Figure provided by Dr. Richard Zager.
Ischemia is a significant loss of blood supply that can affect any tissue. Such decrease in the supply of blood results in oxygen deprivation preventing effective cellular metabolism and can bring about cellular senescence or necrosis to cause irreversible tissue damage. Ischemia can be observed in the event of atherosclerosis or embolism, but also during major surgeries such as cardiac intervention. “3% of individuals are at risk for ischemic injury but this rate rises to 30% in intensive care units and can affect 30-40% of newborns undergoing major surgery” explained Dr. Richard Zager, a principal investigator (Clinical Research Division). Therefore, these groups of individuals are considered at high risk for tissue failure and research is in progress to lower this risk.
Dr. Zager’s laboratory studies pre-conditioning as a protective therapy for clinically induced kidney injury by administration of pre-conditioning agents injected 24 to 48 hours before the surgery. Cytoprotective effects can be seen in animal models as early as 12-24 hours post-infusion as observed by increased detection of cytoprotective proteins such as heat shock proteins or bilirubin reductase, which exert multifunctional effects to protect the tissue from ischemia. Hence, when there is a significant loss of blood, the pathways are already activated by the pre-conditioning, protecting the tissue from damage.
However, the preconditioning agents can have both beneficial and deleterious effects. Dr. Zager and staff scientist Dr. Ali Johnson recently published a study in the journal Nephrology, Dialysis, Transplantation comparing different preconditioning agents for acute kidney injury and their impact on renal toxicities in mice. Despite cytoprotective effects, p21-inducing ischemic preconditioning (IPC) is also associated with renal cell senescence and renal failure, which can be fatal. As an alternative conditioning method, oxidant-induced preconditioning (OIP), exerts p21-independent cytoprotective effects, through the Nrf2 pathway, and is not associated with senescence or renal toxicities.
p21 is induced by IPC, but is also known as a cyclin dependent kinase inhibitor promoting cell cycle arrest, similarly to p16 and p19, raising concerns about the long-term impact of p21 induction on cell proliferation and senescence. To address this concern, a comparison of the influence of IPC or OIP agents on p21, p16, p19 and Nrf2 pathways was performed in vivo using mice. The preconditioning treatments were administered 18 hours prior to kidney injury. IPC can be induced by transiently occluding blood circulation to one of the kidneys using microvascular clamps. In the OIP group, the animals were administered with the heme oxygenase inhibitor, Tin protoporphyrin (SnPP) alone or in combination with either iron sucrose (FeS) or nitrite Myoglobin (N-Mgb), combinations known to provide greater cytoprotective effects.
While IPC or acute kidney injury without preconditioning induced a significant increase in p21 expression, none of the OIP preconditioning agents led to an increase in p21, p19 or p16 mRNA or protein expression, confirming that OIP and IPC exert their cytoprotective effects through independent pathways. OIP prevented p21 induction and renal failure (detected by blood urea nitrogen and plasma creatinine) following acute kidney injury. These effects were observed as early as 18 hours following ischemic injury but were also persistent as they were still detected two weeks later, reinforcing the initial concerns about the potential harmful effects of a long-lasting p21 induction.
Indeed, while OIP induced cell proliferation as shown by immunohistochemistry staining and quantification of Ki67 and PHH3 proteins, IPC induced tubular senescence, NGAL (Neutrophil gelatinase-associated lipocalin) mRNA expression, a biomarker of tissue damage and a 25% decrease of the renal mass, two weeks after the induced injury. These data clearly indicate the long-term benefits of using OIP over IPC.
Nrf2 protein is also called the “Master regulator of endogenous cellular defenses”. Nrf2 pathway activation mediates OIP cytoprotective effects through induction of NQO1 (NAD(P)H Quinone Dehydrogenase 1), GCLC (Glutamate-Cysteine Ligase Catalytic Subunit) and SXRN1 (sulfiredoxin) mRNA, whose expressions are known as Nrf2- specific. NQO1, GCLC and SXRN1 proteins also have diverse cytoprotective functional properties such as involvement in detoxification or metabolism pathways. However, their upregulation following OIP was abolished in Nrf2-/- mice, demonstrating the key role played by Nrf2.
While these data are exciting news, “it remains unclear what signs are onset indicator or consequences of kidney injury and how these could be used to prevent the tissue damages ”, explained Dr. Zager. “Senescence might be one characteristic and our future experiments will focus on new ways to monitor senescence in the blood by measuring the levels of p21, p16 and p19 in the plasma, with the goal to block the injury from happening.”
Funding for this study was provided by Renibus Therapeutics.
Johnson ACM, Zager RA. 2018. Mechanisms and consequences of oxidant-induced renal preconditioning: an Nrf2-dependent, P21-independent, anti-senescence pathway. Nephrology Dialysis Transplantation. [Epub ahead of print].