Biomedical subjects

Michael Gekle

Publications and source records attributed to Michael Gekle.

At least 19 recordsLinked to original sources

PubMed2007

Role of the tumor microenvironment in the activity and expression of the p-glycoprotein in human colon carcinoma cells.

The metabolic microenvironment of solid tumors is characterized by an oxygen deficiency and increased anaerobic glycolysis leading to extracellular acidosis and ATP depletion, which in turn may affect other energy-dependent cellular pathways. Since many tumors overexpress active drug transporters (e.g. the p-glycoprotein) leading to a multidrug-resistant phenotype, this study analyzes the impact of the different aspects of the extracellular microenvironment (hypoxia and acidosis) on the activity and expression of the p-glycoprotein (pGP) in the human colon carcinoma cell line LS513. For up to 24 h cells were exposed to hypoxia (pO2<0.5 mmHg), an acidic extracellular environment (pH 6.6), or the combination of hypoxia and acidosis. Under hypoxic conditions (at a normal pH), the pGP activity (measured by the daunorubicin efflux) and the pGP expression were not markedly altered. Under acidic conditions, however, the pGP-mediated drug efflux was increased, an effect which was even more pronounced when the cells were exposed to hypoxia and acidosis simultaneously (increasing the pGP-activity by 70%). The cellular pGP expression remained almost constant under these conditions, indicating that the increased transport rate results from a functional modulation. The findings of the present study indicate that the parameters of the tumor microenviroment (especially extracellular acidosis) can increase the pGP-mediated drug efflux, an effect which may explain the reduced cytotoxicity of chemotherapeutic agents in hypoxic/acidic tumors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

Long-term effects of ochratoxin A on fibrosis and cell death in human proximal tubule or fibroblast cells in primary culture.

Ochratoxin A (OTA) is a mycotoxin produced by several fungi which grow on human food source material. Consumption of OTA is almost unavoidable. The consumption leads to low but detectable amounts of OTA in human blood. Risk assessment of OTA is based on studies performed either in animals or cultured cells. So far, mainly cell lines of different origin were used. To be as close as possible to the situation in humans with respect to the experimental setup, we studied the effect of OTA in human proximal tubule cells (RPTEC) and human fibroblasts in primary culture. OTA was administered at concentrations ranging from 0.3 nmol/l up to 10 micromol/l for time periods up to 14 days. Apoptotic and necrotic cell death, collagen I, III, IV and fibronectin secretion as well as NF-kappaB activation were studied. Under our experimental conditions OTA exerted comparable effects on caspase-3 activity and necrosis in both cell types, however RPTEC were more sensitive (order of 10). Surprisingly, very low concentrations of OTA (0.3-10nM) led to cell hypertrophy during prolonged exposure (14 days). RPTEC but not fibroblasts responded with an increase of NF-kappaB activity and collagen III as well as fibronectin secretion underlining the profibrotic action of OTA in the kidney. Collagen I and IV secretion was only slightly changed. The results presented here give good reasons to re-asses the risk of OTA consumption leading to low blood concentrations which have so far been considered harmless.

Apoptosis↗

PubMed2006

Ca2+ but not H2O2 modulates GRE-element activation by the human mineralocorticoid receptor in HEK cells.

The mineralocorticcoid receptor (MR) plays an important role in salt and water homeostasis as well as during cardiovascular and renal fibrosis but little is known regarding its modulation by other signaling pathways. To investigate a possible modulation under controlled conditions we used human embryonic kidney (HEK) cells (devoid of endogenous MR) transfected with the human MR and measured transactivation with a GRE-SEAP-reporter construct. MR was compared to the glucocorticoid receptor (GR) as well as to MR lacking the N-terminal domains AB (MR(CDEF)). Chelation of cytosolic Ca2+ enhanced MR activity and SGK1-expression, whereas elevation of cytosolic Ca2+ with ionomycin or thapsigargin reduced MR activity. GR activity was not affected by ionomycin or thapsigargin. MR(CDEF) activity was not affected by chelation or elevation of cytosolic Ca2+. Inhibition of ERK1/2 activation by U0126 or activation of PKA by cAMP, previously shown to modulate MR and GR activity, did not affect MR(CDEF) activity either. H2O2<500micromol/l did not affect basal nor hormone-induced reporter activity. Higher concentrations exerted the same relative inhibitory effect on GRE-SEAP-activity under basal conditions as in the presence of aldosterone-stimulated MR and elicited cytotoxic effects. Our data indicate that the genomic function of MR can be modulated by cytosolic Ca2+, PKA and ERK1/2 via an interaction with the AB-domain. H2O2 seems not to affect relative MR activity directly under our experimental conditions.

Calcium↗

PubMed2006

Chloroacetaldehyde as a sulfhydryl reagent: the role of critical thiol groups in ifosfamide nephropathy.

Chloroacetaldehyde (CAA) is a metabolite of the alkylating agent ifosfamide (IFO) and putatively responsible for renal damage following anti-tumor therapy with IFO. Depletion of sulfhydryl (SH) groups has been reported from cell culture, animal and clinical studies. In this work the effect of CAA on human proximal tubule cells in primary culture (hRPTEC) was investigated. Toxicity of CAA was determined by protein content, cell number, LDH release, trypan blue exclusion assay and caspase-3 activity. Free thiols were measured by the method of Ellman. CAA reduced hRPTEC cell number and protein, induced a loss in free intracellular thiols and an increase in necrosis markers. CAA but not acrolein inhibited the cysteine proteases caspase-3, caspase-8 and cathepsin B. Caspase activation by cisplatin was inhibited by CAA. In cells stained with fluorescent dyes targeting lysosomes, CAA induced an increase in lysosomal size and lysosomal leakage. The effects of CAA on cysteine protease activities and thiols could be reproduced in cell lysate. Acidification, which slowed the reaction of CAA with thiol donors, could also attenuate effects of CAA on necrosis markers, thiol depletion and cysteine protease inhibition in living cells. Thus, CAA directly reacts with cellular protein and non-protein thiols, mediating its toxicity on hRPTEC. This effect can be reduced by acidification. Therefore, urinary acidification could be an option to prevent IFO nephropathy in patients.

Acetaldehyde↗

PubMed2006

Transient activation and delayed inhibition of Na+,K+,Cl- cotransport in ATP-treated C11-MDCK cells involve distinct P2Y receptor subtypes and signaling mechanisms.

In C11-MDCK cells, which resemble intercalated cells from collecting ducts of the canine kidney, P2Y agonists promote transient activation of the Na+,K+,Cl- cotransporter (NKCC), followed by its sustained inhibition. We designed this study to identify P2Y receptor subtypes involved in dual regulation of this carrier. Real time polymerase chain reaction analysis demonstrated that C11-MDCK cells express abundant P2Y1 and P2Y2 mRNA compared with that of other P2Y receptor subtypes. The rank order of potency of agents (ATP approximately UTP >> 2-(methylthio)-ATP (2MeSATP); adenosine 5'-[beta-thio]diphosphate (ADPbetaS) inactive) indicated that P2Y2 rather than P2Y1 receptors mediate a 3-4-fold activation of NKCC within the first 5-10 min of nucleotide addition. NKCC activation in ATP-treated cells was abolished by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin (CaM) antagonists trifluoroperazine and W-7, and KN-62, an inhibitor of Ca2+/CaM-dependent protein kinase II. By contrast with the transient activation, 30-min incubation with nucleotides produced up to 4-5-fold inhibition of NKCC, and this inhibition exhibited a rank order of potency (2MeSATP > ADPbetaS > ATP >> UTP) typical of P2Y1 receptors. Unlike the early response, delayed inhibition of NKCC occurred in 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-loaded cells and was completely abolished by the P2Y1 antagonists MRS2179 and MRS2500. Transient activation and delayed inhibition of NKCC in C11 cell monolayers were observed after the addition of ATP to mucosal and serosal solutions, respectively. NKCC inhibition triggered by basolateral application of ADPbetaS was abolished by MRS2500. Our results thus show that transient activation and delayed inhibition of NKCC in ATP-treated C11-MDCK cells is mediated by Ca2+/CaM-dependent protein kinase II- and Ca2+-independent signaling triggered by apical P2Y2 and basolateral P2Y1 receptors, respectively.

Adenosine Triphosphate↗

PubMed2006

Aldosterone stimulates activity and surface expression of NHE3 in human primary proximal tubule epithelial cells (RPTEC).

The steroid hormone aldosterone is a major regulator of extracellular volume and blood pressure. Aldosterone effectors are for example the epithelial Na(+) channel (ENaC), the Na(+)-K(+)-ATPase and the proximal tubule Na(+)/H(+) exchanger isoform 3 (NHE3). The aim of this study was to investigate whether aldosterone acts directly on proximal tubule cells to stimulate NHE3 and if so whether the EGF-receptor (EGFR) is involved. For this purpose, primary human renal proximal tubule cells were exposed to aldosterone. NHE3 activity was determined from Na(+)- dependent pH-recovery, NHE3 surface expression was determined by biotinylation and immunoblotting. EGFR-expression was assessed by ELISA. pH(i)- measurements revealed an aldosterone-induced increase in NHE3 activity, which was inhibited by the mineralocorticoid receptor blocker spironolactone and by the EGFR-kinase inhibitor AG1478. Immunoprecipitation and immunoblot analysis showed an aldosterone-induced increase in NHE3 surface expression, which was also inhibited by spironolactone and AG1478. Furthermore, aldosterone enhanced EGFR-expression. In conclusion, aldosterone stimulates NHE3 in human proximal tubule cells. The underlying mechanisms include AG1478 inhibitable kinase and are paralleled by enhanced EGFR expression, which could be compatible with EGF-receptor-pathway-dependent surface expression and activity of NHE3 in human primary renal proximal tubule epithelial cells.

Aldosterone↗

PubMed2006

Impact of extracellular acidity on the activity of P-glycoprotein and the cytotoxicity of chemotherapeutic drugs.

The expression and activity of P-glycoprotein (pGP) play a role in the multidrug resistance of tumors. Because solid-growing tumors often show pronounced hypoxia or extracellular acidosis, this study attempted to analyze the impact of an acidic environment on the expression and activity of pGP and on the cytotoxicity of chemotherapeutic agents. For this, prostate carcinoma cells were exposed to an acidic extracellular environment (pH 6.6) for up to 24 hours. pGP activity was more than doubled after 3 to 6 hours of incubation in acidic medium, whereas cellular pGP expression remained constant, indicating that increased transport rate is the result of functional modulation. In parallel, the cytotoxic efficacy of daunorubicin showed pronounced reduction at low pH, an effect that was reversible on coincubation with a pGP inhibitor. A reduction of intracellular Ca2+ concentration by 35% under acidic conditions induced a higher transport rate of pGP, an effect comparable to that found on inhibition of protein kinase C (PKC). These data indicate that pGP activity is increased by low extracellular pH presumably as a result of lowered intracellular calcium levels and inhibition of PKC. These findings may explain the reduced cytotoxicity of chemotherapeutic agents in hypoxic/acidic tumors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

Prostaglandin E2 inhibits its own renal transport by downregulation of organic anion transporters rOAT1 and rOAT3.

Prostaglandin E2 (PGE2) is the principal mediator of fever and inflammation. Recently, evidence emerged that during febrile response, PGE2 that is generated in the periphery enters the hypothalamus and contributes to the maintenance of fever. In a rat model of fever generation, peripheral PGE2 is increased, whereas clearance by metabolism of peripheral PGE2 is downregulated. The major route of PGE2 excretion is via the renal proximal tubular organic anion secretory system, where basolateral uptake that is mediated by renal organic anion transporter 1 (rOAT1) and rOAT3 is rate limiting. Therefore, it was hypothesized that PGE2 itself will abolish its excretion by rOAT1 or rOAT3. Fluorescein was used as a prototypic organic anion, and NRK-52E cells from rat served as a proximal tubular model system. PGE2 time-dependently downregulates basolateral organic anion uptake, without affecting cell volume or cell protein, recirculation of counter ions, or proximal tubular transport systems in general. In addition, PGE2 diminishes expression of both rOAT1 and rOAT3. Both organic anion uptake and expression of rOAT1 and rOAT3 are dose-dependently downregulated by PGE2. These findings suggest that during fever or inflammation, renal secretory transport of PGE2 is reduced, contributing to elevated PGE2 levels in blood. These data fit into the hypothetical concept of peripheral PGE2's playing a significant role in fever. The described regulatory mechanism may also be of relevance in chronic inflammatory events. Moreover, the data presented could explain why increased plasma urate levels occur in diseases that go along with increased levels of PGE2.

Animals↗

PubMed2005

Chloroacetaldehyde- and acrolein-induced death of human proximal tubule cells.

Ifosfamide (ifo) is a commonly used drug in chemotherapy. It is metabolized to acrolein (acro) and chloroacetaldehyde (CAA), which are thought to be responsible for renal side effects. We studied the effects of ifo and cyclophosphamide (cyclo) as well as their metabolites, acro and CAA, on cellular protein content, necrosis, apoptosis and cytosolic calcium concentration using a human proximal tubule cell line. The protein content decreased during acro or CAA administration (15 to 300 micromol/l), but not during ifo or cyclo exposure over a time period of up to 72 h. Mild apoptosis was induced only by high acro (150, 300 micromol/l) and low CAA concentrations (15, 75 micromol/l) and only in a narrow time window (24 h). Necrosis was increased after exposure to acro or CAA at all concentrations. CAA was more potent than acro. Ifo and cyclo did not induce necrosis or apoptosis. Glutathione abolished CAA-induced cell death. Cytosolic calcium concentrations increased after acro or CAA administration and showed an oscillating pattern. Cytosolic Ca(2+) chelation did not prevent necrosis. We conclude that neither ifo nor cyclo induce cell damage, but that their metabolites acro and CAA induce cell death. This cell death occurs mainly by necrosis and not by apoptosis.

Acetaldehyde↗

PubMed2005

Cl- secretion in ATP-treated renal epithelial C7-MDCK cells is mediated by activation of P 2Y1 receptors, phospholipase A2 and protein kinase A.

This study examines the mechanism of P 2Y-induced Cl- secretion in monolayers of C7-Madin-Darby canine kidney (MDCK) cells triggered by basolateral application of ATP and measured as transcellular short current (I(SC)). Both ATP-induced arachidonic acid (AA) synthesis and I(SC) in ATP-treated cells were abolished by the phosholipase A2 (PLA2) inhibitor, AACOCF3. The cyclo-oxygenase inhibitor indomethacin decreased I(SC) and cAMP production in ATP-treated cells with an IC50 of approximately 0.3 microm. ATP led to rapid activation of cAMP-dependent protein kinase A (PKA), as estimated by phosphorylation of a vasodilator-stimulated phosphoprotein. PKA activity and I(SC) evoked by ATP, as well as by prostaglandin E1 (PGE1), were diminished in the presence of the PKA inhibitor H-89 or an adenovirus-mediated expression of PKA-inhibitor protein, PKI. In contrast, indomethacin completely blocked the increment of PKA and I(SC) triggered by ATP and AA, but did not affect PKA activation and I(SC) detected with PGE1. The kinetics of [Ca2+]i elevation in ATP- and thapsigargin-treated cells were similar and suppressed by the Ca(2+)i chelator BAPTA. Neither baseline nor maximal increment of ATP-induced I(SC) was affected by thapsigargin and BAPTA. Real-time PCR showed that C7 cells express more mRNA for P 2Y1 and P 2Y2 than for other P 2Y receptor subtypes. The rank order of potency (2MeSATP > ATP > ADP >> UTP) indicates that P 2Y1 rather than P 2Y2 receptors contribute to PKA and I(SC) activation. Viewed collectively, these data show that Cl- secretion in C7-MDCK monolayers treated with basolateral ATP is triggered by P 2Y1 receptors and is mediated by subsequent [Ca2+]i-independent activation of PLA2 and PKA.

Adenosine Triphosphate↗

PubMed2005

Human mineralocorticoid receptor expression renders cells responsive for nongenotropic aldosterone actions.

The steroid hormone aldosterone is important for salt and water homeostasis as well as for pathological tissue modifications in the cardiovascular system and the kidney. The mechanisms of action include a classical genomic pathway, but physiological relevant nongenotropic effects have also been described. Unlike for estrogens or progesterone, the mechanisms for these nongenotropic effects are not well understood, although pharmacological studies suggest a role for the mineralocorticoid receptor (MR). Here we investigated whether the MR contributes to nongenotropic effects. After transfection with human MR, aldosterone induced a rapid and dose-dependent phosphorylation of ERK1/2 and c-Jun NH2-terminal kinase (JNK) 1/2 kinases in Chinese hamster ovary or human embryonic kidney cells, which was reduced by the MR-antagonist spironolactone and involved cSrc kinase as well as the epidermal growth factor receptor. In primary human aortic endothelial cells, similar results were obtained for ERK1/2 and JNK1/2. Inhibition of MAPK kinase (MEK) kinase but not of protein kinase C prevented the rapid action of aldosterone and also reduced aldosterone-induced transactivation, most probably due to impaired nuclear-cytoplasmic shuttling of MR. Cytosolic Ca2+ was increased by aldosterone in mock- and in human MR-transfected cells to the same extend due to Ca2+ influx, whereas dexamethasone had virtually no effect. Spironolactone did not prevent the Ca2+ response. We conclude that some nongenotropic effects of aldosterone are MR dependent and others are MR independent (e.g. Ca2+), indicating a higher degree of complexity of rapid aldosterone signaling. According to this model, we have to distinguish three aldosterone signaling pathways: 1) genomic via MR, 2) nongenotropic via MR, and 3) nongenotropic MR independent.

Aldosterone↗

PubMed2005

Cisplatin-induced apoptosis is enhanced by hypoxia and by inhibition of mitochondria in renal collecting duct cells.

Cisplatin is a widely used chemotherapeutic agent. Here we show that cisplatin induces apoptosis in renal collecting duct-derived cells (MDCK-C7 cells, resembling principal cells) in a dose-dependent manner. Additionally, we studied the role of mitochondria in this process by inhibition of the mitochondrial respiratory chain, the F1F(o)-ATP synthase or by uncoupling. The role of intra- and extracellular pH in apoptosis induction was investigated. Activation of caspase-3 and DNA ladder formation were used to monitor the apoptotic response. When cells were incubated with inhibitors of the mitochondrial respiratory chain or an inhibitor of the ATP-synthase, cisplatin-induced apoptosis was markedly enhanced. Mitochondrial blockade led to enhanced production of lactic acid. Also, anoxia potentiated the cisplatin-induced caspase-3 activation. Neither intra- nor extracellular pH had an influence on caspase-3 activation at low cisplatin concentrations. Acidic conditions (pH 6.8) potentiated the caspase-3 activation when high (100 microM) cisplatin concentrations were used. We demonstrate that intact mitochondria are important to prevent cisplatin-induced apoptosis in MDCK-C7 cells and that acidic conditions can aggravate the toxic effects of cisplatin.

Animals↗

PubMed2005

Effect of the mycotoxin, ochratoxin A, on hormone-stimulated ion transport in a cultured cell model of the renal principal cell.

The mycotoxin ochratoxin A (OTA) is a common contaminant of many foodstuffs and, consequently, is present in a large proportion of tested populations of humans and commercial animals. The predominant effects of OTA are manifested in the kidney where the severity varies from salt wasting to renal carcinoma formation in a concentration-dependent fashion. The MDCK-C7 renal cell culture model responds to various hormones known to regulate electrolyte and fluid balance and was used as a model to study the chronic effects of an acute exposure to low dose OTA. The natriferic hormones aldosterone and insulin-like growth factor 1 (IGF1) both stimulate Na(+) flux in a reabsorptive direction via activation of the epithelial Na(+) channel (ENaC). In contrast, anti-diuretic hormone (ADH) stimulates three separate and temporally distinct ion transport responses, one of which is Na(+) reabsorption. Treatment of MDCK-C7 cells with OTA (100 nM) for 48 h selectively and irreversibly inhibits hormone-stimulated Na(+) reabsorption via ENaC. This effect was retained for 48 cell passages after the removal of the toxin and mimics the OTA-induced salt-wasting that has been documented in clinical studies. These studies indicate that the effect of the toxin is genomic and therefore, likely to be long lasting in exposed animals and humans.

Animals↗

PubMed2005

Exposure to nephrotoxic ochratoxin A enhances collagen secretion in human renal proximal tubular cells.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin. There is evidence that OTA leads to cortical interstitial nephropathies in humans, associated with fibrosis. No data are available on the effect of OTA-induced collagen secretion from renal cortical cells. As kidney cortex mainly consists of proximal tubules, we investigated the effect of OTA on particular collagens (I, III, IV) in a well-established proximal tubular cell line (opossum kidney (OK) cells) and in primary cultured human renal proximal tubular epithelial cells (RPTECs). In fibroblasts, OTA neither exerted toxic effects nor induced collagen secretion, most probably due to the absence of suitable uptake mechanisms. OTA exerted time- and dose-dependent toxicity in both OK cells and human RPTECs. Moreover, OTA induced collagen secretion in a time- and dose-dependent manner in both cell types. In opposite to transforming growth factor beta1 (TGF-beta1), OTA incubation induced increased apical secretion of the basement membrane collagen IV. This might be evidence for a loss of cellular polarity after OTA incubation. We conclude that in proximal tubular cells, OTA is able to induce extracellular matrix deposition. As collagen secretion was also inducible in primary cultured human RPTECs, we hypothesize that OTA-induced extracellular matrix deposition by proximal tubular cells may be of importance in generation of renal diseases in humans which are under suspicion of being induced by OTA.

Animals↗

PubMed2005

Ochratoxin A at nanomolar concentrations: a signal modulator in renal cells.

Ochratoxin A (OTA) is a ubiquitous fungal metabolite with nephrotoxic, carcinogenic, and apoptotic potential. Toxicokinetics make the kidney the primary target organ for OTA. Due to its widespread occurrence in improperly stored foodstuff the complete and safe avoidance of OTA for humans is impossible. There are several reports showing a significant correlation between OTA exposure and certain forms of nephropathies. At nanomolar concentrations OTA leads to specific changes of function and phenotype in renal cells. The toxin interacts with certain cellular "key-molecules" (e. g., mitogen-activated protein (MAP) kinases, Ca2+), thereby disturbing cellular signalling and regulation events as well as mitochondrial function. Moreover, OTA has the ability to modulate physiological signals (e. g., angiotensin II or TNFalpha) and thereby influences cell function and cell growth and may even stable re-program the cells (e. g., altered distribution of chromosomes). This review concentrates on the effects of OTA in the nanomolar range and its interactions with cellular signalling networks in different renal cells proposing OTA to act as a signal modulator.

Animals↗

PubMed2005

A rapid screening method to test apoptotic synergisms of ochratoxin A with other nephrotoxic substances.

The body may be exposed simultaneously to more than one nephrotoxic substance and to measure the effects of the great number of possible combinations of nephrotoxins will rapidly become a great challenge when using the traditional methods. Therefore, we developed a rapid and cost-efficient method to screen the apoptotic potential of combinations of known cell- or nephrotoxic substances as ochratoxin A (OTA), cisplatin, cadmium, H(2)O(2), and amphotericin B on renal epithelial cell lines. The cells were seeded in 96-well plates and the apoptotic and necrotic potential of different combinations of nephrotoxins was determined. We found different results for the combinations used: depending on the concentrations of the various substances, antagonistic, additive, or potentiating effects on caspase-3 activity were found after co-exposure to OTA. 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, the concentration and on the cell line investigated. A "harmless" substance can thus convert to a potent cell toxic substance when combined with OTA or vice versa. The underlying mechanisms of the synergistic effects remain unknown.

Apoptosis↗

PubMed2005

Disturbed Ca2+-signaling by chloroacetaldehyde: a possible cause for chronic ifosfamide nephrotoxicity.

BACKGROUND: Renal damage following chemotherapy with ifosfamide is attributed to the metabolic activation of the drug and the generation of chloroacetaldehyde (CAA). Little is known about the mechanism by which CAA impairs renal function. In this study the effect of CAA on intracellular Ca(2+) homeostasis in human renal proximal tubule cells (RPTEC) in primary culture was investigated. METHODS: Intracellular Ca(2+) was measured using the Ca(2+)-sensitive dye fura-2. Cell viability was determined by protein content and cell number. Oncotic and apoptotic cell death was assayed using trypan blue exclusion, caspase-3 activity, and 4',6-diamino-2-phenylindole (DAPI) staining. RESULTS: CAA (1.5 to 150 micromol/L) induced sustained elevations of intracellular free calcium ([Ca(2+)](i)) from 75 +/- 3 nmol/L to maximal 151 +/- 6 nmol/L. This effect was dependent on extracellular Ca(2+), but not Ca(2+) entry. The rise in [Ca(2+)](i) mediated by CAA could be attributed to inhibition of Na(+)-dependent extrusion of intracellular Ca(2+), indicating an inhibitory action of CAA on Na(+)/Ca(2+) exchange. Modulation of protein kinase A (PKA), but not protein kinase C (PKC) blunted the effect of CAA. Thus, CAA seems to inhibit Na(+)/Ca(2+) exchange by interaction with cyclic adenosine monophosphate (cAMP)-PKA-signaling. A 48-hour exposure to 15 micromol/L CAA significantly reduced cell number and protein content of RPTEC by induction of necrosis. This effect of 15 micromol/L CAA could be overcome by coadministration of the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM). CONCLUSION: First, CAA inhibits the Na+/Ca2+-exchanger. Second, this effect is dependent on PKA. Third, CAA induces necrotic rather than apoptotic cell death. Finally, disturbed Ca(2+) homeostasis via Na(+)/Ca(2+) exchange contributes to the nephrotoxic action of CAA in RPTEC.

Acetaldehyde↗

PubMed2005

Renal tubule albumin transport.

Albumin is the most abundant protein in serum and contributes to the maintenance of oncotic pressure as well as to transport of hydrophobic molecules. Although albumin is a large anionic protein, it is not completely retained by the glomerular filtration barrier. In order to prevent proteinuria, albumin is reabsorbed along the proximal tubules by receptor-mediated endocytosis, which involves the binding proteins megalin and cubilin. Endocytosis depends on proper vesicle acidification. Disturbance of endosomal acidification or loss of the binding proteins leads to tubular proteinuria. Furthermore, endocytosis is subject to modulation by different signaling systems, such as protein kinase A (PKA), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3-K) and transforming growth factor beta (TGF-beta). In addition to being reabsorbed in the proximal tubule, albumin can also act as a profibrotic and proinflammatory stimulus, thereby initiating or promoting tubulo-interstitial diseases.

Animals↗