Fig. 6.

Inhibition of p38 MAPK decreases β-arrestin2 mediated nephrin endocytosis in vitro and in vivo. a Biotinylation assay in HEK293T under normal (5.5 mM) and high (30 mM) glucose conditions. Immunoprecipitation of nephrin was performed, and the biotinylated fraction of nephrin was analyzed (WB: streptavidin). Staining of nephrin in the lysate and immunoprecipitation (WB nephrin) and actin (WB: actin) in the lysate served as loading controls. The results were quantified by densitometry and graphed as the ratio of biotinylated signal intensity to total nephrin signal intensity – 5.5 mM glucose group was normalized to 100%. A total of 30 mM glucose (n = 3) vs. 30 mM glucose + SB202190 (n = 3): 62.4 ± 5.0 vs. 109.3 ± 5.2% (*p < 0.05). A total of 5.5 mM glucose (n = 3) vs. 30 mM glucose + SB202190 (n = 3):100.0 ± 14.1 vs. 109.3 ± 5.2% (p = ns). Data represent means ± SEM. Statistical analysis: unpaired t-test with Welch’s correction. b Representative immunofluorescence staining of murine kidney sections of normoglycemic (control), hyperglycemic (STZ), and hyperglycemic mice treated with p38 MAPK inhibitor (STZ + SB202190) (day 5, n = 3). Staining was performed with an anti-nephrin antibody (red) and nuclear DNA with DAPI (blue). c Representative immunofluorescence images of colocalization of nephrin with early endosomal antigen (EEA1) in immunofluorescence staining of murine kidney sections of healthy mice (control), untreated hyperglycemic mice (STZ), and hyperglycemic mice treated with p38 MAPK inhibitor (STZ + SB202190) (day 5, n = 3). Staining was performed with an anti-nephrin antibody (red), an anti-EEA1-antibody (green), and nuclear DNA with DRAQ5 (blue). White arrows indicate colocalization of nephrin with EEA1-positive vesicles. d Inhibition of p38 MAPK decreases β-arrestin2 mediated nephrin endocytosis in diabetic mice in a biotinylation assay of murine glomerular lysates of non-diabetic (control), diabetic (STZ), and diabetic mice treated with p38 MAPK inhibitor (STZ + SB202190) (day 5). Immunoprecipitation of nephrin was performed, and the biotinylated fraction of nephrin was analyzed (WB: streptavidin). Staining of nephrin in the lysate and immunoprecipitation (WB: nephrin) and actin (WB: actin) in the lysate served as loading controls. The results were quantified by densitometry and graphed as the ratio of biotinylated signal intensity to total nephrin signal intensity – the control group was normalized to 100%. Control (n = 4) vs. STZ (n = 4): 100.0 ± 0.7 vs. 74.2 ± 3.2% (**p < 0.01). STZ (n = 4) vs. STZ + SB202190 (n = 4): 74.2 ± 3.2 vs. 88.0 ± 4.5% (*p < 0.05). Control (n = 4) vs. STZ + SB202190 (n = 4): 100.0 ± 0.7 vs. 88.0 ± 4.5% (p = NS). Data represent means ± SEM. Statistical analysis: unpaired t-test with Welch’s correction