Renal Histopathology and Transcriptomic Analysis in Hepatorenal Syndrome During Liver Transplantation
Phase: NA
Conditions: Hepatorenal Syndrome, Liver Regeneration, AKI - Acute Kidney Injury, Liver Transplant
Sponsor: The University of Hong Kong

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Renal Histopathology and Transcriptomic Analysis in Hepatorenal 
Syndrome during Liver Transplantation 
 
STUDY PROTOCOL 
 
 
 
 
 
 
 
 
 
Principal Investigator 
 
Professor Tiffany Wong 
 
Clinical Associate Professor 
Department of Surgery, The University of Hong Kong 
Tel: 2255-3025     Fax: 2817-5475      Email: wongtcl@hku.hk 
 
 
 
Co-Investigators 
 
Prof. Albert Chan1, Dr. Wing Chiu Dai1, Dr. Kin Pan Au 1 
 
Dr. James Fung2, Prof. Walter Seto2, Prof. Man Fung Yuen2, Prof. Desmond Yap2, Prof. Tak Mao Chan2 
 
1 Department of Surgery, The University of Hong Kong 
2 Department of Medicine, The University of Hong Kong 
 
 
This study is supported by General Research Fund (GRF), University Grants Committee (UGC) in 2025/2026 (Ref 
no.17117825). 
 
 
Registration on Clinicaltrials.gov - NCT no: xxxxxxxxxxx 
 
 
 

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Background 
 
Hepatorenal Syndrome (HRS) is a severe complication of cirrhosis characterised by functional renal impairment in the 
absence of structural kidney disease.  The diagnostic criteria for HRS have evolved significantly over time, with the 
International Club of Ascites (ICA) revising the criteria in 2019 and harmonizing with the definition of acute kidney injury 
(AKI) established by the Kidney Disease: Improving Global Outcomes (KDIGO).  Type 1 HRS was renamed as HRS AKI. 
 
There are two key notions about HRS AKI in the literature: (1) it is a functional disorder that typically occurs in the context 
of structurally normal kidneys, and (2) it is reversible with timely liver transplantation (LT). However, no one has ever 
documented renal histology in HRS AKI patients.  The conclusion was only supported by post-mortem histological 
examination showing near-normal renal histology despite severe renal impairment in cirrhotic patients. Post-mortem studies 
have significant limitations, as they only provide a snapshot of the kidney's condition after death. 
 
Our preliminary study from December 2017 to December 2023 recruited 54 liver transplant recipients for renal biopsy at 
the time of LT.  We found a high prevalence of structural abnormalities in LT recipients, with up to 96.2% having 
abnormal renal histology.  This challenges the conventional understanding that HRS AKI occurs in structurally normal 
kidneys.  Hence, we would like to examine the actual structural changes in the kidneys of patients with HRS through 
tissue biopsy and advanced molecular analysis, and hope that may help us better understand the disease mechanism and 
potentially improve treatment approaches.  We believe that this study will provide valuable insights into the 
pathophysiology of HRS and may lead to improved diagnostic and treatment strategies in the future. 
 
Objectives 
 
This study aims to examine the actual structural changes in kidneys of patients with HRS through tissue biopsy and 
advanced molecular analysis, and hope that may help us better understand the disease mechanism and potentially improve 
treatment approaches. 
 
Primary Objective 
 
To refute the null hypothesis that HRS AKI typically develops in the context of structurally normal kidneys. 
 
Secondary Objectives 
 
1. To assess if the histological findings on renal biopsy in HRS AKI patients are reversible following liver 
transplantation 
 
2. To define the single-cell spatial transcriptomic landscape in HRS AKI 
 
3. To compare and delineate the differential alterations in transcriptomic profiles between HRS AKI and non-HRS 
AKI cohorts 
 
 
Study Design 
 
This is a 3-year single center prospective, non-randomised, open label study at Queen Mary Hospital, The University of 
Hong Kong.  All consecutive patients accepted on the liver transplant waiting list will be invited to participate.  Patient 
will undergo several procedures related to liver transplant and kidney assessment, and receive liver tranplantation and 
renal biopsy. 
 
Procedures 
 
1. Before Liver Transplantation 
 
Patients will receive standard evaluation and preparation for liver transplant.  Blood and urine samples will be 
collected to measure kidney function markers, and kidney function will be monitored regularly through blood 
tests. 
 

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2. During Liver Transplantation 
 
All transplant procedures will be performed in standard manner.  Intraoperative renal biopsy will be performed by 
operating surgeons following liver graft implantation and reperfusion.  Two cores of renal tissue with minimal 
length of 8mm will be obtained using a 14-gauge automated biopsy needle.  This additional procedure will be 
performed as part of the transplantation and is expected to add minimal extra risk. 
 
3. After Liver Transplantation 
 
Following transplantation, patients will receive standard post-operative care and monitoring.  Regular blood test 
will be performed to monitor liver and renal function.  Blood and urine samples will also be collected periodically 
for research purposes.  Standardized immunosuppression protocol including induction agent (Basiliximab), 
perioperative hydrocortisone, and maintenance regimen with mycophenolate mofetil and calcineurin inhibitors 
will be applied and montior closely. 
 
4. 3 Months After Liver Transplantation 
 
a. Second Renal biopsy 
 
Ultrasound-guided renal biopsy will be performed by radiologists using a 14-gauge automated needle to 
obtain two cores of renal tissue.  Two small tissue samples will be taken for comparison with the first 
biopsy. 
 
b. Laboratory Analysis 
 
The kidney tissue samples will be examined under a microscope to assess structural changes. Advanced 
molecular analyses will be performed to understand cellular changes, and blood and urine samples will be 
analysed for renal function markers. 
 
Histopathological and Transcriptomic Analysis 
 
Histopathological Assessment 
 
• 
Quantification of glomeruli and scoring of scarring and fibrosis 
• 
Assessment of glomerular, tubular, and interstitial changes 
• 
Specialized stains including PAS, silver stain, trichrome stains 
• 
Immunohistochemical and immunofluorescence studies 
• 
Electron microscopic examination 
 
Transcriptomic Analysis 
 
High-resolution single-cell spatial transcriptomic analysis using 10x Visium HD technology will be 
performed on formalin-fixed paraffin-embedded tissue samples. Sequential Fluorescence In Situ 
Hybridization (seqFISH) and uniform manifold approximation and projection (UMAP) will be used to 
identify distinct cellular populations and spatial gene expression patterns. 
 
 
Biomarker Monitoring 
• 
Serum Cystatin C for precise measurement of renal function 
• 
Neutrophil Gelatinase-Associated Lipocalin (NGAL) - elevated in acute tubular necrosis but lower 
in HRS AKI 
• 
Interleukin-18 (IL-18) - elevated in acute tubular necrosis but lower in HRS AKI 
 
 
 
 

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Outcome 
 
Primary Outcome 
 
• 
Proportion of patients with HRS AKI who have normal, reversible or permanent injury on renal histopathology 
• 
Histopathological changes and their association with clinical parameters 
 
Secondary Outcomes 
 
• 
Incidence and duration of HRS AKI 
• 
Response to standard treatment (Terlipressin and albumin) 
• 
Perioperative morbidity including need and duration for renal replacement therapy 
• 
Post-transplant renal recovery rates 
• 
Transcriptomic signature differences between HRS AKI and non-HRS AKI patients 
 
Selection and Withdrawal of Subjects 
 
42 patients will be recruited to this study over a 2-year period.   
 
According to our preliminary study, 50 out of 54 patients (92.6%) had abnormal renal biopsy results at the time of LT.  
The difference in proportion between abnormal and normal histology was 85.2% (= 92.6% – 7.4%).  To ensure our study 
will be adequately powered, we will assume the difference to be 80%, with alpha (α) at 0.05 and power (1-β) at 0.9, based 
on: 
𝑛𝑛=
൬𝑍𝑍𝛼𝛼
2 + 𝑍𝑍𝛽𝛽൰
2
[𝑝𝑝1(1 −𝑝𝑝1) + 𝑝𝑝2(1 −𝑝𝑝2)]
(𝑝𝑝1 −𝑝𝑝2)2
 
 
The sample size is around 31.7 i.e. 32 patients will be needed.  Assuming 30% dropout rate, 42 patients will be recruited 
in the study. 
 
Subject enrollment criteria 
 
Inclusion Criteria 
• 
Patient must be able to understand and provide informed consent 
• 
Age ≥ 18 years at time of study entry 
• 
Accepted for LT 
• 
Fulfill the diagnostic criteria of HRS AKI according to International Club of Ascites (ICA) guidelines 
 
Exclusion Criteria 
• 
Inability or unwillingness to give written informed consent 
• 
Patient with known pre-existing renal disease 
• 
Patient with solitary kidney 
• 
Re-transplantation 
• 
ABO-incompatible LT 
• 
Fail to provide 3-month post transplant renal biopsy 
 
3-month Post-transplant Renal Biopsy 
 
Inclusion Criteria 
• 
Patient with adequate intraoperative renal biopsy 
• 
Patient must be able to understand and provide informed consent 
 
Exclusion Criteria 
• 
Graft failure after LT 
• 
Any condition deemed inappropriate by the Principal Investigator 

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Diagnosis and Definition of HRS AKI 
 
The definition of HRS AKI is adopted from the latest diagnostic criteria by the International Club of Ascites (ICA): 
 
Diagnostic Criteria 
1. Presence of cirrhosis with ascites 
2. Diagnosis of AKI: increase in serum creatinine by ≥ 0.3 mg/dL (≥ 26.5 µmol/L) within 48 hours, or percentage 
increase in serum creatinine of ≥ 50% from baseline within prior 7 days, or urine output < 0.5ml/kg/hr in 6 hours 
3. Absence of shock 
4. No current or recent nephrotoxic drug use 
5. No improvement after volume expansion with albumin (1g/kg body weight per day up to maximum 100g/day for 2 
consecutive days) 
6. Exclusion of other renal disease through urinary analysis and kidney imaging 
 
 
Assessment of Safety and potential risks 
 
Standard liver transplantation procedures are employed.  Intraoperative renal biopsy is a well-established procedure with 
minimal additional risk. Post-transplant renal biopsy carries standard procedural risks including bleeding, infection, and 
pain, with overall complication rates < 1%. 
 
Close monitoring for complications will be performed, including assessment using the Clavien-Dindo classification 
system. All adverse events will be documented and reported according to institutional guidelines. 
 
Liver Transplantation 
 
Liver transplantation is a standard, life-saving procedure with a success rate over 90%. Common risks include surgical 
complications, infection, rejection, and side effects of immunosuppressive medications etc.  These risks will be minimized 
through experienced surgical teams, sterile techniques, and close monitoring. 
 
Intraoperative Renal Biopsy 
 
Obtaining kidney tissue during liver transplantation carries minimal additional risk beyond the standard transplant 
procedure.  Potential complications include pain at the biopsy site, minor bleeding (which usually stops on its own), and 
infection (rare).  These will be minimised by performing the biopsy with sterile equipment and experienced surgeons. 
 
Post-transplant Renal Biopsy 
 
The renal biopsy under ultrasound guidance may cause risks such as pain at the biopsy site, bleeding requiring 
hospitalisation (less than 1%), infection (rare), or very rarely, the need for blood transfusion etc.  These risks will be 
reduced by using experienced radiologists and proper monitoring during and after the procedure. 
 
Blood and Urine Collection 
 
Routine blood draws may cause temporary discomfort, bruising, or rarely, infection at the needle site.  These risks will be 
minimised by using sterile techniques and skilled personnel. 
 
 
 
 

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Statistical Analysis 
 
Descriptive characteristics of all patients will be described using median (IQR) and/or proportions. Categorical data will 
be analyzed with Chi-square or Fisher’s exact test where appropriate. Continuous variables will be analyzed with Mann 
Whitney-U test. All P values are two-sided, and statistical significance is defined as P value <0.05. All analyses are 
performed by R version 4.4.1. Proportion of different renal histology findings and comparison will be made at time of LT 
and 3 months post-LT between HRS AKI and non-HRS AKI cohorts, and their correlation with pre-LT, intraoperative and 
post-LT variables. Incidence of functional and histological renal recovery will be described, and their correlation with 
clinical parameters will be made between patients who have renal recovery and who do not 
 
 
Duration of Study and Data/Samples Handling 
 
The study duration is approximately 3 years, with 2 years for recruitment and 1 year for follow-up and analysis.  Collected 
personal data and study data will be stored on password protected, encrypted database in the Department of Surgery, with 
the study team permanently as it is possible to be reviewed again in future.  Collected samples will also be stored 
permanently in the department for future use.  Unused tissue samples will be destroyed and discarded upon request, 
however, the results will remain as a part of the overall research data if part of the tissue has been proceeded. 
 
 
Consent 
 
Written voluntary consent will be obtained after full explanation of the study to patients before liver transplantation. 
Additional consent will be obtained for the 3-month post-transplant renal biopsy. 
 
 
Conflicts of Interest 
 
All investigators have no financial interests in the materials used in the study. This study will not receive any financial 
support from organizations that have any relationship with the materials used in this study. 
 
 
Ethics 
 
The study will be performed in accordance with ethical principles that have their origin in the Declaration of Helsinki, and 
applicable regulatory requirements Patient data protection.