Activating AMP-activated protein kinase (AMPK) slows renal cystogenesis

Abstract

Renal cyst development and expansion in autosomal dominant polycystic kidney disease (ADPKD) involves both fluid secretion and abnormal proliferation of cyst-lining epithelial cells. The chloride channel of the cystic fibrosis transmembrane conductance regulator (CFTR) participates in secretion of cyst fluid, and the mammalian target of rapamycin (mTOR) pathway may drive proliferation of cyst epithelial cells. CFTR and mTOR are both negatively regulated by AMP-activated protein kinase (AMPK). Metformin, a drug in wide clinical use, is a pharmacological activator of AMPK. We find that metformin stimulates AMPK, resulting in inhibition of both CFTR and the mTOR pathways. Metformin induces significant arrest of cystic growth in both in vitro and ex vivo models of renal cystogenesis. In addition, metformin administration produces a significant decrease in the cystic index in two mouse models of ADPKD. Our results suggest a possible role for AMPK activation in slowing renal cystogenesis as well as the potential for therapeutic application of metformin in the context of ADPKD.

Fig. 1.

Metformin activates AMPK in vitro and in vivo. (A) MDCK cells were incubated with 1.0 mM metformin for the number of hours stated. Cells lysates were blotted for pAMPK, the activated form of AMPK. (B) Quantitation of pAMPK band density normalized to β-actin. Comparisons of the mean (±SEM) are shown for each time point (**P = 0.00002 at 2 h, P = 0.001 at 6 h, P = 0.0005 at 24 h; Tukey's test relative to vehicle-treated control for that set of wells; n = 3 wells for each condition). (C) MDCK cells were treated as in A and blotted for pACC, a downstream target of pAMPK. (D) Comparisons of the mean band density relative to β-actin (±SEM) are shown for each time point. There is no significant change in protein expression between 0 and 2 h (**P = 0.0306 at 6 h, P = 0.005 at 24 h; Tukey's test relative to vehicle-treated control for that set of wells; n = 3 for each condition). (E) C57BL/6 mice (8 wk old) were treated i.p. with metformin or with vehicle for 3 d. Western blot analysis of kidney homogenates using anti-pAMPK demonstrates increasing activation of AMPK with increasing metformin dosing. (F) Quantitation of Western blot of in vivo pAMPK levels by normalized band density to β-actin. Comparisons of the mean (±SEM) are shown for each time point; n = 3 mice for each dose.

Fig. 2.

Metformin inhibits I_sc in an AMPK-dependent manner. (A) MDCK cells stably expressing empty vector or shRNA plasmids directed against either the catalytic α1 or α2 subunits of AMPK (AMPK-α1–KD and AMPK-α2–KD cells, respectively) were blotted with antibodies against phosphorylated Thr¹⁷² (pThr¹⁷²), AMPKα2, or AMPKα1 to measure the level of AMPK expression. (B) A representative I_sc trace of cells with or without 1 mM metformin pretreatment. Mock-transduced or NH₂-terminally GFP-tagged, CFTR-transduced MDCK empty vector control cells, AMPK-α1–KD cells, or AMPK-α2–KD cells were treated with 1 mM metformin or vehicle for 2–4 h before Ussing chamber measurements of I_sc. A representative I_sc trace of vehicle-pretreated CFTR-expressing empty vector control MDCK cells treated with IBMX and forskolin (Fsk) and then with CFTR-Inh₁₇₂ at the indicated times is shown. (C) A similar representative trace of mock-transduced empty vector control cells shows no response to these cAMP agonists or to CFTR-Inh₁₇₂. There also was no significant change in I_sc following addition of 10 μM amiloride, indicating that the epithelial Na⁺ channel does not contribute significantly to I_sc in these MDCK cells. (D) Comparisons of the normalized mean (±SEM) CFTR-dependent I_sc in empty vector control, AMPK-α1–KD, and AMPK-α2–KD cells with (dark gray bars) or without (white bars) metformin pretreatment (*P = 0.002, ^#P = 0.022; unpaired t test relative to vehicle-treated controls for that cell type; n = 6–9 filters for each condition).

Fig. 3.

Metformin inhibits phosphorylation of the mTOR downstream target, p70 S6K, and slows cellular proliferation in an AMPK-dependent manner. A subconfluent monolayer of MDCK cells was incubated with 1.0 mM metformin for the indicated time. Cells lysates were blotted for the downstream marker of mTOR activity. (A) p70 S6K. (B) Total S6K. (C) Quantitation of phospho-S6K Western blot band density normalized to β-actin. Comparisons of the mean (±SEM) are shown for each time point. (**P = 0.00005 at 6 h, P = 0.009 at 12 h, P = 0.00009 at 24 h; one-way ANOVA with Tukey's analysis relative to vehicle-treated control for that set of wells; n = 3 wells for each condition). (D) Effect of metformin on proliferation of control MDCK cells and MDCK cells stably transfected with shRNA against AMPK, graphed relative to control. The y axis represents cell number at each concentration of metformin, normalized to the control value measured for the same cell type at the same time point without metformin treatment. (**P = 0.0008 at 0.5 mM, P = 0.009 at 1.0 mM, P = 0.004 at 5 mM; unpaired t tests between both cell lines, comparing rates of cell proliferation with n = 3 per metformin concentration).

Fig. 4.

Metformin reduces cyst size in vitro and ex vivo. (A) Representative light micrographs of MDCK cell cysts grown in collagen gels. Cysts were treated with forskolin (Forsk) and IBMX to enhance apical fluid secretion with (Lower) or without (Upper) 1.0 mM metformin for 20 d. Gels were melted, and the cysts were allowed to precipitate to the bottom for imaging. (B) Metformin treatment reduces cyst size in an ex vivo model of renal cystogenesis. Embryonic kidneys were placed in culture at E12 and maintained for 5 d in the continued presence of 100 μM 8-Br-cAMP. Representative light microscopic images from one mouse are shown. Each row shows the same kidney. The contralateral kidney (Lower) was treated with metformin for 4 d and then switched to normal medium, illustrating that the embryonic kidney remains viable and capable of cystogenesis.

Fig. 5.

Metformin treatment reduces the cystic index in two mouse models of ADPKD. (A–C) Representative midsagittal sections from the kidneys of (A) a PKD1^+/+;Ksp-Cre mouse, (B) a metformin-treated PKD1^flox/-;Ksp-Cre mouse, and (C) a vehicle-treated PKD1^flox/-;Ksp-Cre mouse at P7. The metformin- and vehicle-treated mice were given daily weight-adjusted i.p. injections from P4 until P6. (D and E) Representative images from PKD^flox/-;pCX-CreER mice treated with vehicle (D) or metformin (E) from P7–P17, with Cre induction at P9 or P10.