Assessment of binding of 2,4,4-trimethyl-2-pentanol to low-molecular-weight proteins isolated from kidneys of male rats and humans.
A number of chemicals cause a nephrotoxic syndrome in male rats, referred to as protein droplet or alpha 2u-globulin (alpha 2u) nephropathy. To evaluate the potential human risk for developing a similar chemically-induced protein-mediated nephrotoxic response, the low-molecular-weight protein fraction (LMWPF) was isolated from the kidneys of male rats and humans to compare protein concentrations and chemical binding capabilities. Kidney cytosol, prepared from control male rats, was applied to a G-75 gel filtration column and LMWPF (containing proteins from 10,000 to 30,000 Da) were collected and concentrated. 2,4,4-Tri[3H]methyl-2-pentanol ([3H]TMP-2-OH), a chemical known to bind alpha 2u and cause it to accumulate in the kidneys of treated male rats, was incubated with the LMWPF for 1 hr at 25 degrees C. This mixture was applied to a PD-10 column to separate total protein (free and chemically bound) from free [3H]TMP-2-OH. The total protein fraction was injected onto a DEAE anion exchange column and proteins were eluted with a NaCl gradient. [3H]TMP-2-OH was found to coelute with alpha 2u. The alpha 2u protein peaks were identified using SDS-PAGE and Western blot analysis. The LMWPF from human kidneys was then prepared and analyzed identically to that from the male rat kidneys. Human kidney had a concentration of proteins lower than that of the male rat kidney, with a smaller percentage of low-molecular-weight (LMW) proteins. Unlike the results obtained using rat kidney LMWPF, [3H]TMP-2-OH was not found to coelute with the human kidney LMWPF. Taken together, these findings indicate that humans do not possess a protein similar to alpha 2u in relative abundance or binding characteristics and that humans may not be at risk of developing a chemically induced protein-mediated nephrotoxic response.
Borghoff
S J
SJ
Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709.
Lagarde
W H
WH
D000328:Adult; D000368:Aged; D000510:Alpha-Globulins / Q000032:analysis; D000818:Animals; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D008970:Molecular Weight; D000439:Pentanols / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344
D000510:Alpha-Globulins; D000439:Pentanols; D011506:Proteins; C013803:alpha 2u globulin; C067352:2,4,4-trimethyl-2-pentanol