S-(1,2-dichlorovinyl)-L-cysteine-induced nephrotoxicity in the New Zealand white rabbit: characterization of proteinuria and examination of the potential role of oxidative injury.

S-(1,2-dichlorovinyl)-L-cysteine (DCVC)-induced nephrotoxicity in vivo was investigated in New Zealand White rabbits. A primary emphasis in these studies was further characterization of DCVC-induced nephrotoxicity using a variety of serum and urinary analytes, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, the role of oxidative injury was assessed to address the dichotomy between reports indicating that such a mechanism is important in vivo and those indicating that such mechanisms do not contribute substantially to the mechanism of effects observed in vitro. Urine was collected prior to and at 8 and 24 hr after iv administration of DCVC. Serum was collected 15 min prior to and 24 hr after DCVC administration. Rabbits were euthanized 24 hr post-DCVC administration, and kidneys were fixed in formalin and further processed for light microscopic examination. DCVC (10 mg/kg, iv) induced a 45-50-fold increase in total urinary protein excretion, a 10-15-fold increase in urinary N-acetyl-beta-D-glucosaminidase concentration, plus a marked glucosuria by 24 hr postadministration. Additionally, DCVC increased serum creatinine levels by about 2-fold, with a trend toward increased blood urea nitrogen. SDS-PAGE analysis of rabbit urine confirmed the clinical finding of marked proteinuria in DCVC-treated animals, which in contrast to previously reported data was due to the presence of both low and high molecular weight proteins. Antioxidants had no significant effect on DCVC-dependent renal injury, nor was there evidence for DCVC-induced lipid peroxidation, as measured by either thiobarbituric acid-reactive substances or a commercial assay for malondialdehyde and hydroxalkenals.(ABSTRACT TRUNCATED AT 250 WORDS)

A non-radioactive iothalamate and p-aminohippuric acid high-performance liquid chromatographic method for simultaneously measuring glomerular filtration rate and renal blood flow in the rat.

A high-performance liquid chromatographic (HPLC) assay method has been developed for the quantitative determination of iothalamate and p-aminohippuric acid (PAH) concentrations in serum and urine samples in the male rat. Glomerular filtration rate (GFR) was measured as clearance of iothalamate, while effective renal blood flow (ERBF) was measured as clearance of PAH. The method is simple, rapid and sensitive and detects iothalamate and PAH in rat serum and urine following administration of bolus doses and continuous infusions of iothalamate and PAH. Samples of serum and urine were deproteinized with two volumes of acetonitrile containing the internal standard, and an aliquot chromatographed on a C18 reversed-phase column. The mobile phase was comprised of 0.1 M sodium phosphate with 1.2 mM tetrabutylammonium phosphate: methanol, 85:15 (v/v), at a flow rate of 1.0 mL/min. The analytical column eluate was monitored with a UV detector at 254 nm with quantitation achieved using peak-height ratios. The precision of the method was 6.6 and 3.6% for iothalamate in serum and urine, and 5.6 and 4.9% for PAH in serum and urine, respectively. The lower limit of quantitation was 0.63 microgram/mL for iothalamate and 1.25 microgram/mL for PAH in serum, and 3.1 microgram/mL for iothalamate and 1.5 microgram/mL for PAH in urine. Recovery of iothalamate from serum and urine was 99.9 and 93.5%, respectively. Recovery of PAH from serum and urine was 99.8 and 92.6%, respectively. The present study demonstrated that non-radioactive iothalamate and PAH can be measured simultaneously using a HPLC assay to measure GFR and ERBF in the male rat.

Renal papillary necrosis and urinary protein alterations induced in Fischer-344 rats by D-ormaplatin.

D-ormaplatin (previously called tetraplatin) produced dose-related renal papillary necrosis when given intravenously to Fischer-344 rats at doses of 2, 4, and 9 mg/kg. The lesions were most severe at 4 days postdosing and had repaired by day 9 in the 2- and 4-mg/kg dose groups. Blood urea nitrogen and the N-acetyl-beta-glucosaminidase (NAG): creatinine ratio were slightly elevated at day 4 while creatinine clearance was decreased. Body weight was reduced in a dose-related manner while kidney weights increased. Total protein excretion in male and female rats was elevated at day 4 postdosing. The evaluation of urinary proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed an increase, primarily in high molecular weight proteins at 4 days postdosing, indicating an increase in glomerular filtration of albumin and transferrin. The morphologic appearance of the glomeruli was normal by light microscopy. At day 4 postdosing, alpha 1-microglobulin was elevated. This correlated with an increase in the NAG: creatinine ratio also seen at this time and the morphologic appearance of the kidney, indicating that the proximal tubules were affected but were not a major site of toxicity. Although the change in urinary proteins occurred at the same time as morphologic alterations in the renal papilla, these findings were not considered to be related. SDS-PAGE provided a useful method for detecting and localizing renal toxicity when used in conjunction with morphologic and clinical chemistry methods.

The use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis to detect renal damage in Sprague-Dawley rats treated with gentamicin sulfate.

Renal toxicity is a common manifestation to the exposure of laboratory animals and humans to a wide range of xenobiotics. Traditional methods for evaluating renal damage by clinical chemistry such as blood urea nitrogen (BUN) and serum creatinine are not sensitive to early, mild changes. The use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to measure the molecular weight spectrum of urinary proteins allows for an evaluation of the functional changes associated with renal damage. The ability of the kidney to filter and reabsorb proteins is related to the functional ability of glomeruli and the proximal tubules. Gentamicin sulfate produces injury to the S-1 and S-2 segments of the proximal tubule in laboratory animals and humans. While severe damage to the tubules is associated with increased BUN, serum creatinine, and N-acetyl-beta-glucosiminadase (NAG), mild injury is not detected by these means. The evaluation of urinary proteins by SDS-PAGE demonstrated renal toxicity at a dose of 6 mg/kg after 2 days of sc treatment. The NAG: creatinine ratio was shown to be elevated after 2 days of treatment at 63 mg/kg. The use of SDS-PAGE as described in this paper provides a sensitive method for detecting renal injury.

Biochemical, metabolic and morphological characteristics of human neutrophil activation with pepstatin A.

Pepstatin A, a chemotactic pentapeptide, elicited a concentration-dependent extracellular release of granule-associated beta-glucuronidase and lysozyme from, and generation of superoxide anion (O2-) by, cytochalasin B (CB)-treated human neutrophils. Prior exposure of neutrophils to pepstatin A before the addition of CB, suppressed, in a time-dependent fashion, the subsequent production of O2- and exocytotic response. The rate and amount of enzymes released and O2- generated by pepstatin A-activated neutrophils were significantly enhanced in the presence of extracellular calcium. Pepstatin A-elicited degranulation and O2- production were suppressed by the intracellular calcium antagonist, 8-(N,N-diethylamino)-octyl-(3, 4, 5-trimethoxy) benzoate hydrochloride (TMB-8). Granule exocytosis and O2- generation by pepstatin A-treated neutrophils were suppressed by the sulphydryl reagents, N-ethylmaleimide (NEM) and iodoacetic acid (IA), and by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG). Sodium cyanide was inactive. Preincubation of neutrophils with pepstatin A "desensitized' the cells to a subsequent exposure to pepstatin A or the chemotactic tripeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). Pepstatin A-induced desensitization of granule enzyme release and O2- generation appears to be stimulus-specific in that phorbol myristate acetate (PMA) was capable of eliciting normal responses from pepstatin A-pretreated cells. The morphological changes observed in pepstatin A-treated neutrophils are reminiscent of those seen in cells exposed to FMLP.

Alloxan-induced hyperglycemia in rats is reduced by 16,16-dimethyl-PGE2.

Female rats were treated with subcutaneous 16,16-dimethyl-PGE2 (1-75 micrograms/kg) for 24, 18, and 0.5 h prior to and 6, 24, and 48 h after intravenous beta cell destruction. Protection was assessed by morphological examination of beta cells and the level of fasting hyperglycemia seen 72 h after alloxan treatment. Prostaglandin reduced the degree of alloxan-induced hyperglycemia in a dose-dependent fashion but had no demonstrable effect on morphological assessment of beta cell destruction. However, prostaglandin treatment by itself induced transient (0-2 h) hyperglycemia that could be correlated inversely with the level of fasting blood glucose observed 72 h after alloxan treatment. Administration of oral glucose, which mimics the prostaglandin-induced hyperglycemia, afforded protection against alloxan challenge comparable to that produced by the prostaglandin. Thus, it appears that reduction of alloxan-induced hyperglycemia by 16,16-dimethyl-PGE2 may be linked to the transient hyperglycemia produced prior to alloxan administration.

Renal lesions in MRL mice.

Female MRL-Mp-lpr/lpr mice spontaneously develop autoimmune disease at three to five months of age and die most commonly from immune complex glomerulonephritis. Kidneys of two-month-old females appeared nearly normal by electron microscopy, and glomerular deposits of IgG an complement component 3 (C3) barely were detectable. In five-month-old females, immunofluorescence revealed numerous deposits of IgG and C3; glomerular mesangial cells were hypertrophic and hyperplastic and contained electron-dense material. There were subepithelial and subendothelial deposits of electron-dense material with swelling of epithelial cell cytoplasm. This disease has many features similar to the immune complex glomerulonephritis observed in New Zealand Black and White hybrid mice and in man.

Corneal lesions induced by antidepressants: a selective effect upon young Fischer 344 rats.

Within a few days of dosing with various structurally unrelated antidepressants, young Fischer 344 rats display punctate lesions on the posterior surface of the cornea. Similar lesions are not seen after treatment with stimulants, an anticholinergic, a serotonin uptake blocker or an alpha adrenergic antagonist. These lesions are peculiar to the Fischer strain; four other strains displayed no such effects. Within the Fischer strain, sensitivity to the lesion-inducing effects of antidepressants is progressively lost with increasing age and adults are insensitive. Examination of the corneal lesions with transmission and scanning electron microscopy revealed that the lesion involved focal eruptions of the endothelium containing fibroblasts, necrotic cells, collagen and mineralized deposits. The latter were confirmed by energy-dispersive X-ray analysis to contain calcium. Thus the reported corneal lesions are unusual in their morphology as well as in their strain, age and drug specificity.

Protective effects of 16,16-dimethyl PGE2 on the liver and kidney.

The ability of subcutaneous 16,16-dimethyl PGE2 to protect the liver and the kidney against damage induced by carbon tetrachloride and ANIT (alpha-napthylisothiocyanate) was examined. Rats were given 5-75 micrograms/kg of 16,16-dimethyl PGE2 24 and 0.5 hrs before challenge with 1 ml of oral carbon tetrachloride with an additional prostaglandin dose 6 hrs later. Twenty-four hrs after carbon tetrachloride animals was sacrificed by decapitation. 16,16-Dimethyl PGE2 partially prevented fat accumulation and necrosis in the liver with complete or partial reduction in the SGPT caused by the hepatotoxin. Higher doses of carbon tetrachloride (1.5 ml) caused elevation in BUN and uric acid also; these changes were prevented by 16,16-dimethyl PGE2 even when doses of the prostaglandin were too low to protect against liver necrosis. Elevated serum bilirubin observed 48 hrs after oral ANIT (30 mg/kg) was prevented by 100 micrograms/kg of 16,16-dimethyl PGE2 given 24 and 0.5 hrs prior to the challenge with additional doses 6 and 24 hrs after ANIT. Higher doses of oral ANIT (200 mg/kg) when combined with small doses of carbon tetrachloride (0.25 ml per rat) resulted in elevated BUN and uric acid levels in the serum although neither compound produced these changes when given alone. 16,16-Dimethyl PGE2 (75 micrograms/kg) administered by the same schedule as used for protection against ANIT resulted in normalization of these parameters in the absence of significant liver protection. Thus, it appears that 16,16-dimethyl PGE2 can protect the liver against necrosis induced by moderate amounts of carbon tetrachloride and ANIT. At higher doses of these hepatotoxins, the liver is not protected by prostaglandins. Elevation of BUN and uric acid is observed under these conditions, however, and can be prevented by 16,16-dimethyl PGE2.

Effects of DDT on eggshell quality and calcium adenosine triphosphatase.

Adult mallard ducks were fed a diet containing 50 ppm DDT for 6 months. Eggs laid during this period were collected and eggshell weight, thickness, and calcium were determined. Chronic ingestion of DDT resulted in production of eggshells that were significantly thinner and lighter than those of controls. Total calcium of thinned eggshells was also reduced; however, calcium per gram of eggshell was not altered, indicating that other eggshell constituents were not incorporated as well. Calcium adenosine triphosphatase activity in the microsomal fraction of eggshell gland epithelium was assayed in control and DDT-fed ducks. Enzyme activity in DDT-fed ducks was reduced to 65% of control values. Since Ca-ATPase has been shown to be associated with calcium transport, enzyme inhibition may be responsible for decreased eggshell weight and thickness. Electron microscopic evaluation of microsomal fractions showed elements of the plasma membrane, including cilia and microvilli, as well as rough and smooth endoplasmic reticulum. Inhibition of calcium transport at the plasma membrane of mucosal epithelium is proposed as a possible mechanism of DDT-induced eggshell thinning.