng/ml. At this same dose the drug significantly increased in vitro albumin permeability through a podocyte monolayer. Interestingly, Sema3a, while increasing in a dose dependent manner albumin permeability, induced a significant reduction in the protein expression of podocyn and CD2AP, two key slit-diaphragm-associated proteins. Finally, rapamycin at 20ng/ml caused in the long term the phosphorylation at ser473 of akt, suggesting the activation of the mTOR complex-2. Interestingly, akt specific inhibition caused a significant reduction in rapamycin-induced Sema3a.

In conclusion, our data would suggest that rapamycin-induced proteinuria is dose dependent and might be mediated by the overexpression of Sema3a through the activation of the mTOR complex 2.