Identification of agents that reduce renal hypoxia–reoxygenation injury using cell-based screening: Purine nucleosides are alternative energy sources in LLC-PK1 cells during hypoxia

Abstract

Acute tubular necrosis is a clinical problem that lacks specific therapy and is characterized by high mortality rate. The ischemic renal injury affects the proximal tubule cells causing dysfunction and cell death after severe hypoperfusion. We utilized a cell-based screening approach in a hypoxia-reoxygenation model of tubular injury to search for cytoprotective action using a library of pharmacologically active compounds. Oxygen-glucose deprivation (OGD) induced ATP depletion, suppressed aerobic and anaerobic metabolism, increased the permeability of the monolayer, caused poly(ADP-ribose) polymerase cleavage and caspase-dependent cell death. The only compound that proved cytoprotective either applied prior to the hypoxia induction or during the reoxygenation was adenosine. The protective effect of adenosine required the coordinated actions of adenosine deaminase and adenosine kinase, but did not requisite the purine receptors. Adenosine and inosine better preserved the cellular ATP content during ischemia than equimolar amount of glucose, and accelerated the restoration of the cellular ATP pool following the OGD. Our results suggest that radical changes occur in the cellular metabolism to respond to the energy demand during and following hypoxia, which include the use of nucleosides as an essential energy source. Thus purine nucleoside supplementation holds promise in the treatment of acute renal failure.

The Hypoxia-Reoxygenation Injury Model

“…Most cells can survive in culture if the cellular ATP concentration will be reduced by less than 75-80% the normal ATP level [1][2][3]5]. Following an OGD injury that does not reduce the cellular ATP concentration below 20% of the initial baseline value full recovery is expectable if optimal culture conditions are provided.…”

“…During apoptosis PARP is inactivated by caspase cleavage that dissociates the DNA binding and catalytic domains of PARP and prevents PARP activation by DNA strand breaks. Apart from caspases, various proteases (cathepsin, calpain, granzyme B) may inactivate PARP by proteolytic cleavage following OGD or hypoxia injury [2]. PARP also catalyzes its self-PARylation and this auto-modification reduces the catalytic activity of the enzyme, thus, it also serves as a control of its activity.…”

“…Since the cells try to maintain normal ATP level and use all resources that can be utilized for energy production during the OGD phase, the recovery process is time-consuming: all precursor molecules need to be resynthesized in the cells. A more robust injury that decreases the cellular ATP concentration below 20% will initiate severe viability loss in the cell population [2] ( Figure 1). The cellular energy production remains impaired following an OGD injury: the cellular ATP production is slow even if the energy sources are provided in liberal amounts.…”

“…Drug treatments may be administered before the hypoxia induction to test preventive effects or following the hypoxic period to test the therapeutic potential in ischemic diseases [1][2][3]. For gene silencing small interfering RNAs may be added 48 hours prior to the hypoxia exposure to effectively reduce RNA and protein levels of the gene of interest at the time of the hypoxia experiment [4,5].…”

“…In vivo ischemia-reperfusion models are technically simple and reproduce many aspects of ischemic diseases, but in vitro models are equally important, because they allow detailed study of the mechanism of cellular damage and make it possible to test large chemical libraries or sets of human small interfering RNAs (siRNAs) that are essential for early phase drug discovery [1][2][3][4][5]. Chemical hypoxia models that use mitochondrial uncoupling agents or respiration blockers reproduce the rapid onset of ischemia but there is no way to study the recovery processes that occur during reperfusion [6][7][8][9].…”