Phenotypic analysis of conditionally immortalized cells isolated from the BPK model of ARPKD.

To study the pathophysiology of autosomal recessive polycystic kidney disease (ARPKD), we sought to develop conditionally immortalized control and cystic murine collecting tubule (CT) cell lines. CT cells were isolated from intercross breedings between BPK mice (bpk(+/-)), a murine model of ARPKD, and the Immorto mice (H-2K(b)-ts-A58(+/+)). Second-generation outbred offspring (BPK x Immorto) homozygous for the BPK mutation (bpk(-/-); Im(+/+/-); cystic BPK/H-2K(b)-ts-A58), were phenotypically indistinguishable from inbred cystic BPK animals (bpk(-/-)). Cystic BPK/H-2K(b)-ts-A58 mice developed biliary ductal ectasia and massively enlarged kidneys, leading to renal failure and death by postnatal day 24. Principal cells (PC) were isolated from outbred cystic and noncystic BPK/H-2K(b)-ts-A58 littermates at specific developmental stages. Epithelial monolayers were under nonpermissive conditions for markers of epithelial cell polarity and PC function. Cystic and noncystic cells displayed several properties characteristic of PCs in vivo, including amiloride-sensitive sodium transport and aquaporin 2 expression. Cystic cells exhibited apical epidermal growth factor receptor (EGFR) mislocalization but normal expression of ZO-1 and E-cadherin. Hence, these cell lines retain the requisite characteristics of PCs, and cystic BPK/H-2K(b)-ts-A58 PCs retained the abnormal EGFR membrane expression characteristic of ARPKD. These cell lines represent important new reagents for studying the pathogenesis of ARPKD.

A novel inhibitor of tumor necrosis factor-alpha converting enzyme ameliorates polycystic kidney disease.

BACKGROUND: Transforming growth factor-alpha (TGF-alpha) expression is abnormal in polycystic kidney disease. We previously demonstrated that blockade of the epidermal growth factor receptor (EGFR), the receptor for TGF-alpha, significantly slowed disease progression in the bpk murine model of autosomal-recessive kidney disease (ARPKD). In the present study, kidney TGF-alpha expression in this model is characterized, and the therapeutic potential of inhibiting TGF-alpha in ARPKD is examined using a novel inhibitor of tumor necrosis factor-alpha converting enzyme (TACE), the metalloproteinase that cleaves membrane-bound TGF-alpha to release the secreted ligand. METHODS: Immunohistochemistry (IH) and Western analysis were performed on kidneys from cystic bpk mice and noncystic littermates at postnatal days 7, 14, and 21. Bpk mice and normal controls were treated with WTACE2, a competitive inhibitor of TACE, from day 7 until day 21, and the effects on kidney histology and renal function were assessed. RESULTS: Increased TGF-alpha expression by IH was demonstrated in the proximal tubules (PT) at postnatal day 7 and collecting tubules (CT) by day 21. A parallel increase in kidney TGF-alpha expression was demonstrated by Western analysis. Treatment of cystic bpk mice with WTACE2 resulted in a 43% reduction in kidney weight to body weight ratio (11.2 vs. 19.7%), improved cystic index (3.2 vs. 4.8), reduced cystic CT to PT ratio (1.2 vs. 8), and a greater than 30% reduction in BUN and serum creatinine. CONCLUSIONS: These findings support the pathophysiological role of the TGF-alpha/EGFR axis in murine ARPKD and demonstrate that inhibition of TGF-alpha secretion has therapeutic potential in PKD.

Can progression of autosomal dominant or autosomal recessive polycystic kidney disease be prevented?

Data from animal and human studies suggest that the rate of progression of renal insufficiency can be retarded with careful control of blood pressure, institution of a low-protein diet, and the use of lipid-lowering agents. These therapeutic interventions become important when managing patients with renal insufficiency secondary to autosomal dominant polycystic kidney disease (PKD) and autosomal recessive polycystic kidney disease, in which end-stage renal disease is present in nearly 17,000 individuals per year. Several dietary and pharmacologic intervention strategies including blood pressure control, dietary modification, and the use of antioxidants as well as lipid-lowering agents have been studied in humans and animals with PKD in an effort to slow the rate of renal progression. This article reviews the current understanding of the effectiveness of these conventional therapies, as well as novel therapies that specifically target the mediators of cyst formation in PKD using tyrosine kinase inhibitors and gene therapy in an effort to identify potential strategies for retarding cyst formation and parenchymal injury in PKD. Current pharmacologic and dietary strategies fail to show any consistent benefits in preserving renal function and reducing renal injury in human PKD. The therapeutic potential for exciting new gene therapies and pharmacologic agents designed to target the pathophysiologic pathways involved in cyst formation are promising. Randomized, controlled trials in children and adults with early PKD are necessary to evaluate the effectiveness of these therapeutic interventions.

Expression of the orpk disease gene during kidney development and maturation.

Further analysis of the orpk mouse model of human autosomal recessive polycystic kidney disease is providing more insight into the function of the Tg737 gene and the pathobiology of renal cystic disease. Here we have determined the temporal-spatial profile of Tg737 expression and ascertained the profile of disease pathology utilizing Tg737delta2-3betaGal/+ and Tg737delta2-3betaGal/ Tg737orpk compound heterozygotes from embryonic day 13.0 (E13.0) to postnatal day 270 (D270). This has allowed us to correlate disease progression and Tg737 expression in the context of the mutant orpk phenotype. These data reveal that Tg737 is dynamically regulated during kidney development and during postnatal kidney maturation in normal and in orpk mutants. This expression pattern correlates with the pathology of the disease, such that tubular segments with the highest expression levels are most protected from cystic disease. These data indicate that kidney tubules require a threshold level of Tg737 function for normal tubular development, structure, and function. In addition, these data demonstrate that the timing of cyst formation and severity of cyst progression is modulated differently in different regions of the nephron in this model.

Renal dysfunction but not cystic change is ameliorated by neonatal epidermal growth factor in bpk mice.

BALB/c mice homozygous for the bpk gene exhibit a form of autosomal recessive (AR) polycystic kidney disease (PKD) with massive collecting duct cysts, common bile duct dilation and chaotic intrahepatic bile ducts/portal triads. The combined renal and biliary pathology mimics much of the pathology seen in human ARPKD. Murine models of ARPKD generally have a reduced renal expression of epidermal growth factor (EGF) and an increased expression of EGF receptors (EGF-R). However, the role that EGF and EGF-R play in the progression of PKD has been unclear. Evidence from various model systems/ages of treatment produces conflicting results. Treating neonatal C57BL/6J-cpk mice with EGF ameliorates the renal pathology and dysfunction while treating 2- and 3-week-old bpk mice with an EGF-receptor tyrosine kinase inhibitor also ameliorates ARPKD. Therefore, to determine whether neonatal EGF treatment would accelerate or inhibit the progression of the PKD in bpk mice, we administered exogenous EGF (1 microgram/g body weight subcutaneously) daily from postnatal days 3-9 (a critical period for tubule maturation). Neonatal EGF treatment but not sham treatment retarded the development of azotemia and common bile duct dilation and the chaotic hepatic triad changes in cystic mice. However, EGF treatment neither reduced the severity of the renal cystic pathology nor reduced the degree of cystic enlargement of the kidneys. Cystic mice treated past 9 days of age died prior to their scheduled termination at 21 days of age. The role of EGF in the progression of polycystic kidney disease in bpk mice is relatively complicated, with neonatal treatment being associated with some amelioration of the renal dysfunction and extrarenal pathology without an effect on the renal pathology. Continuation of treatment beyond 9 days increased morbidity. Therefore, in discussing the role of EGF or EGF receptor in mediating the pathophysiology of PKD, the stage of development may be an important consideration.

Generation and phenotype of cell lines derived from CF and non-CF mice that carry the H-2K(b)-tsA58 transgene.

Tracheal, renal, salivary, and pancreatic epithelial cells from cystic fibrosis [CF; cystic fibrosis transmembrane conductance regulator (CFTR) -/-] and non-CF mice that carry a temperature-sensitive SV40 large T antigen oncogene (ImmortoMouse) were isolated and maintained in culture under permissive conditions (33 degrees C with interferon-gamma). The resultant cell lines have been in culture for >1 year and 50 passages. Each of the eight cell lines form polarized epithelial barriers and exhibit regulated, electrogenic ion transport. The four non-CF cell lines (mTEC1, mCT1, mSEC1, and mPEC1) express cAMP-regulated Cl(-) permeability and cAMP-stimulated Cl(-) secretion. In contrast, the four CFTR -/- cell lines (mTEC1-CF, mCT1-CF, mSEC1-CF, and mPEC1-CF) each lack cAMP-stimulated Cl(-) secretory responses. Ca(2+)-activated Cl(-) secretion is retained in both CF and non-CF cell lines. Thus we have generated genetically well-matched epithelial cell lines from several tissues relevant to cystic fibrosis that either completely lack CFTR or express endogenous levels of CFTR. These cell lines should prove useful for studies of regulation of epithelial cell function and the role of CFTR in cell physiology.

Treatment of polycystic kidney disease with a novel tyrosine kinase inhibitor.

UNLABELLED: Treatment of polycystic kidney disease with a novel tyrosine kinase inhibitor. BACKGROUND: We have previously demonstrated an essential role for increased epidermal growth factor receptor (EGFR) activity in mediating renal cyst formation and biliary epithelial hyperplasia in murine models of autosomal recessive polycystic kidney disease (ARPKD). This study was designed to determine whether or not treatment with a newly developed inhibitor of EGFR tyrosine kinase activity (EKI-785) would reduce renal and biliary abnormalities in murine ARPKD. METHODS: Balb/c-bpk/bpk (BPK) litters were treated with EKI-785, an EGFR-specific tyrosine kinase inhibitor. Animals were treated by intraperitoneal injection beginning at postnatal day 7 and were treated until postnatal day 24 or 48. EKI-785's effectiveness was measured by a reduction in the renal cystic index, an increased life span, and maintenance of normal renal function. RESULTS: Treatment of BPK mice with EKI-785 resulted in a marked reduction of collecting tubule (CT) cystic lesions, improved renal function, decreased biliary epithelial abnormalities, and an increased life span. Untreated cystic animals died of renal failure at postnatal day 24 (P-24) with a CT cystic index of 4.8, a maximal urine osmolarity of 361 mOsm, and moderate to severe biliary abnormalities. Cystic animals treated with EKI-785 to postnatal day 48 (P-48) were alive and well with normal renal function, a reduced CT cystic index of 2.0 (P < 0.02), a threefold increased in maximum urinary concentrating ability (P < 0.01), and a significant decrease in biliary epithelial proliferation/fibrosis (P < 0.01). CONCLUSION: This study demonstrates that EKI-785 has therapeutic effectiveness in improving histopathologic abnormalities and decreasing mortality in murine ARPKD.

Cellular pathophysiology of cystic kidney disease: insight into future therapies.

Polycystic kidney disease (PKD) is a developmental kidney disorder which can be inherited as either an autosomal dominant trait, with an incidence of 1:50 to 1:1000, or as an autosomal recessive trait with an incidence of 1:6,000 to 1:40,000. Three different genes have now been cloned that are associated with mutations that cause PKD. Two of these are linked to the most common forms of the dominant disease while the third is associated with the orpk mouse model of recessive polycystic kidney disease. Advances in understanding the molecular genetics of PKD have been paralleled by new insights into the cellular pathophysiology of cyst formation and progressive enlargement. Current data suggest that a number of PKD proteins may interact in a complex, which when disrupted by mutations in PKD genes may lead to altered epithelial proliferative activity, secretion, and cell matrix biology. The identification of a unique cystic epithelial phenotype presents new opportunities for targeted therapies. These include targeted gene therapy, gene complementation, and specific immunological or pharmacological interruption of growth factor pathways.

In vitro modulation of cyst formation by a novel tyrosine kinase inhibitor.

BACKGROUND: Recessively transmitted polycystic kidney disease (PKD) in many murine models is characterized by the initial formation of proximal tubular cysts (stage 1), followed by growth and enlargement of renal collecting tubule (CT) cysts (stage 2). Previous studies have reported that stage 1 cyst formation and growth could be manipulated in vitro by using embryonic kidney explants and newborn explant microslices in organ culture. METHODS: Microslices of postnatal kidneys cultured on Transwell tissue culture inserts allow experimental manipulation of stage 2 CT cyst development and growth. This system was used to test a potential therapeutic compound for treatment of PKD. This compound, EKI-785, modulates altered epidermal growth factor receptor (EGFR) expression in CT cysts by inhibition of EGFR autophosphorylation. RESULTS: These studies demonstrate that: (a) minor modifications of the previously described organ culture system permit successful culture of more mature renal tissue, and (b) cystic explants treated with EGF and EKI-785 demonstrated a marked reduction in CT cystic lesions compared with cystic explants treated with EGF alone. CONCLUSIONS: This study suggests that pharmacological strategies can be used to decrease EGFR tyrosine kinase activity and CT cyst formation and enlargement in murine PKD.

New insights into the molecular pathophysiology of polycystic kidney disease.

Polycystic kidney diseases are characterized by the progressive expansion of multiple cystic lesions, which compromise the function of normal parenchyma. Throughout the course of these diseases, renal tubular function and structure are altered, changing the tubular microenvironment and ultimately causing the formation and progressive expansion of cystic lesions. Renal tubules are predisposed to cystogenesis when a germ line mutation is inherited in either the human PKD1 or PKD2 genes in autosomal dominant polycystic kidney disease (ADPKD) or when a homozygous mutation in Tg737 is inherited in the orpk mouse model of autosomal recessive polycystic kidney disease (ARPKD). Recent information strongly suggests that the protein products of these disease genes may form a macromolecular signaling structure, the polycystin complex, which regulates fundamental aspects of renal epithelial development and cell biology. Here, we re-examine the cellular pathophysiology of renal cyst formation and enlargement in the context of our current understanding of the molecular genetics of ADPKD and ARPKD.

Morphological and functional characterization of a conditionally immortalized collecting tubule cell line.

A conditionally immortalized collecting tubule cell line, mCT1, was derived from the H-2Kb-ts A58 transgenic mouse (ImmortoMouse), which harbors a temperature-sensitive mutant of the SV40 large T antigen oncogene. Cells maintained under permissive conditions [33 degreesC with interferon-gamma (IFN-gamma)] form epithelial monolayers, express large T antigen, and proliferate (>50 passages). The cells retain properties characteristic of the renal collecting tubule (CT) including: vasopressin (VP)-stimulated cAMP accumulation, aquaporin-2 expression, high transepithelial electrical resistance, VP-stimulated ion transport, and amiloride-sensitive sodium absorption. When the cells are transferred to nonpermissive conditions (39 degreesC without IFN-gamma), the steady-state level of large T antigen protein declines (>95% decrease) and cell proliferation is arrested. This conditionally immortalized, murine renal cell line should prove useful for studies of CT physiology and large T antigen biology.

Functional activity of epidermal growth factor receptors in autosomal recessive polycystic kidney disease.

Evidence from a number of laboratories suggests a potential role for the epidermal growth factor (EGF)-transforming growth factor-alpha-epidermal growth factor receptor (EGF-R) axis in promoting epithelial hyperplasia and cyst formation in autosomal recessive polycystic kidney disease (ARPKD). As previously reported, in the C57BL-6Jcpk/cpk (CPK), BALB/c-bpk/bpk (BPK), and C3H-orpk/orpk (ORPK) murine models of ARPKD, as well as in human ARPKD and human ADPKD, the EGF-R is mislocated to the apical surface of cystic collecting tubule (CT) epithelial cells. The present studies demonstrate that cells from cystic and control CTs can be isolated and that these cells maintain their in vivo EGF-R phenotype in vitro. Domain-specific high-affinity ligand binding was assessed by standard Scatchard analysis, and selective ligand stimulation of apical vs. basolateral EGF-R in these cells was followed by measurement of receptor autophosphorylation and determination of cell proliferation. These studies demonstrate that in vitro apically expressed EGF-Rs exhibit high-affinity binding for EGF, autophosphorylate in response to EGF, and transmit a mitogenic signal when stimulated by the appropriate ligand.

The role of hepatocyte growth factor (HGF) at progressive stages of metanephric development.

Several lines of evidence suggest that hepatocyte growth factor (HGF), a soluble protein secreted by mesenchymal cells, may elicit a morphogenic response in the developing metanephros. We investigated the role of HGF at three different stages of murine metanephric development utilizing serum-free organ culture. Cultures were initiated at E-13, E-15, and E-17; treated with exogenous HGF or antibodies to HGF (to block endogenous HGF) for 120 h in vitro; and evaluated for growth and differentiation in comparison to control explants cultured for 120 h in basal medium. HGF treatment of E-13 explants resulted in a reduction of growth and differentiation compared to control explants. Treatment of E-13 explants with antibodies to HGF produced explant growth and differentiation indistinguishable from control explants. In contrast to the results of E-13 cultures, explants initiated at E-15 and E-17 demonstrated an increased growth and differentiation profile when treated with HGF compared to controls. Treatment of E-15 and E-17 explants with antibodies to HGF resulted in a decrease growth and differentiation profile compared to control or HGF-treated explants. These data demonstrate that HGF has differential effects on renal morphogenesis at progressive developmental stages of metanephric development.

Epidermal growth factor receptor activity mediates renal cyst formation in polycystic kidney disease.

A consistent phenotype observed in both human patients and several different mouse models of autosomal recessive polycystic kidney disease (ARPKD) is an increased activity of the epidermal growth factor receptor (EGFR) in the affected kidneys. To determine whether this increased activity of the EGFR is a functional event that is directly part of the disease pathway of renal cyst formation, we used a genetic approach to introduce a mutant EGFR with decreased tyrosine kinase activity into a murine model of ARPKD. We found that the modified form of the EGFR could block the increase in EGFR-specific tyrosine kinase activity that normally accompanies the development of renal cysts, and this correlated with an improvement in kidney function and a substantial decrease in cyst formation in the collecting ducts. These results suggest that changes in the expression of the EGFR contribute to the formation of cysts in the collecting ducts, and that drugs that target the tyrosine kinase activity of the EGFR may potentially be therapeutic in ARPKD.

Differential rescue of the renal and hepatic disease in an autosomal recessive polycystic kidney disease mouse mutant. A new model to study the liver lesion.

Autosomal recessive polycystic kidney disease (ARPKD) is characterized by biliary and renal lesions that produce significant morbidity and mortality. The biliary ductual ectasia and hepatic portal fibrosis associated with ARPKD have not been well studied even though such lesions markedly affect the clinical course of patients after renal replacement therapy such as dialysis or transplantation. Here we describe the generation of a new mouse model to study the hepatic lesions associated with polycystic kidney disease. This model was generated by differentially rescuing the renal pathology in the orpk mutant mouse that displays a hepatorenal pathology that is similar to that seen in human patients with ARPKD. This was accomplished by expressing, as a transgene in the mutant animals, the cloned wild-type version of the gene associated with the mutant locus in this line of mice. Although renal function in the rescue animals is normal, the liver still exhibits biliary and ductular hyperplasia along with varying degrees of hepatic portal fibrosis that is indistinguishable from that in the mutant animals. Most important, the rescue animals survive significantly longer than mutants and will permit a more detailed analysis of the clinical and cellular pathophysiology of the hepatic defect associated with this disease.

Efficacy of taxol in the orpk mouse model of polycystic kidney disease.

Polycystic kidney disease (PKD) a is common disease in the human population that can lead to renal failure and death. Taxol has recently been reported to be of therapeutic benefit in the cpk mouse model of PKD. To determine whether these results also apply to other models of PKD, we studied the effects of taxol treatment on the development of renal cysts and biliary hyperplasia/dysplasia/fibrosis in the orpk mouse mutant, a unique murine model for human autosomal recessive PKD. We report no significant differences between the treatment and control groups with respect to weight gain, survival, urine to serum osmolality ratio, and serum concentration of liver enzymes. Moreover, renal cystic development was not affected by taxol treatment in the orpk mutant animals. This was confirmed by lectin staining and morphometric analysis of the renal cysts, which indicated no significant differences between treatment groups. Therefore, while taxol has a positive effect on the cystic kidney disease in cpk mutant mice, this effect is not applicable to all forms of PKD.

Functional correction of renal defects in a mouse model for ARPKD through expression of the cloned wild-type Tg737 cDNA.

Autosomal recessive polycystic kidney disease (ARPKD) is characterized by the formation of large collecting tubule and ductular cysts that often result in renal insufficiency within the first decade of life. Understanding the process leading to cyst formation will require the identification and characterization of genes involved in the etiology of this disease. In this regard, we previously described the generation of a mouse model (TgN737Rpw) for ARPKD and the cloning of a candidate gene. Here we show direct involvement of the Tg737 gene in collecting duct cyst formation by expressing the wild-type Tg737 cDNA as a transgene in TgN737Rpw mutants. In contrast to TgN737Rpw mutants, the "rescued" animals survive longer, have normal renal function and normal localization of the EGFr to the basolateral surfaces of collecting duct epithelium.

Novel Madin Darby canine kidney cell clones exhibit unique phenotypes in response to morphogens.

Novel Madin Darby Canine Kidney cell clones were isolated. These cell clones exhibit differential responsiveness to inducers of tubule or cyst formation in collagen gel culture: hepatocyte growth factor or inducers of intracellular cAMP formation, respectively. In gel culture, clone OR93.22.D6 forms cysts and responds with morphological transformation to both hepatocyte growth factor and prostaglandin E1, and is most typical of a previously described cell type except for its higher transepithelial electrical resistance. OR55.25.II20 forms tubules in culture, is unresponsive to hepatocyte growth factor, and forms prostaglandin-induced spherical cysts. OR55.28.V2 forms dense cell spheres under control conditions, is induced to form tubules by hepatocyte growth factor, and is unresponsive to prostaglandin. OR55.29 forms only cysts, and is the only clone to form domes in monolayer culture. Tubule formation induced by hepatocyte growth factor, in all clones except OR55.25.II20, is blocked by a neutralizing antibody. In defined medium, without hepatocyte growth factor or prostaglandin, OR55.25.II20 forms spontaneous tubules. This finding indicates that a tubulogenic serum factor is not responsible for the observed phenotype. Increasing prostaglandin concentrations lead to inhibition of tubule formation and increased cyst formation. This observation suggests that induction of intracellular cAMP formation negatively regulates tubule formation in these cells, and implies that cystogenesis may represent a "default pathway" for impaired tubulogenesis. These observations demonstrate that some facets of renal tubulogenesis may be independent of hepatocyte growth factor, and that care must be exercised when comparing biological studies utilizing different clones.

Insertional mutagenesis and molecular analysis of a new gene associated with polycystic kidney disease.

We have identified a new insertional mutation in the mouse (TgN737Rpw) that causes a phenotype that closely resembles human autosomal recessive polycystic kidney disease. The renal pathology in these mutants first presents itself as a dilation of the proximal tubules, which is quickly followed by the development of cystic lesions in the collecting ducts. The livers in the mutant animals develop a variable lesion depending upon the genetic background. We have cloned the mutant locus and have isolated and characterized a gene, Tg737, whose expression is disrupted in the mutant animals. Expression of the Tg737 gene can normally be detected using the Northern blot analysis at low levels in a variety of tissues, including the kidney and liver. Using the in situ hybridization procedure, expression of the Tg737 mRNA can be detected in the collecting ducts of adult kidneys and in portions of the embryonic day 15.5 kidney. Most important, we have corrected the defective kidney trait by expressing the wild-type cDNA as a transgene in the mutant animals. The human homologue of the Tg737 gene has also been cloned and mapped to human chromosome 13.

1 alpha, 25-dihydroxyvitamin D3 receptor ontogenesis in fetal renal development.

We used immunohistochemical techniques to examine the distribution of 1 alpha, 25-dihydroxyvitamin D3 receptors (VDR) in developing rat and mouse kidneys and murine metanephric organ culture. In vivo, the patterns of expression in the two species were similar despite the slight difference in gestational periods (rat, 22 days; mouse, 19 days). Starting at gestational day 15, epitopes for VDR were found in cells of branching ureteral buds and in surrounding mesenchyme and at later developmental stages in glomerular visceral and parietal epithelial cells and proximal and distal tubules (DT). Epitopes for the 28-kDa calcium-binding protein (calbindin D28k) were found exclusively in DTs starting at gestational day 19. The pattern of VDR expression during in vitro nephrogenesis in serum-free murine metanephric organ culture paralleled that seen in vivo. At the time of explantation into organ culture (gestational day 13), VDR epitopes were not detected. By 3 days of in vitro development, VDR expression was identical to that found in gestational day 15 metanephroi in vivo. VDR expression after 5 days of in vitro development mirrored the pattern of gestational day 17 metanephroi in vivo. No calbindin D28k epitopes were seen at any in vitro developmental stage studied. We demonstrate for the first time that VDR are present in specific areas of the developing rat and mouse kidney early in gestation. Calbindin D28k appears later in developing rat and mouse kidney and is distributed differently than the VDR. Metanephric organ culture may be a useful model for studying the regulation and function of VDR during early renal development.