Diabetes Metab J.  2024 May;48(3):405-417. 10.4093/dmj.2023.0196.

Reducing Oxidative Stress and Inflammation by Pyruvate Dehydrogenase Kinase 4 Inhibition Is Important in Prevention of Renal Ischemia-Reperfusion Injury in Diabetic Mice

• Khang AR
• Kim DH
• Kim MJ
• Oh CJ
• Jeon JH
• Choi SH
• Lee IK

• KMID: 2555772
• DOI: http://doi.org/10.4093/dmj.2023.0196

Abstract

Background
Reactive oxygen species (ROS) and inflammation are reported to have a fundamental role in the pathogenesis of ischemia-reperfusion (IR) injury, a leading cause of acute kidney injury. The present study investigated the role of pyruvate dehydrogenase kinase 4 (PDK4) in ROS production and inflammation following IR injury.
Methods
We used a streptozotocin-induced diabetic C57BL6/J mouse model, which was subjected to IR by clamping both renal pedicles. Cellular apoptosis and inflammatory markers were evaluated in NRK-52E cells and mouse primary tubular cells after hypoxia and reoxygenation using a hypoxia work station.
Results
Following IR injury in diabetic mice, the expression of PDK4, rather than the other PDK isoforms, was induced with a marked increase in pyruvate dehydrogenase E1α (PDHE1α) phosphorylation. This was accompanied by a pronounced ROS activation, as well as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1) production. Notably, sodium dichloroacetate (DCA) attenuated renal IR injury-induced apoptosis which can be attributed to reducing PDK4 expression and PDHE1α phosphorylation levels. DCA or shPdk4 treatment reduced oxidative stress and decreased TNF-α, IL-6, IL-1β, and MCP-1 production after IR or hypoxia-reoxygenation injury.
Conclusion
PDK4 inhibition alleviated renal injury with decreased ROS production and inflammation, supporting a critical role for PDK4 in IR mediated damage. This result indicates another potential target for reno-protection during IR injury; accordingly, the role of PDK4 inhibition needs to be comprehensively elucidated in terms of mitochondrial function during renal IR injury.

Keyword

Acute kidney injury; Diabetes mellitus; Inflammation; Ischemia; Pyruvate dehydrogenase kinase 4; Reactive oxygen species; Reperfusion

Figure

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  Fig. 1. Pyruvate dehydrogenase kinase 4 (PDK4) is induced in ischemia-reperfusion (IR) kidney injury in diabetic mice. (A) Hematoxylin and eosin (H&E) staining in mouse kidneys (original magnification ×200; scale bar, 100 μm; arrows, damaged tubules). (B) Relative mRNA level of Pdk isoforms in mice kidney tissues. (C) Protein expression and quantitative graph of PDK isoforms in mice kidney tissues. (D) Immunohistochemical image of p-pyruvate dehydrogenase E1α (p-PDHE1α) expression in mice kidney tissues (original magnification: ×200; scale bar, 100 μm; arrow, positive regions). (E) Protein expression and quantitative graph of p-PDHE1α in mice kidney tissues. Data are the mean±standard error of the mean. STZ, streptozotocin. aP<0.01 vs. Control, bP< 0.05, cP<0.01, dP<0.001 vs. STZ.

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  Fig. 2. Sodium dichloroacetate (DCA) attenuates ischemia-reperfusion (IR) injury in diabetic mice. (A) Hematoxylin and eosin (H&E) staining, periodic acid-Schiff (PAS) staining, neutrophil gelatinase-associated lipocalin (NGAL) staining, and kidney injury molecule-1 (KIM-1) staining of mice kidney tissues (original magnification ×200; scale bar, 100 μm; arrows, damaged tubules). (B) Serum blood urea nitrogen (BUN) and creatinine in mice. (C) Relative mRNA expression of pyruvate dehydrogenase kinase (Pdk) isoforms in mice kidney tissues. (D) Protein expression of PDK isoforms in mice kidney tissues. (E) Immunohistochemical image of p-pyruvate dehydrogenase E1α (p-PDHE1α) expression in mice kidney tissues (original magnification ×200; scale bar, 100 μm; arrow, positive regions). (F) Protein expression of p-PDHE1α in mice kidney. Data are the mean±standard error of the mean. STZ, streptozotocin. aP<0.01 vs. Control, bP<0.01 vs. STZ+IR, cP<0.01 vs. STZ, dP<0.05 vs. STZ+IR, eP<0.05 vs. STZ.

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  Fig. 3. Sodium dichloroacetate (DCA) and pyruvate dehydrogenase kinase (PDK) knockdown attenuates ischemia-reperfusion (IR)-induced apoptosis in mice and NRK-52E cells. (A) TdT-mediated dUTP nick end labeling (TUNEL) staining in mice kidney tissues (original magnification ×200; scale bar, 200 μm; arrows, TUNEL positive tissues). (B) Protein expression and quantitative graph of cleaved caspase-3 in mice. (C) Representative Annexin V/propidium iodide (PI) staining in NRK-52E cells. (D) TUNEL staining in mouse primary tubular cells (original magnification ×200; scale bar, 100 μm; arrows, TUNEL positive cells). Protein expression of cleaved caspase-3 in NRK-52E cells (E) and mouse primary tubule cells (F). (G) Protein expression of cleaved caspase-3 in NRK-52E cells infected with adenoviral short hairpin RNA targeting green fluorescent protein (shGFP) or small hairpin PDK4 (shPDK4). (H) Protein expression of cleaved caspase-3 in mouse primary tubular cells isolated from wild-type (WT) and PDK4 knockout (KO) mice. Data are the mean±standard error of the mean. STZ, streptozotocin; PE, phycoerythrin; FITC, fluorescein isothiocyanate; HR, hypoxia-reoxygenation. aP<0.01 vs. Control, bP<0.01 vs. STZ, cP<0.01 vs. STZ+IR, dP<0.01 vs. HR.

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  Fig. 4. Sodium dichloroacetate (DCA) attenuates ischemia-reperfusion (IR)-induced oxidative stress and inflammation in diabetic mice and NRK-52E cells. (A) 4-Hydroxynenenal (4-HNE) staining in mice kidney tissues (original magnification ×200; scale bar, 100 μm; arrows, 4-HNE positive areas). (B) Nitrotyrosine (NT) staining in mice kidney tissues (original magnification ×100; scale bar, 50 μm; arrows, NT positive areas). (C) Inflammatory markers in mice. (D) Inflammatory markers in NRK-52E with or without DCA. (E) Inflammatory markers in NRK-52E cells infected with adenoviral short hairpin RNA targeting green fluorescent protein (shGFP) or small hairpin pyruvate dehydrogenase kinase 4 (shPDK4). Data are the mean±standard error of the mean. STZ, streptozotocin; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; IL-1β, interleukin-1β; MCP-1, monocyte chemoattractant protein-1; HR, hypoxia-reoxygenation. aP<0.05, bP<0.01 vs. Control, cP<0.05, dP<0.01 vs. STZ+IR, eP<0.05, fP<0.01 vs. HR, gP<0.05, hP<0.01 vs. shGFP, iP<0.05, jP<0.01 vs. HR+shGFP.

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