| Abstract |
Abstract: The effect of rapamycin in the structure and function of glomerular epithelial cells in normal mice.
Introduction: In the normal kidney, rapamycin is considered to be non-nephrotoxic based on initial studies in some animal species. In the diseased kidney, rapamycin may be beneficial or detrimental, depending on the type of renal injury. One explanation for the conflicting results maybe either the dose used, or the duration of rapamycin administration. The current study was undertaken a) to examine whether or not sirolimus is indeed non-nephrotoxic in normal Balb/c mice b) to define the clinical manifestations of the nephrotoxicity, proteinuria or decline of GFR c) in case that rapamycin is nephrotoxic to determine whether the toxicity is dose or time dependent d) in case that rapamycin induces glomerular proteinuria to study the molecular mechanisms of podocyte dysfunction
Methods: Two months old Balb/c mice were given three different doses of rapamycin i.p for 1 week (LD:1, ID:1.5, and HD:3mg/kgBW/day, 4 mice in each group - dose model). An intermediate dose of rapamycin (1.5mg/kg) was given i.p for three different periods 1, 4 and 8 weeks (4 mice in each group,-time model). Six Balb/c mice were injected with the rapamycin solvent (DMSO) and served as controls.
The 24h urine protein excretion and BW were estimated weekly. The 24h urine albumin excretion was measured at the beginning and the end of the study. Rapamycin and creatinine blood levels (Cr) were measured at sacrifice. Kidney morphology was examined by light microscopy, electron microscopy (EM) and immunofluorescence (IF for nephrin and podocin). WB & RT-PCR were performed to examine the expression of the slit diaphragm proteins nephrin and podocin. Glomerular Akt kinase expression and phosphorylation was examined by WB.
Results: All treated animals reduced urine protein excretion throughout the course of the study while urine albumin was increased statistically significantly in the HD and 4-weeks groups. Renal function and body weight showed a mild to moderate decline especially in the HD group (Cr: 0.34±0.03 vs. 0.24±0.01mg.dl in controls, p<0.01). By EM, we noticed an increase in the mean foot process width on the first week (controls 335nm, LD 385nm, ID 420nm, HD 420nm), which was further deteriorated on the 4th week (456nm) and finally improved on week 8 (401nm). Podocin and Nephrin mRNA levels both showed a significant (40-60% lower vs. control, all
p<0.001) and dose-independent decrease on the first week, which improved on the 4th week and was restored to normal on week 8. Nephrin and podocin protein levels showed a more delayed and less pronounced decline (relative to mRNA levels) while on IF they were normally expressed in the periphery of the capillary loops. Total Akt levels remained stable in both models but Ser473phospho-Akt increased slightly in the HD group and significantly (p<0.005) in the 4 and 8-weeks groups.
Conclusions: In both models, significant alterations were noticed in kidney function, podocyte architecture, slit diaphragm proteins transcription and glomerular Akt activation. These alterations occured mainly during the first month of treatment while they were completely restored in the long term, indicating that a rapamycin “escape phenomenon” may exist. The observed increase in Akt phosphorylation may be related to the concomitant alterations of the slit diaphragm proteins or the induction of a member of the PDK2s family of kinases. It is not clear though, if it is part of the pathogenetic mechanism for the observed glomerular alterations especially in podocytes or it represents a compensatory mechanism eventually leading to resolution of the lesions.
Abstract: The effect of rapamycin in the structure and function of glomerular epithelial cells in mice with lupus nephritis
Introduction: The mammalian target of rapamycin (mTOR) inhibitor, rapamycin, inhibits the progression of murine lupus nephritis by virtue of its potent immunosuppressive properties. The phosphoinositol-3-kinase (PI3K)/Akt pathway is a major upstream activator of mTOR and has been implicated in the propagation of cancer and autoimmunity. However, the activation status of the PI3K/Akt/mTOR pathway in lupus nephritis has not been studied so far.
Methods: In NZBW/F1 female mice the glomerular expression of Akt and mTOR was examined by immunofluorescence and western blot. We also searched for specific phosphorylations of these kinases known to ensue during activation of the PI3K/Akt/mTOR pathway. In parallel, we examined the therapeutic role of rapamycin either before or after the development of overt lupus nephritis.
Results: In untreated mice, as opposed to healthy controls, Akt and mTOR were over-expressed and phosphorylated at key activating residues. Rapamycin prolonged survival, maintained normal renal function, normalized proteinuria, restored nephrin and podocin levels, reduced anti-dsDNA titers, ameliorated histological lesions and reduced Akt and mTOR glomerular expression-activation.
Conclusions: The PI3K/Akt/mTOR pathway is upregulated in murine lupus nephritis, supporting the role of rapamycin in the treatment of this disease. Rapamycin not only blocks mTOR, but also negatively regulates the PI3K/Akt/mTOR pathway and is an effective treatment of murine lupus nephritis.
Investigation of the PI3K/Akt/mTOR pathway may offer new insights into the pathogenesis of lupus nephritis in humans and may lead to more individualized and less toxic treatment.
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