Oxygen Regulates Vascular Endothelial Growth Factor-Mediated Vasculogenesis and Tubulogenesis

AbstractTo determine whether low oxygen is a stimulus for endothelial cell differentiation and vascular development in the kidney, we examined the effect of low oxygen on rat metanephric organ culture, a model known to recapitulate nephrogenesis in the absence of vessels. After 6 days in culture in standard (20% O2) or low oxygen (1–3% O2) conditions, metanephric kidney growth and morphology were assessed by DNA measurement, and light and electron microscopy. DNA content was higher in 3% O2-treated explants (2.5 ± 0.17 μg/kidney,n= 9) than in 20% O2explants (1.5 ± 0.09 μg/kidney,n= 9),P< 0.05. Low oxygen induced proliferation of tubular epithelial cells, resulting in enhanced number of tubules of similar size. Endothelial cells forming capillaries were localized in 3% O2explants by light and electron microscopy and by immunocytochemistry using endothelial cell markers. Flt-1, Flk-1, and ACE-containing cells were detected in 3% O2-treated explants, whereas 20% O2explants were virtually negative. VEGF mRNA levels were 10-fold higher in 3% O2-treated explants than in 20% O2-treated explants. Addition of anti-VEGF antibodies to 3% O2-treated explants prevented low oxygen-induced growth and endothelial cell differentiation and proliferation. Our data indicate that low oxygen stimulates growth by cell proliferation and induces tubulogenesis, endothelial cell differentiation, and vasculogenesis in metanephric kidneys in culture. Upregulation of VEGF expression by low oxygen and prevention of low oxygen-induced tubulogenesis and vasculogenesis by anti-VEGF antibodies indicate that these changes were mediated by VEGF. These data suggest that low oxygen is the stimulus to initiate renal vascularization

Effect of oxygen on the expression of renin-angiotensin system components in a human trophoblast cell line

During the first trimester, normal placental development occurs in a low oxygen environment that is known to stimulate angiogenesis via upregulation of vascular endothelial growth factor (VEGF). Expression of the placental renin-angiotensin system (RAS) is highest in early pregnancy. While the RAS and oxygen both stimulate angiogenesis, how they interact within the placenta is unknown. We postulated that low oxygen increases expression of the proangiogenic RAS pathway and that this is associated with increased VEGF in a first trimester human trophoblast cell line (HTR-8/SVneo). HTR-8/SVneo cells were cultured in one of three oxygen tensions (1%, 5% and 20%). RAS and VEGF mRNA expression were determined by qPCR. Prorenin, angiotensin converting enzyme (ACE) and VEGF protein levels in the supernatant, as well as prorenin and ACE in cell lysates, were measured using ELISAs. Low oxygen significantly increased the expression of both angiotensin II type 1 receptor (AGTR1) and VEGF (both P < 0.05). There was a positive correlation between AGTR1 and VEGF expression at low oxygen (r = 0.64, P < 0.005). Corresponding increases in VEGF protein were observed with low oxygen (P < 0.05). Despite no change in ACE1 mRNA expression, ACE levels in the supernatant increased with low oxygen (1% and 5%, P < 0.05). Expression of other RAS components did not change. Low oxygen increased AGTR1 and VEGF expression, as well as ACE and VEGF protein levels, suggesting that the proangiogenic RAS pathway is activated. This highlights a potential role for the placental RAS in mediating the proangiogenic effects of low oxygen in placental development

Reduced renal length and volume 20 years after very preterm birth

Intrauterine growth retardation is presumed to be associated with decreased renal size and impaired renal function as a result of stunted kidney development and nephron deficit. To study whether very preterm birth also affects renal size at young adulthood, we sonographically measured bipolar kidney length and volume in 51 very premature individuals (<32 weeks of gestation), either small (SGA) or appropriate (AGA) for gestational age (22 SGA and 29 AGA), and 30 full-term controls 20 years after birth. Relative kidney length and volume were calculated. Both absolute and relative left kidney length and volume were significantly lower in SGA and AGA individuals, notably in women. Renal size did not differ between SGA and AGA individuals. In 70% of controls, the left kidney was larger than the right one compared with 40.9% in SGA [relative risk (RR) 1.7; 95% confidence interval (CI) 1.0−3.0] and 48.3% in AGA (RR 1.5; 95% CI 0.9−2.3) individuals. Renal structural anomalies were present in eight prematurely born participants only. Our data suggest that kidney growth is stunted after preterm birth, especially on the left side, and in the female gender

Reduced angiotensinogen expression attenuates renal interstitial fibrosis in obstructive nephropathy in mice

A novel approach was employed to assess the contribution of the renin-angiotensin system (RAS) to obstructive nephropathy in neonatal mice having zero to four functional copies of the angiotensinogen gene (Agt). Two-day-old mice underwent unilateral ureteral obstruction (UUO) or sham operation; 28 days later, renal interstitial fibrosis and tubular atrophy were quantitated. In all Agt genotypes, UUO reduced ipsilateral renal mass and increased that of the opposite kidney. Renal interstitial collagen increased after UUO linearly with Agt expression, from a fractional area of 25% in zero-copy mice to 54% in two-copy mice. Renal expression of transforming growth factor-β1 was increased by ipsilateral UUO in mice expressing Agt, but not in zero-copy mice. However, the prevalence of atrophic tubules due to UUO did not vary with Agt expression. Blood pressure was not different in all groups, except for a reduction in sham zero-copy mice. We conclude that a functional RAS is not necessary for compensatory renal growth. This study demonstrates conclusively that angiotensin regulates at least 50% of the renal interstitial fibrotic response in obstructive nephropathy, an effect independent of systemic hemodynamic changes. Angiotensin-induced fibrosis likely is a mechanism common to the progression of many forms of renal disease