Good morning everyone.

My name is Nutwara Sukhtungmun and I’m pleased to be here today to

talk to you about the journal that I’m interested.
That’s “HLA-DQA1, -DQB1, and -DRB1 Alleles Associated with Acute
Tubulointerstitial Nephritis in a Chinese Population: A Single-Center
Cohort Study”

Public in The Journal of Immunology with Impact factor 4.718

Firstly, I divided my presentation into four main parts
I will start with Introduction
I will start with some general information on Kidney

The kidneys are the primary functional organ of the renal system.
They are essential in homeostatic functions such as the regulation of
electrolytes, maintenance of acid–base balance, and the regulation of
blood pressure (by maintaining salt and water balance).
And they serve the body as a natural filter of the blood and remove
wastes that are excreted through the urine. 
Kidney have three main regions: Renal cortex, Renal medulla and Renal
pelvis
The disease that involve to kidney are the general term for damage that
reduces function of the kidney
The kidney disease are classified into acute kidney injury and chronic
kidney disease

On this presentation I will focus on Acute kidney injury

Acute kidney injury or AKI is characterized by sudden impairment in
kidney function resulting in the retention of nitrogenous and other waste
products normally cleared by the kidneys lead to dysregulation of
extracellular volume and electrolytes and reduced urine output.
The causes of acute kidney injury can be divided into three categories:
- Prerenal (caused by decreased renal perfusion, often because of
volume depletion)
- Intrinsic renal (caused by a process within the kidneys)
- Postrenal (caused by inadequate drainage of urine distal to the kidneys)

Intrinsic renal that caused by damage to process within the kidneys can
divide into four compartments. Glomerular, Tubular, Interstitial and
Vascular
This journal focus on the Tubulointerstitial compartment
Acute tubulointerstitial nephritis is the common cause of acute kidney injury (AKI) that
Associated with an immune-mediated infiltration of the kidney tubular and interstitial
by inflammatory cells

From this picture, the first one is The histology of Normal kidney
The second is kidney of patient with Acute tubular nephritis and the last is kidney of
patient with acute interstitial nephritis

In ATN patients, the histology of their kidney will display a dilated and cystic tubules,
loss of brush border and found the debris in tubule lumen

And in AIN patients, their kidney histology shows the infiltration of inflammatory cells
into the renal interstitial and edema of interstitial compartment with tubulitis
The diagnosis of ATIN based on clinical features and histology

The clinical features criteria have the three factors

- First is Abnormal renal function such as the elevation of serum creatinine
- Second is Abnormal urinalysis
- And the third is Systemic illness lasting more than or equal to 2 wks
Sign and symptoms of illness such as Fever, weight loss, anorexia, malaise, fatigue,
rash, abdominal or flank pain and arthralgias
and Laboratory findings such as the evidence of anemia, abnormal liver function,
eosinophilia

If the three factors listed are present It defines as “complete criteria” but If less than
three factors listed are present defines as “incomplete criteria”
Besides the clinical features, The renal histopathological is required

This picture shows the renal histology of patient with ATIN with various cause

In picture A show the most common renal histopathology of patient with ATIN. In the renal
interstitial indicated the Infiltration of predominantly lymphocytic cells associated with tubular
damage and tubulitis like you can see at the arrow marked

Picture B show the renal histopathology of patient with Drug-induced tubular injury
The (star: *) indicated the interstitial infiltrate of inflammatory cells
The (Hash sign: #) show the edema of renal interstitial
And the (arrows) marked the tubular regenerative changes

And the last one, when Inflammatory cells infiltrating the tubulointerstitium can form granulomas.
The presence of granulomas on renal biopsy defines Granulomatous tubulointerstitial nephritis
ATIN has multiple etiologies including Infections
Idiopathic
Drug-induced e.g. non-steroid anti-inflammatory drugs (NSAIDs) and
proton pump inhibitors (PPIs)
Systemic inflammatory condition such as
Tubulointerstitial nephritis and uveitis (TINU) syndrome
Sjogren’s syndrome (SS)
IgG4-related ATIN (IgG4-RD)

The previous studies found that have HLA alleles conferring genetic
susceptibility to TINU syndrome
Tubulointerstitial nephritis and uveitis or as known as TINU syndrome is a rare
disorder that present both Tubulointerstitial nephritis and uveitis

Diagnosis of TIN is the same as I mentioned previously

And for Uveitis characteristic, can divide into 2 part
First, Typical defines as bilateral anterior uveitis with or without intermediate
uveitis or posterior uveitis and Onset of uveitis is less than or equal to two
month before of more than or equal to 12 month after TIN

Second is, Atypical defines as Unilateral anterior uveitis or intermediate uveitis

or posterior uveitis or a combination and Onset of uveitis is more than two
months before or more than twelve months after TIN
The picture show the uveitis characteristics
In picture A indicated the anterior uveitis that complicated by posterior synechiae
which the arrow marked

Synechiae is an eye condition where the iris adheres to either the cornea (defines as
anterior synechiae) or lens (defines as posterior synechiae)

In picture B show the Panuveitis that define as the inflammation of all layers of the
uvea eyes. The arrows marked the endothelial precipitates and chronic anterior
synechiae

And Picture C show the fundus photograph of patient with panuveitis demonstrating
the retinal infiltration of inflammatory cell
As previously mentioned,
Small case series found that HLA alleles conferring genetic susceptibility
to TINU syndrome in different population such as
Finland, Spain, Japan and china

From these small case series provided the evidence for relevance of
HLA-DQA1, -DQB1, -DRB1 to TINU syndrome
• The histocompatibility complex gene group provides instructions for
making a group of related proteins known as the human leukocyte antigen
(HLA) complex
• HLA is the human version of the major histocompatibility complex (MHC)

HLA genes are located on the short arm of chromosome 6 and encode
numerous immunologically functional molecules, including HLA class I and II
molecules. HLA genes have extremely high levels of polymorphism and
heterozygosity and are associated with most autoimmune disorders

In this study they focus on HLA class II region

• Those previous studies provided evidence for the relevance of HLA-
DQA1, -DQB1, and -DRB1 to TINU syndrome in different populations.
• However, no studies have focused on HLA genetic susceptibility in
ATIN because of other causes and the specificity of the reported
TINU-related HLA susceptibility alleles has not been well evaluated

• so
The objective of this study are for examination of the HLA-DQA1, -
DQB1 and –DRB1 alleles frequencies of Chinese Han cohort with ATIN
various causes and Analyzed the associations between specific HLA
alleles/haplotypes and drug-induced ATIN (D-ATIN) and TINU syndrome
Now I’m going to move on to the second part, Method
Patients were from a prospective cohort of adults (more than or equal to 18 y) with ATIN that
had been clinicopathologically diagnosed in the Renal Division of Peking University First
Hospital

The enrollment criteria included the following:

1) consent to the study
2) availability of DNA samples from peripheral WBCs
and 3) availability for follow-up for at least twelve month after biopsy.

Patients who had glomerular diseases or hereditary renal diseases were excluded from the
study.

Those who had ATIN secondary to malignancy infiltration or deposition, such as lymphoma,
leukemia, L chain disease, and multiple myeloma, were also excluded
The final enrollment of patient with ATIN was one hundred and fifty-
four then all patient were divided by the cause of ATIN into six
subgroup
Then Clinical parameters and laboratory data were documented.
Estimated glomerular filtration rate (eGFR) was calculated by the Chronic
Kidney Disease Epidemiology Collaboration equatio

Renal dysfunction was evaluated by the levels of serum creatinine (Scr).

AKI was defined and staged according to the Kidney Disease: Improving
Global Outcomes criteria

And Patients were scheduled for monthly follow-ups for six month and
then every three month until at least 1 year after renal biopsy
After that, renal pathology and immunofluorescence was examine.
The Standard processing of kidney biopsy specimens included light
microscopy, immunofluorescence, and electron microscopy.

For light microscopy, all cases were stained with H&E, periodic acid–Schiff,
Masson’s trichrome, and Jones methenamine silver

A pathologic diagnosis of ATIN required both the presence of prominent

interstitial inflammation in the nonfibrotic cortex and tubulitis.
They use Semiquantitative scores for classifying tubular injuries and
interstitial injuries referred to the criteria of the Banff working
classification

For tubular injuries including

- tubular brush border loss, necrosis, and atrophy

- For interstitial injuries including

- interstitial edema, inflammation, and fibrosis

- Then applied a 0–4+ scale as follows:

Immunofluorescence staining was used to identify interstitial-infiltrating
inflammatory cells and expression of HLA-DR and -DQ in renal tubules
and interstitial cells.

The Antibodies were obtained commercially

Antibodies against CD3, CD4, CD8, CD68(sixty-eight), and CD20 were

from Zsbio (Beijing, China)

and Antibodies against CD38, neutrophil elastase, HLA-DR, and HLA-DQ

were from Abcam (Cambridge, U.K.)
Immunostaining results were evaluated using the single-blind method.

All nonoverlapping microscopic fields (x400) of the cortical interstitial

area without glomeruli and vessels were selected.

The number of tubules that were positively stained with HLA-DR/DQ

was counted under 3400 magnification and expressed as the number of
positive tubular cells/3400.
Then, HLA genotyping
Peripheral blood samples with EDTA anticoagulant were collected from patients with ATIN and
healthy controls.

Genomic DNA was extracted using a Gentra Puregene Blood Core Kit C (QIAGEN,
Germantown, MD) and stored at -80 ̊C.

All HLA genotypings were performed in the same laboratory

For HLA-DQA1, the alleles were typed using electrophoresis.

HLA-DQB1 alleles were typed by bidirectional sequencing of exons 2 and 3,

and HLA-DRB1 alleles were typed by bidirectional sequencing of exon 2, using SeCore SBT Kits
Moving on to the next part, results
Altogether, one hundred and fifty four patients who were
clinicopathologically diagnosed with ATIN were enrolled in this study

The causes of ATIN included D-ATIN in seventy-six cases (49.4%), TINU

syndrome in thirty-eight cases (24.7%), SS in twenty-five cases (16.2%),
IgG4-RD in seven cases (4.5%), and other causes in eight cases (5.2%)
The clinical features of ATIN patients are shown in this Table S2
Let’s focus on the confirmatory analysis
to confirm the HLA-B*5701 (five-seven-O-one) in an independent
dataset of one-thousand and two patients who received pazopanib at
various doses, either as monotherapy or in combination with other
agent for solid tumour
It indicated that HLA-B*5701 was borderline significantly associated
with MaxALT and a statistically significant replication of the association
with time to ALT more than 3 and 5 times of ULN event
In combined datasets HLA-B*5701 (five-seven-O-one) was significantly
associated with ALT elevation in pazopanib-treated patients for all three
ALT endpoints

In HLA-B*5701 (five-seven-O-one) carriers and non-carriers, the median

values at twenty-five to seventy-five percentile plot in log-scale of the
MaxALT were 1.7 times of ULN and 1.2 times pf ULN respectively
consistent with 1.4-fold increase carriers estimated by regression
Fig.2 show Cumulative incidence of ALT more than 3 time of ULN in
graph A and ALT more than 5 time of ULN in graph B

At the left side, The frequency of ALT > 3x ULN was thirty-one
percentage in HLA-B*5701 (five-seven-O-one) carriers and nineteen
percentage in non-carriers

And the right, The frequency of ALT > 5x ULN was eighteen percentage
in HLA-B*5701 (five-seven-O-one) carriers and ten percentage in non-
carriers
Within combined data, ALT > 3x ULN and > 5x ULN events occurred in
20% and eleven percentage of pazopanib-treated patients, respectively
with HLA-B*5701 (five-seven-O-one) carriers having higher risk of
experiencing ALT elevation than non-carriers
As a predictor of ALT elevation in pazopanib-treated patients (ever or
never had ALT more than 3 times of ULN,
HLA-B*5701 (five-seven-O-one) carriage improves the positive
predictive value for thirty-one percentage compare with twenty
percentage in all patient but has little impact on the negative predictive
value for eighty-one percentage compare with background eighty
percentage.
The predictive performance was similar for ever or never had ALT more
than 5 times of ULN
And The fraction of patients with ALT elevation potentially attributable
to HLA-B*5701 (five-seven-O-one) in modest: ten percentage for ALT
more than 3 times of ULN and ten percentage for ALT more than 5
times of ULN
In post hoc sensitivity analyses, the excluded one-hundred and ninety-
two patients with baseline ALT more than ULN and observed Slightly
stronger associations between HLA-B*5701 (five-seven-O-one) and ALT
elevation compared with the main analyses that modeled the effect of
baseline ALT as a covariate
Then, A key liver safety signal
• Hy’s law case is assessment of a drug’s potential to cause
severe DILI
• In this study includes 2 components:
• Causality of at least probable in DILI assessment
• Bilirubin fractionation (>30% direct bilirubin) or
absence of Gilbert’s UGT1A1 genotype
• In addition: bilirubin elevation needed to occur at time
of ALT increase and R value (ALT/ALP as fold of ULN) ≥ 5
reflecting hepatocellular injury
And DILIN is The Drug-Induced Liver Injury Network which was
established to advance understanding and research into DILI by
prospective study
- Causality assessment is determined by a panel of hepatologists who
independently.
- Causality score ranging from 1 (definite >95%) to 5 (unlikely <25%)
- Severity score ranging from 1 (mild) to 5 (fatal)
From Hy’s law and DILIN, they characterized twenty-six patients with
laboratory ALT more than 3x ULN and total bilirubin more than 2x ULN

8 patients (33%) had the Gilbert UGT1A1 genotypes

3 patients (%12) were HLA-B*5701 carriers

No patients carried both Gilbert UGT1A1 genotypes and HLA-

B*5701
For all twenty-six cases, This assessment adjudicated 4 cases that meet
criteria for Hy’s Law; of these, two carries HLA-B*5701, the point
estimate 50% is notably greater than 10% of isolated ALT elevations
potentially attributable to HLA-B*5701 (five-seven-O-one)
Then Molecular mechanism
Figure 3 show the model of pazopanib in light pink carbon and abacavir in
orange carbon. Blue represent Nitrogen atom and water molecule retained in
the model represent as red spheres.

Pyrimidine ring of pazopanib can make a pi-methyl stacking

interaction with Val97 And make a pi-edge stack interaction with
Trp147

N1 pyrimidine nitrogen is predicted to form hydrogen bond with

the side chain of Asp114 Similar to the observed interactions of
the purinyl group of abacavir in the E pocket of HLA-B*5701
In the binding mode
the N-methyl of pazopanib occupies a similar region as the cyclopropyl of abacavir
whereas the indazole of pazopanib fills more of the F pocket relative to abacavir
by making additional hydrophobic contacts with Asn77, Ile80, Tyr84, Thr143, and
Trp147.
Finally, the phenyl group of pazopanib is predicted to bind in a similar region as
the cyclopentene group of abacavir in the D pocket of HLA-B*57:01and make
hydrophobic contacts with Tyr9, Tyr99, Leu156, and Tyr159
The sulfonamide group of pazopanib is directed toward solvent and is
predicted to form a water-mediated interaction with the side chain
hydroxyls of Tyr9 and Ser70
An exploratory GWAS meta-analysis for time to ALT more than 3 times of ULN
event using data from all patients in the combined dataset

Did not reveal any common variant associations at genome-wide significance

The most significant GWAS signal was at SNP rs1800625 (one-eight-zero-zero-

six-two-five)

Further evaluated using the combined dataset in a pooled analysis

This SNP maps to the MHC class III region is weakly correlated with HLA-B*5701
carriage
A joint analysis of rs1800625 and HLA-B*5701 indicates that the two
associations are most independent

Then, Discussion