Induction of apoptosis in cultured human proximal tubule cells by fumonisins and fumonisin metabolites.

Fumonisin B1 (FB1) causes apoptosis in a variety of cell types and tissues but the apoptotic potential of other fumonisins and fumonisin metabolites has not been determined and the underlying mechanisms are not completely understood. In our studies we exposed human proximal tubule-derived cells (IHKE cells) to FB1, fumonisin B2 (FB2), fumonisin B3 (FB3), hydrolyzed fumonisin B1 (HFB1) and N-palmitoyl-hydrolyzed fumonisin B1 (N-Pal-HFB1) and investigated caspase-3 activation, chromatin condensation and DNA fragmentation. Exposure to 10 micromol/L FB1 for 24 h led to a significant increase in caspase-3 activity, chromatin condensation and to DNA fragmentation. All other tested compounds did not show any significant activation of caspase-3 activity nor chromatin condensation and DNA-fragmentation. Furthermore, we examined if a sphinganine accumulation is correlated with an induction of apoptosis in IHKE cells. Therefore we used a liquid chromatography/electrospray ionization-mass spectrometry(LC/ESI-MS)-method using phytosphingosine as an internal standard to determine sphinganine and sphingosine concentrations in IHKE cells. Whereas a significant increase of sphinganine (up to 7000% compared to control cells) was observed with all fumonisin-derivates, sphingosine levels nearly remained unchanged indicating that all substrates inhibited ceramide synthase effectively. These results demonstrate that all compounds let to increased sphinganine levels in IHKE cells but only FB1 was able to induce apoptosis. We conclude that the inhibition of the ceramide synthase is not per se a predictor whether or not fumonisins induce apoptosis.

Programmed cell death by interaction of ochratoxin A with other nephrotoxins.

We determined the effects of co-exposure of OTA and other (potentially) nephrotoxic substances on renal tubule cells (IHKE, from the proximal tubule; MDCK-C7 cells, representing principal cells of the collecting duct) by measuring the activation of caspase-3, an enzyme with key roles in the process of programmed cell death or apoptosis.Substances used in combination with OTA included: cadmium, a known nephrotoxin with apoptotic effects, cisplatin (anticancer drug, also a known nephrotoxin), cyclosporin A (an immunosuppresive agent), H2O2 (generated during oxidative cell stress), amphotericin B (antifungal agent), and fumonisin B1. Because of the great number of possible combinations, a method based on the 96-well format was developed which allowed a fast and cost-efficient succesive measurement of caspase-3 activity and protein content in one well as well as necrotic effects in parallel.We found that cells responded differently to the various combinations: for example, 1 µM OTA combined with 100 µM cisplatin had an antagonizing effect on caspase-3 activation in IHKE cells but potentiating effects in MDCK-C7. DNA ladder formation confirmed the results. Necrotic effects were very small and additive. We conclude that the co-exposure of renal cells to OTA with other substances can enhance or reduce the apoptotic potential of one substance alone depending on the substance and on the cell line investigated. A "harmless" substance can thus convert to a potent cell toxic substance when combined with OTA or other mycotoxins.These first findings show the necessity to further investigate the combined effects of OTA or other mycotoxins with other substances or of mycotoxins with each other.

Liquid chromatography/electrospray ionisation-mass spectrometry method for the quantification of sphingosine and sphinganine in cell cultures exposed to fumonisins.

Fumonisins, mycotoxins produced by Fusarium verticillioides, are potent inhibitors of the de novo sphingolipid biosynthesis via inhibition of the key enzyme ceramide synthase. The cellular response to a fumonisin exposure is obvious as an alteration of the ratio of the sphingoid bases sphingosine (SO) and sphinganine (SA). We developed a new column liquid chromatography/electrospray ionisation-mass spectrometry (LC-ESI-MS) method for the rapid, simultaneous and quantitative determination of these bases in cell cultures of immortalised human kidney epithelial cells (IHKE cells). For sample preparation, cell lysates were only diluted, centrifuged and directly used for LC-MS measurements. Quantification was carried out using phytosphingosine (PSO) as an internal standard. Detecting the protonated molecule [M+H](+) signals of SO (m/z 300) and SA (m/z 302) in the selected ion monitoring (SIM) mode, detection limits of 10 pg for SO (signal-to-noise ratio S/N=3:1) and 25 pg for SA (S/N=3:1) were established. The average recovery for SO and SA was higher than 90% for control IHKE-cells, respectively. The developed LC-ESI-MS method allows the sensitive, selective and rapid monitoring of sphingosine and sphinganine in cell matrices with a drastically reduced time for sample preparation.

Determination of basolateral Na(+)/H(+) exchange activity in MDCK cells using a multiwell-multilabel reader.

Na(+)/H(+) exchangers (NHE) are important membrane transport proteins involved in transepithelial transport and cellular pH homeostasis. NHE-1, important for cellular pH and volume homeostasis, is expressed in the basolateral membrane of epithelial cells. We evaluated the use of a multiwell-multilabel reader to investigate basolateral NHE-1 in confluent MDCK cells and compared the results with data obtained using an imaging system equipped with a filter perfusion system. Using the multiwell-multilabel reader we obtained virtually the same values for steady-state pH and NHE-1 activity under control conditions compared to the imaging system. With both setups Na(+)-dependent pH recovery after an acid load occurred virtually only after basolateral addition of Na(+). Furthermore, Na(+)-dependent pH recovery was reduced by >80% in the presence of the NHE-1 inhibitor HOE642. In addition, we detected an almost identical increase of NHE-1 activity with the two methods after stimulation of protein kinase C using phorbol myristate acetate. In summary, our data indicate that multiwell-multilabel readers are suitable to investigate physiology and regulation of basolateral NHE. Thus, multiwell-multilabel readers offer a valid and convenient alternative to investigate basolateral transporters.

Rapid activation of Na+/H+-exchange in MDCK cells by aldosterone involves MAP-kinase ERK1/2.

The mineralocorticoid aldosterone is essential for the adequate regulation of electrolyte homeostasis, extracellular volume and blood pressure. As a steroid hormone it influences cellular functions by genomic actions. Previously it has been shown that aldosterone can activate Na+/H+-exchange (NHE) by a rapid, nongenomic mechanism. Because (1) NHE can be regulated by ERK1/2 (extracellular signal-regulated kinase) and (2) steroids have been reported to rapidly activate ERK1/2, we tested the hypothesis that activation of NHE by aldosterone involves ERK1/2, using MDCK-C11 cells. We show that nanomolar concentrations of aldosterone induce a rapid, non-genomic activation of NHE, which is characterized by an increased affinity for H+ with minor changes in the maximum transport rate. Accordingly, aldosterone led to an increase of cytosolic steady-state pH. The aldosterone-induced activation of NHE was prevented by the two specific inhibitors of ERK1/2 activation, PD 98059 (2.5 x 10(-5) mol/l) and U0126 (10(-5) mol/l). Furthermore, in the presence of U0126 there was no aldosterone-induced increase of steady-state pH. Finally, aldosterone induced a rapid phosphorylation of ERK1/2, indicating its ability to activate ERK1/2. The data presented here support the hypothesis that the rapid activation of NHE by aldosterone at nanomolar concentrations involves ERK1/2. Thus, in certain cell types, the MAPK cascade may represent an additional pathway mediating rapid aldosterone effects.

Inhibition of Na+-H+ exchanger-3 interferes with apical receptor-mediated endocytosis via vesicle fusion.

1. Receptor-mediated endocytosis in epithelial cells is a crucial mechanism for transport of macromolecules and regulation of cell-surface protein expression. Na+-H+ exchanger type 3 (NHE3) has been shown to cycle between the apical plasma membrane and the early endosomal compartment and to interfere with endocytosis. 2. In the present study we investigated in detail the NHE3-dependent step of apical endocytosis in an epithelial cell line (opossum kidney cells). 3. Inhibition of NHE3 led to a rapid dose-dependent inhibition of apical albumin endocytosis but did not affect basolateral transferrin endocytosis. Re-exocytosis of albumin was not increased by NHE3 inhibition. 4. NHE3 dependency of albumin endocytosis was still observed at 20 degrees C or when microtubules had been disrupted. This was not the case for inhibition of vacuolar H+-ATPase. 5. NHE3 inhibition rapidly blocked internalisation of pre-bound albumin and attenuated degradation of internalised albumin without changing general protein degradation. 6. Furthermore, NHE3 inhibition reduced the rate of endocytic vesicle fusion significantly. 7. In summary, our data indicate that NHE3 is important for the early phase of the apical endocytic pathway, located between the plasma membrane and early endosomes, at least in part due to its involvement in endocytic vesicle fusion.

[Induction of apoptosis in cultured human proximal tubule cells by fumonisins and fumonisin metabolites].

It is well-known that fumonisin B1 (FB1) stimulates apoptosis in a variety of cell types and tissues but the apoptotic potential of other fumonisins and fumonisin metabolites has not been investigated. In our studies we exposed human proximal tubule-derived cells (IHKE cells) to FB1, fumonisin B2 (FB2), hydrolyzed fumonisin B1 (HFB1) and N-palmitoyl-hydrolyzed fumonisin B1 (N-Pal-HFB1) and investigated caspase 3 activation and DNA fragmentation. Only exposure to 10 µmol/L FB1 for 24h led to a significantly increased activity of caspase 3 and to DNA fragmentation. All other compounds tested did not show any significant activation of caspase 3 activity. Further we examined wether a sphinganine accumulation is correlated with the induction of apoptosis in IHKE cells. Therefore we developed a liquid chromato-graphy/electrospray ionization-tandem-mass spectrometry(HPLC-MS/MS)-method using phytosphingosine as an internal standard to determine sphinganine- and sphingosine concentrations in incubated IHKE cells. Whereas a significant increase of sphinganine was observed with all substrates, sphingosine levels remained unchanged. This shows that FB1 exposure leads to apoptosis in a sphinganine-independent mechanism.

Cubilin- and megalin-mediated uptake of albumin in cultured proximal tubule cells of opossum kidney.

BACKGROUND: Reabsorption of albumin from the glomerular filtrate occurs via receptor-mediated endocytosis in the proximal tubule. This process is initiated by binding of albumin in apical clathrin-coated pits, followed by endocytosis and degradation in lysosomes. Although binding sites have been characterized by kinetic studies, the receptors responsible for the binding of albumin have not been fully identified. Two giant glycoproteins, cubilin and megalin, constitute important endocytic receptors localized to the kidney proximal tubule. METHODS: In the present study, we examined the colocalization of cubilin and megalin in the endocytic pathway and the relationship between the uptake of albumin and the expression of cubilin and megalin in opossum kidney (OK) proximal tubule cells by immunocytochemistry and immunoblotting. RESULTS: OK cells expressed both cubilin and megalin. The light microscope labeling patterns for cubilin and megalin were almost identical and were mainly located at the surface area of the cells. Cubilin and megalin were also shown to colocalize on cell surface microvilli, in coated pits, and in endocytic compartments at the electron microscope level. Endocytosed bovine serum albumin (BSA) was identified exclusively in cells expressing megalin and cubilin. Uptake of BSA-FITC was saturable and inhibited by receptor-associated protein (RAP) and by intrinsic factor-vitamin B12 complex (IF-B12) at high concentrations. Significant inhibition was also observed by specific antibodies to cubilin, and megalin and cubilin antisense oligonucleotides likewise significantly reduced albumin uptake. Egg albumin did not affect the uptake of BSA. CONCLUSION: The present observations suggest that the two receptors cubilin and megalin are both involved in the endocytic uptake of albumin in renal proximal tubule cells.

Nephritogenic ochratoxin A interferes with mitochondrial function and pH homeostasis in immortalized human kidney epithelial cells.

The ubiquitous nephritogenic and carcinogenic fungal metabolite ochratoxin A (OTA) has been shown to interact with renal cell function at low nanomolar concentrations. This is possibly brought about through changes in cellular pH (pHc) homeostasis and mitochondrial function. We assessed the effect of nanomolar concentrations of OTA on pHc homeostasis and the possible involvement of mitochondria using immortalized human kidney epithelial (IHKE1) cells. Within seconds OTA evoked a decrease of pHc with a threshold concentration of 0.1 nmol/l, followed by a sustained alkalinization. Acidification was the same in bicarbonate and non-bicarbonate Ringer solution. When Ca2+ entry across the plasma membrane was prevented, virtually no OTA-induced pH changes could be observed. Inhibition of Na+/H+-exchange (NHE, Na+-free solution) and H+-ATPase (bafilomycin A1) did not reduce the OTA-induced acidification. By contrast, determination of NHE activity as a function of pHc revealed that OTA stimulates NHE (maximal flux increases) in a Ca2+-dependent manner. OTA exposure did not increase lactic acid production, indicating that anaerobic glycolysis was not enhanced. Inhibiting complexes I, III and IV of the mitochondrial electron transport chain (ETC) with rotenone, antimycin A and CN- prevented the OTA-induced acidification almost completely. Completely inhibiting F1FO-ATPsynthase with oligomycin reduced the effect of OTA by approximately equal 50%. In addition, OTA induced a hyperpolarization of the mitochondrial membrane potential (psim) in a Ca2+-dependent manner. Furthermore, OTA exposure resulted in a mitochondria-dependent increase of the cellular ATP content. We conclude that OTA activates mitochondria and NHE by interfering with cellular Ca2+ homeostasis. Stimulation of mitochondrial metabolism leads to enhanced "proton production". Anaerobic glycolysis is not enhanced.

Ochratoxin A induces JNK activation and apoptosis in MDCK-C7 cells at nanomolar concentrations.

Ochratoxin A (OTA) is a ubiquitous fungal metabolite with nephritogenic, carcinogenic, and teratogenic action. Epidemiological studies indicate that OTA may be involved in the pathogenesis of different forms of human nephropathies. Previously we have shown that OTA activates extracellular signal-regulated kinases 1 and 2, members of the mitogen-activated protein kinases (MAPK) family, in the C7-clone but not in the C11-clone of renal epithelial Madin-Darby canine kidney (MDCK) cells. Here we show that nanomolar concentrations of OTA lead to activation of a second member of the MAPK family, namely, c-jun amino-terminal-kinase (JNK) in MDCK-C7 cells but virtually not in MDCK-C11 cells, as determined by kinase assay and Western blot. Furthermore, OTA potentiated the effect of tumor necrosis factor-alpha on JNK activation. In parallel to its effects on JNK, nanomolar OTA induced apoptosis in MDCK-C7 cells but not in MDCK-C11 cells, as determined by DNA fragmentation, DNA ladder formation, and caspase activation. In addition, OTA potentiated the proapoptotic action of tumor necrosis factor-alpha. Our data provide additional evidence that OTA interacts in a cell type-specific way with distinct members of the MAPK family at concentrations where no acute toxic effect can be observed. Induction of apoptosis via the JNK pathway can explain some of the OTA-induced changes in renal function as well as part of its teratogenic action.

Apoptosis in cultured renal epithelial cells caused by ochratoxin A.

Apoptosis induction in renal epithelial cells caused by exposure to ochratoxin A (OTA) was studied. Nanomolar concentrations of OTA led to activation of caspase 3 with subsequent chromatin condensation and DNA ladder formation, both characteristic for cells undergoing apoptosis. Inhibition of the mitochondrial ATP/ADP carrier prevented OTA induced caspase activation showing that intact mitochondria are necessary for OTA induced apoptosis.

Inhibition of Na+-H+ exchange impairs receptor-mediated albumin endocytosis in renal proximal tubule-derived epithelial cells from opossum.

1. Receptor-mediated endocytosis is an important mechanism for transport of macromolecules and regulation of cell-surface receptor expression. In renal proximal tubules, receptor-mediated endocytosis mediates the reabsorption of filtered albumin. Acidification of the endocytic compartments is essential because it interferes with ligand-receptor dissociation, vesicle trafficking, fusion events and coat formation. 2. Here we show that the activity of Na+-H+ exchanger isoform 3 (NHE3) is important for proper receptor-mediated endocytosis of albumin and endosomal pH homeostasis in a renal proximal tubular cell line (opossum kidney cells) which expresses NHE3 only. 3. Depending on their inhibitory potency with respect to NHE3 and their lipophilicity, the NHE inhibitors EIPA, amiloride and HOE694 differentially reduced albumin endocytosis. The hydrophilic inhibitor HOE642 had no effect. 4. Inhibition of NHE3 led to an alkalinization of early endosomes and to an acidification of the cytoplasm, indicating that Na+-H+ exchange contributes to the acidification of the early endosomal compartment due to the existence of a sufficient Na+ gradient across the endosomal membrane. 5. Exclusive acidification of the cytoplasm with propionic acid or by removal of Na+ induced a significantly smaller reduction in endocytosis than that induced by inhibition of Na+-H+ exchange. 6. Analysis of the inhibitory profiles indicates that in early endosomes and endocytic vesicles NHE3 is of major importance, whereas plasma membrane NHE3 plays a minor role. 7. Thus, NHE3-mediated acidification along the first part of the endocytic pathway plays an important role in receptor-mediated endocytosis. Furthermore, the involvement of NHE3 offers new ways to explain the regulation of receptor-mediated endocytosis.

Ochratoxin A-binding proteins in rat organs and plasma and in different cell lines of the kidney.

In order to detect cellular proteins which bind the mycotoxin ochratoxin A (OTA) we coupled OTA covalently to horseradish peroxidase (HRP). The peroxidase activity of the conjugate was used to detect these proteins in Western (ligand) blot analysis. Only signals caused by OTA binding to proteins were viewable. HRP alone detected no proteins and OTA-HRP binding could be inhibited by free OTA. Several proteins from the rat intestine, liver, spleen, and kidney were detected by OTA. Also rat plasma proteins bind OTA which confirms previous findings. In all renal cell lines investigated (MDCK-C11, OK, LLC-PK1, IHKE, and SKPT) there are several proteins which bind OTA. Comparison of the PonceauS stain on the nitrocellulose sheet with the signal obtained from OTA-HRP unveiled proteins with high specific OTA binding. Especially, proteins with molecular masses between 55 and 60 kDa, 40 and 45 kDa and 25 and 30 kDa showed OTA binding in all samples. OTA was partially displaced by aspartame and phenylalanine from some but not all proteins. Binding to cytosolic and organellar proteins was comparable in all investigated cell lines. In the OK cell organellar compartment a 62 kDa protein is preferentially detected by OTA-HRP although virtually no protein band is detectable. In conclusion we have found a method to clearly detect proteins which bind OTA. With this new method we proved that OTA has the potential to bind to several proteins yet specific binding differs dramatically. Thus, highly specific binding of OTA possibly makes certain proteins a preferential target of OTA toxicity. Furthermore, binding contributes to intracellular accumulation of OTA, thus leading to a prolonged half life in the mammalian body and emphasises the toxic potential of this fungal metabolite.

The nephrotoxin ochratoxin A induces apoptosis in cultured human proximal tubule cells.

To test the apoptotic potential of the nephrotoxic mycotoxin ochratoxin A (OTA), we exposed human proximal tubule-derived cells (IHKE cells) for various times to OTA concentrations close to those occurring during dietary exposure (from 2 to 100 nmol/L) and investigated caspase 3 activation, chromatin condensation, and DNA fragmentation. OTA induced a time- and concentration-dependent activation of caspase 3: concentrations as low as 5 nmol/L OTA caused a slight but significant increase in caspase 3 activity after 7 days of OTA exposure. Exposure to 10 nmol/L OTA for 72 or 24 h led to a significantly increased activity of caspase 3 in human proximal tubule-derived cells. Radical scavengers such as N-acetylcysteine had no effect on OTA-induced caspase 3 activation. Chelation of intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethylester) (BAPTA-AM) also showed no effect. Exposure to 30 nmol/L or more OTA led to DNA fragmentation and chromatin condensation in IHKE cells. Cultured renal epithelial MDCK-C7 and MDCK-C11 or OK cells also showed increased caspase 3 activity after OTA exposure. We conclude that exposure to low OTA concentrations can lead to direct or indirect caspase 3 activation and subsequently to apoptosis in cultured human proximal tubule cells and in other renal epithelial cell lines of different origins.

Characterization of an ochratoxin-A-dedifferentiated and cloned renal epithelial cell line.

Ochratoxin A (OTA) is a ubiquitous fungal metabolite with predominant nephrotoxic action. OTA impairs postproximal renal electrolyte handling and increases the incidence of renal adenoma and carcinoma. Furthermore, it is supposed to be involved in the pathogenesis of different forms of human renal diseases. Previously we have shown that OTA activates extracellular signal-regulated kinase 1 (ERK1) and ERK2 in the C7 clone but not in the C11 clone of renal epithelial MDCK cells. Here we show that nanomolar concentrations of OTA lead to stable and irreversible phenotypical and genotypical alterations, resulting in sustained dedifferentiation of MDCK-C7 cells but not of MDCK-C11 cells. Dedifferentiated MDCK-C7 cells (OTA-C7 cells) display a distinct morphology from the parent cell line (spindle-shape, pleiomorphic, narrow intercellular spaces, increased cell size) and show a reduced proliferation rate and numerical chromosomal aberrations. Functionally, OTA-C7 cells are characterized by a dramatic reduction of transepithelial electrolyte transport and the complete loss of responsiveness to the mineralocorticoid hormone aldosterone. Our data provide further evidence that OTA can lead to cell dedifferentiation and eventually to transformation of cloned quiescent cells. The changes in phenotype due to this dedifferentiation could explain some of the OTA-induced changes in renal function.

Long-term protein exposure reduces albumin binding and uptake in proximal tubule-derived opossum kidney cells.

To avoid renal loss of large amounts of proteins, filtered proteins are reabsorbed by endocytosis along the proximal tubule. However, although protein reabsorption is a task of proximal tubular cells, it is also a threat because it may cause cell injury. This study determines whether exposure to bovine serum albumin (BSA) leads to regulatory changes in endocytosis of FITC-BSA in proximal tubule-derived opossum kidney cells. Preincubation with BSA led to a decrease of FITC-BSA endocytosis with an IC50 value of 0.58 g/L. Specific binding of FITC-BSA to the apical membrane was also reduced (IC50 = 0.69 g/L). Kinetic analyses revealed that maximal uptake rate and maximal binding capacity were decreased with no change in affinity. Similar effects were observed after preincubation with equimolar amounts of other proteins (lactalbumin, transferrin, and conalbumin), but not after preincubation with dextran. The effect of preincubation with BSA could be mimicked by preincubation with some amino acids. Preincubation with L-Ala, L-Gln, or NH4Cl, but not with L-Leu, L-Glu, or L-Asp, reduced FITC-BSA endocytosis and binding. Preincubation with BSA, but not with dextran, reduced protein degradation and increased ammonia production, vesicular pH, as well as the rate of lactate dehydrogenase release. Apical fluid-phase endocytosis and apical uptake of neutral amino acids were not reduced. It is concluded that proximal tubular cells reduce the uptake rate for proteins, but not for other substrates, in response to increased protein load. This reduction is achieved by reducing the number of apical binding sites, partially in response to increased ammoniagenesis with deranged vesicular pH and enzyme activities. Thus, increased protein filtration could result in reduced protein reabsorption, thereby enhancing proteinuria.

Apical uptake of radiolabelled ochratoxin A into Madin-Darby canine kidney cells.

Uptake of ochratoxin A (OTA) across the apical cell membrane of collecting duct cells is the first step in reabsorption and partly mediated by proton-dipeptide cotransport. As the remaining part of apical OTA uptake remained unclear, we studied the characteristics of apical uptake of tritium-labelled OTA (3H-OTA) in MDCK-C11 cells in detail. Uptake of 3H-OTA was pH- and temperature-dependent and led to intracellular accumulation of OTA. Lowering pH led to an increase and lowering temperature (4 degrees C) to a decrease of OTA uptake. Besides dipeptides, the beta-lactam antibiotics cephalexin and ceftibuten inhibited the 3H-OTA uptake also confirming the role of the proton dipeptide cotransporter. In addition, substrates of organic anion transporter, taurocholate and methotrexate, inhibited 3H-OTA uptake in part. Aspartylphenylalanine methyl ester (aspartame) had no inhibitory effect on 3H-OTA uptake. Uptake of OTA was not dependent on sodium. Sixty minutes of preincubation with phorbol 12-myristate 13-acetate (PMA) led to increased apical uptake of OTA. The PMA effects were inhibited by ethylisopropylamilorid (EIPA). We conclude that apical uptake of OTA occurs by Na+-independent transport. One part of the uptake is mediated by proton-dipeptide cotransport (30%, dipeptide-inhibitable), by organic anion transporter (20%, taurocholate-inhibitable) and by diffusion (20%, responsible for uptake at 4 degrees C). The remaining part occurs by as yet unidentified but pH-dependent transport mechanisms. An acidic urine in distal parts of the nephron provides thus the main risk for OTA uptake leading to its reabsorption and consequently alkalinisation of the urine should help to prevent this reabsorption.

Albumin endocytosis in OK cells: dependence on actin and microtubules and regulation by protein kinases.

We used proximal tubule-derived opossum kidney (OK) cells to determine the dependence of albumin endocytosis on regulation by protein kinases and on the cytoskeleton. Uptake was observed only across the apical but not the basolateral membrane and exceeded uptake in collecting duct-derived Madin-Darby canine kidney cells 14-fold. Inhibition of endocytosis via clathrin-coated vesicles but not via caveolae abolished uptake. Cytochalasin D reduced uptake to < 5% of control, and inhibition of microtubule polymerization by nocodazole reduced uptake to approximately 55% of control. Activation of protein kinase A (PKA) by adenosine 3',5'-cyclic monophosphate, forskolin, or parathyroid hormone (PTH) reduced uptake to approximately 65% of control. Protein kinase C (PKC) activation did affect uptake to a similar extent as PKA activation but with a certain delay. Stimulation of PKG by guanosine 3',5'-cyclic monophosphate did not affect albumin endocytosis. The inhibitor of tyrosine kinases (TRK), genistein, induced an increase of uptake to approximately 160% of control. Reexocytosis of albumin was enhanced by PKC activation but not by PKA activation. TRK inhibition reduced the rate of reexocytosis. We conclude that albumin endocytosis in OK cells requires the integrity of the actin cytoskeleton. Microtubules facilitate endocytosis. Uptake is regulated by PKA, PKC, and TRK, yet with different time course and by different mechanisms, e.g., reexocytosis. Possibly TRK activity serves in a negative feedback loop to limit albumin endocytosis via a stimulation of reexocytosis.

Apical-to-basolateral transepithelial transport of Ochratoxin A by two subtypes of Madin-Darby canine kidney cells.

In this study we investigated the transepithelial transport of Ochratoxin A (OTA), a potent nephrotoxin, across monolayers of two collecting duct-derived cells clones (Madin-Darby canine kidney cells (MDCK)-C7 and MDCK-C11 cells, resembling principal and intercalated cells, respectively) either from the apical to the basolateral side or vice versa. We cultured cells on permeable supports and compared the transport rates of OTA, p-aminohippuric acid (PAH) and fluorescein-labelled inulin. Monolayers of both cell clones translocated OTA from the apical to the basolateral side but not in the opposite direction. Transport rate across MDCK-C11 cell monolayers was 2.9-fold the transport rate across MDCK-C7 cell monolayers. OTA transport was temperature-dependent being reduced from 77.5 pmol/cm2 per h to 10.1 pmol/cm2 per h in MDCK-C11 and from 27.0 pmol/cm2 per h to 7.6 pmol/cm2 per h in MDCK-C7 cells when temperature was decreased from 37 degrees C to 4 degrees C. In both cell clones, the dipeptides carnosine and glycylsarcosine but not the amino acids glycine or phenylalanine had an inhibitory effect on OTA transport. In both cell clones, transepithelial transport of OTA was dependent on the apical pH (pK(a) of OTA = 7.1). In an environment mimicking the transepithelial in vivo pH gradient to some extent with more acidic pH on the apical side than on the basolateral side, transport was 4-fold higher in both cell clones as compared to conditions when pH was 7.4 in both bath solutions. In the absence of a pH gradient, transport rates were similar to that at 4 degrees C. Apical uptake of [3H]OTA was inhibited by carnosine and by glycylsarcosine and the uptake of [3H]carnosine was inhibited by OTA. Our results indicate that OTA is transported across the apical membrane of MDCK cells by both non-ionic diffusion and by a H+-dipeptide cotransporter. Thus, reabsorption of OTA in the collecting duct contributes to the observed long half life of OTA in the mammalian body.

Ochratoxin A-induced stimulation of extracellular signal-regulated kinases 1/2 is associated with Madin-Darby canine kidney-C7 cell dedifferentiation.

The kidneys represent one of the main targets of ochratoxin A (OTA), a secondary fungal metabolite that is produced by certain species of Aspergillus and Penicillium. OTA has the ability to disturb Madin-Darby canine kidney (MDCK) cell pH homeostasis, leading to intracellular alkalinization and morphological alterations resembling those that occur when MDCK cells are exposed to transient alkaline stress. Because alkali-induced epithelial dedifferentiation of MDCK-C7 cells is associated with an increase in the activity of extracellular signal-regulated kinases (ERK), we performed experiments that investigated a possible role for ERK1 and ERK2 as intracellular signaling molecules mediating some of the mycotoxin's effects on renal epithelia. We studied the effects of OTA on ERK1/2 phosphorylation and activation, as well as on cell morphology by using cloned MDCK-C7 and MDCK-C11 cells. In MDCK-C7 cells, but not in MDCK-C11 cells, OTA led to a time-dependent and concentration-dependent increase in ERK1/2 phosphorylation. OTA-induced ERK1/2 phosphorylation in MDCK-C7 cells occurred at concentrations of 500 nM, started after 2 hr and was maximal after 8 hr. Furthermore, after 8 hr of incubation, 500 nM and 1 microM OTA significantly increased ERK1/2 activity in MDCK-C7 but not in MDCK-C11 cells. This OTA-stimulated ERK1/2 phosphorylation and ERK1/2 activation in MDCK-C7 cells was partially inhibited by the synthetic mitogen-activated protein kinase kinase (MKK or MEK) inhibitor PD098059. Transepithelial resistance and lactate dehydrogenase release remained unaltered after incubation in the presence of 1 microM OTA for 8 hr or of 100 nM OTA for 24 hr, so it is unlikely that these OTA effects on ERK1/2 are due to secondary toxic effects of the mycotoxin. Interestingly, OTA-induced long-term activation of ERK1/2 in MDCK-C7 cells was associated with epithelial dedifferentiation, as assessed by analysis of vectorial solute and water transport as well as cell morphology. In contrast, MDCK-C11 cells, which do not show significant increases in ERK1/2 phosphorylation and ERK1/2 activity in response to OTA, retained their epithelial phenotype under identical experimental conditions. Taken together, our data demonstrate an epithelial dedifferentiation of MDCK-C7 cells, but not of MDCK-C11 cells, after long-term incubation in the presence of OTA, a result associated with the ability of this mycotoxin to stimulate ERK1/2 in MDCK-C7 cells but not in MDCK-C11 cells. We conclude that OTA-induced activation of ERK1/2 could be an important intracellular signaling pathway that mediates some of the mycotoxin's effects on renal epithelia.