Growth characteristics of cultured human proximal tubule cells exposed to aminoglycoside antibiotics.

It is well known that the nephrotoxic lesions that occur during aminoglycoside-induced nephrotoxicity are both dose- and time-dependent. It was the purpose of this study to determine if a cell culture model based on the human proximal tubule would exhibit similar dose- and time-dependent relationships when exposed to aminoglycosides of various nephrotoxic potential. For this determination, the human proximal tubule (HPT) cells were exposed to increasing concentrations of streptomycin, kanamycin, gentamicin, and neomycin and monitored for cell growth and toxicity over an 18-day period of exposure. Both actively-dividing and resting cells were assessed with regard to aminoglycoside exposure. At high levels of aminoglycoside exposure, linear regression analysis disclosed that the rank order of toxicity of the aminoglycosides to be: neomycin greater than kanamycin greater than gentamicin greater than streptomycin. Both actively-dividing and resting cultures of HPT cells displayed both dose- and time-dependency with regard to toxicity and the ability of the cells to regenerate in the continued presence of aminoglycoside exposure. This pattern of dose- and time-dependency was unique for each aminoglycoside and varied depending on the replicative state of the cells. With the exception of neomycin, clear evidence was obtained that toxicity and cell regeneration were occurring simultaneously throughout the time course of aminoglycoside exposure; the equilibrium between the two processes determining overall cell toxicity or regeneration. In addition, the HPT cells exposed to gentamicin displayed a unique pattern of toxicity and cell regeneration when compared to the other aminoglycosides tested, with gentamicin having an increased ability to stimulate cell proliferation. While the results obtained are in excellent agreement with that known from the clinical experience with the aminoglycosides, the dose- and time-dependency of the responses will require careful attention to growth state during employment in experimental protocols.

Cultured human proximal tubule cells as a model for aminoglycoside nephrotoxicity.

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.

Effects of substituted dithiocarbamates on the testicular toxicity of cadmium.

Five substituted dithiocarbamates were evaluated for effectiveness in protecting mouse testes following administration of an LD100 dose of CdCl2 X 2.5 H2O (Cd). Toxicologic responses were assessed by light microscopic, electron microscopic, and computerized image analysis techniques. Diethyldithiocarbamate (DDTC) was the most effective antagonist. N-ethyl-N-hydroxyethyldithiocarbamate (EHDC), N-methyl-N-hydroxyethyldithiocarbamate (MHDC), and dimethyldithiocarbamate (DMDC) were moderately to minimally effective, while di-(hydroxyethyl)dithiocarbamate (DHDC) afforded no protection. Degenerative changes increased progressively in severity as the time interval between administration of Cd and DDTC was increased, but remained minimal when DDTC was given as late as two hours after Cd.

Diabetic glomerulonephropathy: histopathologic, immunofluorescent, and ultrastructural studies of 16 cases.

Sixteen cases of diabetic glomerulopathy are reported. Direct immunofluorescent and ultrastructural studies of renal biopsy tissues demonstrated that two patients had linear deposits of IgM and C'3 in the absence of IgG, four diabetic patients had sclerosis-induced entrapment of immunoglobulins and complement, and one patient had granular immune complexes in the subepithelial and intramembranous portion of the glomerular basement membrane. In one patient, who had nodular glomerular lesions, diffuse fibrillar deposits of electron-dense material were observed in the mesangium. In this mesangial infiltrate, light microscopy revealed the absence of amyloid and direct immunofluorescence revealed the absence of all immunoglobulins, complement components, and fibrinogen. Our study suggests that the morphologic alterations observed in diabetic glomerulopathy might be mediated by either immune mechanism or by abnormal biochemical or functional factors, such as impairment of the mesangial IgA clearance mechanism.

Intratubular calcium phosphate deposition in acute analgesic nephropathy in rabbits.

Earlier chronic studies using both animal and human autopsy material have suggested that the initial lesion of analgesic nephropathy is papillary necrosis with secondary cortical interstitial nephritis. The present study was designed to define ultrastructural changes in renal tubules exposed to high levels of analgesics. Female New Zealand White rabbits were given 5-7 g APC and sacrificed after periods of 6 to 36 hours. As early as 6 hours after treatment, hydroxyapatite crystallite aggregates were seen impacted in Segment III (straight segment) of the proximal tubule at the cortico-medullary junction. Ultrastructural changes included selective calcification of the brush border, mitochondrial calcification, and peroxisomal changes. It was concluded, on the basis of ultrastructural changes, that calcification of altered tubular cells may be an initial event in analgesic nephropathy.

Wolman's disease: report of a case, with multiple studies.

We studied a case of Wolman's disease that is apparently the first to be reported in a black infant. Culture of the skin fibroblasts showed massive accumulation of neutral lipids, accompanied by decreased esterolytic activity as measured by fluorometric assay. Electrophoresis showed decreased activity of an esterase allotype, with low electrophoretic mobility.

Gold nephropathy. Ultrastructural, fluorescent, and energy-dispersive x-ray microanalysis study.

The nephrotic syndrome developed in a patient receiving therapy with gold for rheumatoid arthritis. The results of a histopathological examination of the renal biopsy specimen were unremarkable. Immunofluorescent studies showed deposits of immunoglobulins and C3 in a granular pattern in the glomerular basement membranes. Ultrastructurally, the discrete osmiophilic immune complexes were epimembranous. By x-ray microanalysis, gold that was complexed with sulfur was present in proximal tubular cytoplasmic vacuoles and nuclei. Gold and sulfur could not be demonstrated in glomerular epimembranous deposits. The results of these studies suggest that immune complex deposition does not involve gold and sulfur acting as haptens. Gold-salt therapy may result in damage to proximal tubules that leak renal tubular antigens, which in turn complex with autoantibody and produce an autoimmune membranous nephropathy. The evidence for this mechanism is not convincing. Although the data indicate an immune-complex cause for gold-salt nephropathy, the incident antigen (or antigens) and mechanism of action remain unidentified.

Membranous glomerulonephritis in a child asymptomatic for hepatitis B virus. Concomitant seropositivity for HBsAG and anti-HBs.

The presence of hepatitis B surface antigen (HBsAg) in association with immunoglobulins and complement components within the glomerular basement membranes of adults having chronic active hepatitis has been well documented. In addition, investigators in Poland have demonstrated HBsAg immune complexes in glomeruli of children who did not have clinical evidence of hepatitis. More recently, a single case of childhood membranous glomerulonephritis in an asymptomatic carrier of hepatitis B virus was cited by observers in Canada. Reported here is the deposition of HBsAg immune complexes in the glomerular basement membranes of a 13-year-old black boy who had membranous glomerulopathy but not clinical evidence of hepatitis. This may be the first reported case in the United States of HbsAg-associated membranous glomerulonephritis in a child asymptomatic for hepatitis B virus, and only the second such case in North America. However, unlike previous studies of childhood glomerulopathy in association with hepatitis B virus, this patient is seropositive for both HBsAg and anti-HBs (antibody for hepatitis B surface antigen). Similar "rare" serologic findings were found for the patient's eldest male sib.

Effects of prolonged exposure to dietary DDT and PCB on rat liver morphology.

Livers from mature female rats exposed for up to 36 weeks to dietary levels of Aroclor 1242 (75 or 150 ppm) and/or commercial grade DDT (75 to 150 ppm) were compared to those from animals receiving basal diets. In earlier studies, reproductive effects of the test substances were assessed. Moreover, the markedly abnormal gross appearance of the livers led to examination of the hepatic effects of PCB and DDT in more detail, at both the light microscope (LM) and electron microscope (EM) levels. Light microscopy revealed focal liver cell necrosis in rats fed PCB, DDT, and PCB-DDT combinations. Higher levels of PCB (150 ppm) increased the severity of necrosis. Feeding both DDT and PCB produced similar effects at 75 ppm, and caused atypical centrolobular regeneration, occasionally forming nodules, resembling small tumors. The experimentally induced injury was associated with the marked accumulation of iron-containing pigment in hepatocytes and Kupffer cells. Electron micrographs demonstrated the presence of whorl structures (myelin figures) within liver cell cytoplasm, and for the first time clearly illustrated the endocytotic expulsion of these membranous whorls from hepatocytes into the bile canaliculi and sinusoids. Other ultrastructural changes were similar to those previously reported in rats livers injured by several hepatotoxic substances. Mitochondria enclosed by or projecting into large non-lipid vacuoles were present in several experimental groups. The electron micrographs provide the most convincing evidence to date to support the hypothesis that myelin figures may be the vehicle whereby the cells rids itself of specific hepatotoxic substances.

Drug-induced granulomatous hepatitis.

Granulomatous hepatitis is a generic histopathologic diagnosis seen in approximately 5 to 10% of liver biopsy specimens. In the past, tuberculosis and sarcoidosis have been most frequently incriminated, although numerous other infectious and noninfectious etiologies have been reported. We have studied 95 cases of granulomatous hepatitis representing 6% of 1500 liver biopsies performed over a period of 10 years. Although sarcoidosis accounted for approximately one-third of these cases, probable and possible associations with medicinal compounds were detected in 29%. Highly suspect drugs include antihypertensive, antirheumatic and analgesic, anticonvulsant, and antimicrobial agents, but any drug may act as a hapten by covalent binding with macromolecular protein. The morphologic features of drug-induced, immunologic granuloma have not been described in detail. In our experience, eosinophils are prominent in the early granulomatous reaction to medicinal compounds and under continued antigenic stimulation are accompanied by plasma cells. Eosinophils are rare to absent in tuberculous hepatic granulomas and, when present in significant numbers, militate strongly against sarcoidosis. Drug-induced granulomas are consistently noncaseous. Although Kupffer cell granulomas have many causes, they are not uncommon hypersensitivity reactions to medicinal drugs and may give rise to clinical illness. Our review suggests that the previous literature does not reflect the magnitude of drug-induced granulomatous hepatic disease and that many cases reported as "granulomatous hepatitis consistent with sarcoidosis," as well as many "undiagnosed" cases, have a drug etiology.

Dialysis enhances renal epithelial proliferations.

Study of 115 kidneys from 60 patients with chronic renal failure maintained by dialysis for two months to five years revealed an unexpected number and variety of epithelial proliferative processes, several types of which are hitherto unreported. Proliferative activity was defined either by the presence of epithelial structures in ectopic situations, continuity with existing structures being demonstrable by serial sections, or by mitotic figures, or by both. The tendency for renal carcinoma development may relate to these dialysis-related epithelial proliferations originating in both glomerular and tubular epithelia. Enhanced renal epithelial proliferative capacity in dialysis may be employable in the experimental study of renal regeneration and in the therapy of patients with preterminal renal disease.

Atypical cysts, acquired renal cystic disease, and renal cell tumors in end stage dialysis kidneys.

Hyperplasia and tumors of epithelium are found in "end stage" dialysis kidneys. Epithelial hyperplasia is most conspicuous within "atypical cysts" in which the lining cells are multilayered and occasionally papillary. These features were studied in the kidneys of 66 renal failure patients by means of multiple tissue blocks and serial histologic sections. Atypical cysts were observed in 20 of the 66 cases. Solid or cystic renal cell adenomas were found in nine cases. Six of the cases having adenomas were among the 20 cases having atypical cysts. Tumors occurred in kidneys having atypical cysts, as dintinguished from kidneys without such cysts, with a frequency greater than would be expected to be due to chance alone (P = 0.0106). Renal cell adenomas are found at a younger age in dialysis patients (mean = 41.2 years) than in a control group of autopsies and surgical cases that had not received chronic dialysis (mean = 61.8 years). These observations provide histologic evidence that renal cell neoplasms are prone to develop in relatively young renal failure patients when their uremia is treated by long term dialysis. The studies further indicate that the stimulus for neoplastic growth accompanies a cystic transformation of the kidneys. Kidneys in five cases in the series, although much smaller than normal, were grossly multicystic, corresponding to the recently recognized acquired renal cystic disease. Hyperplastic cells, like those observed in atypical cysts, were present focally along the cyst walls. This form of epithelial hyperplasia, common to both atypical cysts and the multicystic dialysis kidney, may give rise to the renal cell tumors that are reported to occur with increased frequency in acquired cystic disease.

Angiomatoid neuroblastoma with cytoplasmic glycogen: a case report and histogenetic considerations.

A case of angiomatoid neuroblastoma with intracytoplasmic glycogen demonstrable by both light and electron microscopy is presented. A review of the histogenesis of the adrenal medulla indicates that glycogen-containing tumors of the adrenal medulla originate from a more primitive cell than their non-glycogen-containing counterparts.

Selective mitochondrial degeneration in renal tubules following hyperbaric oxygen exposure.

To study the ultrastructural effects of hyperoxia on the kidney, young adult Sprague Dawley rats were exposed to 3 atmospheres absolute (ATAs) of pure oxygen for 5 hours and were killed in a time sequence varying from immediately to 30 days after exposure. Their renal cortices were processed for electron microscopy. Selective mitochondrial changes were observed within sublethally and transiently altered proximal tubular epithelial cells. The most consistent finding was the accumulation of 0.08 mu to 0.5 mu round to ovoid homogeneous matrical inclusions which frequently formed larger confluent amorphous masses. The inclusions stained intensely with lead and uranium but appeared homogeneously electron-lucent in unstained sections. Energy-dispersive x-ray analysis revealed that they did not contain calcium or phosphorus. The inclusions were different from the innately electron-opaque flocculent densities commonly found in pathologically altered mitochondria. Since the mitochondria containing them were removed by autophagocytosis, it is suggested that the inclusions were associated with selective mitochondrial degeneration induced by hyperoxia. No glomerular lesions were found.

Hemochromatosis caused by excessive vitamin iron intake.

Rare cases of hemochromatosis have been reported in patients who underwent prolonged oral iron therapy for hemolytic anemia or prolonged self-treatment with iron pills. A proportionately large segment of the South African Bantu tribe, who ingest large quantities of an alcoholic beverage brewed in iron pots, are found to have the disease. Reports of health fadists developing hemochromatosis due to excessive dietary iron intake, however, are extremely rare. This report presents clinical considerations and pathologic findings in a compulsive health fadist who consumed large numbers of vitamins containing iron. Clinical findings included the development and progression of cirrhosis of the liver, bronzing of the skin, and diabetes mellitus, all consistent with a diagnosis of hemochromatosis. Light microscopy of liver biopsies taken late in the course of the disease revealed a massive buildup of iron in the hepatocytes, less in the Kupffer cells, and sparse deposition in the epithelial cells of the bile duct. Minimal periportal fibrosis was noted. Electron microscopy showed numerous pleomorphic siderosomes with varying degrees of crystallization and ferritin attached at uniform intervals to the membranes of residual bodies. Abundant free ferritin was observed in most cells. The aggregated and membrane-associated ferritin was verified by non-dispersive x-ray analysis. An additional finding, noted only by electron microscopy, was the presence of many fat-storing cells of Ito, which are thought to be involved in the onset of fibrosis.

Forensic pathology--the hidden specialty: A survey of forensic pathology training available to medical students and residents.

It is no wonder that some people refer to forensic pathology as the "hidden specialty." This paper shows evidence that the only exposure given to most medical students is a brief, all-too-explicit demonstration ("horror show") of interesting cases that can only attract the bizarre of mind into the field. It is further pointed out that even residents in pathology receive scanty, if any, formal training in most medical school residency programs, in spite of the fact that two thirds of the medical schools responding have one or more forensic pathologists on their faculty. The obvious answer is a change in the curriculum if this increasingly important subspecialty is to be saved.