Constitutive activation of the mTOR signaling pathway within the normal glomerulus

Abstract

Agents that target the activity of the mammalian target of rapamycin (mTOR) kinase in humans are associated with proteinuria. However, the mechanisms underlying mTOR activity and signaling within the kidney are poorly understood. In this study, we developed a sensitive immunofluorescence technique for the evaluation of activated pmTOR and its associated signals in situ. While we find that pmTOR is rarely expressed in normal non-renal tissues, we consistently find intense expression in glomeruli within normal mouse and human kidneys. Using double staining, we find that the expression of pmTOR co-localizes with nephrin in podocytes and expression appears minimal within other cell types in the glomerulus. In addition, we found that pmTOR was expressed on occasional renal tubular cells within mouse and human kidney specimens. We also evaluated mTOR signaling in magnetic bead-isolated glomeruli from normal mice and, by Western blot analysis, we confirmed function of the pathway in glomerular cells vs. interstitial cells. Furthermore, we found that the activity of the pathway as well as the expression of VEGF, a target of mTOR-induced signaling, were reduced within glomeruli of mice following treatment with rapamycin. Collectively, these findings demonstrate that the mTOR signaling pathway is constitutively hyperactive within podocytes. We suggest that pmTOR signaling functions to regulate glomerular homeostasis in part via the inducible expression of VEGF.

Figure 1. pmTOR is expressed at high levels in normal mouse and human glomeruli

Kidneys were harvested from C57BL/6 mice, and immunofluorescence was performed on 3μm cryostat sections. Illustrated are low power (Panel A) and high power (Panel B) micrographs of the renal cortex showing intense expression of pmTOR within glomeruli and minimal expression on occasional tubules in extra-glomerular tissues. Panels C and D illustrate a similar pattern of expression of pmTOR in a human kidney transplant biopsy (Panel C) and in normal human kidney tissue (Panel D). In Panel E, glomeruli were isolated from C57BL/6 mice using magnetic beads (as described in Methods), and the expression of pmTOR, pS6 kinase and GAPDH was evaluated by Western blot analysis. Illustrated is the expression of pmTOR and pS6 kinase in the glomerular isolates (Glom) and in non-glomerular (Non-G) renal tissue.

Figure 2. pmTOR is primarily expressed by the podocyte

Double immunofluorescence staining of normal mouse kidneys was performed using anti-pmTOR and either anti-CD31 or anti-nephrin. Panel A, immunofluorescence staining for pmTOR (left panel, red), CD31 (mid panel, green) and the merged image (right panel) showing a lack of co-localization (yellow) of pmTOR with CD31. Panel B, immunofluorescence staining for pmTOR (left panel, red), nephrin (mid panel, green) and the merged image (right) showing co-localization (yellow) of pmTOR with podocyte-specific nephrin in the glomerulus.

Figure 3. The treatment of mice with rapamycin inhibits glomerular expression of pmTOR and pS6 kinase

C57BL/6 mice were treated with 1.5mg/kg/day or 4mg/kg/day of rapamycin i.p. or vehicle as a control. After 4 days, the kidneys were harvested and processed for light microscopy, electron microscopy and Western blot analysis. Panel A illustrates the histological appearance by light microscopy, and Panels B and C show the typical electron microscopy appearance of the renal cortex following treatment with rapamycin (4mg/kg). No histological changes are evident. Panels D-G show expression of pmTOR (Panels D, F), and pS6 kinase (Panels E, G) as evaluated by Western blot analysis in magnetic bead isolated glomeruli from control vehicle-treated and rapamycin-treated mice. Representative blots are illustrated in Panels D-E, and the mean densitometric analysis (+/- 1SD) from three different experiments are shown in Panels F and G. As illustrated, we found a dose-dependent reduction of glomerular expression of pmTOR and pS6 kinase in treated mice (vs. controls).

Figure 4. The effect of rapamycin on glomerular expression of VEGF

Kidneys were harvested from C57BL/6 mice following treatment with rapamycin 1.5mg/kg/day or 4mg/kg/day for 4 days. Panel A, illustrates a representative pattern of expression of VEGF mRNA by in situ hybridization within glomeruli of these treated mice (upper panel). The sense control is shown in the lower panel. The expression was found to be identical to that observed in normal untreated mice (not shown). Panels B and C, illustrate quantitative VEGF expression in glomerular isolates by Western blot analysis. A representative blot and densitometry from one experiment is shown in Panel B and the mean densitometry from 3 different blots is shown in Panel C. VEGF protein levels were reduced in kidneys harvested from the mice that were treated with rapamycin.