Coordination of the cell cycle is an important determinant of the syndrome of acute renal failure

Judit Megyesi, Lucia Andrade, Jose M. Vieira Jr., Robert L. Safirstein, and Peter M. Price

Department of Internal Medicine, University of Arkansas for Medical Sciences, and Department of Veterans Affairs Medical Center, Little Rock, Arkansas 72205


    ABSTRACT
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INTRODUCTION
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Recovery from injury is usually accompanied by cell replication, in which new cells replace those irreparably damaged. After acute renal failure, normally quiescent kidney cells enter the cell cycle, which in tubule segments is accompanied by the induction of cell cycle inhibitors. We found that after acute renal failure induced by either cisplatin injection or renal ischemia, induction of the p21 cyclin-dependent kinase (cdk) inhibitor is protective. Mice lacking this gene developed more widespread kidney cell death, more severe renal failure, and had reduced survival, compared with mice with a functional p21 gene. Here, we show induction of 14-3-3sigma , a regulator of G2-to-M transition, after acute renal failure. Our findings, using both in vivo and in vitro models of acute renal failure, show that this protein likely helps to coordinate cell cycle activity to maximize recovery of renal epithelial cells from injury and reduce the extent of the injury itself. Because in terminally differentiated cells, these proteins are highly expressed only after injury, we propose that cell cycle coordination by induction of these proteins could be a general model of tissue recovery from stress and injury.

kidney failure; acute; p21 cyclin kinase inhibitor; cisplatin; ischemia


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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AFTER INJURY, MANY TYPES OF quiescent, terminally differentiated cells enter the cell cycle (38). In the kidney after acute renal failure, these cell types primarily include epithelial cells of proximal and distal tubules and collecting ducts (22, 34). This progression is controlled in part by the upregulation of genes coding for cyclin-dependent kinase (cdk) inhibitors, such as p21 (25), that check the cell cycle, primarily at the G1-to-S and G2-to-M transitions. We found that p21 induction is protective because mice lacking this gene, after either cisplatin injection or renal ischemia, developed more widespread kidney cell death, more severe renal failure, and had reduced survival, compared with mice with a functional p21 gene (21, 24).

Recently, it was shown that 14-3-3sigma , another cell cycle inhibitor, may also determine cell fate after injury (4, 5, 15). After DNA damage, both p21 and 14-3-3sigma were induced in cells in vitro, which led to cell cycle arrest. Without either inhibitor, injury caused an uncoordinated cell cycle, resulting in increased cell death (15). We now show 14-3-3sigma mRNA induction after both cisplatin- and ischemia-induced acute renal failure in vivo. This mRNA is expressed in kidney tubules previously described as inducing p21 mRNA (25). After acute renal failure, the increased mortality of this syndrome in mice lacking the p21 gene was most likely caused by the inability of the cells to induce both p21 and 14-3-3sigma cell cycle inhibitors, which resulted in an uncoordinated cell cycle. The uncoordinated cell cycle in kidney of p21(-/-) mice was characterized by increased cell cycle activity, increased nuclear size and cellular DNA content, and ultimately more widespread cell death (Refs. 21 and 24 and this study).

To explore the roles of p21 and 14-3-3sigma in relevant in vitro models of renal cell injury, we determined the effects of cisplatin and hydrogen peroxide exposure on cells in which one or both genes were deleted. Our results show that compared with wild-type cells, cells with the gene deletions had much decreased survival after cisplatin or peroxide exposure.

These studies are compatible with the idea that cell stress induces pathways that compete between cell death and cell cycle arrest. In wild-type cells, stress results in induction of cell cycle inhibitors that lead to arrest, whereas in p21- and/or 14-3-3sigma -deleted cells, similar stress causes cell death pathways to predominate. Our results show that coordinated cell cycle control, initially manifested as cell cycle inhibition, is necessary for optimum recovery from acute renal failure and is a likely paradigm for other types of epithelial cell injuries.


    METHODS
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INTRODUCTION
METHODS
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Mice and induction of acute renal failure. Mice carrying a deletion of a large portion of the p21 gene in which neither p21 mRNA nor p21 protein is expressed were obtained from Dr. Philip Leder (Harvard Medical School). Mice that were homozygous for the p21 deletion were selected from the offspring of heterozygous matings using Southern blotting of tail DNA as described (6). Wild-type homozygous littermates were selected as controls. Male mice from 8 to 15 wk of age were studied as described. The animals used in these studies were housed at the Veterinary Medical Unit at the John L. McClellan Memorial Veterans' Hospital, Little Rock, AR. Survival surgery protocols were approved by the Animal Care and Use Committee, Department of Veterans Affairs. When appropriate, animals were painlessly killed according to methods of euthanasia approved by the Panel on Euthanasia of the American Veterinary Medical Association.

Ischemia was induced in anesthetized (pentobarbital sodium, 50 mg/kg ip) male mice by exposure of the kidneys through a midline incision under sterile conditions. The kidneys were decapsulated, and both renal hila were clamped with small arterial clamps for 50 min and released. During the operation, the mice received ~1 ml of saline intraperitoneally. Sham operations were also performed, in which the kidneys were manipulated as described without induction of ischemia. After surgery, the animals were returned to their cages and allowed free access to food and water. Cisplatin was administered by a single intraperitoneal injection to mice at 20 mg/kg.

Morphological assessment. At various times after ischemia or cisplatin treatment, kidneys were removed, immersed in 4% neutral-buffered formaldehyde, and fixed for 48-72 h. Tissues were embedded in paraffin, and sections were cut with a microtome and placed on slides. The tissues were used as described below or were stained with hematoxylin-eosin or periodic acid-Schiff.

mRNA isolation and Northern blotting. Poly[A]+ RNA was isolated from kidney using a RiboSep kit (Becton-Dickinson), electrophoresed, and blotted as described (25, 33). Briefly, tissue was lysed in the presence of SDS and proteinase K. After digestion of the lysate for 2 h at 45°C, it was applied to an oligo(dT) column. After being washed with a buffer containing 0.5 M NaCl, poly[A]+ RNA was eluted with 10 mM Tris, pH 7.5, 1 mM EDTA, and 0.05% sarkosyl. The polyadenylated RNA (5 µg) was denatured by heating in a buffer containing formamide and formaldehyde and electrophoresed through 1% agarose in denaturing buffer. After electrophoresis, the gel was washed with 20× SSC (1× SSC = 0.15 M NaCl, 0.015 M sodium citrate), and the RNA was transferred to nitrocellulose membranes by upward diffusion. After prehybridization, the RNA was hybridized with a 32P-radiolabeled probe (14-3-3sigma cDNA; courtesy of Dr. Bert Vogelstein). After hybridization, the filters were washed, air-dried, and autoradiographed. Hybridization to a probe specific for GAPDH mRNA was used as an internal control. We have previously shown that the levels of this mRNA do not vary appreciably after acute renal failure (22, 33) when measured either by Northern hybridization or RT-PCR.

In situ localization of 14-3-3sigma mRNA. In situ hybridization of 14-3-3sigma mRNA on kidney sections was performed as previously described (22, 25), using paraffin-embedded tissue. The specificity of the hybridization was controlled by hybridization with "sense" probes, which did not result in any color reaction on the sections. Probes for in situ hybridization were labeled with digoxigenin using in vitro transcription of a linearized murine 14-3-3sigma plasmid in which the 14-3-3sigma cDNA had been inserted in pBluescript KS(+) between T7 and T3 promoters.

Nuclei preparation and in situ hybridization for DNA quantification. Kidneys were homogenized with a Potter-Elvehjem grinder in buffer (10 ml for 2 kidneys) containing 10 mM HEPES, pH 7.6, 25 mM KCl, 1 mM EDTA, 10% glycerol, and 1.8 M sucrose. The homogenate was layered on 2.2 ml of the above buffer and centrifuged with an SW60 rotor for 30 min at 24,000 rpm and 2°C. The nuclear pellets were combined in 0.1 ml PBS for 2 kidneys. Fixative (3:1 methanol-acetic acid) was added, and the nuclei were pelleted at 500 rpm for 10 min. The nuclei were resuspended in fixative and applied to silanized glass slides. The dried nuclei were treated with thiocyanate for 10 min at 80°C, digested with 75 µg/ml pepsin for 15 min at 37°C, and postfixed in formaldehyde. The slides were hybridized with a biotinylated P1 mouse ES probe (locus D15 MIT 13; Genome Systems) for 16 h at 37°C in buffer containing 60% formamide, 2× SSC, 10% dextran sulfate, and 0.25 µg/ml denatured salmon DNA. Slides were washed and incubated with avidin-conjugated peroxidase in 4× SSC and 5% nonfat dry milk. The reaction was developed with 3,3'-diaminobenzidine as a substrate and washed with water, and the nuclei were stained with hematoxylin. The slides were dehydrated through ethanol, transferred into xylene, and mounted under coverslips.

Tissue culture cells and treatments. HCT116 cells containing either wild-type p21 and 14-3-3sigma genes, or with p21(-/-), and 14-3-3sigma (-/-) deletions (5) were obtained from Dr. B. Vogelstein. The cells were cultured in McCoy's medium containing 10% fetal bovine serum. Cells were treated at ~75% confluence with 0-50 µM cisplatin or 0-400 µM hydrogen peroxide for 24 h. Cell survival after treatments was determined by trypan blue exclusion. Statistical analyses of survival curves were evaluated using the F-probability distribution with GraphPad Prism 3.02.


    RESULTS
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INTRODUCTION
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Induction of 14-3-3sigma mRNA. Induction of 14-3-3sigma after acute renal failure was determined by using mRNA isolated from mice (Fig. 1). There was no 14-3-3sigma mRNA present in untreated kidney (lanes 1 and 2), but it was induced to high levels by both renal ischemia (lanes 3 and 4) and cisplatin injection (lanes 5 and 6). Also, the persistence of its induction is seen by its presence 3 days after cisplatin injection (lane 6).


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Fig. 1.   Northern blot analysis for 14-3-3sigma mRNA transcripts in mouse kidney cells. Poly(A)+ RNA was isolated from untreated mouse kidney (lanes 1 and 2); kidney 4 (lane 3) and 24 h (lane 4) after 50-min ischemia; kidney 24 (lane 5) and 72 h (lane 6) after cisplatin (20 mg/kg) injection. Uniformity of loaded RNA was assessed by hybridization to a probe specific for constitutively expressed GAPDH mRNA (not shown).

In situ hybridization for localization of 14-3-3sigma mRNA. In situ hybridization before (Fig. 2, A and C) and 24 h after cisplatin injection (Fig. 2, B and D) in both wild-type (Fig. 2, A and B) and p21(-/-) (Fig. 2, C and D) mouse kidney sections showed induction of 14-3-3sigma mRNA in tubules of the distal nephron. A similar distribution was found in sections after ischemia (data not shown). There were no quantitative differences in 14-3-3sigma mRNA induction when wild-type and p21(-/-) mouse kidney are compared using Northern blot analysis (data not shown).


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Fig. 2.   In situ hybridization for localization of 14-3-3sigma mRNA. Hybridization of an antisense 14-3-3sigma cRNA probe to cells of either wild-type (A and B) or p21-/- (C and D) mouse kidney before (A and C) or 24 h after (B and D) cisplatin injection.

Polyploidy determination by nuclear morphology and in situ hybridization. Many nuclei in kidney sections from p21(-/-) mouse kidney after acute renal failure were larger than nuclei in similar sections from p21(+/+) mice or from mice without renal failure (Fig. 3). To confirm that this represented polyploidy of the DNA, we performed in situ hybridization (Fig. 4) using a probe specific for mouse chromosome 15 on isolated interphase nuclei from kidney of untreated and cisplatin-injected mice, both p21(+/+) and p21(-/-). Nuclei isolated from untreated mouse kidney (Fig. 4A) showed two areas of hybridization, indicative of diploid chromosome number. Similar results were obtained using nuclei isolated from kidney of untreated p21(+/+) and p21(-/-) mice and from cisplatin-injected p21(+/+) mice. Nuclei isolated from kidney of cisplatin-treated p21(-/-) mice (Fig. 4B) frequently showed many areas of hybridization, indicative of polyploidy of the genome.


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Fig. 3.   Nuclear morphology in p21-/- mouse kidney. Periodic acid-Schiff-stained section (A) from kidney 2 days after ischemia and hematoxylin-eosin-stained section (B) from kidney 7 days after ischemia are shown. Note many enlarged nuclei, especially in A, some of which are indicated by arrows.



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Fig. 4.   In situ hybridization analysis of kidney nuclei for ploidy determination. Representative nuclei isolated from kidney of mice before cisplatin injection, wild-type mice after cisplatin injection (A), or p21 -/- mice 4 days after cisplatin (B) are shown.

Tissue culture model of cisplatin- or oxidant injury-induced acute renal failure. Cells were grown to ~75% confluence and exposed to either cisplatin (10-50 µM) or hydrogen peroxide (50-400 µM). The numbers of surviving cells, as measured by trypan blue exclusion, were compared with numbers of surviving cells in control cultures (0 µM cisplatin or peroxide) after 24-h exposure (Fig. 5). Comparison of the survival curves by F-probability distribution showed that survival of p21(-/-) cells or 14-3-3sigma (-/-) cells was significantly lower than wild-type cells (P = 2.0 × 10-8, cisplatin, p21 deletion; P = 0.0070, peroxide, p21 deletion; P = 0.00010, cisplatin, 14-3-3sigma deletion; P = 0.047, peroxide, 14-3-3sigma deletion). However, the effect of either deletion was not significantly different from each other (P = 0.154, cisplatin; P = 0.124, peroxide).


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Fig. 5.   Survival curves of tissue culture cells 24 h after exposure to either cisplatin (A) or hydrogen peroxide (B). Analysis was performed using parental wild-type HCT116 (), or derived p21(-/-) (black-triangle) and 14-3-3sigma (-/-) (). Errror bars, SE.


    DISCUSSION
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INTRODUCTION
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DISCUSSION
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Cell cycle regulation. The control of the cell cycle involves a complex interaction of both positive and negative factors controlling mRNA transcription and translation, protein activity and stability, and compartmentalization of enzymes and substrates. This cell cycle control primarily occurs at the G1-to-S and G2-to-M transitions. In a DNA-damage model, it is dependent on the induction of two genes, p21 and 14-3-3sigma (9, 15). In the replication of cells in vitro, expression of one of these genes in the absence of the other had different effects on cell survival. Whereas p21 expression caused cell cycle inhibition, expression of 14-3-3sigma in the absence of p21 expression caused uncoordinated replication, an increase in DNA content, and eventual cell death (15). In DNA-damaged cells, the absence of either gene resulted in increased cell death.

p21 (9, 14, 42), a 21-kDa protein, is overexpressed before terminal differentiation (13, 16, 20, 30, 36, 37, 43) and in senescent cells (29). In addition, it is induced by p53 (9) and is necessary for p53-dependent cell cycle arrest after DNA damage (2, 6, 40). Induction of p21 is associated with cell cycle interruption (7-9, 12, 14, 41), and the protein has been shown to inhibit one or more cyclin-cdk kinase activities (7, 14, 41) and PCNA (10, 18, 39). The role of p21 in differentiation is probably redundant, because mice lacking p21 develop normally (6).

The 14-3-3 family of proteins was originally discovered as abundant acidic proteins in the brain (reviewed in Ref. 1). They are expressed in a wide range of eukaryotes, including yeast, plants, nematodes, and humans. At least seven different isoforms have been described in mammalian cells. These proteins bind to several proteins involved in cell cycle regulation and signal transduction, specifically to phosphoserine residues (28). A major role of these proteins in cell cycle regulation occurs at the G2-to-M checkpoint, where they bind to phosphorylated cdc25B (3) and cdc25C (31) and either sequester them into the cytoplasm (19) or otherwise inhibit their activity (3). The cdc25s are dual-specificity protein phosphatases whose activity in the G2-to-M transition is to dephosphorylate cdc2 (26), a kinase required for entry into mitosis. During G2, active cdc25 is dephosphorylated and localized in the nucleus, but during interphase or after several types of cellular stress it is phosphorylated and localized in the cytoplasm (3, 11, 31, 35). Chan et al. (4) have shown that after DNA damage, 14-3-3sigma is bound to phosphorylated cdc2 and that a different 14-3-3 protein(s) is bound to phosphorylated cdc25.

Cell cycle control in acute renal failure. Acute renal failure caused by disparate injuries, i.e., ischemia or nephrotoxic drugs (e.g., cisplatin), results in severe and acute reduction in the glomerular filtration rate and in clearance of serum nitrogenous wastes, such as creatinine and blood urea nitrogen (BUN). There are also varying degrees of kidney cell death, especially involving the S3 segment of the proximal tubules in the deep cortex. We have previously shown a rapid and high level induction of a cell cycle inhibitory protein, p21, in murine kidney after acute renal failure (25). This induction was beneficial because after both cisplatin and ischemic acute renal failure (21, 24) there was increased and more widespread cell death in kidney of p21(-/-) mice compared with p21(+/+) mice. Along with the more severe cytotoxicity, we found increased cell cycle activity in kidney of p21(-/-) mice and speculated that this increased cell cycle activity could have detrimental effects.

We investigated whether, besides p21, other cell cycle regulators were induced in acute renal failure and, as shown in Fig. 1, 14-3-3sigma is induced in both models, as well as in ureteral obstruction-induced injury (data not shown). The 14-3-3sigma mRNA was localized primarily to tubules of the distal nephron, including thick ascending limbs, distal convoluted tubules, and collecting ducts (Fig. 2), which was similar to that of p21 mRNA (25). The localization of p21 mRNA, however, was more indicative of its higher concentration in the distal nephron, because p21 protein was found in nuclei of cells throughout both distal and proximal nephrons (21). It remains to be seen whether 14-3-3sigma protein is also distributed throughout both distal and proximal kidney segments. If a similar cooperation exists in vivo between p21 and 14-3-3sigma induction to inhibit the cell cycle effectively and to maintain cell cycle coordination as had been described in vitro (5), the two proteins should be colocalized after acute renal failure. The absence of 14-3-3sigma induction in damaged proximal tubule cells would make this kidney segment more susceptible to cell death after injury, which is the case in the S3 segment of the proximal tubules.

It is likely that the nature of the injury to the cell in combination with the induction of a cell cycle inhibitor(s) is an important determinant of cell fate. In undamaged cells, p21 is a cell cycle inhibitor, being induced during terminal differentiation in several cell types (13, 16, 20, 30, 36, 37, 43) and blocking the growth of cultured cells (9, 14). Similarly, 14-3-3sigma is expressed during differentiation in epithelial cells (17, 32). On the other hand, expression of 14-3-3sigma in the absence of p21 causes an uncoordinated cell cycle in and death (15) of undamaged cells. The expression of p21 reduces the severity of acute renal failure (21, 24), and its induction may contribute to resistance to repeated doses of cisplatin (27). However, with the use of a chronic renal failure model in which a large part of the kidney is physically removed, mice lacking the p21 gene are not as sensitive to injury as are their p21(+/+) littermates (41).

We have noted the increased size of many nuclei in p21(-/-) mice after renal failure (Fig. 3), and one hallmark of an uncoordinated cell cycle is the presence of cells with increased DNA content. As confirmed by the polyploidy of many kidney nuclei from these mice after acute renal failure (Fig. 4B), this was caused by increased DNA content. Neither the large percentage of nuclei with increased size nor genomic polyploidy was seen in kidneys of untreated mice or in kidney of p21(+/+) mice even after renal failure.

We next investigated whether, as had been proposed after treatment of cells with DNA-damaging agents (5), the pathways of protection from p21 and 14-3-3sigma gene activation can be seen in vitro using cisplatin or hydrogen peroxide treatment of cultured cells in which these genes are deleted. As seen in Fig. 5, the activation of both genes is protective because any single gene deletion increased the toxicity of either cisplatin or peroxide. We have also seen this increased sensitivity caused by either p21 or 14-3-3sigma gene deletion in cells exposed to cisplatin for up to 3 days (data not shown). The similar sensitivity of these cells suggests that a functional deletion of the 14-3-3sigma gene in vivo would, as we have reported for p21 gene-deleted mice (21, 24), likely cause increased kidney cell death and mortality from acute renal failure.

We propose (Fig. 6) that after acute renal failure, in which epithelial cells are damaged, normally quiescent cells enter the cell cycle. In kidneys of wild-type animals, cell cycle inhibitors (p21 and 14-3-3sigma ) are also induced, and their combined activities check the cell cycle at G1 and G2. As extrapolated from the in vitro results, the presence of both p21 and 14-3-3sigma is necessary to coordinate the cell cycle, and the absence of either of these factors will result in increased cell death and increased mortality from acute renal failure. In this model, cell cycle arrest is a prerequisite for renal cell repair and/or regeneration after injury, and the inhibition of the cell cycle allows the repair of cellular damage to occur before cell replication.


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Fig. 6.   Proposed mechanism for the interaction of cell cycle inhibitors with the course of acute renal failure.


    ACKNOWLEDGEMENTS

We thank Dr. Philip Leder (Harvard Medical School) for providing several heterozygous mice carrying the p21 gene deletion and for providing a probe for screening. We thank Dr. Bert Vogelstein (Johns Hopkins University School of Medicine) for providing the probe for 14-3-3sigma mRNA and HCT116 cells and derivatives with p21 and 14-3-3sigma gene deletions.


    FOOTNOTES

This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-54471 and with resources of and the use of facilities at the John L. McClennan Memorial Veterans Hospital, Little Rock, AR.

Address for reprint requests and other correspondence: P. M. Price, Univ. of Arkansas for Medical Sciences, 4300 West 7th St., Mail Route 151, Little Rock, AR 72205 (E-mail: PricePeterM{at}uams.edu).

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.

May 29, 2002;10.1152/ajprenal.00078.2002

Received 25 February 2002; accepted in final form 14 May 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 283(4):F810-F816