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 (1). 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 (2, 3). Administration of recombinant Sema3a to wild-type mice induces foot process effacement and fusion and reversible albuminuria (4).
To investigate whether rapamycin may modulate Sema3a expression, in the attempt to evaluate the role of Sema3a in mTOR inhibition-induced proteinuria.
Materials and Methods
Rapamycin effect on Sema3a protein expression was evaluated in vitro, by confocal microscopy and western blotting, using an immortalized human podocyte cell line. To this purpose, 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. Rapamycin effect on rictor protein expression and Akt phosphorylation on ser473 were evaluated by western blotting in podocytes under the same cultured conditions. To investigate the effect of Sema3a on slit-diaphragm-associated proteins, cultured podocytes were incubated for 48 hours with different Sema3a concentrations (20, 50, 100 ng/ml). CD2AP and podocyn protein expression were studied by western blotting. The permeability test was performed on podocyte grown to confluency on trans-well collagen permeable (3 mm) PTFE membrane and exposed to rapamycin or Sema3a for 48 hours. Albumin (40mg/ml) added in the upper channel was then measured on both sides of podocytes monolayer.
- Rapamycin increased Sema3a protein expression in a dose dependent manner with a peak at 20 ng/ml (Figure 1, 2).
- At this same dose the drug significantly increased in vitro albumin permeability through a podocyte monolayer (Figure 3).
- Interestingly, Sema3a, while increasing albumin permeability (Figure 3), induced a significant reduction in the protein expression of podocyn and CD2AP (Figure 4).
- Finally, rapamycin induced in the long term the expression of rictor (Figure 5) and the phosphorylation at ser473 of akt (Figure 6), suggesting the activation of the mTOR complex-2.
Our data would suggest that rapamycin-induced proteinuria is dose dependent and is associated with the overexpression of Sema3a and the activation of the mTOR complex 2.
- G. Stallone et al. Sirolimus and proteinuria in renal transplant patients: evidence of a dose-dependent effect on slit diaphragm-associated proteins. Transplantation, 2011
- K.J. Reidy et al. Semaphorin3a regulates endothelial cell number and podocyte differentiation during glomerular development. Development, 2009
- F. Guan et al. Autocrine class 3 semaphorin system regulates slit diaphragm proteins and podocyte survival. Kidney International, 2006
- R. Tapia et al. Semaphorin3a disrupts podocyte foot processes causing acute proteinuria. Kidney International, 2008