Partial Purification and Characterisation of a Renal Growth Factor from Plasma of Uninephrectomised Rats

Abstract

Renal growth factor activity was extracted from plasma of adult uninephrectomised rats and partially purified by gel filtration and anion-exchanger FPLC. It induced a maximal stimulation of mouse DNA synthesis in vivo at 1.75 micrograms/mouse. In addition, renal growth factor was found to maximally stimulate DNA synthesis in LLC-PK1 cells at 150 ng/ml. This maximal response was then found to decrease with higher doses of renal growth factor, in vivo and in vitro. The apparent molecular weight of renal growth factor was estimated to be 17K-22 K by gel filtration. It was found to be resistant to heat and to trypsin, but labile to reduction with dithiothreitol.

Biological Properties of a Renotropic Protein Present in Plasma of Uninephrectomized Rats

“…Therefore, KGF has a specific activity of 1.5 x 104 units/mg protein injected into mouse. It represents about 20-fold purification as compared to that at the Mono O step or about 500-fold relative to that at the first gel filtration step in the purification procedure [6]. Due to possible existence of inhibitors, specific activity of plasma was not tested.…”

“…The effect of test samples on DNA synthesis was examined in cell cultures which were serum-depleted 24 h befoie the addition of ( 'H)thymidinc, as previously reported [6].…”

“…After 90 min of incubation, the tissue was washed 3 times with ice-cold KHB and 3 times with ice-cold 7% perchloric acid. Incorporated (3H)thymidine was measured after DNA extraction as described [6].…”

“…Flow-cytometric analysis of cellular DNA content was carried out in subeonfluent LLC-PK, cells which had been treated with or without KGF (1.5 ng/ml) for 20 h as described [6], Firstly, the cells were removed from the culture dishes by incubation with 0.05% tryp Esbrit/Garcia-Ocafia/Garcia-Caiiero/Manzano/Jim6ncz-Clavero sin-0.02% EDTA for 5-10 min and processed for cellular DNA analysis according to the method of Taylor and Milthorpe [9]. Briefly, 2-5 xlO5 cells were washed 3 times with saline and fixed with 70% ethanol at 4°C overnight.…”

“…1988, Montreal. Canada. sitivity to allow further characterization of the renotropic activity since the possible existence of circulating inhibitors of renal growth might interfere with such an activity [4,5], By using chromatographic techniques, we recently partially purified a renal growth factor activity from plasma of uninephrectomized rats [6]. This activity was found to increase, dose-dependently, ( 'U)thymidine incorpora tion into mouse kidney DNA in vivo as well as into DNA of LLC-PKi cells in vitro.…”