We have identified a new type of growth factor called Ngal (lipocalin-2) which is a member of the lipocalin-superfamily. It is known that lipocalins bind and transport small chemicals ligands, but little has been known about Ngal, and some of its presumptive ligands have been disproved. We purified Ngal from liters of media conditioned by an embryonic cell line, which revealed a growth factor with a reddish color. The cloned protein was also colored and x-ray crystallography of the cloned protein (R. Strong, Fred Hutchinson Cancer Research Center) showed that it contained a siderophore: Fe complex that it had bound during cloning. Using recombinant Ngal, we could induce growth and conversion of mesenchyme into epithelia, tubule and nephrons, but only when rNgal was loaded with this siderophore and iron. This suggested that Ngal's inductive action must have been due to its ligand, and we speculate that perhaps a mammalian version of the siderophore is present in vivo. Ngal is 1 of the most overexpressed proteins in ischemic disease of the kidney (>1000 fold increases). It is also the earliest known protein to be increased in the urine and in the serum of ischemic and toxic diseases of the kidney. In situ hybridization shows that Ngal is massively expressed by TALH and collecting ducts. In this grant, we propose to study the activity of the endogenous Ngal protein. We will do this by completing an isolation of a small organic molecule that binds Ngal and binds iron. The Ngal ligand is found in mammalian urine. We will identify the structure of this compound using IR, NMR, Mass spectroscopy and x-ray crystallography. To further examine the function of Ngal in the kidney, we have deleted its gene in a tissue specific manner. Because Ngal previously had a protective function in an ischemic model of the kidney, we will examine whether the Ngal knockout is more sensitive to ischemia reperfusion injury. We will test the hypothesis that the Ngal:urine siderophore complex removes catalytic iron and conversely the Ngal knockout contains more catalytic iron and iron mediated cell damage. The knockout will then be rescued by exogenous Ngal or iron chelators. This work is important because it characterizes a novel growth factor that is massively expressed by damaged renal epithelia and it evaluates a novel iron transport pathway that appears critical to Ngal function. ? ? ?
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