A mutant Ahr allele protects the embryonic kidney from hydrocarbon-induced deficits in fetal programming

Abstract

Background: The use of experimental model systems has expedited the elucidation of pathogenetic mechanisms of renal developmental disease in humans and the identification of genes that orchestrate developmental programming during nephrogenesis.

Objectives: We conducted studies to evaluate the role of AHR polymorphisms in the disruption of renal developmental programming by benzo(a)pyrene (BaP).

Methods: We used metanephric cultures of C57BL/6J (C57) mice expressing the Ahr(b-1) allele and B6.D2N-Ahr(d)/J (D2N) mice expressing a mutant allele deficient in ligand binding (Ahr(d)) to investigate molecular mechanisms of renal development. Deficits in fetal programming were evaluated in the offspring of pregnant mice treated with BaP during nephrogenesis.

Results: Hydrocarbon challenge of metanephri from C57 mice altered Wilms' tumor suppressor gene (Wt1) mRNA splice variant ratios and reduced mRNAs of the Wt1 transcriptional targets syndecan-1 (Sdc1) paired box gene 2 (Pax2), epidermal growth factor receptor (Egfr), and retinoic acid receptor, alpha (Rarα). These changes correlated with down-regulation of effectors of differentiation [secreted frizzled-related sequence protein 1 (Sfrp1), insulin-like growth factor 1 receptor (Igf1r), wingless-related MMTV-integration site 4 (Wnt4), Lim homeobox protein 1 (Lhx1), E-cadherin]. In contrast, metanephri from D2N mice were spared hydrocarbon-induced changes in Wt1 splice variant ratios and deficits of differentiation. We observed similar patterns of dysmorphogenesis and progressive loss of renal function at postnatal weeks 7 and 52 in the offspring of pregnant C57 but not D2N mice gavaged with 0.1 or 0.5 mg/kg BaP on gestation days 10-13.

Conclusions: These findings support a functional link between AHR and WT1 in the regulation of renal morphogenesis and raise important questions about the contribution of human AHR polymorphisms to the fetal origins of adult-onset kidney disease.

Figure 1

BaP inhibits nephrogenesis via an Ahr allele–specific mechanism, as shown by metanephric cultures treated with 3 µM BaP or DMSO without or with α-NF (see “Materials and Methods” for details). (A) Photomicrographs of C57 and D2N metanephri stained with H&E. BaP-exposed C57 metanephri display less morphologically distinct differentiated structures compared with the C57 DMSO control. Expression of the Ahr^d allele in D2N mice abrogates BaP-induced deficits; co‑treatment with the competitive inhibitor α-NF also inhibited BaP effects. Abbreviations: C, comma-shaped bodies; g, glomeruli; s, S-shaped bodies. Bars = 100 µm. (B and C) Quantification (mean ± SD) of glomeruli (B) and comma and S‑shaped bodies (C) normalized to area from serial sections (n ≥ 6 metanephri/group). *p < 0.05 compared with the corresponding DMSO control, by ANOVA and LSD post hoc tests.

Figure 2

AHR expression correlates with nephrogenesis, as shown by immunohistochemical analysis of metanephric cultures treated with 3 µM BaP or DMSO without or with α-NF (see “Materials and Methods” for details). Exposure to BaP decreases AHR protein levels, as indicated by DAB staining (A) and density (mean ± SD) of AHR normalized to total area (B). Bars = 100 µm. Protein expression was similar in DMSO-treated C57 and D2N metanephri, but D2N and α-NF–co-treated C57 metanephri were not sensitive to BaP-induced deficits in AHR protein expression. *p < 0.05 compared with the corresponding DMSO control, by ANOVA and LSD post hoc tests.

Figure 3

BaP exposure down-regulates markers of renal cell differentiation, as shown by qRT‑PCR analysis of BaP-treated C57 (A) and D2N (B) metanephric cultures (see “Materials and Methods” for details). Values (mean ± SD) represent 2^−∆∆CT normalized fold change relative to the DMSO CF7 control (dashed line); n ≥ 6 metanephri/group. At 4 days of BaP exposure, C57 metanephri (A) but not in D2N metanephri (B) showed significant decreases in differentiation markers. *p < 0.05, by Wilcoxon rank sum test.

Figure 4

WT1 dysregulation by BaP correlates with loss of known WT1 targets in C57 metanephri, as shown by qRT‑PCR analysis (see “Materials and Methods” for details). Values (mean ± SD) represent 2^−∆∆CT normalized fold change relative to the DMSO control (shown as a dashed line in E); n ≥ 6 metanephri/group. (A–D) Abundance of splice variants from BaP-treated and DMSO control samples. After 4 days of BaP exposure, significant increases were seen in –KTS (B) compared with +KTS (A) isoforms but not in +17aa (C) or –17aa (D) isoforms. No changes were seen in total Wt1 mRNA expression. (E) Down-regulation of known WT1 targets correlated with changes in WT1 isoform abundance. *p < 0.05, by Wilcoxon rank sum test.

Figure 5

WT1 dysregulation by BaP is Ahr allele specific in D2N-Ahr^d/d metanephri, as shown by qRT‑PCR analysis (see “Materials and Methods” for details). Values (mean ± SD) represent 2^−∆∆CT normalized fold change relative to the DMSO D2N control (shown as a dashed line in E); n ≥ 6 metanephri/group. (A–D) Abundance of splice variants [(A) +KTS, (B) –KTS, (C) +17aa, and (D) –17aa)] from BaP-treated and DMSO CF7 control samples. Expression of the Ahr^d/d allele abrogated BaP-induced modulation of Wt1 mRNA isoforms in D2N mice. No changes were seen in total Wt1 mRNA expression. (E) Down-regulation of known WT1 targets correlated with changes in WT1 isoform abundance. 18S, β‑actin, and GADPH were run as internal controls for all qRT-PCR reactions. While all controls tested demonstrated tolerable variability, 18S provided the optimal reproducibility in our assays. *p < 0.05, by Wilcoxon rank sum test.

Figure 6

BaP inhibits nephrogenesis via an Ahr allele–specific mechanism, as shown by glomerular number normalized per area (mean ± SD; A) and photomicrographs of eosin-stained cross sections (bars = 200 μm; B) from kidneys resected from 7‑day old C57^b1/b1 and D2N-AHR^d/d mice exposed to 0.1 or 0.5 mg/kg BaP or MCT oil vehicle in utero (see “Materials and Methods” for details). g, glomeruli. Bars = 200 μm. *p < 0.05 compared with MCT, by ANOVA and LSD post hoc tests.

Figure 7

BaP exposure induces glomerular-specific injury, as shown by albumin (A,B), podocyte number (C), and total WT1 signal (D) in urine collected from 52‑week-old C57^b1/b1 and D2N-AHR^d/d mice exposed to 0.1 or 0.5 mg/kg BaP or MCT oil in utero (see “Materials and Methods” for details). (A) Expression of the Ahr^d/d allele abrogates BaP-induced alterations in albumin urinary levels in C57 mice compared with D2N mice. (B) Silver stain visualization of mouse urinary albumin in C57 mice exposed in utero to 0.5 mg/kg MCT or BaP. (C) Immunohistochemical analysis of podocyte numbers quantified using WT1 signal filtered for intensity, color, and size normalized to glomerular density. (D) Immunohistochemical analysis of total WT1 signal quantified using WT1 normalized to podocyte numbers. For A, C, and D, data are mean ± SD. *p < 0.05 compared with the corresponding MCT control, by ANOVA and LSD post hoc tests.

Figure 8

BaP exposure does not induce collecting duct or distal tubular injury, as shown by absorbances from enzyme immunoassay detection of RPA1 (A) and GSTYb1 (B) in urine collected from 52‑week-old C57^b1/b1 and D2N-AHR^d/d mice exposed to 0.1 or 0.5 mg/kg BaP or MCT oil in utero (see “Materials and Methods” for details). Absorbances were normalized to internal controls and expressed as relative units (mean ± SD). *p < 0.05 compared with the corresponding MCT control, by ANOVA and LSD post hoc tests.