Peter M. Price

Induction of p21WAF1/CIP1/SDI1 in kidney tubule cells affects the course of cisplatin-induced acute renal failure.

The p21 protein is found in the nucleus of most cells at low levels and is induced to elevated levels after DNA damage, causing cell-cycle arrest. We have reported that p21 mRNA is rapidly induced to high levels in murine kidney after acute renal failure. The function(s) in the kidney of p21 induction in cisplatin-induced acute renal failure was studied with mice that are homozygous for a p21 gene deletion. After drug administration, as compared with their wild-type littermates, p21(-/-) mice display a more rapid onset of the physiologic signs of acute renal failure, develop more severe morphologic damage, and have a higher mortality. Therefore, the induction of p21 after cisplatin administration is a protective event for kidney cells. Using both bromodeoxyuridine incorporation and nuclear proliferating cell nuclear antigen detection, we found that cisplatin administration caused kidney cells to start entering the cell-cycle. However, cell-cycle progression is inhibited in wild-type mice, whereas kidney cells in the p21(-/-) mice progress into S-phase. We propose that p21 protects kidneys damaged by cisplatin by preventing DNA-damaged cells from entering the cell-cycle, which would otherwise result in death from either apoptosis or necrosis.

The cell cycle and acute kidney injury

Acute kidney injury (AKI) activates pathways of cell death and cell proliferation. Although seemingly discrete and unrelated mechanisms, these pathways can now be shown to be connected and even to be controlled by similar pathways. The dependence of the severity of renal-cell injury on cell cycle pathways can be used to control and perhaps to prevent acute kidney injury. This review is written to address the correlation between cellular life and death in kidney tubules, especially in acute kidney injury.

Dependence of Cisplatin-Induced Cell Death In Vitro and In Vivo on Cyclin-Dependent Kinase 2

Cisplatin is one of the most effective chemotherapeutics, but its usefulness is limited by its toxicity to normal tissues, including cells of the kidney proximal tubule. The purpose of these studies was to determine the mechanism of cisplatin cytotoxicity. It was shown in vivo that cisplatin administration induces upregulation of the gene for the p21 cyclin-dependent kinase (cdk) inhibitor in kidney cells. This protein is a positive effector on the fate of cisplatin-exposed renal tubule cells in vivo and in vitro; adenoviral transduction of p21 completely protected proximal tubule cells from cisplatin toxicity. Herein is reported that cdk2 inhibitory drugs protect kidney cells in vivo and in vitro, that transduction of kidney cells in vitro with dominant-negative cdk2 also protected, and that cdk2 knockout cells were resistant to cisplatin. The cdk2 knockout cells regained cisplatin sensitivity after transduction with wild-type cdk2. It is concluded that cisplatin cytotoxicity depends on cdk2 activation and that the mechanism of p21 protection is by direct inhibition of cdk2. This demonstrated the involvement of a protein that previously was associated with cell-cycle progression with pathways of apoptosis. It also was demonstrated that this pathway of cisplatin-induced cell death can be interceded in vivo to prevent nephrotoxicity.

64Cu metabolism in Menkes and normal cultured skin fibroblasts.

Summary: The amount of radioactivity accumulated in normal skin fibro-blasts cultured in the presence of 64Cu increased during the first few hours of incubation, and reached a plateau after 18 hr. Fibroblasts from patients with Menkes syndrome continued to accumulate 64Cu and after 48 hr, the mutant cells contained over 3 times more radioactivity than the normal cells. Normal skin fibroblasts grown in the presence of 64Cu for 24 hr and chased for 6 and 24 hr with 64Cu-free medium released 78% and 91% of the radioactivity, respectively, whereas Menkes fibroblasts grown under similar conditions released only 22% and 51%, respectively. The amount of radioactivity incorporated by fibroblasts increased with increasing concentrations of 64Cu in the culture medium, but the Menkes fibroblasts incorporated more 64Cu than normal fibroblasts. A slight reduction in the incorporation and efflux of 64Cu was seen with several metabolic inhibitors, but there was no difference between normal and mutant cells. Treatment of labeled cells with trypsin did not affect the amount of 64Cu picked up by the cells. Most of the radioactive copper incorporated by the cells was not precipitated by trichloroacetic acid or phosphotungstic acid, although the percentage of precipitated 64Cu was consistently higher in normal than in Menkes fibroblasts. Most of the 64Cu was bound to a molecule with a molecular weight of about 10,000, whereas a small fraction, proportionally higher in normal cells, was bound to a large molecular weight component(s). The amount of 64Cu bound to the small molecular weight species was significantly greater in Menkes fibroblasts than in normal cells. Menkes fibroblasts were more sensitive to high nonphysiologic levels of nonradioactive copper than were normal cells. These findings demonstrate pronounced metabolic differences between normal and Menkes fibroblasts and indicate the need for further studies before proper treatment of this disease can be instituted.Speculation: Copper picked up by cultured skin fibroblasts is preferentially bound to a molecule with a molecular weight of about 10,000. The increased copper accumulation in Menkes fibroblasts may be caused by the deficiency of an enzyme that catalyzes the cleavage of this bond, by the presence of an increased amount of this small molecular weight molecule in the mutant cells, or by an increased binding capacity of this molecule for copper.

Cytoplasmic initiation of cisplatin cytotoxicity.

The mechanism of action of cisplatin as a chemotherapeutic agent has been attributed to DNA binding, while its mechanism of action as a nephrotoxin is unresolved. Only approximately 1% of intracellular cisplatin interacts with DNA, primarily forming intrastrand cross-linked adducts, and many studies have implicated both nuclear and cytoplasmic causes of cisplatin-induced death in cultured cells. We have demonstrated that cisplatin cytotoxicity depends on cdk2 activity, which is at least partly through the cdk2-E2F1 pathway. The mechanism of the dependency on cdk2, and whether cdk2 activation of E2F1 represents the only cell death pathway involved, is still unclear. Our previous work showed that deletion of the nuclear localization signal from p21 WAF1/CIP1, a cdk2 inhibitor, did not alter its protective action against cisplatin cytotoxicity. Active cdk2-cyclin complexes are localized in both the nucleus and cytoplasm, and it was reported that cdk2 translocated to the cytoplasm after an apoptotic stimulus. Herein, we show that cisplatin caused cell death in enucleated mouse kidney proximal tubule cells (TKPTS), which was prevented by cdk2 inhibition. Also, we localized cytoplasmic cdk2 to both the endoplasmic reticulum (ER) and Golgi compartments, and ER stress was blocked by specific cdk2 inhibition. We conclude that cisplatin can induce nuclear independent apoptosis, cisplatin cytotoxicity can be initiated by cytoplasmic events, and cytoplasmic cdk2 plays an important role in apoptosis signaling.

Cell cycle regulation: repair and regeneration in acute renal failure

Research into mechanisms of acute renal failure has begun to reveal molecular targets for possible therapeutic intervention. Much useful knowledge into the causes and prevention of this syndrome has been gained by the study of animal models. Most recently, investigation of the effects on acute renal failure of selected gene knock-outs in mice has contributed to our recognition of many previously unappreciated molecular pathways. Particularly, experiments have revealed the protective nature of 2 highly induced genes whose functions are to inhibit and control the cell cycle after acute renal failure. By use of these models we have started to understand the role of increased cell cycle activity after renal stress and the role of proteins induced by these stresses that limit this proliferation.

Identification of protein-binding sites in the hepatitis B virus enhancer and core promoter domains

We have investigated the role of liver-specific trans-acting factor(s) in the regulation of hepatitis B virus (HBV) gene expression. A recorder plasmid (pEcoAluCAT; HBV nucleotides 1 through 1878) was constructed containing the HBV enhancer and the promoter region of the pregenomic RNA, which was ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene. Upon transfecting this plasmid into various cell lines, the CAT gene was expressed only in cells of liver origin. Moreover, competition cotransfections with pEcoAluCAT and plasmids containing HBV enhancer sequences in human hepatoblastoma-derived HepG2 cells indicated the presence of titratable trans-acting factor(s) in these cells. Gel mobility shift assays using HBV enhancer and core promoter domains confirmed the existence of sequence-specific DNA-binding proteins in liver cell nuclear extract which bound to these regions. These binding sites encompass 17- and 12-nucleotide palindromes in the HBV enhancer and core promoter domains, respectively, when mapped by the methylation interference assay.

Proximal tubule PPARα attenuates renal fibrosis and inflammation caused by unilateral ureteral obstruction.

We examined the effects of increased expression of proximal tubule peroxisome proliferator-activated receptor (PPAR)α in a mouse model of renal fibrosis. After 5 days of unilateral ureteral obstruction (UUO), PPARα expression was significantly reduced in kidney tissue of wild-type mice but this downregulation was attenuated in proximal tubules of PPARα transgenic (Tg) mice. When compared with wild-type mice subjected to UUO, PPARα Tg mice had reduced mRNA and protein expression of proximal tubule transforming growth factor (TGF)-β1, with reduced production of extracellular matrix proteins including collagen 1, fibronectin, α-smooth muscle actin, and reduced tubulointerstitial fibrosis. UUO-mediated increased expression of microRNA 21 in kidney tissue was also reduced in PPARα Tg mice. Overexpression of PPARα in cultured proximal tubular cells by adenoviral transduction reduced aristolochic acid-mediated increased production of TGF-β, demonstrating PPARα signaling reduces epithelial TGF-β production. Flow cytometry studies of dissociated whole kidneys demonstrated reduced macrophage infiltration to kidney tissue in PPARα Tg mice after UUO. Increased expression of proinflammatory cytokines including IL-1β, IL-6, and TNF-α in wild-type mice was also significantly reduced in kidney tissue of PPARα Tg mice. In contrast, the expression of anti-inflammatory cytokines IL-10 and arginase-1 was significantly increased in kidney tissue of PPARα Tg mice when compared with wild-type mice subjected to UUO. Our studies demonstrate several mechanisms by which preserved expression of proximal tubule PPARα reduces tubulointerstitial fibrosis and inflammation associated with obstructive uropathy.

Gender differences control the susceptibility to ER stress-induced acute kidney injury.

Endoplasmic reticulum (ER) stress contributes to acute kidney injury induced by several causes. Kidney dysfunction was shown to be influenced by gender differences. In this study we observed differences in the severity of kidney injury between male and female mice in response to tunicamycin, an ER stress agent. Tunicamycin-treated male mice showed a severe decline in kidney function and extensive kidney damage of proximal tubules in the kidney outer cortex (S1 and S2 segments). Interestingly, female tunicamycin-treated mice did not show a decline in kidney function, and their kidneys showed damage localized primarily to proximal tubules in the inner cortex (S3 segment). Protein markers of ER stress, glucose-regulated protein, and X-box binding protein 1 were also more elevated in male mice. Similarly, the induction of apoptosis was higher in tunicamycin-treated male mice, as measured by the activation of Bax and caspase-3. Testosterone administered to female mice before tunicamycin resulted in a phenotype similar to male mice with a comparable decline in renal function, tissue morphology, and induction of ER stress markers. We conclude that kidneys of male mice are much more susceptible to ER stress-induced acute kidney injury than those of females. Moreover, this sexual dimorphism could provide an interesting model to study the relation between kidney function and injury to a specific nephron segment.

The effect of 5-bromodeoxyuridine on messenger RNA production in cultured cells.

Cells of clone B(5)59, a derivative of the murine B16 melanoma, are highly tumorigenic, and produce both melanin and plasminogen activator. Cells of clone C(3)471, a line obtained by continued growth and maintenance of B(5)59 cells in medium containing 5-bromodeoxyuridine are nontumorigenic, amelanotic, and have no plasminogen activator. Differences in the mRNA complement of these two syngenic mouse melanoma clones have been determined by hybridization kinetics between complementary DNA transcribed from mRNA of either B(5)59 OR C(3)471 cells, and mRNA isolated from both sources. To detect the presence of unique mRNA sequences produced in B(5)59 cells, complementary DNA produced using B(5)59 mRNA as a template was exhaustively hybridized to C(3)471 mRNA. The results indicate that (i) polyadenylated mRNA produced by either clone can be divided into three main groups based upon the relative complexity of mRNA species within each group, (ii) more than 98% of the mRNA species produced by the B(5)59 cells are also produced by the cells of the C(3)471 clone, (iii) approximately 25% of the mRNA species produced by the cells of the C(3)471 clone in moderate abundance (500 molecules per cell) are not produced by the B(5)59 clone. Despite the fact that at least two of the proteins synthesized by B(5)59 cells are not detectable in C(3)471 cells, our results support the hypothesis that the major effect of BrdUrd incorporation into DNA is the induction rather than the repression of transcription of polyadenylated mRNA sequences.