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*
Department of Pediatrics University of Michigan, Ann Arbor,
Michigan
Department of Medicine, Vanderbilt University, Nashville,
Tennessee
Department of Cell and Developmental Biology, University of Michigan, Ann
Arbor, Michigan
§
Max Delbrück Center for Molecular Medicine,
Berlin-Buch, Germany.
Correspondence to Dr. William E. Smoyer, Pediatric Nephrology, University of Michigan Medical Center, 8220E MSRB III, Box 0646, 1150 W. Medical Center Drive, Ann Arbor, MI 48109. Phone: 734-763-9524; Fax: 734-615-1386; E-mail: wsmoyer{at}umich.edu
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B-crystallin and heat shock protein (hsp) 25 are
structurally and functionally related small stress proteins induced by a
variety of insults, including heat and ischemia. Cytoprotection by these two
hsp is thought to result from molecular chaperoning and/or cytoskeletal
stabilization. Because renal ischemia is characterized by disruption of the
renal tubular cell actin cytoskeleton, this study was conducted to determine
the localization and quantify the expression and phosphorylation of both hsp
in renal cortex, isolated glomeruli, outer medulla, and inner medulla of rats
after bilateral renal ischemia. Sham-operated kidneys had similarly small
amounts of hsp25 and B-crystallin in cortex and glomeruli, with
substantially greater amounts of B-crystallin versus hsp25 in
outer and inner medulla. Ischemia resulted in significantly increased hsp25
(and hsp70i) but variable B-crystallin levels in cortex and outer
medulla, and progressively decreased glomerular hsp25 phosphorylation. In
sham-operated kidneys, hsp25 localized to glomeruli, vessels, and collecting
ducts, with B-crystallin primarily in medullary thin limbs and
collecting ducts. After ischemia, hsp25 accumulated in proximal tubules in
cortex and outer medulla, while B-crystallin labeling became
nonhomogeneous in outer medulla, and increased in Bowman's capsule. It is
concluded that: (1) There is striking differential expression of
hsp25 and B-crystallin in various renal compartments; and (2)
Renal ischemia results in differential accumulation of hsp25 and
B-crystallin, with hsp25 part of a generalized stress response in renal
proximal tubular cells, which may play a role in recovery from
ischemia-induced actin filament disruption. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ischemia is also known to be a strong stimulus for the induction and
accumulation of stress proteins (i.e., heat shock proteins [hsp]) in
the kidney (4,
5,
6) as well as other organs
(7), and these proteins have
been reported to have a protective effect against ischemia-induced injury in
both the myocardium (8,
9,
10) and kidney
(11). Two of the low molecular
weight (small) stress proteins, B-crystallin and
hsp25a, share
significant sequence and structural characteristics and both have been shown
to function as chaperones in vitro
(12,
13,
14). In addition, hsp25 has
been reported to have a potentially important role in the organization of
microfilaments (15,
16,
17), while B-crystallin
has been reported to interact with both microfilaments and intermediate
filaments (18,
19,
20). Despite their common
features, differential tissue expression of these hsp has been reported. High
concentrations of hsp25 have been reported in cardiac and skeletal muscle
(21,22),
and its abundance and distribution have been studied in a variety of cell
types, including Sertoli cells
(23) and podocytes in the
renal glomerulus (24). The
highest concentrations of B-crystallin have been reported in eye lens
fiber cells (25), cardiac
muscle
(22,26),
striated muscle (26), lung
(27), and in the renal
medulla, where it is primarily in the thin limbs of Henle's loop and
collecting ducts (28).
Because disruption of the renal tubular epithelial cell actin cytoskeleton
is thought to play a critical role in the structural and functional
alterations that characterize renal ischemia, and B-crystallin and
hsp25 have been reported to interact with cytoskeletal proteins, we
hypothesized that the accumulation of these proteins after ischemia may have
an important role in the stabilization of cytoskeletal structures and/or their
recovery from disruption. To begin to address this question, we designed
studies to quantitatively determine the distribution, accumulation, and
phosphorylation of hsp25 and B-crystallin in various renal compartments
of rats at several time points following acute renal ischemia versus
sham operation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Renal Expression of Hsp25 and B-Crystallin
Renal Tissue Preparation. Kidneys were harvested from ischemic and
sham-operated rats 6 h, 24 h, and 5 d after surgery and snap-frozen and stored
at -80°C. Kidneys were later thawed in ice-cold phosphate-buffered saline
(PBS), and a transverse full thickness slice containing both cortex and
medulla was snap-frozen for later immunofluorescence microscopy. Several
2-mm3 pieces of tissue from the cortex, outer medulla, and inner
medulla were harvested and snap-frozen for later protein extraction. Finally,
for glomerular isolation cortical tissue from both kidneys of each animal was
minced with a razor blade in ice-cold PBS, rinsed with PBS, pressed through a
No. 140 stainless steel sieve (W. S. Tyler, Mentor, OH), wetted with ice-cold
PBS, and collected on a No. 200 sieve as described previously
(30). Glomeruli were then
pelleted by centrifugation for 10 min at 2000 x g, suspended in
1 ml of ice-cold PBS, pelleted again by centrifugation at 14,000 x
g for 3 min, and then snap-frozen. Glomerular preparations were
assessed by microscopic examination and routinely contained >95%
glomeruli.
Quantitative Western Blotting. Frozen tissues (stored at -80°C after snap freezing) were homogenized at room temperature in extraction solution (8.5 M urea, 2% [wt/vol] NP-40 [Sigma Chemical Co., St. Louis, MO], 2% 2-mercaptoethanol, 67 mM ß-D glycerophosphate, 50 mM sodium fluoride, 5 mM pyrophosphate, 1 mM ethylenediaminetetra-acetic acid, 1 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, and 50 µg/ml of pepstatin, leupeptin, and aprotinin, pH 7.4) at 150 µl per 2-mm3 tissue piece or glomerular preparation in an Eppendorf tube using a plastic pestle. After 30 min, homogenates were centrifuged at 14,000 x g for 5 min, and the supernatant was divided into aliquots and frozen at -80°C.
Aliquots of each sample containing 20 µg of total protein, in addition
to serially diluted standard proteins (hsp25, hsp70i, and B-crystallin;
StressGen Biotechnologies, Victoria, British Columbia, Canada), were resolved
by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on 13%
acrylamide gels (31) and
transferred to polyvinylidene difluoride membranes (Immobilon P, Millipore,
Bedford, MA). The membranes were briefly washed in PBST (0.05% Tween 20 in
PBS) and incubated at room temperature for 2 h each with: (1)
blocking solution (5% nonfat dry milk in PBST); (2) primary antibody
in blocking solution, and after repeated washing in PBST (3 x 10 min);
and (3) secondary antibody conjugated with horseradish peroxidase.
The primary antibodies used were: (1) 1:5000 dilution of rabbit
polyclonal anti-hsp25 antibody; (2) 1:4000 dilution of rabbit
polyclonal anti-B-crystallin antibody; and (3) 1:2000 dilution
of mouse monoclonal anti-hsp70i antibody (all from StressGen Biotechnologies).
Membranes were washed again, and antibody binding was detected on x-ray film
(Kodak X-OMAT AR) by the enhanced chemiluminescence Western blotting detection
system (Amersham Life Science, Cleveland, OH). Densitometry of developed film
was performed using a digital camera, and analysis of scanned images was
performed using NIH Image version 1.61. The protein content of extracts was
determined by the method of Bradford
(32). Quantification of the
amount of hsp per microgram of total protein for each tissue sample was
performed by analyzing six standard protein amounts with each set of samples
and linear regression to a standard curve of known quantities of each standard
protein.
To verify the accuracy of our protein quantification results, we performed
preliminary studies to verify that protein solubilization using this
urea-based buffer resulted in efficient extraction of both hsp25 and
B-crystallin with minimal protein degradation. Protein solubilization
with this buffer resulted in essentially complete extraction of both hsp25 and
B-crystallin, with neither protein detectable in the remaining pellet
after solubilization with SDS sample buffer (data not shown). In contrast,
protein solubilization in a Tris/NaF/ethylenediaminetetra-acetic acid-based
buffer reported in a previous analysis of small stress proteins
(6) resulted in less efficient
protein extraction, with the majority of hsp25 and B-crystallin
remaining in the pellet. Solubilization of this remaining pellet by boiling in
a urea/Triton X-100-based buffer
(6) resulted in loss of
antibody-reactive hsp25 and B-crystallin when compared with a similar
solubilization of the pellet in SDS sample buffer (data not shown). These
findings confirmed that the protein solubilization technique and protein
quantification in the present study were accurate.
Immunofluorescence Microscopy. For immunohistochemistry,
cryosections of 5 µm thickness were prepared from snap-frozen tissue
sections (see above) with a Jung cryostat (Frigocut 2800N; Leica, Germany).
Fixation of cryosections was performed with 4% formaldehyde in 0.1 M phosphate
buffer, pH 7.4, for 15 min at room temperature and for 5 min in acetone at
-20°C. To suppress nonspecific labeling, cryosections were preincubated
with a solution containing 20 mM Tris-HCl, 130 mM NaCl, 0.05% Tween 20, 0.02%
NaN3, and 1% BSA, pH 8.2 (1% BSA-Tris), for 30 min at room
temperature. Immunolabeling was done with a polyclonal goat anti-hsp25
antibody (33) diluted to a
protein concentration of 40 µg/ml, and with a polyclonal rabbit
anti-B-crystallin peptide serum (StressGen Biotechnologies) diluted
1:2000. In preliminary studies (data not shown), identification of different
cell types was performed using the following antibodies: mouse anti-smooth
muscle actin antibody (clone asm-1; Boehringer Mannheim, Mannheim, Germany),
mouse anti-rat endothelial cell antibody (clone OX-43; Dianova, Hamburg,
Germany), mouse anti-villin antibody (clone 20/24; Biogenesis, Poole, United
Kingdom) to label proximal tubules, goat anti-Tamm Horsfall protein antibody
(Biotrend Chemikalien, Cologne, Germany) to label Henle's loop, and goat
anti-vimentin antibody (kind gift of G. Giese and R. Traub, Ladenburg,
Germany) to label cells expressing vimentin after ischemia-induced remodeling
(34). In double-labeling
experiments, primary antibodies were visualized with DTAF
(dichlorotriazinylaminofluorescein)- and Cy3-labeled species-specific
secondary antibodies (Dianova, Hamburg, Germany) diluted to a protein
concentration of 2 µg/ml and 0.5 µg/ml, respectively. Dilutions of
primary and secondary antibodies were carried out with 1% BSA-Tris, while
washing steps were carried out with 1% BSA-Tris, pH 7.4, containing 630 mM
NaCl instead of 130 mM NaCl. Incubation with primary and secondary antibodies
was performed overnight at room temperature and 90 min at 37°C,
respectively. Immunolabeled cryosections were evaluated with an Axioplan
fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Micrographs were
taken with an automatic camera (Carl Zeiss) using Kodak TMax 400 film.
Renal Phosphorylation of Hsp25 and B-Crystallin
Isoelectric Focusing Gel Electrophoresis and Analysis. Isoelectric
focusing (IEF) gel electrophoresis was performed using the 111 Mini IEF Cell
(Bio-Rad, Hercules, CA). IEF gels were composed of 5.5 M urea, 1.5% Biolytes
3/10, 0.5% Biolytes 5/7 (Bio-Rad), 5% glycerol, 5% acrylamide, and 0.2%
N,N'-methylene-bis-acrylamide, and polymerization was initiated
by the addition of 0.015% ammonium persulfate, 0.0005% riboflavin
5'-phosphate, and 0.03%
N,N,N',N'-tetramethylethylenediamine essentially as
described previously (24). The
amount of each sample loaded corresponded to approximately 1.5 ng of hsp25.
The conditions for running and transfer of the proteins onto the
polyvinylidene difluoride membrane have been described previously
(24), and the immunologic
procedures to detect hsp25 and B-crystallin were as described above.
Densitometric values for the unphosphorylated, monophosphorylated, and
diphosphorylated isoforms of hsp25 were used to generate relative percentages
of each isoform within a single sample.
Statistical Analyses
Data are expressed as the mean ± SEM. Statistics were performed
using unpaired, two-tailed t tests to compare results from
sham-operated and ischemic animals for each kidney compartment at each time
point. Results represent data collected from three sham-operated control and
three ischemic animals at each time point. Differences between groups were
considered statistically significant if P < 0.05.
|
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B-Crystallin, and Hsp70i
in Sham-Operated KidneysB-crystallin, and hsp70i in
various renal compartments of sham-operated rat kidneys.
Figure 1 shows the combined
results from the 6-h, 24-h, and 5-d sham-operated rats (n = 9) for
each protein in the cortex, glomeruli, outer medulla, and inner medulla. The
inner medulla contained the greatest amount of each of the three stress
proteins, with decreasing amounts of each protein in the outer medulla and
cortex, respectively. Within each renal compartment, hsp70i was present in
equal or greater amounts than hsp25, but B-crystallin was present in
greater quantity than either hsp70i or hsp25 in the outer medulla and in
vastly greater quantity in the inner medulla. Direct comparison of the molar
amounts of each protein in the various renal compartments (cortex, glomeruli,
outer medulla, and inner medulla, respectively) revealed the following
results: (1) hsp25: 2.0, 9.6, 6.2, and 36 pmol/mg; (2)
B-crystallin: 4.6, 4.7, 69, and 356 pmol/mg; and (3) hsp70i:
12, 8.9, 24, and 75 pmol/mg. These values correspond to the following molar
ratios (cortex, glomeruli, outer medulla, and inner medulla, respectively):
(1) hsp25: 1.0 : 4.8 : 3.1 : 18; (2) B-crystallin:
1.0 : 1.0 : 15 : 78; and (3) hsp70i: 1.0 : 0.7 : 1.9 : 6.0.
Determination of the corresponding approximate ratios of each of the stress
proteins (hsp25 : B-crystallin : hsp70i) in cortex (1 : 2 : 6),
glomeruli (1 : 0.5 : 1), outer medulla (1 : 11 : 4), and inner medulla (1 : 10
: 2) revealed that each renal compartment has a characteristic pattern of
expression of the stress proteins.
|
Renal Ischemia Results in Acute Renal Failure
Figure 2 shows the serum BUN
levels over the 5 d after induction of renal ischemia with 60 min of bilateral
complete renal artery occlusion in a typical experiment. Serum BUN levels
increased significantly from the normal value of 10.4 ± 1 to a maximum
of 88 ± 7 mg/dl at 24 h after ischemia, and gradually returned to
normal values by 5 d, confirming induction of ischemic acute renal
failure.
|
Renal Ischemia Results in Disparate Accumulation of hsp25 in Various
Renal Compartments
Figure 3 shows the absolute
quantities of hsp25 in cortex (A), glomeruli (B), outer medulla (C), and inner
medulla (D) in rats subjected to renal ischemia and in paired sham-operated
control rats at several time points following renal ischemia. In both the
cortex and outer medulla, the amount of hsp25 was greater after ischemia than
in sham control tissues at all time points, with the maximal accumulation
occurring at 24 h in both cortex (7.2x; 0.05 ng/µg sham
versus 0.37 ischemic) and outer medulla (5.9x; 0.19 ng/µg
sham versus 1.14 ischemic). In contrast, hsp25 amounts in glomeruli
and inner medulla did not undergo significant changes after ischemia. These
findings are all consistent with the known increased susceptibility of tubular
cells of the cortex and outer medulla to ischemia compared with glomeruli
(2,3,37).
|
Renal Ischemia Results in Highly Variable Alterations of
B-Crystallin in Various Renal Compartments
Figure 4 shows the absolute
quantities of B-crystallin in cortex (A), glomeruli (B), outer medulla
(C), and inner medulla (D) in rats subjected to renal ischemia and in paired
sham-operated control rats at several time points following renal ischemia. In
the cortex, B-crystallin tended to accumulate after ischemia, reaching
a maximum increase over controls at 5 d (2.0x; 0.08 ng/µg sham
versus 0.16 ischemic), while in the outer medulla a significant
reduction in B-crystallin was noted at 5 d (0.2x; 1.42 ng/µg
sham versus 0.33 ischemic). In glomeruli and inner medulla, no
differences in B-crystallin in ischemic versus paired controls
were noted at any time point. In the inner medulla, the determined amounts of
B-crystallin appeared to be more variable than in the other kidney
compartments.
|
Renal Ischemia Results in Disparate Accumulation of hsp70i in Various
Renal Compartments
Because the accumulation of hsp25 and B-crystallin following renal
ischemia were clearly disparate, we also compared their responses to that of
the well-characterized stress-inducible cytosolic hsp, hsp70i, in an effort to
determine whether the pattern of accumulation of either hsp25 or
B-crystallin was consistent with a generalized cellular stress
response. Figure 5 shows the
absolute quantities of hsp70i in cortex (A), glomeruli (B), outer medulla (C),
and inner medulla (D) in rats subjected to renal ischemia and in paired
sham-operated control rats at several time points following renal ischemia.
After ischemia, the amounts of hsp70i increased in all tissues at the majority
of time points compared with sham control tissues. Maximal hsp70i accumulation
occurred at 24 h after ischemia in both cortex (3.9x; 0.91 ng/µg sham
versus 3.57 ischemic) and outer medulla (3.7x; 1.67 ng/µg
sham versus 6.24 ischemic), while it occurred at 5 d in inner medulla
(2.5x; 5.19 ng/µg sham versus 13.2 ischemic) and glomeruli
(1.4x; 0.63 ng/µg sham versus 0.86 ischemic). Within the
cortex and outer medulla, where ischemic renal injury is the most severe,
these results thus revealed a highly similar time course and pattern of
accumulation of hsp70i and hsp25 in response to ischemia. In contrast,
B-crystallin amounts increased in cortex and decreased in outer medulla
following ischemia. This similar pattern of response for hsp25 and hsp70i
after ischemia suggests that in this setting hsp25 and hsp70i are likely under
similar regulatory control, which is distinct from that of
B-crystallin.
|
Phosphorylation of Hsp25 or B-Crystallin in Various Renal
Compartments
In rats, hsp25 can be phosphorylated at either of two serine residues
(38), and thus can exist in
one of three isoforms: unphosphorylated, monophosphorylated, or
diphosphorylated. These isoforms have distinct isoelectric points, allowing
them to be resolved by isoelectric focusing. Evaluation of the relative
abundance of the three isoforms of hsp25 in the various kidney compartments of
rats subjected to renal ischemia and in paired sham-operated control rats
revealed that at the time points measured, hsp25 existed primarily in the
unphosphorylated isoform and underwent no notable alterations in
phosphorylation after ischemia in the renal cortex, outer medulla, or inner
medulla (data not shown). In contrast, in glomeruli hsp25 underwent a
progressive decrease in phosphorylation during the 5 d after ischemia
(Figure 6). In addition, in
sham-operated control animals the cortex contained a relatively high
percentage of the monophosphorylated isoform of hsp25 compared with the other
renal compartments (data not shown).
|
B-crystallin can also be phosphorylated on serine residues
(39). Analysis of
B-crystallin phosphorylation in sham-treated versus ischemic
kidneys revealed that essentially all B-crystallin was in the
unphosphorylated state at all time points examined (data not shown). It should
be noted that this study was not designed to primarily analyze phosphorylation
events involving small stress proteins that may have occurred in the time
period before 6 h.
Renal Ischemia Results in Disparate Alterations in the Distribution
of Hsp25 or B-Crystallin
Figure 7 shows
representative double-labeled immunofluorescence-stained sections (Panels A
and B, overviews; Panels C through K, selected areas) of hsp25 (A, C, E, G,
and I) and B-crystallin (B, D, F, H, and K) localization in renal
cortex (C and D), outer stripe of outer medulla (E and F), inner stripe of
outer medulla (G and H), and inner medulla (I and K) from sham-operated
control rats 5 d after recovery. Labeled structures were identified by their
morphology and location in different compartments of the kidney as well as
double labeling with cell-type-specific antibodies as described in Materials
and Methods (data not shown). Hsp25 labeling was found primarily in glomeruli
(A and C), endothelial and smooth muscle cells of vessel walls (A), and
endothelial cells of capillaries in the interstitium (A, C, E, and G). Weak
hsp25 labeling was found in collecting ducts, with increasing intensity toward
the inner medulla (A, G, and I). In accordance with the previous results of
other groups
(28,40),
B-crystallin labeling was found primarily in the medulla (B, F, H, and
K). As demonstrated at higher magnification, prominent labeling was seen in
the proximal straight tubules in the outer stripe of the outer medulla (F), in
the thin descending limbs of Henle's loop in the inner stripe of the outer
medulla (H), and in the thin descending and ascending limbs of Henle's loop in
the inner medulla (K). The collecting ducts had minimal labeling in the outer
medulla (F and H) with slightly greater labeling in the inner medulla (K).
Weak punctate B-crystallin labeling was also found in glomeruli (D),
and proximal and distal tubules in the cortex (D). No differences in labeling
patterns were observed in tissues prepared from sham-operated kidneys 24 h or
5 d after surgery. In sham-operated kidneys prepared 6 h after surgery,
however, B-crystallin labeling was seen in Bowman's capsule, a finding
previously seen only in embryonic kidney
(28). With the exception of
B-crystallin labeling of Bowman's capsule, the localization of hsp25
and B-crystallin described above for sham-operated animals was similar
to untreated control animals (data not shown). Interestingly, colocalization
of both small hsp was seen only in some areas of the collecting ducts.
|
In staining experiments analogous to those in
Figure 7, ischemic kidney
tissues were evaluated at 6 h, 24 h, and 5 d after surgery. At 6 h after
ischemia, intense hsp25 labeling of individual cells developed in proximal
tubules in the outer medulla, whereas no difference was seen in
B-crystallin labeling compared with sham-operated animals (data not
shown). At 12 h after ischemia, more diffuse hsp25 labeling was seen in some
proximal tubules, but the most notable changes in labeling occurred at 5 d
after ischemia. Figure 8 shows
representative double-labeled immunofluorescence-stained sections (Panels A
and B, overviews; Panels C through K, selected areas) of hsp25 (A, C, E, G,
and I) and B-crystallin (B, D, F, H, and K) localization in renal
cortex (C and D), outer stripe of outer medulla (E and F), inner stripe of
outer medulla (G and H), and inner medulla (I and K) from rats subjected to
renal ischemia 5 d after recovery (I and K from 24 h after ischemia). Strong
hsp25 labeling appeared in a nonhomogeneous pattern in proximal tubular cells
in the cortex (A and C) and the outer stripe of the outer medulla (E), where
it was not detectable in sham-operated animals. No apparent increase in hsp25
labeling was seen in glomeruli (C), the inner stripe of the outer medulla (G),
or the inner medulla (I). These findings corresponded well with the increased
amounts of hsp25 observed in cortex and outer medulla shown in
Figure 3. In comparison,
B-crystallin labeling underwent a dramatic change from a homogeneous
pattern to a more irregular pattern in the proximal straight tubules in the
outer stripe of the outer medulla (F), suggestive of intracellular
redistribution. No major changes were observed in the B-crystallin
distribution in the inner stripe of the outer medulla or the inner medulla.
Furthermore, B-crystallin labeling was also seen prominently in the
parietal epithelial cells lining Bowman's capsule (D).
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|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B-crystallin, and hsp70i in various renal compartments of
sham-operated rats. In addition, the accumulation and phosphorylation of hsp25
and B-crystallin were quantified, and their intrarenal localization was
determined following bilateral renal ischemia.
In the sham-operated rats, we found that each renal compartment had a
characteristic expression pattern of the stress proteins hsp25,
B-crystallin, and hsp70i, with some striking differences between the
compartments. All three stress proteins were present in highest amounts in the
inner medulla, but B-crystallin was generally found in greater molar
amounts than hsp25 in all renal compartments except glomeruli. The dramatic
differences in the expression of hsp25 and B-crystallin in the various
renal compartments were also readily apparent in double-labeling
immunofluorescence microscopic studies. Hsp25 in the cortex localized
primarily to glomeruli and blood vessels, whereas in the medulla it localized
primarily to the collecting ducts. In comparison, B-crystallin in the
cortex was present only in small amounts, with minimal glomerular and blood
vessel labeling. These findings are in partial agreement with previous studies
regarding the renal abundance and localization of hsp
(28,35,41,42),
although we did not detect hsp25 in the brush border of proximal tubules in
the outer medulla or the juxtaglomerular mesangium, and we did find weak
labeling for B-crystallin in the tubules and glomeruli in the cortex,
which was not observed by Iwaki et al.
(28). Such differences in
labeling may have resulted from differences in antibodies and/or fixation
procedures.
Differential expression and localization of both small hsp, as demonstrated
here in various compartments of the kidney, have also been observed in other
experimental systems, including rat heart
(22) and rabbit skeletal
muscle (43). With regard to
possible specific functions of the small hsp in different renal compartments,
few data are available. In adult organisms, B-crystallin (outside of
the lens) has been reported to be largely restricted to tissues with high
rates of oxidative metabolism (heart, oxidative type I and IIa skeletal muscle
fibers, and kidney)
(22,26,28,43),
and its increased expression in muscle and kidney during development has been
reported to correlate well with an increase in oxidative marker enzymes
(28). Furthermore, expression
of B-crystallin has been reported to correlate directly with increases
in oxidative metabolism in skeletal muscle
(43). Within the kidney,
increased expression of B-crystallin in Henle's loops in the medulla
correlated temporally with the acquisition of normal physiologic function
(i.e., with the appearance of the medullary osmotic gradient) in
early postnatal life in rats, indicating that it may be necessary for normal
functional development (28).
Interestingly, markedly increased expression of the other small hsp, hsp25 (as
well as hsp70i), has also been reported in the inner medulla (versus
cortex) of normal adult kidneys
(35). These differences were
attributed to the greatly increased osmolality and presumed osmotic stress
within the inner medulla. These findings suggest that the high levels of the
small hsp in the medulla might render medullary cells permanently
stress-tolerant, while the relatively lower levels in the cortex might provide
an explanation for its high sensitivity to ischemia.
Ischemic acute renal failure is characterized by marked structural and
functional disruption of the proximal tubular epithelial cells in the renal
cortex, with less injury to cells of the ascending limb of the loop of Henle
and intercalated cells of the outer medullary collecting duct
(2,3,37,44).
Many of the observed functional abnormalities have been linked to structural
changes within the epithelial cells, the most prominent of which is disruption
of the actin cytoskeleton
(3,44,
45,
46). Such changes are critical
to tubular cell function because actin filaments comprise the structural
backbone of both the microvilli and terminal web of the brush border, and
attach directly to the tight junctions, which form the cell-cell barrier to
paracellular solute movement
(3). Because of the importance
of actin filament disruption to ischemia-induced damage and the reported
induction of heat shock (stress) proteins by ischemia, we sought to analyze
the response to renal ischemia of the two small hsp, hsp25 and
B-crystallin. These proteins have been reported to colocalize with
actin
(23,47,48)
and regulate microfilament dynamics in vitro
(15,
16,
17,49,50).
The results of our quantitative studies revealed that hsp25 accumulates above
control levels only in the renal cortex and outer medulla following prolonged
renal ischemia, with no significant changes occurring in either glomeruli or
the inner medulla. These changes were most pronounced at 24 h after
reperfusion but remained increased for at least 5 d. Immunohistochemical
localization of hsp25 demonstrated dramatically increased nonhomogeneous
labeling of proximal tubular cells in the cortex and outer medulla. A similar
but not identical pattern of accumulation of hsp70i in cortex and outer
medulla was observed, which was also maximal at 24 h after reperfusion. Late
increases in hsp70i at 5 d were also noted in glomeruli and inner medulla.
These findings suggest that hsp25 and hsp70i may be under similar regulatory
control in response to renal ischemia, and that induction of hsp25 may thus be
part of a generalized stress (heat shock) response by renal tubular epithelial
cells. The low baseline levels of hsp25 and the restriction of significant
increases in the amount of hsp25 to only the cortex and outer medulla are
consistent with the known high degree of susceptibility to ischemia of
proximal tubular epithelial cells, which are located primarily in these renal
compartments. Because enhanced expression and phosphorylation of hsp25 has
been reported to stabilize actin microfilaments, we speculate that the marked
disruption in tubular epithelial cell actin filaments that characterizes renal
ischemia may stimulate hsp25 expression, and possibly phosphorylation, as part
of the cellular response to injury.
Induction of hsp25 may thus have an important role in the recovery from ischemia-induced renal epithelial cell actin filament damage. Support for this hypothesis comes from in vitro studies in which enhanced expression and phosphorylation of hsp25 have been reported to stabilize actin filaments and to protect cells against both toxic and thermal stresses (15, 16, 17,51, 52, 53). Although the present study examined bilateral renal ischemia, relative increases in hsp25 in the cortex and outer medulla at 24 h after unilateral ischemia have also been reported by Schober et al. and Aufricht et al. (6,42). Although we found persistently increased amounts of hsp25 and hsp70i in both cortex and outer medulla at 5 d after ischemia, Schober et al. noted minimal to no increase in water-soluble hsp25 and in hsp70i at 7 d after ischemia. Such differences may be due to a continued decline in hsp25 and hsp70i levels, which peaked at 24 h after ischemia, between days 5 and 7. These investigators also reported an immediate and prolonged decrease in water-soluble hsp25 and in hsp70i in the inner medulla, and an increase in both proteins in a water-insoluble fraction, leading them to the conclusion that hsp are redistributed intracellularly in response to ischemia. However, a boiling step in the presence of 6 M urea, a treatment known to cause protein degradation, was included in their extraction procedure for the water-insoluble pellet, which may have resulted in artificially low amounts of hsp. In Aufricht's report, ischemia resulted in increased hsp25 labeling of selected proximal tubules in the cortex, a finding supported by our immunohistochemical analysis, as well as transient redistribution of hsp25 into a detergent-insoluble cell fraction (42). Despite different experimental settings, each of these studies suggests that renal ischemia results in induction of a generalized stress response by the kidney, which is most prominent in the cortex and outer medulla.
The change in the amount of B-crystallin in response to ischemia in
the various renal compartments was different from the pattern observed with
hsp25 (and also from hsp70i), suggesting that both small hsp, despite their
structural and functional similarities, undergo differential regulation within
the kidney, and therefore may have different functions in response to renal
ischemia. Of particular interest, the finding of B-crystallin labeling
of Bowman's capsule in the glomerulus after ischemia is similar to that
reported for the embryonic kidney by Iwaki et al.
(28). This suggests that
ischemic kidneys may share at least some features with embryonic kidneys. A
similar response was observed when the abundance and localization of both
small hsp were studied in diseased human hearts
(22).
As mentioned, both hsp25 and B-crystallin can undergo
posttranslational phosphorylation on serine residues. In the unphosphorylated
state, hsp25 has been reported to act as an actin-capping protein in
vitro, with phosphorylation resulting in inhibition of its
polymerization-inhibiting activity
(49). Phosphorylation of hsp25
has also been reported to be associated with enhanced microfilament stability
and accelerated recovery in response to actin microfilament disruption
(17). In addition, in cultured
endothelial cells subjected to ATP depletion as a model of ischemia and actin
filament disruption, dephosphorylation of hsp25 was noted to occur within 2 h,
and was associated with hsp25 insolubilization and nuclear translocation,
suggesting that early changes in hsp25 phosphorylation may have a role in the
actin cytoskeletal changes resulting from ischemia
(54). For B-crystallin,
even less is known about the possible function of phosphorylation. Based on
comparison with other hsp, such as GroEL (the bacterial homologue of hsp60),
however, it has been suggested that phosphorylation may play a role in
altering the chaperone function of B-crystallin
(12). In the present study, we
found a progressive decrease in glomerular hsp25 phosphorylation during the 5
d following renal ischemia, but no other changes in hsp25 or
B-crystallin phosphorylation. Although this is the first study to our
knowledge that analyzes small hsp phosphorylation in response to renal
ischemia, the study design was such that ischemia-induced changes in hsp25
and/or B-crystallin phosphorylation occurring before the initial 6-h
time point would not be detected. Because the alterations in actin filaments
during and after renal ischemia are rapid, such early changes in
phosphorylation might play an important role in regulating the extent of actin
cytoskeletal damage and/or the degree of recovery of epithelial cell structure
and function.
In conclusion, we performed a quantitative analysis of the distribution,
accumulation, and phosphorylation of the small hsp hsp25 and
B-crystallin in various renal compartments of rats at several time
points after acute renal ischemia versus sham operation. In
sham-operated kidneys, we found only small amounts of hsp25 and
B-crystallin in cortex and glomeruli, with substantially greater
amounts of B-crystallin versus hsp25 in outer and inner
medulla. Ischemia resulted in differential accumulation of hsp25 and
B-crystallin, with hsp25 accumulation part of a generalized stress
response specifically in cortex and outer medulla. In contrast, the changes in
the quantity of B-crystallin were highly variable and clearly distinct
from that of hsp25, with increased amounts in cortex and decreased amounts in
outer medulla. The specific accumulation of hsp25 in only the cortex and outer
medulla after renal ischemia suggests that hsp25 may play a role in recovery
from ischemia-induced actin filament disruption in ischemic acute renal
failure.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-Crystallin can function as a molecular
chaperone. Proc Natl Acad Sci USA 89: 10449
-10453, 1992-crystallins
modulates intermediate filament assembly. EMBO J 13
: 945-953, 1994[Medline]
B-crystallin from human
skeletal muscle. J Biol Chem 267: 7718
-7725, 1992B-crystallin in rat and human heart. Circulation 96
: 3466-3476, 1997B
crystallin in the rat determined with a sensitive immunoassay system. Biochim Biophys Acta
1074: 201
-208, 1991[Medline]
B-crystallin gene is not restricted to the lens. Mol Cell
Biol 9: 1083-1091, 1989B-crystallin in the developing rat kidney. Kidney Int 40
: 52-56, 1991[Medline]
B-crystallin in response to various types of stress. J Biol
Chem 272: 29934-29941, 1997B-crystallin in non-lenticular tissues. J Histochem
Cytochem 38: 31-39, 1990[Abstract]
B-crystallin and HSP27 in skeletal muscle during continuous contractile
activity: Relationship to myogenic regulatory factors. J Biol
Chem 271: 24089-24095, 1996B-crystallin in
skeletal muscle: Purification and localization. J Biochem 110
: 812-822, 1991B-crystallin in cardiac
tissue: Association with actin and desmin filaments. Circ Res 71
: 288-294, 1992This article has been cited by other articles:
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