Expression of Notch pathway proteins correlates with albuminuria, glomerulosclerosis, and renal function

Abstract

Recent studies indicate that the Notch signaling pathway plays an important role in the development of diabetic kidney disease and focal segmental glomerulosclerosis (FSGS). Here we analyzed the degree of expression and localization of Notch ligands (Jagged1 and Delta1) and activated (cleaved) receptors (Notch1 and Notch2) in healthy human kidneys and in renal biopsies from a wide variety of kidney diseases. These included patients with minimal change disease, membranous nephropathy, lupus nephritis ISN/RPS classes III/IV/V, hypertensive nephrosclerosis, crescentic glomerulonephritis, tubulointerstitial fibrosis, IgA nephropathy, diabetic kidney disease, and FSGS. We found that cleaved Notch1, Notch2, and Jagged1 are expressed on podocytes in proteinuric nephropathies and their level of expression correlated with the amount of proteinuria across all disease groups. The degree of glomerulosclerosis correlated with podocyte expression of cleaved Notch1, while the severity of tubulointerstitial fibrosis and the estimated glomerular filtration rate correlated with expression of cleaved Notch1 in the tubulointerstitium. Hence, our results raise the possibility that Notch pathway activation is a common mechanism in the pathophysiology of a wide range of acquired renal diseases.

Figure 1. Expression of Notch molecules in control human kidneys

Double immunostaining of a single control (healthy) human kidney sample with WT-1 (A, B, C, D) (FITC;green) and cleaved Notch1 (A), and Notch2 (B), Jagged1 (C) or Delta1 (D) (Cy3;red), nuclear stain with DAPI(blue) or their combination shown as indicated on the figure.

Figure 2. Expression of Notch molecules in diseased human kidney biopsies

Double immunostaining from a single diseased human kidney samples with WT-1 (FITC; green) and cleaved Notch1 (A), and Notch2 (B), Jagged1 (C) or Delta1 (D) (Cy3;red) nuclear stain with DAPI (blue) as indicated on the figure. The pictures were taken from a single case of membranous nephropathy.

Figure 3. Glomerular specific expression of cleaved Notch1, 2 and Jagged1 in different renal disease groups

The relative expression of active Notch1 (blue bar) Notch2 (red bar) and Jagged1 (yellow bar) in the glomerulus was scored in each biopsy sample. The graph shows the mean and SD of the expression of each molecules. * denotes statistically significant differences (p<0.05) when compared to controls.

Figure 4. Tubulointerstitial expression of cleaved Notch1, 2, Jagged1 and Delta1 in different renal disease groups

The relative expression of activeNotch1 (blue bar), Notch2 (red bar), Jagged1 (yellow bar) and Delta1 (light blue bar) in the tubulointerstitium was scored in each biopsy sample. The graph shows the mean and SD of the expression of each molecules. *denotes statistically significant differences (p<0.05) when compared to controls.

Figure 5. Result of the linear regression analysis of Notch expression and histological and functional renal parameters

Results of linear regression analysis (significance and correlation coefficient) of Notch pathway protein expression (Podocyte and tubulointerstitial active Notch1, Notch2, Jagged1 and Delta1) and proteinuria (log transformed) (A) estimated GFR (B) glomerulosclerosis (log transformed) (C), tubulointerstitial fibrosis (D). The Bonferroni adjusted p-value for this analysis is 0.002 or the unadjusted p-value 0.05 were considered statistically significant.

Figure 6. Correlation plots of PodocyteNotch1 expression and glomerulosclerosis and tubulointerstitial fibrosis and tubulointerstitial expression of active Notch1

Scatter-plot with fitted values (red line) and 95% confidence (green line) intervals for (A) percent glomerulosclerosis and podocyte expression of Notch1 and (B) and tubular expression of Notch1 and tubulointerstitial fibrosis.