In Vivo Detection of Deposition of Radiolabeled Lupus Antikidney Antibody and Its Inhibition by Soluble Antigen

MATERIALS AND METHODS

Kidney deposition of nephritogenic iodinated R4A antibody was examined after injection of an iodinated antibody into 7- to 9-wk-old female BALB/c mice (Albert Einstein College of Medicine or The Jackson Laboratory, Bar Harbor, ME). R4A is an immunoglobulin G2b (IgG2b) murine anti-DNA antibody and was obtained from a cell culture supernatant or from ascites (11). The antibody was purified on a G protein column (Gamma Bind; Pharmacia, Peapack, NJ). Isotype-matched IgG2b with no DNA binding (Sigma, St. Louis, MO) was used as a control antibody. Peptide DWEYS (blocking peptide) was purchased as a d isoform (Research Genetics, Huntsville, AL).

Forty-five mice (body weight range, 17–27 g) were studied. They were given food ad libitum, and iodinated drinking water was added 24 h before the study to block thyroid ¹²⁵I accumulation. R4A and control antibody were iodinated using the standard iodogenic method (yield, 35%–60%). Fifty to 100 μg DNA-binding R4A or control antibody in 0.1 mL phosphate-buffered saline were mixed with 60–100 μCi sodium ¹²⁵I and incubated in test tubes coated with 20 μg Iodo-Gen (Pierce Chemical Co., Rockford, IL) for 30–50 min at room temperature.

Iodinated R4A antibody and control antibody were fractionated by chromatography using a G-25M column (Sephadex; Pharmacia Fine Chemicals, Piscataway, NJ) after equilibration with phosphate-buffered saline. Eight fractions were collected. ¹²⁵I activity and antibody concentration in each fraction were determined using, respectively, a gamma counter (model CRC-12; Capintec, Inc., Ramsey, NJ) and a spectrophotometer at 280 μm. The three fractions with both highest ¹²⁵I activity and highest antibody concentration were pooled and used for this study. Labeling yields less than 40% were not used. The antibody was shown to maintain antigenic specificity after iodination.

Group 1 mice received injections of 3–7 μg ¹²⁵I R4A (4–6 μCi) through the tail vein. The R4A antibody is an IgG2b anti-DNA antibody that has previously been shown to bind to renal glomeruli in vivo (6,8,9). Group 2 mice simultaneously received tail-vein injections of 10–19 μg ¹²⁵I R4A (2–5 μCi) and 100–240 μg peptide with the sequence DWEYS (blocking peptide). Group 3 mice received injections of 5–10 μg ¹²⁵I R4A (2–8 μg) iodinated control antibody. R4A deposition in the kidney was measured 48 h and 8 d after administration. The animals were anesthetized with 0.1 mg in 0.1 mL Nembutal sodium solution (Abbott Laboratories, North Chicago, IL) by intramuscular injection and were then exsanguinated before removal of organs to minimize the effect on organ counting rates. The percentage of the dose accumulated in kidney, spleen, liver, and lung was determined in a well scintillation counter (CompuGamma CS; LKB Walloc, Turkee, Finland). All analyses for ¹²⁵I tissue concentration were expressed as mean ± SE of percentage injected dose per gram of tissue. Statistical analysis was performed using t test comparisons of sample means and SE analysis. The study was approved by the Animal Institute Care and Use Committee of the Albert Einstein College of Medicine.

RESULTS

As seen in Table 1, mice given R4A retained 5.21% of the injected dose per gram of tissue antibody in the kidneys after 48 h, whereas mice given control antibody retained only 2.50% (P < 0.0004) of the injected antibody in the kidneys. When mice were given R4A antibody and DWEYS peptide that was bound by the antibody, they retained 1.53% (P < 0.00001) of the injected antibody in the kidneys at 48 h. The amount of antibody in the total blood volume after 48 h was 22.31% in mice given R4A, 10.69% (P < 0.030) in mice given control antibody, and 3.42% (P < 0.0005) in mice given both R4A antibody and peptide.

Seven to 8 d after injection (Table 2), R4A antibody was still sequestered in the kidneys (1.44% of the injected dose) compared with control antibody (0.20% of the injected dose, P < 0.00004) and compared with R4A antibody plus peptide (0.64% of the injected dose, P < 0.0017). Likewise, R4A in whole blood was greater (6.19%) than control antibody (1.14%, P < 0.0011) and R4A plus peptide (0.40%, P < 0.003).

DISCUSSION

These studies show that the sequestration of an anti-DNA antibody in renal tissue can be detected using iodinated antibody. Antigenic peptide will inhibit renal sequestration of antibody. These data suggest the feasibility of using antigen blockade therapeutically to protect kidneys from antibody-mediated damage and to evaluate that effect using nuclear medicine techniques. It seems highly probable that imaging technology can detect potentially pathogenic anti-DNA antibodies and can determine if inhibitors successfully prevent renal sequestration. Furthermore, it is reasonable to expect that nuclear imaging can monitor this intervention. Noninvasive monitoring will be critical if peptide inhibition becomes a therapeutic modality, because the amount of peptide needed to provide blockade will differ among patients.

CONCLUSION

Although most lupus patients with renal disease display high serum titers of anti-DNA antibodies, some patients in whom nephritis develops have no detectable anti-DNA activity. We are now able to ask whether imaging renal deposition of serum immunoglobulin from such individuals can help identify their risk for renal disease and permit early therapy. Although little attention has been directed at the kidney in immunoimaging, these data show the potential of this approach in renal nuclear imaging.