Renal protection in chronic kidney disease: hypoxia-inducible factor activation vs. angiotensin II blockade

Abstract

The 5/6(th) nephrectomy or ablation/infarction (A/I) preparation has been used as a classic model of chronic kidney disease (CKD). We observed increased kidney oxygen consumption (Q(O2)) and altered renal hemodynamics in the A/I kidney that were normalized after combined angiotensin II (ANG II) blockade. Studies suggest hypoxia inducible factor as a protective influence in A/I. We induced hypoxia-inducible factor (HIF) and HIF target proteins by two different methods, cobalt chloride (CoCl(2)) and dimethyloxalyglycine (DMOG), for the first week after creation of A/I and compared the metabolic and renal hemodynamic outcomes to combined ANG II blockade. We also examined the HIF target proteins expressed by using Western blots and real-time PCR. Treatment with DMOG, CoCl(2), and ANG II blockade normalized kidney oxygen consumption factored by Na reabsorption and increased both renal blood flow and glomerular filtration rate. At 1 wk, CoCl(2) and DMOG increased kidney expression of HIF by Western blot. In the untreated A/I kidney, VEGF, heme oxygenase-1, and GLUT1 were all modestly increased. Both ANG II blockade and CoCl(2) therapy increased VEGF and GLUT1 but the cobalt markedly so. ANG II blockade decreased heme oxygenase-1 expression while CoCl(2) increased it. By real-time PCR, erythropoietin and GLUT1 were only increased by CoCl(2) therapy. Cell proliferation was modestly increased by ANG II blockade but markedly after cobalt therapy. Metabolic and hemodynamic abnormalities were corrected equally by ANG II blockade and HIF therapies. However, the molecular patterns differed significantly between ANG II blockade and cobalt therapy. HIF induction may prove to be protective in this model of CKD.

Fig. 1.

Changes in renal hemodynamics in rats one week after renal ablation and infarction (A/I) under different treatments. A: 1-wk after renal A/I the untreated kidney demonstrated a markedly decreased renal blood flow (RBF). This reduction of RBF was significantly prevented by both dual ANG II blockade with captopril and losartan (C&L) and cobalt chloride (Co). B: untreated 1-wk A/I kidney incurred a significant reduction in glomerular filtration rate (GFR). This decreased GFR was significantly corrected not only by dual ANG II inhibition (C&L) but also by cobalt chloride. Data are means ± SE. *P < 0.05 vs. normal; **P < 0.05 vs. A/I.

Fig. 2.

Changes in renal metabolic efficiency in rats 1-wk postrenal A/I under different conditions. Despite significant reduction in GFR, total oxygen consumption (Q_O2) was not significantly reduced in untreated A/I. Therefore, Q_O2 when factored with total amount of sodium reabsorbed (Q_O2 /T_Na) was significantly elevated, indicating decreased renal metabolic efficiency. Both cobalt chloride and ANG II inhibition with captopril and losartan increased GFR to normal levels without a parallel increase in Q_O2, resulting in a normal values of Q_O2/T_Na. Data are means ± SE. *P < 0.05 vs. normal; **P < 0.05 vs. A/I.

Fig. 3.

Western blot analysis of activation of hypoxia inducible factor (HIF)-1 by cobalt chloride in murine cortical proximal tubular (MCT) cells. MCT cells were cultured in DMEM with low glucose and supplemented with 10% FCS. To activate HIF, cobalt chloride was added to the confluent culture at 200 μM for 12 h. Proteins (70 ug) from cytosol and nuclei were electrophorertically separated on 7% glycine-Tris gel, transferred to PVDF membrane, and detected with ECL plus. Both HIF-1α (nuclear extraction) and its prototypical target protein, carbonic anhydrase IX (CN9, cytosol extraction), were clearly detected in MCT cells treated with cobalt chloride.

Fig. 4.

Immunoprecipitation and Western blot analysis of induction of HIF-1α by both HIFα stabilizers, cobalt chloride and dimethyloxalyglycine (DMOG), in renal cortex from rats 1 wk after renal A/I HIF-1α is detectable by Western blot in renal cortex after immunoprecipitation of 500 μg of proteins with anti-HIF-1α. When compared with either normal (lane 1) or untreated 1-wk A/I (lane 2), ANG II blockade (lane 3) significantly reduced HIF-1α expression and both cobalt (lane 4) and DMOG (lane 5) increased HIF-1α protein expression. Immunoblot represents 4 repeated detections.

Fig. 5.

Western blot analysis of induction of carbonic anhydrase IX (CN9) by cobalt chloride in renal cortex from rats at early renal A/I. Expression of carbonic anhydrase IX (CN9), a HIF-1 protoypical target, can been seen in 18-h A/I kidneys treated with cobalt, suggesting an early HIF pathway activation in vivo by cobalt chloride.

Fig. 6.

Western blot analysis of VEGF in proteins extracted from renal cortex of normal rats and untreated, cobalt chloride-treated, and captopril and losartan-treated rats 1-wk postrenal A/I. VEGF was constitutively expressed in normal renal cortex and tended to increase (2-fold) in untreated A/I but did not reach statistical significance. VEGF protein was significantly increased (3-fold) by ANG II inhibition with captopril and losartan and substantially increased (>7 fold) by cobalt chloride. Data are means ± SE. *P < 0.01 vs. normal.

Fig. 7.

Western blot analysis of heme oxygenase-1 (HO-1) induction in renal cortex of normal rats and untreated, cobalt chloride-treated, and captopril and losartan-treated rats 1-wk postrenal A/I. Untreated 1-wk A/I kidney cortex showed signs of HO-1 induction but was not statistically significant from the normal. HO-1 was not induced in the renal cortex from A/I rats receiving captopril and losartan. However, HO-1 was dramatically upregulated (54-fold) in the A/I kidney treated with cobalt chloride. Data are means ± SE. *P < 0.01 vs. normal; **P < 0.05 vs. A/I.

Fig. 8.

Western blot analysis of GLUT1. Induction of GLUT1 expression in renal cortex of normal rats and untreated, cobalt-treated, and captopril and losartan-treated rats 1-wk postrenal A/I. GLUT1 in A/I + Co was significantly different from A/I but not from A/I + C&L. A/I + C&L was not significantly different from A/I. Data are means ± SE. *P < 0.05 vs. A/I.

Fig. 9.

Real-time PCR analysis of erythropoietin (Epo) gene expression in renal cortex from normal, untreated, cobalt chloride-treated, and captopril and losartan-treated rats 1-wk postrenal A/I. Real-time reactions were performed in triplicate for both the target gene and GAPDH as a housekeeping control. Relative expression was calculated using delta ΔC_T method. Numbers show the fold changes relative to the normal mRNA expression levels normalized to GAPDH. Data are means ± SE (n = 8 for each group). *P < 0.01 vs. normal. In untreated A/I kidney, Epo mRNA was slightly but significantly decreases at day 3 and day 7. Cobalt chloride greatly increases Epo mRNA by 20-fold at day 3 and >2-fold at day 7. ANG II blockade (C&L) dramatically reduced Epo mRNA levels when compared with the normal.

Fig. 10.

Real-time PCR analysis of GLUT1 gene expression in renal cortex from normal, untreated, cobalt chloride-treated, and captopril and losartan-treated rats 1-wk postrenal A/I. Quantification of PCR was done using the comparative CT method and reported as fold difference relative to the calibrator cDNA (GAPDH). Data are means ± SE (n = 8 for each group). GLUT1 mRNA was unchanged at either day 3 or day 7 in untreated A/I kidney cortex. Cobalt chloride significantly increased GLUT1 mRNA by 2-fold at day 3 and returned to normal levels at day 7. ANG II inhibition (C&L) significantly decreased GLUT1 mRNA at day 7. *P < 0.01 vs. normal.

Fig. 11.

Renal cortical cell proliferation shown by bromodeoxyuridine (BrdU) incorporation. Rats received BrdU (50 mg/kg ip daily) for the last 5 days. BrdU was stained using a Zamed kit. Means of BrdU-positive cells were calculated using twenty ×10 fields of higher counting number from each group. Data are means ± SE. *P < 0.05 vs. A/I; **P < 0.01 vs. A/I and A/I + C&L.

Fig. 12.

Western blot analysis of proliferating cell nuclear antigen (PCNA) in renal cortex from normal, untreated 1-wk renal A/I, A/I treated with cobalt chloride, and A/I treated with captopril and losartan. PCNA protein expression tended to increase at 1 wk in A/I kidney cortex relative to normal kidney but was not significant. A/I + C&L levels were not different than normal kidneys. PCNA expression in the A/I + Co group was significantly different from A/I and A/I + C&L. Data are means ± SE. *P < 0.01.