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, 350 (2), 257-64

Agonism of the 5-hydroxytryptamine 1F Receptor Promotes Mitochondrial Biogenesis and Recovery From Acute Kidney Injury

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Agonism of the 5-hydroxytryptamine 1F Receptor Promotes Mitochondrial Biogenesis and Recovery From Acute Kidney Injury

Sara M Garrett et al. J Pharmacol Exp Ther.

Abstract

Many acute and chronic conditions, such as acute kidney injury, chronic kidney disease, heart failure, and liver disease, involve mitochondrial dysfunction. Although we have provided evidence that drug-induced stimulation of mitochondrial biogenesis (MB) accelerates mitochondrial and cellular repair, leading to recovery of organ function, only a limited number of chemicals have been identified that induce MB. The goal of this study was to assess the role of the 5-hydroxytryptamine 1F (5-HT1F) receptor in MB. Immunoblot and quantitative polymerase chain reaction analyses revealed 5-HT1F receptor expression in renal proximal tubule cells (RPTC). A MB screening assay demonstrated that two selective 5-HT1F receptor agonists, LY334370 (4-fluoro-N-[3-(1-methyl-4-piperidinyl)-1H-indol-5-yl]benzamide) and LY344864 (N-[(3R)-3-(dimethylamino)-2,3,4,9-tetrahydro-1H-carbazol-6-yl]-4-fluorobenzamide; 1-100 nM) increased carbonylcyanide-p-trifluoromethoxyphenylhydrazone-uncoupled oxygen consumption in RPTC, and validation studies confirmed both agonists increased mitochondrial proteins [e.g., ATP synthase β, cytochrome c oxidase 1 (Cox1), and NADH dehydrogenase (ubiquinone) 1β subcomplex subunit 8 (NDUFB8)] in vitro. Small interfering RNA knockdown of the 5-HT1F receptor blocked agonist-induced MB. Furthermore, LY344864 increased peroxisome proliferator-activated receptor coactivator 1-α, Cox1, and NDUFB8 transcript levels and mitochondrial DNA (mtDNA) copy number in murine renal cortex, heart, and liver. Finally, LY344864 accelerated recovery of renal function, as indicated by decreased blood urea nitrogen and kidney injury molecule 1 and increased mtDNA copy number following ischemia/reperfusion-induced acute kidney injury (AKI). In summary, these studies reveal that the 5-HT1F receptor is linked to MB, 5-HT1F receptor agonism promotes MB in vitro and in vivo, and 5-HT1F receptor agonism promotes recovery from AKI injury. Induction of MB through 5-HT1F receptor agonism represents a new target and approach to treat mitochondrial organ dysfunction.

Figures

Fig. 1.
Fig. 1.
Htr1f is expressed in RPTC (A), and the 5-HT1F receptor protein is expressed in freshly isolated renal proximal tubules (T), tubule membranes (TM), RPTC, and RPTC membranes (RM) (B). (C) Various concentrations of α-methyl 5-HT (αm5-HT), a nonselective 5-HT receptor agonist, and selective 5-HT1F receptor agonists LY334370 and LY344864 stimulate FCCP-OCR in RPTC at 24 hours. Data are X + S.E.M., n ≥ 3. *Significantly different from vehicle (P < 0.05). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; M, marker.
Fig. 2.
Fig. 2.
5-HT1F receptor agonists increase mitochondrial proteins in RPTC at 24 hours. Representative immunoblots and quantification of concentration response for LY334370 (A) and LY344864 (B). Data are X + S.E.M., n ≥ 5. *Significantly different from vehicle (P < 0.05). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Fig. 3.
Fig. 3.
Knockdown of 5-HT1F receptor protein in RPTC by siRNA transfection at 72 hours (A) Representative immunoblot and quantification of 5-HT1F receptor protein knockdown. (B) Mitochondrial protein levels after 5-HT1F receptor knockdown. (C) Decreased 5-HT1F receptor levels block LY334370- and LY344864-induced MB. RPTC were treated with scramble or siRNA for 72 hours and treated with LY334370 or LY344864 for 24 hours. Data are X + S.E.M., n ≥ 3. *Significantly different from scrambled or 5-HT1F receptor agonist treatment (P < 0.05). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Fig. 4.
Fig. 4.
Gene expression of PGC1α, Cox1, NDUFB8, and mitochondrial copy number after selective 5-HT1F receptor agonist LY344864 (2 mg/kg) in the kidney cortex at 1, 8, and 24 hours (A) and after selective 5-HT1F receptor agonist LY344864 at 1 mg/kg every 8 hours (1 mg/kg × 3), 2 mg/kg, and 10 mg/kg in the kidney cortex at 24 hours (B). Data are X + S.E.M., n ≥ 3. *Significantly different from vehicle (P < 0.05).
Fig. 5.
Fig. 5.
(A) Htr1f is expressed in murine heart (HT) and liver (LV). Gene expression of PGC1α, Cox1, NDUFB8, and mtDNA copy number after 5-HT1F receptor agonist LY344864 (2 mg/kg) in heart (B) and liver (C) at 1, 8, and 24 hours. Data are X + S.E.M., n ≥ 3. *Significantly different from vehicle (P < 0.05). M, marker.
Fig. 6.
Fig. 6.
Blood urea nitrogen (24 and 144 hours) (A), renal cortical KIM-1 (144 hours) (B), and renal mtDNA copy number (144 hours) (C) in a murine I/R-AKI model. Data are X + S.E.M., n ≥ 4. *P < 0.05 versus sham; #P < 0.05 versus I/R + vehicle.

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