The occurrence of acute kidney injury (AKI) in humans is associated with a poor long-term prognosis including higher morbidity and increased mortality. Our recent studies showed that the inactivation of apoptotic DNases/endonucleases provided protection against AKI induced by ischemic or toxic insults. These observations led to the conclusion that endonucleases are involved in premortem DNA fragmentation, which precedes and causes cell death. This proposal is a continuation of the previous accomplished study, which was focused on the role of DNase I in ischemic acute renal failure. This study resulted in two important conclusions. One of them is that the inactivation of DNase I has much broader implications than it was initially thought, and it can be applied to the toxic kidney injury induced by cisplatin. Another observation was that DNase I may act in concert with other endonucleases. Our preliminary studies showed that another DNase, endonuclease G (EndoG), is induced in the tubular epithelium by cisplatin in vitro and in vivo. These studies showed that DNase I is necessary for EndoG induction in several in vitro and in vivo models. The regulation of EndoG expression by DNase I is a previously unknown pathway. The hypothesis of the current proposal is that (a) during cisplatin kidney injury, premortem enzymatic DNA damage is induced by EndoG which acts along the pathway initiated by DNase I, and (b) the inactivation of EndoG may protect the kidney against injury induced by cisplatin. This hypothesis is supported by the preliminary data, which showed that the genetic inactivation of EndoG in mice or primary tubular epithelial cells, or the silencing of EndoG in tubular epithelial cells provided protection against DNA damage and cell death induced by cisplatin.
In Specific Aim 1, we plan to determine the role of DNase I in the regulation of EndoG expression, DNA damage and apoptosis.
Specific Aim 2 will be focused on examining the effects of EndoG activation or inactivation on tubular epithelial cell injury.
In Specific Aim 3, we are going to define the intermediate mechanisms of EndoG regulation by apoptosis, DNA damage, membrane permeability and others during cisplatin injury. DNase I and EndoG null mice, RNA interference, overexpression of DNase I and EndoG, dominant-negative mutant and inhibitors will be used to interrupt specific pathways in vivo or in vitro and thus address the goals in a mechanistic and cause-effect relationship manner. Our endpoints will include: expression of endonucleases quantified by real-time RT-PCR, Western blotting and activity, immunolocalization of EndoG, the assessment of DNA fragmentation by TUNEL or Comet assays, and the analysis of cell viability. It is very likely that the identification of the role and regulation of EndoG in the mechanisms of premortem DNA damage and kidney cell death pathways will provide new modalities for the prevention and treatment of AKI in humans.

Public Health Relevance

The proposal is focused on the DNA damage mechanisms induced during toxic kidney injury, and the role of two enzymes which are responsible for this DNA damage: DNase I and endonuclease G. We propose that these two enzymes are linked in a pathway, in which DNase I activates endonuclease G, and that inactivation of any of these endonuclease can protect kidney from toxic injury.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
3R01DK078908-02S1
Application #
7992611
Study Section
Pathobiology of Kidney Disease Study Section (PBKD)
Program Officer
Kimmel, Paul
Project Start
2009-12-08
Project End
2010-12-07
Budget Start
2009-12-08
Budget End
2010-12-07
Support Year
2
Fiscal Year
2010
Total Cost
$63,990
Indirect Cost
Name
University of Arkansas for Medical Sciences
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
122452563
City
Little Rock
State
AR
Country
United States
Zip Code
72205
Jang, Dae Song; Penthala, Narsimha R; Apostolov, Eugene O et al. (2015) Novel cytoprotective inhibitors for apoptotic endonuclease G. DNA Cell Biol 34:92-100
Zhdanov, Dmitry D; Fahmi, Tariq; Wang, Xiaoying et al. (2015) Regulation of Apoptotic Endonucleases by EndoG. DNA Cell Biol 34:316-26
Jang, Dae Song; Penthala, Narsimha R; Apostolov, Eugene O et al. (2015) Novel high-throughput deoxyribonuclease 1 assay. J Biomol Screen 20:202-11
Nima, Zeid A; Mahmood, Meena; Xu, Yang et al. (2014) Circulating tumor cell identification by functionalized silver-gold nanorods with multicolor, super-enhanced SERS and photothermal resonances. Sci Rep 4:4752
Singh, Mandeep; Odeniyi, Dolapo T; Apostolov, Eugene O et al. (2013) Protective effect of zinc-N-acetylcysteine on the rat kidney during cold storage. Am J Physiol Renal Physiol 305:F1022-30
Apostolov, Eugene O; Ok, Ercan; Burns, Samuel et al. (2013) Carbamylated-oxidized LDL: proatherosclerotic effects on endothelial cells and macrophages. J Atheroscler Thromb 20:878-92
Ronis, Martin J J; Hennings, Leah; Stewart, Ben et al. (2011) Effects of long-term ethanol administration in a rat total enteral nutrition model of alcoholic liver disease. Am J Physiol Gastrointest Liver Physiol 300:G109-19
Apostolov, Eugene O; Soultanova, Izoumroud; Savenka, Alena et al. (2009) Deoxyribonuclease I is essential for DNA fragmentation induced by gamma radiation in mice. Radiat Res 172:481-92