Han et al. F-00009-2006.R1 (Final Accepted Version)

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Differential regulation of B/K protein expression in proximal and distal tubules of

rat kidneys with ischemia-reperfusion injury

Ki-Hwan Han1, U-Young Lee2,5, Yoon-Seong Jang3,5, Yoon Mi Cho3,5, Young Min

Jang3,5 In-A Hwang1, Jung Yeon Ghee4,5, Sun-Woo Lim4,5, Wan-Young Kim2,5, Chul

Woo Yang4,5, Jin Kim2,5, and Oh-Joo Kwon3,5

1Department of Anatomy, College of Medicine, Ewha Womans University, Seoul,

Korea; 2Departments of Anatomy, 3Biochemistry and 4Internal Medicine, and 5MRC for

Cell Death Disease Research Center, College of Medicine, The Catholic University of

Korea, Seoul, Korea

Running Head: B/K protein in the kidney

Address Correspondence to:

Jin Kim, M.D., Ph.D., Department of Anatomy, College of Medicine, The Catholic

University of Korea, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea, Phone: 82-2-

590-1153, Fax: 82-2-536-3110, e-mail: jinkim@catholic.ac.kr

Address Co-correspondence to:

Oh-Joo Kwon, M.D., Ph.D., Department of Biochemistry, College of Medicine, The

Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea,

Phone: 82-2-590-1179, FAX: 82-2-596-4435, e-mail: ojkwon@catholic.ac.kr

Page 1 of 27Articles in PresS. Am J Physiol Renal Physiol (August 8, 2006). doi:10.1152/ajprenal.00009.2006

Copyright © 2006 by the American Physiological Society.

Han et al. F-00009-2006.R1 (Final Accepted Version)

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ABSTRACT

Brain/kidney (B/K) protein is a novel double C2-like-domain protein that is highly

expressed in rat brain and kidney, but its cellular localization and functional role in the

kidney are still undetermined. We examined the cellular localization of B/K protein in

the rat kidney under normal and ischemic conditions. Ischemia-reperfusion (IR) injury

was induced by clamping both renal arteries for 45 minutes, and animals were

sacrificed at 1 and 6 hours and 1, 2, 3, 5, 7, 14, and 28 days after the reperfusion.

Kidney tissues were processed for immunohistochemistry and immunoblot analyses

using rabbit anti-B/K polyclonal antibodies. In control kidneys, B/K protein was

expressed primarily in the distal tubules including the thick ascending limb, distal

convoluted and connecting tubules, and collecting duct. Notably, B/K protein was also

expressed in the straight portion (S3 segment), but not in the S1 or S2, of the proximal

tubules, and podocytes of the glomerulus. In rat kidneys with IR injury, expression of

B/K protein was differentially regulated according to anatomical location. In the distal

tubules, overall expression of B/K protein was markedly decreased. On the other hand,

IR injury significantly increased B/K protein expression in the S3 segment of the outer

medulla as well as in rat proximal tubular epithelial cell line NRK-52E in vitro.

Furthermore, B/K protein was strongly expressed in many exfoliated cells in the lumen

and urine. These findings suggest that B/K protein is closely associated with cell death

of the proximal tubules which are vulnerable to IR injury in kidney.

Key words: brain/kidney (B/K) protein, ischemia-reperfusion (IR) injury, cell death

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INTRODUCTION

Acute renal failure represents a significant and persistent problem in clinical medicine

with serious consequences. Renal ischemia-reperfusion (IR) injury is a major cause of

acute renal failure in the native kidney and an invariable occurrence in the transplanted

kidney. Understanding the molecular mechanisms underlying renal cell injury is critical

for the prevention or treatment of acute renal failure, and considerable attention and

efforts have been directed towards identification of the therapeutic targets or

biomarkers involved in the tubular cell damage following IR injury. Global changes in

renal gene expression by the ischemic renal injury have been recently reviewed (7).

However, the intracellular and molecular mechanisms leading to acute renal failure by

IR injury remain incompletely understood.

We previously isolated a novel protein with two C2-like domains from the rat brain and

named it “B/K” based on its predominant expression in the brain and kidney (18). B/K is

a 474 amino acid protein and has several unique features. Although B/K protein does

not contain a transmembrane domain, it can bind to the plasma membrane via the N-

terminal cysteine cluster (9). Three negatively charged amino acids in the C2a domains

that have been suggested to be necessary for calcium binding (28) are substituted.

Moreover, B/K has three consensus sequences for protein kinase A (PKA). We

recently reported the distribution pattern of B/K protein. In the brain, B/K protein is

prominently expressed in neurosecretory areas such as the hypothalamic neurons, the

circumventricular organs, and some endocrine cells of the adenohypophysis (20). B/K

protein is expressed in most ganglion cells, a few amacrine cells, and the retinal fibers

of Müller cells in the rat retina (19). Interestingly, expression of B/K protein was induced

in the vulnerable regions of the hippocampus by kainate-induced excitotoxic injury (13)

as well as in the retina by IR injury (14). These findings suggest the possibility that B/K

protein may play a role in the mechanism of cell death in some pathological conditions

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including IR injury.

Besides the brain, B/K protein is most abundantly expressed in the kidney. Recently,

we reported the vasopressin-induced and PKA-dependent phosphorylation of B/K

protein in LLC-PK1, a porcine renal proximal tubule cell line (3). However, in spite of its

very high level of expression, its candidature as a substrate for PKA, and its possible

involvement in vasopressin-mediated signaling, the role of B/K protein in the kidney

has remained unknown.

In the present study, we investigated the expression and the cellular localization of B/K

protein in the rat kidney under normal and IR injury conditions.

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MATERIALS AND METHODS

Animals

Male Sprague Dawley rats (250-300 g, n = 6 per each group) were housed at 21 , 12℃ -

hour light/dark cycle, and allowed free access to food and water. Animals were

anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg body

weight). After opening the abdomen through a midline incision, both renal pedicles

were exposed and cleaned by blunt dissection. Microvascular clamps were placed on

both renal arteries to completely block renal blood flow, core body temperature was

maintained by placing the animals on a homeothermic table (29). After 45 minutes, the

clamps were removed and blood flow returned to the kidneys. Animals were sacrificed

at 1, and 6 hours and 1, 2, 3, 5, 7, 14, and 28 days after reperfusion. Control animals

received sham treatment. The sham operation was performed in a similar manner,

except for the clamping of the renal vessels.

Tissue Preservation

The kidneys were first perfused briefly through the abdominal aorta with phosphate-

buffered saline (PBS) to rinse out all blood and subsequently perfused with the fixative

solution, periodate-lysine-2% paraformaldehyde (PLP), for 5 min. The kidneys were

removed and cut transversely into 1- to 2-mm thick slices that were immersed in PLP

overnight at 4 . After being rinsed in PBS, sections of tissue were cut transversely ℃

through the entire kidney on a vibratome (Lancer Vibratome Series 1000; Technical

Products International, St. Louis, MO) at a thickness of 50 µm and processed for

immunohistochemical studies using a horseradish peroxidase preembedding technique.

Immunohistochemistry

Fifty-micrometer vibratome sections were processed for immunohistochemistry using

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an indirect preembedding immunoperoxidase method, as previously described (10, 11).

All sections were washed with 50 mM NH4Cl in PBS three times for 15 min. Before

incubation with the primary antibody, the all tissue sections were incubated for 3 hours

with PBS containing 1% BSA, 0.05% saponin, and 0.2% gelatin (solution A). The tissue

sections were then incubated overnight at 4 with the antibody against B/K protein℃

(20) diluted 1:1,000 in 1% BSA-PBS (solution B). Control incubations were performed

in solution B without primary antibody. After several washes with solution A, the tissue

sections were incubated for 2 hours in peroxidase-conjugated goat anti-rabbit IgG Fab

fragment (Jackson ImmunoResearch Laboratories, West Grove, PA), diluted 1:50 in

solution B. The tissues were then rinsed, first in solution A and subsequently in 0.05 M

Tris buffer, pH 7.6. For the detection of horseradish peroxidase, the sections were

incubated in 0.1% 3,3’-diaminobenzidine in 0.05 M Tris buffer for 5 min, after which

H2O2 was added to a final concentration of 0.01% and the incubation was continued for

10 min. After washing with 0.05 M Tris buffer three times, the sections were dehydrated

in a graded series of ethanol and embedded in Epon-812. From all animals, 50-µm-

thick vibratome sections through the entire kidney were mounted in Epon-812 between

polyethylene vinyl sheets. Sections were examined and photographed on an Olympus

photomicroscope equipped with differential-interference contrast (DIC) optics.

In Vitro IR Injury

A normal rat proximal tubular epithelial cell line (NRK-52E) was obtained from

American Type Culture Collection (Manassas, VA), and maintained in DMEM

supplemented with 5% fetal bovine serum (FBS) containing 0.2% gentamycin at 37°C

in a humidified atmosphere of 5% CO2 / 95% air. In vitro ischemia-reperfusion injury

was induced as previously described (30) with some modifications. In brief, when the

density of the cells (5 × 105 cells in 100 mm culture dishes) reached to around 70%

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confluency, the culture media was replaced with low serum media (DMEM/0.5% FBS)

and further incubated for 1 day. Then, the dishes were transferred to the anaerobic

chamber (ThermoForma model 1025, Marietta, OH) which was saturated with 85%

N2/10% H2/5% CO2, and incubated for 6 hours at 37°C. At the end of the ischemic

incubation, the dishes were removed from the anaerobic chamber and the culture

media was replaced with 10 ml complete media, and further incubated in a humidified

incubator with 5% CO2 at 37°C for additional 0, 12 and 24 hours.

For the cytotoxicity analysis, the cells were harvested at the indicated time and treated

with reagents containing Alexa Fluor 488-labeled annexin Ⅴ and propidium iodide (PI)

(Vybrant Apoptosis Assay Kit, Molecualr Probes, Eugene, OR) for 15 min at room

temperature. The apoptotic cells were analyzed by fluorescence-activated cell sorting

analysis (FACSCalibur; Becton Dikinson, Franklin Lakes, NJ). The total apoptotic cells

positive to both PI and annexin V were counted.

Immunoblot Analysis

For immunoblot analysis of in vivo experiments, the kidneys were isolated from rats

with IR injury at 1, and 6 hours and 1, 3, 5, 7, 14, and 28 days after the reperfusion and

perfused with PBS. The kidneys from sham operated rat were used as a control. The

cortex and medulla of the kidney were dissected carefully and homogenized in 10

volumes of 20 mM Tris-HCl (pH 7.4) containing 1% Triton X-100, 150 mM NaCl, 0.5%

sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 0.02% sodium azide, 1 µM leupeptin,

and 1 mM phenylmethylsulfonylfluoride (Sigma, St. Louis, MO). After centrifugation

(12,000 xg, 10 min, 4℃), the supernatant was isolated and the protein concentration

was determined using Bradford method (Pierce, Rockford, IL). Protein concentrations

were measured by bicinchonic acid assay kit (Bio-Rad, Hercules, CA), and 25 µg of

each protein was separated by 7.5% SDS-PAGE and transferred onto a nitrocellulose

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membrane (Schleicher & Schell, Keene, NH). The membrane were blocked with 5%

skim milk for 1 hour, and incubated with the rabbit anti-B/K polyclonal antiserum

(1:5,000 dilution) overnight at 4 . Antibo℃ dy binding was detected using horseradish

peroxidase-conjugated goat anti-rabbit IgG (1:1,000 dilution) (Sigma) and the

immunoreactive bands were visualized with ECL method (Amersham Pharmacia

Biotech, Upsala, Sweden). For in vitro IR injury samples, the cell lysates from treated

or untreated cells were resuspended in RIPA cell lysis buffer (25 mM Tris-HCl, pH 7.5,

0.1% SDS, 0.1% Triton X-100, 1% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA,

10% glycerol, 1 mM orthovanadate, 5 mM NaF, 1 mM phenylmethanesulfonyl fluoride,

and proteinase inhibitor). After incubation on ice for 5 min, the homogenate was

centrifuged at 13,200 xg for 30 min at 4°C, and 4 µg of total protein of the clarified

supernatants was separated by 12% SDS-PAGE.

Urine Cytology

Fresh urine samples from control or IR injury model were obtained by bladder puncture

and centrifuged at 4℃ for 10 min at 1,500 xg and fixed in PLP. The resuspended

aliquots were cytocentrifuged (Cytospin 2, Shandon, Frankfurt, Germany) onto

microscope slides for 5 min at 500 xg, and processed for immunocytochemistry using a

commercially available kit (Dako LSAB2Kit, Dako Corporation, Carpinteria, CA). B/K

protein antibody was used at 1:500 dilution.

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RESULTS

In control animals, B/K protein was immunostained in the renal cortex and outer

medulla (Fig. 1a). In the cortex, it was mainly located in distal tubules including the

cortical thick ascending limb, distal convoluted tubule and cortical collecting duct.

Some interstitial cells and podocytes of the glomerulus also showed positive

immunostaining. However, B/K protein immunoreactivity was not observed in proximal

tubules (S1 and S2 segments) in the cortex (Fig. 1b).

In the outer medulla, B/K protein immunostaining was strong in the medullary thick

ascending limb and the outer medullary collecting duct (Figs. 1c-e). Interestingly, the

labeling intensity appeared more prominent in type A intercalated cells than in principal

cells in the outer medullary collecting duct and the initial portion of the inner medullary

collecting duct (Figs. 1d and f). Moderate immunoreactivity was also observed in the

cells of proximal straight tubules (S3 segment) in the outer stripe of the outer medulla

(Fig. 1c). B/K protein was not immunostained in vascular bundles (Fig. 1e).

After IR injury, there was a notable change of B/K protein immunostaining in both the

cortex and the outer medulla (Fig. 2). In the cortex, the overall intensity of

immunostaining was decreased in distal tubules including the cortical thick ascending

limb, distal convoluted tubule and the cortical collecting duct, and it reached the lowest

level at day 1 after IR injury (Figs. 2c and 3a). The moderately decreased

immunostaining was also observed in podocytes of the glomerulus (Fig. 3a). Although

B/K protein immunoreactivity was not observed in proximal tubules in the cortex, some

damaged cells of the S2 segments showed B/K protein immunolabeling (Fig. 3a).

In the outer medulla, B/K protein immunostaining was rapidly decreased in the

medullary thick ascending limb and outer medullary collecting duct at 6 hours after IR

injury, which was easily recognizable in the inner stripe of the outer medulla (Fig. 2b).

The decreased immunoreactivity in the distal nephron segments began to be restored

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between 5 and 7 days and recovered fully at 14 days after the reperfusion (Fig. 2). On

the other hand, there was a marked increase of B/K protein expression in proximal

tubules (S3 segment) located in the outer stripe of the outer medulla (Fig. 2c). In

injured proximal tubules, B/K protein immunostaining was strongest in the many

exfoliated cells in the lumen at day 1 after IR injury and gradually decreased until 5

days after IR injury (Figs. 3b-f). Although B/K protein immunoreactivity increased in

severely damaged cells, it was very weak in intact tubular epithelial cells of the S3

segment (Fig. 4). Results from immunoblot analysis showed the early reduction of B/K

protein expression at 6 hours after IR injury, and rapid recovery at day 1 in the medulla

(Fig. 5). However, there were no significant changes of B/K protein expression in the

cortex (not shown). IR injury-induced increase of B/K protein expression in proximal

tubules was confirmed by in vitro experiments. As shown in Fig. 6a, B/K protein

expression in a rat proximal tubular epithelial cell line NRK-52E was strongly induced

by the 6 hour-ischemia, and further increased time-dependently by the reperfusion. In

the same experimental condition, IR strongly induced the apoptosis of NRK-52E cells

(Fig. 6b). Fraction of total apoptotic cells which were reactive to both propidium iodide

and annexin V were increased three-fold by 6 hour-ischemia (3.41%), and were

significantly and time-dependently increased by reperfusion (12 hour reperfusion,

17.54%; 24 hour reperfusion, 30.52%).

Cells immunopositive to B/K protein was also detected in the urine after IR injury. The

immunocytological investigations showed the strongest B/K protein immunostaining in

many urinary renal cells at day 1 and a gradual decrease until 5 days after IR injury.

However, few or no positive cells were found in the urine from control animal (Fig. 7).

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DISCUSSION

Renal ischemia-reperfusion (IR) injury is the major cause of acute renal failure, and

attempts at unraveling the molecular basis of cell damages induced by IR injury have

been facilitated by recent advances in functional genomics. Expression of many genes

has been found to be changed by IR injury (21, 22, 26), and recently, novel biomarkers

for early renal IR injury were identified by cDNA microarray analysis (7). In this study,

we examined the cellular localization of B/K protein in the rat kidney under normal

conditions and observed striking changes after IR injury, and suggest B/K protein as a

candidate marker for renal cell death.

In normal kidney, B/K protein was localized primarily in distal tubules including the thick

ascending limb, distal convoluted tubule and collecting duct. Moderate immunostaining

was also observed in the glomerulus, interstitium, and proximal straight tubule (S3

segment), suggesting a possible involvement of B/K protein in the cAMP-mediated

signaling mechanism. In the kidney, isoforms of adenylyl cyclase (AC) are expressed in

many nephron segments. To date, at least five AC isoforms (AC6, AC5, AC4, AC7, and

AC9) have been demonstrated in the rat kidney (2, 6, 12). Among them, AC4 was

expressed exclusively in the glomerulus, and both AC5 and AC6 were abundant in

some cells probably podocytes of glomeruli, and in the interstitium. AC6 was highly

expressed in the thick ascending limbs and weakly in the S3 segment, while AC5

mRNA was not detected in proximal tubules or the thick ascending limb. In collecting

ducts, AC5 was expressed only in intercalated cells, and AC6 was observed in both

intercalated and principal cells. Although we did not examine the expression of AC and

B/K proteins by coimmunostaining, it is possible that B/K protein is colocalized with

specific AC isoforms, especially AC5 and AC6, in the kidney.

cAMP produced by AC activates protein kinase A (PKA), and vasopressin is one of the

most important ligands for cAMP-PKA-mediated signaling mechanisms in the kidney.

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Vasopressin upregulates the expression of apical Na+-K+-2Cl−-cotransporter (BSC-1) in

the thick ascending limb (15, 16), and it increases water reabsorption in collecting duct

principal cells through a redistribution of the water channel aquaporin 2 by a

mechanism involving cAMP increase and PKA activation (23, 24). Apical K+ secretion

by ROMK in the thick ascending limb and the collecting duct segments also occurred

via a vasopressin-stimulated PKA pathway (8, 16). In our data, a significantly high level

of B/K protein expression was observed in the thick ascending limb, collecting duct and

distal convoluted tubules where vasopressin-stimulated cAMP-PKA pathways are well

established. Moreover, complete conservation of the consensus sequence for PKA

phosphorylation in B/K proteins among rat, mouse, and human genes, and

vasopressin- and PKA-dependent phosphorylation of B/K protein in a porcine kidney

cell line LLC-PK1 (3) strongly suggest the involvement of B/K protein in the

vasopressin-mediated cAMP-PKA pathways.

The S3 segment of the proximal tubule is a major target of hypoxic injury (21, 27). In

our experiments, IR injury induced B/K protein expression in many exfoliating cells but

not in intact epithelial cells in the S3 segment. Moreover, cells strongly positive to B/K

protein were detected in urine after IR injury. In addition, results from in vitro studies

showed that IR injury significantly induced B/K protein expression in a rat proximal

tubular cell line (NRK-52E), and also resulted in the apoptosis of the cells in the

manner nearly proportional to the level of B/K protein in the cells. These findings

strongly support our hypothesis that B/K protein may play a role in the cellular damage

of this segment after IR injury. Consistent with this result, our previous findings showed

a high level of expression of B/K protein in the most vulnerable regions of the

hippocampus in the kainate seizure model (13).

Many cases of IR-induced cellular damage are explained by apoptosis for which the

mitochondria play a crucial role in inducing apoptosis. Recently, the endoplasmic

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reticulum (ER)-mediated apoptosis mechanism has been suggested (25). Prolonged or

more substantial stress to ER (ER stress) may lead to cell death via apoptosis, and

many reports has also suggested ER stress as mechanisms for pathogenesis of

various diseases (1). In the kidney, ER stress responses also have been characterized

in experimental models of glomerulonephritis (5) and IR injury (17). For example,

phosphorylation of PERK and eIF2, important mediators for ER stress response, has

been reported in IR-dependent glomerular epithelial cell injury (4). In relation to ER

stress response, we also reported the ER-specific localization and the ER stress-

induced expression of B/K protein (13). These findings indicate the possible

involvement of B/K protein in an ER-mediated cell death mechanism in the IR injury

model of rat kidney.

In summary, B/K protein is expressed mainly in distal tubules in the normal kidney, and

IR injury remarkably changed the expression of B/K protein in both proximal and distal

tubules. We suggest a possible role for B/K protein in renal tubular cells under

conditions of cellular adaptation or damage after IR injury. However, the functional

significance of B/K protein expression in relation to the cAMP-mediated signaling

pathway or ER stress response remains to be established.

ACKNOWLEDGEMENTS

This work was supported by Korea Science & Engineering Foundation (KOSEF)

through the MRC for Cell Death Disease Research Center at the Catholic University of

Korea (R13-2002-005-01001-0) to J Kim, CW Yang and OJ Kwon.

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Physiol Renal Physiol 289: F289-F297, 2005.

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FIGURE LEGENDS

Figure 1. Light micrographs of immunostaining for B/K protein in the normal kidney.

Fifty micrometer sections of the kidney were cut on a vibratome and immunostained

with rabbit anti-rat B/K protein antibody as described in the Materials and Methods. In

the low magnification view (a), B/K protein is strongly expressed in outer and inner

stripes of the outer medulla and weakly in the cortex. In high-magnification views (b-f),

B/K protein is highly expressed in the podocyte (b, arrow), distal convoluted tubule (b,

DT), S3 segment of proximal tubule (c, S3), cortical and medullary thick ascending limb

(d, mTAL), and collecting duct (d and f). Note the strong immunoreactivity for B/K

protein in the intercalated cells (arrowheads) in the outer and inner medullary collecting

duct (d and f). No B/K immunoreactivity is observed in the proximal convoluted tubule

(b, PT) or vascular bundle (e, VB). Cx, cortex; OSOM, outer stripe of outer medulla;

ISOM, inner stripe of outer medulla; IM, inner medulla; IMi, initial part of inner medulla.

Magnifications: a, ⅹ20; b-f, ⅹ320.

Figure 2. IR injury-induced change of B/K protein expression in the cortex (Cx), outer

stripe of outer medulla (OSOM), and inner stripe of outer medulla (ISOM).

Fifty micrometer sections of the kidney from the rats with sham operation (a) or IR-

injury for 6 hours (b) and 1 (c), and 14 days (d) were cut on a vibratome and

immunostained with rabbit anti-rat B/K protein antibody as described in Materials and

Methods. Immunostaining for B/K protein was decreased by IR injury in the distal

nephron including thick ascending limb from 6 h, reached the lowest level at day 1, but

was restored at 14 days after the injury. Magnifications: ⅹ20.

Figure 3. High-magnification views of light micrographic findings showing IR injury-

induced B/K protein expression in the cortex (Cx) and the outer stripe of the outer

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medulla (OSOM).

IR injury was induced for 1 (a and b), 2 (c), 3 (d), 5 (e), and 7 days (f), and the

expression of B/K protein in the cortex (a) or OSOM (b-f) was examined using

immunohistochemical staining methods. Immunoreactivity to B/K protein was reduced

in the podocyte (arrow) and distal convoluted tubule (DT) in the cortex (a). In contrast,

B/K expression was significantly increased in many exfoliating cells of the proximal

tubule S3 segment in the OSOM between 1 and 3 days after the injury (b-d). Note that

B/K protein in the S3 segment of the proximal tubule was restored from 5 days after the

reperfusion (e and f). Magnifications: ⅹ320.

Figure 4. Light micrograph of IR injury-induced B/K immunoreactivity in the outer stripe

of outer medulla.

IR injury was induced for 3 days, and the expression of B/K protein in the outer stripe of

outer medulla was examined using immunohistochemical staining methods in higher

magnification view. Note the cells detached in the tubular lumen, which were heavily

stained with B/K antibody. Magnification: ⅹ960.

Figure 5. Temporal pattern of IR injury-induced B/K protein expression in the renal

medulla.

IR injury was induced in the rat kidney, and the medulla was dissected at the indicated

times after the reperfusion. The kidneys from sham-operated rats were used as a

control (Sham). Protein was extracted from each tissue, and the expression of B/K

protein was examined using immunoblot analysis as described in the Materials and

Methods. B/K protein immunoreactivity in the medulla was reduced immediately after

the reperfusion, reached the lowest level at 6 hours, and then returned to the control

level after 1 day. Beta actin was used as a loading control.

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Figure 6. IR inury-induced increase of B/K protein expression in NRK-52E cells.

In vitro IR injury was induced in a rat proximal tubular epithelial cell line (NRK-52E) by

6-hour ischemia and followed by the reperfusion for the indicated times. (a) The

expression of B/K protein in each sample was examined using immunoblot analysis.

Control cells (Con) showed very low level of B/K protein expression, and it was strongly

induced by the exposure of the cells to 6-hour ischemia and further increased time-

dependently by the reperfusion. Alpha-tubulin was used as a loading control. (b) IR

injury-induced apoptosis of NRK-52E cells were analyzed using FACS methods, and

expressed as total apoptotic cells as described in Materials and Methods. Fraction of

total apoptotic cells was increased by ischemia and reperfusion, and it was almost

proportional to the level of B/K protein in the cells.

Figure 7. Immunocytochemical staining of B/K protein-positive cells in urine after IR

injury.

Fresh urine samples from control or IR injury model were obtained at the indicated

times by bladder puncture, and the cells immunopositive to B/K protein were stained as

described in Materials and Methods. Cells positive to B/K protein in urine samples were

strongly increased by IR injury, reached the peak at day 1 and then gradually

decreased until day 5. Urine collected from control animals showed few or no B/K

protein positive cells.

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Figure 1. Han et al.

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Figure 2. Han et al.

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Figure 3. Han et al.

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Figure 4. Han et al.

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Figure 5. Han et al.

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Figure 6. Han et al.

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Figure 7. Han et al.

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