Generation of NZM.C57Lc1 and NZM.C57Lc4 Congenic Lines.
A microsatellite marker-assisted protocol was used to facilitate the generation of both NZM.C57Lc1 and NZM.C57Lc4. A panel of 91 informative markers was used for the selection of the breeding pairs. As shown in , the C57L/J alleles were maintained throughout the genomic interval flanked by D1Mit15 (87.9 cM) and D1Mit155 (112.0 cM) for the derivation of NZM.C57Lc1 and D4Mit91 (15.6 cM) and D4Mit72 (59.9 cM) for NZM.C57Lc4. Each of these genetic intervals contains the 95% confidence interval for the loci linked to acute and chronic glomerulonephritis (Agnz1 and Cgnz1) on chromosome 1 and to ANA and anti-dsDNA Ab production (Adnz1) on chromosome 4.
Figure 1. NZM.C57Lc1 and NZM.C57Lc4 congenic lines were derived by replacing the genetic intervals in NZM2328 with those from C57L/J (hatched bars). The genetic intervals with SLE susceptibility genes in NZM2328 delineated by informative microsatellite markers (more ...)
Severe Proteinuria and Chronic Glomerulonephritis in NZM.C57Lc4 but Not in NZM.C57Lc1 Female Mice.
Cohorts of NZM.C57Lc1 and NZM.C57Lc4 females were observed for the development of severe proteinuria and acute and chronic glomerulonephritis. As shown in A, 70% (24/34) of NZM.C57Lc4 female mice developed severe proteinuria and only 3 of the 41 NZM.C57Lc1 females (7.3%) had severe proteinuria by 12 mo of age. The incidence of severe proteinuria for NZM.C57Lc4 was similar to that of parental stain NZM2328. The incidence of severe proteinuria in NZM.C57Lc1 female mice was markedly reduced. The kinetics of proteinuria development was similar between NZM2328 and its congenic NZM.C57Lc4. There was a delay of 4 mo in the development of severe proteinuria in the three NZM.C57Lc1 mice.
Development of severe proteinuria in females of NZM2328 and NZM.C57Lc4 but not in those of NZM.C57Lc1. (A) Incidence of severe proteinuria in each strain of mice. (B) Kinetics of proteinuria development.
The kidneys from 14 females in each congenic line and from 44 females of the parental line NZM2328 were evaluated for histological evidence of glomerulonephritis. Acute glomerulonephritis was defined by the presence of hypercellularity, mesangial proliferation, mononuclear cellular infiltration, and thickening of the capillary loops. Chronic glomerulonephritis was scored by the presence of glomerulosclerosis tubular atrophy and interstitial fibrosis (16
). The incidences of acute and chronic glomerulonephritis in NZM2328 at 12 mo of age were 81.8 (36/44) and 52.2% (23/44), respectively. These incidences were not significantly different from those for NZM.C57Lc4 female cohort, which had 78.5 (11/14) and 50.0% (7/14) incidences for acute and chronic glomerulonephritis, respectively. The three NZM.C57Lc1 females, who developed severe proteinuria, had both acute and chronic glomerulonephritis. Three of the other NZM.C57Lc1 female mice had acute glomerulonephritis without chronic glomerulonephritis. Thus, there was marked reduction in renal disease in the congenic line NZM.C57Lc1. At the age of 12 mo, no significant glomerulonephritis was detected in C57L/J (19
Histological, immunofluorescence, and EM studies of representative kidneys of the NZM2328 congenic lines are shown in . The glomeruli of NZM.C57Lc1 are normal in size without cellular infiltration and similar to those seen in kidneys of C57L/J ( A). In contrast, the glomeruli of the NZM.C57Lc4 congenic female were enlarged with hypercellularity, mesangial proliferation, thickened capillary loops, and segmental glomerulosclerosis ( B). Although not shown, there were tubular atrophy and interstitial fibrosis in the diseased NZM.C57Lc4 kidneys. Immunofluorescence studies on NZM.C57Lc4 kidneys showed intense staining for IgG and C3 (). The thickened capillary loops were more evident with anti-IgG staining. Immunofluorescence staining of NZM.C57Lc1 kidneys showed limited staining for IgG in the mesangia and some C3 deposit in some mesangia and the Bowman capsule (). These limited staining patterns were often seen in aged C57L/J mice and are considered of no pathological consequences. The IgG and C3 staining patterns of NZM.C57Lc4 kidneys were suggestive of immune complex–mediated glomerulonephritis. This was confirmed by the EM study, showing electron-dense deposits in the subendothelial and subepithelial areas ( H). In contrast, a representative EM of NZM.C57c1 kidneys did not show such electron-dense deposits ( G).
Figure 3. Histological, immunofluorescence, and EM studies of representative kidneys from NZM.C57Lc1 and NZM.C57Lc4 female mice. (A) Normal glomeruli (hematoxylin and eosin staining, ×200) are seen in NZM.C57Lc1. (B) In contrast, in the NZM.C57Lc4 congenic, (more ...)
Lack of Circulating ANA, Anti-dsDNA, and Antinucleosome Ab in Both NZM2328 Congenic Lines.
Sera from the terminal bleeds of female NZM2328 and its two congenic lines were analyzed for ANA, anti-dsDNA, and antinucleosome Ab. As shown in the top of , ANAs were readily detected in the sera of NZM2328 and not in those of NZM.C57Lc1 and NZM.C57Lc4. The quantitative data of this study are summarized in the bottom of . At 1/50 dilution, 19.2% (5/26) of NZM.C57Lc1 sera and 4.5% (1/22) of NZM.C57Lc4 sera were positive for ANAs. 12% of the C57L/J sera at 12 mo of age were positive for ANAs. For comparison, 53.3% (24/45) of NZM2328 sera were positive for ANAs. Thus, there were marked reductions of ANAs in both NZM congenics. Similar marked reductions in circulating anti-dsDNA and antinucleosome (histone–DNA complexes) Abs were also detected in these two congenics. It is important to note that the incidences of these three autoantibodies were not significantly different from those in the nonlupus–prone strain C57L/J. In addition, the serum IgG concentrations of C57L/J, NZM2328, and its two congenics were similar.
Figure 4. Marked reduction of circulating anti-dsDNA, antinuclear, and antinucleosome Ab in NZM.C57Lc1 and NZM.C57Lc4 congenic lines in comparison with NZM2328. Staining of HeLa cell nuclei by DAPI are seen in A, C, and E. The right side of the figure shows the (more ...)
RF and Other Autoantibody Activities in Sera of NZM2328 and Its Congenic Lines.
The sera from NZM2328, NZM.-C57Lc1, and NZM.C57Lc4 females were screened for RF. As shown in , there were little anti-IgG Ab activities in the sera of NZM2328 and its congenic lines. The lack of RF in C57L/J was also demonstrated. In contrast, RF activities were readily demonstrated in sera from MRL/lpr+/+females. These results were obtained with either rabbit IgG or human IgG as the substrate and with sera either at 1/50 or 1/250 dilutions.
Figure 5. Lack of RF in sera of NZM2328 and its congenics. Human IgG was used as the substrate in A and C to detect anti-IgG activities. Mouse sera were used at a dilution of 1:50 for A and 1:250 for C. Rabbit IgG was used as the substrate in B and D with mouse (more ...)
The presence of autoantibodies against cytoplasmic constituents in sera of NZM2328 and its congenics was studied by Western blot analysis with WEHI 7.1 lymphoma cell extract as the substrate. Individual serum was used at a 1/50 dilution. By the ages of 4–5 mo, autoantibodies of multiple specificities were readily detected in NZM2328 females ( , top, lanes 16–22), whereas autoantibodies of limited reactivities were found in NZM.C57Lc4 (, top, lanes 9–15). Fewer autoantibodies were detected in the sera of NZM.C57Lc1 (, top, lanes 1–8). By the time the mice developed fatal glomerulonephritis, fewer circulating autoantibodies were detected in NZM2328 (, bottom, lanes 14–20). The sera of NZM.C57Lc4 remained limited in their autoreactivties (, bottom, lanes 7–13). By the age of 12 mo, sera of some of the NZM.C57Lc1 and C57L/J showed autoreactivities, whereas others remained nonautoreactive (, bottom, lanes 1–6 and 21–24). In , a MRL/lpr+/+ serum was used as the positive control to show that both top and bottom panels were developed to a similar extent in chemiluminescence.
Figure 6. Western blot analysis to detect autoantibodies to cellular constituents in sera of NZM2328 and its congenics. Cell lysate of WEHI 7.1 lymphoma cell line was used as the substrate with sera at the dilution of 1:50. On the top, sera from mice at 4–5 (more ...)
In , some of the sera from both congenic lines and NZM2328 reacted with proteins of molecular weights between 30 and 46 kD. Histone H1 with molecular weights in low 30 kD might be a candidate protein for the target Ag as revealed by the Western blot analysis. However, in an ELISA specific for histone H1, no specific anti-H1 Abs were detected in the sera of NZM2328 and its congenics as well as in those of C57L/J (unpublished data).
Lack of Anti-dsDNA Abs in the Eluates from NZM.C57Lc4 Kidneys Afflicted with Glomerulonephritis.
The lack of detectable circulating autoantibodies to dsDNA and other nuclear Ags in NZM.C57Lc4 despite the presence of immune complex–mediated nephritis is not a definitive proof that these Abs do not play a pathological role because these autoantibodies may have been fixed in the kidneys, rendering their absence in the blood. To rule out this possibility, NZM2328, NZM.C57Lc1, NZM.C57Lc4, and C57L/J females 10–12 mo old were killed. At death, sera were collected and their kidneys were frozen in liquid nitrogen and stored for elution. Many of the NZM2328 and NZM.C57Lc4 mice have moderate to severe proteinuria. The kidneys were homogenized and acid eluates were collected and neutralized. NZM2328 and NZM.C57Lc4 kidney eluates had comparable IgG levels, whereas those of NZM.C57Lc1 and C57L/J had significantly lower levels of IgG (unpublished data). Anti-dsDNA Ab activity was expressed as units/microgram of IgG. As shown in , anti-dsDNA Abs were detected in the majority of the NZM2328 sera. There was a concentration of these anti-dsDNA Abs in the NZM2328 kidneys. The enrichment of these Abs in the kidneys was ~20-fold. As expected, few anti-dsDNA Abs were detected in the sera of NZM.C57Lc1, NZM.C57Lc4, and C57L/J. More importantly, the kidney eluates of NZM.C57Lc4 rarely had demonstrable anti-dsDNA Abs. It is of interest to note that there was little correlation of anti-dsDNA Ab concentration in the kidney eluate with serum dsDNA Ab titer in an individual mouse. In addition, the kidney eluates of NZM.C57Lc1, NZM.C57Lc4, and C57L/J did not show antinuclear reactivity on HeLa cells. There was staining for ANA with NZM2328 kidney eluates when they were used without dilution. As with sera, the sera and their paired kidney eluates were also studied regarding RF. The kidney eluates of NZM2328, NZM.C57Lc1, NZM.C57Lc4, and C57L/J did not have detectable RF. These results support our hypothesis that ANA and RF play no significant role in the kidney disease in NZM2328 and its congenic NZM.C57Lc4.
Figure 7. Anti-dsDNA Ab in sera and kidney eluates from NZM2328, NZM.C57Lc1, NZM.C57Lc4, and C57L/J females at 11–12 mo. Abs to dsDNA were present in sera of NZM2328 and they were enriched in their kidney eluates. These Abs were rarely detected in the sera (more ...)
Eluates from NZM.C57Lc4 Diseased Kidneys Identified Several Target Ags.
Kidney and liver from 8-wk-old NZM2328 were perfused with cold PBS. Extracts of them were used as substrates for Western blot analysis. Kidney eluates from NZM2328 and NZM.C57Lc4 were used at either 30 or 10 μg/ml for assaying Ab activity to these extracts. The results are summarized in . Both eluates from NZM2328 and NZM.C57Lc4 kidneys stained several bands in the region between molecular weights of 25 and 40 kD in the kidney extract. The eluate from NZM2328 also had activities against proteins of higher molecular weights. Although the bands were not distinct, proteins of a lower molecular weight of <21 kD were also reactive with the NZM2328 eluate. It appears that the reactive Ags are also present in the liver extract. Similar results were obtained in another experiment.
Figure 8. Abs eluted from nephritic kidneys of NZM2328 and NZM.C57Lc4 mice are reactive with proteins within the kidney and liver extracts. Proteins in the kidney (left) and liver (right) extracts were separated on a 12% SDS-PAGE, transferred to nitrocellulose (more ...)