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【摘要】 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 (I/R) injury was induced by clamping both renal arteries for 45 min, and animals were killed at 1 and 6 h 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 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 proximal tubules, and podocytes of the glomerulus. In rat kidneys with I/R injury, expression of B/K protein was differentially regulated according to the anatomic location. In distal tubules, overall expression of B/K protein was markedly decreased. On the other hand, I/R injury significantly increased B/K protein expression in the S3 segment of the outer medulla as well as in the 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 in proximal tubules, which are vulnerable to I/R injury in the kidney.
【关键词】 brain/kidney protein cell death
ACUTE RENAL FAILURE REPRESENTS a significant and persistent problem in clinical medicine with serious consequences. Renal ischemia-reperfusion (I/R) 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 toward identification of the therapeutic targets or biomarkers involved in the tubular cell damage following I/R 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 I/R 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 NH 2 -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 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 I/R 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 including I/R 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-PK 1, a porcine renal proximal tubule cell line ( 3 ). However, despite its very high level of expression, its candidacy 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 I/R injury conditions.
MATERIALS AND METHODS
Animals. Male Sprague-Dawley rats (250-300 g, n = 6/group) were housed at 21°C, a 12:12-h light-dark cycle, and allowed free access to food and water. Animal experiments were performed with the full approval by the Animal Care and Use Committee of the Catholic University of Korea. Animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt). After the abdomen was opened 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, and core body temperature was maintained by placing the animals on a homeothermic table ( 29 ). After 45 min, the clamps were removed and blood flow returned to the kidneys. Animals were killed at 1 and 6 h 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 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°C. 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 an indirect preembedding immunoperoxidase method, as previously described ( 10, 11 ). All sections were washed with 50 mM NH 4 Cl in PBS three times for 15 min. Before incubation with the primary antibody, all tissue sections were incubated for 3 h with PBS containing 1% BSA, 0.05% saponin, and 0.2% gelatin ( solution A ). The tissue sections were then incubated overnight at 4°C 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 h 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 H 2 O 2 was added to a final concentration of 0.01% and the incubation was continued for 10 min. After being washed 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 I/R 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% CO 2 -95% air. In vitro I/R injury was induced as previously described ( 30 ) with some modifications. In brief, when the density of the cells (5 x 10 5 cells in 100-mm culture dishes) reached 70% 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% N 2 -10% H 2 -5% CO 2, and incubated for 6 h 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 of complete media and further incubated in a humidified incubator with 5% CO 2 at 37°C for an additional 0, 12, and 24 h.
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, Molecular Probes, Eugene, OR) for 15 min at room temperature. The apoptotic cells were analyzed by fluorescence-activated cell-sorting analysis (FACSCalibur; Becton Dickinson, 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 I/R injury at 1 and 6 h and 1, 3, 5, 7, 14, and 28 days after the reperfusion and perfused with PBS. The kidneys from sham-operated rats 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 phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO). After centrifugation (12,000 g, 10 min, 4°C), the supernatant was isolated and the protein concentration was determined using the Bradford method (Pierce, Rockford, IL). Protein concentrations were measured with a bicinchonic acid assay kit (Bio-Rad, Hercules, CA), and 25 µg of each protein were separated by 7.5% SDS-PAGE and transferred onto a nitrocellulose membrane (Schleicher & Schell, Keene, NH). The membrane was blocked with 5% skim milk for 1 h, and incubated with rabbit anti-B/K polyclonal antiserum (1:5,000 dilution) overnight at 4°C. Antibody binding was detected using horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1,000 dilution, Sigma), and the immunoreactive bands were visualized with the ECL method (Amersham Pharmacia Biotech, Uppsala, Sweden). For in vitro I/R 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 phenylmethylsulfonyl fluoride, and proteinase inhibitor). After incubation on ice for 5 min, the homogenate was centrifuged at 13,200 g for 30 min at 4°C, and 4 µg of total protein of the clarified supernatants were separated by 12% SDS-PAGE.
Urine cytology. Fresh urine samples from control or the I/R injury model were obtained by bladder puncture and centrifuged at 4°C for 10 min at 1,500 g and fixed in PLP. The resuspended aliquots were cytocentrifuged (Cytospin 2, Shandon, Frankfurt, Germany) onto microscope slides for 5 min at 500 g and processed for immunocytochemistry using a commercially available kit (Dako LSAB2Kit, Dako, Carpinteria, CA). B/K protein antibody was used at 1:500 dilution.
RESULTS
In control animals, B/K protein was immunostained in the renal cortex and outer medulla ( Fig. 1 a ). 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. 1 b ).
Fig. 1. Light micrographs of immunostaining for brain/kidney (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 MATERIALS AND METHODS. In the low-magnification view ( a ), B/K protein is strongly expressed in outer (OSOM) and inner stripes of the outer medulla (ISOM) and weakly in the cortex (Cx). In high-magnification views ( b - f ), B/K protein is highly expressed in the podocyte ( b, arrow), distal convoluted tubule (DT; b ), the S3 segment of proximal tubule (S3; c ), cortical and medullary thick ascending limb (mTAL; d ), 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 (PT; b ) or vascular bundle (VB; e ). IM, inner medulla; IMi, initial part of inner medulla. Magnifications: x 20 ( a ); x 320 ( b - f ).
In the outer medulla, B/K protein immunostaining was strong in the medullary thick ascending limb and the outer medullary collecting duct ( Fig. 1, c - 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 ( Fig. 1, d 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. 1 c ). B/K protein was not immunostained in vascular bundles ( Fig. 1 e ).
After I/R 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 I/R injury ( Figs. 2 c and 3 a ). The moderately decreased immunostaining was also observed in podocytes of the glomerulus ( Fig. 3 a ). 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. 3 a ).
Fig. 2. Ischemia-reperfusion (I/R) injury-induced change in B/K protein expression in the Cx, OSOM, and ISOM. Fifty-micrometer sections of the kidney from rats with sham operation ( a ) or I/R-injury for 6 h ( 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 I/R 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. Magnification: x 20.
Fig. 3. High-magnification views of light micrographic findings showing I/R injury-induced B/K protein expression in the Cx and the OSOM. I/R 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 Cx ( a ) or OSOM ( b - f ) was examined using immunohistochemical staining methods. Immunoreactivity to B/K protein was reduced in the podocyte (arrow) and DT in the Cx ( a ). In contrast, B/K expression was significantly increased in many exfoliating cells of the PT 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 PT was restored from 5 days after the reperfusion ( e and f ). Magnifications: x 320.
In the outer medulla, B/K protein immunostaining was rapidly decreased in the medullary thick ascending limb and outer medullary collecting duct at 6 h after I/R injury, which was easily recognizable in the inner stripe of the outer medulla ( Fig. 2 b ). The decreased immunoreactivity in the distal nephron segments began to be restored 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 in B/K protein expression in proximal tubules (S3 segment) located in the outer stripe of the outer medulla ( Fig. 2 c ). In injured proximal tubules, B/K protein immunostaining was strongest in the many exfoliated cells in the lumen at day 1 after I/R injury and gradually decreased until 5 days after I/R injury ( Fig. 3, b - 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 h after I/R injury, and rapid recovery at day 1 in the medulla ( Fig. 5 ). However, there were no significant changes in B/K protein expression in the cortex (not shown). I/R injury-induced increase of B/K protein expression in proximal tubules was confirmed by in vitro experiments. As shown in Fig. 6 a, B/K protein expression in a rat proximal tubular epithelial cell line NRK-52E was strongly induced by the 6-h ischemia, and further increased time dependently by the reperfusion. In the same experimental condition, I/R strongly induced the apoptosis of NRK-52E cells ( Fig. 6 b ). A fraction of total apoptotic cells that were reactive to both propidium iodide and annexin V were increased threefold by 6-h ischemia (3.41%) and were significantly and time dependently increased by reperfusion (12-h reperfusion, 17.54%; 24-h reperfusion, 30.52%).
Fig. 4. Light micrograph of I/R injury-induced B/K immunoreactivity in the OSOM. I/R injury was induced for 3 days, and the expression of B/K protein in the OSOM was examined using immunohistochemical staining methods at high magnification. Note the cells detached in the tubular lumen, which were heavily stained with B/K antibody. Magnification: x 960.
Fig. 5. Temporal pattern of I/R injury-induced B/K protein expression in the renal medulla. I/R 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 MATERIALS AND METHODS. B/K protein immunoreactivity in the medulla was reduced immediately after reperfusion, reached the lowest level at 6 h, and then returned to the control level after 1 day. -Actin was used as a loading control.
Fig. 6. I/R injury-induced increase in B/K protein expression in NRK-52E cells. In vitro I/R injury was induced in a rat proximal tubular epithelial cell line (NRK-52E) by 6-h ischemia, followed by reperfusion for the indicated times. A : expression of B/K protein in each sample was examined using immunoblot analysis. Control cells (Con) showed a very low level of B/K protein expression, and it was strongly induced by the exposure of the cells to 6-h ischemia and further increased time dependently by reperfusion. -Tubulin was used as a loading control. B : I/R injury-induced apoptosis of NRK-52E cells was analyzed using FACS methods and expressed as total apoptotic cells, as described in MATERIALS AND METHODS. The fraction of total apoptotic cells was increased by I/R, and it was almost proportional to the level of B/K protein in the cells.
Cells immunopositive to B/K protein were also detected in the urine after I/R 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 I/R injury. However, few or no positive cells were found in the urine from control animal ( Fig. 7 ).
Fig. 7. Immunocytochemical staining of B/K protein-positive cells in urine after I/R injury. Fresh urine samples from control or I/R injury model were obtained at the indicated times by bladder puncture, and the cells immunopositive for B/K protein were stained as described in MATERIALS AND METHODS. Cells positive for B/K protein in urine samples were strongly increased by I/R injury, reached a 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. Magnification: x 320.
DISCUSSION
Renal I/R injury is the major cause of acute renal failure, and attempts at unraveling the molecular basis of cell damages induced by I/R injury have been facilitated by recent advances in functional genomics. Expression of many genes has been found to be changed by I/R injury ( 21, 22, 26 ), and recently novel biomarkers for early renal I/R 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 I/R 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 PKA, and vasopressin is one of the most important ligands for cAMP-PKA-mediated signaling mechanisms in the kidney. Vasopressin upregulates the expression of the apical Na + -K + -2Cl - cotransporter 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 the porcine kidney cell line LLC-PK 1 ( 3 ), strongly suggests 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, I/R 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 I/R injury. In addition, results from in vitro studies showed that I/R 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 I/R 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 I/R-induced cellular damage are explained by apoptosis for which the mitochondria play a crucial role in inducing apoptosis. Recently, the endoplasmic 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 have also suggested ER stress as a mechanism for pathogenesis of various diseases ( 1 ). In the kidney, ER stress responses also have been characterized in experimental models of glomerulonephritis ( 5 ) and I/R injury ( 17 ). For example, phosphorylation of PERK and eIF2, important mediators for ER stress response, has been reported in I/R-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 I/R injury model of rat kidney.
In summary, B/K protein is expressed mainly in distal tubules in the normal kidney, and I/R 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 I/R 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.
GRANTS
This work was supported by the 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, C. W. Yang and O.-J. Kwon).
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作者单位：1 Department of Anatomy, College of Medicine, Ewha Womans University, and 2 Department of Anatomy, 3 MRC for Cell Death Disease Research Center, and Departments of 4 Biochemistry and 5 Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea