Inhibition of the protein kinase MK-2 protects podocytes from nephrotic syndrome-related injury - PubMed
Inhibition of the protein kinase MK-2 protects podocytes from nephrotic syndrome-related injury
Ruma Pengal et al. Am J Physiol Renal Physiol. 2011 Sep.
Abstract
While mitogen-activated protein kinase (MAPK) activation has been implicated in the pathogenesis of various glomerular diseases, including nephrotic syndrome (NS), its specific role in podocyte injury is not known. We hypothesized that MK-2, a downstream substrate of p38 MAPK, mediates the adverse effects of this pathway and that inhibition of MK-2 would protect podocytes from NS-related injury. Using cultured podocytes, we analyzed 1) the roles of MK-2 and p38 MAPK in puromycin aminonucleoside (PAN)-induced podocyte injury; 2) the ability of specific MK-2 and p38 MAPK inhibitors to protect podocytes against injury; 3) the role of serum albumin, known to induce podocyte injury, in activating p38 MAPK/MK-2 signaling; and 4) the role of p38 MAPK/MK-2 signaling in the expression of Cox-2, an enzyme associated with podocyte injury. Treatment with protein kinase inhibitors specific for both MK-2 (C23, a pyrrolopyridine-type compound) or p38 MAPK (SB203580) reduced PAN-induced podocyte injury and actin cytoskeletal disruption. Both inhibitors reduced baseline podocyte p38 MAPK/MK-2 signaling, as measured by the degree of phosphorylation of HSPB1, a downstream substrate of MK-2, but exhibited disparate effects on upstream signaling. Serum albumin activated p38 MAPK/MK-2 signaling and induced Cox-2 expression, and these responses were blocked by both inhibitors. Given the critical importance of podocyte injury to both NS and other progressive glomerular diseases, these data suggest an important role for p38 MAPK/MK-2 signaling in podocyte injury and identify MK-2 inhibition as a promising potential therapeutic strategy to protect podocytes in various glomerular diseases.
Figures

Schematic diagram of the p38 MAPK/MK-2 signaling upstream of HSPB1, a downstream substrate of MK-2, and the site of action of the protein kinase inhibitors C23 and SB203580. MK-2, a downstream substrate of p38 MAPK, phosphorylates 2 sites in mouse (Ser15, Ser86) and 3 sites in human HSPB1 (Ser15, Ser78, Ser82). While both inhibitors show strong inhibitory effects at the level of HSPB1, they can also affect the signaling upstream of their target kinases (cf. Fig. 6). TGF, transforming growth factor.

Phosphorylation of HSPB1 and efficacy of C23 and SB203580 on p38 MAPK/MK-2 signaling in long-term podocyte cultures. A: phosphorylation of HSPB1 in podocytes in response to anisomycin, as analyzed by isoelectric focusing (IEF)-PAGE/Western blotting (using an anti-HSPB1 antibody that recognizes all isoforms; top) and by SDS-PAGE/Western blotting (using phosphorylation site-specific antibodies; bottom). Cells were treated for 1 h with different concentrations of anisomycin as indicated, or were left untreated, and processed for electrophoresis. The IEF-PAGE analysis shows a gradual shift in the distribution of the HSPB1 isoforms from the predominantly unphosphorylated (0p) isoform in untreated cells via the singly phosphorylated (1p) isoform toward the doubly phosphorylated (2p) isoform, in response to increasing concentrations of anisomycin. The SDS-PAGE analysis shows the corresponding increase in phosphorylation at both MK-2 phosphorylation sites, Ser15 and Ser86. GAPDH served as a loading control. B: efficacy of C23 and SB203580 on inhibition of p38 MAPK/MK-2 signaling in long-term podocyte cultures, as analyzed by IEF-PAGE/Western blotting. Cultured podocytes were treated with different concentrations of C23 or SB203580 for 3 or 5 days as indicated. One hour before harvest, cells were stimulated with 10 or 50 ng/ml anisomycin as indicated. The blots indicate a dose-dependent inhibition of HSPB1 phosphorylation by C23 and SB203580. +/−, Acidic and basic sides, respectively, of the IEF gels.

Viability of puromycin aminonucleoside (PAN)-injured podocytes following treatment with C23 or SB203580. Cells were injured with 5 or 10 μg/ml PAN alone (white bars of the PAN sample groups) or cotreated with different concentrations of C23 or SB203580 (black bars of the PAN sample groups). For controls, cells were left untreated (white bars of the control sample group) or treated with different concentrations of C23 or SB203580 alone (black bars of the control sample groups). After 3 or 5 days of treatment, cell viability was determined by MTT assay. Inhibition of MK-2 (with C23) and p38 MAPK (with SB203580) improved the viability of PAN-injured podocytes. Inhibitor concentrations: white bars, no inhibitor; horizontally striated bars, 0.3 μM C23 or SB203580; vertically striated bars, 3 μM C23 or SB203580; black bars, 10 μM C23 or 30 μM SB203580. Values are means ± SD of quadruplicate samples of 1 representative experiment out of 4 independent experiments. Viability of untreated cells was set at 100%. Significant differences between samples within each sample group (*), or between the corresponding samples of both other sample groups (+), are indicated.

Organization of the actin cytoskeleton of PAN-injured podocytes following treatment with C23 or SB203580. Cells were injured with 5 or 10 μg/ml PAN for 3 or 5 days and cotreated with varying concentrations of C23 or SB203580 as indicated and processed for fluorescence microscopy. The actin cytoskeleton was visualized using Texas Red-labeled phalloidin. Inhibition of MK-2 (with C23) and p38 MAPK (with SB203580) protected the podocyte actin cytoskeleton from PAN-induced damage.

Phosphorylation of HSPB1 following long-term treatment of podocytes with C23 or SB203580. A: cells were treated for 1, 3, or 5 days with different concentrations of PAN and C23, alone or in combination, or were left untreated (negative controls). B: treatment was as in A but using SB203580 instead of C23. Additionally, cells were treated for 1 h with 50 ng/ml anisomycin before harvest (positive controls). At the days indicated, cells were processed for analysis of HSPB1 isoforms by IEF-PAGE/Western blotting. C and D: semiquantitative evaluation of the blots shown for 1-day treatments in A and B, respectively. The ratios of the phosphorylated and nonphosphorylated HSPB1 isoforms (RP values) were calculated from densitometric measurements of the corresponding bands and plotted as relative RP values (control = 1). The blots and graphs demonstrate that long-term treatment of podocytes with C23 or SB203580 resulted in reduced baseline activity of p38 MAPK/MK-2 signaling downstream of MK-2 which was most pronounced after 1 day of treatment. Cell injury by PAN had no major effect on the phosphorylation of HSPB1. The designation of the HSPB1 isoforms and of the orientation of the IEF gels is as in Fig. 2.

Disparate effects of C23 and SB203580 on the protein kinases upstream of HSPB1 following short-term treatments of podocytes. A: cells were serum-starved overnight before treatment with C23 or SB203580 for various time periods up to 2 h, or were left untreated (negative control). After harvesting, cells were processed for SDS-PAGE/Western blotting using specific antibodies recognizing phosphorylated or total p38 MAPK and MK-2. In addition, cells were processed for IEF-PAGE/Western blotting to detect the various HSPB1 isoforms. B: densitometric evaluation of the blots shown in A following treatment with C23. The graph shows relative changes in phosphorylation (activation) of p38 MAPK and MK-2 and in phosphorylation of HSPB1 (expressed as relative RP values). C: corresponding densitometric evaluation of the blots shown in A following treatment with SB203580. In these short-term treatments, C23 and SB203580 showed activating and inhibiting effects, respectively, on both upstream protein kinases. p-p38 MAPK, p38 MAPK: activated and total p38 MAPK, respectively; p-MK-2, MK-2: activated and total MK-2, respectively. The designation of the HSPB1 isoforms and of the orientation of the IEF gels is as in Fig. 2.

Effect of serum albumin (SA) on p38 MAPK/MK-2 signaling in podocytes. Cells were serum-starved overnight, treated for 1 h with SA (50 mg/ml), or pretreated with C23 (10 μM) or SB203580 (30 μM) 30 min before treatment with SA (50 mg/ml) as indicated. In addition, cells were treated with anisomycin (50 ng/ml; positive control) or were left untreated (negative control). A: SDS-PAGE/Western blotting shows activation of p38 MAPK by SA, C23, and anisomycin, as measured by phosphorylation of p38 MAPK. Activation by SA was inhibited by SB203580, but not by C23. B: IEF-PAGE/Western blotting shows activation of p38 MAPK/MK-2 signaling by SA and anisomycin, as measured by the isoform distribution of HSPB1. The effect of SA was inhibited by both C23 and SB203580. C: semiquantitative determination of the relative RP values of the blot shown in B. The RP value of the negative control was set at 1. The designations of the p38 MAPK forms, of the HSPB1 isoforms, and of the orientation of the IEF gels are as in Figs. 2 and 6.

Involvement of p38 MAPK/MK-2 signaling in cyclooxygenase (Cox)-2 mRNA expression in podocytes. A: decrease in baseline expression of Cox-2 mRNA in podocytes upon inhibition of p38 MAPK/MK-2 signaling. Cells were left untreated (control), or were treated with 10 μM C23 or 10 μM SB203580 for 24 h before RNA extraction. B: induction of Cox-2 by SA and its inhibition by C23 or SB203580. Serum-starved cells were left untreated (control), or were pretreated with 10 μM C23 or 30 μM SB203580 for 1 h followed by treatment with 5 or 50 mg/ml SA for 2 h before RNA extraction. Both C23 and SB203580 significantly reduced the induction of Cox-2 by either concentration of SA. Cox-2 mRNA was quantified by SYBR green RT-PCR, normalized to β-actin mRNA, and plotted as fold-change. Values are means ± SD of triplicate samples of 1 representative experiment of 3 independent experiments. Significant differences compared with the control values are indicated with asterisks (*), and significant differences compared with the respective SA-alone treatment are indicated by plus signs (+). SA 5 = 5 mg/ml; SA 50 = 50 mg/ml SA.
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