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Lymphangiogenesis Is Upregulated in Kidneys of Patients With Multiple Myeloma.

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THE ANATOMICAL RECORD 293:1497–1505 (2010)
Lymphangiogenesis Is Upregulated in
Kidneys of Patients With Multiple
Myeloma
JULIA K. ZIMMER,1 SUZAN DAHDAL,2 CHRISTIAN MÜHLFELD,3
IVO P. BERGMANN,1 MATHIAS GUGGER,2 AND UYEN HUYNH-DO1*
1
Department of Nephrology and Hypertension, Inselspital, University of Bern,
Bern, Switzerland
2
Institute of Pathology, University of Bern, Bern, Switzerland
3
Institute of Anatomy, University of Bern, Bern, Switzerland
ABSTRACT
Neolymphangiogenesis has recently been demonstrated in transplanted kidneys as well as in chronic interstitial nephritis and IgA nephropathy. However, its significance in kidney disease remains to be defined and
a systematic study of renal lymphangiogenesis is warranted. We investigated patients with multiple myeloma (MM) presenting in the great majority with acute renal insufficiency. Controls were allograft kidney donors and
patients with renal insufficiency due to acute renal failure (ARF). Lymph
vessel length density (LVD) was quantified immunohistochemically by
means of antipodoplanin staining followed by computer-assisted stereology.
The mean LVD in kidneys of patients with MM (23.19 mm2) was higher
when compared with allograft donors (7.42 mm2, P ¼ 0.0003) and patients
with ARF (6.78 mm2, P ¼ 0.0002). The higher LVD was significantly associated with interstitial inflammation, and the newly formed lymph vessels
were accompanied by diffuse and nodular interstitial infiltrates composed
mainly of CD20þ B cells and CD27þ plasma cells. The infiltrates in patients
with MM also displayed a higher expression of the B-cell chemoattractant
CXCL13. These results demonstrate for the first time that lymphangiogenesis is a prominent feature in MM kidneys and that it is associated with a significant accumulation of macrophages, CD20þ and CD27þ B lymphocytes.
Further studies should clarify whether these changes represent a beneficial
or detrimental factor in the progression of the myeloma-related kidney damC 2010 Wiley-Liss, Inc.
age. Anat Rec, 293:1497–1505, 2010. V
Key words: lymphangiogenesis; multiple myeloma; cast nephropathy; B cells; chemokines
Multiple myeloma (MM) belongs to the group of
plasma cell dyscrasias and around four new cases per
100,000 members of the general population are diagnosed every year (Kyle et al., 2004), with a peak at the
age of 68 years. It is one of the main reasons for unexplained renal failure in elderly people (Haas et al.,
2000). About 50% of patients with MM show increased
serum creatinine at diagnosis and 15%–20% already
have significantly impaired renal function (serum creatinine 177–221 lmol/L or 2.0–2.5 mg/dL). The most frequent histological changes in kidney biopsies of patients
with MM are cast nephropathy (40%–63%), light chain
deposition disease (LCDD, 19%–26%), and amyloidosis
C 2010 WILEY-LISS, INC.
V
These data were presented as a poster at the 40th Annual
Meeting of the ASN in San Francisco.
Grant sponsor: Swiss National Science Foundation; Grant
number: 3100A0-105871.
*Correspondence to: Uyen Huynh-Do, MD, MME, Department
of Nephrology and Hypertension, Inselspital, University of Bern,
CH-3010 Bern, Switzerland. Fax: þ41-31-632-9734. E-mail:
uyen.huynh-do@insel.ch
Received 15 October 2009; Accepted 18 March 2010
DOI 10.1002/ar.21189
Published online 20 July 2010 in Wiley Online Library
(wileyonlinelibrary.com).
1498
ZIMMER ET AL.
(7%–30%; Montseny et al., 1998). Renal affection and
remaining function have an important impact on the
prognosis of the patient. Patients with cast nephropathy
have an especially poor survival outcome once renal
insufficiency has progressed to end-stage renal disease
requiring dialysis. Therefore, it is crucial to identify
MM-related renal diseases as early as possible to initiate
adequate treatment. Potential therapeutic strategies for
improving renal function are chemotherapy, with or
without subsequent autologous stem cell transplantation, and extended dialysis with protein-leaking dialyzers (Hutchison et al., 2007). According to in vitro and
in vivo data (Arimura et al., 2006), the application of the
pituitary adenylate cyclase-activating polypeptide could
be a promising alternative in the future.
Angiogenesis has become a major topic in tumor
research due to its role in metastatic spread and its
potential prognostic value. List (2001) reported an upregulation of vascular endothelial growth factor (VEGF)
in MM, while others have found an increased microvessel density in bone marrow of patients with MM induced
by VEGF or hepatocyte growth factor (Alexandrakis
et al., 2004). These observations are the rationale behind
targeting MM with antiangiogenic drugs such as thalidomide (Du et al., 2004).
In contrast, lymphangiogenesis has only very recently
become the topic of investigation following the discovery
of reliable and specific lymphatic endothelial markers
(Sleeman et al., 2001). The lymphatic endothelial cells of
these thin-walled vessels can be detected with the following antibodies: anti-Prox-1, anti-LYVE-1, and antipodoplanin (Breiteneder-Geleff et al., 1999). Prox-1 is a
key transcription factor for the lymphatic phenotype,
and LYVE-1 is a hyaluronate receptor that first appears
during lymphatic development, while the mucoprotein
podoplanin is probably responsible for the shape of podocytes (Matsui et al., 1999).
Besides well-established functions such as fluid balance and immune cell transport, little is known about
the role of lymphatic vessels in the kidney and other
parenchymal organs. Lymph vessels originate from embryonic veins, and except for the eye and the brain, they
can be found physiologically in several organs as a characteristic feature of higher vertebrates. This ‘‘second
circulation’’ network is also present in the kidney, where
the lymph vessels are confined to the area around largeand middle-sized arteries (McIntosh and Morris, 1971)
in the cortex, but do not appear in the peritubular space
(Kerjaschki et al., 2004). The medulla of a healthy person rarely possesses any lymphatics (Cuttino et al.,
1989). Just a few years ago, the Kerjaschki group
showed that neolymphangiogenesis occurs in allograft
kidney transplants, a finding confirmed by another
report 3 years later (Adair et al., 2007; Stuht et al.,
2007). In another study, Kerjaschki et al. (2006) demonstrated that lymphatic endothelial cells in renal transplants originated from recipient-derived lymphatic
progenitor cells. These studies on lymphangiogenesis in
renal transplants were followed by a recent report demonstrating neolymphangiogenesis in chronic interstitial
nephritis as well as in IgA nephropathy (Heller et al.,
2007). Those lymph vessels were associated with nodular
lymphatic infiltrates. To date however, and to the best of
our knowledge, none of these studies have used morphometric methods to quantify renal neolymphangiogenesis.
De novo formation of tertiary lymphoid organs
(‘‘lymphoid neogenesis’’) with typical B lymphocyte accumulation is also present in multiple autoimmune diseases such as rheumatoid arthritis (Takemura et al.,
2001) or inflammatory bowel disease (Kaiserling, 2001).
Martin and Chan (2006) pointed out that B cells are
involved in tissue injury via chemokine release and Tcell response triggering.
MM can induce several non-MM specific as well as
specific lesions (i.e., amyloidosis, cast nephropathy,
LCDD) in the kidney depending on the stage of the disease. Cast nephropathy or amyloidosis is mainly displayed in the full-blown picture of late myeloma phases.
The aim of this retrospective study was to use rigorous
morphometric methods to determine whether an upregulation of lymphangiogenesis is present in MM kidneys
and if there is a difference between the early and late
disease stages. A further hypothesis was that kidneys of
patients with MM are associated with immunological
alterations such as tissue infiltrations of immunocompetent cells. By characterizing the composition of these
infiltrates, we looked for potential triggers of renal
lymphangiogenesis.
MATERIALS AND METHODS
Study Population
Archival renal biopsies from patients with MM who
presented in the great majority with an acute decline of
kidney function were used (n ¼ 37). The diagnosis of typical myeloma signs in the kidney, that is, cast nephropathy, LCDD, or amyloidosis, was based on light
microscopy, immunohistochemistry (IHC), immunofluorescence, and/or electron microscopy. Fifteen biopsies
from healthy kidney allograft donors taken prior to transplantation and 12 biopsies from patients with acute renal
failure (ARF) due to other causes served as controls. Data
collection and usage were performed with informed
patient consent and in accordance with the Declaration of
Helsinki.
Immunohistochemistry and
Immunofluorescence
IHC and immunofluorescence were performed on 3lm-thick formalin-fixed and paraffin-embedded tissue
samples. Slides were dewaxed and hydrated in xylole
and ethanol before being microwaved in 10 mM Tris–
1 mM EDTA buffer, pH ¼ 9.0 (410 W, 2 min 5 min).
Lymphatic vessels were identified by staining with
monoclonal mouse anti-human podoplanin IgG (D2-40,
1:1,000; Covance, Lausen, Switzerland). Since the first
description of its use in 1997 (Breiteneder-Geleff et al.,
1997), this marker has been shown in numerous studies
to allow a specific discrimination between lymphatic and
vascular vessels in a very reliable manner. Furthermore,
it works very well for IHC on paraffin sections (Fogt
et al., 2004; Galambos and Nodit, 2005; Kerjaschki
et al., 2006). Characterization of infiltrating cell populations and chemokines was performed using the following
antibodies: monoclonal mouse anti-human CD68 (PGM1, 1:1,000) and CD20 (L26, 1:1,000; Dako, Baar, Switzerland), CD27 (137B4, 1:50; Dianova, Hamburg, Germany), CD3 (F2.2.38, 1:100; Antikoerper-online, Aachen,
Germany), and goat anti-human CXCL13 (polyclonal,
RENAL LYMPHANGIOGENESIS IN MULTIPLE MYELOMA
1:100; R&D, Abingdon, UK). For IHC, a biotin-streptavidin-alkaline phosphatase-based method was applied
according to manufacturer’s instructions (LSABþ kit,
New Fuchsin, Dako). Immunofluorescence labeling was
done with mouse anti-human podoplanin (monoclonal,
1:50, Dako) and monoclonal mouse anti-Ki-67 (SP6,
1:100, Thermo RM-9106-S1). Appropriate secondary
antibodies labeled with fluorochromes Alexa 488 and
Alexa 546 (Molecular Probes, Luzern, Switzerland) were
used, and the stainings were visualized with a Zeiss Axiophot 2 microscope.
Analysis of Lymph Vessel Length Density
Length density of lymph vessels was quantified at an
objective lens magnification of 20 using a light microscope equipped with a camera and a computer-assisted
stereology tool (CAST, Olympus Denmark). Systematic
uniform random sampling was applied to ensure that every part of the section had an equal chance of being
included in the analysis. An unbiased counting frame
with an area of 21668.7 lm2 was projected onto each
test field. Lymph vessels were recognized by positive immunostaining against podoplanin, and their profiles
were counted if they were lying inside the counting
frame, but not touching the exclusion line or its extensions. Length density of lymph vessels was calculated
from the number of lymph vessel profiles, the number of
counting frames, and the area of the counting frame
according to the following formula (Weibel, 1979):
Lv ðlymph vessels=biopsyÞ
2 Q Possible cornersCF
¼
ACF CornersCF on tissue
where Q is the lymph vessel profiles; possible cornersCF
¼ 4; CF is the counting frame; and A is the area of CF.
Length density refers to the length of a structure contained within a unit of the reference volume and is
therefore expressed in mm2. As length density estimations may change, either due to the length of a structure
or to variations in the reference volume, they should
ideally be multiplied by the reference volume, which
gives rise to the total length. However, it was not possible to gather data on the reference volume (i.e., total
kidney volume) of the patients. To ensure that there had
been no significant changes in the reference volume, the
kidney ultrasound data of the different groups were collected. The craniocaudal pole diameter is generally used
to describe kidney size. As we found no alteration in this
parameter between patients with MM and the control
group, we concluded that changes in the length density
were due to changes in lymph vessel length and not due
to a different reference volume. Therefore, length density provided a suitable parameter for comparing lymph
vessel length between the groups.
Assessment of Interstitial Fibrosis
and Inflammation
All biopsies were screened for interstitial fibrosis in
the Trichrom Masson and for inflammation in the hematoxylin and eosin staining at an objective lens magnifica-
1499
tion of 10. The evaluation was performed
semiquantitatively in a blind manner by two independent investigators (J.Z. and S.D.) according to the score
previously used by Heller et al. (2007); for fibrosis, 1:
fibrotic tissue <1/3 of the biopsy, 2: fibrotic tissue 1/3< <2/3 of the biopsy, 3: fibrotic tissue >2/3 of the biopsy;
for inflammation, 1: few cells, diffusely spread, 2: at
least one infiltrate, 3: confluent infiltrates.
Analysis of Cellular Infiltrates
The occurrence of different cell populations and chemokine expression, based on immunostaining with the
markers CD20, CD27, CD68, CD3, and CXCL13, was
assessed at an objective lens magnification of 10 in a
blind manner by two independent investigators (J.Z. and
C.M.). The following semiquantitative score was used: (no positively stained cells), þ (few positively stained
cells, diffusely spread), þþ (aggregation of positively
stained cells), and þþþ (many aggregations of positively
stained cells, partially confluent; Adair et al., 2007).
Statistical Analysis
Data between groups were analyzed with the KruskalWallis test and the Mann-Whitney U-test as there was
no Gaussian distribution. A value of P < 0.05 was considered to be statistically significant.
RESULTS
Increased Lymph Vessel Length Density in
Kidneys of Patients With MM
Between 1994 and 2007, 37 patients with MM, presenting in the great majority with acute worsening of
kidney function, underwent renal biopsy after the treating physicians had excluded reversible causes such as
dehydration, hypercalcemia, exposure to nonsteroidal
anti-inflammatory drugs (NSAIDS) or contrast media, or
postrenal obstruction. Their average creatinine was 359
lmol/L, and the average creatinine clearance added up
to 30 mL/min. The clinical features of these patients are
depicted in Table 1. In 22 patients, histological changes
were typical for MM, that is, cast nephropathy (n ¼ 14),
LCDD (n ¼ 4), both (n ¼ 1), or amyloidosis (n ¼ 3),
whereas only non-MM-specific alterations were found in
15 biopsies. Fifteen healthy kidney allograft donors
(mean creatinine 81 lmol/L) and 12 patients with ARF
(mean creatinine 604 lmol/L) served as controls (histological diagnosis in Table 2).
By immunostaining for podoplanin expressing LVs,
the LV length density was determined in these biopsies,
and a higher frequency of small caliber LV in patients
with MM versus kidney allograft donor controls was
found (Fig. 1). The MM group had a mean LV length
density of 23.19 mm2 when compared with 7.42 mm2
in the allograft donor samples (P ¼ 0.0003).
To ensure that these findings were not just due to renal insufficiency, a second control group was chosen consisting of patients with ARF due to a variety of
etiologies such as acute postinfectious glomerulonephritis, drug-induced interstitial nephritis, or tubular necrosis (creatinine 604 lmol/L vs. 359 lmol/L in MM). With
a mean value of 6.78 mm2, LV length density of the
ARF group was in the same range as the allograft
1500
ZIMMER ET AL.
TABLE 1. Clinical features of patients with multiple myeloma
No
Gender/Age
MM with specific signs
1
W/56
2
M/80
3
W/61
4
M/77
5
M/63
6
M/73
7
W/75
8
W/62
9
M/80
10
M/61
11
M/74
12
W/58
13
M/68
14
M/60
15
W/78
16
M/72
17
M/61
18
M/72
19
M/58
20
M/47
21
M/43
22
W/54
MM but no specific signs
23
M/33
24
M/42
25
W/53
26
M/74
27
M/70
28
M/51
29
W/63
30
W/43
31
M/57
32
M/52
33
M/69
34
M/73
35
M/79
36
M/63
37
M/67
Lv (mm2)
Histological
diagnosis
Creatinine
(lmol/L)
Creatinine
clearance
(mL/min)
Pole distance
(ultrasound,
in cm)
27.2
53.5
60.9
10.9
12.2
19.5
3.8
19.9
26.5
16.1
22.2
33.4
37.2
37.2
10.2
4.5
7.5
37.6
16.1
11.4
6.0
12.4
CAST
Amyloidosis
LCDD
Amyloidosis
CAST
CAST
LCDD
LCDD
CAST
CAST
Amyloidosis
CAST
CAST
CAST
LCDDþCAST
LCDD
CAST
CAST
CAST
CAST
CAST
CAST
215
–
335
61
976
800
189
142
622
160
478
408
278
234
339
135
854
350
–
1360
390
145
–
–
16
–
3
–
29
–
12
42
–
–
8
–
44
59
7
22
14
–
11
45
–
11.0
10.2
–
–
11.8
–
11.0
10.0
11.0
–
13.0
10.5
12.6
–
12.4
12.0
10.0
13.1
13.8
14.0
11.1
46.6
6.7
44.5
37.9
56.4
24.5
28.9
11.3
7.2
16.6
0.0
24.8
25.9
28.5
11.9
Minor tubular lesions
Glomerulosclerosis
Minor glomerular changes
Membranoprolif.GN
Mesangial sclerosis
Ischemic damage
Glomerulosclerosis
Minor arteriosclerosis
Tubulointerstitial nephritis
Endocapillary proliferation GN
Tubulointerstitial nephritis
Chronic GN
Hypertensive nephropathy
Nephangiosclerosis
Nephangiosclerosis
450
658
–
200
285
161
183
–
170
164
413
280
176
82
168
–
–
–
–
–
66
–
47
50
54
6
3
25
60
–
14.0
12.5
–
–
–
–
10.4
–
12.8
–
10.0
11.6
–
–
–
donors (ARF versus allograft donors, P ¼ 0.30) and significantly lower than that of patients with myeloma (P ¼
0.0002; Fig. 2).
Renal fibrosis was assessed semiquantitatively as an
additional means of ruling out that higher LV length
density in patients with MM could only be due to interstitial fibrosis. We found that the higher lymph vessel
length density (LVD) in MM samples was not associated
with a higher degree of fibrosis (P ¼ 0.215; Fig. 3A).
However, the higher LVD in patients with MM was significantly associated with interstitial inflammation, for
example, lymphocyte infiltrates (P ¼ 0.019; Fig. 3B).
Because the histopathological spectrum of the MM biopsies reached from typical myeloma signs such as cast nephropathy (Fig. 4; ‘‘ARF with typical MM lesions,’’ n ¼
23) to changes not specific for MM (Fig. 4; ‘‘MM with
ARF but no specific lesions,’’ n ¼ 11; ‘‘MM with chronic
renal failure,’’ n ¼ 3), a comparison of LV length density
was made in these three subpopulations. Interestingly,
the different clinical types of the disease showed no difference in LV length density. Patients with MM-specific
affection of the kidney had a mean value of 23.19 mm2
vs. 22.10 mm2 in the group with chronic renal failure
and versus 23.47 mm2 among the population with nonMM-specific alterations (P ¼ 0.872 and P ¼ 1.00; Fig. 4).
In each population studied, LV length density showed
a certain variation, a finding also observed in the study
by Stuht et al. (2007) and which is due to a widespread
distribution of lymph vessels in humans. Nevertheless,
the overlap of the LV length densities in the different
groups was minor.
Proliferating Lymph Vessels in
Patients With MM
The next step was to examine whether the higher LV
length density was associated with ongoing lymphangiogenesis or whether the lymphatic beds were already quiescent at the time of investigation. In kidney
transplants, Kerjaschki et al. (2006) demonstrated proliferating lymphatics, but this finding could not be confirmed in the later study of Adair et al. (2007). We
performed immunofluorescence staining for podoplanin
and Ki-67. Ki-67 is a protein that is solely expressed in
the active phases of mitosis but not in the G0 phase and
therefore represents a specific marker for cell
1501
RENAL LYMPHANGIOGENESIS IN MULTIPLE MYELOMA
TABLE 2. Clinical features of control group
No
Gender/Age
Allograft Donors
1
W/38
2
M/14
3
M/23
4
M/40
5
M/42
6
M/44
7
M/49
8
M/21
9
M/55
10
M/30
11
M/62
12
M/53
13
M/70
14
M/48
15
M/68
ARF
1
M/75
2
W/55
3
W/61
4
W/50
5
W/55
6
M/27
7
W/70
8
W/82
9
W/65
10
M/70
11
M/58
12
M/49
Lv (mm2)
Histological diagnosis
2.1
16.5
7.6
0.0
5.5
0.0
0.7
20.4
23.3
0.7
0.7
5.4
27.0
0.0
1.4
2.0
17.0
4.6
6.1
7.6
1.7
8.7
10.0
5.0
5.0
4.2
9.5
Healthy
Healthy
Healthy
Healthy
Healthy
Healthy
Healthy
Healthy
Healthy
Healthy
Healthy
Healthy
Healthy
Healthy
Healthy
kidney
kidney
kidney
kidney
kidney
kidney
kidney
kidney
kidney
kidney
kidney
kidney
kidney
kidney
kidney
Postinfectious endocapillary GN
Postinfectious endocapillary GN
Postinfectious endocapillary GN
Postinfectious endocapillary GN
Postinfectious tubulointerstitial inflammation
Postinfectious exsudative GN
Drug-induced acute interstitial nephritis
Drug-induced acute interstitial nephritis
Drug-induced acute interstitial nephritis
Acute tublar necrosis
Acute tublar necrosis
Acute tublar necrosis
Creatinine
(lmol/L)
Creatinine
clearance
(mL/min)
Pole distance
(ultrasound,
in cm)
109
74
73
105
68
68
78
119
50
82
98
–
94
70
40
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
12.5
–
12.5
–
–
10.2
10.5
9.5
10.8
10.3
–
10
10
164
149
419
260
481
876
1000
998
814
878
602
–
44
47
–
29
–
–
–
–
–
–
–
–
–
9.8
13.3
11.5
9.8
–
–
–
–
13.6
13.0
–
Fig. 1. Lymph vessel staining (!) in patients with multiple myeloma
(MM) and control group. Immunohistochemistry was performed with
antipodoplanin (D2-40) on formalin-fixed and paraffin-embedded tissue of MM (A) and kidney allograft donors (B). New Fuchsin staining
and hematoxylin counterstaining. Magnification, 20.
proliferation (Gerdes et al., 1983; Scholzen and Gerdes,
2000). By colocalization of these two markers, active proliferation of LVs in our myeloma samples was shown
(Fig. 5), which was comparable in magnitude with the
findings shown by Kerjaschki et al. (2006).
Higher Occurrence of B Lymphocytes
in MM Kidneys
As previously described by Kerjaschki et al. (2004) in
transplanted kidneys, we found nodular cellular infiltrates within the regions of higher LV count. To further
characterize the infiltrating cell populations, IHC stain-
Fig. 2. Increased lymph vessel length density in patients with MM
when compared with allograft donors and patients with acute renal
failure (ARF). Quantitative analysis of lymph vessel length is shown as
scatter plot. The horizontal bar represents the mean. P ¼ 0.0003
between MM and allograft donors; P ¼ 0.0002 between MM and ARF.
No significant difference between the allograft donor and the ARF
group (P ¼ 0.30).
ings for B lymphocytes, that is, CD20 for B cells and
CD27 for plasma cells, were performed. Figure 6(A,C)
clearly demonstrates aggregations of CD20þ and CD27þ
B lymphocytes in patients with MM, whereas the control
samples showed weaker or even no signals (Fig. 6B,D).
1502
ZIMMER ET AL.
Fig. 4. Comparable lymph vessel length density in different subgroups of patients with MM: ARF with typical myeloma-related lesions,
ARF with non-MM-specific lesions and chronic renal failure. Quantitative analysis of lymph vessel length is shown as scatter plot. The horizontal bar represents the mean. There was no significant difference
between the three groups (P ¼ 1.00, P ¼ 0.872, and P ¼ 0.876).
Fig. 3. MM samples with increased LVD do not display increased
renal fibrosis but a higher grade of inflammatory infiltrates. Evaluation
of interstitial fibrosis and inflammation was performed by two investigators in a blind manner using a semiquantitative score. The horizontal
bar in the box plot represents the mean. While the fibrosis level was
the same (P ¼ 0.215, SD for MM 0.56 and for control 0.43), inflammatory cells were more significantly present in patients with myeloma
when compared with the control group (P ¼ 0.019, SD for MM 0.50
and for control 0.35).
Higher Occurrence of Macrophages
in MM Kidneys
Macrophages are able to trigger lymphangiogenesis
via two pathways: either by release of VEGF-C or by
metamorphosis into lymphatic endothelial cells (Maruyama et al., 2005). The paraffin slides were immunolabeled with anti-CD68 to determine the number of
infiltrating macrophages. CD68þ cells were more abundant in patients with MM when compared with controls
(Fig. 7, A vs. B).
Higher Level of CXCL 13 in MM Kidneys
Another mediator that macrophages are capable of
producing is the chemokine CXCL13, which has been
shown to be a strong chemoattractant for B lymphocytes
(Legler et al., 1998). Immunohistochemical staining for
CXCL13 confirmed the hypothesis of a higher CXCL13
chemokine level in MM kidneys when compared with
controls (Fig. 7, C vs. D).
Slight Increase in CD31 Lymphocytes
in MM Kidneys
To complete the different subsets of cell populations, a
semiquantitative analysis of T lymphocytes by means of
Fig. 5. Proliferating lymph vessels: colocalization of podoplanin and
Ki-67. Immunofluorescence staining for podoplanin (red) and Ki-67
(green) in a patient with MM. The subpart (A) shows a longitudinal section while (B) represents a cross-sectional staining. Magnification, 40.
anti-CD3 was performed. The number of CD3þ cells only
slightly increased in MM samples versus controls (data
not shown).
An overview of all analyzed cell populations and the
chemokine CXCL13 is given in Table 3 (patients with
MM, n ¼ 34; allograft donors, n ¼ 15).
DISCUSSION
Research on renal lymphangiogenesis under pathological conditions started only very recently following the
discovery of specific lymphatic markers. In this study,
lymph vessels in kidneys of patients with MM were
detected by immunohistochemical staining for podoplanin. A pronounced increase in LVD in this patient group
was found, accompanied by a prominent tissue infiltration by B lymphocytes and macrophages. The demonstration that this higher LVD was not associated with a
1503
RENAL LYMPHANGIOGENESIS IN MULTIPLE MYELOMA
TABLE 3. Semiquantitative analysis of cell
populations and chemokine expression in patients
with MM(n 5 34) and controls (allograft donors,
n 5 15)
Marker
MM
Allograft Donors
CD20
CD27
CXCL13
CD68
CD3
þþ/þþþ
þþ/þþþ
/þ
þþ/þþþ
/þ
/þ
/þ
/þ
/þ
/þ
Evaluation was performed by two investigators in a blind
manner using a semiquantitative score (, þ, þþ, þþþ).
Fig. 6. Immunostaining for CD20 (A) and CD27 (C) in patients with
MM versus controls (B, D). New Fuchsin staining and hematoxylin
counterstaining. CD20þ resp. CD27þ lymphocytes are stained as red
dots (arrows !). Magnification, 20.
Fig. 7. Immunostaining for CD68 (A, !) and CXCL 13 (C) in
patients with MM versus controls (B, D). New Fuchsin staining and hematoxylin counterstaining. CD68þ macrophages are stained as red
dots (arrow !). Magnification, 20.
more advanced degree of interstitial fibrosis suggests
that these findings are not just a ‘‘paraphenomenon’’ secondary to interstitial scarrings but that they could be
specifically caused by the disease itself and the associated inflammation processes.
Most data about renal lymphangiogenesis have so far
been obtained in kidney transplant recipients with either acute graft rejection or chronic allograft nephropathy (Kerjaschki et al., 2004; Adair et al., 2007; Stuht
et al., 2007). In both cases, the number of lymph vessels
was much higher when compared with native nephrectomy tissue; however, there was no significant difference
in LV density between different Banff rejection grades
and chronic allograft nephropathy (Stuht et al., 2007).
The significance and clinical relevance of lymph vessel
presence is still not completely elucidated. It is considered as a detrimental factor in cancer because metastatic dissemination and early lymph node metastasis
are associated with a poor prognosis (Morice et al., 2003;
Schoppmann et al., 2004). In contrast, the relevance of
neolymphangiogenesis in diseases such as asthma or
inflammatory arthritis (Zhang et al., 2007) remains
unclear. This uncertainty is reflected in the ongoing
post-transplant discussion on whether increased LVD in
the kidney allograft predicts a higher risk of organ loss
or a better graft survival. Both an ongoing maintenance
of immune response through lymphocyte invasion and a
clearing function of infiltrates can be attributed to
lymph vessels (Kerjaschki et al., 2004). Thus, neolymphangiogenesis could be detrimental if the immune process is perpetuated and augmented in the inflammed
tissues, but it may be beneficial if cytotoxic cells are
removed more efficiently. In kidney grafts, the newly
formed lymphatics reach deep into the tubulointerstitial
space, unlike in normal kidneys (Cuttino et al., 1989),
and are associated with nodular infiltrates that can be
found in 10%–15% of transplant biopsies. As apoptosis
can only partially remove infiltrates at a rate similar to
the one in the thymus, lymph vessels could be responsible for the residual evacuation (Paavonen et al., 2000).
Their function as so-called exit routes could explain an
even better kidney graft function if lymphatics are present within nodular infiltrates. This hypothesis is supported by the work from Stuht et al. (2007), who showed
that kidney graft function 1 year after transplantation
was better in cases with increased lymphangiogenesis.
More recently, Adair et al. (2007) also demonstrated new
interstitial lymph vessels in nephrectomized end-stage
allografts. The authors mentioned that no correlation
between lymph vessel number and time to graft failure
was found. However, the fact that their study of transplant nephrectomy specimens had its limitations as they
represented the end stage of a disease process that was
multifactorial in origin was acknowledged.
However, what about the presence and role of neolymphangiogenesis in the native kidney? To date, there has
been only one study addressing this question. As B cells
have recently emerged as potential players and therapeutic targets in inflammatory diseases of the kidney,
Heller et al. (2007) evaluated biopsies from patients
1504
ZIMMER ET AL.
with IgA nephropathy and chronic interstitial nephritis.
They found an increased number of CD20þ lymphocytes
in these kidneys, together with a striking clustering of
neolymphatics around the aggregates of inflammatory
cells. Control group with acute interstitial nephritis also
displayed an increased infiltration by CD20þ cells, but
neolymphangiogenesis was absent. Obviously, an acute
inflammatory process alone is not sufficient to trigger renal lymphangiogenesis. Our results confirm this because
lymph vessel length of patients with ARF due to postinfectious glomerulonephritis or acute interstitial nephritis
did not differ from the allograft donor group. In our
patient collective, there was no direct correlation
between the number of lymphatics and renal function at
presentation.
We recognize that our population was heterogenous;
however, this has been and will be the problem of all
clinicopathological studies dealing with kidney diseases
in patients with myeloma. As our knowledge about this
subject improves, we realize that ‘‘myeloma kidney" is
no longer the sole manifestation of renal involvement in
plasma cell dyscrasias, although the common denominator of these seemingly heterogenous clinicopathological
pictures remains the detrimental effect of deposited free
light chains and the ensuing inflammation.
The MM collective was further divided into three subgroups for analysis: one subgroup with ARF associated
with typical myeloma-related changes in the kidney, a
second subgroup with ARF but no myeloma specific
changes, and a third category with MM and chronic renal failure presenting with non-MM-specific histological
alterations. Interestingly, we observed comparable LVD
in all three groups. This raises the question of whether
the observed changes could be specific for myeloma. However, two arguments speak against this hypothesis. First,
according to our internal guidelines, renal biopsies were
performed only when an elevated creatinine persisted after all reversible causes for MM-associated renal insufficiency had been ruled out or treated (i.e., treatment of
dehydration and/or hypercalcemia, withdrawal from
NSAIDS, supportive therapy for acute tubular necrosis
(ATN), etc.). Second, as shown in Table 1, the group of
patients with MM with non-MM-specific renal histologies
encompassed a wide range of histopathologic diagnoses
ranging from minor lesions (n ¼ 3) to predominantly glomerular (n ¼ 6), tubular damage (n ¼ 3), or nephroangiosclerosis (n ¼ 3)—the only common denominator being
active MM. In aggregate, this observation suggests that
neolymphangiogenesis could be an early feature of MMassociated renal disease. The colocalization experiments
with podoplanin and Ki-67 present evidence that this process is due to ongoing lymph vessel proliferation.
The factors potentially influencing neolymphangiogenesis in the kidney were then assessed. Besides the soluble growth factors VEGF-C and VEGF-D (Mandriota
et al., 2001), fibroblast growth factor (Kubo et al., 2002),
or platelet-derived growth factor (Cao et al., 2004) neolymphangiogenesis can also be influenced by several cell
populations such as B lymphocytes. Angeli et al. (2006)
depicted a close interaction between antigen-presenting
dendritic cells, B cells, and lymph vessels. Together with
T cells, these three components are the main characteristics of the so-called tertiary lymphoid organs, whose
occurrence has been described in chronic inflammatory
processes such as rheumatoid arthritis (Manzo et al.,
2005) or in infectious diseases caused by Helicobacter
pylori or hepatitis C virus (Freni et al., 1995). Renal
lymphoid neogenesis has been observed in kidney allografts underlying chronic rejection (Thaunat et al.,
2005). In native kidneys, B lymphocytes have been found
more frequently in a number of glomerulopathies, such
as IgA or membranous nephropathy (Heller et al., 2007).
Our analysis of the cellular infiltrates accompanying
newly formed lymphatics revealed a clear dominance of
B lymphocytes and plasma cells in patients with myeloma when compared with controls. The accumulation
of these B lymphocytes was possibly triggered by
CXCL13, a chemokine produced by parenchymal cells
and macrophages and which has been shown to be a
strong B-cell chemoattractant (Legler et al., 1998).
Although CXCL13 was nearly absent in control biopsies,
an abundance was observed in lymphatic infiltrates of
MM biopsies. As a higher occurrence of macrophages in
the myeloma samples was also detected, we hypothesize
a dual role for these cells in renal neolymphangiogenesis: as a consequence of myeloma-related tissue injury,
macrophages invade the renal interstitium and release
CXCL13. Thereby, B lymphocytes are attracted and, after activation, produce VEGF-A (Angeli et al., 2006),
which in turn can also recruit new macrophages. On the
other hand, macrophages can also directly stimulate
lymphangiogenesis through the release of VEGF-C or
metamorphose themselves into lymphatic endothelial
cells (Kerjaschki, 2005).
In summary, this study shows for the first time that
lymphangiogenesis is a striking feature in kidneys of
patients with myeloma. It is accompanied by a local
accumulation of likely triggers such as CD20þ, CD27þ B
lymphocytes, and macrophages as can be encountered in
chronic processes like interstitial nephritis. Interestingly, LVD was significantly higher in MM samples even
before disease-specific kidney damage appeared. This observation, if confirmed by further studies encompassing
a larger number of patients, would suggest that lymph
vessel staining could be a useful tool for early identification of myeloma-related kidney injury. As a consequence,
further prospective investigations, including serial biopsies before and after initiation of chemotherapy, will be
needed to clarify whether renal neolymphangiogenesis
represents a beneficial or a detrimental factor in the progression of the myeloma-related kidney injury.
ACKNOWLEDGMENTS
The authors thank B.M. Frey and F.J. Frey (Department of Nephrology and Hypertension, Inselspital, University of Bern, Switzerland) for their support and
fruitful input. They also thank S. Moll (Renal Pathology,
University of Geneva, Switzerland) for some biopsies of
patients with multiple myeloma.
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