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 environmental pollutants that cause nephrotoxicity has increased, it has
become clear that chemicals with very diverse chemical structures produce nephrotoxicity. For example, the heavy metal HgCl2, the mycotoxin 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 different toxicants. Nevertheless, there are similarities between chemicalinduced acute tubular injury and ischemia/reperfusion injury.
The tubular cells of the kidney are particularly vulnerable to toxicant-mediated injury due to their disproportionate exposure to circulating chemicals and transport processes that result in high intracellular 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 macromolecule(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
stress both lipid peroxidation and protein oxidation have been shown
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 dependence of renal function on aerobic metabolism. The loss of ATP leads
CHAPTER
15
15.2
Acute Renal Failure
to disruption of cellular ion homeostasis with decreased cellular
K+ content, increased Na+ content and membrane depolarization. Increased cytosolic free Ca2+ concentrations can occur in
the early or late phase of cell injury and plays a critical role leading to cell death. The increase in Ca2+ can activate calcium activated 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, followed 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.
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-mediated renal cell injury and death that leads to the loss of tubular
epithelial cells, tubular obstruction, “backleak” of the glomerular filtrate and a decreased glomerular filtration rate. The recovery from the structural and functional damage following chemical exposures is dependent on the repair of sublethally-injured
and regeneration of noninjured cells.
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.
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 15-1
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
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
Aminoglycosides (gentamicin, tobramycin,
amikacin, netilmicin)
Amphotericin B
Cephalosporins
Ciprofloxacin
Demeclocycline
Penicillins
Pentamidine
Polymixins
Rifampin
Sulfonamides
Tetracycline
Vancomycin
Chemotherapeutic agents
Adriamycin
Cisplatin
Methotraxate
Mitomycin C
Nitrosoureas
(eg, streptozotocin, Iomustine)
Radiocontrast media
Ionic (eg, diatrizoate, iothalamate)
Nonionic (eg, metrizamide)
Immunosuppressive agents
Cyclosporin A
Tacrolimus (FK 506)
Antiviral agents
Acyclovir
Cidovir
Foscarnet
Valacyclovir
Heavy metals
Cadmium
Gold
Mercury
Lead
Arsenic
Bismuth
Uranium
Organic solvents
Ethylene glycol
Carbon tetrachloride
Unleaded gasoline
Vasoactive agents
Nonsteroidal anti-inflammatory
drugs (NSAIDs)
Ibuprofen
Naproxen
Indomethacin
Meclofenemate
Aspirin
Piroxicam
Angiotensin-converting
enzyme inhibitors
Captopril
Enalopril
Lisinopril
Angiotensin receptor antagonists
Losartan
Other drugs
Acetaminophen
Halothane
Methoxyflurane
Cimetidine
Hydralazine
Lithium
Lovastatin
Mannitol
Penicillamine
Procainamide
Thiazides
Lindane
Endogenous compounds
Myoglobin
Hemoglobin
Calcium
Uric acid
Oxalate
Cystine
FIGURE 15-4
Exogenous and endogenous chemicals that cause acute renal failure.
Proximal convoluted tubule
(S1/S2 segments)
Aminoglycosides
Cephaloridine
Cadmium chloride
Potassium dichromate
Renal vessels
NSAIDs
ACE inhibitors
Cyclosporin A
Papillae
Phenacetin
Glomeruli
Interferon–α
Gold
Penicillamine
Proximal straight tubule
(S3 segment)
Cisplatin
Mercuric chloride
Dichlorovinyl–L–cysteine
Interstitium
Cephalosporins
Cadmium
NSAIDs
FIGURE 15-5
Nephrotoxicants may act at different sites in the kidney, resulting
in altered renal function. The sites of injury by selected nephrotoxicants are shown. Nonsteroidal anti-inflammatory drugs (NSAIDs),
angiotensin-converting enzyme (ACE) inhibitors, cyclosporin A,
and radiographic contrast media cause vasoconstriction. Gold,
interferon-alpha, and penicillamine can alter glomerular function
and result in proteinuria and decreased renal function. Many
nephrotoxicants damage tubular epithelial cells directly.
Aminoglycosides, cephaloridine, cadmium chloride, and potassium
dichromate affect the S1 and S2 segments of the proximal tubule,
whereas cisplatin, mercuric chloride, and dichlorovinyl-L-cysteine
affect the S3 segment of the proximal tubule. Cephalosporins, cadmium chloride, and NSAIDs cause interstitial nephritis whereas
phenacetin causes renal papillary necrosis.
15.4
Acute Renal Failure
Prerenal azotemia
Renal vasoconstriction
Intravascular
volume
Increased tubular pressure
n
e
p
h
r
o
t
o
x
i
t c
o a
n
t
E
x
p
o
s
u
r
e
Tubular obstruction
Intratubular
casts
Sympathetic
tone
"Back-leak" of glomerular filtrate
Functional
abnormalties
GFR
Capillary permeability
Endothelial injury
Tubular damage
Persistent medullary hypoxia
Physical constriction
of medullary vessels
Hemodynamic
Glomerular
hydrostatic
alterations
pressure
Intrarenal
vasoconstriction Perfusion pressure
Efferent tone
Afferent tone
Glomerular factors
Hypertension
Endothelin
Nitric oxide
Thromboxane
Prostaglandins
Renal and systemic
vasoconstriction
Intrarenal factors
Obstruction
Vascular smooth muscle
sensitivity to vasoconstrictors
Cyclosporin A
Angiotensin II
Tubular cell injury
Glomerular ultrafiltration
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 hydrostatic pressure. Some nephrotoxicants may decrease glomerular
function, leading to proteinuria and decreased renal function.
Striped interstitial
fibrosis
GFR
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 endothelium 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 vasculature to vasoconstrictors, activate the renin-angiotensin system, and
increase angiotensin II levels. All of these changes lead to vasoconstriction and hypertension. Vasoconstriction in the kidney contributes 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
Nephrotoxic insult
to the nephron
Uninjured cells
Compensatory
hypertrophy
Cellular
adaptation
Injured cells
Cellular
proliferation
Re-epithelialization
Cell death
Cellular
repair
Cellular adaptation
Differentiation
Structural and functional recovery of the nephron
FIGURE 15-8
The nephron’s response to a nephrotoxic insult. After a population
of cells are exposed to a nephrotoxicant, the cells respond and ultimately the nephron recovers function or, if cell death and loss is
extensive, nephron function ceases. Terminally injured cells undergo cell death through oncosis or apoptosis. Cells injured sublethally undergo repair and adaptation (eg, stress response) in response
to the nephrotoxicant. Cells not injured and adjacent to the injured
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
injury. Finally the uninjured cells may also undergo adaptation in
response to nephrotoxicant exposure.
Pathophysiology of Nephrotoxic Acute Renal Failure
Loss of polarity, tight junction
integrity, cell–substrate adhesion,
simplification of brush border
Intact tubular epithelium
Cell death
Toxic injury
Necrosis
α
β
Cast formation
and tubuler
obstruction
Na+/K+=ATPase
β1 Integrin
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
Migrating
spreading cells
Cell
proliferation
Basement
membrane
Toxicant inhibition
of cell repair
Apoptosis
Sloughing of viable
and nonviable cells
with intraluminal
cell-cell adhesion
Cytoskeleton
Extracellular matrix
Sublethally
injured cells
15.5
Toxicant inhibition
of cell migration/spreading
Toxicant inhibition
of cell proliferation
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
Potential sites where nephrotoxicants can interfere with the structural and functional recovery of nephrons.
15.6
Acute Renal Failure
140
Percent of control
120
100
Oncosis
Apoptosis
80
60
Cell number/confluence
Mitochondrial function
Active Na+ transport
+
Na -coupled glucose transport
GGT activity
40
20
0
0
1
2
3
4
5
Blebbing
Budding
6
Time after exposure, d
FIGURE 15-11
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 mitochondrial 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 physiologic functions are diminished after oxidant injury and that a hierarchy 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].)
Necrosis
Phagocytosis
inflammation
Phagocytosis
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 morphologically by cell and organelle swelling, plasma membrane blebbing, 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 breakdown and cell debris. (From Majno and Joris [4]; with permission.)
Mechanisms of Toxicant-Mediated Cellular Injury
Transport and biotransformation
Toxicant whose primary
mechanism of action is
ATP depletion
Toxicants in general
Apoptosis
Oncosis
Cell death
Cell death
Oncosis
Apoptosis
Toxicant concentration
Toxicant concentration
FIGURE 15-13
The general relationship between oncosis and apoptosis after
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
nephrotoxicant is ATP depletion, oncosis may be the predominant
cause of cell death with limited apoptosis occurring.
Pathophysiology of Nephrotoxic Acute Renal Failure
GSH-Hg-GSH
GSH-Hg-GSH
CYS-Hg-CYS
GSH-Hg-GSH
γ-GT
?
GLY-CYS-Hg-CYS-GLY
Acivicin
CYS-Hg-CYS
Lumen
Dipeptidase
+
CYS-Hg-CYS Na
Neutral amino
acid transporter
–
R-Hg-R–
CYS-Hg-CYS
GSH-Hg-GSH
Na+ α-Ketoglutarate α-Ketoglutarate
Dicarboxylate
Organic anion
transporter
transporter
Proximal
tubular cell
Blood
Urine
CYS-Hg-CYS Na
+
Organic anions
(PAH or
probenecid)
α-Ketoglutarate
Na+
Dicarboxylic
acids
α-Ketoglutarate
Biotransformation
Altered activity of
critical macromolecules
FIGURE 15-14
The importance of cellular transport in mediating toxicity.
Proximal tubular uptake of inorganic mercury is thought to be the
result of the transport of mercuric conjugates (eg, diglutathione
mercury conjugate [GSH-Hg-GSH], dicysteine mercuric conjugate
[CYS-Hg-CYS]). At the luminal membrane, GSH-Hg-GSH appears
to be metabolized by (-glutamyl transferase ((-GT) and a dipeptidase to form CYS-Hg-CYS. The CYS-Hg-CYS may be taken up by
an amino acid transporter. At the basolateral membrane, mercuric
conjugates appear to be transported by the organic anion transporter. (-Ketoglutarate and the dicarboxylate transporter seem to
play important roles in basolateral membrane uptake of mercuric
conjugates. Uptake of mercuric-protein conjugates by endocytosis
may play a minor role in the uptake of inorganic mercury transport. PAH—para-aminohippurate. (Courtesy of Dr. R. K. Zalups.)
–
R-Hg-R–
CYS-Hg-CYS
GSH-Hg-GSH
Toxicant
High-affinity binding
to macromolecules
15.7
Reactive intermediate
Redox cycling
Covalent binding
to macromolecules
Increased reactive
oxygen species
Damage to critical
macromolecules
Oxidative damage to
critical macromolecules
FIGURE 15-15
Covalent and noncovalent binding versus oxidative stress mechanisms of cell injury. Nephrotoxicants are generally thought to produce cell injury and death through one of two mechanisms, either
alone or in combination. In some cases the toxicant may have a
high affinity for a specific macromolecule or class of macromolecules that results in altered activity (increase or decrease) of these
molecules, resulting in cell injury. Alternatively, the parent nephrotoxicant may not be toxic until it is biotransformed into a reactive
intermediate that binds covalently to macromolecules and in turn
alters their activity, resulting in cell injury. Finally, the toxicant may
increase reactive oxygen species in the cells directly, after being biotransformed into a reactive intermediate or through redox cycling.
The resulting increase in reactive oxygen species results in oxidative damage and cell injury.
Cell injury
Cell repair
Cell death
Plasma RSG
Plasma RSG
R-SG
R + SG
1.
R-SG
6.
Glomerular filtration
2.
R-SG 3.
4. γ-Glu
Na+
Plasma
R-Cys
7.
R-Cys
Na+
Plasma
R-NAC
8.
R-NAC
Na+
5.
R-Cys
12. NH3+H3CCOCO2H
10. 11. R-SH 13.
Covalent binding
Cell injury
R-NAC
Basolateral
membrane
9.
Brush border
membrane
R-Cys
R-NAC
Gly
FIGURE 15-16
This figure illustrates the renal proximal tubular uptake, biotransformation, and toxicity of glutathione and cysteine conjugates and mercapturic acids of haloalkanes and haloalkenes (R). 1) Formation of a
glutathione conjugate within the renal cell (R-SG). 2) Secretion of the
R-SG into the lumen. 3) Removal of the -glutamyl residue (-Glu)
by -glutamyl transferase. 4) Removal of the glycinyl residue (Gly) by
a dipeptidase. 5) Luminal uptake of the cysteine conjugate (R-Cys).
Basolateral membrane uptake of R-SG (6), R-Cys (7), and a mercapturic acid (N-acetyl cysteine conjugate; R-NAC)(8). 9) Secretion of
R-NAC into the lumen. 10) Acetylation of R-Cys to form R-NAC.
11) Deacetylation of R-NAC to form R-Cys. 12) Biotransformation
of the penultimate nephrotoxic species (R-Cys) by cysteine conjugate
-lyase to a reactive intermediate (R-SH), ammonia, and pyruvate.
13) Binding of the reactive thiol to cellular macromolecules (eg, lipids,
proteins) and initiation of cell injury. (Adapted from Monks and Lau
[5]; with permission.)
15.8
Acute Renal Failure
A
B
Representative
starting
material
Submitochondrial fractions
A. Untreated
B. TFEC (30 mg/kg)
Mr (kDa)
228
109
P99
P84
P66
P52
P42
70
Inter
Outer
Matrix
Inner
Inter
Outer
C
Matrix
Inner
44
FIGURE 15-17
Covalent binding of a nephrotoxicant
metabolite in vivo to rat kidney tissue, localization of binding to the mitochondria, and
identification of three proteins that bind to
the nephrotoxicant. A, Binding of tetrafluoroethyl-L-cysteine (TFEC) metabolites in vivo
to rat kidney tissue detected immunohistochemically. 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
fractionation of renal cortical mitochondria
from untreated and TFEC treated rats and
immunoblot analysis revealed numerous proteins that bind to the nephrotoxicant (innerinner membrane, matrix-soluble matrix,
outer-outer membrane, inter-intermembrane
space). The identity of three of the proteins
that bound to the nephrotoxicant: P84,
mortalin (HSP70-like); P66, HSP 60; and
P42, aspartate aminotransferase. Mr—relative molecular weight. (From Hayden et al.
[6], and Bruschi et al. [7]; with permission.)
Lipid peroxidation and mitochondrial dysfunction
HH
HO•
•H
R
Lipid
H 2O
Hydrogen abstraction
R
Lipid radical
Diene conjugation
R
•
H
O2
R
•O–O H
LH
O O
O
O
HOO H
Fe(II)
Fe(III)
Malondialdehyde
•O H
Lipid radical, conjugated diene
Oxygen addition
R
Lipid peroxyl radical
Hydrogen abstraction
L•
R
Lipid hydroperoxide
Fenton reaction
HO•
R
Lipid alkoxyl radical
Fragmentation
H
H
•
H
LH •
L
R
H
O
Lipid aldehyde
H
H
H
H
Ethane
FIGURE 15-18
A simplified scheme of lipid peroxidation. The first step, hydrogen
abstraction from the lipid by a radical (eg, hydroxyl), results in the
formation of a lipid radical. Rearrangement of the lipid radical
results in conjugated diene formation. The addition of oxygen
results in a lipid peroxyl radical. Additional hydrogen abstraction
results in the formation of a lipid hydroperoxide. The Fenton reaction produces a lipid alkoxyl radical and lipid fragmentation,
resulting in lipid aldehydes and ethane. Alternatively, the lipid peroxyl radical can undergo a series of reactions that result in the formation of malondialdehyde.
15.9
Pathophysiology of Nephrotoxic Acute Renal Failure
50
Control
DCVC
DCVC + DEF (1 mM)
DCVC + DPPD (50µM)
80
LDH release, %
40
LDH release, %
100
Control
TBHP (0.5 mmol)
TBHP + DEF (1 mM)
TBHP + DPPD (2 µM)
30
20
10
60
40
20
0
0
0
1
2
3
A
4
5
6
0
1
2
B
Time, h
1.2
3
4
5
6
Time, h
2.0
+1 mM DEF
Lipid peroxidation,
nmol MDA•mg protein–1
Lipid peroxidation,
nmol MDA•mg protein–1
1.0
0.8
0.6
0.4
0.2
0.0
C
Control
TBHP
+1 mM DEF
+2 µM DPPD
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-butylhydroperoxide (TBHP) and the halocarbon-cysteine conjugate
dichlorovinyl-L-cysteine (DCVC) caused extensive lipid peroxidation after 1 hour of exposure and cell death (lactate dehydrogenase (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
ALTERATION OF RENAL TUBULAR CELL
ENERGETICS AFTER EXPOSURE TO TOXICANTS
Decreased oxygen delivery secondary to vasoconstriction
Inhibition of mitochondrial respiration
Increased tubular cell oxygen consumption
1.6
+50 µM DPPD
1.2
0.8
0.4
0.0
D
Control
DCVC
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-mediated 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
Mechanisms by which nephrotoxicants can alter renal tubular
cell energetics.
15.10
Acute Renal Failure
FIGURE 15-21
Some of the mitochondrial targets of nephrotoxicants: 1) nicotinamide adenine dinucleotide (NADH) dehydrogenase; 2) succinate dehydrogenase; 3) coenzyme
Q–cytochrome C reductase; 4) cytochrome
C; 5) cytochrome C oxidase; 6) cytochrome
Aa3; 7) H+-Pi contransporter; 8) F0F1ATPase; 9) adenine triphosphate/diphosphate
(ATP/ADP) translocase; 10) protonophore
(uncoupler); 11) substrate transporters.
Substrates
11
Cephaloridine
Atractyloside
Ochratoxin A
TCA
cycle
ADP
Bromohydroquinone
9
ATP
Dichlorovinyl–L–cysteine
Tetrafluoroethyl–L–cysteine
Pentachlorobutadienyl–L–cysteine
Citrinin
Ochratoxin A
Hg2+
CN–
1
H+
ATP
H+
3
H+
Oligomycin
4
5
H+
Pi
6
O2
8
2
Pi
H+
7
Matrix
H 2O
Ochratoxin A
10
Pentachlorobutadienyl–L–cysteine
H+
Citrinin
FCCP
Inner membrane
Outer membrane
Disruption of ion homeostasis
Na+
Na+
ATPase
–
–
–
ATP
Na+
Na+
ATPase
ATP
–
Cl–
Cl–
–
Cl
–
–
–
+
K
K+ –
K+
A
Cl–
B
Antimycin A
K+
Na+
Na+
ATPase
Na+
ATPase
ATP
ATP
Cl–
K+
Cl–
Cl–
Cl–
K+
A Antimycin A
100
90
80
70
60
50
40
30
20
10
0
Na+
K+
B Antimycin A
K+
H 2O
Membrane
potential
QO2
K+
H 2O
ATP
0
FIGURE 15-22
Early ion movements after mitochondrial dysfunction. A, A control
renal proximal tubular cell. Within minutes of mitochondrial inhibition (eg, by antimycin A), ATP levels drop, resulting in inhibition of
the Na+, K+-ATPase. B, Consequently, Na+ influx, K+ efflux, membrane depolarization, and a limited degree of cell swelling occur.
Na+
Relative cellular changes
H 2O
Na+
5
Antimycin A
10
15
20
25
30
Time, min
FIGURE 15-23
A graphic of the phenomena diagrammed in Figure 15-22.
FIGURE 15-24
The late ion movements after mitochondrial dysfunction that leads
to cell death/lysis. A, Cl- influx occurs as a distinct step subsequent
to Na+ influx and K+ efflux. B, Following Cl- influx, additional
Na+ and water influx occur resulting in terminal cell swelling.
Ultimately cell lysis occurs.
Relative cellular changes
Pathophysiology of Nephrotoxic Acute Renal Failure
100
90
80
70
60
50
40
30
20
10
0
Na
15.11
FIGURE 15-25
A graph of the phenomena depicted in Figures 15-22 through 1524, illustrating the complete temporal sequence of events following
mitochondrial dysfunction. QO2—oxygen consumption.
+
Cl–
Membrane
potential
QO2
H 2O
Ca++
K+
ATP
0
5
10
Antimycin A
15
20
25
30
Time, min
Disregulation of regulatory enzymes
er
Ca2+
BIOCHEMICAL CHARACTERISTICS OF CALPAIN
ATP
2+
Ca
(1 mM)
Ca2+
(100 nM)
Mitochondria
ATP
Ca2+
FIGURE 15-26
A simplified schematic drawing of the regulation of cytosolic
free Ca2+.
Endopeptidase
Heterodimer: 80-kD catalytic subunit, 30-kD regulatory subunit
—Calpain and -calpain are ubiquitously distributed cytosolic isozymes
—Calpain and -calpain have identical regulatory subunits but distinctive catalytic
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 15-27
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 cytosolic substrates. Calpains may also undergo Ca2+-mediated translocation 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.)
Acute Renal Failure
35
40
30
35
LDH release, %
LDH release, %
15.12
25
20
15
30
25
20
15
10
10
5
5
0
A
0
CON
TFEC
+C12
BHQ
+C12
TBHP
B
+C12
FIGURE 15-29
A, B, Dissimilar types of calpain inhibitors block renal proximal
tubular toxicity of many agents. Renal proximal tubular suspensions 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
CON
TFEC
+PD
BHQ
+PD
TBHP
+PD
domain on the enzyme. The toxicants used were the haloalkane
cysteine conjugate tetrafluoroethyl-L-cysteine (TFEC), the alkylating quinone bromohydroquinone (BHQ), and the model oxidant tbutylhydroperoxide (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 depletion. 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
Biochemical characteristics of several identified phospholipase A2s.
PROPERTIES OF PHOSPHOLIPASE A2 GROUP
Characteristics
Secretory
Cytosolic
Localization
Molecular mass
Arachidonate preference
Ca2+ required
Ca2+ role
Secreted
~14 kDa
mM
Catalysis
Cytosolic
~85 kDa
(M
Memb. Assoc.
Ca2+-Independent
Cytosolic
~40 kDa
None
None
Membrane
unknown
None
None
15.13
Pathophysiology of Nephrotoxic Acute Renal Failure
50
80
LLC-cPLA2
LLC-vector
LDH release, % total
AA release, %
40
LLC-cPLA2
LLC-PK1
LLC-vector
70
30
20
10
60
50
40
30
20
10
0
0
30
60
A
90
120
0.0
LLC-cPLA2
LLC-sPLA2
LLC-vector
50
40
30
20
10
0
0.0
C
0.1
0.2
0.3
[H2O2], mmol
0.4
0.5
0.5
FIGURE 15-33
Potential role of caspases in cell death in LLC-PK1 cells exposed to
antimycin A. A, Time-dependent effects of antimycin A treatment on
caspase activity in LLC-PK1 cells. B, C, The effect of two capase
inhibitors on antimycin A–induced DNA damage and cell death, respectively. Antimycin A is an inhibitor of mitochondrial electron transport.
r II
bito
rI
bito
Con
trol
C
Inhi
B
Inhi
0
Ant
imy
cin A
Cell death, %
0
r II
30
10
20
Time of antimycin A treatment,
min
bito
0
20
10
Con
trol
0
30
25
Inhi
50
50
rI
100
40
75
bito
150
100
Ant
imy
cin A
Residual double-stranded DNA, %
∆ Increase in caspase activity,
units/mg protein
0.4
50
200
A
0.3
[H2O2], mmol
Inhi
LDH release, % total
60
0.2
FIGURE 15-32
The importance of the cytosolic phospholipase A2 in oxidant
injury. A, Time-dependent release of arachidonic acid (AA)
from LLC-PK1 cells exposed to hydrogen peroxide (0.5 mM).
B and C, The concentration-dependent effects of hydrogen peroxide on LLC-PK1 cell death (using lactate dehydrogenase [LDH]
release as marker) after 3 hours’ exposure. Cells were transfected
with 1) the cytosolic PLA2 (LLC-cPLA2), 2) the secretory PLA2
(LLC-sPLA2), 3) vector (LLC-vector), or 4) were not transfected
(LLC-PK1). Cells transfected with cytosolic PLA2 exhibited
greater AA release and cell death in response to oxidant exposure
than cells transfected with the vector or secretory PLA2 or not
transfected. These results suggest that activation of cytosolic
PLA2 during oxidant injury contributes to cell injury and death.
(From Sapirstein et al. [15]; with permission.)
80
70
0.1
B
Time, min
Inhibitor 1 is IL-1 converting enzyme inhibitor 1 (YVAD-CHO) and
inhibitor II is CPP32/apopain inhibitor (DEVD-CHO). These results
suggest that caspases are activated after mitochondrial inhibition and
that caspases may contribute to antimycin A–induced DNA damage
and cell death. (From Kaushal et al. [16]; with permission.)
15.14
Acute Renal Failure
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