Liver transplantation (LT) is the treatment of choice for end-stage liver disease (ESLD), with recent advances in surgical techniques and anti-hepatitis virus C (HCV) therapy having led to improved survival of LT recipients. However, the development of liver steatosis and steatohepatitis after LT might affect clinical outcomes and recipient survival.1-3 It is known that improvement in nutritional status and the use of immunosuppressive drug, such as corticosteroid, can induce liver steatosis after LT.
The prevalence of liver steatosis in western countries is estimated at 18% to 70%,1-5 with nonalcoholic fatty liver disease (NAFLD) being one of the most common causes of chronic liver disease worldwide.6 In Japan, the prevalence of NAFLD has increased with the rise in obesity and metabolic syndrome associated with lifestyle changes.7 Obesity, pre- and post-LT, diabetes, hyperlipidemia, and administration of tacrolimus are risk factors for liver steatosis after LT.1-4 Recently, we have also reported Patatin-like phospholipase domain-containing 3 (PNPLA3) 738409 GG donor genotype as a further risk factor for liver steatosis and steatohepatitis after living-donor LT in patients with HCV infection.8 However, it is controversial that donor factors, including PNPLA3 genotype, could be risk factors for liver steatosis after LT. Moreover, the risk factors and clinical course of steatosis after LT have not been clearly evaluated in Asian countries. Therefore, our aim in this study was to clarify the significant recipient- and donor-specific risk factors for liver steatosis or steatohepatitis after living-donor LT in Japan, and to describe the clinical course of liver steatosis.
MATERIALS AND METHODS
We conducted a retrospective analysis of prospectively collected data for 100 recent recipients, who underwent LT at our institution, between January 2007 and June 2015, with subsequent ultrasound-guided liver biopsy performed at 1 year post-LT. The data of 43 of these cases have previously been reported.8 Relevant recipient and donor characteristics are summarized in Table 1. Written informed consent was obtained from all patients, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by our institution’s research ethics board.
Steatosis in each donor was graded as none (normal ultrasound liver structure), mild (slight increase of echogenicity, normal visualization), moderate (diffuse increase of echogenicity, slight impaired visualization), or severe (marked increase of echogenicity, poor or no visualization). Preoperative liver biopsy was performed selectively in 8 of 100 donors with abnormal liver enzyme or a moderate degree of steatosis on imaging studies. Donors with mild hepatic steatosis (<33%) were considered acceptable candidates for living donation.
Metabolic syndrome was defined by obesity in combination with any 2 of the following abnormalities: dyslipidemia, hypertension, and hyperglycemia.
Assessment of Liver Histopathology and Steatosis
All recipients underwent ultrasound-guided liver biopsies at 1 year post-LT, and on a yearly basis, thereafter, and as needed, when abnormal liver enzyme levels were detected. Donor steatosis was assessed from biopsies performed at the time of graft harvesting. Biopsy specimens were fixed in 10% formalin, embedded in paraffin, cut into 4-μm sections, and exposed to hematoxylin and eosin and azan staining. All liver tissue specimens were evaluated by 2 pathologists, blinded to the clinical condition of the patient. Donor steatosis was assessed by a zero biopsy. Steatosis was defined as a greater than 5% presence of steatosis hepatocyte and nonalcoholic steatohepatitis (NASH), defined by a NAFLD activity score (NAS) of 5 or greater.9 In terms of clinical course, progression was defined as an increase in the NAS.
Univariate analysis (the χ2 or t test as appropriate for the data distribution) was performed to evaluate each factor, with significant factors entered into a multivariate analysis. Multivariate analysis was performed using the multiple logistic regression model. All analyses were performed using SPSS Statistics (version 20.0; IBM Corp., Armonk, NY).
Risk Factors for Steatosis Development in Recipients After LT
Over the median follow-up after LT of 4 years (range, 2~10 years), liver steatosis was identified in 33 cases (33%), with steatohepatitis identified in 9 of these 33 steatosis cases (Tables 2 and 3). The average time to steatosis development after LT was 3.81 ± 2.46 years. With regard to recipient characteristics, patients with steatosis were younger and had a higher body mass index (BMI) at the time of biopsy than those without steatosis (age, 53.4 ± 9.5 vs 57.6 ± 9.9 years, respectively, P = 0.045; BMI, 24.5 ± 4.3 vs 22.6 ± 4.2 kg/m2, respectively; P = 0.046). In contrast, sex, the Child-Pugh score, and diabetes were not associated with steatosis development. The prevalence of steatosis after LT was also greater among patients who received a graft from a donor with steatosis than without (60% vs 23%, respectively, P = 0.001). The prevalence of metabolic syndrome after LT was higher in recipients with steatosis than without (27% vs 8%, respectively, P = 0.019). On multivariate analysis, age (P = 0.023) and donor steatosis (P = 0.005) were retained as independent predictors for steatosis after LT. Moreover, among the 8 donors who had undergone predonation liver biopsy, 3 developed liver steatosis, and 3 developed steatohepatitis.
Risk Factors in Steatohepatitis in Recipients After LT
Among the 9 patients with steatohepatitis, the level of steatosis was 5%-33% in 1 case and 31% to 65% the other 9 cases (Table 4). Among these 9 patients, a grade 1 inflammation was identified in 5 patients and a grade 2 in 4 patients, with 3 patients showing a grade 1 ballooning and 6 a grade 2 ballooning. Moreover, the prevalence of donor steatosis was greater among recipients with steatohepatitis than those with steatosis (88% vs 50%, P = 0.05). There was no influence of age, sex, BMI, the Child-Pugh score, and diabetes on steatohepatitis.
Clinical Course of Steatosis in the Recipients After LT
Among the 33 recipients forming our study group, 26 underwent serial liver biopsies every 2 years, with 6 (19%) showing a progression of the NAS. Among these 6 patients, progression from simple steatosis to steatohepatitis was identified in all cases, with 2 progressing to stage 3 fibrosis. The change in body weight after LT was greater among patients who progressed from simple steatosis to steatohepatitis than those without progression (6.0 ± 3.7 kg vs −1.0 ± 6.2 kg, P = 0.005).
In this study, we clarified the risk factors for liver steatosis and steatohepatitis, as well as describing the clinical course of steatosis after LT. The inclusion of yearly or biyearly biopsies post-LT is a strength of our study.
The average time to steatosis development after LT was 3.81 ± 2.46 years, which was relatively short and might be related to the rapid improvement in nutritional status after LT, as well as the use of immunosuppressive agents, such as steroids and calcineurin inhibitors, which can increase glucose tolerance.10,11 As pretreatment factors, we identified younger age recipient and donor steatosis as risk factors for steatosis after LT. In terms of age, the prevalence of steatosis was higher among younger than older recipients. Various complications, including infectious disease, among older patients could limit improvement in nutritional status after LT, which would lower the risk for steatosis after LT.
Donor steatosis was associated with both steatosis and steatohepatitis. Considering our recent finding that donor PNPLA3 genotype was associated with steatosis, NASH and fibrosis progression after LT,12-15 it is possible that this could also be a risk factor among LT recipients with HCV.8 Moreover, the prevalence of steatosis in donors whose recipient progressed to steatosis was significantly higher than among donors whose recipients did not progress to steatosis (37% vs 8%, P = 0.002). These results indicate that the phenotype in the liver could affect steatosis and steatohepatitis after LT. In studies with populations from western countries, donor steatosis was not found to be associated with steatosis after LT.3,4 This difference might be attributed to between-race differences in genotype. Of note, the prevalence rate of PNPLA3 rs738409 GG genotype ranges between 13 and 19% among Asian populations, compared with 4% among Caucasians and 2% among African Americans.16 Differences in the cause of ESLD could also influence this differing result. In western countries, alcoholic liver disease and NASH are more common etiologies of ESLD than in Asian countries.17-19 Among our study group, alcoholic liver disease was the cause of ESLD in 14% of patients, with NASH being the causative etiology in 2%.
Although BMI before LT was associated with steatosis after LT, an increase in BMI post-LT was also a risk factor of steatosis after LT. Therefore, preventing weight gain after LT would be an important factor to improve clinical outcomes after LT.
About 20% of patients with steatosis among our study group showed a histological progression, with two thirds of these patients progressing from simple steatosis to steatohepatitis, with 2 patients progressing to stage 3 fibrosis. Of note, all patients with steatosis progression had gained weight after LT. The importance of body weight control after LT is supported by evidence (derived from meta-analysis) of weight loss via lifestyle intervention, such as dietary caloric restriction and exercise, as being the first-line treatment for NAFLD.20,21 Therefore, dietary caloric restriction and exercise also could be important for lowering the risk of progression of steatosis after LT.
The limitations of our study need to be acknowledged. Foremost, our study sample was from a single center and was relatively small. Therefore, to validate our conclusions, a large study is needed.
In summary, we identified recipient age and donor steatosis as significant risk factors for liver steatosis after LT. Weight gain after LT was a significant risk factor for the progression of steatosis after LT. The clinical course of steatosis after LT was relatively benign in our study group, with 19% of recipients developing NAS and 7.6 % stage 3 fibrosis. Taken together, our findings indicate that body weight should be carefully monitored after LT in younger recipients and in those who received a graft from a donor with steatosis.
1. Lim LG, Cheng CL, Wee A, et al. Prevalence and clinical associations of posttransplant fatty liver disease. Liver Int
2. Seo S, Maganti K, Khehra M, et al. De novo nonalcoholic fatty liver disease after liver transplantation. Liver Transpl
3. Dumortier J, Giostra E, Belbouab S, et al. Non-alcoholic fatty liver disease in liver transplant recipients: another story of “seed and soil”. Am J Gastroenterol
4. Hejlova I, Honsova E, Sticova E, et al. Prevalence and risk factors of steatosis after liver transplantation and patient outcomes. Liver Transpl
5. Malik SM, Devera ME, Fontes P, et al. Recurrent disease following liver transplantation for nonalcoholic steatohepatitis cirrhosis. Liver Transpl
6. Chalasani N, Younossi Z, Lavine JE, et al. The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Association for the Study of Liver Diseases, American College of Gastroenterology, and the American Gastroenterological Association. Hepatology
7. Kojima S, Watanabe N, Numata M, et al. Increase in the prevalence of fatty liver in Japan over the past 12 years: analysis of clinical background. J Gastroenterol
8. Miyaaki H, Miuma S, Taura N, et al. PNPLA3 as a liver steatosis risk factor following living-donor liver transplantation for hepatitis C. Hepatol Res
9. Kleiner DE, Brunt EM, Van Natta M, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology
10. Chakkera HA, Mandarino LJ. Calcineurin inhibition and new-onset diabetes mellitus after transplantation. Transplantation
11. Hakkera HA, Kudva Y, Kaplan B. Calcineurin inhibitors: pharmacologic mechanisms impacting both insulin resistance and insulin secretion leading to glucose dysregulation and diabetes mellitus. Clin Pharmacol Ther
12. Romeo S, Kozlitina J, Xing C, et al. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nat Genet
13. Kawaguchi T, Sumida Y, Umemura A, et al. Genetic polymorphisms of the human PNPLA3 gene are strongly associated with severity of non-alcoholic fatty liver disease in Japanese. PLoS One
14. Dongiovanni P, Donati B, Fares R, et al. PNPLA3 I148M polymorphism and progressive liver disease. World J Gastroenterol
15. Miyaaki H, Nakao K. Significance of genetic polymorphisms in patients with nonalcoholic fatty liver disease. Clin J Gastroenterol
16. Fan JG, Kim SU, Wong VW. New trends on obesity and NAFLD in Asia. J Hepatol
17. Adam R, Karam V, Delvart V, et al. Evolution of indications and results of liver transplantation in Europe. A report from the European Liver Transplant Registry (ELTR). J Hepatol
18. Alqahtani SA, Larson AM. Adult liver transplantation in the USA. Curr Opin Gastroenterol
19. Shukla A, Vadeyar H, Rela M, et al. Liver transplantation: east versus west. J Clin Exp Hepatol
20. Promrat K, Kleiner DE, Niemeier HM, et al. Randomized controlled trial testing the effects of weight loss on nonalcoholic steatohepatitis. Hepatology
21. Keating SE, Hackett DA, George J, et al. Exercise and non-alcoholic fatty liver disease: a systematic review and meta-analysis. J Hepatol