The Journal of Toxicological Sciences
Online ISSN : 1880-3989
Print ISSN : 0388-1350
ISSN-L : 0388-1350
Original Article
Activation of the arylhydrocarbon receptor through maternal beta-naphthoflavone exposure in the neonatal kidney
Wataru YoshiokaKanta Kikutake
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2025 Volume 50 Issue 4 Pages 161-170

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Abstract

The kidneys of neonates are vulnerable to stressors due to their immature structure and function. Excess activation of the transcription factor arylhydrocarbon receptor (AhR) in the kidneys of neonates can cause severe hydronephrosis, as shown previously using 2,3,7,8-tetrachlorodibenzo-p-dioxin, an AhR agonist. In this study, we aimed to clarify the conditions under which AhR activation leads to hydronephrosis using beta-naphthoflavone (BNF), another potent agonist of AhR. Mouse dams were fed a BNF-containing diet, and the kidneys of their pups were examined. Maternal BNF exposure on postnatal day 1 (PND 1) significantly activated AhR, as evidenced by the increased mRNA levels of the target genes. However, AhR activation was hardly detectable on PND 2 or subsequent days although the mice were continually fed the BNF-containing diet. Further, no hydronephrosis or a related alteration was observed. Similarly, maternal BNF exposure from PND 6 induced significant AhR activation on PND 6 but not on PND 14. The overproduction of prostaglandin E2 (PGE2), which is a pivotal mechanism in the development of neonatal hydronephrosis, was not observed, and no hydronephrosis was observed. These results suggested that the intense activation of AhR on PND 1 or 6 is insufficient to induce overproduction of PGE2 or hydronephrosis. Together with findings from previous studies, we conclude that the development of neonatal hydronephrosis depends on the duration and intensity of AhR activation.

INTRODUCTION

Mammalian kidneys continue to develop after birth. The structural and functional changes occurring in postnatal kidneys (McMahon, 2016; Bueters et al., 2020) enable the transition from intrauterine to extrauterine physiology. As neonatal kidneys are immature, they are vulnerable to exogenous stressors, such as drugs and environmental substances (Sekine and Endou, 2009).

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the hazardous substances to neonatal kidney (Moore et al., 1973; Nishimura et al., 2006). TCDD administration to dams of mice or rats on the day after parturition results in its distribution in the kidneys of pups. This leads to hydronephrosis, a kidney abnormality characterized by dilation of the renal pelvis and calyces in mild cases and destruction of renal parenchyma in severe cases. The point of action of TCDD is the arylhydrocarbon receptor (AhR), a transcription factor activated upon the binding of agonists, such as TCDD (Nebert, 2017), and then it triggers downstream mechanisms that induce hydronephrosis (Yoshioka and Tohyama, 2019). Therefore, AhR activation in the neonatal kidneys is a significant risk factor for hydronephrosis. However, little is known about the risks associated with other AhR agonists, except for TCDD.

The overproduction of prostaglandin E2 (PGE2) is a pivotal downstream mechanism in the pathogenesis of neonatal hydronephrosis via AhR activation. This was evidenced by the genetic ablation of an enzyme responsible for the PGE2 production, which completely prevented hydronephrosis in pups that were exposed to TCDD during lactation (Yoshioka et al., 2012). PGE2 is a lipid mediator important for renal function and development (Nørregaard et al., 2015), and its overproduction can disrupt these functions. Cytosolic phospholipase A2α, (cPLA2α), cyclooxygenase-2 (COX-2), and microsomal prostaglandin E synthase-1 (mPGES-1) are the enzymes that have roles in the process of converting membrane phospholipids to PGE2. The mRNA levels of these enzymes were elevated in neonatal kidneys exposed to TCDD, and they are significantly involved in the incidence of hydronephrosis (Yoshioka and Tohyama, 2019). However, the conditions under which AhR activation upregulates PGE2 synthesis are still unclear. The key problem is determining the period during kidney development when AhR activation leads to the upregulation of PGE2 synthesis because the period is most likely to correspond to the critical window for the development of neonatal hydronephrosis (Couture-Haws et al., 1991).

In this study, we utilized beta-naphthoflavone (BNF), a potent agonis of AhR (Seidel et al., 2000), to elucidate the effects of AhR activation on the mechanisms of hydronephrosis development in neonatal mice. To this end, we developed an experimental model in which maternal BNF exposure elicited an intense AhR activation in the neonatal kidney. Using the model, we first administered BNF the day after birth (PND 1) because AhR activation by TCDD on the same day was shown to cause a high incidence of hydronephrosis (Couture-Haws et al., 1991). Next, we focused on a later day (PND 6) when the PGE2 synthesis is physiologically upregulated (Frölich et al., 2012).

MATERIALS AND METHODS

Materials

Unless otherwise stated, all chemicals and reagents were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). BNF was purchased from Tokyo Chemical Industry (Tokyo, Japan) and Nacalai Tesque (Kyoto, Japan). The radiation-sterilized powder diet was purchased from CLEA (CE-2; CLEA Japan, Tokyo, Japan). BNF was added directly into this powder and mixed thoroughly.

Animals and treatment

The protocols for animal experiments were approved by the Ethical Committee for Vertebrate Experiments at Azabu University (ID#220517-3). Pregnant C57BL/6N mice were purchased from SLC (Shizuoka, Japan). The mice were housed at 24°C ± 2°C and 55% ± 10% humidity under a 12/12 hr light-dark cycle. Parturition was checked daily around noon, and the day of birth was designated postnatal day 0 (PND 0). Dams were included in either of the experimental groups when the number of the pups were from 5 to 7. A powder diet without BNF (control diet) or with BNF and water was provided ad libitum. The percentage of BNF (100 x BNF mas/BNF-containing diet mas) was 1.5%, which was empirically determined to induce intense expression of the AhR target genes, Cyp1a1 and Ahrr, in the kidney of pups. Male and female pups were not discriminated in the analyses because similar incidence and severity of neonatal hydronephrosis as well as basal and induced levels of PGE2 synthesis system in male and female pups were previously reported (Yoshioka et al., 2012; Yoshioka et al., 2014).

Histology

The right kidneys were fixed in 10% neutral-buffered formalin for a week and then kept in 2-propanol. The fixed kidneys were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Hydronephrosis severity scores were assigned as described (Bryant et al., 2001; Theobald and Peterson, 1997; Yoshioka et al., 2016): “0 = no hydronephrosis,” “+1 = slight dilation of the renal pelvis,” “+2 = reduced papilla size and noticeable dilation of the pelvic space,” “+3 = very short papilla and compressed renal tissue,” and “+4 = extremely severe hydronephrotic kidney.” Based on previous studies, the incidence of hydronephrosis in the pups was calculated as the percentage of pups with a severity score ≥ 2.

Urine analyses

Urine was collected from the bladder using a 29-gauge syringe (SS-10M2913A: TERUMO, Tokyo, Japan) and measured using an analytical balance. Urine density was determined based on the mass of 20 μL urine for each specimen. Urine volume was calculated using the mass of total urine and urine density. The data for urine volume included uncertainty because neonatal mice are prone to incontinence. In biochemical assays and osmolality measurement, urine specimens were diluted ten-fold with ultrapure water. Urinary PGE2 (uPGE2) and creatinine (uCre) concentrations were measured using a Prostaglandin E2 EIA kit and a Creatinine assay kit (Cayman Chemical, MI, USA). Urine osmolality was measured using the freezing point depression method with a Fiske 210 Micro-Sample Osmometer (Advanced Instruments, Norwood, MA, USA).

RNA extraction and mRNA quantification

The left kidneys were snap-frozen in liquid nitrogen and stored at −80°C until RNA extraction. Total RNA was isolated from the kidneys using the miRNeasy Mini Kit (Qiagen, Tokyo, Japan). cDNA was synthesized using SuperScript IV VILO Master Mix (Thermo Fisher Scientific, Tokyo, Japan). Real-time PCR was performed using MyGo Pro (IT-IS International, North Yorkshire, UK) with Thunderbird SYBR qPCR Mix (Toyobo, Osaka, Japan). Primers for the targets (Table 1) were designed from the respective mRNA sequences. Amplification efficiencies for the targets were comparable. No-template controls were analyzed in every qPCR to monitor contamination. Melting curve analysis was used to verify the amplification specificity of the products. The mRNA abundance was calculated with the ΔCt method (Schmittgen and Livak, 2008) using the Ct value of a housekeeping gene cyclophilin B for normalization. The Ct values of cyclophilin B were similar among groups: 16.0 ± 0.2 and 15.9 ± 0.4 for the control and BNF groups, respectively, in Experiment 1 and 15.6 ± 0.2 and 15.7 ± 0.2 for the control and BNF groups, respectively, in Experiment 2.

Table 1. Sequences of primers used for qPCR.

Target name Primer sequence (5′ to 3′)
Cyclophilin B Forward GAC TTC ACC AGG GGA GAT GG
Reverse TGT GAG CCA TTG GTG TCT TTG
Cyp1a1 Forward GGC ACC TCT GTT CAC CCT A
Reverse GAA TCT CTC CCT CTG TTC TTG
Cyp1a2 Forward AGT GGA CTT CTT CCC GGT CCT
Reverse GGC ACT TGT GAT GTC TTG GAT ACT
AhRR Forward CAG GGC AGA CAT TGT GGT TA
Reverse CTC CAT TGC TCT TTC CTG CT
cPLA2α Forward AGC ATT CAA AAG GCT TCA CG
Reverse GGG AAA CAG AGC AAC GAG AT
COX-2 Forward TGT GAA CAA TCA AAC AAA ATG ATG
Reverse GCG TAA ATT CCA ACA GCC TAA GT
mPGES-1 Forward CTC AAG CCC TGC TAC CAC A
Reverse GGC CTC AGA CAA GAG ACC AT

Statistical analysis

To minimize the possible litter effects, measured values for individual pups were averaged within a litter. The averaged values were used for statistical analyses. Data are expressed as mean ± standard deviation. Differences in means were analyzed by Welch’s t-test. The statistical significance level was set at p < 0.05.

RESULTS

Experiment 1

To examine the potential impact of maternal BNF exposure on the kidneys of neonatal mice, dams were fed a diet containing 1.5% BNF from PND 1 to PND 14 (Fig. 1A). The body weights of the BNF-exposed and control dams were similar to each other on PND 1 (27.2 ± 0.8 g and 27.2 ± 0.8 g for the control and BNF groups, respectively), on PND 2 (29.2 ± 1.6 g and 28.4 ± 1.4 g for the control and BNF groups, respectively), on PND 3 (28.5 ± 1.5 g and 29.2 ± 0.7 g for the control and BNF groups, respectively), and on PND 14 (31.6 ± 1.2 g and 31.8 ± 1.7 g for the control and BNF groups, respectively). The body weights of the pups in the BNF groups tended to be slightly lower than those in the control groups (Fig. 2A) without any statistically significant difference. The stomachs of all the pups examined in the experiment were filled with white milk. These results suggested that the BNF-exposed dams breast-fed the pups similarly to the control dams.

Fig. 1

Schedules of experiments in this study. PND 0 was the day of parturition. Mouse dams were fed a BNF-containing diet from (A) PND 1 and (B) PND 6, which marks the beginning of Experiments 1 and 2, respectively.

Fig. 2

Effects of maternal BNF exposure on pups in Experiment 1. (A) Body weights of pups. The mRNA levels of (B) Cyp1a1, (C) Cyp1a2, and (D) Ahrr in the kidneys of pups. Data are presented as mean ± standard deviation, and were analyzed using Welch's t-test (3-4 dams/group at each time point, *: p < 0.05).

The activation of AhR in the kidneys of neonates was evaluated by assessing the induction levels of the AhR target genes, Cyp1a1, Cyp1a2, and Ahrr (Nukaya et al., 2009; Mimura et al., 1999; Tijet et al., 2006). Between 3–9 hr during the experiment (Fig. 1A), the lowest abundances of Cyp1a1 mRNA in the BNF groups were between 23-fold and 231-fold higher than the highest abundances in the time-matched control groups. The difference in the abundance between the BNF and control groups was statistically significant at 6 hr (Fig. 2B). Similarly, the Cyp1a2 mRNA abundances in the BNF groups exceeded those in the control groups between 3-24 hr, and the difference was statistically significant at 9 hr. (Fig. 2C). The Ahrr mRNA levels in the BNF groups were significantly higher than those in the control groups at 6 hr and 9 hr (Fig. 2D). The induction levels of the AhR target genes declined after 24 hr although the mice were continually fed the BNF-containing diet.

AhR activation in the neonatal kidneys upregulates the expression of PGE2 synthesis enzymes (Nishimura et al., 2008), which, in turn, inhibits water reabsorption to produce dilute urine (Yoshioka et al., 2016). Therefore, we investigated the potential effects of BNF on the expression of these enzymes. We observed that BNF did not upregulate cPLA2α, COX-2, or mPGES-1 expression at 6 hr (Figs 3A–C), although the expression levels of the AhR target genes were intensely upregulated (Figs. 2B–D). Moreover, the mRNA levels of these enzymes were not elevated at other times. In accordance with the observation, no sign of urine dilution or polyuria was detected on PND 14; the osmolality of the urine in the two groups were similar (1.03 ± 0.10 Osm/kg and 0.97 ± 0.09 Osm/kg for the control and BNF groups, respectively), and the urine volume in the BNF group (31 ± 12 μL) was not larger than that in the control group (110 ± 30 μL). We further confirmed that the kidney of pups on PND 14 in the BNF group had no obvious tissue damage (Fig. 4A, B), including hydronephrosis (Table 2).

Fig. 3

Effects of maternal BNF exposure on prostaglandin synthesis enzymes in Experiment 1. The mRNA levels of (A) cPLA2α, (B) COX-2, and (C) mPGES-1 in the kidneys of pups. Data are presented as mean ± standard deviation, and were analyzed using Welch's t-test (3-4 dams/group at each time point, *: p < 0.05).

Fig. 4

Representative images of the kidneys of pups on PND 14. The kidneys of the (A) control and (B) BNF-exposed groups in Experiment 1 and those of the (C) control and (D) BNF-exposed groups in Experiment 2. See Table 2 for the number of pups examined. An arrow and arrowheads indicate a renal pelvis and a papilla, respectively. Scale bars = 500 μm.

Table 2. Hydronephrosis severity and incidence

Group N a Severity b Incidence c (%)
0 1 ≥ 2
Experiment 1 Cnt 3 3 0 0 0
(PND 1–14) BNF 9 9 0 0 0
Experiment 2 Cnt 4 3 1 0 0
(PND 6–14) BNF 6 5 1 0 0

a The number of pups in each group (3-4 dams/group)

b pups of each severity score

c Severity ≥ 2 was regarded as hydronephrosis.

Experiment 2

As the intense AhR activation on PND 1 was not sufficient to induce the expression of prostaglandin synthesis enzymes (Figs. 1–3), we hypothesized that these enzymes have a critical window to respond to AhR activation. Since the enzymes are highly expressed during PND 4–11 in the developing kidneys (Frölich et al., 2012), we examined the potential effects of AhR activation during this window. Mouse dams were fed a diet containing 1.5% BNF from PND 6 to PND 14 (Fig. 1B). The body weights of the dams were 29.7 ± 0.8 g and 28.3 ± 1.1 g in the control and BNF groups, respectively, on PND 6, and 30.5 ± 0.8 g and 29.2 ± 0.8 g in the control and BNF groups, respectively, on PND 14. The body weights of the pups were 3.79 ± 0.30 g and 3.92 ± 0.27 g in the control and BNF groups, respectively, on PND 6, and 8.75 ± 0.35 g and 8.55 ± 0.48 g in the control and BNF groups, respectively, on PND14. The stomachs of all the pups were filled with white milk, suggesting that BNF-exposed dams breast-fed their pups similarly to the control dams.

At 6 hr on PND 6 (Fig. 1B), the mRNA levels of Cyp1a1 and Cyp1a2 in the neonatal kidneys in the BNF group were significantly higher than those in the control group (Fig. 5A, B). The lowest abundance of Ahrr mRNA in the BNF group exceeded the highest abundance in the control groups (Fig. 5C). Since there was an upper outlier in the BNF group, logarithmic transformation was applied in the statistical analysis. On PND 14, the mRNA abundance of Cyp1a1, Cyp1a2, and Ahrr was at a similar level between the control and BNF groups. These results indicated that AhR was activated at 6 hr but was not on PND 14 despite the continuous feeding of the BNF-containing diet during the experiment.

Fig. 5

Effects of maternal BNF exposure on pups in Experiment 2. The mRNA levels of (A) Cyp1a1, (B) Cyp1a2, (C) Ahrr, (D) cPLA2α, (E) COX-2, and (F) mPGES-1 in the kidneys of pups. (G) The concentration of PGE2 normalized to that of creatinine in urine. (H) Urine osmolality. Data are presented as mean ± standard deviation (3-5 dams/group at each time point), and were analyzed using Welch's t-test (*: p < 0.05, **: p < 0.01, and ***: p < 0.001). In the analysis of the mRNA level of Ahrr, Welch's t-test was applied for logarithmic transformed data (#: p < 0.05).

Neither the expression of prostaglandin synthesis enzymes nor PGE2 excretion into urine was elevated by BNF at 6 hr (Fig. 5). On PND 14, the mRNA levels of cPLA2α and COX-2 were unaltered by BNF exposure, but that of mPGES-1 was significantly higher in the BNF group than the control group. However, this BNF-induced increase was only 1.1-fold, and PGE2 levels were not increased in the urine (Fig. 5G). Urine was not diluted (Fig. 5H), and no hydronephrosis was observed (Fig. 4C, D and Table 2).

DISCUSSION

In this study, we examined the potential effects of BNF, an AhR agonist, on the developing kidneys. Mouse dams were fed a BNF-containing diet, and the AhR activation in the kidneys of the pups was evaluated based on the increase in the mRNA levels of AhR target genes. While this activation was evident 6–9 hr after BNF administration on PND 1, it was hardly detectable after 24 hr despite the continuous feeding of the BNF-containing diet. The decline in AhR activation might be due to the unresponsiveness of AhR in the pups on PND 2 and later. However, contradictorily, AhR activation was observed on PND 6 in the second experiment, in which a BNF-containing diet was given from PND 6. In addition, AhR activation was reported in the kidneys of pups on PND 7–21 and adult mice (Nishimura et al., 2008; Yoshioka et al., 2016). These results suggest that AhR remains responsive to agonists throughout kidney development. Another potential mechanism may be that BNF was cleared by drug-metabolizing enzymes induced by activated AhR, resulting in suppression of AhR activation due to the depletion of the agonist. This mechanism is plausible since the rapid clearance of BNF in the rats continuously infused with BNF was reported (Adedoyin et al., 1993; Chen et al., 2010). In these rat studies, the plasma concentration of BNF reached the maximum around 100 min post initiation of BNF infusion, and then rapidly declined during 100–300 min. Such a decline in the later phase suggested an increase in BNF clearance. Consistently, BNF is a substrate of cytochrome P450 enzymes (Vyas et al., 1983; Lee et al., 2022), which are the drug-metabolizing enzymes induced by AhR activation (Nukaya et al., 2009). BNF could be metabolized in the body of the dam as well as in the body of the pups, which is thought to have a potential to lower the level of BNF-exposure via milk to the pups in the experiments. Although the precise mechanisms are still unknown, maternal BNF exposure was shown to induce transient activation of AhR in the neonatal kidneys.

While AhR activation in the neonatal period is known to induce hydronephrosis (Moore et al., 1973; Nishimura et al., 2008), the conditions that elicit neonatal hydronephrosis are unclear. There is a critical window for neonatal hydronephrosis. Couture-Haws et al. found that TCDD administration to mouse dams on PND 1 induced severe hydronephrosis at a high incidence in the pups, but TCDD administration on PND 4 barely induced hydronephrosis (Couture-Haws et al., 1991). This indicated that the critical window resides between PND 1 and PND 4. The exact span of the window is unclear because TCDD persists in the bodies of animals (Gasiewicz et al., 1983; Birnbaum, 1986). The current findings reveal that AhR activation during 3–9 hr on PND 1 is not sufficient to induce the development of hydronephrosis. A non-persistent AhR agonist could be useful in narrowing down the span of the critical window for hydronephrosis development. However, AhR activation by BNF is too transient for this purpose. Using other AhR agonists that activate AhR for intermediate durations might clarify the relationship between the duration of AhR activation and hydronephrosis development.

The extent of AhR activation in the kidneys of neonates was prominent. The average Cyp1a1 mRNA level in the BNF groups was 645-fold higher than in the control group on PND 1. This fold increase was comparable to those in the kidneys of neonates lactationally exposed to TCDD, the most potent agonist of AhR; the fold increases by TCDD exposure were 435-fold (Yoshioka et al., 2014), 351-fold (Yoshioka et al., 2012), 174-fold (Aida-Yasuoka et al., 2014), and 900-fold (Yoshioka et al., 2016). In these TCDD studies, the doses ranged 10–20 μg/kg body weight and hydronephrosis incidences ranged from 58% to 100%. The Cyp1a1 induction did not reach a plateau at 20 μg/kg body weight, for a higher level of Cyp1a1 induction was observed at 80 μg/kg body weight (Yoshioka et al., 2016). Contrastingly, no hydronephrosis was observed in this study despite strong AhR activation (Table 2). This suggests that the intensity of AhR activation is not solely responsible for causing neonatal hydronephrosis, and at least one more factor should be required for this. A candidate factor is the duration of AhR activation because AhR activation can be detected for 3 weeks after TCDD administration on PND 1 (Nishimura et al., 2008), while it disappeared 24 hours after BNF administration on PND 1 (Fig. 2). This significant difference in the duration of AhR activation could explain the difference in the adverse effects. It should be noted that intense AhR activation is a necessary condition, if not a sufficient condition, because no sign of hydronephrosis is observed in the kidney of mice expressing mutant AhR, which is continuously but mildly active (Brunnberg et al., 2006).

PGE2 is a major prostaglandin in the kidney (Nørregaard et al., 2015). COX-2 and mPGES-1 are prostaglandin synthesis enzymes inducible in response to physiological and pathophysiological stimuli, and have important roles in PGE2 production. Either genetic ablation or pharmacological inhibition of COX-2 causes defects in renal development (Slattery et al., 2016). The importance of PGE2 is evident by the defects observed in the development and functions of kidneys of mice deficient in the PGE2 receptors (Frölich et al., 2012; Fuchs et al., 2022). In the kidneys of neonatal mice, the expression of COX-2 and mPGES-1 are upregulated around PND 6, when elevated PGE2 levels are observed in the urine (Frölich et al., 2012; Kömhoff et al., 2000). The elevated urinary PGE2 level on PND 6 was confirmed in this study (Cnt 6 hr vs Cnt 2 wks in Fig. 5G: p = 0.011). During this stage of kidney development, AhR activation by TCDD induces abnormal upregulation of the COX-2/mPGES-1/PGE2 axis, which is critical for hydronephrosis (Yoshioka and Tohyama, 2019). Importantly, AhR activation in the adult kidneys does not upregulate the COX-2/mPGES-1/PGE2 axis or cause hydronephrosis. Thus, it is thought that inducibility of this axis determines the critical window for neonatal hydronephrosis, and that the genes encoding COX-2 and mPGES-1 are temporally accessible by transcription factors during the critical window. However, AhR is unlikely to have a direct role in the transcription of COX-2 and mPGES-1 since BNF-induced AhR activation did not augment the expression of COX-2 and mPGES-1 on PND 6. A potential key factor is cPLA2α because the genetic ablation of cPLA2α suppresses the TCDD-induced increases in COX-2 and mPGES-1 mRNAs (Yoshioka et al., 2014), and cPLA2α can be enzymatically activated and/or transcriptionally upregulated by AhR (Kinehara et al., 2009; Dong and Matsumura, 2008). Currently, the molecular mechanism in which cPLA2α is involved in developing hydronephrosis has not been elucidated, and the exact pathway that links AhR activation and PGE2 overproduction is still an enigma.

In conclusion, maternal BNF exposure resulted in strong AhR activation in the kidneys of mouse neonates. The AhR activation was transient and did not cause an incidence of hydronephrosis in the neonates, suggesting that the intensity of AhR activation alone is not sufficient to induce hydronephrosis in the neonates and that the duration of AhR activation is also required.

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES
 
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