This R01 application, over its funded cycles, articulated and substantiated the thesis that the inducible heme- degrading enzyme, heme oxygenase-1 (HO-1), protects against AKI induced by ischemia-reperfusion (IR), heme proteins, and inflammation; this R01 also demonstrated that HO-1 and its products protect against hypertensive and vascular injury. Evidence is emerging that 1) intracellular and extracellular levels of heme, a nephrotoxin, are increased in AKI, thereby providing one of the final common pathways for AKI; and 2) the paradigm exemplified by HO-1 applies to other heme-related proteins: namely, HO-1 is the forerunner for a family of proteins that are not only of pathophysiologic significance beyond heme metabolism, but may provide new therapeutic strategies for AKI. This renewal focuses on 3 such heme-related proteins: hemopexin (HPX), the high-affinity, heme-binding protein; HO-2, the constitutive heme-degrading protein; and haptoglobin (Hp), the high-affinity, hemoglobin-binding protein.
Aim #1 tests the hypothesis that HPX is an inducible, heme- binding protectant against AKI. The effects of HPX protein and genetic deficiency of HPX, singly and in combination, will be examined in: 1) cisplatin-induced and IR-induced AKI; 2) heme-induced apoptosis and inhibition of cell proliferation, and cisplatin-induced apoptosis; and 3) heme-induced inflammation, the latter involving TLR4 signaling.
Aim #2 hypothesizes that constitutive HO-2 is an unrecognized protectant against AKI. Using HO-2-/- mice, this aim will examine whether the absence of the restraining effect of HO-2 exaggerates heme/NF-kB-dependent, inflammatory responses, and the extent to which TLR4 is involved. The role of HO-2 in suppressing heme-driven discharge of the inflammatory and vasoactive constituents of the Weibel-Palade Bodies (WPBs) will be examined.
This aim will also determine whether HO-2 deficiency exacerbates vascular behavior in AKI, and whether a product of HO, namely CO, attenuates such effects.
Aim #3 hypothesizes that haptoglobin (Hp) is an inducible protectant against AKI. Hp is now known to possess antioxidant effects, besides its binding of hemoglobin.
Aim #3 will examine whether Hp-/- mice exhibit increased sensitivity to IR-induced AKI and the capacity of Hp protein to reduce such sensitivity, focusing on the antioxidant actions of Hp; the efficacy of Hp in interrupting the prooxidant effects of heme in vitro will also be testd. Finally, this aim will examine whether mutant Hp 2-2 mice, expressing a disease-prone human genotype, exhibit increased sensitivity to IR-induced AKI due to impaired antioxidant actions of Hp 2-2. In summary, this application explores 3 novel, renal protective pathways and elucidates the importance of: heme as a common pathway for AKI and the role of heme-binding as a protective strategy; constitutive heme- degrading mechanisms in protecting against AKI; heme, by activating TLR4, as a novel damage-associated molecular pattern (DAMP); heme as a agonist for release of WPBs in AKI; and Hp, a protein in use or considered for use in human disease, as a novel, broadly applicable, but as yet untested therapy for AKI.
Heme proteins are essential proteins that contain a chemical structure known as heme. Examples include hemoglobin, but there are many others, and heme proteins number in the hundreds. These proteins are necessary for storing and transporting oxygen; enabling cells to consume oxygen and to perform their usual metabolism; control of vascular function; detoxifying potentially toxic substances, among other functions. However, in multiple instances, these heme proteins can actually cause kidney and other diseases. A central and long-standing focus in kidney research has been how these heme proteins cause kidney disease, and how the kidney can protect itself against such injury. In prior cycles of this grant application evidence was generated that a special enzyme known as heme oxygenase-1 can defend the kidney and other tissues from these potentially damaging heme proteins and, indeed, these observations have led to interest in the therapeutic application of these findings. This renewal application extends this line of investigation to involve three proteins that are related to heme, heme proteins, and heme oxygenase-1, and each of which may exert protective functions in the kidney. These proteins include the heme-binding protein called hemopexin, another heme-degrading enzyme called heme oxygenase-2, and a third protein known as haptoglobin which binds hemoglobin, but is now known to be a potent antioxidant protein; haptoglobin is used in certain countries to treat severe hemolysis. These proposed studies may provide evidence and direction for new therapeutic treatments for human acute kidney injury.
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