The Role of Hypoxia-Inducible Factor/Prolyl Hydroxylation Pathway in Deoxycorticosterone Acetate/Salt Hypertension in the Rat

Abstract

KKidney disease could result from hypertension and ischemia/hypoxia. Key mediators of cellular adaptation to hypoxia are oxygen-sensitive hypoxia inducible factor (HIF)s which are regulated by prolyl-4-hydroxylase domain (PHD)-containing dioxygenases. However, HIF activation can be protective as in ischemic death or promote renal fibrosis in chronic conditions. This study tested the hypothesis that increased HIF-1α consequent to reduced PHD expression contributes to the attendant hypertension and target organ damage in deoxycorticosterone acetate (DOCA)/salt hypertension and that PHD inhibition ameliorates this effect. In rats made hypertensive by DOCA/salt treatment (DOCA 50 mg/kg s/c; 1% NaCl orally), PHD inhibition with dimethyl oxallyl glycine (DMOG) markedly attenuated hypertension (P<0.05), proteinuria (P<0.05) and attendant tubular interstitial changes and glomerular damage (P<0.05). Accompanying these changes, DMOG blunted the increased expression of kidney injury molecule (KIM)-1 (P<0.05), a marker of tubular injury and reversed the decreased expression of nephrin (P<0.05), a marker of glomerular injury. DMOG also decreased collagen I staining (P<0.05), increased serum nitrite (P<0.05) and decreased serum 8-isopostane (P<0.05). However, the increased HIF-1α expression (P<0.01) and decreased PHD2 expression (P<0.05) in DOCA/salt hypertensive rats was not affected by DMOG. These data suggest that reduced PHD2 expression with consequent increase in HIF-1α expression probably results from hypoxia induced by DOCA/salt treatment with the continued hypoxia and reduced PHD2 expression evoking hypertensive renal injury and collagen deposition at later stages. Moreover, a PHD inhibitor exerted a protective effect in DOCA/salt hypertension by mechanisms involving increased nitric oxide production and reduced production of reactive oxygen species.

Keywords: Collagen I; DOCA hypertension; Hypoxia inducible factor (HIF); KIM-1; Nitric oxide; Prolyl-4-hydroxylase domain (PHD); Reactive oxygen species; ephrin.

Figure 1

Mean arterial blood pressure (a), proteinuria (b), kidney weight- (c) and left ventricular/body weight indices (d) in uninephrectomized rats placed on 1% NaCl (CONTROL, n=8) treated with DOCA (25 mg/kg s.c. twice weekly) in the absence (DOCA, n=9) or presence of DMOG (15 mg/kg i.p; DOCA+ DMOG; n=6). Animals that underwent uninephrectomy and received ordinary tap water were treated with DMOG (DMOG, n=5) for comparison. #P< 0.05 versus Control @P<0.05 vs DOCA ##P<0.01) versus Control @@P<0.05 versus DOCA Figure 1a represents data from 5 animals per group. The other n values apply to Figure 1 b, c, and d.

Figure 2

Expression of KIM-1 (a) or nephrin (b) in whole kidneys of uninephrectomized rats placed on 1% NaCl in drinking water (CONTROL, n=6; Lane 1) treated with DOCA (25 mg/kg s.c. twice weekly) in the absence (DOCA, n=9; Lane 3) or presence of DMOG (DOCA+DMOG; n=6; Lane 4). A set of animals that underwent uninephrectomy were placed on tap water and treated with DMOG alone (DMOG; n=5; Lane 2). A representative blot of the expression of KIM-1 or nephrin is shown with internal loading control (β-actin). Graphs depict the quantitation by densitometry. #P<0.05 (##P<0.01) versus Control @P<0.05 (@@P<0.01) versus DOCA

Figure 3

Histopathological changes (a, b) or immunohistochemical staining for collagen I (c) in kidneys of uninephrectomized rats placed on 1% NaCl in drinking water (CONTROL, n=8) treated with DOCA (25 mg/kg s.c. twice weekly) in the absence (DOCA, n=9) or presence of DMOG (DOCA+ DMOG; n=6). A set of animals that underwent uninephrectomy were placed on tap water and treated with DMOG alone (DMOG; n= 6). Figure shows sections stained with hematoxylin and eosin (H & E; a) or immunoperoxidase staining for collagen I (c). Magnifications are shown at ×200 (×400 for the arteriole). Arrows show lesions in DOCA/salt hypertensive rats (DOCA) as described fully in the text: glomerular enlargement, hypercellularity, inflammatory infiltrates, fibrinoid deposition (a, upper panel), thickening of arterial wall, periarterial fibrosis, mural disorganization (a, middle panel), interstitial inflammation, fibrosis, tubular atrophy or dilatation (a, lower panel). Quantitation of glomerular and tubular lesions are shown in Figure 3b and that of collagen deposition are shown in Figure 3c. Normal deposition of collagen I (in rectangle inset). #P<0.05 versus Control @P<0.05 versus DOCA

Figure 4

PHD2 (a) or HIF-1α (b) expression in whole kidneys of uninephrectomized rats placed on 1% NaCl in drinking water (CONTROL, n=8) treated with DOCA (25 mg/kg s.c. twice weekly) in the absence (DOCA, n=9) or presence of DMOG (DOCA+ DMOG; n=6). A set of animals that underwent uninephrectomy were placed on tap water and treated with DMOG alone (DMOG; n=5). A representative blot of the expression of PHD2 or HIF-1α is also shown with internal loading control (β -actin). Graphs depict quantitation of immunoblot by densitometry. #P<0.05 versus Control @P<0.05 versus DOCA

Figure 5

Serum creatinine (a), nitrite (b), or 8-Isoprostane (c) in rats after three weeks of treatment with DOCA/1% NaCl (DOCA, n= 5) or with DMOG (DOCA+DMOG, n=6). Serum levels in rats placed on tap water treated with DMOG alone (DMOG, n=5) or in uninephrectomized rats placed on 1% NaCl drinking water (Control, n= 6) are also shown. #P<0.05 versus Control @P<0.05 versus DOCA