Generation of podocyte-specific Crb2 knockout mice. (a) Two loxP sites were inserted into introns 6 and 8 of the Crb2 gene to skip exons 7 and 8 in the Crb2 floxed allele. (b) While there was a 353-bp band with Crb2 primers in Crb2^fl/fl mice and Crb2^fl/flpod-Cre^Tg/+ mice, there was a 153-bp product in Crb2^+/+pod-Cre^Tg/+ mice. There was a 102-bp band with pod-Cre primers in Crb2^+/+pod-Cre^Tg/+ mice and Crb2^fl/flpod-Cre^Tg/+ mice. (c) There were a strong band between 140–180 kDa and a weak band between 180–245 kDa markers with CRB2 antibody in the glomeruli from Crb2^+/+pod-Cre^Tg/+ mice or Crb2^fl/fl mice, while these bands were invisible in those of Crb2^fl/flpod-Cre^Tg/+ mice at 2 months of age. The band of β-actin was similar among the three groups. (d) Synpo, a podocyte marker, was co-localized with Crb2 in Crb2^+/+pod-Cre^Tg/+ or Crb2^fl/fl mice. No co-localization of Crb2 and Synpo was observed in Crb2^fl/flpod-Cre^Tg/+ mice. Scale bars 20 μm.

Podocyte-specific Crb2 knockout mice develop massive albuminuria and microhematuria at 2 months of age. (a) Massive albuminuria was observed at 2 months of age in Crb2^fl/flpod-Cre^Tg/+ mice compared to Crb2^+/+pod-Cre^Tg/+ or Crb2^fl/fl mice. Microhematuria was observed at 2 months of age in Crb2^fl/flpod-Cre^Tg/+ mice compared to Crb2^+/+pod-Cre^Tg/+ or Crb2^fl/fl mice. (b) The blood urea nitrogen and serum creatinine levels were comparable among the three groups at 2 months of age. (c) The sclerotic indices were comparable among the three groups at 2 months of age. Scale bars 50 μm. (d) The fibrotic indices were comparable among the three groups at 2 months of age. Scale bars 100 μm. Data are presented as mean ± SD. Statistical analysis by one-way analysis of variance, followed by Scheffe's multiple comparison test; n = 6 in each group. **P < 0.01, ****P < 0.0001.

Podocyte-specific Crb2 knockout mice develop FSGS at 6 months of age. (a) Massive albuminuria was observed at 6 months of age in Crb2^fl/flpod-Cre^Tg/+ mice compared to Crb2^+/+pod-Cre^Tg/+ or Crb2^fl/fl mice. Microhematuria was observed at 6 months of age in Crb2^fl/flpod-Cre^Tg/+ mice compared to Crb2^+/+pod-Cre^Tg/+ or Crb2^fl/fl mice. (b) The blood urea nitrogen and serum creatinine levels were comparable among the three groups at 6 months of age. (c) The sclerotic indices in Crb2^fl/flpod-Cre^Tg/+ mice were significantly higher than those in Crb2^+/+pod-Cre^Tg/+ or Crb2^fl/fl mice. Scale bars 50 μm. (d) The fibrotic indices in Crb2^fl/flpod-Cre^Tg/+ mice were significantly higher than those in Crb2^+/+pod-Cre^Tg/+ or Crb2^fl/fl mice. Scale bars 100 μm. Data are presented as mean ± SD. Statistical analysis by one-way analysis of variance, followed by Scheffe's multiple comparison test; n = 6 in each group. ***P < 0.001, ****P < 0.0001.

Hemosiderin accumulation and CD68-positive cells in the interstitium of podocyte-specific Crb2 knockout mice at 6 months of age and electron microscopic study. (a) The blue area was significantly larger in Crb2^fl/flpod-Cre^Tg/+ mice than in Crb2^+/+pod-Cre^Tg/+ or Crb2^fl/fl mice. Scale bars 100 μm. (b) The brown cells in the interstitium of Crb2^fl/flpod-Cre^Tg/+ mice were positive for CD68, suggesting that they were macrophages. CD68-positive cells were significantly more frequent in the interstitium of Crb2^fl/flpod-Cre^Tg/+ mice than in that of Crb2^+/+pod-Cre^Tg/+ or Crb2^fl/fl mice. HPF high-power field. Scale bars 50 μm. Data are presented as mean ± SD. Statistical analysis by one-way analysis of variance, followed by Scheffe's multiple comparison test; n = 5 in each group. ***P < 0.001, ****P < 0.0001. (c) Transmission electron microscopy showed foot process effacement in Crb2^fl/flpod-Cre^Tg/+ mice compared to Crb2^fl/fl mice. Scale bars 1 μm. The mean width of the foot processes in Crb2^fl/flpod-Cre^Tg/+ mice was significantly greater than that in Crb2^fl/fl mice. Data are presented as mean ± SD. Statistical analysis by Student's t-test; n = 3 in each group. **P < 0.01. (d) Scanning electron microscopy showed damage to the foot processes' urinary sides in Crb2^fl/flpod-Cre^Tg/+ mice compared to Crb2^fl/fl mice. Scale bars 1 μm. (e) Hemosiderin-laden macrophages were observed in the interstitial area of Crb2^fl/flpod-Cre^Tg/+ mice, but not in that of Crb2^fl/fl mice, at 6 months of age. Scale bars 10 μm.

The protein expressions of Nphs2, Podxl, and Nphs1 and the number of Wt1 positive cells in the glomeruli were decreased in podocyte-specific Crb2 knockout mice. (a) An immunofluorescence study showed that Nphs2, Podxl, and Nphs1 expression significantly decreased in Crb2^fl/flpod-Cre^Tg/+ mice compared to Crb2^fl/fl mice. Scale bars 50 μm. (b) The protein expressions of Nphs2, Podxl, and Nphs1 were significantly decreased in Crb2^fl/flpod-Cre^Tg/+ mice compared to Crb2^fl/fl mice. (c) Wt1-positive cells were significantly decreased in the glomeruli of Crb2^fl/flpod-Cre^Tg/+ mice compared to those of Crb2^fl/fl mice. Data are presented as mean ± SD. Statistical analysis by Student's t-test; n = 5 in each group. *P < 0.05, **P < 0.01, ****P < 0.0001.

The circulating permeability factors in the mouse serum samples at 6 months of age. (a) The serum levels of CLCF1 were comparable among the three groups. (b) The serum levels of TNF-α were comparable among the three groups. (c) The serum levels of suPAR were significantly elevated in the Crb2^fl/flpod-Cre^Tg/+ mice compared to the Crb2^fl/fl mice while there was no significant difference between serum levels of the Crb2^fl/flpod-Cre^Tg/+ mice and the Crb2^+/+pod-Cre^Tg/+ mice. Data are presented as the mean ± SD. The statistical analysis was performed by a one-way analysis of variance, followed by Scheffe's multiple comparison test; n = 5 in each group. *P < 0.05. CLCF1 cardiotrophin like cytokine factor 1, TNF-α tumor necrosis factor-α, suPAR soluble urokinase-type plasminogen activator receptor.

Generation of CRB2 knockout human immortalized podocytes. (a) The endogenous expression of CRB2 in differentiated human culture podocytes was weakly observed in the nucleus and cytoplasm. Scale bar 20 μm. (b) In differentiated human podocytes electroporated with carboxy-terminal FLAG-tagged mouse full-length Crb2, the staining with rabbit anti-CRB2 antibody and mouse anti-FLAG antibody was co-localized in the cytoplasm and was weak in the plasma membrane. Scale bars 20 μm. (c) While there was a 627-bp product in wild-type podocytes, there were 614- and 388-bp products in CRB2 knockout t17 clone and 628- and 464-bp products in CRB2 knockout t32 clone. The left lane was the 100-bp marker. (d) The CRB2 knockout t17 clone possessed mutations in an allele with c.336_348del TGCATCCCGGCCG, which caused a frameshift mutation and premature stop codon due to a 13-bp deletion, and in the other allele with c.114–352del, which caused a frameshift mutation and premature stop codon due to a 239-bp deletion. The CRB2 knockout t32 clone possessed mutations in an allele with c.195insC, which caused a frameshift mutation and premature stop codon due to a 1-bp insertion, and in the other allele with c.188–350del, which caused a frameshift mutation and premature stop codon due to a 163-bp deletion. (e) There were weak bands above and below 140 kDa marker with CRB2 antibody in the lysates of wild-type podocytes, while these bands were invisible in those of CRB2 knockout t17 and t32 clones. The positive control was a lysate from human embryonic kidney 293 cells transfected with CRB2-DYK. β-actin served as a loading control. (f) The protein expressions of NPHS2, PODXL, and NPHS1 were comparable among wild-type, CRB2 knockout t17, and t32 clones. (g) The F-actin positive area in CRB2 knockout t17 or t32 clones significantly decreased compared to wild-type human podocytes. Scale bars 50 μm. Data are presented as mean ± SD. Statistical analysis by one-way analysis of variance, followed by Scheffe's multiple comparison test; n = 6 in each group. *P < 0.05, **P < 0.01. CRB2KO CRB2 knockout, WT wild-type.

Apoptosis and adhesion assay. (a) Apoptotic assay demonstrated that CRB2 knockout t17 and t32 clones were more susceptible to apoptosis under serum starvation than wild-type human podocytes. (b) CRB2 knockout t17 clone was more susceptible to apoptosis under high glucose conditions than wild-type human podocytes, while there was no significant difference between CRB2 knockout t32 clone and wild-type podocytes. Data are presented as mean ± SD. Statistical analysis by one-way analysis of variance, followed by Scheffe's multiple comparison test; n = 4 in each group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (c) Adhesion assay showed decreased adhesive ability in CRB2 knockout t32 clone compared to wild-type podocytes or CRB2 knockout t17 clone while there was no significant difference between CRB2 knockout t17 and wild-type podocytes. Data are presented as mean ± SD. Statistical analysis by one-way analysis of variance, followed by Scheffe's multiple comparison test; n = 5 in each group. *P < 0.05, **P < 0.01. CRB2KO CRB2 knockout, FBS fetal bovine serum, HG high glucose, LG low glucose, WT wild-type.