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.     

Radiocontrast-induced DNA fragmentation of renal tubular cells in vitro: role of hypertonicity

Background: Radiocontrast-induced nephropathy is a clinically important complication of invasive cardiological procedures. It has been associated with DNA fragmentation of renal tubular cells, which is a hallmark feature of programmed cell death (apoptosis). We investigated the mechanism of this DNA fragmentation in an in vitro model of radiocontrast cytotoxicity on renal epithelial cells. Methods: Madin Darby canine kidney (MDCK) cell monolayers were incubated (for 2-8 h) with isoiodine doses (27-111 mg iodine/ml) of the highly hyperosmolal, ionic radiocontrast agent diatrizoate or of the less hyperosmolal, non-ionic substance iopamidol. Mannitol, urea, and NaCl control media of corresponding hyperosmolality were used to evaluate the contribution of hypertonicity, hyperosmolality and/or ionic strength to radiocontrast toxicity. DNA fragmentation was assessed using fluorescence-activated cell sorting (FACS), agarose gel electrophoresis and terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end labelling (TUNEL), cell morphology was analysed in Giemsa-stained cytospins. Results: Diatrizoate induced concentration- and time-dependent DNA fragmentation of MDCK cells which was associated with morphological signs of apoptosis. Cycloheximide (1 &mgr;g/ml) did not prevent diatrizoate-induced DNA fragmentation, indicating that it is not dependent on protein synthesis. Diatrizoate-mediated cell death was associated with cell detachment from the tissue culture matrix. However, the DNA fragmentation is not a consequence of cell detachment since the prevention of cell attachment on agarose-coated dishes induced significantly less DNA fragmentation than diatrizoate. Iopamidol caused no detectable DNA breakdown. In contrast, hypertonic mannitol and sodium chloride, but not hyperosmolal urea, induced DNA fragmentation in MDCK cells, albeit less than diatrizoate. Conclusions: The DNA fragmentation of MDCK cells induced by diatrizioate is related to its hypertonicity in this in vitro model of radiocontrast cytotoxicity. Nuclear disintegration with subsequent cell death may contribute to the pathophysiology of radiocontrast-induced nephropathy, particularly in the hypertonic/hypoxic environment of the renal medulla. The present results underscore the importance of avoiding hyperosmolal urine states in patients at high risk of radiocontrast-induced nephropathy. Key words: apoptosis; cytotoxicity; hypertonicity; MDCK; radiocontrast; renal failure      

Ionic radiocontrast media disrupt intercellular contacts via an extracellular calcium-independent mechanism

Direct cytotoxic effects of radiocontrast (RC) agents have been implicated in radiocontrast nephropathy (RCIN). The interaction between extracellular calcium, which plays a central role in intercellular contacts, and the in vitro toxicity of RC was tested in Madin-Darby canine kidney (MDCK) cell monolayers grown on permeable supports. Cell viability was determined by trypan blue exclusion. The function of intercellular junctions was assessed by measuring the electrical transmonolayer resistance (TMR). The cell contacts were examined with indirect immunofluorescence microscopy using antibodies against the junctional proteins E-cadherin, ZO-1 and occludin. The ionic RC agents diatrizoate and ioxaglate (74 mg iodine/ml), but not the nonionic compounds iohexol or iodixanol, decreased ionized calcium (Ca2+) in the incubation media from 1.48 +/- 0.04 mM (control) to 0.89 +/- 0.06 mM (diatrizoate), respectively to 1.05 +/- 0.08 mM (ioxaglate). Diatrizoate, and to a lesser extent ioxaglate, reduced the number of viable MDCK cells and showed a redistribution of the E-cadherin, ZO-1 and occludin immunofluorescence signal with a parallel decrease of the TMR indicating an impaired monolayer integrity. A similar reduction of extracellular Ca2+ through EGTA failed to reproduce these effects. Conversely, raising Ca2+ in diatrizoate-containing media to control levels did not abrogate its toxicity. In conclusion, the ionic RC agents diatrizoate and ioxaglate, but not the nonionic compounds iohexol or iodixanol, reduce extracellular Ca2+ in vitro. However, this reduction of Ca2+ does not explain their cytotoxic effects which could contribute to the pathogenesis of RCIN in vivo by opening intercellular junctions.     

Comparative cytotoxicity of ionic and non-ionic radiocontrast agents on MDCK cell monolayers in vitro.

BACKGROUND: Intravascular radiocontrast agents may cause acute renal failure, particularly in patients with pre-existing renal insufficiency. Direct cytotoxic effects of radiocontrast agents on renal tubular cells may contribute to the pathogenesis of radiocontrast-induced nephropathy. METHODS: We analysed the cytotoxicity of the ionic radiocontrast agents diatrizoate (monomeric) and ioxaglate (dimeric), as well as of the non-ionic radiocontrast agents iohexol (monomeric) and iodixanol (dimeric) on the renal epithelial Madin Darby Canine Kidney (MDCK) cell line grown on permeable supports. The toxicity assays assessed cell viability, transmonolayer resistance and inulin permeability between the apical and basal cell culture compartment. In addition, the distribution of the tight-junction-associated membrane proteins ZO-1 and occludin was analysed using immunofluorescence microscopy. RESULTS: In all assays the high osmolal ionic compound diatrizoate had significant cytotoxic effects that included the partial redistribution of the tight-junction-associated membrane proteins into a cytoplasmic compartment. To a lesser extent this redistribution also occurred with the dimeric ionic compound ioxaglate, but not with the non-ionic radiocontrast agents. With regards to cell viability, transmonolayer resistance and inulin permeability the radiocontrast agents with reduced osmolality were significantly less toxic than diatrizoate, independent of their ionic strength. CONCLUSIONS: Physicochemical factors contribute to the cytotoxicity of radiocontrast agents in vitro. The redistribution of tight-junction-associated membrane proteins by the ionic radiocontrast agents corresponds with the loss of the barrier function of the epithelial cell monolayer, which is a major pathophysiological mechanism in acute renal failure. The radiocontrast agents with reduced osmolality are less cytotoxic than diatrizoate, independent of their ionicity. Hyperosmolality appears to be a more important determinant of the cytotoxicity of diatrizoate than ionic strength.     

Hydrogen peroxide mediates FK506-induced cytotoxicity in renal cells

BACKGROUND: The nephrotoxicity induced by immunosuppressant FK506 remains a serious clinical problem, and the underlying mechanism has not been completely understood. The present study was undertaken to determine the role of hydrogen peroxide in FK506-mediated cytotoxicity in a porcine renal proximal tubular cell line, LLC-PK1 cells, and human embryonic kidney (HEK293) cells. METHODS: Cytotoxicity was estimated by crystal violet and lactate dehydrogenase release assays. The activity of reactive oxygen species (ROS) was detected by flow cytometry. FK506-induced cell death was examined in the presence of the hydrogen peroxide scavenger, catalase, or a scavenger of hydroxyl radicals, sodium benzoate. As a control, FK506-induced cell death was also measured in the presence of superoxide anion inhibitor, 4,5-dihydroxy-1,2-benzene disulfonic acid (Tiron), TEMPO, or overexpressed human manganese superoxide dismutase (MnSOD). Catalase was also used in tumor necrosis factor-alpha (TNF-alpha)-induced cell injury to determine whether the enzyme specifically protected cells against FK506-mediated cytotoxicity. RESULTS: FK506 induced cell death in a dose-dependent manner and coincided with a dose-dependent increase in ROS activity. Abrogation of FK506-mediated ROS by catalase and N-acetylcysteine blunted FK506-induced cell death. Furthermore, overexpression of catalase, sodium benzoate, and deferoxamine inhibited the cytotoxic effect of FK506. In contrast, Tiron, TEMPO, or overexpression of human MnSOD failed to show cytoprotection. In fact, TEMPO or expression of MnSOD enhanced the effect of FK506. Catalase did not significantly affect TNF-alpha-induced cell injury. CONCLUSION: Catalase is uniquely required in cellular protection against FK506 cytotoxicity, which suggests an important role for hydrogen peroxide in the cellular actions of FK506.     

Role of reactive oxygen metabolites in organophosphate-bidrin-induced renal tubular cytotoxicity.

Due to low toxicity to nontarget species and rapid degradation after its application, organophosphate (OP) remains a widely used class of pesticide. Suicidal or accidental overdose of OP can result in acute tubular necrosis. Experimental evidence shows little correlation between the renal tubular necrosis and the degree of OP-induced acetylcholinesterase inhibition, the main mechanism of OP's toxicity, suggesting the involvement of alternate mechanisms. Since reactive oxygen species (ROS) are known mediators of many toxin-induced renal injuries, this study was conducted to investigate whether ROS play a role in Bidrin (BD)-induced renal tubular epithelial cell (LLC-PK1) toxicity. BD is an OP insecticide formulation with dicrotophos as the active ingredient. LLC-PK1 cell death, determined by lactate dehydrogenase (LDH) release (% of total), rose concentration- and time-dependently after exposure of the cells to 1000, 1250, 1500, 1750, and 2000 ppm of BD for 6, 12, 24, and 48 h. Antioxidants 2-methylaminochroman (2-MAC; 0.3 to 2.5 microM) and desferrioxamine (DFO; 0.25 to 2 mM) reduced cell damage induced by 1250 ppm of BD over a 24-h incubation in a concentration-related manner. The greatest reductions in % LDH were produced by DFO 2 mM and 2-MAC 2.5 microM, both significantly lower than BD alone. H2O2 levels (micromol/mg protein per h) were significantly elevated after exposure to 1250 ppm of BD. Significantly increased malondialdehyde formation (nmol/mg protein) compared with control was also found in BD-exposed cells indicating enhanced lipid peroxidation. Malondialdehyde generation was significantly suppressed by 2-MAC and DFO. These results demonstrate that the organophosphate BD can cause direct tubular cytotoxicity, and implicate, at least in part, a role for ROS and accompanying lipid peroxidation in cytotoxicity. Based on these direct in vitro findings, it is hypothesized that, besides hypotension that often accompanies OP intoxication, OP-induced oxidative stress at the tubular level may play a role in the pathogenesis of acute tubular necrosis.     

Reactive oxygen molecule-mediated injury in endothelial and renal tubular epithelial cells in vitro

To investigate renal tubular epithelial cell injury mediated by reactive oxygen molecules and to explore the relative susceptibility of epithelial cells and endothelial cells to oxidant injury, we determined cell injury in human umbilical vein endothelial cells and in four renal tubular epithelial cell lines including LLC-PK1, MDCK, OK and normal human kidney cortical epithelial cells (NHK-C). Cells were exposed to reactive oxygen molecules including superoxide anion, hydrogen peroxide and hydroxyl radical generated by xanthine oxidase and hypoxanthine. We determined early sublethal injury with efflux of 3H-adenine metabolites and a decline in ATP levels, while late lytic injury and cell detachment were determined by release of 51chromium. When the cells were exposed to 25, 50, and 100 mU/ml xanthine oxidase with 5.0 mM hypoxanthine, ATP levels were significantly lower (P less than 0.001) in LLC-PK1, NHK-C and OK cells compared to MDCK cells while ATP levels were significantly lower (P less than 0.01) in endothelial cells compared to all tubular cell lines. A similar pattern of injury was seen with efflux of 3H-adenine metabolites. When the cells were exposed to 50 mU/ml xanthine oxidase with 5.0 mM hypoxanthine for five hours, total 51chromium release was significantly (P less than 0.001) greater in LLC-PK1, NHK-C and OK cells compared to MDCK cells, while total 51chromium release was significantly (P less than 0.001) greater in endothelial cells compared to all tubular cells. However, lytic injury was the greatest in LLC-PK1 cells and NHK-C cells while cell detachment was the greatest in endothelial cells. MDCK cells were remarkably resistant to oxidant-mediated cell detachment and cell lysis. In addition, we determined ATP levels, 3H-adenine release and 51chromium release in LLC-PK1, NHK-C and endothelial cells in the presence of superoxide dismutase to dismute superoxide anion, catalase to metabolize hydrogen peroxide, DMPO to trap hydroxyl radical and DMTU to scavenge hydrogen peroxide and hydroxyl radical. We found that catalase and DMTU (scavengers of hydrogen peroxide) provided significant protection from ATP depletion, prevented efflux of 3H-adenine metabolites and cell detachment while DMPO (scavenger of hydroxyl radical) prevented lytic injury. In addition, we found that the membrane-permeable iron chelator, phenanthroline, and preincubation with deferoxamine prevented cell detachment and cell lysis, confirming the role of hydroxyl radical in cell injury.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hypoxia-mediated impaired differentiation by LLC-PK1 cells: evidence based on the protein kinase C profile.

We recently reported that mild hypoxia in LLC-PK1 cells, grown in standard fashion under a still layer of overlying medium at 5% CO2/18% O2 environment, result in decreased oxidative metabolism and impaired differentiated functions in comparison to adequately oxygenated cultures maintained either under a higher oxygen (36% O2) environment or conditions of continuous rocking of the media fluid. In the present study, subcellular distribution of a regulatory enzyme protein kinase C (PKC) was examined between hypoxic still and normoxic rocked LLC-PK1 cells. Subconfluent cultures of hypoxic LLC-PK1 cells exhibited significantly lower and predominantly membrane-bound PKC activity in comparison to mostly cytosolic localization of this enzyme in normoxic rocked cells. One hour of exposure of adequately oxygenated-rocked LLC-PK1 cells with the phorbol ester TPA, a dedifferentiating agent that did not effect the cell ATP content, resulted in significant inhibition of dome formation and sodium-dependent glucose transport activity, a partial loss of pH-responsive ammoniagenesis, and almost complete translocation of protein kinase C activity from cytosol to the membrane pool; all of which resembles the behavior of hypoxic still cultured cells. In addition, acute re-oxygenation of hypoxic still cultures by rocking the media fluid for one hour resulted in an increase in cell ATP content to the cellular levels of ATP observed in normoxic rocked cells. However, all the parameters of differentiation were unaffected by re-oxygenation. These studies support the notion that hypoxia can act in some primary fashion, independent of its effects on energy metabolism, to impair cellular differentiation in LLC-PK1 cells. They also raise the possibility that activation of protein kinase C may act as an important mediator in this process.     

A role for sodium in hypoxic but not in hypothermic injury to hepatocytes and LLC-PK1 cells

BACKGROUND: Hypothermia is considered to be responsible for sodium influx during cold hypoxic incubation. However, we have previously shown that hypothermia alone leads to a pronounced decrease in cellular sodium content when liver endothelial cells or hepatocytes are incubated under such conditions. In the research described here, we therefore studied the effects of hypothermia and hypoxia, alone or combined, on cellular sodium homeostasis and assessed the role sodium plays in the pathogenesis of hypoxic and hypothermic injury to cultured liver and kidney cells. METHODS: Isolated hepatocytes and LLC-PK1 cells were incubated in Krebs-Henseleit buffer or a sodium-free modification thereof under normoxic and hypoxic conditions at 4 degrees C as well as at 37 degrees C. Cytosolic sodium concentration was determined in isolated hepatocytes under both warm and cold conditions using digital fluorescence microscopy and the Na+-sensitive dye sodium-binding benzofuran isophthalate. RESULTS: When hepatocytes were incubated under cold normoxic conditions the cellular sodium concentration decreased. However, it increased strongly under hypoxic conditions at 4 degrees C and at 37 degrees C. When either hepatocytes or LLC-PK1 cells were incubated under hypoxic conditions at 4 degrees C or 37 degrees C, sodium-free medium provided protection. In contrast, sodium-free medium did not alleviate the hypothermic injury observed when cells were incubated under cold normoxia. CONCLUSIONS: The sodium influx observed during cold hypoxia is triggered by hypoxia and not by hypothermia. Sodium plays a prominent role in hypoxic injury to cultured liver and kidney cells, although hypothermic injury of these cells is independent of sodium homeostasis.     

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.     

Renal vasoconstriction after low and high osmolar contrast agents in ischemic and non ischemic canine kidney

We have assessed the effect of contrast media on renal blood flow before and after inducing renal ischemia. Diatrizoate, iopamidol and ioxaglate were injected within 15 seconds at 20 min intervals, at the dose of 1 ml/kg during a control period and 15 min after applying an aortic clamp to reduce the renal perfusion pressure to 70 mmHg. During the control period iopamidol, ioxaglate (17 +/- 13%) and diatrizoate (16 +/- 2%) induced a comparable decrease in renal blood flow (RBF). During the ischemic period the effects of diatrizoate on renal hemodynamic were dramatically enhanced. Ioxaglate andiopamidol induced a 20 +/- 12 and a 32 +/- 9% decrease in RBF at 1 minute, respectively. Iopamidol induced an increase in renal vascular resistance (RVR) from 0.8 +/- 0.08 to 1.46 +/- 0.26 mmHg min/ml (p less than 0.05). Ioxaglate induced an increase in RVR from 0.8 +/- 0.09 to 1.36 +/- 0.38 (p less than 0.05). Diatrizoate induced a 77 +/- 10% decrease in RBF and a maximum increase in RVR at 1 minute from 0.9 +/- 0.09 to 26 +/- 12 mmHg min/ml. There was still a 36 +/- 14% and a 23 +/- 13% decrease in RBF 10 and 20 min after diatrizoate administration. These changes were significantly higher than those observed with all contrast media during the control period and low osmolar contrast media during the ischemic period. We have thus shown that ischemia potentiates the renal vascular effect of contrast media.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of glycosaminoglycans in tubular epithelial cells: calcium oxalate and oxalate ions effects

BACKGROUND: The interaction between tubular epithelial cells and calcium oxalate crystals or oxalate ions is a very precarious event in the lithogenesis. Urine contains ions, glycoproteins and glycosaminoglycans that inhibit the crystallization process and may protect the kidney against lithogenesis. We examined the effect of oxalate ions and calcium oxalate crystals upon the synthesis of glycosaminoglycans in distal [Madin-Darby canine kidney (MDCK)] and proximal (LLC-PK1) tubular cell lines. METHODS: Glycosaminoglycan synthesis was analyzed by metabolic labeling with (35)S-sulfate and enzymatic digestion with specific mucopolysaccharidases. Cell death was assessed by fluorescent dyes and crystal endocytosis was analised by flow cytometry. RESULTS: The main glycosaminoglycans synthesized by both cells were chondroitin sulfate and heparan sulfate most of them secreted to the culture medium or present at cellular surface. Exposition of MDCK cells to oxalate ions increased apoptosis rate and the incorporation of (35)S-sulfate in chondroitin sulfate and heparan sulfate, while calcium oxalate crystals were endocyted by LLC-PK1, induced necrotic cell death, and increased (35)S-sulfate incorporation in glycosaminoglycans. These effects seem to be specific and due to increased biosynthesis, since hydroxyapatite and other carboxylic acid did not induced cellular death or glycosaminoglycan synthesis and no changes in sulfation degree or molecular weight of glycosaminoglycans could be detected. Thapsigargin inhibited the glycosaminoglycan synthesis induced by calcium oxalate in LLC-PK1, suggesting that this effect was sensitive to the increase in cytosolic calcium. CONCLUSION: Tubular cells may increase the synthesis of glycosaminoglycans to protect from the toxic insult of calcium oxalate crystals and oxalate ions, what could partially limit the lithogenesis.     

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)     

Citrate provides protection against oxalate and calcium oxalate crystal induced oxidative damage to renal epithelium

PURPOSE: Oxalate and calcium oxalate (CaOx) crystals are injurious to renal epithelial cells. The injury is caused by the production of reactive oxygen species (ROS). Citrate is a well-known inhibitor of CaOx crystallization and as such it is one of the major therapeutic agents prescribed. Since citrate increases cellular reduced nicotinamide adenine dinucleotide phosphate and glutathione (GSH), we hypothesized that exogenously administered citrate should act as an antioxidant and protect cells from oxalate induced injury. MATERIALS AND METHODS: We exposed LLC-PK1 and MDCK cells to 500 microM/ml oxalate or 150 mug/cm calcium oxalate crystals for 30, 60 and 180 minutes with or without 3 mg/ml citrate in the medium. We determined cell viability by lactate dehydrogenase release and trypan blue exclusion, ROS involvement by changes in hydrogen peroxide and GSH, and lipid peroxidation by quantifying 8-isoprostane. RESULTS: The presence of citrate was associated with significant decrease in lactate dehydrogenase release (p <0.001) and staining with trypan blue (p <0.05). In addition, there was a significant increase in GSH (p <0.005) and a decrease in the production of hydrogen peroxide (p <0.05) and 8-isoprostane (p <0.0005) secretion into the culture medium when citrate was present in the medium. CONCLUSIONS: Citrate protects cells from oxalate and CaOx crystal induced injury by preventing lipid peroxidation through a decrease in ROS production. The results provide additional data for the beneficial role of citrate therapy for CaOx nephrolithiasis.     

Molecular mechanism of oxalate-induced free radical production and glutathione redox imbalance in renal epithelial cells: effect of antioxidants

BACKGROUND: Peroxidation of renal cells is a critical event in the nucleation and formation of calcium oxalate crystals under hyperoxaluric conditions. We previously demonstrated that oxalate-induced peroxidative injury is one of the major mechanisms in promoting crystal attachment to renal epithelial cells. METHODS: In this study we have demonstrated that the mechanism of oxalate-induced peroxidative injury is through the induction of TGF-beta1 and glutathione (GSH) redox imbalance in LLC-PK1 cells. RESULTS: LLC-PK1, renal epithelial cells exposed to oxalate had significantly higher reactive oxygen species (ROS) production; higher TGF-beta1 levels, as measured by ELISA (1.89 +/- 0.035 fold increase) or Western blot (1.65 +/- 0.01 fold increase); increased malondialdehyde formation; increased LDH release, and loss of cell viability. In addition, oxalate exposure significantly decreased GSH content, glutathione reductase, glucose-6-phosphate dehydrogenase activities, and increased oxidized GSH content. Treatment with vitamin E, neutralizing anti-TGF-beta antibody, or diphenylene iodium, an inhibitor of NAD(P)H oxidase, significantly inhibited oxalate-induced ROS production and prevented peroxidative injury and cytolysis. Vitamin E, catalase, or desferoxamine treatment also significantly restored the oxalate-induced cellular GSH redox status toward the control level, and vitamin E treatment significantly attenuated the oxalate-mediated increase in TGF-beta1 protein in cultured LLC-PK1 cells. CONCLUSIONS: This is the first study to demonstrate that the mechanism of oxalate-induced free radical production in renal tubular epithelial cells is through the activation of NAD(P)H oxidase via cytokine TGF-beta1 induction. These results also provide direct evidence that antioxidant therapy might prevent calcium oxalate nucleation and kidney stone formation by preventing oxalate-mediated peroxidative injury and GSH redox imbalance.     

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.     

Calcium oxalate crystals induces tight junction disruption in distal renal tubular epithelial cells by activating ROS/Akt/p38 MAPK signaling pathway

Tight junction plays important roles in regulating paracellular transports and maintaining cell polarity. Calcium oxalate monohydrate (COM) crystals, the major crystalline composition of kidney stones, have been demonstrated to be able to cause tight junction disruption to accelerate renal cell injury. However, the cellular signaling involved in COM crystal-induced tight junction disruption remains largely to be investigated. In the present study, we proved that COM crystals induced tight junction disruption by activating ROS/Akt/p38 MAPK pathway. Treating Madin–Darby canine kidney (MDCK) cells with COM crystals induced a substantial increasing of ROS generation and activation of Akt that triggered subsequential activation of ASK1 and p38 mitogen-activated protein kinase (MAPK). Western blot revealed a significantly decreased expression of ZO-1 and occludin, two important structural proteins of tight junction. Besides, redistribution and dissociation of ZO-1 were observed by COM crystals treatment. Inhibition of ROS by N-acetyl-l-cysteine (NAC) attenuated the activation of Akt, ASK1, p38 MAPK, and down-regulation of ZO-1 and occludin. The redistribution and dissociation of ZO-1 were also alleviated by NAC treatment. These results indicated that ROS were involved in the regulation of tight junction disruption induced by COM crystals. In addition, the down-regulation of ZO-1 and occludin, the phosphorylation of ASK1 and p38 MAPK were also attenuated by MK-2206, an inhibitor of Akt kinase, implying Akt was involved in the disruption of tight junction upstream of p38 MAPK. Thus, these results suggested that ROS-Akt-p38 MAPK signaling pathway was activated in COM crystal-induced disruption of tight junction in MDCK cells.     

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.     

HIF activation protects from acute kidney injury

The contribution of hypoxia to cisplatin-induced renal tubular injury is controversial. Because the hypoxia-inducible factor (HIF) pathway is a master regulator of adaptation to hypoxia, we measured the effects of cisplatin on HIF accumulation in vitro and in vivo, and tested whether hypoxic preconditioning is protective against cisplatin-induced injury. We found that cisplatin did not stabilize HIF-1alpha protein in vitro or in vivo under normoxic conditions. However, hypoxic preconditioning of cisplatin-treated proximal tubular cells in culture reduced apoptosis in an HIF-1alpha-dependent fashion and increased cell proliferation as measured by BrdU incorporation. In vivo, rats preconditioned with carbon monoxide before cisplatin administration had significantly better renal function than rats kept in normoxic conditions throughout. Moreover, the histomorphological extent of renal damage and tubular apoptosis was reduced by the preconditional treatment. Therefore, development of pharmacologic agents to induce renal HIF might provide a new approach to ameliorate cisplatin-induced nephrotoxicity.