Effect of gentamicin on the lysosomal system of cultured human proximal tubular cells. Endocytotic activity, lysosomal pH and membrane fragility.

Gentamicin treatment results in significant changes in lysosomal morphology and enzyme activity in renal tubular epithelium both in vivo and in vitro. In this study, cultured human proximal tubular cells (PTC) were treated with gentamicin (0, 0.01, 0.1, and 1.0 mg/ml) for 3, 7, 10 and 14 days, and the endocytotic activity, pH, and membrane fragility of the lysosomal system were examined. Fluorescein isothiocyanate-labeled dextran (FITC-dextran) was used to estimate endocytotic activity and intralysosomal pH. The fragility of isolated lysosomes was estimated by the release of N-acetyl-beta-glucosaminidase (NAG, EC3.2.1.30) into the medium. Gentamicin content was measured and correlated with the changes seen in lysosomal function. Gentamicin treatment caused a slight decrease in the rate with which human PTC accumulated FITC-dextran and a slight increase in intralysosomal pH. Treatment of human PTC with NH4Cl, a lysosomotropic compound, significantly increased the lysosomal pH; the NH4Cl-induced increase in the lysosomal pH of gentamicin-treated PTC, however, was not significantly different from control (0 mg gentamicin/ml). Lysosomes isolated from human PTC cultures released NAG upon incubation for 60 min at 37 degrees. There was no significant effect on the fragility of lysosomes isolated from cultures exposed to gentamicin for less than or equal to 7 days. Significantly increased fragility was seen, however, after 10 days of treatment with 1.0 mg gentamicin/ml and especially after a 14-day exposure to 0.01, 0.1, and 1.0 mg gentamicin/ml. Human PTC accumulated 0.47, 2.05 and 10.30 micrograms gentamicin/mg protein with 10 days of exposure to 0.01, 0.1 and 1.0 mg gentamicin/ml medium respectively. Gentamicin treatment associated with increased numbers of morphologically altered lysosomes, i.e. myeloid bodies, did not affect significantly the endocytotic activity and pH of lysosomes in cultured human PTC. Prolonged exposure (14 days) of human PTC to gentamicin, however, did increase the fragility of lysosomes after isolation. The increased numbers of morphologically altered lysosomes with increased fragility were not associated with any significant in vitro cell death. Therefore, it would appear that these lysosomal alterations are not directly responsible for the in vivo nephrotoxicity.

Biochemical and morphological studies on human kidneys preserved for transplantation.

Seven human kidneys that had been preserved for transplantation by pulsatile perfusion were studied to correlate the biochemical data with morphologic changes. Metabolite concentrations in mumol/g wet tissue were ATP = 0.26; ADP = 0.34; AMP = 0.45; lactate = 15.21; pyruvate = 0.23; 3-phosphoglycerate = 0.05; fructose-1,6-bisphosphate = 0.06; and hexose-6-phosphate = 0.03. Enzyme activities in mumol/min . mg protein found in the microsomal fraction were alkaline phosphatase = 0.049 and gamma-glutamyl transpeptidase = 0.844. Morphologically, none of the kidneys showed irreversible cell injury in the renal tubules, but some glomeruli showed areas where the endothelial cells appeared stripped off of the capillary basement membranes, indicating possible perfusion injury. The data suggest that it is the resynthesizing ability, as opposed to the absolute concentration of ATP, which determines the recovery and the subsequent viability of the tissue.

Marijuana and alcohol use among 1023 trauma patients. A prospective study.

Marijuana use prior to injury was determined prospectively in 1023 patients injured as the result of vehicular (67.6%) and nonvehicular (32.4%) trauma. Most were men (72.8%); most were 30 years of age or younger (58.4%). All were admitted directly from the scene of injury. Serum delta-9-tetrahydrocannabinol activity was ascertained using a radioimmunoassay. Activity of 2 ng/mL or more was detected in 34.7% of subjects. Blood alcohol determinations were made in 1006 patients; 33.5% were positive. Marijuana use among vehicular and nonvehicular trauma victims was not significantly different. Marijuana use was higher among those 30 years of age or younger and among men. Vehicular crash victims consumed alcohol more frequently. Use of marijuana and alcohol in combination (16.5%) was highly significant compared with marijuana alone (18.3%), alcohol alone (16.1%), or neither drug (49.1%).

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.

Cytolethality of hemolytic Escherichia coli to primary human renal proximal tubular cell cultures obtained from different donors.

OBJECTIVES: In earlier experiments, we confirmed epidemiologic studies demonstrating the prominence in acute pyelonephritis of Escherichia coli expressing P fimbriae and hemolysin, produced the disease with pyelonephritogenic strains in an animal model, and developed in vitro assays using human renal proximal tubular cells that demonstrated bacterial adherence by P fimbriae and killing of the renal cells by hemolysin. In the present series of experiments, we sought to determine whether P-fimbriated hemolytic E coli killed human renal proximal tubular epithelial cells obtained from different human donors. METHODS: Human renal proximal tubular cells, putative target cells for bacteria causing acute pyelonephritis, were cultured from 9 donors and cell death was measured by two methods. RESULTS: We showed that the E coli strain was significantly more cytolethal for renal cells of all donors than its hemolysin-negative mutant. CONCLUSIONS: This work suggests that the pathogenesis of acute pyelonephritis by P-fimbriated hemolytic E coli, characteristics of the causative organism in about 50% of human cases, may be at least in part through killing of human renal epithelial cells by hemolysin.

Cytotoxic effects of FK506 on human renal proximal tubule cells in culture.

FK506 has been used as the primary immunosuppressive agent administered after a variety of organ transplants, with less reported nephrotoxicity than that of cyclosporine. This study examined in vitro cytotoxicity of FK506 on normal human renal proximal tubule cells. Cytotoxicity was assessed by neutral red inclusion and trypan blue exclusion; morphology was assessed by light and transmission electron microscopy. Neutral red inclusion decreased to less than 10% of the control after 3 days exposure to 200 micrograms/ml FK506. Forty microgram per milliliter FK506 caused a decrease in neutral red inclusion to 61% of the control on Day 7, with recovery to 86% on Day 12. Similarly, trypan blue exclusion decreased to 66% of the control following 7 days exposure to 40 micrograms/ml FK506, and confluency of the monolayer was reduced to 50% as evidenced by phase contrast microscopy. After a 12-day exposure, treated monolayers became more confluent. On ultrastructural examination, FK506-treated cells exhibited increased cytoplasmic vacuolation and lipid inclusion. These data suggest that FK506 is reversibly and mildly toxic to monolayers of human renal proximal tubule cells and are consistent with clinical reports of reversible nephrotoxicity.

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.

Isolation, culture and characterization of human renal proximal tubule and collecting duct cells.

The complexity and heterogeneity of the human nephron with regard to cell types make well-defined in vitro systems of renal cells valuable for studies of the pathogenetic mechanisms involved in nephrotoxicity. In our laboratory renal proximal tubule cells (PTC) and collecting duct cells (CDC) have been isolated, cultured and characterized from cadaver kidneys (postmortem time <24 h) for use in studies of renal cytotoxicity induced by therapeutics and bacteria. PTC seeded at 10(6) cells/ml formed confluent monolayers within 7 days. Histochemical markers were used to determine the origin of the cell cultures. Cells were negative for factor VIII, positive for cytokeratin and gamma-glutamyltransferase (GGT), and bound winged pea lectin. CDC, isolated from the renal papillae, formed monolayers within 14 days of seeding. CDC were negative for factor VIII and GGT, positive for cytokeratin and bound peanut lectin. PTC and CDC isolates and cultures exhibited typical epithelial cell ultrastructure: cell junctions, intermediate filaments, microvilli, and numerous mitochondria. The morphological and histochemical evidence confirms that PTC and CDC can be isolated and cultured for use in in vitro studies.

Characterization of an in vitro system of human renal papillary collecting duct cells.

We have developed an in vitro model of human papillary collecting duct cells isolated from cadaver kidneys using methods similar to those we previously reported for the isolation of human proximal tubule cells. To date we have isolated papillary collecting duct cells from 100 normal human kidneys. Papillae were dissected and digested in Cellgro containing 400 U/ml collagenase. Cells were plated on fibronectin-coated culture flasks at a density of 10(4) live cells/ml in Cellgro supplemented with insulin and 10% fetal bovine serum. Confluent monolayers, which were able to withstand 600 mOSM for 8 h, were obtained within 10 to 15 d. Cells of primary isolates and first passages exhibited epithelial cell ultrastructure including cell junctions, microvilli, and cilia. A dark-brown reaction product was observed in these cells when stained by the immunoperoxidase method with peroxidase-labeled peanut lectin (Arachis hypogaea), which binds specifically to human distal tubule and collecting duct cells. These cells were negative for Factor-VIII (a marker for endothelial cells) and gamma-glutamyltransferase (a marker for proximal tubule cells). High activities of the glycolytic enzyme pyruvate kinase and arginine vasopressin-stimulated cAMP production in these cells are consistent with a distal nephron origin. The results indicate that human collecting duct cells can be isolated and cultured to provide an in vitro system to probe pathogenetic mechanisms of potential nephrotoxins.

Effects of cyclosporine on human endothelial cell cultures.

Reports of vascular changes in renal biopsies of transplant patients treated with cyclosporine prompted review of our own renal biopsies and examination of human endothelial cell cultures exposed to cyclosporine in vitro. Endothelial cells were isolated from human umbilical cords by collagenase digestion and cultured in Medium 199 with Earle's salts plus 20% pooled human serum in the absence of antibiotics. Cultures exposed to cyclosporine (0, 0.4, 1.0, 5.0, 10.0 micrograms/ml) for 0, 3, 7, 10, and 14 days were subsequently fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer. Vascular thrombosis was seen in renal biopsies of cyclosporine- and azathioprine-treated patients but the incidence was the same in both groups. No change in the morphology of endothelial cell cultures was observed until 7 days when an increase in size and number of cytoplasmic inclusions became apparent in both control and cyclosporine-treated cultures. By electron microscopy, these inclusions were identified as secondary lysosomes. Their number and size increased with the length of time in culture but did not appear to correlate with the concentration of cyclosporine in the medium. No other morphologic change was identified. It is concluded that the appearance of increased numbers of secondary lysosomes in human endothelial cell cultures is a function of culture age as opposed to cyclosporine exposure. Furthermore, the data indicate that small vessel thrombosis is not specific to treatment with cyclosporine.

The effect of gentamicin on human renal proximal tubular cells.

Human renal proximal tubular cells were exposed to gentamicin (0, 0.1, 0.5, 1.0, 5.0, 10.0 mg/ml medium) for 3, 7, 10, and 14 days. Cells were counted and cell viability was estimated by lactate dehydrogenase release. In addition, brush border-associated (gamma-glutamyltransferase) and lysosomal (acid phosphatase, N-acetyl-beta-glucosaminidase, and sphingomyelinase) enzyme activities were measured (0.01, 1.0, 3.3 mg/ml gentamicin for 3, 7, 10 and 14 days). The number of cells did not change significantly after gentamicin treatment. Cell viability, however, significantly decreased after 3 and 7 days exposure to 5 and 10 mg/ml gentamicin. Total lactate dehydrogenase activity was significantly decreased at 7, 10, and 14 days exposure to gentamicin greater than or equal to 5.0 mg/ml. gamma-Glutamyltransferase, acid phosphatase, and sphingomyelinase were decreased at 3, 7, and 10 days exposure to gentamicin greater than or equal to 1.0 mg/ml. Continued exposure to gentamicin less than or equal to 1.0 mg/ml appeared to have little or no effect on the activity of these enzymes at 14 days. N-Acetyl-beta-glucosaminidase activity, in contrast, was elevated (120-140% control) in the gentamicin-treated (greater than or equal to 1.0 mg/ml) groups at all time periods studied. Thus, gentamicin exposure resulted in changes in some of the enzyme activities in human renal proximal tubular cell cultures, but longer exposure (14 days) to gentamicin (less than or equal to 1.0 mg/ml) resulted in a return to control levels of some activities.

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.

Use of human renal proximal tubule cell cultures for studying foscarnet-induced nephrotoxicity in vitro.

Foscarnet is an antiviral agent used for the treatment of cytomegalovirus retinitis and acyclovir-resistant herpes simplex virus infections in AIDS patients. Renal impairment has been reported for many patients treated with foscarnet. We have studied the effects of foscarnet on the viability (estimated by neutral red inclusion) and ultrastructure of cultures of human renal proximal tubule cells (HRPTC) isolated from the kidneys of five cadavers and cultured. The degree of foscarnet-induced toxicity was dose dependent and varied among the HRPTC cultures. The data obtained by using the in vitro system of HRPTC mimic the data of the clinical trials in that there is a dose-dependent individual variation among human cases in response to foscarnet treatment. Thus, these cultures are extremely well-suited to investigations of the mechanism of toxicity at the subcellular level.

Biochemical and morphological effects of gentamicin in human proximal tubular cells: effects on lipid and lipoprotein metabolism.

The effects of gentamicin, an antibiotic used extensively for antimicrobial therapy on the ultrastructure, binding, internalization, degradation, and cholesterol esterification of low-density lipoproteins, were investigated in cultured human proximal tubular cells. Cells were incubated with 0.3 mM gentamicin for 21 days with the following observations. Cells treated with gentamicin contained numerous "myeloid bodies." The binding, internalization, and degradation of 125I-labeled low-density lipoproteins ([125I]LDL) in cells treated with gentamicin was twofold lower than control cells. Pulse-chase experiments demonstrated that gentamicin did not impair the internalization of receptor-bound LDL and their subsequent transport to the lysosome. The relative amounts of [125I]LDL displaced by increasing concentrations of unlabeled LDL were the same in both gentamicin-treated and control cells. This pattern was reflected in the cell surface binding, internalization, and degradation of [125I]LDL. Gentamicin did not alter the degradation of [125I]LDL in cell homogenates at 4.0. The data suggest that gentamicin decreases the receptor-mediated endocytosis of LDL and subsequent lipid metabolism.

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.

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 (Hunner's 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.

Internalization of Proteus mirabilis by human renal epithelial cells.

Proteus mirabilis, a common agent of bacteriuria in humans, causes acute pyelonephritis and bacteremia. Renal epithelium provides a barrier between luminal organisms and the renal interstitium. We have hypothesized that P. mirabilis may be internalized into renal epithelium. To test this hypothesis, we added suspensions of three P. mirabilis strains (10(8) CFU) to confluent monolayers of primary cultures of human renal proximal tubular epithelial cells (HRPTEC) and, after 3 h, found the bacteria internalized within membrane-bound vacuoles by light and electron microscopy. Internalization of bacteria by HRPTEC was corroborated by using the gentamicin protection assay. Cytolysis of HRPTEC by the HpmA hemolysin, however, was a confounding factor in this assay, and therefore a hemolysin-negative hpmA mutant was used in subsequent experiments. The nonhemolytic mutant WPM111 did not disrupt the monolayer and was recovered in numbers that were 10- to 100-fold higher than those of the hemolytic parent BA6163. Cytochalasin D (20 micrograms/ml) inhibited internalization of Salmonella typhimurium but not that of P. mirabilis, suggesting that the latter species enters HRPTEC by a mechanism that is not dependent on actin polymerization. We suggest that HpmA hemolysin-mediated cytotoxicity and internalization of bacteria by HRPTEC may play a role in the development of Proteus pyelonephritis.