Anna L. Trifillis

Isolation, culture and characterization of human renal tubular cells.

Tubular cells have been isolated, characterized and cultured from more than 70 adult cadaver kidneys (postmortem time less than or equal to 12 hr.). Confluent monolayers were observed at 7 days after seeding (10(6) cells/ml.) and cells demonstrating normal human karyotypes have been passaged up to 6 times. Primary isolates and monolayer cultures were negative for Factor VIII activity, and strongly positive for gamma-glutamyltransferase activity and keratin. Ultrastructurally primary isolates consisted of cells with numerous mitochondria, microvilli, cytoplasmic filaments and well-developed endocytotic apparati. Monolayer cultures examined at 7, 14, 21 and 72 days demonstrated less prominent microvilli and the additional structures of desmosomes and cell junctions. Membrane-associated and cytosolic enzyme activities were measured up to 28 days in culture. The membrane-associated enzymes gamma-glutamyltransferase and alkaline phosphatase both exhibited approximately 10-fold decreases in activity during the 1st 7 days in culture. There was an approximately 5-fold increase in pyruvate kinase activity during the same time period, while fructose-1,6-bisphosphatase activity exhibited a 5-fold decrease. Glucose-6-phosphatase activity did not change during the 28 day culture period examined. From 7 to 28 days no further changes were noted in any of the enzyme activities measured. Decreased membrane-associated enzyme activity corresponded to the ultrastructural observation of less prominent microvilli. Increases in glycolytic enzyme activity and decreases in gluconeogenic enzyme activity may reflect the presence of glucose in the culture medium. The morphologic and biochemical evidence suggests that primary isolates and cultures are proximal tubule cells which should provide a well-defined in vitro human system for future studies.

Decreased sup 3 H-Thymidine Incorporation By Human Bladder Epithelial Cells Following Exposure To Urine From Interstitial Cystitis Patients

PURPOSE Interstitial cystitis (IC) is a chronic bladder disease of unknown etiology. We sought to determine whether a cytotoxin is present in the urine of IC patients that could cause the epithelial damage seen in this disease. MATERIALS AND METHODS Evidence for a cytotoxin was sought using both a neutral red uptake viability assay in T24 bladder epithelial cells, and a 3H-thymidine incorporation assay in primary normal adult bladder epithelial cells and FHS 738 Bl human fetal bladder cells. RESULTS The neutral red assay in T24 cells indicated the presence of a cytotoxin in 2 of 9 IC patient urine specimens. However, the more sensitive assay of cell proliferation (3H-thymidine incorporation) in normal adult human bladder epithelial cells revealed antiproliferative activity in urine from 10 of 13 (77%) IC patients vs. 3 of 19 (16%) controls (two-way analysis of variance, p = .019). The antiproliferative activity of IC urine specimens was confirmed using FHS 738 Bl human fetal bladder cells. The antiproliferative urinary substance(s) appeared to be a low molecular weight (< 10,000 Da), heat stable, trypsin-sensitive factor(s). CONCLUSIONS Because a denuded or damaged bladder epithelium is a central finding in IC, it is possible that the antiproliferative protein(s) contributes to the pathogenesis of this disease.

Marijuana and other drug use among automobile and motorcycle drivers treated at a trauma center

Serum from injured automobile and motorcycle drivers treated at a trauma center was tested for delta-9-tetrahydrocannabinol activity to determine precrash marijuana use. From June 1990 to March 1991, samples from approximately 20 automobile drivers per month and all motorcycle drivers were available for testing. Also, toxicology screens were performed for ethyl alcohol, cocaine, and phencyclidine (PCP) among the driver groups. Six (2.7%) of the 225 automobile (AUT) drivers and 34 (32.0%) of the 106 motorcycle (MTC) drivers were THC+ (p < .001). Compared with a prior study, the THC+ rate decreased significantly from 31.8% among AUT drivers (p < .001) but had not changed significantly from the 38.6% rate among MTC drivers. Positive toxicology rates were higher among the 261 MTC drivers compared to the 1,077 AUT drivers tested for ETOH, CO, and PCP, being 47.1% vs 35.2% (p < .001), 5.0% vs 8.0% (p < .08), and 1.5% vs 3.1% (NS), respectively.

Renal cysteine conjugate β-lyase-mediated toxicity studied with primary cultures of human proximal tubular cells

The beta-lyase pathway has been shown to mediate the nephrotoxicity of S-cysteine conjugates of a variety of haloalkenes in a number of animal models in vitro and in vivo. However, there is no information available concerning this mechanism of bioactivation in human tissues. In this investigation a well-characterized model of human proximal tubule epithelial cells, the presumed target cell, was used to investigate the toxicity of a series of glutathione and cysteine conjugates of nephrotoxic haloalkenes. Both S-(1,2-dichlorovinyl)-glutathione (DCVG) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) caused dose-dependent toxicity over a range of 25 to 500 microM. DCVC was consistently found to be more toxic than DCVG, but the inclusion of gamma-glutamyltransferase (0.5 U/ml) increased the toxicity of DCVG to that observed with an equimolar concentration of DCVC, indicating that metabolism to the cysteine conjugate is an important rate-limiting step in this in vitro model. S-(1,2,3,4,4-Pentachlorobutadienyl)-L-cysteine, S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine, and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine were also found to be toxic to human proximal tubular cells. Incubation with [35S]DCVC resulted in covalent binding of 35S-label, which increased linearly to a final level of 1.05 nmol/mg protein at 6 hr. Aminooxyacetic acid (250 microM), an inhibitor of pyridoxal phosphate-dependent enzymes such as beta-lyase, protected the cells from the toxicity of all of the cysteine conjugates and inhibited the covalent binding of 35S-label from [35S]DCVC to cellular macromolecules. The results of the present study provide the first evidence that human proximal tubular cells are sensitive to the toxicity of glutathione and/or cysteine conjugates of a variety of chloro- and fluoroalkenes which are activated via the beta-lyase pathway. The implications for human health are discussed.

Urine Autoantibodies in Interstitial Cystitis

Interstitial cystitis is a chronic bladder disease with certain features that suggest autoimmunity may play a role in initiating or maintaining the disease process. We therefore determined whether immunoglobulin fractions from 14 IC patient and 19 control urine specimens bound in vitro to primary cultures of human bladder epithelial cells, as well as epithelial cells from a variety of other tissues. Urine autoantibodies that bound to normal human bladder epithelial cells were present in 8 of 14 IC specimens (from 6 of 9 IC patients) as compared to 3 of 23 control specimens (from 2 of 17 control patients). These antibodies, which were usually also present at low titers in sera from these persons, bound to at least four nuclear or cytoplasmic antigens, with the specificity of autoantibodies from a given individual varying over time. The autoantibodies were not specific for normal or malignant bladder epithelial cells, but bound to epithelial cells from a variety of tissues. These data show that anti-epithelial cell autoantibodies are present in the urine of IC patients, but suggest that these antibodies are not likely to be a primary cause of this disease.

A prospective study of microorganisms in urine and bladder biopsies from interstitial cystitis patients and controls

OBJECTIVES Interstitial cystitis (IC) is a chronic inflammatory condition of the bladder of unknown etiology. We tested the hypothesis that a microorganism would be found at higher prevalence in urine or bladder tissue from women with IC than from control women. METHODS Urine and bladder tissue were obtained at cystoscopy from 11 IC patients and 7 control subjects. These specimens were cultured for a variety of fastidious and nonfastidious bacteria, mycobacteria, fungi, and viruses. In addition, special staining techniques were used to examine biopsy specimens and cytospun urine, and tissue sections and outgrowths of explanted bladder cells were examined by electron microscopy. RESULTS Cultures of urine from 6 of 11 IC patients grew five different bacteria (Corynebacterium sp. Klebsiella pneumoniae, Lactobacillus sp, Streptococcus constellatus, and Streptococcus morbillorum), human cytomegalovirus, or Torulopsis glabrata; one of these organisms (Lactobacillus sp) was found in urine from 2 patients. Although contamination by urethral organisms is possible, the prevalence of microorganisms in urine of IC patients (6 of 11) was significantly greater than in urine of control subjects (0 of 7) (P < 0.05). Acridine orange staining revealed rods with appropriate morphology in urine from 4 of the 5 IC patients who had positive bacterial cultures and yeastlike organisms in urine and bladder tissue specimens that grew Torulopsis. Additionally, rodlike organisms were seen in urine from 2 IC patients with negative bacterial cultures and cocci were seen in the urine of 1 control patient. Biopsy specimens from 2 IC patients grew Torulopsis sp or Lactobacillus sp, in agreement with the results of acridine orange staining and culture of urine from these patients; in contrast, specimens from 3 control subjects grew small numbers of Pseudomonas sp or Staphylococcus epidermidis, but no organisms were cultured from urine or seen in acridine orange-stained tissue smears. All other cultures and stains were negative. CONCLUSIONS These data do not provide evidence that IC is associated with infection or colonization by a single microorganism. However, they do generate the hypothesis that the prevalence of microorganisms, especially bacteria at low concentrations, is greater in the urine of IC patients than of control subjects. If these results are confirmed by other controlled studies, the question of whether the presence of these organisms is a cause or a result of IC should be addressed.

Culture of Bladder Epithelium From Cystoscopic Biopsies of Patients With Interstitial Cystitis

Interstitial cystitis is a chronic disease of unknown etiology characterized by bladder pain and urinary frequency and urgency. The epithelium may be critical in its pathogenesis; the hallmarks of the disease are visible epithelial defects (Hunners ulcers and epithelial ruptures). Areas denuded of epithelium are commonly seen, and defects in epithelial permeability are characteristic. We report here the culture and characterization of epithelial cells from cystoscopic bladder biopsies obtained from 7 female patients with interstitial cystitis. Within 4 to 14 days cellular outgrowths appeared from explants incubated in cell medium. Monolayers reached confluence after 6 weeks. Cells of the monolayer were cytokeratin-positive and smooth muscle actin-negative, confirming their epithelial origin. They exhibited epithelial cell ultrastructure including intermediate filaments and junctional complexes. Vesicles bounded by a trilaminar plasma membrane and lateral interdigitations were also present. This is the first report of the culture of bladder epithelium from interstitial cystitis patients. Epithelial cells may be targets for initiating agents and inflammatory effects of interstitial cystitis and should be useful for studies of the pathogenesis of this disease.

Metabolic studies of postischemic acute renal failure in the rat.

Postischemic acute renal failure was induced by 1 hr of clamping of the renal vasculature. Adenine nucleotide (ATP, ADP, AMP) and lactate (Lac) levels were measured after 0, 0.25, 1, 6, 24, and 48 hr of reflow to determine the time necessary for recovery to control levels. After 1 hr of ischemia with no reflow, [ATP] was 18% and [Lac] was 10-fold control levels. Control levels were restored after 24 hr of reflow. Variable ischemic times (5, 15, 30, 60, 90, and 120 min) followed by (1) no reflow or (2) 24 hr of reflow were also studied. [ATP] decreased to 25 and 13% of controls after 5 and 120 min of ischemia, respectively, and [Lac] increased to 5- and 13-fold controls after 5 and 120 min. Five to ninety minutes of ischemia followed by 24 hr of reflow resulted in a trend toward restoration of ATP and Lac levels; whereas, 120 min of ischemia followed by 24 hr of reflow resulted in death. The results indicate that: (1) In vivo ischemia results in a drastic and rapid shift in the ATP-ADP-AMP equilibrium; (2) the absolute concentration of ATP is not a reliable criterion of cell viability, but the ability to resynthesize ATP may be determinant in the reversibility of the lesion; (3) 1 hr of ischemia is reversible with respect to restoration of [ATP] and [Lac], but 24 hr of reflow are needed for restoration; and (4) ischemia for 90 min results in a metabolic derangement which is partially reversible in that metabolite levels are partially restored after 24 hr of reflow. However, 90 min of vascular clamping is not functionally reversible since the majority of animals exhibit severe azotemia and do not survive.

Metabolic studies of HgCl2-induced acute renal failure in the rat

HgCl2 was used to produce acute renal failure (ARF) in rats. It caused significant decreases (P < 0.001) in ATP, ADP, and total adenine nucleotide levels to 50% of controls at 48 hr with no change in AMP levels. Lactate increased to threefold control levels at 6 hr and remained significantly elevated (P < 0.001) at 48 hr. At 24 hr, there was widespread necrosis in the pars convoluta and pars recta. At 48 hr, necrosis was accompanied by some regeneration, and creatinine levels exceeded 5 mg/dl. Dithiothreitol (30.8 mg/kg, ip, 30 min after HgCl2) partially ameliorated the functional lesion (creatinine levels ⩽3.6 mg/dl) and resulted in reduced necrosis in the pars convoluta, but did not significantly alter the metabolic pattern. The results indicate an early metabolic disturbance and are consistent with the idea that HgCl2 exerts a direct action on the enzymes of the mitochondrial electron transport chain. The failure of dithiothreitol to alter the metabolic pattern of HgCl2-induced ARF indicated that its protective effect was probably not mediated directly through the maintenance of adenine nucleotide levels.

Culture and characterization of normal epithelium from cystoscopic biopsies of human bladder

Dear Editor: Cultures of bladder epithelium have been obtained from neoplastic bladder tissue (1,2,5), embryonic bladders (4) and postmortem adult bladders (3). Our goal was to culture normal human bladder epithelium from the tissues obtained at cystoscopy, a commonly performed procedure in which a scope is passed through the urethra into the bladder for diagnostic or therapeutic reasons. We believe this to be the first report of the culture of normal human bladder epithelial cells from the small amounts of tissue available from cystoscopic biopsy material. Initially, to develop suitable culture conditions, we obtained normal areas of bladder tissues (3-5 em 3) which had been resected during open surgery from three patients with bladder carcinoma. Two different methods of culture were attempted on the resected bladder material: 1) enzymatic digestion of the tissue prior to culture and 2) direct culture of the explant. Enzymatic digestion was used to separate the mucosal epithelium from the submucosa prior to culture to minimize contamination with fibroblasts. A portion of the tissue was minced and digested at 37 ° C for 30 rain with 100 U collagenase/ml Cellgro TM (Eagles minimum essential medium + L-glutamine; Mediatech, Herudon, VA) in a spinner culture tlask. Digested tissue was allowed to settle at room temperature for 5 min and the supernate was centrifuged (72 g, 5 min). The resultant cell pellet was resuspended, washed in Cellgro TM and filtered through 100 # mesh. The filtrate was subsequently centrifuged (72 g, 5 min) and resuspended in complete cell medium which is composed of Cellgro TM supplemented with the following constituents obtained from GIBCO (Grand Island, NY): insulin (1.0 U/ml), heat inactivated fetal bovine serum (10%), amphotericin B (1.25 Ug/ml), penicillin (100 U/ml), streptomycin (100 #g/ml), and HEPES buffer (10 mM). The cell suspension was plated for attachment and growth in tissue culture flasks in an atmosphere of 95% air:5% CO z. In the direct culture method, tissues were minced into 1/2 mm 3 pieces to increase the surface area and placed into tissue culture dishes containing complete cell medium. Glass or Thermanox TM coverslips placed on top of the biopsy were covered with a glass slide to anchor the explant to the bottom of the culture dish and prevent floating of the tissue. In this fashion cellular outgrowths of the mucosa attached to the Tbermanox TM coverslips could be processed for electron microscopy (EM); outgrowths onto the tissue culture dishes or glass coverslips could be used for histochemical characterization. The tissues were incubated at 37 ° C in an atmosphere of 95% air:5% CO2. Within 5 days, cells grew out from both the bladder explants and the digested tissue and monolayers were confluent after 6 weeks of culture. Since similar growth patterns were observed with both methods of culture, we chose the direct culture method for culture of cystoscopic biopsies. Cystoseopic biopsies (2-4 mm 3) obtained