The abundance of c-mip increases in the glomeruli of patients with MCNS. (A) Representative immunohistochemical analysis of serial sections from normal human kidney (NHK) and kidney biopsy specimens from patients with MCNS relapse. Scale bars, 20 μM (B) Confocal microscopy analysis of nephrin (red) and c-mip (green) expression in normal human kidney tissues (upper panel) and MCNS biopsies (lower panel). The basal amount of c-mip was below the limit of detection, whereas c-mip was visualized along the peripheral capillary loops in patients with MCNS. The c-mip and nephrin were colocalized in the glomerulus. Scale bars, 10 μM.

Stable overexpression of c-mip in podocytes induces phenotypic alterations. (A) Confocal microscopy analysis of phalloidin staining. c-mip–overexpressing podocytes display a loss of stress fibers, whereas the actin network is well preserved in non-induced stable c-mip transfectants. Scale bars, 10 μM. (B) Western blot of protein lysates from non-induced (−) and induced (+) c-mip stable transfectants. The induction of c-mip inhibits the phosphorylation of nephrin and triggers the inactivation of Fyn and Akt. Data from two independent experiments are shown.

c-mip transgenic mice develop nephrotic proteinuria without inflammatory lesions or cell infiltrations. (A) Proteinuria was assessed by the proteinuria/creatinine ratio (Upr/Ucr). Upr/Ucr was 3.5 ± 0.75 in eight-week-old female heterozygotes (Tg+/−, n=26 mice), 61 ± 12.5 in male hemizygote Tg(+) transgenic mice (n=34 mice) and 2.6 ± 0.5 wild-type (WT) mice (n=12 mice). (B) Urine samples (5 μl) and BSA (15 μg) were resolved by SDS-PAGE and gels stained with Coomassie Blue. (C) Kidney sections from eight-week-old c-mip transgenic mice were examined. Tg(+) mice display podocyte flattening and the effacement of foot processes. The effacement of foot processes is associated with the disappearance of slit diaphragms, which are replaced by occluding junctions (arrow). Scale bars, Tg(+): upper and middle micrographs, 0.5 μM; lower micrograph, 0.1 μM. (D); The foot processes are evenly spaced in wild-type mice. Scale bars, upper micrograph, 1 μM; lower microgaph, 0.1 μM. (E) Morphometric analysis of foot process effacement indicating that the width of the podocyte sole in Tg(+) mice is twice that in the wild-type. N=3 mice per genotype were analyzed. Data are means ± SEM. Mann-Whitney test, * P <0.05.

Podocytes of c-mip transgenic mice have an abnormal phenotype. (A) Immunoprecipitation of nephrin from two different Tg(+) glomerular lysates, followed by Western blotting for phosphorylated nephrin and total nephrin. The amount of phosphorylated nephrin is reduced (right panel). Data from two independent experiments are shown. (B) Western-blot analysis of glomerular protein lysates from 10-week-old wild-type mice and Tg(+) mice. Fyn is mostly present in the inactive form, whereas the amount of pSer⁴⁷³-Akt is reduced. Data from two independent experiments are shown. (C) Lower abundance of phosphorylated Akt (pSer⁴⁷³-Akt) in the glomeruli (G) of patients with MCNS disease and in Tg(+) mice than in normal human kidney (NHK) and wild-type mice, respectively. Scale bars, 10 μM.

Interactions between nephrin, Nck and N-WASP are altered in c-mip Tg(+) mice. Immunoprecipitation (IP) of Fyn, N-WASP, and Nck from glomerular lysates of wild-type and Tg(+) mice. (A) c-mip coimmunoprecipitates with Fyn in Tg(+) glomerular lysates. (B) The amount of nephrin present in Fyn complexes is lower in glomerular lysates from Tg(+) mice than in those from wild-type mice. (C) The interactions of N-WASP with Fyn is decreased in Tg(+) mice compared to wild-type mice. (D) Immunoprecipitation of Nck from wild-type and Tg(+) glomerular lysates, followed by Western blotting with an anti-nephrin antibody. The interactions of Nck with nephrin are decreased in Tg(+) mice compared to wild-type mice. (E) Immunoprecipitation of nephrin from three different Tg(+) glomerular lysates, followed by Western blotting with anti-Nck antibody. The interaction of nephrin with Nck is reduced in Tg(+) mice compared to wild-type mice. Reprobing with an antibody against nephrin showed that equal amounts of nephrin were immunoprecipitated.

Knockdown of c-mip prevents the development of nephrotic proteinuria. (A) Detection of c-mip in LPS-treated Balb/c mice. Immunohistochemistry analysis showing a greater abundance of c-mip in podocytes (upper panel). Scale bars, 20 μM. Immunoprecipitation of glomerular extracts from LPS treated mice with anti-Fyn or mouse IgG (lower panel). (B) Increased abundance of c-mip in the podocytes of LPS-treated SCID mice than in those of untreated SCID mice. Scale bars, 20 μM. (C) Effects of c-mip siRNA injection in LPS-treated Balb/c mice. Balb/c mice (n=10 mice) received a single injection of c-mip siRNA (10 mg/kg) coupled to Alexa Fluor 647 into the internal jugular vein, and after 30 min, were injected intraperitoneally with LPS (200 μg). Controls include mice injected with LPS (n=5 mice) or Invivofectamine (n=5 mice) and non-injected mice (NI, n=5 mice). Urine was collected over a 24-hour period and the kidneys were harvested and processed for immunohistochemistry. Alexa Fluor 647 staining of kidney sections demonstrated that c-mip-siRNA duplexes were delivered to podocytes (upper panel). Scale bars, 10 μM. Immunohistochemistry analysis showed that mice treated with LPS and c-mip siRNA contained less c-mip than mice treated with LPS only (middle panel). Scale bars, 10 μM. RT-qPCR analysis of c-mip expression in mouse kidney (lower panel, left). mRNA abundance is expressed with respect to glyceraldehyde-3-phosphate deshydrogenase (GAPDH). Data are means ± SEM (p<0.05, Kruskal-Wallis test). Proteinuria was significantly lower in c-mip siRNA-treated mice than in mice treated with LPS alone (p=0.0017, Kruskal-Wallis test) (lower panel, left).

(A) Confocal microscopy analysis of phosphorylated nephrin (green) and total nephrin (red) in kidney sections. The relative abundances of nephrin and phosphorylated nephrin were assessed by quantifying the specific glomerular fluorescence intensity (lining the capillary loops) in three-dimensional stacks of images taken by confocal microscopy and normalized with respect to total glomerular area. Twenty glomeruli were analyzed for each set of conditions. The data presented are means ± SEM. The amount of phosphorylated nephrin was lower in LPS-treated mice than in siRNA-treated mice. The decrease in nephrin abundance is less marked in these mice. Scale bars, 10 μM. (B) Confocal microscopy analysis of phosphorylated Akt (pSer⁴⁷³-Akt) in kidney sections from mice treated with LPS alone or with LPS and siRNA. Scale bars, 10 μM. Data are means ± SEM. The amount of pSer⁴⁷³-Akt is significantly lower in LPS-treated mice (n=3 mice), than in LPS- and siRNA-treated mice (n=3 mice) (20 glomeruli were analyzed per mouse, p< 0.05, Kruskal-Wallis test).