TIGAR regulates glycolysis in ischemic kidney proximal tubules

Abstract

Tp53-induced glycolysis and apoptosis regulator (TIGAR) activation blocks glycolytic ATP synthesis by inhibiting phosphofructokinase-1 activity. Our data indicate that TIGAR is selectively induced and activated in renal outermedullary proximal straight tubules (PSTs) after ischemia-reperfusion injury in a p53-dependent manner. Under severe ischemic conditions, TIGAR expression persisted through 48 h postinjury and induced loss of renal function and histological damage. Furthermore, TIGAR upregulation inhibited phosphofructokinase-1 activity, glucose 6-phosphate dehydrogenase (G6PD) activity, and induced ATP depletion, oxidative stress, autophagy, and apoptosis. Small interfering RNA-mediated TIGAR inhibition prevented the aforementioned malevolent effects and protected the kidneys from functional and histological damage. After mild ischemia, but not severe ischemia, G6PD activity and NADPH levels were restored, suggesting that TIGAR activation may redirect the glycolytic pathway into gluconeogenesis or the pentose phosphate pathway to produce NADPH. The increased level of NADPH maintained the level of GSH to scavenge ROS, resulting in a lower sensitivity of PST cells to injury. Under severe ischemia, G6PD activity and NADPH levels were reduced during reperfusion; however, blockade of TIGAR enhanced their levels and reduced oxidative stress and apoptosis. Collectively, these results demonstrate that inhibition of TIGAR may protect PST cells from energy depletion and apoptotic cell death in the setting of severe ischemia-reperfusion injury. However, under low ischemic burden, TIGAR activation induces the pentose phosphate pathway and autophagy as a protective mechanism.

Keywords: ATP depletion; Tp53-induced glycolysis and apoptosis regulator; glycolytic inhibition; ischemic renal injury; phosphofructokinase; proximal straight tubules.

Fig. 1.

Ischemia-reperfusion injury (IRI) increases Tp53-induced glycolysis and apoptosis regulator (TIGAR) expression in proximal straight tubule (PST) cells. A: TIGAR expression in the outer medulla in kidneys derived from wild-type or p53 knockout (KO) mice at 6 h of reperfusion after sham operation or 30 min of ischemia using immunohistochemistry. C, cortex; D, distal tubule; im, inner medulla (n = 3). B: TIGAR expression in MCT cells exposed to normoxia or hypoxia for 3 or 5 h using immunohistochemistry (n = 3). Magnification: ×400. C: expression of kidney TIGAR in wild-type or p53 KO mice at 6 h of reperfusion after sham operation or 30 min of ischemia using Western blot analysis (n = 3).

Fig. 2.

Renal dysfunction, tubular damage, and TIGAR expression after mild or severe IRI. A: concentrations of plasma creatinine and blood urea nitrogen (BUN) at various time points of reperfusion after sham operation or 20 or 30 min of renal ischemia. B: tubular damage in the outer medulla (left) represented by periodic acid-Schiff (PAS) stain on kidney sections at 48 h after sham operation or 20 or 30 min of renal ischemia. Scale bars = 50 μm. Right, tubular injury score measured on PAS-stained kidney sections. C: TIGAR expression in whole kidneys at various time points of reperfusion after sham operation or 20 or 30 min of renal ischemia using Western blot analysis. Values are means ± SD; n = 4. *P < 0.05 vs. sham.

Fig. 3.

Phosphofructokinase (PFK)-1 activity and ATP level in the renal outer medulla correlated with TIGAR expression after IRI. A: PFK-1 activity in the outer medullary region of kidneys at various time points of reperfusion after sham operation or 20 or 30 min of renal ischemia. B: ATP concentration in the outer medullary region of kidneys at various time points of reperfusion after sham operation or 20 or 30 min of ischemia. Values are means ± SD; n = 4. *P < 0.05 vs. sham.

Fig. 4.

Glucose 6-phosphate dehydrogenase (G6PD), NADPH, oxidative stress, and glutathione in kidneys after mild or severe IRI. Mice were subjected to either 20 or 30 min of bilateral renal ischemia followed by 1, 3, 6, 12, 24, or 48 h of reperfusion. A: G6PD activity in kidneys. B: ratio of NADPH to NADPH + NADP⁺ in kidneys. C: lipid hydroperoxide levels in kidneys. D: ratio of GSSG to GSSG + GSH in kidneys. Values are means ± SD; n = 4. *P < 0.05 vs. sham.

Fig. 5.

Light chain 3 (LC3) expression in kidneys after mild or severe IRI. Mice were subjected to either 20 or 30 min of bilateral renal ischemia followed by 1, 3, 6, 12, 24, or 48 h of reperfusion. LC3-I (top bands) and LC3-II (bottom bands) expression was assessed in kidneys using Western blot analysis. Values are means ± SD; n = 4. *P < 0.05 vs. sham.

Fig. 6.

TIGAR inhibition reduces renal dysfunction and tubular damage after severe IRI. Small interfering (si)RNA for TIGAR (siTIGAR) or control siRNA (siControl) (50 μg in 1 ml PBS) was injected within 10 s into mouse tail veins at 48 and 24 h before ischemia. Mice were subjected to 30 min of bilateral renal ischemia or sham operation followed by 6 or 24 h of reperfusion (n = 6). A: TIGAR expression in kidneys at 6 h after IRI or sham operation. B: plasma creatinine and BUN concentration. C: tubular damage in the outer medulla (left) represented by PAS stain on kidney sections at 24 h after 30 min of renal ischemia. Scale bars = 50 μm. Right, tubular injury score measured on PAS-stained kidney sections. Values are means ± SD. *P < 0.05 vs. sham; #P < 0.05 vs. siControl.

Fig. 7.

Protective effect of TIGAR inhibition on PFK-1 activity, ATP concentration, G6PD activity, NADPH level, lipid hydroperoxide level, and GSH level after severe IRI. siTIGAR or siControl (50 μg in 1 ml PBS) was injected within 10 s into mouse tail veins at 48 and 24 h before ischemia. Mice were subjected to 30 min of bilateral renal ischemia or sham operation followed by 6 or 24 h of reperfusion (n = 6). A: PFK-1 activity in kidneys. B: ATP concentration in kidneys. C: G6PD activity in kidneys. D: ratio of NADPH to NADPH + NADP⁺ in kidneys. E: lipid hydroperoxide level in kidneys. F: ratio of GSSG to GSSG + GSH in kidneys. Values are means ± SD. *P < 0.05 vs. sham; #P < 0.05 vs. siControl.

Fig. 8.

Renal injury aggravated by TIGAR inhibition on PFK-1 activity, G6PD activity, NADPH level, lipid hydroperoxide level, and BUN concentration after mild IRI. siTIGAR or siControl (50 μg in 1 ml PBS) was injected within 10 s into mouse tail veins at 48 and 24 h before ischemia. Mice were subjected to 20 min of bilateral renal ischemia or sham operation followed by 6 or 24 h of reperfusion (n = 6). A: PFK-1 activity in kidneys. B: G6PD activity in kidneys. C: ratio of NADPH to NADPH + NADP⁺ in kidneys. D: lipid hydroperoxide level in kidneys. E: BUN concentration in mice. Values are means ± SD. *P < 0.05 vs. sham; #P < 0.05 vs. siControl.

Fig. 9.

Preventive effect of TIGAR inhibition on tubular apoptosis and autophagy after severe IRI. siTIGAR or siControl (50 μg in 1 ml PBS) was injected within 10 s into mouse tail veins at 48 and 24 h before ischemia. Mice were subjected to 30 min of bilateral renal ischemia or sham operation followed by 24 h of reperfusion (n = 6). A: TUNEL assay on kidney sections (left). Scale bars = 50 μm. Apoptosis in tubular cells represented by TUNEL-positive tubular cells were counted on kidney sections (right). B: LC3-I (top bands) and LC3-II (bottom bands) expression in kidneys using Western blot analysis. Values are means ± SD. *P < 0.05 vs. sham; #P < 0.05 vs. siControl.