Free radical scavengers, catalase and superoxide dismutase provide protection from oxalate-associated injury to LLC-PK1 and MDCK cells

PURPOSE: Current studies have provided evidence that exposure of renal epithelial cells to oxalate and calcium oxalate crystals induces lipid peroxidation and injures the cells. Since oxidant/antioxidant balance is likely to play a critical role, we determined the effect of antioxidant scavengers on production of free radicals and injury to LLC-PK1 and MDCK cells from exposure to oxalate (Ox) or Ox + calcium oxalate monohydrate (COM) crystals. MATERIALS AND METHODS: LLC-PK1 and MDCK cells were grown in monolayers and exposed to 1.0 mmol. Ox or 1.0 mmol. Ox + 500 microg. /ml. COM crystals for 120 or 240 minutes. We measured the release of lactate dehydrogenase (LDH) as a marker for cell injury and malondialdehyde (MDA) as a marker of lipid peroxidation. Superoxide and hydroxyl radicals were measured in the presence or absence of 400 U/ml. catalase, or superoxide dismutase (SOD). RESULTS: Exposure of LLC-PK1 cells to Ox resulted in a significant increase in MDA and release of LDH, which was further elevated when COM crystals were added. MDCK cells responded similarly to both challenges, but showed significantly less impact when compared with LLC-PK1 cells. Both treatments were associated with significant increase in the generation of hydroxyl and superoxide radicals by both cell types. In both cell lines, the addition of catalase or SOD significantly reduced the increase of MDA and release of LDH. CONCLUSIONS: Results of the present study indicate that both Ox and COM crystals are injurious to renal epithelial cells and the injury is associated with generation of free radicals. Cells of proximal tubular origin are more susceptible than those of distal tubules and collecting ducts. Free radical scavengers, catalase and SOD provide significant protection.     

Overexpression of the human 72 kDa heat shock protein in renal tubular cells confers resistance against oxidative injury and cisplatin toxicity.

BACKGROUND: Recent studies have shown that the 72-kDa heat shock protein (HSP72) can be induced in renal tubular cells by a variety of stress conditions, and suggested its cytoprotective function. We have tested this hypothesis directly by transfection studies. METHODS: LLC-PK1 cells (porcine renal tubular epithelial cells) were stably transfected with pBK-CMV or pBK-CMV containing the human HSP72 gene (pBK-CMV-HSP72). These cells were then treated with various concentrations of hydrogen peroxide or cisplatin. The cell viability and lytic cell damage were determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and lactate dehydrogenase release assay. RESULTS: Immunoblot and immunocytochemical analyses showed the high level expression of HSP72 in LLC-PK1 cells transfected with pBK-CMV-HSP72. In addition, the expression of other major HSPs (HSP90, HSP73, HSP60 and HSP27) was not affected by transfection. LLC-PK1 cells overexpressing HSP72 were significantly more resistant to hydrogen peroxide and cisplatin treatments than control cells. CONCLUSION: These results indicate that overexpressed HSP72 plays a direct role in protecting renal tubular cells against oxidative injury and cisplatin toxicity.     

氯离子通道阻断剂对氧化剂诱导的肾小管上皮细胞损伤的保护作用

目的研究氯离子（Cl-）通道阻断剂在氧化剂诱导的肾小管上皮细胞损伤中的作用。方法以H2O2诱导肾小管上皮细胞株（LLC-PK1）损伤，观察Cl-通道阻断剂对受损细胞LDH释放量、ATP含量和DNA降解程度的影响。结果Cl-通道阻断剂可使受损细胞的LDH释放量下降、ATP含量回升和DNA降解程度减轻。结论Cl-通道参与了氧化剂对细胞损伤的病理过程，Cl-通道阻断剂对肾小管上皮细胞具有保护作用。     

Differential role of reactive oxygen species in chemical hypoxia-induced cell injury in opossum kidney cells and rabbit renal cortical slices

This study was undertaken to evaluate the role of reactive oxygen species (ROS) and lipid peroxidation in chemical hypoxia in opossum kidney (OK) cells and rabbit renal cortical slices. Chemical hypoxia was induced by incubating cells or slices with antimycin A, an inhibitor of mitochondrial electron transport. Exposure of OK cells to chemical hypoxia resulted in a time-dependent cell death and parallel depletion of intracellular ATP. In OK cells subjected to chemical hypoxia, the generation of ROS was increased, and this was prevented by the H(2)O(2) scavenger catalase, but not by the hydroxyl radical scavenger dimethylthiourea (DMTU). Catalase prevented OK cell death induced by chemical hypoxia, but [Cu, Zn]-superoxide dismutase (SOD) and DMTU were not effective. The iron chelators deferoxamine and phenanthroline prevented chemical hypoxia-induced OK cell death, but the potent antioxidants N,N'-diphenyl-p-phenylenediamine (DPPD) and butylated hydroxyanisole (BHA) showed no beneficial effect. Antimycin A in OK cells increased lipid peroxidation, which was prevented by DPPD and phenanthroline. In rabbit renal cortical slices, antimycin A caused an increase in LDH release and lipid peroxidation, and these effects were prevented by ROS scavengers (SOD, catalase, and DMTU), iron chelator (deferoxamine), and antioxidants (DPPD and BHA). However, in primary cultured rabbit proximal tubular cells the antimycin A-induced cell death was not altered by antioxidants. The extent of ATP depletion was similar in renal cortical slices and primary cultured cells treated with antimycin A. These results indicate that chemical hypoxia-induced cell injury is not directly resulted from lipid peroxidation in OK cells, but this cell injury is mediated by lipid peroxidation in rabbit renal cortical slices. This discrepancy may be due to the difference in cell preparation (freshly prepared tubules and cultured cells).     

Renal tubular epithelial cells express osteonectin in vivo and in vitro.

Osteonectin (SPARC, culture shock protein, BM-40) is a widely distributed glycoprotein which binds calcium and several extracellular matrix proteins, including interstitial collagens and thrombospondin, but whose physiologic role remains undefined. In the present studies, we have demonstrated that immunoreactive osteonectin is present in the distal cortical tubule and medullary tubules of murine kidney. We surveyed the renal epithelial cell lines LLC-PK1, MDCK, and OK for the expression of mRNA encoding osteonectin. We found that osteonectin mRNA is expressed by LLC-PK1 and OK cells but not by MDCK cells, as well as by adult kidney from several species. Calcitonin and vasopressin, agents which increase cAMP in these cells, were found to decrease steady-state osteonectin mRNA concentrations. We found that LLC-PK1 cells produced osteonectin protein, that the protein was localized to intracellular granules, and that the protein bound hydroxyapatite in vitro. Pulse-chase analysis revealed that osteonectin was secreted from the cell layer to the medium after a lag time of four to six hours and was secreted preferentially from the basolateral domain of the cell. The preferential secretion of the calcium-binding protein osteonectin from the renal epithelial cell is consistent with several possible functions, including a structural extracellular matrix protein, a participant in transepithelial ion transport, and an inhibitor of extracellular calcification.     

The primary localization of free radical generation after anoxia/reoxygenation in isolated endothelial cells

While free radical-mediated reperfusion injury is clearly important in a variety of disparate organs, the particular cellular source of these radicals is unclear. To address this question, we subjected relatively pure (92% +/- 3% by factor VIII immunoassay) cultures of rat pulmonary artery endothelial cells to 0 to 45 minutes of anoxia (95% N2, 5% CO2), followed by reoxygenation (95% air, 5% CO2), to simulate ischemia/reperfusion. Cell injury was assayed after reoxygenation by the release of previously incorporated 51chromium and/or lactate dehydrogenase, and viability was determined by means of trypan blue exclusion. These three end points correlated closely. Without anoxia, the cells remained viable, with minimal evidence of injury for the entire experimental period, while 45 minutes of hypoxia followed by 30 minutes of reoxygenation produced substantial evidence of cell injury in 71% +/- 6% of the cells. This injury was reduced to 21% +/- 2% by treatment with the highly specific free radical scavengers superoxide dismutase and catalase together, either before anoxia or after anoxia, but just before reoxygenation. Similar protection was provided by xanthine oxidase inhibition with allopurinol. The injury was mimicked (without anoxia) by the exogenous generation of superoxide radicals with xanthine and xanthine oxidase. These experiments establish the essential components of free radical generation at reperfusion to be localized within the isolated endothelial cell in the absence of neutrophils or parenchymal cells.     

Meprin, a brush-border enzyme, plays an important role in hypoxic/ischemic acute renal tubular injury in rats

BACKGROUND: It has been shown that non-congenic mice strains with lower levels of renal meprin develop less renal injury following renal ischemia and reperfusion. We have demonstrated that following ischemia-reperfusion renal injury, there is a rapid shift of meprin localization and intensity from the brush border to the cytoplasmic compartment, tubular lumens and the tubular basement membranes. Radical shifts in the localization of an activated enzyme to potentially sensitive areas of the tubule suggest a toxic role for meprin in ischemia-reperfusion injury. Though meprin degrades extracellular matrix components and other substrates, to our knowledge meprin cytotoxicity has never been examined. Therefore, the first objective of this study was to determine if meprin is directly cytotoxic to renal cells in vitro. The second objective was to determine if inhibition of meprin is protective against hypoxia-reoxygenation injury in vitro and ischemia-reperfusion injury in vivo. METHODS: The immortalized porcine epithelial cell line (LLC-PK1) and Madin-Darby canine kidney (MDCK) cells in culture were exposed to meprin in various concentrations and for various times. Cell death was determined by Trypan Blue exclusion, lactate dehydrogenase (LDH) release and the 3-[4,5] dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) assay. Renal slices were used to examine the effect of the meprin inhibitor, actinonin, on hypoxic injury in vitro. Male Sprague-Dawley rats were used in ischemia-reperfusion injury studies to determine the effect of actinonin on renal function as measured by plasma urea nitrogen, creatinine and renal histology. RESULTS: Meprin is cytotoxic to LLC-PK1 and MDCK cells in a concentration and time dependent manner. The meprin inhibitor 1,10-phenanthroline completely abolished the cytotoxic effect. Renal slices exposed to hypoxia and hypoxia followed by reoxygenation showed marked cell death. Pre-treatment with the actinonin was markedly protective while not interfering with the hypoxia-induced fall in adenosine 5'-triphosphate (ATP) levels. In in vivo studies, rats exposed to ischemia/reperfusion injury were markedly protected against acute renal failure by IP treatment with actinonin. CONCLUSIONS: Meprin is cytotoxic to cultured renal tubular epithelial cells in vitro. Renal slices are protected from hypoxia-reoxygenation injury in vitro by the meprin inhibitor actinonin. Meprin inhibition is protective against rat renal hypoxia-reoxygenation injury. These data strongly support the concept that meprin is cytotoxic and may play a key role in renal ischemia-reperfusion induced renal injury.     

Reactive oxygen species independent cytotoxicity induced by radiocontrast agents in tubular cells (LLC-PK1 and MDCK)

PURPOSE: Radiocontrast agents (RAs) cause renal tubular damage by hemodynamic imbalance, which could cause hypoxic stimulus and direct cytotoxicity. However, reactive oxygen species (ROS) could be an important factor in RAs' direct cytotoxicity. This study investigated the involvement of ROS in deleterious effects produced by RAs on normoxic and hypoxic renal tubular cells. MATERIALS AND METHODS: LLC-PK1 and MDCK were exposed to diatrizoate and ioxaglate in normoxic and hypoxic conditions. Apoptotic and necrotic cell death were assessed by acridine orange/ethidium bromide and annexin V methods. Hydrogen peroxide, superoxide anion, and malondialdehyde levels were analyzed by, respectively, 2',7'-dichlorofluorescein, luminal, and thiobarbituric acid. Antioxidant agents were used to prevent cellular RAs damage. RESULTS: Diatrizoate and ioxaglate decreased cellular viability in both cells, and this effect was enhanced by hypoxic conditions. Diatrizoate induced more injury than ioxaglate to both cell lines. LLC-PK1 underwent necrosis, while MDCK cells underwent apoptosis when exposed to diatrizoate. These results could not be attributed to an increase in osmolality. RAs did not increase hydrogen peroxide, superoxide anion or malondialdehyde levels in both cells. Additionally, N-acetyl-L-cysteine (NAC), ascorbic acid, alpha-tocopherol, glutathione, beta-carotene, allopurinol, cimetidine, and citric acid did not protect cells against RAs damage. Surprising, NAC increased the cellular damage induced by ioxaglate in the both cell lines. CONCLUSION: The present study shows that RAs induce damage in cultured tubular cells, especially in hypoxic conditions. ROS were not involved in the observed RAs' cytotoxicity, and NAC increased ioxaglate-induced tubular damage.     

Successful Treatment of Focal Segmental Glomerulosclerosis in Association with Mantle Cell Lymphoma

We have examined the role of reactive oxygen metabolites (ROM) in gentamicin nephrotoxicity and in glycerol-induced acute renal failure, a model for myoglobinuric acute renal failure. Several agents which affect mitochondrial respiration have been shown to enhance the generation of hydrogen peroxide. Based on gentamicin s ability to alter mitochondrial respiration both in vitro and in vivo we postulated that gentamicin may enhance the generation of ROM by renal com'cal mitochondria. Gentamicin, in a dose-dependent farhion, enhanced hydrogen peroxide production by rat renal cortical mitochondria as measured by the decrease in scopoletin jluorescence. At the highest concentration of gentamicin tested (4.0 mM), the rate of hydrogen peroxide generation w s markedly increased from 0. I7 f 0.02 to 6.21 f 0.67 nmol/mg/m*n. We next demonstrated that hydroxyl radical scavengers and an iron chelator provide a marked functional and histological protection in gentamicin-induced acute renal failure in rats. Hydroxyl radical scavengers and the iron chelator deferoxamine also protected renal function in glycerol-injected rats, a model for acute renal failure due to muscle injury. Although these data suggest that ROM may be important mediators of toxic renal injury, in vivo generation of ROM by kidney in normal and pathological states has not been previously examined. Aminotriazole (An irreversibly inactivates catalase only in the presence of hydrogen peroxide and previous studies have shown that AT-medated inhibition of catalase in a sensitive measure of in vivo changes in the hydrogen peroxide generation. Using this method, we have demonstrated the in vivo generation of hydrogen peroxide under normal conditions and enhanced generation of hydrogen peroxide in rats treated with gentamicin or glycerol. Finally, in in vitro studies we have shown that iron and intracellular calcium play a critical role in hydrogen peroxide-mediated cytotoxicity to LLC-PK, cells, a renal tubular epithelial cell line. Taken together our data provide evidence for a role of ROM in gentamicin and glycerol-induced acute renal failure and provide evidence for the role of iron and calcium in oxidant injury to renal tubular epithelial cells.     

Urokinase synthesis and binding by glomerular epithelial cells in culture

Fibrin deposits are frequently observed in the course of proliferative extracapillary glomerulonephritis and could be related to a defective local fibrinolysis. We studied human glomerular epithelial cells in culture which were found to release mainly a urokinase-type plasminogen activator (u-PA) identified on zymography by its molecular weight (53 kD), its plasminogen activator activity, and its neutralization by specific polyclonal anti-u-PA IgG. Trace amounts of tissue-type plasminogen activator (t-PA) complexed to a plasminogen activator inhibitor type 1 (PAI-1) were identified with specific antibodies. Specific binding sites were found at the surface of glomerular epithelial cells (kD: 2.10(-9) M), partially occupied by secreted u-PA. The spontaneous u-PA activity of the culture medium conditioned by glomerular epithelial cells was very low, suggesting that u-PA was released in its inactive single chain proenzyme form (SC-u-PA). After activation of SC-u-PA by plasmin, u-PA activity of the culture medium was found to increase in a time- and dose-dependent manner when cells were incubated with phorbol myristic acetate (PMA). This effect was inhibited by H7, a protein kinase C inhibitor. Stimulation of u-PA synthesis by PMA was also observed in two different epithelial tubular cell lines. LLC-PK1 and MDCK cells. However, 8 bromo cyclic AMP which increased u-PA release by LLC-PK1 cells was found to inhibit u-PA release by PMA-stimulated glomerular epithelial cells and MDCK cells. By Northern blot analysis we found that PMA induced an increase of u-PA mRNA level in glomerular epithelial cells and that cyclic AMP had an opposite effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein uptake disturbs collagen homeostasis in proximal tubule-derived cells

BACKGROUND: Interstitial fibrosis is of major importance for the deterioration of renal function, leading to uremia. Interaction of filtered proteins with proximal tubular cells is important for the onset and development of tubulointerstitial damage. METHODS: We investigated the effects of protein endocytosis on collagen homeostasis and signaling pathways of proximal tubule-derived cells (OK cells, LLC-PK1 cells), which express the endocytic machinery typical for the proximal tubule (megalin and cubilin), and compared it to renal epithelial cells with low endocytic activity (MDCK, IHKE1, NHE3-deficient OK cells). Collagen homeostasis was assessed by proline incorporation, ELISA, and Western blot. Matrix metalloproteinase (MMP) activity was assessed by gelatinase assay. Signaling pathways were monitored by reporter gene assay. RESULTS: Albumin, glycated albumin, fatty acid-free albumin, or globulins led to an increase of secreted collagen (types I, III, and IV) in OK and LLC-PK1 cells. In cells with low protein uptake activity, albumin exposure inhibited collagen secretion. Western blot analysis showed an increase of cellular collagen. MMP activity was significantly decreased by albumin exposure. Furthermore, albumin exposure led to activation of the NF-kappa B-, AP1-, NFAT-, SRE-, and CRE-pathways. Inhibition of NF-kappa B, PKC, or PKA partially reversed the effects of albumin. In addition, inhibition of albumin endocytosis reduced collagen secretion and activation of the signaling pathways. Discussion. The data show that endocytic uptake of proteins disturbs collagen homeostasis in proximal tubular cells. This disturbed matrix homeostasis probably supports the progression of interstitial fibrosis, which is of importance for the development of renal insufficiency.     

Reactive Oxygen Species Independent Cytotoxicity Induced by Radiocontrast Agents in Tubular Cells (LLC-PK1 and MDCK)

Purpose. Radiocontrast agents (RAs) cause renal tubular damage by hemodynamic imbalance, which could cause hypoxic stimulus and direct cytotoxicity. However, reactive oxygen species (ROS) could be an important factor in RAs' direct cytotoxicity. This study investigated the involvement of ROS in deleterious effects produced by RAs on normoxic and hypoxic renal tubular cells. Materials and Methods. LLC-PK1 and MDCK were exposed to diatrizoate and ioxaglate in normoxic and hypoxic conditions. Apoptotic and necrotic cell death were assessed by acridine orange/ethidium bromide and annexin V methods. Hydrogen peroxide, superoxide anion, and malondialdehyde levels were analyzed by, respectively, 2′,7′-dichlorofluorescein, luminal, and thiobarbituric acid. Antioxidant agents were used to prevent cellular RAs damage. Results. Diatrizoate and ioxaglate decreased cellular viability in both cells, and this effect was enhanced by hypoxic conditions. Diatrizoate induced more injury than ioxaglate to both cell lines. LLC-PK1 underwent necrosis, while MDCK cells underwent apoptosis when exposed to diatrizoate. These results could not be attributed to an increase in osmolality. RAs did not increase hydrogen peroxide, superoxide anion or malondialdehyde levels in both cells. Additionally, N-acetyl-L-cysteine (NAC), ascorbic acid, α-tocopherol, glutathione, β-carotene, allopurinol, cimetidine, and citric acid did not protect cells against RAs damage. Surprising, NAC increased the cellular damage induced by ioxaglate in the both cell lines. Conclusion. The present study shows that RAs induce damage in cultured tubular cells, especially in hypoxic conditions. ROS were not involved in the observed RAs' cytotoxicity, and NAC increased ioxaglate-induced tubular damage.     

Role of cytochrome P4502E1 activation in proximal tubular cell injury induced by hydrogen peroxide

BACKGROUND: There is now good evidence to suggest that cytochrome P450 (CYP450) may act as an iron-donating catalyst for the production of hydroxyl ion (OH*), which contributes to proximal tubular cell injury. However, it remains unclear which isoform of CYP450 is involved in this process. Cytochrome P4502E1 (CYP2E1) is a highly labile isoform which is not only involved in free radical generation, but has also been shown to be a source of iron in cisplatin-induced renal injury. This study investigates the role of CYP2E1 in the proximal tubular cell injury induced by hydrogen peroxide (H2O2). METHODS: Porcine proximal tubular cells (LLC-PK1) were incubated with H2O2 (1 mM) for 4 h in the presence or absence of 0.1 mM of two CYP2E1 inhibitors; diallyl sulfide (DAS), or disulfiram (DSF), desferrioxamine (DFO) (0.1-0.4 mM), or catalase (CT) (78, 150, 300 U/mL). Cell death was determined by measuring LDH release. CYP2E1 activity was determined by p-nitrophenol hydroxylation after 2 h incubation with H2O2. RESULTS: Exposure of LLC-PKI to H2O2 significantly increased cell death. CT, DFO, DAS and DSF significantly reduced H2O2-mediated cell death. Incubation with H2O2 increased CYP2EI activation in time- and dose-dependent manner, which was significantly reduced by CT, DFO, DAS and DSF. CONCLUSION: We propose that CYP2E1 activation occurs possibly due to OH* and contributes to H2O2-mediated LLC-PK1 cell necrosis by acting as a source of iron and perpetuating the generation of OH* via the Fenton reaction. Inhibition of CYP2E1 may be a novel approach for the prevention of tubular injury caused by oxidative stress.     

Hydrogen peroxide cytotoxicity in LLC-PK1 cells: a role for iron.

Reactive oxygen metabolites have been postulated to play an important role in both toxic and ischemic forms of acute renal tubular epithelial injury. In the present study, we examined the effect of enzymatically generated hydrogen peroxide on LLC-PK1 cells, a renal proximal tubule cell line. Exposure of LLC-PK1 cells to glucose and glucose oxidase (GO; which generates hydrogen peroxide) resulted in cytotoxicity (as measured by trypan blue exclusion) which was dose dependent and increased linearly over time to 81 +/- 5% at 180 minutes (8 +/- 1% at time 0; mean +/- SEM, N = 3 to 7). Catalase (which decomposes hydrogen peroxide) completely prevented the cytotoxicity, confirming that the toxicity was due to hydrogen peroxide production. To assess whether the hydrogen peroxide toxicity was a direct effect or mediated by other toxic oxygen metabolites, several scavengers of reactive oxygen metabolites and iron chelators were used. Superoxide dismutase (a scavenger of superoxide) had no effect. Deferoxamine (DFO), an iron chelator, provided marked protection (GO alone 45.9 +/- 4.4%; GO + DFO 13.0 +/- 2.0%; control 7.1 +/- 1.2%; N = 15 to 17, P less than 0.001). Pretreatment with DFO (1 hr, then 2 washes to remove DFO before GO addition) also markedly inhibited the cytotoxicity, suggesting that DFO's effect was due to iron chelation. Two other metal chelators (dihydroxybenzoic acid and 1,10-phenanthroline) also significantly decreased the GO-induced cytotoxicity. However, three of four hydroxyl radical scavengers used (mannitol, dimethyl sulfoxide, sodium benzoate) did not significantly decrease cell death. Only dimethylthiourea provided protection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Minimal role of xanthine oxidase and oxygen free radicals in rat renal tubular reoxygenation injury

The role of xanthine oxidase and oxygen free radicals in postischemic reperfusion injury in the rat kidney remains controversial. Proximal tubules, the focal segment affected by ischemic renal injury, were isolated in bulk, assayed for xanthine oxidase activity, and subjected to 60 min of anoxia or hypoxia and 60 min of reoxygenation to evaluate the participation of xanthine oxidase and oxygen radicals in proximal tubule reoxygenation injury. The total xanthine oxidase in isolated rat proximal tubules was 1.1 mU/mg of protein, approximately 30% to 40% of the activity found in rat intestine and liver. Lactate dehydrogenase release, an indicator of irreversible cell damage, increased substantially during anoxia (39.8 +/- 2.3 versus 9.8 +/- 1.8% in controls) with an additional 8 to 12% release during reoxygenation. Addition of 0.2 mM allopurinol, a potent xanthine oxidase inhibitor, and dimethylthiourea, a hydroxyl radical scavenger, failed to protect against the reoxygenation lactate dehydrogenase release. Analysis of xanthine oxidase substrate levels after anoxia and flux rates during reoxygenation indicates that hypoxanthine and xanthine concentrations are in a 15-fold excess over the enzyme Km and 0.3 mU/mg of protein of xanthine oxidase activity exists during reoxygenation. Hypoxic tubule suspensions had a minimal lactate dehydrogenase release during hypoxia and failed to demonstrate accelerated injury upon reoxygenation. In conclusion, although xanthine oxidase is present and active during reoxygenation in isolated rat proximal tubules, oxygen radicals did not mediate reoxygenation injury.     

Protection against hydrogen peroxide induced injury in renal proximal tubule cell lines by inhibition of poly(ADP-ribose) synthase

Radicals including superoxide anions, hydrogen peroxide or hydroxyl radicals and NO or peroxynitrite cause the breakage of DNA strands and activation of poly-(ADP-ribose) synthase (PARS). Recent studies showed that inhibition of PARS activity reduces the tissue injury after exposure to oxidative stress. However, the role of PARS in renal injury by oxidants has not been examined. In this study effect of a PARS inhibitor, 3-aminobenamide (3-AB), on injury of opossum kidney or LLC-PK(1) cells by hydrogen peroxide or tert-butyl hydroperoxide (t-BHP) was examined. The exposure of opossum kidney cells to hydrogen peroxide activated PARS and decreased cellular adenosine triphosphate levels in a concentration-dependent manner. Inhibition of PARS with 3-AB prevented the cell death induced by hydrogen peroxide and also prevented adenosine triphosphate depletion. 3-AB did not have hydroxyl radical scavenging effect. In contrast, t-BHP did not affect the PARS activity. The decrease in cellular adenosine triphosphate levels by t-BHP was less than that by hydrogen peroxide. 3-AB failed to prevent the cell death induced by t-BHP. PARS activation after exposure of hydrogen peroxide was inhibited by addition of t-BHP. However, t-BHP showed an additive effect on cell death with hydrogen peroxide. These results indicate that activation of PARS plays an important role in hydrogen peroxide induced injury in opossum kidney cells and that hydrogen peroxide and t-BHP induce cell injury by different mechanisms.     

热休克蛋白72抑制ATP耗竭时细胞色素C释放所诱导的肾小管上皮细胞凋亡

目的 研究热休克蛋白(HSP)72对ATP耗竭时细胞色素C释放所导致的肾小管上皮细胞凋亡的保护作用及其分子机制。方法 应用代谢抑制剂暂时性阻断细胞内ATP的生成,引起细胞凋亡。应用热处理细胞或编码HSP72 RNA的腺病毒感染细胞,诱导HSP72的表达。以Western印迹检测释放于胞浆内的细胞色素C。荧光肽法测定半胱氨酸天冬氨酸蛋白酶(caspase)3活性。Hoechst33342染色观察细胞凋亡的发生情况。结果 肾小管上皮细胞内ATP耗竭时,释放至胞浆内的细胞色素C的含量增多,caspase 3活性增强;细胞内ATP再恢复时,细胞色素C的释放和caspase 3活性进一步增加,细胞体积缩小,核浓染、固缩,形成凋亡小体。预先热处理后,各组细胞色素C的释放明显减少,caspase 3活性显著抑制(P<0.05,n=3)。高表达HSP72时,各时间点caspase 3活性的抑制程度与热处理组相似,细胞体积缩小,核浓染、固缩,凋亡小体的形成明显减少。结论 HSP72可抑制ATP耗竭时细胞色素C所导致的肾小管上皮细胞凋亡,其机制是抑制凋亡通路中细胞色素C的释放和caspase 3的激活。     

Effect of nitric oxide donors on renal tubular epithelial cell-matrix adhesion.

BACKGROUND: Nitric oxide (NO) and its metabolite, peroxynitrite (ONOO-), are involved in renal tubular cell injury. We postulated that if NO/ONOO- has an effect to reduce cell adhesion to the basement membrane, this may contribute to tubular obstruction and may be partially responsible for the harmful effect of NO on the tubular epithelium during acute renal failure (ARF). METHODS: We examined the effect of the NO donors (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate (DETA/NO), spermine NONOate (SpNO), and the ONOO- donor 3-morpholinosydnonimine (SIN-1) on cell-matrix adhesion to collagen types I and IV and fibronectin using three renal tubular epithelial cell lines: LLC-PK1, BSC-1, and OK. RESULTS: In LLC-PK1 cells, DETA/NO (500 microM) had no effect, and SpNO (500 microM) had a modest effect on cell adhesion compared with controls. Exposure to SIN-1 caused a dose-dependent impairment in cell-matrix adhesion. Similar results were obtained in the different cell types and matrix proteins. The effect of SIN-1 (500 microM) on LLC-PK1 cell adhesion was not associated with either cell death or alteration of matrix protein and was attenuated by either the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, the superoxide scavenger superoxide dismutase, or the ONOO- scavenger uric acid in a dose-dependent manner. CONCLUSIONS: These results therefore support the possibility that ONOO- generated in the tubular epithelium during ischemia/reperfusion has the potential to impair the adhesion properties of tubular cells, which then may contribute to the tubular obstruction in ARF.     

The effects of gamma-glutamylcysteine ethyl ester, a prodrug of glutathione, on ischemia-reperfusion-induced liver injury in rats.

This study was designed to clarify the effects of changes in liver tissue glutathione (GSH) concentration on postischemic liver injury together with the effects of gamma-glutamylcysteine ethyl ester (GCE), a prodrug of GSH, and GSH. Rats were pretreated with GSH (50 mg/kg, i.v.), or GCE (50 mg/kg, i.v.), or untreated. In each rat, liver was isolated, and liver mitochondria were prepared after 2 h of ischemia or 1 h of reperfusion following 2 h of ischemia. Mitochondrial function was measured polarographically. Liver adenine nucleotide concentrations were also determined using high-performance liquid chromatography. Liver tissue GSH, an oxidized form of glutathione (GSSG) concentrations, and activities of GSH peroxidase and GSSG reductase were determined enzymatically. Liver hypoxanthine and xanthine concentrations were determined by HPLC. Liver tissue concentration of lipid peroxide was measured. Leakages of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and adenine nucleotides into the hepatic vein after reperfusion were also measured. Administration of GCE improved the recovery of mitochondrial function and maintained tissue GSH concentration concomitantly. Increases in liver lipid peroxide concentration after reperfusion, and leakage of liver cell enzymes and adenine nucleotides were mitigated by administration of GCE. Administration of GSH itself failed to maintain tissue GSH concentration and had no protective effects. From these results, it is concluded that in the postischemic process, free radical formation might be enhanced, and the radical scavenging system deteriorated. To enhance the radical scavenging system is a possible maneuver to prevent radical-related cell damage associated with reperfusion, because pharmacological reduction of breakdown of ATP to hypoxanthine and xanthine seems to be difficult. GCE maintained liver GSH concentrations and mitigated postischemic liver injury, concomitantly. Clinical use of GCE might be recommended.