Meeting Report

Abstract

Urinary system toxicity is a significant concern to pathologists in the hazard identification, drug and chemical safety evaluation, and diagnostic service industries worldwide. There are myriad known human and animal urinary system toxicants, and investigatory renal toxicology and pathology is continually evolving. The system-specific Research Triangle Park (RTP) Rodent Pathology Course biennially serves to update scientists on the latest research, laboratory techniques, and debates. The Sixth RTP Rodent Pathology Course, Urinary Pathology, featured experts from the government, pharmaceutical, academic, and diagnostic arenas sharing the state of the science in urinary pathology. Speakers presented on a wide range of topics including background lesions, treatment-related non-neoplastic and neoplastic lesions, transgenic rodent models of human disease, diagnostic imaging, biomarkers, and molecular analyses. These seminars were accompanied by case presentation sessions focused on usual and unusual lesions, grading schemes, and tumors.

Since 2002, the biennial Research Triangle Park (RTP) Rodent Pathology Course has sought to provide valuable information about organ system–specific current topics and techniques in rodent pathology and toxicology to pathologists, trainees, and other involved colleagues of the scientific community. This course uniquely bridges the rodent toxicologic pathology and the rodent phenotyping communities. The 2012 program featured experts, mostly from the RTP, presenting on various contemporary and emerging subjects regarding the pathology of the urinary system. The first 5 RTP Rodent Pathology Courses focused on reproductive pathology, neuropathology, cardiopulmonary pathology, immunopathology, and hepatobiliary pathology, respectively.

In keeping with tradition, the reputation of the speakers attracted a diverse audience from local institutes such as the University of North Carolina at Chapel Hill, Duke University, North Carolina State University, the National Toxicology Program (NTP), National Institute of Environmental Health Sciences (NIEHS), GlaxoSmithKline (GSK), Experimental Pathology Labs (EPL), Environmental Protection Agency (EPA), the Hamner Institutes of Health Sciences (previously CIIT), and constituents from other parts of the nation and world. Normal anatomy and histology of the urinary system were initially presented, followed by 16 presentations on topics including, but not limited to, renal transporters, neuroendocrine control of renal function, imaging techniques, biomarkers, carcinogenesis, translational pathology, and clinical applications. A case presentation intermission, coordinated by Dr Jerrold Ward (Global Vet Pathology), gave the participants and speakers an opportunity to opine on difficult cases and discuss popular renal pathology grading schemes.

Dr Susan Elmore (NTP/NIEHS) opened the conference with an overview of normal kidney structure and function. Dr Elmore’s lecture was an educational review from the level of the budding pathology resident to that of the experienced pathologist. Using diagrams and photomicrographs, she reviewed the kidney’s role in osmoregulation, waste excretion, hormonal and metabolic functions, and the subtopographical compartments of glomeruli, tubules, collecting system, vasculature, and interstitium, all of which should be examined independently as well as collectively. Particularly instructive were beautiful schematics differentiating segments P1, P2, and P3 (Fig. 1) and more detailed examination of the corticomedullary renal structure and segment characterization (Fig. 2). Dr Elmore reminded the audience to remain cognizant of sexual dimorphisms, such as that found in the Bowman’s capsular epithelium. An androstenedione-treated female mouse that recapitulated the cuboidal epithelial morphology found in the male was provided as an example. ²²

Figure 1.P1, P2, and P3 localization within the proximal convoluted tubule and pars recta (also known as S1, S2, and S3). Figure 2. Corticomedullary organization and tubular segment distribution within the nephron.

Dr Jeff Engelhardt (Experimental Pathology Laboratories) expanded on Dr Elmore’s introductory lecture with his sessions, collectively titled Comparative Toxicopathology of the Urinary System. The kidney is often a target of xenobiotics simply due to its receipt of approximately 20% of cardiac output, despite comprising a mere 0.5% to 1% of the body weight in rats. Rats, dogs, and nonhuman primates are generally apposite comparative species to humans. However, differences in renal pathology due to strains may be seen, such as the increased sensitivity of Fisher F344 rats to aminoglycoside toxicity when compared with age-matched Sprague-Dawley rats. ²⁶ Dr Engelhardt reviewed the structure and function of the nephron, emphasizing species variation. As natives to arid environments, hamsters and gerbils have comparatively long loops of Henle, which help with urine concentration and water conservation.

Familiarity with common background lesions in experimental species (eg, spontaneous glomerular lipid vacuolation in the Beagle dog; renal amyloidosis in rodents, the most common cause of death in the hamster) is essential for the toxicologic pathologist. Segment awareness, as thoroughly reviewed by Dr Elmore, is important when evaluating specific toxicities such as that observed with cisplatin in the S3 segment. ²⁴ Identification of these segments can be accomplished using various methods, including the periodic acid-Schiff (PAS) reaction for staining the prominent brush border of tubules within the S1 segment.

Dr Engelhardt discussed contributory aspects of chronic progressive nephropathy (CPN) in rats, such as its amelioration by diet modification and the uniquity of hypercholesterolemia due to decreased mevalonic acid feedback to the liver in the CPN-affected rat. ²³ Interestingly, it is known that tubular epithelial cells that have undergone epithelial to mesenchyme transition and migrated to the interstitium secrete cytokines that drive matrix deposition and consequent interstitial fibrosis. ²⁰ Of note is that most agents that exacerbate CPN do not necessarily cause nephrotoxicity in humans. However, certain components of CPN may be aggravated by chemical administration, such as increased intratubular mineralization in the case of selective estrogen receptor modulators.

Dr Joseph Polli (GlaxoSmithKline) discussed the importance of drug transporters in absorption, distribution, metabolism, and excretion (ADME). Drug transporters may act as effective barriers to drug exposure or, through their induction or inhibition, may lead to drug-drug interactions, as in the antiretroviral dolutegravir’s potential to inhibit organic cation transporter (OCT) activity of other drugs. ²⁵ Dr Polli presented an exposure paradigm using whole-body autoradiography (WBA). By identifying the distribution ratios of a compound (eg, liver: blood, brain: blood), it can be determined where the compound accumulates and, to some extent, potential transport mechanisms. Despite such tools, however, drug transporters present challenges to toxicologic pathologists. Drug exposure, as measured by WBA, does not necessarily reflect its route or toxicity target. Not only are numerous transporters involved in drug ADME, but they also have tissue-specific distribution patterns and sexual dimorphic expression patterns, and several transporters in rodents lack human orthologs altogether. ¹ It is therefore important for drug development to identify key drug transporters and their genetic variants, elucidate their role in the disposition of a compound, and establish robust transport assays. In presenting a renal toxicity case study for GSK1, Dr Polli illustrated that the renal organic anion (Oat) and cation (Oct) transporters, known to be responsible for uptake of drugs in the kidney, are not the primary drivers of the renal disposition of GSK1. This was elegantly demonstrated using the cimetidine assay, autoradiography, and Oct knockout mice, which suggested that active transport is likely not affecting GSK1’s disposition, and that some other cellular transport mechanism (ie, phagocytosis) may be at play. Multiple transporters can influence the disposition and resulting toxicity of a compound, underscoring the challenge of transporter identification for drug discovery and development programs.

Dr Peter Little (Experimental Pathology Laboratories) dis-cussed the renal neuroendocrine cross-chatter in blood pressure, fluid, and ion homeostasis. In describing the function and microanatomical location of the less known and infrequently addressed circumventricular organs (CVOs) of the brain, he demonstrated the interplay between juxtaglomerular secretion of renin, plasma osmolality detectors in the brain, and vascular baroreceptors reporting through the cerebral medullary centers. ¹¹ Dr Little highlighted the subfornical organ (SFO), which contains one of the highest concentrations of atrial natriuretic factor receptors as well as angiotensin II receptors in the brain. The SFO has long been implicated in the central control of body fluid homeostasis, blood pressure, and thirst regulation. Dr Little also described the supraoptic nucleus, which produces antidiuretic hormone for release into the posterior pituitary gland, causing vasodilation and subsequent blood pressure elevation. After functional descriptions and physiology reviews, Dr Little gave morphological and pathological correlates to these organs and systems. ²⁸ With light microscopy, secreted antidiuretic hormone is often visible as oval eosinophilic bodies (Herring bodies) coursing down dendrites. In addition, the audience was alerted to the fact that the CVO is devoid of a blood-brain barrier and, as Dr Little stated, an “open physiologic window” to the brain.

Dr Kennita Johnson (University of North Carolina, Chapel Hill) gave an overview of rodent imaging systems useful for renal analysis. Techniques discussed included those that provide anatomical and functional information (magnetic resonance imaging, micro–computed tomography, and ultrasound) and those that reveal functional information only (positron emission tomography and single photon emission computed tomography). Of particular interest was a cost-effective, quick, and noninvasive method for quantifying renal perfusion, including blood flow velocity and blood volume, using microbubble-based contrast media for ultrasound. With this technique, approximately 6μ bubbles of protein, polymer, or lipid encapsulating air or a gas are injected into the test subject. With an ultrasound pulse, these microbubbles are safely destroyed, and the rate at which they refill the ultrasound plane is measured as a function of perfusion. ²¹

The next presentation, by Dr Sam Cohen (University of Nebraska Medical Center), discussed the normal structure, toxicity, and carcinogenesis of the lower urinary tract. Dr Cohen shared comparative urothelial (no longer referred to as “transitional epithelium”) insight, including the varying thicknesses, lymphocyte populations, and keratohyaline granules of the primate urothelium. The presence of mucosal lymphocytes in rodents suggests that it previously suffered ulceration, as erosion alone will not elicit this response. Dr Cohen also suggested that rodent urothelial papillomatosis may be a sequel of diffuse ulceration and may resolve in 3 to 4 weeks upon cessation of the stimulus. Human urothelial tumor classification describes invasive urothelial neoplasia and noninvasive urothelial neoplasia, the latter further categorized into carcinoma in situ, papilloma, and papillary neoplasms. ⁵ Urinary bladder tumors may morphologically and molecularly be roughly divided into 2 variants: hyperplasia is often the result of deletions on both arms of chromosome 9, whereas dysplasia and carcinoma in situ result from TP53 alterations. Mutations in FGFR3, TSC1, and cyclin D have been associated with transformation of hyperplastic lesions to low-grade papillary urothelial tumors. ¹⁹ High-grade and invasive urothelial tumors, on the other hand, have been shown to harbor inactivating mutations of the Rb gene and deletions on chromosome 8p. Dr Cohen also discussed his research with 2 urothelial tumor–causing agents in rats: sodium saccharin and melamine. ^6,9 Dr Cohen concluded by sharing his recommended diagnostic techniques, which include in situ instillation of Bouin’s fixative, BrdU, Ki-67 or proliferating cell nuclear antigen staining, and scanning electron microscopy to detect subtle superficial urothelial changes.

Dr Greg Travlos, a clinical pathologist at the NTP and NIEHS, gave an overview on renal clinical pathology and biomarkable targets to evaluate the approximately 35 000 nephrons in the rat kidney. Dr Travlos discussed several urine collection systems and shared studies showing that β₂-microglobulin and γ-glutamyl transferase, among other analytes, were not stably stored refrigerated or frozen over long periods. For analytes not affected by refrigeration or freezing for short periods (eg, N-acetyl glucosaminidase), urine must be brought to room temperature to ensure dissolution of potential precipitates. Regarding biomarking glomerular filtration rate (GFR), cystatin C has been shown to be more sensitive than creatinine, as its levels are perturbed with approximately 20% loss of nephron function. ¹⁵ For segment-specific injury localization, μGST and RPA-1 are indicative of injury to the distal tubule and collecting duct, respectively, whereas the proximal tubule has many biomarkers, including albumin, β₂-microglobulin, α₁-microglobulin, RBP, glucose, Kim-1, and αGST. ¹³ For tubular epithelial injury localization, Dr Travlos indicated that isoforms of alkaline phosphatase, aspartate aminotransferase, and N-acetyl-β-D-glucosaminidase were his preferred analytes to evaluate injury to the brush-border, cytosolic, and lysosomal compartments, respectively. Dr Travlos concluded with an example demonstrating the critical importance of timed volume- and creatinine-normalized calculations by comparing raw and corrected data from control and propylene glycol monobutyl ether (PGMBE)–administered animals.

No rodent pathology conference on the urinary system would be complete without a discussion of spontaneous background lesions, including chronic progressive nephropathy (CPN). Dr Kendall Frazier (GlaxoSmithKline) provided an excellent review of the familiar lesion that is characterized by tubular basophilia, ensuing thickened basement membranes, nuclear crowding, and various other sequela. ¹⁰ Tubular hypertrophy and hyperplasia may both be seen along with CPN. However, tubular hypertrophy, unlike tubular hyperplasia, is not considered preneoplastic. Dr Frazier indicated that simple and atypical tubular hyperplasias occur spontaneously as well as with xenobiotic administration, although atypical tubular hyperplasia is a preneoplastic finding more commonly seen as test article related. Regarding α_2U nephropathy, he cautioned the audience that although a Mallory Heidenhain stain may differentiate α_2u-globulin from other material, α_2u-globulin is rarely irregular or angular. Thus, positively staining material with such morphology should be investigated further.

Dr Frazier also discussed conventional rodent renal tumor diagnoses. As malignant renal tumors of the kidney are rare in rats and mice, any increase in numbers in test article–administered animals is troublesome in a preclinical toxicity study. Typically, males tend to exhibit greater numbers of renal tumors in a carcinogenicity study, with the exception of the amphophilic-vacuolar (AV) tumor type. ¹² Females exhibit a higher incidence of AV tumors because of their longer average life expectancy; however, when corrected for days on study, these tumors generally also follow the paradigm of males being more affected.

Dr Frazier concluded his set of lectures by sharing his tiered approach to mechanistic assays in the urinary system. End points commonly used in toxicity studies (eg, histopathology, organ weights, routine clinical pathology) comprise the first tier. The second tier is composed of a combination of electron microscopy, immunohistochemistry, novel urinary system biomarkers, and WBA. Dr Frazier emphasized that the lack of effective routine clinical biomarkers for glomerular insult (other than proteinuria) makes special staining techniques and immunohistochemistry (IHC) particularly useful. For example, IHC antibodies to synaptopodin, nephrin, von Willebrand factor, and CD90/thy-1 identify podocytes, podocyte slit diaphragms, endothelial cells, and rat mesangial cells, respectively. Tier III assays include GFR and renal blood flow measurement, transporter studies such as those shared by Dr Polli, and imaging studies similar to those presented by Dr Johnson.

Dr Rachel Cianciolo (University of North Carolina, Chapel Hill) opened a set of lectures regarding animal models of human renal disease. She shared her evaluation of tubules and interstitium in a doxorubicin nephropathy model using transcriptomics and stereology. Stereologic evaluation of organs requires strict adherence to sectioning and analysis protocols to re-create a 3-dimensional evaluation from 2-dimensional tissue sections. ² Using her rat doxorubicin model to study tubulointerstitial renal disease, Dr Cianciolo discovered altered transcription pathways that led her to understand the interplay between proteinuria, tubular disease, and peritubular capillary compromise.

Dr Wei Qu (NTP) challenged the current dogma surrounding the carcinogenesis of metals. It has long been thought that although considered a potent human carcinogen, arsenic does not cause cancer in rodents. ⁸ Dr Qu’s lab has been modeling metal-induced carcinogenesis and has used in vitro and in vivo models to study arsenic, lead, nickel, and cadmium carcinogenesis. ²⁹ Using a developmental exposure paradigm consisting of fetal exposure in utero via maternal drinking water coupled with postweaning exposure to dimethylarsinic acid (a potent promoter), Dr Qu was able to demonstrate renal cell carcinomas in arsenic-exposed animals. ²⁹

The keynote address was given by the Chair of Pathology and Laboratory Medicine and Chief of Service at the University of North Carolina Hospitals Clinical Laboratories, Dr J. Charles Jennette. Dr Jennette enthralled participants and attendees alike with his elegant investigations into the pathogenesis of antineutrophil cytoplasmic antibody (ANCA) glomerulonephritis and vasculitis. ¹⁶ Dr Jennette described numerous experiments using a variety of mouse models from Rag2 mice immunized against myeloperoxidase (MPO) to mice deficient in critical complement components. ¹⁷ These experiments were used to posit the following critical criteria for this devastating human disease: anti-MPO IgG alone may cause disease, the Fcγ receptor is important in the pathogenesis, neutrophils and the alternative complement pathway are required for disease, cytokines may exacerbate ANCA, and T cells do not incite the acute inflammatory component.

Dr Jeffrey Everitt (GlaxoSmithKline) discussed the importance of studies of hereditary renal cancer for elucidating the signaling pathways associated with cancer development and opportunities for interventional therapies. He reviewed the current state of rodent models of renal carcinogenesis and the challenges inherent in the phenotyping of genetically modified animals. The talk stressed the importance of understanding genotype, spontaneous lesions, and genetic manipulations when considering phenotypic alterations in rodent models, as well as the need for a complete necropsy with a thorough macroscopic examination. Dr Everitt reviewed phenotyping issues using Eker rats that have alteration in Tsc2 as an example. ¹⁸ He reviewed how the Tsc2 product tuberin, an mTOR-dependent protein, is involved in signaling pathways implicated in renal cell carcinoma. ¹⁴

Dr Susan Gurley (Duke University) continued on the topic of mouse models of human disease, specifically diabetic nephropathy (DN). For a DN model, criteria including specific declines in GFR, magnitudes of increase in albuminuria, and characteristic histopathologic lesions within glomeruli must be present. ³ The familiar streptozotocin (STZ)–induced model was compared with the Akita mouse, the latter of which harbors a mutation leading to an amino acid substitution in mature insulin. ⁴ In addition to lacking the need for toxin administration, the Akita model developed more pronounced hyperglycemia and albuminuria than did the STZ-treated mice on the same background. In the human DN population, inhibition of the renin-angiotensin-aldosterone system prolongs renal function, which Dr Gurley modeled by adding a renin transgene to the mouse.

Dr Jim Swenberg (University of North Carolina, Chapel Hill) closed the Sixth RTP Rodent Pathology Conference with a historical perspective on α_2u-globulin induction and nephropathy. Dr Swenberg shared years of research, spanning from trimethylpentane to d-limonene. ⁷ There are many tenets of α_2u-globulin in comparative pathology: humans do not produce it, and the globulins in the human α_2u family do not bind chemicals that induce hyaline droplets in rodents, and this syndrome in male rats should not indicate a risk of hyaline droplet nephropathy or renal carcinogenesis. However, to definitively determine a chemical is solely a male rat hyaline droplet nephropathogen, several criteria must be met: the chemical must be neither directly or indirectly genotoxic, the effect must be limited to the male rat, and protein droplets must be identified in short-term studies and be positively identified as α_2u-globulin. ²⁷

Sixth RTP Rodent Pathology Course Committee

• Richard A. Peterson II, Program Co-Chair; GlaxoSmith-Kline

• Amera K. Remick, Program Co-Chair; WIL Research

• Molly H. Boyle, Integrated Laboratory Systems

• Rachel Cianciolo, University of North Carolina, Chapel Hill

• Samuel M. Cohen, University of Nebraska Medical Center

• Torrie A. Crabbs, Experimental Pathology Laboratories

• John M. Cullen, North Carolina State University College of Veterinary Medicine

• Susan A. Elmore, National Institute of Environmental Health Sciences/National Toxicology Program

• Jeff Engelhardt, Experimental Pathology Laboratories

• Jeffrey Everitt, GlaxoSmithKline

• Kendall Frazier, GlaxoSmithKline

• Virginia Godfrey, University of North Carolina, Chapel Hill

• Susan B. Gurley, Duke University Medical Center

• Georgette Hill, Integrated Laboratory Systems

• Mark J. Hoenerhoff, National Institute of Environmental Health Sciences/National Toxicology Program

• J. Charles Jennette, University of North Carolina, Chapel Hill

• Kennita Johnson, University of North Carolina, Chapel Hill

• Peter B. Little, Experimental Pathology Laboratories

• Andrew Marias, Experimental Pathology Laboratories

• Rodney A. Miller, Experimental Pathology Laboratories

• Joseph W. Polli, GlaxoSmithKline

• Wei Qu, National Institute of Environmental Health Sciences/National Toxicology Program

• Arlin Rogers, University of North Carolina, Chapel Hill

• Brian Short, Allergan

• James A. Swenberg, University of North Carolina, Chapel Hill

• Gregory S. Travlos, National Institute of Environmental Health Sciences/National Toxicology Program

• Jerrold M. Ward, Global Vet Pathology

• Gabrielle Willson, Experimental Pathology Laboratories