5-HT1F receptor regulates mitochondrial homeostasis and its loss potentiates acute kidney injury and impairs renal recovery

Abstract

Our laboratory previously reported that agonists of the 5-hydoxytryptamine 1F (5-HT_1F) receptor induce renal mitochondrial biogenesis (MB) and that stimulation of the 5-HT_1F receptor following ischemia/reperfusion (I/R)-induced acute kidney injury (AKI) accelerated the recovery of renal function in mice. The goal of this study was to examine the contribution of the 5-HT_1F receptor in the regulation of renal mitochondrial homeostasis and renal function in naïve and injured mice. Although 5-HT_1F receptor knockout (KO) mice were healthy and fertile, and did not exhibit renal dysfunction, renal mitochondrial DNA copy number and mitochondrial fission gene expression increased at 10 wk of age. The 5-HT_1F receptor KO mice exhibited greater proximal tubular injury and diminished renal recovery after I/R-induced AKI compared with wild-type mice. These findings were associated with persistent suppression of renal cortical MB and ATP levels after injury. In summary, the 5-HT_1F receptor is a component of physiological MB regulation in the kidney, and its absence potentiates renal injury and impedes recovery.

Keywords: acute kidney injury; gene expression; mitochondria; serotonin.

Fig. 1.

Absence of the 5-HT_1F receptor gene does not induce renal injury. Expression of renal cortical 5-HT_1F receptor was measured at the mRNA level in WT, HET, and KO mice (A). Immunoblot analysis of renal cortical KIM-1 and NGAL expression was used to assess tubular injury in 10-wk old male 5-HT_1F receptor WT and KO mice (B). Data are reported as mean ± SE, n = 6. ^a,b,cStatistically significant differences (P < 0.05). 5-HT, 5-hydroxytryptamine; HET, heterozygous; KIM-1, kidney injury molecule-1; KO, knockout; NGAL, neutrophil gelatinase-associated lipocalin; WT, wild type.

Fig. 2.

Absence of the 5-HT_1F receptor potentiates I/R-induced AKI at 24 h. Serum creatinine was assessed 24 h after renal I/R injury in WT and 5-HT_1F receptor KO mice (A). Immunoblot analysis of renal cortical KIM-1 and NGAL expression was used to assess tubular injury (B). Densitometry analysis of KIM-1 (C) and NGAL (D). Total RNA was harvested from renal cortex of WT and 5-HT_1F receptor KO mice and expression of 5-HT_1F receptor mRNA was measured (E). Data are reported as mean ± SE; Sham: n = 4; KO: n = 6. ^a,b,cStatistically significant differences (P < 0.05). 5-HT, 5-hydroxytryptamine; AKI, acute kidney injury; I/R, ischemia/reperfusion; KIM-1, kidney injury molecule-1; KO, knockout; NGAL, neutrophil gelatinase-associated lipocalin; WT, wild type.

Fig. 3.

Absence of the 5-HT_1F receptor has no effect on renal necrosis in I/R-induced AKI at 24 h. Representative slides of the outer stripe of the outer medulla stained with periodic acid-Schiff (PAS) at 24 h (magnification, ×100) following sham or I/R-induced AKI in WT and 5-HT_1F receptor KO mice (A). Tubular necrosis scoring (B). Loss of brush border scoring (C). Data are reported as mean ± SE; WT I/R: n = 5; KO I/R: n = 6. 5-HT, 5-hydroxytryptamine; AKI, acute kidney injury; I/R, ischemia/reperfusion; KO, knockout; WT, wild type.

Fig. 4.

Absence of the 5-HT_1F receptor reduces the recovery of renal function. Serum creatinine (A) and Δ serum creatinine (B) were assessed 144 h after renal I/R injury in WT and 5-HT_1F receptor KO mice. Immunoblot analysis of renal cortical KIM-1 and NGAL expression was used to assess tubular injury (C). Densitometry analysis of KIM-1 (D) and NGAL (E). Data are reported as mean ± SE; Sham: n = 4; I/R: n = 6. ^a,b,cStatistically significant differences (P < 0.05). 5-HT, 5-hydroxytryptamine; I/R, ischemia/reperfusion; KIM-1, kidney injury molecule-1; KO, knockout; NGAL, neutrophil gelatinase-associated lipocalin; WT, wild type.

Fig. 5.

Absence of the 5-HT_1F receptor has no effect on renal necrosis in I/R-induced AKI at 144 h. Representative slides of the outer stripe of the outer medulla stained with periodic acid-Schiff (PAS) at 144 h (magnification, ×100) following sham or I/R-induced AKI in WT and 5-HT_1F receptor KO mice (A). Tubular necrosis scoring (B). Loss of brush border scoring (C). Data are reported as mean ± SE; WT I/R: n = 5; KO I/R: n = 6. 5-HT, Δ serum creatinine, change in serum creatine; 5-hydroxytryptamine; AKI, acute kidney injury; I/R, ischemia/reperfusion; KO, knockout; WT, wild type.

Fig. 6.

Absence of the 5-HT_1F receptor suppresses MB in I/R-induced AKI. Total protein was harvested from the renal cortex of WT and 5-HT_1F receptor KO mice following I/R-induced AKI. Immunoblot analysis of renal cortical PGC-1α, NDUFS1, COX1, and TFAM protein expression was used to assess MB at 24 h (A) and 144 h (B). Densitometry analysis of PGC-1α (C), NDUFS1 (D), COX1 (E), and TFAM (F) at 24 h and PGC-1α (G), NDUFS1 (H), COX1 (I), and TFAM (J) at 144 h following I/R injury. Data are reported as mean ± SE; Sham: n = 4; I/R: n = 6. ^a,b,cStatistically significant differences (P < 0.05). 5-HT, 5-hydroxytryptamine; AKI, acute kidney injury; COX1, cytochrome c oxidase subunit 1; I/R, ischemia/reperfusion; KO, knockout; MB, mitochondrial biogenesis; NDUFS1, NADH dehydrogenase (ubiquinone) FE-S protein 1; PGC-1α, peroxisome proliferator-activated receptor γ coactivator-1α; TFAM, mitochondrial transcription factor A; WT, wild type.

Fig. 7.

Absence of the 5-HT_1F receptor disrupts mitochondrial homeostasis following I/R-induced AKI. Total RNA was extracted from renal cortical tissue. Gene expression of PGC-1α (A), NDUFS1 (B), COX1 (C), TFAM (D) at 24 h and PGC-1α (E), NDUFS1 (F), COX1 (G), TFAM (H) at 144 h were measured to assess mitochondrial homeostasis following I/R-induced renal injury. Data are reported as mean ± SE; Sham: n = 4; I/R: n = 6. ^a,b,cStatistically significant differences (P < 0.05). 5-HT, 5-hydroxytryptamine; AKI, acute kidney injury; COX1, cytochrome c oxidase subunit 1; I/R, ischemia/reperfusion; KO, knockout; MB, mitochondrial biogenesis; NDUFS1, NADH dehydrogenase (ubiquinone) FE-S protein 1; PGC-1α, peroxisome proliferator-activated receptor γ coactivator-1α; TFAM, mitochondrial transcription factor A; WT, wild type.

Fig. 8.

5-HT_1F receptor KO mice exhibit persistent decreased ATP following I/R-induced renal injury. Renal cortical ATP was measured in WT and 5-HT_1F receptor KO mice 24 h (A) and 144 h (B) following renal I/R injury and then normalized to protein. Data are reported as mean ± SE; Sham: n = 4; I/R: n = 6. ^a,b,cStatistically significant differences (P < 0.05). 5-HT, 5-hydroxytryptamine; I/R, ischemia/reperfusion; KO, knockout; WT, wild type.

Fig. 9.

Altered renal mitochondrial homeostasis in 5-HT_1F receptor KO mice. Total RNA was harvested from renal cortical tissue of 5-HT_1F receptor WT and KO mice. Gene expression of key regulators of mitochondrial biogenesis, fission, and autophagy were measured at 10 wk of age (A–D). Relative mitochondrial DNA content in the renal cortex was determined by qRT-PCR analysis (A). Data are reported as mean ± SE, n = 6. *P < 0.05 vs. WT controls. 5-HT, 5-hydroxytryptamine; COX1, cytochrome c oxidase subunit 1; Drp1, dynamin-related protein 1; KO, knockout; ND1, NADH dehydrogenase 1; NDUFS1, NADH dehydrogenase (ubiquinone) FE-S protein 1; Nrf, nuclear respiratory factor; PGC-1α, peroxisome proliferator-activated receptor γ coactivator-1α; PINK1, PTEN-induced putative kinase 1; qRT-PCR, quantitative real-time PCR; TFAM, mitochondrial transcription factor A; WT, wild type.

Fig. 10.

5-HT₁ and 5-HT₂ Receptors are expressed in mouse and human kidney and are altered in renal cortex in 5-HT_1F receptor in KO mice. Total RNA was extracted from human and mouse renal tissue. Gene expression of renal 5-HT_1B, 5-HT_1D, 5-HT_1F, 5-HT_2A, 5-HT_2B, and 5-HT_2C receptors were measured in human and mouse (A). n = 1. Data reported as Δ Cq values. Total RNA was harvested from renal cortical tissue of 5-HT_1F receptor WT and KO mice at 10 wk of age. Gene expression of 5-HT_1B, 5-HT_1D, 5-HT_2A, 5-HT_2B, and 5-HT_2C receptors were measured (B). Data are reported as mean ± SE, n = 6. *P < 0.05 vs. WT controls. 5-HT, 5-hydroxytryptamine; KO, knockout; WT, wild type.