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Regular Articles |
From the Department of Medicine,*
Fred Hutchinson Cancer
Center, University of Washington, Seattle, Washington; and the
Department of Solid Organ and Cellular
Transplantation,
Legacy/Good Samaritan
Hospital, Portland, Oregon
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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0.99) between FC/CE and blood-urea nitrogen levels were observed. The
FC/CE increases were specific to damaged kidney (glycerol did not raise
hepatic FC/CE; unilateral renal ischemia did not alter contralateral
renal FC/CE levels). Overnight dehydration raised renal CE
levels, most notably in the medulla. Conclusions: FC/CE
accumulation is a hallmark of the maintenance phase of ischemic and
nephrotoxic ARF, and can reflect its severity. That
cholesterol accumulation can result from glomerular injury and
dehydration suggests that it is a generic renal stress
response, with potential relevance extending beyond just the
phenomenon of acquired cytoresistance.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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18 to 24 hours after
the first renal insult must transpire before acquired resistance is
expressed;7,9-11
2) the protection is broad-based, such
that diverse toxic, physical, or ischemic injuries can induce
nonspecific protection against a broad variety of secondary
insults;9-11
3) it is expressed directly at the proximal
tubule cell level, as evidenced by the fact that proximal tubules
extracted from cytoresistant kidneys are resistant to in
vitro damage;9-11
4) the protection may not be
dependent on de novo protein synthesis, given that protein
synthesis inhibition does not prevent the emergence of
cytoresistance;12
and 5) the protection is expressed at
the plasma membrane and mitochondrial levels. This is indicated by the
fact that plasma membranes obtained from cytoresistant isolated tubules
are relatively resistant to oxidant- and
PLA2-mediated injury,11
and that
corresponding mitochondria generate less free radicals during oxidant
stimulated attack.13
In search of potential mechanism(s) for renal cytoresistance, this
laboratory has found that within 18 to 24 hours after heterogeneous
forms of in vivo or in vitro renal tubular cell
damage, increased cellular cholesterol content
develops.14,15
Linking these increases to acquired
cytoresistance are the following observations: 1) if cholesterol levels
are reduced to normal in cytoresistant tubules by chemical extraction,
cytoresistance is lost;14
2) lowering cholesterol by
20% in normal cells via differing mechanisms (eg, statins,
enzymatic attack) exaggerates cellular vulnerability to toxic or
hypoxic damage;14
3) chemical modification of cellular
cholesterol, either via cholesterol oxidase or cholesterol esterase,
causes profound mitochondrial dysfunction and cell
death;16
and 4) the emergence of elevated cholesterol
levels and cytoresistance temporally correlate, each being first
apparent at 18 hours after the initial renal insult.14
In
sum, then, considerable data indicate that cholesterol accumulation is
a generic response to acute tubular injury, and can contribute to the
cytoresistant state.
To date, cholesterol elevations after injury have only been documented at one time point: 18 to 24 hours after ischemic or toxic injury. However, it is well known that the state of acquired cytoresistance can persist for days to several weeks after the induction of acute renal damage.1,5 If cholesterol levels are, in fact, mechanistically linked to acquired cytoresistance, then one would assume that renal cortical/tubular cholesterol elevations are a durable response. Conversely, if previously documented cholesterol increases rapidly dissipate, eg, with 24 to 48 hours, this would suggest that cholesterol accumulation is only an acute stress reaction with little or no long-term implications for a postinjury state.
To shed light on this issue, in this study we have now performed a time course experiment in which renal cortical cholesterol profiles have been monitored from 3 days to 2 weeks after the induction of acute myohemoglobinuric acute renal failure (ARF), and 3 to 6 days after ischemic ARF. Additionally, we have addressed whether more insidious models of tubular injury [cyclosporine A (CSA) or tacrolimus nephrotoxicity], and a nontubular model of renal injury [acute nephrotoxic serum (NTS), anti-glomerular basement membrane, glomerulonephritis] also evoke cholesterol accumulation. Finally, we have sought to address whether a physiological, and not simply pathological, stress might also impact renal cholesterol expression. To this end, the impact of overnight dehydration on renal cholesterol levels has been determined. The result of these investigations forms the basis for this report.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male CD-1 mice, weighing 25 to 35 g (Charles River
Laboratories, Wilmington, MA), and maintained under normal vivarium
conditions with free access to food and water, were used for all
experiments. On the day of experimentation, they were lightly and
momentarily anesthetized with isoflurane, and then injected with 50%
glycerol (total, 8.5 ml/kg) into each upper hind limb in equally
divided doses. The mice were then returned to their cages and allowed
continued free access to food and water. Either 3, 5, 7, or 14 days
later (n = 6, 6, 5, and 4, respectively), the
mice were deeply anesthetized with pentobarbital (2 mg/kg;
intraperitoneally). They were subjected to a midline abdominal
incision, blood was withdrawn from the inferior vena cava for blood
urea nitrogen (BUN) analysis, and then the kidneys were removed. A
piece of renal cortex was obtained from each kidney for subsequent free
cholesterol (FC) and cholesterol ester (CE; a cholesterol storage form)
analysis (see below). In addition, phospholipid profiles were assessed
(see below). An equal number of normal kidneys, obtained from
sham-treated normal mice, were processed simultaneously to provide
control FC/CE and BUN concentrations.
Hepatic Cholesterol Levels after Glycerol-Induced ARF
The following experiment was undertaken to ascertain whether cholesterol changes after renal injury were relatively renal-specific, or whether they were part of a generalized, systemic response to ARF. To this end, hepatic tissues were collected 72 hours after glycerol injection (described above). Three normal mice provided normal hepatic tissues. These tissues were processed for FC and CE, as described below.
Postischemic Renal Damage
Although cholesterol increments have been noted during the early
maintenance phase (<24 hours after injury) of postischemic
ARF,14,17
whether these changes persist into the late
period after injury has not been determined. To this end, eight mice
were anesthetized with pentobarbital (2 mg intraperitoneally), a
midline abdominal incision was made, and then half of the mice were
subjected to 40 minutes of left renal pedicle vascular occlusion (the
right kidney was left alone to prevent subsequent death from uremia).
The remaining four mice were subjected only to sham left renal pedicle
occlusion. The animals were then sutured and allowed to recover from
anesthesia. At either 3, 4, 5, or 6 days after surgery, one
experimental (unilateral ischemia) and one control mouse were
re-anesthetized, and both kidneys were removed. The renal cortices of
the postischemic left kidneys, the contralateral right kidneys, and the
left kidneys from the sham-operated mice were removed, the cortices
isolated, and analyzed for FC and CE levels.
CSA- and Tacrolimus-Mediated Nephrotoxicity
Previously well-described models of CSA18,19 and tacrolimus20 nephrotoxicity, performed with adult male Sprague-Dawley rats (Charles River, Wilmington, MA, 200 to 250 g), were used. They were housed in individual cages under standard vivarium conditions and fed a low-salt diet (0.05% Na; Teklad Premier, WI) with free access to water. After 7 days on this diet, weight-matched pairs of rats were randomized into four groups: 1) CSA treatment: 15 mg/kg [times[ 10 days, n = 6 rats (Sandimmune oral solution; Novartis Pharmaceuticals, East Hanover, NJ; dissolved in olive oil, 15 mg/ml); 2) CSA vehicle-treated controls (n = 3 rats); 3) tacrolimus treatment: 1 mg/kg x 10 days, n = 6 rats (Prograf injection; Fujisawa Health Care; Deerfield IL; dissolved in sterile water 1 mg/ml); and 4) tacrolimus controls (n = 3). After 10 days of treatment, the rats were anesthetized with intraperitoneal ketamine. A blood sample was obtained for BUN and then the kidneys were removed. One kidney was saved for FC/CE and phospholipid analysis, as noted below. For statistical analysis, the two control groups were combined (as no significant differences between them).
NTS Nephritis
Previous studies from this8
and another
laboratory3
have demonstrated that acquired
cytoresistance, as expressed at the tubular cell level, can be induced
by acute anti-glomerular basement membrane-mediated glomerulonephritis.
However, it has not previously been ascertained whether this form of
cytoresistance is associated with altered cholesterol expression. To
address this issue, three Sprague-Dawley rats (250 g) were lightly
anesthetized with ether and injected via tail vein with 0.2 ml/kg of
anti-glomerular basement membrane NTS.21
Three additional
rats injected with an equal amount of nonimmune serum served as
controls. From 36 to 48 hours after serum injection, a timed urine
collection was completed to assess proteinuria, using the
sulfosalicylic acid method.22
At the completion of the
experiment, the rats were deeply anesthetized with pentobarbital, both
kidneys were resected, and the cortices from each rat were dissected,
combined, and frozen (1 sample per animal). The tissues were then
processed for cholesterol analysis, as noted below.
Cortical/Medullary Cholesterol and CE Analysis
The following experiment was undertaken to assess whether a physiological, rather than a pathophysiological, stress might also alter renal FC/CE profiles. To this end, 14 mice were divided into two groups: normal mice (n = 7) and mice subjected to overnight dehydration (withdrawal of water, but not food; n = 7). The next morning, the mice were anesthetized with pentobarbital, and both kidneys removed. Cortical and medullary tissues were dissected from each kidney. They were then processed for FC and CE to address two questions: 1) does a cortical-medullary cholesterol gradient exist within the kidney, consistent with the normally occurring osmotic gradient? and 2) if so, does increasing medullary osmolality increase medullary cholesterol profiles?
Tissue Cholesterol Analysis
The kidneys were placed on an iced plate and cortical tissue samples were dissected using a razor blade. The samples were weighed, added to four parts cold methanol, homogenized, and extracted in chloroform:methanol (1:2) as previously described in detail.15,16 Hepatic tissues were treated in the same manner. The extracts were dried under N2 and saved for FC and CE analysis by gas chromatography.15 In brief, the dried lipid extracts obtained from the above experiments were reconstituted in 1 to 2 ml of hexane, followed by sonication and vortexing to dissolution. To analyze FC, a 100-µl sample of each extract was transferred to a glass culture tube containing 50 µl of an internal standard solution (stigmasterol, 100 µg/ml in ethyl acetate, EtOAc). The sample was dried under N2 and reconstituted in 100 µl of bis-(trimethylsilyl)trifluoroacetamide [BSTFA; Sigma (St. Louis, MO); 25% v/v EtOAc]. They were then transferred to an injection vial, sealed, and heated for 1.0 hours at 60°C. After completion of BSTFA derivatization, a 1-µl sample was applied to a Hewlett Packard 5890 Series II gas chromatograph fitted with a flame ionization detector and a 30 m x 0.32 mm DB-5 (0.25 µm) column (J&W Scientific, Folsom, CA). The initial temperature (100°C) was maintained for 3 minutes, after which it was increased by 40°C/minute to 290°C, and thereafter by 5°C/minute to 300°C for 5 minutes. The trimethylsilyl ether of cholesterol eluted at 12.5 minutes and that of the internal standard at 13.6 minutes. To quantify CEs, they were first separated from the FC pool. This was achieved by adding 400-µl samples of the hexane aliquots to an amino solid-phase extraction column (3 ml, 500 mg; Varian Bond Elut, Varian, Harbor City, CA) previously washed with hexane. The column eluant, containing CE, but not FC, was saved.15 After two subsequent column washings, all of the eluants were combined, the internal standard was added, and the sample was evaporated to dryness. The esters were hydrolyzed in EtOH/30% KOH at 55°C for 45 minutes.15 The samples were added to 2.0 ml of water, and then 3.0 ml of hexane were added. The hexane phase was dried under N2 and then assayed for FC, as above. Previously performed validation studies confirmed the complete absence of any contaminating FC within the CE eluant, and 100% efficiency in CE recovery.15 Hence, the amount of recovered FC resulting from CE hydrolysis was taken as the amount of CE present in the original sample. FC and CE results were each expressed as nmol/µmol phospholipid phosphate (Pi) in the initial lipid extract.15
Phospholipid Analysis
To assess whether changes in membrane phospholipids accompanied changes in cholesterol expression, renal cortical lipid extracts from all glycerol-treated mice, postischemic mice, tacrolimus/CSA-treated rats, NTS nephritis rats, and each group’s corresponding controls were analyzed for phospholipids by two-dimensional thin layer chromatography, as previously described in detail.23 In brief, the five dominant plasma membrane phospholipids (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, or sphingomyelin) were assessed. Individual phospholipids were quantified by scraping the individual spots from the silica thin-layer chromatography plates and measuring their phosphate content.23 Results were expressed as the percent to which each phospholipid contributed to the total phospholipid mass (the sum of phosphatidylcholine plus phosphatidylserine plus phosphatidylethanolamine plus phosphatidylinositol plus sphingomyelin). By so doing, relative changes in membrane phospholipids could be assessed and contrasted with changes in cortical cholesterol content.
Calculations and Statistics
All values are given as means ± 1 SEM. Statistical comparisons were made by either paired or unpaired Student’s t-test: paired analysis was used if left versus right kidney values from a given set of animals was used; unpaired analysis was used for comparison between values obtained from different animals. If more than one comparison with any set of data were made, the Bonferroni correction was applied.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As shown in Figure 1
,
glycerol-induced myohemoglobinuria induced severe azotemia, as first
assessed 3 days after glycerol injection. With each subsequent time
point, the severity of azotemia decreased, but remained significantly
elevated compared to simultaneous controls. There was considerable
variability in the severity of azotemia in the postglycerol-treated
mice, with coefficients of variation being 60, 58, 90, and 28% at 3,
5, 7, and 14 days, respectively, after the induction of ARF. These
differences allowed for a comparison between the degree of azotemia
versus the extent of FC/CE changes, as discussed immediately
below.
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At each assessed time point, both CE (Figure 2)
, and in most instances FC (Figure 3)
, were dramatically higher in the
postglycerol versus the control renal tissues. The greatest
relative increase was in CE, which for the first 7 days reached values
that were 10 times higher than was observed in control tissues. The
CE/FC elevations persisted almost completely unabated for the first 7
days. At day 14, CE levels still remained elevated, whereas FC had
returned to near normal values.
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). This relationship was particularly noteworthy at 7 and 14 days,
where r values of 
0.90 were observed. This relationship is
further presented in Table 1
(individual
postglycerol BUN, FC, and CE values at day 7 and day 14 are presented).
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At 3 days after glycerol injection, the BUNs for the three test
mice were 76, 98, and 220 mg/dL (compared to BUNs of 25, 28,and 34 for
their normal controls). Despite the severe ARF in the postglycerol
mice, no significant elevation in hepatic FC or CE was observed (FC:
120 ± 8 versus132 ± 21; CE: 12.5 ± 2.7
versus 13.3 ± 1.3; control versus
postglycerol group, respectively). This was despite the fact that there
was a dramatic elevation in renal cholesterol pools in these same
postglycerol-treated animals (FC, 213 ± 7 versus
319 ± 25; P < 0.025; CE: 4.8 ± 1
versus 39.3 ± 11; P < 0.025; controls
versus postglycerol, respectively). It was noteworthy that
normal kidney and liver had differing FC/CE profiles: liver had 50%
lower (P < 0.01) FC, and an approximate
fourfold higher (P = 0.05) CE content (values
given above). This indicates that in liver, a relatively greater amount
of cholesterol is stored in the CE pool.
Cholesterol/CE Values after Unilateral Renal Ischemia
As shown in Figure 4
, both FC and CE
were dramatically elevated in the left, postischemic kidneys that were
harvested 3 to 6 days after surgery (all values at each time point are
presented as a single postischemia group). This was true whether the
postischemic values were contrasted to their own right (Rt) nonischemic
kidney, or to kidneys harvested from sham-operated control (cont) mice.
Each individual FC and CE value was elevated irrespective of whether
the kidneys were harvested at 3, 4, 5, or 6 days postischemia (ie, each
>99% confidence band for control values).
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Cholesterol/CE Values after CSA and Tacrolimus Treatment
Ten days of CSA or tacrolimus each caused only slight, but
significant, increases in FC content (Figure 5
, left). Far more dramatic CE elevations
were observed with each agent, reaching values that were 8 to 10x
those observed in control tissues (Figure 5
, right). (Note: this is
consistent with the fact that a large increase in cholesterol storage,
ie, ester formation, can be observed under conditions of only slight
increases in FC levels.) Both CSA and tacrolimus induced mild azotemia
during the course of the experiments (controls: 17 ± 1 mg/dL;
CSA, 31 ± 1 mg/dL; tacrolimus, 29 ± l mg/dL; each group,
P < 0.01, versus the control group).
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As shown in Figure 6
, injection of
NTS caused a profound CE increase, reaching values that were
approximately four times those seen in control rats. Conversely, only a
very small, and nonsignificant (P = 0.08),
increase in FC levels was observed. NTS injection caused marked
proteinuria, with protein excretion rates of 1.25 ± 0.25 and
23.5 ± 0.6 mg/hour being observed in control and NTS-treated
rats, respectively (P < 0.0001).
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Glycerol-Induced ARF in the Mouse
At 3 days after glycerol injection, a significant increase in
percent sphingomyelin content was observed (Table 2)
. This was associated with reciprocal
decreases in phosphatidylserine and phosphatidylethanolamine content.
The relative increase in sphingomyelin was also noted at 5 days after
glycerol injection (Table 3)
, and
dissipated thereafter (Tables 4 and 5)
.
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Neither CSA nor tacrolimus induced any detectable change in
phospholipid expression (Table 6)
. Thus,
the cholesterol changes were observed in the absence of any other
discernible change in membrane lipids (although it is recognized that
small, but significant differences might have been observed had a
larger number of samples been assessed).
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As with the tacrolimus- and CSA-treated rats, the NTS
nephritis-induced cholesterol increments were unaccompanied by any
discernible changes in membrane phospholipids (Table 7)
. (As noted above, it remains possible
that small, but significant, differences might have been observed had a
larger number of samples been assessed).
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Despite dramatic cholesterol increments, no major changes in
phospholipid profiles were observed. The only significant difference
was a slight, but significant, decrease in percent phosphatidylserine
expression, relative to the other phospholipids (see Table 8
).
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As shown in Figure 7, a
cortical-medullary CE gradient was apparent in normal
mouse kidney, with medullary values being 40% higher than those
observed in renal cortex (P < 0.001). With
overnight dehydration, both cortical and medullary CE levels increased,
compared to control values (P < 0.001).
However, there was a preferential increase within the medulla: in the
normal kidneys, the absolute difference between medullary
versus cortical CE values was 1.67 ± 0.3 nmol/µ mole
Pi; in contrast, the difference in values for the dehydrated mice was
2.7 ± 0.2 nmol/µ mole Pi; (P < 0.01).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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35% increase in membrane
ceramide content within cytoresistant renal tissues.26-28
However, ceramide, an effector molecule within the sphingomyelin
signaling pathway, is generally considered to be a pro-apoptotic, not a
survival, factor.29,30
Furthermore, when exogenous
ceramide has been added to either isolated tubules or to cultured human
proximal tubule (HK-2) cells, increased, or in some instances
decreased, susceptibility to superimposed hypoxic and toxic injuries
have been observed.26-29
These considerations caused us
to also reject ceramide accumulation as a prime determinant of the
cytoresistant state.
Although the above considerations make it unlikely that ceramide is a
key determinant of cytoresistance, we have considered its consistent
elevation in cytoresistant tissues as a possible clue to more
pathogenetically relevant factor(s). Because ceramide is derived from
sphingomyelin, and because sphingomyelin tightly associates with
cholesterol in membrane microdomains (DRMs, caveolae, rafts),
31-35
we questioned if cholesterol accumulation might
accompany the ceramide increases, and potentially mediate the
cytoresistant state. Indeed, in each model of cytoresistance tested to
date, a 20 to 40% increase in total cholesterol content has been
observed.14
However, unlike ceramide, cholesterol does
seem to be linked to acquired cytoresistance, based on the following
observations: 1) decreasing cholesterol content in normal cells (either
by statin-induced synthesis blockade, enzymatic degradation, or
chemical extraction) sensitizes them to superimposed hypoxic and toxic
injuries14
; and 2) lowering cholesterol levels to normal
values in cytoresistant tubules restores normal cellular resistance to
attack.14
It is notable that cholesterol exists within
cells either as FC or as CEs. The latter, which normally comprises only
1 to 2% of total renal cortical cholesterol,15
has
shown the greatest percent increase in cytoresistant tissues, rising as
much as 10-fold more than basal values.15
That specific
reductions in cellular CE levels results in profound ATP depletion and
lethal cell injury16
suggest that the CE pool may have
prime importance in mediating the cytoresistant state. The mechanism(s)
by which increased cholesterol pools enhance cellular resistance to
damage remains incompletely defined. However, previous studies from
this laboratory suggest that cholesterol’s ability to increase plasma
membrane rigidity is at least partly responsible.14
The results of the present studies extend on our understanding of cholesterol accumulation after injury in a number of important ways, as follows:
First, we have documented for the first time that cholesterol increments after injury represent a durable, and not simply, transitory event. The data demonstrate that they persist for at least 2 weeks after glycerol-induced injury, and for at least 6 days after unilateral renal ischemic damage. That the cytoresistant state can persist for days to weeks after injury1,5,7 suggests the potential mechanistic importance of these durable cholesterol increments.
Second, the present results are the first to demonstrate a striking
correlation between the severity of tissue injury/organ failure and the
extent of cholesterol accumulation. This is indicated by the
extraordinarily tight correlation between BUN and cholesterol/CE
elevations in the postglycerol ARF model (eg, r 0.90
at 7 to 14 days after injury).
Third, the cholesterol elevations seem to be a direct result of tubular injury, and not simply a result of the uremic state. Stated differently, cholesterol accumulation after injury seems to be an organ-specific, injury-induced, phenomenon. This is underscored by observations that: 1) hepatic cholesterol levels remained normal in uremic (postglycerol-treated) mice that manifested massive renal cholesterol accumulation; and 2) postischemic cholesterol accumulation in mouse kidney was unaccompanied by any cholesterol increase in the contralateral control kidney. Previous observations that unilateral ureteral obstruction also raises cholesterol levels in the absence of uremia10 further illustrates the dissociation between uremia and tissue cholesterol increments.
Fourth, this is the first study to demonstrate that insidious renal tubular injury (in this case induced by either tacrolimus or CSA) can, like acute massive renal damage (eg, ischemia/reperfusion, rhabdomyolysis), induce cholesterol/ester accumulation. These observations thereby extend the potential importance of altered cholesterol metabolism to a much broader range of renal insults than previously recognized (eg, possibly involving aminoglycosides, radiocontrast agents, NSAIDS, and so forth). Whether cholesterol accumulation during the course of insidious nephrotoxic injury might serve as a defense mechanism that helps stem further tissue damage remains an intriguing, but unresolved, issue as of this time.
Fifth, although both our previous14 and present data document cholesterol accumulation in response to a variety of direct proximal tubular insults, the present study is the first to demonstrate that glomerular injury can also induce this result. Noteworthy in this regard are previous observations from Nath and colleagues3 and from this laboratory8 that the NTS (anti-glomerular basement membrane) nephritis model induces a tubular cytoresistant state. Nath and colleagues3 have ascribed this cytoresistance to heme oxygenase-1 (HO-1) induction. The present results suggest that an up-regulation of cholesterol expression may also be involved. The mechanistic link between glomerular injury and renal cortical cholesterol accumulation, and whether the cholesterol increments are glomerular and/or tubular in location, remain to be defined.
Sixth, the present study underscores that after injury cholesterol increments typically occur in the absence of any obvious, or uniform, change in membrane phospholipid composition, at least as assessed by two dimensional-thin layer chromatography. Although we have previously noted a dissociation of cholesterol accumulation from altered phospholipid expression at 18 to 24 hours after injury, that this same lack of relationship also exists during the maintenance phase of renal injury underscores the relative specificity of the cortical cholesterol changes. Thus, it can now be stated that a hallmark of the cytoresistant state, and the maintenance phase of ARF, is not simply a cholesterol elevation; rather there is an increase in the ratio of cholesterol to individual phospholipids. This would be expected to confer increased membrane rigidity at a time of superimposed tubular cell attack.14,36
Seventh, the present study provides the first evidence that perturbations in cholesterol/CE expression may extend to physiological, and not just pathophysiological, stress. This is evidenced by observations that overnight water deprivation was a sufficient renal challenge to induce a significant increase in both cortical and medullary CE content. It is notable that within the kidney, a cortical-medullary CE gradient exists, and it is enhanced by overnight water deprivation. These two observations suggest that the balance between free and esterified cholesterol in renal tissues may be partly under osmoregulation. Although purely speculative, it is interesting to hypothesize that an increase in CE content might confer medullary cytoprotection, helping medullary cells to withstand hyperosmotic stress. That dehydration increased medullary CE content is at least consistent with this concept.
Finally, the mechanism(s) responsible for cholesterol/CE accumulation after cellular stress remain to be completely defined. At least three possibilities exist: increased synthesis, decreased cellular efflux, and/or increased extraction from blood via the low-density lipoprotein receptor.37-39 To date, we have demonstrated that statins can completely block cholesterol accumulation in the aftermath of cell injury in cell culture experiments.15 This indicates an important role for increased synthesis. However, statin therapy does not completely abrogate cholesterol accumulation in vivo (unpublished data from this laboratory). This suggests that decreased cellular cholesterol efflux, and/or increased low-density lipoprotein cholesterol uptake, are also likely to be involved. Multiple pathways exist by which of these processes may occur, and their relevance to the present results are subjects of ongoing investigations. However, one possibility that has been excluded is a loss of ABCA-1, a key cholesterol efflux protein.40 This is because pilot data indicate that ABCA-1 is not normally expressed in normal mouse renal cortex, as assessed by Western blot.40 This makes it very difficult to ascribe increased cholesterol accumulation to a specific derangement in this cholesterol efflux pathway. Clearly, more work is required to both define the operative cholesterol accumulation pathway(s), and the molecular species that trigger them (eg, a specific cytokine, free radical, and so forth) after tissue damage.
In conclusion, the present studies indicate the following: 1) cholesterol/CE accumulation represents a durable adaptive response to either acute or subacute forms of renal tubular cell injury; 2) the degree of cholesterol elevations can closely mirror the extent of renal damage; 3) the cholesterol accumulation is directly caused by tissue damage, rather than being a secondary consequence of uremia; and 4) heterogeneous insults, eg, acute glomerulonephritis, or modest dehydration, can trigger renal cholesterol accumulation. This implies that altered cholesterol/CE expression, and presumably concomitant changes in the underlying mevalonate synthetic/signaling pathway,41 likely have broad-reaching biological implications, potentially extending well beyond acute tubular necrosis and its attendant cytoresistant state.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grants DK38432, RO1 54200, and RO1 DK 37652.
Accepted for publication May 3, 2001.
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R. A. Zager, V. O. Shah, H. V. Shah, P. G. Zager, A. C. M. Johnson, and S. Hanson The Mevalonate Pathway during Acute Tubular Injury : Selected Determinants and Consequences Am. J. Pathol., August 1, 2002; 161(2): 681 - 692. [Abstract] [Full Text] [PDF]
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, P < 0.05 versus
controls).
