Regulation of Transporters for Organic Cations by High Glucose.

Endogenous positively charged organic substances, including neurotransmitters and cationic uremic toxins, as well as exogenous organic cations such as the anti-diabetic medication metformin, serve as substrates for organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs). These proteins facilitate their transport across cell membranes. Vectorial transport through the OCT/MATE axis mediates the hepatic and renal excretion of organic cations, regulating their systemic and local concentrations. Organic cation transporters are part of the remote sensing and signaling system, whose activity can be regulated to cope with changes in the composition of extra- and intracellular fluids. Glucose, as a source of energy, can also function as a crucial signaling molecule, regulating gene expression in various organs and tissues. Its concentration in the blood may fluctuate in specific physiological and pathophysiological conditions. In this work, the regulation of the activity of organic cation transporters was measured by incubating human embryonic kidney cells stably expressing human OCT1 (hOCT1), hOCT2, or hMATE1 with high glucose concentrations (16.7 mM). Incubation with this high glucose concentration for 48 h significantly stimulated the activity of hOCT1, hOCT2, and hMATE1 by increasing their maximal velocity (Vmax), but without significantly changing their affinity for the substrates. These effects were independent of changes in osmolarity, as the addition of equimolar concentrations of mannitol did not alter transporter activity. The stimulation of transporter activity was associated with a significant increase in transporter mRNA expression. Inhibition of the mechanistic target of rapamycin (mTOR) kinase with Torin-1 suppressed the transporter stimulation induced by incubation with 16.7 mM glucose. Focusing on hOCT2, it was shown that incubation with 16.7 mM glucose increased hOCT2 protein expression in the plasma membrane. Interestingly, an apparent trend towards higher hOCT2 mRNA expression was observed in kidneys from diabetic patients, a pathology characterized by high serum glucose levels. Due to the small number of samples from diabetic patients (three), this observation must be interpreted with caution. In conclusion, incubation for 48 h with a high glucose concentration of 16.7 mM stimulated the activity and expression of organic cation transporters compared to those measured in the presence of 5.6 mM glucose. This stimulation by a diabetic environment could increase cellular uptake of the anti-diabetic drug metformin and increase renal tubular secretion of organic cations in an early stage of diabetes.

An integrative approach to cisplatin chronic toxicities in mice reveals importance of organic cation-transporter-dependent protein networks for renoprotection.

Cisplatin (CDDP) is one of the most important chemotherapeutic drugs in modern oncology. However, its use is limited by severe toxicities, which impair life quality after cancer. Here, we investigated the role of organic cation transporters (OCT) in mediating toxicities associated with chronic (twice the week for 4 weeks) low-dose (4 mg/kg body weight) CDDP treatment (resembling therapeutic protocols in patients) of wild-type (WT) mice and mice with OCT genetic deletion (OCT1/2-/-). Functional and molecular analysis showed that OCT1/2-/- mice are partially protected from CDDP-induced nephrotoxicity and peripheral neurotoxicity, whereas ototoxicity was not detectable. Surprisingly, proteomic analysis of the kidneys demonstrated that genetic deletion of OCT1/2 itself was associated with significant changes in expression of proinflammatory and profibrotic proteins which are part of an OCT-associated protein network. This signature directly regulated by OCT consisted of three classes of proteins, viz., profibrotic proteins, proinflammatory proteins, and nutrient sensing molecules. Consistent with functional protection, CDDP-induced proteome changes were more severe in WT mice than in OCT1/2-/- mice. Laser ablation-inductively coupled plasma-mass spectrometry analysis demonstrated that the presence of OCT was not associated with higher renal platinum concentrations. Taken together, these results redefine the role of OCT from passive membrane transporters to active modulators of cell signaling in the kidney.

Tetraspanin CD63 controls basolateral sorting of organic cation transporter 2 in renal proximal tubules.

CD63 is a ubiquitously expressed member of the tetraspanin superfamily. Using a mating-based split-ubiquitin-yeast 2-hybrid system, pull-down experiments, total internal reflection fluorescence microscopy, Förster resonance energy transfer, and biotinylation assays, we found that CD63 interacts with human organic cation transporter 2 (hOCT2), which transports endogenous and exogenous substrates, such as neurotransmitters and drugs in several epithelial cells. CD63 overexpression affects cellular localization of hOCT2 expressed in human embryonic kidney (HEK)293 cells. Studies with CD63-knockout mice indicate that in renal proximal tubules, CD63 determines the insertion of the mouse ortholog of the transporter into the proper membrane domain and mediates transporter regulation by trafficking processes. In polarized Madin-Darby kidney canine kidney (MDCK) epithelial cells, CD63 and hOCT2 colocalize with the small GTPase Rab4, which controls the rapid recycling from sorting endosomes back to the cell surface. Suitable negative and positive control experiments were performed for each experimental approach. Empty vector transfected cells and wild-type mice were used as control. CD63 seems to play a role in the recycling of hOCT2 from endosomes to the basolateral membrane in polarized epithelia. These data indicate that CD63 has a previously uncharacterized function in regulating trafficking of specific membrane proteins in polarized cells.-Schulze, U., Brast, S., Grabner, A., Albiker, C., Snieder, B., Holle, S., Schlatter, E., Schröter, R., Pavenstädt, H., Herrmann, E., Lambert, C., Spoden, G. A., Florin, L., Saftig, P., Ciarimboli, G. Tetraspanin CD63 controls basolateral sorting of organic cation transporter 2 in renal proximal tubules.

Mitigation of acute kidney injury by cell-cycle inhibitors that suppress both CDK4/6 and OCT2 functions.

Acute kidney injury (AKI) is a potentially fatal syndrome characterized by a rapid decline in kidney function caused by ischemic or toxic injury to renal tubular cells. The widely used chemotherapy drug cisplatin accumulates preferentially in the renal tubular cells and is a frequent cause of drug-induced AKI. During the development of AKI the quiescent tubular cells reenter the cell cycle. Strategies that block cell-cycle progression ameliorate kidney injury, possibly by averting cell division in the presence of extensive DNA damage. However, the early signaling events that lead to cell-cycle activation during AKI are not known. In the current study, using mouse models of cisplatin nephrotoxicity, we show that the G1/S-regulating cyclin-dependent kinase 4/6 (CDK4/6) pathway is activated in parallel with renal cell-cycle entry but before the development of AKI. Targeted inhibition of CDK4/6 pathway by small-molecule inhibitors palbociclib (PD-0332991) and ribociclib (LEE011) resulted in inhibition of cell-cycle progression, amelioration of kidney injury, and improved overall survival. Of additional significance, these compounds were found to be potent inhibitors of organic cation transporter 2 (OCT2), which contributes to the cellular accumulation of cisplatin and subsequent kidney injury. The unique cell-cycle and OCT2-targeting activities of palbociclib and LEE011, combined with their potential for clinical translation, support their further exploration as therapeutic candidates for prevention of AKI.

GlucoCEST magnetic resonance imaging in vivo may be diagnostic of acute renal allograft rejection.

Acute cellular renal allograft rejection (AR) frequently occurs after kidney transplantations. It is a sterile T-cell mediated inflammation leading to increased local glucose metabolism. Here we demonstrate in an allogeneic model of Brown Norway rat kidneys transplanted into uninephrectomized Lewis rats the successful implementation of the recently developed glucose chemical exchange saturation transfer (glucoCEST) magnetic resonance imaging. This technique is a novel method to assess and differentiate AR. Renal allografts undergoing AR showed significantly increased glucoCEST contrast ratios of cortex to medulla of 1.61 compared to healthy controls (1.02), syngeneic Lewis kidney to Lewis rat transplants without rejection (0.92), kidneys with ischemia reperfusion injury (0.99) and kidneys affected by cyclosporine A toxicity (1.10). Receiver operating characteristic curve analysis showed an area under the curve value of 0.92, and the glucoCEST contrast ratio predicted AR with a sensitivity of 100% and a specificity of 69% at a threshold level over 1.08. In defined animal models of kidney injuries, the glucoCEST contrast ratios of cortex to medulla correlated positively with mRNA expression levels of T-cell markers (CD3, CD4, CD8a/b), but did not correlate to impaired renal perfusion. Thus, the glucoCEST parameter may be valuable for the assessment and follow up treatment of AR.

The kidney-specific expression of genes can be modulated by the extracellular osmolality.

With this study, we wanted to prove the hypothesis that the unique extracellular osmolality within the renal medulla modulates a specific gene expression pattern. The physiologic functions of the kidneys are mediated by the segment-specific expression of key proteins. So far, we have limited knowledge about the mechanisms that control this gene expression pattern. The hyperosmolality in the renal medullary interstitium is of major importance as a driving force for urine concentration. We made use of primarily cultured rat renal inner medullary collecting-duct cells and microarray analysis to identify genes affected by the environmental osmolality of the culture medium. We identified hundreds of genes that were either induced or repressed in expression by hyperosmolality in a time- and osmolality-dependent fashion. Further analysis demonstrated that many of them, physiologically, showed a kidney- and even collecting-duct-specific expression, including secreted proteins, kinases, and transcription factors. On the other hand, we identified factors, down-regulated in expression, that have a diuretic effect. In conclusion, the kidney is the only organ that has such a hyperosmotic environment, and study provides an excellent method for controlling tissue-specific gene expression.-Schulze Blasum, B., Schröter, R., Neugebauer, U., Hofschröer, V., Pavenstädt, H., Ciarimboli, G., Schlatter E., Edemir, B. The kidney-specific expression of genes can be modulated by the extracellular osmolality.

Mouse organic cation transporter 1 determines properties and regulation of basolateral organic cation transport in renal proximal tubules.

The proximal tubule of mouse kidney expresses mouse organic cation transporter 1 (mOCT1), mOCT2, and much less mOCT3. Therefore, mOCT-mediated transport across the basolateral membrane of proximal tubules reflects properties of at least mOCT1 and mOCT2. Here, we unraveled substrate affinities and modulation of transport activity by acute regulation by protein kinases on mOCT1 and mOCT2 separately and compared these findings with those from isolated proximal tubules of male and female mOCT2−/− mice. These data are also compared to our recent reports on isolated tubules from wild-type and mOCT1/2 double knockout (mOCT1/2−/−) mice. OCT-mediated transport in proximal tubules of mOCT2−/− mice was only 20 % lower compared to those isolated from wild-type mice. While mOCT1 was regulated by all five pathways examined [protein kinase A (PKA), protein kinase C (PKC), p56lck, phosphoinositide 3-kinase (PI3K), and calmodulin (CaM)], mOCT2 activity was modulated by PKA, p56lck, and CaM only, however, in the same direction. As mOCT-mediated transport across the basolateral membrane of mOCT2−/− mice expressing only mOCT1 and to a small amount mOCT3 was identical to that observed for tubules isolated from wild-type mice and to that observed for human embryonic kidney 293 (HEK293) cells stably expressing mOCT1, mOCT1 represents the relevant paralog for OCT-dependent organic cation transport in the mouse kidney. Gender does not play a major role in expression and activity of renal OCT-mediated transport in the mouse. Properties of mouse OCT considerably differ from those of rat or human origin, and thus, observations made in these rodents cannot directly be transferred to the human situation

The organic cation transporter 3 (OCT3) as molecular target of psychotropic drugs: transport characteristics and acute regulation of cloned murine OCT3.

The organic cation transporter 3 (OCT3) is a widely expressed transporter for endogenous and exogenous organic cations. Of particular interest is OCT3 expression and function in the brain, where it plays a role in serotonin clearance and influences mood and behavior. Protein kinase signaling mediates rapid modulation of cerebral processes, but little is known about acute regulation of OCT3 by protein kinases. Therefore, we cloned mouse OCT3 (mOCT3) and generated a human embryonic kidney cell line stably expressing the transporter to study transport characteristics, acute regulation by protein kinases, and interaction with psychotropic drugs. Uptake measurement was performed using the fluorescent cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+), 1 µM) as a substrate. The translational value of these findings was determined by comparing results obtained with cloned mouse and human OCT3. mOCT3-mediated transport is membrane potential dependent and pH independent. ASP(+) uptake by mOCT3 and human OCT3 (hOCT3) was efficiently inhibited by 1-methyl-4-phenylpyridinium, tetrapentylammonium (TPA(+)), corticosterone, serotonin, and histamine and by the drugs ketamine, fluoxetine, and diazepam. The half maximal inhibitory concentrations of mOCT3 and hOCT3 for TPA(+), serotonin, diazepam, and ketamine are significantly different. Diazepam is a non-transported inhibitor. Furthermore, the activities of mOCT3 and hOCT3 are acutely regulated by the p56 (lck) tyrosine kinase by decreasing their V max. Studies with freshly isolated renal proximal tubules from mOCT1/2(-/-) mice, in which mOCT3 is the only OCT present, confirmed this regulation pathway. Only the activity of hOCT3 is regulated by calmodulin. These findings suggest that even though many transport properties of mOCT3 and hOCT3 are similar, there are also species-specific aspects of OCT3 function.

The calcium-activated chloride channel Anoctamin 1 contributes to the regulation of renal function.

The role of calcium-activated chloride channels for renal function is unknown. By immunohistochemistry we demonstrate dominant expression of the recently identified calcium-activated chloride channels, Anoctamin 1 (Ano1, TMEM16A) in human and mouse proximal tubular epithelial (PTE) cells, with some expression in podocytes and other tubular segments. Ano1-null mice had proteinuria and numerous large reabsorption vesicles in PTE cells. Selective knockout of Ano1 in podocytes (Ano1-/-/Nphs2-Cre) did not impair renal function, whereas tubular knockout in Ano1-/-/Ksp-Cre mice increased urine protein excretion and decreased urine electrolyte concentrations. Purinergic stimulation activated calcium-dependent chloride currents in isolated proximal tubule epithelial cells from wild-type but not from Ano1-/-/Ksp-Cre mice. Ano1 currents were activated by acidic pH, suggesting parallel stimulation of Ano1 chloride secretion with activation of the proton-ATPase. Lack of calcium-dependent chloride secretion in cells from Ano1-/-/Ksp-Cre mice was paralleled by attenuated proton secretion and reduced endosomal acidification, which compromised proximal tubular albumin uptake. Tubular knockout of Ano1 enhanced serum renin and aldosterone concentrations, probably leading to enhanced compensatory distal tubular reabsorption, thus maintaining normal blood pressure levels. Thus, Ano1 has a role in proximal tubular proton secretion and protein reabsorption. The results correspond to regulation of the proton-ATPase by the Ano1-homolog Ist2 in yeast.

The cysteines of the extracellular loop are crucial for trafficking of human organic cation transporter 2 to the plasma membrane and are involved in oligomerization.

Human organic cation transporter 2 (hOCT2) is involved in transport of many endogenous and exogenous organic cations, mainly in kidney and brain cells. Because the quaternary structure of transmembrane proteins plays an essential role for their cellular trafficking and function, we investigated whether hOCT2 forms oligomeric complexes, and if so, which part of the transporter is involved in the oligomerization. A yeast 2-hybrid mating-based split-ubiquitin system (mbSUS), fluorescence resonance energy transfer, Western blot analysis, cross-linking experiments, immunofluorescence, and uptake measurements of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium were applied to human embryonic kidney 293 (HEK293) cells transfected with hOCT2 and partly also to freshly isolated human proximal tubules. The role of cysteines for oligomerization and trafficking of the transporter to the plasma membranes was investigated in cysteine mutants of hOCT2. hOCT2 formed oligomers both in the HEK293 expression system and in native human kidneys. The cysteines of the large extracellular loop are important to enable correct folding, oligomeric assembly, and plasma membrane insertion of hOCT2. Mutation of the first and the last cysteines of the loop at positions 51 and 143 abolished oligomer formation. Thus, the cysteines of the extracellular loop are important for correct trafficking of the transporter to the plasma membrane and for its oligomerization.

Kidney transplantation down-regulates expression of organic cation transporters, which translocate ß-blockers and fluoroquinolones.

Kidney transplanted patients are often treated with immunosuppressive, antihypertensive, and antibiotic drugs such as cyclosporine A (CsA), ß-blockers, and fluoroquinolones, respectively. Organic cation transporters (OCT) expressed in the basolateral membrane of proximal tubules represent an important drug excretion route. In this work, the renal expression of OCT after syngeneic and allogeneic kidney transplantation in rats with or without CsA immunosuppression was studied. Moreover, the interactions of CsA, ß-blockers (pindolol/atenolol), and fluoroquinolones (ofloxacin/norfloxacin) with rOCT1, rOCT2, hOCT1, and hOCT2 in stably transfected HEK293-cells were studied. Kidney transplantation was associated with reduced expression of rOCT1, while rOCT2 showed only reduced expression after allogeneic transplantation. All drugs interacted subtype- and species-dependently with OCT. However, only atenolol, pindolol, and ofloxacin were transported by hOCT2, the main OCT in human kidneys. While CsA is not an OCT substrate, it exerts a short-term effect on OCT activity, changing their affinity for some substrates. In conclusion, appropriate drug dosing in transplanted patients is difficult partly because OCT are down-regulated and because concomitant CsA treatment may influence the affinity of the transporters. Moreover, drug-drug competition at the transporter can also alter drug excretion rate.

Regulation of organic cation transport in isolated mouse proximal tubules involves complex changes in protein trafficking and substrate affinity.

This study characterizes the complex mechanisms of acute regulation of organic cation (OC) transport across the basolateral membrane of isolated mouse proximal tubules. The fluorescent substrate ASP(+), 4-(-4-(dimethylamino) styryl-N-methylpyridinium, was used to quantify OC transport using a microtiter plate based fluorescence reader method. Inhibition of phosphatidylinositol-3-kinase, of p56 tyrosine kinase, stimulation of PKC and inhibition of PKA reduced ASP(+)-uptake. ASP(+)-kinetic and Dixon plot analyses revealed effects on transporter trafficking as explanation for the inhibition of ASP(+)-uptake by these pathways. Angiotensin II (AII) via stimulation of Ca(2+)/calmodulin increased ASP(+)-uptake. This effect aroused from an altered substrate affinity. Bafilomycin, an inhibitor of the vacuolar H(+)-ATPase and thus endosomal and lysosomal function, reduced ASP(+)-uptake, but did not prevent the AII effect on ASP(+)-uptake. Bafilomycin seemed to diminish the recycling rate of OCTs and hence to reduce the amount of transporters in the membrane. AII via Ca(2+)/calmodulin increased the substrate affinity of the remaining OCTs. The involvement of the cytoskeleton in acute regulation of OCTs became obvious as colchicine induced inhibition of microtubule polymerisation reduced ASP(+)-uptake. Acute regulation of mouse OCTs mostly involves changes in trafficking from and to the plasma membrane and only in the case of AII/CaM changes in substrate affinity.

Mechanisms of cell polarity and aquaporin sorting in the nephron.

The kidneys participate in whole-body homeostasis, regulating acid-base balance, electrolyte concentrations, extracellular fluid volume, and regulation of blood pressure. Many of the kidney's functions are accomplished by relatively simple mechanisms of filtration, reabsorption, and secretion, which take place in the nephron. The kidneys generate 140-180 l of primary urine per day, while reabsorbing a large percentage, allowing for only the excretion of approximately 2 l of urine. Within the nephron, the majority of the filtered water and solutes are reabsorbed. This is mainly facilitated by specialized transporters and channels which are localized at different segments of the nephron and asymmetrically localized within the polarized epithelial cells. The asymmetric localization of these transporters and channels is essential for the physiological tasks of the renal tissues. One family of these proteins are the water-permeable aquaporins which are selectively expressed in cells along the nephron and localized at different compartments. Here, we discuss potential molecular links between mechanisms involved in the establishment of cell polarity and the members of the aquaporin family. In the first part of this review, we will focus on aspects of apical cell polarity. In the second part, we will review the motifs identified so far that are involved in aquaporin sorting and point out potential molecular links.

Different effects of CsA and FK506 on aquaporin-2 abundance in rat primary cultured collecting duct cells.

Calcineurin (Cn) inhibitors (CnI) such as cyclosporine A (CsA) and FK506 are nephrotoxic immunosuppressant drugs, which decrease tubular function. Here, we examined the direct effect of CnI on aquaporin-2 (AQP2) expression in rat primary cultured inner medullary collecting duct cells. CsA (0.5-5 µM) but not FK 506 (0.01-1 µM) decreased expression of AQP2 protein and messenger RNA (mRNA) in a concentration and time dependent manner, without affecting mRNA stability. This effect was observed despite similar inhibition of Cn activity by both CnI, thereby suggesting that the CsA-dependent decrease in AQP2 expression was Cn independent. Another inhibitor of cyclophilin A, the primary intracellular target of CsA, had no effect on AQP2 expression. In order to investigate the mechanism of decreased AQP2 transcription, we studied activation status of two suggested transcriptional regulators of AQP2, cAMP-responsive element binding protein (CREB), and tonicity enhancer binding protein (TonEBP). Localization of TonEBP, as well as TonEBP-mediated gene transcription, was not affected by CsA. Phosphorylation of CREB at an activating phosphorylation site (S133) was decreased by CsA, but not by FK506. However, both CnI did not affect cellular cAMP levels. We show that CsA decreases transcription of AQP2, a process that is in part independent of Cn or cyclophilin A and suggests dependence on decreased activity of CREB.

Properties and regulation of organic cation transport in freshly isolated mouse proximal tubules analyzed with a fluorescence reader-based method.

The main elimination site of organic cations (OCs) is the renal proximal tubule (PT). OC transporters (OCT) accept endogenous and exogenous substances and xenobiotics. As transgenic mouse models are increasingly used in translational medicine, functional properties with special focus on regulation of OCT of isolated mouse PTs were studied with a new fluorescence reader-based method, which allows studying larger numbers of tubules per kidney. OC transport across the basolateral membrane of PTs from male mice was measured as initial uptake of the fluorescent dye 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP). A microtiter plate fluorescence reader was used to semi-automatically analyze OC transport in freshly isolated tubules. Relative mRNA expression of OCT1/OCT2/OCT3 in PTs was 1/0.3/0.01 and did not vary from S1 to S3 segments. ASP was transported by PTs with a K (m) of 6 µM. It was inhibited by TEA, TPA, or cimetidine (IC(50)=5, 19, or 53 µM, respectively). Angiotensin II stimulated ASP uptake (+63%), while stimulation of PKC reduced (-37%) OC transport. Inhibition of p56(lck) tyrosine kinase (-60%), of PI3K (-36%), of Ca(2+)/calmodulin (-25%), or of PKA (-33%) reduced OC transport. In PTs from OCT1/2(-/-) mice ASP uptake was reduced to ~20%. Using this fluorescence reader-based method, we report substrate specificities and a complex pattern of acute regulation of OC transport in isolated mouse PTs. Compared to isolated human PTs or rat and human OCT isoforms expressed in HEK293-cells, OC transport across the basolateral membrane of freshly isolated mouse PTs shows similarities but also specific differences.

Organic cation transporter 2 mediates cisplatin-induced oto- and nephrotoxicity and is a target for protective interventions.

The use of the effective antineoplastic agent cisplatin is limited by its serious side effects, such as oto- and nephrotoxicity. Ototoxicity is a problem of special importance in children, because deafness hampers their language and psychosocial development. Recently, organic cation transporters (OCTs) were identified in vitro as cellular uptake mechanisms for cisplatin. In the present study, we investigated in an in vivo model the role of OCTs in the development of cisplatin oto- and nephrotoxicity. The functional effects of cisplatin treatment on kidney (24 hours excretion of glucose, water, and protein) and hearing (auditory brainstem response) were studied in wild-type and OCT1/2 double-knockout (KO) mice. No sign of ototoxicity and only mild nephrotoxicity were observed after cisplatin treatment of knockout mice. Comedication of wild-type mice with cisplatin and the organic cation cimetidine protected from ototoxicity and partly from nephrotoxicity. For the first time we showed that OCT2 is expressed in hair cells of the cochlea. Furthermore, cisplatin-sensitive cell lines from pediatric tumors showed no expression of mRNA for OCTs, indicating the feasibility of therapeutic approaches aimed to reduce cisplatin toxicities by competing OCT2-mediated cisplatin uptake in renal proximal tubular and cochlear hair cells. These findings are very important to establish chemotherapeutical protocols aimed to maximize the antineoplastic effect of cisplatin while reducing the risk of toxicities.

New clues for nephrotoxicity induced by ifosfamide: preferential renal uptake via the human organic cation transporter 2.

Anticancer treatment with ifosfamide but not with its structural isomer cyclophosphamide is associated with development of renal Fanconi syndrome leading to diminished growth in children and bone problems in adults. Since both cytotoxics share the same principal metabolites, we investigated whether a specific renal uptake of ifosfamide is the basis for this differential effect. First we studied the interaction of these cytotoxics using cells transfected with organic anion or cation transporters and freshly isolated murine and human proximal tubules with appropriate tracers. Next we determined changes in membrane voltage in proximal tubular cells to understand their differentiated nephrotoxicity. Ifosfamide but not cyclophosphamide was significantly transported into cells expressing human organic cation transporter 2 (hOCT2) while both did not interact with organic anion transporters. This points toward a specific interaction of ifosfamide with hOCT2, which is the main OCT isoform in human kidney. In isolated human proximal tubules ifosfamide also interacted with organic cation transport. This interaction was also seen in isolated mouse proximal tubules; however, it was absent in tubules from OCT-deficient mice, illustrating the biological importance of this selective transport. Ifosfamide decreased the viability of cells expressing hOCT2, but not that of control cells. Coadministration of cimetidine, a known competitive substrate of hOCT2, completely prevented this ifosfamide-induced toxicity. Finally, ifosfamide but not cyclophosphamide depolarized proximal tubular cells. We propose that the nephrotoxicity of ifosfamide is due to its selective uptake by hOCT2 into renal proximal tubular cells, and that coadministration of cimetidine may be used to prevent ifosfamide-induced nephrotoxicity.

Unexpected localization of AQP3 and AQP4 induced by migration of primary cultured IMCD cells.

Aquaporin-2-4 (AQP) are expressed in the principal cells of the renal collecting duct (CD). Beside their role in water transport across membranes, several studies showed that AQPs can influence the migration of cells. It is unknown whether this also applies for renal CD cells. Another fact is that the expression of these AQPs is highly modulated by the external osmolality. Here we analyzed the localization of AQP2-4 in primary cultured renal inner medullary CD (IMCD) cells and how osmolality influences the migration behavior of these cells. The primary IMCD cells showed a collective migration behavior and there were no differences in the migration speed between cells cultivated either at 300 or 600 mosmol/kg. Acute increase from 300 to 600 mosmol/kg led to a marked reduction and vice versa an acute decrease from 600 to 300 mosmol/kg to a marked increase in migration speed. Interestingly, none of the analyzed AQPs were localized at the leading edge. While AQP3 disappeared within the first 2-3 rows of cells, AQP4 was enriched at the rear end. Further analysis indicated that migration induced lysosomal degradation of AQP3. This could be prevented by activation of the protein kinase A, inducing localization of AQP3 and AQP2 at the leading edge and increasing the migration speed.

Cyclosporin-A induced toxicity in rat renal collecting duct cells: interference with enhanced hypertonicity induced apoptosis.

BACKGROUND/AIMS: Rat renal inner medullary collecting duct (IMCD) cells are physiologically exposed to a wide range of ambient tonicity. To maintain their function upon changes in osmolality, IMCD cells induce expression of osmoprotective and antiapoptotic genes, mainly mediated by the transcription factor Tonicity Enhancer Binding Protein (TonEBP). Some drugs like Cyclosporin-A (CsA) are discussed to interfere with the activity of TonEBP and thereby mediate their nephrotoxic effects. The aim of our study was to further understand CsA toxicity during elevation of ambient osmolality. METHODS: First we examined cytotoxicity of CsA in IMCD exposed to elevated tonicity. Employing microarray analysis of gene expression, real-time PCR and immunoassays, we scrutinized pathways contributing to this effect. RESULTS: We show that in IMCD cells CsA but not FK506 increases apoptosis upon an increase in tonicity. This effect is independent of cellular TonEBP localization or activity and reactive oxygen species. Microarray studies revealed marked quantitative differences in gene expression. Functional analysis showed overrepresentation of genes associated with cell death in presence of CsA. This correlated with increased mRNA expression of genes associated with the death receptor pathway and detection of TNFα in culture medium of cells treated with CsA. CONCLUSION: Our results show that CsA cytotoxicity is induced under elevated ambient osmolality and that death receptor signaling probably contributes to CsA cytotoxicity.

IF/TA-related metabolic changes--proteome analysis of rat renal allografts.

BACKGROUND: Chronic allograft nephropathy, now more specifically termed interstitial fibrosis and tubular atrophy without evidence of any specific aetiology (IF/TA), is still an important cause of late graft loss. There is no effective therapy for IF/TA, in part due to the disease's multifactorial nature and its incompletely understood pathogenesis. METHODS: We used a differential in-gel electrophoresis and mass spectrometry technique to study IF/TA in a renal transplantation model. Dark Agouti (DA) kidneys were allogeneically transplanted to Wistar-Furth (DA-WF, aTX) rats. Syngeneic grafts (DA-DA, sTX) served as controls. Nine weeks after transplantation, blood pressure, renal function and electrolytes were studied, in addition to real-time PCR, western blot analysis, histology and immunohistochemistry. RESULTS: In contrast to sTX, the aTX developed IF/TA-dependent renal damage. Ten differentially regulated proteins were identified by 2D gel analysis and mass spectrometry, whereupon five proteins are mainly related to oxidative stress (aldo-keto reductase, peroxiredoxin-1, NAD(+)-dependent isocitrate dehydrogenase, iron-responsive element-binding protein-1 and serum albumin), two participate in cytoskeleton organization (l-plastin and ezrin) and three are assigned to metabolic functions (creatine kinase, ornithine aminotransferase and fructose-1,6-bisphosphatase). CONCLUSION: The proteins related to IF/TA and involved in oxidative stress, cytoskeleton organization and metabolic functions may correspond with novel therapeutic targets.