Immunolocalization of H⁺-ATPase subunit α4 and AE1 proteins in the <i>PRR</i>^UB−/− and control kidneys on E18.5.

<p>A–F: Sections were co-stained with anti-H⁺-ATPase subunit α4 (red) and anti-Aqp2 (green) antibodies. The number of H⁺-ATPase subunit α4-positive collecting duct cells appears to be reduced in mutant (D–F) compared to control (A–C) kidneys. G–L: Sections were co-stained with anti-AE1 (red) and anti-Aqp2 (green) antibodies. The number of AE1-positive collecting duct cells appears to be reduced in mutant (J–L) compared to control (G–I) kidneys. CD- collecting duct.</p

Effect of targeted genetic inactivation of the <i>PRR</i> in the UB on <i>Gdnf</i>, <i>Ret</i>, <i>Wnt11</i>, <i>Etv4</i> and <i>Etv5</i> mRNA expression in the mouse metanephros on E14.5.

<p>A–J’: Representative images of E14.5 section <i>in situ</i> hybridization. <i>PRR</i>^UB−/− kidneys (B, D, F, H, J) are smaller and have decreased mRNA expression intensity compared with <i>PRR</i>^UB+/+ kidneys (A, C, E, G, I). High power images of areas shown by dashed line insets appear to show a reduction in <i>Gdnf</i> expression in the mesenchyme (B’ vs. A’) and of <i>Ret</i>, <i>Wnt11</i>, <i>Etv4</i> and <i>Etv5</i> expression at the tips of ureteric buds in <i>PRR</i>^UB−/− (D’, F’, H’, J’) compared with <i>PRR</i>^UB+/+ kidneys (C’, E’, G’, I’), respectively. K: Bar graph showing decreased <i>Gdnf</i>, <i>Ret</i>, <i>Wnt11</i>, <i>Etv4</i> and <i>Etv5</i> mRNA levels in whole E12.5 <i>PRR</i>^UB−/− metanephroi as determined by qRT-PCR. *p<0.001. <i>PRR</i>^UB+/+ value is defined as 1.</p

Immunolocalization of PRR protein in the mouse kidney on embryonic days E13.5 and P1.

<p>A–D: Sections were co-stained with anti-PRR (red staining) and anti-pancytokeratin (green staining) antibodies. PRR is detectable using antibody concentrations of 1/100 in the ureteric bud (UB) (arrows) and glomeruli (G). E: E13.5 wild-type mouse kidney (Kid), intact UBs (iUB) isolated from E11.5 wild-type mouse kidneys and immortalized UB cells (UBc) express PRR mRNA (521 bp). F–H: In <i>PRR</i>^UB+/+ kidney, PRR (red staining) is detected in the cortex (Co) and collecting ducts (CD). CDs are visualized with anti-Aqp-2 antibody (green staining). I–K: In <i>PRR</i>^UB−/− kidney, PRR immunoreactivity is present in the cortex (Co) only and is not detected above the background level in Aqp-2-positive collecting ducts.</p

Effect of targeted genetic inactivation of the <i>PRR</i> in the UB on UB cell apoptosis and proliferation on E12.5.

<p>UBs (A–H) are visualized with anti-pancytokeratin antibody (green). A–D: Proliferating cells are identified by anti-phospho-histone H3 (pH3) antibody (red). E–H: Apoptotic cells are identified by anti-caspase 3 (Casp3) antibody staining (red). All cells in B, D, F and H are identified by DAPI stain (blue). I, J: Bar graphs show the effect of targeted genetic inactivation of the <i>PRR</i> in the UB on the ratio of pH3/DAPI-positive (I) and caspase 3/DAPI- positive (J) cells. *p<0.001.</p

Bar graphs showing water intake, 24-hour urine volume, urine osmolality and urine pH in <i>PRR</i>^UB−/− and <i>PRR</i>^UB+/+ mice on postnatal day P30.

<p>Despite no difference in water intake (A), <i>PRR</i>^UB−/− mice have increased urine volume (B), decreased urine osmolality (C) and increased urine pH after acidic load compared to <i>PRR</i>^UB+/+ mice.</p

Effect of targeted genetic inactivation of the <i>PRR</i> in the ureteric bud (UB) on UB branching and Erk1/2 phosphorylation in E12.5 metanephroi.

<p>A, B: Metanephroi were co-stained with anti-pancytokeratin antibody to visualize the UB (green) and anti-WT1 antibody to visualize metanephric mesenchyme (red). The number of UB tips is reduced in mutant <i>PRR</i>^UB−/− compared with <i>PRR</i>^UB+/+ kidneys. C: Bar graph showing the effect of <i>PRR</i> deletion in the UB on the number of UB tips. D–I: Sections of E12.5 kidneys show that phospho Erk1/2 (pErk1/2) immunostaining (red) is reduced in the UB of mutant (G, I) compared with control (D, F) mice. J. Whole E12.5 kidney lysates were subjected to Western blotting with anti-pErk1/2 antibody. After stripping, the membrane was reprobed with anti-total Erk1/2 antibody (Erk1/2). Erk1/2 phosphorylation appears to be reduced in <i>PRR</i>^UB−/− compared with <i>PRR</i>^UB+/+ kidneys.</p

Effect of targeted genetic inactivation of the <i>PRR</i> in the UB on <i>Aqp2, Foxi1, AE1</i> and H⁺-ATPase subunit <i>α4</i> mRNA expression in the mouse metanephros on E18.5.

<p>A–H: Representative images of E18.5 section <i>in situ</i> hybridization. <i>PRR</i>^UB−/− kidneys show apparent decrease in <i>Aqp2</i> (C, D) and <i>Foxi1</i> (G, H) mRNA expression compared with <i>PRR</i>^UB+/+ kidneys (A, B, E, F). I: Bar graph showing decreased <i>Aqp2, Foxi1, AE1</i> and H⁺-ATPase subunit <i>α4</i> mRNA levels in whole E18.5 <i>PRR</i>^UB−/− metanephroi as determined by qRT-PCR. J: Bar graph showing decreased <i>Wnt2b, Wnt7b</i> and <i>Wnt9b</i> mRNA levels in whole E18.5 <i>PRR</i>^UB−/− metanephroi as determined by qRT-PCR. <i>PRR</i>^UB+/+ value is defined as 1. *p<0.001 vs. <i>PRR</i>^UB+/+.</p

Effect of targeted genetic inactivation of the <i>PRR</i> in the UB on collecting duct (CD) cell apoptosis and proliferation on E18.5.

<p>A–H: CDs are visualized with anti-pancytokeratin antibody (green). A–D: Proliferating cells are identified by anti-phospho-histone H3 (pH3) antibody (red). E–H: Apoptotic cells are identified by anti-caspase 3 (Casp3) antibody staining (red). All cells in B, D, F and H are identified by DAPI stain (blue). I, J: Bar graphs show the effect of targeted genetic inactivation of the <i>PRR</i> in the UB on the ratio of pH3/DAPI-positive (I) and caspase 3/DAPI- positive (J) cells. *p<0.001.</p

Gross images of P1 urogenital blocks and histological sections of P1 kidneys from control (A–D, H, I) and <i>PRR</i>^UB−/− (E–L, J–M) mice.

<p>Mutant kidneys (E, F, K) are smaller than control kidneys (A, B, H). Numbers in panels B and F show kidney length. Hematoxylin and eosin-stained sections show the presence of collecting duct cysts (H, arrows), dilated tubules (G, arrow), thin cortex (Co, brace), and decreased number of nephrons (G, arrowhead) in mutant kidneys. H, I, K, L: Kidney sections co-stained with anti-Wilms tumor 1 (WT1, red) and anti-pancytokeratin (green) antibodies show apparent reduction in WT1 staining in <i>PRR</i>^UB−/− (K, L) compared with <i>PRR</i>^UB+/+ (H, I) mice. J, M: Kidney sections of <i>PRR</i>^UB−/− mice co-stained with anti-Lotus Tetragonolobus Lectin (LTL, green) and anti-pancytokeratin (red) antibodies show that some of the dilated tubules (M, arrow) are from a proximal tubule origin. I, L, M: High power images of areas shown by dashed line insets in H, J and K. N = 3 mice per genotype. Scale bars (A, B, E, F)- 500 µm.</p

Cytotoxicity and gene expression profiling of two hydroxylated polybrominated diphenyl ethers in human H295R adrenocortical carcinoma cells.

Polybrominated diphenyl ethers (PBDEs) are commonly used as flame retardants in a variety of commercial and household products. They have been detected in the environment and accumulate in mammalian tissues and fluids. PBDE toxicity is thought to be associated with endocrine disruption developmental neurotoxicity and changes in fetal development. Although humans are exposed to PBDEs, our knowledge of the effects of PBDE metabolites on human cells with respect to health risk is insufficient. Two hydroxylated PBDEs (OHPBDEs), 2-OH-BDE47 and 2-OH-BDE85, were investigated for their effects on cell viability/proliferation, DNA damage, cell cycle distribution and gene expression profiling in H295R adrenocortical carcinoma cells. We show that the two agents are cytotoxic in a dosedependent manner only at micromolar concentrations, with 2-OH-BDE85 being more toxic than 2-OH-BDE47. However, no DNA damage was observed for either chemical, suggesting that the biological effects of OH-PBDEs occur primarily via non-genotoxic routes. Furthermore, no evidence of aryl hydrocarbon receptor (AHR)-mediated, dioxin-like toxicity was observed. Instead, we report that a micromolar concentration of OH-PBDEs induces transcriptional changes associated with endoplasmic reticulum stress and the unfolded protein response. We discuss whether OH-PBDE bioaccumulation could result in impairment of the adrenocortical secretory function