1. Volume 83, Issue 6, Pages 1065-1075 (June 2013)
Aberrant Notch1-dependent effects on glomerular parietal epithelial cells promotes collapsing focal segmental glomerulosclerosis with progressive podocyte loss 
Toshiharu Ueno, Namiko Kobayashi, Makiko Nakayama, Yasutoshi Takashima, Takamoto Ohse, Ira Pastan, Jeffrey W. Pippin, Stuart J. Shankland, Noriko Uesugi, Taiji Matsusaka, Michio Nagata 
Kidney International 
Volume 83, Issue 6, Pages (June 2013)
DOI: /ki
Copyright © 2013 International Society of Nephrology Terms and Conditions

2. Figure 1
Podocyte-selective injury induces progressive proteinuria and histological features of collapsing focal segmental glomerulosclerosis associated with hyperplastic parietal epithelial cells. (a) Total proteinuria was determined from the urinary protein/urinary creatinine ratio. NEP25 transgenic mice that received immunotoxin (LMB2 mice, n=6) showed progressive proteinuria beginning at days 8–12 compared with vehicle-treated NEP25 mice (controls, n=6). Periodic acid-silver methenamine (PAM) staining showed normal morphology in (b) controls, whereas (c) global tuft collapse was observed along with epithelial hyperplasia on day 12 in LMB2 mice. (d) Double labeling for β-galactosidase (β-gal) and claudin-1 showed podocyte-limited β-gal staining (blue) in controls. (e) In LMB2 mice, marked decrease of podocytes on β-gal staining and expression of claudin-1 (brown) in the hyperplastic lesions were observed. Bars=15μm. (f) The number of WT1-positive cells per glomerulus was significantly and progressively reduced in LMB2 mice on days 8 and 12 compared with day 0 (day 0: 10.24±0.22; day 8: 5.17±0.1; day 12: 2.4±0.05 cells per glomerulus). Cleaved caspase 3–positive cells were rarely observed throughout the observation period (day 0: 0±0; day 8: 0.009±0.006; day 12: 0.03±0.006 cells per glomerulus). (g) The PEC score on day 12 for LMB2 mice was significantly higher than that for controls (0.40±0.04 vs. 0.03±0.01 per glomerulus; P<0.001). *P<0.001 for day 0 versus day 8 or 12; †P<0.001 for controls versus LMB2 mice. Double labeling for (h) β-gal (blue) and cleaved caspase 3 and (i) Ki-67 (brown) did not reveal colocalization. Mirror images of claudin-1 with (j, j′) cleaved caspase 3 and (k, k′) Ki-67 showed that these markers were colocalized (arrowheads) (bars=15μm). For electron microscopy, ultrathin sections were stained with uranyl acetate and lead citrate. (l) A low-magnification view of an early glomerular lesion in an LMB2 mice showed initial PEC migration toward the glomerular tuft (original magnification × 200; bar=28μm). (l′) Higher magnification view of the inset in l showing adhesion of a podocyte-lost glomerular capillary with a migrated PEC, and remnant podocytes on the glomerular tuft revealed severe foot process effacement, pseudocyst, and villous transformation representing severe podocyte damage (original magnification × 1000; bar=12μm).
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

3. Figure 2
Aberrant Notch expression is observed in hyperplastic parietal epithelial cells (PECs) in LMB2 mice. (a) Only minimal cleaved Notch1 immunostaining (green) and abundant expression of synaptopodin (a’, red) were observed in the glomeruli of controls, even in a merged image (a′′). (b) In contrast, cleaved Notch1 expression was apparent both in podocytes and migrating PECs in an early glomerular lesion of LMB2 mice, and coexpression with decreasing podocyte marker podocalyxin (b′, red) was observed in a merged image (b′′, white arrows: podocytes, white arrowhead: PECs). (c) Cleaved Notch1 expression was more obvious in an advanced glomerular lesion and the coexpression of cleaved Notch1 and the podocyte marker podocalyxin (nearly disappeared) (c′, red) was rarely observed in a merged image. Cleaved Notch1 was observed predominantly in extracapillary hyperplastic cells, presumably PECs (c′′, white arrowheads), and in the same glomerulus with periodic acid–Schiff (PAS) staining (c′′′, black arrowheads). On serial sections, cleaved Notch1 (d, brown dots) expression was colocalized with claudin-1 (d′, brown) expression, similar to Jagged1 (e, brown) and Hes1 (e′, brown) expression. (f) The relative mRNA expression of Notch pathway genes from isolated glomeruli on day 12 was scored for each group (n=6). Notch1, Notch4, Jagged1, Delta1, and Hes1 mRNAs were significantly increased in LMB2 mice (17.99±5.85, 11.21±4.00, 3.07±0.56, 6.25±1.11, and 2.73-fold respective increases vs. controls; P=0.016, 0.029, 0.005, 0.001, and 0.003, respectively). Both Notch3 and HeyL mRNAs appeared to be increased, but the differences were not statistically significant. Claudin-1 mRNA levels were markedly increased in LMB2 mice (24.43±12.68-fold increases vs. controls). All results are expressed in arbitrary units and standard errors (*P<0.05, controls vs. LMB2 mice). DAPI, 4′,6-diamino-2-phenylindole. Bars=15μm.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

4. Figure 3
Aberrant Notch expression is observed in hyperplastic parietal epithelial cells (PECs) in human collapsing focal segmental glomerulosclerosis. (a) Periodic acid-silver methenamine (PAM) staining showed severely collapsed glomerular capillaries with extracapillary hyperplasia. On serial sections, cleaved Notch1 staining (a’, brown dots) was observed in hyperplastic epithelial cells but was almost completely absent in glomerular tufts. (b) Extracapillary hyperplastic epithelial cells expressed claudin-1 (red) but were negative for Nestin (violet). On serial sections, Jagged1 (c) and Hes1 (c’) colocalized in hyperplastic PECs (brown), as shown with periodic acid–Schiff (PAS) counterstaining. Bars=40μm.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

5. Figure 4
Transforming growth factor (TGF)-β1 induces Notch signaling mRNAs in cultured parietal epithelial cells (PECs) and Notch inhibition effectively suppresses mesenchymal phenotypic change–related gene expressions. (a) Relative Notch signaling mRNA expression at 1, 3, 6, and 12h with or without (control) TGF-β1 treatment. TGF-β1 treatment resulted in significantly increased Notch1, Jagged1, and Hes1 mRNA expression (P<0.001,=0.004, and <0.001, respectively). (b) Relative Hes1 mRNA levels in control, TGF-β1-treated, and TGF-β1+dibenzazepine (DBZ)-treated cells showed that TGF-β1-induced Hes1 mRNA expression was significantly suppressed by DBZ at 6 and 12h. (c) Relative mRNA expression of E-cadherin, smooth muscle actin (α-SMA), vimentin, and Snail1 were significantly changed 3, 6, and 12h after TGF-β1 treatment. (d–g) Time course changes in phenotypic marker mRNAs in control, TGF-β1-treated, and TGF-β1+DBZ-treated cells. TGF-β1-induced mRNA expression of mesenchymal markers were significantly suppressed by DBZ at 6 and 12h. All results are expressed in arbitrary units and standard errors (n=3 for each group; *P<0.05 vs. control; †P<0.05 for TGF-β1-treated group vs. TGF-β1+DBZ-treated group).
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

6. Figure 5
Inhibition of Notch signaling suppresses transforming growth factor (TGF)-β1-induced cell migration, but does not affect cultured parietal epithelial cell (PEC) proliferation or apoptosis. (a–c) Representative images from a Boyden chamber motility assay (original magnification × 40): (a) control (no treatment), (b) TGF-β1, (c) TGF-β1+ dibenzazepine (DBZ). (d) Quantitative analysis showed that the accelerated cell migration due to TGF-β was effectively inhibited by DBZ (27.44±5.71, ±2.44, and 29.22±2.03 cells per field, respectively; *P<0.001). DBZ treatment did not significantly affect (e) cell proliferation or (f) apoptosis of the cultured PEC line. Results are averages with standard errors (n=3 for each treatment group). NS, not significant. Bars=200μm.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

7. Figure 6
Notch inhibition suppresses parietal epithelial cell (PEC) lesions but worsens proteinuria and histopathology. (a) Time course for the urinary protein/urinary creatinine ratio. Proteinuria was not different in both groups until day 3, but became significantly higher on days 8 and 12 in dibenzazepine (DBZ)-treated LMB2 mice. (b) PEC scores were significantly reduced in DBZ-treated LMB2 mice; the frequency and severity of PEC lesion were lower in the DBZ-treated group (0.33±0.03 vs. 0.19±0.02; P=0.028, LMB2 vs. LMB2+DBZ mice). (c) The numbers of WT1-positive cells per glomerulus were not significantly different between these groups (2.87±0.19 vs. 2.69±0.19 cells per glomerulus; P=0.54). A low-magnification view of the renal cortex of a nontreated LMB2 mouse showed a few urinary casts by (d) periodic acid–Schiff (PAS) staining, whereas far more abundant urinary casts were observed in (e) DBZ-treated LMB2 mice. (f) Planimetry of urinary casts in the renal cortex revealed significantly more protein casts in the DBZ-treated LMB2 mice (8.01±0.81 vs ±4.17%; P<0.001, LMB2 vs. LMB2+DBZ mice). In each group, n=5; *P<0.05 and **P<0.001 for LMB2 vs. LMB2+DBZ mice. NS, not significant. Bars=150μm.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

8. Figure 7
Transmission electron micrographs of glomeruli in LMB2 and dibenzazepine (DBZ)-treated LBM2 mice. (a) A low-magnification view of a glomerulus in an LMB2 mouse showed a collapsed glomerular tuft with segmental hyperplastic parietal epithelial cells (PECs; original magnification × 200; bar=38μm). PECs migrated onto the glomerular tuft from the presumed entry site (arrowhead), and clusters or single PECs had attached to the collapsed tuft surface. (b) Higher magnification view of the inset in a showing the collapsed tuft covered by migrated PECs (original magnification × 1000; bar=12μm). (c) A low-magnification view of a glomerulus in a DBZ-treated LMB2 mouse showed a segmentally collapsed glomerular tuft and no PEC lesion (original magnification × 200; bar=38μm). (d) Higher magnification view of the inset in c (original magnification × 600; bar=12μm). Podocytes on the collapsed glomerular tuft exhibited severe foot process effacement, a pseudocyst, and villous transformation.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions