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

Abstract

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.

This article is a synopsis of the first two presentations from Session 2: Acute Kidney Injury: Toxicologic Pathologist’s Constant Companion from the 37th Annual Symposium of the Society of Toxicologic Pathology (STP) held in Indianapolis, Indiana, on June 18, 2018. The focus of this session was acute kidney injury (AKI), and the first two presentations provided a review of the fundamentals of renal tubule and interstitial anatomy and physiology, in addition to an overview of the International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) initiative.

Fundamentals of Renal Tubule and Interstitial Anatomy and Physiology

Dr. Kevin McDorman from Charles River Laboratory, Inc., gave a brief introductory presentation on the “Fundamentals of Renal Tubule and Interstitial Anatomy and Physiology.” The audience was reminded that the kidney is a complex organ, with multiple distinct anatomic components and a variety of physiologic functions and organism homeostasis that include, but are not limited to, the regulation of body water, electrolytes, and protein; maintenance of extracellular fluid volume; regulation of blood pressure; maintenance of acid–base balance; and excretion of waste products of metabolism, including elimination of foreign substances such as xenobiotics and their metabolites. The audience was reminded that the kidney is often exposed to higher concentrations of foreign substances than other organs/tissues, due to relatively high blood flow volume, in addition to its primary functions of filtration and waste product excretion.

Anatomically, the nephron is the microscopic functional unit of the kidney which is composed of numerous subunits that are of significance to toxicologic pathologists. These subunits include the glomerulus, proximal convoluted tubule (PCT), pars recta (PR), loop of Henle, thick ascending tubule, juxtaglomerular apparatus (JGA), and distal convoluted tubule (DCT; Young and Heath 2002); each subunit has region-specific roles that involve the exchange, transport, and diffusion of region-specific ions.

There are various immunohistochemical stains that can help identify/differentiate the kidney nephron segments in commonly used laboratory animal species (Bauchet et al. 2011). Those which were discussed include the following:

Aquaporin-1—PCTs and thin descending limbs of Henle’s loops in dogs, rats, mice, and nonhuman primates (NHPs);

Aquaporin-2—Connecting and collecting ducts in dogs, rats, mice, and NHPs;

Alpha-glutathione-S-transferase—PCTs and straight part of the PR in rats and dogs;

Tamm-Horsfall protein—Thick ascending limbs of Henle’s loops in mice, rats, dogs, and NHPs and stains scattered cells in cortical connecting/collecting ducts of dogs;

Calbindin-D28 K—DCTs and cortical connecting and collecting ducts of dogs, rats, and mice and DCTs; connecting ducts; and cortical and medullary collecting ducts of NHPs.

In addition, there are many useful markers that can assist in the diagnosis and differentiation of renal neoplasia: cytokeratins, vimentin, PAX2, PAX8, renal cell carcinoma (RCC) marker, CD10, E-cadherin, kidney-specific cadherin, parvalbumin, claudin-7, claudin-8, a-methylacyl coenzyme A racemase, CD117, transcription factor E3 (TFE3), thrombomodulin, uroplakin III, p63, CD57, and carbonic anhydrase IX (Shen et al. 2012).

The JGA, which is a collection of physiologically active cells located adjacent to each renal glomerulus, consists of a portion of the DCT referred to as the macula densa, segments of the afferent and efferent arterioles closest to that glomerulus, and the cells lying between these structures. The JGA is involved in the regulation of blood pressure via the secretion of renin, which is released either following (1) a decrease in blood pressure which is sensed by pressure receptors in the afferent arteriolar wall and/or (2) a decrease in sodium ion concentrations in the DCT (due to a decreased glomerular filtration rate [GFR]), which is sensed by the macula densa. Renin results in the conversion of angiotensinogen to angiotensin I, which ultimately gets converted to angiotensin II via angiotensin-converting enzyme secreted by the lungs. Angiotensin II is a potent vasoconstrictor, which results in increased blood pressure via direct constriction of peripheral blood vessels. It also induces release of aldosterone from the adrenal glands, which causes increased absorption of sodium ions, and ultimately water, in the DCT. Ultimately, plasma volume is increased, which further aids to increase blood pressure.

The interstitium includes the extravascular intertubular spaces or the renal parenchyma, with their attendant cellular elements and extracellular substances and fluid; it is bounded on all sides by tubular and vascular basement membranes. The interstitium harbors dendritic cells, macrophages, lymphocytes, lymphatic endothelial cells, and various types of fibroblasts, which produce a variety of local (autocoid) and systemic hormones. The interstitium is responsible for mediating and modulating almost all exchange among the tubular and vascular elements of the renal parenchyma and influencing glomerular filtration through its effects on tubuloglomerular feedback. It impacts growth and differentiation of parenchymal cells and determines the compliance of the peritubular microvasculature. It is important to remember that alterations in the interstitium contribute to the clinical manifestations of renal disease.

Dr. McDorman spent several minutes discussing three background findings commonly seen in toxicity models: inflammatory cell infiltrates in the interstitium of the cortex, hyaline casts, and tubular mineralization. Spontaneous lesions, normal physiologic variations, and concurrent natural diseases or pathologic changes can be commonly observed in kidneys from toxicology models. These can be influenced by age, genetics, the supplier, and husbandry practices. Distinguishing normal clinical and anatomic/morphologic variability from test article–induced changes is critical because the former is expected. Balanced and accurate interpretations require experience with the test system (animal model) and integration of all available data, including historical control data (HCD).

HCD can serve as an invaluable resource for any organization, but the relevance and usefulness of HCD is dependent on the quality and consistency of the histopathology, particularly with regard to thresholds, lumping versus splitting diagnoses, and usage of standardized terminology. The varying use of thresholds and/or threshold levels by pathologists can dilute HCD, differences between pathologists and/or studies with regard to lumping versus splitting diagnoses can confound HCD, and the use of standardized terminology will strengthen HCD. The use of different, even if synonymous, diagnostic terms for the same lesion makes accurate retrieval of HCD a challenge; therefore, ideally standardized terminology should be used whenever possible. However, numerous challenges exist, especially with regard to nonneoplastic lesions. The INHAND for lesions was created to address these issues.

Renal INHAND, Standard for Exchange of Nonclinical Data (SEND), and Drug-induced Kidney Injury (DIKI) Nomenclature Review

Dr. Torrie A. Crabbs (Experimental Pathology Laboratories, Inc.) gave a brief overview of the INHAND initiative, walked the audience through how to use the global open Registry Nomenclature Information System (goRENI) website (www.goreni.org) to access the most up-to-date INHAND terminology, and explained how/why SEND is applicable to toxicologic pathologists. In addition, she covered the diagnostic features of numerous renal INHAND terminologies (Frazier et al. 2012), which included the three most common renal tubular lesions associated with DIKI, in addition to several potentially confusing/controversial lesions.

For regulatory agencies to appropriately interpret and utilize histopathologic data, it is critical that nomenclature and diagnostic criteria for histologic findings be clear, consistent, and accurate. The harmonization of terminology has long been a goal for toxicologic pathologists. In the latter part of the 20th century, combined efforts between STP and the Registry of Industrial Toxicology Animal (RITA) data base group in Europe resulted in several internationally recognized publications: Standardized System of Nomenclature and Diagnostic Criteria: Guides for Toxicologic Pathology and the World Health Organization/International Agency for Research on Cancer International Classification of Rodent Tumors. INHAND, a collaborative effort between the STP, the European Society for Toxicologic Pathology (ESTP), and RITA was initiated in 2005. In 2006, the British Society of Toxicologic Pathology (BSTP) and Japanese Society of Toxicologic Pathology (JSTP) joined the effort, establishing a truly global initiative to harmonize diagnostic terminology. The primary goal of INHAND is to establish and publish internationally accepted standardized nomenclature and diagnostic criteria for proliferative and nonproliferative lesions in each organ system for laboratory animals commonly used in toxicity and carcinogenicity studies. The accepted nomenclature and diagnostic criteria is published both on goRENI (www.goreni.org) and in the official journals of the participating Societies: Toxicologic Pathology (STP, BSTP, and ESTP) and the Journal of Toxicologic Pathology (JSTP).

goRENI is a web-based platform where final INHAND nomenclature is published, draft INHAND nomenclature can be reviewed, and requests for new and/or modifications of existing INHAND nomenclature can be made. While access to goRENI is available to all members of the organizations participating in the INHAND effort, in addition to government regulators, individuals must apply and set up an account. This is easily done by going to www.goRENI.org, clicking on the membership tab along the top, filling out the required information, and clicking the submit data button. Once an account is set up, members can search for diagnoses via Organ Systems, Organs, or the Index; the introductions for each published organ system can also be accessed. For each diagnostic term, the following information is provided: biological behavior, rodent species in which the term applies (i.e., rat, mouse, or both), pathogenesis/cell of origin, diagnostic histologic features, differential diagnoses, and references. In addition, the following sections are present when applicable: sublocations, synonyms, modifiers, special techniques for diagnostics, and/or comments. The following biological behavior options are available: M—malignant tumor, B—benign tumor, H—preneoplastic lesion, and N—nonproliferative and nonpreneoplastic proliferative lesion. The synonym section lists terms previously used in literature that describe the same morphologic lesion and is provided for appropriate review of historical data. For risk assessment, it is important to interpret data based on current terminology; however, sometimes review of historical data is required. Names of lesions can evolve over time, despite the fact that their morphologic appearance has remained the same. Given that historical data could have been collected/published years or decades prior, familiarity with these changes in terminology is necessary. In addition to text, variable numbers of representative digital images for each histologic lesion are present along the right-hand side.

The Global Executive Steering Committee (GESC) oversees all activities of the INHAND project and is made up of representatives from each of the participating organizations, in addition to several technical consultants. The Organ Working Groups (OWGs) are the core of INHAND. They are composed of expert toxicologic pathologists that develop the preferred nomenclature and diagnostic criteria for each organ system. Once the initial draft nomenclature has been developed, the GESC completes an initial review. This is followed by a review period during which all members of participating organizations are requested to review the proposed nomenclature. The OWG then finalizes the nomenclature based upon the comments it receives from the GESC and the general membership. While currently there is only published nomenclature for rodents, there are OWGs for dogs, rabbits, mini-pigs, fish, nonhuman primates, apoptosis/necrosis, and nonrodent ocular.

While the individual organ system publications provide information on specific diagnostic entities and differential diagnoses, there are certain key principles and processes that are common across the various organ systems. Dr. Crabbs briefly reviewed several of these principles; more detailed information can be found in Mann et al. (2012). Some of the key principles are as follows: descriptive terminology is preferred over diagnostic terminology as it allows for tabulation and comparison of group effects; modifiers such as topography, distribution, character of change, and duration are not always necessary but provide additional information and clarity; combination terms and slash terms such as cardiomyopathy, nephropathy, and degeneration/regeneration are appropriate in certain circumstances as they can reduce the number of entries for common findings that involve a combination of morphologic diagnoses; establishing a universal system for severity grading will be difficult; therefore, consistency is key; severity grading system should reflect the extent of the process, distribution, and actual degree of severity; some processes such as autolysis, neoplasms, cysts, and congenital anomalies do not require a severity grade; and the use of thresholds creates a potential to miss exacerbation of background findings, while reading a study with no threshold can result in overly complex tables.

Both SEND and Study Data Tabulation Model (SDTM) are required standards for data submission to the Food and Drug Administration (FDA). SEND specifies a way to collect and present nonclinical data in a consistent format. It enables the implementation of SDTM, which is a standard for organizing and formatting data; it streamlines the collection, management, analysis, and reporting of data. Since the FDA has indicated INHAND terminology as its preference, SEND will predominately utilize INHAND terminology. Therefore, it is crucial that toxicologic pathologists be familiar and comfortable using INHAND terminology and be familiar with the goRENI website so they can access the most up to date terms and diagnostic criteria.

Understanding the appropriate use of diagnostic criteria in renal toxicologic pathology is particularly important when interpreting AKI. AKI has three primary causes: ischemia, hypoxia, and administration of nephrotoxic agents/contrast media. The underlying feature in all causes is a rapid decline in GFR, which ultimately results in a decrease in renal blood flow. If there is mild injury, an adaptive repair response initiates, which results in regeneration. With more severe injury, there is incomplete regeneration; nephron mass gets replaced by scar tissue, which can result in progression of chronic kidney disease.

In animals, AKI caused by the administration of nephrotoxic agents/contrast media is referred to as DIKI; in human literature, this disease is often referred to as drug-induced kidney disease (DIKD). DIKI/DIKD toxicity can result from direct injury, hemodynamic changes, and/or urinary obstruction and can affect the glomerulus, interstitium, and/or renal tubules. The renal tubules, however, are particularly sensitive because their role in concentrating/reabsorbing glomerular filtrate exposes them to high levels of circulating toxins. In addition, renal tubular cells have relatively high Cytochrome P450 levels, which can result in the formation of reactive metabolites.

Three of the most common renal tubular changes associated with DIKI are vacuolation, degeneration, and necrosis. Vacuolation is characterized by the intracellular accumulation of fluid, lipid, or other material, giving the cytoplasm a swollen, pale, or granular appearance. Discrete clear or translucent spaces of variable size are present. While vacuolation may precede degeneration and/or necrosis, it can also be seen in control animals. The term vacuolation is best reserved for situations when it is the primary or sole degenerative process present. One of the main differential diagnoses for vacuolation is glycogen accumulation, which has a slightly different morphologic appearance and pathogenesis. Glycogen accumulation is associated with hyperglycemia and, histologically, there is a clearing and/or frothy appearance to the cytoplasm. Tubular degeneration is characterized by one or more of the following features: vacuolation, tinctorial change, blebbing, cell sloughing, and repair/regeneration. When there is concurrent evidence of regenerative changes, some pathologists will use the “slash” term degeneration/regeneration. The main differential diagnosis for degeneration is necrosis, which is histologically characterized by one or more of the following features: cytoplasmic eosinophilia, nuclear pyknosis or karyorrhexis, cell sloughing, epithelial thinning/attenuation, and the presence of cellular casts and/or amorphous luminal debris. Inflammation and other degenerative processes, such as tubular dilation, epithelial vacuolation, and crystalluria may be variably present. With repeated injury, there may be evidence of regenerative processes. Chronic injury can result in loss of the basal lamina which can further result in tubular atrophy and/or interstitial fibrosis.

Renal tubular lesions can provide problems in recognizing and interpreting lesions due to the fact that toxic insult often results in a constellation of changes, such as vacuolation, degeneration, regeneration, necrosis, cellular proliferation, dilation, and casts. In addition, there are several morphologies that overlap between spontaneous findings and those induced by toxicants. The most complicated findings include differentiating between renal tubular basophilia, hyperplasia, regeneration, and chronic progressive nephropathy (CPN).

Renal tubular basophilia is characterized by slightly enlarged epithelial cells that have basophilic cytoplasm with otherwise normal profiles. Cells may exhibit an increased nuclear: cytoplasmic ratio and mitoses may be present. The main differential diagnoses are hyperplasia, regeneration, and CPN. With simple hyperplasia, the cytoplasm is basophilic, but the cells are crowded and increased in number; however, the cells do not extend into the lumen or beyond a single layer. Regenerative tubules also have basophilic cytoplasm, but the cells are often flat or low cuboidal with a rudimentary brush border. Increased numbers of mitoses are common. With CPN, tubular epithelium is basophilic, but there is often nuclear crowding and/or a thickened basement membrane. Hyaline casts are also common. Additional features of CPN include tubular atrophy, tubular dilation, focal glomerular sclerosis, and mononuclear cell infiltrates; interstitial fibrosis and epithelial hyperplasia of the renal pelvis can be seen in advanced cases. It is important to note that there are numerous histologic findings that can be increased secondary to CPN. These findings should not be recorded separately unless they are believed to represent a separate process. Some of these changes include basophilia, tubular dilation, casts, epithelial hypertrophy, inflammation, fibrosis, and epithelial hyperplasia of the renal pelvis.

In summary, basophilic cytoplasm is a morphologic feature of more than one lesion; it can be seen as a component of a spontaneous process (CPN), a degenerative process (toxic insult), or a reparative process (regeneration). Tubular basophilia should not be used interchangeably with regeneration, hyperplasia, or CPN. According to Seely and Frazier (2015), tubular basophilia is the preferred term when there is an absence of any evidence of regeneration, degeneration, or additional features of CPN. When basophilia is a component of a complex renal change, an overarching term, such as CPN, obstructive/retrograde nephropathy, or pyelonephritis, is preferred; in applicable cases where there is no standard/arching term, degeneration may be the preferred term. Degeneration is preferred when one or more of the following features is evident: tubular dilation, cell swelling, cytoplasmic vacuolation, nuclear pyknosis, and/or cell sloughing. Regeneration is preferred if one or more of the following is present: flattened cells, increased mitoses, nuclear crowding, cellular heterogeneity, and/or an increased nuclear: cytoplasmic ratio. With regard to CPN, advanced cases pose few issues; however, in young rats, tubular basophilia is occasionally the only feature present. In these cases, basophilia may be the appropriate diagnosis.

Traditional renal tubular adenomas are often basophilic and composed of well-differentiated cells with variable cellular and nuclear pleomorphism. Growth patterns can be solid, tubular, cystic, lobular, papillary, cystopapillary, or mixed. Mini-lumens may be present; however, areas of necrosis and hemorrhage are lacking. Basophilic tubular adenomas can occur spontaneously or be chemically induced, are primarily present in older rats, and are more common in males. An amphophilic vacuolar (AV) variant of renal tubule tumors exists that is often included diagnostically with these more traditional basophilic renal tubule tumors. AV tumors exhibit a distinct morphologic phenotype composed of uniform lobules of cells separated by a fibrovascular stroma. As the name implies, cytoplasm is amphophilic and vacuolated. AV tumors have been demonstrated to be spontaneous, nontreatment-related tumors that are likely of familial origin, have been recorded in rats as young as seven to ten weeks of age, and may occur more often in females (Crabbs et al. 2013). Currently, a distinct INHAND term for AV tumors does not exist; however, the renal INHAND OWG recommends separately recording these spontaneous tumors to prevent complications with data interpretation of potential test article–related tumors.

The last controversial finding that was discussed was urothelial hyperplasia. While the renal pelvis is lined by urothelium, it has been demonstrated that the epithelial lining of the papilla is not urothelium. A recent study by Souza et al. (2018) confirmed this finding by demonstrating a lack of uroplakin immunostaining. This study also demonstrated that in proliferative appearing lesions of the renal papilla of rats with CPN, there was a lack of nuclear staining for Ki-67 and that mitotic figures were lacking. Based on these results, the authors recommended that the epithelium lining the renal papilla should not be referred to as urothelium and that CPN-associated “proliferative” lesions of the renal papilla be designated as “vesicular alteration of the lining of the papilla” as opposed to “hyperplasia.” These recommendations are currently under consideration by the renal INHAND OWG.