Transgenic overexpression of p23 induces spontaneous hydronephrosis in mice

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1 Int. J. Exp. Path. (), 9, 5 59 ORIGINAL ARTICLE Transgeni overexpression of p indues spontaneous hydronephrosis in mie Jaehoon Lee,, Hye Jin Kim, Jung Ah Moon, Young Hoon Sung, In-Jeoung Baek, Jae-il Roh, Na Young Ha, Seung-Yeon Kim, Young Yil Bahk à, Jong Eun Lee, Tae Hyun Yoo and Han-Woong Lee Department of Biohemistry, College of Life Siene and Biotehnology and Yonsei Laboratory Animal Researh Center, Yonsei University, Seoul, Department of Moleular Cell Biology, Sungkyunkwan University Shool of Mediine, Suwon, à Department of Integrated OMICS for Biomedial Sienes, Graduate Shool, Yonsei University, Department of Anatomy, Yonsei University College of Mediine and Division of Nephrology, Department of Internal Mediine, Institute of Kidney Disease, Yonsei University College of Mediine, Seoul, Korea INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY doi:./j x Reeived for publiation: June Aepted for publiation: January Correspondene: Han-Woong Lee Department of Biohemistry College of Life Siene and Biotehnology Yonsei University Seoul -79 Korea Tel.: Fax: hwl@yonsei.a.kr Summary p is a ohaperone of heat shok protein 9 and also interats funtionally with numerous steroid reeptors and kinases. However, the in vivo roles of p remain unlear. To explore its in vivo funtion, we generated the transgeni () mie ubiquitously overexpressing p. The p mie spontaneously developed kidney abnormalities losely resembling human hydronephrosis. Consistently, kidney funtions deteriorate signifiantly in the p mie ompared to their wild-type () littermates. Furthermore, the expression of target genes for aryl hydroarbon reeptor (AhR), suh as ytohrome P5, family, subfamily A, polypeptide (CypA) and ytohrome P5, family, subfamily B, polypeptide (CypB), were indued in the kidneys of the p mie. These results indiate that the overexpression of p ontributes to the development of hydronephrosis through the upregulation of the AhR pathway in vivo. Keywords aryl hydroarbon reeptor, hydronephrosis, kidney, p, mouse Jaehoon Lee and Hye Jin Kim ontributed equally to this study. p was first identified as a ohaperone of heat shok protein 9 (Hsp9) and has important roles in mediating various ellular signalling events, espeially those triggered by nulear hormone reeptors (Huthison et al. 995; Johnson & Toft 995; Freeman et al. 996, ). The p protein is expressed ubiquitously and is highly onserved from yeast to human, and its defiieny is linked to perinatal lethality with retarded lung development in mie (Bohen 998; Muñoz et al. 999; Grad et al. 6). Moreover, the Hsp9 haperone omplex affets a number of ellular targets and is upregulated in aner ells; this heteroomplex onsisting of Hsp9 and p is one of the potential targets for aner therapy (Nekers et al. 999). In vitro experiments further revealed that p regulates various ellular signal transdution pathways through the regulation of lient proteins suh as estrogen reeptor, Fes tyrosine kinase, heat shok fator (Hsf), aryl hydroarbon reeptor and telomerase (Nair et al. 996; Akalin et al. ). Hydronephrosis, the pathologial ondition where there is dilation of the kidney aused by abnormal urine disharge, often results in irreversible parenhymal damage leading to severe renal failure and death (Gulmi et al. ; Kumar Ó The Authors. International Journal of Experimental Pathology Ó International Journal of Experimental Pathology 5

2 5 J. Lee et al. et al. 5). It is a ommon and serious disease in human, ourring approximately in live births (Belarmino & Kogan 6). Geneti studies suggest that abnormalities of kidney funtions inluding injury to the eletrolyte transporter, the water hannel and pump, the renin angiotensin system and renal ell differentiation and proliferation, an all indue hydronephrosis (MDill et al. 6). In rodent models, treatment with,,7,8-tetrahlorodibenzo-(p)-dioxin (TCDD) is known to indue hydronephrosis through the ativation of aryl hydroarbon reeptor (AhR) signalling, leading to progressive atrophy of the kidney (Abbott et al. 987; Mimura et al. 997). Although reent studies indiate that p is important for the poteny and effiay of ligand-indued AhR signalling (Kazlauskas et al. 999, ; Mar et al. ; Cox & Miller ), the in vivo funtions of p remained to be eluidated. We hypothesized that the dysregulation of p may indue hydronephrosis through the ativation of the AhR signalling pathway. In this study, therefore we used mie overexpressing p as our model to test whether p overexpression promotes the hydronephroti kidney via the AhR signalling. Methods Animals and genotyping To generate p mie, we inserted the DNA enoding hemagglutinin (HA)-tagged p downstream of the hiken (Ck) b-atin promoter and ytomegalovirus (CMV)-IE enhaner of the pcaggs mammalian expression vetor (Figure a). The plasmid was linearized with restrition enzymes SalI and NotI and then miroinjeted into the fertilized mouse eggs of a FVB N mouse strain (Taoni Farms, Germantown, NY, USA). The genotype of the mie was determined by PCR, and gene expression was onfirmed by Western blot and immunohistohemial analyses. The primers designed from the onstrut (Figure a) for genotyping were 5 -CGCCACCACAGCCCTTCCAAAG- (forward for HA-p), 5 -GTATTAGCCAGCGC AT- (reverse for HA-p), 5 -GCTATACATC TACCACT- (forward for endogenous p) and 5 - AAGGTTAGTCATCCCAGAC- (reverse for endogenous p). All mie were housed in groups in the speifi pathogen-free (SPF) faility of the Laboratory Animal Researh Center (LARC) in Yonsei University, whih is operated in aordane with the Institutional Animal Care and Use Committee (IACUC) and international guidelines on the ethial use of animals. All animals were maintained on a :-h light:dark yle with aess to food and water ad libitum. Ink injetion experiment To observe possible anatomial obstrutions of the urinary trat under anaesthesia, dark blue ink was injeted into the pyeloalieal spae of mie using a -gauge needle as desribed previously (Nishimura et al. 8). All photographs were taken at the same magnifiation immediately after ink injetion. Miroomputed tomography imaging Mie were anaesthetized by intraperitoneal (i.p.) injetion of avertin solution. Computed tomography (CT) images were obtained with 6-setion CT sanners (Somatom Sensation 6; Siemens Healthare, Marburg, Germany). After obtaining unenhaned CT images, ml of iopromide (Ultravist 7; Shering, Berlin, Germany) was administered via the tail vein and 5-min delayed images were obtained. All sanned images were sent to the Piture Arhiving and Communiation System (PACS; GE Healthare, Freiburg, Germany) and reonstruted. Haematoxylin and eosin (H&E) staining and immunohistohemistry Kidneys were fixed in % formalin (Sigma, St. Louis, MO, USA), embedded in paraffin and setioned at -lm intervals. For histologial examination, the setions were stained with H&E as desribed previously (Cheong et al. 6). To perform the immunohistohemial study, the paraffin setions were inubated with.% H O for min to eliminate endogenous peroxidases, followed by several PBS rinses. The setions were bloked by normal goat serum for min and inubated in a humid hamber with primary antibody against HA (Rokland, Gilbertsville, PA, USA) for h at C and then for h with seondary antibody (biotinylated anti-rabbit antibody, Vetor Laboratories, Burlingame, CA, USA). To amplify signals, the setions were inubated with an avidin biotin omplex (ABC, Vetor Laboratories) and tissues were visualized by DAB (Vetor Laboratories). Blood urea nitrogen and reatinine test Serum was obtained from wild-type () and p mie at 9 months of age (males, n = 7 for and n = 8 for ; females, n = 7 for and n = for ). Analysis of blood urea nitrogen (BUN) and reatinine onentration was performed in the Eone Referene Laboratory ( Republi of Korea). Reverse transription (RT)-PCR Kidney mrna was isolated using ml of Trizol solution aording to manufaturer s proedures (Invitrogen, Carlsbad, CA, USA). Reverse transription (RT) was performed with Supersript III (Invitrogen) for 6 min at 7 C using an oligo-dt primer. All RT reations were performed using lg mrna. The PCR yling onditions were 9 C for min, followed by yles of 9 C for s, 55 6 C for s, 7 C for s. All RT reations and PCRs were performed on a MyCyler Ô Themal Cyler (Bio-Rad, Herules, CA, USA). All signals were normalized to Gapdh. The primer sequenes are listed in Table S. Ó The Authors. International Journal of Experimental Pathology Ó International Journal of Experimental Pathology, 9, 5 59

3 p overexpression indues hydronephrosis in mie 5 Western blot analysis Whole ell lysates were obtained from eah organ using CHAPs buffer [ mm Tris Cl (ph 7.5), 5 mm NaCl, mm EDTA, % CHAPS, mm PMSF and proteinase inhibitor oktail (Calbiohem)], and Western blot analyses were performed using antibodies against p (originally from Dr. Toft; Beton Dikinson, Cokeysville, MD, USA), ytohrome P5, family, subfamily A, polypeptide (CYPA) (Santa Cruz, Santa Cruz, CA, USA), ytohrome P5, family, subfamily B, polypeptide (CYPB) (Lifespan Siene, Seattle, WA, USA), ylooxygenase- (COX-) (BD Biosiene), b-atin (Sigma) and GAPDH (Labfrontier, Seoul, Republi of Korea). Results p mie develop spontaneous hydronephrosis To investigate the in vivo effet of p overexpression, we designed a mouse strategy ubiquitously overexpressing HA-p (Figure a). Four independent lines were established, and their transgene expression in various tissues was examined by Western blot (Figure b, ). Marked expression of HA-p protein was ubiquitously deteted from all mouse lines with variable degrees (Figure b, ). All mie were always maintained the hemizygous for p transgene and did not show developmental defets and reprodutive problems. The transgeni mouse lines C and G showing relatively higher renal expression of HA-p than others were used for the subsequent experiments. In addition to the kidney, HA-p was highly expressed in ureter and bladder (Figure S). Even though the p transgene was highly expressed, the phenotypi manifestation was not lear at an early age exept for slight loss of body weight (data not shown). However, the adult p mie frequently showed spontaneous distension and dilatation of the renal pelvis and alyes (Table and Figure a), losely resembling the pathologial onditions of human hydronephrosis. The expanded pelvi region in the kidney had been filled with the urine. In its extreme ase, the kidneys of p mie showed a fluid- SalI EoRI EoRI NotI L W T CMV Ckβ HA-p pa HA-p (56 bp) p (5 bp) (b) Lu Sp Br He Li Mu Th Ki In St Te p-ha p GAPDH () Figure Generation of mie overexpressing p. Shemati representation of the p transgeni () onstrut and a result of PCR genotyping are shown. The positions of restrition sites for loning and linearization are indiated. Primers used for genotyping are represented by blak arrows. Cytomegalovirus (CMV); CMV-IE enhaner; Ckb, hiken b-atin promoter; pa, poly A signal. (b) Western blot analysis with p antibody shows a representative expression of endogenous and transgeni p in mouse tissues (p line G). Proteins were isolated from the lung (Lu), spleen (Sp), brain (Br), heart (He), liver (Li), musle (Mu), thymus (Th), kidney (Ki), intestine (In), stomah (St) and testis (Te) of and p transgeni () mie. GAPDH is used as a loading ontrol. The representative data shown were obtained from the line G mie. () The p transgene expression patterns of four lines (lines B, C, D and G) were ompared in various organs as indiated. The relative expression of transgene hemagglutinin (HA)-p was normalized to endogenous p. Ó The Authors. International Journal of Experimental Pathology Ó International Journal of Experimental Pathology, 9, 5 59

4 5 J. Lee et al. Table Inidene of spontaneous hydronephrosis in and p mie (Line G). Similar to the epidemiologial onditions observed in human hydronephrosis Month C 8 9 Total 8 9 Total Male Female , wild type;, transgeni. M C M Vertial Horizontal md md p (b) (b) () Figure Transgeni () mie overexpressing p exhibit hydronephrosis. Kidney of p mie showed distension and dilatation of renal pelvis (white arrowheads) and alyes (white arrows). (b) p mie exhibited swollen kidneys with perinephri pseudoysts (blue arrowhead). yellow arrowhead, blood vessel; red arrowhead, ureter. HA md rp rp HA filled avity (perinephri pseudoyst) between the renal apsule and remnant kidney, as well as the dilated renal pelvis (Figure b). These mie showed no aberrant blood vessels and the dilated ureter onneted orretly to the lower pole of the kidney (Figure b). There was no evidene of kidney stone or mass induing ureteral obstrution in the urinary trat (data not shown). However, the pathologial ondition progressively deteriorate with age, and the inidene was higher in the males (9.6%) than in the females (%, Table ). The inidene and symptoms were more severe in p mie of line G than in those of line C, possibly refleting the higher level of transgene expression in the line G than in the line C (Figure and Table S). Although total p expression in the kidney was the same in both sexes (Figure S), the female mie were resistant to hydronephrosis (Tables and S), whih is similar Figure Histologial analysis of hydronephroti kidney of p transgeni () mie. Normal and hydronephroti kidney setions from and p mie. The renal pelvis was grossly dilated in p mie. The dilatation regions of the renal pelvis in hydronephroti kidney of p mie. (b) Kidney setions from -month-old and p mie were stained with H&E. The ortex regions from normal and hydronephroti kidneys of and p mie, respetively, are shown. Detailed images of setions from and hydronephroti p kidney do not show detetable strutural hanges in the glomeruli (yellow arrowheads). () Immunohistohemial staining using an anti-ha antibody showing representative expression of the p protein in the kidney (Sale bar = lm). yellow arrows, glomeruli; red arrows, distal onvoluted tubules; blue arrows, proximal tubules;, ortex; md, medulla; rp, renal pelvis; p, papilla. Ó The Authors. International Journal of Experimental Pathology Ó International Journal of Experimental Pathology, 9, 5 59

5 p overexpression indues hydronephrosis in mie 55 5 BUN (mg/dl) 5 Male Female (b).5 (b) Creatinine (mg/dl)..5. a b Male Female d e f Figure Histologial analysis of kidney and dynami ontrast omputed tomography (CT) in (a ) and p transgeni () (d f) mie. Representative images after ink injetion into the pyeloalieal spae of kidney. Higher magnifiation images of the ureter are shown as insets. (b) Preontrast image (a, d). Coronal CT image s after ontrast injetion via tail vein showed olletion of the ontrast dye in the renal pelvis without signifiant dilatation (b). At 8 s after ontrast injetion, most of the ontrast dye was washed out in the renal pelvis and filled in the bladder (). Coronal CT images s after ontrast injetion showed dye aumulation along the marked dilated renal pelvis (red arrowheads) (e). CT image 6 se after ontrast injetion showed that the ontrast dye still remained in the renal pelvis (f). Contrast emptying in the bladder was inomplete even though it ourred over a prolonged time. White arrowhead, kidney. Figure 5 Hydronephroti kidneys of p transgeni () mie show renal failure. Blood urea nitrogen (BUN) and reatinine levels in serum from and p mie are shown. BUN level in male p mie ( ± mg dl, n = 8) was signifiantly higher than that of ontrols ( ± 5 mg dl, n = 7). This differene was not observed in female mie. (b) The reatinine level in male p mie (. ±. mg dl, n = 8) was markedly higher than that of ontrols (. ±. mg dl, n = 7). This differene was not observed in female mie. P <.5; P <.6. to human disease influened by oestrogen (Wright & Lay 988; Weide & Lay 99; Beishlag & Perdew 5). These results indiate that overexpression of p spontaneously indues hydronephrosis, and that both prevalene and severity are progressively inreased with age. Histologial analysis of hydronephrosis As hydronephrosis leads to progressive atrophy of the kidney, the hydronephroti kidneys of p mie were histologially analysed. Conspiuous dilatation of the pelvis and alyes was observed, and the renal parenhyma in the medullary region was thinner ompared with that of mie (Figure a). However, there were no apparent morphologial hanges in the ortex, the outer part of the kidney ontaining the glomerulus, proximal and distal onvoluted tubules (Figure a). The glomerulus and renal tubules of these regions were histologially intat (Figure b), even though prominent HA-p expression was Ó The Authors. International Journal of Experimental Pathology Ó International Journal of Experimental Pathology, 9, 5 59

6 56 J. Lee et al CypB Aqp Avpr Id p Gapdh Relative expression to Cypb 5 5 Avpr Aqp 5 5 Id 5 p (b) CypA CypB COX- p-ha p β-atin Relative expression to Cypa 8 6 Cypb Cox- 5 Total p 8 6 Figure 6 Altered expression of hydronephrosis-related genes in kidneys of and p transgeni () mie at 9 months of age. mrna transripts of CypB, Aqp, Avpr, Id- and p in kidneys from (lanes ) and p (lanes 5 8) mie were quantified by RT-PCR. Eah lane represents transripts from an individual mouse. The transript level was normalized to Gapdh values. A representative graph is shown (, n = ;, n = ). (b) The protein expression of ytohrome P5, family, subfamily A, polypeptide (CypA), ytohrome P5, family, subfamily B, polypeptide (CypB) and ylooxygenase- (Cox-) in kidneys from (lanes ) and p (lanes 5 8) mie was analysed by Western blotting using the indiated antibodies. The expression level was normalized to b-atin values. A representative graph is shown (, n = ;, n = ). P <.; P =.7; P =.. immunohistohemially deteted in the glomerulus and distal onvoluted tubules (Figure ). Expression pattern of the HA-p is similar to that of endogenous p in the kidney of mie (Figures and S). These results indiate that hydronephrosis in p mie an result from the funtional proesses interrupting the flow of the urine. Funtional analysis of hydronephrosis Beause obstrution of the free urine flow from the kidney to the bladder usually auses hydronephrosis (MDill et al. 6), dark blue ink was infused into the pyeloalieal spae of the kidney to diagnose the possible anatomial obstrutions of the urinary trak. The injeted ink ran to the bladder and made the ureter visible in mie (Figure a). However, the ureter of p mie displayed the thinning of urethral lining and stenosis of the ureter with dilation and or distortion (Figure a). These results were onfirmed by miroomputed tomography using a onventional extraellular ontrast agent, iopromide that is readily eliminated by funtionally normal glomerular filtration (Hakstein et al. ). In mie, the ontrast agent was deteted in the renal pelvis s after ontrast injetion and learly disappeared 8 s after ontrast injetion (Figure b, a ). Although the ontrast agent similarly appeared s after ontrast injetion in the p mouse, the renal pelvis revealed muh brighter signal, indiating onentration of the ontrast agent and distension of the renal pelvis (Figure b,e). In ontrast to mie, the strong signal was still deteted 6 s after ontrast injetion in the p mie (Figure b,f). These results suggest that the defet in the urine flow elevating the internal pressure on the kidney may ause the distention of kidney, hydronephrosis, in p mie. As hydronephrosis frequently auses progressive atrophy of the kidney and irreversible parenhymal damage assoiated with severe renal failure (Gulmi et al. ; Kumar et al. 5), we evaluated whether p transgene expression results in renal failure by measuring the BUN and serum reatinine levels as markers of kidney funtion (Gagnon & Duguid 98; MDill et al. 6). The levels of BUN (P <.5) and serum reatinine (P <.6) were signifiantly inreased in male p mie, but not in females, ompared with those of their littermate ontrols (Figure 5), further supporting its similarity to the gender disparity observed in human hydronephrosis. Therefore, the Ó The Authors. International Journal of Experimental Pathology Ó International Journal of Experimental Pathology, 9, 5 59

7 p overexpression indues hydronephrosis in mie 57 hydronephrosis of p mie is potentially harmful to normal kidney funtion. Altered gene expression in hydronephroti kidneys of p mie A number of genes are involved in generating hydronephrosis in mie. Hydronephrosis is observed in mie laking aquaporin (Aqp; MDill et al. 6), inhibitor of DNA binding (Id; Aoki et al. ), arginine vasopressin reeptor (Avpr; Yun et al. ) and angiotensin type reeptor (Agtr; Nishimura et al. 999). In addition, TCDDindued hydronephrosis mouse models suggested the importane of AhR and its well-known target genes, ylooxygenase- (Cox-), CypA and ytohrome P5, family, subfamily B, polypeptide (CypB) in hydronephrosis (Nishimura et al. 999; Nebert et al. ). The expression of these markers was assessed in the kidneys from and p mie. While expressions of Avpr and Id- were not hanged, Aqp transript was unexpetedly inreased (Figure 6a). The level of CypB mrna was upregulated in the hydronephroti kidneys of p mie ompared with those of mie (Figure 6a). As CypB is a well-known AhR target gene and its ativation is impliated in TCDDindued hydronephrosis, we examined the expression of AhR target genes inluding CypA, CypB and Cox- by Western blot analyses. As these expressions were signifiantly upregulated in p- kidneys (Figures 6b and SB), hydronephrosis in the p- kidneys may require AhR signalling. Taken together, the overexpression of p in vivo ontributes to the development of hydronephrosis by regulating AhR ativity and its target genes inluding CypA, CypB and Cox-. Moreover, this result shows signifiant orrelations between the expression of Cypa and Cox- among eah individual p mouse. Disussion Here, we have shown that two independent lines of p mie develop spontaneous hydronephrosis. Our results show that the prevalene and severity of hydronephrosis in p mie leads to progressive deterioration with age. Previous mouse models have shown that hydronephrosis is aused by mutations in the genes involved in normal kidney homoeostasis. However, there have been no reports linking p to the development of hydronephrosis. For example, the mie defiient for arginine vasopressin reeptor (Avpr) or angiotensin type reeptor (Agtr), whih belongs to the subfamily of G protein-oupled reeptors in the renin angiotensin system, develop hydronephrosis, hypoplasti and multiysti kidneys and or megaureter (Nishimura et al. 999). The mie defiient for the integral membrane protein aquaporin (Aqp), whih forms pores in the membrane and plays a vasopressin (VP)-regulated role in water reabsorption by the kidney, have been reported to develop hydronephrosis beause of the obstrution in the ureteropelvi juntion (MDill et al. 6). The mie defiient for NaK Cl otransporter (NKCC) (Takahashi et al. ) or renal outer medullary potassium hannel (ROMK) (Lorenz et al. ; Lu et al. ) were shown to have the symptoms of polyuria and eletrolyte imbalane, resulting in hydronephrosis of varying severities. In addition, the xenobioti hemial TCDD has been used to indue hydronephrosis in rodents, and it upregulates Cox- and prostaglandin E (PGE )inan AhR-dependent manner (Abbott et al. 987; Mimura et al. 997; Nishimura et al. 8). These lines of evidene suggest that the disruption of normal kidney funtion ontributes to the indution of hydronephrosis; however, the evidene regarding hydronephrosis inluding the effet of p remained equivoal. Here, we have shown that p overexpression an indue spontaneous hydronephrosis. AhR target genes, CypA, CypB and Cox-, were upregulated in the kidneys of p mie (Figures 6 and S), onsistent with the TCDDindued hydronephrosis model. Although the exat moleular mehanism is unknown, these evidenes suggest that AhR ativation is required for the indution of hydronephrosis in the kidneys of p mie. In addition, p is ruial for PGE prodution (Grad et al. 6) and effiient AhR signalling through the stabilization of AhR (Kazlauskas et al. 999, ; Mar et al. ; Cox & Miller ). Therefore, the moleular mehanism underlying the phenotype of p mie appears to be linked to AhR ativation or to inrease the sensitivity of AhR binding to unknown endogenous ligands (Seidel et al. ). As the expression of AhR target genes is known to be repressed by oestrogen signalling (Beishlag & Perdew 5), the resistane of p females to hydronephrosis is expeted to be a potential output of the estrogen signalling. Hydronephrosis is indued not only by anatomial obstrution in the ureter, but also by abnormal peristalti movement of the ureter (Aoki et al. ; Zeidel & Pirtskhalaishvili ; Mahoney et al. 6). For example, hydronephrosis is aused by the defiieny of peristalti ontrations of the smooth musle ells surrounding the ureter (Zeidel & Pirtskhalaishvili ). Furthermore, prostaglandins affet the smooth musle ontratility of the upper urinary trat (Cole et al. 998), and TCDD-exposed hydronephroti mie showed an inreased prodution of PGE (Nishimura et al. 8). Although further study is still required, the present findings suggest that p overexpression an lead to hydronephrosis in anatomial obstrution and or abnormal peristalti movement of the ureter. In addition, fibroti lesions assoiated with hydronephrosis are onsidered to be aused seondarily by the severe lesions seen in the kidney (Yao et al. ). The absene of fibroti lesions in the p kidneys (data not shown) indiates that p overexpression may not indue the type of severe lesions whih result in renal fibrosis. Apart from the individual genotypi differenes, the geneti bakground an be an important fator affeting the inidene of hydronephrosis. Spontaneous hydronephrosis was observed in >6% of male C57BL KsJ mie by 5 weeks of age (Wright & Lay 988; Weide & Lay Ó The Authors. International Journal of Experimental Pathology Ó International Journal of Experimental Pathology, 9, 5 59

8 58 J. Lee et al. 99) and in 6% of male and 9% of female C57BL 6 mie (Zurher et al. 98). In the present study, p mie were generated and maintained in a FVB NJ geneti bakground, and the hydronephroti kidneys were not found in littermates of this bakground (Table ). Therefore, our mouse model also provides the first evidene for the effet of p overexpression in vivo on the development of ongenital and spontaneous hydronephrosis. Taken together, it is likely that p is important for the normal kidney funtion and that p overexpression may indue interruption in the urine flow through the AhR signalling events, finally leading to hydronephrosis and loss of kidney funtion that is refleted by the elevated BUN and reatinine levels in the blood. Although more detailed studies are required to eluidate the exat mehanism governing these phenomena, our mouse model will provide inreased omprehensions of the role of p in the normal kidney funtion and will also provide insights into the diagnosis and therapy of ongenital hydronephrosis in the future. Aknowledgements We greatly appreiate the gift of p antibody from Dr. David Toft (Department of Biohemistry and Moleular Biology, Mayo Clini College of Mediine, Rohester, USA). The work was supported by the National Researh Foundation of Korea (NRF) grants funded by the Korea government (MEST) (9-865, 9-877, 8-585, 9K8) and National R&D Program for Caner Control, Ministry for Health, Welfare and Family Affairs (MoHWFA), Republi of Korea (5). Referenes Abbott B.D., Birnbaum L.S., Pratt R.M. (987) TCDD-indued hyperplasia of the ureteral epithelium produes hydronephrosis in murine fetuses. Teratology 5, 9. Akalin A., Elmore L.W., Forsythe H.L. et al. 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9 p overexpression indues hydronephrosis in mie 59 and Cd in the mitoti ontrol of fission yeasts. Genetis 5, Nair S.C., Toran E.J., Rimerman R.A., Hjermstad S., Smithgal T.E., Smith D.F. (996) A pathway of multi-haperone interations ommon to diverse regulatory proteins: estrogen reeptor, Fes tyrosine kinase, heat shok transription fator Hsf, and the aryl hydroarbon reeptor. Cell Stress Chaperones, 7 5. Nebert D.W., Dalton T.P., Okey A.B., Gonzalez F.J. () Role of aryl hydroarbon reeptor-mediated indution of the CYP enzymes in environmental toxiity and aner. J. Biol. Chem. 79, Nekers L., Mimnaugh E., Shulte T.W. (999) Hsp9 as an antianer target. Drug Resist. Updates, Nishimura H., Yerkes E., Hohenfellner K. et al. (999) Role of the angiotensin type reeptor gene in ongenital anomalies of the kidney and urinary trat, CAKUT, of mie and men. Mol. Cell,. Nishimura N., Matsumura F., Vogel C.F. et al. (8) Critial role of ylooxygenase- ativation in pathogenesis of hydronephrosis aused by latational exposure of mie to dioxin. Toxiol. Appl. Pharmaol., 7 8. Seidel S.D., Winters G.M., Rogers W.J. et al. () Ativation of the Ah reeptor signaling pathway by prostaglandins. J. Biohem. Mol. Toxiol. 5, Takahashi N., Chernavvsky D.R., Gomez R.A., Igarashi P., Gitelman H.J., Smithies O. () Unompensated polyuria in a mouse model of Bartter s syndrome. Pro. Natl Aad. Si. USA 97, Weide L.G. & Lay P.E. (99) Hereditary hydronephrosis in C57BL KsJ mie. Lab. Anim. Si., 5 8. Wright J.R. & Lay P.E. (988) Spontaneous hydronephrosis in C57BL KsJ mie. J. Comp. Pathol. 99, 9 5. Yao Y., Zhang J., Ye D.F. et al. () Left-right determination fator is down-regulated in fibroti renal tissue of human hydronephrosis. BJU International DOI:./j.6-X..95.x Yun J., Shöneberg T., Liu J. et al. () Generation and phenotype of mie harboring a nonsense mutation in the V vasopressin reeptor gene. J. Clin. Invest. 6, 6 7. Zeidel M.L. & Pirtskhalaishvili G. () Urinary trat obstrution. In: Brenner and Retor s The Kidney, pp (ed. B.M. Brenner), Philadelphia: Saunders. Zurher C., Van Zwieten M.J., Solleveld H.A., Hollander C.F. (98) Aging researh in the mouse in biomedial researh: volume IV. In: Experimental Biology and Onology, pp. 5 (eds H.L. Foster, J.D. Small, J.G. Fox), New York, NY: Aademi Press. Supporting Information Additional Supporting Information may be found in the online version of this artile: Figure S. The p transgene expression patterns in male and female mie. Western blot analysis showed the representative expression of endogenous and transgeni p in various organs as indiated. Proteins were isolated from indiated organs of p and male and female mie. GAPDH was used as a loading ontrol. The representative data were obtained from the line G mie. M, male; F, female. Figure S. Expression patterns of endogenous p in the kidney. Immunohistohemial staining using an anti-p antibody showed the representative expression of endogenous p protein in the kidney of mie (sale bar = lm). Figure S. Altered expression of hydronephrosis-related genes in the kidneys of line C mie. (A) mrna transripts of Aqp, Avpr, ROMK, NKCC and p in the kidneys from (lanes ) and p (lanes 5 8) mie were quantified by RT-PCR. The transript level was normalized to Gapdh. (B) The protein expression of CypA, CypB and Cox- in the kidneys from (lanes ) and p (lanes 5 8) mie was analysed by Western blotting using the indiated antibodies. The expression level was normalized to b-atin. Table S. Inidene of spontaneous hydronephrosis in and p mouse (line C). Similar to the epidemiologial onditions observed in human hydronephrosis. Table S. Primer pairs used for RT-PCR analysis. Please note: Wiley-Blakwell are not responsible for the ontent or funtionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be direted to the orresponding author for the artile. Ó The Authors. International Journal of Experimental Pathology Ó International Journal of Experimental Pathology, 9, 5 59