1Division of Nephrology and Hypertension, Department of Pediatrics, Children Hospital Medical Center, Cincinnati, Ohio 45229; 2Division of Nephrology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21218; 3Division of Nephrology and Hypertension, Department of Medicine, University of Cincinnati, Cincinnati 45267; and 4Veterans Affairs Medical Center, Cincinnati, Ohio 45220
Submitted 6 October 2003 ; accepted in final form 29 December 2003
ABSTRACT
Ischemic renal injury can be classified into the initiation and extension phase followed by the recovery phase. The recovery phase is characterized by increased dedifferentiated and mitotic cells in the damaged tubules. Suppression subtractive hybridization was performed by using RNA from normal and ischemic kidneys to identify the genes involved in the physiological response to ischemia-reperfusion injury (IRI). The expression of stathmin mRNA increased by fourfold at 24 h of reperfusion. The stathmin mRNA did not increase in sodium-depleted animals or in animals with active, persistent injury secondary to cis-platinum. Immunofluorescent labeling demonstrated that the expression of stathmin increased dramatically at 48 h of reperfusion. Labeling with antibodies to stathmin and proliferating cell nuclear antigen (PCNA) indicates that the expression of stathmin was induced before the upregulation of PCNA and that all PCNA-positive cells expressed stathmin. Double immunofluorescent labeling demonstrated the colocalization of stathmin with vimentin, a marker of dedifferentiated cells. Stathmin expression was also significantly enhanced in acute tubular necrosis in humans. On the basis of its induction profile in IRI, the data indicating its enhanced expression in proliferating cells and regenerating organs, we propose that stathmin is a marker of dedifferentiated, mitotically active epithelial cells that may contribute to tubular regeneration and could prove useful in distinguishing the injury phase from recovery phase in IRI.
acute renal failure; acute tubular necrosis; ischemia-reperfusion injury; cis-platinum nephrotoxicity; sodium depletion; tubule regeneration
The microtubule network is important in mitosis and in the maintenance of cell polarity (1). In the kidney, epithelial cells undergo depolarization and become mitotically active as a part of their response to IRI (52). The microtubule structure is disrupted as early as 1 h after reperfusion. This loss of microtubule integrity may contribute to the changes in cell polarity and structure observed in the kidneys subjected to IRI (1). In later stages of IRI, at time points that coincide with increased mitotic activity (2448 h after reperfusion), the microtubule structures become visible in renal tubular epithelium and may signal the onset of the tubular repair process and the recovery phase of IRI (1).
During a gene discovery process to identify novel therapeutic and diagnostic targets in renal IRI using suppression subtractive hybridization (SSH), we observed a major increase in stathmin mRNA levels (see RESULTS) at 2448 h of reperfusion. Stathmin is a ubiquitously expressed, 19-kDa cytosolic protein encoded by a single gene on human chromosome 10 (10). It is a member of a group of proteins that bind to and destabilize the microtubule networks (37, 39, 40). Other than stathmin, which is expressed in a variety of cell types, the remaining members of this family, SCG19, SCLIP, and RB3 variants, are only expressed in the nervous system (22, 37, 40, 41, 49). The ability of stathmin to bind to tubulin and disrupt the microtubule structure is modulated through its phosphorylation on multiple serine residues by protein kinases such as p34Cdc2 kinase, cAMP-dependent protein kinase, mitogen-activated protein kinase (MAPK), and calcium/calmodulin-dependent kinaseGr (2730). It is possible that the expression levels and phosphorylation status of stathmin regulate cell division by increasing the instability of interphase microtubules, leading to their depolymerization at the onset of mitosis followed by repolymerization of microtubules to form the mitotic spindle (16, 19). Stathmin is expressed at high levels in some leukemias, lymphomas, and a variety of solid tumors derived from prostate, breast, and ovary (2, 5, 8, 11, 48). Stathmin expression is increased in proliferating, transformed, and nontransformed cells (2, 5, 8, 11, 48). The expression of this protein is also enhanced during neuronal and hepatic regeneration (2, 5, 8, 11, 48). Stathmin expression is regulated by the activity of the p53 tumor suppressor, where stathmin levels are reduced in cells subjected to p53-mediated growth inhibition (9, 21). Recent studies indicate that stathmin functions in the G2-to-M transition during the cell cycle and plays an important role in the regulation of the mitotic spindle (16, 19, 24). Cells overexpressing stathmin or expressing mutated forms of stathmin that cannot be phosphorylated undergo growth arrest at G2-to-M transition (21, 24, 25). These observations suggest that stathmin is important in the regulation of cell proliferation and tissue regeneration after injury.
The association of the stathmin increase with recovery from tubular injury was confirmed in kidney biopsies from a patient recovering from acute tubular necrosis. To assess potential diagnostic specificity to recovery from tubular injury, we compared stathmin levels at time-matched intervals (72 h) in IRI and in a model of persistent injury by cis-platinum; stathmin expression levels increased in IRI but remained unchanged with cis-platinum treatment. The significance of stathmin expression in models of renal failure and its prognostic significance are discussed.
METHODS
Materials
[32P]dCTP was purchased from New England Nuclear (Boston, MA). Nitrocellulose filters and chemicals were purchased from Sigma (St. Louis, MO). RadPrime DNA labeling kit was purchased from GIBCO-BRL (Rockville, MD).
Animal Models
Ischemic reperfusion injury. IRI was induced as previously described (45, 46). Briefly, bilateral IRI was induced in male Sprague-Dawley rats (200250 g) or Swiss mice (2530 g) by occluding the renal pedicles with microvascular clamps (15, 30, or 45 min) under ketamine-xylazine anesthesia. Completeness of ischemia was verified by blanching of the kidneys, signifying the stoppage of blood flow. The blood flow to the kidneys was reestablished by removal of the clamps (reperfusion) with visual verification of blood return. Animals subjected to sham operation (identical treatment except that the renal pedicles were not clamped) were used as controls. During the procedure, animals were well hydrated and their body temperature was controlled around 94°F with an adjustable heating pad. After ischemia, animals were kept under the veterinarian's observation. At 12, 24, and 48 h postischemia, animals were killed, and their kidneys were harvested and snap frozen in liquid nitrogen or fixed in paraformaldehyde for immunohistochemical studies. For mice, animals were killed at 2, 12, 24, 48, and 72 h of reperfusion after 30 min of ischemia.
Sodium depletion. Sodium depletion was induced by placing rats (80120 g) on a sodium-free diet for 5 days. The purpose of the sodium depletion was to induced "prerenal' acute renal failure, where there would be a rise in serum creatinine without tubular injury.
Cis-platinum treatment. To induce cis-platinum injury, rats (80120 g) were administered a single intraperitoneal injection of cis-platinum (5 mg/kg body wt) and killed at 1, 3, and 7 days. In this model there is significant tubular injury without an early rise in serum creatinine. The onset of ARF in these animals was established by the measurement of blood urea nitrogen (BUN) and serum creatinine levels and were previously reported (53).
RNA Isolation and SSH
Cortex and medulla were separated and snap frozen in liquid nitrogen. Total cellular RNA was extracted from renal cortex or medulla by using the Tri reagent method (MRC, Cincinnati, OH) following the manufacturer's protocol. Total RNA from control rats and rats subjected to IRI were used to make driver and tester cDNAs. SSH was performed by using the PCR-Select cDNA subtraction kit according to the manufacturer's instructions (Clontech, Palo Alto, CA). Subtracted PCR products were ligated into the pGEM-T easy vector (Promega, Madison, WI), and ligation mixtures were transformed into the DH-5 strain of Escherichia coli (Invitrogen Life Technologies, Gaithersburg, MD). This approach provided us with subtracted libraries containing differentially expressed genes in the kidneys of control animals but not in the kidneys of animals subjected to ischemic injury after 24 and 48 h of reperfusion, as well as subtraction libraries representing the genes differentially expressed in the kidneys subjected to IRI after 24 and 48 h of reperfusion. Differentially expressed products were selected by using the PCR-Select differential screening kit (Clontech). The cloned products were sequenced, and the results were compared with GenBank database sequences by using the BLAST homology search program (National Institutes of Health, Bethesda, MD).
Northern Hybridization
Total cellular RNA (30 µg/lane) was size fractionated on a 1.2% agarose-formaldehyde gel and transferred to nylon membranes by capillary transfer by using 10x SSPE buffer. Membranes were crosslinked by UV light or baked. Hybridization was performed according to Gilbert and Church (13). Membranes were washed, blotted dry, exposed to a PhosphorImager screen at room temperature for 2472 h, and scanned with a PhosphorImager. A 32P-labeled cDNA fragment of the mRNA encoding the region spanning nucleotides 28 to 393 of stathmin (accession no. NM_017166 [GenBank] ) and a cDNA probe that identifies the spermidine/spermine N1-acetyltransferase (SSAT) transcript previously described by Zahedi et al. (53) were used to identify stathmin or SSAT mRNAs.
Preparation of Kidney Extracts
Briefly, the tissue samples (cortex or medulla) were homogenized in ice-cold isolation solution (250 mM sucrose and 10 mM triethanolamine, pH 7.6) containing protease inhibitors (0.1 mg/ml phenazinemethylsulfonyl fluoride and 1 µg/ml leupeptine) by using a Polytron homogenizer. The homogenate was centrifuged at low speed (1,000 g) for 10 min at 4°C to remove nuclei and cell debris.
Western Blot Analysis of Stathmin Expression
Western blot analyses were performed as previously described by Peschanski et al. (42). Briefly, 50 µg of protein from each extract were loaded onto an SDS-12% polyacrylamide gel. After size fractionation, proteins were transferred to a polyvinylidene difluoride membrane in buffer containing 20 mM Tris, 192 mM glycine, 20% methanol, and 0.1% SDS (pH 8.3) with the use of a transfer apparatus (Idea Scientific, Minneapolis, MN) at a constant power of 400 mA for 2 h. Blocking reaction was performed overnight in 5% nonfat dry milk in Tris-buffered saline (TBS; 137 mM NaCl and 20 mM Tris, pH 7.4). Exposure to the anti-stathmin primary antibody (Calbiochem, San Diego, CA), diluted 1:1,000, was carried out in 5% dry milk in TBS for 4 h at room temperature. After being washed twice in TBS containing 2.5% dry milk and 0.1% Tween 20 (TTBS) for 10 min each and then once in TBS, the membrane was incubated with a secondary antibody (goat anti-rabbit IgG-peroxidase conjugate diluted 1:2,000 in 2.5% dry milk in TTBS) for 1 h at room temperature. After two 10-min washes in 2.5% dry milk in TTBS and two 10-min washes in TBS, the membrane was developed by using peroxidase detection reagents (ECL kit; Amersham, Piscataway, NJ) and exposed for 530 s to an X-ray film. The stathmin antibody is highly specific and detects the stathmin as an 19-kDa band (42).
Immunofluorescent Staining of Kidney Sections
Paraformaldehyde-fixed paraffin-embedded sections were washed twice in PBS (pH 7.4) and blocked with 10% rabbit serum-0.3% Triton X-100 in PBS for 1 h. For double labeling, tissue sections were incubated with rabbit anti-stathmin polyclonal antibody (Calbiochem) and mouse anti-proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-vimentin (Sigma) monoclonal antibodies. Sections were washed and then incubated with appropriate secondary antibodies (rabbit anti-goat IgG conjugated with Oregon Green 488 for anti-stathmin antibody and goat anti-mouse IgG conjugated with Alexa Fluor 568 dye for anti-PCNA or anti-vimentin antibodies) for 2 h at room temperature. Sections were examined and images acquired on a Nikon PCM 2000 laser confocal scanning microscope as 0.5-µm "optical sections" of the stained cells. The 488-nm line of the argon laser, isolated with the standard argon laser exciter filter supplied with PCM 2000, was used for the green dye excitation. The PCM 2000 standard 515/30-nm emission filter was used for the green emitting dye. Red dye was excited with the 543.5-nm single line output of the helium-neon laser. The PCM 2000 standard red channel long-pass 565-nm filter was used as the emission filter for the red dye. Digital images of the green and red dyes were simultaneously acquired through a single illumination and detection pinhole. The images were discretely resolved into two channels and separately analyzed.
Immunofluorescent Staining of Stathmin in Human Kidneys
Acquiring and processing of human kidney tissues were performed according to the institutional and Health Insurance Portability and Accountability Act of 1996 guidelines.
RESULTS
Identification of Stathmin by SSH As a Transcript That Is Upregulated During Renal IRI
Total RNA from the cortical regions of control rat kidneys and rat kidneys subjected to IRI (30 min of ischemia; 12, 24, or 48 h of reperfusion) were used to identify differentially expressed transcripts with the use of SSH (occurrence of tissue damage was determined by the measurement of BUN and serum creatinine levels). Subtracted cDNAs were cloned to develop four condition- and time-specific libraries. A total of fifty colonies from the four subtracted libraries were isolated and sequenced. Comparison of the sequence of the isolated clones to those available in the BLAST database identified a number of previously known transcripts including SSAT, membrane-associated protein 17 (MAP 17), dynactin (p50), adenosine receptor type 3, and stathmin as being differentially regulated in response to IRI. Other genes identified by SSH included mitochondrial transcripts such as those involved in the oxidative reduction pathway, the electron transport chain, and three previously unidentified transcripts. To confirm the results of SSH, total RNA from the renal cortex and medulla of control animals and animals subjected to IRI (30 min of ischemia; 12, 24, or 48 h of reperfusion) was size fractionated and subjected to Northern blot analysis. Our data thus far indicate that SSAT, MAP 17, dynactin, and two of the three novel transcripts are differentially regulated in response to IRI. Furthermore, Northern blot analyses indicated that stathmin mRNA expression was unchanged at 12 h but increased by approximately fourfold (P < 0.01 vs. sham or 12-h reperfusion, n = 3) and fivefold (P < 0.01 vs. sham or 12-h reperfusion, n = 3) in the cortex (Fig. 1A) and medulla (Fig. 1B) after 24 h of reperfusion in kidneys subjected to IRI. A longer time-course analysis demonstrated that stathmin expression was increased at 24 h and remained elevated at 48 h of reperfusion in both the cortex and medulla (Fig. 2A). To determine whether the increase in stathmin mRNA also reflects an increase in stathmin protein abundance, we subjected kidney extracts harvested at various reperfusion intervals (0, 12, 24, and 48 h) to Western blot analysis using a polyclonal rabbit anti-stathmin antibody. As shown in Fig. 2B, stathmin protein levels peaked at 48 h after reperfusion. The time course of the increase in stathmin levels correlates with the tissue repair and recovery phase of IRI.
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Stathmin mRNA Expression in Kidneys of Animals Subjected to Varying Intervals of Renal Ischemia
To determine whether the expression of stathmin correlates with the extent of tissue damage induced by the ischemic insult, we subjected rats to 15, 30, and 45 min of renal ischemia and examined the expression of stathmin mRNA at 24 h of reperfusion. Total RNA (30 µg/lane) from each animal was size fractionated on a denaturing agarose gel and subjected to Northern blot analysis with a stathmin cDNA probe. Our results indicate that the expression of stathmin mRNA increases in all IRI samples and that the magnitude of this response is greater in the RNA obtained from the kidneys that were subjected to more prolonged (30 or 45 min) ischemia (Fig. 3A).
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Stathmin Expression in a Model of Kidney Failure Without Tubular Injury
Distinguishing tubular injury from volume depletion as the cause of renal failure is a common clinical dilemma. To determine whether enhanced expression of stathmin was due to tissue damage secondary to IRI or accumulation of nitrogenous wastes because of renal dysfunction, we subjected rats to sodium depletion for 5 days. Occurrence of acute renal failure was verified by increases in serum BUN and creatinine at the time of euthanasia. The urine sodium excretion decreased by >95% after 24 h of sodium deprivation and remained very low throughout the experiments. The blood levels on BUN and creatinine in sodium-depleted animals were recently published by our laboratories (53). As shown in Fig. 3B, the stathmin mRNA levels did not change in the kidneys of rats subjected to sodium depletion. These results indicate that renal failure per se does not lead to increased expression of stathmin.
Stathmin Expression in a Model of Nephrotoxic Tubular Injury
Nephrotoxic injuries are major causes of kidney failure in the U.S. population. One common cause of nephrotoxic injury is cis-platinum treatment in the patients with solid tumors such as breast or lung cancer. Nephrotoxic injury mediated by cis-platinum involves the proximal tubule and the medullary thick ascending limb of Henle and causes acute renal failure. We recently demonstrated that a single dose of cis-platinum injection causes persistent renal injury for at least 72 h (53); therefore, we entertained the possibility that the regenerative response in kidneys of cis-platinum-treated animals is delayed due to the persistence of tubular cell injury compared with IRI. Northern hybridizations (Fig. 3C) indicated that the kidney expression of stathmin decreased at 1 and 3 days after cis-platinum treatment. Interestingly, the kidney expression of SSAT, which is a marker of active tubular cell injury, showed sustained elevation at both 1 and 3 days after cis-platinum treatment. These results strongly suggest that at the time of ongoing cell injury (enhanced SSAT expression) stathmin expression is decreased in cis-platinum nephrotoxicity. This completely contrasts with the same time points (24 and 72 h of reperfusion) in kidney IRI, which show abundant expression of stathmin (see below).
Immunocytochemical Analysis of Stathmin Expression and Its Correlation with Cell Proliferation in Renal IRI
To demonstrate the transferability of these findings to other species, we used a mouse model of renal IRI. Accordingly, mice were subjected to 30 min of kidney ischemia followed by 24, 48, and 72 h of reperfusion. Northern hybridizations demonstrated that stathmin mRNA expression in mice with kidney IRI displayed a pattern very similar to that in rats, with stathmin mRNA levels increasing at 24 h and remaining elevated at 48 and 72 h after reperfusion (data not shown).
To examine the cellular distribution and regulation of stathmin, we performed immunocytochemical staining. Paraffin-embedded sections from control kidneys and kidneys from mice subjected to IRI were harvested at timed intervals (0, 24, and 48 h after reperfusion) and examined for the expression of stathmin by immunofluorescent microscopy. Our results indicate that in the kidneys of control animals (time 0), stathmin is expressed by a very limited number of renal tubular epithelial cells (<0.1%) and not at all in the glomeruli (Fig. 4a). However, the expression of stathmin increased significantly at 48 h after reperfusion (Fig. 4c). At this time point, both the number of the renal tubular epithelial cells expressing stathmin and the intensity of their staining increased significantly (Fig. 4c). Expression of stathmin at 24 h of reperfusion was greater than that of the control but considerably less than that at 48 h of reperfusion (Fig. 4b). Stathmin staining in kidney sections after 48 h of reperfusion indicates considerable variation, with some tubules demonstrating no labeling of any cells, whereas in some tubules all cells expressed stathmin (Fig. 4c). To identify the site of expression of stathmin, we also performed examination of the stathmin expression in the kidney after 48 h of reperfusion at low magnification. Figure 4d depicts the pattern of fluorescent staining for stathmin under low magnification in kidneys subjected to IRI. The results indicate that stathmin-positive cells are primarily located in the inner cortical and outer medullary regions (corticomedullary junction) of the kidney, a region that contains the S3 segment of the proximal tubules.
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IRI involves both the loss of epithelial cell polarity and the onset of proliferative response; therefore, we decided to examine the proliferative status of renal tubular epithelial cells that express stathmin. Immunofluorescent double staining of kidney sections (Fig. 5) with antibodies against stathmin and PCNA, a marker of cell proliferation, indicated that stathmin (green) and PCNA (red) are expressed, albeit at very low levels (<0.1% of the renal epithelial cells) in the kidneys of sham-operated animals (Fig. 5, ac). Examination of multiple fields in control kidneys indicated that only a fraction (<20%) of stathmin-positive cells also express PCNA. The expression of stathmin and PCNA increased at 24 (Fig. 5, df) and 48 h after reperfusion (Fig. 5, gi). The merged images (Fig. 5, b, e, h, and k) clearly demonstrate coexpression of stathmin and PCNA at both 24 and 48 h of reperfusion in the tubular epithelium of ischemic kidneys. A closer analysis of the merged images in Fig. 5, e and h, demonstrates that whereas all PCNA-expressing cells express stathmin, a substantial fraction (>75%) of cells expressing stathmin do not express PCNA at 24 and 48 h of reperfusion. At 72 h after reperfusion (Fig. 5, jl), the expression of both stathmin and PCNA in the tubular epithelium increased dramatically (Fig. 5, j and l). At 72 h, all PCNA-positive epithelial cells also expressed stathmin (Fig. 5k), and only a minor fraction (<10%) of stathmin-positive cells did not express PCNA.
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It has been previously reported that dedifferentiation of renal proximal tubular cells is a component of the repair process after IRI (52). To determine whether the stathmin-expressing cells represent a dedifferentiated population of proximal tubule cells, we examined kidney sections from control animals and animals subjected to ischemia followed by 72 h of reperfusion for coexpression of stathmin and vimentin, a marker of cellular dedifferentiation. As shown in Fig. 6, ac, cells that express stathmin also express vimentin. In addition, these results lend further support to the observation that cells that express stathmin are located in the S3 segment of renal tubular epithelial cells, because previous studies indicate that vimentin expression in IRI is primarily in the epithelial cells of the S3 segment of the proximal tubule (52). The colocalization of stathmin with PCNA (Fig. 5) and vimentin (Fig. 6) also indicates that stathmin is expressed by dedifferentiated cells that have reentered the cell cycle.
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Immunocytochemical Analysis of Stathmin Expression in Human Kidneys Recovering from Acute Tubular Necrosis
Increased expression of stathmin at 48 and 72 h after reperfusion (Figs. 4, 5, 6) corresponds with stabilization and improvement of kidney function, respectively, in kidneys subjected to IRI. The purpose of the next series of experiments was to determine whether enhanced expression of stathmin could be detected in human kidneys recovering from acute ischemic renal failure. Accordingly, a kidney biopsy from a human patient recovering from acute tubular necrosis (declining serum creatinine and improving urine output) and sections from an otherwise normal kidney that was removed for treatment of cancer were examined for the expression of stathmin. Figure 7, left, is an immunofluorescent microscopic image of a normal human kidney and demonstrates very low expression levels of stathmin. Figure 7, right, is an image from a kidney recovering from acute tubular necrosis and demonstrates significant expression of stathmin. These results demonstrate that enhanced stathmin expression occurs in the recovery phase of IRI and may be useful in distinguishing the kidneys that are recovering from those that are not recovering from IRI.
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DISCUSSION
Acute renal failure caused by IRI is among the major causes of morbidity in hospitalized patients and indicates poor prognosis in patients with multisystem organ failure. Identification of the genes that contribute to the pathology of renal IRI is of great importance in developing diagnostic tools and therapeutic approaches for the treatment of this injury. Using SSH, we identified stathmin as one of the transcripts that is induced in the kidneys after IRI. Stathmin is a member of a family of proteins that includes SCG10, SCLIP, and RB3 variants (38). The stathmin family proteins share extensive structural homology and are involved in the regulation of microtubule assembly (12). Stathmin is a ubiquitously expressed cytosolic phosphoprotein (42, 48). In its nonphosphorylated form, stathmin binds to tubulin and causes microtubule catastrophe (12, 17). Stathmin is the phosphorylation target for a number of serine/threonine kinases such as protein kinase A, MAPK, and p34Cdc2 (3, 4, 7, 26, 27, 29, 30). The ability of stathmin to bind to tubulin is abrogated upon its phosphorylation (31, 32).
The initial injury phase of IRI (the first 24 h after reperfusion) involves initiation and progression of tissue injury and is characterized by loss of cell polarity, increased cell swelling, loss of adherence to extracellular matrix, and cell death. The injury phase is followed by the recovery phase, which at the cellular level is manifested by the increased presence of dedifferentiated and mitotic cells in the damaged tubules. The continuation of the recovery phase is associated with the recovery of tubular epithelium and reestablishment of cell polarity. Recent studies indicate that polarity of the actin cytoskeleton is reconstituted before the reorganization of the microtubule network in proximal tubule cells (51). The expression of stathmin does not correlate with the progression of tissue injury because its expression in both IRI- and cis-platinum-induced renal damage is delayed compared with other established markers of tissue injury such as SSAT and kidney injury molecule 1 (15, 53). On the other hand, the time course of stathmin expression (2472 h after reperfusion) parallels the onset of cellular dedifferentiation, mitosis, and tubular regeneration response in the kidneys subjected to IRI (52). In addition, it cannot be ruled out that the expression of stathmin may play a role in the delayed reestablishment of the microtubule network and cell polarity after IRI (51).
Microtubules play an indispensable role in vesicular transport, regulation of cell polarity, and mitosis. Previous studies have demonstrated that stathmin is expressed in mitotically active cells such as cancer cells, crypt cells in the intestinal epithelium, and hepatic epithelial cells after partial hepatectomy (20, 23, 47, 48). Increased expression of stathmin has been shown to be associated with the reentry of the cells into the cell cycle and the onset of cell proliferation (33, 34). Once expressed, stathmin function (i.e., its ability to bind to tubulin) is regulated via its phosphorylation on four serine residues (16, 25, 38 and 63) in its carboxy-terminal regulatory domain in a cell cycle-dependent manner (3, 4, 7, 26, 27, 29, 30). The role of stathmin in the regulation of cell division is supported by recent studies indicating that the downregulation of stathmin by antisense RNA leads to disruption of spindle structure and difficulties in completing mitosis (19). Taken together, these results indicate that the primary role of stathmin is in the regulation of spindle structure during cell division and that its phosphorylation by kinases such as MAPKs and Cdc2 is necessary for the modulation of its activity and completion of mitosis (4, 27).
Immunofluorescence studies show that only a minor fraction of cells (0.1%) in the normal kidney express stathmin (Figs. 4 and 5). In early IRI (2448 h postreperfusion), the vast majority of PCNA-positive cells also express stathmin, whereas only a fraction of stathmin-positive cells are also PCNA positive (Fig. 5). Stathmin expression increases dramatically in the kidneys subjected to IRI, and by 72 h after reperfusion, the majority of cells that express stathmin also express PCNA, indicating that they are actively proliferating. These observations as well as those demonstrating enhanced expression of stathmin in mitotically active cells suggest that this protein is a marker of cell proliferation and may play an important role in renal tubular regeneration after IRI. The lack of expression of stathmin in differentiated renal epithelial cells that are in G0 arrest and coexpression of stathmin with vimentin and PCNA in renal epithelial cells indicate that stathmin is expressed by dedifferentiated renal tubular epithelial cells that have reentered the cell cycle and may participate in the tubular repair process associated with the recovery phase of IRI.
Stathmin expression increased at 72 h of reperfusion in kidney IRI (Fig. 5) but decreased at 72 h after cis-platinum treatment (Fig. 3). Interestingly, markers of injury (i.e., SSAT expression) were increased at 72 h after cis-platinum treatment (Fig. 3) but were decreased at the same time point in kidney IRI (53). The differential expression of stathmin in kidneys of animals subjected to IRI at 72 h of reperfusion (which corresponds to recovery from injury) vs. those treated with cis-platinum (which have active injury) indicates that this protein does not play a significant role in the induction of cell injury and ARF but is involved in the regulation of cell proliferation as a component of the tubular repair process.
Stathmin expression was significantly enhanced in a human kidney recovering from acute tubular necrosis as determined by increased urine output and the decline in serum creatinine (Fig. 7). On the basis of studies indicating enhanced expression of stathmin in rodent kidneys recovering from IRI (Figs. 1, 2, 4, and 5) and its decreased expression in kidneys with active injury (cis-platinum treatment, Fig. 3C), we propose that stathmin expression may be used to distinguish kidneys recovering from injury vs. those not recovering from injury. In addition, and on the basis of data indicating that the downregulation of stathmin decreases cell mitosis, we suggest that stathmin contributes to tubular regeneration following IRI.
The current studies establish stathmin as a marker of the proliferative phase of IRI; however, they do not examine the potential role of this molecule in the pathophysiology of nephrotoxic or ischemic renal injuries. On the basis of the time course of induction of stathmin and its biological function, stathmin may play a role in either the regulation of the reestablishment of the microtubule network or the proliferative response of the renal tubular epithelial cells in the injured kidneys. Examination of the effect of stathmin deficiency on the outcome of acute renal tubular injuries is currently underway and will help clarify the role of stathmin in the kidney's ability to recover from acute tubular necrosis.
ACKNOWLEDGMENTS
We thank Dr. Prasad Devarajan and Dr. John J. Bissler for critical review of this manuscript.
GRANTS
These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54220 and DK-66589 (to M. Soleimani) and DK-54770 (to H. Rabb), a Merit Review Award (to M. Soleimani), and grants from Dialysis Clinic Incorporated (to M. Soleimani).
Address for reprint requests and other correspondence: K. Zahedi, Division of Nephrology and Hypertension, Children's Hospital Research Foundation, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: Zahek0{at}cchmc.org) or M. Soleimani, Division of Nephrology and Hypertension, Dept. of Medicine, Univ. of Cincinnati, 231 Albert Sabin Way, MSB 259G, Cincinnati OH 45267-0585 (E-mail: Manoocher.soleimani{at}uc.edu).
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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