Gram negative sepsis remains a formidable clinical problem with an unacceptably elevated mortality despite decades of intensive research in the field. The multi organ failure that frequently sets in during sepsis often ushers a grim outcome that so far has been difficult to alter. The pathophysiology of acute kidney injury (AKI) in sepsis is thought to result from uncontrolled inflammation and a procoagulant state triggered by stimulation of Toll-like receptors (TLRs) on cells of the immune system. Nevertheless, interference with systemic inflammatory cytokines, anticoagulation therapy and vigorous hemodynamic support all met with little success in preventing AKI and improving overall mortality. It is the purpose of this proposal to explore a novel hypothesis regarding the pathophysiology of ARF and AKI in gram negative sepsis. The hypothesis proposes that AKI in systemic gram negative sepsis results in part from a direct interaction between endotoxin and renal tubular cells.
In specific aim 1, we will measure the magnitude and distribution of tubule- endotoxin interactions in mice and rat models of systemic sepsis. Using 2 photon microscopy in live animals, along with novel fluorescent probes, we will measure tubular oxidative stress and apoptosis that result from direct tubular endotoxin uptake. To examine the pathophysiology of endotoxin-tubule interactions in the absence of systemic cytokines, we will generate chimera mice lacking systemic TLR4. Finally, the role of p53 in mediating apoptosis and oxidative stress will be examined through protein expression, co localization and inhibitory studies.
In specific aim 2, we will determine the roles of TLR4, CD14 and receptor-independent fluid-phase endocytosis in tubular endotoxin uptake with the powerful tool of targeted live delivery of receptor-specific siRNA. The impact of acute TLR4 or CD14 knockdown on endotoxin uptake, oxidative stress and apoptosis will be determined with live 2 photon imaging studies as well as traditional ex vivo techniques. Chimera mice lacking renal TLR4 or CD14 will provide an additional approach to examine the role of these molecules in tubular endotoxin uptake.
These specific aims will determine the magnitude, impact and mechanism of the interaction between endotoxin and tubular cells. We believe they will establish a novel and so far unexplored mechanism underlying sepsis-induced renal injury. By exposing such a new mechanism, our studies could be at the basis of future therapies that aim at interfering with the direct detrimental effects of endotoxin on renal cells.

Public Health Relevance

Acute kidney failure and sepsis represent a major healthcare burden in the US with a cost exceeding millions of dollars yearly.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK080067-05
Application #
8450638
Study Section
Pathobiology of Kidney Disease Study Section (PBKD)
Program Officer
Kimmel, Paul
Project Start
2009-05-15
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
5
Fiscal Year
2013
Total Cost
$312,314
Indirect Cost
$97,145
Name
Indiana University-Purdue University at Indianapolis
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
603007902
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Hato, Takashi; El-Achkar, Tarek M; Dagher, Pierre C (2013) Sisters in arms: myeloid and tubular epithelial cells shape renal innate immunity. Am J Physiol Renal Physiol 304:F1243-51
Sutton, Timothy A; Hato, Takashi; Mai, Erik et al. (2013) p53 is renoprotective after ischemic kidney injury by reducing inflammation. J Am Soc Nephrol 24:113-24
Kalakeche, Rabih; Hato, Takashi; Rhodes, Georges et al. (2011) Endotoxin uptake by S1 proximal tubular segment causes oxidative stress in the downstream S2 segment. J Am Soc Nephrol 22:1505-16
Sutton, Timothy A; Dagher, Pierre C (2011) Fueling the fire in acute kidney injury: endothelial cells collect their Toll. Kidney Int 79:267-9