Hypoxia-inducible factor-1α (HIF-1α) and autophagy in polycystic kidney disease (PKD)

Abstract

Cyst expansion in polycystic kidney disease (PKD) results in localized hypoxia in the kidney that may activate hypoxia-inducible factor-1α (HIF-1α). HIF-1α and autophagy, a form of programmed cell repair, are induced by hypoxia. The purposes were to determine HIF-1α expression and autophagy in rat and mouse models of PKD. HIF-1α was detected by electrochemiluminescence. Autophagy was visualized by electron microscopy (EM). LC3 and beclin-1, markers of autophagy, were detected by immunoblotting. Eight-week-old male heterozygous (Cy/+) and 4-wk-old homozygous (Cy/Cy) Han:SPRD rats, 4-wk-old cpk mice, and 112-day-old Pkd2WS25/- mice with a mutation in the Pkd2 gene were studied. HIF-1α was significantly increased in massive Cy/Cy and cpk kidneys and not smaller Cy/+ and Pkd2WS25/- kidneys. On EM, features of autophagy were seen in wild-type (+/+), Cy/+, and cpk kidneys: autophagosomes, mitophagy, and autolysosomes. Specifically, autophagosomes were found on EM in the tubular cells lining the cysts in cpk mice. The increase in LC3-II, a marker of autophagosome production and beclin, a regulator of autophagy, in Cy/Cy and cpk kidneys, followed the same pattern of increase as HIF-1α. To determine the role of HIF-1α in cyst formation and/or growth, Cy/+ rats, Cy/Cy rats, and cpk mice were treated with the HIF-1α inhibitor 2-methoxyestradiol (2ME2). 2ME2 had no significant effect on kidney volume or cyst volume density. In summary, HIF-1α is highly expressed in the late stages of PKD and is associated with an increase in LC3-II and beclin-1. The first demonstration of autophagosomes in PKD kidneys is reported. Inhibition of HIF-1α did not have a therapeutic effect.

Fig. 1.

Hypoxia-inducible factor-1α (HIF-1α) immunofluorescence. To demonstrate that HIF-1α is present in the cells lining the cysts, immunofluorescence was performed. There is HIF-1α staining (red) in tubular epithelial cells of normal controls. There is HIF-1α staining (red) in cells lining the cysts in Cy/+ and Cy/Cy rats. HIF-1α is known to be a nuclear protein. Nuclei are represented by blue (DAPI) staining. Superimposed red and blue staining indicates localization of HIF-1α staining to the nucleus.

Fig. 2.

Electron microscopy of autophagy. A–C: +/+ rats. Magnification ×2,500 (A), ×12,000 (B), and ×20,000 (C). D–G: Cy/+ rats. Magnification ×2,500 (D), ×40,000 (E), ×40,000 (F), and ×60,000 (G). H and I: Cy/Cy rats. Magnificaion ×2,500 (H) and ×8,000 (I). J and K: +/+ mice. Magnification ×2,500 (J) and ×6,000 (K). L–N: cpk mice. Magnification ×2,500 (L), ×12,000 (M), and ×12,000 (N). Autophagosomes (arrows) are demonstrated in kidneys of +/+ rats (A–C), Cy/+ rats (D–G), +/+ mice (J and K), and cpk mice (L–N). Lysosomes (L) fusing to autophagosomes are shown in F and G. Mitochondria (M) within a autophagosome (mitophagy) are shown in C and F. Autophagosomes were not seen in epithelial cells lining cysts in Cy/Cy rats (arrowheads, H and I). Autophagosomes in epithelial cells lining cysts are shown in J–L.

Fig. 2.

Electron microscopy of autophagy. A–C: +/+ rats. Magnification ×2,500 (A), ×12,000 (B), and ×20,000 (C). D–G: Cy/+ rats. Magnification ×2,500 (D), ×40,000 (E), ×40,000 (F), and ×60,000 (G). H and I: Cy/Cy rats. Magnificaion ×2,500 (H) and ×8,000 (I). J and K: +/+ mice. Magnification ×2,500 (J) and ×6,000 (K). L–N: cpk mice. Magnification ×2,500 (L), ×12,000 (M), and ×12,000 (N). Autophagosomes (arrows) are demonstrated in kidneys of +/+ rats (A–C), Cy/+ rats (D–G), +/+ mice (J and K), and cpk mice (L–N). Lysosomes (L) fusing to autophagosomes are shown in F and G. Mitochondria (M) within a autophagosome (mitophagy) are shown in C and F. Autophagosomes were not seen in epithelial cells lining cysts in Cy/Cy rats (arrowheads, H and I). Autophagosomes in epithelial cells lining cysts are shown in J–L.

Fig. 3.

Immunoblotting for LC3-II. LC3-II specifically associates with autophagosomes and not with any other vesicular structures. The intensity of the LC3-II bands was increased in cpk mice compared with wild-type (+/+) mice (A). In +/+ mice treated with bafilomycin A1, there was an increase in LC3-II (A). In cpk mice treated with bafilomycin A1, LC3-II was not increased (A). LC3-II was increased in Cy/Cy rats compared with +/+ and Cy/+ rats (B) compared with respective normal littermate controls. β-Actin, used as a loading control, was not different among the groups. *P < 0.05 vs. controls; n = 4/group.

Fig. 4.

Immunoblotting for beclin-1. Beclin-1 participates in the initiation and elongation processes of autophagosome and has a key role in autophagy. The intensity of the beclin-1 bands was increased in cpk mice (A) and Cy/Cy rats (B) compared with respective normal littermate controls. β-Actin, used as a loading control, was not different between the groups. *P < 0.05 vs. controls; n = 4/group.

Fig. 5.

Immunofluorescence for LC3. To demonstrate that LC3 is present in the cells lining the cysts, immunofluorescence was performed. There is LC3 staining (red) in cells lining the cysts in Cy/+ and Cy/Cy rats. Nuclei are represented by blue (DAPI) staining. LC3 staining is cytosolic.

Fig. 6.

2-Methoxyestradiol (2ME2) treatment in Cy/Cy rats. Cy/Cy rats were treated with the HIF-1α inhibitor 2ME2 or vehicle from 14 to 28 days of age. Kidneys were removed at death for determination of HIF-1α protein, kidney size as determined by 2K/TBW ratio, and cyst volume density (CVD). 2ME2 treatment (2 mg·kg⁻¹·day⁻¹ ip) resulted in inhibition of HIF-1α as determined by immunoblotting. There was no significant difference in 2-kidney-to-total body weight (2K/TBW) ratio and CVD between vehicle- and 2ME2-treated Cy/Cy rats. NS, not significant; n = 5/group.

Fig. 7.

2ME2 treatment in cpk mice. Cpk mice were treated with the HIF-1α inhibitor 2ME2 or vehicle from 14 to 21 days of age. Kidneys were removed at death for determination of HIF-1α protein, kidney size as determined by 2K/TBW ratio, and CVD. 2ME2 treatment (2 mg·kg⁻¹·day⁻¹ ip) resulted in inhibition of HIF-1α as determined by immunoblotting. There was no significant difference in 2K/TBW ratio and CVD between vehicle- and 2ME2-treated cpk mice; n = 3/group.