Exogenous Gene Transmission of Isocitrate Dehydrogenase 2 Mimics Ischemic Preconditioning Protection

Abstract

Ischemic preconditioning confers organ-wide protection against subsequent ischemic stress. A substantial body of evidence underscores the importance of mitochondria adaptation as a critical component of cell protection from ischemia. To identify changes in mitochondria protein expression in response to ischemic preconditioning, we isolated mitochondria from ischemic preconditioned kidneys and sham-treated kidneys as a basis for comparison. The proteomic screen identified highly upregulated proteins, including NADP +-dependent isocitrate dehydrogenase 2 (IDH2), and we confirmed the ability of this protein to confer cellular protection from injury in murine S3 proximal tubule cells subjected to hypoxia. To further evaluate the role of IDH2 in cell protection, we performed detailed analysis of the effects of Idh2 gene delivery on kidney susceptibility to ischemia-reperfusion injury. Gene delivery of IDH2 before injury attenuated the injury-induced rise in serum creatinine (P,0.05) observed in controls and increased the mitochondria membrane potential (P,0.05), maximal respiratory capacity (P,0.05), and intracellular ATP levels (P,0.05) above those in controls. This communication shows that gene delivery of Idh2 can confer organ-wide protection against subsequent ischemiareperfusion injury and mimics ischemic preconditioning.

Intravital imaging of real-time endogenous actin dysregulation in proximal and distal tubules at the onset of severe ischemia-reperfusion injury

“…These ex vivo studies have also determined that the expression of eGFP-actin can affect cell behavior. Fortunately, this concern as the plasmid titer and period of expression were previously shown to support the stable expression of fluorescent/exogenous proteins that did not significantly alter cellular function in vivo 13 , 14 . It was suitable to consider this type of expression vector for our initial studies, as both forms of actin are essential cytoskeletal components.…”

“…Animals were anesthetized with inhaled isoflurane (5% in oxygen, Webster Veterinary Supply, Devens, MA) and then given intraperitoneal injections of 50 mg/kg of pentobarbital (Hospira, Inc., Lake Forest, IL). The details of the HGD process are outlined in the literature 13 , 14 . Briefly, for this process, 1–3 µg of EGFP-actin plasmid DNA per gram of body weight were suspended in 0.5 ml of saline for retrograde renal vein injections.…”

“…By altering hydrodynamic fluid pressures, it has been shown that it is possible to transiently increase intravascular pressure within peritubular capillary networks and induce exogenous gene expression in the surrounding tubular epithelium 12 . This method, which is known as hydrodynamic gene delivery (HGD), is capable of producing widespread genetic alterations in various segments of the kidney, namely the proximal and distal convoluted tubules, with minimal effect to the organ 13 , 14 . Therefore, to improve the current understanding of renal IRI, we extended this approach to investigate real-time changes in tubular structures that are altered in response to IRI, using a sophisticated imaging tool.…”

Intravital imaging of real-time endogenous actin dysregulation in proximal and distal tubules at the onset of severe ischemia-reperfusion injury

“…These ex vivo studies have also determined that the expression of eGFP-actin can affect cell behaviour. Fortunately, this concern as the plasmid titer and period of expression were previously shown to support the stable expression of fluorescent/exogenous proteins that did not significantly alter cellular function in vivo [13, 14]. It was suitable to consider this type of expression vector for our initial studies, as both forms of actin are essential cytoskeletal components.…”

Correlation of occludin protein mobility with paracellular leak pathway permeability in renal epithelia

“…Full-length EGFP-occludin construct in a viral expression construct was introduced into rat renal epithelial cells by retrograde pressure transduction (Corridon et al, 2013). Renal ischemia/reperfusion injury was produced by the renal single clamp model (Kolb et al, 2018). The unclamped kidney was used as a control.…”