Silencing of hypoxia-inducible factor-1α gene attenuates chronic ischemic renal injury in two-kidney, one-clip rats

Abstract

Overactivation of hypoxia-inducible factor (HIF)-1α is implicated as a pathogenic factor in chronic kidney diseases (CKD). However, controversy exists regarding the roles of HIF-1α in CKD. Additionally, although hypoxia and HIF-1α activation are observed in various CKD and HIF-1α has been shown to stimulate fibrogenic factors, there is no direct evidence whether HIF-1α is an injurious or protective factor in chronic renal hypoxic injury. The present study determined whether knocking down the HIF-1α gene can attenuate or exaggerate kidney damage using a chronic renal ischemic model. Chronic renal ischemia was induced by unilaterally clamping the left renal artery for 3 wk in Sprague-Dawley rats. HIF-1α short hairpin (sh) RNA or control vectors were transfected into the left kidneys. Experimental groups were sham+control vector, clip+control vector, and clip+HIF-1α shRNA. Enalapril was used to normalize blood pressure 1 wk after clamping the renal artery. HIF-1α protein levels were remarkably increased in clipped kidneys, and this increase was blocked by shRNA. Morphological examination showed that HIF-1α shRNA significantly attenuated injury in clipped kidneys: glomerular injury indices were 0.71 ± 0.04, 2.50 ± 0.12, and 1.34 ± 0.11, and the percentage of globally damaged glomeruli was 0.02, 34.3 ± 5.0, and 6.3 ± 1.6 in sham, clip, and clip+shRNA groups, respectively. The protein levels of collagen and α-smooth muscle actin also dramatically increased in clipped kidneys, but this effect was blocked by HIF-1α shRNA. In conclusion, long-term overactivation of HIF-1α is a pathogenic factor in chronic renal injury associated with ischemia/hypoxia.

Keywords: chronic kidney diseases; collagen; renal fibrosis; α-smooth muscle actin.

Fig. 1.

Changes in mean arterial blood pressure (MAP). Angiotensin-converting enzyme-1 (ACEI) indicates the start of enalapril treatment. *P < 0.001 vs. other 2 groups by 2-way repeated measures ANOVA (n = 7).

Fig. 2.

Effects of hypoxia-inducible factor (HIF)-1α short hairpin (sh) RNA on HIF-1α accumulation in clipped kidneys. A: representative enhanced chemiluminescence (ECL) gel documents of Western blot analyses depicting protein levels of HIF-1α. B: summarized intensities of HIF-1α blots (normalized to Sham+Ctrl). *P < 0.001 vs. other 2 groups by 1-way ANOVA with Tukey's post hoc test (n = 7).

Fig. 3.

Effect of HIF-1α shRNA on morphological changes in the glomeruli in clipped kidneys. A: representative photomicrographs showing glomerular structures [periodic acid-Schiff (PAS) staining, ×400]. B: summarized glomerular damage index by semiquantitation of scores in different groups. *P < 0.001 vs. Clip+Ctrl and P = 0.003 vs. Sham+Ctrl by 1-way ANOVA with Tukey's post hoc test (n = 7).

Fig. 4.

Effect of HIF-1α shRNA on the percentage of globally damaged glomeruli in clipped kidneys. A: representative photomicrographs showing glomeruli (PAS staining, ×100). B: calculated percentage of globally damaged glomeruli. *P < 0.001 vs. other 2 groups by 1-way ANOVA with Tukey's post hoc test (n = 7).

Fig. 5.

Effect of HIF-1α shRNA on collagen I/III staining in clipped kidneys. A: representative photomicrographs showing staining of collagens in outer medulla (red color). B: calculated percentage of positively stained area. *P = 0.009 vs. Clip+Ctrl and P = 0.004 vs. Sham+Ctrl by 1-way ANOVA with Tukey's post hoc test (n = 6).

Fig. 6.

Effect of HIF-1α shRNA on α-smooth muscle actin (SMA) staining in clipped kidneys. A: representative photomicrographs showing staining of α-SMA in outer medulla (brown color). B: calculated percentage of positively stained area. *P < 0.001 vs. Clip+Ctrl and P = 0.029 vs. Sham+Ctrl by 1-way ANOVA with Tukey's post hoc test (n = 7).

Fig. 7.

Effect of HIF-1α shRNA on protein levels of collagen I/III and α-SMA in clipped kidneys. A: representative ECL gel documents of Western blot analyses depicting the protein levels. B: summarized blot intensities (normalized to Sham+Ctrl). *P = 0.001 vs. Sham+Ctrl and P = 0.01 vs. Clip+shRNA. #P < 0.001 vs. other 2 groups by 1-way ANOVA with Tukey's post hoc test (n = 7).