1. Volume 3, Issue 1, Pages 1-10 (January 1999)
Role of the Angiotensin Type 2 Receptor Gene in Congenital Anomalies of the Kidney and Urinary Tract, CAKUT, of Mice and Men 
Hideki Nishimura, Elizabeth Yerkes, Katharina Hohenfellner, Yoichi Miyazaki, Ji Ma, Tracy E Hunley, Hiroaki Yoshida, Toshihiro Ichiki, David Threadgill, John A Phillips, Brigid M.L Hogan, Agnes Fogo, John W Brock, Tadashi Inagami, Iekuni Ichikawa 
Molecular Cell 
Volume 3, Issue 1, Pages 1-10 (January 1999)
DOI: /S (00)

2. Figure 1
Mild Vesicoureteral Reflux in Agtr2 Null Mutant Mice (Agtr2−/Y)
Wild-type (n = 10) and Agtr2−/Y (n = 9) anesthetized males from line 3 that lacked gross anomalies were subjected to cystometrogram at 4–8 weeks of age. Intravesical pressure was progressively raised by continuous infusion of 1% lissamine green B solution while the level of intravesical pressure was monitored. The minimum pressure required for backflux of dye from the bladder to the ureter was recorded. This minimum pressure was lower in Agtr2−/Y mice. VUR was seen in 7 out of 9 Agtr2−/Y mice (4 were on the right side only; 3 were on both sides). Closed circles represent backflux; open circles represent void or rupture (i.e., a higher, undetermined pressure is required to cause backflux).
Molecular Cell 1999 3, 1-10DOI: ( /S (00) )

3. Figure 2
Apoptotic Activity of the Undifferentiated Mesenchymal Cells around the Ureter and Metanephros of Wild-Type (Agtr2+/Y) and Agtr2 Null Mutant Embryos (Agtr2−/Y) Studied In Vivo and In Vitro
(a) The insert presents apoptotic cells identified by ISNT (shown by arrows; the black bar indicates 50 μm; counter staining is methyl green). The density of ISNT-positive cells in UMCs was determined by the number of positive cells/mm2 in each section. ISNT-positive cells are present in significantly less abundance (P < 0.05) in E16.5 Agtr2−/Y embryos when compared with Agtr2+/Y littermates.
(b) The effect of exogenous Ang II on the cell apoptosis of E14.5-old ureteral tissues cultured in vitro. The number of apoptotic cells was quantitated as a percentage of the ISNT-positive cells against the total number of UMCs surrounding the ureter. In tissues from wild-type animals, Ang II caused a significant (P < 0.001) increase in the number of apoptotic cells, whereas in tissues from Agtr2 null mutant embryos, Ang II treatment had no effect.
Molecular Cell 1999 3, 1-10DOI: ( /S (00) )

4. Figure 3
Overview of the Phenotypes of Mice in Line 2 Agtr2 Null Mutant Mice
Line 2 was founded by a male hemizygote and female homozygote pair so that no offspring within this line carries a normal, wild-type, Agtr2 allele. The numbers within symbols represent the number of mice with normal gross appearance. Affected males (squares) and females (circles) are shown in black. Unaffected mice are shown by open symbols. The asterisk shows F6 hemizygotes whose parents were different. It is apparent that the pattern of anomalies is markedly diverse, and there appears to be no rule for the appearance of any of the specific anomalies. They occur predominantly in males. The mode of penetrance is not typical Mendelian, but incomplete, with an average frequency of 21%. Note that minor phenotypes, such as mild VUR, double pelvis and/or ureter, kidney malrotation, and abnormality of kidney position, are not indicated in this chart. When more than one abnormal phenotype appears on one side, only the single most prominent feature is listed. Those with two features indicate bilateral involvement of CAKUT. UPJ, hydronephrotic kidneys with a stenosis or atresia at the level of ureteropelvic junction; UVJ, both hydronephrotic kidney and hydroureter with ureterovesical junction obstruction or vesicoureteral reflux; MD, multicystic dysplastic kidney; HK, hypoplastic kidney; MU, megaureter; AK, aplastic kidney.
Molecular Cell 1999 3, 1-10DOI: ( /S (00) )

5. Figure 4
Gross Anatomy and Light Microscopic Patterns of a Few Forms of the Congenital Anomalies of Kidney and Urinary Tract Found in Agtr2 Null Mutant Pups and Embryos
The schematic drawing on the top shows the spectrum of CAKUT found in Agtr2−/Y mice. (a) UPJ; (b) UPJ with atretic ureter; white arrows indicate ureteral atresia. (c) UVJ; (d) HK with microscopically dysplastic kidney; (e) MD with polycystic lesions within the kidney; a black arrow indicates a multicystic dysplastic lesion. (f) AK, that is, no trace of renal tissue despite a presence of the ureter. The histology of affected kidneys is characterized by either of two distinct patterns. One is “hydronephrosis” with thinning of renal parenchyma without interstitial fibrosis or cellular infiltration (g). The other is “cystic dysplastic” renal parenchyma, which includes cysts of various sizes, premature tubules, and glomeruli surrounded by undifferentiated mesenchymal cells (h). An E18.5 Agtr2 null mutant embryo has an apparent vesicoureteral junction stenosis or ureterovesical reflux (i). The affected ureter is markedly dilated and tortuous. (j) Double ureters, obtained from an E17.5 embryo. Upper pole of the kidney appears to have hydronephrosis attached to hydroureter (white arrow), while the remaining kidney tissue appears normal with nonhydrotic ureter (black arrow). Agtr2 null mutant embryos often carry subtle abnormalities that are similar to those shown in (k), which is taken from an E17.5 embryo. The left ureter is tortuous and winding (arrow), encased within the undifferentiated mesenchymal tissue. This pattern of tortuous ureter closely resembles the appearance of hydroureter typically found in Agtr2 null mutants after birth, including the one shown in (l) (1 week of age). White or black bars indicate 1 cm, 200 μm, and 1 mm for (a)–(f), (g) and (h), and (i)–(l), respectively. (g) H and E staining; (h) Masson trichrome staining.
Molecular Cell 1999 3, 1-10DOI: ( /S (00) )

6. Figure 5
Tissue Distribution of Agtr2 mRNA and Localization of Undifferentiated Mesenchymal Cells within the Kidney and Urinary Tract System According to the Embryonic Stages (Wild-Type)
Two adjacent transverse sections of embryonic tissues (all wild-type) are simultaneously shown on the left (H and E) and right (in situ hybridization, Agtr2 mRNA). At E11.0 stage, that is, prior to the onset of first ureteral budding, the Wolffian duct is densely encased by undifferentiated mesenchymal cells (a), which already express Agtr2 mRNA intensely (b). An E11.5 embryo (c and d) shows signals for Agtr2 mRNA around the Wolffian duct and ureteric bud (d), which are occupied by undifferentiated mesenchymal cells. By contrast, there is little expression in the metanephric blastema. Transverse sections of an E17.5 embryonic tissue at the level of the uretero-pelvic junction (e and f). Both ureter and metanephros are seen (e). The undifferentiated mesenchymal cells surrounding the ureter (e) strongly express Agtr2 mRNA (f). By contrast, there is sparse expression in ureteral epithelial cells, or in mesenchymal cells that have already differentiated into the smooth muscle. UB, ureteric bud; MN, metanephros; WD, Wolffian duct. White bars indicate 100 μm.
Molecular Cell 1999 3, 1-10DOI: ( /S (00) )

7. Figure 6
DNA Sequence and RFLP Analyses of the Human AGTR2 Gene from CAKUT Patients and Control American and German Populations
(a) Direct DNA sequence analysis of PCR amplicons of a 5′ portion of the AGTR2 gene, containing the A→G transition within the intron 1. An A→G transition was found in the mutated allele at position −1,332 shown by an arrow but not in the normal allele.
(b) Due to the modification (A→C) at position −1327, together with the mutation (A→G) at position −1332, PCR amplification products from mutated allele are predicted to have an EcoRI site (GAATTC), whereas those from normal allele are not. For convenience of discussion, the sense strand complementary to the antisense (reverse) primer used in the study is presented as the reverse primer in this figure.
(c) The actual PCR products from males with A and G genotypes. Unlike A allele, G allele shows, instead of the undigested fragment of 120 bp, digested fragments of 91 bp and 29 bp. P34 and P65 are from patients with CAKUT while C71, C59, and C29 are from controls.
(d) Whereas A genotype predominates in the control populations of both Americans (n = 31) and Germans (n = 24), G genotype predominates in patients with CAKUT in both Americans (n = 13) and Germans (n = 23). The imbalance between the control and patient populations is significant for both Americans and Germans.
Molecular Cell 1999 3, 1-10DOI: ( /S (00) )

8. Figure 7 RT-PCR and RNase Protection Assays for Human AGTR2 mRNA
(a) Right, the RT-PCR product from full-length AGTR2 mRNA including all of exons 1–3 is expected to have a length of 366 bp, whereas the product that includes only exons 1 and 3, but not 2, is 305 bp. Left, the actual size of RT-PCR products from males with A or G AGTR2 genotype are 366 bp and 305 bp, respectively. The difference in the intensities of these two product sizes reflects the decreased amounts of products obtained from G compared to A alleles.
(b) Sequencing analysis of RT-PCR products of A or G genotype shows that the transcripts from A alleles contain exon 1, 2, and 3, whereas the transcripts from G alleles contain only exons 1 and 3, that is, exon 2 is spliced out.
(c) RNase protection assay of mRNA from human female uterine samples, demonstrating differences in the quality and quantity of transcripts found in samples from individuals with A/A or G/G genotype. The probe, which includes exons 1, 2, and 3, was hybridized with yeast RNA either with (lane 2) or without RNase digestion (lane 1) and with human myometrium RNA (lanes 3–5, A/A; 6–8, G/G genotypes). In lane 1, discrete bands can be seen at their predicted length of 495 (AGTR2) and 195 nucleotides (cyclophilin), whereas lane 2 shows complete digestion of unprotected RNA. The AGTR2 mRNA from A/A genotype females (lanes 3–5) produces a discrete band at its predicted length of 366 nucleotides (exons ), whereas the mRNA from females with G/G genotype (lanes 6–8) shows a band pattern that is dim and indistinct. The panel presents the quantitative intensity of AGTR2 mRNA (366 nucleotides) normalized for cyclophilin (103 nucleotides), showing that the mRNA of A/A genotype females (n = 9) is some six times higher than that of G/G genotype females (n = 6).
Molecular Cell 1999 3, 1-10DOI: ( /S (00) )