Electrophysiological evidence for the presence of cystic fibrosis transmembrane conductance regulator (CFTR) in mouse sperm

Mammalian sperm must undergo a maturational process, named capacitation, in the female reproductive tract to fertilize the egg. Sperm capacitation is regulated by a cAMP/protein kinase A (PKA) pathway and involves increases in intracellular Ca²⁺, pH, Cl^?, protein tyrosine phosphorylation, and in mouse and some other mammals a membrane potential hyperpolarization. The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl^? channel modulated by cAMP/PKA and ATP, was detected in mammalian sperm and proposed to modulate capacitation. Our whole‐cell patch‐clamp recordings from testicular mouse sperm now reveal a Cl^? selective component to membrane current that is ATP‐dependent, stimulated by cAMP, cGMP, and genistein (a CFTR agonist, at low concentrations), and inhibited by DPC and CFTR_inh‐172, two well‐known CFTR antagonists. Furthermore, the Cl^? current component activated by cAMP and inhibited by CFTR_inh‐172 is absent in recordings on testicular sperm from mice possessing the CFTR ΔF508 loss‐of‐function mutation, indicating that CFTR is responsible for this component. A Cl^? selective like current component displaying CFTR characteristics was also found in wild type epididymal sperm bearing the cytoplasmatic droplet. Capacitated sperm treated with CFTR_inh‐172 undergo a shape change, suggesting that CFTR is involved in cell volume regulation. These findings indicate that functional CFTR channels are present in mouse sperm and their biophysical properties are consistent with their proposed participation in capacitation. J. Cell. Physiol. 228: 590–601, 2013. © 2012 Wiley Periodicals, Inc.     

cAMP/Protein Kinase A Activates Cystic Fibrosis Transmembrane Conductance Regulator for ATP Release from Rat Skeletal Muscle during Low pH or Contractions

We have shown that cystic fibrosis transmembrane conductance regulator (CFTR) is involved in ATP release from skeletal muscle at low pH. These experiments investigate the signal transduction mechanism linking pH depression to CFTR activation and ATP release, and evaluate whether CFTR is involved in ATP release from contracting muscle. Lactic acid treatment elevated interstitial ATP of buffer-perfused muscle and extracellular ATP of L6 myocytes: this ATP release was abolished by the non-specific CFTR inhibitor, glibenclamide, or the specific CFTR inhibitor, CFTR_inh-172, suggesting that CFTR was involved, and by inhibition of lactic acid entry to cells, indicating that intracellular pH depression was required. Muscle contractions significantly elevated interstitial ATP, but CFTR_inh-172 abolished the increase. The cAMP/PKA pathway was involved in the signal transduction pathway for CFTR-regulated ATP release from muscle: forskolin increased CFTR phosphorylation and stimulated ATP release from muscle or myocytes; lactic acid increased intracellular cAMP, pCREB and PKA activity, whereas IBMX enhanced ATP release from myocytes. Inhibition of PKA with KT5720 abolished lactic-acid- or contraction-induced ATP release from muscle. Inhibition of either the Na⁺/H⁺-exchanger (NHE) with amiloride or the Na⁺/Ca²⁺-exchanger (NCX) with SN6 or KB-R7943 abolished lactic-acid- or contraction-induced release of ATP from muscle, suggesting that these exchange proteins may be involved in the activation of CFTR. Our data suggest that CFTR-regulated release contributes to ATP release from contracting muscle in vivo, and that cAMP and PKA are involved in the activation of CFTR during muscle contractions or acidosis; NHE and NCX may be involved in the signal transduction pathway.     

The liver X receptor agonist TO901317 protects mice against cisplatin-induced kidney injury

Liver X receptors are in the nuclear receptor superfamily and are contained in the regulation of lipid and cholesterol metabolism. Besides, liver X receptors are considered crucial regulators of the inflammatory response and innate immunity. The current study evaluates the in vivo effects that the synthetic liver X receptor agonist TO901317 protects against cisplatin-induced kidney injury in mice. Mice received cisplatin administration through a single intraperitoneal injection (20 mg/kg in saline). And then the mice were treated with the TO901317 by daily gavage (10 mg/kg/day) 12 h postcisplatin administration, and cisplatin nephrotoxicity was evaluated. At 72 h after cisplatin treatment, elevated plasma urea and creatinine levels (P < 0.05) were evidenced which indicates the renal dysfunction of the vehicle-treated mice, consistent with tubular necrosis, protein cast, dilation of renal tubules, and desquamation of epithelial cells in renal tubules. In contrast, the severity of renal dysfunction and histological damage was reduced in TO901317 treated mice (P < 0.05). In accordance, circulating tumor necrosis factor alpha levels, renal tumor necrosis factor alpha, p47^phox, gp91^phox, and protein expression levels and COX-2 mRNA, renal monocyte chemoattractant protein 1, VACAM-1 mRNA and intercellular adhesion molecule-1 contents, and renal prostaglandin E₂ amounts, were higher in samples from cisplatin-treated mice in comparison with controls (P < 0.05) but attenuated in the TO901317 treatment group (P < 0.05). Taken together, treatment with the liver X receptor agonist TO901317 ameliorated the inflammatory response and oxidative stress in cisplatin-induced kidney injury in mice.     

Autophagy delays apoptosis in renal tubular epithelial cells in cisplatin cytotoxicity

One of the major side effects of cisplatin chemotherapy is toxic acute kidney injury due to preferential accumulation of cisplatin in renal proximal tubule epithelial cells and the subsequent injury to these cells. Apoptosis is known as a major mechanism of cisplatin-induced cell death in renal tubular cells. We have also recently demonstrated that autophagy induction is an immediate response of renal tubular epithelial cell exposure to cisplatin. Inhibition of cisplatin-induced autophagy blocks the formation of autophagosomes and enhances cisplatin-induced caspase-3, -6, and -7 activation, nuclear fragmentation, and apoptosis. The switch from autophagy to apoptosis by autophagic inhibitors suggests that autophagy induction was responsible for a pre-apoptotic lag phase observed on exposure of renal tubular cells to cisplatin. Our studies provide evidence that autophagy induction in response to cisplatin mounts an adaptive response that suppresses and delays apoptosis. The beneficial effect of autophagy has a potential clinical significance in minimizing or preventing cisplatin nephrotoxicity.

Addedum to: Yang C, Kaushal V, Shah SV, Kaushal GP. Autophagy and apoptosis are associated in cisplatin injury to renal tubular epithelial cell injury. Am J Physiol Renal Physiol 2008; 294:F777-87.     

Lubiprostone activates CFTR, but not ClC-2, via the prostaglandin receptor (EP4)

The goal of this study was to determine the mechanism of lubiprostone activation of epithelial chloride transport. Lubiprostone is a bicyclic fatty acid approved for the treatment of constipation [1]. There is uncertainty, however, as to how lubiprostone increases epithelial chloride transport. Direct stimulation of ClC-2 and CFTR chloride channels as well as stimulation of these channels via the EP₄ receptor has been described [2], [3], [4] and [5]. To better define this mechanism, two-electrode voltage clamp was used to assay Xenopus oocytes expressing ClC-2, with or without co-expression of the EP₄ receptor or β adrenergic receptor (βAR), for changes in conductance elicited by lubiprostone. Oocytes co-expressing CFTR and either βAR or the EP₄ receptor were also studied. In oocytes co-expressing ClC-2 and βAR conductance was stimulated by hyperpolarization and acidic pH (pH = 6), but there was no response to the β adrenergic agonist, isoproterenol. Oocytes expressing ClC-2 only or co-expressing ClC-2 and EP₄ did not respond to the presence of 0.1, 1, or 10 μM lubiprostone in the superperfusate. Oocytes co-expressing CFTR and βAR did not respond to hyperpolarization, acidic pH, or 1 μM lubiprostone. However, conductance was elevated by isoproterenol and inhibited by CFTR_inh172. Co-expression of CFTR and EP₄ resulted in lubiprostone-stimulated conductance, which was also sensitive to CFTR_inh172. The EC₅₀ for lubiprostone mediated CFTR activation was ∼10 nM. These results demonstrate no direct action of lubiprostone on either ClC-2 or CFTR channels expressed in oocytes. However, the results confirm that CFTR can be activated by lubiprostone via the EP₄ receptor in oocytes.     

CFTR inhibition by glibenclamide requires a positive charge in cytoplasmic loop three

The sulfonylurea glibenclamide is widely used as an open-channel blocker of the CFTR chloride channel. Here, we used site-directed mutagenesis to identify glibenclamide site of interaction: a positively charged residue K978, located in the cytoplasmic loop 3. Charge-neutralizing mutations K978A, K978Q, K978S abolished the inhibition of forskolin-activated CFTR chloride current by glibenclamide but not by CFTR_inh-172. The charge-conservative mutation K978R did not alter glibenclamide sensitivity of CFTR current. Mutations of the neighbouring R975 (R975A, R975S, R975Q) did not affect electrophysiological and pharmacological properties of CFTR. No alteration of halide selectivity was observed with any of these CFTR mutant channels. This study identifies a novel potential inhibitor site within the CFTR molecule, and suggests a novel role of cytoplasmic loop three, within the second transmembrane domain of CFTR protein. This work is the first to report on the role of a residue in a cytoplasmic loop in the mechanism of action of the channel blocker glibenclamide.     

Autophagy delays apoptosis in renal tubular epithelial cells in cisplatin cytotoxicity

One of the major side effects of cisplatin chemotherapy is toxic acute kidney injury due to preferential accumulation of cisplatin in renal proximal tubule epithelial cells and the subsequent injury to these cells. Apoptosis is known as a major mechanism of cisplatin-induced cell death in renal tubular cells. We have also recently demonstrated that autophagy induction is an immediate response of renal tubular epithelial cell exposure to cisplatin. Inhibition of cisplatin-induced autophagy blocks the formation of autophagosomes and enhances cisplatin-induced caspase-3, -6, and -7 activation, nuclear fragmentation and apoptosis. The switch from autophagy to apoptosis by autophagic inhibitors suggests that autophagy induction was responsible for a pre-apoptotic lag phase observed on exposure of renal tubular cells to cisplatin. Our studies provide evidence that autophagy induction in response to cisplatin mounts an adaptive response that suppresses and delays apoptosis. The beneficial effect of autophagy has a potential clinical significance in minimizing or preventing cisplatin nephrotoxicity.     

SLC26A9 stimulates CFTR expression and function in human bronchial cell lines

We investigated the possible functional‐ and physical protein‐interactions between two airway Cl^? channels, SLC26A9 and CFTR. Bronchial CFBE41o‐ cell lines expressing CFTR_WT or CFTR_ΔF508 were transduced with SLC26A9. Immunoblots identified a migrating band corresponding to SLC26A9 present in whole‐cell lysates as on apical membrane of cells grown on polarized filters. CFTR levels were increased by the presence of SLC26A9 in both CFTR_WT and CFTR_ΔF508 cell lines. In CFBE41o‐ cells and CFBE41o‐/CFTR_WT cells transduced with SLC26A9, currents associated to the protein expression were not detected. However, the forskolin (FK)‐stimulated currents were enhanced in SLC26A9‐transduced cells compared to control cells. Therefore, the presence of SLC26A9 resulted in an increase in CFTR activity (same % of CFTR_(inh)‐172 or GlyH‐101 inhibition in both groups). In CFBE41o‐/CFTR_ΔF508 cells transduced with SLC26A9 (at 27°C), a current associated to the protein expression was also lacking. FK‐stimulated currents and level of CFTR_(inh)‐172 inhibition were not different in both groups. The presence of SLC26A9 in Xenopus oocytes expressing CFTR also enhanced the FK‐stimulated currents as compared to oocytes expressing CFTR alone. This stimulation was mostly linked to CFTR. An enhancement of FK‐stimulated currents was not found in oocytes co‐expressing SLC26A9 and CFTR_ΔF508. In conclusion, in both protein expression systems used, SLC26A9 stimulates CFTR activity but not that of CFTR_ΔF508. Our co‐immunoprecipitation studies demonstrate a physical interaction between both anion channels. We propose as an alternative hypothesis (not exclusive) to the known SLC26A9‐STAS domain/CFTR interaction, that SLC26A9 favors the biogenesis and/or stabilization of CFTR, leading to stimulated currents. J. Cell. Physiol. 226: 212–223, 2010. © 2010 Wiley‐Liss, Inc.     

CFTR mediates cadmium-induced apoptosis through modulation of ROS level in mouse proximal tubule cells

The aim of this study was to characterize the role of CFTR during Cd²⁺-induced apoptosis. For this purpose primary cultures and cell lines originated from proximal tubules (PCT) of wild-type cftr^+/+ and cftr^?/? mice were used. In cftr^+/+ cells, the application of Cd²⁺ (5 μM) stimulated within 8 min an ERK1/2-activated CFTR-like Cl^? conductance sensitive to CFTR_inh-172. Thereafter Cd²⁺ induced an apoptotic volume decrease (AVD) within 6 h followed by caspase-3 activation and apoptosis. The early increase in CFTR conductance was followed by the activation of volume-sensitive outwardly rectifying (VSOR) Cl^? and TASK2 K⁺ conductances. By contrast, cftr^?/? cells exposed to Cd²⁺ were unable to develop VSOR currents, caspase-3 activity, and AVD process and underwent necrosis. Moreover in cftr^+/+ cells, Cd²⁺ enhanced reactive oxygen species (ROS) production and induced a 50% decrease in total glutathione content (major ROS scavenger in PCT). ROS generation and glutathione decrease depended on the presence of CFTR, since they did not occur in the presence of CFTR_inh-172 or in cftr^?/? cells. Additionally, Cd²⁺ exposure accelerates effluxes of fluorescent glutathione S-conjugate in cftr^+/+ cells. Our data suggest that CFTR could modulate ROS levels to ensure apoptosis during Cd²⁺ exposure by modulating the intracellular content of glutathione.     

Potentiation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl− currents by the chemical solvent tetrahydrofuran

The chemical solvent tetrahydrofuran (THF) increases short-circuit current (I_sc) in renal epithelia endogenously expressing the cystic fibrosis transmembrane conductance regulator (CFTR). To understand how THF increases I_sc, we employed the Ussing chamber and patch-clamp techniques to study cells expressing recombinant human CFTR. THF increased I_sc in Fischer rat thyroid (FRT) epithelia expressing wild-type CFTR with half-maximal effective concentration (K_D) of 134?mM. This THF-induced increase in I_sc was enhanced by forskolin (10 µM), inhibited by the PKA inhibitor H-89 (10 µM) and the thiazolidinone CFTR_inh-172 (10 µM) and attenuated greatly in FRT epithelia expressing the cystic fibrosis mutants F508del- and G551D-CFTR. By contrast, THF (100?mM) was without effect on untransfected FRT epithelia, while other solvents failed to increase I_sc in FRT epithelia expressing wild-type CFTR. In excised inside-out membrane patches, THF (100?mM) potentiated CFTR Cl^? channels open in the presence of ATP (1?mM) alone by increasing the frequency of channel openings without altering their duration. However, following the phosphorylation of CFTR by PKA (75?nM), THF (100?mM) did not potentiate channel activity. Similar results were obtained with the ?R-S660A-CFTR Cl^? channel that is not regulated by PKA-dependent phosphorylation and using 2′deoxy-ATP, which gates wild-type CFTR more effectively than ATP. Our data suggest that THF acts directly on CFTR to potentiate channel gating, but that its efficacy is weak and dependent on the phosphorylation status of CFTR.     

Carvedilol protects against cisplatin-induced oxidative stress, redox state unbalance and apoptosis in rat kidney mitochondria

Cisplatin is a highly effective chemotherapeutic agent which causes severe nephrotoxicity. Studies have suggested that reactive oxygen species, mainly generated in mitochondria, play a central role in cisplatin-induced renal damage. A wide range of antioxidants have been evaluated as possible protective agents against cisplatin-induced nephrotoxicity; however a safe and efficacious compound has not yet been found. The present study is the first to evaluate the protective potential of carvedilol, a beta-blocker with strong antioxidant properties, against the mitochondrial oxidative stress and apoptosis in kidney of rats treated with cisplatin. The following cisplatin-induced toxic effects were prevented by carvedilol: increased plasmatic levels of creatinine and blood urea nitrogen (BUN); lipid peroxidation, oxidation of cardiolipin; oxidation of protein sulfhydryls; depletion of the non-enzymatic antioxidant defense and increased activity of caspase-3. Carvedilol per se did not present any effect on renal mitochondria. It was concluded that carvedilol prevents mitochondrial dysfunction and renal cell death through the protection against the oxidative stress and redox state unbalance induced by cisplatin. The association of carvedilol to cisplatin chemotherapy was suggested as a possible strategy to minimize the nephrotoxicity induced by this antitumor agent.     

Amelioration of cisplatin-induced nephrotoxicity in mice by oral administration of diphenylmethyl selenocyanate

Cisplatin is one of the most potent and active cytotoxic drug in the treatment of cancer. However, side-effects in normal tissues and organs, notably nephrotoxicity in the kidneys, limit the promising efficacy of cisplatin. The present study was designed to ascertain the possible in vivo protective potential of a synthetic organoselenium compound diphenylmethyl selenocyanate (3 mg/kg.b.w.) against the nephrotoxic damage induced by cisplatin (5 mg/kg.b.w. for 5 days) in Swiss albino mice. Treatment with diphenylmethyl selenocyanate markedly reduced cisplatin-induced lipid peroxidation, serum creatinine and blood urea nitrogen levels. Renal antioxidant defense systems, such as glutathione-S-transferase, glutathione peroxidase, superoxide dismutase, catalase, activities and reduced glutathione level, depleted by cisplatin therapy, were restored to normal by the selenium compound. The selenium compound also reduced renal tubular epithelial cell damage, nitric oxide levels and expression of COX-2, and iNOS in kidneys injured by cisplatin. These results demonstrate the protective effect of diphenylmethyl selenocyanate against cisplatin-induced nephrotoxicity in mice.     

Protection against cisplatin-induced nephrotoxicity by Cu(II)2(3,5-diisopropylsalicylate)4

The ability of Cu(II)(2)(3,5-diisopropylsalicylate)(4), CuDIPS, which exhibits superoxide dismutase (SOD)-like activity, to prevent cisplatin-induced nephrotoxicity was examined in rats. Rats were divided into four groups and treated as follows: (i) vehicle control; (ii) cisplatin (16 mg/kg, intraperitoneally); (iii) CuDIPS (10 mg/kg, intraperitoneally); and (iv) cisplatin plus CuDIPS. Rats were sacrificed 3 days post-treatment. Cisplatin alone resulted in significantly increased plasma creatinine and urea. Administration of 10 mg/kg CuDIPS prevented the cisplatin-induced elevation of plasma creatinine and urea and protected against kidney damage. Relative to controls, rats that received cisplatin treatment displayed a decrease of reduced glutathione (GSH) and elevated platinum and thiobarbituric acid reactive substances (TBARS) levels in the kidney. In comparison with controls, activities of antioxidant enzymes (SOD, CAT, GSH-Px and GSH-Rd) were also reduced in the kidney of rats treated with cisplatin. Administration of 10 mg/kg CuDIPS prevented cisplatin-induced alterations in renal platinum, GSH, TBARS, and antioxidant enzyme activities. This study suggests that the protection offered by CuDIPS against cisplatin-induced nephrotoxicity is partly related to maintenance of renal antioxidant systems.     

PSTPIP2 inhibits cisplatin-induced acute kidney injury by suppressing apoptosis of renal tubular epithelial cells

Cisplatin (CP) is an effective chemotherapeutic agent widely used in the treatment of various solid tumours. However, CP nephrotoxicity is an important limitation for CP use; currently, there is no method to ameliorate cisplatin-induced acute kidney injury (AKI). Recently, we identified a specific role of proline–serine–threonine phosphatase-interacting protein 2 (PSTPIP2) in cisplatin-induced AKI. PSTPIP2 was reported to play an important role in a variety of diseases. However, the functions of PSTPIP2 in experimental models of cisplatin-induced AKI have not been extensively studied. The present study demonstrated that cisplatin downregulated the expression of PSTPIP2 in the kidney tissue. Administration of AAV-PSTPIP2 or epithelial cell-specific overexpression of PSTPIP2 reduced cisplatin-induced kidney dysfunction and inhibited apoptosis of renal tubular epithelial cells. Small interfering RNA-based knockdown of PSTPIP2 expression abolished PSTPIP2 regulation of epithelial cell apoptosis in vitro. Histone acetylation may impact gene expression at the epigenetic level, and histone deacetylase (HDAC) inhibitors were reported to prevent cisplatin-induced nephrotoxicity. The UCSC database was used to predict that acetylation of histone H3 at lysine 27 (H3K27ac) induces binding to the PSTPIP2 promoter, and this prediction was validated by a ChIP assay. Interestingly, an HDAC-specific inhibitor (TSA) was sufficient to potently upregulate PSTPIP2 in epithelial cells. Histone acetylation-mediated silencing of PSTPIP2 may contribute to cisplatin nephrotoxicity. PSTPIP2 may serve as a potential therapeutic target in the prevention of cisplatin nephrotoxicity.Subject terms: Cancer, Cancer prevention     

MicroRNA-34a suppresses invasion and metastatic in esophageal squamous cell carcinoma by regulating CD44

Cisplatin is a widely used antineoplastic agent in the treatment of head and neck cancer. However, it is highly nephrotoxic. Oxidative stress is the main mechanism responsible for cisplatin-induced nephrotoxicity. The aim of this study was to characterize cisplatin-induced nephrotoxicity, oxidative stress in peripheral blood mononuclear cells, and the relationship between them. Twenty-four patients were included in the study. Patients had their blood collected prior to cisplatin administration, and 5 and 20 days after initiating therapy, to assess renal function and to determine oxidative stress with MitoSOX?Red, H₂DCF-DA, and Amplex^® Red tests. Renal function was assessed by measuring serum creatinine, creatinine clearance, and blood urea nitrogen (BUN). Serum creatinine and creatinine clearance were used to grade nephrotoxicity using Common Terminology Criteria for Adverse Events (CTCAE) v4.0. Compared to baseline values, the mean BUN and serum creatinine increased 135 and 100%, respectively, 5 days after cisplatin infusion. Mean creatinine clearance showed a 43% decrease compared to baseline value. Non-statistically significant changes in superoxide anion (O ₂ ^?? ), hydrogen peroxide (H₂O₂), and general reactive oxygen species production occurred. A higher production of H₂O₂ was correlated with variation in serum creatinine, and was associated with higher grades for serum creatinine increases and creatinine clearance reductions. Linear regression analyses showed an association between H₂O₂ production and serum creatinine, creatinine clearance, and BUN levels. These results were observed for 5 days following cisplatin administration. In conclusion, H₂O₂ production was significantly related to changes in all renal parameters that were evaluated, following the cisplatin infusion.     

Protection by ebselen against cisplatin-induced nephrotoxicity: Antioxidant system

This study was designed to investigate the cisplatin-induced alteration in renal antioxidant system and the nephroprotection with ebselen. Male Wistar rats were injected with (1) vehicle control; (2) cisplatin; (3) ebselen; and (4) cisplatin plus ebselen. Rats were sacrificed three days post-treatment and plasma as well as kidney were isolated and analyzed. Plasma creatinine increased 598% following cisplatin administration alone which decreased by 158% with ebselen pretreatment. Cisplatin-treated rats showed a depletion of renal glutathione (GSH) levels (52% of control), while cisplatin plus ebselen injected rats had GSH values close to the controls. Antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities decreased 38, 75 and 62% of control, respectively, and malondialdehyde (MDA) levels increased 174% of control following cisplatin administration, which were restored to control levels after ebselen treatment. The renal platinum level did not significantly change with ebselen pretreatment. This study suggests that the protection offered by ebselen against cisplatin-induced nephrotoxicity is partly related to the sparing of antioxidant system.     

Sodium-dependent glucose transporter reduces peroxynitrite and cell injury caused by cisplatin in renal tubular epithelial cells

Cisplatin causes nephropathy accompanied by two types of cell death, necrosis and apoptosis, according to its dosage. The mechanisms of necrosis are still unclear. In this study, we examined how high doses of cisplatin induce cell injury and whether a high affinity sodium-dependent glucose transporter (SGLT1) has a cytoprotective function in renal epithelial LLC-PK₁ cells. Cisplatin decreased in transepithelial electrical resistance (TER) and increased in the number of necrotic dead cells in a time dependent manner. Phloridzin, a potent SGLT1 inhibitor, enhanced both TER decrease and increase of necrotic dead cells caused by cisplatin. Cisplatin increased in the intracellular nitric oxide, superoxide anion and peroxynitrite productions. Phloridzin enhanced the peroxynitrite production caused by cisplatin. The intracellular diffusion of ZO-1 and TER decrease caused by cisplatin were inhibited by N-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor. Protein kinase C was not involved in the cisplatin-induced injury. 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrinato iron (III) and reduced glutathione, peroxynitrite scavengers, inhibited the cisplatin-induced ZO-1 diffusion, TER decrease, and increase of necrotic dead cells. These results suggest that peroxynitrite is a key mediator in the nephrotoxicity caused by high doses of cisplatin. SGLT1 endogenously carries out the cytoprotective function by the reduction of peroxynitrite production.