Download Dooley MA.Clinical and laboratory features of lupus nephritis

Significance of intrarenal B and T lymphocytic infiltrates in lupus nephritis
Nashwa M. Emara, Mona A. Abou Elmahasen, Eman Saad Omar, Adel Zaki El-saidy, Zeinab I.
El shawarby and Samia A.Youssef
Abstract:
Lupus nephritis (LN) is one of the most serious complications of systemic
lupus erythematosus (SLE). The true prevalence of clinical lupus nephritis in
persons with SLE is probably around 50%, being higher in certain ethnic groups
and in children.
It was hypothesized that intrarenal B cells form part of a local system with
pivotal involvement in the pathogenesis of lupus nephritis. It enhances
immunological responses and exaggerates the local immune response to persisting
autoimmune damage in the tubulointerstitium. T cells cause tissue injury by
activating and providing help to nephritogenic antibody-producing B cells,
recruiting macrophages and dendritic cells (DCs), and producing cytokines.
Aim: This retrospective study aims to assess intrarenal B and T lymphocytic
infilterates in cases of lupus nephritis using CD20 and CD3and determine their
possible value in lupus nephritis and their possible relation to clinical outcome.
Results: Intrarenal B &T cells were more likely to be associated with class IV
LN. LN activity index (AI), chronicity index (CI), proteinuria and serum creatinin
had a significant statistical correlation with intrarenal B and T cell infiltrates.
Conclusion: The current study concludes that interstitial inflammatory infiltrates
and CI, but not AI could be used as prognostic factors in lupus nephritis and also
may concludes that the presence of intrarenal B:T cell infiltrates and its grading
could be used as a prognostic factor in patients of lupus nephritis. It shall provide a
hope for management and improvement of prognosis of lupus nephritis patients
especially the resistant cases by trials of anti CD20 drugs.
Page 1
Introduction:
Nephritis is a major cause of morbidity and mortality in patients with lupus.
Nephritis occurs in approximately 50% of lupus patients, but rates vary
significantly between genders (with female to male ratio 9:1) (Sada & Makino;
2009). The pathogenesis of lupus nephritis involves extrarenal and intrarenal
pathogenic mechanisms. The extrarenal etiology of systemic lupus and lupus
nephritis is based on multiple combinations of genetic variants that compromise
those mechanisms normally assuring immune tolerance to nuclear autoantigens.
The intrarenal etiology of lupus nephritis involves antibody binding to multiple
intrarenal autoantigens rather than the deposition of circulating immune complexes
(ICs) (Lech & Anders; 2013).
Following the formation and/or deposition of ICs in the kidney, interactions
between resident renal cells and infiltrating inflammatory cells promote tissue
injury. Local cytokine, chemokine and adhesion molecule production leads to
further influx of inflammatory cells and production of proinflammatory cytokines,
ultimately resulting in renal inflammation, tissue injury and fibrosis. Lupus T cells
express increased levels of molecules necessary for homing to the kidney (Wang et
al; 2010). Mechanisms by which T cells contribute to tissue injury include
activating and providing help to nephritogenic antibody-producing B cells,
recruiting macrophages and dendritic cells (DCs), and producing cytokines
(Apostolidis; 2011).
Till now the role of pathogenic B cells were supposed to have a variety of
functions that contribute to lupus nephritis. As they produce autoantibodies that
can cause renal damage via disruption of cellular functions, cytotoxicity mediated
by interactions with complement and release of inflammatory mediators. Presence
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of germinal center-like structures and T cell-B cell aggregates in the kidney
suggest in situ secretion of pathogenic antibodies, including nephritogenic
antibodies, and ICs in human lupus patients (Chang et al; 2011, Hutloff et al;
2004).
Material and Methods:
Tissue samples
Formalin-fixed, paraffin-embedded 57 renal biopsy specimens were obtained
from Urology and Nephrology Center -Mansura University (UNC) and department
of pathology, Faculty of Medicine, Benha University, during the years from 2009
to 2010. The studied biopsies were divided into control group formed of 6 biopsies
of morphologically normal renal tissue (obtained postmortem) and the study group
which was formed of 57 biopsies of different classes of lupus nephritis.
All cases fulfilled the American College of Rheumatolgy criteria for
diagnosis of SLE (Crow; 2013). Clinical evidence of LN was obtained in all cases
and LN diagnosis was confirmed by examination of renal biopsy specimen. The
following demographic, clinical and laboratory data were collected at time of renal
biopsy: age, sex, 24 hr proteinuria, levels of serum creatinine, and presence or
absence of anti-nuclear antibodies (ANA) (all the studied cases were positive for
ANA). In addition to follow up of eighteen cases of the whole study group whose
clinicopathologic data were available. The follow up was for one year from the
diagnosis by clinical, laboratory and renal biopsy.
Histopathological study:
All patients underwent ultrasound-guided renal needle biopsy. Renal tissues
obtained by biopsy were fixed in 10% neutral-buffered formalin, dehydrated
gradually, and embedded in paraffin. Six serial tissue sections were cut at 3-4
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micrometer. The first two slides were used for immunostaining and the remaining
four slides were used for Hematoxylin and Eosin, Periodic Acid-Schiff, Masson’s
Trichrome, and Periodic Acid-Silver Methenamine.
The biopsy specimens were classified using the International Society of
Nephrology/Renal Pathology Society (ISN/RPS) 2003 classification of LN
(Weening et al., 2004). Activity and Chronicity Indices of renal tissue injury were
scored according to National Institutes of Health system (Austin; et al; 1983 ).
Renal tissue injury was evaluated on the basis of activity and chronicity indices
according to methods reported by Austin et al 1983. Activity index was calculated
as the sum of the scores (on a scale of 1–3) of endocapillary proliferation,
karyorrhexis, fibrinoid necrosis (the score was multiplied by 2), cellular crescents
(the score was multiplied by 2), hyaline deposits, leukocyte exudation, and
interstitial inflammation. The score of the chronicity index was the sum of the
scores (on a scale of 1–3) for glomerular sclerosis, fibrous crescents, tubular
atrophy, and interstitial fibrosis.
Each activity and chronicity factor is graded on a scale of 0, 1, 2, and 3
depending on the percentage of involvement of glomeruli present in sections;
where 0 (absence of lesions), 1 (lesions involving up to 25%), 2 (lesions involving
25–50%), and 3 (lesions involving >50%) (Gomaa et al ., 2014).
Intervals of Activity and chronicity indices
For the activity index, the biopsy specimens were classified in three
intervals according to Makni et al., 2009 as follow:
1- AI of 5 or less was described as moderately active lupus nephritis.
2- AI of 5–9 was described as active lupus nephritis.
3- AI of >9 was described as extremely active lupus nephritis.
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Chronicity index was classified in two intervals according to Makni et al.,
2009 as follow:
1- CI of 3 or less was described when the specimen showed weak and limited
chronic changes.
2- CI of more than 3 was described when the specimen showed widespread
chronic changes.
Scoring of tubulointerstitial inflammatory infilterate
- 0 (nil), normal.
- 1+ (mild), <25% of the acreage of interstitium affected.
- 2+ (moderate), 25–50% of the acreage of interstitium affected.
- 3+ (severe), >50% of the acreage of interstitium affected in each specimen
(Yu et al., 2010).
Levels of proteinuria was estimated according to Ivanyi., 2001:
(+1) Proteinuria: less than 0.5 g /24 h urine, (+2) Proteinuria: 0.5-1g/24 h urine,
(+3) Proteinuria: 1-2 g/24 h urine, (+4) Proteinuria: more than 2 g/24 h urine.
Serum creatinin
The typical human reference ranges for serum creatinine are 0.5 to 1.0 mg/dl for
women and 0.5 to 1.5 mg/dl for men, above these levels creatinin was considered
elevated or high serum creatinin (Faull., 2007).
Division into progressors and non progressors was based on the slope of
serum Creatinin according to Makni et al; 2009: Progressors were defined when
creatinin increased by two fold or more from the lowest creatinine level reached
under treatment, while The non-progressors were defined when serum creatinin
level of patients was stable or improved.
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Immunohistochemistry and quantification:
Immunohistochemical staining of serial sections was performed for each LN
patient using the following antibodies: CD20 (Labvision, cat, NO.MS-340-S0,
USA) and CD3 (Labvision, cat, NO. MS401SO, USA) using DAKO envision
flex method to detect the presence & distribution of B & T lymphocytes
respectively through their membranous expression for such immunohistochemical
markers. Sections were cut at 4 micrometer and mounted on positively charged
slides. Sections were deparaffinized in xylene and rehydrated through descending
grades of ethyle alcohol. Slides were washed with distilled water three times for 2
minutes each and placed in pressure cooking slide dish and covered with sufficient
antigen retrieval solution and
placed in pressure cooking at +125C0 in
TRIS/EDTA buffer PH 8.5 for 5 minutes for antigen retrieval. Pressure cooking
was continued at 80% power for 5 minutes then 60% for 5 minutes. Endogenous
peroxidase activity was blocked by covering the sections with Envision TMFlex
Peroxidase-Blocking reagent (DAKO, cat. NO. SM801) (which contains 3%
H2O2) and incubated for 5 minutes. The sections were covered with primary
mouse monoclonal anti-human CD3 (Labvision, cat, NO. MS401SO, USA) and
CD20 (Labvision, Cat, NO.MS-340-S0, USA) at dilution of 1:50 then slides
were incubated at room temperature for one hour. Slides were rinsed with buffer
solution and were incubated at room temperature with Envision TMFlex/HRP
(DAKO, cat. NO. SM802) for 30 minutes. The sections were covered with
prepared DAB and incubated at room temperature for 10 minutes then washed
with distilled water. Slides were counterstained was with mayer's hematoxylin
(DAKO S3309) for 10 seconds. Slides were rinsed with tap water and dehydrated
by dipping them several times in three changes of 95% ethanol followed by three
changes of 100% ethanol. Slides were cleared by dipping several times in three
changes of xylene and coverslippeded with mounting media (DPX).
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Sections were screened on low power to show areas stained with CD20 and CD3,
then examined using x400 objective and screened for both CD20 and CD3.
Results of renal biopsies were classified according to organizational stage of
intrarenal inflammatory infilterates descriped by Steinmetz et al.; 2008 and Shen
et al.; 2012 who classified renal biopsies in four different groups as follow:
Grade 0: shows no B cells but scattered or cluster like structure of T cells.
Grade 1: consists of infiltrate of scattered T- and B-cells.
Grade 2: consists of infiltrate have cluster-like structure but no T- or B-cell zones
are evident .
Grade 3: consists of infiltrates of clearly distinguishable separate T- and B-cell
areas.
Grade 4: consists of infiltrates of a highly organized structure with a central
network of FDCs.
All renal biopsies were scored with no prior knowledge of the clinical and
laboratory analysis of patients.
Statistical analysis:
SPSS (version 16.0 software) statistical program was used, on a personal
computer. The Pearson correlation coefficient was used to correlate different
clinicopathologic variables to the median values of defined patient groups. Values
were expressed as mean and standard deviation. P value of 0.05 or less was
considered to be statistically significant.
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Results:
Clinical and histological results:
The age of all studied cases ranges between 13 - 49 years with mean age (
28.05 , SD+ 9.01). Out of fivety seven cases, fivety one (89.5%) were female, six
(10.5%) cases were male with nearly female to male ratio 9:1 .
-Distribution of of the ISN/RPS classification of the 57 patients was as follows:
seven cases(12.3%) are class II, nine cases (15.8%) are class III, twenty seven
cases (47.4%) are class IV ,seven cases (12.3%) are class V, seven cases (12.3%)
are class VI.
-Regarding the activity index: Fourteen cases (26.4%) are moderately active LN
(0-5/24), twenty (35.1%) cases are active LN (5-9/24), twenty three (40.4%) cases
are extremely active LN (>9/s24).
-Regarding the chronicity index: Six cases (10.5%) show no obvious chronic
changes, thirty one (54.5%) cases show weak and limited chronic changes (03/12), twenty (35.1%) cases show wide spread chronic changes(>3/12).
-Regarding interstitial inflammtory infiltrate: Twenty six cases (45.6%) show mild
(1+) inflammatory infiltrate, twenty four cases (42.1%) show moderate (2+)
inflammatory infiltrate, seven cases (12.3%) show severe (+3) inflammatory
infiltrate.
-Regarding proteinuria: Two cases (3.5%) have (+1) proteinuria (<0.5 gm/24 hr
urine), eight cases (14%) have (+2) proteinuria (0.5-1 gm/24hr urine), two case
(3.5%) have (+3) proteinuria (1-2 gm/24 hr urine), fourty five cases (79%) have
(+4) proteinuria (>2 gm /24 hr urine).
-Regarding serum creatinin level: Twenty two cases (38.6%) have normal serum
creatinine levels, thirty five cases (61.4%) have elevated serum creatinine levels.
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Table (1): shows correlation between classes of LN with different clinicopathologic variables:
Classes of lupus nephritis
Clinicopathologic variable
Total
Significance
P >0.05 (Insig)
II
III
IV
V
VI
Moderately active LN
6(85.7%)
1(11.1%)
1(3.7%)
2(28.6%)
4(57.1%)
14
Active LN
1(14.3%)
6(66.7%)
6(22.2%)
4(57.1%)
3(42.9%)
20
Extremely active LN
0(0%)
2(22.2%)
20(74.1%)
1(14.3%)
0(0%)
23
No chronic changes
2(28.6%)
0(0%)
3(11.1%)
1(14.3%)
0(0%)
6
Weak chronic changes
5(71.4%)
6(66.7%)
13(48.1%)
5(71.4%)
2(28.6%)
31
Widespread chronic changes
0(0%)
3(33.3%)
11(40.7%)
1(14.3%)
5(71.4%)
20
Mild(+1)
5(71.4%)
7(77.8%)
10(37%)
4(57.1%)
0(0%)
26
Moderate(+2)
2(28.6%)
2(22.2%)
12(44.4%)
3(42.9%)
5(71.4%)
24
Severe(+3)
0(0%)
0(0%)
5(18.5%)
0(0%)
2(28.6%)
7
+1
2(28.6%)
0(0%)
0(0%)
0(0%)
0(0%)
2
+2
2(28.6%)
1(11.1%)
4(14.8%)
1(14.3%)
0(0%)
8
+3
1(14.3%)
0(0%)
1(3.7%)
0(0%)
0(0%)
2
+4
2(28.6%)
8(88.9%)
22(81.5%)
6(85.7%)
7(100%)
45
Normal
5(71.4%)
7(77.8%)
6(22.2%)
4(57.1%)
0(0%)
22
Elevated
2(28.6%)
2(22.2%)
21(77.8%)
3(42.9%)
7(100%)
35
Total
7
9
27
7
7
57
Activity index
Chronicity index
P=0.031(Sig)
Inflammmatory infiltrate
P=0.004(Sig)
Proteinuria
P=0.003(Sig)
S.creatinin
P=0.003(Sig)
Page 9
- No significant statistical correlation was detected between lupus nephritis classes
and activity index, as it was found that most of cases of class II (85.7%) are
moderately active lupus nephritis, in class III (66.7%) are active lupus nephritis, in
class IV 74.1% are extremely active lupus nephritis. In class V most of cases
57.1% are active LN, whereas class VI was divided into (57.1%) moderately
active and (42.9%) active LN (Table 1).
- There was a significant statistical correlation between LN classes and chronicity
index, as it was found that (71.4%) of class II and (66.7%) of class III have weak
chronic changes. In class IV (48.1%) show weak chronic changes, (40.7%) show
widespread chronic changes. In class V (71.4%) show weak chronic changes and
in class VI (71.4%) show widespread chronic changes (Table 1).
- There was a significant statistical correlation between LN classes and interstitial
inflammatory infiltrate, as it was found that 71.4% of class II and 77.8% of class
III have (+1) inflammatory infiltrate. While 44.4% of class IV have (+2)
inflammatory infiltrate and 18.5% have (+3) infiltrate. In class V, 57.1% have (+1)
inflammatory infiltrate, in class VI (71.4%) of cases have (+2) & (28.6%) have
(+3) inflammatory infiltrate (Table 1).
-There was a significant statistical correlation between LN classes and proteinuria,
as cases in class II have different levels of proteinuria with nearly close percentage,
but in class III 88.9% have (+4) proteinuria. In Class IV, 81.5% have (+4)
proteinuria. In class V, 85.7% have (+4). All cases (100%) of class VI had +4
proteinuria (Table 1).
-There was a significant statistical correlation between LN classes and serum
creatinine , as most cases (71.4) of class II and (77.8%) of class III have normal
serum creatinine level. In class IV, (77.8%) of cases have elevated serum
creatinine. (57.1%) of class V have normal serum creatinine. All cases (100%) of
class VI have elevated serum creatinine (Table 1).
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Immunohistochemical results and their correlations:
Upon staining of CD20 & CD3 of the same cases to show microanatomical
organization of intrarenal lymphocytic infiltrate, renal biopsies were classified into
four different groups as follow: eleven cases (19.3%) were negative for CD20 but
showed scattered positive CD3 T cells and this group named non B cell group
(Figure 1). In the B cell group twenty cases (35.1%) were grade 1 (Figure 2),
twenty one cases (36.8%) were grade 2 (Figure 3), five cases (8.8%) were grade 3
(Figure 4; 5). No patients are identified in grade 4 (Table 2).
- There was a significant statistical correlation between grades of B:T cell infitrates
and LN classes, as it was shown that 63.7% of cases of grade 0 (non B cell group)
were between class II &III (36.4% in class II, 27.3% in class III) . Sixty percent
(60%) of cases of grade 1 were between class III & IV, (25% in class III, 35% in
class IV) and 61.9% of grade 2 were in class IV and 80% of grade 3 were in class
IV (Table 2).
- There was a significant statistical correlation between grades of B:T cell
infiltrates and activity index, as it was found that 54.5% of grade 0 (non B cell
group) were moderately active LN (low AI), while the majority of grade 1 cases
showed higher AI; as 45% were active LN and 25% were extremely active LN.
Also in grade 2 about 95% showed higher AI and 80% of grade 3 were extremely
active LN (Table 2).
- There was a significant statistical correlation between B: T cell infiltrates and
chronicity index as most cases (90.9%) of grade 0 (non B cell group) showed low
CI (18.2% shows no obvious chronic changes and 72.7% shows weak chronic
changes), while (85%) of grade 1 showed higher CI (60% shows weak chronic
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changes and 25% shows widespread chronic changes), whereas 47.6% of grade 2
and 80% showed widespread chronic changes (Table 2).
- There was a significant statistical correlation between B:T cell infiltrates and
interstitial inflammatory infiltrate, as all cases(100%) of grade 0 (non B cell group)
had (+1) interstitial inflammatory infiltrate. Sixty percent (60%) of grade 1 had
(+1) inflammatory infiltrate. In grade 2 (71.4%) had (+2) inflammatory infiltrate.
While in grade 3, 40% had (+2) inflammatory infiltrate and 60% had (+3)
inflammatory infiltrate (Table 2).
- There was a significant statistical correlation between B:T cell infiltrates and
proteinuria , as 54.5% of grade 0 (non B cell group), 80% of grade 1, 90.4% of
grade 2 and 80% of grade 3 have (+4) proteinuria (Table 2).
- There was a significant statistical correlation B &T cell infiltrates and serum
creatinin level , as it was found that 81.8% of grade0 (non B cell group) had
normal serum creatinin while 55% of grade 1,81% of grade 2, 100% of grade 3 had
elevated serum creatinin level (Table 2).
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Table (2): shows correlation between B and T cell grades with clinicopathologic variables:
B and T cell grades
B cell group
Total
Significance
Classes
II
III
IV
Non B cell
group
Grade 0
4(36.4%)
3(27.3%)
3(27.3%)
Grade 1
2(10%)
5(25%)
7(35%)
Grade 2
1(4.8%)
1(4.8%)
13(61.9%)
Grade 3
0(0%)
0(0%)
4(80%)
7
9
27
V
VI
1(9%)
0(0%)
4(20%)
2(10%)
2(9.5%)
4(19%)
0(0%)
1(20%)
7
7
6(30%)
1(4.8%)
1(20%)
14
9(45%)
5(25%)
7(33.3%)
13(61.9%)
0(0%)
4(80%)
20
23
2(18.2%)
3(15%)
1(4.8%)
0(0%)
6
8(72.7%)
12(60%)
10(47.6%)
1(20%)
31
1(9.1%)
5(25%)
10(47.6%)
4(80%)
20
11(100%)
0(0%)
0(0%)
12(60%)
7(35%)
1(5%)
3(14.3%)
15(71.4%)
3(14.3%)
0(0%)
2(40%)
3(60%)
26
24
7
P =0.0001(Sig)
2(18.2%)
3(27.3%)
0(0%)
6(54.5%)
0(0%)
3(15%)
1(5%)
16(80%)
0(0%)
1(4.8%)
1(4.8%)
19(90.1%)
0(0%)
1(20%)
0(0%)
4(80%)
2
8
2
45
P=0.019 (Sig)
9(81.8%)
2(18.2%)
9(45%)
11(55%)
4(19%)
17(81%)
0(0%)
5(100%)
22
45
p= 0.0001(Sig)
11
20
21
5
57
Activity index
Moderately
6(54.5%)
active
Active
4(36.4%)
Extremely
1(9.1%)
active
Chronicity
index
No chronic
changes
Weak chronic
changes
Widespread
chronic
changes
Inflammator
y infiltrate
Mild (+1)
Moderate (+2)
Severe (+3)
Proteinuria
+1
+2
+3
+4
Serum
creatinin
Normal
Elevated
Total
P=0.003(Sig)
P=0.0001(Sig)
P=0.002(Sig)
Page 13
- There was no significant statistical correlation between progression and activity
index of lupus nephritis. As it had been found that 50% of non progressors and
40% of progressors were extremely active LN. So, progression is not associated
with increase in the AI (P value>0.05).
- There was a significant statistical correlation between progression and chronicity
index of LN .As seventy five percent (75%) of non progressors were without
obvious chronic changes while all
progressors (100%) showed
widespread
chronic changes. (P value=0.0001) (Table 3).
- There was a borderline significant statistical correlation between progression of
the disease and the interstitial inflammatory infiltrate, as 75% of non progressors
have mild (+1) infiltrate while the progressors shows more infiltrate as 60% have
(+2) infiltrate and 20% have +3 infiltrate (P value=0.079) (Table 3).
- There was no significant statistical correlation between progression of lupus
nephritis and proteinuria. As seventy five percent (75%) of non progressors and
90% of progressors have (+4) proteinuria (P value >0.05) (Table 3).
- There was a significant statistical correlation between progression of lupus
nephritis and grades of B:T cell infiltrates as 25% of non progressor were grade 0
(non-B cell group) and 62.5% of the same group were grade (1) while all
prorgressors were in the B &T cell group (B- cell group) ( P value=0.006) (Table
3).
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Table (3): progression of lupus nephritis in relation to clinicopathologic variables and B & T
cell grades:
Variable
LN classes
Class III
Class IV
Class V
AI
Moderately
active
Active
Extremely active
Progression of LN
Non
Progressor
progressor
Significance
Total
2(25%)
6(75%)
0(0%)
2(20%)
7(70%)
1(10%)
4(22.2%)
13(72.2%
1(5.6%)
P>0.05(Insig)
0(0%)
2(20%)
2(11.1%)
P>0.05(Insig)
4(50%)
4(50%)
5(40%)
4(40%)
8(44.4%)
8(44.4%)
6(75%)
2(25%)
0(0%)
0(0%)
6(33.3%)
2(11.1%)
0(0%)
10(100%)
10(55.6%)
6(75%)
1(12.5%)
1(12.5%)
2(0%)
6(60%)
2(20%)
8(44.4%)
7(38.9%)
3(16.7%)
P=0.079(borderline
sig)
1(12.5%)
1(12.5%)
6(75%)
1(10%)
0(0%)
9(90%)
2(11.1%)
1(5.6%)
15(83.3%)
P >0.05(Insig)
2(25%)
0(0%)
2(11.1%)
P =0.006(Sig)
5(62.5%)
1(12.5%)
0(0%)
3(30%)
4(40%)
3(30%)
8(44.4%)
5(27.8%)
3(16.7%)
8
10
18
CI
No changes
Weak chronic
changes
Widespread
chronic changes
Inflammatory
infiltrate
Mild(+1)
Moderate(+2)
Severe(+3)
Proteinuria
+2
+3
+4
B & Tcell
grades
Non B cell
group
B cell group
Grade 1
Grade 2
Grade 3
Total
P=0.0001(Sig)
Page 15
Discussion:
Lupus nephritis is an immune complex GN & it is considered one of the
most and serious complication of SLE (Lech & Anders., 2013). Up to 60% of SLE
patients develop lupus nephritis (Hseih et al., 2011). The pathogenesis of lupus
nephritis involves a variety of pathogenic mechanisms. Although the pathogenesis
of lupus nephritis is not clear, production of pathogenic antibodies is traditionally
viewed as the principle contribution of B cells to the pathogenesis of immunemediated glomerulonephritis. However, it is increasingly appreciated that B cells
have a much broader role in such diseases, functioning as antigen-presenting cells,
regulators of T cells, macrophages, and dendritic cells, as well as being involved in
the formation of local lymphatic expansion (Shen et al., 2012).
In this study the mean age of studied cases was 28.05 and SD was ±9.01,
and the majority of cases was in the second and third decades. This agrees with
Rus et al (2007) and Brent et al (2014); who reported that LN patients typically
occurs in patients aged 20-40 years.
In agreement with our results Sada & Makino (2009), Arfaj et al (2009),
Brent et al (2014) and Satirapoj et al (2015) found that female to male ratio was
nearly 9:1 and this may be explained by that endogenous sex hormones have a role
in disease predisposition.
Currently; very few publications are available concerning the demographic,
clinical, and pathological features of LN and their correlations in developing
countries (Shariati-Sarabi et al; 2013, Chen et al; 2014 and Satirapoj et al.;
2015).
Page 16
In this study; the activity index of LN was increasing from class II to class
IV, while 57.1% of class V were active LN, whereas, 57.1% and 42.9% of class VI
were moderately active and active LN respectively, and although the results were
statistically insignificant; yet from the table 66.7% of class III were active and
74.1% of class IV were extremely active. Similar to our results; Satirapoj et al
(2015) found that class III and IV had a higher activity index. This may be
explained by the fact that Class V showed no residual activitiy unless it is
combined with III or IV and class VI is advanced sclerosing lesions.
This study showed that CI was progressing with higher classes, as 71.4% of
class II and 66.7% of class III showed weak chronic changes; while 71.4% and
71.4% of class V and VI respectively showed widespread chronic changes and the
results were statistically significant. This result was parallel to that of Nasri et al
(2014) & Satiroapoj et al (2015) & Fugo et al (2014). This could be explained
by; as the lupus nephritis disease progress from class I to class VI the activity
index decrease while chronicity index increase to be the highest in class VI which
is termed advancing sclerosing lupus nephritis.
However Hseih et al (2011) study showed that ISN/RPS Class III or IV
proliferative nephritis was somewhat more common in subjects with severe
interstitial nephritis (which had +3 inflammatory infiltrate), our study revealed that
the score of interstitial inflammatory infiltrate was increasing with higher classes
as 71.4% of class II showed +1 inflammatory infiltrate while 100% of class VI
showed +3 inflammatory infiltrate (P value = 0.004). Thus, the study concluded
that severe interstitial nephritis may be widespread regardless of ISN/RPS lupus
nephritis class and this could be explained by the pathogenesis of glomerular and
interstitial pathology in lupus nephritis had different mechanisms (Chan et al.,
2007).
Page 17
This study revealed that proteinuria may be high regardless the class of
lupus nephritis as patients in class II,III,IV,V,VI were 42.9%, 88.9%, 85.5%, 85.9,
100% respectively had +3 & +4 proteinuria (significant statistical correlation P
value=0.003) ,and this also had been observed by Makni et al (2009). This finding
could be explained by that podocytes are highly specialized epithelial cells that
form part of the filtration barrier in the kidney, and their loss reflects a malfunction
in glomerular filtration and result in loss of protein in urine (Bollian-y-Gotia et al;
2011).
In agreement with Nezhad & Sepashkah (2008) and Satirapoj et al.
(2015) studies; our study revealed significant statistical correlation between lupus
nephritis classes and serum creatinine (P value= 0.003) as 71.4% of class II had
normal serum creatinine and all the seven cases (100%) of class VI had elevated
serum creatinine. This could be explained by predominant chronicity of these cases
in form of diffuse global glomerulosclerosis, tubular atrophy and interstitial
fibrosis is often will be associated with loss of renal function (elevated serum
creatinine). So, we can concluded that serum creatinin and histologic evaluation of
LN patients are important prognostic factors.
There was significant statistical correlation between different classes of
lupus nephritis and intrarenal B &T cells ((P value=0.003); as high grades of B&T
cell infiltrate (grade 2&3) were mainly in class IV) agrees with Shen et al (2012)
and Sun et al (2013) studies who noted that the B cell infiltration was more likely
to be associated with class IV LN (the most aggressive class). This could be
explained by that pathogenic B cells have a variety of functions such as producing
autoantibodies that can cause renal damage via disruption of cellular functions,
cytotoxicity mediated by interactions with complement and release of
inflammatory mediators. And MRL/lpr lupus-prone mice that have B cells unable
Page 18
to secrete antibodies still develop nephritis, this may indicate that there are
additional B-cell functions, such as antigen presentation and activation of
pathogenic T cells and proinflammatory cytokine production which contribute to
kidney injury (Nowling & Gilkeson., 2011).
Expression of B:T cell infiltrates was increasing in ascending manner with
activity and chronicity indices (P value = 0.0001,0.002 respectively); as 54.5% of
non B –cell group had low AI (Active LN) and 72.7% of non B-cell group showed
weak chronic changes while 80% of grade 3 had a higher AI (Extremely active
LN) and widespread chronic changes and this result was in line with Shen et al
(2012) study. This finding suggest the role of B & T cell infiltrates in increasing
the inflammatory process in lupus nephitis as Apostolidis et al (2011) reported
that mechanisms by which T cells contribute to tissue injury include activating and
providing help to nephritogenic antibody-producing
B cells, recruiting
macrophages and dendritic cells (DCs), and producing cytokines.
It was not a striking finding when we found that B:T cell
infiltrates
correlated with the extent of inflammatory infiltrate (as it is a part of a whole) (p
value = 0.0001). Hseih et al (2011) study confirms this finding and this increases
the suggestion of the dominant roles of B and T cell infiltrates in pathogenesis and
progression of lupus nephritis.
Although there was a significant statistical correlation between B:T cell
infiltrates and proteinuria (p value = 0.019), it may not be considered a true
significancy as 54.5% of the non B-cell group and 80% of grade 3 had (+4)
proteinuria. This result against Sun et al (2013) study, which found that the level
of proteinuria (g/24 hours) in grade 0 was lower than other grades. It could be
explained by that proteinuria is highly variable and reversible clinical presentation.
This finding may suggest the role of B and T cells in prtoteinuria as recruitment of
Page 19
B & T cells occur parallel to binding of podocytes to dsDNA which cause
widespread, segmental effacement of podocyte foot processes that leads to
proteinuria (Nowling & Gilkeson; 2011 , Yung & Chan; 2012).
About eighty two percent of non B- cell group had normal serum creatinine,
while all cases of grade 3 had elevated serum creatinine. There was a significant
statistical correlation between B:T cell aggregates and serum creatinine level (p
value = 0.0001). Shen et al (2012), Sun et al (2013) studies were in line with us. It
may be explained by that B cells serve as a local immune system & have an
important role in deteriorating renal function in lupus nephritis & in progression of
renal diseases; Shen et al (2012) was beside my suggession and reported that
intrarenal B cells form part of a local system that enhances the immunological
response by functioning as antigen presenting cells, and as a source of cytokines
promoting T cell proliferation and lymphatic neoangiogenesis. In this way,
intrarenal B cells could aggravate the local immune response to persisting
autoantigens in the tubulointerstitium, resulting in persistence and chronicity of
renal inflammation. And this explanation could be confirmed by Sarwal et al
(2003) and Heller et al (2007) studies which confirm the role of B cells in
deteriorating renal function by demonstrating that the accumulation of CD 20
positive B cells in the tubulointerstitium was associated with steroid resistant acute
allograft rejection and graft loss.
B cells are recruited in the kidney via specific chemokines such as BCA1/CXCL13 and its specific receptor CXCR5, and local expression of B-cell
survival factors such as APRIL and BLyS (which are important factors for
intrarenal B cells in lupus nephritis patients). Antagonists of these chemokines,
such as anti-BAFF/BCA-1 antibodies or corresponding receptors may have
Page 20
therapeutic efficacy in patients with autoimmune disorders of this type (Neusser et
al., 2011).
In agreement with Kiremitci and Ensari (2014) who reported that the
ISN/RPS classification provided significant advances in the handling of renal
biopsies of SLE patients, but does not sufficiently stress the involvement of
nonglomerular compartments such as the tubulointerstitium which may also be
related to the prognosis, as 90% of progressors and all (100%) non progressors
were in class III & IV. There was no significant statistical correlation between
class and progression, and this suggests that class could not predict renal outcome
in lupus nephritis patients and another factor is needed. Also Dolff et al (2010) and
Hseih et al (2011) reported that Class III and IV LN have several pathogenic and
histopathological similarities and patients with these types of LN share a poor long
term renal survival due to deterioration of kidney function. So; the aim of clinical
& the first therapeutic goal is to reduce disease activity and to restore renal
function or to prevent deterioration in both classes of lupus nephritis.
Our result revealed that AI provided no prognostic value in LN progression;
as there was no significant statistical correlation between AI and progression of
lupus nephritis (P>0.05). (50%) of non progressors and (40%) of progressors
showed extremely active LN. This result agrees with Hseih et al (2011) and
Jaipaul (2013) studies but against Kammoun et al (2011) study which revealed
that high AI has been shown to predict poor renal outcome in LN patients. This
could be explained by that the activity score is based on the degree of glomerular
inflammation, which is more reversible with treatment.
The chronicity index was increasing with progression of lupus nephritis (P =
0.0001); as 75% of non progressors showed no obvious chronic changes and all
progressors 100% had widespread chronic changes. Parallel to us Hseih et al
Page 21
(2011) and Jaipaul (2013) reported that the chronicity index predicts progression
of lupus nephritis to renal failure; and this could be explained by the fact that
crescent formation, glomerular sclerosis, tubular atrophy and interstiatial fibrosis
are entities of CI will impair creatinine clearance with subsequent elevated serum
creatinine. Nasri et al (2014) confirm this suggestion by finding that the serum
creatinine (which progression in our study is based on) positively correlated with
proportion of glomeruli with crescents. The progression occurs by contribution of
anti-ds DNA antibody (main antibody in LN) into tubulo-interstitial fibrosis in
lupus nephritis by induction of
both cell-associated and soluble fibronectin
secretion in (proximal tubular epithelial cells) PTEC, the former adds to
extracellular matrix deposition while the latter amplifies the fibrotic process
through induction of TGF-β1 and collagen type I (Yung et al., 2015).
Parallel to Makni et al (2009); our result revealed no significant statistical
correlation between proteinuria and progression of lupus nephritis (p>0.05),so
proteinuria could not used as an indicator for progression of LN. However Falk et
al (2014) reported that proteinuria may have a role in pathogensis of progression
of renal diseases based on response of the renal tubules to proteinuria, as it causes
increased tubular synthesis of macrophagic and other chemokines, with increased
tubular cellular proliferation and apoptosis, (apoptosis more than proliferation)
leading to interstitial inflammation and fibrosis. So, medical controlling and
diminution of proteinuria may leads to slowing of progression.
Grades of B:T cell aggregates and interstitial inflammatory infiltrate was
increasing with progression of LN cases (P value = 0.006, 0.079 respectively), as
25% of the non progressor group was (grade 0) (non B-cell group) and 62.5%was
grade 1, while 70% of the progressor group were grade 2 and grade 3(a significant
statistical correlation). This result runs parallel to Yu et al (2010) and Bagavant
Page 22
& Fu (2009) studies. The current study suggets importance of intrarenal B cells in
progression of lupus nephritis as it can be used as a predictor for progression.
This study suggested that B cells may have a role in interstitial fibrosis;
However; little was known about the role of B cells in the process of renal
interstitial fibrosis, as Heller et al (2007) study support our hypothesis by
demonstrating that a model of CCI4-induced liver fibrosis approximately half of
the infiltrating cells in the liver were found to B cells. Eliminating B cells
significantly improved liver injury with decreases collagen deposition. This
improvement was not T-cell or immunoglobulin-dependent. It may be explained by
that B cells might be involved in interstial injury through release of cytokines and
chemokines, as antigen-presenting cells, via augmentation of T-cell responses and
through neolymphogenesis. Regarding kidney, Sun et al (2013) described the high
prevalence of intrarenal B cells in immune mediated diseases, including renal
transplant rejection and glomerulonephritis, local B-cell infiltrates could play a
role in tissue injury such as fibrosis, neolymphangiogenesis, and ectopic
lymphomagenesis by deposition of immune complexes and/or autoantibodies
which induce the secretion of cytokines in the renal resident cells, which promote
the infiltration and activation of leukocytes (T & B cells) which also produce
cytokines that leads to the further immune response and disease progression .
Conclusion:
The present work reported that the presence of intrarenal B & T cell
infiltrates and their classification into grades according to microanatomical
organization in patients suffered from lupus nephritis could suggest the role of
intrarenal B & T lymphocytic infiltrates in pathogenesis and progression of lupus
nephritis.
Page 23
The current study revealed that the non B-Cell group (grade 0) had lower
histopathological and clinical parameters, while different grades of the B-Cell
group had elevated histopathological and clinical parameters in ascending manner
with CD20 & CD3 expression. This finding may suggest the role of B cell in
deteriorating the histopathological and clinical conditions of the patient.
This finding suggests that trials of treatment with anti CD20 may provide a
new strategy in management of LN especially in resistant cases to the ordinary
medical protocol.
Figure (1): shows negative immunohistochemical staining of CD20 in LN,
grade 0 (non-B cell group). (CD20 ×400).
Page 24
Figure (2): shows immunohistochemical staining of CD3 in previous case of
LN: scattered positive T cells (grade 0) (CD3 ×400).
Figure (3): shows immunohistochemical staining of CD20 in LN, cluster like
structure of CD20 positive B cells (grade 2) (CD20 ×400).
Page 25
Figure (4): shows immunohistochemical staining of CD20 in LN: clearly
distinguishable zone of CD20 positive B cells (grade 3)(CD20 ×400).
Figure (5): shows immunohistochemical staining of CD3 in previous case of LN,
clearly distinguishable zone of CD3 positive T cells (grade 3) (CD3 ×400).
Page 26
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