Candidate gene associated with a mutation causing recessive polycystic kidney disease in mice.

A line of transgenic mice was generated that contains an insertional mutation causing a phenotype similar to human autosomal recessive polycystic kidney disease. Homozygotes displayed a complex phenotype that included bilateral polycystic kidneys and an unusual liver lesion. The mutant locus was cloned and characterized through use of the transgene as a molecular marker. Additionally, a candidate polycystic kidney disease (PKD) gene was identified whose structure and expression are directly associated with the mutant locus. A complementary DNA derived from this gene predicted a peptide containing a motif that was originally identified in several genes involved in cell cycle control.

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.

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.

Immunocytochemical localization of gamma-glutamyltranspeptidase during fetal development of mouse kidney.

In the fully developed kidney, gamma-glutamyltranspeptidase is localized predominantly to the apical plasma membrane of the proximal tubules. The appearance of this activity during murine fetal nephrogenesis was quantitated using a sensitive fluorometric assay, and development of membrane polarity was assessed by immunocytochemistry. Specific activity of the transpeptidase in 13-day fetal kidney was approximately 1 mU/mg protein. Between 13-21 days of gestation, total transpeptidase activity increased 7500-fold, whereas specific activity increased 50-fold. At 13 days of gestation, gamma-glutamyltranspeptidase immunoreactivity is localized to the apical surfaces of developing renal vesicles and the proximal segment of the S-shaped tubules. The organized cell structures have tight tubular junctions but lack a well-defined brush-border membrane. By 15 days of gestation, immunostaining of the apical surface of developing proximal segments is more prominent, and slight reactivity of the basolateral membrane is evident. By 17 days of gestation, the kidney is organized into discrete zones. The large increase in gamma-glutamyltranspeptidase activity correlates with the appearance of increased immunostaining of the developing brush-border membranes of the proximal tubules contained in the inner cortex. A very similar although somewhat delayed pattern of appearance of transpeptidase activity and immunostaining was observed in metanephric organ culture. Induction of proximal tubular cyst formation had no effect on the increase in transpeptidase activity that occurred during organotypic nephrogenesis.

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.

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.

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.

A new murine model of autosomal recessive polycystic kidney disease.

We describe the renal cystic disease occurring in a new inbred strain of mice which developed as a spontaneous mutation in otherwise healthy Balb/c mice. The disease displays characteristics of an autosomal recessive polycystic kidney disease. Affected animals develop massive cystic enlargement of the kidneys and die of renal failure at the age of 4 weeks. During postnatal development, there is a gradual shift in site of the lesions. At birth, cystic dilations are localized almost exclusively in proximal tubular segments, whereas in the terminal stages of the disease, 80% of the cysts are localized in collecting tubular segments as defined by segment specific lectin binding. The composition of the basement membrane of the cystic tubular walls during postnatal development as analyzed by immunocytochemistry is essentially normal during the earliest stage of cyst formation. However, with disease progression, the cystic tubular basement membrane demonstrates a decreased immunoreactivity to anti-laminin and anti-entactin antibodies. This indicates a shift in cyst localization during disease progression in this model, and suggests that basement membrane abnormalities are not a primary feature of the early cyst formation and progressive enlargement.

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.

Polypeptide growth factors in metanephric growth and segmental nephron differentiation.

Although the developing nephron expresses receptors for various polypeptide growth factors, the specific roles of such factors in renal organogenesis are unknown. Therefore, the effects of epidermal growth factor (EGF) (8.2 x 10(-11) M-1.6 x 10(-8) M), multiplication stimulating activity (MSA) (6.6 x 10(-10) M-1.3 x 10(-8) M) and transforming growth factor beta (TGF-beta) (1 x 10(-12) M-1 x 10(-9) M) on organotypic renal growth and segmental nephron differentiation were studied in a serum-free hormone-supplemented, murine metanephric organ culture system. Following culture in control or growth-factor-supplemented medium, explant growth was assessed, and explant growth and differentiation were determined morphometrically in four defined neprhon segments which were identified morphologically or immunohistologically with segment-specific antibodies and/or lectins: glomeruli, proximal tubules, thick ascending limb-early distal tubules, and collecting tubules. Results showed that EGF increased overall renal growth and specific differentiation of distal elements, but retarded differentiation of glomeruli and proximal tubules. EGF also induced hyperplastic cystic malformation in proximal tubules. MSA stimulated explant growth and promoted segmental differentiation of all tubular segments. TGF-beta globally retarded in vitro nephrogenesis. Such data demonstrate that polypeptide growth factors have multiple and often disparate effects on overall renal growth in relation to differentiation of discrete nephron segments and provide insight into the factors which may regulate normal and abnormal renal embryogenesis.

In vitro modulation of tubular cyst regression in murine polycystic kidney disease.

Recent studies in a murine model of genetically-determined polycystic kidney disease, the CPK mouse, have suggested that alterations in renal Na-K ATPase activity in concert with tubular epithelial hyperplasia have pathogenic import in proximal tubular cyst formation. In the current study, we therefore studied the relative roles of Na-K ATPase activity, tubular epithelial hyperplasia, and basal lamina alterations during in vitro modulation of proximal tubular cyst regression during serum-free organ culture of newborn CPK kidneys. Under basal in vitro conditions, regression of CPK proximal tubular cysts was demonstrated in association with progressive decreases in Na-K ATPase activity and tubular epithelial hyperplasia. The pattern of proximal tubular cyst regression was modified by: a) Na-K ATPase activity induction with triiodothyronine, which promoted proximal tubular cystogenesis; and b) Na-K ATPase activity inhibition with ouabain, which blocked the effects of T3 on the process of cyst formation. Modulation of proximal tubular cystogenesis by Na-K ATPase induction and inhibition were accomplished without significant changes in proximal tubular epithelial hyperplasia or expression of basal lamina components. We conclude that increased Na pump activity may have a significant role in proximal tubular cyst formation and progressive enlargement in the CPK mouse.

Cyst formation in metanephric organ culture induced by cis-dichlorodiammineplatinum (II).

A new experimental model of renal tubular cyst formation has been developed in metanephric organ culture. The addition of cis-dichlorodiammineplatinum (II), 50 micrograms/ml, to culture medium induces cystic changes during in vitro nephrogenesis. The model has particular utility in the study of basic mechanisms underlying renal tubular cystic changes, as well as the mechanisms by which nephrotoxins may mediate renal tubular injury.

Abnormal sodium pump distribution during renal tubulogenesis in congenital murine polycystic kidney disease.

Congenital polycystic kidney disease is characterized by the formation of large fluid-filled cysts in kidney tubules. It has been postulated that increased epithelial cell proliferation and altered transtubular fluid transport are necessary for cyst formation. To address the latter problem, we have studied the plasma membrane distribution of the alpha 1 and beta 1 subunits of Na+/K(+)-ATPase during progressive stages of proximal and collecting tubular cyst formation in the CPK mouse, a murine model of autosomal recessive polycystic kidney disease. In both control and cystic proximal tubules, Na+/K(+)-ATPase distribution was restricted to the basal-lateral membrane of cells. However, in newborn through day 5 kidney tissue, 16% of control vs. 47% of cystic outer cortical, 6% of control vs. 46% of cystic inner cortical, and 2% of control vs. 63% of cystic medullary collecting tubules demonstrated apical and lateral membrane distribution of Na+/K(+)-ATPase. In all nephrogenic zones, the percentage of control or cystic collecting tubules demonstrating apical membrane distribution of Na+/K(+)-ATPase decreased over time, but the percentage of cystic collecting tubules with apical membrane Na+/K(+)-ATPase remained significantly greater than in developmentally matched controls. No alterations in the normal distributions of other apical or basal-lateral membrane marker proteins were noted at any stage of control or cystic proximal or collecting tubule development. We conclude that apical-lateral membrane Na+/K(+)-ATPase expression is a normal transient feature of early collecting tubule development. However, apical membrane Na+/K(+)-ATPase persists in cystic kidneys, suggesting that such expression may be a manifestation of the relatively undifferentiated phenotype of epithelial cells lining collecting tubule cysts. The persistence of apical membrane Na+/K(+)-ATPase, if the enzyme is functional, may have pathogenic important in abnormal transtubular fluid transport in polycystic kidney disease.

Tyrosine kinase activity of the EGF receptor in murine metanephric organ culture.

Epidermal growth factor (EGF) and its fetal form, transforming growth factor alpha (TGF-alpha) are renal mitogens which induce epithelial hyperplasia, proximal tubular cyst formation (TC), and accelerated distal nephron differentiation in metanephric organ culture. To delineate the intracellular mechanisms mediating these growth factor effects, we studied the specific role of the epidermal growth factor receptor (EGF-R), the common receptor for both ligands, as an activated tyrosine kinase in TC formation and nephrogenesis. Fetal murine metanephric explants were incubated for 120 hours in control, and EGF (15 ng/ml)/TGF-alpha (10 ng/ml) supplemented medium with and without EGF-R blocking monoclonal antibody (50 mg/ml), or tyrosine kinase inhibitor. EGF-R tyrosine kinase inhibition was achieved by incubation with a synthetic tyrphostin (TP B42) (0.1 microM) or genestein (5.5 micrograms/ml). The following parameters were assessed: (a) segment-specific nephron development using morphometry and immunohistology; (b) tubular epithelial hyperplasia by protein content and BrdU uptake; and (c) TC formation by morphometric cystic index. Both growth factors produced hyperplastic proximal TC, significantly increased explant growth, and significantly increased distal nephron differentiation. Inhibiting the ligand-EGF-R interaction with EGF-R blocking monoclonal antibody abolished all growth factor-induced effects and resulted in increased amounts of undifferentiated mesenchyme and decreased distal nephron differentiation. Inhibition of EGF-R tyrosine kinase activity with either Tyrphostin B42 or genestein blocked TC formation and produced nodular blastemal hyperplasia and decreased distal nephron differentiation.

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.

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.

Sodium-potassium ATPase activity mediates cyst formation in metanephric organ culture.

To study the possible role of altered transtubular transport in renal tubular cyst formation, the ontogeny of renal Na-K ATPase was studied during glucocorticoid-induced cystic metanephric tubular development in serum-free, murine organ culture (SFMOC). Utilizing an enzyme-linked kinetic microassay, a developmental profile of total ATPase and specific Na-K ATPase activity was established for control (CON) and glucocorticoid-induced cystic organ culture (CY) explants. During 120 hr of CON and CY organ culture nephrogenesis total Na-K ATPase activity, specific Na-K ATPase activity, and the Na-K ATPase: total ATPase ratio progressively increased, simulating normal in vivo murine enzyme development. However, from 48 to 120 hr of organ culture, CY showed significant increases in Na-K ATPase activity when compared to CON at similar stages of development. Na-K ATPase activity (expressed as nmoles . min-1 . mg protein -1, mean +/- SD) was, at: 48 hr, CY 13.1 +/- 0.7 vs. CON 11.0 +/- 0.9 (P less than 0.01); 72 hr, CY 16.4 +/- 1.1 vs. CON 12.2 +/- 0.7 (P less than 0.001); 96 hr, CY 35.4 +/- 4.9 vs. CON 13.7 +/- 0.4 (P less than 0.001); and 120 hr, CY 26.1 +/- 1.4 vs. CON 16.3 +/- 0.9 (P less than 0.001). The initial differences in CY enzyme activity preceded the earliest ultrastructural evidence of cyst formation by 18 to 24 hr, while subsequent increases in Na-K ATPase activity in CY paralleled progressive tubular cyst formation. Tubular cyst formation in CY could be largely prevented by daily incubation of explants with ouabain, 0.2 mM (final concentration) X 120 min, without deleterious effects on overall metanephric development.(ABSTRACT TRUNCATED AT 250 WORDS)

Congenital murine polycystic kidney disease. II. Pathogenesis of tubular cyst formation.

In the current study, the pathogenesis of proximal tubular cyst formation was studied in an animal model of polycystic kidney disease, the CPK mouse. The specific roles of (a) sodium-potassium adenosine triphosphatase (Na-K ATPase) activity, determined by an enzyme-linked kinetic microassay, (b) proximal tubular epithelial hyperplasia, determined by calculation of mitotic indices, and (c) altered proximal tubular basal lamina formation, determined by immunohistological localization of basal lamina glycoproteins, were investigated at progressive developmental stages of CPK proximal tubular cyst formation. Increases in renal Na-K ATPase were present at the earliest fetal stages of proximal tubular cyst formation, and subsequently paralleled the course of proximal tubular cyst progression. Proximal tubular epithelial hyperplasia, although not present at the earliest stages of cyst formation, was a consistent feature of progressive proximal tubular cystic enlargement. Abnormalities in basal lamina glycoprotein expression were not present at any stage of proximal tubular cyst development. We conclude that increased Na-K ATPase and tubular epithelial hyperplasia are significant features of proximal tubular cyst formation in the CPK mouse.

A new model of glucocorticoid-induced metanephric maldevelopment.

A new experimental model of glucocorticoid-induced tubular cyst formation has been developed in metanephric organ culture. The addition of cortisol (1.4 X 10(-5) M) to chemically defined serum-free culture medium produces cystic changes during in vitro nephrogenesis . The model isolates the role of glucocorticoids in experimental cyst formation.

Abundance of mRNAs encoding urea cycle enzymes in fetal and neonatal mouse liver.

The relative abundances of mRNAs encoding the five urea cycle enzymes during development of mouse liver have been determined and compared with those of mRNAs encoding four other liver-specific proteins (phosphoenolpyruvate carboxykinase, tyrosine aminotransferase, alpha-fetoprotein, and albumin). Urea cycle enzyme mRNAs in fetal liver are expressed at 2-14% of the abundance in adult liver as early as 6 days before birth. Expression of the urea cycle enzyme mRNAs is not coordinate during the fetal and neonatal period. However, profiles of three urea cycle enzyme mRNAs are quite similar to that of alpha-fetoprotein mRNA, suggesting the possibility of a common response to regulatory signals during fetal development. With the exception of ornithine transcarbamylase mRNA, the urea cycle enzyme mRNAs have been shown previously to be inducible by cAMP and glucocorticoids. However, only argininosuccinate lyase mRNA exhibits any significant change in abundance at birth, resembling postnatal expression of tyrosine aminotransferase mRNA. The results indicate that the urea cycle enzyme mRNAs are potentially useful markers for elucidating various features of hepatocyte differentiation in mammals.