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Dexamethasone-dependent modulation of cyclic GMP synthesis in podocytes.

Lewko B, Waszkiewicz A, Maryn A, Gołos M, Latawiec E, Daca A, Witkowski JM, Angielski S, Stępiński J - Mol. Cell. Biochem. (2015)

Bottom Line: In the presence of DEX these effects were prevented.We conclude that DEX modulates the production of cGMP in podocytes stimulated with vasoactive factors such as Ang II, ANP, and NO, and the effect is time-dependent. cGMP increases podocyte motility, which is prevented by DEX.This mechanism may account for the antiproteinuric effect of glucocorticoids.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Pathophysiology, Medical University of Gdansk, Debinki 7, 80-211, Gdansk, Poland. blew@gumed.edu.pl.

ABSTRACT
Podocytes may be direct target for glucocorticoid therapy in glomerular proteinuric disease. Permeability of podocytes largely depends on their capacity to migrate which involves the contractile apparatus in their foot processes. In this study, we examined the effect of synthetic glucocorticoid dexamethasone (DEX) on the ability of podocytes to produce cyclic guanosine monophosphate (cGMP) in the presence of vasoactive factors, atrial natriuretic peptide (ANP), nitric oxide (NO), and angiotensin II (Ang II). We investigated also the effects of cGMP and DEX on podocyte motility. Primary rat podocytes and immortalized mouse podocytes were pretreated with 1 µM DEX for 4 or 24 h. Glomerular hypertension was mimicked by subjecting the cells to mechanical stress. Total and subcellular cGMP levels were determined in podocytes incubated with 0.1 µM ANP, 1 µM S-nitroso-N-acetyl penicillamine (SNAP), and 1 µM Ang II. Cell motility was estimated by a wound-healing assay. The ANP-dependent production of cGMP increased after 4 h exposition to DEX, but was attenuated after 24 h. Adversely, a 24-h pretreatment with DEX augmented the NO-dependent cGMP synthesis. Ang II suppressed the ANP-dependent cGMP production and the effect was enhanced by DEX in mechanical stress conditions. Mechanical stress reduced total cGMP production in the presence of all stimulators, whereas extracellular to total cGMP ratio increased. 8-Br cGMP enhanced podocyte migration which was accompanied by F-actin disassembly. In the presence of DEX these effects were prevented. We conclude that DEX modulates the production of cGMP in podocytes stimulated with vasoactive factors such as Ang II, ANP, and NO, and the effect is time-dependent. cGMP increases podocyte motility, which is prevented by DEX. This mechanism may account for the antiproteinuric effect of glucocorticoids.

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Ability of mouse (a and b) and rat (c and d) podocytes to produce cGMP varied depending on the time of exposure to 1 µM DEX. The cells cultured in 12-well plates were stimulated with 0.1 µM ANP or 1 µM SNAP for 15 min. a and c cGMP synthesis increased in ANP-stimulated cells after a 4-h preincubation with DEX. b and d Following a 24-h preincubation with DEX, the ANP- dependent cGMP production was suppressed, while SNAP-induced cGMP was elevated, as compared to the controls. Co-incubation of podocytes with ANP and SNAP did not reverse the inhibitory effect of DEX on ANP-induced synthesis of cGMP. The DEX-dependent effects were abolished in the presence of RU486, a specific GR antagonist. Results are presented as means from 5 to 6 independent experiments performed in duplicate ±SEM. *P < 0.05, **P < 0.01, ***P < 0.001
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Fig1: Ability of mouse (a and b) and rat (c and d) podocytes to produce cGMP varied depending on the time of exposure to 1 µM DEX. The cells cultured in 12-well plates were stimulated with 0.1 µM ANP or 1 µM SNAP for 15 min. a and c cGMP synthesis increased in ANP-stimulated cells after a 4-h preincubation with DEX. b and d Following a 24-h preincubation with DEX, the ANP- dependent cGMP production was suppressed, while SNAP-induced cGMP was elevated, as compared to the controls. Co-incubation of podocytes with ANP and SNAP did not reverse the inhibitory effect of DEX on ANP-induced synthesis of cGMP. The DEX-dependent effects were abolished in the presence of RU486, a specific GR antagonist. Results are presented as means from 5 to 6 independent experiments performed in duplicate ±SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Mentions: A 24-h exposure of mouse podocytes to 1 μM DEX reduced their basal (non-stimulated) cGMP synthesis by almost 50 % (0.61 ± 0.02 vs. 1.19 ± 0.01 nmol/mg protein, P < 0.01). Similar effect was observed in rat cells. DEX affected also the ability of podocytes to produce cGMP in the presence of ANP and SNAP, albeit the effects varied depending on preincubation time (Fig. 1). The ANP-mediated cGMP formation was markedly enhanced by DEX after a short-time (4-h) preincubation (Fig. 1a, c), whereas adverse effect was observed after prolonged (24-h) DEX pretreatment (Fig. 1b, d). Moreover, in both mouse and rat cells, DEX-induced suppression of response to pGC activator ANP was accompanied by increased response to sGC activator, SNAP. Interestingly, when podocytes were incubated with both activators simultaneously, cGMP production remained suppressed in the cells pretreated for 24 h with DEX. This indicates that upregulation of sGC-derived cGMP did not compensate for decreased production of cGMP by pGC (Fig. 1b, d). Pretreatment of podocytes with RU486 abolished the effects of DEX in both, rat and mouse podocytes (Fig. 1). This suggests that modulation of cGMP level in these cells was mediated by glucocorticoid receptors (GR).Fig. 1


Dexamethasone-dependent modulation of cyclic GMP synthesis in podocytes.

Lewko B, Waszkiewicz A, Maryn A, Gołos M, Latawiec E, Daca A, Witkowski JM, Angielski S, Stępiński J - Mol. Cell. Biochem. (2015)

Ability of mouse (a and b) and rat (c and d) podocytes to produce cGMP varied depending on the time of exposure to 1 µM DEX. The cells cultured in 12-well plates were stimulated with 0.1 µM ANP or 1 µM SNAP for 15 min. a and c cGMP synthesis increased in ANP-stimulated cells after a 4-h preincubation with DEX. b and d Following a 24-h preincubation with DEX, the ANP- dependent cGMP production was suppressed, while SNAP-induced cGMP was elevated, as compared to the controls. Co-incubation of podocytes with ANP and SNAP did not reverse the inhibitory effect of DEX on ANP-induced synthesis of cGMP. The DEX-dependent effects were abolished in the presence of RU486, a specific GR antagonist. Results are presented as means from 5 to 6 independent experiments performed in duplicate ±SEM. *P < 0.05, **P < 0.01, ***P < 0.001
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig1: Ability of mouse (a and b) and rat (c and d) podocytes to produce cGMP varied depending on the time of exposure to 1 µM DEX. The cells cultured in 12-well plates were stimulated with 0.1 µM ANP or 1 µM SNAP for 15 min. a and c cGMP synthesis increased in ANP-stimulated cells after a 4-h preincubation with DEX. b and d Following a 24-h preincubation with DEX, the ANP- dependent cGMP production was suppressed, while SNAP-induced cGMP was elevated, as compared to the controls. Co-incubation of podocytes with ANP and SNAP did not reverse the inhibitory effect of DEX on ANP-induced synthesis of cGMP. The DEX-dependent effects were abolished in the presence of RU486, a specific GR antagonist. Results are presented as means from 5 to 6 independent experiments performed in duplicate ±SEM. *P < 0.05, **P < 0.01, ***P < 0.001
Mentions: A 24-h exposure of mouse podocytes to 1 μM DEX reduced their basal (non-stimulated) cGMP synthesis by almost 50 % (0.61 ± 0.02 vs. 1.19 ± 0.01 nmol/mg protein, P < 0.01). Similar effect was observed in rat cells. DEX affected also the ability of podocytes to produce cGMP in the presence of ANP and SNAP, albeit the effects varied depending on preincubation time (Fig. 1). The ANP-mediated cGMP formation was markedly enhanced by DEX after a short-time (4-h) preincubation (Fig. 1a, c), whereas adverse effect was observed after prolonged (24-h) DEX pretreatment (Fig. 1b, d). Moreover, in both mouse and rat cells, DEX-induced suppression of response to pGC activator ANP was accompanied by increased response to sGC activator, SNAP. Interestingly, when podocytes were incubated with both activators simultaneously, cGMP production remained suppressed in the cells pretreated for 24 h with DEX. This indicates that upregulation of sGC-derived cGMP did not compensate for decreased production of cGMP by pGC (Fig. 1b, d). Pretreatment of podocytes with RU486 abolished the effects of DEX in both, rat and mouse podocytes (Fig. 1). This suggests that modulation of cGMP level in these cells was mediated by glucocorticoid receptors (GR).Fig. 1

Bottom Line: In the presence of DEX these effects were prevented.We conclude that DEX modulates the production of cGMP in podocytes stimulated with vasoactive factors such as Ang II, ANP, and NO, and the effect is time-dependent. cGMP increases podocyte motility, which is prevented by DEX.This mechanism may account for the antiproteinuric effect of glucocorticoids.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Pathophysiology, Medical University of Gdansk, Debinki 7, 80-211, Gdansk, Poland. blew@gumed.edu.pl.

ABSTRACT
Podocytes may be direct target for glucocorticoid therapy in glomerular proteinuric disease. Permeability of podocytes largely depends on their capacity to migrate which involves the contractile apparatus in their foot processes. In this study, we examined the effect of synthetic glucocorticoid dexamethasone (DEX) on the ability of podocytes to produce cyclic guanosine monophosphate (cGMP) in the presence of vasoactive factors, atrial natriuretic peptide (ANP), nitric oxide (NO), and angiotensin II (Ang II). We investigated also the effects of cGMP and DEX on podocyte motility. Primary rat podocytes and immortalized mouse podocytes were pretreated with 1 µM DEX for 4 or 24 h. Glomerular hypertension was mimicked by subjecting the cells to mechanical stress. Total and subcellular cGMP levels were determined in podocytes incubated with 0.1 µM ANP, 1 µM S-nitroso-N-acetyl penicillamine (SNAP), and 1 µM Ang II. Cell motility was estimated by a wound-healing assay. The ANP-dependent production of cGMP increased after 4 h exposition to DEX, but was attenuated after 24 h. Adversely, a 24-h pretreatment with DEX augmented the NO-dependent cGMP synthesis. Ang II suppressed the ANP-dependent cGMP production and the effect was enhanced by DEX in mechanical stress conditions. Mechanical stress reduced total cGMP production in the presence of all stimulators, whereas extracellular to total cGMP ratio increased. 8-Br cGMP enhanced podocyte migration which was accompanied by F-actin disassembly. In the presence of DEX these effects were prevented. We conclude that DEX modulates the production of cGMP in podocytes stimulated with vasoactive factors such as Ang II, ANP, and NO, and the effect is time-dependent. cGMP increases podocyte motility, which is prevented by DEX. This mechanism may account for the antiproteinuric effect of glucocorticoids.

No MeSH data available.


Related in: MedlinePlus