Dose- and time-dependent glucocorticoid receptor signaling in podocytes

Abstract

Glucocorticoids (GC) are the primary therapy for idiopathic nephrotic syndrome (NS). Recent evidence has identified glomerular podocytes as a potential site of GC action in this disease. The objectives of this study were to determine the presence of key components of the glucocorticoid receptor (GR) complex and the functionality of this signaling pathway in podocytes and to explore potential opportunities for manipulation of GC responsiveness. Here, we show that cultured murine podocytes express key components of the GR complex, including the GR, heat shock protein 90, and the immunophilins FKBP51 and FKBP52. The functionality of GR-mediated signaling was verified by measuring several GC (dexamethasone)-induced responses, including 1) increases in mRNA and protein levels of selected GC-regulated genes (FKBP51, phenol sulfotransferase 1, αB-crystallin); 2) downregulation of the GR protein; 3) increased phosphorylation of the GR; and 4) translocation of the GR into the nuclear fraction. Dexamethasone-induced phosphorylation and downregulation of GR protein were also demonstrated in isolated rat glomeruli. Podocyte gene expression in response to dexamethasone was regulated at both the transcriptional and posttranscriptional levels, the latter also including protein degradation. Short-term, high-dose GC treatment resulted in similar changes in gene expression and GR phosphorylation to that of long-term, low-dose GC treatment, thus providing a molecular rationale for the known efficacy of pulse GC therapy in NS. Induction of FKBP51 and downregulation of the GR represent negative feedback mechanisms that can potentially be exploited to improve clinical GC efficacy. Collectively, these findings demonstrate the presence of key molecular components of the GR signaling pathway and its functionality in podocytes and identify novel opportunities for improving clinical GC efficacy in the treatment of NS.

Fig. 1.

Expression of selected genes in cultured podocytes. A: RT-PCR demonstrated the mRNA expression of key members of the glucocorticoid receptor (GR) complex, glucocorticoid-regulated genes and of the podocyte differentiation marker synaptopodin (Synpo). +/−, Presence or absence of the reverse transcriptase in RT-PCR. PCR primers were designed to produce amplicons of ∼200 bp (bar). B: SDS-PAGE/Western blotting demonstrated the expression of corresponding proteins in podocytes. The relevant bands are labeled with asterisks. The positions of molecular mass markers are indicated on the left of each group of blots (in kDa).

Fig. 2.

Induction of FKBP51 following dexamethasone (Dex) treatment. A: qRT-PCR demonstrated that long-term treatments with each concentration of Dex strongly induced FKBP51 mRNA, with 1 μM Dex being most effective. B and C: Western blotting demonstrated the accumulation of FKBP51 protein after 1 day of each long-term Dex treatment. D: qRT-PCR demonstrated that 10 and 100 μM Dex short-term treatments induced FKBP51 mRNA after 3 days. E and F: Western blotting demonstrated the accumulation of FKBP51 protein after 1 day short-term treatment, with 10 and 100 μM Dex being most effective. Note that in controls and, in some conditions (1 μM Dex, short-term treatment), the expression of FKBP51 was low and at or below its detection limit, which caused some variability not reflecting actual changes. White bars, analysis at 2 h; horizontally striated bars, analysis at 1 day; vertically striated bars, analysis at 3 days; black bars, analysis at 5 days. Asterisks (★) indicate significant differences between samples taken at different times, either between the specified groups (A and D) or compared with untreated control cells (C and F). Plus signs (+) indicate significant differences to the corresponding samples of both other sample groups, taken at the same time. Detection of GAPDH served as a loading control (data not shown; B and E).

Fig. 3.

Induction of phenol sulfotransferase 1 (PST1) following Dex treatment. A: qRT-PCR demonstrated that long-term treatment with each concentration of Dex strongly induced PST1 mRNA with 1 μM Dex being most effective. B and C: Western blotting demonstrated the accumulation of PST1 protein after 3 days of each long-term Dex treatment. D: qRT-PCR demonstrated that 10 and 100 μM Dex short-term treatments induced PST1 mRNA after 3 days. E and F: Western blotting demonstrated the accumulation of PST1 protein after 3 days of 100 μM Dex short-term treatment. The fills of the columns and the use of the indicators for statistical significance are as in Fig. 2. Detection of GAPDH served as a loading control (data not shown; B and E).

Fig. 4.

Induction of αB-crystallin (αB-Cry) following Dex treatment. A: qRT-PCR demonstrated that long-term treatment with each concentration of Dex moderately induced αB-Cry mRNA within 5 days approximately to the same extent. B and C: Western blotting demonstrated that the accumulation of αB-Cry protein was apparent after 3 days of 1 μM Dex long-term treatment. D: qRT-PCR demonstrated that short-term treatments with 10 and 100 μM Dex slightly induced αB-Cry mRNA within 5 days. E and F: Western blotting demonstrated that the accumulation of αB-Cry protein was apparent after 3 days of 100 μM Dex short-term treatment. The fills of the columns and the use of the indicators for statistical significance are as in Fig. 2. Detection of GAPDH served as loading control (data not shown; B and E).

Fig. 5.

Expression of GR, HSP90, and FKBP52 and phosphorylation of GR in podocytes following Dex treatment. A: qRT-PCR demonstrated that 1 μM Dex long-term treatment for 1 and 3 days had no major effect on mRNA expression. B and C: Western blotting demonstrated that 1 μM Dex long-term treatment resulted in reduced GR protein after 1 day, while increased phosphorylation of the GR was apparent after 2 h. HSP90 and FKBP52 remained constant. D: qRT-PCR demonstrated that 100 μM Dex short-term treatment for 1 and 3 days had no major effects on mRNA expression. E and F: Western blotting demonstrated that 100 μM Dex short-term treatment for 2 h through 5 days resulted in reduced GR protein after 1 day, while increased phosphorylation of the GR was apparent after 2 h. HSP90 and FKBP52 remained constant. The use of the indicators for statistical significance (C and F) are as in Fig.2. Detection of GAPDH served as a loading control (B and E). White bars, analysis at 1 day; gray bars, analysis at 3 days.

Fig. 6.

Effect of RU486 on the expression of FKBP51 and PST1 in podocytes. A: qRT-PCR demonstrated significant inhibition by RU486 of the induction of FKBP51 mRNA after 3 days of either 1 μM Dex long-term or 100 μM Dex short-term treatments. B: qRT-PCR demonstrated that RU486 significantly reduced the induction of PST1 mRNA following 1 μM Dex long-term treatment, yet had only a minor effect on PST1 mRNA following 100 μM Dex short-term treatment. C: Western blotting demonstrated that RU486 significantly reduced accumulation of FKBP51 protein following 3 days of either treatment, yet had no effect on the accumulation of PST1 protein. Brackets with asterisks indicate significant differences (A and B). Detection of GAPDH served as a loading control (C).

Fig. 7.

Effect of Dex on the translocation of GR into the nuclear fraction. Seventy-two hours after 1 μM Dex long-term or 100 μM Dex short-term treatment of podocytes, the distribution of the GR shifted from a predominant cytosolic (C) to a nuclear (N) location. GAPDH and lamin-B1 served as markers for the cytosolic and nuclear fractions, respectively.

Fig. 8.

Response of isolated rat glomeruli to Dex. A: image demonstrates integrity of isolated glomeruli; B: Western blotting of glomerular extracts demonstrated that both 1 μM Dex long-term and 100 μM Dex short-term treatment resulted in reduced GR protein and in its increased phosphorylation after 24 h. Detection of GAPDH served as a loading control (B).