Deletion of the EGF receptor in vascular smooth muscle cells prevents chronic angiotensin II-induced arterial wall stiffening and media thickening.

AIM: In vivo vascular smooth muscle cell (VSMC) EGF receptor (EGFR) contributes to acute angiotensin II (AII) effects on vascular tone and blood pressure. The ubiquitously expressed EGFR has been implicated in vascular remodelling preceding end-organ damage by pharmacological inhibition, and AII signalling in cultured vascular cells is partly EGFR-dependent. However, the role of VSMC-EGFR in vivo during AII-induced pathophysiological processes is not known. METHODS: This study assesses the in vivo relevance of VSMC-EGFR during chronic AII challenge without further stressors, using a mouse model with inducible, VSMC-specific EGFR knock out (VSMC-EGFR-KO). In these mice functional and structural vascular, renal and cardiac effects or biomarkers were investigated in vivo and ex vivo. RESULTS: Vascular smooth muscle cell-EGFR-KO prevented AII-induced media hypertrophy of mesenteric arteries, renal arterioles and the aorta, VSMC ERK1/2-phosphorylation as well as the impairment of vascular compliance. Furthermore, induction of vascular fibrosis, creatinineamia, renal interstitial fibrosis as well as the increase in fractional water excretion was prevented. AII-induced increase in systolic blood pressure was mitigated. By contrast, endothelial dysfunction, induction of vascular inflammatory marker mRNA and albuminuria were not inhibited. Cardiac and cardiomyocyte hypertrophy were also not prevented by VSMC-EGFR-KO. CONCLUSION: Vascular smooth muscle cell-EGFRs are relevant for pathological AII action in vivo. Our data show in vivo and ex vivo the necessity of VSMC-EGFR for AII-induced structural and functional vascular remodelling, not including endothelial dysfunction. Hereby, VSMC-EGFR gains importance for complete AII-induced renal end-organ damage succeeding vascular remodelling.

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.

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.

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.

Nephritogenic ochratoxin A interferes with hormonal signalling in immortalized human kidney epithelial cells.

The ubiquitous nephritogenic and carcinogenic fungal metabolite ochratoxin A (OTA) affects function and growth of renal epithelial cells. We studied the possible contribution of changes in cellular Ca2+ homeostasis to the effects of nanomolar concentrations of OTA on immortalized human kidney epithelial (IHKE-1) cells. The effects of OTA on cellular calcium homeostasis ([Ca2+]i), cell proliferation and viability and its interaction with angiotensin II (Ang II) and epidermal growth factor (EGF) were investigated. OTA potentiated EGF- and Ang II-induced cell proliferation Ca2+ dependently at OTA concentrations of 0.1 or 1 nmol/l. A decrease in cell viability could be observed only after 24 h exposure, with threshold concentrations greater than 10 nmol/l. This reduction of cell viability was independent of Ca2+. Within seconds, OTA evoked reversible and concentration-dependent [Ca2+]i oscillations with a threshold concentration of < or =0.1 nmol/l. These oscillations were abolished by removal of extracellular Ca2+, by the Ca(2+)-channel blocker SKF 96365 and by inhibition of phospholipase C. OTA also stimulated thapsigargin-sensitive Ca(2+)-ATPase activity and increased the filling state of thapsigargin-sensitive Ca(2+)-stores. Exposure to OTA concentration dependently increased cellular adenosine 3',5'-cyclic monophosphate (cAMP) content. In addition, OTA-induced changes of [Ca2+]i were reduced significantly by the protein kinase A inhibitor H-89. Finally, 0.1 or 1 nmol/l OTA potentiated the effects of Ang II and EGF on cellular Ca2+ homeostasis. We conclude that OTA may impair cellular Ca2+ and cAMP homeostasis already at low nanomolar concentrations, resulting in concentration-dependent [Ca2+]i oscillations. OTA interferes also with hormonal Ca2+ signalling, thereby leading to altered cell proliferation. The reduction of cell viability at higher OTA concentrations seems not to depend on Ca2+.

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.

Inhibition of initial transport rate of basolateral organic anion carrier in renal PT by BK and phenylephrine.

The effect of ligands for phospholipase C-coupled receptors and of protein kinase C (PKC) stimulation with phorbol ester [phorbol 12-myristate 13-acetate (PMA)] or 1,2-dioctanoyl-sn-glycerol on the activity of the basolateral organic anion transporter (OAT) in S2 segments of single, nonperfused rabbit proximal tubules (PT) was measured with the use of fluorescein and epifluorescence microscopy. The initial uptake rate (25 s, OAT activity) was measured in real time by using conditions similar to those found in vivo. Stimulation of PKC with PMA or 1,2-dioctanoyl-sn-glycerol led to an inhibition of OAT activity, which could be prevented by 10(-7) mol/l of the PKC-specific inhibitor bisindolylmaleimide. The alpha(1)-receptor agonist phenylephrine as well as the peptide hormone bradykinin induced a reversible decrease of OAT activity, which was prevented by bisindolylmaleimide. The observed effect was not due to a decrease in the concentration of the counterion alpha-ketoglutarate or to impaired alpha-ketoglutarate recycling, because it was unchanged in the continuous presence of alpha-ketoglutarate or methyl succinate. We conclude that physiological stimuli can inhibit the activity of OAT in rabbit PT via PKC. The effect is not mediated by alterations in counterion availability but by a direct action on the OAT.

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.

Acute volume expansion and salt-loading studies in rats. The role of atrial natriuretic peptide and catecholamines.

Differences have been postulated for the mechanism of natriuresis due to atrial natriuretic peptide (ANP), salt loading with high salt diet (HS) and acute volume expansion (AcVE), in particular between AcVE and ANP based on the observed synergism between the two. Therefore the effects of and the interaction between the three were investigated in rats. ANP and AcVE produced the same natriuresis in HS as in normal salt (NS) rats and, in both, the actions of ANP and AcVe were significantly additive showing similarity in mechanisms. Synergism [(AcVE + ANP) - AcVE] was, however, present only in the NS rats. Proximal tubular sodium transport was the same with AcVE and ANP+AcVE suggesting that synergism is a property of more distal nephron segments. In conscious HS rats, plasma ANP was significantly less but natriuresis was higher than in NS rats. ANP therefore probably has some causative role in the natriuresis of AcVE but none in that of HS loading. Urinary dopamine was significantly increased by HS and further increased by AcVE in both NS and HS rats, the relationship between dopamine and natriuresis being significantly positive (r2 = 0.328) reaching equivalent levels in both NS and HS rats. Systemic benserazide prevented the increase in urinary dopamine but only attenuated the natriuresis of AcVE. We conclude that HS does not potentiate the natriuresis of AcVE or ANP, synergism between AcVE and ANP is not a proximal tubule event and dopamine accounts for significant natriuresis of VE in addition to other natriuretic factors.

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.

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.

Functional characterization of albumin binding to the apical membrane of OK cells.

We characterized binding of albumin to the apical membrane of opossum kidney (OK) cells using fluorescein isothiocyanate (FITC)-albumin (i.e., bovine serum albumin, BSA) as substrate. Functional analysis of binding data showed one specific binding site characterized by half-maximal binding (Michaelis constant, (Km) at 20 mg/l (300 nmol/l) and maximal binding capacity (Bmax) of 0.61 microgram/mg cellular protein. Excess of unlabeled albumin (BSA) inhibited binding at low concentrations of FITC-albumin completely but only partially at high concentrations. FITC-albumin binding was reversible and pH dependent. Km increased about sixfold when pH decreased from 7.4 to 5.0. The inhibitory effects of conalbumin, alpha-lactalbumin, and transferrin were significantly smaller compared with BSA. We conclude that OK cells express a high-affinity binding site for albumin on the apical membrane. This binding site is pH sensitive, binds albumin in the physiological range, and could be responsible for the effective receptor-mediated reabsorption of albumin in the proximal tubule.

Kinetics of receptor-mediated endocytosis of albumin in cells derived from the proximal tubule of the kidney (opossum kidney cells): influence of Ca2+ and cAMP.

In this study we investigated the effects of Ca2+ and cyclic adenosine monophosphate (cAMP) on the kinetic of receptor-mediated (RME) and fluid-phase (FPE) endocytosis in opossum kidney (OK) cells, derived from the proximal tubule of the kidney. We used fluorescein isothiocyanate (FITC)-labelled albumin and FITC-labelled dextran as endocytotic substrates for RME and FPE, respectively. Removal of extracellular Ca2+ led to a dramatic decrease of the apparent affinity of RME, but did not influence the maximum endocytotic uptake rate (Jmax). Reduction of extracellular Ca2+ to 1 mumol/1 had no effect. Apparent affinity of specific binding of albumin to the plasma membrane was increased to 200% of control in the absence of extracellular Ca2+, whereas maximum binding capacity was slightly decreased. FPE was not affected by removal of extracellular Ca2+. Additional removal of cytoplasmic Ca2+, using ionomycin, had no further effect on RME and did not affect FPE. Increases of cytoplasmic (using ionomycin at extracellular Ca2+ concentrations of 1 mumol/l or 1.2 mmol/l) or extracellular Ca2+ did not alter the kinetics of RME or FPE. Dibutyryl-cAMP reduced Jmax but left the apparent affinity of RME unchanged. FPE and albumin binding to the plasma membrane were not changed in the presence of cAMP. Removal of extracellular Ca2+ and addition of cAMP led to an alkalinization of endocytotic vesicles. Yet the alkalinization induced by removal of Ca2+ was significantly greater as compared to the alkalinization in the presence of cAMP. Endosomal alkalinization with bafilomycin A1 had no further effect in the absence of Ca2+, but reduced RME in the presence of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Endosomal alkalinization reduces Jmax and Km of albumin receptor-mediated endocytosis in OK cells.

In this study, we investigated the effects of endosomal alkalinization on kinetics of endocytotic uptake in intact proximal tubule-derived opossum kidney cells. We used fluorescein isothiocyanate (FITC)-labeled albumin and FITC-dextran as endocytotic substrates for receptor-mediated endocytosis and fluid-phase endocytosis, respectively. The pH in endosomes labeled with either FITC-albumin or FITC-dextran rose in the presence of the vacuolar-type ATPase inhibitor, bafilomycin A1, and in the presence of NH4Cl. Cytoplasmic pH, decreased in the presence of bafilomycin A1, but was not significantly different from control during prolonged exposure of the cells to NH4Cl. Endocytotic uptake of FITC-dextran was not affected by endosomal pH changes. Endocytotic uptake of FITC-albumin was reduced markedly by bafilomycin A1 (decrease of maximum transport rate and apparent affinity). Selective alkalinization of endosomes using NH4Cl (i.e., with the cytoplasmic pH not different from control) reduced FITC-albumin uptake in a similar way but to a lesser extent than did bafilomycin A1. Intracellular albumin degradation was impaired by bafilomycin A1 and NH4Cl. Prevention of endosome-lysosome fusion (lowering the temperature to 20 degrees C) abolished the effects of endosomal alkalinization. Furthermore, specific binding of albumin to the plasma membrane was reduced after incubation with bafilomycin A1, indicating an impairment of receptor recycling. These data show that endosomal pH is an important determinant for the kinetics of receptor-mediated endocytotic uptake of albumin in the proximal tubule but not for fluid-phase endocytosis. Endosomal alkalinization disturbs intracellular ligand handling and receptor trafficking, leading to a reduction of endocytotic capacity and affinity.

The mycotoxin ochratoxin-A impairs protein uptake in cells derived from the proximal tubule of the kidney (opossum kidney cells).

Proximal tubule-derived opossum kidney (OK) cells are a suitable model to study proximal tubular protein endocytosis by using fluorescein-isothiocyanate-albumin as substrate. We used OK cells to investigate several steps of the endocytotic process in control cells and in ochratoxin A (OTA)-treated cells. OTA is a mycotoxin which causes proteinuria. When OTA was present only during the 15-min period in which uptake was studied, it had no effect on albumin endocytosis. Preincubation of OK cells with OTA (10 mumol/l) for 24 hr led to a reduction of transport capacity (Jmax to approximately 50% of control) and of apparent affinity (Km to approximately 200% of control). Specific binding of albumin to the apical cell surface was reduced also. Maximum binding capacity was reduced to 72% of control. By contrast, endocytotic uptake of the fluid-phase marker dextran was not affected by OTA. Preincubation of OK cells for 24 hr with 10 mumol/l of OTA reduced degradation of fluorescein-isothiocyanate-albumin to trichloroacetic acid-soluble fluorescence to 59% of control. We could not detect any difference in endosomal pH (6.13 +/- 0.05 in controls vs. 6.04 +/- 0.10 in OTA-treated cells). Furthermore, the rate of re-exocytosis of albumin taken up was significantly greater in OTA-treated cells. We conclude that: 1) OK cells are a suitable model to study several steps of the endocytotic process separately and thus to investigate the pathophysiology of reduced tubular protein reabsorption and 2) 24-hr exposure to OTA reduces protein uptake because of a decrease of specific binding sites and of enhanced exocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)