Reversal of Established Nephropathy in New Zealand B/W F₁Mice by 15-Deoxyspergualin

Abstract

The therapeutic effect of 15-deoxyspergualin (DSP) in old New Zealand Black/White F₁ mice (B/W mice) with clinical nephropathy was studied and compared with cyclophosphamide (CY). The mice were treated with 0.05 ml phosphate-buffered saline, subcutaneously, four times/week, with DSP, 6 mg/kg body weight, s.c, four times/week, or with CY, 15 mg/kg, i.p., once a week, starting at the 28th week of age. They were serially semiquantitated for proteinuria, and serum IgG anti-dsDNA antibody was measured by ELISA. Spleen cell surface markers such as L3T4, Lyt 2 and IgG were flow-cytometrically analyzed, and interleukin-2 (IL-2) activity in vitro was measured using CTLL cells. Kidney specimens were studied with light and immunofluorescence microscopy. The mice treated with either CY or DSP survived significantly longer than the control mice. L3T4⁺ cells in the DSP-treated mice at 40 weeks of age were significantly less than those in the 28-week-old control mice (p < 0.05). In contrast, IL-2 generation in the three groups of mice showed no significant variations at 32–40 weeks of age. Serum anti-DNA antibody levels in both of the CY and DSP groups remained low and comparable with that in the 28-week-old mice, and the incidence of significant proteinuria decreased. Likewise, glomerular histology in the treated groups was improved compared with the 28-week-old control mice, and the deposition of IgG and C3 in the treated groups remained unchanged or further decreased. Accordingly, the renal (immuno)histological findings in the DSP group were quite comparable with or even better than those in the CY-treated mice. DSP may have suppressed the abnormal antibody production by modulating the T cell function(s), which is in contrast to the direct action against B cells due to CY.

Effect of 15-Deoxyspergualin on Lupus Nephropathy in New Zealand Black/White F1 Mice

“…Details of the method were already reported [2,3]. In brief, a million splenocytes suspended in 50 pi PBS were incubated with 25 ul rat antimouse L3T4 (Clone YTS 191.1, Sera-Lab, UK) or Lyt 2 (Clone YTS 169.4) at 0°C for 30 min, then were further incubated with 50 pi fluorescein isothiocyanate (FITC)-conjugated goat antirat IgG (Cappel Laboratories, Cochranville, Pa., USA) at 0°C for another 30 min.…”

“…According to the method previously reported [2][3][4], 5 x 106 spleno cytes in 2 ml RPMI-l640(Difco Laboratories. Detroit.…”

“…Serum anti-DNA activity was measured using an enzyme-linked immunosorbent assay (ELISA) [3]. A round-bottomed 96-well plate (Dynatech Laboratories, Plochingen, FRG) was successively coated with 100 pi 0.01% protamine sulfate, and 50 pi dsDNA, which was prepared by digeting calf thymus DNA (Sigma Chemical, St. Louis, Mo., USA) with SI nuclease and fractionating on a hydroxyappatite column.…”

“…According to the methods previously reported [2,3,5], glomerular histopathological findings were evaluated concerning the following three parameters: intracapillary proliferation, extraca pillary proliferation and tuft necrosis, and each was graded from 0 to 3. At least 100 glomeruli of each uncoded specimen were separately studied by three of us (MO.…”

“…In addition, DSP produced a decrease of L3T4+ splenocytes without affecting the number of Lyt 2+ cells, while the level of IL-2 generated in vitro was somewhat elevated. It may be concluded that DSP has a therapeutic range within an order of ten, but its exact mechanism of immunosuppression remains to be determined.15-Deoxyspergualin (DSP), an antibiotic derived from spergualin [1], a secondary bacterial metabolite, was previ ously shown to effectively suppress the development of nephropathy in New Zealand Black/White FI (B/W) mice, when treatment was started either prior to or following the onset of glomerulonephritis [2,3]. However, since a relative ly large dose of DSP was employed in both studies, it may be necessary to determine the minimally effective dose of DSP as well as the lowest maximally effective dose without toxicity, prior to studying the synergistic effect with other drugs and before proceeding to future clinical use.…”