Biomedical subjects

Michael Gekle

Publications and source records attributed to Michael Gekle.

At least 37 records · Page 2Linked to original sources

PubMed2005

The nephrotoxin ochratoxin A induces key parameters of chronic interstitial nephropathy in renal proximal tubular cells.

Ochratoxin A (OTA) is a nephrotoxic and cancerogenic mycotoxin. There is epidemiological evidence that OTA exposition leads to cortical interstitial nephropathies in humans. However, virtually no data are available investigating the effect of OTA on renal cortical cells with respect to induction of nephropathy. Thus, we investigated whether OTA is able to induce changes of cellular properties potentially leading to interstitial nephropathy, using proximal tubular cell lines (OK, NRK-52E). OTA decreased cell number and cell protein time and dose dependently. Accordingly we investigated the effect of 100 nM or 1000 nM OTA. The decline of cell number after OTA exposure is due to necrosis and apoptosis, as measured by LDH release or DNA ladder formation and caspase-3 activation, respectively. OTA incubation of proximal tubular cells also resulted in a loss of epithelial tightness as determined by diffusion of FITC labeled inulin. Inflammation, fibrosis and epithelial-to-mesenchymal transition are described in chronic interstitial renal disease. Therefore, we also investigated the effect of OTA on NFkappaB activity, collagen secretion and generation of alpha smooth muscle actin. OTA alone was sufficient to induce the latter parameters in proximal tubular cells. Finally, OTA is a nephrotoxcic substance and elevated activity of mitogen activated protein kinases (MAPK) is described in nephropathies. As we investigated the effect of OTA on activity of ERK, JNK and p38 by ELISA, we found that OTA activates the MAPK measured dose dependently. In summary, OTA induced phenomena typical for chronic interstitial nephropathy, like loss of cells and epithelial tightness, necrosis and apoptosis as well as markers of inflammation, fibrosis and epithelial-to-mesenchymal transition in proximal tubular cells. Thus, we could show for the first time that OTA is able to induce key parameters of nephropathy in proximal tubular cells in culture. Moreover OTA interacts with MAPK and thus may exert its specific toxic actions.

Apoptosis↗

PubMed2005

Megalin is essential for renal proximal tubule reabsorption of (111)In-DTPA-octreotide.

UNLABELLED: Radiolabeled somatostatin analogs have been shown to be important radiopharmaceuticals for tumor diagnosis and radionuclide therapy. The kidney has appeared to be the critical organ during radionuclide therapy because of peptide reabsorption and retention in the proximal tubules after glomerular filtration. The molecular mechanism of renal reabsorption of these analogs has not been clarified. A possible receptor candidate is megalin, a multiligand scavenger receptor in the renal proximal tubules. The objective of this study was to investigate the role of megalin in tubular reabsorption of radiolabeled somatostatin analogs by using kidney-specific megalin-deficient mice versus mice with normal renal megalin expression. [(111)In-Diethylenetriaminepentaacetic acid (DTPA)]octreotide was used as a practical model of peptide. METHODS: Renal uptake of [(111)In-DTPA]octreotide was determined by animal SPECT scintigraphy at different time points after injection of the tracer and by measurement of radioactivity after isolation of the organs. Furthermore, ex vivo autoradiography of renal sections revealed the zonal distribution of radioactivity in the megalin-deficient and megalin-expressing kidneys. RESULTS: SPECT scintigraphy of [(111)In-DTPA]octreotide at 3 and 24 h after injection clearly showed lower renal radioactivity in megalin-deficient kidneys than in megalin-expressing kidneys, both in male and in female mice, in accordance with counts obtained after isolation of the organ (70%-85% reduction of uptake in the megalin-deficient kidneys, P < 0.001). Renal uptake of [(111)In-DTPA]octreotide was significantly higher in female than in male kidneys (P < 0.001). Ex vivo autoradiograms clearly showed that renal radioactivity was not homogeneously distributed in the megalin-expressing kidneys but localized in the renal cortex. Quantification of the autoradiogram data confirmed the reduced radioactivity in the renal cortex of megalin-deficient kidneys. CONCLUSION: This study revealed the molecular mechanism of [(111)In-DTPA]octreotide uptake in renal proximal tubules involving the receptor megalin. Identification of megalin may be crucial for further research into strategies to reduce renal uptake.

Absorption↗

PubMed2004

NHE3 Na+/H+ exchanger supports proximal tubular protein reabsorption in vivo.

Proximal tubular receptor-mediated endocytosis (RME) of filtered proteins prevents proteinuria. Pharmacological and genetic studies in cultured opossum kidney cells have shown that the apical Na(+)/H(+) exchanger isoform 3 (NHE3) supports RME by interference with endosomal pH homeostasis and endocytic fusion events. However, it is not known whether NHE3 also supports proximal tubular RME in vivo. We analyzed proximal tubular protein reabsorption by microinfusion experiments in rats and investigated renal protein excretion in NHE3 knockout (Nhe3 -/-) mice. Inhibition of NHE3 by EIPA or S-3226 reduced the fractional reabsorption of [(14)C]cytochrome c by approximately 50% during early proximal microinfusion. During early distal microinfusion, no protein reabsorption could be detected. Urinary protein excretion of Nhe3 -/- or heterozygous mutant mice was significantly higher compared with wild-type mice. SDS-PAGE analysis of urinary proteins revealed that Nhe3 -/- animals excreted proteins the size of albumin or smaller. Thus a reduction in NHE3 activity or abundance causes tubular proteinuria. These data show that NHE3 supports proximal tubular RME of filtered proteins in vivo.

Absorption↗

PubMed2004

Evidence for epidermal growth factor receptor as negative-feedback control in aldosterone-induced Na+ reabsorption.

Aldosterone enhances Na(+) reabsorption via epithelial Na(+) channels (ENaC). Aldosterone also stimulates the protein kinase ERK1/2- and the epidermal growth factor (EGF) receptor (EGFR)-signaling pathway. Yet EGF and ERK1/2 are known inhibitors of ENaC-mediated Na(+) reabsorption. In the present study, using the well-established Madin-Darby canine kidney C7 cell line, we tested the hypothesis that EGFR represents a negative-feedback control for chronic aldosterone-induced Na(+) reabsorption [amiloride-inhibitable short-circuit current (I(sc))]. Mineralocorticoid receptor expression was confirmed by RT-PCR and Western blot analysis. Aldosterone enhanced ERK1/2 phosphorylation in an EGFR-dependent way. Furthermore, aldosterone stimulated EGFR expression. Aldosterone (10 nmol/l) induced a small transient increase in I(sc) under control conditions. Inhibition of ERK1/2 phosphorylation with U-0126 (10 micromol/l) stimulated I(sc), indicating constitutive ENaC inhibition. Aldosterone exerted a significantly larger effect in the presence of U-0126 than without U-0126. EGF (10 microg/l) inhibited I(sc), whereas inhibition of EGFR kinase by tyrphostin AG-1478 (100 nmol/l) enhanced I(sc). Aldosterone was more effective in the presence of AG-1478 than without AG-1478. In summary, we propose that the EGFR-signaling cascade can serve as a negative-feedback control to limit the effect of aldosterone-induced Na(+) reabsorption.

Aldosterone↗

PubMed2004

Nephrotoxicity of platinum complexes is related to basolateral organic cation transport.

BACKGROUND: Cisplatin and its analogs oxaliplatin and carboplatin are widely used antitumor drugs. Nephrotoxicity is a common and relevant adverse effect that occurs especially in cisplatin therapy. Cellular and molecular mechanisms of cisplatin-induced nephrotoxicity are not completely understood. The nephrotoxicity of platinum complexes was evaluated by a new in vitro system that utilizes the high Trans Epithelial Electrical Resistance (TEER) of the C7 clone of the MDCK (Madin-Darby canine kidney) cells. By means of this assay system we addressed the question whether the side of application of renal epithelia influences platinum complex toxicity. METHODS: C7 cells were grown in membrane filter cups, and the apical or basolateral membranes were exposed to 100-micromol/L cis-, oxali-, or carboplatin. TEER and caspase-3 activity were determined. Cimetidine was used as an inhibitor of organic cation transporters (OCTs). C7 cell lysates were analyzed for OCT-1 and -2 by Western blot analysis. RESULTS: TEER dropped by 89.5 +/- 9.3% (mean +/- SEM; N= 6) within 24 hours after addition of cisplatin to the basolateral side of C7 cells, while caspase activity increased up to 840.6 +/- 17.4% (mean +/- SEM; N= 6) compared to control cells. Exposure of the apical membrane to cisplatin reduced TEER by only 13.4 +/- 8.7% (mean +/- SEM; N= 6), and increased caspase-3 activity up to 213.9 +/- 7.6% (mean +/- SEM; N= 6). Oxaliplatin and carboplatin reduced TEER to a lesser extent than cisplatin. Oxaliplatin lowered TEER stronger than carboplatin. In general, basolateral application led to higher caspase activities and lower TEERs. The OCT-inhibitor cimetidine inhibited the TEER decrease induced by platinum complexes. Immunoblotting confirmed the presence of OCT-2 in C7 cells. CONCLUSION: Toxic effects of platinum complexes on renal epithelia depend on the platinum complex used and the site of application. We conclude that cell polarity and basolateral transport mechanisms are essential in nephrotoxicity of platinum drugs.

Animals↗

PubMed2004

Inhibition of mitochondria and extracellular acidification enhance achratoxin A-induced apoptosis in renal collecting duct-derived MDCK-C7 cells.

Ochratoxin A (OTA) is a potent nephrotoxin and suspected to be involved in the etiology of Balkan endemic nephropathy. Nanomolar concentrations of this mycotoxin induce apoptosis in renal collecting duct-derived cells (MDCK-C7 cells, resembling principal cells). We studied the role of mitochondria in this process by inhibition of the mitochondrial respiratory chain, the F1FO-ATP synthase or by uncoupling. Also, 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, OTA-induced apoptosis was enhanced dramatically. Also, mitochondrial uncoupling potentiated the effects of OTA. OTA-induced apoptosis was not dependent on a decrease of the mitochondrial potential. Mitochondrial blockade led to medium acidification due to enhanced production of lactic acid. Artificial extracellular acidification potentiated OTA-induced caspase-3 activation. Artificial extracellular alkalization had no influence on caspase-3 activity. Intracellular pH after 24 hours exposure to inhibitors of mitochondria or acidic or alkaline media did not correlate with caspase-3 activity but correlated with caspase-3 activity when OTA was present: acidic intracellular pH (pHin) was associated with higher caspase-3 activity as compared to alkaline pHin. We conclude that extra- and intracellular pH are important factors in OTA-induced apoptosis in MDCK-C7 cells. The physiologically changing pH conditions in the collecting duct can thus alter or even aggravate the toxic effects of OTA.

Acids↗

PubMed2004

Apical expression or expression in a non polarized cell of hOAT1 inverses regulation by epidermal growth factor (EGF) as compared to basolateral hOAT1.

Physiologically, OAT1 is located in the basolateral membrane of proximal tubular cells. During renal damage loss of polarity occurs in renal epithelial cells, leading to missorting of proteins or complete loss of polarity. Missorting or loss of polarity generally leads to disturbance of vectorial transport. In the present study, hOAT1 was expressed in human renal epithelial IHKE cells (IHKE-hOAT1) and in non polarized CHO cells (CHO-hOAT1). Because EGF and its receptor is described to play on important role in recovery from renal damage, we compared the regulation of hOAT1 by EGF in the (a) basolateral and (b) apical membrane of epithelial cells, and in (c) non polarized cells, resembling the above mentioned pathophysiological situations. Expression of hOAT1 was verified by determination of the kinetic parameters (using fluorescein as a substrate) and western blot (CHO-hOAT1) or RT-PCR (IHKE-hOAT1). To investigate the EGF effect on hOAT1, CHO-hOAT1 cells were additionally co-transfected with the human EGF receptor HER1. In agreement with previous publications, incubation of IHKE-hOAT1 cells with EGF increased fluorescein uptake via basolateral hOAT1. In opposite, EGF inhibited hOAT1 mediated fluorescein uptake across the apical membrane of IHKE-hOAT1 cells. Additionally EGF inhibited hOAT1 mediated fluorescein uptake into non polarized CHO-hOAT1-HER1 cells, too. In summary, we confirmed that EGF stimulates basolateral uptake of organic anions (a) in proximal tubular cells mediated by hOAT1. However, EGF inhibits hOAT1 located in the apical membrane (b) or in non polarized cells (c). Renal failure is associated with successive loss of epithelial polarity. Therefore, inverted regulation of hOAT1 falsely located in the apical membrane of proximal tubular cells may be part of a mechanism stabilizing organic anion secretion in pathophysiological situations.

Animals↗

PubMed2003

Transforming growth factor-beta1 reduces megalin- and cubilin-mediated endocytosis of albumin in proximal-tubule-derived opossum kidney cells.

Transforming growth factor (TGF)-beta1 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-beta1-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-beta1 reduces megalin-cubilin-mediated endocytosis. Previously we have shown that albumin endocytosis in OK cells is mediated by megalin/cubulin. TGF-beta1 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-beta1 action does not involve mitogen-activated protein kinases, protein kinase C or A, or reactive oxygen species. In contrast, TGF-beta1-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-beta1. In conclusion our data indicate that enhanced levels of TGF-beta1 occurring in various nephropathies can lead to downregulation of megalin-cubilin-dependent endocytosis. Probably, TGF-beta1 leads to Smad2- and Smad3-dependent expression of negative regulators of receptor-mediated endocytosis.

Albumins↗

PubMed2003

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.

Aldosterone↗

PubMed2003

Aldosterone stimulates surface expression of NHE3 in renal proximal brush borders.

The mineralocorticoid aldosterone is one of the major regulators of extracellular volume and blood pressure. It acts by enhancing Na(+) reabsorption across tight epithelia such as renal collecting ducts and colon. In addition, it has been shown that aldosterone stimulates NaCl and volume reabsorption in renal proximal tubules by an unknown mechanism. To test the hypothesis that the application of aldosterone results in greater activity of the apical Na(+)/H(+) exchanger-3 (NHE3), we investigated the effect of aldosterone on amiloride-sensitive, proximal tubular volume reabsorption and proximal tubular NHE3 abundance in adrenalectomized rats. Aldosterone at physiological concentrations (dosage 36 microg/100 g b.w. per day) increased NHE3-dependent proximal tubular volume reabsorption and the abundance of NHE3 in brush borders without changing the total amount of NHE3 in cortical homogenates. These results indicate that renal proximal tubular NHE3 is a target for aldosterone-mediated regulation resulting in increased Na(+) reabsorption and thus extracellular volume and blood pressure. Further studies are required to determine the precise mechanism of action, especially whether the action of aldosterone on proximal tubular function is direct or indirect.

Aldosterone↗

PubMed2003

Inhibition of mitochondria prevents cell death in kidney epithelial cells by intra- and extracellular acidification.

BACKGROUND: Nephrotoxic substances like cisplatin or ochratoxin A (OTA) induce cell death in human proximal tubule-derived cells (IHKE cells). Mitochondria play a significant role in apoptosis and loss of their function may influence OTA- or cisplatin-induced apoptosis. Extracellular pH also plays an important role in tumor genesis. Therefore, we investigated the role of mitochondria and intra- and extracellular pH on cell death induction by cisplatin or OTA. METHODS: IHKE cells were incubated in the presence of OTA or cisplatin, together with inhibitors of the mitochondrial metabolism, and the activity of caspase-3 was measured and DNA laddering was monitored. Adenosine triphosphate (ATP) content of the cells, lactate release into the media, and glucose consumption was determined. In addition, media and cells were acidified or alkalized artificially to investigate the effect of intra- and extracellular pH on cell death induction. Cytochrome C was immunodetected in cellular compartments. RESULTS: Inhibition of the mitochondrial function reduced OTA- or cisplatin-induced cell death and led to considerable lactic acid production and extracellular acidification. Intra- and extracellular acidification prevented cells from cell death induced by OTA or cisplatin. No cytochrome C release from mitochondria could be detected during 24 hours of exposure to OTA or cisplatin. CONCLUSION: We conclude that OTA- or cisplatin-induced cell death is dependent on functional and intact, ATP-producing mitochondria and that intra- and extracellular pH is crucial for induction of cell death in IHKE cells.

Acidosis↗

PubMed2003

Protein uptake disturbs collagen homeostasis in proximal tubule-derived cells.

BACKGROUND: Interstitial fibrosis is of major importance for the deterioration of renal function, leading to uremia. Interaction of filtered proteins with proximal tubular cells is important for the onset and development of tubulointerstitial damage. METHODS: We investigated the effects of protein endocytosis on collagen homeostasis and signaling pathways of proximal tubule-derived cells (OK cells, LLC-PK1 cells), which express the endocytic machinery typical for the proximal tubule (megalin and cubilin), and compared it to renal epithelial cells with low endocytic activity (MDCK, IHKE1, NHE3-deficient OK cells). Collagen homeostasis was assessed by proline incorporation, ELISA, and Western blot. Matrix metalloproteinase (MMP) activity was assessed by gelatinase assay. Signaling pathways were monitored by reporter gene assay. RESULTS: Albumin, glycated albumin, fatty acid-free albumin, or globulins led to an increase of secreted collagen (types I, III, and IV) in OK and LLC-PK1 cells. In cells with low protein uptake activity, albumin exposure inhibited collagen secretion. Western blot analysis showed an increase of cellular collagen. MMP activity was significantly decreased by albumin exposure. Furthermore, albumin exposure led to activation of the NF-kappa B-, AP1-, NFAT-, SRE-, and CRE-pathways. Inhibition of NF-kappa B, PKC, or PKA partially reversed the effects of albumin. In addition, inhibition of albumin endocytosis reduced collagen secretion and activation of the signaling pathways. Discussion. The data show that endocytic uptake of proteins disturbs collagen homeostasis in proximal tubular cells. This disturbed matrix homeostasis probably supports the progression of interstitial fibrosis, which is of importance for the development of renal insufficiency.

Albumins↗

PubMed2003

Short-term regulation of basolateral organic anion uptake in proximal tubular opossum kidney cells: prostaglandin E2 acts via receptor-mediated activation of protein kinase A.

It was shown previously that EGF induces release of the important prostanoid prostaglandin E(2) (PGE(2)) in proximal tubular opossum kidney (OK) cells and PGE(2) then stimulates initial basolateral uptake of organic anions (OA) dose dependently. PGE(2) is a receptor agonist and a known substrate for the basolateral exchanger mediating OA uptake (OAT1 and/or OAT3). This study investigated the mechanism of short-term PGE(2) action on initial basolateral OA uptake in OK cells. PGE(2) stimulation of OA uptake was abolished by selective inhibition of adenylate cyclase (by MDL-12, 330A) or protein kinase A (PKA; by H89). PGE(2) stimulation of OA uptake persisted after preloading the cells with glutarate and was still abolished by inhibition of PKA. Selective activation of adenylate cyclase by forskolin led to identical results. These data contradicted the hypothesis that PGE(2) action on OA uptake is due to its action as a counter ion. Therefore, we tested whether the PGE(2) receptors (EP1 to 4) are involved in stimulation of OA uptake in OK cells by PGE(2). Because of their intracellular signaling profile, EP1 and EP3 were not taken into account as possible receptors for mediation of PGE(2)-induced OA uptake. With the use of selective agonists (11-deoxy PGE(1) and butaprost), EP4 was pharmacologically identified as the receptor responsible for PGE(2)-mediated stimulation of OA uptake. By reverse transcription-PCR, cloning, and subsequent sequencing, a homologue fragment to EP4 was identified in OK cells. EGF-induced stimulation of basolateral organic anion uptake was abolished by inhibition of adenylate cyclase or PKA. This indicates that EGF action is mediated by generation of PGE(2). The following model is proposed: PGE(2) generated in the cells does not act as a counter ion but activates adenylate cyclase. This is mediated by a homologue of EP4 receptor. cAMP then activates PKA, which stimulates initial basolateral uptake of OA in OK cells by a not-yet-known mechanism. PGE(2) is an organic anion, a potential stimulator of organic anion excretion, and an important mediator of inflammation all at once. Thus, the mechanism presented here may contribute to a limitation of inflammatory events in the kidney cortex interstitium.

Adenylyl Cyclase Inhibitors↗

PubMed2003

Albumin and glucose effects on cell growth parameters, albumin uptake and Na(+)/H(+)-exchanger Isoform 3 in OK cells.

BACKGROUND: The degree of albuminuria, the presence of sodium-dependent hypertension, and histological evidence of both tubular and interstitial pathology correlate with the progression of diabetic nephropathy. The sodium-hydrogen exchanger NHE-3 plays an integral role in both sodium reabsorption and receptor-mediated albumin endocytosis in proximal tubular cells (PTCs). The aim of this study was to investigate the direct effects of hyperglycemia and albumin on cell growth parameters, NHE-3 protein expression and albumin uptake in an IN VITRO model of PTCs. METHODS: Opossum kidney (OK) cells were exposed to 5 mmol/l glucose (control) or 25 mmol/l (high) glucose in the presence or absence of either 0.1 or 1.0 g/l bovine serum albumin (BSA) for up to 72 hrs prior to study. 20 mmol/l mannitol + 5 mmol/l glucose was used as a control for hyperosmolality. The cell number, the degree of cell swelling, cell protein content and NHE-3 protein expression were assessed. Cellular albumin uptake and the role of NHE in both control and high glucose conditions were determined by FITC-BSA +/- NHE-inhibitor ethyl isopropyl amiloride (EIPA). RESULTS: High glucose and the hyperosmolar control induced cellular hypertrophy, which was not modified in the presence of albumin. Cell volume was initially increased by 1.0 g/l BSA, +/-high glucose, which normalized over 48-72 hrs. All experimental conditions induced an early and sustained increase in NHE-3 protein expression. High glucose increased albumin uptake, independent of changes in osmolality. EIPA reduced the albumin uptake in PTCs with kinetics supporting the role of NHE-3 in this process. CONCLUSION: These results suggest that exposure of PTCs to high glucose concentrations promotes osmolality mediated cell hypertrophy and increased tubular albumin reabsorption linked to an increase in NHE-3 expression. It is postulated that this increase in albumin uptake due to high glucose exposure may lead to proinflammatory protein overload of PTCs, ultimately impairing the compensatory increase in tubular albumin reabsorption.

Albumins↗

PubMed2003

Mechanism of cell death of rat cardiac fibroblasts induced by serum depletion.

Serum starvation has recently been shown to cause cell death of cardiac fibroblasts and increased synthesis of extracellular matrix proteins in the surviving cells. In the present study, events occurring in the dying cells were investigated. Cultured adult rat cardiac fibroblasts were exposed to serum-free medium. Cell number was measured using a Coulter Counter Channelyzer. The activity of the extracellular signal-regulated or mitogen-activated protein kinases (ERK1/2, p42/p44(MAPK)), the p38 kinase (p38(MAPK)), the c-Jun N-terminal kinases (p46/p54(JNK)), and Akt kinase was assessed by Western blotting and phospho-specific antibodies. Caspase 7-cleavage was investigated by Western blotting and specific antibodies. Caspase 3 activity was measured by detection of its cleaved substrate. The appearance of necrosis was studied by inclusion of trypan blue. Apoptosis was assessed by DNA ladder formation. The mRNA expression of Bax and Bcl-2 was investigated by quantitative real-time PCR. Serum withdrawal led to the death of 26% of cultured isolated cardiac fibroblasts during the first 5 h. The activity of the p42/ p44(MAPK) as well as of Akt kinase was partially reduced. For p46/p54(JNK) and p38(MAPK), elevated phosphorylation was measured. Inhibition of p46/p54(JNK) and p38(MAPK) activity by SB202190 did not affect the decrease in cell number. Cleavage of caspase 7 was detected after 90 min. However, no activation of caspase 3 was measured. DNA fragmentation was not found after serum depletion. Trypan blue staining, however, was observed in 16% of the cells after 5 h. The mRNA levels of both Bax and Bcl-2 were increased after 30 min. These results indicate the appearance of necrosis during serum starvation in cardiac fibroblasts. However, some processes typical of apoptosis were also detected.

Animals↗

PubMed2002

Human epidermal growth factor receptor-1 expression renders Chinese hamster ovary cells sensitive to alternative aldosterone signaling.

The epidermal growth factor (EGF) regulates cell proliferation, differentiation, and ion transport using ERK1/2 as a downstream effector. Furthermore, the EGF receptor (EGFR) is involved in signaling by G-protein-coupled receptors, growth hormone, and cytokines via transactivation. It has been suggested that steroids interact with peptide hormones. Previously, we have shown that aldosterone modulates EGF responses in Madin-Darby canine kidney cells (Gekle, M., Freudinger, R., Mildenberger, S., and Silbernagl, S. (2002) Am. J. Physiol. 282, F669-F679). Here, we tested the hypothesis that human EGFR-1 can confer alternative aldosterone responsiveness with respect to ERK1/2 phosphorylation to Chinese hamster ovary cells, which do not express EGFR. Wild-type Chinese hamster ovary cells did not respond to EGF or aldosterone. After transfection of human EGFR-1, the cells responded to EGF, but not to aldosterone. However, when submaximal concentrations of EGF were used, nanomolar concentrations of aldosterone potentiated the action of EGF within minutes, resulting in a leftward shift of the EGF dose-response curve. This was not the case in mock-transfected cells. The EGFR kinase inhibitor tyrphostin AG1478 or the MEK1/2 inhibitor U0126 completely prevented the effect. Furthermore, aldosterone enhanced Tyr phosphorylation of c-Src and EGFR, and an inhibitor of cytosolic tyrosine kinases (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyriociaine) prevented the action of aldosterone. Our data show that aldosterone uses the EGF-EGFR-MEK1/2-ERK1/2 signaling cascade to elicit its alternative effects. In the presence of EGF, aldosterone leads to EGFR transactivation via cytosolic tyrosine kinases of the Src family.

Aldosterone↗

PubMed2002

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.

Aldosterone↗

PubMed2002

Angiotensin converting enzyme inhibition and angiotensin II AT1-receptor blockade reduce the levels of asymmetrical N(G), N(G)-dimethylarginine in human essential hypertension.

BACKGROUND: Asymmetrical N(G), N(G)-dimethylarginine (ADMA) is associated with impaired endothelium-dependent vasodilation in humans. METHODS: Twenty young, male, mildly hypertensive subjects were included in a randomized, double-blind, fourfold cross-over study with placebo, enalapril (20 mg/day), eprosartan (600 mg/day), or a combination of both drugs (10 and 300 mg/day, respectively) each over 1 week, followed by a 2-week wash-out phase. After each treatment phase, ADMA concentration was measured. RESULTS: ADMA concentration was 1.69+/-0.59 micromol/L in the placebo phase, and was significantly lower in the enalapril, eprosartan, and combination phases (1.41+/-0.29, 1.42+/-0.43, and 1.38+/-0.30 micromol/L, respectively; all P < 0.05 v placebo). Changes in ADMA levels were independent of the drugs' action on blood pressure (BP). CONCLUSIONS: Levels of ADMA were reduced with enalapril and eprosartan therapy. Our results suggest a specific action of these drugs on ADMA levels that is independent of BP.

Acrylates↗