PMC

The induction of β-catenin and NF-κB signaling pathways in diabetic glomeruli is limited in Pod-ETRKO glomeruli. RT-PCR analysis of Snai1, Vimentin, and Axin2 mRNA expression in glomerular extracts from 20-week-old WT control, WT diabetic, Pod-ETRKO control, and Pod-ETRKO diabetic mice. Values are the mean±SEM from at least six mice. *P<0.05 versus respective nondiabetic mice; **P<0.01 versus respective nondiabetic mice; ^##P<0.01 versus WT diabetic mice.

At basal state, β-catenin and NF-κB signaling pathways are decreased in glomeruli of Pod-ETRKO mice. (A) Western blot analysis of β-catenin and phospho-NF-κB (p-NF-κB) expression in glomerular extracts from 10-week-old WT and Pod-ETRKO mice. Protein concentration is normalized to tubulin expression. (B) Quantification of Western blot bands for β-catenin and p-NF-κB normalized to tubulin band intensity. (C) RT-PCR analysis of Snai1, Vimentin, and Axin2 mRNA expression in glomerular extracts from 10-week-old WT and Pod-ETRKO mice. Values are the mean±SEM from at least six mice. *P<0.05; **P<0.01; ***P<0.001.

Pod-ETRKO mice are protected from diabetes-induced glomerulosclerosis and podocyte loss in a model of uninephrectomy followed by streptozotocin-diabetes induction. (A) Representative images of hematoxylin/eosin-stained sections of renal cortex from 16-week-old WT uninephrectomized diabetic and Pod-ETRKO uninephrectomized diabetic mice. (B) Representative images of the expression of podocalyxin (upper panel), podocin (middle panel), and WT1 (lower panel) in 16-week-old WT uninephrectomized diabetic and Pod-ETRKO uninephrectomized diabetic mice. (C) Quantification of the glomerular WT1-positive cell numbers in 16-week-old WT uninephrectomized diabetic and Pod-ETRKO uninephrectomized diabetic mice. Data represent the mean±SEM from at least four mice. *P<0.05. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 50 μm.

Activation of the Ca²⁺ pathway in podocytes stimulated by thrombin, ET-1, and sarafotoxin 6c. (A) Typical cell images illustrating the change in fluorescence ratio induced after addition of ET-1 (1 µM), sarafotoxin 6c (1 µM), or thrombin (10 nM) compared with the basal fluorescence ratio. (B) Time course of the averaged responses to the compounds. (C) Summary of the maximal fluorescence ratios. Values are the mean±SEM of nine values representing three different experiments each performed in triplicate.

In vitro, endothelin-1 activates β-catenin and NF-κB signaling pathways in glomeruli and its induction is decreased in Pod-ETRKO mice. (A) Western blot analysis of β-catenin and phospho-NF-κB (p-NF-κB) expression in glomeruli extracts from 10-week-old WT and Pod-ETRKO mice, treated or not with ET-1 at 100 nM for 4 hours. Protein concentration is normalized to tubulin expression. (B) Quantification of Western blot bands for β-catenin and p-NF-κB normalized to tubulin band intensity. (C) RT-PCR analysis of Snai1, Vimentin, and Axin2 mRNA expression in glomerular extracts from 10-week-old WT and Pod-ETRKO mice, treated or not with ET-1 at 100 nM for 4 hours. Values are the mean±SEM from at least six mice. *P<0.05 versus respective non-ET-1–treated mice; ^##P<0.01 versus respective WT mice; ^###P<0.001 versus respective WT mice.

Deletions of Ednar and Ednbr specifically in podocytes protect glomeruli from diabetes-induced glomerulosclerosis. (A) Representative images of hematoxylin/eosin-stained sections of renal cortex from 20-week-old WT control, WT diabetic, Pod-ETRKO control, and Pod-ETRKO diabetic mice. (B) Representative images of Masson’s trichrome–stained sections of glomeruli from 20-week-old WT control, WT diabetic, Pod-ETRKO control, and Pod-ETRKO diabetic mice. (C and D) Percentage of glomeruli with mesangial thickening (C) in the renal cortex of 20-week-old WT control, WT diabetic, Pod-ETRKO control, and Pod-ETRKO diabetic mice. (D) RT-PCR analysis of preproendothelin-1 (Edn1 gene), Ctgf, and trpc6 mRNA expression in glomerular extracts from 20-week-old WT control, WT diabetic, Pod-ETRKO control, and Pod-ETRKO diabetic mice. Values are the mean±SEM from at least six mice. *P<0.05 versus respective nondiabetic mice; ^#P<0.05 versus WT diabetic mice; ***P<0.001 versus respective nondiabetic mice; ^###P<0.001 versus WT diabetic mice. Scale bar, 50 μm.

ETAR and ETBR podocyte-specific deletions do not alter glomerular structure. (A) RT-PCR analysis of Ednar and Ednbr mRNA expression in isolated podocytes from 10-week-old WT and Pod-ETRKO mice. (B) Effective deletion of ETBR protein by NPHS2-Cre recombinase confirmed by immunoblotting analysis of isolated glomerular homogenates. Quantification of Western blot bands for ETBR normalized to tubulin band intensity. (C) Representative images of Masson’s trichrome–stained sections of glomeruli from 10-week-old WT and Pod-ETRKO mice. (D) Representative photomicrograph of transmission electron microscopy sections of podocytes from 10-week-old WT and Pod-ETRKO mice. Values are the mean±SEM from four mice. *P<0.05; **P<0.01. Scale bar, 50 μm in C; 1 μm in upper panel in D; 200 nm in lower panel in D.

ETAR and ETBR podocyte-specific deficiency protects podocytes from diabetes-induced podocyte loss. (A) Representative images of the expression of podocalyxin (upper panel) and podocin (lower panel) by immunofluorescence in 20-week-old WT control, WT diabetic, Pod-ETRKO control, and Pod-ETRKO diabetic mice. Images are representative of at least six mice. (B) Representative images of the expression of WT1 by immunohistochemistry in 20-week-old WT control, WT diabetic, Pod-ETRKO control, and Pod-ETRKO diabetic mice. Images are representative of at least six mice. (C) Quantification of the glomerular WT1-positive cell numbers in 20-week-old WT control, WT diabetic, Pod-ETRKO control, and Pod-ETRKO diabetic mice. Data are normalized to WT control and represent the mean±SEM from at least six mice. (D) Representative photomicrographs of transmission electron microscopy sections of podocytes from 20-week-old WT diabetic and Pod-ETRKO diabetic mice showing glomerular basement membrane thickening (asterisk) and foot process effacement (arrow) in WT DM mice. ***P<0.001 versus respective nondiabetic mice; ^###P<0.001 versus WT diabetic mice. Scale bar, 50 μm in A and B; 1 μm in upper panel in D; 200 nm in lower panel in D.