Pegylated interferon-α-2b add-on therapy to renal function for chronic hepatitis B patients who were treated with tenofovir disoproxil fumarate: a retrospective real-world study

Abstract

Background

Long-term tenofovir disoproxil fumarate (TDF) administration has been associated with potential adverse effects on renal function. Pegylated interferon-α2 (PEG-IFN-α2) therapy in chronic hepatitis B (CHB) patients increased estimated glomerular filtration rate (eGFR). Thus, the aim of this study was to analyze the influence of PEG-IFN-α-2b add-on to on-going TDF therapy in renal function in CHB patients.

Methods

This was a retrospective observational study. Ninety-one CHB patients who were treated with TDF for more than 48 weeks, had hepatitis B surface antigen (HBsAg) < 1500 IU/mL, and were hepatitis B e antigen-negative were recruited. Sixty-seven patients continued TDF monotherapy, and twenty-four patients received PEG-IFN-α-2b add-on therapy. Renal function indices were collected at baseline, 12 weeks, 24 weeks, and 48 weeks post-therapy. A linear mixed effects model for repeated measures was employed to analyze the associations between baseline information and serum β2-microglobulin (β2-MG)/urine α1-microglobulin (α1-MG) changes.

Results

HBsAg clearance rate was higher in TDF + PEG-IFN group compared with TDF monotherapy group (20.83% vs. 0.00%). There were no significant changes in blood urea nitrogen (BUN), creatinine (Cr), or eGFR in TDF monotherapy group. BUN and Cr was down-regulated, while eGFR was up-regulated at 48 weeks in TDF + PEG-IFN group. In TDF monotherapy group, serum β2-MG was elevated at 12, 24, and 48 weeks, whereas no significant changes were observed in TDF + PEG-IFN group. Serum β2-MG was higher in TDF monotherapy group than in TDF + PEG-IFN group. Urine α1-MG was increased in both groups at 48 weeks. TDF monotherapy was positive predictor for serum β2-MG increase, while TDF + PEG-IFN therapy negatively affect serum β2-MG level. Both TDF-based therapeutic strategies were positive predictors for urine α1-MG elevation.

Conclusion

PEG-IFN-α-2b might exert a protective effect against TDF-induced glomerular injury in CHB patients.

Background

Hepatitis B virus (HBV) infection affects approximate 296 million people all over the world, with significant renal complications including glomerulonephritis, acute and chronic kidney injury [1]. HBV-associated glomerulonephritis accounts for 2.74%–10.62% of biopsy-proven glomerular diseases in China, predominantly manifesting as membranous nephropathy or membranoproliferative glomerulonephritis [1]. Recent studies confirm HBV antigen deposition in kidneys elevates adverse renal outcomes by 7.49-fold in IgA nephropathy patients [2]. The mechanism of these renal dysfunctions has not been fully understood. The potential risk factors include elder age, co-infection with human immunodeficiency virus-1 (HIV-1), hypertension, diabetes mellitus, end-stage liver diseases [3–5].

HBV-induced kidney diseases usually improves with inhibition of viral replication by antiviral therapy [6]. Currently, there are two main first-line therapeutic recommendations for chronic hepatitis B (CHB) patients: oral nucleos(t)ide analogue (NAs) treatment [including entecavir (ETV), tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, and tenofovir amibufenamide] and of pegylated interferon-α2 (PEG-IFN-α2, including PEG-IFN-α-2a and PEG-IFN-α-2b) subcutaneous injection [7–10]. Long-term TDF administration has been associated with potential adverse effects on renal function, which require careful monitoring in clinical practice [11, 12]. Regarding renal safety, TDF is eliminated primarily via the kidneys, and its accumulation in proximal tubular cells may induce mitochondrial toxicity, leading to Fanconi syndrome, renal tubular dysfunction, or a gradual decline in estimated glomerular filtration rate (eGFR) [13–15]. Clinical studies have shown that elderly patients, those with pre-existing renal impairment, or individuals receiving concurrent nephrotoxic medications are at higher risk of TDF-related renal adverse events [16].

PEG-IFN-α2 could lead to higher rate of hepatitis B surface antigen (HBsAg) clearance with finite therapeutic duration, leading to functional cure in CHB patients [10]. Previous studies have shown that 48-week PEG-IFN-α2 monotherapy or combined with ETV treatment demonstrated a renal protective effect for CHB patients [4, 5]. However, it is still not elucidate the influence of PEG-IFN-α-2b add-on to on-going TDF therapy in renal function in CHB patients. Thus, we conducted a real-world retrospective observational study to investigate the renal function and antiviral efficacy under PEG-IFN-α-2b combined with TDF therapy in CHB patients.

Methods

Institutional review board

The study protocol was approved by the Institutional Review Board of The First Affiliated Hospital of Xinxiang Medical University (No. EC-2022-698). The Ethics Committees waived the requirement of written informed consent for participation from the participants or the participant’s legal guardians/next of kin because this was a retrospective study and only characteristics and laboratory indicators were collected. The study was conformed to the guidelines of the Declaration of Helsinki and the principles of Good Clinical Practice. The data were collected on June 2025. We had access to information that could identify individual enrolled subjects during and after data collection.

Study design

This was a retrospective observational cohort study. Inclusive criteria: (1) Age 18 ~ 65 years; (2) The diagnoses of CHB met the standard of the Chinese Guidelines for the Prevention and Treatment of Chronic Hepatitis B [10]; (3) The patients received TDF therapy for more than 48 weeks. (4) Negative for hepatitis B e antigen (HBeAg); (5) HBsAg level < 1500 IU/mL, because HBsAg less than 1500 IU/mL was associated with high rate of HBsAg loss [17, 18]. Exclusive criteria: (1) Co-infected with other hepatitis viruses or HIV-1; (2) Concurrently afflicted by decompensated liver cirrhosis, liver failure, or hepatocellular carcinoma; (3) Afflicted by alcoholism or drug addiction; (4) Afflicted by autoimmune diseases; (5) Afflicted by solid cancer or leukemia; (6) Pregnant women. The enrolled CHB patients were divided into two groups: TDF monotherapy group and TDF + PEG-IFN group. TDF monotherapy group were continuously treated with TDF (300 mg, orally once daily). TDF + PEG-IFN group received PEG-IFN-α-2b (Y shape, 40 kD; 180 µg, subcutaneous injection weekly; Xiamen Amoytop Biotech Co., Ltd., Xiamen, Fujian Province, China) add-on therapy to TDF (300 mg, orally once daily) for 48 weeks. The dosage of PEG-IFN-α-2b was adjusted to 135 µg weekly if the neutrophil count was < 0.75 × 10⁹/L or the platelet count was < 50 × 10⁹/L, whereas PEG-IFN-α-2b was discontinued if the neutrophil count was < 0.50 × 10⁹/L or the platelet count was < 25 × 10⁹/L or if serious adverse events occurred according to the instructions of manufacturer [19, 20]. The four observation time point was baseline, 12, 24, and 48 weeks post therapy.

Evaluation of virological, serological, and biochemical indices

Serum HBV DNA was quantified by real-time fluorescence quantitative polymerase chain reaction using a commercial HBV detection kit (Xiamen Amplly, Xiamen, Fujian Province, China) with the detection limit of 50 IU/mL. HBsAg, anti-HBs, HBeAg, anti-HBe, and hepatitis B core antibody was quantified using the ARCHITECH HBsAg, anti-HBs, HBeAg, anti-HBe, and anti-HBc reagent kits (Abbott GmbH & Co., KG., Wiesbaden, Germany). The detection limit for HBsAg was 0.05 IU/mL. Other renal function related parameters, including blood urea nitrogen (BUN), creatinine (Cr), cystatin C (Cys C), retinol-binding protein (RBP), serum β2-microglobulin (β2-MG), and urine α1-microglobulin (α1-MG), were collected. The eGFR was estimated using the following formulas based on age and Cr as previously described [4, 5]. The Modification of Diet in Renal Disease (MDRD) calculation for eGFR (mL/min/1.73 m²) = 186 × sCr^{− 1.154} × age^{− 0.203} × 0.742 (if female) [21]. The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) calculation for eGFR (mL/min/1.73 m²) = 141 × min(sCr/κ, 1)^α × max (sCr/κ, 1)^−1.209 × 0.993^age × 1.018 (if female). κ is 0.7 for female and 0.9 for male. α is -0.329 for female and − 0.411 for male [22].

Statistical analysis

SPSS Version 25.0 (IBM SPSS Software, Chicago, IL, USA) and SAS Version 9.4 (SAS Institute Inc., Cary, NC, USA) were used for general statistical analysis. For continuous variables, Shapiro-Wilk test was firstly used for normal distribution assay. The continuous variables following normal distribution were presented as mean ± standard deviation. The statistical significance was determined by one-way analysis of variance followed by Student-Newman-Keuls-q test. The continuous variables following skewed distribution were presented as median (interquartile range). The statistical significance was determined by Krusal-Wallis H test followed by Dunn’s multiple comparison test. For categorical variables, the data were presented as n (%). The Chi-squared test or Fisher’s exact test was used for comparisons. To evaluate the association between several variables and serum β2-MG/urine α1-MG changes over time, a linear mixed effects model for repeated measures was used by SAS Version 9.4 with MIXED procedure. The model considered the baseline age (in years), gender, baseline HBsAg, BUN, Cr, eGFR, Cys C, RBP, and therapeutic strategy as fixed effects and incorporated random effects for individual subjects. P values less than 0.05 were considered as statistically significant.

Results

Baseline characteristics of patients

A total of 91 CHB patients were enrolled in this study. Baseline characteristics were shown in Table 1. The average age of all enrolled patients was approximately 40 years old. Sixty-four patients (70.33%) were male, leading to a male-to-female ratio of 2.37: 1. Seventy-four patients (81.32%) had undetectable serum HBV DNA (< 50 IU/mL) at baseline, and the median HBsAg level was 94.95 (24.92, 201.4) IU/mL. Base on MDRD formula, twelve patients (eight in TDF monotherapy group and four in TDF + PEG-IFN group) had an eGFR less than 90 mL/min/1.73 m². Based on CKD-EPI formula, ten patients (six in TDF monotherapy group and four in TDF + PEG-IFN group) showed an eGFR less than 90 mL/min/1.73 m². No patients revealed a baseline eGFR less than 60 mL/min/1.73 m² based on both formulas.

Table 1.

Baseline characteristics of patients

Characteristic	Total	TDF monotherapy group	TDF + PEG-IFN group	P value (TDF monotherapy vs. TDF + PEG-IFN)
Patients enrolled, n	91	67	24	-
Male gender, n (%)	64 (70.33%)	47 (70.15%)	17 (70.83%)	0.959
Age (years)	40.88 ± 10.56	40.84 ± 10.66	41.00 ± 10.50	0.948
Age < 40 years, n (%)	47 (51.65%)	34 (50.75%)	13 (54.17%)	0.774
Age ≥ 40 years, n (%)	44 (48.35%)	33 (49.25%)	11 (45.83%)
HBV DNA undetectable (< 50 IU/mL), n (%)	74 (81.32%)	58 (86.57%)	16 (66.67%)	0.032
HBV DNA detectable (> 50 IU/mL), n (%)	17 (18.68%)	9 (13.43%)	8 (33.33%)
HBsAg (IU/mL)	94.95 (24.92, 201.4)	94.95 (21.76, 186.7)	99.81 (29.24, 222.3)	0.797
HBsAg < 10 IU/mL	17 (18.68%)	12 (17.91%)	5 (20.83%)	0.753
HBsAg 10 ~ 100 IU/mL	29 (31.87%)	22 (32.84%)	7 (29.17%)
HBsAg 100 ~ 1000 IU/mL	39 (42.86%)	28 (41.79%)	11 (45.83%)
HBsAg 1000 ~ 1500 IU/mL	6 (6.59%)	5 (7.46%)	1 (4.17%)
BUN (mmol/L)	4.69 ± 1.21	4.61 ± 1.18	4.90 ± 1.30	0.316
Cr (µmol/L)	65.77 ± 12.69	64.09 ± 11.84	70.46 ± 14.03	0.034
eGFR MDRD (mL/min/1.73 m2)	119.8 ± 30.83	123.2 ± 31.74	110.2 ± 26.45	0.077
eGFR CDK-EPI (mL/min/1.73 m2)	110.7 ± 16.23	112.4 ± 15.68	106.0 ± 17.13	0.098
Cys C (mg/L)	1.42 ± 0.39	1.46 ± 0.38	1.30 ± 0.40	0.072
RBP (mg/L)	59.41 ± 15.78	59.33 ± 15.82	59.65 ± 16.00	0.933
Serum β2-MG (mg/L)	0.55(0.34, 0.86)	0.53(0.31, 0.88)	0.58(0.37, 0.80)	0.722
Urine α1-MG (mg/L)	12.49 ± 3.14	12.78 ± 3.05	11.70 ± 3.33	0.150

TDF: tenofovir disoproxil fumarate; PEG-IFN: pegylated interferon HBV: hepatitis B virus; HBsAg: hepatitis B surface antigen; BUN: blood urea nitrogen; Cr: creatinine; eGFR: estimated glomerular filtration rate; MDRD: Modification of Diet in Renal Disease; CKD-EPI: Chronic Kidney Disease Epidemiology Collaboration; Cys C: cystatin C; RBP: retinol-binding protein; β2-MG: β2-microglobulin; α1-MG: α1-microglobulin

Sixty-seven patients continuously received TDF monotherapy, while other twenty-four patients received PEG-IFN-α-2b add-on therapy. There were no significant differences in gender ratio, average age, or baseline HBsAg level between TDF monotherapy group and TDF + PEG-IFN group (Table 1). The percentage of HBV DNA undetectable rate was lower in patients with TDF + PEG-IFN-α-2b therapy compared with TDF monotherapy (66.67% vs. 86.57%; P = 0.032, Table 1). Patients with TDF + PEG-IFN-α-2b therapy had a slightly higher baseline Cr level compared with TDF monotherapy (P = 0.034, Table 1), but the differences in baseline eGFR levels failed to achieve statistical significances (P = 0.077 and P = 0.098, respectively, Table 1). There were also no remarkable differences in BUN, Cys C, RBP, serum β2-MG, or urine α1-MG levels between two groups (Table 1).

Antiviral efficacy and adverse events

At 48 weeks of therapy, HBV DNA remained detectable in three patients (two in TDF monotherapy group and on e in TDF + PEG-IFN group). No patients in TDF monotherapy group achieved HBsAg clearance or seroconversion. Five patients (20.83%) in TDF + PEG-IFN group achieved HBsAg clearance, and two patients (8.33%) achieved HBsAg seroconversion.

All patients completed therapy during 48 weeks observation. No end-stage diseases (such as decompensated cirrhosis, liver failure, or hepatocellular carinoma) occurred during treatment. No severe adverse events were observed in TDF monotherapy group. In TDF + PEG-IFN group, all patients developed symptoms such as fever and myalgia during treatment, particularly in the early phase, which resolved without specific intervention. Alanine aminotransferase (ALT) elevation was observed in 70.83% (17/24) of patients, with only one patient experiencing ALT level higher than 5× upper limit of normal. The ALT level of this patient returned to normal after hepatoprotective therapy. Neutrophil counts dropped below 1.5 × 10⁹/L in 95.83% (23/24) of patients in TDF + PEG-IFN group. Seventeen patients received granulocyte colony-stimulating factor to elevate leukocyte counts. None of adverse events compromised the continuation of treatment in TDF + PEG-IFN group.

Changes in renal function in response to TDF monotherapy and TDF + PEG-IFN-α-2b therapy

There were no significant differences in serum BUN, Cr, eGFR MDRD, or eGFR CKD-EPI levels over 48 weeks therapy in patients with TDF monotherapy (P > 0.05, Fig. 1A, B and C, and 1D). Serum BUN and Cr levels were remarkably down-regulated, while eGFR MDRD and eGFR CKD-EPI levels were notably up-regulated 48 weeks post PEG-IFN-α-2b add-on therapy to TDF monotherapy (P < 0.05, Fig. 1A, B and C, and 1D). There were also no remarkable differences in serum Cys C or RBP levels over 48 weeks therapy in both groups (P > 0.05, Fig. 1E and F). Serum β2-MG level was elevated at 12 weeks post TDF monotherapy compared with baseline [0.78 (0.51, 1.25) mg/L vs. 0.53 (031, 0.88) mg/L; P < 0.05, Fig. 1G], and continuously keep in high level at 24 weeks [0.89 (0.55, 1.70) mg/L] and 48 weeks post TDF monotherapy [1.25 (0.71, 2.06) mg/L] compared with baseline (P < 0.05, Fig. 1G). However, there was no statistical difference in serum β2-MG level over 48 weeks in patients with TDF + PEG-IFN-α-2b therapy (P = 0.101, Fig. 1G). Importantly, serum β2-MG level at 48 weeks post therapy was also higher in patients with TDF monotherapy compared with in patients with TDF + PEG-IFN-α-2b therapy [1.25 (0.71, 2.06) mg/L vs. 0.67 (0.53, 1.20) mg/L; P < 0.001, Fig. 1G]. Urine α1-MG level was notably elevated at 48 weeks post therapy in both groups (P < 0.05, Fig. 1H).

Fig. 1.

Evolution of renal function by TDF monotherapy or TDF + PEG-IFN-α-2b therapy over 48 weeks. (A) Changes of blood urea nitrogen (BUN). (B) Changes of creatinine (Cr). (C) Changes of estimated glomerular filtration rate (eGFR) calculated by Modification of Diet in Renal Disease (MDRD) formula. (D) Changes of eGFR calculated by Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula. (E) Changes of cystatin C (Cys C). (F) Changes of retinol-binding protein (RBP). (G) Changes of serum β2-microglobulin (β2-MG). (H) Changes of urine α1-microglobulin (α1-MG). Statistical analysis was performed using one-way analysis of variance followed by Student-Newman-Keuls-q test, or using Krusal-Wallis H test followed by Dunn’s multiple comparison test. Black ^# indicates P < 0.05 compared with baseline in patients with TDF monotherapy therapy. Black ^### indicates P < 0.001 compared with baseline in patients with TDF monotherapy therapy. Red ^# indicates P < 0.05 compared with baseline in patients with TDF + PEG-IFN-α-2b therapy. ^& indicates P < 0.05 compared with patients with TDF + PEG-IFN-α-2b therapy at the same time point. ^&&& indicates P < 0.001 compared with patients with TDF + PEG-IFN-α-2b therapy at the same time point

Predictors for significant renal function changes in response to TDF monotherapy and TDF + PEG-IFN-α-2b therapy

In TDF monotherapy group, there were no significant differences in BUN (Fig. 2A), Cr (Fig. 2B), eGFR MDRD (Fig. 2C), eGFR CKD-EPI (Fig. 2D), Cys C (Fig. 2E), or RBP levels (Fig. 2F) over 48 weeks therapy (P > 0.05). The differences of eGFR MDRD and eGFR CKD-EPI in each time point between different genders and ages (Fig. 2C and D) were due to the enrolled factors of the formulas. There were also no remarkable differences in serum β2-MG or urine α1-MG levels between difference genders, ages, baseline HBV DNA, and baseline HBsAg level at each investigation time point (P > 0.05, Fig. 2G and H). In TDF + PEG-IFN-α-2b therapy group, serum BUN level was notably down-regulated at 48 weeks post therapy in patients with baseline HBsAg > 100 IU/mL (P < 0.05, Fig. 3A), while serum Cr level was remarkably reduced in patients with baseline HBsAg < 100 IU/mL (P < 0.001, Fig. 3B). Both eGFR MDRD and eGFR CKD-EPI levels at 48 weeks post therapy were notably higher in patients with baseline HBsAg < 100 IU/mL compared with those who with baseline HBsAg > 100 IU/mL (P < 0.05, Fig. 3C and D). There no significant differences in Cys C or RBP levels over 48 weeks therapy (P > 0.05, Fig. 3E and F). There were also no remarkable differences in serum β2-MG or urine α1-MG levels between difference genders, ages, baseline HBV DNA, and baseline HBsAg level at each investigation time point (P > 0.05, Fig. 3G and H).

Fig. 2.

Evolution of renal function by TDF monotherapy over 48 weeks under different factors. (A) Changes of blood urea nitrogen (BUN) between different genders, between different ages (< 40 years and ≥ 40 years), between different baseline HBV DNA (< 50 IU/mL and > 50 IU/mL), and between different HBsAg (< 100 IU/mL and > 100 IU/mL). (B) Changes of creatinine (Cr) between different genders, between different ages (< 40 years and ≥ 40 years), between different baseline HBV DNA (< 50 IU/mL and > 50 IU/mL), and between different HBsAg (< 100 IU/mL and > 100 IU/mL). (C) Changes of estimated glomerular filtration rate (eGFR) calculated by Modification of Diet in Renal Disease (MDRD) formula between different genders, between different ages (< 40 years and ≥ 40 years), between different baseline HBV DNA (< 50 IU/mL and > 50 IU/mL), and between different HBsAg (< 100 IU/mL and > 100 IU/mL). (D) Changes of eGFR calculated by Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula between different genders, between different ages (< 40 years and ≥ 40 years), between different baseline HBV DNA (< 50 IU/mL and > 50 IU/mL), and between different HBsAg (< 100 IU/mL and > 100 IU/mL). (E) Changes of cystatin C (Cys C) between different genders, between different ages (< 40 years and ≥ 40 years), between different baseline HBV DNA (< 50 IU/mL and > 50 IU/mL), and between different HBsAg (< 100 IU/mL and > 100 IU/mL). (F) Changes of retinol-binding protein (RBP) between different genders, between different ages (< 40 years and ≥ 40 years), between different baseline HBV DNA (< 50 IU/mL and > 50 IU/mL), and between different HBsAg (< 100 IU/mL and > 100 IU/mL). (G) Changes of serum β2-microglobulin (β2-MG). (H) Changes of urine α1-microglobulin (α1-MG) between different genders, between different ages (< 40 years and ≥ 40 years), between different baseline HBV DNA (< 50 IU/mL and > 50 IU/mL), and between different HBsAg (< 100 IU/mL and > 100 IU/mL). Statistical analysis was performed using one-way analysis of variance followed by Student-Newman-Keuls-q test, or using Krusal-Wallis H test followed by Dunn’s multiple comparison test. ^# indicates P < 0.05 compared with baseline. ^### indicates P < 0.001 compared with baseline. ^& indicates P < 0.05 compared with the level at the same time point. ^&&& indicates P < 0.001 compared with the level at the same time point

Fig. 3.

Evolution of renal function by TDF + PEG-IFN-α-2b therapy over 48 weeks under different factors. (A) Changes of blood urea nitrogen (BUN) between different genders, between different ages (< 40 years and ≥ 40 years), between different baseline HBV DNA (< 50 IU/mL and > 50 IU/mL), and between different HBsAg (< 100 IU/mL and > 100 IU/mL). (B) Changes of creatinine (Cr) between different genders, between different ages (< 40 years and ≥ 40 years), between different baseline HBV DNA (< 50 IU/mL and > 50 IU/mL), and between different HBsAg (< 100 IU/mL and > 100 IU/mL). (C) Changes of estimated glomerular filtration rate (eGFR) calculated by Modification of Diet in Renal Disease (MDRD) formula between different genders, between different ages (< 40 years and ≥ 40 years), between different baseline HBV DNA (< 50 IU/mL and > 50 IU/mL), and between different HBsAg (< 100 IU/mL and > 100 IU/mL). (D) Changes of eGFR calculated by Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula between different genders, between different ages (< 40 years and ≥ 40 years), between different baseline HBV DNA (< 50 IU/mL and > 50 IU/mL), and between different HBsAg (< 100 IU/mL and > 100 IU/mL). (E) Changes of cystatin C (Cys C) between different genders, between different ages (< 40 years and ≥ 40 years), between different baseline HBV DNA (< 50 IU/mL and > 50 IU/mL), and between different HBsAg (< 100 IU/mL and > 100 IU/mL). (F) Changes of retinol-binding protein (RBP) between different genders, between different ages (< 40 years and ≥ 40 years), between different baseline HBV DNA (< 50 IU/mL and > 50 IU/mL), and between different HBsAg (< 100 IU/mL and > 100 IU/mL). (G) Changes of serum β2-microglobulin (β2-MG). (H) Changes of urine α1-microglobulin (α1-MG) between different genders, between different ages (< 40 years and ≥ 40 years), between different baseline HBV DNA (< 50 IU/mL and > 50 IU/mL), and between different HBsAg (< 100 IU/mL and > 100 IU/mL). Statistical analysis was performed using one-way analysis of variance followed by Student-Newman-Keuls-q test, or using Krusal-Wallis H test followed by Dunn’s multiple comparison test. ^# indicates P < 0.05 compared with baseline. ^## indicates P < 0.01 compared with baseline. ^### indicates P < 0.001 compared with baseline. ^& indicates P < 0.05 compared with the level at the same time point

Moreover, we entered all variables as fixed effects and incorporated random effects in the linear mixed model accounting for repeated measures [4, 5]. TDF monotherapy was a notable positive predictor for increase serum β2-MG (estimate value: 2.446, P = 0.017, Table 2), but TDF + PEG-IFN-α-2b therapy were observed to be negatively affect serum β2-MG level at 48 weeks of treatment (estimate value: -1.334, P = 0.043, Table 2). Both TDF monotherapy and TDF + PEG-IFN-α-2b therapy showed positive predictor values for urine α1-MG elevation at 48 weeks of treatment (P < 0.05, Table 2). However, the changes of serum β2-MG or urine α1-MG were not significantly associated with gender, age, baseline HBV DNA status, baseline HBsAg, baseline Bun, Cr, eGFR, Cys C, or RBP (P > 0.05, Table 2).

Table 2.

Predictors for serum β2-MG or urine α1-MG levels at 48 weeks of therapy

	Serum β2-MG			Urine α1-MG
	Estimate	Standard error	P value	Estimate	Standard error	P value
Male gender	0.034	0.028	0.673	0.038	0.072	0.356
Age	2.201	1.918	0.536	2.325	2.834	0.448
Baseline HBV DNA detectable	0.024	0.026	0.687	0.039	0.032	0.707
Baseline HBsAg	0.017	0.019	0.743	0.004	0.006	0.639
BUN	-0.023	0.018	0.138	-0.101	0.092	0.102
Cr	-0.221	0.231	0.286	-0.413	0.590	0.559
eGFR MDRD	1.135	2.720	0.452	1.991	1.504	0.141
eGFR CDK-EPI	1.102	2.121	0.378	2.374	2.409	0.287
Cys C	0.002	0.004	0.815	0.015	0.013	0.740
RBP	0.021	0.031	0.679	0.042	0.039	0.583
TDF monotherapy	2.446	0.028	0.017	1.042	0.124	< 0.001
TDF + PEG-IFN-α-2b therapy	-1.334	0.039	0.043	1.104	0.493	0.027

Discussion

Most CHB patients require long-term NAs therapy. Since NAs and their metabolites are primarily excreted by the kidney, renal function is one of the crucial considerations when selecting antiviral treatment regimens for CHB patients. Kidney is an important excretory organ in the human body, responsible for metabolizing waste products, regulating water-electrolyte balance, and maintaining systemic homeostasis. The core functions of kidney include glomerular filtration, tubular re-absorption and secretion, and renal endocrine function. Owing to the robust compensatory capacity, abnormalities in traditional renal function indicators, such as BUN and Cr, may not manifest until the eGFR declines by more than 50%. Therefore, although BUN, Cr, and eGFR are widely used for renal function assessment, they are insufficient for the early detection of renal impairment. In recent years, various novel biomarkers, especially several low-molecular-weight proteins (LMWPs), including Cys C, RBP, α1-MG, and β2-MG, have emerged as promising indicators for evaluating early renal injury [23, 24]. NAs-associated renal impairment primarily affects the renal tubules [25, 26]. LMWPs are largely re-absorbed by the tubular cells after glomerular filtration. Therefore, the serum levels of Cys C, RBP, and β2-MG were elevated in the early stages of glomerular dysfunction. Persistent decompensated renal injury may subsequently lead to the increase in BUN and Cr levels, accompanied by a reduction in eGFR level. When tubular damage is persistent, the urinary level of LMWPs and specific tubular enzymes (including α1-MG, Cys C, and N-acetyl-β-D-glucosaminidase) are increased, reflecting impaired tubular re-absorption capacity and direct tubular cell injury [27, 28].

TDF, while highly effective as an antiviral agent for HIV-1 and HBV, carries a well-documented risk of nephrotoxicity, characterized by a dual impact on both glomerular filtration and proximal tubular re-absorption function. The sequence of this injury can be effective monitored through serial changes in LMWPs in blood and urine. The primary insult often occurs at the level of the proximal tubule. Longitudinal studies demonstrated that initiation of TDF-based therapy was independently associated with a significant elevation in urinary α1-MG and β2-MG levels in patients living with HIV-1, confirming the direct role of TDF in impairing tubular re-absorptive capacity [29–31]. This tubular dysfunction can precede and contribute to a decline in eGFR. While serum Cr is a late marker, serum Cys C serves as a more sensitive indicator of early glomerular function impairment. A large international cohort study reported that administration of TDF was associated with a significantly greater annual decline in eGFR, particularly when eGFR was estimated using Cys C compared to Cr, highlighting the superior sensitivity in detecting TDF-related glomerular hemodynamic changes [32, 33]. The clinical trajectory often begins with subclinical tubular injury with increased urinary LMWPs, which may progresses to a measurable decline in glomerular filtration with a rising serum Cys C and a failing eGFR. Ultimately, persistent injury leads to overt renal dysfunction, marked by elevated BUN and Cr. Therefore, the combined monitoring of urinary LMWPs for tubular health and serum Cys C for glomerular function provides a robust strategy for the early detection of TDF-associated nephrotoxicity, allowing for timely intervention in clinical practice. However, research on the impact of PEG-IFN-α2 on renal function in CHB patients has primarily focused on glomerular filtration. Studies have consistently reported an improvement in eGFR during PEG-IFN-α2 treatment. For instance, in treatment-naïve CHB patients receiving PEG-IFN-α-2a monotherapy for 48 weeks, eGFR levels were robustly increased compared to baseline [5]. Similarly, the addition of PEG-IFN-α-2b to ETV in experience CHB patients led to a remarkable elevation in eGFR as early as 12 weeks into treatment, which remained stable thereafter [4]. However, investigations into the effects of PEG-IFN-α2 on renal tubular function in CHB patients are notably scarce and lack comprehensive reporting.

Based on the availability of clinical laboratory tests at our institution, the current study selected a comprehensive panel of renal biomarkers, including BUN, Cr, eGFR, serum Cys C, serum RBP, serum β2-MG, and urine α1-MG, to evaluate renal injury in CHB patients. We conducted a retrospective analysis to investigate the impact PEG-IFN-α-2b add-on therapy on renal function in patients previously treated with TDF. The study cohort consisted of patients with essentially normal baseline renal function who had received TDF monotherapy for over 48 weeks, presented with HBsAg < 1500 IU/mL, and were HBeAg-negative, representing an optimal population for pursuing functional cure [18]. PEG-IFN-α-2b combined with TDF treatment demonstrated decreased BUN and Cr levels and increased eGFR at 48 weeks post therapy. In contrast, patients who continued on TDF monotherapy showed no significant changes in these parameters during the observation period. The TDF + PEG-IFN group had a significantly higher baseline serum Cr (P = 0.034), but eGFR (by MDRD and CKD-EPI) was not statistically different (P = 0.077 and P = 0.098, respectively). This baseline difference might slightly confound the final renal function comparison but was unlikely to alter the conclusion, for two reasons. On the one hand, the linear mixed-effects model included baseline Cr, eGFR, and other covariates as fixed effects, ecplicitly accounting for baseline differences. The model confirmed that TDF + PEG-IFN therapy independently reduced serum β2-MG regardless of baseline Cr. On the other hand, despite higher baseline Cr, the TDF + PEG-IFN group showed a reduction in Cr and increase in eGFR at 48 weeks, whereas the monotherapy group had stable Cr and eGFR. If baseline Cr were a confounding factor, we would expect the TDF + PEG-IFN group to have worse renal outcomes, but the opposite was observed, strengthening the conclusion that PEG-IFN-α-2b had a protective effect.

Serum β2-MG level exhibited a progressive increasing trend in TDF monotherapy group. However, no significant change in serum β2-MG was observed in PEG-IFN-α-2b add-on group. At 48 weeks post therapy, serum β2-MG level was notably lower in PEG-IFN-α-2b add-on group compared to that in TDF monotherapy group. These findings suggested that the addition of PEG-IFN-α-2b may confer a protective effect on glomerular filtration function in TDF-experienced patients over the 48-week observation period. This aligned with previous findings indicating that TDF affects both glomerular and tubular function [34–36], while PEG-IFN-α2 has a protective effect on glomerular function in NAs-experienced patients [4, 5]. Crucially, urine α1-MG level increased at 48 weeks compared to the baseline in both TDF monotherapy and PEG-IFN-α-2b add-on group. There was also no statistical difference in urine α1-MG level between two groups at 48 weeks post therapy. This indicated that while PEG-IFN-α-2b might not protect against TDF-associated tubular function injury, it did not appear to exacerbate the risk of TDF-induced tubular damage either. To further elucidate these dynamics, we employed a linear mixed-effects model for repeated measures to analyze changes in serum β2-MG and urine α1-MG. The model confirmed that TDF treatment was a positive predictor for changes in urine α1-MG, regardless of PEG-IFN-α-2b administration, reinforcing the association between TDF and tubular injury in CHB patients. Although TDF treatment was also a positive predictor for serum β2-MG changes, the PEG-IFN-α-2b add-on therapy was found to reverse this effect, revealing that PEG-IFN-α-2b might specifically counteract the glomerular injury potentially induced by TDF.

Although the exact mechanisms by which PEG-IFN-α-2b add-on therapy ameliorates glomerular function remain incompletely understood, several potential reasons might be considered. Firstly, PEG-IFN-α-2b enhanced HBV-specific immune responses and promotes HBsAg clearance, as shown that TDF + PEG-INF group achieved higher HBsAg clearance than TDF monotherapy group (20.83% vs.0%). The reduction in viral antigen load might reduce immune complex deposition in the glomeruli, thereby mitigating immune-mediated glomerular injury [2]. Secondly, IFNs have been shown to modulate vascular tone and improve endothelial function, potentially through the up-regulation of nitric oxide synthesis, which could enhance glomerular filtration and reduce serum β2-MG levels [4, 5]. Thirdly, the anti-inflammatory and anti-fibrotic properties of interferons might attenuate glomerular basement membrane thickening and mesangial matrix expansion, thereby preserving glomerular integrity [7, 18]. Conversely, the persistence of elevated urinary α1-MG in both groups suggested that tubular injury induced by TDF was not reversed by the addition of PEG-IFN-α-2b. TDF is known to accumulate in proximal tubular cells and cause mitochondrial toxicity through inhibition of DNA polymerase-γ, leading to impaired energy metabolism and cell apoptosis [13, 26]. This direct tubular toxicity may be structurally less responsive to immunomodulatory agents like interferons. Moreover, α1-MG is a sensitive marker of tubular reabsorptive function, and its elevation reflects ongoing tubular dysfunction despite improvements in glomerular filtration [29, 30]. Therefore, while PEG-IFN-α-2b might protect glomerular function through immunomodulatory and hemodynamic effects, it appeared to have limited impact on TDF-induced tubular damage. Future studies should investigate whether longer treatment durations or switching to renal-safer NAs could further ameliorate tubular injury in such patients [16].

Importantly, it was seemed that the apparent paradox in the endothelial injury and immune activation of PEG-IFN-α2 vs. observed renal benefit. However, PEG-IFN-α2 improved eGFR via mechanisms that outweigh potential endothelial injury and immune activation. Enhanced viral suppression and HBsAg seroclearance reduced HBV-induced renal inflammation. PEG-IFN-α2 might also increase nitric oxide production or reduce renal vascoconstriction, improving glomerular filtration. Furthermore, the endothelial injury of PEG-IFN-α2 is typically transient and mild in patients without pre-existing vascular disease. The 48-week observation period might have captured the beneficial effects without long-term adverse outcomes.

This study has several limitations. On the one hand, this was a retrospective design and the limited number of enrolled subjects might introduce inherent biases. The observation period was set at 48 weeks, which might be insufficient to capture the long-term renal effects. We also did not enrolled fibrosis stages and had small sized of patients with detectable HBV DNA, leading to the unaddressed association between detectable HBV DNA post-therapy and fibrosis severity. On the other hand, the study only evaluated urine α1-MG as tubular injury marker. α1-MG does not assess urinary phosphate, urate, glucose, or bicarbonate, which served as key indicators of proximal tubular dysfunction caused by TDF-induced mitochondrial injury. Moreover, no renal biopsies were performed. The study only relied on serum and unrinary biomarkers to infer plomerular/tubular function. Therefore, future investigations with larger sample size and prospective, randomized controlled designs (with targeted biopsis) are necessary to validate the current findings, to explore the association of viral clearance and advanced fibrosis, and to clarify the effect of PEG-IFN-α-2b on tubular histology.

Conclusion

In summary, the present data indicated that long-term TDF therapy might lead to both glomerular and tubular dysfunction in CHB patients. The PEG-IFN-α-2b add-on therapy did not reverse TDF-induced tubular injury but might provide a protective effect against TDF-associated glomerular damage.

Abbreviations

ALT

Alanine aminotransferase

BUN

Blood urea nitrogen

CHB

Chronic hepatitis B

CKD-EPI

Chronic Kidney Disease Epidemiology Collaboration

Cr

Creatinine

Cys C

Cystatin C

eGFR

Estimated glomerular filtration rate

ETV

Entecavir

HBeAg

Hepatitis B e antigen

HBsAg

Hepatitis B surface antigen

HBV

Hepatitis B virus

LMWPs

Low-molecular-weight proteins

PEG-IFN-α2

Pegylated interferon-α2

MDRD

Modification of Diet in Renal Disease

MG

Microglobulin

NAs

Nucleos(t)ide analogue

RBP

Retinol-binding protein

TDF

Tenofovir

Author contributions

XHW performed study conception and design. TBW, AHL, YZ, HJW, MQY, YPW, and XHW performed data collection and analysis. TBW and XHW performed statistical analysis. TBW, AHL, and XHW wrote the first draft of the manuscript. YZ, HJW, MQY, and YPW made critically review for the manuscript. All authors read and approved the final manuscript.

Funding

None.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The study protocol was approved by the Institutional Review Board of The First Affiliated Hospital of Xinxiang Medical University (No. EC-2022-698). The Ethics Committees waived the requirement of written informed consent for participation from the participants or the participant’s legal guardians/next of kin because this was a retrospective study and only characteristics and laboratory indicators were collected. The study was conformed to the guidelines of the Declaration of Helsinki and the principles of Good Clinical Practice. The data were collected on June 2025. We had access to information that could identify individual enrolled subjects during and after data collection.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.