A small-molecule inhibitor of hypoxia-inducible factor prolyl hydroxylase improves obesity, nephropathy and cardiomyopathy in obese ZSF1 rats

Abstract

Prolyl hydroxylase (PH) enzymes control the degradation of hypoxia-inducible factor (HIF), a transcription factor known to regulate erythropoiesis, angiogenesis, glucose metabolism, cell proliferation, and apoptosis. HIF-PH inhibitors (HIF-PHIs) correct anemia in patients with renal disease and in animal models of anemia and kidney disease. However, the effects of HIF-PHIs on comorbidities associated with kidney disease remain largely unknown. We evaluated the effects of the HIF-PHI FG-2216 in obese ZSF1 (Ob-ZSF1) rats, an established model of kidney failure with metabolic syndrome. Following unilateral nephrectomy (Nx) at 8 weeks of age, rats were treated with 40 mg/kg FG-2216 or vehicle by oral gavage three times per week for up to 18 weeks. FG-2216 corrected blood hemoglobin levels and improved kidney function and histopathology in Nx-Ob-ZSF1 rats by increasing the glomerular filtration rate, decreasing proteinuria, and reducing peritubular fibrosis, tubular damage, glomerulosclerosis and mesangial expansion. FG-2216 increased renal glucose excretion and decreased body weight, fat pad weight, and serum cholesterol in Nx-Ob-ZSF1 rats. Additionally, FG-2216 corrected hypertension, improved diastolic and systolic heart function, and reduced cardiac hypertrophy and fibrosis. In conclusion, the HIF-PHI FG-2216 improved renal and cardiovascular outcomes, and reduced obesity in a rat model of kidney disease with metabolic syndrome. Thus, in addition to correcting anemia, HIF-PHIs may provide renal and cardiac protection to patients suffering from kidney disease with metabolic syndrome.

Fig 1. Experimental study design.

Study design of renal (A) and cardiac (B) studies.

Fig 2. FG-2216 corrected hemoglobin levels in Nx-Ob-ZSF1 rats.

Hemoglobin levels were determined from blood collected every four weeks during treatment. Values represent mean ± SEM (n = 6–12). *P < 0.05 vs. Nx-Ob-ZSF1 Vehicle (Bonferroni test).

Fig 3. FG-2216 improved kidney function in Nx-Ob-ZSF1 rats.

Glomerular filtration rate (GFR) was determined by inulin clearance assay after 18 weeks of treatment (A). GFR was normalized to total renal mass (B). Urine was collected for 24 hours every four weeks during the treatment period to measure proteinuria (C), glycosuria (D) and volume (F). After 17 weeks of treatment, water consumption was measured over 48 hours encompassing a dosing and a non-dosing day (E). Values represent mean ± SEM (n = 7–12). *P < 0.05 vs. Nx-Ob-ZSF1 Vehicle (Dunnett’s test for A, B, and E; Bonferroni test for C, D and F).

Fig 4. FG-2216 improved kidney morphology in Nx-Ob-ZSF1 rats.

Kidneys were harvested after 18 weeks of treatment and sectioned for histology. Picrosirius red (PSR) staining of fibrillar collagen showed tubular interstitial fibrosis (A). FG-2216 reduced PSR staining (B). Immunohistochemistry for Kidney Injury Molecule-1 (KIM-1) indicated tubular damage (C). FG-2216 decreased KIM-1 signal intensity (D). Periodic acid-Schiff (PAS) staining showed mesangial expansion in the glomerulus (E). FG-2216 reduced fractional mesangial expansion (F) without affecting glomerulus tuft area (G). Glomerulosclerosis quantified on sections stained with methenamine silver-trichrome showed less focal global glomerulosclerosis (FGGS) in Nx-Ob-ZSF1 FG-2216 animals than in Nx-Ob-ZSF1 Vehicle animals (H). The right kidney increased in mass in the Nx-Ob-ZSF1 animals to compensate for nephrectomy of the left kidney (I). Values represent mean ± SEM (n = 8–12). *P < 0.05 vs. Nx-Ob-ZSF1 Vehicle (Dunnett’s test).

Fig 5. FG-2216 did not affect glucose tolerance in Nx-Ob-ZSF1 rats.

Blood glucose levels during oral glucose tolerance tests after 6 weeks (A) and 14 weeks (B) of treatment. Values represent mean ± SEM (n = 8–12). *P < 0.05 vs. Nx-Ob-ZSF1 Vehicle (Bonferroni test).

Fig 6. FG-2216 reduced obesity in Nx-Ob-ZSF1 rats.

Rats were weighed at regular intervals during the treatment period (A). Fat pads were harvested after 18 weeks of treatment and weighed (B). After 17 weeks of treatment, food consumption was measured over 48 hours encompassing a dosing and a non-dosing day (C). Blood was collected every four weeks during the treatment period to determine serum cholesterol (D). Values represent mean ± SEM (n = 8–12). *P < 0.05 vs. Nx-Ob-ZSF1 Vehicle (Bonferroni test for A and D; Dunnett’s test for B and C).

Fig 7. FG-2216 prevented hypertension in Nx-Ob-ZSF1 rats.

Systolic (A) and diastolic (B) blood pressures (BP) were measured and mean BP (C) was calculated using a tail-cuff at baseline and three hours after dose administration at 12 and 17 weeks of treatment. Systolic (SBP) and diastolic (DBP) blood pressures were also measured 24 hours following dosing after 17 weeks of treatment and mean BP (MBP) was calculated (D). Values represent mean ± SEM (n = 8–12). *P < 0.05 vs. Nx-Ob-ZSF1 Vehicle (Bonferroni test).

Fig 8. FG-2216 reduced cardiac hypertrophy in Nx-Ob-ZSF1 rats.

Hearts were harvested and weighed after 18 weeks of treatment. FG-2216 normalized heart weight in Nx-Ob-ZSF1 rats (A). Blood was collected from rats to measure plasma levels of NT-proBNP after 16 weeks of treatment. FG-2216 normalized NT-proBNP concentration in Nx-Ob-ZSF1 rats (B). Cardiac histology with picrosirius red (PSR) staining for fibrillar collagen showed left ventricular fibrosis (C). FG-2216 decreased PSR staining in Nx-Ob-ZSF1 rats (D). Values represent mean ± SEM (n = 8–12). *P < 0.05 vs. Nx-Ob-ZSF1 Vehicle (Dunnett’s test).

Fig 9. FG-2216 decreased plasma markers of cardiac injury in Nx-Ob-ZSF1 rats.

Blood was collected after 16 weeks of treatment to measure plasma concentrations of cardiac troponin I (cTnI) (A), fatty acid binding protein 3 (FABP3) (B), and myosin light chain 3 (MLC3) (C). Values represent mean ± SEM (n = 8–12). *P < 0.05 vs. Nx-Ob-ZSF1 Vehicle (Dunnett’s test).