International Journal of Toxicology

1-13
Kidney Pathophysiology, Toxicology, and ª The Author(s) 2019
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Drug-Induced Injury in Drug Development DOI: 10.1177/1091581819831701
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Zaher A. Radi1

Abstract
Anatomically, the kidneys are paired, bean-shaped (in most mammals), excretory organs that lie in the retroperitoneum. High
blood flow to the kidneys, together with high oxygen consumption, makes them more vulnerable to exposure, via the circulation,
and subsequent injury related to high concentrations of xenobiotics and chemicals. In preclinical drug development and safety
assessment of new investigational drugs, changes in kidney structure and/or function following drug administration in experi-
mental laboratory animals need to be put in context with interspecies differences in kidney functional anatomy, physiology,
spontaneous pathologies, and toxicopathological responses to injury. In addition, translation to human relevance to avoid pre-
mature drug termination from development is vital. Thus, detection and characterization of kidney toxicity in preclinical species
and human relevance will depend on the preclinical safety testing strategy and collective weight-of-evidence approach including
new investigational drug mechanism of action (MOA), preclinical and clinical interspecies differences, and MOA relevance to
humans. This review describes kidney macroscopic and microscopic functional anatomy, physiology, pathophysiology, toxicology,
and drug-induced kidney toxicities in safety risk assessment and drug development.

Keywords
kidney, pathophysiology, toxicology, drug-induced injury, drug development

Introduction emerging technologies bridging the preclinical to clinical gap.

Understanding the science of kidney injury with respect to
functional anatomy, pathophysiology, mechanisms and bio-
markers of nephrotoxicity, and alternative models that allow
human translation of preclinical data is very critical for phar-
maceutical drug development, chemical hazard risk assess-
ment, and consumer health industries. In this review, the
basic anatomy of the normal kidney tubular, interstitial, vascu-
lar, and glomerular structures and their function are discussed.
In addition, the basic general pathological terms applicable to
kidney commonly used in toxicologic pathology are presented.
Examples of toxicologically relevant interspecies physiologic
and developmental differences are discussed. In addition, some
examples of common drug-induced tubular, interstitial, vascu-
lar, and glomerular pathologies and toxicities encountered in
drug development along with preclinical to clinical translation
and human relevance of some of these toxicities are presented.
This review is based on a symposium entitled “The Brilliant
Kidney in Drug Development: From Basics to Chips” at the
Annual Meeting of the American College of Toxicology (West
Palm Beach, Florida, 2018). The objective of this symposium
was to provide current state, innovation, and anticipated reg-
ulatory changes as it relates to kidney efficacy and safety risk
assessment. The symposium covered basic, yet relevant kidney
general physiology and drug-induced pathologies and case
examples to illustrate recent advances in biomarkers and
Specifically, 4 talks highlighted: (1) basic kidney histologic
anatomy, physiology, and toxicologic pathology; (2) mechan-
isms of drug-induced kidney toxicity and their interpretations;
(3) translational biomarkers for early detection of kidney
injury; and (4) application of kidney-on-a-chip microphysiolo-
gical system for kidney safety and regulatory assessment.
Kidney Macroscopic and
Microscopic Anatomy
Kidney Macroscopic Anatomy
The renal system is comprised of 2 kidneys, 2 ureters, a urinary
bladder, and a urethra. The kidneys are paired, bean-shaped (in
most mammals), excretory organs that lie in the retroperito-
neum and are situated in the posterior part of the abdomen
on each side of the vertebral column. The right kidney is lower
than the left kidney. Kidneys weight varies with body surface
1
Pfizer Worldwide Research and Development, Drug Safety R&D, Cambridge,
MA, USA
Corresponding Author:
Zaher A. Radi, DVM, MBA, PhD, DABT, DACVP, Pfizer Worldwide Research,
Development, and Medical, Drug Safety Research Development, One Portland
Street, Cambridge, MA 02139, USA.
Email: zaher.radi@Pfizer.com
2 International Journal of Toxicology XX(X)

area, age, and sex, and both kidneys weigh approximately

0.51% to 1.08% (a mean of 0.65%) of the body weight.1,2
Despite this low weight relative to body weight, the kidneys
receive 25% of the cardiac output, with 85% of renal blood
flow in the cortex, 14% in the outer medulla, and only 1% in the
inner medulla.1,2 Such significant kidney microanatomic dif-
ferences in perfusion and blood flow and oxygen consumption
makes the kidneys more vulnerable to exposure, via the circu-
lation, and subsequent injury related to high concentrations of
xenobiotics and chemicals.2 The main renal artery, which ori-
ginates from the abdominal aorta, branches between kidney
parenchymal lobes to form the interlobar arteries and these
arteries extend to the corticomedullary junction to form the
arcuate arteries (ie, these arteries arch between the cortex and
the medulla), which run parallel to the capsule. Interlobular
arteries give rise to afferent arterioles that give rise to glomer-
ular capillaries (ie, capillary loops). When these capillaries exit
the glomerulus, they exit as efferent arterioles that further
branch to form peritubular (ie, interstitial) capillaries. Efferent
arterioles extend into the medulla as vasa recta and supply the
outer and inner medullae. The vessels of the venous system run
parallel to the arterial vessels and form the interlobular vein,
arcuate vein, interlobar vein, and renal vein. The vasa recta and
peritubular capillaries drain into interlobular veins3; the veins
leave the kidneys as renal veins, which empty into the inferior
vena cava. Lymphatics run parallel only to the cortical
vasculature.3
Each kidney is surrounded by a fibrous capsule, which is
smooth and can be easily removed at autopsy in healthy kid-
neys. Surrounding the renal capsule is a thick layer of perineph-
ric fat to provide protection from trauma. Macroscopically on
cut surface at autopsy, the kidney parenchyma has 4 major
anatomical regions: (1) the superficial cortex (a pale outer
region) that contains renal corpuscles, (2) the deep medulla
(a darker inner region) with conical medullary pyramids, (3)
renal papilla, and (4) renal pelvis. The medulla is further sub-
divided into the outer medulla and the inner medulla. The outer
medulla is divided into the outer stripe of the outer medulla and
the inner stripe of the outer medulla (Figure 1). The outer stripe
contains parts of the proximal tubule (thick descending limbs
of Henle’s loop), straight parts of the distal tubule (thick
ascending limbs), and collecting ducts. The inner stripe com-
prises descending thin limbs, ascending thick limbs, and col-
lecting ducts. The inner medulla is established by thin limbs
(descending and ascending) and collecting ducts.4 In the lower
urinary tract, ureters transport urine from the kidneys to the
urinary bladder. The urinary bladder serves as a temporary
storage reservoir for urine before the urethra transports urine
out of the body. The urethra of females is short and is longer in
males. The structure and function of the lower urinary tract is
generally similar in all mammals.1,2
Kidney Microscopic Anatomy
The urine-producing functional and structural units of the kid-
ney that are present in the cortex and medulla are called
Figure 1. Microscopic anatomical regions of the rat kidney. Note
inner stripe of the outer medulla (ISOM), outer stripe of the outer
medulla (OSOM), very well-developed OSOM, and unipapillary, single
renal pyramid. Hematoxylin and eosin stain. Source: Reprinted from
Khan, Hard, and Radi (2012), ©2012.
nephrons. The nephron consists of glomeruli, renal tubules
(proximal tubules, loop of Henle, distal tubules, connecting
segment, collecting ducts), interstitium, and juxtaglomerular
apparatus (JGA). The initial plasma filtering portion of the
nephron is the glomerulus, which is contained within Bowman
capsule. Microscopically, the glomerulus is composed of capil-
lary network lined by a thin layer of fenestrated endothelial
cells, glomerular basement membrane (GBM), a central region
composed of mesangial cells and matrix, visceral (inner)
epithelial cells (podocytes), and parietal (outer) epithelial cells.
Afferent arterioles supply blood to the glomerulus, while effer-
ent arterioles exit the glomerulus. The glomerular filtration
barrier (size, shape, and charge selective) has 3 layers: the
fenestrated endothelium, a negatively charged GBM, and the
podocytes. During the filtration process through glomerular
capillaries, it is important not to overload such process as this
might lead to glomerular lesions and proteinuria such as the
case with glomerular calcification that might be seen after
administration of dibasic sodium phosphate solution in rats.5
The glomerulus opens into the proximal convoluted tubules,
and in mature male mice, the proximal convoluted tubulars can
extend into Bowman capsule.2 The JGA is located at the vas-
cular pole of the glomerulus. The components of the JGA
include the macula densa of the thick ascending limb, afferent
and efferent arterioles, granular cells of the afferent arteriole
that produce renin and angiotensin II (AII), and the extraglo-
merular mesangial cells. The JGA is important in tubuloglo-
merular feedback control of renin secretion produced by the
granular cells.2
Radi
Cortical proximal convoluted tubular epithelial cells are
cuboidal cells with well-developed, dense surface microvilli
(brush border).3 The first segment of the proximal tubule is
convoluted and is called pars convolute, and the subsequent
part that passes down toward the medulla to become the des-
cending limb of the loop of Henle is straight and is called pars
recta. Thus, the segments (S) of proximal tubules can be sub-
divided into the S1 and S2 (convoluted) and the S3 (pars recta
or straight) in experimental laboratory animals (rats, mice, and
monkeys).2 These segments have different energy and oxygen
requirements and can be distinguished using various markers
via immunohistochemical techniques. The first part of the
proximal convoluted tubules, S1 segment, is short, connects
with the glomerular filtration space, contains numerous mito-
chondria with highest oxidative metabolic rate, and its brush
borders and phagosomal system are well developed as this
segment is involved in reabsorption and degradation of macro-
molecules. The S2 segment represents most of the proximal
tubules, has a shorter brush border with fewer mitochondria
but has more lysosomal bodies, and is the primary site for
low-molecular-weight protein resorption. The S3 segment has
a tall brush border and peroxisomes and is a primary site for
cytochrome P450 metabolizing enzymes. This is important as
some drugs might cause tubular hypertrophy in the S3 segment
that is related to peroxisome proliferation. Mixed-function oxi-
dase activity occurs primarily in pars recta in the rat. The
cortical distal convoluted tubules have very few microvilli and
connect the nephron with the collecting duct. In rodents, the
distal convoluted tubules contain Tamm-Horsfall protein, a
mucoprotein.2
The spaces among various kidney microscopic anatomic
structures (the intertubular, extraglomerular, extravascular
spaces) are defined as the interstitium. These spaces in the
cortex and medulla (ie, cortical and medullary interstitium)
contain different cellular constituents, extracellular matrix, and
interstitial fluid. The interstitial space accounts for approxi-
mately 7% to 8% of the total parenchymal volume in the cortex
and is thicker and accounts for approximately 29% to 40% in
the inner medulla. The cells in the interstitium include dendritic
cells (DCs), macrophages, lymphocytes, lymphatic endothelial
cells, and fibroblasts. The cells in the interstitium of the papilla
include stellate cells, monocytes, and pericytes. The proportion
of interstitium in the inner medulla increases with age and
ischemia. The papillary interstitium is rich in mucopolysac-
charides matrix.2,6
The inner stripe of the outer zone of the medulla consists of
Henle loops, thick ascending tubules (thick ascending seg-
ment or straight portion of the distal tubule), and collecting
ducts.2 The inner zone of the medulla consists of Henle loops
and collecting ducts. The thick ascending limb of the loop of
Henle (TAL) is one of the 3 structural segments of the distal
tubules. The TAL is located in the outer zone of the medulla
and in the cortex. Cells lining the TAL are cuboidal with
eosinophilic cytoplasm and round central nucleus. Ultrastruc-
turally, cells of the TAL have prominent mitochondria, rough
endoplasmic reticulum, and Tamm-Horsfall protein, which
3
cover the luminal surface of the cells lining the TAL. The
collecting duct system (cortical collecting ducts, the outer
medullary collecting ducts, and the inner medullary collecting
ducts) extends from the connecting segment in the cortex
through the outer and the inner medullae to the tip of the
papilla. The 2 cell types in this system are the intercalated
cell (a dark cell) and the principal cell with amphophilic or
clear cytoplasm. In the inner medulla, the intercalated cell
is absent. In the papilla, collecting ducts form the ducts of
Bellini, which empty into the pelvis.2
Kidney Interspecies Macroscopic and Microscopic
Anatomical Differences
The interspecies and sex differences in kidney macroscopic
and microscopic anatomy among experimental laboratory
animals used in preclinical safety testing versus humans
need to be considered when evaluating and interpreting test
article–related kidney changes and pathologies. For exam-
ple, the kidney medulla in humans is multipapillary (multi-
lobular) because it is divided into 8 to 18 conical masses
(pyramids; lobes). Similarly, minipigs have multilobular
kidney. Rat, mice, dogs, and cynomolgus monkeys have a
unipapillary, single renal pyramid (Figure 1; Table 1).2,7 In
addition, simple (ie, has vascular bundles consisting only of
descending and ascending vasa recta) renal medulla anato-
mical structure is present in most mammals, including
humans. However, a complex (ie, descending thin limbs
of short loops that are integrated into vascular bundles)
renal medulla is present in mammals (eg, rat and mouse)
with a high urine concentrating ability with thickest renal
medulla 2,4 (Table 1). Therefore, while humans have an
osmolality approximately up to 300 mOsm/kg, rats and mice
osmolality can be up to 4,300 mOsm/kg and this would, for
example, affect drug-related kidney crystalluria formation, a
finding in rodents that is not relevant to humans. Mice have
long loops of Henle and associated vasa recta in the
medulla. In rats, the outer stripe is very well developed and
makes up about one-third of the outer medulla (Figure 1)7,
very thin in humans and nonhuman primates, and is almost
completely absent in dogs.2,4,7 In dogs, all nephrons are of
the long-looped variety, whereas in rats and mouse, long-
looped nephrons account for 28% of the total nephrons.2
Normal sexual dimorphism can be noted in both male and
female mice (eg, larger kidney corpuscles noted in males
than females). The epithelium lining Bowman capsule in the
kidney of male mice is cuboidal, while in females the
epithelium is simple squamous. This should not be confused
with drug-induced epithelial hyperplasia. Also, unlike other
species, mice excrete large amounts of protein in the urine
under normal physiologic conditions; their urine contains
taurine and lacks tryptophan; and they also excrete creati-
nine in urine. Adult rabbits normally have cloudy urine
because most of their dietary calcium is absorbed and they
excrete it via the kidneys.
4 International Journal of Toxicology XX(X)
Table 1. Kidney Comparative Anatomy and Physiology.
Glomerular filtration
Species Nephron number (000s) rate (GFR; mL/min/kg)
Human *300-1,100 1.78
Monkey *103-123 2.08
Dog *340-490 6.13
Rat *30 5.24
Mouse *10 14.0
Kidney Physiology
Kidney Glomerular Physiology
The primary functions of kidneys are (1) maintenance and
regulation of body’s fluid and electrolytes, (2) maintenance
of extracellular fluid volume, (3) endocrine such as elaboration
of hormones, (4) regulation of blood pH and pressure, (5)
excretion of the waste products of metabolism, and (6) meta-
bolic activities.7 Adequate blood flow and perfusion affects
normal kidney function and about 10% of oxygen consumption
occurs in the kidneys. Measurement of kidney function is deter-
mined by glomerular filtration rate (GFR) that varies across
species (Table 1) and pathologies in afferent and efferent arter-
ioles (eg, drug-induced vasoconstriction) can affect GFR. The
plasma is filtered via the glomerulus to produce glomerular
ultrafiltrate. Ultrafiltration movement across the glomerulus
blood capillary network barrier into the urinary Bowman cap-
sule space involves crossing 3 layers: fenestrated endothelium
of glomerular capillaries, GBM, and slit pores between the foot
processes of visceral epithelial cells of the Bowman capsule.
The fenestrated (ie, have pores) endothelium allows the pas-
sage of fluid, blood plasma solutes, and low-molecular-weight
proteins and prevents passage of blood cells and platelets. Fac-
tors that affect GBM glomerular filtration include molecular
size, shape, and charge. Only small-molecular-weight mole-
cules can pass through the glomerular filter and small mole-
cules (eg, amino acids, glucose, sodium, potassium, water) pass
through the glomerular ultrafiltrate. Podocytes provide struc-
tural support to the capillary loop with their actin cytoskeleton,
can express Toll-like receptor-4 (TLR4), and maintain GBM.
There are structural proteins (eg, nephrin, podocin, and P cad-
herin) involved in maintaining the structure of the foot pro-
cesses, and mutations in these proteins can lead to
proteinuria. Mesangial cells are phagocytic and may prevent
basement membrane accumulation of macromolecules that
escaped from the capillaries. The plasma ultrafiltrate passes
down the nephron tubule to form urine.2
Kidney Tubular Physiology
Physiologically, the proximal tubules account for 70% of all
reabsorptive activity. The glomerular ultrafiltrate undergoes
reabsorption process of sodium (65%), water (65%), potassium
(65%), calcium (90%), phosphate, bicarbonate, magnesium,
amino acids (99%), glucose (100%), vitamins, sulfate, and
Outer stripe of the
Medullary structure outer medulla Number of papillae
Simple Very thin Multiple
Simple Thin Single
Simple Almost absent Single
Complex Well developed Single
Simple Well developed Single
low-molecular-weight proteins in the proximal tubules. These
are returned to the circulation by the peritubular and vasa recta
capillaries. Sodium reabsorption by the proximal tubule is an
active process that is driven by Naþ/Kþ ATPase at the baso-
lateral cell surface membranes. Reabsorption of NaCl accounts
for most of the energy expenditure in the kidney. Obligatory
(ie, water is obligated to follow Naþ) water reabsorption is a
passive process. Approximately two-thirds of calcium reab-
sorption is passive in exchange for sodium. Approximately
two-thirds of phosphate is reabsorbed in the proximal tubules.
In the dog, 85% of bicarbonate reabsorption takes place in the
proximal tubules. Reabsorption and degradation of macromo-
lecules in the ultrafiltrate takes place in the pars convoluta of
the proximal tubule due to its well-developed endocytic-
lysosomal apparatus.2
Maintaining nearly constant blood plasma osmolality and
sodium concentration is essential for survival. The urine con-
centrating mechanism plays an essential role in regulating
water and sodium excretion. The descending and ascending
portions of the loop of Henle enable recovery of much of the
Naþ and water that were filtered by the glomerulus. The urine
concentrating and urine forming process in the kidney starts in
the descending loop of Henle and depends on the generation of
an interstitial osmotic gradient from the corticomedullary bor-
der down to the papilla tip. This osmotic gradient is generated
by a countercurrent (the descending and ascending loops fluid
movement in opposite directions) multiplication (solute pump-
ing leading to multiplication of solutes concentration in the
deep medulla) system within the descending and ascending
loops of Henle, and passive diffusion in the vasa recta, the
countercurrent exchanger. Cells in the descending thick loop
(DTL) have aquaporin (AQP) channel proteins (upper 40% of
each DTL expresses AQP1) to allow for the high permeability
and movement of water from the descending loop lumen into
the surrounding interstitium, leading to osmolality increases
from approximately 300 mOsmol/kg (isosmotic with blood)
in the cortex to about 1,200 mOsmol/kg (hypertonic solution)
in the papillary tip region, resulting in hypertonic
interstitium.3,8
There are important species differences in such urine osmol-
alities with toxicological implications and human risk assess-
ment if there are solubility issues with a test article. For
example, rats can concentrate to approximately 3,000 mOsm/kg
H2O and mice to approximately 4,300 mOsm/kg H2O. These
changes in urine osmolalities are accomplished by osmosis in
Radi
the descending limb and active epithelial cell transport in the
ascending limb and lead to the reabsorption of up to 20% of the
water and 10% of Naþ entering this segment of the nephron.
Solutes and water recovered from these loops are returned to the
circulation via the vasa recta. The ascending loop of Henle lacks
AQP proteins and is impermeable to water but is permeable to
sodium and other solutes which are actively pumped out of the
loop via the Naþ/Kþ ATPase pump. This leads to the hyper-
osmotic environment in the kidney medulla. The interstitium is
rich in osmotically active urea because of reabsorption by the
collecting ducts.8
The distal convoluted tubule plays a critical role in sodium,
potassium, and cation homeostasis. It reabsorbs approximately
5% to 10% of the filtered sodium, 7% to 10% of filtered cal-
cium, 10% of filtered magnesium, and 10% to 15% of water.
Water reabsorption in the distal tubules is under the influence
of antidiuretic hormone (ADH; vasopressin). In addition, this
distal part of the nephron is aldosterone sensitive, a hormone
that responds to volume depletion or hyperkalemia.9 Aldoster-
one increases the amount of Naþ/Kþ ATPase in the basal mem-
brane of distal tubules and Na/Cl reabsorption. Distal tubules
have receptors for parathyroid hormone to facilitate Caþþ
recovery. Hydrogen ions, creatinine, and some drugs such as
penicillin are actively secreted and move from the blood into
the distal tubules.
The collecting ducts regulate urine volume and osmolarity
and contain 2 distinct cell types: principal cells and interca-
lated cells. Principal cells help maintain body water and salt
balance. Plasma osmolarity is regulated by the posterior pitui-
tary hormone ADH. Aldosterone is secreted by the adrenal
cortex in response to AII, a vasoconstrictor, stimulation. Prin-
cipal cells have receptors for ADH and aldosterone to regulate
water and Naþ recovery. In fact, the principal cell plays a
central role in salt and water transport with transporters such
as the epithelial Naþ channel (ENaC), the renal outer medul-
lary Kþ channel, and the AQP2 water channel.10 Control of
plasma Naþ and Kþ concentrations, extracellular fluid vol-
ume, and blood pressure is achieved via the coordinated reg-
ulation of ENaC by aldosterone and AQP2 by arginine
vasopressin in principal cells.10 Intercalated cells secrete or
absorb acid or bicarbonate and play an important role in reg-
ulating blood pH. Intercalated cells reabsorb Kþ and HCO3
and secrete Hþ. This function lowers the acidity of the plasma
while increasing the acidity of the urine.
Kidney Endocrine Functions
The kidneys can be hormonally sensitive as they have high
levels of sex hormone receptor expression. Kidney endocrine
function involves secreting 3 key hormones: erythropoietin,
calcitriol (1,25-dihydroxycholecalciferol), and renin. In addi-
tion, kidneys synthesize prostaglandins, growth factors, and
arachidonate metabolites.2,6,11 Erythropoietin is produced by
renal cortical peritubular interstitial cells in response to
hypoxic conditions. This stimulates erythropoiesis and the pro-
duction of red blood cells in bone marrow. Hypoxia-inducible
5
factor (HIF) is a key regulator of adaptive responses in renal
hypoxic conditions to regulate renal oxygen homeostasis, espe-
cially in ischemia–reperfusion injury of acute kidney injury
(AKI).12 Renin is produced within juxtaglomerular (JG) cells
after processing and cleavage of circulating prorenin, which is
produced in the liver. Reduced renal perfusion pressure and
hyponatremia are the primary stimuli for renin release from
JG cells that act as sensors of blood pressure. Renin is released
by the sympathetic nervous system and AII, ADH, endothelin,
and prostaglandins. Renin activates AII, which causes secretion
of aldosterone by the adrenal cortical zona glomerulosa. The
net effect is systemic vasoconstriction, intrarenal vasoconstric-
tion, and increased aldosterone release. Thus, due to the
reduced negative feedback on renin production by JG cells,
angiotensin-converting enzyme (ACE) inhibitors can cause
hypertrophy and hyperplasia of JG cells. In addition, the kidney
is a site of degradation of hormones, such as insulin and aldos-
terone, and is also responsible for the production of the active
form of vitamin D derived from vitamin D3 under the influence
of parathyroid hormone.2 Kidney interstitial cells such as stel-
late cells, in conjunction with pericytes, act as sensor and have
endocrine effector functions for the control of blood pressure,
salt balance, and oxygen delivery.13
Kidney Metabolic Physiology
The kidney is the primary route of xenobiotic excretion and
contains xenobiotic biotransformation enzymes. This is
because: (1) xenobiotics present in the glomerular ultrafiltrate
are concentrated and present at high concentrations, (2) the
cortex receives about 80% of total renal blood flow and thus
is exposed to the high concentration of xenobiotics coming
from the bloodstream, and (3) such high concentrations of
xenobiotics may accumulate in proximal tubulars following
reabsorption or secretion processes. Xenobiotic-metabolizing
enzymes activities such as mixed-function oxidases are present
in the proximal tubules and the activity varies along various
segments of the nephron.14 Mixed-function oxidase activity
occurs primarily in pars recta region of the proximal tubules
in the rat. The kidney plays an important role in the hydroxyla-
tion and glucuronidation of fatty acids, arachidonic acid (AA),
and 25-hydroxyvitamin D3.15 Epoxidation of AA is catalyzed
by the cytochrome P4502C (CYP2C) and CYP4F subfamilies
and these enzymes play an important role in the renal regula-
tion of salt and water balance and in inflammation. The highest
activities of cytochromes P450 are present in the proximal
tubules and involved in conjugation of glutathione, glucuronic
acid, and sulfate.16 The interspecies and sex differences in
kidney physiology among experimental laboratory animals in
preclinical safety testing versus humans need to be considered
when evaluating and interpreting test article–related kidney
changes and pathologies. S1, S2, and S3 tubular toxicological
manifestation vary due to differences in kidney metabolic
activities, and there is little to no induction of cytochrome
P450s following administration of phenobarbital due to the
absence of the CYP2B subfamily. In human kidney, CYP2D6
6 International Journal of Toxicology XX(X)
and CYP2E1 are absent. The activity of aldehyde oxidase in
humans is higher than that in rats and dogs. Mitochondria in the
proximal tubules are the site of production of circulating 1a,25-
dihydroxyvitamin D3.15 Because the blood flow in the renal
medulla is much lower than the cortex, the levels of xenobiotic-
metabolizing enzymes are lower, and this region have more
anaerobic metabolism. Some isoenzymes of P450 and glu-
tathione S-transferases are localized in the thick ascending limb
and distal tubules, while prostaglandin H-synthase is localized
in the collecting ducts in the medulla.17 Renal enzyme activi-
ties can be different between sexes and cytochrome P450 activ-
ity in rodents is often polymorphic by sex and sex hormones
(eg, drug-induced acetaminophen toxicity) might influence
proximal tubules metabolizing enzyme activity. For example,
male mice have more P450 activity than female mice. Induc-
tion of kidney metabolizing enzymes activities can lead to
increases in kidney absolute weights.2
In addition, kidney transporters are important consideration
because interference with transporters by a drug candidate (eg,
inhibition of tubular secretion of creatinine without affecting
glomerular function or causing any microscopic changes in the
kidney; drug is a substrate for a transporter; transporter affect-
ing drug disposition) might allow for mechanistic understand-
ing of kidney safety signal risk assessment. For example, sex
differences in transporter expression were observed with high-
est in the mouse followed by rat and dog. Organic anion trans-
porter 2 was higher in male mouse than the female. Organic
cation transporter 2 was higher in male rat and mouse. Multi-
drug resistance protein 4 is predominantly expressed in male
mouse kidney.18
Kidney Pathophysiology and Toxicology
Kidney Pathophysiologic Responses
The kidney is a common target organ of toxicity and injury as a
result of its exposure to drugs, xenobiotics, or chemicals. This
is because: (1) the high blood flow rate as they receive more
blood flow per gram of tissue (approximately 25% of cardiac
output) than any other organ, (2) high doses of test articles
typically administered in preclinical toxicity and safety studies,
(3) kidney’s key role in excretion through urine, (4) high renal
metabolic biotransformation and high oxygen consumption of
renal epithelium, (5) high transporter activity of the renal
epithelium, and (6) kidney’s role in reabsorption and its ability
to concentrate fluids.2,7 The specific pattern of kidney toxicity
and injury is dependent on the physiochemical properties of the
drugs (eg, low intrinsic solubility) and their dose, xenobiotics,
or chemicals, toxicokinetic properties, renal clearance profile
and metabolic attributes, and local kidney tissue concentration
and length of time of exposure. In addition, the kidney par-
enchyma is especially vulnerable to the pro-inflammatory
response to infection and subsequent kidney tissue damage.
This is because damage-associated molecular patterns
(DAMPs), pathogen-associated molecular patterns (PAMPs),
and pro-inflammatory mediators gain access to kidney
parenchyma and its various compartments via glomerular fil-
tration or peritubular capillaries microcirculation.12,19,20 For
example, lipopolysaccharides (LPS) are filtered into the tubular
fluid and can directly interact with renal tubular epithelial cells
(RTECs) via a TLR4-dependent mechanism.19,21 As a conse-
quence, various compartments of the kidney, including RTECs,
glomerular, and endothelial, are injured. This is because the
filtered PAMPs and DAMPs, also called “danger signals,” bind
to and activate RTECs, renal endothelial, and glomerular cells
and can lead to endothelial cell activation and structural
changes in the glomerular and peritubular capillaries.20 There-
fore, exogenous and endogenous PAMPs and DAMPs are inte-
gral to AKI pathogenesis.12
Clinical and Anatomic Pathology in Kidney
Safety Assessment
Assessment for test article–related kidney pathology and toxi-
city can include standard clinical pathology parameters such as
clinical chemistries for blood urea nitrogen (BUN) and serum
creatinine (sCr), along with disturbances in electrolytes and
water balance, urine excretion of protein (eg, proteinuria, urine
protein to creatinine ratio, and microalbuminuria in nonrodents
with glomerular pathologies) and electrolytes, urinalysis (urine
volume [eg, oliguria in AKI], appearance, specific gravity, pH,
quantitation for microalbumin, and absolute and relative kid-
ney weight determination), and macroscopic and microscopic
examination of the kidney tissue.7,12 Microscopic examination
of urine sediment for presence of RBC, glucose, ketones, bilir-
ubin, and crystals can be used to assess for drug-induced
changes such as crystalluria and it is important to consider
species differences in kidney drug handling. For example,
drug-induced crystalluria can be noted in rats without histo-
pathological evidence of kidney injury. In addition, urinary
bladder tumors if noted in rodents due to crystalluria mechan-
ism of action (MOA) are not relevant to human cancer risk, and
rodent urothelial proliferative lesions secondary to crystalluria
are not predictive of human cancer risk. Furthermore, rats are
sensitive to the development of irritation-related urothelial
tumors as a result of crystalluria, while mice are much less
likely to develop urothelial tumors secondary to crystals or
uroliths. Drug-induced kidney glomerular pathological
changes can manifest clinically as nephrotic syndrome due to
albumin loss (ie, plasma proteins leaks into the urine [protei-
nuria] through abnormally permeable GBM). Such reduction in
plasma osmotic pressure leads to edema, pleural effusions,
reduced intravascular volume, renal hypoperfusion, and/or sec-
ondary hyperaldosteronism. Drug-induced kidney glomerular
pathological changes can also lead to nephritic syndrome,
which usually presents with hematuria, red cell casts in the
urine, azotemia, and/or oliguria. Toxicologists need to be
familiar with this language to appropriately communicate pre-
clinical kidney findings with nephrologists and clinicians.
For urine biomarkers, it is essential to collect urine speci-
mens over a 16- to 24-hour period, ideally in metabolism cages
on cold packs. For investigative toxicological assessment of
Radi 7

Table 2. Kidney Spontaneous/Incidental Pathologies in Experimental Laboratory Animals.

Species Glomerular Tubular Interstitial Vascular

Monkey Fatty metaplasia, immature glomerulus, Mineralization Cell infiltrates Arteritis, polyarteritis
sclerosis nodosa
Minipig Glomerulonephritis, hemorrhage, and Hyaline casts, acute tubular hemorrhage, Cell infiltrates Arteritis and vasculitis
sclerosis hyaline droplets
Dog Focal/segmental lipidosis/foam cells, focal Mineralization, hyaline casts, vacuolation Cell infiltrates Idiopathic necrotizing
glomerulitis, immature glomerulus polyarteritis
Rat Focal/segmental glomerular sclerosis Mineralization, hyaline casts, dilatation, Lipomatous Mineralization,
hydronephrosis and cysts (juvenile rats) metaplasia polyarteritis nodosa
Mouse Amyloidosis Hyaline casts, dilatation, vacuolation Lipomatous Polyarteritis
metaplasia

rodent crystalluria, it is important not to fast the animals before
urine collection. Historical control data from the testing facility
might be used to help distinguish test article-related effects
from background changes and in assessing individual animal
variability. This is especially important in rats and mice
because renal function tests may become more variable due
to development of spontaneous age-related, species-specific
kidney pathologies with no human counterpart such as chronic
progressive nephropathy (CPN) as early as 12 weeks of age.2
Rats with CPN may show increases in kidney weights;
increased urine excretion of protein and calcium; decreased
urinary excretion of sodium and chloride; decreased creatinine
clearance; increased serum concentration of BUN, sCr,
sodium, and chloride; and decreased serum concentration of
albumin. Castration can result in a lower incidence and severity
of CPN. The physiologic status of experimental laboratory ani-
mals, diet (eg, imbalance of calcium/phosphorus leading to
mineralization in rats), and various physiologic parameters
such as age, sex, and interspecies differences in kidney anat-
omy and physiology are important when assessing kidney
responses to injurious test articles and/or chemicals.2,7
Kidney Spontaneous Pathologies
Histopathology is recognized as the single most appropriate
screen for evidence of kidney injury. Familiarity with sponta-
neous diseases occurring in laboratory animals is essential for
the establishment of the nature and cause of morbidity or mor-
tality in an individual animal. For example, rats, especially
Sprague Dawley strain, have vault-like folds called fornices
in the upper portion of kidney pelvis that extend into the renal
medulla and as a result can develop age-related and species-
specific spontaneous, random (ie, no dose–response relation-
ship), mineralization, inflammation, and/or hyperplasia in
males and/or females in this unique anatomic location.
Mechanisms for the development of urothelial proliferative
lesions in rodents (ie, genotoxicity, mitogenicity, cytotoxicity)
need to be ruled out in the risk assessment paradigm. Most
common spontaneous/incidental light microscopic changes in
control laboratory animals include CPN in rodents, glomerular
amyloidosis in CD-1 mice, glomerular lipidosis/foam cells in
beagle dogs with no effects on glomerular function, immature
glomeruli in the cortex of dogs2 and monkeys, corticomedul-
lary mineralization or nephrocalcinosis in female rats, kidney
pelvis dilatation in rats, kidney pelvis minimal epithelial hyper-
plasia and mineralization in rats, increased hyaline droplet
accumulation in rats, tubular epithelial vacuolation in mice,
mineral deposits, and interstitial mononuclear cell infiltrates
in multiple species (Table 2). Mineral deposits, for example,
occur at the corticomedullary junction in female rats due to
shedding of microvilli and microvesicles from S1 proximal
tubules22 and accumulation in the outer stripe of medulla.7 In
beagle dogs, such deposits can be noted in papillary collecting
duct region and in the interstitium of renal papilla in cynomol-
gus monkeys. The CPN is a spontaneous disease of laboratory
rats and mice that represents a significant confounder for inter-
pretation of kidney histopathology in toxicology studies after
test article administration. Male gender is a primary risk factor
for developing CPN. The CPN is an age-related disease in rats
and mice influenced by numerous extrinsic factors such as diet
and chemical exposures.2 Dietary restriction prevented or
delayed the development of glomerulosclerosis, tubulointersti-
tial damage, functional changes, morbidity, and mortality asso-
ciated with CPN in rats by controlling initial body and kidney
growth, glomerular size, and nephron hypertrophy.2,23 Micro-
scopically, there is tubular atrophy, interstitial fibrosis, tubular
dilation, casts, hyperplasia, dilated Bowman space, glomerulo-
sclerosis, glomerular atrophy, casts, and interstitial inflamma-
tory infiltrates.2,7
Mature male rats spontaneously develop prominent hyaline
droplets in the cytoplasm of the S2 segment of proximal
tubules. Ultrastructurally, these hyaline bodies consist of
alpha2m globulin and represent glomerular filtration and
resorption and catabolism of low-molecular-weight proteins
(eg, albumin and immunoglobulins) in secondary lysosomes.
Alpha2m-globulin is androgen regulated and synthesized in the
liver of male rats only, which is rapidly cleared in the glomer-
ular filtrate. Alpha2m-globulin is lost in the urine in the normal
mature male rat, creating a physiologic proteinuria. Thus,
alpha2m-globulin nephropathy is a sex- and species-specific
entity that can be exacerbated by test article administration and
is not relevant to humans.2,7 Xenobiotics or their metabolites
can reversibly bind to alpha2m-globulin and decrease
8 International Journal of Toxicology XX(X)
effectiveness of lysosomal degradation leading to accumula-
tion. The hyaline droplets can be readily visualized with histo-
chemical stains including the Mallory-Heidenhain stain. In
addition, a wear-and-tear lipofuscin pigment can be noted in
the RTECs in rats and increases with age without kidney func-
tional sequela or clinical significance. Idiopathic necrotizing
polyarteritis (beagle pain syndrome) is a spontaneous condition
that can be seen in beagle dogs. Clinically, it can be associated
with fever, weight loss, and cervical pain (“beagle pain
syndrome”). Pathologically, there is involvement of medium-
sized muscular arteries including those in the kidney leading to
thickened, tortuous vasculature with fibrinoid necrosis, mixed
cell infiltrates, and thrombosis. Beagle pain syndrome is pre-
sumed to be an immune-mediated condition. Other presenta-
tions of vasculitis in dogs might include dermatomyositis and
similar disorders with ischemic cutaneous and vascular lesions
(ie, dermatopathy).
Drug-Induced AKI and Kidney Toxicology
Acute Kidney Injury
Acute kidney injury is one of the most common causes of
kidney toxicity. In AKI, impairment in kidney function (eg,
rise in sCr and/or sustained oliguria) leads to retention of nitro-
genous waste products. Pathophysiologically, the classification
of AKI can be divided into 3 categories: (1) prerenal, (2) intrin-
sic [renal], and (3) postrenal.12 In prerenal AKI, kidney perfu-
sion decreases as a result of hypovolemia (eg, hemorrhage,
vomiting, or diarrhea), decreased cardiac output (eg, heart fail-
ure or pulmonary embolism), impairment of kidney vasomo-
dulation (eg, nonsteroidal anti-inflammatory drugs [NSAIDs],
ACE inhibitors, or cyclosporine), and/or vasodilation (eg, sep-
sis or hepatorenal syndrome).12 Intrinsic AKI etiopathogenesis
could be related to specific insults in kidney vascular, glomer-
ular, tubular, or interstitial compartments. Cellular insults in
intrinsic AKI can lead to various cell death modalities such
as apoptosis, autophagy, or regulated and genetically con-
trolled cell death (necroptosis).24 Vascular causes of AKI
include vascular stenosis or vasculitis (eg, disseminated intra-
vascular coagulation or drug induced). Glomerular AKI
includes immune complex diseases (ICD), glomerulonephritis,
and postinfectious glomerulonephritis. Tubular and interstitial
AKI can be caused by ischemia (shock), inflammation (eg,
sepsis, infections), rhabdomyolysis, hemolysis, crystals, ethy-
lene glycol (EG), cisplatin toxicity, or medications (eg, amino-
glycosides, rifampin, NSAIDs, prednisone).12,25 Postrenal AKI
can be caused by bladder obstruction or urinary stones.
Drug-Induced Kidney Glomerular Pathologies
As the initial plasma filtering portion of the nephron, the glo-
merulus plays an integral role in AKI and drug-induced injury
such as ICD. The glomerulus selective barrier to filtered mole-
cules based on their size, shape, and/or charge is facilitated by
its structural anatomical features of fenestrated endothelium, a
negatively charged GBM, and podocytes. These features and
the presence of complement regulatory proteins are contribut-
ing factors in ICD. For example, the presence of in situ granular
deposits of immunoglobulins and/or complement at the site of
tissue injury in kidney sections, using immunohistochemical
staining, even in few animals, is generally consistent with ICD.
In the kidney of humans and/or rodents, cell surface and soluble
complement regulatory proteins (eg, decay accelerating factor
[CD55], membrane cofactor protein [CD47], complement
receptor type 1 (CR1), CD59, clusterin, and Factor H) are
expressed by various microanatomic kidney compartments,
including glomerular capillaries, peritubular capillaries, prox-
imal tubules, collecting ducts, medullary interstitium, and glo-
merular cells (endothelial, epithelial, and mesangial).12,26-28
Thus, immune complex-mediated type 3 hypersensitivity reac-
tions and ICD are relatively common in toxicology studies,
especially with biotherapeutics after repeated dose administra-
tion to monkeys.29 The reader is strongly encouraged to refer to
comprehensive reviews on ICD pathophysiology, especially as
it relates collective weight-of-evidence approach, time course
of complement formation, and antidrug antibody (ADA) for-
mation and its correlation with exposure in toxicity studies.
Immune complex-mediated glomerular pathology related to
immunogenicity in animals is generally not considered relevant
for predicting immunogenicity in humans. For example,
administration of obinutuzumab, anti-CD20 monoclonal anti-
body, in a 26-week toxicity study in cynomolgus monkeys
resulted in dose-independent ICD-mediated hypersensitivity
and chronic inflammation in tissues including the kidneys.
Kidney glomerulus pathological changes included mesangial
hypercellularity and thickening, proliferation of parietal
epithelial cells in Bowman space with crescents formation,
and periglomerular mononuclear infiltrates. Subepithelial
electron-dense deposits in glomerular capillary basement
membrane consistent with ICD were noted by transmission
electron microscopy.30
In dogs, glomerulopathy and/or proteinuria has been
observed in dogs with Cushing syndrome, a syndrome caused
by excessive adrenocorticotropic hormone production (hyper-
adrenocorticism) and in dogs treated with prednisone. 25
Increased urine volume, decreased urine specific gravity, pro-
teinuria, and glomerulopathy were observed in 10 male beagle
dogs after oral administration of prednisone at 2.2 mg/kg,
twice daily, for 6 weeks. Similarly, drug-induced glomerulo-
pathy was observed microscopically in dogs in a 9-month
toxicity study after oral administration of fosdagrocorat, a
dissociated agonist of the glucocorticoid receptor, to beagle
dogs. It is interesting to note that obesity resulting from ad
libitum feeding exacerbated the severity of the kidney find-
ings observed in both the fosdagrocorat- and prednisone-
treated dogs, with the prednisone-treated dogs showing
greater overall glucocorticoid-induced renal effects than those
dosed with fosdagrocorat.25
It is important to note that drug-induced minimal change
disease (MCD) can be noted in various experimental laboratory
animals. The MCD is by characterized by normal glomerular
Radi 9

Table 3. Examples of Drugs That Induce Kidney Pathologies in Experimental Laboratory Animals.

Species Glomerular Tubular Interstitial Vascular

Monkey Human biotherapeutics, VEGFR2 NSAIDs, antisense oligonucleotide, NSAIDs Antisense deoxyoligonucleotide,
inhibitor, antisense recombinant IL-18 human biotherapeutics, oral
oligonucleotide, angiotensin II phosphodiesterase inhibitors
receptor antagonists
Minipig Cyclosporine Single-stranded oligonucleotide Cyclosporine Betamethasone, hydrocortisone
Dog Hydrocortisone, ampicillin, ACE inhibitors, tenofovir Cisplatin, estrogen Glucocorticoid agonists
growth hormone diphosphonate,
NSAIDs
Rat 5-lipooxygenase, human basic ACE inhibitors, aminoglycoside Cisplatin, NSAIDs Cyclosporine, oral
fibroblast growth factor, H2 antibiotics, cyclosporine, glycoprotein phosphodiesterase inhibitors,
receptor antagonists IIb-IIIa antagonists, fibrans, siRNA angiotensin II antagonist
oligonucleotide therapeutics
Mouse Antisense oligonucleotide Cisplatin, methotrexate, antisense ACE inhibitor ACE inhibitor, ephedrine
oligonucleotide
Abbreviations: ACE, angiotensin-converting enzyme; H2 receptor antagonists, histamine 2 receptor antagonists; IL, interleukin; NSAIDs, nonsteroidal
anti-inflammatory drugs; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

morphology by light microscopic examination and lack of
tubulointerstitial or vascular pathologies. However, animals
can be proteinuric with nephrotic syndrome and glomerular
changes of podocyte foot process effacement and protein
resorption droplets, and cell lysis can be noted only by electron
microscopic examination. 5-Lipooxygenase inhibitors in rats
and masitinib mesylate (tyrosine-kinase inhibitor) in dogs are
examples of drug-induced MCD31,32 (Table 3). Toxicologists
need to consider alternative assessment methodologies such as
electron microscopy if kidney microscopic examination is
within normal limits, yet there is evidence of drug-induced
proteinuria.
Drug-induced segmental glomerulosclerosis is rare in dogs
but has been noted in preclinical safety studies with adminis-
tration of growth hormone and synthetic progesterone.33 Focal
segmental glomerulosclerosis (FSGS) is a kidney disease in
humans characterized by progressive glomerular scarring and
a clinical presentation of nephrotic syndrome. In primary
FSGS, immunologic podocyte injury takes place. In secondary
FSGS, pathologic glomerular hyperfiltration/hypertension
takes place in patients with an underlying etiology (eg, obesity,
diabetes mellitus). Examples of drug-induced FSGS-like
pathologies include Adriamycin, puromycin, and streptozoto-
cin. In the early stages of FSGS-like pathologies, glomerular
hypertrophy/enlargement and/or mesangial hypercellularity
might be noted morphologically. Interestingly, glomerular tufts
collapse and extracapillary proliferation with reticular inclu-
sions can be noted by electron microscopy with some viral
infections (eg, Simian immunodeficiency virus) or some drugs
[anabolic steroids and interferons]). Differentiating primary
versus secondary FSGS-like glomerular pathologies in experi-
mental laboratory animals administered a test article is impor-
tant in understanding drug MOA and differentiating primary
versus secondary glomerular insults.
Drug-induced glomerular vacuolation and proteinuria has
been noted after intravenous administration of human basic
fibroblast growth factor to rats and monkeys. Histopathologically,
enlargement, vacuolation, and karyomegaly of podocytes in glo-
meruli, hypertrophy/hyperplasia of the parietal epithelium of
Bowman capsule in the glomeruli, tubular dilatation and cast
formation, thickening of the media in the lobular arteries, arterio-
pathy, and/or hyperplasia of the epithelium of the papilla and
collecting ducts were noted.34
Anti-vascular endothelial growth factor (VEGF) therapies
can induce glomerular thrombotic microangiopathy, mild pro-
teinuria, and hypertension in humans.35 Strong VEGF expres-
sion in mice is demonstrable using immunohistochemistry
in glomerular podocytes.36 In addition, VEGF plays a role in
glomerular endothelial cell fenestration formation, at least in
vitro.37 The VEGF is released by podocytes and binds to its
receptors on endothelial cells (eg, VEGFR-2). This helps main-
tain podocyte health and slit diaphragm integrity because inhi-
bition of VEGF signaling downregulates the expression of
nephrin, a protein involved in the maintenance of the glomer-
ular slit diaphragm. Reduction in VEGF after administration of
some anti-VEGF drugs (eg, VEGFR-2 inhibitor) can lead to
dose-dependent, yet reversible, glomerular filtration barrier
injury in rats and/or monkeys after test article administration
(Table 3). Histopathologically, eosinophilic, hyaline droplets
can be noted in podocytes, mesangial, and/or endothelial cells
and podocyte and parietal epithelial cell hypertrophy and
mesangial cell hyperplasia. Similar droplets might be seen in
the S3 segment of proximal tubules.
Kidney Drug-Induced Tubular, Interstitial, and
Vascular Pathologies
The immunopathogenesis of AKI is an important consideration
as the kidneys contribute to the immune system homeostasis by
removing circulating cytokines and bacterial toxins (eg, LPS)
and by continuously sampling blood-borne proteins.38 Emer-
ging evidence supports the involvement of RTECs and the
10
innate and adaptive arms of the immune system in the patho-
genesis of intrinsic AKI. Pro-inflammatory DAMPs, PAMPs,
TLRs, oxidative stress, HIF, complement system, adhesion
molecules, cell death, resident renal DCs, neutrophils, T and
B lymphocytes, macrophages, natural killer T cells, secreted
cytokines, and chemokines contribute to the immunopathogen-
esis of AKI. Thus, loss of the immune system homeostasis can
have direct or indirect effects on kidney pathology. If the
immunopathologic processes in AKI continue, this can lead
to renal fibrosis and/or chronic kidney disease.12
Sepsis is one of the most common causes of AKI.39 In
sepsis, there is a systemic inflammatory response, triggered
by the innate immune system, to attack foreign bacterial patho-
gens (eg, LPS) and toxins to limit their spread.20 Such systemic
inflammatory response leads to the release of PAMPs and
DAMPs into the circulation.20 The PAMPs are released from
pathogens and DAMPs are released from damaged and injured
tissues at sites of infection, and both PAMPs and DAMPs
prime and signal the immune system to fight back the invading
pathogens and toxins. Interactions of PAMPs and DAMPs with
sensors of the innate immune system called pattern recognition
receptors (eg, TLRs, nucleotide-binding oligomerization
domain-like receptors, and retinoic-acid-inducible-gene-I
[RIGI]-like receptors) amplify the systemic inflammatory
response.12,19,20 For example, there is TLR4-dependent LPS
recognition mechanism in the RTECs in the S1 segment of the
proximal tubules.21 Kidney-specific DAMPs (eg, uromodulin
[Tamm-Horsfall protein]) are synthesized by distal epithelial
tubules and selectively released into the tubular lumen and leak
into the interstitium after renal tubular injury. This activates
renal DCs via TLR4, and NACHT, LRR, and PYD domains-
containing protein 3 inflammasome potentially leading to ster-
ile kidney inflammation.40,41
The kidney parenchyma is especially vulnerable to the pro-
inflammatory response to infection and subsequent kidney
tissue damage because it receives approximately 25% of the
cardiac output. This is because the toxin-rich blood, PAMPs,
DAMPs, and pro-inflammatory mediators gain access to kid-
ney parenchyma and its various compartments via glomerular
filtration or peritubular capillaries microcirculation.19,20 For
example, LPS is filtered into the tubular fluid and can directly
interact with RTECs via a TLR4-dependent mechanism.19,21
As a consequence, various compartments of the kidney includ-
ing RTEC, glomerular, and endothelial are injured. This is
because the filtered PAMPs and DAMPs, also called “danger
signal,” bind to and activate RTECs and kidney endothelial and
glomerular cells and can lead to endothelial cell activation and
structural changes in the glomerular and peritubular capil-
laries.20 It is important to distinguish adaptive kidney response
(eg, drug-induced medullary tubular hypertrophy, tubular
hypertrophy secondary to increased GFR, potassium depletion
[chronic hypokalemia], high dietary sodium chloride, increased
sodium loss, testosterone treatment, adrenocorticotropic
hormone-mediated hyperadrenocorticism1,2) from direct injur-
ious tubular damage. Collectively, exogenous and endogenous
PAMPs and DAMPs are integral to AKI pathogenesis.
International Journal of Toxicology XX(X)
Examples of drug-induced tubular injury include aminogly-
coside antibiotics (eg, gentamicin), cephalosporins, cisplatin,
antiviral agents, and calcineurin (CNI) inhibitors (Table 3).
Pathophysiologic factors in aminoglycoside kidney toxicology
include alterations in tubular cells reabsorption, tubular
obstruction and tubular malfunction, reduced glomerular filtra-
tion, tubuloglomerular feedback activation, renal vasoconstric-
tion, and mesangial contraction.42 Local kidney factors (eg,
variability in P-glycoprotein and CYP3A4/5 expression or
activity, older age, salt depletion, use of NSAIDs, and genetic
polymorphisms in genes) are important for susceptibility CNI
kidney toxicity.43
Unless associated with morphologic evidence of tubular cell
injury of necrosis and/or degeneration, antisense oligonucleo-
tides (ASO) can cause nonadverse and adaptive kidney changes
in rats evident histologically by accumulation of basophilic or
eosinophilic and granular, amorphous, or vacuolar material in
the cytoplasm or nuclei of renal epithelial cells. This is con-
sistent with lysosomal accumulation and endosomal localiza-
tion of ASO. Oligonucleotides can be absorbed rapidly and
distributed in tissues, most commonly in the kidneys, after
systemic or local administration. In rats, exacerbation of CPN
and proteinuria might be observed after ASO chronic adminis-
tration. Pathophysiologically, once oligonucleotides interact
with and taken by the brush boarder of proximal tubules via
polyanion transporter, they are internalized by endocytosis,
leading to incorporation into endosomes and eventually lyso-
somes and phagolysosomes. Some reports suggest that
although there are high concentrations of ASO and cytoplasmic
vacuolation, no evidence of kidney functional abnormalities
(eg, sCr or BUN) were noted, but low incidence of reversible
increase in urine protein to creatinine ratio was also noted in
monkeys.44
Drugs that can induce crystal nephropathy in humans and/or
experimental animals include quinolone antibiotics (eg, ampi-
cillin, norfloxacin, and ciprofloxacin), sulfonamides and fluor-
oquinolones, glycoprotein IIb-IIIa antagonists, ganciclovir,
sulfadiazine, and dopamine autoreceptor agonist (Table 3).
Intratubular precipitation of xenobiotics/crystals can cause or
promote AKI and chronic kidney injury. A number of xenobio-
tics/drugs are known to precipitate within renal tubules, due to
their insolubility (parent drug or its metabolite) within urine.
Risk factors for xenobiotic/crystal precipitation within the kid-
ney tubules include intravascular volume depletion, underlying
kidney disease, and metabolic disturbances that promote
changes in urine pH. EG-mediated crystal nephropathy
requires metabolic transformation of EG to oxalic acid that
takes place primarily in the liver via alcohol dehydrogenase.
When oxalic acid is concentrated in the urine, calcium oxalate
crystals form, leading to crystalluria, nephrolithiasis, metabolic
acidosis, low urine specific gravity, and proteinuria. The EG-
induced crystal deposition in kidney proximal tubules was
detected unbound in the lumen and attached to the surface of
epithelial cells.45 Crystal formation can lead to chronic inter-
stitial nephritis with glomerulosclerosis, tubular atrophy, and
interstitial fibrosis. 46 Sulfonamides have caused crystal
Radi
nephropathy, affecting the distal nephron in animals and
humans. As the pH of murine urine generally varies between
5 and 8, and is greatly influenced by diet, this would influence
crystal nephropathy formation. In addition, a major difference
between murine and human urine is the overall osmolality.
Murine urine is usually highly concentrated, with male urine
having higher protein concentrations than females. It is impor-
tant to note that for human risk assessment, murine urinary
crystalluria MOA is not relevant to human urinary bladder
cancer risk.
Cyclooxygenase-mediated prostaglandins are involved in
renal functions and their inhibition is associated with potential
adverse renal effects.11 Analgesic nephropathy is characterized
by chronic interstitial nephritis and renal papillary necrosis
(RPN) or calcifications. Some laboratory animals (eg, rats,
dogs) are unusually susceptible to RPN. Kidney changes in
monkeys associated with administration of NSAIDs-
containing carboxylic acid, such as naproxen, and carboxylic
acid moiety (eg, fibrans) were shown to induce formation of
casts with crystalline material that precipitates in the kidneys.29
This can lead to obstructive nephropathy in rats because of
accumulation of intratubular crystal precipitates (eg, glycopro-
tein IIb-IIIa antagonists).46 Obstructive nephropathy can be
seen secondary to co-precipitation of administered IL-18 and
Tamm-Horsfall protein in the distal nephron (Table 3).47
Drug-induced inclusion bodies can be noted in rats with
norepinephrine/serotonin reuptake inhibitors and propiverine
(anticholinergic drug). Such inclusion bodies can be related
to D-amino acid oxidases (DAO). The physiologic function of
DAO, present in the kidney, liver, and brain, is to act as a
detoxifying agent and to metabolize D-amino acids. The patho-
physiologic mechanism of DAO nuclear and cytosolic accumu-
lation in kidney tubules in the form of inclusion bodies is likely
related to peroxisome proliferation and enzyme induction
mediated through peroxisome proliferator activated receptor-
a (PPARa).48 The PPARa is found predominately in the renal
cortex proximal convoluted tubules49 and increased DAO
activity has been demonstrated in kidneys of rats exposed to
ciprofibrate, a PPARa agonist.50 The kidney finding of tubular
inclusion bodies is species-specific, reversible, and not relevant
to human safety risk assessment.48 Other examples of drug-
induced kidney (vascular and interstitial) injury are outlined
in Table 3. Also, there are species differences in response to
drug-induced injury. For example, while rats and monkeys are
sensitive to the development of kidney pathology after puro-
mycin administration, dogs, mice, and rabbits are relatively
insensitive. Finally, various investigative techniques (eg,
immunohistochemistry, histochemical stains; in situ hybridi-
zation, polarized light microscopic examination to visualize
crystals in kidney sections, monocyte chemoattractant
protein-1 and C-reactive protein biomarkers for vascular
pathologies, complement levels, inulin clearance, endogenous
creatinine clearance, kidney microautoradiography, confocal
Raman microspectroscopy, matrix-assisted laser desorption/
ionization mass spectrometry imaging).2,7 might need to be
11
employed on a case-by-case basis to further dissect a drug-
induced kidney finding.
Conclusions
Detection and characterization of direct and indirect kidney
changes and/or toxicity in preclinical species and human rele-
vance will depend on the preclinical safety testing strategy and
collective weight-of-evidence approach including new investi-
gational drug MOA, drug physiochemical properties, distin-
guishing effects related to the parent drug or its metabolite,
changes in sCr related to renal transporters, preclinical and
clinical interspecies, and sex differences in kidney macro- and
micro-anatomy, physiology, and metabolic and hormonal
activities, background pathologies, drug-induced, yet sex-
and/or species-specific kidney toxicity, and MOA relevance
to humans. Various investigative toxicology and pathology
tools can be employed on a case-by-case scientific basis to
better characterize pharmaceutical drug-induced kidney
toxicities.
Author Contributions
Z. Radi contributed to conception and design, contributed to acquisi-
tion, analysis, and interpretation, drafted the manuscript, critically
revised the manuscript, gave final approval, and agrees to be accoun-
table for all aspects of work ensuring integrity and accuracy.
Declaration of Conflicting Interests
The author(s) declared the following potential conflicts of interest
with respect to the research, authorship, and/or publication of this
article: The author is a Pfizer Inc. employee.
Funding
The author received no financial support for the research, authorship,
and/or publication of this article.
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