Ruth Freudinger

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.

Aldosterone Stimulates Epidermal Growth Factor Receptor Expression

The steroid hormone aldosterone plays an important role during pathological tissue modifications, similar to cardiovascular or renal fibrosis. The underlying mechanisms for the pathological actions are not understood. Interaction of aldosterone with the epidermal growth factor (EGF) receptor is an attractive hypothesis to explain pathological tissue remodeling elicited by aldosterone, because (i) mineralocorticoids can sensitize cells for EGF, (ii) mineralocorticoid receptor (MR)-antagonists reduce EGFR-mRNA expression, (iii) EGFR itself supports the development of cardiovascular or renal fibrosis, and (iv) signaling elements involved in the pathological action of aldosterone (similar to ERK1/2 or NFkB) are typical downstream modules during EGF signaling. In addition, an interaction of aldosterone and EGF with respect to ERK1/2 activation has been described. Here we show that aldosterone stimulates EGFR expression in renal tissue of adrenalectomized rats and in human renal primary cell cultures. Furthermore, Chinese hamster ovary (CHO) cells normally devoid of EGFR or MR express EGFR after transfection with human MR (CHO-MR cells) but not after transfection with human glucocorticoid receptor (CHO-GR cells). In CHO-MR cells, EGFR-expression is up-regulated by aldosterone and inhibited by spironolactone. CHO-MR cells but not CHO-GR cells respond with ERK1/2 phosphorylation to EGF exposure. The responsiveness to other peptide hormones was virtually not affected. These data suggest that EGFR is an aldosterone-induced protein and is involved in the manifold (patho)biological actions of aldosterone.

Transforming growth factor‐β1 reduces megalin‐ and cubilin‐mediated endocytosis of albumin in proximal‐tubule‐derived opossum kidney cells

Transforming growth factor (TGF)‐β1 is a member of a superfamily of multifunctional cytokines involved in several pathological processes of the kidney, including fibrogenesis, apoptosis and epithelial‐mesenchymal transition. These events lead to tubulointerstitial fibrosis and glomerulosclerosis. Less is known about TGF‐β1‐induced alterations of cell function. An important function of proximal tubular cells is reabsorption of filtered proteins, including albumin, via megalin‐cubilin‐dependent receptor‐mediated endocytosis. In this study we used a well established cell culture model (proximal‐tubule‐derived opossum kidney (OK) cells) in order to test the hypothesis that TGF‐β1 reduces megalin‐cubilin‐mediated endocytosis. Previously we have shown that albumin endocytosis in OK cells is mediated by megalin/cubulin. TGF‐β1 led to a time‐ and dose‐dependent downregulation of megalin‐cubilin‐mediated endocytosis without affecting two other transport systems tested. Binding, internalization and intracellular trafficking of the ligand albumin were affected. Decreased binding resulted from reduced cubilin and megalin expression in the 200 000 g membrane fraction. The underlying mechanism of TGF‐β1 action does not involve mitogen‐activated protein kinases, protein kinase C or A, or reactive oxygen species. In contrast, TGF‐β1‐induced downregulation of megalin‐cubilin‐mediated endocytosis was sensitive to inhibition of translation and transcription and was preceded by Smad2 and 3 phosphorylation. Dominant negative Smad2/3 constructs prevented the effect of TGF‐β1. In conclusion our data indicate that enhanced levels of TGF‐β1 occurring in various nephropathies can lead to downregulation of megalin‐cubilin‐dependent endocytosis. Probably, TGF‐β1 leads to Smad2‐ and Smad3‐dependent expression of negative regulators of receptor‐mediated endocytosis.

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 °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.

Abstract Fumonisin B 1 (FB 1 ) 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 FB 1 , fumonisin B 2 (FB 2 ), fumonisin B 3 (FB 3 ), hydrolyzed fumonisin B 1 (HFB 1 ) and N-palmitoyl-hydrolyzed fumonisin B 1 (N-Pal-HFB 1 ) and investigated caspase-3 activation, chromatin condensation and DNA fragmentation. Exposure to 10 μmol/L FB 1 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 FB 1 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.

EF Domains Are Sufficient for Nongenomic Mineralocorticoid Receptor Actions

The mineralocorticoid receptor (MR) is important for salt homeostasis and reno-cardiovascular pathophysiology. Signaling mechanisms include, besides classical genomic pathways, nongenomic pathways with putative pathophysiological relevance involving the mitogen-activated protein kinases ERK1/2. We determined the MR domains required for nongenomic signaling and their potential to elicit pathophysiological effects in cultured cells under defined conditions. The expression of full-length human MR or truncated MR consisting of the domains CDEF (MRCDEF), DEF (MRDEF), or EF (MREF) renders cells responsive for the MR ligand aldosterone with respect to nongenomic ERK1/2 phosphorylation, whereas only full-length MR and MRCDEF conferred genomic responsiveness. ERK1/2 phosphorylation depends on the EGF receptor and cSRC kinase. MREF expression is sufficient to evoke the aldosterone-induced increase of collagen III levels, similar to full-length MR expression. Our data suggest that nongenomic MR signaling is mediated by the EF domains and present the first proof of principle showing that nongenomic signaling can be sufficient for some pathophysiological effects. The minimum amino acid motif required for nongenomic MR signaling and its importance in various effects have yet to be determined.

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.

Albumin Induces NF-κB Expression in Human Proximal Tubule-Derived Cells (IHKE-1)

Background: Chronic renal diseases with enhanced glomerular protein filtration are accompanied by tubulointerstitial inflammation and progression to renal function deterioration. Filtered proteins, like albumin, seem to be a pathogenic factor per se in the progression of renal diseases. There is evidence that the nuclear factor ĸB (NF-ĸB) is involved in protein-overload stimulated renal inflammatory pathomechanisms. The aim of this study was to investigate albumin-induced NF-ĸB expression as well as NF-ĸB activity upon long long term exposure to albumin in human proximal tubular cells as only acute albumin-induced NF-ĸB activity has been reported so far.Methods: To investigate the hypothesis, that NF-ĸB may be involved in protein-induced renal inflammatory pathomechanisms, we exposed human renal proximal tubule-derived cells (IHKE-1) to bovine serum albumin (BSA: 50 and 500 µg/ml). The NF-ĸB and TNF-α specific mRNA expression was detected by RT-PCR. NF-ĸB specific protein expression was analysed by Western blot. Reporter gene assays were performed to determine the NF-ĸB specific activity.Results: Albumin-exposure induced an increase in NF-ĸB specific mRNA expression, NF-ĸB protein expression and activity. These effects are decreased by the protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM) and the tyrosine kinase inhibitor herbimycin A. An albumin-induced increase in TNF-α specific mRNA expression as biological, inflammatory parameter associated with the albumin-induced NF-ĸB activity was detectable.Conclusion: We suggest, that albumin-exposure induces an increase in NF-ĸB and TNF-α specific mRNA expression, NF-ĸB specific protein expression and protein activity in renal proximal tubule cells in culture, which is at least in part PKC and tyrosine kinase dependent.

Rapid actions of aldosterone on cells from renal epithelium: the possible role of EGF-receptor signaling.

It has been suggested that steroids interact with peptide hormones in part by rapid, potentially non-genomic, mechanisms. The peptide hormone epidermal growth factor (EGF) regulates cell proliferation and ion transport using ERK1/2 as downstream signal. Furthermore, the EGF-receptor (EGF-R) is involved in signaling by G-protein-coupled receptors, growth hormone and cytokines via transactivation. We show that aldosterone modulates Na(+)/H(+)-exchange in renal collecting duct-derived Madin-Darby canine kidney (MDCK) cells via ERK1/2 in a similar way as compared to growth factors. Furthermore, we tested the hypothesis that aldosterone uses the EGF-R as heterologous signal transducer in MDCK cells. Aldosterone induces a rapid increase of ERK1/2 phosphorylation and cytosolic Ca(2+)-concentration of similar extend as compared to EGF. Furthermore, aldosterone stimulates EGF-R Tyr-phosphorylation. Inhibition of EGF-R kinase abolished aldosterone-induced signaling. Aldosterone-induced Ca(2+)-influx seems to be mediated by the activation of ERK1/2, whereas ERK1/2 activation does not depend on Ca(2+)-influx. Our data show that aldosterone uses the EGF-R-ERK1/2 signaling cascade to elicit its rapid effects in MDCK cells.

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.