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, 347 (3), 626-34

cGMP-selective Phosphodiesterase Inhibitors Stimulate Mitochondrial Biogenesis and Promote Recovery From Acute Kidney Injury

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cGMP-selective Phosphodiesterase Inhibitors Stimulate Mitochondrial Biogenesis and Promote Recovery From Acute Kidney Injury

Ryan M Whitaker et al. J Pharmacol Exp Ther.

Abstract

Recent studies demonstrate that mitochondrial dysfunction is a mediator of acute kidney injury (AKI). Consequently, restoration of mitochondrial function after AKI may be key to the recovery of renal function. Mitochondrial function can be restored through the generation of new, functional mitochondria in a process called mitochondrial biogenesis (MB). Despite its potential therapeutic significance, very few pharmacological agents have been identified to induce MB. To examine the efficacy of phosphodiesterase (PDE) inhibitors (PDE3: cAMP and cGMP activity; and PDE4: cAMP activity) in stimulating MB, primary cultures of renal proximal tubular cells (RPTCs) were treated with a panel of inhibitors for 24 hours. PDE3, but not PDE4, inhibitors increased the FCCP-uncoupled oxygen consumption rate (OCR), a marker of MB. Exposure of RPTCs to the PDE3 inhibitors, cilostamide and trequinsin, for 24 hours increased peroxisome proliferator-activated receptor γ coactivator-1α, and multiple mitochondrial electron transport chain genes. Cilostamide and trequinsin also increased mRNA expression of mitochondrial genes and mitochondrial DNA copy number in mice renal cortex. Consistent with these experiments, 8-Br-cGMP increased FCCP-uncoupled OCR and mitochondrial gene expression, whereas 8-Br-cAMP had no effect. The cGMP-specific PDE5 inhibitor sildenafil also induced MB in RPTCs and in vivo in mouse renal cortex. Treatment of mice with sildenafil after folic acid-induced AKI promoted restoration of MB and renal recovery. These data provide strong evidence that specific PDE inhibitors that increase cGMP are inducers of MB in vitro and in vivo, and suggest their potential efficacy in AKI and other diseases characterized by mitochondrial dysfunction and suppressed MB.

Figures

Fig. 1.
Fig. 1.
PDE3 inhibitors, but not PDE4 inhibitors, increase FCCP-induced uncoupled mitochondrial respiration in RPTCs. RPTCs were treated with cilostamide (A), trequinsin (B), (R)-(−)-rolipram (C), or Ro 20-1724 (D) for 24 hours. FCCP-uncoupled mitochondrial respiration was measured using the Seahorse XF-96 instrument. Data are presented as the mean ± S.E.M. (n ≥ 3). *P < 0.05 vs. vehicle control.
Fig. 2.
Fig. 2.
PDE3 inhibitors cilostamide or trequinsin induce mitochondrial protein gene expression in RPTCs. RPTCs were exposed to cilostamide (25 nM) or trequinsin (30 nM) for 24 hours and evaluated for changes in mRNA expression of PGC-1α, ND6, and NDUFβ8 relative to dimethylsulfoxide controls. Data are presented as the mean ± S.E.M. (n ≥ 4). *P < 0.05 vs. vehicle control.
Fig. 3.
Fig. 3.
PDE inhibitor–induced increases in cGMP, but not cAMP, stimulate MB in RPTCs. cAMP (A) and cGMP (B) levels were measured in RPTCs by enzyme-linked immunosorbent assay 20 minutes after treatment with dimethylsulfoxide, cilostamide (25 nM), trequinsin (30 nM), rolipram (0.5 μM), or sildenafil (10 nM). (C) FCCP-uncoupled mitochondrial respiration was measured using the Seahorse XF-96 instrument after 24-hour treatment with 8-Br-cAMP or 8-Br-cGMP. (D) RPTCs were exposed to 8-Br-cAMP (10 μM) or 8-Br-cGMP (10 μM) for 24 hours and evaluated for changes in mRNA expression of PGC-1α, ND6, and NDUFβ8 relative to dimethylsulfoxide controls. Data are presented as the mean ± S.E.M. (n ≥ 3). *P < 0.05 vs. vehicle control.
Fig. 4.
Fig. 4.
The PDE5 inhibitor sildenafil stimulates MB in RPTCs. Sildenafil increases FCCP-uncoupled mitochondrial respiration at various doses (A) and mitochondrial gene expression at 10 nM (B) in RPTCs. mRNA expression of PGC-1α, ND6, and NDUFβ8 is presented as the mean ± S.E.M. of at least three biologic replicates. *P < 0.05 vs. vehicle control.
Fig. 5.
Fig. 5.
PDE3 inhibitors cilostamide and trequinsin induce mitochondrial gene expression and mtDNA copy number in mouse renal cortex. mRNA expression and mtDNA copy number were evaluated in the renal cortex of mice 24 hours after a single intraperitoneal injection of cilostamide (A and C) or trequinsin (B and D). Values indicate fold change relative to dimethylsulfoxide controls. Data are presented as the mean ± S.E.M. (n ≥ 4). *P < 0.05 vs. vehicle control.
Fig. 6.
Fig. 6.
Sildenafil induces mitochondrial gene expression, mtDNA copy number, and ATP levels in mouse renal cortex. mRNA expression (A), mtDNA copy number (B), and ATP levels (C) were evaluated in the renal cortex of mice 24 hours after a single intraperitoneal injection of sildenafil. Values indicate fold change relative to dimethylsulfoxide controls. Data are presented as the mean ± S.E.M. (n ≥ 4). *P < 0.05 vs. vehicle control.
Fig. 7.
Fig. 7.
Sildenafil stimulates MB after FA-induced AKI. AKI was induced in C57BL/6 by a single intraperitoneal injection of FA. Mice received daily injections of sildenafil (0.3 mg/kg) or saline vehicle beginning 24 hours after FA. Mice were killed and kidneys were collected 7 days after FA administration. mRNA expression (A) and mtDNA copy number (B) were evaluated in the renal cortex. Immunoblotting was performed for renal cortical assessment of KIM-1 expression (C) and quantified via densitometry (D). Data are presented as the mean ± S.E.M. (n ≥ 3). *P < 0.05 versus vehicle control; #P < 0.05 vs. FA.

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