Pathophysiology of

Nephrotoxic Acute
Renal Failure
Rick G. Schnellmann
Katrina J. Kelly

H
umans are exposed intentionally and unintentionally to a
variety of diverse chemicals that harm the kidney. As the list
of drugs, natural products, industrial chemicals and environ-
mental pollutants that cause nephrotoxicity has increased, it has
become clear that chemicals with very diverse chemical structures pro-
duce nephrotoxicity. For example, the heavy metal HgCl2, the myco-
toxin fumonisin B1, the immunosuppresant cyclosporin A, and the
aminoglycoside antibiotics all produce acute renal failure but are not
structurally related. Thus, it is not surprising that the cellular targets
within the kidney and the mechanisms of cellular injury vary with dif-
ferent toxicants. Nevertheless, there are similarities between chemical-
induced acute tubular injury and ischemia/reperfusion injury.
The tubular cells of the kidney are particularly vulnerable to toxi-
cant-mediated injury due to their disproportionate exposure to circu-
lating chemicals and transport processes that result in high intracellu-
lar concentrations. It is generally thought that the parent chemical or
a metabolite initiates toxicity through its covalent or noncovalent
binding to cellular macromolecules or through their ability to produce
reactive oxygen species. In either case the activity of the macromole-
cule(s) is altered resulting in cell injury. For example, proteins and
lipids in the plasma membrane, nucleus, lysosome, mitochondrion and
cytosol are all targets of toxicants. If the toxicant causes oxidative
CHAPTER
stress both lipid peroxidation and protein oxidation have been shown

15
to contribute to cell injury.
In many cases mitochondria are a critical target and the lack of
adenosine triphosphate (ATP) leads to cell injury due to the depen-
dence of renal function on aerobic metabolism. The loss of ATP leads
15.2 Acute Renal Failure
to disruption of cellular ion homeostasis with decreased cellular
K+ content, increased Na+ content and membrane depolariza-
tion. Increased cytosolic free Ca2+ concentrations can occur in
the early or late phase of cell injury and plays a critical role lead-
ing to cell death. The increase in Ca2+ can activate calcium acti-
vated neutral proteases (calpains) that appear to contribute to
the cell injury that occurs by a variety of toxicants. During the
late phase of cell injury, there is an increase in Cl- influx, fol-
lowed by the influx of increasing larger molecules that leads to
cell lysis. Two additional enzymes appear to play an important
role in cell injury, particularly oxidative injury. Phospholipase A2
consists of a family of enzymes in which the activity of the
cytosolic form increases during oxidative injury and contributes
to cell death. Caspases are a family of cysteine proteases that are
activated following oxidative injury and contribute to cell death.
Clinical Significance of Toxicant-Mediated
Acute Renal Failure
CLINICAL SIGNIFICANCE OF
TOXICANT–MEDIATED RENAL FAILURE
Nephrotoxins may account for approximately 50% of all cases of acute and chronic
renal failure.
Nephrotoxic renal injury often occurs in conjunction with ischemic acute renal failure.
Acute renal failure may occur in 2% to 5% of hospitalized patients and 10% to 15% of
patients in intensive care units.
The mortality of acute renal failure is approximatley 50% which has not changed
significantly in the last 40 years.
Radiocontrast media and aminoglycosides are the most common agents associated
with nephrotoxic injury in hospitalized patients.
Aminoglycoside nephrotoxicity occurs in 5% to 15% of patients treated with
these drugs.
FIGURE 15-1
Clinical significance of toxicant-mediated renal failure.
FACTORS THAT PREDISPOSE THE
KIDNEY TO TOXICANT INJURY
Preexisting renal dysfunction
Dehydration
Diabetes mellitus
Exposure to multiple nephrotoxins
Following exposure to a chemical insult those cells sufficiently
injured die by one of two mechanisms, apoptosis or oncosis.
Clinically, a vast number of nephrotoxicants can produce a
variety of clinical syndromes-acute renal failure, chronic renal
failure, nephrotic syndrome, hypertension and renal tubular
defects. The evolving understanding of the pathophysiology of
toxicant-mediated renal injury has implications for potential
therapies and preventive measures. This chapter outlines some
of the mechanisms thought to be important in toxicant-mediat-
ed renal cell injury and death that leads to the loss of tubular
epithelial cells, tubular obstruction, “backleak” of the glomeru-
lar filtrate and a decreased glomerular filtration rate. The recov-
ery from the structural and functional damage following chemi-
cal exposures is dependent on the repair of sublethally-injured
and regeneration of noninjured cells.
REASONS FOR THE KIDNEY’S
SUSCEPTIBILITY TO TOXICANT INJURY
Receives 25% of the cardiac output
Sensitive to vasoactive compounds
Concentrates toxicants through reabsorptive and secretive processes
Many transporters result in high intracellular concentrations
Large luminal membrane surface area
Large biotransformation capacity
Baseline medullary hypoxia
FIGURE 15-2
Reasons for the kidney’s susceptibility to toxicant injury.
FIGURE 15-3
Factors that predispose the kidney to toxicant injury.
 Pathophysiology of Nephrotoxic Acute Renal Failure 15.3

EXOGENOUS AND ENDOGENOUS CHEMICALS THAT CAUSE ACUTE RENAL FAILURE

Antibiotics Immunosuppressive agents Vasoactive agents Other drugs

Aminoglycosides (gentamicin, tobramycin, Cyclosporin A Nonsteroidal anti-inflammatory Acetaminophen
amikacin, netilmicin) Tacrolimus (FK 506) drugs (NSAIDs) Halothane
Amphotericin B Antiviral agents Ibuprofen Methoxyflurane
Cephalosporins Acyclovir Naproxen Cimetidine
Ciprofloxacin Cidovir Indomethacin Hydralazine
Demeclocycline Foscarnet Meclofenemate Lithium
Penicillins Valacyclovir Aspirin Lovastatin
Pentamidine Heavy metals Piroxicam Mannitol
Polymixins Cadmium Angiotensin-converting Penicillamine
Rifampin Gold enzyme inhibitors Procainamide
Sulfonamides Mercury Captopril Thiazides
Tetracycline Lead Enalopril Lindane
Vancomycin Arsenic Lisinopril Endogenous compounds
Chemotherapeutic agents Bismuth Angiotensin receptor antagonists Myoglobin
Adriamycin Uranium Losartan Hemoglobin
Cisplatin Organic solvents Calcium
Methotraxate Ethylene glycol Uric acid
Mitomycin C Carbon tetrachloride Oxalate
Nitrosoureas Unleaded gasoline Cystine
(eg, streptozotocin, Iomustine)
Radiocontrast media
Ionic (eg, diatrizoate, iothalamate)
Nonionic (eg, metrizamide)

FIGURE 15-4
Exogenous and endogenous chemicals that cause acute renal failure.

FIGURE 15-5
Proximal convoluted tubule Nephrotoxicants may act at different sites in the kidney, resulting
(S1/S2 segments) in altered renal function. The sites of injury by selected nephrotoxi-
Aminoglycosides cants are shown. Nonsteroidal anti-inflammatory drugs (NSAIDs),
Cephaloridine Glomeruli angiotensin-converting enzyme (ACE) inhibitors, cyclosporin A,
Cadmium chloride Interferon–α and radiographic contrast media cause vasoconstriction. Gold,
Potassium dichromate Gold interferon-alpha, and penicillamine can alter glomerular function
Penicillamine and result in proteinuria and decreased renal function. Many
Proximal straight tubule nephrotoxicants damage tubular epithelial cells directly.
Renal vessels (S3 segment) Aminoglycosides, cephaloridine, cadmium chloride, and potassium
NSAIDs Cisplatin dichromate affect the S1 and S2 segments of the proximal tubule,
ACE inhibitors Mercuric chloride whereas cisplatin, mercuric chloride, and dichlorovinyl-L-cysteine
Cyclosporin A Dichlorovinyl–L–cysteine affect the S3 segment of the proximal tubule. Cephalosporins, cad-
mium chloride, and NSAIDs cause interstitial nephritis whereas
phenacetin causes renal papillary necrosis.

Interstitium
Papillae Cephalosporins
Phenacetin Cadmium
NSAIDs
15.4 Acute Renal Failure

Renal vasoconstriction Prerenal azotemia

Intravascular Sympathetic
Increased tubular pressure volume tone

n Tubular obstruction "Back-leak" of glomerular filtrate

E e
Intratubular Functional Capillary permeability Hypertension
x p GFR
h casts abnormalties Endothelin
p
o r Nitric oxide
o Tubular damage Endothelial injury Thromboxane Renal and systemic
s
t Prostaglandins vasoconstriction
u
r o Intrarenal factors Persistent medullary hypoxia
e x Physical constriction
i of medullary vessels Vascular smooth muscle
Hemodynamic Glomerular sensitivity to vasoconstrictors
t c hydrostatic
alterations
o a pressure
n
t Intrarenal Striped interstitial
Cyclosporin A Angiotensin II fibrosis
vasoconstriction Perfusion pressure
Efferent tone
Afferent tone
Glomerular factors Tubular cell injury GFR

Glomerular ultrafiltration
Obstruction Postrenal failure
FIGURE 15-6
Mechanisms that contribute to decreased glomerular filtration rate
(GFR) in acute renal failure. After exposure to a nephrotoxicant,
one or more mechanisms may contribute to a reduction in the
GFR. These include renal vasoconstriction resulting in prerenal
azotemia (eg, cyclosporin A) and obstruction due to precipitation
of a drug or endogenous substances within the kidney or collecting
ducts (eg, methotrexate). Intrarenal factors include direct tubular
obstruction and dysfunction resulting in tubular backleak and
increased tubular pressure. Alterations in the levels of a variety of
vasoactive mediators (eg, prostaglandins following treatment with
nonsteroidal anti-inflammatory drugs) may result in decreased
renal perfusion pressure or efferent arteriolar tone and increased
afferent arteriolar tone, resulting in decreased in glomerular hydro-
static pressure. Some nephrotoxicants may decrease glomerular
function, leading to proteinuria and decreased renal function.
FIGURE 15-7
Renal injury from exposure to cyclosporin A. Cyclosporin A is one
example of a toxicant that acts at several sites within the kidney.
It can injure both endothelial and tubular cells. Endothelial injury
results in increased vascular permeability and hypovolemia, which
activates the sympathetic nervous system. Injury to the endotheli-
um also results in increases in endothelin and thromboxane A2
and decreases in nitric oxide and vasodilatory prostaglandins.
Finally, cyclosporin A may increase the sensitivity of the vascula-
ture to vasoconstrictors, activate the renin-angiotensin system, and
increase angiotensin II levels. All of these changes lead to vasocon-
striction and hypertension. Vasoconstriction in the kidney con-
tributes to the decrease in glomerular filtration rate (GFR), and
the histologic changes in the kidney are the result of local ischemia
and hypertension.

Renal Cellular Responses to Toxicant Exposures

FIGURE 15-8
Nephrotoxic insult The nephron’s response to a nephrotoxic insult. After a population
to the nephron of cells are exposed to a nephrotoxicant, the cells respond and ulti-
mately the nephron recovers function or, if cell death and loss is
Uninjured cells Injured cells Cell death
extensive, nephron function ceases. Terminally injured cells under-
go cell death through oncosis or apoptosis. Cells injured sublethal-
ly undergo repair and adaptation (eg, stress response) in response
Compensatory Cellular Cellular Cellular to the nephrotoxicant. Cells not injured and adjacent to the injured
hypertrophy adaptation proliferation repair area may undergo dedifferentiation, proliferation, migration or
spreading, and differentiation. Cells that were not injured may also
undergo compensatory hypertrophy in response to the cell loss and
Re-epithelialization Cellular adaptation
injury. Finally the uninjured cells may also undergo adaptation in
response to nephrotoxicant exposure.
Differentiation

Structural and functional recovery of the nephron

 Pathophysiology of Nephrotoxic Acute Renal Failure 15.5

Intact tubular epithelium Loss of polarity, tight junction

integrity, cell–substrate adhesion,
simplification of brush border

Toxic injury Cell death

Necrosis Apoptosis

α
β Sloughing of viable
and nonviable cells
with intraluminal
cell-cell adhesion

Cytoskeleton
Extracellular matrix
Cast formation
Na+/K+=ATPase
and tubuler
β1 Integrin obstruction
RGD peptide

FIGURE 15-9
After injury, alterations can occur in the cytoskeleton and in
the normal distribution of membrane proteins such as Na+, K+-
ATPase and 1 integrins in sublethally injured renal tubular
cells. These changes result in loss of cell polarity, tight junction
integrity, and cell-substrate adhesion. Lethally injured cells
undergo oncosis or apoptosis, and both dead and viable cells
may be sloughed into the tubular lumen. Adhesion of sloughed
cells to other sloughed cells and to cells remaining adherent to
the basement membrane may result in cast formation, tubular
obstruction, and further compromise the glomerular filtration
rate. (Adapted from Fish and Molitoris [1], and Gailit et al. [2];
with permission.)

FIGURE 15-10
Sublethally Migrating Cell
Potential sites where nephrotoxicants can interfere with the struc-
injured cells spreading cells proliferation
tural and functional recovery of nephrons.

Basement
membrane
Toxicant inhibition Toxicant inhibition Toxicant inhibition
of cell repair of cell migration/spreading of cell proliferation
15.6 Acute Renal Failure

140

120
100
Percent of control

Oncosis Apoptosis
80

60
Cell number/confluence
40 Mitochondrial function
Active Na+ transport
20 +
Na -coupled glucose transport
GGT activity Blebbing Budding
0
0 1 2 3 4 5 6
Time after exposure, d

FIGURE 15-11 Necrosis

Inhibition and repair of renal proximal tubule cellular functions
after exposure to the model oxidant t-butylhydroperoxide.
Approximately 25% cell loss and marked inhibition of mitochon-
drial function active (Na+) transport and Na+-coupled glucose
transport occurred 24 hours after oxidant exposure. The activity
of the brush border membrane enzyme -glutamyl transferase
(GGT) was not affected by oxidant exposure. Cell proliferation
and migration or spreading was complete by day 4, whereas active
Na+ transport and Na+-coupled glucose transport did not return to
control levels until day 6. These data suggest that selective physio-
logic functions are diminished after oxidant injury and that a hier-
archy exists in the repair process: migration or spreading followed
by cell proliferation forms a monolayer and antedates the repair of
physiologic functions. (Data from Nowak et al. [3].)
Phagocytosis Phagocytosis
inflammation by macrophages
or nearby cells
FIGURE 15-12
Apoptosis and oncosis are the two generally recognized forms of
cell death. Apoptosis, also known as programmed cell death and
cell suicide, is characterized morphologically by cell shrinkage, cell
budding forming apoptotic bodies, and phagocytosis by
macrophages and nearby cells. In contrast, oncosis, also known as
necrosis, necrotic cell death, and cell murder, is characterized mor-
phologically by cell and organelle swelling, plasma membrane bleb-
bing, cell lysis, and inflammation. It has been suggested that cell
death characterized by cell swelling and lysis not be called necrosis
or necrotic cell death because these terms describe events that
occur well after the cell has died and include cell and tissue break-
down and cell debris. (From Majno and Joris [4]; with permission.)

Mechanisms of Toxicant-Mediated Cellular Injury

Transport and biotransformation
FIGURE 15-13
Toxicant whose primary
Toxicants in general mechanism of action is The general relationship between oncosis and apoptosis after
ATP depletion nephrotoxicant exposure. For many toxicants, low concentrations
cause primarily apoptosis and oncosis occurs principally at higher
concentrations. When the primary mechanism of action of the
Oncosis Oncosis nephrotoxicant is ATP depletion, oncosis may be the predominant
cause of cell death with limited apoptosis occurring.
Cell death

Cell death

Apoptosis

Apoptosis

Toxicant concentration Toxicant concentration

 Pathophysiology of Nephrotoxic Acute Renal Failure 15.7

FIGURE 15-14
GSH-Hg-GSH The importance of cellular transport in mediating toxicity.
GSH-Hg-GSH Proximal tubular uptake of inorganic mercury is thought to be the
GSH-Hg-GSH result of the transport of mercuric conjugates (eg, diglutathione
CYS-Hg-CYS γ-GT Urine
GLY-CYS-Hg-CYS-GLY ? mercury conjugate [GSH-Hg-GSH], dicysteine mercuric conjugate
CYS-Hg-CYS Acivicin [CYS-Hg-CYS]). At the luminal membrane, GSH-Hg-GSH appears
Dipeptidase to be metabolized by (-glutamyl transferase ((-GT) and a dipepti-
Lumen +
CYS-Hg-CYS Na
dase to form CYS-Hg-CYS. The CYS-Hg-CYS may be taken up by
Neutral amino an amino acid transporter. At the basolateral membrane, mercuric
–
R-Hg-R–
acid transporter conjugates appear to be transported by the organic anion trans-
Proximal +
tubular cell CYS-Hg-CYS Na CYS-Hg-CYS porter. (-Ketoglutarate and the dicarboxylate transporter seem to
GSH-Hg-GSH
play important roles in basolateral membrane uptake of mercuric
Na+ α-Ketoglutarate α-Ketoglutarate conjugates. Uptake of mercuric-protein conjugates by endocytosis
Dicarboxylate Organic anion
transporter transporter may play a minor role in the uptake of inorganic mercury trans-
port. PAH—para-aminohippurate. (Courtesy of Dr. R. K. Zalups.)
α-Ketoglutarate Organic anions
Na+
Blood (PAH or
Dicarboxylic α-Ketoglutarate probenecid)
acids –
R-Hg-R–
CYS-Hg-CYS
GSH-Hg-GSH

High-affinity binding
to macromolecules
Altered activity of
critical macromolecules
FIGURE 15-15
Toxicant Covalent and noncovalent binding versus oxidative stress mecha-
nisms of cell injury. Nephrotoxicants are generally thought to pro-
duce cell injury and death through one of two mechanisms, either
Biotransformation
alone or in combination. In some cases the toxicant may have a
high affinity for a specific macromolecule or class of macromole-
Reactive intermediate Redox cycling cules that results in altered activity (increase or decrease) of these
molecules, resulting in cell injury. Alternatively, the parent nephro-
toxicant may not be toxic until it is biotransformed into a reactive
Covalent binding Increased reactive intermediate that binds covalently to macromolecules and in turn
to macromolecules oxygen species alters their activity, resulting in cell injury. Finally, the toxicant may
increase reactive oxygen species in the cells directly, after being bio-
transformed into a reactive intermediate or through redox cycling.
Damage to critical Oxidative damage to
critical macromolecules
The resulting increase in reactive oxygen species results in oxida-
macromolecules
tive damage and cell injury.

Cell injury

Cell repair Cell death

FIGURE 15-16
Plasma RSG Plasma RSG
This figure illustrates the renal proximal tubular uptake, biotransfor-
R + SG Glomerular filtration mation, and toxicity of glutathione and cysteine conjugates and mer-
1. 2. capturic acids of haloalkanes and haloalkenes (R). 1) Formation of a
6.
R-SG R-SG R-SG 3. glutathione conjugate within the renal cell (R-SG). 2) Secretion of the
Na+ R-SG into the lumen. 3) Removal of the -glutamyl residue (-Glu)
4. γ-Glu by -glutamyl transferase. 4) Removal of the glycinyl residue (Gly) by
Plasma 7. 5. Gly a dipeptidase. 5) Luminal uptake of the cysteine conjugate (R-Cys).
R-Cys R-Cys R-Cys
R-Cys 12. NH3+H3CCOCO2H Basolateral membrane uptake of R-SG (6), R-Cys (7), and a mercap-
Na+ turic acid (N-acetyl cysteine conjugate; R-NAC)(8). 9) Secretion of
10. 11. R-SH 13.
R-NAC into the lumen. 10) Acetylation of R-Cys to form R-NAC.
Covalent binding
11) Deacetylation of R-NAC to form R-Cys. 12) Biotransformation
8. Cell injury
Plasma of the penultimate nephrotoxic species (R-Cys) by cysteine conjugate
R-NAC R-NAC R-NAC
R-NAC 9. -lyase to a reactive intermediate (R-SH), ammonia, and pyruvate.
Na+
Basolateral Brush border 13) Binding of the reactive thiol to cellular macromolecules (eg, lipids,
membrane membrane proteins) and initiation of cell injury. (Adapted from Monks and Lau
[5]; with permission.)
15.8 Acute Renal Failure

FIGURE 15-17
Covalent binding of a nephrotoxicant
metabolite in vivo to rat kidney tissue, local-
ization of binding to the mitochondria, and
identification of three proteins that bind to
the nephrotoxicant. A, Binding of tetrafluo-
roethyl-L-cysteine (TFEC) metabolites in vivo
to rat kidney tissue detected immunohisto-
chemically. Staining was localized to the S3
segments of the proximal tubule, the segment
that undergoes necrosis. B, Immunoreactivity
in untreated rat kidneys. C, Isolation and
A B fractionation of renal cortical mitochondria
from untreated and TFEC treated rats and
Representative immunoblot analysis revealed numerous pro-
starting Submitochondrial fractions teins that bind to the nephrotoxicant (inner-
material A. Untreated B. TFEC (30 mg/kg) inner membrane, matrix-soluble matrix,
Mr (kDa) outer-outer membrane, inter-intermembrane
space). The identity of three of the proteins
228
that bound to the nephrotoxicant: P84,
P99 109 mortalin (HSP70-like); P66, HSP 60; and
P84 P42, aspartate aminotransferase. Mr—rela-
P66 70
tive molecular weight. (From Hayden et al.
P52
P42 44 [6], and Bruschi et al. [7]; with permission.)
Matrix
Inner

Outer

Matrix
Inter

Inner

Outer

Inter

Lipid peroxidation and mitochondrial dysfunction

HH R
FIGURE 15-18
A simplified scheme of lipid peroxidation. The first step, hydrogen
HO• Lipid abstraction from the lipid by a radical (eg, hydroxyl), results in the
H 2O formation of a lipid radical. Rearrangement of the lipid radical
Hydrogen abstraction results in conjugated diene formation. The addition of oxygen
•H R
results in a lipid peroxyl radical. Additional hydrogen abstraction
Lipid radical results in the formation of a lipid hydroperoxide. The Fenton reac-
Diene conjugation tion produces a lipid alkoxyl radical and lipid fragmentation,
R resulting in lipid aldehydes and ethane. Alternatively, the lipid per-
• oxyl radical can undergo a series of reactions that result in the for-
H Lipid radical, conjugated diene mation of malondialdehyde.
O2 Oxygen addition
R R
•O–O H Lipid peroxyl radical
O O
LH Hydrogen abstraction
L•
R
O O Lipid hydroperoxide
HOO H
Fe(II) Fenton reaction
Malondialdehyde
Fe(III) HO•
R
•O H Lipid alkoxyl radical
Fragmentation
H R
• H
H
H O Lipid aldehyde
LH • H
L H
H Ethane
H
 Pathophysiology of Nephrotoxic Acute Renal Failure 15.9

50 100
Control Control
TBHP (0.5 mmol) DCVC
40 TBHP + DEF (1 mM) 80 DCVC + DEF (1 mM)
TBHP + DPPD (2 µM) DCVC + DPPD (50µM)
LDH release, %

LDH release, %
30 60

20 40

10 20

0 0
0 1 2 3 4 5 6 0 1 2 3 4 5 6
A Time, h B Time, h

1.2 2.0
+1 mM DEF
1.0 +50 µM DPPD
1.6
nmol MDA•mg protein–1

nmol MDA•mg protein–1

Lipid peroxidation,

Lipid peroxidation,
0.8
1.2
0.6
0.8
0.4

0.4
0.2

0.0 0.0
C Control TBHP +1 mM DEF +2 µM DPPD D Control DCVC

FIGURE 15-19
A–D, Similarities and differences between oxidant-induced and
halocarbon-cysteine conjugate–induced renal proximal tubular
lipid peroxidation and cell death. The model oxidant t-butylhy-
droperoxide (TBHP) and the halocarbon-cysteine conjugate
dichlorovinyl-L-cysteine (DCVC) caused extensive lipid peroxi-
dation after 1 hour of exposure and cell death (lactate dehydro-
genase (LDH) release) over 6-hours’ exposure. The iron chelator
deferoxamine (DEF) and the antioxidant N,N’-diphenyl-1,
4-phenylenediamine (DPPD) completely blocked both the lipid
peroxidation and cell death caused by TBHP. In contrast,
while DEF and DPPD completely blocked the lipid peroxidation
caused by DCVC, cell death was only delayed. These results
suggest that the iron-mediated oxidative stress caused by TBHP
is responsible for the observed toxicity, whereas the iron-mediat-
ed oxidative stress caused by DCVC accelerates cell death. One
reason that cells die in the absence of iron-mediated oxidative
stress is that DCVC causes marked mitochondrial dysfunction.
(Data from Groves et al. [8], and Schellmann [9].)

FIGURE 15-20
ALTERATION OF RENAL TUBULAR CELL Mechanisms by which nephrotoxicants can alter renal tubular
ENERGETICS AFTER EXPOSURE TO TOXICANTS cell energetics.

Decreased oxygen delivery secondary to vasoconstriction

Inhibition of mitochondrial respiration
Increased tubular cell oxygen consumption
15.10 Acute Renal Failure

FIGURE 15-21
Substrates Some of the mitochondrial targets of nephro-
11 toxicants: 1) nicotinamide adenine dinu-
Cephaloridine Atractyloside cleotide (NADH) dehydrogenase; 2) succi-
TCA Ochratoxin A nate dehydrogenase; 3) coenzyme
cycle Q–cytochrome C reductase; 4) cytochrome
ADP C; 5) cytochrome C oxidase; 6) cytochrome
Bromohydroquinone 9
ATP Aa3; 7) H+-Pi contransporter; 8) F0F1-
Dichlorovinyl–L–cysteine ATPase; 9) adenine triphosphate/diphosphate
1
Tetrafluoroethyl–L–cysteine H+ ATP (ATP/ADP) translocase; 10) protonophore
Pentachlorobutadienyl–L–cysteine 8
2 H+ (uncoupler); 11) substrate transporters.
Citrinin 3
Ochratoxin A H+
Hg2+ CN– 4 Oligomycin
5
H+
Pi Pi
6 7
H+
Matrix
O2 H 2O Ochratoxin A
10
Pentachlorobutadienyl–L–cysteine
H+ Inner membrane
Citrinin
FCCP Outer membrane

Disruption of ion homeostasis

Na+ H 2O
100
Na+ 90
Relative cellular changes

80 Na+
Na+ Na+ ATPase
Na+ ATPase 70
ATP 60
– – Cl–
ATP – Cl– 50 QO2 Membrane
– potential
40
Cl– Cl –
– K+ – 30 K+
– H 2O
K + – 20
10
ATP
A K+ B Antimycin A K+ 0
0 5 10 15 20 25 30
Antimycin A Time, min
FIGURE 15-22
Early ion movements after mitochondrial dysfunction. A, A control
renal proximal tubular cell. Within minutes of mitochondrial inhibi- FIGURE 15-23
tion (eg, by antimycin A), ATP levels drop, resulting in inhibition of A graphic of the phenomena diagrammed in Figure 15-22.
the Na+, K+-ATPase. B, Consequently, Na+ influx, K+ efflux, mem-
brane depolarization, and a limited degree of cell swelling occur.

FIGURE 15-24
Na+
The late ion movements after mitochondrial dysfunction that leads
Na+
to cell death/lysis. A, Cl- influx occurs as a distinct step subsequent
ATPase
Na+
to Na+ influx and K+ efflux. B, Following Cl- influx, additional
Na+ ATPase Na+ and water influx occur resulting in terminal cell swelling.
ATP Ultimately cell lysis occurs.
ATP Cl– Cl–
Cl– Cl–

K+
K+
H 2O
A Antimycin A K+ B Antimycin A K+
 Pathophysiology of Nephrotoxic Acute Renal Failure 15.11

FIGURE 15-25
100
A graph of the phenomena depicted in Figures 15-22 through 15-
90 24, illustrating the complete temporal sequence of events following
Relative cellular changes

80 +
Na mitochondrial dysfunction. QO2—oxygen consumption.
70
60 Cl–
H 2O
50 QO2 Membrane
potential
40
30 K+ Ca++
20
10
ATP
0
0 5 10 15 20 25 30
Antimycin A Time, min

Disregulation of regulatory enzymes

BIOCHEMICAL CHARACTERISTICS OF CALPAIN

er
ATP Ca 2+
Ca2+
(1 mM)
Ca2+ Endopeptidase
(100 nM) Heterodimer: 80-kD catalytic subunit, 30-kD regulatory subunit
—Calpain and -calpain are ubiquitously distributed cytosolic isozymes
ATP —Calpain and -calpain have identical regulatory subunits but distinctive catalytic
Mitochondria Ca2+ subunits
—Calpain requires a higher concentration of Ca2+ for activation than -calpain
Phospholipids reduce the Ca2+ requirement
Substrates: cytoskeletal and membrane proteins and enzymes

FIGURE 15-26
A simplified schematic drawing of the regulation of cytosolic
free Ca2+. FIGURE 15-27
Biochemical characteristics of calpain.

FIGURE 15-28
Calpain translocation. Proposed pathways of calpain activation
and translocation. Both calpain subunits may undergo calcium
(Ca2+)-mediated autolysis within the cytosol and hydrolyze cytoso-
lic substrates. Calpains may also undergo Ca2+-mediated transloca-
tion to the membrane, Ca2+-mediated, phospholipid-facilitated
autolysis and hydrolyze membrane-associated substrates. The
autolyzed calpains may be released from the membrane and
hydrolyze cytosolic substrates. (From Suzuki and Ohno [10], and
Suzuki et al. [11]; with permission.)
15.12 Acute Renal Failure

35 40
30 35
30
LDH release, %

LDH release, %
25
25
20
20
15
15
10 10
5 5
0 0
A CON TFEC +C12 BHQ +C12 TBHP +C12 B CON TFEC +PD BHQ +PD TBHP +PD

FIGURE 15-29
A, B, Dissimilar types of calpain inhibitors block renal proximal
tubular toxicity of many agents. Renal proximal tubular suspen-
sions were pretreated with the calpain inhibitor 2 (CI2) or
PD150606 (PD). CI2 is an irreversible inhibitor of calpains that
binds to the active site of the enzyme. PD150606 is a reversible
inhibitor of calpains that binds to the calcium (Ca2+)-binding
domain on the enzyme. The toxicants used were the haloalkane
cysteine conjugate tetrafluoroethyl-L-cysteine (TFEC), the alkylat-
ing quinone bromohydroquinone (BHQ), and the model oxidant t-
butylhydroperoxide (TBHP). The release of lactate dehydrogenase
(LDH) was used as a marker of cell death. CON—control. (From
Waters et al. [12]; with permission.)

FIGURE 15-30
One potential pathway in which calcium (Ca2+) and calpains play a role in renal proximal
tubule cell death. These events are subsequent to mitochondrial inhibition and ATP deple-
tion. 1) -Calpain releases endoplasmic reticulum (er) Ca2+ stores. 2) Release of er Ca2+
stores increases cytosolic free Ca2+ concentrations. 3) The increase in cytosolic free Ca2+
concentration mediates extracellular Ca2+ entry. (This may also occur as a direct result of er
Ca2+ depletion.) 4) The influx of extracellular Ca2+ further increases cytosolic free Ca2+
concentrations. 5) This initiates the translocation of nonactivated m-calpain to the plasma
membrane (6). 7) At the plasma membrane nonactivated m-calpain is autolyzed and
hydrolyzes a membrane-associated substrate. 8) Either directly or indirectly, hydrolysis of
the membrane-associated substrate results in influx of extracellular chloride ions (Cl-). The
influx of extracellular Cl- triggers terminal cell swelling. Steps a–d represent an alternate
pathway that results in extracellular Ca2+ entry. (Data from Waters et al. [12,13,14].)

FIGURE 15-31
PROPERTIES OF PHOSPHOLIPASE A2 GROUP Biochemical characteristics of several identi-
fied phospholipase A2s.

Characteristics Secretory Cytosolic Ca2+-Independent

Localization Secreted Cytosolic Cytosolic Membrane
Molecular mass ~14 kDa ~85 kDa ~40 kDa unknown
Arachidonate preference    
Ca2+ required mM (M None None
Ca2+ role Catalysis Memb. Assoc. None None
 Pathophysiology of Nephrotoxic Acute Renal Failure 15.13

50 80
LLC-cPLA2 LLC-cPLA2
70
LLC-vector LLC-PK1
40
60

LDH release, % total

LLC-vector
AA release, %

30 50
40
20 30
20
10
10
0 0
30 60 90 120 0.0 0.1 0.2 0.3 0.4 0.5
A Time, min B [H2O2], mmol

FIGURE 15-32
80
LLC-cPLA2 The importance of the cytosolic phospholipase A2 in oxidant
70 injury. A, Time-dependent release of arachidonic acid (AA)
LLC-sPLA2
60 from LLC-PK1 cells exposed to hydrogen peroxide (0.5 mM).
LDH release, % total

LLC-vector
50
B and C, The concentration-dependent effects of hydrogen perox-
ide on LLC-PK1 cell death (using lactate dehydrogenase [LDH]
40 release as marker) after 3 hours’ exposure. Cells were transfected
30 with 1) the cytosolic PLA2 (LLC-cPLA2), 2) the secretory PLA2
20 (LLC-sPLA2), 3) vector (LLC-vector), or 4) were not transfected
(LLC-PK1). Cells transfected with cytosolic PLA2 exhibited
10
greater AA release and cell death in response to oxidant exposure
0 than cells transfected with the vector or secretory PLA2 or not
0.0 0.1 0.2 0.3 0.4 0.5 transfected. These results suggest that activation of cytosolic
C [H2O2], mmol PLA2 during oxidant injury contributes to cell injury and death.
(From Sapirstein et al. [15]; with permission.)

50
200
100
Residual double-stranded DNA, %
∆ Increase in caspase activity,

40
150 75
Cell death, %
units/mg protein

30

100 50
20

50 25 10

0 0
0
cin A

cin A
r II

r II
rI

rI
trol

trol

0 10 20 30
bito

bito
bito

bito
Con

Con
imy

imy

Time of antimycin A treatment,

Inhi

Inhi
Inhi

Inhi
Ant

Ant

A min B C

FIGURE 15-33
Potential role of caspases in cell death in LLC-PK1 cells exposed to Inhibitor 1 is IL-1 converting enzyme inhibitor 1 (YVAD-CHO) and
antimycin A. A, Time-dependent effects of antimycin A treatment on inhibitor II is CPP32/apopain inhibitor (DEVD-CHO). These results
caspase activity in LLC-PK1 cells. B, C, The effect of two capase suggest that caspases are activated after mitochondrial inhibition and
inhibitors on antimycin A–induced DNA damage and cell death, respec- that caspases may contribute to antimycin A–induced DNA damage
tively. Antimycin A is an inhibitor of mitochondrial electron transport. and cell death. (From Kaushal et al. [16]; with permission.)
15.14 Acute Renal Failure
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