ARTHRITIS & RHEUMATISM
Vol. 56, No. 5, May 2007, pp 1569–1578
© 2007, American College of Rheumatology
Involvement of Renal Tubular Toll-like Receptor 9 in the
Development of Tubulointerstitial Injury in Systemic Lupus
Ariela Benigni,1Cristina Caroli,1Lorena Longaretti,1Elena Gagliardini,1Carla Zoja,1
Miriam Galbusera,1Daniela Moioli,1Paola Romagnani,2Angela Tincani,3
Laura Andreoli,3and Giuseppe Remuzzi4
Objective. Toll-like receptor 9 (TLR-9), a receptor
for CpG DNA, has been implicated in the activation of
immune cells in lupus. We undertook this study to
determine whether the expression of TLR-9 in resident
renal cells in lupus nephritis is related to the develop-
ment of tubulointerstitial injury.
Methods. TLR-9 was analyzed in selectively re-
trieved renal tissue from (NZB ? NZW)F1mice at
different stages of disease by laser capture microdissec-
tion combined with real-time quantitative reverse
transcriptase–polymerase chain reaction, and in renal
biopsy specimens from lupus nephritis patients by im-
munohistochemistry. We investigated for the molecular
component responsible for TLR-9 activation by cultured
proximal tubular cells in serum from patients with
Results. Renal tissue from NZB ? NZW mice
displayed robust TLR-9 expression localized to proxi-
mal tubular cells. TLR-9 levels correlated with protein-
uria and tubulointerstitial injury to the extent that a
cyclin-dependent kinase inhibitor, while reducing pro-
teinuria and renal structural damage, prevented tubu-
lar TLR-9 generation in lupus mice. Consistently, exag-
gerated TLR-9 staining was found in proximal tubular
cells of lupus patients, which correlated with tubulo-
interstitial damage. DNA-containing immune complexes
purified from sera of patients with lupus induced TLR-9
in cultured proximal tubular cells. This was prevented
by CCGG-rich short oligonucleotides, specific antago-
nists of CpG DNA, indicating that the DNA component
of immune complexes was required for TLR-9 stimula-
Conclusion. These findings suggest that tubular
TLR-9 activation has a pathogenetic role in tubulo-
interstitial inflammation and damage in experimental
and human lupus nephritis, and they indicate a novel
target for future therapies.
Lupus nephritis is a severe organ manifestation
of systemic lupus erythematosus (SLE) (1) and may
influence morbidity and mortality. In SLE, abnormali-
ties of immune regulation lead to loss of self tolerance,
which in turn triggers autoimmune responses. Those
include T and B cell dysfunction and activation of
autoreactive B cells that produce antibodies against
nuclear antigens. Immune complexes are detected in the
circulation, and their deposition, together with comple-
ment activation in glomeruli and along the tubular
basement membrane, plays a major role in the pleomor-
phic histopathology of lupus nephropathy. The initiating
event may be local binding of nuclear or other antigens
to glomerular and tubular sites followed by in situ
immune complex formation. Antigens contain nucleic
acids, and antinuclear antibody (ANA) formation occurs
as a consequence of stimulation of invariant receptors
that recognize nucleic acid determinants (2).
Toll-like receptors (TLRs) are a family of trans-
membrane proteins that recognize conserved molecular
patterns shared by a wide variety of microorganisms
Dr. Caroli is recipient of a fellowship from Fondazione
ARMR (Aiuti per la Ricerca sulle Malattie Rare), Bergamo, Italy, in
memory of Dr. Fausto Radici. Dr. Moioli is recipient of a fellowship
from Fondazione Cariplo, Milan, Italy.
1Ariela Benigni, PhD, Cristina Caroli, ChemPharmD, Lorena
Longaretti, BiolSciD, Elena Gagliardini, PhD, Carla Zoja, PhD,
Miriam Galbusera, BiolSciD, Daniela Moioli, BiolSciD: Mario Negri
Institute for Pharmacological Research, Bergamo, Italy;2Paola Ro-
magnani, MD, PhD: University of Florence, Florence, Italy;3Angela
Tincani, MD, Laura Andreoli, MD: Brescia Hospital and University,
Brescia, Italy;4Giuseppe Remuzzi, MD, FRCP: Mario Negri Institute
for Pharmacological Research, and Ospedali Riuniti di Bergamo,
Address correspondence and reprint requests to Ariela Be-
nigni, PhD, Mario Negri Institute for Pharmacological Research, Via
Gavazzeni 11, 24125 Bergamo, Italy. E-mail: firstname.lastname@example.org.
Submitted for publication October 24, 2006; accepted in
revised form January 16, 2007.
(3,4). Among TLRs, TLR-9 specifically binds CpG
DNA, a hypomethylated form of DNA typical of bacte-
ria and virus (5). CpG DNA activates potentially auto-
reactive B cells and plasmacytoid dendritic cells to
secrete Th1-like cytokines (6). Aberrant TLR-9 activa-
tion and release of cytokines and chemokines occurs in
plasmacytoid dendritic cells after exposure to immune
complexes containing DNA, rich in CpG motifs, isolated
from the serum of patients with active lupus nephritis
Lesions of lupus nephritis involve the renal glo-
merulus and tubulointerstitium. Whereas the glomerular
lesions have been studied extensively, the pathophysiol-
ogy of tubulointerstitial inflammation and damage re-
mains ill-defined. Yet tubulointerstitial changes are
prominent and contribute prominently to the unfavor-
able long-term prognosis (8). Proximal tubular epithelial
cells play an active role in tubulointerstitial inflamma-
tion and fibrotic lesions, to the extent that when these
cells are exposed to anti–double-stranded DNA (anti-
dsDNA) antibodies from patients with active lupus, they
produce cytokines that promote local recruitment of
inflammatory cells (9). DNA-containing autoantibody
complexes found in the serum of patients with lupus
activate dendritic cells to generate cytokines and chemo-
kines via TLR-9 (7).
Considering that proximal tubular cells have
antigen-presenting capacity as dendritic cells under cer-
tain circumstances (10), we wondered whether TLR-9
could be involved in the development of tubular damage
and interstitial inflammation in lupus nephritis. To this
end, we investigated renal TLR-9 expression in (NZB ?
NZW)F1lupus-prone mice at different stages of the
disease, both when animals had elevated serum levels of
anti-dsDNA antibodies and later on, when proteinuria
and tubulointerstitial damage ensued. TLR-9 expression
in resident renal cells was assessed by laser capture
microdissection combined with TaqMan real-time quan-
titative reverse transcriptase–polymerase chain reaction
(RT-PCR). The finding that increased tubular TLR-9
generation was associated with proteinuria provided the
rationale for using seliciclib, a cyclin-dependent kinase
(CDK) 2, 7, and 9 inhibitor, which effectively reduced
proteinuria and ameliorated renal injury in this model
(11). To assess the relevance of animal data to the
pathophysiology of interstitial lesions in human lupus,
we evaluated the expression of TLR-9 in renal biopsy
specimens from patients with lupus nephritis. Based on
the previously reported finding that endogenous DNA-
containing autoantibody complexes in the serum of SLE
patients activated dendritic cells through TLR-9 (7), we
finally assessed whether DNA-containing immune com-
plexes purified from SLE serum stimulated cultured
human proximal tubular cells to express TLR-9.
MATERIALS AND METHODS
Animal experiments. Female (NZB ? NZW)F1mice
(Harlan, Milan, Italy) and CD-1 mice (Charles River, Calco,
Italy) were used. Animal care and treatment were conducted
according to the institutional guidelines in compliance with
national (Decreto Legislativo n.116, Gazzetta Ufficiale suppl
40, 18 febbraio 1992, Circolare n.8, Gazzetta Ufficiale 14 luglio
1994) and international (European Community [formerly, the
European Economic Community] Council Directive 86/609,
OJL358-1, December 1987; Guide for the Care and Use of
Laboratory Animals, US National Research Council, 1996)
laws and policies. NZB ? NZW mice were divided into the
following groups: group 1 (n ? 5), killed at age 2 months,
before the onset of renal disease (12,13); group 2 (n ? 5),
killed at age 5 months, when immune complex deposition
occurs but renal structure and function are not yet severely
impaired (14); and group 3 (n ? 10), killed at age 8 months. An
additional group of NZB ? NZW mice (n ? 10) was treated
with 200 mg/kg seliciclib (R-Roscovitine, CYC202; Cyclacel,
Dundee, UK) (11), given daily by gavage from age 2 months to
age 8 months. Eight-month-old normal CD-1 mice (n ? 5)
were used as controls for TLR-9 expression analyses.
At the end of the experimental period, urinary protein
excretion and serum blood urea nitrogen (BUN) levels were
determined as described previously (14), and renal tissue
specimens were removed. Serum levels of anti-dsDNA auto-
antibodies were evaluated by enzyme immunoassay (Diastat
anti-dsDNA kit; Bouty Laboratory, Milan, Italy) (15).
Renal morphology. Duboscq-Brazil–fixed and paraffin-
embedded renal cortex sections (3 ?m, Microtome V; LKB,
Bromma, Sweden) were stained with hematoxylin and eosin,
Masson’s trichrome, and periodic acid–Schiff reagent. Glomer-
ular and tubulointerstitial lesions were assessed using light
microscopy. Glomerular intracapillary hypercellularity was
given a score ranging from 0 to 3 (0 ? absent; 1 ? mild; 2 ?
moderate; 3 ? severe). Extracapillary proliferation was also
graded (0 ? absent; 1 ? ?25%; 2 ? 25–50%; 3 ? ?50% of
glomeruli involved). Deposits were graded from 0 to 3 (0 ? no
deposits; 1 ? ?25%; 2 ? 25–50%; 3 ? ?50% of glomeruli
involved). At least 100 glomeruli were examined for each
biopsy specimen. Glomerular damage was expressed as a single
mean value of the intracapillary hypercellularity, extracapillary
proliferation, and deposits scores.
Tubular changes (atrophy, casts, and dilatation) and
interstitial inflammation were graded from 0 to 3 (0 ? no
changes; 1 ? ?25%; 2 ? 25–50%; 3 ? changes affecting
?50% of the sample). At least 10 fields per sample were
examined at low magnification (10?) for scoring of the
interstitium. All biopsy specimens were analyzed by the same
pathologist in a blinded manner.
Immunohistochemical analysis. Paraffin kidney sec-
tions (3 ?m) were hydrated, retrieved, and immunostained
with a monoclonal antibody against F4/80 antigen of mouse
monocyte/macrophages (Caltag, Burlingame, CA) and a poly-
clonal antibody recognizing mouse and human TLR-9 (Santa
1570BENIGNI ET AL
cytokine expression: implications on tubulointerstitial inflamma-
tion in lupus nephritis. J Am Soc Nephrol 2005;16:3281–94.
10. Kelley VR, Singer GG. The antigen presentation function of renal
tubular epithelial cells. Exp Nephrol 1993;1:102–11.
11. Zoja C, Casiraghi F, Conti S, Corna D, Rottoli D, Cavinato RA, et
al. Cyclin-dependent kinase inhibition limits glomerulonephritis
and extends lifespan of mice with systemic lupus. Arthritis Rheum
12. Howie JB, Helyer BJ. The immunology and pathology of NZB
mice. Adv Immunol 1968;9:215–66.
13. Theofilopoulos AN, Dixon FJ. Etiopathogenesis of murine SLE.
Immunol Rev 1981;55:179–216.
14. Zoja C, Corna D, Rottoli D, Zanchi C, Abbate M, Remuzzi G.
Imatinib ameliorates renal disease and survival in murine lupus
autoimmune disease. Kidney Int 2006;70:97–103.
15. Zoja C, Corna D, Benedetti G, Morigi M, Donadelli R, Gugliel-
motti A, et al. Bindarit retards renal disease and prolongs survival
in murine lupus autoimmune disease. Kidney Int 1998;53:726–34.
16. Morigi M, Buelli S, Angioletti S, Zanchi C, Longaretti L, Zoja C,
et al. In response to protein load podocytes reorganize cytoskele-
ton and modulate endothelin-1 gene: implication for permselective
dysfunction of chronic nephropathies. Am J Pathol 2005;166:
17. Romagnani P, Lazzeri E, Mazzinghi B, Lasagni L, Guidi S, Bosi A,
et al. Nephrotic syndrome and renal failure after allogeneic stem
cell transplantation: novel molecular diagnostic tools for a chal-
lenging differential diagnosis. Am J Kidney Dis 2005;46:550–6.
18. Lasagni L, Francalanci M, Annunziato F, Lazzeri E, Giannini S,
Cosmi L, et al. An alternatively spliced variant of CXCR3 medi-
ates the inhibition of endothelial cell growth induced by IP-10,
Mig, and I-TAC, and acts as functional receptor for platelet factor
4. J Exp Med 2003;197:1537–49.
19. Weening JJ, D’Agati VD, Schwartz MM, Seshan SV, Alpers CE,
Appel GB, et al. The classification of glomerulonephritis in
systemic lupus erythematosus revisited. J Am Soc Nephrol 2004;
20. Ryan MJ, Johnson G, Kirk J, Fuerstenberg SM, Zager RA,
Torok-Storb B. HK-2: an immortalized proximal tubule epithelial
cell line from normal adult human kidney. Kidney Int 1994;45:
21. Donadelli R, Zanchi C, Morigi M, Buelli S, Batani C, Tomasoni S,
et al. Protein overload induces fractalkine upregulation in proxi-
mal tubular cells through nuclear factor ?B- and p38 mitogen-
activated protein kinase-dependent pathways. J Am Soc Nephrol
22. Harris EN, Gharavi AE, Tincani A, Chan JK, Englert H, Mantelli
P, et al. Affinity purified anti-cardiolipin and anti-DNA antibod-
ies. J Clin Lab Immunol 1985;17:155–62.
23. Lenert P. Inhibitory oligodeoxynucleotides—therapeutic promise
for systemic autoimmune diseases? Clin Exp Immunol 2005;140:
24. Anders HJ, Vielhauer V, Eis V, Linde Y, Kretzler M, Perez de
Lema G, et al. Activation of toll-like receptor-9 induces progres-
sion of renal disease in MRL-Fas(lpr) mice. FASEB J 2004;18:
25. Patole PS, Pawar RD, Lech M, Zecher D, Schmidt H, Segerer S,
et al. Expression and regulation of Toll-like receptors in lupus-like
immune complex glomerulonephritis of MRL-Fas(lpr) mice.
Nephrol Dial Transplant 2006;21:3062–73.
26. Patole PS, Zecher D, Pawar RD, Grone HJ, Schlondorff D,
Anders HJ. G-rich DNA suppresses systemic lupus. J Am Soc
27. Wu X, Peng SL. Toll-like receptor 9 signaling protects against
murine lupus. Arthritis Rheum 2006;54:336–42.
28. Lartigue A, Courville P, Auquit I, Francois A, Arnoult C, Tron F,
et al. Role of TLR9 in anti-nucleosome and anti-DNA antibody
production in lpr mutation-induced murine lupus. J Immunol
29. Brunn GJ, Platt JL. The etiology of sepsis: turned inside out.
Trends Mol Med 2006;12:10–6.
30. Sultzer BM. Genetic control of leucocyte responses to endotoxin.
31. Pippin JW, Qu Q, Meijer L, Shankland SJ. Direct in vivo inhibition
of the nuclear cell cycle cascade in experimental mesangial prolif-
erative glomerulonephritis with Roscovitine, a novel cyclin-depen-
dent kinase antagonist. J Clin Invest 1997;100:2512–20.
32. Gherardi D, D’Agati V, Chu TH, Barnett A, Gianella-Borradori
A, Gelman IH, et al. Reversal of collapsing glomerulopathy in
mice with the cyclin-dependent kinase inhibitor CYC202. J Am
Soc Nephrol 2004;15:1212–22.
33. Bauer S. Toll-erating self DNA. Nat Immunol 2006;7:13–5.
34. Latz E, Schoenemeyer A, Visintin A, Fitzgerald KA, Monks BG,
Knetter CF, et al. TLR9 signals after translocating from the ER to
CpG DNA in the lysosome. Nat Immunol 2004;5:190–8.
35. Leifer CA, Kennedy MN, Mazzoni A, Lee C, Kruhlak MJ, Segal
DM. TLR9 is localized in the endoplasmic reticulum prior to
stimulation. J Immunol 2004;173:1179–83.
36. Gaulin JF, Fiset A, Fortier S, Faure RL. Characterization of
Cdk2-cyclin E complexes in plasma membrane and endosomes of
liver parenchyma: insulin-dependent regulation. J Biol Chem
37. Brentjens JR, Sepulveda M, Baliah T, Bentzel C, Erlanger BF,
Elwood C, et al. Interstitial immune complex nephritis in patients
with systemic lupus erythematosus. Kidney Int 1975;7:342–50.
38. Sano H, Morimoto C. Dna isolated from DNA/anti-DNA antibody
immune complexes in systemic lupus erythematosus is rich in
guanine-cytosine content. J Immunol 1982;128:1341–5.
39. Richardson B, Scheinbart L, Strahler J, Gross L, Hanash S,
Johnson M. Evidence for impaired T cell DNA methylation in
systemic lupus erythematosus and rheumatoid arthritis. Arthritis
40. Birn H, Christensen EI. Renal albumin absorption in physiology
and pathology. Kidney Int 2006;69:440–9.
41. Kobayashi N, Suzuki Y, Tsuge T, Okumura K, Ra C, Tomino Y.
FcRn-mediated transcytosis of immunoglobulin G in human renal
proximal tubular epithelial cells. Am J Physiol Renal Physiol
1578 BENIGNI ET AL