Effect of reversible and irreversible ischemia on marker
enzymes of BBM from renal cortical PT subpopulations
Syed Jalal
Khundmiri,
Mohammed
Asghar,
Farah
Khan,
Samina
Salim, and
Ahad Noor Khan
Yusufi
Department of Biochemistry, Faculty of Life Sciences, Aligarh
Muslim University, Aligarh-202 002, India
 |
ABSTRACT |
The effect of the reversible and relatively irreversible
ischemia induced acute renal failure (ARF) in the activities of
alkaline phosphatase (AlkPase) and
-glutamyltransferase (GGTase)
after early (15-30 min) and prolonged (45-60 min) ischemia in
the homogenates, and the brush-border membranes (BBM) from rat renal
whole, superficial (SC), and juxtamedullary (JMC) cortices were
studied. The enzyme activities declined progressively in proportion to
the duration of ischemia. Early blood reflow of 15 min to the ischemic
rats caused a further decrease in the enzyme activities. However,
prolonged reflow (up to 120 min) resulted in partial reversal of the
ischemic effect in the early but not in the prolonged ischemic rats.
The decrease in the enzyme activities was due to the loss of
membrane-bound enzyme components from the damaged BBM into the
supernatant fraction as membrane-free enzymes. The activities of
AlkPase and GGTase were significantly more decreased by the ischemia in
the brush-border membrane vesicles (BBMV)-JMC than in
BBMV-SC. The rate of recovery due to reflow for AlkPase
was greater in BBMV-SC than apparently for GGTase in BBMV-JMC in early
ischemic (15-30 min) rats.
proximal tubule; acute renal failure; superficial cortex; juxtamedullary cortex; alkaline phosphatase;
-glutamyltransferase
 |
INTRODUCTION |
RENAL ISCHEMIA AND TOXIC insults are known to produce
profound alterations in the structure and excretory function of the kidney and, depending on the severity of the damage caused, lead to
reversible or permanent acute renal failure (ARF) (4, 23, 28). In general, ARF is manifested by a steady rise in the
plasma concentrations of creatinine and urea (21, 24). Morphological studies have shown that renal proximal tubule (PT) in general and its
brush-border membrane (BBM) in particular are major targets for
ischemic injury (3, 4, 19, 27). It is supported by ischemia-induced
decrease in the specific activities of certain marker enzymes of renal
cortical BBM (14, 17, 18), accompanied by an increase of enzyme
activities in the urine (1, 9). According to the recent concept, the
deep nephrons in general and pars recta
(S3 subsegment, proximal straight
tubule) of the proximal tubules in particular have been shown to be
more greatly affected by ischemia-induced ARF (16, 20) than the
superficial nephrons (S1
subsegment, proximal convoluted tubules). Studies with
blood reflow showed partial morphological and biochemical recovery (17,
18, 28).
The present study was undertaken to determine in greater detail the
structural damage caused by the ischemia to various subpopulations of
the proximal tubules of whole, superficial, and deep cortices under
reversible and relatively irreversible conditions. The effect of
ischemia was further studied in response to blood reflow to the
ischemic rats for variable time periods. The results demonstrate differential effects of ischemia and blood reflow on various
biochemical components, including BBM marker enzymes of proximal tubule
subpopulations.
 |
METHODS |
Young male Wistar rats weighing 150-200 g, fed a standard rat diet
and water ad libitum, were used in the study. On the day of experiment,
the rats were anesthetized by an injection of pentobarbital sodium (50 mg/kg body wt ip). The abdomen was opened by a left flank incision, and
the left renal artery was separated from the renal vein. Ischemia was
produced by clamping the left renal artery for the required time, as
specified in RESULTS, with a stainless steel microaneurysm clip (1.5 × 10 mm). After occlusion of the renal artery, the abdominal viscera were covered with 0.9% NaCl-soaked gauze. Sham-operated rats, subjected to the same surgical procedure except that the renal artery was not clamped, were used as controls. For reflow studies, renal artery was declamped and the blood was allowed to reflow for variable time periods (15-120 min). After the designated time of ischemia and/or blood reflow, the
kidneys were removed, decapsulated, and kept in ice-cold buffered
saline. The cortex was carefully separated from medulla to get either the whole cortex and/or superficial (SC) and juxtamedullary
(JMC) cortex as described by Yusufi et. al. (31).
Brush-border membrane preparation.
Brush-border membrane vesicles (BBMV) were prepared at 4°C, using
MgCl2 precipitation method, exactly as described by Yusufi et. al. (32). Briefly, freshly minced
cortical slices were homogenized in 50 mM mannitol and 5 mM
tris(hydroxymethyl)aminomethane-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Tris-HEPES), pH 7.0 (20 ml/g), in a glass Teflon homogenizer with
four complete strokes. The homogenate was then subjected to high-speed
[20,500 revolutions/min (rpm)] homogenization in an
Ultra-Turex Kunkel homogenizer for three strokes of 15 s each with an
interval of 15 s between each stroke.
MgCl2 was added to the homogenate
to a final concentration of 10 mM and slowly stirred for 20 min. The
homogenate was spun at 2,000 g in
J2-21 Beckman centrifuge using JA-17 rotor. The supernatant was
recentrifuged at 35,000 g for 30 min.
The pellet was resuspended in 300 mM mannitol and 5 mM Tris-HEPES, pH
7.4, with four passes by a loose-fitting Dounce homogenizer (Wheaton,
IL) and centrifuged at 35,000 g for 20 min in 15-ml Corex tubes, using JA-20 rotor. The outer white fluffy
pellet was resuspended carefully in small volume of buffered 300 mM
mannitol. Aliquots of homogenates were also saved for enzyme analysis
together with BBM preparations.
BBM(s) purity was checked by analyzing the activities of BBM marker
enzymes,
Na+-K+-adenosinetriphosphatase
(Na+-K+-ATPase)
(basolateral membrane enzyme) and acid phosphatase (lysosomal enzyme).
It was observed that the membrane preparations were severalfold purified as the activities of BBM enzymes were increased (5- to 8-fold), whereas those of
Na+-K+-ATPase
and acid phosphatase declined compared with their activities in the
homogenates (data not shown), and the BBM-to-homogenate ratio was
always <1. The purity of BBMV-SC and BBMV-JMC was determined on the
basis of higher
-glutamyltransferase (GGTase) activity in BBMV-JMC
(31), as also observed in the present study (see Fig. 5).
Enzyme assays. The activities of
marker enzymes in the homogenate and BBMV fraction were determined by
usual methods described elsewhere. The activity of alkaline phosphatase
(AlkPase) was measured by the method of Yusufi et al. (30). GGTase
activity was determined by the method of Glossmann and Neville (7) as reported by Yusufi et al. (31). Acid phosphatase was determined according to Verjee (29), whereas
Na+-K+-ATPase
activity was determined by the method described by Szczepanska-Konkel et al. (25). Protein was estimated by the modified method of Lowry et
al. (13) as described previously by Yusufi et al. (30).
Analysis of serum parameters. The
serum samples were deproteinated with 3% trichloroacetic acid in the
ratio of 1:3. The samples were centrifuged at 2,000 g (4,000 rpm) (Remi Centrifuge) for 10 min. The protein-free supernatant was used to estimate creatinine (11)
and Pi (26), whereas the pellet
was used for phospholipid estimation (15). Total serum cholesterol was
estimated directly in serum samples by the method of Zlatkis et al.
(33).
Statistical analysis. All experiments
were repeated at least three to four times to document reproducibility.
In each experiment, tissue from five to six animals was pooled to
prepare BBM in each group. All data are expressed as means ± SE. Where appropriate, statistical evaluation was conducted by group
t-test.
 |
RESULTS |
General. Renal ischemia in rats was
produced by occlusion of left renal artery for different time periods
as specified in Tables 1-5 or Figs. 1-9, followed by a brief
reflow of blood for 2 min to clear the kidney tissue before harvesting
the kidney for subsequent analysis. This procedure was applied
throughout the study and was considered as the baseline for observing
the damage caused by ischemia on various biochemical
parameters.
In one set of experiments, ischemia was produced by clamping the left
renal artery for 15, 30, 45, and/or 60 min. The results summarized in Table 1 indicate that, after
15 min of ischemia, serum creatinine was significantly increased (25%)
compared with sham-operated controls. A linear increase in serum
creatinine was observed when ischemia was produced for 30 min (56%) or
for a longer duration, i.e., for 60 min (80%). Because the values for
creatinine after 0-60 min in sham-operated controls were not significantly different, the data for control group was therefore expressed from pooled mean values observed at various time points. Similar to creatinine, the serum levels of
Pi, phospholipids, and cholesterol
were also increased significantly but to a different extent with respect to the duration of ischemia (Table 1).
The reflow of blood to ischemic rats (15 and 30 min ischemia) for
different time periods (15-120 min) showed a reversal in the
effect caused by the ischemia, and the serum creatinine,
Pi, and cholesterol
levels were significantly decreased both in 15 and 30 min
ischemic rats (Tables 2 and
3) and tended back toward normal values
after 120 min of reflow.
Effect of ischemia on activities of BBM marker
enzymes: whole cortex data. The effect of ischemia was
first determined on the activities of BBM marker enzymes in the
homogenates and in BBMV fractions isolated from whole cortex. The
specific activities of both AlkPase and GGTase were significantly but
differentially decreased with increase in the duration of
ischemia in BBMV (Fig. 1) but insignificantly
decreased in the homogenates (data not shown) of ischemic rats
compared with sham-operated controls. The maximum decrease was
observed after 60 min of ischemia.

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Fig. 1.
Specific activity (in µmol · mg
protein 1 · h 1)
of alkaline phosphatase (AlkPase) and -glutamyltransferase (GGTase)
in brush-border membrane vesicles (BBMV) from whole cortex after
different durations of ischemia. Values are means ± SE of 4 different experiments. * P < 0.05, significantly different from control values by group
t-test.
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Effect of ischemia and reflow on activities of BBM
marker enzymes from whole cortex. The effect of blood
reflow was determined in 30 and 60 min ischemic rats as shown in Fig.
2. Initial reflow of blood for 15 min
caused additional decrease compared with ischemic (with 2-min blood
reflow) and control rats in the activities of both AlkPase and GGTase
(Fig. 2). The decrease in the activities, however, was much greater in
60 min compared with 30 min ischemic rats (54 vs. 36% for AlkPase; 70 vs. 30% for GGTase). A prolonged blood reflow for 120 min showed a
sharp reversal of the effect of ischemia in the activities of both
AlkPase and GGTase. The activities were restored up to 86 and 92%,
respectively, of the control values in 30 min ischemic rats. However,
the recovery was much less (50-60% of the control values) in 60 min ischemic rats. The specific activities (enzyme unit/mg protein) of
both the enzymes in the cortical homogenate in control, contralateral, and ischemic kidneys were not significantly different (data not shown).

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Fig. 2.
Specific activity (µmol · mg
protein 1 · h 1)
of AlkPase and GGTase in BBMV from whole cortex after 30 ( ) and 60 ( ) min of ischemia and different durations of blood reflow. Values
are means ± SE of 4 different experiments.
* P < 0.05, significantly
different by group t-test from control
values. ** P < 0.05, significantly different by group
t-test from 15-min blood reflow
group.
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Because enzyme-specific activities (activity/mg protein) were found to
be altered only in BBM fractions and not in the homogenates, further
analysis showed that total enzyme activities (expressed as enzyme
units) greatly declined in the membrane-bound fractions, while
simultaneously increasing in the supernatant fractions (free enzymes
released due to ischemia). The changes observed were found to be in
proportion to the duration of the ischemia, i.e., the decrease in the
membrane-bound enzyme, as well as corresponding increase in supernatant
fraction, was much greater in 60 min (plus 15-min reflow) compared with
30 min (plus 15-min reflow) ischemic rats (Fig. 3). In
contrast, blood reflow for 120 min after 30 min of ischemia resulted in
a significant increase in the membrane-bound enzyme, while, at the same
time, the supernatant enzyme was decreased. However, in 60 min ischemic
rats, blood reflow for 120 min did not produce any change in the
membrane-bound enzyme activity, while free supernatant enzyme fractions
were further lowered (Fig. 3).

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Fig. 3.
Total enzyme activity (in µmol/h) of AlkPase and GGTase, membrane
bound (open bars) and free (solid bars), in whole cortical homogenates
after 30 and 60 min of ischemia, followed by 15 and 120 min of blood
reflow. Values are means ± SE of 3 different experiments.
* P < 0.05, significantly
different from respective controls by group
t-test.
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Effect of ischemia on BBM marker enzymes isolated from
superficial and deep cortices. To localize the effect
of ischemia, BBMV were isolated from superficial (BBMV-SC) and
juxtamedullary (BBMV-JMC) cortex, and the damage caused by the ischemia
was studied. As shown in Fig. 4, the activity of AlkPase
was decreased in ischemic rats both in BBMV-SC and BBMV-JMC compared
with respective control (or contralateral; data not shown) values. The
decrease in the activity was always linearly proportional to the time
(15-60 min) of ischemia. The activity of GGTase was similarly
declined (Fig. 5). However, the
decrease in the activity of both the enzymes was greater in BBMV-JMC
compared with BBMV-SC, at least in early ischemic conditions (15 and 30 min). The decrease in the enzyme activities was attributed mainly to
decrease in the maximal velocity (Vmax) of the
enzyme activities rather than in the Michaelis constant (Km) values
(Tables 4 and
5). Similar to the whole cortex, the activities of both enzymes were not significantly different in SC and
JMC homogenates of control, contralateral, or ischemic kidneys (data not shown).

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Fig. 4.
Specific activity of AlkPase in BBMV from superficial (SC) and
juxtamedullary (JMC) cortices after different time durations of
ischemia. Values are means ± SE of 4 different experiments.
* P < 0.05, significantly
different from controls by group
t-test.
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Fig. 5.
Specific activity of GGTase in BBMV from SC and JMC after different
durations of ischemia. Values are means ± SE of 4 different
experiments. * P < 0.05, significantly different from controls by group
t-test.
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Effect of ischemia and blood reflow on BBM enzymes
from superficial and deep cortices. The blood reflow
for 15 to 15 or 30 min ischemic rats resulted in further
decrease in the activities of AlkPase (Fig. 6) and
GGTase (Fig. 7) in both BBMV-SC and BBMV-JMC, as was
observed in BBMV whole cortex. Further blood reflow for up to 120 min
caused reversal of ischemia-induced decrease in the activities of both
the enzymes in BBMV-SC and BBMV-JMC (Figs. 6 and 7). However, the
reversal of AlkPase appeared to be slower in BBMV-JMC than in BBMV-SC,
whereas that of GGTase was slower in BBMV-SC than in BBMV-JMC
(Figs. 6 and 7). In comparision with 15-min reperfusion values (the
most affected time point by the ischemia), the activities of AlkPase
and GGTase increased by 60 or 120 min of blood reflow. However, the
recovery rates of enzyme activities were different in BBMV-SC and
BBMV-JMC (Figs. 6 and 7). In 15 min ischemic rats, the activity of
AlkPase was recovered to a much greater extent in BBMV-SC both at 60 (+27%) or 120 (+38%) min of reflow than in BBMV-JMC (+11 and +30%,
respectively), whereas the activity of GGTase was greatly recovered in
BBMV-JMC (+42%) compared with BBMV-SC (+22%). Similar recovery
patterns were also obtained in 30 min ischemic rats after 60 or 120 min
of blood reflow for both AlkPase and GGTase. The activities of both the enzymes in the contralateral kidneys were not significantly different from sham-operated normal kidneys (data not shown). The activities of
both the enzymes were not changed significantly in cortical homogenate
of control, contralateral, or ischemic kidneys after blood reflow
also (data not shown).

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Fig. 6.
Specific activity of AlkPase in BBMV from SC ( ) and JMC ( ) after
15 (left) and 30 (right) min of ischemia and
different durations of blood reflow. Values are means ± SE of 4 different experiments. * P < 0.05 and ** P < 0.05, significantly different from controls by group
t-test.
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Fig. 7.
Specific activity of GGTase in BBMV from SC ( ) and JMC ( ) after
15 (left) and 30 (right) min of ischemia and
different durations of blood reflow. Values are means ± SE of 4 different experiments. * P < 0.05 and ** P < 0.05, significantly different from controls by group
t-test.
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The activities of both AlkPase (Fig. 8) and GGTase (Fig.
9) in the homogenates were further fractionated. Similar
to whole cortex, the total activities of the membrane-bound fractions
(enzyme units) significantly declined, whereas the supernatant (free or released) enzymes were found to be increased after 30 min of ischemia and 15 min of blood reflow in both SC and JMC. However, after 120 min
of blood reflow, the activities of membrane-bound enzymes were
significantly increased with a compensatory decrease in the supernatant. In contrast, the enzyme activities decreased in the membrane-bound fractions to a much greater extent after 60 min of
ischemia and 15 min of reflow (Figs. 8 and 9) but did not change significantly after 120 min of reflow in the 60 min ischemic rats. The
decline in the membrane-bound enzymes and increase in the supernatant
enzymes were greater in JMC compared with SC. It appears that the
changes in the enzyme activities were reversible to some extent after
30 min of ischemia but mostly irreversible after 60 min of ischemia.

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Fig. 8.
Total enzyme activity (µmol/h) of bound (open bars) and free (solid
bars) AlkPase in SC (left) and JMC
(right) after 30 and 60 min of
ischemia, followed by 15 and 120 min of blood reflow. Values are means ± SE of 3 different experiments.
* P < 0.05, significantly
different from respective controls by group
t-test.
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Fig. 9.
Total enzyme activity (µmol/h) of bound (open bars) and free (solid
bars) GGTase in SC (left) and JMC
(right) after 30 and 60 min of
ischemia, followed by 15 and 120 min of blood reflow. Values are means ± SE of 3 different experiments.
* P < 0.05, significantly
different from respective controls by group
t-test.
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|
The activities of the marker enzymes of the other organelles like acid
phosphatase (lysosome) and
Na+-K+-ATPase
(basolateral membrane) were determined in the BBM and cortical
homogenate samples. The results indicate that the activities of above
enzymes were also lowered in response to ischemia, indicating an
overall effect of ischemia on the proximal tubular cells (data not
shown).
 |
DISCUSSION |
The proximal tubular segment is considered to be the chief nephron site
for the damage that occurs because of ischemic or toxic insult
(4-6, 27). Histological evidence shows that the damage due to ARF
primarily occurs in the pars recta (the
S3 segment) or in the deep
nephrons in animal models of ischemic injury (2, 27). The present
research was aimed at determining the effect of reversible and
irreversible ischemia on renal proximal tubules isolated from SC and
JMC and from whole rat renal cortex after different durations of
ischemia and blood reflow. The activities of BBM marker enzymes,
namely, AlkPase and GGTase, were determined to examine the structural
and functional integrity of proximal tubules under ischemic and reflow
conditions. The serum concentrations of creatinine,
Pi, phospholipids, and cholesterol
(Table 1) were increased progressively with increased durations of
ischemia and brought back toward normal values after 120 min of blood
reflow (Tables 2 and 3). Increase or decrease in serum creatinine
levels reflects the degree of the damage caused to the kidney by
ischemia and its reversal by subsequent blood reflow.
The activities of AlkPase and GGTase (Fig. 1) in the BBMV isolated from
whole cortex declined markedly with 15 to 60 min of ischemia and were
in partial agreement with the earlier studies (17, 18). Because the
specific activities (activity/mg protein) were not changed
significantly in the homogenates, as also reported by other studies
(17), we assert that the activities of AlkPase and GGTase were actually
decreased in the pellet of cortical homogenate (membrane-bound enzyme),
whereas the dissociated enzymes were traced in the supernatant where
the activities were increased (Fig. 3). This implies that BBM might
have been severely damaged during ischemia, and the enzyme and other
proteinic components after dissociation from the BBM released in the
supernatant and later can be excreted in the urine, as has been
observed by Herminghuysen et al. (9) and Desmouliere and Cambar
(1).
The distribution of the enzyme pattern in BBMV isolated from SC and JMC
showed that the activities of these marker enzymes decreased due to
ischemia to a greater extent in BBMV-JMC than in BBMV-SC (Figs. 4 and
5). As observed in the whole cortical homogenates, the activities of
AlkPase and GGTase were declined only in the membrane-bound fraction of
SC and JMC, with the corresponding increase in the supernatant as free
or dissociated enzymes (Figs. 8 and 9). The decrease in membrane-bound
enzyme and the corresponding increase in the free enzyme was greater in
JMC than in SC regions of the cortex. The data of the present study
clearly uphold the earlier findings of morphological and some
biochemical studies suggesting greater ischemic damage to the nephrons
in the deep cortex and especially pars recta (S3 subsegment) of
the proximal tubule (6, 17, 18, 28), as indicated by greater reductions of both AlkPase and GGTase in BBMV-JMC, and, in particular, GGTase, a
marker enzyme for this subsegment of the nephron (8, 31). Kinetic
studies further strengthen this viewpoint, because the decrease in the
activities of both AlkPase and GGTase was largely due to decrease in
Vmax, with little
or no effect on
Km values (Tables
4 and 5). This indicates that the decrease was largely due to the loss
of active enzyme molecules bound per unit of BBM isolated from ischemic
kidneys compared with nonischemic control preparations.
It has been demonstrated (12, 17, 18) that the damage caused to the
renal BBM due to ischemia is reversible and is associated with
reversible decrease in the membrane-associated enzyme-specific activities and morphological changes in the proximal tubule segment on
reflow of blood (18, 27). The reversibility of ischemic acute renal
failure depends on renal epithelial cell regeneration to reconstruct
normal nephronal architecture so as to reestablish normal functioning
of the kidney (10). It has also been reported that both the
degeneration as well as regeneration of tubular cells depends on the
duration of ischemia and blood reflow (10). Significant recovery of the
specific activities of both AlkPase and GGTase was observed after 120 min of reflow in 15 and/or 30 min ischemic BBMV isolated from
whole cortex (Fig. 2) and in SC and JMC (Figs. 6 and 7). However, only
small and insignificant recovery was observed in the specific
activities of both the marker enzymes in 60 min ischemic rats, even
after 120 min of blood reflow (Fig. 2). These results suggest that 15 to 30 min of ischemia causes, to some extent, reversible damage,
whereas 60 min of ischemia causes greater and relatively irreversible
damage to proximal tubular membrane components, as supported by the
observations of membrane-bound and unbound enzymes during ischemia and
reflow (Fig. 3).
The results also indicate that early reflow of 15 min under all
ischemic conditions showed maximum derease in enzyme activities as
reported earlier (17, 18). The fall in enzyme activity from 2-15
min of reperfusion may be caused either by continued tissue damage or
by sloughing and washing out of already damaged membranes
and/or their components from the proximal tubular cells (17,
18). The rate of recovery of AlkPase was relatively greater in BBMV-SC
compared with BBMV-JMC (Fig. 6) under early ischemic conditions
(15-30 min), whereas the rate of recovery of GGTase appears to be
greater in BBMV-JMC than BBMV-SC, at least in early (15-30 min)
ischemic conditions (Fig. 7). Differential localization and
organization of AlkPase and GGTase in the BBM and differential susceptibility to ischemic injury may be the cause of different effects
(8, 22, 27). Although AlkPase is located deep in the cytoplasmic site
of the membrane, GGTase is located in the middle of the BBM (8, 31).
Moreover, GGTase together with leucine aminopeptidase is considered to
be the marker enzyme of pars recta
(S3 subsegment) of the proximal
tubules, especially of the JMC region (31). Thus it can be envisioned
that BBMV of deep proximal tubular regions are greatly damaged because
of ischemia and regenerate at a slower rate than BBMV-SC.
In summary, the results clearly indicate that the activities of BBM
enzymes decreased linearly in a time-dependent manner. The decreases
because of ischemia in AlkPase and GGTase activities were relatively
greater in BBMV-JMC than in BBMV-SC. However, the rate of recovery due
to blood reflow for AlkPase was relatively greater in BBMV-SC and for
GGTase in BBMV-JMC. The effects of early ischemia (15-30 min) were
largely reversible, whereas prolonged ischemia (60 min) caused
relatively irreversible changes in the enzyme activities.
 |
ACKNOWLEDGEMENTS |
We thank Fazlur Rahman Khan for technical assistance.
 |
FOOTNOTES |
This work was supported by a grant (SP/SO/B-93/89) from Department of
Science and Technology, Government of India. S. J. Khundmiri, M. Asghar, and F. Khan are recipients of National Entrance Test fellowships from University Grants Commission, New Delhi,
India.
Part of this work was presented at the Federation of American Societies
for Experimental Biology Meeting, New Orleans, LA, March 28 to April 1, 1993.
Address for reprint requests: A. N. K. Yusufi, Dept. of Biochemistry,
Faculty of Life Sciences, Aligarh Muslim Univ., Aligarh-202 002, India.
Received 1 August 1996; accepted in final form 16 July 1997.
 |
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