Noncanonical TGF-beta pathways, mTORC1 and Abl, in renal interstitial fibrogenesis

Abstract

Renal interstitial fibrosis is a major determinant of renal failure in the majority of chronic renal diseases. Transforming growth factor-beta (TGF-beta) is the single most important cytokine promoting renal fibrogenesis. Recent in vitro studies identified novel non-smad TGF-beta targets including p21-activated kinase-2 (PAK2), the abelson nonreceptor tyrosine kinase (c-Abl), and the mammalian target of rapamycin (mTOR) that are activated by TGF-beta in mesenchymal cells, specifically in fibroblasts but less in epithelial cells. In the present studies, we show that non-smad effectors of TGF-beta including PAK2, c-Abl, Akt, tuberin (TSC2), and mTOR are activated in experimental unilateral obstructive nephropathy in rats. Treatment with c-Abl or mTOR inhibitors, imatinib mesylate and rapamycin, respectively, each blocks noncanonical (non-smad) TGF-beta pathways in the kidney in vivo and diminishes the number of interstitial fibroblasts and myofibroblasts as well as the interstitial accumulation of extracellular matrix proteins. These findings indicate that noncanonical TGF-beta pathways are activated during the early and rapid renal fibrogenesis of obstructive nephropathy. Moreover, the current findings suggest that combined inhibition of key regulators of these non-smad TGF-beta pathways even in dose-sparing protocols are effective treatments in renal fibrogenesis.

Fig. 1.

Transforming growth factor-β (TGF-β) expression and Smad2/3 phosphorylation. A: expression of TGF-β is increased in obstructed kidneys (gray bars) compared with contralateral, nonobstructed kidneys (open bars). This ∼4-fold increase is not affected by treatment with imatinib (IM or I) or rapamycin (Rapa or R) alone or in combination at 2 different dosages (n = 6 each). B: in obstructed kidneys smad2/3 phosphorylation is increased compared with nonobstructed kidneys. Smad2/3 phosphorylation is not changed by treatment with imatinib or rapamycin. C: quantitative depiction of pSmad2/3 in control (open bars) and obstructed kidneys (gray bars) in each of the 5 treatment groups (n = 4 each).

Fig. 2.

A: non-smad TGF-β targets p21-activated kinase-2 (PAK2) and c-Abl are activated in obstructive nephropathy. Kinase activity and levels of PAK2 and c-Abl in the right (nonobstructed) and the contralateral (obstructed) kidney were determined as described previously (17) and under materials and methods. Groups consisted of vehicle (control), imatinib (IM or I), rapamycin (Rapa or R), or I + R at the indicated drug concentrations (mg/kg). In obstructive nephropathy, both PAK2 and c-Abl are activated and PAK2 activation is not affected by any of the treatment regimens. At the higher dose (50 mg/kg), imatinib (IM) blocks Abl activation. At the lesser dose of 25 mg/kg, IM inhibition of Abl activity by imatinib is submaximal. B: quantitative assessment of Abl activity in right, control kidneys (open bars) and left, obstructed kidneys (gray bars); n = 3 each. *P < 0.05 vs. control kidneys. **P < 0.05 vs. obstructed kidneys in vehicle-treated rats. C: Akt phosphorylation and mammalian target of rapamycin (mTOR) signaling are induced in obstructive nephropathy. Akt phosphorylation is substantially increased in the obstructed kidney and, as expected, is not modified by treatment with imatinib or rapamycin. Similarly, TSC2 (a substrate of Akt and an inhibitor of mTORC1 which is inactivated by phosphorylation) is phosphorylated to a similar degree in unilateral ureteral obstruction (UUO) rats irrespective of treatment with imatinib (IM or I) or rapamycin (Rapa or R). In contrast, P70S6K, a downstream effector of mTORC1, is activated in obstructive nephropathy and not affected by IM, but very effectively reduced by Rapa even at the lower dose. Thus, mTORC1 is activated during obstructive nephropathy, presumably through TGF-β, and its activation is blocked by Rapa, 1.5 or 0.5 mg/kg. D: quantitative assessment of mTOR activity which was measured as the ratio of phosphorylated over total P70S6K in right, control kidneys (open bars) and left, obstructed kidneys (gray bars); n = 3 each. *P < 0.05 vs. control kidneys. **P < 0.05 vs. obstructed kidneys in vehicle-treated rats.

Fig. 3.

A: quantitative assessment of myofibroblast accumulation by immunoreactive interstitial α-smooth muscle actin (α-SMA) in UUO rats. Animals were treated as described in Fig. 1 legend and the expression of interstitial α-SMA was determined. L (gray bars) indicates left, obstructed kidney; R (open bars) indicates right, nonobstructed kidney; n = 6 each. *P < 0.05 vs. control. **P < 0.05 vs. imatinib, 50 mg/kg. B–D: matrix gene expression in UUO rats. mRNA levels encoding Col3A1 (B), Col1A2 (C), and fibronectin (D) in the left (obstructed) kidney (gray bars) and the right (nonobstructed) kidney (open bars); n = 6 each. *P < 0.05 vs. control. **P < 0.05 vs. 50 mg/kg imatinib or 1.5 mg/kg rapamycin.

Fig. 4.

Levels of fibronectin in obstructive nephropathy. Representative sections of control and obstructed kidneys stained with anti-fibronectin antibody. Animals were treated as indicated and fibronectin expression was determined by immunohistochemistry. The bar graph displays quantification of immunoreactive renal interstitial fibronectin; n = 6 each. *P < 0.05 vs. control. **P < 0.05 vs. 50 mg/kg imatinib or 1.5 mg/kg rapamycin.

Fig. 5.

Non-smad TGF-β signaling is required for in vivo collagen III accumulation. Representative immunofluorescence sections of control and UUO kidneys stained with anti-collagen III. The bar graph displays quantification of immunoreactive renal interstitial collagen III; n = 6 each. *P < 0.05 vs. control. **P < 0.05 vs. 50 mg/kg imatinib or 1.5 mg/kg rapamycin.

Fig. 6.

Working model of TGF-β actions in renal interstitial fibrogenesis. The canonical pathway through smad2/3 contributes primarily to the transition of fibroblasts into myofibroblasts and to the transcriptional activation of various extracellular matrix proteins in myofibroblasts as well as in epithelial and nonepithelial glomerular and tubular cells. The noncanonical (smad-independent) pathways are initiated by activation of PI3K which is a branch point for the activation of PAK2-Abl. The latter induces fibroblast proliferation, thereby increasing the number of myofibroblast precursors. PI3K also activates Akt. One of its downstream targets is TSC2 which becomes phosphorylated and thereby inactivated. Active (nonphosphorylated) TSC2 functions as a GTPase-activating protein (GAP) protein converting mTORC1-activating Rheb-GTP to Rheb-GDP. Thus, active TSC2 is an inhibitor of mTORC1, and loss of TSC2 activity by phosphorylation increases mTORC1 activity. This activates downstream substrates including P70S6 kinase, a translational activator of many proteins including cell cycle proteins and hence, proliferation. The Akt → mTORC1 → P70S6K branch pathway contributes also to fibroblast proliferation. Thus, in fibroblasts, the noncanonical and canonical TGF-β pathways closely collaborate: the noncanonical pathway induces fibroblast proliferation and the canonical smad2/3 pathway induces transition of the increased number of fibroblasts into active myofibroblasts and acts in these latter cells as transcriptional activator of multiple extracellular matrix proteins as well as regulators of matrix accumulation.