Human renal proximal tubular cells will be investigated as a model system to understand the molecular basis of renal proximal tubular cell growth. The objective of this proposal is to investigate the basic mechanism involved in maintaining human renal proximal tubular cell integrity for preservation of function. We have developed techniques for measuring the proliferative activity of growth factors on human renal proximal tubular cells. A growth which is mitogenic for human renal proximal tubular cells have been identified and partially purified from a human renal adenocarcinoma clear cell line. In this proposal, we will purify the growth factor utilizing Sephadex CM-50, heparin- Sepharose affinity chromatography plus a combination of reverse phase, gel filtration, ion exchange or hydrophobic high performance liquid chromatography. The growth factor will be characterized as to size, isoelectric point and the terminal amino acid sequence determined on a gas phase microsequencer. Further studies will be performed to measure the potential mitogenic activity against a large number of diverse cell types. The presence of the growth factor in normal human kidneys will be evaluated. Human renal proximal tubular cells exhibit limited proliferative potential in culture. The serial propagation of human renal proximal tubular cells with retention of differentiated properties will provide a useful model for the investigation of renal proximal tubular cell function. We will attempt to serial propagate human renal proximal tubular cells utilizing conditioned media from the human renal adenocarcinoma clear cell line and the partially purified growth factor. Biochemical markers, morphological criteria and proliferative capacity will be measured following subculturing of the proximal tubular cells. Finally, we will initiate a pilot study to investigate the role of the renal proximal tubular cell growth factor on cell adhesion and heparan sulfate proteoglycan deposition. Thus, the proposal will identify, isolate, purify and characterize growth factors directly affecting the kidney.
|Effective start/end date
|12/31/89 → 8/31/91
- Cell Biology
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