Figure 1.
HIF-1 α expression in an in vitro model mimicking I/R.
Akt/mTOR signaling mediates HIF-1 α induction during reoxygenation. (a) Scheme of in vitro protocol of Hypoxia/Reoxygenation (H/R) HK-2 cells were subjected to H/R protocol which includes oxygen and nutrients deprivation and replenishment. (b) HIF-1α protein expression was determined by western blot. Actin expression was used as loading control. Representative western blot are shown. (c) qRT-PCR analysis of HIF-1 α mRNA, expressed as mean ±SEM of HIF-1α levels using b-actin mRNA as internal control. No significant alterations on mRNA levels of HIF-1 α during H/R were found in four different experiments. (d) Activation of Akt estimated by western blot of 473Ser phosporylation in HK2 cells subjected to H/R. Total Akt expression was used as control. (e) Effect of LY294002 on HIF-1 α inductions: 50 mM LY294002 applied during hypoxia (+h) did not have any effect but during reoxygenation (+r) it reduced HIF-1α expression. Representative western blots are shown. (f) Control of Akt inhibition by LY294002. 50 mM of LY294002 efficiently inhibits Akt phosphorylation when added during reoxygenation (+r). (g) Effect on the HIF-1α inductions of 20 nM rapamycin applied during reoxygenation, estimated by western blot. Phosphorylation of p70S6K was used as control of rapamycin efficiency.
Figure 2.
In vitro, HIF-1 α induction during reoxygenation is not due to low oxygen levels.
(a) Scheme of in vitro protocol of Depletion/Replenishment (D/R). HK-2 cells were subjected to D/R which only includes nutrients deprivation and replenishment, maintaining normal oxygen tension. (b) Expression of HIF-1 α during H/R and D/R protocol, estimated by western blot. (c) Akt is also activated (p473Ser) during D/R protocol.
Figure 3.
Hypoxia/reoxygenation and nutrient replenishment all induced HIF-1 α activity, promoting HIF-1 α target genes expression.
(a) Luciferase reporter assays in HK2 cells subjected to H/R (grey bars) or D/R (striped bars), by transfecting 800 ng/well of 9xHRE-luc reporter and 4 ng/well of renilla-luc reporter. Data are represented as mean ±SEM of the ratio firefly/renilla luciferase of six independent experiments, relative to control condition (ratio = 1). (b) qRT-PCR to estimate mRNA expression of HIF-1α target genes EPO, PHD3, and VEGF during H/R (black bars) or D/R protocols (striped bars). Data are represented as mean ±SEM of three independent experiments, using 28 s mRNA levels as internal control. All the genes are induced during reoxygenation in both protocols. Statistical significance was found in comparison to control (H/R), p≤0.05.
Figure 4.
HIF-1 α is induced unexpectedly during reperfusion in rat kidney, with normal oxygen levels in renal parenchyma.
(a) Immunohistochemitry to determine HIF-1 α expression in paraffin-embedded renal tissue sections from SD rats during I/R. Ischemia of 45 min and different times of reperfusion: 24 hours or 3, 5 or 7 days (R-24h, R-3d, R-5d, R-7d). HIF-1 α is detected in the nucleus of proximal tubule cells after ischemia and in reperfusion (3-5-7days). Magnification: ×400 (b) Immunostaining for pimonidazol-protein and HIF-1α adducts in renal tissue sections of rats during I/R. Ischemia of 45 min and 5 days of reperfusion. Notice positive pimonidazole immunostaining exclusively after ischemia. Magnification: ×200.
Figure 5.
HIF-1α is transcriptionaly active in vivo promoting gene expression.
PHD3, VEGF and EPO mRNAs levels were determined by qRT-PCR in total renal tissue lysates and 28 mRNA levels were used as internal control. Statistical significance was found in comparison to sham condition.
Figure 6.
HIF-1α is required for proximal epithelial cell survival in response to oxygen and nutrient alterations.
Quantification of cell death, by propidium iodide staining and flow cytometry in HK2 cells subjected to H/R and D/R, (a) previously transfected with specific HIF-1α siRNA (100 nM) and scramble (100 nM) or (b) treated with 10 mM YC-1 added during hypoxia (hyp) or during reoxygenation (reox). Statistical significance was found in comparison to scramble or control respectively, p≤0.05. (c) 100 nM of siRNA (sc-44225) efficiently prevents both HIF-1α inductions (hypoxia and R-3h). (d) 10 mM YC-1 inhibits HIF-1α inductions when added during hypoxia and reoxygenation or (e) added separately during hypoxia (+h) or reoxygenation (+r). Representative blots are shown and actin was used as control. (f) Akt activation in HK-2 cells subjected to H/R protocol, HK-2 cells treated with 10 mM YC-1 or 100 nM siRNA for HIF-1α, all estimated by western blot of 473Ser phosphorylation.
Figure 7.
(a) Scheme of siRNA and scramble treatments in vivo. SD Rats were injected with 100 nM of specific siRNA against HIF-1α or scramble through the tail vein at indicated times. (b) Percentage of HIF-1α interference estimated by qRT-PCR in total renal lysates from rats treated with HIF-1 α siRNA in comparison to rats treated with scramble in each condition.
Figure 8.
HIF-1α interference in vivo exacerbates I/R-induced renal injury.
(a) PAS staining in paraffin-embedded renal tissue sections from SD rats during I/R, injected with scramble or with specific siRNA against HIF-1α. Note increased renal damage at 3 and 5 days of reperfusion in siRNA treated rats. Representative images are shown. Magnification: ×200. (b) Renal function estimated by serum creatinine and urea levels. Statistical significance was found compared to sham scramble condition, p≤0.05.
Table 1.
Human postransplant Renal Biopsies features.
Figure 9.
HIF-1α is expressed exclusively in non-damaged proximal tubules of human post-transplant renal biopsies.
(a) PAS staining for renal structure and immunohistochemistry for HIF-1α in paraffin-embedded human renal biopsies. HIF-1 α is expressed in non-damaged proximal tubules (biopsy n°4). Images of representative biopsies are presented: severe ATN (biopsies n°6 and n°14) and ATN regeneration (biopsy n°4). Magnification: ×400. (b) Spearman Rho-Correlation coefficient between ATN grade and HIF-1α expression in all biopsies, with statistical significance p≤0.01.
Table 2.
Quantification of ATN development vs recovery HIF-1α expression in renal biopsies, by PAS staining and immunohistochemistry detection.
Table 3.
Primer sequences used for qRT-PCR in vivo and in vitro.