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Wilms' tumor, or nephroblastoma, is a developmental malignancy of the kidney that affects approximately 1 in 10,000 children between 1 and 6 years of age. Typically, the histology of nephroblastoma reveals a disorganized renal developmental process showing blastema and epithelia randomly interspersed in varying amounts of stroma. This developmental disruption is associated with the loss of function of the tumor suppressor gene WT-1. This gene, located on chromosome 11 at band p13, codes for a zinc finger protein that may act as a transcriptional repressor. Familial cases of Wilms' tumor fit Knudson's "two hit" model, according to which a germ line mutation of one WT-1 allele predisposes to the tumor while an additional somatic mutation of the other allele causes malignant transformation. Originally proposed for retinoblastoma, this model defines the nature of the tumor suppressor gene as a gene that is tumorigenic when inactivated. However, not all Wilms' tumor cases fit this model because the majority of Wilms' tumors do not show a mutation of WT-1. For Wilms' tumor, the loss of tumor suppression appears to be more complex than for retinoblastoma. Some of the mechanisms recognized to date involve dominant negative WT-1 mutations, interaction of the WT-1 gene product with other mutated transcription factors such as p53, loss of imprinting, and mutations of other tumor suppressor genes at 11p15 or other loci. Although classic Wilms' tumor is associated with good prognosis (85% survival), its anaplastic form is often fatal. Despite the plethora of knowledge gained in recent years, Wilms' tumor remains the center of attention for further investigation because it offers opportunities for studying normal kidney development, for understanding the molecular basis for clinically important anaplastic forms, as well as for elucidating the molecular mechanisms of tumor suppressor genes. To facilitate this task, Wilms' tumor heterotransplants have been established in nude mice. This provides an indefinite source of tumor tissue and a means to test their growth properties in response to drug treatments or molecular genetic manipulations. Furthermore, the establishment of stable Wilms' tumor cell lines is essential to investigating further the molecular basis of tumorigenesis using recombinant DNA technology.

Clinically the anaplastic variant of Wilms' tumor differs from the classical Wilms' tumor by its poor prognosis. To begin to understand and characterize the distinctive biology of this rare form of Wilms' tumor, a study of the histology, ultrastructure, and mRNA expression was performed on the anaplastic tumor and its associated cell line. The anaplastic tumor generated mouse heterotransplants that were readily used to establish epithelial cell cultures. The epithelial cultures, in turn, produced tumors when reinjected into nude mice. Microscopic evaluation revealed that the anaplastic epithelial cells were less differentiated than their epithelial counterpart in classical Wilms' tumors. In general the molecular profile of the anaplastic tumor was more consistent with that of an epithelial-rich classic Wilms' tumor than with the classic triphasic Wilms' tumor. Unlike the classic triphasic Wilms' tumor that contains blastema, stroma, and epithelial tubules, the anaplastic tumor expressed only marginal levels of insulin-like growth factor 2 (IGF-2) mRNA and imperceptible levels of the Wilms' tumor gene (WT-1), Pax-2, and Pax-8 mRNA. In common with the classic Wilms' tumor, the anaplastic variant retained the expression of the N-myc gene while failing to express C-myc. A comparison of cultures derived from an epithelial-rich, classic Wilms' tumor and the anaplastic Wilm's tumor indicated that both lacked IGF-2 and WT-1 mRNA expression. However, the well-differentiated epithelial cell culture derived from the classic Wilms' tumor expressed C-myc, Pax-8, and Pax-2 mRNA, none of which were expressed by the anaplastic epithelial cells. Furthermore, the well-differentiated epithelial cell component failed to express N-myc, which was expressed by both the primary triphasic Wilms' tumor and the anaplastic tumor. Overall, the findings indicate that patterns of gene expression within a single component do not correlate with the aggressive clinical behavior of the anaplastic Wilms' tumor.

Humans have a complex expression of metallothionein (MT) genes which involves many MT isoforms encoded by a family of genes containing an upper limit of 12 possible functional genes, in contrast to most animals which have one or two functional MT genes. In the present study, human proximal tubule (HPT) cells were exposed to cadmium (Cd) to determine if these cultures might serve as a model system to study MT gene expression in the renal proximal tubule. Three independent isolates of HPT cells were shown to repeatably induce MT protein when exposed continually to a non-toxic dose of 1 microgram/ml of Cd administered as CdCl2. Accumulation of MT protein was noted within 3 h and persisted over the 16-day time course. The expression of mRNA for the MT-IIA, MT-IA, B, E, F and G genes was also assessed through 16 days of exposure to 1 microgram/ml of Cd versus control media. Of these, the mRNA for the MT-IIA, MT-IE, MT-IF and MT-IG genes were detected in the cells exposed to 1 microgram/ml of Cd. Overall, the results were supportive that the HPT cells can provide a valuable model system to study the regulation of MT gene expression as it applies to the human renal proximal tubule.

Eleven separate isolates of human renal proximal tubule cells (HPT) were analyzed for toxic response to ionic cadmium exposure over a 16-day period in vitro. This study demonstrates a heterogeneous response to Cd2+ exposure in isolates from different individuals with some individuals nearly 3-times more sensitive to ionic cadmium exposure than others. There was no apparent correlation to the race, sex or age of the individuals in the response to cadmium. In addition, readily identifiable culture artifacts, i.e., culture age, passage number, had no influence on the response to Cd2+ exposure and the different isolates had homogeneous baseline HPT properties and morphology. This difference in response to Cd2+ may reflect a heterogeneous response within the human population to cadmium exposure.

Cell cultures retaining properties of the human proximal tubule were utilized to determine whether or not the aminoglycoside antibiotics alter paracellular transport. The transepithelial resistance (RT) of the human proximal tubule (HPT) cell monolayers was determined by Ussing chamber analysis of cells grown on permeable supports. This analysis revealed that RT was reduced as a result of aminoglycoside exposure and that the reduction corresponded to the known clinical nephrotoxicities of the aminoglycosides. Variation in the aminoglycoside concentration necessary to elicit this response was documented using 14 individual cell isolates. Ultrastructural analysis provided evidence indicating that the alterations in RT were not associated with general damage to the HPT cells. An examination of the structure of the tight junctions by freeze-fracture analysis demonstrated only minimal alteration of the sealing strands as a result of aminoglycoside exposure. Consequently, the reductions in RT were not directly associated with discernible tight junction structural alterations. Alteration in the paracellular route of transport, as indicated by altered RT values, was clearly documented as a result of aminoglycoside exposure. In addition, this alteration was accompanied by an increased density of intramembrane particles within the apical cell membrane.

A human Wilms tumor cell line (RM1) was developed to test the tumor suppressor activity of WT1, a zinc finger transcription factor that is expressed in the developing human kidney and is mutationally inactivated in a subset of Wilms tumors. Transfection of each of four wild-type WT1 isoforms suppressed the growth of RM1 cells. The endogenous WT1 transcript in these cells was devoid of exon 2 sequences, a splicing alteration that was also detected in varying amounts in all Wilms tumors tested but not in normal kidney. Production of this abnormal transcript, which encodes a functionally altered protein, may represent a distinct mechanism for inactivating WT1 in Wilms tumors.

Nephrogenic rests consist of foci of primitive renal cells, typically microscopic, that are found within the normal kidney tissue of children with Wilms' tumour. To study the relationship between nephrogenic rests and the associated tumours, we screened these lesions for mutations in the 11p13 Wilms' tumour suppressor gene, WT1. In two cases in which the Wilms' tumour contained a somatic WT1 mutation, the nephrogenic rest had the identical mutation. Nephrogenic rests and Wilms' tumours are therefore topographically distinct lesions that are clonally derived from an early renal stem cell. Inactivation of WT1 appears to be an early genetic event which can lead to the formation of nephrogenic rests, enhancing the probability that additional genetic hits will lead to Wilms' tumour.

We previously reported that cell cultures of human proximal tubule (HPT) cells respond to ionic cadmium in a manner consistent with well-defined Cd(2+)-elicited responses reported for in vivo systems. However, one unique finding was that the transepithelial electrical resistance and tight junction sealing strands were altered as a result of Cd2+ exposure at micromolar concentrations. These alterations are reexamined in detail in the present report to determine whether the Cd(2+)-induced alterations are specific alterations in the tight junction structure or reflect a general alteration in the cell membrane. Exhaustive analysis of tight junction sealing strands demonstrated no significant alterations due to Cd2+ exposure, even at the concentration that elicited a significant reduction in transepithelial resistance. Further analysis of intramembrane particle distribution demonstrated a significant increase in apical intramembrane particles, indicating that Cd2+ exposure altered the characteristics of the apical cell membrane. Overall, the results were consistent with evidence of Cd(2+)-induced alteration in the apical cell membrane of the HPT cell.

Increased routing of glucose through the hexosamine-biosynthetic pathway has been implicated in the development of glucose-induced insulin resistance of glucose transport in cultured adipocytes. Because both glucosamine and glucose enter this pathway as glucosamine-6-phosphate, we examined the effects of preincubation with glucosamine in isolated rat diaphragms and in fibroblasts overexpressing the human insulin receptor (HIR-cells). In muscles, pre-exposure to glucosamine inhibited subsequent basal and, to a greater extent, insulin-stimulated glucose transport in a time- and dose-dependent manner and abolished the stimulation by insulin of glycogen synthesis. Insulin receptor number, activation of the insulin receptor tyrosine kinase in situ and after solubilization, and the total pool of glucose transporters (GLUT4) were unaffected, and glycogen synthase was activated by glucosamine pretreatment. In HIR-cells, which express GLUT1 and not GLUT4, basal and insulin-stimulated glucose transport were unaffected by glucosamine, but glycogen synthesis was markedly inhibited. Insulin-stimulated activation of protein kinases (MAP and S6) was unaffected, and the fractional velocity and apparent total activity of glycogen synthase was increased in glucosamine-treated HIR-cells. In pulse-labeling studies, addition of glucosamine during the chase prolonged processing of insulin proreceptors to receptors and altered the electrophoretic mobility of proreceptors and processed alpha-subunits, consistent with altered glycosylation. Glucosamine-induced insulin resistance of glucose transport appears to be restricted to GLUT4-expressing cells, i.e., skeletal muscle and adipocytes; it may reflect impaired translocation of GLUT4 to the plasmalemma. The glucosamine-induced imbalance in UDP sugars, i.e., increased UDP-N-acetylhexosamines and decreased UDP-glucose, may alter glycosylation of critical proteins and limit the flux of glucose into glycogen.

This study determined the sensitivity of 12 independent isolates of cultured human proximal tubule (HPT) cells to gentamicin. The TD50 (dose [microgram per ml]) for each isolate was determined by linear regression analysis of cells exposed to a wide concentration range of gentamicin over a 16-day period of exposure. The results of this determination clearly showed that within a series of HPT cell cultures there was significant diversity in sensitivity to gentamicin. Three groups of toxic response were delineated. A single isolate, isolate I, was extremely resistant to gentamicin toxicity and was the sole member of group 1. The majority (9 of 12) of the isolates demonstrated intermediate sensitivity to gentamicin and were classified as group 2. Group 3 included two isolates that were very sensitive to gentamicin, with relatively low doses producing cell death. These results provide initial evidence that a portion of the variability seen in nephrotoxicity among patients receiving the aminoglycoside antibiotics might be mediated by factors intrinsic to each individual's proximal tubule cells.

Wilms tumor is a pediatric neoplasm that arises from the metanephric blastema. The expression of the gene encoding insulin-like growth factor II (IGF-II) is often elevated in these tumors. Since many of the actions of IGF-II are mediated through activation of the IGF-I receptor (IGF-IR), we have measured the levels of IGF-IR mRNA in normal kidney and in Wilms tumor samples using solution hybridization/RNase protection assays. IGF-IR mRNA levels in the tumors were 5.8-fold higher than in adjacent normal kidney tissue. Among the tumors themselves, the levels of IGF-IR mRNA in those containing heterologous stromal elements were 2-fold higher (P < 0.01) than in tumors without these elements. IGF-IR gene (designated IGF1R) expression in the tumors was inversely correlated with the expression of the Wilms tumor suppressor gene WT1, whose inactivation appears to be a key step in the etiology of Wilms tumor. Cotransfection of Chinese hamster ovary cells with rat and human IGF-IR gene promoter constructs driving luciferase reporter genes and with WT1 expression vectors showed that the active WT1 gene product represses IGF-IR promoter activity in a dose-dependent manner. These results suggest that underexpression, deletion, or mutation of WT1 may result in increased expression of the IGF-IR, whose activation by IGF-II may be an important aspect of the biology of Wilms tumor.

Monolayers of human proximal tubule (HPT) cells, when grown on permeable supports and mounted in Ussing chambers, spontaneously display a transepithelial potential difference (PD), short-circuit current (Isc), and transepithelial specific resistance (RT). These electrical parameters were used to determine the degree of heterogeneity among independent isolates of human proximal tubule cell cultures. Seventeen independent isolates of cells were assessed, totaling 260 individual determinations of spontaneous electrical properties. On average, these cell monolayers displayed an apical-negative PD of 1.5 +/- 0.1 mV, an Isc of 2.7 +/- 0.2 microA/cm2, and an RT of 480 +/- 19 ohms x cm2. Each independent cell isolate, however, displayed electrical values within a narrow range, in some cases allowing isolates to be distinguished from one another. The individual isolates were also assessed for Na-coupled glucose transport, Na+,K(+)-ATPase activity, cAMP stimulation by parathyroid hormone (PTH), forskolin stimulation of Isc, and ouabain inhibition. With the exception of a strong correlation between Na+,K(+)-ATPase activity and Isc, these parameters, in contrast to electrical properties, were found to be consistent and did not reveal distinctions among the isolates. HPT cell cultures seem to consistently retain important features of proximal tubule differentiation while maintaining the variability, as demonstrated by electrical properties, that might be expected of cells isolated from a random population.

The ability to establish cell cultures representing the epithelial component of Wilms' tumor was determined for 18 cases of classic Wilms' tumors. From these 18 cases only two resulted in the culture of epithelial cells. Although the tumors from both cases were composed of a prominent epithelial component, other classic tumors not producing epithelial cell cultures also possessed appreciable epithelial components. Likewise, heterotransplants of these two primary tumors failed to give rise to epithelial cell cultures, although cultures of the blastemal element were produced. This suggests that Wilms' tumors may be prone to differentiate in different directions at varying times during tumor growth, possibly dependent on local tumor environment. Epithelial cells from these two classic cases were grown in culture in basal medium composed of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium, supplemented with selenium, insulin, transferrin, hydrocortisone, tri-iodothyronine, and epidermal growth factor, on a collagen type I matrix with absorbed fetal calf serum proteins. One of the two cases also required the addition of bovine pituitary extract, ethanolamine, prostaglandin E1, and putrescine for optimum growth. Morphological analysis disclosed that the cultured cells were very similar to normal renal tubular cells in culture, except that the cells displayed little evidence for differentiated active ion transport and tended to grow in a multilayered arrangement. The culture of the epithelial cells from classic Wilms' tumors provides a model system for the study of tumor differentiation and progression.

The growth rate of primary tumors and derived cell lines is an important identifying trademark to researchers studying the growth and differentiation of pediatric neoplasms. For the in vivo tumor specimen, the mitotic index is utilized to assess growth potential while, in vitro, the determination of the growth curve and doubling time of the cells is employed. Because of the importance of these parameters this laboratory has developed a technique for the simultaneous determination of cell number and mitotic index for adherent cultured cells, as well as solid tumors, using the polycationic dye DAPI (4',6-diamino-2-phenylindole). DAPI, when bound to nuclear DNA, fluoresces, and this property has been utilized to develop an image analysis based technique to determine cell number automatically based on gray scale discrimination. In addition, DAPI staining clearly delineates mitotic figures and this has allowed the simultaneous manual determination of mitotic index for each automatic cell count. This technique was first tested using four different human cell lines and was shown to determine cell growth and mitotic index simultaneously. Lastly, employing an atypical mesoblastic nephroma, it was shown that the mitotic index of the primary tumor could easily be obtained and compared to both the mitotic index of tumor heterotransplant in nude mice and the mitotic index of the tumor-derived cell culture. This technique should be useful in studies assessing the effects of various factors on the growth of adherent cultured cells as well as the accurate determination of the mitotic index in solid tumors.

The histology, ultrastructure, and messenger RNA expression of heterotransplants derived from the G401 cell line (American Type Culture Collection) have been characterized by comparison with Wilms' and rhabdoid tumors of the kidney. This analysis illustrates that the properties of G401 heterotransplant were consistent with a rhabdoid phenotype rather than that of a Wilms' tumor. The G401 cell line has been utilized in recent experiments to demonstrate the central role of chromosome 11 in Wilms' tumor. However, the present results suggest that these experiments may be more relevant to define the involvement of chromosome 11 in rhabdoid tumor of the kidney, a malignancy distinct from Wilms' tumor. This is clinically relevant since the rhabdoid tumor of the kidney is very aggressive and associated with an extremely poor prognosis.

The localization of Thy-1, a surface membrane lipoglycoprotein, was investigated using a monoclonal antibody specific for human Thy-1 (HB-2S-1). The localization of Thy-1 during development was established in a series of five fetal, three childhood, and two adult normal kidneys. In this series, Thy-1 immunolocalization progressed from mesangial and endothelial cell staining in the 16- to 17-week fetuses to similar staining along with staining of the parietal epithelium of the capsule and proximal tubule staining in the 20- to 24-week fetuses. Glomerular mesangial cell and endothelial cell staining was absent by 9 months postnatally when the adult pattern of staining was apparent. The localization of Thy-1 during development was also compared with a series of pediatric renal tumors including 14 Wilms' tumors, 3 congenital mesoblastic nephromas, 1 clear cell sarcoma, and 1 pediatric renal cell carcinoma. Thy-1 staining was demonstrated in epithelial tubules of Wilms' tumors and in the spindle-shaped cells of congenital mesoblastic nephroma correlating with Thy-1 immunoreactivity in the kidney proximal tubule and fetal medullary stroma, respectively. Thy-1 staining was absent in the anaplastic epithelial Wilms' tumor, the renal cell carcinoma, and the clear cell sarcoma. This staining pattern fails to provide evidence that these tumors may arise from the medullary mesenchyme or the differentiated proximal convoluted tubule. These results show that Thy-1 is a renal differentiation marker and is useful in the characterization of tumors of renal development.

Initial attempts to model urinary folate reabsorption using cultures of HPT cells on porous filter inserts produced disappointing results in that large amounts of 5M were transported across the epithelial monolayer in a nonspecific manner. Since the impermeable molecule inulin was also transported, there apparently existed a significant leakage pathway in the way that the cultured cells were used for transport studies. 5M was bound to the AP membrane and taken up into the HPT cell by specific processes, while inulin was excluded, suggesting that the HPT cells were nevertheless operating functionally. TER values from cultured HPT cells plateaued at a high level when cells became confluent, suggesting an epithelial layer with functional tight junctions. However, when growth media were removed and replaced with transport buffers, there was an immediate loss of TER that fully recovered if the transport buffers were preincubated for 60 min. Under these conditions, transport studies showed the expected results--no movement of inulin through the cell layer and much reduced transfer of folate through the paracellular pathway. These results suggest that a transient opening of tight junctions occurs when growth media are replaced with biological buffers (or with fresh growth media), but that recovery of tight junction function occurs with time.

Because mechanisms for the renal regulation of folate excretion are poorly understood, a cell culture system representative of the human proximal tubule (HPT) was used for studies of renal folate transport. After confluent cultures of HPT cells were incubated with 3H-labeled folic acid (PteGlu), binding to the apical membrane was determined by an acid removal procedure, and transport was subsequently measured in solubilized cells. Although PteGlu binding was almost all specific (suppressed by excess unlabeled PteGlu), HPT cells transported PteGlu by specific and nonspecific processes. Specific PteGlu binding and transport were both saturable processes, reaching maxima of 0.5 and 0.1 pmol/mg protein, respectively, with half-maximal constants of 12 and 50 nM, respectively. The PteGlu analogues methotrexate and 5-methyltetrahydrofolic acid (5-CH3-H4PteGlu) inhibited both the binding and transport of PteGlu, with 5-CH3-H4PteGlu being more potent (lower half-maximal inhibitory concentration). In contrast, 5-formyltetrahydrofolic acid significantly reduced PteGlu transport at concentrations (100-250 nM) that had no effect on binding. These data suggest that the HPT cells will serve as a good model for studies of renal folate reabsorption. Initial characterization of the transport of folate by HPT cells suggests two distinct processes, binding to a high-affinity membrane folate-binding protein followed by a structurally specific transfer into the cell.

The polyol pathway is present in tissues of several organs where its activation may participate in the development of diabetic complications. We measured the accumulation of polyol-pathway intermediates in HPT cells isolated from 21 different human kidneys from nondiabetic individuals. When exposed to 27.5 mM glucose in the growth media, cells isolated from approximately 75% of individuals (accumulators) accumulated sorbitol within 1-4 days, whereas 25% (nonaccumulators) accumulated only negligible amounts, even when the period of exposure was extended to 2 wk. Surprisingly, measurement of the activities of the polyol-pathway enzymes showed no difference in the levels of either AR or SDH between accumulators and nonaccumulators, even when the conversion of galactose to galactitol was used to measure AR activity in intact cells independently of SDH. Measurement of sorbitol in the growth media indicated that nonaccumulators were not releasing sorbitol into the growth media. Fructose levels in the conditioned growth media were 4 times higher in the sorbitol-accumulating cells. Together, these results indicate that the tendency of cells from an individual to accumulate significant amounts of sorbitol may reflect the cells' ability to metabolize sorbitol in steps subsequent to the polyol pathway.

The growth of cultured epithelial cells on permeable supports allows increased cell differentiation and the assessment of a variety of transcellular and paracellular transport processes. The need to assess the corresponding ultrastructural characteristics of these cells under identical conditions prompted this laboratory to develop a reliable method for producing freeze-fracture replicas of these cultures. Sections of filter inserts with the cell-side facing up are placed between layers of polyvinyl alcohol with a strip of mylar positioned on the layer of polyvinyl alcohol. Following freezing, the monolayer is fractured by lifting the mylar strip from the assembly. The result is a consistent fracture of the apical membrane sufficient for analysis of tight junction sealing strands, microvilli distribution, and intramembranous particle (IMP) distribution between apical and lateral membrane domains. This method utilizes standard equipment and readily available materials and, most importantly, allows the freeze-fracture and replication of an undisturbed cell monolayer.

Homozygous inactivation of WT1, a Wilms' tumor gene located on chromosome 11 at p13, is believed to predispose to Wilms' tumor and therefore may be a common occurrence in this cancer. The expression of this gene in primary Wilms' tumors was examined by northern and quantitative RNA slot blot analysis and compared with clinical, histologic, and molecular features of each case. The characteristic 3.2 kb RNA was readily detected in most primary tumors although there was marked variation in the level of WT1-specific transcripts. No abnormal-sized RNA products were detected and expression of WT1 was not coordinated with that of several other oncofetal genes: N-myc, insulinlike growth factor 2 (IGF-2), and c-myc. The relative abundance of WT1-specific RNA did correlate with the histologic category of Wilms' tumor such that those tumors with heterologous differentiation have, in general, lower relative levels of WT1 transcripts than those tumors without heterologous differentiation. These results further establish the heterogeneity of Wilms' tumor with respect to the expression of tumor-associated oncofetal genes and suggest a relationship between WT1 expression and cellular differentiation.

Monolayers of human proximal tubule (HPT) cells, when grown on permeable supports and mounted in Ussing chambers, spontaneously display a transepithelial potential difference (PD) and short-circuit current (Isc). These electrical parameters were used in the present study to determine if aminoglycoside exposure altered electrogenic sodium transport by HPT cells. The results of this determination demonstrated that exposure to gentamicin, at levels below that producing cell necrosis, caused a marked reduction in Isc and that this reduction followed the known in vivo nephrotoxicities of the aminoglycosides streptomycin, gentamicin, and neomycin. It was concluded through a similar analysis on a total of 14 isolates of HPT cells that the aminoglycosides repeatably reduced the electrogenic sodium transport of HPT cells. It was further determined that this alteration in electrogenic transport by gentamicin was mediated through exposure of the drug to the basolateral cell surface and that apical exposure had little effect. Evidence was obtained against the involvement of Na+, K(+)-ATPase, adenosine 3',5'-cyclic monophosphate, and sodium-coupled substrate transport in this alteration in electrogenic transport by the aminoglycosides. The basolaterally located Na+: CO3(-2):HCO3(-1) symporter is a possible site for aminoglycoside-induced nephrotoxicity.

The human proximal tubule (HPT) is the characteristic site within the kidney that mediates absorption of glucose. This study was designed to determine whether cultured HPT cells would respond to a hyperglycemic environment through activation of the polyol pathway. The results of this study clearly indicate that exposure of the HPT cells to an extracellular glucose concentration greater than or equal to 11 mM results in substantial intracellular accumulation of sorbitol. This accumulation is inhibited by approximately 70% by treatment with 100 microM sorbinil. When cells growing 24 h on 27.5 mM glucose were changed to medium containing 5.5 mM glucose, sorbitol concentration returned to the control level within 12 h. The activity of aldose reductase was increased by a factor of 1.6 by exposure to elevated glucose concentrations, and the relative reactivity of the enzyme with glucose as substrate was approximately 0.1 compared with that of glyceraldehyde as substrate. Together, these results indicate that cultured cells derived from the HPT undergo activation of the polyol pathway when exposed to a hyperglycemic environment.

Insulinlike growth factors (IGF) and epidermal growth factor (EGF) are produced in renal tissue, as are specific receptors for these hormones. To evaluate the significance of these observations to regulation of renal tubular cell proliferation, we have examined the interaction of IGF and EGF with cultured human proximal tubular epithelial cells (HPT). HPT cells showed specific binding of IGF-1, insulin, and EGF. IGF-1 binding was inhibited by antibody to the type 1 IGF receptor (alpha-IR3). Insulin receptors and type 1 IGF receptors were identified by bifunctional cross-linking. IGF-1, insulin, and EGF stimulated [3H]thymidine incorporation by 77, 73, and 87%, respectively. Half maximal stimulation by IGF-1, insulin, and EGF were produced with 4 X 10(-9) M, 2.5 X 10(-8) M, and 8 X 10(-10) M concentrations of these hormones. Alpha-IR3 inhibited stimulation of thymidine incorporation by IGF-1 and insulin but had no effect in EGF-stimulated thymidine incorporation. EGF and high concentrations of insulin both stimulated cell proliferation by 83 and 79%, respectively. These data are consistent with regulation of tubular epithelial proliferation by IGF-1, insulin, and EGF and suggest that the mitogenic activity of both insulin and IGF-1 is mediated by the type 1 IGF receptor.

The growth of NIL and NILpy hamster embryo fibroblasts was determined in the presence and absence of pyruvate as a component of the growth medium. It was demonstrated that NIL cells respond to the presence of pyruvate by decreasing cell doubling time, glucose utilization, glutamine utilization, and increasing lactate production with the effects being more pronounced at low inoculum densities. Polyoma-virus transformed NIL cells (NILpy) demonstrate none of the above effects upon pyruvate addition regardless of initial cell inoculum density.

To determine if endothelial cell metabolism is affected by the elevated glucose concentrations known to occur in diabetes mellitus, we measured cellular Factor VIIIR:Ag in endothelial cells grown under conditions of increased glucose concentration. The results of this study illustrate that, under cell culture conditions, an increase in glucose concentration results in increased cellular levels of Factor VIIIR:Ag. Specifically, a glucose concentration of 300 mg/dl resulted in a 23% increase in Factor VIIIR:Ag levels while a glucose concentration of 600 mg/dl resulted in a 54% increase in Factor VIIIR:Ag levels. These in vitro findings may relate to the increase in plasma Factor VIIIR:Ag levels known to occur in diabetic patients.

The presence of high concentrations of sodium in the sweat of patients with cystic fibrosis is widely accepted by all investigators as one of the most important manifestations of the genetic defect. However, the simple means of sweat collection utilized for diagnostic purposes has been extended to the biochemical and physical analysis of the macromolecular components of human sweat. The most popular technique for the attainment of sweat has been collection of sweat droplets from the skin surface following heat stimulation. In this report, it is demonstrated that a far superior means of sweat collection exists based on a new collection device called the MACRODUCT. Utilizing this device, in conjunction with thin-layer isoelectric focusing, demonstrated that human sweat contains sex-specific proteins expressed in adult males and absent or greatly decreased from females and prepubertal males. Additionally, results are presented which suggest that the presence of epidermal keratins are an excellent marker for sweat contaminated with nonsweat gland-derived macromolecules.

Two alpha-fucose-binding lectins, Ulex europaeus agglutinin I (UEA I) and Lotus tetragonolobus agglutinin, were employed to compare and contrast the distribution of fucosubstance in normal human kidneys and a variety of renal tumors. The study employed a total of 31 kidneys surgically removed for the presence of a variety of tumors, including 11 unilateral Wilms' tumors, two cases of bilateral Wilms' tumors, 13 renal cell carcinomas, two congenital mesoblastic nephromas, one renal oncocytoma, one neuroblastoma metastatic to the kidney, and one clear cell sarcoma of the kidney. The results show that UEA I-reactive fucosubstance is detected in vascular endothelium of all kidneys and tumors, except bilateral Wilms' tumors. The presence of UEA I-reactive alpha-fucose in the vasculature of unilateral but not bilateral Wilms' tumors defines a unique histochemical distinction between the two groups of tumors. Conceivably, this property might be exploited as a screening procedure for the more aggressive bilateral neoplasms. Other findings detail histochemical differences between UEA I and L tetragonolobus agglutinin, as evidenced by the ability of one lectin to stain a particular cell type that is not reactive with the other lectin.

In order to facilitate studies on human salivary glands, a method was developed for the culture of minor salivary gland duct cells from tissues obtained from oral surgery protocols. Minor salivary glands were isolated from such tissues, and a serum-free growth medium was developed which supported the growth of the ductal component of these glands. The ductal origin of these cells was confirmed through immunohistochemical localization of replicating nuclei through incorporation of BrdU. The presence of epidermal keratin in replicating cells and the absence of smooth muscle myosin further substantiated the ductal origin of cells. Using normal growth medium calcium concentrations (1.05 mM), these cells produced a keratinized multilayer of cells unable to undergo routine subculture procedures. A reduction in calcium ion concentration to 0.1 mM resulted in a cell monolayer, without evidence of terminal keratinization, which could undergo at least eight serial passages (1:3 ratio) under cell culture conditions. It is advanced that these minor salivary gland duct cell cultures will be of use to those studying diseases and disorders of the salivary glands.

Thrombin activation of factor VIII results in a marked, but transient, increase in factor VIII procoagulant activity. This proteolytic process has been examined by immobilizing factor VIII in a solid phase system using mouse monoclonal antibody specific for the factor VIII related antigen. These studies demonstrate that thrombin activation is the result of proteolytic cleavage from the factor VIII:C protein of a peptide fragment which contains both the factor VIII:C procoagulant and VIII:C antigen sites recognized by human antibodies.

The urine from patients with cystic fibrosis (CF) was screened for the presence of epithelial cells that could be serially propagated in vitro. The urine from two newborn infants with meconium ileus was an excellent source of epithelial cells, and over 400 cultures were initiated in each case. The urine of older CF patients hospitalized for pulmonary complications also yielded cells but in fewer numbers. No cells were obtained from older CF patients without pulmonary complications or from healthy volunteers. Hypoxia may be the factor stimulating cell exfoliation in the urine of sick CF patients and in a larger percentage of newborn infants.

Paraffin sections of seven cases of nephroblastoma and one case of clear cell sarcoma were stained with a battery of eleven lectins conjugated to horseradish peroxidase. Lectin staining revealed similarities between blastema and stroma with respect to their content of glycoconjugates whereas blastema and epithelial cells exhibited major differences. In general, blastema and stroma contained glycoconjugates with terminal or penultimate beta-galactose, glycoconjugates having either biantennary or triantennary N-linked sugar chains or both, sialoglycoconjugates, and occasionally glycogen. Epithelial cells also showed these complex carbohydrates but stained additionally for terminal disaccharide galactose-(beta 1----3)-N-acetylgalactosamine, terminal alpha-galactose and terminal alpha-N-acetylgalactosamine. Furthermore, staining with three fucose-binding lectins revealed that the linkage between terminal alpha-fucose residues to the constituent oligosaccharide chains varied between epithelial cells, blastema and stroma. In general, the distribution and content of glycoconjugates in tumour cells comprising clear cell sarcoma resembled that in blastema and stroma of nephroblastoma. Other findings included differences in content of glycosubstance between cuboidal and columnar cells within the same tumour. Also observed were variations between a primary tumour and its metastasis with respect to the occurrence of certain complex carbohydrates.

The in vitro culture of human kidney epithelial cells of defined nephronal origin would prove valuable in a variety of studies defining the factors and mechanisms responsible for diseases and disorders of the kidney. In this study we have tested the hypothesis that employing a serum-free growth medium allows the selective cultivation of human kidney epithelial cells. It is demonstrated that human kidney cortex, explanted into serum-free hormonally defined growth medium gives rise to a primary culture of kidney epithelial cells of homogeneous morphology capable of hemicyst formation. While these cells were able to proliferate to confluency as an explant culture, they were unable to undergo stable subculture. Subsequent manipulation of the culture vessel surface (an initial coat of bovine type I collagen followed by the absorption of macromolecules from fetal calf serum) yielded cultures able to be subcultured with growth to at least 30 cell generations. These cells were identified to be of proximal tubule origin by employment of enzyme histochemistry, immunohistochemistry, and ultrastructural examination.

The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, increases after a protein meal. The effect of amino acid mixtures on hepatic and renal ODC activity and polyamine content was studied in postabsorptive and 72-hour fasted rats. Fasting decreased ODC activity in liver and in kidney by approximately 50%. Hepatic ODC activity increased tenfold 4 hours after intraperitoneal injection of either 1 g/kg of a synthetic mixture of 17 amino acids or of casein hydrolysate to fed rats and about 20-fold in fasted rats. Renal ODC activity increased four- and tenfold respectively. A mixture of glutamate, aspartate, and alanine at concentrations given in the hydrolysate reproduced the full amino acid effect. No amino acid was effective when given alone, nor were mixtures of the other amino acid constituents of the hydrolysate. Glutamate + alanine was ineffective as were glucose or various combinations of arginine, ornithine, aspartate and NH3. Ornithine + glutamate or aspartate + glutamate were active but stimulated less than aspartate + glutamate + alanine. Hepatic and renal putrescine content increased in parallel with ODC activity. The data suggest that specific amino acids possess the full ODC-stimulating capability of a high quality protein and that polyamine synthesis is linked to urea cycle activity.

A lectin histochemical and immunohistochemical approach was used to compare the different histologic elements of an unusual case of Wilms' tumor with normal kidneys. This case was stained with ten lectin-horseradish peroxidase conjugates; immuno-stained for Tamm-Horsfall glycoprotein, epidermal keratin, and vimentin; and compared with 19 control kidneys. The morphologic and cytochemical properties of various tumor elements in this specimen served to identify them as tumor analogs of all segments of the normal kidney tubules except distal tubules. Evidence for Wilms' tumor differentiation is provided by this case, whose epithelium histochemically resembles normal human epithelial cells.

Ten Wilms' tumors (WT) were heterotransplated into athymic (nude) mice. Eight of the tumors (80%) grew and were serially passaged as many as 20 times. The histology of the primary heterotransplants resembled that of the surgically excised tumors (seven classical and one anaplastic). Histological examination of serial passages of the classical WT demonstrated the tendency of the stromal and tubular components to disappear. The anaplastic tumor, however, maintained histological features identical to the primary tumor through all passages examined. One WT cell line that exhibited a prominent skeletal muscle component failed to grow beyond the third passage. Spontaneous glomerular differentiation was noted in several heterotransplants. The site of transplantation (subcutaneous, peritoneal, or renal capsule) had no effect on the differentiation of the tumors, and attempts to produce intravenous metastases were unsuccessful. Unilateral nephrectomy of WT-bearing mice gave a transient increase in pulse labeling of the tumors with bromodeoxyuridine, a thymidine analogue, compared with sham-operated controls or mice bearing Ewings' sarcoma heterotransplants. The increased labeling of tumor nuclei reached a maximum at 48 h. Similar increased labeling was observed in the remaining kidney following unilateral nephrectomy. These data show that although WT is a malignant neoplasm, its cells retain the capacity to respond to physiological signals resulting from nephrectomy and that differentiation cannot be modulated by the site of heterotransplantation or serial passage in athymic mice.

The recognized need for epithelial cell culture models for cystic fibrosis (CF) research has resulted in ongoing efforts to improve normal and CF submandibular duct cell culture capabilities. The duct is most likely the site of the CF defect in this and other exocrine glands. In a previous report conditions required for the successful primary explant culture of normal and CF submandibular glands were outlined; however, terminal keratinization and involution of these cultures were recognized as severe limiting factors to their utilization in CF research. This report explores the effects of calcium concentrations in the medium, growth factor supplements, and matrix components on growth and differentiation of these cultures. Results of the study further confirm the ductal origin of cells in the outgrowth and demonstrate that progressive keratinization is initiated only after cells proliferate beyond the environment of the explant fragment. Keratinization with subsequent multilayering, desmosome formation, and involution in the cell outgrowth are governed in degree by the calcium concentration of the growth medium. Upon reduction of medium calcium to 0.1 mM concentration, the cells proliferate as a monolayer and subculture through 8 to 9 passages and retain the capacity to undergo ductlike differentiation.

Current evidence indicates that the defect in cystic fibrosis (CF) involves chloride transport in various epithelial cells. The sweat gland, one site of altered chloride transport in CF, was examined immunocytochemically for localization of a chloride-channel membrane protein, designated band 3 protein. Immunoreactivity was observed in sweat duct cell membranes of both normal and CF samples, whereas secretory coil regions were entirely unreactive. No difference was observed in the pattern or intensity of immunoreactivity between the two groups at the light microscopic (LM) level of resolution.

Despite numerous clinical and animal studies, the initial injury and pathogenesis of aminoglycoside nephrotoxicity remains unclear. To compliment and extend existing research avenues, a cell culture model system representative of the human proximal tubule (HPT) was tested to determine its applicability for use in studies assessing aminoglycoside-induced cellular toxicity. For this determination, the proximal tubule cell cultures were exposed to increasing concentrations of streptomycin and monitored for cell death and light and electron microscopic changes under both confluent (resting) and subconfluent (actively-dividing) culture conditions. Confluent cultures exposed to streptomycin were also assessed for possible alterations in transport activities by monitoring the electrical properties of the cells through Ussing chamber analysis. Both the confluent and subconfluent cultures demonstrated concentration-dependent toxicity to streptomycin. Ultrastructural analysis disclosed that both actively-dividing and stationary cultures contained "myeloid bodies" within the cytoplasm, consistent with those known to occur in vivo. In studies relating cell numbers to the dosage and time of exposure to streptomycin, the confluent cultures demonstrated and "insult-recovery" period at toxic, but sub-lethal, concentration, again correlating to the known in vivo experience with this class of antibiotics. The subconfluent cultures demonstrated increased resistance to the toxic effects of streptomycin, again mimicking the clinical experience with aminoglycoside toxicity. Chamber analysis, at a streptomycin dose well below the toxic level, indicated changes in the transport activities of these cultured cells. It is proposed that the use of cultured proximal tubule cells could be a useful model system to extend current research avenues assessing the mechanism of aminoglycoside nephrotoxicity.

Wilms' tumor has been proposed to originate from a developmental abnormality of the metanephric blastema. This undifferentiated component of Wilms' tumors has previously eluded efforts for in vitro growth. Blastema from a "classical" Wilms' tumor was transplanted into nude mice and passaged through 12 generations of heterotransplantation. Tumors from heterotransplants were grown for 12 serial passages in a serum-free growth medium supplemented with hormones and conditioned media from human kidney proximal tubule cells. The blastema initially grew on a collagen-fetal calf serum matrix as multicellular spheroids, and the cells proliferating from the rim of the spheroids had a flattened shape. Pulse-labeling with bromodeoxyuridine (BrdU) identified the proliferating cell population as blastemal in origin. Except for a loss of extracellular matrix, ultrastructural studies demonstrated morphologic similarities in the cultured cells, compared with the primary tumor and heterotransplants. Lectin histochemical stains for the peanut lectin (PNA) and immunohistochemical stains for cytokeratin (CYTO), vimentin (VIM), and epithelial membrane antigen (EMA) were performed on the original tumor, successive heterotransplants, and cells grown in vitro. The PNA stained the surface of the blastemal cells after sialidase digestion in the original tumor, heterotransplants, and cultured cells. The blastema of the original tumors was negative for CYTO and EMA but reactive for vimentin. This lack of differentiation was maintained in heterotransplants through 12 passages. However, blastemal cells demonstrated coexpression of CYTO and VIM intermediate filaments when grown in a serum-free medium on a matrix material. These studies demonstrate that the blastemal component of Wilms' tumor can be successfully grown in culture, passaged in nude mouse heterotransplants, and shown to undergo early stages of blastemal differentiation in vitro by growth in serum-free medium. This in vitro system provides a model for testing the factors that influence the growth and differentiation of the blastemal component of Wilms' tumors.

The renal kallikrein-kinin system is thought to participate in blood pressure regulation and displays abnormalities in human hypertension, as well as in many animal models of hypertension. Urinary excretion and tissue levels of renal kallikrein were measured in streptozocin (STZ)-diabetic rats in relation to blood pressure, glycemia, and insulin treatment. In study 1, STZ-diabetic rats with marked hyperglycemia showed reduced kallikrein-like esterase excretion, compared with control rats, when first measured after 7 days of diabetes (9.9 +/- 2.5 versus 17.5 +/- 2.4 EU/24 h, P less than 0.05). This difference increased with time and, after 210 days, urinary esterase excretion in diabetic and control rats was 6.7 +/- 2.1 and 39.0 +/- 6.0 EU/24 h, respectively (P less than 0.001). Urine kallikrein, measured by radioimmunoassay, was similarly reduced in diabetic rats (40.4 +/- 8.0 versus 88.0 +/- 6.5 micrograms/24 h, at 30 days, P less than 0.001). At 120 days, systolic blood pressures were elevated in diabetic rats (P less than 0.05), and at 180 days over 60% of the diabetic rats had pressures above the highest pressures of control rats. In study 2, STZ-diabetic rats were treated with insulin for 2 wk (2 U NPH at 0800 h, or 2 U NPH at 0800 and 1600 h). In the single-dose group, with hyperglycemia similar to that of diabetic rats in study 1, kallikrein excretion was reduced as early as day 2, compared with nondiabetic rats (56.0 +/- 6.1 versus 109 +/- 9.4 micrograms/24 h, respectively, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

The role of the type I insulinlike growth factor (IGF) receptor in regulating growth of Wilms' tumor (WT) was evaluated by examining the effect of antibody-mediated inhibition of this receptor on tumor growth in cell cultures and as heterotransplants in athymic mice. An antibody to the human type I IGF receptor (alpha IR-3) inhibited 125I-IGF-1 binding and prevented stimulation of thymidine incorporation by IGF-1 in vitro. Intraperitoneal administration of alpha IR-3 to nude mice bearing WT heterotransplants prevented tumor growth for 4 weeks and resulted in partial regression of established tumors. These data indicate the importance of IGF action in control of WT growth in vivo, and suggest potential therapeutic application using antigrowth factor receptor antibodies to block growth factor action.

Skeletal muscle differentiation within a Wilms' tumor is a well-documented histopathologic entity thought to occur at a relatively low incidence and influence prognosis. A serum-free hormonally defined growth medium has been developed, allowing the long-term growth of the skeletal muscle component of Wilms' tumors. Eight Wilms' tumors have been grown under these conditions. Three cases grew a homogeneous population of cells which ultrastructurally displayed all stages of myogenesis through myotubule formation. They also possessed immunoreactivity for skeletal muscle myosin and myoglobin and synthesized the M and B subunits of creatine kinase. Of interest was the finding that the ability to yield skeletal muscle cultures was limited to those cases which exhibited skeletal muscle fibers in vivo. This technique is also a very sensitive marker for identifying Wilms' tumors possessing a myoid component. A second serum-free hormonally defined medium has also been developed that supports the long-term culture of a unique cell type from Wilms' tumors which contain a myoid component. These cells are spindle-shaped and exhibit all of the characteristics of early myoblasts.

The amount of glucose consumed by chick embryo fibroblasts in primary culture is strongly influenced by the presence of bicarbonate ion in the culture medium. Cells grown on glucose at physiologic concentration (5.5 mm) and in the absence of bicarbonate ion have a reduced rate of glucose utilization when compared to their counterparts cultivated in medium containing the usual 25 mM bicarbonate. The presence or absence of bicarbonate is without effect on chick embryo fibroblast proliferation over a 6-day growth period. Both lactic acid accumulation per mole of glucose consumed and the utilization of glutamine increase as a function of bicarbonate ion in the growth medium.

The elucidation of the underlying defect in fluid secretion by cystic fibrosis (CF) sweat glands is hindered by the unavailability of an experimental model for investigating this disease. As a potential model system, a serum-free growth medium was developed that supports the explant growth of epithelial cells from fragments of human skin. Immunohistochemical analysis demonstrated that these epithelial cell outgrowths originated from the duct of the sweat gland. By electron microscopy, the cells were demonstrated to possess keratinocyte-like morphology as noted by the presence of a multilayered outgrowth of cells containing well-defined keratin bundles. Identical outgrowths from skin biopsies of CF patients were compared to normal outgrowths and alterations were noted to occur in dome formation and in the number of intercellular spaces between cells. Doming alterations were also noted to occur in the CF heterozygous state. No differences in cell fine structure or in growth factor requirements for cell proliferation were noted between normal and CF cells. The potential use of this system as a model for CF research is discussed.

As an approach to investigating the disease cystic fibrosis, attempts were undertaken to culture from human submandibular glands epithelial cells with a potential for manifesting the cystic fibrosis genetic defect. To initiate the culture of submandibular gland epithelial cells, tissue fragments from glands were explanted as a function of both the composition of the serum-free growth medium and of the matrix utilized to coat the culture vessel growth surface. A morphologically homogeneous growth of submandibular gland epithelial cells, uncontaminated by fibroblasts, was obtained, once optimum culture conditions were defined. Light microscopic examination of these explant cultures in a transverse plane of section demonstrated variation in the outgrowth according to distance from the explant. At its outer margin, the outgrowth consisted of one or two layers of viable low cuboidal cells, and more centrally, it was multilayered. Mitotic figures were observed in the periphery of the outgrowth. In the region, a few cells removed from the periphery where the outgrowth consisted of about three to six cell layers, dilated intercellular spaces, indicative of secretion of fluid and ions into the spaces, separated the basal cuboidal cells. Overlying cells were increasingly flattened toward the culture surface and devoid of nuclei. Centrally, near the explant, the multilayer appeared completely involuted throughout. Ultrastructural examination in a transverse plane of the multilayered region with viable basal cells confirmed these observations showing wide spaces separating the cuboidal basal cells, keratinization of midstratum cells, and complete involution of the upper layer of ghost-like cells. These cells cultured from the submandibular gland reacted positively to immunochemical staining for keratin.