Pathophysiology of Ischemic

Acute Renal Failure:

Cytoskeletal Aspects
Bruce A. Molitoris
Robert Bacallao

I
schemia remains the major cause of acute renal failure (ARF) in the
adult population [1]. Clinically a reduction in glomerular filtration
rate (GFR) secondary to reduced renal blood flow can reflect
prerenal azotemia or acute tubular necrosis (ATN). More appropriate
terms for ATN are acute tubular dysfunction or acute tubular injury,
as necrosis only rarely is seen in renal biopsies, and renal tubular cell
injury is the hallmark of this process. Furthermore, the reduction in GFR
during acute tubular dysfunction can now, in large part, be related to
tubular cell injury. Ischemic ARF resulting in acute tubular dysfunc-
tion secondary to cell injury is divided into initiation, maintenance,
and recovery phases. Recent studies now allow a direct connection to
be drawn between these clinical phases and the cellular phases of
ischemic ARF (Fig. 13-1). Thus, renal function can be directly related
to the cycle of cell injury and recovery.
Renal proximal tubule cells are the cells most injured during renal
ischemia (Fig. 13-2) [2,3]. Proximal tubule cells normally reabsorb 70%
to 80% of filtered sodium ions and water and also serve to selectively
reabsorb other ions and macromolecules. This vectorial transport across
the cell from lumen to blood is accomplished by having a surface mem-
brane polarized into apical (brush border membrane) and basolateral
membrane domains separated by junctional complexes (Fig. 13-3) [4].
Apical and basolateral membrane domains are biochemically and CHAPTER
functionally different with respect to many parameters, including
enzymes, ion channels, hormone receptors, electrical resistance,

13
membrane transporters, membrane lipids, membrane fluidity, and
cytoskeletal associations. This epithelial cell polarity is essential for
normal cell function, as demonstrated by the vectorial transport of
sodium from the lumen to the blood (see Fig. 13-3). The establishment
13.2 Acute Renal Failure
and maintenance of this specialized organization is a dynamic
and ATP dependent multistage process involving the formation
and maintenance of cell-cell and cell-substratum attachments
and the targeted delivery of plasma membrane components to
the appropriate domains [5]. These processes are very dependent
on the cytoskeleton, in general, and the cytoskeletal membrane
interactions mediated through F-actin (see Fig. 13-2, 13-3), in
particular.
Ischemia in vivo and cellular ATP depletion in cell culture
models (“chemical ischemia”) are known to produce character-
istic surface membrane structural, biochemical, and functional
abnormalities in proximal tubule cells. These alterations occur
in a duration-dependent fashion and are illustrated in Figures
13-2 and 13-3 and listed in Figure 13-4. Ischemia-induced
alterations in the actin cytoskeleton have been postulated to
mediate many of the aforementioned surface membrane
changes [2,6,7]. This possible link between ischemia-induced
actin cytoskeletal alterations and surface membrane structural
and functional abnormalities is suggested by several lines. First,
the actin cytoskeleton is known to play fundamental roles in
surface membrane formation and stability, junctional complex
formation and regulation, Golgi structure and function, and
cell–extracellular membrane attachment [2,4,5,8]. Second,
proximal tubule cell actin cytoskeleton is extremely sensitive to
ischemia and ATP depletion [9,10]. Third, there is a strong
correlation between the time course of actin and surface mem-
brane alterations during ischemia or ATP depletion [2,9,10].
Finally, many of the characteristic surface membrane changes
RELATIONSHIP BETWEEN THE CLINICAL AND CELLULAR
PHASES OF ISCHEMIC ACUTE RENAL FAILURE
Clinical Phases Cellular Phases
Prerenal azotemia Vascular and cellular adaptation
↓ ↓
Initiation ATP depletion, cell injury
↓ ↓
Maintenance Repair, migration, apoptosis, proliferation
↓ ↓
Recovery Cellular differentiation
induced by ischemia can be mimicked by F-actin disassembly
mediated by cytochalasin D [11]. Although these correlations
are highly suggestive of a central role for actin alterations in the
pathophysiology of ischemia-induced surface membrane dam-
age they fall short in providing mechanistic data that directly
relate actin cytoskeletal changes to cell injury.
Proximal tubule cell injury during ischemia is also known to
be principally responsible for the reduction in GFR. Figure 13-5
illustrates the three known pathophysiologic mechanisms that
relate proximal tubule cell injury to a reduction in GFR.
Particularly important is the role of the cytoskeleton in mediating
these three mechanisms of reduced GFR. First, loss of apical
membrane into the lumen and detachment of PTC result in sub-
strate for cast formation. Both events have been related to actin
cytoskeletal and integrin polarity alterations [12–15]. Cell
detachment and the loss of integrin polarity are felt to play a
central role in tubular obstruction (Fig. 13-6). Actin cytoskeletal-
mediated tight junction opening during ischemia occurs and
results in back-leak of glomerular filtrate into the blood. This
results in ineffective glomerular filtration (Fig. 13-7). Finally,
abnormal proximal sodium ion reabsorption results in large
distal tubule sodium delivery and a reduction in GFR via tubu-
loglomerular feedback mechanisms [2,16,17].
In summary, ischemia-induced alterations in proximal tubule
cell surface membrane structure and function are in large part
responsible for cell and organ dysfunction. Actin cytoskeletal
dysregulation during ischemia has been shown to be responsi-
ble for much of the surface membrane structural damage.
FIGURE 13-1
Relationship between the clinical and cellular phases of ischemic
acute renal failure. Prerenal azotemia results from reduced renal
blood flow and is associated with reduced organ function (decreased
glomerular filtration rate), but cellular integrity is maintained
through vascular and cellular adaptive responses. The initiation
phase occurs when renal blood flow decreases to a level that results
in severe cellular ATP depletion that, in turn, leads to acute cell
injury. Severe cellular ATP depletion causes a constellation of cellular
alterations culminating in proximal tubule cell injury, cell death, and
organ dysfunction [2]. During the clinical phase known as mainte-
nance, cells undergo repair, migration, apoptosis, and proliferation in
an attempt to re-establish and maintain cell and tubule integrity [3].
This cellular repair and reorganization phase results in slowly
improving cell and organ function. During the recovery phase,
cell differentiation continues, cells mature, and normal cell and
organ function return [18].
 Pathophysiology of Ischemic Acute Renal Failure: Cytoskeletal Aspects 13.3

A B C

D E F
FIGURE 13-2
Ischemic acute renal failure in the rat kidney. Light A, B, trans-
mission electron, C, D, and immunofluorescence E, F, micro-
MV scopy of control renal cortical sections, A, C, E, and after mod-
erate ischemia induced by 25 minutes of renal artery occlusion,
B, D, F. Note the extensive loss of apical membrane structure,
B, D, in proximal (PT) but not distal tubule cells. This has been
shown to correlate with extensive alterations in F-actin as shown
ZO by FITC-phalloidin labeling, E, F. G, Drawing of a proximal
tubule cell under physiologic conditions. Note the orderly
arrangement of the actin cytoskeleton and its extensive interac-
tion with the surface membrane at the zonula occludens (ZO,
ZA tight junction) zonula adherens (ZA, occludens junction), inter-
actions with ankyrin to mediate Na+, K+-ATPase [2] stabilization
MT
x and cell adhesion molecule attachment [5,8]. The actin cyto-
N skeleton also mediates attachment to the extracellular matrix
(ECM) via integrins [12,15]. Microtubules (MT) are involved in
the polarized delivery of endocytic and exocytic vesicles to the
surface membrane. Finally, F-actin filaments bundle together via
x actin-bundling proteins [19] to mediate amplification of the api-
HD cal surface membrane via microvilli (MV). The actin bundle
attaches to the surface membrane by the actin-binding proteins
ECM myosin I and ezrin [19,20].

G
13.4 Acute Renal Failure

FIGURE 13-3
Proximal tubule cell Fate of an injured proximal tubule cell.
The fate of a proximal tubule cell after an
ischemic episode depends on the extent and
ADP ATP duration of the ischemia. Cell death can
+
+P
1 K occur immediately via necrosis or in a more
programmed fashion (apoptosis) hours to
Ischemia Necrosis days after the injury. Fortunately, most cells
Death recover either in a direct fashion or via
+ Na+
d Na d an intermediate undifferentiated cellular
ate ure
nti Inj
+ Recovery +
re K ADP K ADP Apoptosis pathway. Again, the severity of the injury
D iffe ATP +P ATP +P
1 1 determines the route taken by a particular
ECM cell. Adjacent cells are often injured to
Na+ varying degrees, especially during mild to
moderate ischemia. It is believed that the
rate of organ functional recovery relates
directly to the severity of cell injury during
d the initiation phase. ECM—extracellular
ate
enti
iffe
r membrane; Na+—sodium ion; K+—potassi-
d
Un um ion; P1—phosphate.

FIGURE 13-4
ISCHEMIA INDUCED PROXIMAL TUBULE CELL ALTERATIONS Ischemia induced proximal tubule cell
alterations.

Alterations References
Surface Membrane Alterations
1. Microvilli fusion, internalization, fragmentation and luminal shedding resulting in loss of [21]
surface membrane area and tubular obstruction
2. Loss of surface membrane polarity for lipids and proteins [2,22,23]
3. Junctional complex dissociation with unregulated paracellular permeability (backleak) [6,24–27]
4. Reduced PTC vectorial transport [28]
Actin Cytoskeletal Alterations
1. Polymerization of actin throughout the cell cytosol [6,16,29]
2. Disruption and delocalization of F-actin structures including stress fibers, cortical actin [2,7,16]
and the junctional ring
3. Accumulation of intracellular F-actin aggregates containing surface membrane pro- [20,30]
teins—myosin I, the tight junction proteins ZO-1, ZO-2, cingulin
4. Disruption and dissociation of the spectrin cytoskeleton [31,32]
5. Disruption of microtubules during early reflow in vivo [33]
6. The cytoskeleton of proximal tubule cells, as compared to distal tubule cells, is more [6,16,34]
sensitive to ischemia in vivo and ATP depletion in vitro
 Pathophysiology of Ischemic Acute Renal Failure: Cytoskeletal Aspects 13.5

FIGURE 13-5
A Normal
Mechanisms of proximal tubule cell—medi-
Afferent arteriole Efferent arteriole ated reductions in glomerular filtration rate
(GFR) following ischemic injury. A, GFR
Glomerular depends on four factors: 1) adequate blood
plasma flow flow to the glomerulus; 2) an adequate
glomerular capillary pressure as determined
by afferent and efferent arteriolar resis-
Glomerular tance; 3) glomerular permeability; and
hydrostatic 4) low intratubular pressure. B, Afferent
pressure
Glomerular arteriolar constriction diminishes GFR by
filtration reducing blood flow—and, therefore,
Intratubular glomerular capillary pressure. This occurs
pressure in response to a high distal sodium delivery
and is mediated by tubular glomerular
feedback. C, Obstruction of the tubular
B Afferent C D
arteriolar Obstruction Backleak lumen by cast formation increases tubular
constriction pressure and, when it exceeds glomerular
capillary pressure, a marked decrease or no
filtration occurs. D, Back-leak occurs when
the paracellular space between cells is open
for the flux of glomerular filtrate to leak
back into the extracellular space and into
Glomerular Obstructing Leakage of the blood stream. This is believed to occur
pressure cast filtrate through open tight junctions.

RG
RG

RGD

RG
D
RGD RG RG
D
D

FIGURE 13-6
Overview of potential therapeutic effects of cyclic integrin-binding
peptides. A, During ischemic injury, tubular obstruction occurs as a
result of loss of apical membrane, cell contents, and detached cells
released into the lumen. B, Also, basolateral integrins diffuse to the
apical region of the cell. Biotinylated cyclic peptides containing the
sequence cRGDDFV bind to desquamated cells in the ascending
limb of the loop of Henle and in proximal tubule cells in ischemic
A rat kidneys. The desquamated cells can adhere to injured cells or
aggregate, causing tubule obstruction.
(Continued on next page)
13.6 Acute Renal Failure

FIGURE 13-6 (Continued)

cRGDDFLG cRDADFV C, When cyclic peptides that contain the RGD canonical binding
1400
x x cRGDDFV Control site of integrins are perfused intra-arterially, the peptides amelio-
1200 rate the extent of acute renal failure, as demonstrated by a higher
glomerular filtration rate (GFR) in rats receiving peptide containing
1000 *** **
x the RGD sequence. B, Proposed mechanism of renal protection by
cyclic RGD peptides. By adhering to the RGD binding sites of the
GFR, µl/min

x x integrins located on the apical plasma membrane or distributed

800
randomly on desquamated cells, the cyclic peptide blocks cellular
* aggregation and tubular obstruction [12–15]. (Courtesy of MS
600 **
x Goligorski, MD.)
400

200

0
C 0 Pre-Op Day 1 Day 2 Day 3

TER vs. Time

80
ATP depleted
ATP depleted
70
Control

Repletion buffer added

60

50
TER, Ω -cm2

40

30

20

10

A 0
0 10 20 30 40 60 90 120 150
C Time,min

FIGURE 13-7
Functional and morphologic changes in tight junction integrity asso-
ciated with ischemic injury or intracellular ATP depletion. A and B,
Ruthenium red paracellular permeability in rat proximal tubules.
A, In control kidneys, note the electron-dense staining of the brush
border, which cuts off at the tight junctions (tj, arrows). B, Sections
from a perfusion-fixed kidney after 20 minutes of renal artery cross-
clamp [35]. The electron-dense staining can be seen at cell contact
sites beyond the tight junction (arrows). The paracellular pathway
is no longer sealed by the tight junction, permitting backleak of
the electron-dense ruthenium red. C, Changes in the transepithelial
resistance (TER) versus time during ATP depletion and ATP reple-
tion [36]. Paracellular resistance to electron movement
(Continued on next page)

B
 Pathophysiology of Ischemic Acute Renal Failure: Cytoskeletal Aspects
D E
Acknowledgment
These studies were in part supported by the National Institute
of Diabetes and Digestive and Kidney Diseases Grants DK
41126 (BAM) and DK4683 (RB) and by an American Heart
13.7
FIGURE 13-7 (Continued)
(the TER falls to zero with ATP depletion).
The cellular junctional complex that controls
the TER is the tight junction. When the TER
falls to zero, this suggests that tight junction
structural integrity has been compromised. D
and E, Staining of renal epithelial cells with
antibodies that bind to a component of the
tight junction, ZO-1 [37]. D, ZO-1 staining in
untreated Mardin-Darby carnine kidney
(MDCK) cells. ZO-1 is located at the periph-
ery of cells at cell contact sites, forming a con-
tinuous linear contour. E, In ATP–depleted
cells the staining pattern is discontinuous.
F and G, Ultrastructural analysis of the tight
junction in MDCK cells. In untreated MDCK
cells, electron micrographs of the tight junc-
tion shows a continuous ridge like structure in
freeze fracture preparations [38]. In ATP
depleted cells the strands are disrupted, form-
ing aggregates (arrows). Note that the contin-
uous strands are no longer present and large
gaps are observable.
Association Established Investigator Award (BAM), a VA
Merrit Review Grant (BAM), and a NKF Clinical Scientist
Award (RB).
13.8 Acute Renal Failure
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