Endothelial Endothelin Receptor A Expression Is Associated With Podocyte Injury and Oxidative Stress in Patients With Focal Segmental Glomerulosclerosis

Abstract

Introduction: The podocyte is thought to be the mainly affected cell type in focal segmental glomerulosclerosis (FSGS). However, recent studies have also indicated a role for glomerular endothelial cells and podocyte-endothelial crosstalk in FSGS development. An experimental model for podocyte injury showed that increased endothelin-1 (ET-1) signaling between podocytes and endothelial cells induces endothelial oxidative stress and subsequent podocyte loss. In the current study, we investigated endothelial endothelin receptor A (ET_AR) expression in patients with FSGS and its association with podocyte injury and glomerular oxidative stress.

Methods: We selected 39 biopsy samples of patients with FSGS and 8 healthy control subjects, and stained them for ET_AR, nephrin and 8-oxo-guanine, a DNA lesion caused by oxidative damage. Glomeruli with ET_AR-positive endothelium and with nephrin loss were scored, and the 8-oxo-guanine-positive glomerular area was measured.

Results: The mean percentage of glomeruli with ET_AR-positive endothelial cells in patients with FSGS was higher compared to that in healthy control subjects (52% vs. 7%; P < 0.001). The presence of glomerular ET_AR-positive endothelium was strongly associated with nephrin loss both on the biopsy level (rho = 0.47; P < 0.01), as on the level of individual glomeruli (odds ratio = 2.0; P < 0.001). Moreover, glomeruli with ET_AR-positive endothelium showed more 8-oxo-guanine-positive staining (1.9% vs. 2.4%; P = 0.037). Finally, 8-oxo-guanine positivity in glomeruli was associated with increased levels of proteinuria.

Conclusion: Taking together our findings, we show that ET_AR is increased in glomerular endothelial cells of patients with FSGS and associated with podocyte damage and glomerular oxidative stress. These findings support the hypothesis that ET-1 signaling in glomerular endothelial cells contributes to disease development in patients with FSGS.

Keywords: crosstalk; endothelin receptor A; focal segmental glomerulosclerosis; glomerular endothelial cells; oxidative stress; podocytes.

Graphical abstract

Figure 1

Increased endothelial endothelin receptor A (ET_AR) expression in glomeruli of patients with focal segmental glomerulosclerosis (FSGS). (a) ET_AR staining (green) colocalizes with endothelial CD31 (red), indicating that ET_AR is expressed on glomerular endothelial cells (enlarged in inset). (b) Representative examples of glomerular ET_AR expression in control kidneys (left) and biopsy samples with FSGS (right). Insets highlight capillaries without ET_AR-positive staining (control) and with ET_AR-positive staining (FSGS). (c) Quantification of the percentage of glomeruli with ET_AR-positive endothelium. The mean percentage of glomeruli with ET_AR-positive endothelial cells per patient was higher in patients with FSGS compared to healthy control subjects (52% vs. 7%; P < 0.001). (d) The percentage of glomeruli with ET_AR-positive endothelium per patient correlated with the percentage of glomeruli with nephrin loss in patients with FSGS. (e) Nephrin loss and ET_AR-positive endothelium colocalized within the same glomeruli. Areas of nephrin loss were positive for an endothelial pattern of ET_AR, whereas areas with normal linear nephrin expression did not show ET_AR positivity. Analyzing all individual glomeruli for nephrin loss and ET_AR expression in sequential sections of the same glomerulus showed that the odds of having nephrin loss was higher for glomeruli with ET_AR-positive endothelium compared to ET_AR-negative glomeruli (OR = 2.0; P < 0.001). Bars = 50 μm.

Figure 2

8-oxo-guanine (8-oxoG) Staining in glomeruli of patients with focal segmental glomerulosclerosis (FSGS) indicates increased oxidative stress and correlates with ET_AR expression. (a) Representative examples of 8-oxoG staining in (left) a transplant control kidney sample and (right) a biopsy sample of a patient with FSGS. (b) Quantification of glomerular 8-oxoG as measured by ImageJ. Graph shows the summary data of the mean 8-oxoG−positive area per patient. Although the median 8-oxoG expression was not different in patients with FSGS compared to controls, there was a subset of FSGS patients with increased 8-oxoG levels (as indicated by the higher spread in the upper quadrants). (c) In patients with FSGS, glomeruli with ET_AR-positive endothelium showed more 8-oxoG−positive staining. (d) Occasionally, we observed colocalization of 8-oxoG−positive cells within areas of nephrin loss (arrowheads). Bars = 50 μm.

Figure 3

8-oxoG−Positive staining in podocytes and endothelial cells of patients with FSGS. (a) Wilms tumor 1−positive staining (red) colocalized with 8-oxoG−positive staining (green) in glomeruli of patients with FSGS, indicating that oxidative stress levels are increased in podocytes (arrowheads) (enlarged in inset; with DAPI). (b, c) von Willebrand factor−positive staining (red) also colocalized with 8-oxoG (green) positive staining, indicating that endothelial cells also had increased oxidative stress levels (arrows). Part b shows nuclear 8-oxoG staining enclosed by cytoplasmic von Willebrand Factor staining (enlarged in inset; with DAPI). Part c shows cytoplasmic 8-oxoG staining in yellow, colocalizing with cytoplasmic von Willebrand factor staining (enlarged in inset; with DAPI). Bars = 50 μm. Asterisks in parts b and c illustrate examples of autofluorescence of erythrocytes present in glomerular capillaries, which is commonly observed.^,