Neoplasms of the urinary tract in fish.

The veterinary literature contains scattered reports of primary tumors of the urinary tract of fish, dating back to 1906. Many of the more recent reports have been described in association with the Registry of Tumors in Lower Animals, and most of the spontaneous neoplasms of the kidney and urinary bladder are single case reports. In rare instances, such as described in nephroblastomas of Japanese eels and tubular adenomas/adenocarcinomas of Oscars, there is suggestion of a genetic predisposition of certain populations to specific renal neoplasms, environmental carcinogenesis, or potentially an unknown infectious etiology acting as a promoter. Hematopoeitic neoplasms have been infrequently described as primary to the kidney of a variety of fish species, and therefore those case reports of renal lymphoma and plasmacytic leukemia are addressed within the context of this review.

Methyl isobutyl ketone (MIBK) induction of alpha2u-globulin nephropathy in male, but not female rats.

Male F-344 rats were administered corn oil (vehicle control), d-limonene (positive control, 300mg/kg), or MIBK (1000mg/kg) and female F-344 rats corn oil (vehicle control) or MIBK for 10 consecutive days by oral gavage. Approximately 24h after the final dose the kidneys were excised and the left kidney prepared and evaluated for histological changes including protein (hyaline) droplet accumulation, immunohistochemical staining for alpha2u-globulin (alpha2u), and proliferating cell nuclear antigen (PCNA) to quantitate renal cell proliferation. The right kidney was prepared for quantitation of total protein and alpha2u using an ELISA. MIBK elicited an increase in protein droplets, accumulation of alpha2u, and renal cell proliferation in male, but not female rats, responses characteristic of alpha2u-mediated nephropathy. MIBK produced identical histopathological changes in the male rat kidney when compared to d-limonene, an acknowledged inducer of alpha2u-nephropathy except that the grade of severity tended to be slightly lower with MIBK. MIBK did not induce any effects in female rats. Therefore, renal histopathology, along with the other measures of alpha2u accumulation, provides additional weight of evidence to support the inclusion of MIBK in the category of chemicals exerting renal effects through a alpha2u-nephropathy-mediated mode-of-action.

Re-evaluation of kidney histopathology from 13-week toxicity and two-year carcinogenicity studies of melamine in the F344 rat: morphologic evidence of retrograde nephropathy.

The histopathologic changes induced in F344 rat kidney by oral administration of melamine for 13-week and 2-year periods in studies conducted by the National Toxicology Program, NIH,(25) from 1976 to 1983 have been re-evaluated and described in detail. A constellation of tubule changes extending from papilla to cortex consistently included tubule dilatation and tubule basophilia as salient features at the subchronic time point. By 2 years, these lesions had usually resolved into fibrotic scars, in which tubule loss and collagen deposition were prominent, running from superficial cortex into the medulla. These fibrotic lesions required discrimination from chronic scars resulting from infarcts and foci of chronic progressive nephropathy (CPN). A case is presented here for interpreting the constellation of histologic changes induced in rats by melamine as representing an ascending form of nephropathy. The term retrograde nephropathy is considered to be the appropriate nomenclature for both the acute and chronic lesions. The cause for the reflux, emanating from the lower urinary tract, appeared not to be infection as an inflammatory response was not prominent. It can be speculated that melamine precipitation in the lower urinary tract created pressure effects through transient obstruction leading to the renal changes. These changes were different from those involved in a major US outbreak of renal disease and death in cats and dogs associated with triazine-contaminated pet food, in which crystalluria from insoluble melamine/cyanuric acid complexes occurred in the kidney. However, the rat findings may be relevant to melamine-associated kidney disease recently reported in infants in China.

Ochratoxin A as a potential etiologic factor in endemic nephropathy: lessons from toxicity studies in rats.

Various reports suggest that chronic dietary exposure to ochratoxin A (OTA), a mycotoxin frequently detected in various food items may be linked to the pathogenesis of endemic nephropathy, a chronic tubulointerstitial kidney disease which occurs in geographically limited areas of the Balkan region. OTA is a potent nephrotoxin and renal carcinogen. However, the pathological lesions observed in kidneys of rats treated with OTA appear be rather different from the clinical and pathological characteristics of endemic nephropathy. Moreover, increasing evidence suggests that OTA does not bind to DNA but induces tumors by an epigenetic, thresholded mechanism. This implies that there is a dose below which no adverse health effects are expected to occur. Based on food consumption data and OTA serum concentrations, it appears that human exposure - even in areas with relatively high dietary exposure to OTA such as endemic villages - is several orders of magnitude below doses known to cause nephrotoxicity and tumor formation in laboratory animals. While it is undoubtedly important to encourage prevention of food contamination by OTA and other mycotoxins, these observations suggest that OTA is not likely to be an etiological factor involved in BEN and indicate a need to search for new clues for the etiology of this endemic kidney disease.

Nephroblastoma in the rat: histology of a spontaneous tumor, identity with respect to renal mesenchymal neoplasms, and a review of previously recorded cases.

The histology of a spontaneously occurring neoplasm of the rat kidney conforming to a classification of nephroblastoma is described and compared with that of N-nitrosodimethylamine-induced renal mesenchymal tumors. This rat nephroblastoma was an encapsulated epitheloid neoplasm with a uniform histologic pattern. Clumps of densely crowded, hyperchromatic cells frequently associated with central, well-differentiated ducts were supported by a less cellular, interconnecting stroma of loose areolar or mature fibrous connective tissue. Neoplastic cells were organized into primitive, ill-defined tubular formations. The neoplastic cell component strongly resembled metanephrogenic blastema. In contrast, the renal mesenchymal tumor was nonencapsulated and consisted of a heterogeneous mixture of connective tissue elements including fibroblast-like spindle cells, smooth muscle, and embryonic mesenchyme that engulfed and sequestered preexisting renal tubules and glomeruli. The separate morphologic identities and apparently unrelated existence of rat nephroblastoma and renal mesenchymal tumor were stressed. The rat nephroblastoma morphologically resembled the malignant epithelial component of human Wilms' tumor, whereas rat renal mesenchymal tumor appeared to have counter-parts in the mesenchymal component of Wilms' tumor and in congenital mesoblastic nephroma (leiomyomatous hamartoma) of infancy. The histologic descriptions of previously recorded occurrences of spontaneous and experimentally induced rat neoplasma classified as nephroblastoma or its synonyms were reevaluated in comparison to the present case. In all but four instances, in which sufficient histologic detail was provided in previous reports, a consistent histologic pattern emerged for this neoplasm in the rat.

Morphologic character of transforming renal cell cultures derived from Wistar rats given dimethylnitrosamine.

The morphologic character of kidney cells during serial subculture following isolation from Wistar rats treated several hours to 1 week previously with a carcinogenic dose of dimethylnitrosamine (DMN; 60 mg/kg body weight following protein deprivation) was compared with the appearance of cultures derived from normal control rats. Apart from early signs of cell toxicity, cultures from DMN-treated rats appeared similar to those from untreated rats for the first four passages. Control cells underwent senescence usually by subculture 4, whereas the test cultures survived to express morphologic transformation (usually at subculture 5) as dense macroscopic colonies of piled up cells. In 18 of the 20 test cultures, the cell populations that persisted in continuous culture following expression of morphologic transformation were exclusively mesenchymal, closely resembling DMN-induced renal mesenchymal tumor cells in continuous culture. In the remaining 2 test cultures from DMN-treated rats, a persisting population of abnormal epithelium was present in addition to morphologically transformed mesenchymal cells. The occurrence of populations of altered mesenchymal and epithelial cells characterized by prolonged survival in vitro following isolation from rats shortly after treatment with a carcinogenic dose of DMN was believed to be related to the long-term induction in the rat kidney of a high incidence of mesenchymal tumors and a lower incidence of cortical epithelial tumors by the same dose schedule.

In vitro culture of cells isolate from dimethylnitrosamine-induced renal mesenchymal tumors of the rat. I. Qualitative morphology.

The in vitro morphology of cells isolated from renal mesenchymal tumors induced in rats by a single ip injection of dimethylnitrosamine was followed in short- and long-term culture. The morphology of cells isolated from normal rat-kidney cortex was studied for comparison. Primary tumor-cell isolates consisted of a range of mesenchymal cell forms interspersed between discrete, homogeneous islands of cohesive epithelium-like cells. The latter were presumably derived from renal parenchyma that had been engulfed within the in vivo neoplasm by proliferating tumor cells, since this epithelial component did not survive serial subculture. Established tumor cell lines consisted of a pleomorphic range of mesenchymal forms resembling the descriptions of various cell types in vitro, including fibroblasts, smooth muscle cells, and endothelial cells. A range of mesenchymal cell forms in culture therefore correlated with the heterogeneous histologic nature of the in vivo neoplasm in which fibroblasts, smooth muscle fibers, and endothelium-like cells were also represented. Multinucleate or polymorphonuclear giant cells were characteristic of tumor cell cultures, whereas large, expanded polygonal cells with longitudinal striations were characteristic of normal kidney cell cultures. Some tumor cell cultures were typified by cells with cytoplasmic vacuolation and some by the acquisition of an epithelioid form at high cell density, a change not preceded by islands of cohesive, epithelium-like cells at preconfluent stages.

Carcinogenesis induced by 7,12-dimethylbenz[a]anthracene in c3H-A vyfB mice: influence of different dietary fats.

The effect of a diet containing either sunflower-seed oil (polyunsaturated fat diet) or tallow (saturated fat diet) on 7,12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis in C3H-A vyfB mice was examined. After receiving either diet for 28 days, some of the mice were given an intragastric dose of 5 mg DMBA. To identify the stage of carcinogenesis that might be influenced by dietary fat, the diets of half of the mice were then interchanged so that those previously fed the saturated fat diet were fed the polyunsaturated fat diet and vice versa. The cumulative incidence of tumor-bearing mice was significantly greater among the females fed the polyunsaturated fat diet compared to those fed the saturated fat diet. This enhancement of carcinogenesis was observed only when the mice were fed the polyunsaturated fat diet after DMBA administration. Similar trends were observed in the male mice, but these mice developed fewer tumors and none of the differences between the tumor incidences were statistically significant. The most common sites for tumors in the male mice were the liver, lungs, and skin, whereas those for tumors in the females were the mammary glands and ovaries. The differences in tumor incidence suggest that carcinogenesis was enhanced by the polyunsaturated fat diet during the promotion stage of carcinogenesis.

von Hippel-Lindau gene mutations in N-nitrosodimethylamine-induced rat renal epithelial tumors.

BACKGROUND: Mutations in the von Hippel-Lindau (VHL) gene are common in human clear cell kidney cancers. Carcinogens in cigarette smoke, especially nitrosamines, are known to induce kidney tumors of a variety of histologic types in rodents--but with no evidence of VHL mutations; however, none of these tumors resembled human clear cell carcinomas. We examined N-nitrosodimethylamine-induced kidney tumors of the clear or mixed clear/granular cell type in Wistar rats to assess the presence of VHL mutations. METHODS: Sections of eight clear or mixed clear/granular cell kidney tumors that had been formalin fixed and paraffin embedded were microdissected. DNA was extracted from the microdissected tissue, and exons 1-3 of the rat VHL gene were examined by use of polymerase chain reaction and cycle sequencing techniques. RESULTS: Four VHL gene mutations (three G:C to A:T and one A:T to G:C) were detected in three of the tumors in contrast to no mutations in 40 previously reported rat kidney tumors of other histologic types (three of eight tumors versus none of 40; two-sided Fisher's exact test; P=.003). Only tumors showing prominent swollen clear cell cytology with a signet-ring appearance had VHL mutations. CONCLUSIONS: To our knowledge, this is the first report of VHL mutations in kidney tumors after direct chemical exposure and provides a possible molecular pathway linking tobacco smoking to kidney cancer.

Histological conformity of implantation tumors produced by kidney cell lines derived from dimethylnitrosamine-treated rats, with dimethylnitrosamine-induced renal mesenchymal tumors.

The histology of five implantation tumors induced in rats by the deposition of cultured cell lines derived from dimethylnitrosamine (DMN)-treated rats is described and compared with the morphology of the predominant kidney neoplasm induced in vivo by a single high dose of DMN. The cell lines leading to growth upon implantation were long-established, continuously growing cultures obtained either from a DMN-induced renal mesenchymal tumor or from rats treated shortly before with a carcinogenic dose of DMN. The latter cultures had expressed morphological transformation at subcultures 5 or 6. All of the implantation tumors were of mesenchymal type, comprising variously a range of cell forms including fibroblast-like spindle cells, smooth muscle fibers, and "giant" cells, which resembled common aspects of the parent mesenchymal tumors induced in the rat kidney by DMN. Deposition of cells intrarenally illustrated the survival of remnants of preexisting nephrons as epithelial profiles scattered through the proliferating malignant tissue, a feature most characteristic of the parent tumor. The results confirmed the malignant nature of the various cell lines tested, in keeping with their altered behavior in vitro, and they were consistent also with the premise that the in vivo-in vitro system is selecting cells in culture that represent the same target population from which the renal mesenchymal tumors are derived in vivo.

Autoradiographic analysis of proliferative activity in rat kidney epithelial and mesenchymal cell subpopulations following a carcinogenic dose of dimethylnitrosamine.

In a system that yields 100% incidence of renal mesenchymal tumors and a 30 to 40% incidence of renal cortical epithelial neoplasms, the proliferative activity of renal epithelial and mesenchymal cell subpopulations following a single dose of dimethylnitrosamine (DMN) was traced by autoradiographic analysis of [methyl-3H]thymidine uptake during the 3 weeks immediately posttreatment. The initial response to DMN was a depression in DNA synthesis and mitosis to near 0 levels in all segments of the nephron and in attendant mesenchymal cells for a period of 1 to 3 days. Following the period of inhibition, increased DNA synthetic activity was observed in certain subpopulations of both epithelium and mesenchyme and these patterns were matched by equivalent mitotic activity. A stimulation of DNA synthesis was observed in cells of the proximal and distal tubules of Zones 1 and 2 but in no other epithelial segments. The increased activity was most intense in Zone 1 epithelium reaching a peak at the 10th day after DMN injection 4 days after epithelial cell necrosis had commenced. In renal mesenchyme, the major response involved only the interstitial cells of Zones 1 and 2. At Day 3, there was a wave of increased DNA-synthetic and mitotic activity in the free interstitial cells of the cortex, followed by a 2nd, more intense peak of activity at Day 6. The cells responding at Day 3 appeared to involve the resident population of cortical fibrocytes while the major contribution to the Day 6 peak came from infiltrating mononuclear inflammatory cells, although resident fibrocytes and capillary endothelium also contributed. A significant wave of increased activity involved the intestitial cells of Zone 2, but the peak, although of equivalent intensity to the response in Zone 1, was single and occurred 3 days later at Day 9. Apart from a small, brief, and variable wave of activity in interstitial cells of Zone 3 from Days 8 to 10, no toerh mesenchymal cell populations in the kidney were stimulated by the injection of DMN.

Identification of a high-frequency model for renal carcinoma by the induction of renal tumors in the mouse with a single dose of streptozotocin.

In order to develop a high-frequency, single-dose model of renal epithelial tumor induction in the mouse, streptozotocin (250 mg/kg) was administered i.v. to 6-week-old males and females of the CBA/H/T6J strain. In groups of 26 males and 30 females representing the effective number of survivors eligible for tumor estimates, renal cell tumors were found in 73% of males and 97% of females, including incidences of lesions diagnosed as renal carcinoma in 31 and 60%, respectively. The difference in renal tumor frequency between the sexes was not considered a real effect owing to the significantly decreased survival of the males. The neoplastic lesions ranged from small papillary or cystopapillary adenomas with a benign appearance to large, solid carcinomas with a low potential for metastasis. In the females, 22% of the larger tumors metastasized to distant sites, mainly the lungs. Intermediate lesions incorporating both papillary and solid adenomatous profiles suggested a sequential development of carcinomas along this pathway from the smaller papillary foci. The data establish the administration of a single dose of streptozotocin in the female CBA/H/T6J as a suitable high incidence model for the study of renal epithelial carcinogenesis in the mouse.

Immunofluorescent characterization of rat kidney tumors according to the distribution of actin as revealed by specific antiactin antibody.

A series of 15 mesenchymal and 10 cortical epithelial tumors induced in the rat kidney by dimethylnitrosamine was investigated for immunofluorescent reactivity with a human antiactin antibody. Cells of epithelial tumors showed staining restricted to peripheral sites, corresponding to the brush border region. All various neoplastic cells forms comprising renal mesenchymal tumor were characterized by cytoplasmic staining in pattersn that varied with cell type. Epithelial profiles in the form of tubules and islands of epithelium showed staining patterns, or absence of them, consistent with their identity as sequestered segments of preexisting nephrons. It is suggested that the difference in actin distribution within the cytoplasm of cells of the two types of renal neoplasm, mesenchymal and epithelial, might reflect their difference in local invasive growth.

Differentiated features of a transformed epithelial cell line (TRKE-1) derived from dimethylnitrosamine-treated rat kidney.

TRKE-1 is a pure line of epithelium-like neoplastic cells derived from the kidney of a rat treated 48 hr previously with a carcinogenic dose of dimethylnitrosamine. Using light microscopy, the line was characterized by cohesive growth behavior typical for epithelium and the formation of hemicysts (domes) at postconfluence. Enhancement of dome formation by dibutyryl cyclic adenosine 3':5'-monophosphate and dimethyl sulfoxide and inhibition by ouabain established these structures as a manifestation of differentiated cellular function, namely, transepithelial fluid transport. Structurally, TRKE-1 cells in monolayer culture were characterized by apical distribution of microvilli, cilia, and endocytic vesicles, ordered sequence of junctional components at the apical lateral border including tight junction and desmosomes, basolateral cellular interdigitations below the junctional complex, basal location of microfilament bundles, and a conspicuous content of mitochondria. Each of these features typifies mammalian renal tubule epithelium in vivo. The occasional profusion of microvilli; the prominent, apically distributed endocytic vesicles; and the well-developed basal microfilament tracts suggest, in particular, that the proximal segment of the nephron may represent the site of origin of this transformed cell line. The various morphological aspects of renal epithelial differentiation were also expressed in multicellular tumor spheroids grown in suspension, with an accentuation of junctional complexes, endocytic vesicles, and intracytoplasmic lumina. In addition, this three-dimensional culture mode supported cellular organization into acinar profiles suggestive of primitive tubule formation. In confirming the epithelial nature of TRKE-1 and a possible identity with the proximal tubule, this study provides an in vitro animal model representative of chemically transformed renal epithelium which may be analogous to human renal cell carcinoma.

Selective mutation of codons 204 and 213 of the p53 gene in rat tumors induced by alkylating N-nitroso compounds.

Kidney and esophageal tumors induced by alkylating N-nitroso compounds in rats contain a high incidence (75-100%) of G----A transition mutations in the p53 gene. These are almost selectively (89%) located in the first base of codon 204 and the second base of 213, leading to amino acid substitutions Glu----Lys and Arg----Gln, respectively. In contrast to human neoplasms, a considerable fraction of rat kidney and esophageal tumors carries multiple p53 mutations. All nephroblastomas induced by transplacental exposure to N-nitrosoethylurea and 56% of esophageal tumors induced by N-nitrosomethylurea showed double mutations in codons 204 and 213 of exon 6. The selective targeting of p53 codons by alkylating nitrosamines may provide a basis for molecular epidemiological studies on this class of chemical carcinogens.

Major difference in the hepatocarcinogenicity and DNA adduct forming ability between toremifene and tamoxifen in female Crl:CD(BR) rats.

The hepatoproliferative effects of 2 antiestrogens, tamoxifen and toremifene, were compared in a sequential 15-month study in which 2 doses of each compound were administered by daily gavage to female Sprague-Dawley rats for up to 12 months. The doses were 11.3 and 22.6 mg/kg for tamoxifen and 12 and 24 mg/kg for toremifene. There were scheduled sacrifices at 3, 6, 12, and 15 months, the latter including a 3-month recovery period from the 12th through the 14th month. In the chronic toxicity study, tamoxifen at 22.6 mg/kg produced 100% incidence of hepatocellular carcinoma at the 12- and 15-month sacrifice intervals and 67% and 71% incidences at the 11.3-mg/kg dose. Sequential observations showed an increased incidence of glutathione S-transferase-positive foci of hepatocellular alteration by 3 months with tamoxifen in the absence of hepatotoxicity, with the first liver carcinoma appearing by 6 months of treatment. Unscheduled deaths occurring beyond 7.5 months in the tamoxifen treated groups were due in almost all cases to liver cancer. In striking contrast, toremifene did not produce any hepatoproliferative effects at 12- and 24-mg/kg dose levels, nor in a pilot study at 48 mg/kg. The 24-mg/kg dose of toremifene exerted an inhibiting effect on foci of hepatocellular alteration in rat liver detectable by glutathione S-transferase immunohistochemistry at 3 months and by conventional histology at 12 months. An antiproliferative effect was also evident in mammary gland and anterior pituitary where both toremifene and tamoxifen suppressed tumor incidence in comparison to the control group. The ability of these drugs to modify rat liver DNA after p.o. administration was investigated using the 32P-postlabeling assay. Administration of tamoxifen at 45 mg/kg for 7 days produced liver DNA nucleoside modifications represented by 7 spots on the autoradiogram. Unlike tamoxifen, toremifene did not produce any modified bases in rat liver DNA detectable by the 32P-postlabeling technique. The dose levels of tamoxifen that are strongly hepatocarcinogenic in the rat are compared with doses used in humans in various applications. Taking internal drug exposure into account, we conclude that the margin of safety for use of tamoxifen as an endocrine prophylactic agent for healthy, but breast cancer prone, women is questionable.

Effects of tea, decaffeinated tea, and caffeine on UVB light-induced complete carcinogenesis in SKH-1 mice: demonstration of caffeine as a biologically important constituent of tea.

Oral administration of green or black tea inhibited UVB light-induced complete carcinogenesis in the skin of SKH-1 mice. Green tea was a more effective inhibitor than black tea. Oral administration of decaffeinated green or black tea resulted in substantially less inhibitory activity than did administration of the regular teas, and in one experiment, administration of a high-dose level of the decaffeinated teas enhanced the tumorigenic effect of UVB. Oral administration of caffeine alone had a substantial inhibitory effect on UVB-induced carcinogenesis, and adding caffeine to the decaffeinated teas restored the inhibitory effects of these teas on UVB-induced carcinogenesis. In additional studies, topical application of a green tea polyphenol fraction after each UVB application inhibited UVB-induced tumorigenesis. The results indicate that caffeine contributes in an important way to the inhibitory effects of green and black tea on UVB-induced complete carcinogenesis.

Inhibition of 7,12-dimethylbenz(a)anthracene-induced skin papillomas and carcinomas by dehydroepiandrosterone and 3-beta-methylandrost-5-en-17-one in mice.

Topical application of the adrenal steroid, dehydroepiandrosterone, or the synthetic steroid, 3-beta-methylandrost-5-en-17-one, which unlike dehydroepiandrosterone is not demonstrably uterotrophic, inhibits 7,12-dimethylbenz(a)anthracene-induced skin papillomas and carcinomas in the CD-1 mouse.

Deregulation and overexpression of c-fos proto-oncogene in rat renal cell-lines and primary tumors induced by dimethylnitrosamine.

Rat renal mesenchymal tumors induced by the chemical carcinogen N-dimethylnitrosamine (DMN) and cell-lines derived from kidneys of rats after DMN treatment were found to express abnormal steady state levels of c-fos RNA. This overexpression was not found to arise by gene amplification or rearrangement, but the c-fos gene appeared to be deregulated resulting in increased transcription. The consistent nature of this observation suggests a function for c-fos gene overexpression in tumorigenesis of the rat kidney by DMN.

Methyl isobutyl ketone exposure-related increases in specific measures of α2u-globulin (α2u) nephropathy in male rats along with in vitro evidence of reversible protein binding.

Chronic exposure to methyl isobutyl ketone (MIBK) resulted in an increase in the incidence of renal tubule adenomas and occurrence of renal tubule carcinomas in male, but not female Fischer 344 rats. Since a number of chemicals have been shown to cause male rat renal tumors through the α2u nephropathy-mediated mode of action, the objective of this study is to evaluate the ability of MIBK to induce measures of α2u nephropathy including renal cell proliferation in male and female F344 rats following exposure to the same inhalation concentrations used in the National Toxicology Program (NTP) cancer bioassay (0, 450, 900, or 1800ppm). Rats were exposed 6h/day for 1 or 4 weeks and kidneys excised approximately 18h post exposure to evaluate hyaline droplet accumulation (HDA), α2u staining of hyaline droplets, renal cell proliferation, and to quantitate renal α2u concentration. There was an exposure-related increase in all measures of α2u nephropathy in male, but not female rat kidneys. The hyaline droplets present in male rat kidney stained positively for α2u. The changes in HDA and α2u concentration were comparable to d-limonene, an acknowledged inducer of α2u nephropathy. In a separate in vitro study using a two-compartment vial equilibration model to assess the interaction between MIBK and α2u, the dissociation constant (Kd) was estimated to be 1.27×10(-5)M. This Kd is within the range of other chemicals known to bind to α2u and cause nephropathy. Together, the exposure-related increase in measures of α2u nephropathy, sustained increase in renal cell proliferation along with an indication of reversible binding of MIBK to α2u, support the inclusion of MIBK in the category of chemicals exerting renal effects through a protein droplet α2u nephropathy-mediated mode of action (MoA).