Healed experimental renal papillary necrosis and cortical scarring in the rat from 2-bromoethylamine hydrobromide

Summary Male Sprague-Dawley rats were each given a single subcutaneous injection of an aqueous solution of bromoethylamine hydrobromide (BEA) at dose levels of 80 mg/kg (16 rats), 125 mg/kg (15 rats) and 250 mg/kg (16 rats) or a single subcutaneous injection of water (controls, 15 rats). The dose levels were chosen so as to cause renal papillary injury varying from minor necrotic foci to necrosis and subsequent sloughing of the entire papilla. The animals were killed after 5 months and the kidneys were weighed and examined macroscopically and microscopically for the presence of RPN and cortical scarring. Macroscopically evident RPN occurred in 18 of 43 surviving BEA-treated rats, bilaterally in 16 and unilaterally in 2. Bilateral asymmetry of the extent of sloughing was evident. All kidneys with macroscopic RPN exhibited cortical scarring. Asymmetry of the extent of atrophy and scarring in some animals resulted in a significant unilateral reduction in renal weight and a significant contralateral compensatory hypertrophy. Twenty-three of the 25 BEA-treated rats without macroscopically evident RPN exhibited minor, histologically visible lesions of the renal papillae including necrosis of loops of Henle in the presence of intact collecting ducts. Only 2 of these animals exhibited tiny, unilateral cortical scars, and renal weights did not differ significantly from those of controls. It may therefore be concluded, contrary to certain published proposals: that experimental RPN may be followed by severe renal cortical scarring, reduction in renal size and (in the presence of asymmetrical lesions) compensatory renal hypertrophy, and that necrosis of thin limbs of loops of Henle does not appear to lead to frequent or severe cortical scarring.     

Acute toxicity of citrinin in turkeys and ducklings

Citrinin, a naturally occurring mycotoxin, was dissolved in dimethyl-sulphoxide - 70% ethanol (3:1, v/v) and administered orally in two trials to 7-day-old male turkey poults and male white Pekin ducklings. The single dose LD50 value in 7-day-old male turkey poults was 56 mg/kg and in 7-day-old male white Pekin ducklings it was 57 mg/kg. The mycotoxin was nephrotoxic in both species, but the renal lesions were more severe in turkeys and were characterised by degeneration and necrosis of renal tubular epithelium. In turkeys, lesions were found in the liver and included hepatic cell necrosis and biliary hyperplasia. Lymphoid necrosis with depletion involved the thymus and cloacal bursa of turkeys and ducklings. These latter lesions were the most prominent histopathological alterations in citrinin-treated ducklings.     

Glomerular lesions in experimental renal papillary necrosis. I. Ultrastructural aspects and some pathogenic considerations.

Papillary necrosis was induced in rats by a single intravenous injection of bromoethylamine hydrobromide (BEA). From 7 days on glomerular lesions were recognized. They consisted of electron dense deposits mainly subepithelial in location; mild mesangial hypercellularity and matrix increase. Immunofluorescence with anti-rat gammaglobulin was positive, showing granular fluorescence in relation with basement membrane and mesangium. The possibility is raised that these lesions are due to the pathogenic action of immune complexes, the antigen being one arising during the necrosis of the renal papilla. It is also suggested that this mechanism can be operative in ths human being in cases of papillary necrosis of the kidney.     

2-Bromoethanamine nephrotoxicity in the nude mouse: an atypical targetting for the renal cortex

Male MF1-nu/nu/Ola/Hsd nude mice, maintained in a gnotobiotic environment, were dosed i.p. with either 50, 100 or 200 mg/kg 2-bromoethanamine (BEA) hydrobromide to induce a model papillary necrosis. Renal histological changes were examined in semithin glycolmethacrylate resin sections at 24, 48 and 72 h after BEA treatment. The sequence of medullary changes included pyknosis of interstitial cell nuclei, increased staining of the interstitial mucopolysaccharide matrix, platelets adhering to capillary endothelium, necrosis of collecting duct epithelial cells and denudation of the covering epithelium of the papilla. This was similar to that previously described in the Wistar rat, but the time course was extended. There was also a concomitant and extensive cortical necrosis of the P2 and P3 segments of the proximal tubule, which was evident prior to the onset of renal papillary necrosis at the higher doses of BEA. Nude mice show an atypical response to BEA compared to several mouse and rat strains, the hamster and pig, that suggests unique characteristics in this athymic murine mutant.     

Experimental renal papillary necrosis. Effects of beta adrenergic receptor blockade, heparin and hydrocortisone.

Renal papillary necrosis was induced in rats by daily subcutaneous injection of 15 mg 2-bromoethylamine hydrobromide (BEA) per 100 g of body weight for 2 successive days. This dose was 50% higher than that reported previously. Beta adrenergic receptor blockade with oxyprenolol did not influence the kidney damage.The administration of heparin did not show any effect. The doses applied did not induce the incoagulability for a sufficient period of time. On the contrary, treatment with hydrocortisone decreased papillary necrosis without inducing increased diuresis.     

Experimental Papillary Necrosis of the Kidney: IV. Medullary Plasma Flow

To test the thesis that vasoconstriction plays a significant role in the pathogenesis of papillary necrosis caused by bromoethylamine hydrobromide (BEA), medullary plasma flow was determined in rats treated with BEA. Medullary blood flow was normal ½ to 1 hour after BEA treatment, and was actually elevated 6 hours after BEA. There was no increase in plasma levels of prostaglandins A and E, which would have been expected if there had been medullary ischemia. Pretreatment with reserpine, which inhibited the development of papillary necrosis, had little effect on medullary plasma flow. These observations do not support the notion that vasoconstriction is the mechanism by which BEA causes papillary necrosis.     

The induction of renal papillary necrosis in Gunn rats by analgesics and analgesic mixtures.

Homozygous members of the mutant Gunn strain of Wistar rats genetically lack the enzyme uridine diphosphate glucuronyl transferase. "High" and "low" dose gavage feeding for 18-34 days of an analgesic mixture containing aspirin, phenacetin and caffeine (APC) confirmed the previously reported susceptibility of these animals to analgesic induced renal papillary necrosis. Heterozygotes do not share the gross enzyme deficiency of homozygotes and, when treated with APC under identical conditions, failed to develop renal papillary necrosis. Groups of homozygotes were dosed by gavage with aspirin, phenacetin and paracetamol for 4 weeks. Renal papillary necrosis was produced by all 3 drugs, the lowest frequency of lesions occurring with phenacetin. It is postulated that the enzyme deficiency of homozygous Gunn rats influences the metabolism of analgesics to favour the excretion of nephrotoxic metabolites. The renal papillary necrosis appearing in these experiments is essentially an acute lesion, differing both in natural history and morphology from the renal papillary necrosis of analgesic nephropathy, suggesting that the pathogeneses of the experimental and human lesions differ.     

The effects of chronic papillary necrosis on acid excretion

Complete papillary necrosis in rats can be induced within 1 month following a single injection of 2-bromoethylamine hydrobromide (BEA) (50 mg, i.v.). Utilizing a combination of clearance and balance techniques the effects of complete absence of the papilla was examined as regards urinary acidification, whole kidney glomerular filtration rate (GFR), single nephron GFR, and morphology. Whole kidney GFR was not different from control, however, the percent filtering juxtamedullary nephrons was markedly diminished (87.2±2.1 vs. 31.5±3.6% filtering, control vs. BEA, respectively,P<0.001) and significantly reduced in the superficial nephrons (80.6±3.6 vs. 62.2±6.1% filtering, control vs. BEA, respectively,P<0.05). There was a significant decrease in juxtamedullary single nephron GFR and an increase in the superficial single nephron GFR as assessed by the quantitative Hanssen's technique in the animals with chronic papillary necrosis. Complete papillary necrosis was associated with normal arterial bicarbonate concentration, pH, and plasma electrolyte concentrations. At the same degree of acidemia (induced by NH₄Cl administration) minimal urinary pH, ammonium excretion, and titratable acid excretion were not different than seen in age matched controls. The response to Na₂SO₄ infusion and phosphate infusion was the same in both groups of animals. The urineblood (U-B)pCO₂, an index of urinary acidification, was identical in BEA and control animals. Scanning electron microscopy showed scarring of the juxtamedullary glomeruli one month after BEA. The papilla was sloughed and lying free in the renal pelvis in every experimental animal. These data demonstrate that complete papillary necrosis is not associated with acidosis nor a defect in urinary acidification.     

Interactions between cyclosporin A, indomethacin and 16,16-dimethyl prostaglandin E2: effects on renal, hepatic and gastrointestinal toxicity in the rat

Rats were treated for 3 or 14 days with cyclosporin A (CsA, 50 mg/kg) or indomethacin (2 or 5 mg/kg) either alone or in combination, or with CsA plus 16,16-dimethylprostaglandin E2 (DMPGE2, 0.25 mg/kg). Hepatic and renal function were unaffected by treatment with indomethacin at either dose and only at the higher dose was severe intestinal ulceration observed. CsA caused renal and hepatic toxicity, evidenced by increased urine N-acetyl-beta-D-glucosaminidase activity, serum urea, creatinine and bilirubin and decreased serum albumin and total protein. In rats cotreated with CsA and either dose of indomethacin the increases in serum urea and creatinine and decreases in serum albumin and total protein were accentuated, but serum bilirubin was not further increased. Intestinal lesions were present in rats treated for 14 days with CsA plus the lower dose of indomethacin, but not in rats treated with either drug alone. In rats treated with DMPGE2 plus CsA, serum urea and creatinine were normal and urine N-acetyl-beta-D-glucosaminidase activity was reduced compared to rats treated with CsA alone, but DMPGE2 cotreatment had no effect on the CsA induced hyperbilirubinaemia. Hepatic microsomal cytochrome P-450 concentration and aminopyrine N-demethylase activity were lower in rats treated with CsA plus indomethacin than in untreated rats or those treated with either drug alone. Coadministration of indomethacin or DMPGE2 had no effect on serum trough CsA levels. The results are interpreted as showing an exacerbation by CsA of the intestinal toxicity of indomethacin, an increase by indomethacin in the renal toxicity of CsA and a protection by DMPGE2 against CsA renal toxicity. Possible mechanisms involving drug interactions and either hepatic cytochrome P-450, renal cyclooxygenase or other renal sites are discussed.     

Strain-related susceptibility to nephrotoxicity induced by aspirin and phenylbutazone in rats

Single doses of aspirin induce scattered foci of necrosis of proximal tubules in some strains of rats, whereas acute or sub-acute administration of phenylbutazone causes renal papillary necrosis. Initially, using Sprague-Dawley rats of CFY and CD strains, it became clear that these rats were not as susceptible to these drugs as the literature suggested. Aspirin-induced necrosis was apparently sex-related, being seen in females but could be induced by hormone treatment in males. Male Wistar and Sprague-Dawley rats had reacted differently to administration of phenylbutazone for two weeks. Two experiments were performed with four rat strains: Wistar, Lister-Hooded, Sprague-Dawley, and Fischer-344. The rats were six weeks old at the start of the experiments. Five males and five females of each strain were gavaged with either a single dose of 1,000 mg/kg of aspirin or 200 mg/kg phenylbutazone once daily for four weeks. The drugs were suspended in methylcellulose, which was given to equal numbers of control male and female rats in each experiment. The rats were maintained under standard conditions. Blood and 18-hour overnight urine samples were collected prior to sacrifice. There were no strain-related differences in the types of renal lesions seen, however there were differences in the degrees of responses to the two drugs. With aspirin the female Fischer-344 rats were the most susceptible showing necrosis of proximal tubules of both kidneys and markedly elevated urinary protein concentration and gamma-GT activity. Other females showed less change. Male rats were affected only slightly and males of the Wistar strain were not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biomarkers of collecting duct injury in Han-Wistar and Sprague-Dawley rats treated with N-phenylanthranilic Acid

N-phenylanthranilic acid is a chloride channel blocker that causes renal papillary necrosis in rats. Studies were conducted in two strains of male rats to evaluate novel biomarkers of nephrotoxicity. Han-Wistar rats were given daily oral doses of 50, 350, or up to 700 mg/kg/day of NPAA, and Sprague-Dawley rats were given 50 or 400 mg/kg/day of NPAA. Rats were euthanized on days 8 and 15. The candidate kidney injury biomarkers renal papillary antigen-1 (RPA-1, for collecting duct injury), clusterin (for general kidney injury), α-glutathione-S-transferase (a proximal tubular marker), and μ-glutathione-S-transferase (a distal tubular marker) were measured in urine by enzyme immunoassay. Characteristic degeneration and necrosis of the collecting duct and renal papilla were observed in Han-Wistar rats at the high dose on day 8 and at the mid and high doses on day 15, and in Sprague-Dawley rats given the high dose on days 8 and 15. Increases in urinary RPA-1, and to a lesser extent urine clusterin, were generally associated with the presence of collecting duct injury and were more sensitive than BUN and serum creatinine. On the other hand, decreases in α-glutathione-S-transferase without proximal tubule lesions in both strains and decreases in μ-glutathione-S-transferase in Sprague-Dawley rats only were not associated with morphological proximal or distal tubule abnormalities, so both were of less utility. It was concluded that RPA-1 is a new biomarker with utility in the detection of collecting duct injury in papillary necrosis in male rats.     

Analgesic nephropathy in Fischer 344 rats: comparative effects of chronic treatment with either aspirin or paracetamol.

This study has compared the relative nephrotoxicity of chronic treatment with aspirin or paracetamol in an animal model. Changes in renal structure and urinary concentrating ability were examined in female Fischer 344 rats after continuous treatment with either aspirin (120-230 mg/kg body wt/day), or paracetamol (140-210 mg/kg body wt/day), and were compared with age-matched untreated control rats. Renal morphological changes were examined after 40-83 weeks of analgesic treatment, using light and electron microscopy. Aspirin caused renal papillary necrosis and a decrease in urinary concentrating ability, whereas paracetamol alone did not cause significant renal damage. Aspirin produced damage to the interstitial cells and matrix, particularly in the mid-papillary region, followed by changes to the thin limbs of the loop of Henle and medullary capillary endothelium. These structural changes were similar to those described previously, when continuous treatment with combined aspirin and paracetamol was studied in the same animal model.     

Experimental renal papillary necrosis in the rat: The selective vulnerability of medullary structures to injury

Summary Acute renal papillary necrosis was produced in rats by the administration of ethyleneimine. Low doses resulted in necrosis of interstitial cells, thin limbs of the loops of Henle and vasa recta, while collecting ducts were spared (subtotal renal papillary necrosis). High doses resulted in necrosis of all elements of the papilla (total renal papillary necrosis). Although the ranges of the doses that produced these two patterns of necrosis overlapped, it is clear that there is a dose dependent selective vulnerability of renal medullary structures to injury by the toxic agent studied.     

Tobramycin-induced changes in renal histology of fetal and newborn Sprague-Dawley rats.

Effects on renal development were studied using tobramycin (TBM) as a model compound. Pregnant Sprague-Dawley rats were injected i.p. with TBM at 30 or 60 mg/kg body weight/day on gestational days (GD) 10-19. Kidneys from dams and conceptuses were examined on GD 20 and on postnatal day (PD) 9. The dosing regimen caused in dams moderate proximal tubular alterations and increased concentrations in serum creatinine. Fetal kidneys showed granularity and swelling of proximal tubule cells at the 30 mg/kg dose, poor glomerular differentiation at the 60 mg/kg dose, increased glomerular density at both doses, and no changes on macroscopic examination at either dose. In newborns were observed a moderate developmental delay and tubular lesions at the higher dose, and dose-related increases of glomerular density and relative medullary area at both doses. All findings were more pronounced in males. A maturational disruption of the tubular structures possibly leading to increased glomerular density was attributed to TBM exposure during renal organogenesis in the rat.     

Pattern of renal cortical scarring after experimental papillary necrosis

Three types of renal cortical damage were found in rats 2 mth after papillary necrosis had been induced by ethylenimine: (1) Circumscribed areas of interstitial nephritis affecting either deep or superficial nephrons. (2) Wedge-shaped or conical scars, extending from capsule to inner medulla. (3) Widespread tubular dilatation and cyst formation with a diffuse increase in interstitial tissue, usually associated with dense fibrous repair of the papillary remnant. The extent and character of the cortical changes did not appear to be determined by the severity of the papillary necrosis, and even the more severe cortical lesions were not accompanied by any major reduction in kidney size. Although these chronic experimental cortical lesions are the products of a less complex and less protracted natural history than end stage cortical damage in analgesic nephropathy, some of the factors influencing their evolution, such as infection, may also determine the natural history of the clinical lesion.     

Effects of reproductive tract glutathione enhancement and depletion on ethyl methanesulfonate-induced dominant lethal mutations in Sprague-Dawley rats.

The effects of altering glutathione (GSH) levels in the male reproductive tract have been studied in an attempt to establish a link between chemical-induced perturbations in glutathione and susceptibility of spermatozoa to chemical insult. Tissue GSH levels were enhanced by a treatment regimen of N-acetylcysteine (NAC) (250 mg/kg, 4 treatments at 2 h intervals). With this treatment, GSH levels in liver, testis, caput epididymis, and cauda epididymis were elevated to 126%, 110%, 178%, and 136% of control values. Sexually mature male rats were then treated with NAC and challenged with a dose of EMS (100 mg/kg) to determine if enhanced tissue GSH would protect against EMS-induced dominant lethal mutations. Pretreatment with NAC significantly decreased the post-implantation loss from 7.05 +/- 0.57 with EMS alone to 5.28 +/- 0.47. Conversely, a dominant lethal assay was conducted using different doses of phorone pretreatment to determine the relative contribution of hepatic versus reproductive tract GSH in protecting against EMS-induced dominant lethal resorptions. Doses of 100 mg/kg and 250 mg/kg phorone significantly lowered both hepatic and reproductive tract GSH while 25 mg/kg lowered only hepatic GSH. These three dose levels were used as pretreatments in a dominant lethal study followed by a challenge administration of EMS (50 mg/kg), which is a threshold dose of EMS for producing dominant lethal mutations. Comparison against controls demonstrated a significant potentiation of fetal resorptions in all groups receiving phorone pretreatment, including the 25 mg/kg pretreatment group which only lowered hepatic GSH prior to EMS challenge. The results of these experiments indicate that GSH reserves in the male reproductive tract are insufficient to protect developing spermatozoa from damage by alkylating agents in the absence of hepatic GSH.     

Papillary necrosis in experimental renal transplantation in the rat

Renal papillary necrosis is a frequent complication of unsuccessful renal transplantation in rats, occurring in both isografts and allografts. Papillary necrosis does not occur alone, but only and inevitably in association with severe cortical damage. The pattern of the lesion is different from other forms of papillary necrosis in that the least severe lesions occur in the outer medulla and the more severe lesions involve both medulla and papilla. The incidence of papillary necrosis is increased in isografts, but not in allografts, by longer preservation times. It is suggested that the principal underlying cause may be damage to medullary capillaries, occurring either during preservation or as a consequence of rejection and leading to medullary ischemia.     

Toxicity of human recombinant interleukin-2 in rats. Pathologic changes are characterized by marked lymphocytic and eosinophilic proliferation and multisystem involvement

To characterize the subacute toxicologic and pathologic effects of human recombinant interleukin-2 (rIL-2) in rats, rIL-2 was administered to rats at doses ranging from 3.0 to 150 X 10(6) units/kg/day for 14 to 16 days, in once or twice daily dosing regimens by intravenous or intraperitoneal dosing routes. The hematologic, clinical chemical, gross pathologic and histopathologic findings were determined. Rats given rIL-2 had dose- and regimen-related incidences of hemolytic anemia, lymphocytosis, neutrophilia, eosinophilia, thrombocytopenia, increased hepatic transaminases, hyperbilirubinemia, hypoalbuminemia, ascites and pleural effusions, and prerenal azotemia. Marked lymphoid infiltration of the liver and eye were associated with hepatocyte necrosis and retinal damage. Eosinophilic infiltration of the adrenal medulla was associated with medullary cell necrosis. Other histopathologic changes included infiltration of multiple tissues by eosinophils and/or lymphocytes, lymphoid hyperplasia and splenic extramedullary erythropoiesis and eosinopoiesis. Hematologic, clinical chemistry, and histopathologic changes were of markedly greater severity in rats receiving rIL-2 twice daily as compared with those receiving the same total dose of rIL-2 once daily. Mortality in severely affected rats was secondary to severe anemia and/or hepatic damage. The results of these studies demonstrate that the major pathologic changes associated with rIL-2 administration in rats are characterized by marked tissue infiltration with lymphocytes and eosinophils. Many of the adverse effects of rIL-2 administration to rats are similar to those reported in humans receiving rIL-2 immunotherapy and the results confirm that the rat is an appropriate species in which to study selected aspects of rIL-2 toxicity.     

Immunolocalization of Kim-1, RPA-1, and RPA-2 in kidney of gentamicin-, mercury-, or chromium-treated rats: relationship to renal distributions of iNOS and nitrotyrosine

Immunohistochemical studies for kidney injury molecule-1 (Kim-1), renal papillary antigen-1 (RPA-1), and renal papillary antigen-2 (RPA-2) were conducted to explore their relationship to inducible nitric oxide synthase (iNOS) and nitrotyrosine expression. Male Sprague-Dawley rats were exposed to gentamicin (100 mg/kg/day Gen, sc, for 3 days), mercury (0.25 mg Hg/kg, iv, single dose), or chromium (5 mg Cr/kg, sc, single dose) and kidney tissue was examined 24 hours or 72 hours after the last dose of the nephrotoxicant. Another group of kidneys was evaluated 24 hours after rats were administered 3 daily doses (50, 100, 150, 200, or 300 mg/kg/day) of Gen. Gen- and Cr-treated rats exhibited increased immunoreactivity of Kim-1, RPA-1, and RPA-2 largely in the S1/S2 segments and to a lesser extent in the S3 segments of the proximal tubule of the kidney, whereas Hg-treated rats showed increased immunoreactivity of Kim-1, RPA-1, and RPA-2 in the S3 segments. Up-regulation of Kim-1, RPA-1, and RPA-2 expression correlated with injured tubular epithelial cells and also correlated with immunoreactivity of iNOS and nitrotyrosine. It is possible that iNOS activation with nitrotyrosine production in injured nephron segments may be involved in the induction of Kim-1, RPA-1, and RPA-2 following exposure to nephrotoxicants.     

Light microscopic and ultrastructural pathology of seminiferous tubules of rats given multiple doses of Pasteurella multocida group D protein toxin.

Male Holtzman rats were given subcutaneous doses of a purified Pasteurella multocida group D heat-labile toxin on alternate days for up to 22 days. Rats were necropsied at 18 days or 36 days (14 days after last dose of toxin) or when moribund, and testicles were taken for histologic and ultrastructural examination. Other selected tissues, including liver and spleen, were taken for histologic examination. Histologically, testicular and splenic lesions occurred more consistently and at much smaller doses when compared with lesions in other target organs such as liver. Testicular and splenic lesions were present in all rats (6/6) given 0.8 micrograms/kg toxin and were seen in some rats (1/6) given as little as 0.2 micrograms/kg toxin. Only 3/6 rats given 0.8 micrograms/kg toxin had hepatic lesions; no hepatic lesions were seen at doses of 0.2 micrograms/kg. Testicles from toxin-treated rats were smaller and weighed less than controls. Seminiferous tubules were moderately dilated and lined by polygonal sertoli cells. The normal spermatogenic maturation sequence and mature spermatids were absent, and many tubules contained multinucleate spermatocytes. Severely affected tubules were necrotic and mineralized. Ultrastructurally, there was necrosis of adluminal spermatocytes, multinucleate cell formation, and spaces between Sertoli cell plasma membranes. Testicular lesions were similar to those described for vitamin D-deficient rats, vitamin A-deficient rats, vasectomized rats, and rats given intravenous tumor necrosis factor; however, rats given lethal doses of toxin did not have elevated levels of TNF alpha activity.