Hyperoxia exposure impairs nephrogenesis in the neonatal rat: role of HIF-1α

Abstract

Preterm neonates are exposed at birth to high oxygen concentrations relative to the intrauterine environment. We have previously shown in a rat model that a hyperoxic insult results in a reduced nephron number in adulthood. Therefore, the aim of this study was to determine the effects of transient neonatal hyperoxia exposure on nephrogenesis. Sprague-Dawley rat pups were raised in 80% O2 or room air from P3 to P10. Pups (n = 12/group, 6 males and 6 females) were sacrificed at P5 (during active nephrogenesis) and at P10 (after the completion of nephrogenesis). Hyperoxia exposure resulted in a significant reduction in both nephrogenic zone width and glomerular diameter at P5, and a significantly increased apoptotic cell count; however, nephron number at P10 was not affected. HIF-1α expression in the developing kidney was significantly reduced following hyperoxia exposure. Systemic administration of the HIF-1α stabilizer dimethyloxalylglycine (DMOG) resulted in enhanced expression of HIF-1α and improved nephrogenesis: kidneys from hyperoxia-exposed pups treated with DMOG exhibited a nephrogenic zone width and glomerular diameter similar to room-air controls. These findings demonstrate that neonatal hyperoxia exposure results in impaired nephrogenesis, which may be at least in part HIF-1α-mediated. Although nephron number was not significantly reduced at the completion of nephrogenesis, early indicators of maldevelopment suggest the potential for accelerated nephron loss in adulthood. Overall, this study supports the premise that prematurely born neonates exposed to high oxygen levels after birth are vulnerable to impaired renal development.

Figure 1. Nephrogenic zone width (A), glomerular diameter (B, D) and cortical width (C) in male and female animals from control (NNI, NH) and hyperoxia-exposed (H) groups at P5 (A-B) and P10 (C-D).

There was a significant effect of hyperoxia exposure (p_H) on nephrogenic zone width and glomerular diameter at P5 (A-B), but no effect at P10 (C-D). There was no effect of sex (p_S) on any parameter. *p<0.05 according to Bonferroni post-hoc analysis.

Figure 2. HIF-1α score (indicating the percentage of cells positive for HIF-1α expression) in the nephrogenic zone (A) and the cortical interstitium (B) in male and female animals from control (NNI, NH) and hyperoxia-exposed (H) groups at P5.

*p<0.05 between groups as indicated. Representative photomicrographs of HIF-1α expression (indicated by brown staining; arrows) in the nephrogenic zone of NNI (C), NH (D) and H animals (E) at P5.

Figure 3. The number of apoptotic cells in the area of nephrogenic zone (A) and glomeruli (B) of male and female animals from control (NNI, NH) and hyperoxia-exposed (H) groups at P5.

There was a significant effect of hyperoxia exposure (p_H) on the number of apoptotic cells, but no effect of sex (p_S). Representative photomicrographs of TUNEL staining of apoptotic cells (indicated by brown staining; arrows) in the nephrogenic zone of NNI (C), NH (D) and H animals (E) at P5.

Figure 4. Nephrogenic zone width (A) and glomerular diameter (B) in NH and H males and females at P5 that were treated with DMOG, compared to saline-treated controls.

There was a significant effect of hyperoxia exposure (pH) and DMOG treatment (pDMOG) on nephrogenic zone width in males and females (A). There was a significant effect of DMOG treatment (pDMOG), but no effect of hyperoxia (pH), on glomerular diameter in both males and females (B). The number of counted glomeruli in 5% of the kidney (C) and glomerular density (D) in in NH and H males and females at P5 that were treated with DMOG, compared to saline-treated controls. There was no significant difference in glomerular number between groups, but glomerular density was significantly increased in NH+DMOG males. *p<0.05, **p<0.01, ***p<0.001 according to Bonferroni post-hoc analysis.

Figure 5. HIF-1α score (indicating the percentage of cells positive for HIF-1α expression) in the nephrogenic zone (A) and the cortical interstitium (B) in NH and H males and females at P5 that were treated with DMOG, compared to saline-treated controls.

*p<0.05 between groups as indicated. Representative photomicrographs of HIF-1α expression (indicated by brown staining; arrows) in the nephrogenic zone of NH Control (C), NH+DMOG (D), H Control (E) and H+DMOG (F) animals at P5.