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Enhanced susceptibility to end-organ disease in the lupus-facilitating NZW mouse strain.

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ARTHRITIS & RHEUMATISM
Vol. 48, No. 4, April 2003, pp 1080–1092
DOI 10.1002/art.10887
© 2003, American College of Rheumatology
Enhanced Susceptibility to End-Organ Disease in
the Lupus-Facilitating NZW Mouse Strain
Chun Xie,1 Xin J. Zhou,2 Xuebin Liu,1 and Chandra Mohan1
kines (monocyte chemoattractant protein 1, RANTES,
KC), and significantly accelerated mortality. Importantly, these changes occurred within a few days after
NTS administration. Finally, (B6 ⴛ NZW)F1 mice were
as susceptible as the NZW parents, which indicates
dominant NZW contributions.
Conclusion. Collectively, these findings support
the notion that a lupus-facilitating genome may contribute to disease susceptibility by modulating the degree of
immune-mediated end-organ damage. The availability
of B6-based congenic strains bearing individual NZWderived lupus susceptibility loci will permit future genetic dissection of end-organ susceptibility in murine
lupus.
Objective. Although the NZW mouse strain is
phenotypically normal, fulminant lupus glomerulonephritis (GN) develops when NZW mice are bred to
several other strains, such as NZB, BXSB, B6.Sle1, and
B6.Yaa. Based on the observation that aging NZW mice
exhibit histologic evidence of GN, we sought to test our
hypothesis that NZW mice may be more susceptible to
immune-mediated renal damage.
Methods. NZW mice, as well as C57BL/6 (B6) and
BALB/c control mice, were challenged with rabbit anti–
glomerular basement membrane nephrotoxic sera
(NTS), to induce renal disease. The different mouse
strains were monitored for the degree of clinical disease,
renal pathology, chemokine profiles, and cellular infiltrates.
Results. Although the NZW and control strains
showed similar glomerular deposits of rabbit Ig and
exhibited similar levels of anti-rabbit xenogeneic immune response, the NZW mice had significantly worse
pathologic changes and disease. Compared with the
control strains, the NTS-injected NZW mice demonstrated significantly increased proteinuria, elevated
blood urea nitrogen levels, more severe histologic GN
and tubulointerstitial nephritis, increased glomerular
crescent formation with macrophage and neutrophil
infiltrates, elevated expression of CC and CXC chemo-
Over the last 4 decades, studies with the New
Zealand strains of mice have contributed significantly to
our understanding of the pathogenesis of lupus. Of
particular interest are the specific genetic and immunologic contributions of the NZW and NZB strains to
disease. The NZW strain is fairly healthy, but contributes to lupus nephritis, in epistasis with several other
strains. When NZW mice are bred to the NZB strain,
either as an F1 hybrid or as a recombinant inbred strain,
this leads to highly penetrant lupus nephritis (1–3).
Likewise, when NZW mice are bred to the BXSB strain,
the F1 offspring exhibit lupus nephritis and coronary
vascular disease (4). Moreover, when NZW mice are
bred to B6 mice, either in the context of the lupus
susceptibility locus Sle1 (5) or the Yaa locus (6), the F1
offspring also exhibit severe proliferative glomerulonephritis (GN) and accelerated mortality. Thus, although
the NZW genome by itself does not lead to the development of clinical nephritis, it clearly has the capacity to
interact with several other genomes to precipitate endorgan disease.
Although NZW mice are clinically healthy, a few
reports have documented subclinical histologic changes
in aging NZW kidneys (7–9). Importantly, these histo-
Supported in part by grants from the NIH (P01-AI-39824),
The Lupus Research Institute, and the National Arthritis Foundation.
Dr. Mohan is the recipient of the Robert Wood Johnson Jr. Arthritis
Investigator Award.
1
Chun Xie, MD, Xuebin Liu, MD, PhD, Chandra Mohan,
MD, PhD: Simmons Arthritis Research Center, University of Texas
Southwestern Medical Center at Dallas; 2Xin J. Zhou, MD: University
of Texas Southwestern Medical Center at Dallas.
Address correspondence and reprint requests to Chandra
Mohan, MD, PhD, Simmons Arthritis Research Center, Department
of Internal Medicine/Rheumatology, University of Texas Southwestern Medical Center, Mail Code 8884, Y8.204, 5323 Harry Hines
Boulevard, Dallas, TX 75390-8884. E-mail: Chandra.mohan@
utsouthwestern.edu.
Submitted for publication July 8, 2002; accepted in revised
form January 3, 2003.
1080
INTRINSIC SUSCEPTIBILITY TO RENAL DISEASE IN NZW MICE
logic changes occur in the absence of any prominent
serologic phenotypes or clinical signs of disease. These
observations raise the possibility that one critical avenue
by which the NZW genome may be contributing to
disease in the (NZB ⫻ NZW)F1, NZM2410, and related
lupus models, is by facilitating immune-mediated endorgan damage. The NZW genome could be effecting this
either by affecting the systemic immune system (e.g., by
promoting the formation of pathogenic antinuclear antibodies) or by facilitating any of the several local renal
processes (e.g., local inflammatory cascades) that lead to
end-organ damage. Certainly, these 2 mechanisms are
not mutually exclusive. One approach to determining
whether the NZW genome does indeed confer increased
susceptibility to end-organ damage is to challenge these
mice with preformed nephrophilic antibodies that are
potentially pathogenic. We aimed to achieve this in the
present study with the use of the nephrotoxic serum
(NTS) nephritis model.
In the nephrotoxic serum nephritis model, also
known as Masugi nephritis (10), preformed rabbit
antiglomerular sera are injected into recipient mice.
Such an abrupt exposure to nephrophilic (or nephrotoxic) antibodies is known to compromise renal function
in a brisk and reproducible manner. Thus, in this model,
one essentially “short-circuits” the chain of pathogenic
events that is otherwise required for the formation of
pathogenic autoantibodies, in order to directly assess
how kidneys of different genotypes might handle the
same immunologic insult. Indeed, this model has been
very useful in demonstrating the essential roles played
by several different molecules in mediating GN. For
instance, studies using this approach have elegantly
dissected out the respective contributions of complement, Fc receptors, cytokines, chemokines, adhesion
molecules, and several other key players in renal disease
(11–19). In the present study, we compared the NZW
strain with 2 normal strains of mice, C57BL/6 (B6) and
BALB/c, with respect to how they handle the same
immunologic insult, in this case, rabbit antiglomerular
nephrotoxic sera.
MATERIALS AND METHODS
Nephrotoxic rabbit sera. NTS was purchased from
Lampire Laboratories (Pipersville, PA). Briefly, renal cortices
of B6 mouse kidneys were minced and then pressed through a
series of sieves of decreasing pore size (250-␮m, 150-␮m, and
75-␮m mesh). The glomeruli were collected on the finest sieve,
washed with cold phosphate buffered saline, and sonicated for
7 minutes. The glomerular sonicates were then used to immunize rabbits (2 mg per rabbit; 3 injections administered 21 days
1081
apart). Sera obtained from these rabbits 50 days following the
primary immunization stained glomeruli strongly by immunofluorescence, and these were the NTS used in the present
study. In addition, preimmune rabbit sera were used as a
negative control (placebo).
Mice and nephrotoxic serum nephritis. B6, BALB/c,
and NZW mice were purchased from The Jackson Laboratory
(Bar Harbor, ME). (B6 ⫻ NZW)F1 mice were bred at our
animal facility. All mice were maintained in a specific
pathogen–free colony. Female mice ages 2–3 months were
used for all studies. Nephrotoxic serum nephritis was induced
as described elsewhere (10), with some modifications.
Briefly, mice were first sensitized on day 0 with an
intraperitoneal injection of rabbit IgG (250 ␮g per mouse) in
adjuvant. On days 4, 5, and 6, the mice received either NTS or
placebo. In both cases, 25 ␮g of total Ig in a 300-␮l volume was
administered intravenously per mouse. The same dosage of
NTS was used for all 3 mouse strains since these strains do not
differ significantly in weight. This dosing regimen was selected
because it was sufficient to induce proteinuria but not mortality in the control strains.
On days 0, 4, 7, 11, 14, 18, and 21, we collected 24-hour
urine samples from all mice with the use of metabolic cages;
mice had free access to drinking water. Urinary protein
concentrations were determined using the Coomassie Plus
protein assay kit (Pierce, Rockford, IL). Sera were collected on
days 0, 4, 7, 14, and 21. Blood urea nitrogen (BUN) levels were
determined using a urea nitrogen kit (Sigma, St. Louis, MO)
according to the manufacturer’s instructions. All surviving
animals were killed on day 21, and the kidneys were harvested
and processed for light microscopy and immunofluorescence
microscopy, as described below.
For the abbreviated 11-day protocol (adopted for the
experiments shown in Figure 9), a modification of the above
approach was used. Briefly, mice were sensitized and challenged with NTS or placebo as described above, but instead of
a 21-day followup, they were killed on day 11 (5 days after the
last injection of NTS). Kidneys were harvested and processed
for histopathologic examination as described below.
Histopathologic examination. Three-micrometer sections of formalin-fixed, paraffin-embedded kidney tissues were
cut and stained with hematoxylin and eosin and with periodic
acid–Schiff. These sections were examined by 2 investigators
(CX and XJZ), in a blinded manner, for any evidence of
pathologic changes in the glomeruli, tubules, or interstitial
areas. The glomeruli were screened for evidence of hypertrophy, proliferative changes, crescent formation, hyaline deposits, fibrosis/sclerosis, and basement membrane thickening. The
severity of GN was graded on a 0–4 scale, where 0 ⫽ normal,
1 ⫽ mild increase in mesangial cellularity and matrix, 2 ⫽
moderate increase in mesangial cellularity and matrix, with
thickening of the glomerular basement membrane (GBM), 3 ⫽
focal endocapillary hypercellularity with obliteration of capillary lumina and a substantial increase in the thickness and
irregularity of the GBM, and 4 ⫽ diffuse endocapillary hypercellularity, segmental necrosis, crescents, and hyalinized endstage glomeruli. Similarly, the severity of tubulointerstitial
nephritis was graded on a 0–4 scale, based on the extent of
tubular atrophy, inflammatory infiltrates, and interstitial fibrosis, as detailed previously (20). The numbers of infiltrating
polymorphonuclear leukocytes were directly enumerated
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XIE ET AL
Table 1. Primer
amplification*
Mouse gene
GAPDH
MCP-1
RANTES
IP-10
KC
sequences
for
polymerase
chain
reaction
Primer sequences
5⬘:
3⬘:
5⬘:
3⬘:
5⬘:
3⬘:
5⬘:
3⬘:
5⬘:
3⬘:
ACC-ACA-GTC-CAT-GCC-ATC-AC
TCC-ACC-ACC-CTG-TTG-CTG-TA
ACT-GAA-GCC-AGC-TCT-CTC-TTC-CTC
TTC-CTT-CTT-GGG-GTC-AGC-ACA-GAC
CCC-TGC-TGC-TTT-GCC-TAC-CTC-TCC
TGG-GTT-GGC-ACA-CAC-TTG-GCG
GGA-TGG-CTG-TCC-TAG-CTC-TG
ATA-ACC-AAT-TGG-GAA-GAT-GG
GGA-TTC-ACC-TCA-AGA-ACA-TCC-AGA-G
CAC-CCT-TCT-ACT-AGC-ACA-GTG-GTT-G
* MCP-1 ⫽ monocyte chemoattractant protein 1; IP-10 ⫽ interferon␥–inducible 10-kd protein.
based on their typical polymorphonuclear morphology, by
examining 50 glomeruli per kidney section per mouse.
Immunohistochemistry and immunofluorescence
studies. For the immunofluorescence studies, 3-␮m–thick cryostat sections of the kidneys obtained from all mice were fixed
for 10 minutes in acetone and air-dried. To detect any deposited rabbit antibodies (from the NTS or preimmune rabbit
sera), the kidney slides were incubated with pretitrated fluorescein isothiocyanate–coupled mouse anti-rabbit Ig and then
quantitated by fluorescence microscopy, as described elsewhere (21).
Briefly, the fluorescence intensity in 10 full-sized glomeruli from each section was measured using the autoexposure feature of the fluorescence microscope imaging system
(Axioskop System; Carl Zeiss Instruments, Dallas, TX). The
duration of exposure, as indicated by the autoexposure system,
was considered to reciprocally reflect staining intensity. Autoexposure units (AU) were calculated by dividing 1,000 by the
autoexposure time (in seconds). On this scale, background
staining uniformly yielded 12–14 AU, whereas maximal fluorescence typically yielded 25–30 AU.
Macrophages were identified and enumerated by serially treating deparaffinized kidney sections with anti–Mac-2
antibody (Cedarlane, Westbury, NY) and rabbit anti-rat Ig
coupled to biotin (Vector, Burlingame, CA), followed by
amplification using a Vectastain ABC kit (Vector), and finally,
with diaminobenzidine (Sigma) substrate. For the negative
control, a monoclonal mouse IgG1 antibody (Bethyl Laboratories, Montgomery, TX) was used at equivalent concentrations.
Analysis of chemokine expression. Total RNA was
isolated from renal cortices using TRIzol reagent (Life Technologies, Grand Island, NY) and quantitated spectrophotometrically. Reverse transcription–polymerase chain reaction
(RT-PCR) was performed using the enhanced avian myeloblastosis virus RT-PCR kit (Sigma), according to the manufacturer’s protocol. The primer sequences used for the RT-PCR
are listed in Table 1. For each pair of primers, PCR was
performed over a range of cycles to define the conditions
under which a linear relationship held between the quantity of
RNA substrate and the final PCR product obtained.
The PCR products were electrophoresed using a 2%
agarose gel and then stained with ethidium bromide. The
density of the bands was ascertained with an AlphaImager
2000 Documentation and Analysis System (Alpha Innotech,
San Leandro, CA), and analyzed using ImageJ software (National Institutes of Health/National Center for Biotechnology
Information, Bethesda, MD; online at: http://rsb.info.nih.gov/
ij/). Results are expressed as relative units (RU), which represent the ratio of the chemokine band intensity to that of
GAPDH assayed in parallel. Urine levels of monocyte chemoattractant protein 1 (MCP-1) and KC were measured using
commercially available enzyme-linked immunosorbent assay
(ELISA) kits (Becton Dickinson, San Jose, CA, and R&D
Systems, Minneapolis, MN, respectively).
ELISA. Mouse antibodies to rabbit Ig were assayed by
ELISA. Briefly, purified rabbit Ig (Sigma) was coated onto
Immulon I plates (Dynatech, Chantilly, CA) and then blocked.
Serially diluted mouse sera were added to these plates, and any
bound anti-rabbit Ig was detected using alkaline phosphatase–
conjugated goat anti-mouse IgG (that was not cross-reactive
with rabbit Ig; Roche, Indianapolis, IN), and paranitrophenyl
phosphate substrate (Sigma). For the measurement of isotypespecific antibodies, enzyme-conjugated anti-mouse IgG1
(PharMingen, San Diego, CA), IgG2a (Zymed, Burlingame,
CA), or IgG2b (PharMingen) was used instead.
All reagents were tested using serum from uninjected
(B6 ⫻ NZW)F1 mice to ensure that the second antibodies
detected both the B6 and NZW Ig allotypes equally well. To
facilitate interstrain comparisons, sera from the different mice
were all assayed within the same ELISA plate. Mouse IgG or
isotype standards were also included on each plate for standardization. Results are expressed as micrograms per milliliter,
based on the standard curves generated.
Statistical analysis. Intergroup comparisons were performed using Student’s t-test, unless indicated otherwise. Results are expressed as the mean ⫾ SEM. All statistical analyses
were performed using SigmaStat (Jandel Scientific, San
Rafael, CA).
RESULTS
Since the goal was to ascertain if NZW mice
exhibited any increased sensitivity to potentially pathogenic (i.e., nephritogenic) autoantibodies, batches of
NZW mice as well as B6 and BALB/c controls were
challenged with rabbit NTS or preimmune sera as
detailed in Materials and Methods. In order to first
establish that kidneys from both strains were exposed to
similar amounts of rabbit NTS, they were examined for
the extent of rabbit Ig deposition in their kidneys. As
indicated in Figures 1A and C, the NZW kidneys and the
kidneys of the control mice exhibited equivalent
amounts of rabbit Ig deposits following exposure to
NTS, as quantitated by fluorescence autoexposure measurements. Intense linear staining of IgG along the
glomerular capillary walls (but not the tubules) was
noted in both the NZW and control kidneys. The
controls that received placebo (i.e., preimmune rabbit
sera) did not exhibit rabbit Ig deposits in their kidneys
INTRINSIC SUSCEPTIBILITY TO RENAL DISEASE IN NZW MICE
Figure 1. Rabbit Ig deposits in NZW and control mouse kidneys. The
presence and specific location of rabbit Ig deposits in A, a nephrotoxic
rabbit sera (NTS)–injected NZW mouse, B, a preimmune rabbit sera
(placebo)–injected NZW mouse, C, an NTS-injected C57BL/6 (B6)
control mouse, and D, a placebo-injected B6 control mouse were
determined by immunofluorescence analysis using fluorescein
isothiocyanate–conjugated anti-rabbit Ig. Images are representative of
kidney sections obtained from 4–6 mice from each of the 4 experimental groups. The immunofluorescence autoexposure times for the
kidneys from the NTS-injected NZW mice (mean ⫾ SEM 47.4 ⫾ 1.6
seconds) did not differ significantly from the corresponding values for
the BALB/c (48.9 ⫾ 2.0 seconds) or the C57BL/6 (46.3 ⫾ 1.7 seconds)
mice.
(Figures 1B and D). In addition, the 3 strains did not
differ in the extent of complement deposition in their
kidneys (data not shown).
Despite receiving the identical immunologic insult and despite equivalent amounts of rabbit Ig deposited in their glomeruli, the NZW strain differed significantly from the normal control strains in responding to
this challenge. First and foremost, the NZW strain
exhibited accelerated mortality, as illustrated in Figure
2. Although all the B6 and BALB/c mice injected with
NTS and all the placebo-injected mice lived for at least
21 days postinjection, NZW mice injected with NTS had
a cumulative mortality rate of 40% by day 21, which was
significantly higher than that in the other NTS-injected
groups (P ⬍ 0.035, by Fisher’s exact test).
1083
To ascertain the likely cause of the increased
mortality, we next examined these mice for evidence of
compromised renal function. In our colony, the control
strains rarely exhibit 24-hour urinary protein excretion
levels in excess of 1 mg (22). With the dose of NTS used,
BALB/c mice did not develop any significant proteinuria
until day 21 (Figure 3A). B6 mice injected with NTS
began excreting significantly more protein from day 18
onward (mean ⫾ SEM 2.38 ⫾ 0.54 mg/24 hours on day
18) compared with the placebo-injected B6 mice (0.77 ⫾
0.26 mg/24 hours on day 18) (P ⬍ 0.027).
The findings were very different in the NZW
strain (Figures 3A and B). NTS-injected NZW mice
exhibited a dramatic (⬃10-fold) increase in urinary
protein excretion between day 7 and day 11. Since NTS
was injected on days 4, 5, and 6, this rapid deterioration
in renal function in the NZW mice was clearly occurring
precipitously, within a couple of days following NTS
administration. At the conclusion of the experiment,
among the mice surviving on day 21, the NTS-injected
NZW mice still excreted significantly more urinary
protein (mean ⫾ SEM 14.93 ⫾ 2.59 mg/24 hours) than
did the placebo-injected NZW mice (1.02 ⫾ 0.13 mg/24
hours) (P ⬍ 0.002), the NTS-injected B6 mice (2.17 ⫾
Figure 2. Accelerated mortality in nephrotoxic rabbit sera (NTS)–
injected NZW mice. NTS-injected NZW mice (n ⫽ 10) and control
mice (n ⫽ 5 preimmune rabbit sera [placebo]–injected NZW mice, 6
NTS-injected C57BL/6 [B6] mice, 8 placebo-injected B6 mice, 5
NTS-injected BALB/c mice, and 5 placebo-injected BALB/c mice)
were monitored for survival (y-axis) over a period of 21 days after
initiation of experiments (x-axis), as detailed in Materials and Methods. The 5 groups of mice that exhibited no mortality are represented
as a single group. The NTS-injected NZW mice had significantly
higher mortality rates compared with the NTS-injected B6 and
BALB/c controls (P ⬍ 0.035, by Fisher’s exact test).
1084
XIE ET AL
0.79 mg/24 hours) (P ⬍ 0.002), and the NTS-injected
BALB/c mice (1.3 ⫾ 0.8 mg/24 hours) (P ⬍ 0.006). Since
the excreted protein in NZW mice was predominantly of
high molecular weight (as determined by sodium dode-
Figure 3. Increased proteinuria in nephrotoxic rabbit sera (NTS)–
injected NZW mice. A, NTS-injected NZW (n ⫽ 10), preimmune
rabbit sera (placebo)–injected NZW (n ⫽ 5), NTS-injected C57BL/6
(B6) (n ⫽ 6), placebo-injected B6 (n ⫽ 8), NTS-injected BALB/c (n ⫽
5), and placebo-injected BALB/c (n ⫽ 5) mice were monitored for
24-hour urinary protein excretion (y-axis) over a period of 21 days
after initiation of experiments (x-axis), as detailed in Materials and
Methods. The NTS-injected NZW mice exhibited significantly elevated proteinuria compared with that in all other groups of mice.
P values are for the differences between the NTS-injected NZW
mice and the NTS-injected control mice (B6 and BALB/c). The
numbers of dead NTS-injected NZW mice shown in parentheses
are not represented at the respective time points. Values are the
mean ⫾ SEM. B, Levels of 24-hour proteinuria in the 10 NZW mice
injected with NTS, as measured at 3–4-day intervals. Broken line
indicates the cutoff for determining positivity, which was set at 2 SD
above the mean levels observed in placebo-injected NZW mice.
Each solid line represents a single NZW mouse; ⴛ indicates death.
Statistical comparison of these data with those in the controls is
summarized in A.
Figure 4. Elevated blood urea nitrogen (BUN) levels in nephrotoxic
rabbit sera (NTS)–injected NZW mice. A, NTS-injected NZW (n ⫽
10), preimmune rabbit sera (placebo)–injected NZW (n ⫽ 5), NTSinjected C57BL/6 (B6) (n ⫽ 6), placebo-injected B6 (n ⫽ 8), NTSinjected BALB/c (n ⫽ 5), and placebo-injected BALB/c (n ⫽ 5) mice
were monitored for BUN levels (y-axis) over a period of 21 days after
initiation of experiments (x-axis), as detailed in Materials and Methods. The NTS-injected NZW mice exhibited significantly elevated
BUN levels compared with those in all other groups of mice. P values
are for the differences between the NTS-injected NZW mice and the
NTS-injected control mice (B6 and BALB/c). The numbers of dead
NTS-injected NZW mice shown in parentheses are not represented at
the respective time points. Values are the mean ⫾ SEM. B, BUN levels
in the 10 NZW mice injected with NTS, as measured at regular
intervals. Broken line indicates the cutoff for determining positivity,
which was set at 2 SD above the mean levels observed in placeboinjected NZW mice. Each solid line represents a single NZW mouse;
ⴛ indicates death. Statistical comparison of these data with those in
the controls is summarized in A.
cyl sulfate–polyacrylamide gel electrophoresis; data not
shown), this most likely reflects glomerular proteinuria.
Whereas increased urinary protein is a relatively
early indication of impaired renal function, elevated
INTRINSIC SUSCEPTIBILITY TO RENAL DISEASE IN NZW MICE
1085
Figure 5. Severe glomerulonephritis (GN) in nephrotoxic rabbit sera (NTS)–injected NZW mice, as
determined histologically. Shown are periodic acid–Schiff–stained, formalin-fixed, paraffin-embedded
renal sections from a, an NTS-injected NZW mouse, b, a preimmune rabbit sera (placebo)–injected NZW
mouse, c, an NTS-injected control C57BL/6 (B6) mouse, and d, a placebo-injected B6 mouse. Shown in
a are some of the typical light microscopic features seen in NTS-injected NZW mice, including
intracapillary hypercellularity, with obliteration of capillary lumina, and tubular dilatation with casts.
Shown in b is mild mesangial proliferative GN typical of the NTS-injected control mice. Both the
placebo-injected NZW and control mice showed essentially normal renal histologic findings, as indicated
in b and d, respectively. Images are representative of sections from at least 4 mice in each study group. The
results are quantitatively summarized in Table 2. (Original magnification ⫻ 400.)
BUN levels appear later, as a consequence of impaired
glomerular clearance, often preceding death. As illustrated in Figure 4, elevated BUN levels were also
prominent in the NTS-injected NZW mice. Thus, on day
14, the NTS-injected NZW mice exhibited significantly
increased BUN levels (mean ⫾ SEM 83.56 ⫾ 23.60
mg/dl), in stark contrast to the placebo-injected NZW
(19.24 ⫾ 2.71 mg/dl) (P ⬍ 0.0003) and the NTS-injected
B6 and BALB/c mice (P ⬍ 0.036, by Mann-Whitney rank
sum test). Although the BUN levels dipped beyond this
time point, the differences remained until the conclusion
of the experiment on day 21 (Figures 4A and B), when
the NTS-injected NZW mice had a mean BUN level of
44.37 ⫾ 3.0 mg/dl, compared with 19.24 ⫾ 2.71 mg/dl in
the placebo-injected NZW mice.
Thus, the 3 strains clearly differed in their clinical
response to the administered NTS. Whereas the BALB/c
strain experienced hardly any proteinuria or mortality,
the B6 strain developed mild proteinuria toward the
conclusion of the experiment. In contrast, the NZW
strain clearly exhibited significantly worse clinical nephritis and mortality rates in response to the same
immunologic insult.
To gain a better understanding of the underlying
renal disease, we next examined the kidneys of these
mice. The kidneys of the NTS-injected NZW mice
exhibited more severe histologic changes compared with
the kidneys of the NTS-injected controls (Figure 5).
Following administration of NTS, the kidneys from the
NZW mice exhibited significantly more severe GN
(characterized by focal to diffuse intracapillary hypercellularity with obliterated capillary lumina and thickened capillary walls), tubulointerstitial nephritis, and
increased glomerular crescent formation, with more
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XIE ET AL
Table 2. Features of nephritis in 3 mouse strains on day 21 after injection of nephrotoxic rabbit sera (NTS) or preimmune rabbit sera (placebo)*
C57BL/6 mice
Glomerulonephritis grade
Tubulointerstitial nephritis
grade
% of 100 glomeruli with
crescents
No. of macrophage
infiltrates
No. of polymorphonuclear
leukocytes
BALB/c mice
NZW mice
Placebo
(n ⫽ 8)
NTS
(n ⫽ 6)
Placebo
(n ⫽ 4)
NTS
(n ⫽ 4)
Placebo
(n ⫽ 5)
NTS
(n ⫽ 6)
0⫾0
0⫾0
1.6 ⫾ 0.2†
0.3 ⫾ 0.2†
0.1 ⫾ 0.1
0⫾0
0.6 ⫾ 0.1‡
0.3 ⫾ 0.1†
0.4 ⫾ 0.2
0.2 ⫾ 0.2
2.3 ⫾ 0.4
1.6 ⫾ 0.4
0⫾0
1.5 ⫾ 0.5§
0⫾0
0 ⫾ 0§
0⫾0
9.2 ⫾ 3.6
20.5 ⫾ 4.7
31.2 ⫾ 4.0§
5.5 ⫾ 1.8
15.7 ⫾ 4.2‡
9.8 ⫾ 0.8
64.4 ⫾ 13.5
11.5 ⫾ 1.0
14.3 ⫾ 1.3‡
13.7 ⫾ 1.2
13.5 ⫾ 1.0‡
16.0 ⫾ 1.4
31.3 ⫾ 2.3
* Glomerulonephritis and tubulointerstitial nephritis were graded on a 0–4 scale (see Materials and Methods). Macrophages were enumerated per
100 glomeruli, after immunohistologic staining. Polymorphonuclear leukocytes were enumerated per 50 glomeruli. Values are the mean ⫾ SEM.
† P ⬍ 0.01, versus NZW kidneys, by Student’s t-test.
‡ P ⬍ 0.001, versus NZW kidneys, by Student’s t-test.
§ P ⬍ 0.05, versus NZW kidneys, by Student’s t-test.
prominent polymorphonuclear leukocyte and macrophage infiltrates compared with the control kidneys
from the B6 and BALB/c mice (Table 2).
Given the prominence of macrophage and neutrophil infiltrates in the NTS-challenged NZW kidneys,
it then became important to determine whether these
differences were driven by coordinate differences in
chemokine profiles. Among the extensive array of chemokines expressed in kidneys, MCP-1, RANTES,
interferon-␥–inducible 10-kd protein (IP-10), and KC
represent key mediators implicated by other investigators as playing a role in end-organ pathology. Therefore,
we next ascertained the expression of these selected
chemokines in NZW kidneys, using B6 kidneys as controls.
Interestingly, the placebo-injected NZW kidneys
(without nephrotoxic immunologic insult) exhibited increased levels of MCP-1 (mean ⫾ SEM 0.40 ⫾ 0.04 RU
versus 0.22 ⫾ 0.05 RU; P ⬍ 0.015), IP-10 (0.90 ⫾ 0.04
RU versus 0.67 ⫾ 0.11 RU; P ⬍ 0.07), and KC (0.92 ⫾
0.11 RU versus 0.41 ⫾ 0.13 RU; P ⬍ 0.01) compared
with the kidneys of the placebo-injected B6 mice (Figure
6). However, the level of RANTES expression was
similar in both of these groups of placebo-injected mice.
NTS administration boosted the levels of most chemokines in the kidneys of both strains. Notably, the kidneys
from the NTS-injected NZW mice exhibited significantly
higher levels of message for RANTES (0.69 ⫾ 0.12 RU
versus 0.37 ⫾ 0.005 RU; P ⬍ 0.028) and KC (1.68 ⫾ 0.10
RU versus 1.04 ⫾ 0.10 RU; P ⬍ 0.001) compared with
the kidneys from NTS-injected B6 mice (Figure 6).
Although an elevation was also noted in the message
levels of MCP-1, no increases were seen in the levels of
Figure 6. Elevated chemokine expression in nephrotoxic rabbit sera
(NTS)–injected NZW mice. Levels of expression of monocyte chemoattractant protein 1 (MCP-1), RANTES, KC, and interferon-␥–
inducible 10-kd protein (IP-10) in the renal cortices of NTS-injected or
preimmune rabbit sera (placebo)–injected NZW and C57BL/6 (B6)
control mice were semiquantitated by reverse transcription–
polymerase chain reaction, as detailed in Materials and Methods. All
kidneys were obtained on day 21, at the conclusion of the experiment,
with 1 exception (the NTS-injected NZW group includes 6 kidneys
harvested on day 21 and 1 kidney rescued from the mouse that died on
day 18). Bars show the mean for each group. Chemokine expression
levels are expressed as relative units, representing the ratio of chemokine band intensity to that of a housekeeping gene, GAPDH. P values
shown at the bottom right of each plot are for the differences between
the NTS-injected NZW mice and the placebo-injected NZW mice (top
value) as well as the NTS-injected B6 mice (bottom value).
INTRINSIC SUSCEPTIBILITY TO RENAL DISEASE IN NZW MICE
Figure 7. Increased urinary excretion of monocyte chemoattractant protein 1 (MCP-1) and
KC over 24 hours in nephrotoxic rabbit sera (NTS)–injected NZW mice, as measured by
enzyme-linked immunosorbent assay on day 21 after NTS injection. Bars show the mean for
each group. P values are for the differences between the NTS-injected NZW mice and the
NTS-injected controls, by Student’s t-test. B6 ⫽ C57BL/6.
Figure 8. Anti-rabbit Ig response in the nephrotoxic rabbit sera (NTS)–injected NZW and
C57BL/6 (B6) control mice. Serum levels of A, IgG1, B, IgG2a, and C, IgG2b
mouse anti-rabbit Ig antibodies (Abs) were quantitated in the NTS-injected NZW mice
(n ⫽ 6) and the NTS-injected control mice on day 21, at the conclusion of the
experiment. D, Serum levels of (total) IgG mouse anti-rabbit Ig and 24-hour urinary
protein excretion in the NTS-injected NZW mice on day 21. The correlation coefficient
for these 2 parameters is indicated by the r value. E, Levels of mouse IgG deposited
in the glomeruli of NTS-challenged mice, expressed in autofluorescence units (AU).
For each kidney section, autofluorescence of 10 glomeruli was quantitated and averaged.
No significant differences were observed between the groups. Broken line indicates
the mean autofluorescence observed in preimmune rabbit sera (placebo)–injected
control mice.
1087
1088
IP-10 (Figure 6) in these 2 groups. These message
differences were paralleled by increased urinary excretion of these chemokines, as depicted for MCP-1 and
KC in Figure 7.
Collectively, the above data indicated that the
NZW mice had worse renal disease than the B6 and
BALB/c controls despite having received the identical
immunologic insult. To exclude the possibility that the
exaggerated disease response seen in the NZW strain
might be the consequence of a stronger systemic immune response to the injected rabbit Ig, we compared
the serum levels of mouse anti-rabbit antibodies in the
NZW mice with those in the B6 controls.
As shown in Figures 8A–C, the NTS-injected
NZW mice did not exhibit a higher anti-rabbit xenogeneic response than that in the controls. Importantly, the
isotype distribution of mouse antibodies to rabbit Ig did
not differ significantly among the 3 groups. Moreover,
when the degree of anti-rabbit immune response in the
NTS-injected mice and the extent of proteinuria at the
same time points were examined in parallel, the correlation between these 2 parameters was poor (Pearson’s
product-moment coefficient r ⫽ –0.51) (Figure 8D). In
addition, the NTS-injected NZW and control mice did
not differ in the extent of mouse IgG deposits in their
kidneys, as quantitated by autofluorescence (Figure 8E).
These observations indicate that the accentuated renal
disease seen in NZW mice is not simply the result of an
exaggerated or altered anti-rabbit (xenogeneic) immune
response against the administered rabbit NTS.
Most of the above data were derived from the
sera, urine, and tissues procured on day 21 of experimentation. Since several of the phenotypes appeared to
dip toward the conclusion of the experiment (Figures 3
and 4), we were concerned that we may not be studying
the pathologic changes in these mice at the peak of their
disease. Hence, we repeated the above studies using an
abbreviated 11-day protocol, where NZW mice and B6
controls were challenged with NTS as before, but were
then killed on day 11 for examination. As illustrated in
Figure 9, and as predicted, the clinical parameters (as
measured by proteinuria and BUN) and the histologic
features (as marked by the GN scores) were even more
pronounced in NZW mice at this earlier time point.
Ongoing studies are focused on evaluating groups of
mice at daily intervals following NTS administration to
elucidate the detailed kinetics of GN onset in these
strains.
Thus, both the conventional and abbreviated
experimental GN induction protocols yielded similar
XIE ET AL
Figure 9. Features of anti–glomerular basement membrane disease at
the peak of pathologic changes. To confirm our findings in the other
studies and to ascertain the degree of pathologic changes at the peak
of the disease, C57BL/6 (B6) and NZW mice were challenged with
nephrotoxic rabbit sera (NTS) or preimmune rabbit sera (placebo)
(n ⫽ 4–7 per group) and killed on day 11, as detailed in Materials and
Methods. Shown are the levels of A, proteinuria, B, blood urea
nitrogen (BUN), and C, glomerulonephritis (GN) scores on day 11.
Although no premature deaths were observed in the NTS-injected B6
mice or the placebo-injected controls, 2 of the 7 NZW NTS-injected
mice died before day 11. Values are the mean and SEM. ⴱ ⫽ P ⬍ 0.05
and ⴱⴱ ⫽ P ⬍ 0.01 versus B6 mice, by Student’s t-test.
INTRINSIC SUSCEPTIBILITY TO RENAL DISEASE IN NZW MICE
Figure 10. Nephrotoxic rabbit sera (NTS)–induced anti–glomerular
basement membrane disease in (B6 ⫻ NZW)F1 mice. To determine if
the enhanced susceptibility to renal disease in the NZW strain was a
dominant or recessive phenotype, (B6 ⫻ NZW)F1 hybrids were bred
and subjected to NTS challenge, using the 21-day protocol. Solid lines
show the urinary protein levels observed in the “negative control”
(C57BL/6 [B6]) and “positive control” (NZW) strains (see Figure 3),
as indicated. Each data point represents an individual F1 mouse. On
days 11, 14, 18, and 21, the mean levels of 24-hour proteinuria in the
F1 mice were significantly higher than those in the B6 controls (P ⬍
0.01). Placebo-injected F1 mice did not develop significant proteinuria
(data not shown).
conclusions: NZW mice were significantly more prone to
renal disease following an immunologic insult than were
B6 and BALB/c controls. As a first step in exploring the
genetic basis of this difference, we next bred (B6 ⫻
NZW)F1 hybrids, and challenged them with NTS and
placebo. As shown in Figure 10, (B6 ⫻ NZW)F1 mice
were clearly as susceptible to renal disease as the NZW
parents were, indicating the contribution of one or more
dominant NZW disease genes toward this phenotype. It
would clearly be important to elucidate the identity of
these culprit genes in future studies.
DISCUSSION
The phenotypically normal NZW strain was first
described by W. H. Hall at the Otago Medical School,
Dunedin, New Zealand (1). Soon after, it was quickly
noted that when this strain was bred to the NZB strain,
the F1 offspring succumbed to lupus nephritis, with
features that were strikingly similar to those seen in
humans with systemic lupus erythematosus (2,3,23).
Indeed, over the last 3 decades, studies with this hybrid
strain and its derivatives have broadened our under-
1089
standing of the pathogenic mechanisms leading to lupus
(3,24,25). Subsequent studies revealed that although the
NZW strain was phenotypically normal, with aging the
strain exhibited histologic changes suggestive of GN
(7–9). However, the relative contributions of the systemic immune components versus the intrinsic renal
factors to these observed pathologic changes have remained unexplored.
In this context, the nephrotoxic serum nephritis
(or, Masugi nephritis) model has been very useful in
discriminating “systemic” factors from intrinsic renal
factors in facilitating renal disease. Indeed, this very
approach has been instrumental in demonstrating the
respective roles of several different molecules in mediating end-organ pathology (11–19). If the NZW mice did
not really differ from the B6 or BALB/c controls with
respect to how they handled and responded to immunologic insults, then one would have expected the NZW
and the control strains to have responded similarly when
challenged with NTS (which clearly target the glomeruli
of these strains in a similar manner, as demonstrated in
Figure 1). This was not the case, however, since the
NTS-injected NZW mice experienced greater renal pathology and higher mortality compared with the NTSinjected control strains.
Indeed, the deterioration in renal function was
noted to occur within a couple of days following NTS
administration. As detailed in Materials and Methods,
NTS administration was completed on day 6. On day 7,
the mice in the different groups did not differ significantly from each other in terms of urinary protein
excretion or BUN level. However, at the next time point
of study 4 days later (day 11), the NTS-injected NZW
mice differed sharply from the NTS-injected controls,
both in the 21-day and the 11-day experiments: they
exhibited significantly worse pathologic changes (Figure
9C), elevated BUN levels (Figure 9B), and higher proteinuria (Figures 3 and 9A). This was accompanied by
death in several of these mice (Figure 2 and legend to
Figure 9). Thus, the immunopathologic pathways triggered by the administration of NTS appear to act very
rapidly, within a couple of days, in a manner sufficient to
induce death.
We believe that the dominant cause of death in
these mice is compromised renal function, for several
reasons. First, most mice that died had sharply diminished urine output but high urine protein concentration,
indicative of acute renal shutdown (Figure 3B). Second,
several histologic features of disease were observed in
the NZW mice that may potentially account for the
1090
worse clinical outcome. This includes the more proliferative nature of the GN (GN scores of 3 or 4) accompanied by the significantly increased numbers of glomerular crescents, neutrophils, and macrophages (Table 2
and Figures 5 and 9). It was also interesting to note that
these mice exhibited tubulointerstitial disease despite
having no Ig deposits on their tubular basement membranes. It remains to be established whether the observed tubulointerstitial disease in the NTS-injected
NZW mice was a secondary consequence of the massive
proteinuria seen in these mice. Finally, no evidence of
disease elsewhere was apparent by morphologic observation or histologic examination of the lungs from these
mice. Thus far, lung involvement in anti-GBM disease
has not been a prominent feature in animal models since
additional pulmonary insults (such as cigarette smoking)
appear to be required (to damage the alveolar endothelial lining) before this phenotype can ensue, as is the
case even in patients with Goodpasture’s disease (26).
Both CC chemokines that chemoattract monocytes and CXC chemokines that chemoattract neutrophils have been implicated by other investigators to play
major roles in spontaneously arising, as well as experimentally induced, models of immune-mediated nephritis
(14,15,27–38). In this study, we therefore ascertained the
expression of 2 CC chemokines (MCP-1 and RANTES)
and 2 CXC chemokines (KC and IP-10). The importance of MCP-1 and RANTES in immune-mediated
nephritis has been demonstrated by several end-organ–
expression studies, as well as by antibody-blocking and
knockout studies (14,33,34,36). KC serves as an important chemoattractant for neutrophils and mesangial
cells, and it also functions to amplify the production of
other chemokines in the end organs (29). IP-10 production is also elevated in experimentally induced nephritis,
and this CXC chemokine plays an important role in
recruiting neutrophils (38).
It is interesting to note that the placebo-injected
NZW mice in the present study exhibited elevated basal
levels of MCP-1, IP-10, and KC compared with the levels
in the B6 controls. This might suggest that the NZW
kidneys exist in a somewhat “triggered” state, even
before they are challenged with nephrotoxic antibodies.
The genetic basis for this increased basal state in the
NZW kidneys remains unexplored. More interestingly,
the NZW kidneys exhibited a further increases in
RANTES and KC levels, compared with those in B6
kidneys, upon NTS administration (Figure 6); this indicates the potential relevance of these chemokines in
inducing the more severe disease seen in these mice. In
contrast, the difference in IP-10 expression between the
XIE ET AL
B6 and the NZW strains did not appear to correlate with
the respective disease outcomes. It is likely, however,
that the chemokine differences we found may represent
only a minor fraction of the molecular differences
between the end organs of these different strains. The
impending challenge is to identify any additional molecular differences that may account for the increased
end-organ disease susceptibility exhibited by the NZW
strain.
One possible explanation for the more severe
disease in the NZW strain may be an exaggerated
systemic immune response to the administered rabbit Ig
as compared with the response of the control strains.
There are several reasons why this is unlikely. First,
previous studies with the nephrotoxic serum–induced
nephritis model have indicated that the host’s humoral
immune response does not contribute significantly to the
observed pathologic features, since host mice devoid of
B cells developed a similar disease (39). Second, the
NZW strain is not known to have heightened immune
responsiveness (40). Third, the NZW mice receiving
preimmune rabbit sera did not develop any pathologic
changes or disease, suggesting that the observed endorgan pathologic changes were absolutely dependent
upon the presence of a nephrotoxic specificity (i.e., NTS
is absolutely required). Fourth, elevated proteinuria was
noted in the NZW mice within a couple of days following the administration of NTS. This makes it unlikely for
an immune response to the rabbit NTS to have been the
cause of the differential pathologic changes observed.
Finally, even when the mouse anti-rabbit Ig (xenogeneic) response following the NTS administration was measured, the NZW mice and the controls both exhibited a
xenogeneic immune response that was quantitatively
and qualitatively similar (Figure 8). It also appeared
unlikely that the observed differences were due to
Th1/Th2 differences between the strains. Both the B6
strain (which is Th1-skewed) and the BALB/c strain
(which is Th2-skewed) developed minimal disease compared with the NZW strain. Furthermore, we could not
discern any differences in the serum levels of IgG1 and
IgG2a mouse anti-rabbit antibody in these different
strains (Figures 8A–C).
Given the above observations, we believe that the
increased disease seen in the NZW strain is most likely
a direct consequence of local pathogenic cascades triggered by the administered NTS in the kidneys. However,
it should be stressed that the present findings do not
conclusively establish whether the NZW genome encodes resident glomerular cells that are intrinsically
abnormal or whether the observed phenotypes reflect
INTRINSIC SUSCEPTIBILITY TO RENAL DISEASE IN NZW MICE
intrinsic abnormalities in the infiltrating leukocytes.
Thus, with respect to one of the phenotypes observed in
NZW mice, increased chemokine expression, for example, 2 non–mutually exclusive scenarios are possible.
First, it is possible that the NZW genome may be
affecting chemokine elaboration by intrinsic glomerular
cells. Alternatively, the NZW genome may be dictating
the responsiveness of infiltrating leukocytes to these
mediators. Renal transplant–based approaches as well
as studies with NZW-derived glomerular cell lines are in
progress to distinguish between these possibilities.
The results of these studies raise the possibility
that a lupus-facilitating genome may contribute to disease susceptibility by encoding end organs that are
potentially more “sensitive” to immune-mediated damage. Indeed, it is becoming increasingly apparent that
enhanced intrinsic susceptibility to other varieties of
end-organ disease may also be genetically determined
(41,42). Given the observation that the NZW genome
confers increased susceptibility to immune-mediated
GN in a dominant manner, it next becomes important to
ascertain which loci/genes within the NZW genome
might actually be responsible for the observed differences. Indeed, the NZW genome is notoriously riddled
with several lupus susceptibility loci, including Sle1,
(proximal part of) Sle2, Sle3, Sle5, and Sle6, many of
which are linked to GN susceptibility and function in a
dose-dependent manner (5,43). Studies of B6 mice
rendered congenic for these different NZW-derived
intervals have recently revealed how several of these loci
have the potential to affect systemic immunity, either by
themselves or in epistasis with other loci (44–51). It
remains to be determined whether any of these loci
might serve to directly modulate susceptibility to endorgan disease. Dissecting out the genetic origins and
molecular mediators of acute experimental nephritis, as
well as spontaneous lupus nephritis, using these novel
NZW-derived genetic models is the challenge that lies
ahead.
ACKNOWLEDGMENTS
The authors would like to acknowledge Drs. Christopher Lu and Michael P. Madaio for critical reading of the
manuscript.
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facilitation, nzw, lupus, end, strait, mouse, enhance, disease, susceptibility, organy
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