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First published online August 18, 2003
doi: 10.1242/10.1242/dev.00666

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Crucial roles of Brn1 in distal tubule formation and function in mouse kidney

Shigeyasu Nakai1,6, Yoshinobu Sugitani1, Hiroshi Sato2, Sadayoshi Ito2, Yukio Miura3, Masaharu Ogawa4, Miyuki Nishi1, Kou-ichi Jishage1, Osamu Minowa1,5 and Tetsuo Noda1,5,6,*

1 Department of Cell Biology, Japanese Foundation for Cancer Research (JFCR) Cancer Institute, 1-37-1 Kami-Ikebukuro, Toshima-Ku, Tokyo 170-8455, Japan
2 Division of Nephrology, Endocrinology, and Vascular Medicine, Tohoku University School of Medicine, Sendai 980-8574, Japan
3 Miyagi Insurance Hospital, Sendai 981-1103, Japan
4 Laboratory for Cell Culture Development, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan
5 Mouse Functional Genomics Research Group, RIKEN Genomic Sciences Center, Kanagawa 244-0804, Japan
6 Division of Molecular Genetics, Center for Translational and Advanced Animal Researches on Human Diseases, Tohoku University School of Medicine, Sendai 980-8575, Japan



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  		Fig. 1. Generation of Brn1 knockout mice and histological changes in their kidneys at birth. (A) Representation of the wild-type allele, targeting vector and targeted allele of Brn1. The Brn1 open reading frame is shown as a black box. The locations of the external 3' and internal 5' probes are indicated. NEO, neomycin-resistance gene driven by phosphoglycerate kinase gene promoter; DTA, diphtheria toxin A-chain gene; E, EcoRI; B, BamHI; X, XhoI; N, NotI; A, ApaI. (B) Western blot analysis of kidney extracts from newborn Brn1 mutants. The specific band of Brn1 detected by a polyclonal antiserum is reduced in Brn1+/- mice and absent from Brn1-/- mice. (C) Electrophoresis mobility-shift assay (EMSA) using brain extracts derived from newborn Brn1 mutants. Cell extracts of Brn1-transfected NIH 3T3 cells (3T3-Brn1) served as a Brn1 protein control. Lysates of P19 cells treated with retinoic acid (P19-RA) were used as a Brn2 protein-positive control. (D,E) Staining of the kidney medulla derived from Brn1+/+ (D) and Brn1-/- (E) mice with Hematoxylin and periodic acid-Schiff (PAS). The collecting ducts (CD) in Brn1-/- kidneys are comparable with those of the Brn1+/+ kidney. The lops of Henle (HL), however, are absent from the Brn1-/- kidney. Interstitial cells (IC) are prominent in the Brn1-/- kidney in comparison with the wild-type kidney. (F,G) Cortices of Brn1+/+ (F) and Brn1-/- (G) kidneys stained with Hematoxylin and Eosin. No significant differences in cortex morphology were observed between Brn1+/+ and Brn1-/- kidneys. Gl, glomerulus. Scale bar: 50 µm.

 


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  		Fig. 2. Brn1 is expressed in the developing loop of Henle (HL) and distal convoluted tubule (DCT) during nephrogenesis. Brn1 immunostaining (A,C,E,G,H) and schematic drawings of the key stages of nephron development (B,D,F,I). Brn1 immunoreactivity is present in sections of the renal vesicle (RV; A,B); within the prospective HL, the DCT and the macula densa of nascent S-shaped bodies (C,D); within the elongating HL (E,F); and within the thick ascending limb (TAL), macula densa (MD) and DCT (G-I) of the newborn kidney. Brn1- positive region of the nephron are indicated by a red outline in the schematic drawings (B,D,F,I). RV, renal vesicle; BC, Bowman's capsule; PCT, proximal convoluted tubule; CNT, connecting tubule; tDL, thin descending limb. Scale bar: 20 µm. (B,D,F,I) Modified, with permission, from Fischer et al. (Fischer et al., 1995Go).

 


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  		Fig. 3. Arrest of loop of Henle (HL) elongation at the primitive loop stage in Brn1-deficient kidney. (A-C) Classification of HL developmental stages; anlage (A), primitive loop (B) and immature loop (C). Each developing HL stage is outlined by arrowheads. (D) Schematic drawing of the anlage, primitive loop and immature loop of Henle. (E,F) Quantitation of the numbers of primitive loops and immature loops of Henle in Brn1-deficient kidneys. All observable independent HLs were counted within one median longitudinal section from each animal at E16.5. The number of primitive loops of Henle in Brn1-/- kidneys increased significantly from the levels seen in Brn1+/+ kidneys (E). However, no immature loops of Henle were identified in Brn1-/- kidneys (F). Data are shown as the mean±s.e.m. (n=3 or 4). *P<0.05 compared with Brn1+/+ kidney (ANOVA). (G-L) BrdU labeling in the anlage and primitive loops of Henle at E16.5. No significant difference in the numbers of BrdU-incorporated cells between Brn1+/+ and Brn1-/- kidneys was detectable at the anlage stage (G-I). At the primitive loop stage, the number of cells incorporating BrdU was significantly decreased in Brn1-/- kidney in comparison with the Brn1+/+ kidney (J-L). Data are shown as the mean±s.e.m. (n=3 or 4). *P<0.05 compared with Brn1+/+ kidney (ANOVA). (M-P) TUNEL analysis of primitive and immature loops of Henle at P0. In the Brn1+/+ kidney, TUNEL-positive cells were detected in the bend of the immature loop of Henle, near the papillary tip of medulla (O, arrow). TUNEL-positive cells were never observed in the primitive loop (M). In the Brn1-/- kidney, TUNEL-positive cells were present near the bend of primitive loop (N, arrows). The HL could not be identified in the papilla of Brn1-/- kidneys (P). (Q-T) Active caspase 3 immunostaining of primitive and immature loops of Henle at P0. In the Brn1+/+ kidney, active caspase 3-positive cells were detected in the bend of the immature loop of Henle, near the papillary tip of medulla (S, arrow). Active caspase 3-positive cells were never observed in the primitive loop (Q). In the Brn1-/- kidney, active caspase 3-positive cells were present near the bend of primitive loop (R, arrow). The HL could not be identified in the papilla of Brn1-/- kidneys (T). Scale bars: 20 µm.

 


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  		Fig. 4. Differentiation of the TAL, MD and DCT was impaired in the Brn1-deficient kidney. (A) In situ hybridization analyses of the TAL in newborn Brn1+/+ and Brn1-/- kidneys. The expression of the Tamm-Horsfall glycoprotein gene (Umod), the prostaglandin E2 receptor subtype EP3 gene (Ptger3) and bumetanide-sensitive Na-K- 2Cl co-transporter gene (Nkcc2/Slc12a1) was detected in the TAL of the Brn1+/+, but not the Brn1-/- kidney, suggesting that TAL differentiation was impaired in Brn1-deficient animals. Scale bar: 50 µm. (B) Histological and in situ hybridization analyses of the MD and the surrounding TAL cells in Brn1+/+ and Brn1-/- kidneys. PAS staining demonstrates that the MD in Brn1+/+ kidneys (arrows) possesses the defining MD features; cells are crowded and protrude into the tubular lumen. The putative MD in Brn1-/- mice (arrowheads) does not display these features. Using in situ hybridization analysis, expression of the constitutive type 1 isoform of nitric oxide synthase gene (Nos1) was detected in the MD of the Brn1+/+, but not the Brn1-/- kidney. Ptger3 expression was detected in both the MD and the surrounding TAL cells of Brn1+/+ kidney but was absent from the Brn1-/- kidney. Scale bar: 20 µm. (C) In situ hybridization analysis of DCT in Brn1+/+ and Brn1-/- kidneys. The expression of the thiazide-sensitive Na-Cl co-transporter gene (Ncc/Slc12a3), a maker for DCT, was detected in Brn1+/+ but not Brn1-/- kidneys. The expression of the Na/Ca exchanger gene (Ncx1/Slc8a1), a maker of the distal part of the DCT and for the CNT, was altered in Brn1-/- kidneys in comparison with that of Brn1+/+ kidneys, suggesting that Ncx1 expression in Brn1-/- kidneys remained only in the CNT. The expression of the amirolide-sensitive epithelial Na+ channel gene (ßEnaC/Scnn1b), a marker for CNT and CD, was indistinguishable in Brn1+/+ kidney from that in Brn1-/- kidneys. Scale bars: 20 µm.

 


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  		Fig. 6. Brn1 gene dosage effects on gene expression levels in Brn1 mutant kidneys. We performed an RNase protection assay (RPA) using total RNA from newborn and adult kidneys. The expression of Umod, Ptger3, Nkcc2 and Kcnj1 (encoding apical K+ channel) were nearly undetectable in the kidneys of newborn Brn1-/- mice. The mRNA level of epidermal growth factor (Egf) in the Brn1-/- kidney was reduced to ~20% to that of the wild type in newborn. The mRNA levels of the basolateral TAL Cl- channel (Clcnk1l) and its ß-subunit (Bsnd) in the Brn1-/- kidney were reduced to about 30% of wild-type levels in newborn. The mRNA levels of Nkcc2, Bsnd and Kcnj1, indispensable regulators of Na+ reabsorption, were significantly reduced in Brn1+/- kidneys in comparison with the levels of Brn1+/+ kidney in both newborn and adult kidneys. The mRNA levels of Umod and Ptger3 in Brn1+/- kidneys were also significantly reduced in comparison with Brn1+/+ kidneys. The mean values and s.e.m. of the ratios of the mutant to the wild-type signals are displayed at the right of the protected band patterns (n=3-4). All data were normalized to the Gapd signal prior to statistical analysis. *P<0.05, **P<0.001 compared with Brn1+/+ kidney (ANOVA).

 


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  		Fig. 5. Transmission electron micrographs of the MD and the surrounding TAL cells of Brn1+/+ (left) and Brn1-/- (right) kidneys. MD cells of the Brn1+/+ kidney exhibit characteristic features, including multiple luminal microvilli and basal and lateral membrane foldings. TAL cells of the Brn1+/+ kidney display characteristic morphology, such as lateral extension, abundant microvilli, basal and lateral membrane foldings and cellular interdigitation. The putative TAL cells and MD cells of the Brn1-/- kidney are indistinguishable from each other, exhibiting immature morphology, including simple cell membranes, sparse luminal microvilli, and small and sparse mitochondria with the cytoplasm. Scale bar: 2 µm.

 

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© The Company of Biologists Ltd 2003