Normal Anatomy, Histology, and Spontaneous Pathology of the Kidney, and Selected Renal Biomarker Reference Ranges in the Cynomolgus Monkey.

To further our understanding of the nonhuman primate kidney anatomy, histology, and incidences of spontaneous pathology, we retrospectively examined kidneys from a total of 505 control Cynomolgus monkeys (Macaca fascicularis; 264 male and 241 females) aged 2 to 6 years, from toxicity studies. Kidney weights, urinalysis, and kidney-related clinical biochemistry parameters were also evaluated. Although the functional anatomy of the monkey kidney is relatively similar to that of other laboratory animals and humans, a few differences and species-specific peculiarities exist. Unlike humans, the macaque kidney is unipapillate, with a relatively underdeveloped papilla, scarce long loops of Henle, and a near-equivalent cortical to medullary ratio. The most common spontaneous microscopic findings were interstitial infiltrates or interstitial nephritis and other tubular lesions, but several forms of glomerulopathy that may be interpreted as drug-induced were occasionally observed. Common incidental findings of little pathological significance included: papillary mineralization, epithelial pigment, multinucleate cells, cuboidal metaplasia of the Bowman's capsule, and urothelial inclusions. Kidney weights, and some clinical chemistry parameters, showed age- and sex-related variations. Taken together, these data will aid the toxicologic pathologist to better evaluate the nonhuman primate kidney and assess the species' suitability as a model for identifying and characterizing drug-induced injury.

Brief Synopsis: Review of Renal Tubule and Interstitial Anatomy and Physiology and Renal INHAND, SEND, and DIKI Nomenclature.

This article provides a synopsis of the first two presentations from the second scientific session of the 37th Annual Symposium of the Society of Toxicologic Pathology in Indianapolis, Indiana, on June 18, 2018; the session focused on acute kidney injury. The first presentation, given by Dr. Kevin McDorman, focused on "Fundamentals of Renal Tubule and Interstitial Anatomy and Physiology." Several common background findings from toxicity studies were additionally discussed. Lastly, factors that impact the relevance and usefulness of historical control data, such as quality and consistency of histopathology, were discussed. The second presentation, given by Dr. Torrie Crabbs, provided a review of International Harmonization of Nomenclature and Diagnostic Criteria (INHAND), Standard for Exchange of Nonclinical Data (SEND), and drug-induced kidney injury (DIKI) nomenclature. INHAND is a global collaborative project that provides internationally accepted standardized nomenclature and diagnostic criteria for proliferative and nonproliferative changes in laboratory animals in toxicity and carcinogenicity studies. SEND is currently a required standard for data submission to the Food and Drug Administration (FDA). Since the FDA has indicated its preference for INHAND nomenclature, SEND will predominately use INHAND terminology; thus, familiarity with INHAND terminology is critical for toxicologic pathologists. The diagnostic features of three common DIKI findings, in addition to several complicated INHAND terminologies, were reviewed.

Tumor penetration and epidermal growth factor receptor saturation by panitumumab correlate with antitumor activity in a preclinical model of human cancer.

BACKGROUND: Successful treatment of solid tumors relies on the ability of drugs to penetrate into the tumor tissue. METHODS: We examined the correlation of panitumumab (an anti-epidermal growth factor [EGFR] antibody) tumor penetration and EGFR saturation, a potential obstacle in large molecule drug delivery, using pharmacokinetics, pharmacodynamics, and tumor growth rate in an A431 epidermoid carcinoma xenograft model of human cancer. To determine receptor saturation, receptor occupancy, and levels of proliferation markers, immunohistochemical and flow cytometric methods were used. Pharmacokinetic data and modeling were used to calculate growth characteristics of panitumumab-treated tumors. RESULTS: Treatment with panitumumab in vivo inhibited pEGFR, Ki67 and pMAPK levels vs control. Tumor penetration and receptor saturation were dose- and time-dependent, reaching 100% and 78%, respectively. Significant tumor inhibition and eradication (p < 0.05) were observed; plasma concentration associated with tumor eradication was estimated to be 0.2 µg/ml. The tumor inhibition model was able to describe the mean tumor growth and death rates. CONCLUSIONS: These data demonstrate that the antitumor activity of panitumumab correlates with its ability to penetrate into tumor tissue, occupy and inhibit activation of EGFR, and inhibit markers of proliferation and MAPK signaling.

Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index.

Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.

Fully human monoclonal antibodies to hepatocyte growth factor with therapeutic potential against hepatocyte growth factor/c-Met-dependent human tumors.

c-Met is a well-characterized receptor tyrosine kinase for hepatocyte growth factor (HGF). Compelling evidence from studies in human tumors and both cellular and animal tumor models indicates that signaling through the HGF/c-Met pathway mediates a plethora of normal cellular activities, including proliferation, survival, migration, and invasion, that are at the root of cancer cell dysregulation, tumorigenesis, and tumor metastasis. Inhibiting HGF-mediated signaling may provide a novel therapeutic approach for treating patients with a broad spectrum of human tumors. Toward this goal, we generated and characterized five different fully human monoclonal antibodies that bound to and neutralized human HGF. Antibodies with subnanomolar affinities for HGF blocked binding of human HGF to c-Met and inhibited HGF-mediated c-Met phosphorylation, cell proliferation, survival, and invasion. Using a series of human-mouse chimeric HGF proteins, we showed that the neutralizing antibodies bind to a unique epitope in the beta-chain of human HGF. Importantly, these antibodies inhibited HGF-dependent autocrine-driven tumor growth and caused significant regression of established U-87 MG tumor xenografts. Treatment with anti-HGF antibody rapidly inhibited tumor cell proliferation and significantly increased the proportion of apoptotic U-87 MG tumor cells in vivo. These results suggest that an antibody to an epitope in the beta-chain of HGF has potential as a novel therapeutic agent for treating patients with HGF-dependent tumors.

Oxidative DNA damage from potassium bromate exposure in Long-Evans rats is not enhanced by a mixture of drinking water disinfection by-products.

Public drinking water treated with chemical disinfectants contains a complex mixture of disinfection by-products (DBPs) for which the relative toxicity of the mixtures needs to be characterized to accurately assess risk. Potassium bromate (KBrO(3)) is a by-product from ozonation of high-bromide surface water for production of drinking water and is a rodent carcinogen that produces thyroid, mesothelial, and renal tumors. The proposed mechanism of KBrO(3) renal carcinogenesis involves the formation of 8-oxoguanine (8-oxoG), a promutagenic base lesion in DNA typically removed through base excision repair (BER). In this study, male Long-Evans rats were exposed via drinking water to carcinogenic concentrations of KBrO(3) (0.4 g/L), 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (0.07 g/L), chloroform (1.8 g/L), bromodichloromethane (0.7 g/L), or a mixture of all these chemicals at the same concentrations for 3 weeks. Half of one kidney was processed for microscopic examination, and the remaining kidney was frozen for isolation of genomic DNA. Levels of 8-oxoG were measured using HPLC with electrochemical detection in DNA samples incubated with formamidopyrimidine-DNA glycosylase. Aldehydic lesions (e.g. abasic sites) in DNA samples were quantitated using an aldehyde-reactive probe slot-blot assay. Treatment with KBrO(3) produced a measurable increase of 8-oxoG in the kidney, and this effect was greater than that produced by treatment with the DBP mixture. No other single chemical treatment caused measurable increases of 8-oxoG. The mixture effect on the amount of 8-oxoG observed in this study suggests an interaction between chemicals that reduced the generation of oxidative DNA damage. No increases in abasic sites were observed with treatment, but a decrease was apparent in the rats treated with the DBP mixture. These data are consistent with previous studies where chronic exposure to this chemical mixture in drinking water resulted in a less than additive carcinogenic response in Tsc2 mutant Long-Evans rats.

Induction of transitional cell hyperplasia in the urinary bladder and aberrant crypt foci in the colon of rats treated with individual and a mixture of drinking water disinfection by-products.

Cancer of the urinary bladder and colon are significant human health concerns. Epidemiological studies have suggested a correlation between these cancers and the chronic consumption of chlorinated surface water containing disinfection by-products (DBPs). The present study was designed to determine if exposure to DBPs would cause preneoplastic or neoplastic lesions in the urinary bladder and colon of rats, and what effect a mixture of DBPs would have on these lesions. Male and female Eker rats were treated via drinking water with low and high concentrations of potassium bromate, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), chloroform, or bromodichloromethane individually or in a mixture for 10 months. The urinary bladders and colons were examined for the presence of preneoplastic lesions. Cell proliferation in the urothelium was examined using immunohistochemical staining for bromodeoxyuridine. Aberrant crypt foci (ACF), as well as the number of individual crypts in each ACF, were identified and counted microscopically after staining with 0.2% methylene blue. Colon crypt cell proliferation and mitotic index were determined using immunohistochemical staining for proliferating cell nuclear antigen. Labeling indexes for the urinary bladder and colon were calculated based on the percentage of positively labeled cells. Treatment with the high dose of MX caused transitional epithelial hyperplasia and cell proliferation in the rat urinary bladder, and this effect was diminished in the high dose mixture animals. Treatment with 4 individual DBPs, as well as a mixture of them, caused the development of ACF, the putative preneoplastic lesion of colon cancer.

Analysis of preneoplastic and neoplastic renal lesions in Tsc2 mutant Long-Evans (Eker) rats following exposure to a mixture of drinking water disinfection by-products.

Disinfection of surface water for human consumption results in the generation of a complex mixture of chemicals in potable water. Cancer risk assessment methodology assumes additivity of carcinogenic effects in the regulation of mixtures. A rodent model of hereditary renal cancer was used to investigate the carcinogenic response to a mixture of drinking water disinfection by-products (DBPs). Rats carrying a mutation in the Tsc2 tumor suppressor gene (Eker rats) readily develop renal preneoplastic and neoplastic lesions, and are highly susceptible to the effects of renal carcinogens. Male and female Eker rats were exposed via drinking water to individual or a mixture of DBPs for 4 or 10 months. Potassium bromate, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), chloroform, and bromodichloromethane were administered at low concentrations of 0.02, 0.005, 0.4 and 0.07 g/l, respectively, and high concentrations of 0.4, 0.07, 1.8 and 0.7 g/l, respectively. Low and high dose mixture solutions were comprised of all four chemicals at either low concentrations or high concentrations, respectively, Following necropsy, each kidney was examined microscopically for preneoplastic lesions (atypical tubules and hyperplasias) and tumors. While some of the mixture responses observed in male rats did fall within the range expected for an additive response, especially at the high dose, predominantly antagonistic effects on renal lesions were observed in response to the low dose mixture in male rats and the high dose mixture in female rats. These data suggest that current default risk assessments assuming additivity may overstate the cancer risk associated with exposure to mixtures of DBPs at low concentrations.

The carcinogenic response of Tsc2 mutant Long-Evans (Eker) rats to a mixture of drinking water disinfection by-products was less than additive.

Cancer risk assessment methods for chemical mixtures in drinking water are not well defined. Current default risk assessments for chemical mixtures assume additivity of carcinogenic effects, but this may not represent the actual biological response. A rodent model of hereditary renal cancer (Eker rat) was used to evaluate the carcinogenicity of mixtures of water disinfection by-products (DBPs). Male and female Eker rats were treated with individual DBPs or a mixture of DBPs for 4 or 10 months. Potassium bromate, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone, chloroform, and bromodichloromethane were administered in drinking water at low concentrations of 0.02, 0.005, 0.4, and 0.07 g/l, respectively, and high concentrations of 0.4, 0.07, 1.8, and 0.7 g/l, respectively. Low and high dose mixture solutions comprised all four chemicals at either the low or the high concentrations, respectively. Body weights, water consumption, and chemical concentrations in the water were measured monthly. All tissues were examined macroscopically for masses and all masses were diagnosed microscopically. Total renal lesions (adenomas and carcinomas) were quantitated microscopically in male and female rats treated for 4 or 10 months. A dose response for renal tumors was present in most treatment groups after 4 or 10 months of treatment. Treatment with the mixture produced on average no more renal, splenic, or uterine tumors than the individual compound with the greatest effect. This study suggests that the default assumption of additivity may overestimate the carcinogenic effect of chemical mixtures in drinking water.

Use of the spontaneous Tsc2 knockout (Eker) rat model of hereditary renal cell carcinoma for the study of renal carcinogens.

The kidney is a frequent site for chemically induced cancers in rodents and among the 10 most frequent sites for cancer in human patients. Renal cell carcinoma (RCC) is the most frequent upper urinary tract cancer in humans and accounts for 80-85% of malignant renal tumors. Hereditary RCC occurs in Eker rats that are heterozygous for an insertion mutation in the Tsc2 tumor suppressor gene. The germline mutation renders heterozygous mutants highly susceptible to renal carcinogens. The utility of this model in studying potential renal carcinogens is due to an ordered progression of proliferative renal lesions that can be identified and counted microscopically. The quantitative nature of the model allows for the production of statistically powerful data to understand the relative degree and potency of chemical effects and allow analysis of genetic alterations that may be chemical specific.

Methods for measuring DNA adducts and abasic sites I: isolation, purification, and analysis of DNA adducts in intact DNA.

The major event involved in the formation of mutations and the initiation and progression of cancer is the induction of DNA damage by reactive intermediates arising from exposure to endogenous and exogenous chemicals. Many electrophilic metabolites of chemicals covalently bind to the bases of DNA causing specific DNA adducts. This unit includes protocols for preparing samples of intact DNA and adduct analysis to quantify the number of adducts that can potentially cause mutagenic or carcinogenic damage.