Impact of the hypoxia-inducible factor-1 α (HIF1A) Pro582Ser polymorphism on diabetes nephropathy

Abstract

Objective: Hypoxia plays a major pathogenic role in diabetic nephropathy (DN). We have investigated in this study the effect of hypoxia-inducible factor 1 α subunit (HIF1A) genetic polymorphisms on the development of DN.

Research design and methods: In 1,165 American type 1 diabetic patients with and without DN selected from the Genetics of Kidneys in Diabetes (GoKinD) study, the HIF1A genetic polymorphisms were genotyped with TaqMan allelic discrimination. The regulation of HIF-1α in the kidneys of diabetic mice was appreciated by immunohistochemistry, and the effect HIF1A Pro582Ser polymorphism on HIF-1α sensitivity to glucose was evaluated in vitro.

Results: We identified a protective association between HIF1A Pro582Ser polymorphism and DN in male subjects. We also provided mechanistic insights that HIF-1α is repressed in the medulla of diabetic mice despite hypoxia and that Pro582Ser polymorphism confers less sensitivity to the inhibitory effect of glucose during a hypoxic challenge.

Conclusions: The current study demonstrates for the first time that HIF1A Pro582Ser polymorphism has an effect on DN, possibly by conferring a relative resistance to the repressive effect of glucose on HIF-1α.

Figure 1

HIF-1α is repressed in diabetic kidneys medulla despite hypoxia. A: The kidney medulla of the diabetic animals (db/db) (A2) is more hypoxic than in normoglycemic heterozygote control mice (A1), as evaluated by pimonidazole adduct formation. B: HIF-1α expression in the medulla of the diabetic mice (db/db) (B2) is lower than in normoglycemic control mice (B1). C: The ratio between areas positive for HIF-1α and hypoxic areas (evaluated by pimonidazole) is lower in the medulla of diabetic animals compared with normoglycemic control mice.*P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)

Figure 2

Effects of HIF1A Pro582Ser polymorphism in glucose levels. A: Wild-type HIF-1α and HIF-1α (P/S) have similar expression level in normoxia and hypoxia. HEK293A cells were transfected with pFLAG/mHIF-1α (wild-type [WT]) or pFLAG/mHIF-1α (P/S), and were exposed to normoxia (N) or hypoxia (H) for 6 h before harvest. The expression of HIF-1α and mutant was detected by immunoblotting using anti-FLAG antibody. B: HIF-1α (P/S) protein is downregulated by 30 mmol/L glucose. HEK293A cells were transfected with pFLAG/mHIF-1α (P/S). The cells were then cultured in media containing 5.5 or 30 mmol/L glucose for 48 h and were exposed to normoxia (N) or hypoxia (H) for 6 h before harvest. The expression of HIF-1α (P/S) was detected by immunoblotting using anti-FLAG antibody. C: HIF-1α (P/S) is more active than wild-type HIF-1α under conditions of combined hyperglycemia and hypoxia. In a dual-luciferase reporter assay, HEK293A cells were transfected with pFLAG/mHIF-1α or pFLAG/mHIF-1α (P/S) together with reporter plasmids. The cells were then cultured in media containing 5.5, 10, 20, and 30 mmol/L glucose and exposed to normoxia (N) or hypoxia (H) for 48 h. The activity of wild-type HIF-1α (WT) and HIF-1α (P/S) is presented as mean ± SD. *P < 0.05 comparing the activity of WT and P/S at indicated concentration of glucose under hypoxic conditions.