Cytokines Convert Epithelial Precursors to Nephrons

Columbia University Health Sciences, New York NY

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Abstract

[unreadable] DESCRIPTION (provided by applicant): When and where do epithelia form in the embryonic kidney? There is an event called "induction", the result of signaling from the ureteric bud that stimulates the mesenchyme to convert into epithelia. But is this 1 event or do multiple signals, working in series or in parallel drive cell conversion? We began looking for inductive factors by making a cell line from the ureteric bud. We grew 35,000 flasks and tested its fractions on mesenchyme taken from the rat when the UB had just invaded. We discovered 1. mesenchyme dies without the UB but 2.can be rescued by UB factors and 3. can be converted when 2 groups of factors are combined. We purified the following survival factors-FGF-2, TlMP-2, SHH and endostatin. The inductive factors included LIF, lipocalin-2 (Ngal, the most highly expressed factor in acute renal failure), a fragment of urokinase (unpublished) and a partially purified, powerful inducer we call ANX. The inducers act like switches--if present for only 24 hours, they drive a single mesenchyme to produce 100 tubules and recognizable nephrons. LIF was the first known purified nephron inducer. To further our search for molecules that control conversion, we isolated 1000 tips from stalks of the in vivo ureteric bud and we identified its secreted factors using 2 different species. Amazingly, 1 of the most expressed tip proteins was a chaperone for LIF-like molecules, known as CLF. In fact, the embryonic kidney likely expresses not only LIF but 2 novel cytokines, and there is good evidence for yet a third. Evaluation of the common receptor KO shows a hypoplastic phenotype that must now be further evaluated. This was an unexpected confirmation of our original work. We need now to define the response of the mesenchyme to the many inducers. We show by comparison, that a common pathway of conversion exists. We find unexpected Wnt regulators, transcription and growth factors downstream of the inducers, which we will analyze by applying new techniques of gene expression and inhibition. Finally as we define the inductive pattern, we ask where do these processes really occur? Is polarity induced uniformly as designed by our bioassays?, or have we only assayed one stage in a long pathway? We apply the basic ideas and tools of cell biology to define the course of polarization and establish a fluorescent polarity mouse to evaluate the entire transition form mesenchyme to epithelia. [unreadable] [unreadable] [unreadable]