Judit Megyesi

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.

Transgenic expression of proximal tubule peroxisome proliferator–activated receptor-α in mice confers protection during acute kidney injury

Our previous studies suggest that peroxisome proliferator-activated receptor-alpha (PPARalpha) plays a critical role in regulating fatty acid beta-oxidation in kidney tissue and this directly correlated with preservation of kidney morphology and function during acute kidney injury. To further study this, we generated transgenic mice expressing PPARalpha in the proximal tubule under the control of the promoter of KAP2 (kidney androgen-regulated protein 2). Segment-specific upregulation of PPARalpha expression by testosterone treatment of female transgenic mice improved kidney function during cisplatin or ischemia-reperfusion-induced acute kidney injury. Ischemia-reperfusion injury or treatment with cisplatin in wild-type mice caused inhibition of fatty-acid oxidation, reduction of mitochondrial genes of oxidative phosphorylation, mitochondrial DNA, fatty-acid metabolism, and the tricarboxylic acid cycle. Similar injury in testosterone-treated transgenic mice resulted in amelioration of these effects. Similarly, there were increases in the levels of 4-hydroxy-2-hexenal-derived lipid peroxidation products in wild-type mice, which were also reduced in the transgenic mice. Similarly, necrosis of the S3 segment was reduced in the two injury models in transgenic mice compared to wild type. Our results suggest proximal tubule PPARalpha activity serves as a metabolic sensor. Its increased expression without the use of an exogenous PPARalpha ligand in the transgenic mice is sufficient to protect kidney function and morphology, and to prevent abnormalities in lipid metabolism associated with acute kidney injury.

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.

Formoterol Restores Mitochondrial and Renal Function after Ischemia-Reperfusion Injury

Mitochondrial biogenesis may be an adaptive response necessary for meeting the increased metabolic and energy demands during organ recovery after acute injury, and renal mitochondrial dysfunction has been implicated in the pathogenesis of AKI. We proposed that stimulation of mitochondrial biogenesis 24 hours after ischemia/reperfusion (I/R)-induced AKI, when renal dysfunction is maximal, would accelerate recovery of mitochondrial and renal function in mice. We recently showed that formoterol, a potent, highly specific, and long-acting β2-adrenergic agonist, induces renal mitochondrial biogenesis in naive mice. Animals were subjected to sham or I/R-induced AKI, followed by once-daily intraperitoneal injection with vehicle or formoterol beginning 24 hours after surgery and continuing through 144 hours after surgery. Treatment with formoterol restored renal function, rescued renal tubules from injury, and diminished necrosis after I/R-induced AKI. Concomitantly, formoterol stimulated mitochondrial biogenesis and restored the expression and function of mitochondrial proteins. Taken together, these results provide proof of principle that a novel drug therapy to treat AKI, and potentially other acute organ failures, works by restoring mitochondrial function and accelerating the recovery of renal function after injury has occurred.

Alteration of renal respiratory Complex-III during experimental type-1 diabetes.

BackgroundDiabetes has become the single most common cause for end-stage renal disease in the United States. It has been established that mitochondrial damage occurs during diabetes; however, little is known about what initiates mitochondrial injury and oxidant production during the early stages of diabetes. Inactivation of mitochondrial respiratory complexes or alteration of their critical subunits can lead to generation of mitochondrial oxidants, mitochondrial damage, and organ injury. Thus, one goal of this study was to determine the status of mitochondrial respiratory complexes in the rat kidney during the early stages of diabetes (5-weeks post streptozotocin injection).MethodsMitochondrial complex activity assays, blue native gel electrophoresis (BN-PAGE), Complex III immunoprecipitation, and an ATP assay were performed to examine the effects of diabetes on the status of respiratory complexes and energy levels in renal mitochondria. Creatinine clearance and urine albumin excretion were measured to assess the status of renal function in our model.ResultsInterestingly, of all four respiratory complexes only cytochrome c reductase (Complex-III) activity was significantly decreased, whereas two Complex III subunits, Core 2 protein and Rieske protein, were up regulated in the diabetic renal mitochondria. The BN-PAGE data suggested that Complex III failed to assemble correctly, which could also explain the compensatory upregulation of specific Complex III subunits. In addition, the renal F0F1-ATPase activity and ATP levels were increased during diabetes.ConclusionIn summary, these findings show for the first time that early (and selective) inactivation of Complex-III may contribute to the mitochondrial oxidant production which occurs in the early stages of diabetes.

Differentiation-dependent expression of signal transducers and activators of transcription (STATs) might modify responses to growth factors in the cancers of the head and neck

Overexpression of the epidermal growth factor receptor (EGFR) in the cancers of the head and neck is well demonstrated. In addition, copy numbers of the EGFR mRNA were significantly higher in poorly differentiated tumors than in tumors that had a differentiated phenotype. Studies by others also showed that the constitutively activated signal transducer and activator of transcription-3 (STAT3), but not STAT1, is required for EGFR-mediated cell growth. Our aim was to reveal if STAT expression is differentiation-dependent and thus, might respond to exogenous stimuli in a differentiation-dependent manner. Both reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry revealed that expression of STAT1 was high in well/moderately differentiated tumors in vivo. In contrast, STAT3 was expressed in poorly differentiated tumors. In vitro experiments showed that differentiated primary oral keratinocytes expressed higher levels of STAT1, but lower levels of STAT3 than did their undifferentiated counterparts. Epidermal growth factor treatment of oral keratinocytes with various degrees of differentiation showed the maximal induction of cyclin D1 in undifferentiated cells. Our findings suggest that the level of differentiation might modulate the outcome of EGFR signaling (i.e. cyclin D1 transcription), due to the differentiation-associated intracellular balance of transcriptional regulators (STAT1 versus STAT3).

Intrahepatic expression and release of vascular endothelial growth factor following orthotopic liver transplantation in the rat.

BACKGROUND Morphological and functional changes to sinusoidal endothelial cells mediated by soluble factors released from activated Kupffer cells, including cytokines, are considered pivotal events in ischemia/reperfusion injury (IRI) to liver grafts. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific cytokine with potent pro-inflammatory and mitogenic effects. We investigated the possible role of VEGF in IRI to liver grafts using a syngeneic rat orthotopic liver transplantation model. METHODS Transplantation was performed in Lewis rats using livers preserved for various periods of time (24-48 hr) in University of Wisconsin solution at 4 degrees C. Systemic VEGF levels were measured by enzyme-linked immunosorbent assay (ELISA). Intrahepatic VEGF expression was analyzed by Northern blotting and in situ hybridization. The effects of anti-VEGF neutralizing antibody treatment on the extent of IRI were assessed by measuring liver function tests, lipid peroxidation, and metalloproteinase activity. RESULTS/CONCLUSION VEGF is expressed and released in a biphasic pattern during the early postoperative period after liver transplantation. Anti-VEGF antibody treatment, administered during reperfusion, decreased the degree of damage, suggesting that VEGF may have a role in IRI to liver grafts.

Sulforaphane protects the heart from doxorubicin-induced toxicity

Cardiotoxicity is one of the major side effects encountered during cancer chemotherapy with doxorubicin (DOX) and other anthracyclines. Previous studies have shown that oxidative stress caused by DOX is one of the primary mechanisms for its toxic effects on the heart. Since the redox-sensitive transcription factor, Nrf2, plays a major role in protecting cells from the toxic metabolites generated during oxidative stress, we examined the effects of the phytochemical sulforaphane (SFN), a potent Nrf2-activating agent, on DOX-induced cardiotoxicity. These studies were carried out both in vitro and in vivo using rat H9c2 cardiomyoblast cells and wild type 129/sv mice, and involved SFN pretreatment followed by SFN administration during DOX exposure. SFN treatment protected H9c2 cells from DOX cytotoxicity and also resulted in restored cardiac function and a significant reduction in DOX-induced cardiomyopathy and mortality in mice. Specificity of SFN induction of Nrf2 and protection of H9c2 cells was demonstrated in Nrf2 knockdown experiments. Cardiac accumulation of 4-hydroxynonenal (4-HNE) protein adducts, due to lipid peroxidation following DOX-induced oxidative stress, was significantly attenuated by SFN treatment. The respiratory function of cardiac mitochondria isolated from mice exposed to DOX alone was repressed, while SFN treatment with DOX significantly elevated mitochondrial respiratory complex activities. Co-administration of SFN reversed the DOX-associated reduction in nuclear Nrf2 binding activity and restored cardiac expression of Nrf2-regulated genes at both the RNA and protein levels. Together, our results demonstrate for the first time that the Nrf2 inducer, SFN, has the potential to provide protection against DOX-mediated cardiotoxicity.

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.