Detection of FS Complexes in the Kidney
Follistatin mRNA is present in the kidnea; therefore, ho-mogenates from rat kidneas were generated and examined ba Western blot analasis using anti-FD antibodies. Identical amounts of total protein were loaded per well. In nonpregnant kidnea protein homogenates, FD proteins were detected at 14-20 kDa, 25 kDa, 30 kDa, and 35-40 kDa (Fig. 3). In the pregnant kidnea, immunoreactive FD bands were detected at 30 kDa, 35-40 kDa, 97 kDa, and 133 kDa, with evidence of larger molecular size bands as well (Fig. 3). buy cheap antibiotics
The smaller molecular size proteins may represent proteolytic cleavage products of the FS monomer.
FS and FS Complexes in Urine
Because FS mRNA and protein are present in the kidney, it was hypothesized that urine may also contain FS or FS complexes. Using Western blot analysis, FS was not detected in nonpregnant female (human) urine. However, urine collected from pregnant women was replete with the proteins previously documented as FS complexes of 66 kDa, 97 kDa, 133 kDa, and 220 kDa. To further clarify the nature of the urine-related FS, total activin A (free activin and activin complexed to binding proteins), total inhibin A (inhibin A or inhibin A complexes), total inhibin B (inhibin B or inhibin B complexes), free FS, and total FS enzyme-linked immunosorbent assays were established to study the urine samples. Inhibin A was detected in urine from pregnant women (62.19 ± 17.6 pg/ml; n = 8) and was not detected in urine from nonpregnant women or from men. The urinary inhibin A diluted linearly and in parallel to the rh-inhibin A standard curve. Neither inhibin B nor activin A was detected in any of the urine samples studied. An FS ELISA was developed that did not detect FS upon addition of activin. Therefore, the assay was designated a free FS assay. FS was not detected by this ELISA using urine from pregnant or nonpregnant women at various dilutions. This implies that there is no free FS in the urine, consistent with the Western blot analysis. However, in an assay that detects FS when it is associated with inhibin or activin (i.e., “bound” FS), FS was detected in the pregnant human urine samples, suggesting that urine contains complexes of FS and inhibin A (7.1 ± 3.2 ng/ml; n = 5).
FIG. 3. Using a Western technique with monoclonal FS antibodies, the following samples were assayed. Note that upon reduction, fewer bands are seen in the urine (97 kDa, 30 kDa); this may represent the loss of a specific conformational epitope for these FS monoclonal antibodies. Pregnant urine samples in the rat and the human contained bands of similar molecular weight. There were larger molecular weight structures in the pregnant kidney as compared with the nonpregnant kidney. Of note are the proteins at approximately 30 kDa, which may represent unbound FS, and at 66 and 97 kDa, which may represent FS complexes. Bands at less than 30 kDa may represent degradation products.