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.

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.

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.

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.

Genomic structure of the gene for the human P1 protein (MCM3) and its exclusion as a candidate for autosomal recessive polycystic kidney disease.

The locus PKHD1 (polycystic kidney and hepatic disease 1) has been linked to all typical forms of the autosomal recessive polycystic kidney disease (ARPKD) and maps to chromosome 6p21.1-p12. We previously defined its genetic interval by the flanking markers D6S1714 and D6S1024. In our current work, we have fine-mapped the gene for the human P1 protein (MCM3), thought to be involved in the DNA replication process, to this critical region. We have also established its genomic structure. Mutation analyses using SSCP were performed in ARPKD patients' cDNA samples, leading to the exclusion of this gene as a candidate for this disorder. We also identified two intragenic polymorphisms that allowed families with critical recombination events to be evaluated. Although neither marker was informative in these individuals, they are the closest yet described for PKHD1 and may help to refine the candidate region.

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.

Safety and efficacy of erythropoietin in children with chronic renal failure.

A prospective randomized study of the use of recombinant human erythropoietin (rHuEPO) in children with chronic renal disease was conducted to assess dosing requirements and side effects. Forty-four children with chronic renal failure, aged 4 months to 21 years, were studied. Twenty-five patients were pre dialysis, 10 on peritoneal dialysis, and 9 on hemodialysis. Patients received either 150 U/kg per week or 450 U/kg per week divided thrice weekly of rHuEPO for 12 weeks or until target hemoglobin (Hb) was attained. Dose was then adjusted to maintain a normal Hb. Eighty-two percent of patients reached target Hb by 7.9+/-5.6 weeks (mean+/-SD); 95% of patients in the high-dose group and 66% in the low-dose group reached target Hb within 12 weeks. The overall median rHuEPO dose at target Hb was 150 U/kg per week. Hemodialysis patients tended to require more rHuEPO to maintain a normal Hb (median 250 U/kg per week). Transfusion requirements and panel-reactive antibody levels decreased during the 12 weeks. Iron deficiency and/or hypertension occurred in 30% of children. In conclusion, rHuEPO at 150 U/kg per week is safe and effective in treating anemia in children with chronic renal disease.

A 1-Mb BAC/PAC-based physical map of the autosomal recessive polycystic kidney disease gene (PKHD1) region on chromosome 6.

The PKHD1 (polycystic kidney and hepatic disease 1) gene responsible for autosomal recessive polycystic kidney disease has been mapped to 6p21.1-p12 to an approximately 1-cM interval flanked by the markers D6S1714/D6S243 and D6S1024. We have developed a sequence-ready BAC/PAC-based contig map of this region as the next step for the positional cloning of PKHD1. This contig comprising 52 clones spanning approximately 1 Mb was established by content mapping of 44 BAC/PAC-end-derived STSs, 3 known genetic markers, 5 YAC-end-derived STSs, 3 random STSs, 1 previously mapped gene, and 1 EST. The average depth per marker is 6.3 clones, and the average STS density is 20 kb. The genomic clone overlaps were confirmed by restriction fragment fingerprint analysis. A high-resolution BAC/PAC-based contig map is essential to the ultimate goal of identifying the PKHD1 gene.

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.

Functional genomics in the post-genome era.

As the biomedical research community enters the post-genome era, studying gene expression patterns and phenotypes in model organisms will be an important part of analyzing the role of genes in human health and disease. New technologies involving DNA chips will improve the ability to evaluate the differential expression of a large number of genes simultaneously. Also, new approaches for generating mutations in mice will significantly decrease the cost and increase the rate of generating mutant lines that model human disease.