The impact of gender on progression of renal disease: potential role of estrogen-mediated vascular endothelial growth factor regulation and vascular protection

Abstract

Male gender is associated with a more rapid progression of renal disease independent of blood pressure, dietary protein intake, or serum lipid levels. Recently, we reported a key role for the intrarenal vasculature in progressive renal disease (Kang D-H, Kanellis J, Hugo C, Truong L, Anderson S, Kerjaschki D, Schreiner GF, Johnson RJ: Role of endothelium in progressive renal disease. J Am Soc Nephrol 2002, 13:806-816). We hypothesized that estrogen-mediated preservation of the renal vasculature could account for the better renal outcome in female rats. We analyzed micro- and macrovascular changes in the 5/6 remnant kidney (RK) models both in male (n = 24) and female (n = 24) Sprague-Dawley rats up to 12 weeks after renal mass reduction. At 12 weeks, male and female RK rats had equivalent blood pressure, glomerular tuft area, and RK/body weight, but male rats showed worse renal function, proteinuria, glomerulosclerosis (%), and tubulointerstitial fibrosis. At 12 weeks peritubular capillary (PTC) EC proliferation and PTC density were higher in female RK rats whereas macrovascular changes in preglomerular vessels (smooth muscle cell proliferation, medial wall thickening, and adventitial fibrosis) were less prominent. The expression of vascular endothelial growth factor (VEGF) and VEGF type 2 receptor (flk-1) in renal cortex assessed by immunostaining were higher in female RK rats. To dissect the mechanism of sex hormone-induced vascular remodeling and VEGF regulation, we investigated the in vitro effect of 17 beta-estradiol (17 beta E, 10 nmol/L) on proliferation and VEGF expression of renal tubular cells (rat proximal tubular cells), vascular smooth muscle cells (VSMCs), and human umbilical vein endothelial cells (HUVECs). 17 beta E directly stimulated the proliferation of HUVECs, whereas it inhibited serum-induced proliferation of VSMCs. 17 beta E stimulated VEGF mRNA expression both in renal tubular cells and VSMCs. However, when cells were pretreated with a nitric oxide donor to simulate the in vivo condition, 17 beta E inhibited VEGF mRNA expression and protein release in VSMCs. In conclusion, female RK rats developed less glomerulosclerosis and renal failure compared to male RK rats in association with greater preservation of PTC and less preglomerular arteriopathy. Estrogen stimulated basal VEGF expression in renal tubular cells. We propose that estrogen may protect female rats in progressive renal disease by stimulating VEGF expression and maintaining a healthy intrarenal vasculature.

Figure 1

Comparison of RKW and percent increase of RKW/body weight (BW) in male and female RK rats. *, P < 0.05 versus baseline; ^#, P < 0.05 versus male RK rats. Data are expressed as mean ± SD.

Figure 2

Changes in systolic blood pressure in male and female RK model. After renal mass reduction, there was a gradual increase in blood pressure in both male and female RK rats that reached statistical significance at 12 weeks. At each time point, there were no differences in blood pressure between groups. *, P < 0.05 versus baseline and other time points of corresponding gender. Data are expressed as mean ± SD.

Figure 3

Comparison of renal scarring and macrophage infiltration in male and female RK rats. Compared to male RK rats (A, PAS; C, ED-1 immunostaining), there is less renal damage and less macrophage infiltration both in glomerular and tubulointerstitial areas in female RK rats (B and D). Original magnifications: ×100 (A and B); ×200 (C and D).

Figure 4

Mircovascular EC proliferation in male and female RK rats. The kinetics of proliferating glomerular (A) and PTC (B) ECs (defined as PCNA+, RECA+ cells) throughout the 12-week period after surgery are shown in male (RK-M) and female RK (RK-F) rats and compared to sham rats. An initial early proliferation of glomerular (week 2) and peritubular (week 1) ECs is not maintained in male RK rats whereas it persists in female RK rats. At week 12, both glomerular and PTC proliferation are lower than sham-operated controls in male RK rats, but they are still comparable to sham and female RK rats. *, p < 0.05 vs. corresponding sham; ^†, p < 0.05 vs. male RK; ^#, p < 0.05 vs. corresponding sham and male RK. Data are expressed as mean ± SD.

Figure 5

Comparison of renal microvasculature in male and female RK rats. Shown are the changes in glomerular and peritubular capillaries in male (A) and female (B) RK rats (RECA) at 12 weeks of RK surgery. Male RK rats had a greater loss of glomerular capillary density and more PTC rarefaction compared to female RK rats. Original magnifications, ×100.

Figure 6

Renal cortical VEGF expression in male and female RK rats. A: Quantification of cortical VEGF expression assessed by immunohistochemistry and computer image analysis is shown for RK and sham rats. In male RK rats, there is a marked decrease in VEGF expression in renal cortex. Data are expressed as mean ± SD. B: Western blotting for VEGF also shows similar findings. Shown is a representative blot of two renal cortical tissue animals in each group at 12 weeks. *, P < 0.05 versus other groups.

Figure 7

VEGFR-2 or KDR expression in male and female RK rats. Immunohistochemistry with VEGFR-2 or KDR (see Material and Methods) shows a better preservation of VEGF receptor staining along the ECs of glomerular and peritubular capillaries in female RK rats (B) compared to male RK rats (A). Original magnification, ×200 (B).

Figure 8

Differential effect of estrogen on VEGF expression in renal tubular cells and VSMCs. 17βE induced an up-regulation of VEGF mRNA and protein both in NRK52E cells and VSMCs assessed by RNase protection assay (A, n = 6) and enzyme-linked immunosorbent assay (B, n = 4), respectively. However, pretreatment with a NO donor resulted in attenuation of 17βE-induced VEGF expression only in VSMCs. A: Shown is a representative RNase protection assay blot. E, 17βE; SNP, sodium nitroprusside; SIN, 3-morpholinosydnonomine. *, P < 0.05 versus control; #, P < 0.05 versus control, E+SNP, and E+SIN. Data are expressed as mean ± SD.

Figure 9

Differential effect of estrogen on the proliferation of HUVECs and VSMCs. 17βE stimulated DNA synthesis of HUVECs time-dependently assessed by ³H-thymidine uptake (n = 6) while 17βE inhibited serum-induced proliferation of VSMCs (n = 6).