Mami Kobayashi

Hydrogen Peroxide Induces Necrosis, Apoptosis, Oncosis and Apoptotic Oncosis of Mouse Terminal Proximal Straight Tubule Cells

Hydrogen peroxide (H₂O₂) has been shown to be an important mediator of ischemic and toxic tubular damage. The purpose of this study was to identify the mode of cell death observed in H₂O₂-exposed cultures of mouse terminal proximal straight tubule (S₃) cells. H₂O₂ induced a dose- and time-dependent decrease in viability of S₃ cells. Morphologically, S₃ cells exposed to H₂O₂ (0.05–0.1 mM) showed features of necrosis, apoptosis, oncosis and apoptotic oncosis, whereas necrosis occurred most frequently in every experimental condition tested. On the other hand, agarose gel electrophoresis of DNA extracted from S₃ cells exposed to H₂O₂ revealed a typical DNA ladder pattern. These data suggest that H₂O₂-induced proximal tubule damages are associated with the induction of various modes of cell death including necrosis, apoptosis, oncosis and apoptotic oncosis, and with the activation of endonuclease.

Cisplatin-induced apoptosis of immortalized mouse proximal tubule cells is mediated by interleukin-1β converting enzyme (ICE) family of proteases but inhibited by overexpression of Bcl-2

Abstract Cisplatin is known to induce serious renal damage including acute renal failure, the major site of renal injury appears to be localized to the third segment of the proximal tubule (S3). Apoptosis occurs during a variety of acute injuries to tubule cell. The purpose of this study was to determine whether cisplatin induces apoptosis of immortalized mouse S3 cells, and to define the intracellular pathways leading to cell death. S3 cells exposed to cisplatin exhibited biochemical, morphological, and flow cytometric changes characteristic of apoptosis associated with slight necrosis. Cisplatin-induced apoptosis could be inhibited by overexpression of crmA, a cowpox virus gene, of which the product is known to suppress activities of the interleukin-1β converting enzyme (ICE) family proteases. On the other hand, overexpression of bcl-2, an antiapoptotic oncogene, rendered S3 cells partially resistant to cisplatin. These results indicate that cisplatin-induced proximal tubule damage is associated with apoptosis, which is positively modulated by the ICE family of proteases and negatively by the product of bcl-2.

Distinct interleukin-1β-converting enzyme family proteases mediate cisplatin- and staurosporine-induced apoptosis of mouse proximal tubule cells

Interleukin-1beta converting enzyme (ICE) family proteases (caspases) are known to be implicated as important effectors of apoptotic pathways. The purpose of this study was to elucidate the role of ICE family proteases in apoptosis of mouse cells derived from the terminal proximal tubule (S3) treated with cisplatin, an anti-tumor drug, or staurosporine, a protein kinase C inhibitor. For this purpose, we measured the activities of ICE family proteases and examined the effects of tetrapeptide and viral ICE family protease inhibitors on the activities of ICE family proteases in and the degree of apoptosis of S3 cells treated with cisplatin and staurosporine. RT-PCR analysis revealed that S3 cells as well as mouse kidney express mRNA for ICE and CPP32, an ICE family protease. Results of enzymatic analysis, determination the degree of DNA fragmentation and cytotoxicity test suggest that CPP32 mediates cisplatin-induced apoptosis of S3 cells, whereas ICE family proteases other than CPP32 mediate staurosporine-induced apoptosis of S3 cells. In conclusion, distinct ICE family proteases mediate apoptosis of mouse proximal tubule cells depending on the stimuli to which the cells are exposed.

ESTABLISHMENT OF A MOUSE CLONAL EARLY PROXIMAL TUBULE CELL LINE AND OUTER MEDULLARY COLLECTING DUCT CELLS EXPRESSING P2 PURINOCEPTORS

The purpose of this study was to establish tubule cells expressing P2 purinoceptors. Use of the polymerase chain reaction coupled with reverse transcription showed that mouse clonal early proximal tubule (S1) cell line (NF‐5) and outer medullary collecting tubule (OMCT) cells expressed mRNA for P2X4, P2Y1 and P2Y2 purinoceptors. ATP and its analogues induced a dose‐dependent increase in cytosolic free calcium concentration ([Ca2+i] in both NF‐5 and OMCT cells. ATP enhanced [3H]‐thymidine uptake by both NF‐5 and OMCT cells in a dose‐dependent manner. In conclusion, NF‐5 and OMCT cells can be used in the further analysis of the function and regulation of activity and expression of P2 purinoceptors in the renal tubule.

Involvement of macromolecule synthesis, endonuclease activation and c-fos expression in cisplatin-induced apoptosis of mouse proximal tubule cells

We have previously demonstrated that cisplatin-induced nephrotoxicity is associated with the induction of apoptosis using mouse renal cells derived from the terminal proximal tubule (S3) which is the major target site of cisplatin-induced injury. The purpose of this study was to elucidate the intracellular mechanisms leading to the cisplatin-induced apoptosis of S3 cells. Actinomycin D (an inhibitor of RNA synthesis), cycloheximide (an inhibitor of protein synthesis) and aurintricarboxylic acid (an endonuclease inhibitor) reduced the extent of DNA fragmentation, a biochemical parameter of apoptosis, in cisplatin-treated S3 cells. Furthermore, cisplatin-induced apoptosis of S3 cells was accompanied by an increase in the level of c-fos mRNA expression, which is inhibited by pretreatment of the cells with actinomycin D, but not with cycloheximide or aurintricarboxylic acid. In contrast, outer medullary collecting duct cells treated with cisplatin exhibited morphological changes characteristic of apoptosis and an increase in the level of c-fos mRNA expression, but no increase in the extent of DNA fragmentation. In conclusion, the synthesis of macromolecules such as RNA and protein, endonuclease activation and c-fos expression appear to be involved in the intracellular pathways leading to the induction of apoptosis in cisplatin-treated S3 cells. In addition, the response to cisplatin may be different in different cells.

ATP‐induced calcium mobilization in glomerular mesangial cells is mediated by P2U purinoceptor

To identify the functional P2 purinoceptor subtype in glomerular mesangial cells (GMC), polymerase chain reaction coupled with reverse transcription (RT‐PCR) were performed, and cytosolic free calcium concentration ([Ca2+]i) was determined. RT‐PCR analysis revealed that the molecular identity of P2 purinoceptor localized to GMC was both P2U and P2Y. The rank order of potency in stimulating [Ca2+]i was ATP≒UTP>2‐methylthio‐ATP>ADP≒adenosine≒AMP. In addition, cross‐desensitization between ATP and UTP occurred. In conclusion, ATP induces increase in [Ca2+]i via P2U purinoceptor in GMC.

Thromboxane A2 mediates cisplatin-induced apoptosis of renal tubule cells

The purpose of this study was to elucidate the role of thromboxane A2 (TXA2) in cisplatin‐induced apoptosis of mouse renal cells derived from the terminal proximal tubule (S3). RT‐PCR analysis revealed that S3 cells express TXA2 receptor mRNA. A TXA2 receptor antagonistKW‐3635, dose‐dependently inhibited cisplatin‐induced apoptosis of S3 cells. Treatment of S3 cells with a TXA2 agonist, STA2, induced a significant decrease in their viability and resulted in the characteristic ladder pattern resulting from intranucleosomal DNA cleavage. Treatment with KW‐3635 resulted in attenuation of the increase in c‐fos mRNA expression level in cisplatin‐treated S3 cells. In conclusion, TXA2 appears to mediate cisplatin‐induced apoptosis of S3 cells by inducing an increase in the level of c‐fos mRNA expression.

Staurosporine-induced apoptosis of immortalized mouse proximal tubule cells is modulated by bcl-2 expression level.

Mouse terminal proximal tubule (S3)cells treated with staurosporine (STS) underwent morphological and biochemical changes characteristic of apoptosis. Treatment of S3 cells with STS, H‐7, PMA, herbimycin A or genistein caused DNA fragmentation. STS inhibited the activity of protein kinase C but not of cdc‐2 kinase in S3 cells. No change in cell cycle distribution was observed in S3 cells treated with STS. However, treatment of S3 cells with STS resulted in a decrease in the level of bcl‐2 mRNA expression in the cells. Overexpression of bcl‐2 inhibited the degree of STS‐induced apoptosis of S3 cells. In conclusion, STS induces apoptosis of mouse S3 cells via inhibition of various protein kinases including protein kinase C, and the apoptosis is modulated by the bcl‐2 expression level.

Cyclosporine Induces Apoptosis of Mouse Terminal Proximal Straight Tubule Cells

Accessible online at: http://BioMedNet.com/karger Dear Sir, Cyclosporine (Cs) is an immunosuppressant which has markedly improved the rate of graft survival of different organ transplants in the last decade. Among its recognized side effects, nephrotoxicity is predominant [1] and often limits the use of Cs. Cs nephrotoxicity encompasses a spectrum of clinical manifestations from precipitous decrease in glomerular filtration rate to chronically progressive scarring. In renal tubules, the major target site of Cs was shown to be the terminal proximal tubule (S3) [2] where isometric vacuolization was reported to occur in response to Cs [3]. In a multicellular organism cell death can occur by two distinct mechanisms, necrosis and apoptosis [4]. Apoptosis is an active form of cell death whereby the cell can be thought of as committing suicide rather than being passively destroyed by the degradative action of proteinases, as in necrosis. Ito et al. [5] have recently demonstrated that apoptosis occurred in a human allografted kidney treated with Cs using an in situ nick end-labeling technique. However, in using this technique it is difficult to discriminate apoptosis and necrosis [6]. To assess the direct effect of Cs on the proximal tubule and to confirm whether Cs induces apoptosis of renal tubules, we determined the effect of Cs on mouse S3 cells derived from transgenic mice harboring the temperature-sensitive simian virus 40 large T-antigen gene. As previously reported by us [7], S3 cells were cultured in RITC 80-7 medium (Kyokuto, Tokyo, Japan) containing 5% fetal bovine serum (Gibco, Gaithersburg, Md., USA), 10 Ìg/ml transferrin (Sigma, St. Louis, Mo., USA), 0.08 U/ml insulin (Shimizu, Shizuoka, Japan), 10 ng/ml recombinant human epidermal growth factor (Wakunaga, Hiroshima, Japan), 100 U/ml penicillin and 100 Ìg/ml streptomycin (Gibco). S3 cells retained hydrolase activities associated with the intact proximal tubule including alkaline phosphatase, Á-glutamyltransferase and leucine-aminopeptidase activities, and the levels of these activities were higher than those in LLC-PK1 cells but lower than those in freshly isolated S3 segments [unpubl. observation]. Cs was kindly provided by Sandoz (Basel, Switzerland). The trypan blue exclusion test revealed that Cs induced a time (24 and 48 h)and dose (10–6 and 10–5 M )-dependent decrease in the cell viability of S3 cells (data not shown). As shown in figure 1, agarose gel electrophoresis of DNA extracted from S3 cells treated with vehicle (methanol) revealed no DNA fragments of low molecular weight. In contrast, DNA from S3 cells treated with 10–6 M Cs for 24 h revealed the characteristic ladder pattern resulting from the internucleosomal chromatin cleavage of 180-bp fragments. As shown in figure 2a, S3 cells treated with vehicle retained normal nucleus features with retaining microvilli. In contrast, as shown in figure 2b, margination and condensation of chromatin, characteristic morphological features of apoptosis, were observed in S3 cells treated with 10–6 M Cs for 24 h. Fig. 1. Agarose gel electrophoresis of DNA extracted from S3 cells exposed to cyclosporine (Cs) or vehicle. Lane 1 = vehicle; lane 2 = 10–6 M Cs for 24 h.

V1a receptor mRNA expression in mouse early proximal tubule (S1) cell line is regulated by protein kinase A

The purpose of this study was to investigate the regulation of V1a receptor gene expression in mouse early proximal tubule (S1) cell line (NF‐5). NF‐5 cells showed arginine vasopressin (AVP)‐induced increase in cytosolic free calcium concentration mediated by V1a receptor. Polymerase chain reaction coupled with reverse transcription analysis revealed that forskolin, but not phorbol‐12‐myristate‐13‐acetate stimulated V1a receptor mRNA expression in NF‐5 cells. Our data suggest that V1a receptor gene expression is regulated by protein kinase A in proximal tubule cell line, and the cell line might be a useful model for the further studies of the function and the regulation of the activity and expression of the V1a receptor in the proximal tubule.