1.                  Introduction

Chronic Hepatitis B Virus (HBV) infection is one of the main causes of chronic liver injury. Around the world about 2 billion people has been infected, and more than 3500 million people were chronic HBV carriers [1,2]. Development of anti-HBV Nucleoside/Nucleotide Analogues(NAs) in the last twenty years have changed the history of Chronic Hepatitis B(CHB) treatment [3,4]. To now five NAs have been approved for CHB antiviral therapy including lamivudine (LAM), adefovir dipivoxil(ADV), telbivudine(LDT), entecavir(ETV) and tenofovir (TDF).

Though NAs can validly inhibit HBV replication and postpone disease progression, these NAs could not eliminate HBV replication template covalently closed circular DNA (cccDNA) and HBsAg clearance is also very rare. As a result, the majority of CHB patients will have to receive long-term or even lifelong NAs treatment to achieve disease remission or serologic endpoints, especially patients with advanced liver disease and cirrhosis[5-6]. As NAs’ wide application, safety data of NAs were of paramount importance in clinical practice. Generally, the security of these NAs is good during registration trials[8-13],but there have been reports of serious adverse events including myopathy, neuropathy, pancreatitis and renal impairment during post marketing surveillance[14-18].

Nephrotoxicity may occur in a small, yet significant, proportion of patients receiving nucleotide analogs. In ADV 5 years registration study, 3%-4% patients developed Serum creatinine (Scr) elevation at average of ≥0.5mg/dL every year and that of controls was 0%[19]. In TDF 5 years registration study, 1% patients also developed similar renal toxicity [20]. Along prolonged ADV application in clinical practice, more and more nephrotoxicity had been reported, including Fanconi syndrome [21-23].

Registration reports of these NAs suggested that the renal injury was largely reversible with dose adjustment or cessation of therapy. But post marketing surveillance found that renal tubular dysfunction is partially reversible with changing to other antivirals in patients received prolonged ADV therapy [24]. In order to evaluate the incidence and outcome of long-term NAs usage related renal tubular dysfunction, 255 CHB patients having been receiving NAs for anti-HBV for more than 6 months were enrolled. Urinary and serum β₂microglobulin (β₂-MG), serum calcium, serum phosphorus and Scr were monitored. Estimated glomerular filtration rate (eGFR) was calculated by using the CKD-EPI equation [25]. Then patients who developed renal injury during antiviral therapy were intervened and followed up prospectively.

2.                  Methods

2.1.              Patients

The observational study was carried out on all patients with CHB who were admitted to the outpatient of the Department of Infectious Diseases of Shanghai Changhai hospital. The study was done during the 3-years period from March 2014 to May 2017.

2.2.              Patient Management and Follow-Up

All patients were subjected to complete history taking by a specialist from the liver diseases center including general social data, antiviral therapy history, viral data, biochemical data, manifestations and renal function data at the start of antiviral treatment. The major inclusion criteria included HBV mono-infection and having been receiving NAs antiviral therapy for more than 6 months at screening.Exclusion criteria included combining with diabetes, chronic renal insufficiency, rheumatic disease, hypertension and other cardiovascular diseases which may cause renal injury.The subjects assigned as disease controls were patients with CHB and first-time disease flare, and all of them did not receive antiviral therapy 6 months before screening and were matched to study groups by age, sex, and clinical diagnosis.

2.3.              Laboratory Tests

Qualitative tests for albuminuria and levels of urine β₂ MG, urine micro-protein, Serum creatinine (Scr), serum calcium, serum phosphorus, HBV DNA load, and alanine aminotransferase (ALT) were conducted. Quantitative analyses of HBsAg, HBeAg, and HBeAb were also performed. Using the data for Scr levels, we calculated the eGFR by using the CKD–EPI equation.

Renal function data at the start of antiviral treatment was determined as the most recent creatinine measurement before antiviral treatment, if available, or the first value obtained on the first three month of antiviral therapy.

2.4.              Treatment Adjustment and Follow-Up

All patients with one or more abnormal renal function indicators at the enrollment were given antiviral strategy adjustment according to the Guidelines for Treatment of Chronic Hepatitis B [26,27] and were followed up for another 52 weeks. In brief, the patients without history of drug-resistant mutation were shifted to other non-cross-resistant antivirals, whereas those with drug-resistant mutations were shifted to ETV 1.0 mg/day or ADV plus LDT.

2.5.              Ethical Aspects

This study was in compliance with Helsinki Declaration and was approved by the Medical Ethics Committee of Shanghai Changhai Hospital. All the enrolled patients gave their written informed consent.

2.6.              Statistical Methods

All data were analyzed with SAS9.4 (SAS Institute Inc., USA). A two-sided p-value < 0.05 was considered as statistically significant. Categorical variables were expressed as number (%), and continuous variables were expressed as mean ± standard deviation (±SD) or median (Q1-Q3). Categorical variables were processed usingx² test. Continuous variables were compared using one-way Kruskal-Wallis or ANOVA analysis in accordance with the normal test result. The logistic regression model or general linear model analysis was used to estimate the risk factors of categorical variables or continuous various respectively.

3.                   Results

3.1.              Demographic and Clinical Data Among Patients with Chronic Hepatitis B

In this study 380 patients with CHB and having been receiving NAs antiviral therapy for more than 6 months were screened. 125 of them were ruled out for being accompanied with hypertension, diabetes, chronic kidney disease or history of liver cancer respectively. At last 255 patients were included in this study. 176 patients were receiving different NAs mono-therapy respectively (ADV, 83 patients; ETV, 39 patients; LAM, 27 patients and LDT, 27 patients) and the other 79 patients were receiving LAM or LDT or ETV plus ADV antiviral therapy at the enrollment. 46/79 of those patients who were receiving NAs combined therapy had drug- resistant mutation history during the long-term antiviral therapy. A total of 25 patients with CHB and first-time disease flare and matched with the treatment groups by age, sex, and clinical diagnosis but did not receive any antiviral therapy prior to the study were included as disease controls.

As shown in Table 1, patients in different treatment groups had good comparability in the gender proportion, liver hardness and Scr level or eGFR value before antiviral treatment. All the treatment groups showed good comparability in age and gender with the disease controls. Patients in LDT therapy group were significantly younger than those in LAM or ADV therapy group (P=0.06, 0.03). Therapy duration of these patients varied from 6 months to 132 months. The median therapy duration of ADV, or LAM or NAs combined therapy group was longer than that of LDT or ETV mono therapy group (all P value <0.05). All patients in therapy groups obtained good viral inhibition and ALT recovery when been enrolled in this study.

3.2.              Renal Function Characteristics of Patients with CHB and Being Receiving Long-Term NAs Therapy

To correct a deviation of age and treatment duration among different therapy groups, general linear model or logistic regression model analysis was used. As showed in Table2, adjusting influence of age and treatment duration, the mean change of eGFR value after long-term NAs antiviral therapy among different therapy groups had no obvious difference(p=0.925). But there were significant difference of serum β₂ MG and urinary β₂ MG abnormal rate among different therapy groups (p<0.0001 Vs p=0.002). Further comparison between any two groups found that there were higher urine β2MG abnormal rate in ADV therapy group than that in LDT therapy group (p=0.032), but there was no significant difference between ADV group and any other therapy groups [LAM(p=0.179), ETV(p=0.203) and NAs combine therapy(p=0.221)]. Unlike urine β₂MG, serum β₂MG mean level in ADV group was higher than both that in LAM therapy group (p=0.041) and ETV group (p=0.007), but there was no significant difference between ADV and LDT therapy group (p=0.45).

3.3.              Relative Risks of Renal Injury During Long-Term NAs Antiviral Therapy in Patients with CHB

Renal injury risk factors for patients with CHB during long-term NAs anti-HBV therapy were analyzed by using general linear model or logistic regression model. The influence factors assessed including age, antiviral strategy and treatment duration.We found that both ADV (OR=1.07,95%CI=0.38-3.04,p=0.0016) and LDT (OR= 0.10,95%CI=0.03-0.40,p=0.0011) therapy were related to urineβ2 MG abnormal rate. But only LDT therapy was related to eGFR value (t=2.33, p=0.0204) (Table 4).Age(OR=1.02，95%CI=1.00-1.05,p=0.0478) was related to both urine β2 MG abnormal rate (Table 3) and eGFR value (t=-4.88, p<0.0001) (Table4).Influence factors included in this study did not show obvious correlation to other renal tubular index, such as serum calcium and serum phosphorus level.

3.4.              Clinical Course of Patients Who Developed Renal Tubular Dysfunction During Long-Term NAs Antiviral Therapy

In this study 135/255(52.94%) patients with CHB developed renal tubular injury and 37 of them (27.40%) were accompanied with mild to moderate eGFR value decrease during long-time antiviral therapy. Only one of all these patients was diagnosed as Fanconi syndrome who with hypophosphatemia and clinical manifestation including fatigue and low limbs ache.117 of the 135 CHB patients finished the 52-weeks’ follow-up and were included into the last data analysis. 59 of these patients continued ADV-including antiviral therapy for another 52 weeks (25 mono therapy and 34 ADV plus LAM or ETV or LDT), and the other 58 patients continued any ADV-excluding NAs antiviral therapy or ceasing antiviral therapy.

As showed in (Table 5), patients in the two antiviral therapy groups had good comparability in the gender, age and liver hardness proportion. Compared to the baseline, urine β₂ MG level of patients in ADV-excluding group decreased at average of 3.94mg/L per year, and eGFR value increased at average of 1.7mL/minute per year. On the contrary, urine β₂ MG level and eGFR value of patents in ADV-including group both deteriorated compared to the baseline (Table 5).

Unfortunately, we found that although the renal tubular function indexes of these CHB patients in ADV-excluding group improved markedly compared to the baseline, but at the end of 52-weeks’ follow-up the urine β₂MG level recovered completely in only 11.36% patients.In this study we found that the renal function of most patients who developed renal injury during long-term anti-HBV therapy could not recover completely during the 52-weeks’ ADV-excluding therapy, and especially in those patients who developed both renal tubular function indexes abnormal and eGFR value decrease.

4.                  Discussion

NAs had become first-line anti-HBV drugs in clinical practice by possessing the advantages of convenient, efficient and well tolerated [26,28-30]. However, because of the rare HBV eradication and high recurrence rate after drug withdrawal, the majority of CHB patients will require long-time antiviral treatment even the lifetime [26,28]. Therefore, the Adverse Drug Reactions Monitoring (ADRM) of these drugs, especially the effect on renal function, are of paramount importance for ensuring long term usage. Drugs such as ADV tend to be more harmful to the tubular than to the glomerular cells, and in this case the decrease in GFR value develops presumably secondarily after tubular injury, being thereby a relatively late event [31] Serum and urine Low-Molecular-Weight Proteins (LMWP) level, such as β₂ MG exhibit renal handling compatibility to that of an “ideal” marker of GFR. Hence, determining the serum concentrations of various LMWPs has been proposed as a useful approach to evaluate GFR impairment [32-36]. This is a study, which evaluated both GFR values estimated with CKD–EPI equation and serum or urine LMWP level. 52.94% CHB patients developed urine β₂ MG increase during long-term NAs therapy but in these patients the abnormal rate of eGFR value was only 27.40%. The results suggested that urine LMWP level such as β₂ MG level was the more sensitive indicator for evaluation of NAs induced renal tubular dysfunction in patients with CHB. Recently Takagi J’s report got the same result [37].In this study we found not only patients who received long-term NAs antiviral therapy, but also those untreated disease controls showed high urine β₂ MG abnormal rate. It indicated that there were subclinical renal dysfunctions in most CHB patients even before NAs therapy. Previous reports also indicated that renal injury was common in patients with CHB because of immune complex glomerulopathy and HBV direct renal injury [38-41]. Unfortunately, in this study we did not obtain the baseline urine β2 MG data of patients with long-term antiviral therapy. Thus, in our study the exact impact of the hepatic disease on renal function and the specific tubular toxicity of NAs cannot be reliably appreciated.

In addition, in this study we found after adjusting influence of age and treatment duration only urine β₂MG abnormal rate in ADV therapy group was significantly higher than that in LDT long-term therapy group. And an increase of more than 10×UNL of urine β₂ MG was only detected in the ADV mono or combine therapy group (Table 2). These results suggested that antivirals may be one of the risk factors for renal injury during long-term NAs therapy. When investigating potential factors associated with urine β₂ MG abnormal and eGFR decrease we identified age as the risk factor for both eGFR decrease and urine β₂ MG increase and ADV was the only uniqueone drug which was related to the urine β₂ MG abnormal during therapy. We also found that LDT had a protect effect on eGFR value and urine β₂ MG, which is also in line with the previous study [42-44]. Although we found patients in ADV mono-therapy group having high risk of urineβ₂MG abnormal, we observed ADV combined therapy did not increase the risk of renal injury compared to ADV monotherapy. The result was in line with previous study [44].

Unfortunately, in our study we observed the majority of those patients with CHB who developed renal injury during long-term antiviral therapy can’t completely recover after adjusting antiviral to ADV-excluding treatment at the end of the 52-weeks’ follow-up. The result was not consistent with the registration study [14,19]. The possible reason was first, most patients in our study had received longer time treatment than those in the registration study and the majority of them in clinical practice have to continue receiving NAs treatment instead of drug withdrawal; second, the kidney injury of these patients was ignored because of low sensitivity of eGFR, and then delayed the drug adjusting time in practice; third, the prolonged NAs treatment may cause renal interstitial fibrosis. When investigating the potential factors associated with renal function improvement, we found that the renal function level before therapy adjustment was the unique significant factor. The result suggested that early detection of injury determined the improvement degree of renal function.

The limitation of this study is the small size and the short time of the prospective observation follow up. As a result, clinical trials with large sample size and long-term follow-up are needed to confirm and investigate the optimal therapy for patients with obvious tubular damage, especially for patients with history of drug resistance mutation and need long-time antiviral therapy.

5.                  Conclusion

In summary, the results of this clinical study suggest that many patients developed renal injury during prolonged ADV-including anti-HBV therapy and urinary β₂ MG are more sensitive to monitor NAs antiviral therapy induced relatedrenal tubular injury. Prolonged ADV-including antiviral therapy maybe causes irreversible renal tubular damage in CHB patients.

6.                   Acknowledgement

We thank all patients for their understanding and participation.

Table 1: Demographic and clinical data of all patients accepting long-term nucleos(t)ide analogue antiviral therapy.

Table 2: Renal function of patients with nucleos(t)ide antiviral therapy for more than 6 months.

Table3: Risk factors for urinary β₂micro globulin abnormality rete in patients with CHB onnucleos(t)ide antiviral therapy.

Table4: Risk factors for estimated Glomerular Filtration Rate (eGFR)inpatients with CHB on nucleos(t)ide antiviral therapy.

Table5:Clinical course of patients developed renal dysfunction during nucleos(t)ide long-term antiviral therapy.

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