5-HT_1F 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.

INTRODUCTION

Mitochondrial dysfunction is a hallmark pathophysiological mediator of many acute and chronic diseases. As such, loss of ATP-dependent cellular functions and increased reactive oxygen species propagate injury and subsequent tissue and organ dysfunction (16, 27). For example, our laboratory demonstrated that persistent mitochondrial dysfunction and suppression of MB corresponds to worsened renal function after ischemia/reperfusion (I/R)- and folic acid-induced acute kidney injury (AKI; 11, 15, 28).

Mitochondrial biogenesis (MB) has received increasing attention as a therapeutic target to promote restoration of normal cellular and organ function, and several studies have reported pharmacological induction of MB as a treatment for acute organ injury and cardiovascular disease (7, 13, 21, 24). MB is defined as the dynamic process of generating new, functional mitochondria and is a highly regulated process controlled by the central mediator and transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) (23). Several groups have developed strategies designed to increase the expression and activity of PGC-1α and subsequent induction of MB, as evidenced by increased mitochondrial number and function (2, 3, 25, 27). For example, we reported that a 5-hydroxytryptamine 1F (5-HT_1F) receptor agonist LY344864 induced MB in vitro and in vivo, restored MB, and accelerated the recovery of renal function following I/R-induced AKI in mice (11). Furthermore, knockdown of the 5-HT_1F receptor in renal proximal tubular cells resulted in decreased mitochondrial proteins, suggesting that the 5-HT_1F receptor may be regulating MB under basal conditions. These findings implicate the 5-HT_1F receptor as a potential regulator of MB in the kidney.

Based on these findings, we utilized a recently developed 5-HT_1F receptor knockout (KO) mouse to understand further the contribution of the 5-HT_1F receptor in the regulation of renal mitochondrial homeostasis. In addition, we tested the hypothesis that the absence of the 5-HT_1F receptor potentiates AKI in mice.

METHODS

I/R-induced AKI model.

Male and female 5-HT_1F receptor KO mice (B6N(Cg)-Htr1f^{tm1.1(KOMP)Vlcg}/J, stock no. 024269) and age-matched wild-type (WT) female and male C57BL/6NJ (WT, stock no. 005304) were purchased from the Jackson Laboratory (Bar Harbor, ME). A 5-HT_1F receptor KO breeding colony was generated from heterozygous mutants and were housed in temperature-controlled conditions under a light/dark photocycle with unrestricted food and water supplied ad libitum. Male and female mice were weighed periodically. Changes in 5-HT_1F KO mice were evaluated at 10 wk of age.

Ten-week-old male WT and KO mice underwent I/R surgery using bilateral renal pedicle clamping for 18 min as described previously (10). Briefly, the renal artery and vein were isolated, and blood flow was occluded with a vascular clamp for 18 min while body temperature was maintained at 36°C–37°C. Sham mice were treated the same as I/R mice without clamping of the renal pedicles. Mice were euthanized 24 h or 144 h after surgery, and blood and kidneys (flash-frozen in liquid nitrogen) were collected for analysis. Studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All protocols were approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina and the University of Arizona, and all efforts were made to minimize animal suffering.

Agarose gel electrophoresis.

Genomic DNA was isolated from mice tail clippings using Viagen Direct PCR DNA Lysis Reagent (Los Angeles, CA) containing 0.4 mg/ml proteinase K. PCR amplification was performed using Promega PCR Master Mix (Madison, WI). PCR products were separated on a 2% agarose gel in 1× TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA) and 0.5 µg/ml ethidium bromide. Gels were visualized using the GE ImageQuant LAS-4000 (GE Life Sciences, Pittsburgh, PA). Primer pairs annealing with the WT allele and the mutant allele were obtained from Jackson Laboratory and are listed in Table 1.

Table 1.

Mouse and human primer pairs used for qRT-PCR

Gene	Primer Sequence	Accession No.
Mouse
PGC-1α	Sense 5′-AGGAAATCCGAGCGGAGCTGA-3′	NM_008904.2
	Antisense 5′-GCAAGAAGGCGACACATCGAA-3′
NDUFS1	Sense 5′-AGATGATTTGGGAACAACAG-3′	NM_001160038.1
	Antisense 5′-TAAGGCTTAGAGGTTAGGGC-3′
ATP Synthase β	Sense 5′-CTATGTGCCTGCTGATGACC-3′	NM_016774.3
	Antisense 5′-GGATAGATGCCCAACTCAGC-3′
COXI	Sense 5′-ACCATCATTTCTCCTTCTCC-3′	ENSMUSG00000064351.1
	Antisense 5′-GGTGGGTAGACTGTTCATCC-3′
ND1	Sense 5′-TAGAACGCAAAATCTTAGGG-3′	ENSMUSG00000064341.1
	Antisense 5′-TGCTAGTGTGAGTGATAGGG-3′
NRF1	Sense 5′-TCGGGCATTTATCCCAGAGATGCT-3′	NM_001164226.1
	Antisense 5′-TACGAGATGAGCTATACTGTGTGT-3′
NRF2	Sense 5′-CCTCTGTCACCAGCTCAAGG-3′	NM_010902.4
	Antisense 5′-TTCTGGGCGGCGACTTTATT-3′
TFAM	Sense 5′-GCTGATGGGTATGGAGAAG-3′	NM_009360.4
	Antisense 5′-GAGCCGAATCATCCTTTGC-3′
DRP1	Sense 5′-GCCTCAGATCGTCGTAGTGG-3′	NM_001276340.1
	Antisense 5′-TCTGCTTCAACTCTCCAGCTC-3′
Pink1	Sense 5′-TTGCAATGCCGCTGTGTATG-3′	NM_026880.2
	Antisense 5′-TGGAGGAACCTGCCGAGATA-3′
SOD2	Sense 5′-CAAGGGAGATGTTACAACTCAGG-3′	NM_013671.3
	Antisense 5′-CTTAGGGCTCAGGTTTGTCCA-3′
UCP2	Sense 5′-GAGATACCAGAGCACTGTCG-3′	NM_011671.5
	Antisense 5′-GTCATCTGTCATGAGGTTGG-3′
5-HT1B	Sense 5′-AGACAGGGGTACCTCTCACCAACC-3′	NM_010482.1
	Antisense 5′-ATGAGCGCCAACAAAGCAACCAGC-3′
5-HT1D	Sense 5′-AAACCAGTCCCTAGAAGGCCTTCC-3′	NM_008309.5
	Antisense 5′-GCCAGTGTGATGACGGACAGCAC-3′
5-HT1F	Sense 5′-GCCGTGATGATGAGTGTGTC-3′	NM_008310.3
	Antisense 5′-ATCATCCGACTCGCTTGTCT-3′
5-HT2A	Sense 5′-GCTGAGCCGACAGCTAATGA-3′	NM_172812.2
	Antisense 5′-CATCCGGTCCATCACACACA-3′
5-HT2B	Sense 5′-TCTTCAATAAGACATTTCGGGA-3′	NM_008311.2
	Antisense 5′-GAATGGTTGAACTTCGGAGC-3′
5-HT2C	Sense 5′-AGATATTTCTGCCCCGTCTG-3′	NM_008312.4
	Antisense 5′-GCCTTAGTCCGCGAATTGAA-3′
β-Actin	Sense 5′-GGGATGTTTGCTCCAACCAA-3′	NM_007393.5
	Antisense 5′-GCGCTTTTGACTCAGGATTTAA-3′
Human
5-HT1B	Sense 5′-ACGCCGTGGAGTACTCAGCTAAAAG-3′	NM_000863.2
	Antisense 5′-GGAGTAGACCGTGTAGAGGATGTGG-3′
5-HT1D	Sense 5′-TGCCGTGGTCCTTTCCGTC-3′	NM_000864.4
	Antisense 5′-GGTGATGGTATAGGCGATGCTG-3′
5-HT1F	Sense 5′-GCTATAGCTTTGGATCGGTATCGAG-3′	NM_001322209.1
	Antisense 5′-CAATCCTACTTGCTTGTCTCTTGTG-3′
5-HT2A	Sense 5′-TCGCCATCCAGAATCCCATCCACC-3′	NM_001165947 0.2
	Antisense 5′-GATGGACTGCATAGTCCTCCTGCC-3′
5-HT2B	Sense 5′-ACGCCTAACATGGTTGACTGTGTC-3′	NM_000867.4
	Antisense 5′-TGAGGCTCTCTGTCCGTTGGAA-3′
5-HT2C	Sense 5′-TAACACTGAAGCAATCATGG-3′	NM_001256761.2
	Antisense 5′-GACTGTGCTGTTCTTTCTCACAC-3′
β-Actin	Sense 5′-CTGGAACGGTGAAGGTGACA-3′	NM_001101.3
	Antisense 5′-GAAGGGACTTCCTGTAACAATGCA-3′

5-HT, 5-hydroxytryptamine; COX1, cytochrome c oxidase subunit 1; Drp1, dynamin-related protein 1; 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; UCP, uncoupling protein.

Complete blood count, blood urea nitrogen, and serum creatinine evaluation.

Following euthanasia by isoflurane inhalation, peripheral blood was collected via cardiac puncture using EDTA as an anticoagulant and complete blood counts were obtained using a HEMAVET Multispecies Hematology Analyzer. Blood was collected via retro-orbital bleed following isoflurane inhalation. Blood urea nitrogen (BUN) was determined using the QuantiChrom Urea Assay kit (BioAssay Systems, Hayward, CA), and serum creatinine was determined using the Creatinine Enzymatic Reagent Kit (Pointe Scientific, Canton, MI) based on the manufacturer’s directions.

Protein isolation and immunoblot analysis.

Mouse kidney cortex tissue was homogenized in protein lysis buffer (1% Triton X 100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4; 1 mM EDTA; 1 mM EGTA; 2 mM sodium orthovanadate; 0.2 mM phenylmethylsulfonylfluoride; 1 mM HEPES pH 7.6; 1 µg/ml leupeptin; and 1 µg/ml aprotinin) using a polytron homogenizer. The homogenate was stored on ice for 10 min and then centrifuged at 7,500 g for 5 min at 4°C. The supernatant was collected and protein determined using a bicinchoninic acid kit (Sigma Chemical Co., St. Louis, MO) with bovine serum albumin as the standard. Proteins (50 µg) were separated on 4%–20% gradient sodium dodecyl sulfate polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked in 5% dried milk in Tris-buffered saline-Tween 20 (0.1% Tween 20 in 1×  Tris-buffered saline) for 1 h and incubated with antibodies for kidney injury molecule-1 (KIM-1) (1:1,000; R&D systems, Minneapolis, MN), neutrophil gelatinase-associated lipocalin (NGAL), PGC-1α, mitochondrial transcription factor A (TFAM), cytochrome c oxidase subunit 1 (COX1), and NADH dehydrogenase (ubiquinone) FE-S protein 1 (NDUFS1), overnight at 4°C. After incubation for 2 h at room temperature with donkey anti-goat secondary antibody (1:2,000; Santa Cruz Biotechnology Inc., Dallas, TX), goat anti-rabbit (1: 2,0000; Abcam), or rabbit anti-mouse (1:20,000, Abcam) conjugated with horseradish peroxidase before visualization using enhanced chemiluminescence (Thermo Scientific, Waltham, MA) and the GE ImageQuant LAS-4000 (GE Life Sciences, Pittsburgh, PA).Optical density was determined using the National Institutes of Health ImageJ software (version 1.46). Each target protein was normalized to β-actin loading control.

Histology.

Kidney sections (5 microns) from WT and 5-HT_1F receptor KO mice at 24 and 144 h after sham or I/R surgery were stained with periodic acid-Schiff staining. Each section was scored based on morphologic changes in a blinded fashion by a renal pathologist (J. M.). Tubular necrosis and brush border damage were chosen as an indication of morphologic damage to the kidney. These measures were evaluated on a scale from 0 to 4, which ranged from not present, 0; mild, 1; moderate, 2; severe, 3; and very severe, 4.

Quantitative real-time polymerase chain reaction analysis of mRNA expression.

Total RNA was isolated from renal cortical tissue with TRIzol reagent (Life Technologies, Grand Island, NY). The iScript Advanced cDNA Synthesis Kit for quantitative real-time polymerase chain reaction (qRT-PCR; Bio-Rad Laboratories, Hercules, CA) was used to produce a cDNA library from 1 μg total RNA according to the manufacturer’s protocol. For human experiments, we obtained human renal cDNA from Takara Bio USA (Mountain View, CA). We performed qRT-PCR with the generated cDNA using the SsoAdvanced Universal SYBR Green Supermix reagent (Bio-Rad Laboratories). The relative mRNA expression of all genes was determined by the 2^−ΔΔCt method, and β-actin was used as a reference gene for normalization. Primer pairs used for PCR are listed in Table 1.

Analysis of mitochondrial DNA content.

Total DNA was isolated from renal cortex tissue using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA) as described by manufacturer’s protocol. Extracted DNA was quantified, and 5 ng was used for qRT-PCR. Relative DNA content was assessed by the mitochondrial-encoded NADH dehydrogenase subunit 1 and was normalized to nuclear-encoded β-actin. Primer sequences are listed in Table 1.

Determination of ATP content.

Renal cortical ATP was measured using the ATP Assay Kit (Abcam). Briefly, mouse renal cortex was homogenized in ice-cold 2 M perchloric acid using a polytron homogenizer. Homogenate was stored on ice for 45 min and then centrifuged at 13,000 × g for 2 min at 4°C. The supernatant was collected and diluted in the ATP Assay buffer. For each sample, pH was measured and adjusted to 6.5–8 using 0.1 M KOH or perchloric acid. Samples were then centrifuged at 13,000 × g for 15 min at 4°C and supernatant was collected. ATP was measured using a Tecan Spark 10M.

Statistics.

Data are expressed as means ± SE for all the experiments. Multiple comparisons of normally distributed data were analyzed using one-way ANOVA with an appropriate post hoc test; group means were compared using Holm-Sidak’s posttest. Single comparisons were analyzed by Student’s t-test where appropriate. The criterion for statistical differences was P ≤ 0.05 for all comparisons.

RESULTS

Absence of the 5-HT_1F receptor gene does not induce renal injury.

To verify the absence of the 5-HT_1F receptor, we measured 5-HT_1F receptor mRNA in the renal cortex. Renal 5-HT_1F mRNA was decreased 50% in heterozygous (HET) mice and absent in 5-HT_1F receptor KO mice (Fig. 1A).

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.

We evaluated BUN in WT and KO mice. There was no difference in BUN levels at 10 wk of age (WT: 26 ± 3 mg/dl, KO: 25 ± 2 mg/dl). To assess further if the absence of the 5-HT_1F receptor induced renal tubular injury, KIM-1 and NGAL were evaluated in the renal cortex of WT and KO mice. At 10 wk of age, KIM-1 was not detected and NGAL was unchanged in KO mice compared with WT mice (Fig. 1B).

It is important to mention that HET × HET matings yielded Mendelian ratios of 1:2:1 of WT, HET, and KO. In addition, KO × KO matings produced healthy, viable mice with similar increases in body weight over time and no differences in blood counts and serum chemistries (data not shown). Taken together, these data suggest that the KO of the 5-HT_1F receptor alone does not cause renal injury at 10 wk of age.

Absence of the 5-HT_1F receptor potentiates I/R-induced AKI at 24 h.

Although the absence of the 5-HT_1F receptor did not cause renal injury, it is possible that under stress the absence of the 5-HT_1F receptor could potentiate renal injury. To test this hypothesis, we subjected 10-wk-old WT and KO mice to renal I/R injury. Renal function was assessed 24 h after I/R-induced AKI and an increase in serum creatinine in WT and KO AKI mice was observed compared with sham controls (Fig. 2A). However, there was no statistical difference in serum creatinine between genotypes. We also measured two markers of renal injury by immunoblot analysis, KIM-1 and NGAL, in the renal cortex at 24 h. KIM-1 and NGAL increased three- and twofold, respectively, in 5-HT_1F receptor KO mice subjected to I/R injury compared with WT mice subjected to I/R injury (Fig. 2, B–D). It is important to note that 5-HT_1F receptor mRNA decreased by ~50% in WT mice subjected to I/R (Fig. 2E).

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.

Renal histopathology was assessed using periodic acid-Schiff staining. Kidneys from both WT and 5-HT_1F receptor KO mice subjected to I/R injury exhibited proximal tubule necrosis and brush border damage. However, the absence of the 5-HT_1F receptor did not potentiate tubular necrosis or the loss of the brush border at 24 h (Fig. 3). These data indicate that I/R-induced AKI 1) is greater in mice lacking the 5-HT_1F receptor, 2) results in depletion of 5-HT_1F receptor mRNA, and 3) suggests that the 5-HT_1F receptor is renal protective under stress.

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.

Absence of the 5-HT_1F receptor reduces the recovery of renal function.

A previous study demonstrated that the stimulation of MB though the activation of the 5-HT_1F receptor accelerates the recovery of renal function after AKI (11). Thus, we postulated that the absence of the 5-HT_1F receptor would reduce recovery of renal function following injury (11). Renal function and tubular injury were assessed at 24 h and 144 h following I/R-induced AKI in WT and KO mice. All I/R mice had equal initial injury with an overall average serum creatinine of 0.7 ± 0.2 mg/dl at 24 h. However, 5-HT_1F receptor KO and WT mice failed to recover renal function as demonstrated by persistent overall elevated serum creatinine of 0.5 ± 0.2 mg/dl at 144 h (Fig. 4, A and C). To explore renal recovery further in 5-HT_1F receptor KO mice, renal function improvement was calculated as change in serum creatinine (Δ serum creatinine) at 144 h compared with 24 h following AKI in each mouse. Using this analysis, serum creatinine decreased ~0.25 mg/dl in AKI WT mice compared with no decrease in serum creatinine in AKI KO mice (Fig. 4B). It is noteworthy that tubular necrosis and loss of brush border remained elevated and to a similar degree at 144 h following I/R injury (Fig. 5).

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.

To assess renal tubular injury, renal cortical KIM-1 and NGAL levels were measured by immunoblot analysis. KIM-1 and NGAL were upregulated two- and threefold, respectively, in 5-HT_1F receptor KO mice subjected to I/R injury compared with injured WT mice (Fig. 4, C–E). Taken together, these findings provide strong evidence that the loss of the 5-HT_1F receptor potentiates AKI and decreases renal recovery.

Absence of the 5-HT_1F receptor suppresses MB during I/R- induced AKI.

To determine whether greater tubular injury in the 5-HT_1F receptor KO mice was associated with suppressed MB, we assessed renal cortical PGC-1α, the master regulator of MB; electron transport chain (ETC) proteins, nuclear-encoded NDUFS1, mitochondrial-encoded COX1, and TFAM protein at 24 and 144 h in mice subjected to I/R. PGC-1α and TFAM protein were reduced ~50%–60% in KO mice after I/R-induced AKI compared with WT and KO sham mice at 24 h (Fig. 6, A, C, F). No difference in PGC-1α and TFAM protein was detected in I/R injured WT mice (Fig. 6, A, C, F). We assessed the same end points at 144 h following I/R injury. Renal cortical PGC-1α, NDUFS1, COX1, and TFAM proteins were reduced ~50%, 75%, 50%, 60%, respectively, in 5-HT_1F receptor KO mice compared with sham animals of both genotypes at 144 h (Fig. 6, B, G–J). It is important to note that NDUFS1 protein was reduced at 144 h in WT mice following injury and to a further extent in 5-HT_1F receptor KO mice subjected with I/R (Fig. 6, B and H). Similar findings were observed following mRNA analysis of these end points (Fig. 7). These findings suggest that MB is persistently suppressed following AKI and to a greater magnitude in the absence of the 5-HT_1F receptor.

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.

5-HT_1F receptor KO mice exhibit a persistent decrease in ATP levels following I/R-induced renal injury.

To assess whether the absence of the 5-HT_1F receptor alters mitochondrial ATP following I/R-induced AKI, cortical ATP levels were measured. ATP was reduced by ~50% following I/R injury in both genotypes at 24 h (Fig. 8A). However, WT mice exhibited partial recovery in ATP content by 144 h, which was absent in the 5-HT_1F receptor KO mice (Fig. 8B).

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.

Renal physiological mitochondrial homeostasis is disrupted in 5-HT_1F receptor KO mice.

Our laboratory demonstrated that knockdown of the 5-HT_1F receptor in renal proximal tubular cells resulted in a decrease in mitochondrial proteins (11). Thus, we hypothesized that the absence of the 5-HT_1F receptor would lead to a reduction in mitochondrial markers in the renal cortex.

We assessed mRNA expression of MB proteins and components of the ETC, NDUFS1, ATP synthase β, and COX1 and NADH dehydrogenase 1 (ND1). Additionally, we assessed PGC-1α and transcription factors, nuclear respiratory factor-1, nuclear respiratory factor-2 and TFAM at 10 wk of age. Transcript levels of nuclear respiratory factor-2, TFAM, NDUFS1, ATP synthase β, and COX1 were increased (1.4-, 1.4-, 1.7-, 1.3-, and 1.6-fold, respectively) at 10 wk and these increases corresponded with elevated mitochondrial DNA copy number (Fig. 9, A–C). These results reveal the loss of the 5-HT_1F receptor leads to increased MB genes in the kidney.

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.

Mitochondrial fission and mitophagy are critical processes in maintaining mitochondrial homeostasis (20, 29). As such, we measured markers of mitochondrial fission, dynamin-related protein 1 (Drp1) and mitophagy, mitochondrial PTEN-induced putative kinase 1 (PINK1). At 10 wk, Drp1 and PINK1 were elevated 1.7- and 1.6-fold, respectively, compared with WT mice (Fig. 9D). These results reveal that mitochondrial fission and mitophagy may be increased in response to the loss of the 5-HT_1F receptor.

5-HT₁ and 5-HT₂ receptors are expressed in mouse and human kidney.

To determine renal 5-HT₁ and 5-HT₂ receptors present in mice and humans, we measured gene expression of 5-HT₁ and 5-HT₂ receptors. 5-HT_1B, 5-HT_1D, 5-HT_1F, 5-HT_2A, 5-HT_2B, and 5-HT_2C receptor gene expression was detected in human and mice renal samples (Fig. 10A).

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.

Altered renal 5-HT receptor gene expression in 5-HT_1F KO mice.

We explored the possibility that the lack of the 5-HT_1F receptor results in altered gene expression of other 5-HT receptors. Absence of the 5-HT_1F receptor decreased renal mRNA of 5-HT_1B, 5-HT_1D, and 5-HT_2A receptors (~30%, 35%, 45%) at 10 wk, whereas 5-HT_2B receptor mRNA increased ~2.6-fold and 5-HT_2C receptor mRNA did not change compared with WT (Fig. 10B). Thus, altered renal 5-HT receptor gene expression may be a compensatory mechanism in response to the absence of the 5-HT_1F gene.

DISCUSSION

Mice lacking the 5-HT_1F receptor gene were viable and physically similar to WT mice. Having demonstrated that 5-HT_1F receptor-deficient mice did not exhibit an obvious phenotype, we investigated the effects of I/R-induced AKI on renal function and MB in 5-HT_1F receptor KO and WT mice. The rationale for these experiments is supported by our previous findings that demonstrated 5-HT_1F receptor stimulation accelerated renal recovery following I/R-induced AKI and rescued mtDNA copy number in mice (11).

An important role for the 5-HT_1F receptor in the onset and recovery of renal injury following AKI was demonstrated by greater renal tubular injury as measured by KIM-1 and NGAL protein expression in 5-HT_1F receptor KO mice 24 after I/R injury. These data are corroborated by the reduction in 5-HT_1F receptor gene expression at peak renal injury following AKI in WT mice at 24 h, providing evidence that the depletion of the 5-HT_1F receptor is linked to greater renal dysfunction following injury.

The absence of the 5-HT_1F receptor also prevented recovery of renal function as demonstrated by persistently increased serum creatinine and potentiated KIM-1 and NGAL at 144 h. Identification of the 5-HT_1F receptor as a mediator of prolonged renal dysfunction is critical for 1) better understanding of the mechanisms leading to chronic renal dysfunction and 2) better patient outcomes by developing novel therapies that target the 5-HT1F receptor to minimize renal tissue damage and tubular dysfunction. We hypothesize that the greater tubular injury is contributing to the prolonged renal dysfunction following I/R-induced AKI in 5-HT_1F receptor KO mice.

The observation that renal injury was worsened in 5-HT_1F receptor KO mice was also associated with persistent suppression of MB. A notable observation was the early suppression in PGC-1α and TFAM, which are both key regulators of MB. These findings were accompanied by tubular injury; thus, we predict that dysregulation of MB is at least, in part, responsible for reduced recovery of renal function following renal I/R injury. Interestingly, numerous markers of mitochondrial homeostasis, including protein expression of ETC components and ATP levels, were continuously suppressed following I/R injury in 5-HT_1F receptor KO mice. These findings are supported by published results that demonstrated that PGC-1α and MB are protective following both folic-acid and I/R-induced AKI (22, 25, 26). In summary, we propose that the loss of 5-HT_1F receptor-dependent MB is the mechanism responsible for diminished PGC-1α, ETC protein expression, and ATP levels, and this suppression prevents the recovery of tubular injury and renal function in the 5-HT_1F receptor KO mice following injury.

Although 5-HT_1F receptor KO mice did not have gross morphological alterations, nor changes in blood counts and serum chemistries, our data provide clear evidence that mitochondrial homeostasis in renal cortical tissue is disrupted following I/R renal injury. We predicted that the absence of the 5-HT_1F receptor altered MB and homeostasis in naïve 5-HT_1F receptor KO mice. Assimilating various mitochondrial markers revealed that the loss of the 5-HT_1F receptor gene resulted in MB and increased mitochondrial DNA copy number in the naïve kidney. Associated with increased markers of MB was an increase in the mitochondrial fission protein Drp1 and mitophagy marker, PINK1. One explanation for these results is that the loss of the 5-HT_1F receptor and its associated basal control of MB results in a compensatory response to increase MB. Consequently, we hypothesize that mitochondrial fission and mitophagy are increased to maintain a given level of healthy mitochondria.

It is also possible that another 5-HT receptor may compensate for the loss of the 5-HT_1F receptor. This hypothesis is supported by a body of evidence indicating functional cross talk as well as direct interaction between different 5-HT receptors. For example, there is functional cross talk between 5-HT_2A and 5-HT_2C receptors, between 5-HT_1B and 5-HT_2C receptors and the 5-HT_1A and 5-HT₇ receptors (5, 8, 18). In this study, examination of other 5-HT receptors revealed altered gene expression in response to the absence of the 5-HT_1F receptor. Only the 5-HT_2B receptor was upregulated at 10 wk in the kidney of the 5-HT_1F receptor KO mouse. Interestingly, overexpression of the 5-HT_2B receptor leads to increased cardiac mitochondrial enzymatic protein expression and cell number and size in transgenic mice (19). Although the 5-HT_1D receptor has not been linked to mitochondria, 5-HT_2A,_2C receptors have been linked to mitochondrial function in the kidney (14). In fact, 5-HT_2A receptor agonists induce MB in renal proximal tubular cells (14). Other possibilities exist, and future studies are needed to identify the mechanism by which MB is induced in the absence of the 5-HT_1F receptor and the potential role of the other 5-HT receptors.

It is important to note that there is limited knowledge regarding the signaling mechanisms linking 5-HT receptors to MB. To this end, our group recently reported that the 5-HT_1F receptor induces MB through dual mechanisms in renal proximal tubule cells (12). Specifically, following activation of the 5-HT_1F receptor, G beta-gamma complex mediates the induction of the Akt, endothelial nitric oxide, cyclic guanosine-monophosphate, protein kinase G, and PGC-1α pathway, while simultaneously repressing the extracellular signal-regulated kinase 1/2 and forkhead box O3 pathway (12). The 5-HT_1F receptor-induced MB pathways provide insight into how MB is regulated downstream of 5-HT receptors. Elucidation of the signaling mechanisms leading to increased gene expression of renal MB markers in the absence of the 5-HT_1F receptor may reveal additional strategies to target MB following acute organ injuries.

This study provides insight for the role of 5-HT_1F receptor in modulating MB and mitochondrial homeostasis. Mitochondrial dysfunction is a critical pathophysiological mediator in various disease states, including acute organ injury and cardiovascular disease (1, 4, 6, 9, 27). The induction of MB to restore mitochondria number and function increases the energy supply needed for tissue repair. Thus, MB is a promising therapeutic strategy for many human pathologies. However, there are few, nontoxic compounds that selectively induce MB (27). This presents an opportunity to target 5-HT receptors for the treatment of human acute organ injuries and cardiovascular diseases because the 5-HT_1F receptor and other 5-HT₁ and 5-HT₂ receptors are present in human renal tissue. Although classical functional roles of serotonin and serotonin receptors in the cardiovascular system include renal vascular dilation, vasoconstriction, and platelet aggregation (17), future studies will be needed to understand the involvement of 5-HT receptors in MB and homeostasis in the kidney, as well as other organs involved in cardiovascular health.

In summary, these results provide the first evidence that the 5-HT_1F receptor regulates mitochondrial homeostasis in renal cortical tissue under physiological conditions. Our data also demonstrate that the 5-HT_1F receptor plays a protective role in I/R-induced AKI and this protection is mediated through mitochondrial homeostasis and biogenesis. This study sets the stage for the use of 5-HT receptors for the treatment of acute organ injuries that are characterized by mitochondrial dysfunction.

GRANTS

This study was funded by National Institutes of Health National Institute of General Medical Sciences: R01-GM-084147 (to R. Schnellmann), the Biomedical Laboratory Research and Development Program of the Department of Veterans Affairs no. BX000851 (to R. Schnellmann), the National Institutes of Health Ruth L. Kirschstein National Research Services Award no. T32-DK-083262 (to W. Gibbs), and Southwest Environmental Health Sciences Center grant no. P30 ES006694.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

W.S.G., C.C.B., and R.G.S. conceived and designed research; W.S.G., J.B.C., and M.M. performed experiments; W.S.G., J.B.C., M.M., and J.M. analyzed data; W.S.G. interpreted results of experiments; W.S.G. prepared figures; W.S.G. drafted manuscript; W.S.G., C.C.B., and R.G.S. edited and revised manuscript; W.S.G., J.B.C., M.M., C.C.B., J.M., and R.G.S. approved final version of manuscript.