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First published online December 22, 2008
doi: 10.1242/10.1242/dev.024463

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Met and the epidermal growth factor receptor act cooperatively to regulate final nephron number and maintain collecting duct morphology

¹ Section of Nephrology, Yale University School of Medicine, New Haven, CT 06510, USA.
² Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA.
³ Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
⁴ Department of Nephrology, Hannover Medical School, Hannover, Germany.

View larger version (43K): [in this window] [in a new window]   Fig. 1. Collecting duct knockout of Met expression. (A) Schematic of the Metfl/fl allele showing the location of the loxP sites flanking exon 16 and the primers (A,B,C) used for genotyping. (B) PCR of tail DNA using primers B and C reveals the expected band at 300 bp in wild-type mice, 380 bp in Metfl/fl mice, and both bands in heterozygotes. All three contain the Cre recombinase gene. (C) PCR of DNA from the heart (H), liver (L) and kidney papilla (KP) of Metfl/fl;HoxB7-Cre mice using primers A and C reveals the selective excision of the floxed allele in the kidney to generate the smaller Met16/16 fragment (M, DNA markers). A faint band is present at the expected size of the Metfl/fl allele in the papilla, presumably owing to the small number of thin limb cells present in this tissue. (D) Western analysis of renal papilla from Metfl/fl;HoxB7-Cre and Met+/+;HoxB7-Cre mice. Aqp2 was used as a loading control and immortalized IMCD cells were included as a positive control. (E) Immunostaining of the renal papilla from Metfl/fl;HoxB7-Cre and Met+/+;HoxB7-Cre mice with -Met and -Aqp2 reveals a strong Met signal in the collecting duct cells of wild-type mice that is markedly diminished in the Metfl/fl;HoxB7-Cre mice.

View larger version (66K): [in this window] [in a new window]   Fig. 2. Metfl/fl;HoxB7-Cre mice have reduced nephron number. (A) Representative images of glomeruli at 12 and 52 weeks from Metfl/fl;HoxB7-Cre and Met+/+;HoxB7-Cre mice, as indicated. (B) Quantification of glomerular surface area (in pixels) from kidney sections as in A (n=15 glomeruli/genotype, *P<0.001 versus Met+/+;HoxB7-Cre glomeruli). (C) Representative image of isolated glomeruli from Metfl/fl;HoxB7-Cre and Met+/+;HoxB7-Cre mice. (D) Quantification of total glomeruli/mouse in Metfl/fl;HoxB7-Cre and Met+/+;HoxB7-Cre mouse (n=5 mice/group, *P<0.001).

View larger version (62K): [in this window] [in a new window]   Fig. 3. Metfl/fl;HoxB7-Cre kidney explants have decreased ureteric bud branching. (A) Representative images of E12.5 kidneys from Metfl/fl;HoxB7-Cre and Met+/+;HoxB7-Cre mice (top panels) and E12.5 kidneys grown for 2 days in explant culture (bottom panels), as indicated, and stained with Dolichos biflorus (DBA). (B) Quantification of kidney surface area (in pixels) from E12.5 (n=7) and E14.5 (n=5) kidneys. (C) Quantification of terminal UB branches from E12.5 kidneys stained as in A (n=7). (D) Quantification of surface area of E12.5 kidneys grown as in A for 2 days in explant culture with or without Egf (n=10, *P<0.03 versus +/+ and P<0.001 versus fl/fl+Egf). (E) Quantification of terminal UB branches from E12.5 kidney explants grown as in A for 2 days in culture with or without Egf (n=12, *P<0.02 versus +/+ and P<0.01 versus fl/fl+Egf). (F) Representative image of E12.5 Metfl/fl;HoxB7-Cre explant as in A cultured in the presence of Egf. (G) Real-time PCR for mRNA levels of the indicated factors in E14.5 Metfl/fl;HoxB7-Cre kidneys expressed relative to Met+/+;HoxB7-Cre E14.5 kidneys. n=3 for each factor. *P<0.05 versus Met+/+;HoxB7-Cre kidneys.

View larger version (54K): [in this window] [in a new window]   Fig. 4. Increased expression and activation of Egf receptor in UB-derived cells of Metfl/fl;HoxB7-Cre mice. (A) Immunostaining of E17 explant cryosections from Met+/+;HoxB7-Cre and Metfl/fl;HoxB7-Cre mice with -Egfr and DBA. Cells from the DBA-positive UB tips (arrow) as well as the adjacent renal vesicle (arrowhead, RV) and metanephric mesenchyme show increased Egfr fluorescence. (B-E) Western blot analyses of renal papilla obtained from 6-week-old Met+/+;HoxB7-Cre and Metfl/fl;HoxB7-Cre mice. (B) Immunoblotted with -Egfr and -aquaporin-2, (C) -pEgfr (Y1068) and -E-cadherin, (D) -pEgfr (Y992) and -E-cadherin, (E) -pErk5 and -E-cadherin.

View larger version (47K): [in this window] [in a new window]   Fig. 5. Embryonic kidneys from Metfl/fl;HoxB7-Cre:wa-2/wa-2 mice have decreased UB branching and fail to respond to exogenous Egf. (A) Representative images of E14.5 kidneys from a Metfl/fl;HoxB7-Cre;Egfr+/+ embryo and three separate Metfl/fl;HoxB7-Cre;wa-2/wa-2 littermates stained with DBA to show UB branches. (B) Quantitation of surface area from images of E14.5 kidneys (n=4, *P<0.005). (C) Representative images of E12.5 kidney explants from Metfl/fl;HoxB7-Cre;Egfr+/+ and Metfl/fl;HoxB7-Cre;wa-2/wa-2 mice. Hoffman images on left and corresponding DBA stain on right. (D) Quantification of surface area from explanted kidneys as in C (n=6, *P<0.03 vs. fl/fl; **P<0.003 vs. fl/fl;wa2/wa2+Egf). (E) Kidneys as in C treated with or without Egf (20ng/µl). (F) Quantification of terminal branch number from explanted kidneys as in A (n=5, *P<0.004 versus fl/fl; **P<0.001 versus fl/fl;wa2/wa2+Egf).

View larger version (56K): [in this window] [in a new window]   Fig. 6. Metfl/fl;HoxB7-Cre:wa-2/wa-2 mice have reduced glomerular number and papillary fibrosis. (A) PCR analysis of genomic DNA from Egfr+/+, wa-2/wa-2 and heterozygous mice. (B) Upper panel shows representative kidneys from Metfl/fl;HoxB7-Cre;Egfr+/+, Metfl/fl;HoxB7-Cre;wa-2/+ and Metfl/fl;HoxB7-Cre;wa-2/wa-2 mice at 21 days. The lower panels show western analysis from renal papillas of these same genotypes immunoblotted with antibodies to detect the activated Egfr (-pY1068) and a loading control (Ecad). (C) Quantification of glomeruli/section in Met+/+;HoxB7-Cre;Egfr+/+ (WT), wa-2/wa-2, Metfl/fl;HoxB7-Cre, Metfl/fl;HoxB7-Cre;wa-2/+ and Metfl/fl;HoxB7-Cre;wa-2/wa-2 mice (n=4 kidneys/genotype, *P<0.04 versus WT; **P<0.04 versus Metfl/fl;wa2/wa2; ***P<0.03 versus Metfl/fl). (D) Representative images of trichrome stained sections of the renal papilla from 3-week-old Metfl/fl;HoxB7-Cre;Egfr+/+ and Metfl/fl;HoxB7-Cre;wa-2/wa-2 mice. (E) Serum BUN values from 3-week-old Metfl/fl;HoxB7-Cre;Egfr+/+, Met+/+;wa-2/wa-2 and Metfl/fl;HoxB7-Cre;wa-2/wa-2 mice (n=6, *P<0.002).

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