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G Friedlander

Publications and source records attributed to G Friedlander.

At least 55 records · Page 3Linked to original sources
PubMed1997

Overexpression of ecto-5'-nucleotidase promotes P-glycoprotein expression in renal epithelial cells.

UNLABELLED: P-glycoprotein (P-gp), responsible for multidrug resistance (MDR) of tumoral cells, is also expressed in apical membranes of normal epithelial cells, among which are proximal tubular cells. Ecto-5'-nucleotidase (5'Nu), co-located with P-gp in renal brush border membranes, could be instrumental in the expression of MDR phenotype. P-gp activity [assessed by rhodamine 123 (R123) and [3H]vinblastine (3H-VBL) accumulation] was evaluated in MDCK cell lines in which human 5'Nu was expressed at different levels after retroviral infection: MDCK-5'NU/- cells with a low 5'Nu activity (Vmax < 2 pmol/mg protein/min) and MDCK-5'NU/+ cells, which expressed a high level of 5'Nu (Vmax 150 +/- 18.5 pmol/mg protein/min). MDCK-5'NU/- cells did not display functional expression of MDR. In MDCK-5'NU/+ cells, R123 and 3H-VBL accumulation was significantly lower than in MDCK-5'NU/- cells and was dramatically enhanced by P-gp inhibitors. This high P-gp activity in MDCK-5'NU/+ cells was confirmed by their resistance to colchicine (measured by LDH release and MTT assay) as compared to MDCK-5'NU/- and was accounted for by increased membrane expression of P-gp assessed by Western blot. Neither AMP nor adenosine, the substrate and the product of 5'Nu, respectively, affected P-gp activity. Inhibition of 5'Nu with alpha beta-methylene-adenosine-diphosphate (alpha beta MADP) or with a blocking anti-5'Nu antibody (1E9) did not blunt MDR expression in MDCK-5'NU/+ cells. Conversely, the anti-5'Nu antibody 5F/F9, which did not block the enzymatic site, induced a decrease of P-gp activity. Further, incubation of MDCK-5'NU/- cells with conditioned medium from MDCK-5'NU/+ cells, which contained significant amounts of released 5'Nu, induced MDR phenotype. IN CONCLUSION: (i) expression of ecto-5'Nu promotes multidrug resistance (MDR) activity in renal epithelial cells by enhancement of P-gp expression; (ii) this effect does not involve enzymatic activity of 5'Nu; (iii) supernatants of cells that express 5'Nu conferred P-gp activity to 5'Nu negative cells.

5'-Nucleotidase↗
PubMed1997

HMG-CoA reductase inhibitors induce apoptosis in mouse proximal tubular cells in primary culture.

Renal cyst formation in polycystic diseases or after nephron reduction is attributed to enhanced tubular cell proliferation with unbalanced cell death. The induction of tubular cell death could be effective to reduce renal cyst formation. In this study, we examined the effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors on apoptosis in mouse proximal tubular (MPT) cells in primary culture. After treatment with HMG-CoA reductase inhibitors, the extracted DNA was analyzed by gel-electrophoresis under ultraviolet light. Apoptosis was evaluated quantitatively by estimating the ratio of fragmented DNA over intact DNA. For morphologic studies, cells were stained with Hoechst 33,258. DNA ladder pattern of 200 kDa typical of apoptosis and significant increase in DNA fragmentation were seen after 24 hours of treatment with lovastatin, a HMG-CoA reductase inhibitor. Staining with the Hoechst dye revealed cleavage of nucleus into pieces under the same condition. Geranylgeranylpyrophosphate (20 microM) and mevalonate (500 microM) completely reversed the effect of lovastatin, while farnesylpyrophosphate (20 microM) partially reversed it. Other products of HMG-CoA pathway such as cholesterol, ubiquinone, dolichol, and isopentenyladenine had no effect. Perillic acid and alpha-hydoxyfarnesylphosphonic acid, isoprenylation inhibitors, induced apoptosis of the cells. A treatment with lovastatin caused actin filament disruption. Cytochalasin D, an inhibitor of actin polymerization, induced apoptosis. Interleukin-1 beta-converting enzyme inhibitor II, a protease inhibitor, had no effect on the apoptosis induced by either HRI or cytochalasin D. The present study suggests that in mouse proximal tubules, HMG-CoA reductase inhibitors induce apoptosis via inhibition of isoprenoid production, and disruption of actin filaments may play a role in the apoptosis induction.

Actins↗
PubMed1997

Lovastatin-induced inhibition of renal epithelial tubular cell proliferation involves a p21ras activated, AP-1-dependent pathway.

Proliferation of tubular epithelial cells underlies the development of cystic lesions and the subsequent impairment of renal function after renal mass reduction. The effect of HMG CoA reductase inhibitors (HRI) on cell proliferation was investigated in rat renal proximal tubular epithelial cells in primary culture. Treatment of renal tubular epithelial cells with three different HRI reduced fetal calf serum (FCS)-induced [3H]-thymidine incorporation (IC50 values were 0.7 microM, 1.7 microM, and 1.6 microM for simvastatin, lovastatin, and compactin, respectively), and lovastatin blocked BrdUrd incorporation, as assessed by immunocytochemical studies. The proliferative effect of epidermal growth factor (EGF) was similarly abolished by lovastatin. The effect of lovastatin (1 microM) was prevented by 100 microM mevalonate, 5 microM farnesyl-pyrophosphate and 5 microM geranylgeranyl-pyrophosphate (in percent of control value, 31% vs. 102%, 60%, and 82%, respectively) while cholesterol and other products of the mevalonate pathway were inactive. Immunoblot analysis showed that lovastatin decreased membrane-bound p21ras and inhibited FCS-induced c-fos and c-jun protein expression. Furthermore, electrophoretic mobility shift assay demonstrated the functional impairement of AP-1 DNA binding activity in lovastatin-treated cells. In conclusion, these results demonstrate that HRI are antiproliferative in epithelial tubule cells and that this effect is exerted, at least in part, via inhibition of the p21ras-activated and AP-1 dependent mitogenic cascade.

Animals↗
PubMed1997

Subtotal nephrectomy alters tubular function: effect of phosphorus restriction.

Few studies have examined tubular function after subtotal nephrectomy (Nx) and conservative treatments. The effects of 70% and 80% Nx (associated with dietary phosphate restriction in the latter case) on the apical brush border membrane (BBM) enzymes 5'-nucleotidase, gamma glutamyl-transferase and alkaline-phosphatase, and one BBM Na-phosphate cotransporter (NaPi-2) were studied in rats after a six week period. Changes in activity and mRNA abundance of the BBM enzymes and in NaPi-2 protein and mRNA abundance were compared with changes in the distal markers of Na,K-ATPase activity and epidermal growth factor (EGF) production. The activity, but not the mRNA of BBM enzymes, was moderately reduced by the 70% Nx. Both the mRNA and activity of gamma glutamyl-transferase and alkaline-phosphatase were decreased in the 80% Nx, and the NaPi-2 mRNA, protein and Na,K-ATPase activities were also reduced. These effects (except for 5'nucleotidase and Na,K-ATPase) were partly reversed by phosphate restriction. Overproduction of EGF occurred after the 70% Nx, was blunted in the 80% Nx, and then partially restored by phosphate restriction. Aggravation of tubular alteration was associated with enhanced renal hyperplasia (increased DNA mass), reduced GFR and hyperphosphatemia, and high PTH levels, but reduced cAMP excretion. Improvement following phosphate restriction was associated with reduced hyperplasia and lowering of phosphatemia and PTH levels. These data demonstrate that Nx selectively affected BBM function through transcriptional changes that were partially reversed by phosphate restriction. Regulatory factors involved in these changes may include intracellular phosphate content and growth factors, but not the PTH effects that are impaired in chronic renal failure.

5'-Nucleotidase↗
PubMed1997

Glucocorticosteroids increase sodium transport in middle ear epithelium.

The effect of glucocorticosteroids on ion transport was investigated on a middle ear cell line with the short-circuit current (Isc) technique. Dexamethasone (DXM) produced a dose- and time-dependent increase in Isc. Concentration of half-maximal stimulation was 2.68 x 10(-8) M. This effect was blunted by the glucocorticoid antagonist RU-38486 and was related to Na+ transport, as evidenced by the inhibition induced by 1) apical addition of the Na+ channel inhibitor benzamil (10(-6) M) or 2) substitution of Na+ with N-methylglucamine in the incubation medium. The increase in Na+ transport resulted from a primary modulation of apical Na+ entry, since 1) the Na(+)-K(+)-ATPase activity of cellular homogenates was not modified by corticosteroids and 2) the DXM-induced increase in the ouabain-sensitive uptake of 86Rb was blunted by benzamil. Ribonuclease protection assay revealed 1) a constitutive expression of the mRNA encoding the alpha-subunit of the epithelial Na+ channel and 2) that DXM increased the expression of this transcript. This increase was dose dependent and paralleled changes in transepithelial Na+ transport. This study suggests that a component of the beneficial effect of steroid therapy for the treatment of otitis media might be related to increased fluid clearance.

Adenosine Triphosphate↗
PubMed1997

Hypoxia downregulates expression and activity of epithelial sodium channels in rat alveolar epithelial cells.

Decrease in alveolar oxygen tension may induce acute lung injury with pulmonary edema. We investigated whether, in alveolar epithelial cells, expression and activity of epithelial sodium (Na) channels and Na,K-adenosine triphosphatase, the major components of transepithelial Na transport, were regulated by hypoxia. Exposure of cultured rat alveolar cells to 3% and 0% O2 for 18 h reduced Na channel activity estimated by amiloride-sensitive 22Na influx by 32% and 67%, respectively, whereas 5% O2 was without effect. The decrease in Na channel activity induced by 0% O2 was time-dependent, significant at 3 h of exposure and maximal at 12 and 18 h. It was associated with a time-dependent decline in the amount of mRNAs encoding the alpha-, beta-, and gamma-subunits of the rat epithelial Na channel (rENaC) and with a 42% decrease in alpha-rENaC protein synthesis as evaluated by immunoprecipitation after 18 h of exposure. The 0% O2 hypoxia also caused a time-dependent decrease in (1) ouabain-sensitive 86Rubidium influx in intact cells, (2) the maximal velocity of Na,K-ATPase on crude homogenates, and (3) alpha1- and beta1-Na,K-ATPase mRNA levels. Levels of rENaC and alpha1-Na,K-ATPase mRNA returned to control values within 48 h of reoxygenation, and this was associated with complete functional recovery. We conclude that hypoxia induced a downregulation of expression and activity of epithelial Na channels and Na,K-ATPase in alveolar cells. Subsequent decrease in Na reabsorption by alveolar epithelium could participate in the maintenance of hypoxia-induced alveolar edema.

Animals↗
PubMed1997

Modulation of middle ear epithelial function by steroids: clinical relevance.

The efficacy of steroid therapy for the treatment of otitis media in children remains controversial, and a putative modulation of the middle ear epithelial function has to be demonstrated. Using the MESV cell line, short-circuit current (ISC) technique was used to evaluate changes in ion transport induced by glucocorticoids. Dexamethasone (DXM) produced a dose- and time-dependent increase in ISC in MESV cells. This effect was inhibited by specific glucocorticoid antagonist (RU-38486) and was related to a sodium transport, since the DXM-induced increase in ISC could be prevented or abolished i) by apical addition of the specific Na+ channel inhibitor benzamil; or ii) by substitution of sodium with N-Methyl-glucamine in the incubation medium. RNase protection assay revealed that DXM increased the expression of the alpha subunit sodium channel mRNA, which changes paralleled the modulation of ion transport. These data demonstrate that steroids up-regulate the trans-epithelial sodium transport in the middle ear epithelium. As far as these experimental data can be extrapolated to the in vivo situation, a component of the beneficial effect of steroid therapy for the treatment of otitis media may result from a corticosteroid-induced improvement in fluid clearance from the middle ear.

Amiloride↗
PubMed1997

Normal tubular regeneration and differentiation of the post-ischemic kidney in mice lacking vimentin.

Proliferation and dedifferentiation of tubular cells are the hallmark of early regeneration after renal ischemic injury. Vimentin, a class III intermediate filament expressed only in mesenchymal cells of mature mammals, was shown to be transiently expressed in post-ischemic renal tubular epithelial cells. Vimentin re-expression was interpreted as a marker of cellular dedifferentiation, but its role in tubular regeneration after renal ischemia has also been hypothesized. This role was evaluated in mice bearing a null mutation of the vimentin gene. Expression of vimentin, proliferating cell nuclear antigen (a marker of cellular proliferation), and villin (a marker of differentiated brush-border membranes) was studied in wild-type (Vim+/+), heterozygous (Vim+/-), and homozygous (Vim-/-) mice subjected to transient ischemia of the left kidney. As expected, vimentin was detected by immunohistochemistry at the basal pole of proximal tubular cells from post-ischemic kidney in Vim+/+ and Vim+/- mice from day 2 to day 28. The expression of the reporter gene beta-galactosidase in Vim+/- and Vim-/- mice confirmed the tubular origin of vimentin. No compensatory expression of keratin could be demonstrated in Vim-/- mice. The intensity of proliferating cell nuclear antigen labeling and the pattern of villin expression were comparable in Vim-/-, Vim+/- and Vim+/+ mice at any time of the study. After 60 days, the structure of post-ischemic kidneys in Vim-/- mice was indistinguishable from that of normal non-operated kidneys in Vim+/+ mice. In conclusion, 1) the pattern of post-ischemic proximal tubular cell proliferation, differentiation, and tubular organization was not impaired in mice lacking vimentin and 2) these results suggest that the transient tubular expression of vimentin is not instrumental in tubular regeneration after renal ischemic injury.

Animals↗
PubMed1996

Locally formed 5-hydroxytryptamine stimulates phosphate transport in cultured opossum kidney cells and in rat kidney.

UNLABELLED: Renal proximal tubular cells have been shown to express aromatic L-amino acid decarboxylase (L-AAAD), which converts L-dopa into dopamine and 5-hydroxytryptophan [(OH)Trp] into 5-hydroxytryptamine (5-HT; serotonin). Because 5-HT receptors have been demonstrated in proximal cells, we hypothesized that 5-HT may act as an autocrine/paracrine modulator of proximal transport. We evaluated this possibility in opossum kidney (OK) cells, a renal epithelial cell line with a proximal phenotype expressing 5-HT1B receptors, and in intact anaesthetized rats. 5-HT synthesis by OK cells increased with incubation time and (OH)Trp concentration, and was abolished by benserazide, an L-AAAD inhibitor. 5-HT reversed parathyroid hormone (PTH)-induced cAMP accumulation in a pertussis toxin-sensitive manner and reduced the PTH inhibition of P(i) uptake without affecting the NaP(i)-4 mRNA level. The effects of 5-HT on cAMP generation and Na-P(i) co-transport were reproduced by (OH)Trp, except in the presence of benserazide, and by L-propranolol and dihydroergotamine, two 5-HT1B receptor agonists. In rats, (OH)Trp and dihydroergotamine decreased fractional P(i) excretion. Benserazide abolished the effect of (OH)Trp but not that of dihydroergotamine. IN CONCLUSION: (i) locally generated 5-HT blunts the inhibitory effect of PTH on Na-P(i) co-transport in OK cells; (ii) endogenous 5-HT decreases P(i) excretion in rats; and (iii) 5-HT is a paracrine modulator involved in the physiological regulation of renal P(i) transport.

5-Hydroxytryptophan↗
PubMed1996

Inhibition of ecto-5'-nucleotidase by nitric oxide donors. Implications in renal epithelial cells.

We evaluated, in renal epithelial cells with a proximal tubule phenotype, the effect of nitric oxide (NO) on ecto-5 -nucleotidase (5'-N U), the underlying mechanism and its functional consequence. Sodium nitroprusside (SNP, 1-1000 microM), a NO donor, inhibited 5'-NU activity in a time- and concentration-dependent manner. Consequently, NO blunted the inhibition by extracellular cyclic AMP (cAMP, 10-1000 microM) of sodium-phosphate cotransport, a pathway which involves degradation of adenosine monophosphate (AMP) by 5'-NU. SNP-induced inhibition of 5'-NU was not mediated by cyclic GMP, since it was not mimicked by atrial natriuretic peptide, and was reproduced by isosorbide dinitrate and sodium nitrate, two NO donors. SNP and genuine NO decreased the activity of 5'-NU in renal homogenates, and the effect of SNP was potentiated by dithiothreitol and glutathione, but not by nicotinamide adenine dinucleotide. In vivo in rats, kidney ischemia/reperfusion, which activates inducible NO-synthase, inhibited renal 5'-NU. This inhibition was prevented by Nomega-nitro-L-arginine methyl ester, a NO-synthase inhibitor. These results indicate that: (i) NO-related activity inhibited the activity of an ecto-enzyme, 5'-NU, most likely through S-nitrosylation of the enzyme; (ii) inhibition of 5'-NU activity by NOx, which can occur in vivo under pathophysiological conditions, affected the extent to which extracellular cAMP inhibited sodium-Pi cotransport.

5'-Nucleotidase↗
PubMed1996

Regulation of renal phosphate handling: recent findings.

Our knowledge of renal phosphate handling and its regulation has expanded in the past 2 years because of the molecular identification of sodium-phosphate cotransport systems. New tools (molecular probes and antibodies) have allowed dissection out of some of the molecular and cellular mechanisms underlying the adaptation of phosphate transport to dietary content, the phosphaturic effect of parathyroid hormone or glucocorticoids and the renal phosphate leak in hypophosphataemic rickets.

Animals↗
PubMed1996

Inhibition of Na-K-ATPase activity after prolonged hypoxia in an alveolar epithelial cell line.

Exposure to alveolar hypoxia may induce acute pulmonary edema. Because the vectorial sodium transport by alveolar epithelium represents an important mechanism for alveolar edema clearance, we examined whether hypoxia affects Na-K-ATPase activity in cultured SV40-transformed rat alveolar type II cells (SV40 ATII cells). Hypoxic exposures (O or 5% O2 for at least 12 h) induced a time- and O2 concentration-dependent decrease in ouabain-sensitive rubidium (osRb) influx. Neither the sensitivity of Rb influx to ouabain nor the maximum velocity of the enzyme measured on crude cell homogenates was affected by hypoxia. The osRb influx decrease was independent of hypoxia-induced ATP depletion. Na-K-ATPase inhibition was most likely related to impaired calcium homeostasis, because 1) calcium influx was increased in hypoxic cells, 2) hypoxia-induced osRb influx decrease was completely prevented by nifedipine (10-5 M), and 3) osRb influx decreased in normoxic cells incubated with ionomycin (10-6 M, 15 min). Furthermore, hypoxia-induced Na-K-ATPase impairment might be due, at least in part, to the endogenous release by hypoxic cells of a lipidic factor in extracellular medium, because incubation of normoxic cells with hypoxic cells conditioned medium (CM), or with the lipidic subphase from hypoxic cells CM, also induced a partial decrease in osRb influx. This decrease was associated with increased calcium influx into normoxic cells and was suppressed either by the removal of external calcium or by nifedipine, suggesting that the lipidic factor exerted its inhibitory action on Na-K-ATPase via an enhancement of calcium entry. These results indicate that prolonged hypoxic exposure impairs Na-K-ATPase activity in SV40 ATII cells and may therefore decrease the vectorial sodium transport by alveolar epithelium.

Adenosine Triphosphate↗
PubMed1996

Expression of alternatively spliced isoforms of the parathyroid hormone (PTH)/PTH-related peptide receptor messenger RNA in human kidney and bone cells.

Using a PCR-based strategy, two variants of the PTH/PTH-related peptide (PTH-rp) receptor mRNA were identified in human kidney, SaOS-2 human osteoblast cells, and rat bone that are produced by alternative splicing of exons coding for the N-terminal portion of the receptor. In the S-N3-E2 isoform, the exon coding the signal peptide (S) is spliced to an alternative 3'-acceptor site, producing a product respecting the reading frame, but in which the E1 exon is replaced by 12 amino acids derived from the N3 intron. In the S-E2 isoform, in which the E1 exon is deleted by cassette exclusion, the reading frame is changed, but a truncated receptor may be produced by reinitiation of translation at an overlapping stop/start codon. After transfection of COS and Chinese hamster ovary cells with the originally described S-E1-E2 isoform and the two splice variants, active transcription of PTH/PTH-rp receptor mRNA was detected by RT-PCR in all cases. Cell lines transfected with the S-E1-E2 and S-N3-E2 isoforms displayed a 15- to 25-fold and 2- to 3-fold increase, respectively, in cAMP content after stimulation with 2.4 x 10(-7) M human PTH(1-34), whereas cells transfected with the S-E2 isoform did not respond. PTH elicited an increase in intracellular calcium only in cells transfected with the S-E1-E2 isoform. Studies evaluating the surface expression of receptors using anti-human PTH/PTH-rp receptor antibodies and the ability of transfected cells to bind [125I]PTH-rp indicated that the low or absent responses to PTH stimulation resulted, at least in part, from low surface expression of the S-N3-E2 and S-E2 isoforms. These studies support the conclusion that exon E1 is extremely important in promoting surface expression of the PTH/PTH-rp receptor but indicate that isoforms lacking this exon can retain the ability to recognize PTH. The possible intracellular expression of these splice variants, which account for 15-20% of total PTH/PTH-rp receptor mRNA, needs to be evaluated.

Alternative Splicing↗
PubMed1996

[Regulation and physiopathology of phosphate transport].

Renal phosphate transport is subjected to a fine tuning which involves hormones and local mediators and results in adaptation of excretion to dietary contents. Recent insights into the molecular structure of phosphate transporters have shed light on the mechanisms which underline the effects of these mediators. Another important repercussion of these advances is the identification of pathological states such as renal phosphate leaks and the elaboration of diagnostic and therapeutic strategies aiming to improve these troubles.

Animals↗
PubMed1995

Locally formed dopamine modulates renal Na-Pi co-transport through DA1 and DA2 receptors.

The involvement of dopamine (DA) receptor subtypes in regulation of renal phosphate transport by DA, either exogenous or locally synthesized from L-dihydroxyphenylalanine (L-dopa) was evaluated in opossum kidney (OK) cells with proximal tubular phenotype. DA synthesis from L-dopa by OK cells was abolished by carbidopa and benserazide, two dissimilar inhibitors of aromatic L-amino acid decarboxylase. L-Dopa stimulated cyclic AMP generation and inhibited Na-dependent Pi uptake, and these effects were abolished by carbidopa and benserazide. The effects of L-dopa or DA on cyclic AMP generation and on Na-Pi co-transport were mimicked by SKF 38393, a DA1 receptor agonist, and were potentiated by S-sulpiride, a DA2 receptor antagonist. Bromocriptine, a DA2 receptor agonist, blunted in a pertussis toxin-dependent manner parathyroid hormone (PTH)-induced cyclic AMP generation and inhibition of Pi uptake. In contrast with PTH, neither L-dopa nor DA affected significantly the cytosolic calcium concentration. These results support the involvement of DA1 and DA2 receptors, positively and negatively coupled into adenylate cyclase respectively, in modulation of renal phosphate transport.

Adenylate Cyclase Toxin↗
PubMed1995

Insulin-like growth factor I, a unique calcium-dependent stimulator of 1,25-dihydroxyvitamin D3 production. Studies in cultured mouse kidney cells.

Previous in vivo and in vitro studies suggest that insulin-like growth factor (IGF-I) could be a regulator of the renal production of 1,25-(OH)2D3. In the present work, the local effect of low nanomolar concentrations of IGF-I on the 25-OH-D3-1 alpha-hydroxylase activity and the mechanism of its action have been investigated. To do so, an in vitro model of mouse proximal tubular cells in primary culture has been developed. These cells bear specific high affinity IGF-I binding sites (apparent Kd = 1.95 +/- 0.46 nM) and express the ability to convert [3H]25-(OH)D3 into [3H]1,25-(OH)2D3 (Km = 139 +/- 15.7 nM). Human recombinant IGF-I (10-100 ng/ml) stimulated both sodium-dependent phosphate uptake and 1,25-(OH)2D3 synthesis by these cells, in a time- and dose-dependent manner. IGF-I did not alter the apparent Michaelis constant but increased the maximum velocity of the 25-OH-D3-1 alpha-hydroxylase activity. This effect required protein synthesis. It was not affected by calphostin or GF109203X, two protein kinase C inhibitors, and was not mimicked by phorbol 12-myristate 13-acetate. In contrast, it was blocked by verapamil, a calcium channel blocker. Calcium depletion of the medium blunted the IGF-I effect but not that of human 1-34 parathyroid hormone 5 x 10(-8) M. IGF-I thus appears to be the first example of a physiological calcium-dependent regulator of the renal metabolism of vitamin D.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗