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ABSTRACT: Filtered proteins play a role in the pathogenesis of renal interstitial inflammation and fibrosis. At least part of these effects are mediated by the nuclear factor kappaB (NF-kappaB). Receptor-mediated endocytosis of proteins like albumin in renal proximal tubular cells is in part dependent on Na superset+/H superset+ exchanger (NHE) isoform 3. We tested the hypothesis that pharmacological inhibition of NHE-3 reduces albumin-induced NF-kappaB activation - and therefore albumin-induced renal interstitial inflammation and fibrosis - using established proximal tubular cell lines (OK and LLC-PK1). 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) or HOE694 were used to inhibit NHE. Albumin endocytosis was determined by fluorometric analysis of FITC-BSA uptake. Electromobility gel shift assays were performed in order to determine the NF-kappaB-specific DNA-binding activity. EIPA reduced albumin uptake in OK and LLC-PK1 cells and HOE694 decreased albumin uptake in LLC-PK1 cells, with IC subset50 values corresponding to NHE-3 inhibition. Furthermore, albumin-induced increases in NF-kappaB DNA-binding activity were partially inhibited by EIPA in OK and LLC-PK1 cells. HOE694 at a concentration of 100 micromol/l similarly decreased albumin-induced NF-kappaB DNA-binding activity. Cytosolic acidification by propionic acid did not prevent BSA-induced activation of NF-kappaB. Inhibition of BSA endocytosis by chlorpromazine decreased NF-kappaB activation. NHE-dependent albumin endocytosis induces an increase in NF-kappaB-specific protein activity in renal proximal tubular cells in culture, which is decreased by EIPA and HOE694. Thus, inhibition of albumin uptake might be a therapeutical strategy to prevent albumin-induced NF-kappaB activation and albumin-associated inflammatory or fibrotic renal pathomechanisms in vivo.
European journal of medical research 11/2001; 6(10):422-32. · 1.13 Impact Factor
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ABSTRACT: Albuminuria represents one of the most unfavourable diagnostic factors for the prognosis of nephropathies. We investigated albumin-induced NF-kappaB activation and the potential contribution of tyrosine kinase and protein kinase C. Therefore we exposed proximal tubule-derived human (IHKE-1), opossum (OK) and porcine (LLC-PK1) cell lines to serum albumin at concentrations of 10 - 500 mg/l. DNA-binding activity of NF-kappaB increased concentration-dependently in the presence of albumin. In OK and LLC-PK1 cells, NF-kappaB activity increased during the first 45 min and reached a plateau thereafter. In IHKE-1, cells NF-kappaB activity reached a plateau after 90 min with a maximum at 180 min exposure to albumin. The albumin-induced increase in NF-kappaB DNA-binding activity was inhibited by herbimycin A (tyrosine kinase inhibitor) and BIM (protein kinase C inhibitor). Reporter gene assays demonstrated that albumin stimulates NF-kappaB mediated reporter gene activation in LLC-PK1 cells, which was partially inhibited by herbimycin A and BIM. Our data indicate, that albumin exposure induces a rapid increase in NF-kappaB protein activity in renal proximal tubule cells of different species via a tyrosine kinase- and protein kinase C-dependent pathway, at concentrations occurring during mild glomerular injury.
European journal of medical research 07/2001; 6(6):247-58. · 1.13 Impact Factor
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ABSTRACT: Ochratoxin A (OTA) is supposed to induce renal diseases in man and animals and a correlation between renal diseases and OTA concentration in blood is suspected. Therefore, we measured OTA concentrations in blood of subjects suffering from various renal diseases as e.g. interstitial nephritis or mesangial proliferating glomerular nephritis (GN) and compared them with the blood concentration of healthy individuals. We found OTA in 87% of all samples. There was no significant difference between OTA concentrations of healthy individuals and patients but some renal diseases (e.g. chronic glomerular nephritis) showed increased numbers of samples containing more than 1.5 nmol/l OTA in sera. In contrast, in samples from patients suffering from membranous or focal-sclerotic glomerular nephritis no concentrations above 1.5 nmol/l were found. Our preliminary results show that OTA is abundant in nearly all serum samples but some renal diseases show increased numbers of samples with high (>1.5 nmol/l) OTA concentrations.
Mycotoxin Research 06/2001; 17 Suppl 2:146-9.
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ABSTRACT: 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.
Mycotoxin Research 06/2000; 16 Suppl 2:154-7.
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ABSTRACT: Amino acids are apparently recycled between loops of Henle and vasa recta in rat papilla in vivo. To examine this process in the absence of metabolism, we performed continuous microinfusions of rat renal papillary ascending thin limbs (ATLs) and vasa recta in vivo, and microperflusions of isolated rat renal papillary descending thin limbs (DTLs) and ATLs in vitro using the nonmetabolizable, synthetic, neutral amino acid cycloleucine. Like naturally occurring amino acids, approximately = 25% of radiolabeled cycloleucine microinfused into ATLs in vivo was reabsorbed by a process that was not saturable or inhibitable. Also, like naturally occurring amino acids, approximately = 47% (relative to inulin) of radiolabeled cycloleucine microinfused into ascending vasa recta in vivo was transferred directly into ipsilateral tubular structures (probably DTLs) by a saturable and inhibitable process. In DTLs perfused in vitro, unidirectional bath-to-lumen fluxes (Jbl) tended to exceed unidirectional lumen-to-bath fluxes (Jlb), whereas in ATLs perfused in vitro Jlb tended to exceed Jbl, but the differences were not statistically significant. Moreover, none of the unidirectional fluxes was saturable or inhibitable, an observation compatible with apparent reabsorption from ATLs in vivo but incompatible with apparent movement from vasa recta to DTLs in vivo. These in vitro observations are like those made previously for the naturally occurring neutral amino acid L-alanine. The lack of saturation and inhibition, like the previous data on L-alanine, suggest that transepithelial movement of amino acids in thin limbs of Henle's loop may occur via a paracellular route and that regulation of amino acid movement in vivo may involve vasa recta, not DTLs. They also suggest that cycloleucine is a good nonmetabolizable surrogate for the study of neutral amino acid transport in the kidney.
Pflügers Archiv - European Journal of Physiology 04/2000; 439(5):517-23. · 4.46 Impact Factor
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ABSTRACT: This study was designed 1) to localize and 2) to characterize betaine reabsorption from the tubular lumen in rat kidney in vivo, and 3) to test whether reabsorption is modulated by the diuretic state. [(14)C]betaine (+ [(3)H]inulin) was microperfused through the proximal convoluted tubule (PCT) and microinfused into late proximal (LP) and early distal (ED) tubules, long loops of Henle (LLH), and vasa recta of the rat in vivo et situ, and the fractional recovery of the (14)C label was determined end proximally (PCT) and in the final urine, respectively. [(14)C]betaine was not reabsorbed during ED microinfusion, whereas fractional reabsorption during LP microinfusion was 82% at 0.06 mM betaine and decreased gradually to 4.8% at 60 mM. L-Proline had lower Michaelis-Menten constant (K(m)) and sarcosine a higher K(m) than betaine. Chronic, but not acute, diuresis inhibited betaine reabsorption in Henle's loops. Fractional [(14)C]betaine reabsorption in PCT was much smaller than that during LP microinfusion. [(14)C]betaine (7.28 mM) microinfused 1) into LLH was reabsorbed to 30% and 2) into vasa recta appeared in the ipsilateral urine to a much higher extent than contralaterally. In both cases, no saturation was detected at 70 mM. We conclude that betaine is reabsorbed by mediated transport from descending limbs of short Henle's loops by a proline-preferring carrier in a diuresis-modulated manner. In the deep medulla, bidirectional blood/urine betaine transport exists.
American journal of physiology. Renal physiology 04/2000; 278(3):F434-9. · 3.68 Impact Factor
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ABSTRACT: Amino acids are apparently recycled between loops of Henle and vasa recta in rat papilla in vivo. To examine this process in the absence of metabolism, we performed continuous microinfusions of rat renal papillary ascending thin limbs (ATLs) and vasa recta in vivo, and microperfusions of isolated rat renal papillary descending thin limbs (DTLs) and ATLs in vitro using the nonmetabolizable, synthetic, neutral amino acid cycloleucine. Like naturally occurring amino acids, ᜥ% of radiolabeled cycloleucine microinfused into ATLs in vivo was reabsorbed by a process that was not saturable or inhibitable. Also, like naturally occurring amino acids, ᜻% (relative to inulin) of radiolabeled cycloleucine microinfused into ascending vasa recta in vivo was transferred directly into ipsilateral tubular structures (probably DTLs) by a saturable and inhibitable process. In DTLs perfused in vitro, unidirectional bath-to-lumen fluxes (Jbl) tended to exceed unidirectional lumen-to-bath fluxes (Jlb), whereas in ATLs perfused in vitro Jlb tended to exceed Jbl, but the differences were not statistically significant. Moreover, none of the unidirectional fluxes was saturable or inhibitable, an observation compatible with apparent reabsorption from ATLs in vivo but incompatible with apparent movement from vasa recta to DTLs in vivo. These in vitro observations are like those made previously for the naturally occurring neutral amino acid l-alanine. The lack of saturation and inhibition, like the previous data on l-alanine, suggest that transepithelial movement of amino acids in thin limbs of Henle's loop may occur via a paracellular route and that regulation of amino acid movement in vivo may involve vasa recta, not DTLs. They also suggest that cycloleucine is a good nonmetabolizable surrogate for the study of neutral amino acid transport in the kidney.
Pflügers Archiv - European Journal of Physiology 01/2000; 439(5):517-523. · 4.46 Impact Factor
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ABSTRACT: 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.
The Journal of Physiology 12/1999; 520 Pt 3:709-21. · 4.72 Impact Factor
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ABSTRACT: 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.
Toxicology 08/1999; 135(1):1-10. · 3.68 Impact Factor
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ABSTRACT: D-Serine normally contributes up to 3% to total plasma serine and up to 23% in chronic renal failure. D-Serine is metabolized by tubular D-amino acid oxidase (D-AAO), and high D-serine plasma levels are nephrotoxic; both events are localized in the straight part of the proximal tubule. We therefore investigated if and how D-serine is reabsorbed there. We microinfused 14C-labeled D- or -L-serine + [3H]inulin into early proximal (EP), late proximal (LP), or early distal (ED) tubule sections of superficial nephrons and into long loops of Henle (LLH) of rats in vivo and in situ. The fractional reabsorption (FR) of the 14C label was determined from the 14C:3H ratio in the final urine. At 0.36 mM, FR of D-[14C]serine was 86% (EP), 90% (LP), and approximately 0 (ED, LLH). FR of D-serine could be saturated and inhibited by L-serine (and vice versa). D-methionine, but not D-glutamate or D-arginine, blocked FR of D-serine (LP). We conlude that filtered D-serine is able to enter the pars recta cells, thereby getting access to D-AAO. The uptake carrier has a very low stereospecificity and is, therefore, different from that in the proximal convolution. The colocalization of exclusive reabsorption and metabolism makes the pars recta the tubule site for the recycling of the carbon structure of D-amino acids and, at the same time, the target of D-serine nephrotoxicity.
The American journal of physiology 07/1999; 276(6 Pt 2):F857-63.
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ABSTRACT: 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.
Cell Biology and Toxicology 02/1999; 15(6):405-15. · 2.51 Impact Factor
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ABSTRACT: 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.
Toxicology 12/1998; 131(2-3):193-202. · 3.68 Impact Factor
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ABSTRACT: Ochratoxin A (OTA) is a widespread mycotoxin, which is nephrotoxic and carcinogenic. Because a decline in net-secretion of para-aminohippuric acid (PAH) was observed after chronic OTA exposition in vivo, we investigated the effect of OTA on proximal-tubule-derived opossum kidney (OK) cells. OTA up to 10(-5) mol/liter had no acute effect on PAH transport when bovine serum albumin (BSA) was present. By contrast, 72-hr incubation of OK cells led to a decrease of PAH transport with half-maximal inhibition at 6 . 10(-7) mol/liter for transepithelial secretion and 6 . 10(-8) mol/liter for basolateral uptake of PAH. Incubation of OK cells with 10(-6) mol/liter OTA for 72 hr reduced the affinity of PAH uptake, and decreased the maximum secretion rate to one-fifth of control values. Apical uptake of amino acids and basolateral uptake of glutarate were not affected. In addition, no signs of general toxic action could be observed. Specific basolateral binding affinity of PAH was reduced to 50% of control. Furthermore, incubation with OTA led to a decrease of PAH efflux across the apical membrane, although efflux across the basolateral membrane and the amount remaining in the cells increased as compared to control. By contrast to control cells, uptake of PAH in OTA-treated cells was not stimulated after preloading with glutarate. Our data show, that 1) long-term incubation with free OTA in the nanomolar range reduces the activity of the organic anion transporter, 2) without influencing general cell function. 3) OTA seems to act preferentially on organic anion transport, by affecting the exchange of organic anions and dicarboxylates. 4) Thereby, OTA reduces its own secretion. 5) The excretion of other xenobiotics and drugs may be also impaired, whereby OTA can exert an indirect toxic action.
Journal of Pharmacology and Experimental Therapeutics 11/1998; 287(1):13-20. · 3.83 Impact Factor
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ABSTRACT: 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.
Toxicology and Applied Pharmacology 10/1998; 152(1):282-91. · 4.45 Impact Factor
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ABSTRACT: 1. The mineralocorticoid aldosterone is essential for the regulation of electrolyte homeostasis, extracellular volume and blood pressure. As a steroid hormone the classical way of action is genomic. Previously we reported a non-genomic action of aldosterone on cytosolic Ca2+ and pH in renal epithelial (MDCK) cells. In parallel, aldosterone induces Zn2+-sensitive cytosolic acidification when extracellular Na+ is absent. 2. We now show that aldosterone (EC50, 7 x 10-11 mol l-1) induces a non-genomic increase in cytosolic sodium in MDCK cells. The membrane-impermeable aldosterone-bovine serum albumin (BSA) conjugate exerted the same effect. The effect of aldosterone was completely abolished by inhibition of Na+-H+ exchange with ethyl-isopropanol amiloride (EIPA). Aldosterone-induced Na+ influx exceeded H+ efflux more than 10-fold. 3. Omission of extracellular Ca2+, inhibition of protein kinase C or pretreatment with pertussis toxin reduced the effect of aldosterone significantly. Zn2+ (IC50, 3.3 x 10-6 mol l-1), but not ouabain, abolished the increase in Na+ almost completely. 4. The aldosterone-induced increase in cytosolic sodium was accompanied by an EIPA- and Zn2+-sensitive cell swelling. 5. Thus, physiological concentrations of aldosterone induce a non-genomic increase in cytosolic sodium concentration by activation of Na+-H+ exchange. Aldosterone exerts its effect, at least in part, at the plasma membrane via interaction with a G-protein-coupled mechanism. 6. The simultaneous activation of the acidification mechanism and Na+-H+ exchange by aldosterone allows a dramatic sodium influx without excessive changes in cytosolic pH and leads to changes in cell volume.
The Journal of Physiology 09/1998; 511 ( Pt 1):255-63. · 4.72 Impact Factor
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ABSTRACT: Ochratoxin A (OTA) is a widespread nephrotoxin excreted to a substantial degree via the kidney. Previously we showed that [3H]OTA can be reabsorbed along the rat nephron in vivo (Zingerle et al., 1997). In this study we investigated in detail the contribution of different nephron segments to [3H]OTA reabsorption and determined the possible mechanisms involved by microinfusion and microperfusion experiments. At pH 6 (approximately 94% of OTA neutral), OTA is reabsorbed in all nephron segments investigated. The estimated fractional reabsorptions (FR) at a tubular load of 20 fmol/min are: proximal convoluted tubule (PCT), 14.8%; proximal straight tubule (PST), 27.4%; ascending limb of Henle's loop (ALH), 13.6%; distal tubule (DT), 11.6%; collecting duct (CD), 24.6%; terminal CD, 22.0%. At pH 8 (approximately 10% of OTA neutral) FR are as follows: PCT, 0%; PST, 25.9%; ALH, 14.0%; DT, 3.2%; CD, 8.2%. Thus, OTA reabsorption in PST and ALH in pH-independent. Reabsorption in PST but not in DT or CD was inhibited by sulfobromophthalein, a substrate of the apical organic anion carrier. L-Phenylalanine did not reduce OTA reabsorption. After intravenous injection of unlabeled OTA, resulting in a plasma concentration of approximately 10(-5) mol/l, the FR of [3H]OTA during early proximal microinfusion was reduced slightly. From our results we conclude: 1) OTA can be reabsorbed in all nephron segments investigated. 2) Under physiological conditions the predominant sites of reabsorption are PST, ALH and terminal CD. 3) Reabsorption in PST and ALH is not pH-dependent. 4) pH-independent reabsorption in PST is mediated by the apical organic anion transporter (OAT-K1), whereas pH-dependent reabsorption in PCT is mediated by H(+)-dipeptide cotransporter(s). 5) Reabsorption also takes place during natural exposure, i.e., when OTA is present in plasma and renal tissue. 6) The high FR in ALH and CD explains, at least in part, the preferential impairment of postproximal functions and the accumulation in renal inner medulla and papila.
Journal of Pharmacology and Experimental Therapeutics 08/1998; 286(1):157-62. · 3.83 Impact Factor
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ABSTRACT: 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.
Journal of the American Society of Nephrology 07/1998; 9(6):960-8. · 9.66 Impact Factor
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Kidney and Blood Pressure Research 02/1998; 21(2-4):277-9. · 1.46 Impact Factor
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ABSTRACT: The mineralocorticoid aldosterone is the most important hormone for the regulation of Na+ and K+ homeostasis in mammals and is thereby involved in the regulation of extracellular volume and blood pressure. Because aldosterone is a steroid hormone, the classical way of action involves transcription, translation, and protein synthesis. We previously reported a rapid, nongenomic, and Zn(2+)-sensitive action of aldosterone on Na+/H+ exchange in renal epithelial [Madin-Darby canine kidney (MDCK)] cells (M. Gekle, N. Golenhofen, H. Oberleithner, and S. Silbernagl. Proc. Natl. Acad. Sci. 93: 10500-10504, 1996). Here we show that, in the absence of Na+ (i.e., with inactive Na+/H+ exchange), aldosterone induces a membrane potential-dependent and Zn(2+)-sensitive cytoplasmic acidification in MDCK cells within 2-4 min. This aldosterone-induced activation of a proton conductance is insensitive to the inhibitor of the classical genomic pathway, spironolactone. Furthermore, the inhibitor of serine/threonine kinases and staurosporine, as well as the specific inhibitor of protein kinase C (PKC), calphostin C, prevented proton conductance activation. Activation of PKC by phorbol esters mimicked the effect of aldosterone. Furthermore, preincubation of the cells with pertussis toxin reduced the effect of aldosterone significantly. We propose a new nongenomic mechanism of action for aldosterone, independently of the intracellular type 1 mineralocorticoid receptor: G protein-dependent stimulation of PKC by aldosterone leads to the activation of a plasma membrane proton conductance that enhances the activity of Na+/H+ exchange. This rapid nongenomic effect could explain the observation that aldosterone may alter renal Na+ and K+ excretion within 5-10 min.
The American journal of physiology 12/1997; 273(5 Pt 1):C1673-8.
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ABSTRACT: This study was designed to elucidate the effects of ochratoxin A (OTA) on pH homeostasis in the kidney. We measured pH in the proximal (PT) and the distal (DT) tubular fluid, the collecting duct urine (CD), the descending and the ascending vasa recta blood (VR), and the renal arterial blood (RA). OTA increased pH significantly in PT, DT, CD as well as in the descending and ascending VR, whereas pH in RA remained unchanged. We further determined CO2 tension (pCO2) and HCO3- in PT, CD as well as in the descending and ascending VR. OTA significantly increased HCO3- in PT, CD and the descending and ascending VR, with no changes in pCO2. Therefore, the increases in pH in PT, CD and the descending and ascending VR result from the increase in HCO3-. Our results suggest that OTA inhibits HCO3- reabsorption in the tubules, leading to the impairment of urinary acidification, and that OTA further leads to the disturbance of the acid-base state (alkalinization) in the interstitium in renal papilla. The impairment of urinary acidification may contribute to the disturbance of pH homeostasis in the renal papilla. The disturbance of pH homeostasis by OTA could be related to its nephrotoxicity.
Pflügers Archiv - European Journal of Physiology 09/1997; 434(4):392-7. · 4.46 Impact Factor
