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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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The influence of 8-Br cGMP and DEX on mouse podocyte motility. Upper panel scrape-wound assay, DAPI staining. The serum-starved podocytes were incubated for 24 h with 100 µM 8-Br cGMP with or without 1 µM DEX, as described in “Materials and methods.” Untreated cells served as the control. Lower panel the results showing that 8-Br cGMP significantly increased the number of migrating podocytes, while DEX inhibited this effect. The data represent the mean ±SEM from three independent experiments performed in duplicate
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Fig5: The influence of 8-Br cGMP and DEX on mouse podocyte motility. Upper panel scrape-wound assay, DAPI staining. The serum-starved podocytes were incubated for 24 h with 100 µM 8-Br cGMP with or without 1 µM DEX, as described in “Materials and methods.” Untreated cells served as the control. Lower panel the results showing that 8-Br cGMP significantly increased the number of migrating podocytes, while DEX inhibited this effect. The data represent the mean ±SEM from three independent experiments performed in duplicate

Mentions: Since the involvement of guanylyl cyclases and glucocorticoids in migration of some cell types has been reported previously [31, 32], we have tested if motility of podocytes is regulated by these factors. Due to phosphodiesterases activity, half-time for cGMP varies between few seconds in vivo to few minutes in vitro. Therefore, in order to perform prolonged incubations, we used a membrane-permeable analog 8-Br cGMP. While no significant effects were observed after a 4-h incubation with 8-Br cGMP, a 24-h incubation resulted in markedly higher (P < 0.01) ability of podocytes to migrate, as compared to the non-treated (control) cells (Fig. 5). This was accompanied by a pronounced disassembly of F-actin fibers (Fig. 6). As shown in Fig. 5, incubation with DEX prevented the cGMP-dependent increase in podocyte motility, while F-actin stress fibers remained well preserved, forming a strong network (Fig. 6).Fig. 5


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)

The influence of 8-Br cGMP and DEX on mouse podocyte motility. Upper panel scrape-wound assay, DAPI staining. The serum-starved podocytes were incubated for 24 h with 100 µM 8-Br cGMP with or without 1 µM DEX, as described in “Materials and methods.” Untreated cells served as the control. Lower panel the results showing that 8-Br cGMP significantly increased the number of migrating podocytes, while DEX inhibited this effect. The data represent the mean ±SEM from three independent experiments performed in duplicate
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4589550&req=5

Fig5: The influence of 8-Br cGMP and DEX on mouse podocyte motility. Upper panel scrape-wound assay, DAPI staining. The serum-starved podocytes were incubated for 24 h with 100 µM 8-Br cGMP with or without 1 µM DEX, as described in “Materials and methods.” Untreated cells served as the control. Lower panel the results showing that 8-Br cGMP significantly increased the number of migrating podocytes, while DEX inhibited this effect. The data represent the mean ±SEM from three independent experiments performed in duplicate
Mentions: Since the involvement of guanylyl cyclases and glucocorticoids in migration of some cell types has been reported previously [31, 32], we have tested if motility of podocytes is regulated by these factors. Due to phosphodiesterases activity, half-time for cGMP varies between few seconds in vivo to few minutes in vitro. Therefore, in order to perform prolonged incubations, we used a membrane-permeable analog 8-Br cGMP. While no significant effects were observed after a 4-h incubation with 8-Br cGMP, a 24-h incubation resulted in markedly higher (P < 0.01) ability of podocytes to migrate, as compared to the non-treated (control) cells (Fig. 5). This was accompanied by a pronounced disassembly of F-actin fibers (Fig. 6). As shown in Fig. 5, incubation with DEX prevented the cGMP-dependent increase in podocyte motility, while F-actin stress fibers remained well preserved, forming a strong network (Fig. 6).Fig. 5

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