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Telbivudine attenuates gentamicin-induced kidney injury in rats
Accepted Manuscript Title: Telbivudine attenuates gentamicin-induced kidney injury in rats Author: Cigdem Kader, Mustafa Sunbul, Yavuz Kursad Das, Murat Yarim, Abdulkerim Bedir, Efe Karaca, Mehmet Celikbilek, Resat Ozaras PII: DOI: Reference:
S0924-8579(17)30102-4 http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.01.015 ANTAGE 5064
To appear in:
International Journal of Antimicrobial Agents
Received date: Accepted date:
20-4-2016 6-1-2017
Please cite this article as: Cigdem Kader, Mustafa Sunbul, Yavuz Kursad Das, Murat Yarim, Abdulkerim Bedir, Efe Karaca, Mehmet Celikbilek, Resat Ozaras, Telbivudine attenuates gentamicin-induced kidney injury in rats, International Journal of Antimicrobial Agents (2017), http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.01.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Telbivudine attenuates gentamicin-induced kidney injury in rats
Cigdem Kader a, Mustafa Sunbul b, Yavuz Kursad Das c, Murat Yarim d, Abdulkerim Bedir e, Efe Karaca d, Mehmet Celikbilek f, Resat Ozaras g,*
a
Departments of Infectious Diseases and Clinical Microbiology, Bozok University,
School of Medicine, Yozgat, Turkey b
Departments of Infectious Diseases and Clinical Microbiology, Ondokuz Mayıs
University, School of Medicine, Samsun, Turkey c
Department of Pharmacology and Toxicology, Ondokuz Mayıs University, School of
Veterinary Medicine, Samsun, Turkey d
Department of Pathology, Ondokuz Mayıs University, School of Veterinary
Medicine, Samsun, Turkey e
Department of Medical Biochemistry, Ondokuz Mayıs University, School of
Medicine, Samsun, Turkey f
Department of Internal Medicine, Unit of Gastroenterology, Bozok University,
School of Medicine, Yozgat, Turkey g
Department of Infectious Diseases and Clinical Microbiology, Istanbul University,
Cerrahpaşa Medical School, Istanbul, Turkey
* Corresponding author. Tel.: +90 212 414 3095; fax: +90 212 414 3095. E-mail address: [email protected] (R. Ozaras).
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ARTICLE INFO Article history: Received 20 April 2016 Accepted 6 January 2017
Keywords: Telbivudine Chronic hepatitis B Cystatin C Glomerular filtration rate Lamivudine
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Highlights Telbivudine (LdT) is not associated with nephrotoxicity in clinical practice. LdT may increase the glomerular filtration rate. Renal function in rats was measured by biochemical and histopathological methods. LdT was associated with significant improvements in renal function in rats in this study.
ABSTRACT Nephrotoxicity has been associated with nucleos(t)ide analogues other than telbivudine (LdT). This study investigated the potential effects of LdT and lamivudine (LAM) on renal function in an experimental rat model of gentamicin-induced acute nephrotoxicity. A total of 28 healthy Wistar albino rats were randomly divided into four experimental groups: negative control; positive control (PC); LdT; and LAM. Nephrotoxicity was induced by gentamicin in the LdT, LAM and PC groups. LdT and LAM were administered to two groups for 6 weeks starting on the ninth day. Blood samples were collected weekly and cystatin C levels were measured by ELISA. Animals were sacrificed on the 50th day and the kidneys were removed for histological examination. Serum cystatin C levels differed significantly between the LdT and LAM groups (P < 0.007) and between the LdT and PC groups (P < 0.001). Renal function was significantly improved in the LdT group at the start of antiviral treatment on Day 8 and at the end of treatment on Day 50 (P = 0.001 and 0.007). Glomerular injury, acute tubular necrosis and total injury score were significantly reduced in the LdT group relative to the PC and LAM groups upon histopathological 3
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examination. LdT was associated with significant improvements in renal function as measured by biochemical and histopathological methods. The acute kidney injury model data should be supported by clinical studies to suggest that LdT treatment may have advantages for patients with underlying chronic kidney disease receiving chronic hepatitis B treatment.
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1. Introduction Nucleos(t)ide analogues (NAs) have been licensed for oral treatment of chronic hepatitis B (CHB) [1] and are the most widely used antiviral drugs in CHB treatment in recent years. NAs inhibit viral DNA polymerase [2]. Although these agents are effective in the suppression of hepatitis B virus (HBV) replication, they cannot fully eliminate the virus from the host. As a result, long-term treatment is required in most patients. NAs are generally safe and well tolerated, although there are concerns of metabolic side effects in some patients with long-term use [3].
Treatment of CHB with NAs is long. Besides renal involvement during the course of hepatitis as an extrahepatic manifestation, long-term use of NAs may be associated with renal failure especially in patients with underlying renal disease, elderly patients and in those using concomitant nephrotoxic drugs. Adefovir (ADV), which nowadays is rarely used, carries a higher risk of nephrotoxicity. Tenofovir (TDF) may induce nephrotoxicity in nearly 15% of patients treated for 2–9 years [2].
NAs are excreted by the kidneys and as a result dosage adjustment according to glomerular filtration rate (GFR) is required [4]. Cohort studies have suggested that renal impairment is more frequently reported in CHB patients treated with ADV. In a longitudinal observational study of 214 patients receiving HBV treatment, the estimated GFR (eGFR) decreased in patients receiving ADV as monotherapy or in a combination but remained stable in patients receiving lamivudine (LAM), TDF or entecavir (ETV) at a median follow-up of 2.4 years. The eGFR decreased in patients with a baseline eGFR of <90 mL/min/1.73 m2 regardless of treatment [5]. Of 641 5
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patients initially randomised in a pivotal study of TDF, 585 patients entered the openlabel phase, of which 10 (1.7%) had an elevation of serum creatinine of ≥0.5 mg/dL above baseline [6]. In another 3-year prospective study of 400 patients receiving TDF treatment, few patients (1.3%) experienced renal adverse reactions, and creatinine clearance (CLCr) remained stable over time. Patients responded favourably when the TDF dose was adjusted for decreased CLCr [7].
In a limited number of clinical studies conducted by different teams worldwide, telbivudine (LdT) has been reported to promote a significant increase in GFR [4,8– 10]. These data suggest that LdT may protect against nephrotoxicity resulting from other conditions. A real-world retrospective study of 587 patients with CHB treated with TDF (n = 170), LdT (n = 184) or ETV (n = 233) showed that eGFR decreased significantly in the TDF group after a mean treatment duration of 17 months, however it increased in the LdT group after a mean treatment duration of 32 months. In the ETV group there was no significant change in eGFR after a mean of 44 months of treatment [11].
In a recent study including 4178 CHB patients treated with NAs, renal functional declined in 706 (16.9%). Age, hypertension, diabetes, history of liver or kidney transplantation, underlying chronic kidney disease and simultaneous administration of diuretics were found to increase the hazard ratio for renal functional decline. The eGFR significantly increased over time in patients receiving LdT or clevudine compared with LAM [12].
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Cystatin C is a low-molecular-weight non-glycosylated protein and a cysteine proteinase inhibitor. It is synthesised continuously at a constant rate in all nucleated cells in the body [13]. Cystatin C is freely filtered through the glomeruli, re-absorbed through tubular epithelial cells and metabolised rapidly in the kidneys [14]. It is used for calculating GFR. More recently, cystatin C has been applied to monitor changes in GFR and has been shown to be more sensitive than traditional measures based on serum creatinine level and CLCr [15]. Gentamicin, an aminoglycoside antibiotic, is frequently used to induce experimental nephrotoxicity [16]. Cystatin C is reported to be the most sensitive test of nephrotoxicity in rats with nephropathy induced by gentamicin and can detect damage earlier than traditional creatinine and blood urea nitrogen assays [17]. Thus, cystatin C was preferred in the current study.
The potential effects of LdT and LAM on renal function were investigated in a model of experimental nephrotoxicity caused by gentamicin exposure. Serum cystatin C levels were measured as an indicator of nephrotoxicity, and histopathological changes in kidney tissue were examined.
2. Materials and methods 2.1. Animals A total of 31 healthy Wistar albino male rats (age 12–16 weeks, weight 260–360 g) were used in the study. Standard chow and water were provided ad libitum during the study period. Animals were housed under a 12-h light/dark cycle.
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2.2. Drugs Gentamicin sulfate was administered by the intraperitoneal (i.p.) route for 8 days at a dose of 80 mg/kg to induce nephropathy [15]. The experimental drugs LdT (Sigma, St Louis, MO) [18] and LAM (Sigma) [19], were dissolved in distilled water and were administered orally by gastric gavage.
2.3. Experimental protocol As a comparator to LdT, LAM was preferred since LAM has sufficient renal safety data, is inexpensive and is easily accessible in many developing and Far East countries. Three rats were used in a preliminary study to verify the nephrotoxicity model [pilot study (PS)]. Rats in the PS group were given 80 mg/kg gentamicin sulfate by i.p. injection once daily for 8 days. Necropsies were performed and nephrotoxicity was determined histopathologically on Day 9.
The principal investigation was initiated after confirming nephrotoxicity in the PS group. The study included four experimental groups as follows: negative control (NC) (i.p. saline only); positive control (PC) (i.p. gentamicin only); LdT (10 mg/kg); and LAM (5 mg/kg).
A total of seven rats were assigned to each group at random. Nephrotoxicity was induced by administering 80 mg/kg gentamicin sulfate to the LdT, LAM and PC groups by i.p. injection for 8 days [16]. Blood samples were collected from the tail vein in all groups at the end of Day 8. A dose of 10 mg/kg LdT was administered to the LdT group [17] and 5 mg/kg LAM was given to the LAM group [19] by gastric 8
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gavage for 6 weeks starting on Day 9. Blood was obtained from the tail vein of rats in the LdT, LAM and PC groups on Days 15, 22, 29, 36, 43 and 50. Blood was collected from the NC group on Day 50. The study protocol is presented in Fig. 1. Rats in all groups were weighed prior to blood collection and LdT and LAM dosing was adjusted according to body weight.
2.4. Serum cystatin C measurement Blood samples were collected into microtubes and were centrifuged at 1000 g for 10 min at room temperature to separate serum for measurement of cystatin C. Serum samples were stored at –80 C for subsequent analysis. Rat serum cystatin C levels were measured quantitatively using a sandwich enzyme-linked immunosorbent assay (ELISA) kit (BioVendor–Laboratorni Medicina a.s., Brno, Czech Republic) according to the manufacturer’s recommendations. Just prior to the assay, serum samples were diluted 500 times with dilution buffer in two steps. The performance of the ELISA kit was validated using low- and high-concentration control materials included by the manufacturer.
2.5. Histopathological examination of kidney tissues PS group rats were sacrificed on Day 8 and rats from the remaining four groups were sacrificed on Day 50. The kidneys from rats in all four groups were excised following necropsy. A semiquantitative evaluation of renal tissues was performed by scoring the degree of damage severity according to previously published criteria [20]. The following parameters were used for the grading of injury: (i) glomerular injury (% 9
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of renal parenchyma involvement): none = 0, <25% of glomeruli exhibiting nonspecific features of injury = +1, 25–50% of glomeruli exhibiting non-specific features of injury = +2, 50–75% of glomeruli exhibiting non-specific features of injury = +3, and >75% of glomeruli exhibiting non-specific features of injury = +4; (ii) acute tubular necrosis (% of renal parenchyma involvement): none = 0, <25% of tubules out of the entire renal parenchyma = +1, 25–50% of tubules out of the entire renal parenchyma = +2, 50–75% of tubules out of the entire renal parenchyma = +3, and >75% of tubules out of the entire renal parenchyma = +4; and (iii) tubulointerstitial inflammatory infiltrates: none = 0, leukocytes confined within the interstitium = +1, and leukocytes infiltrating the interstitium and tubular epithelial cells = +2 [20].
2.6. Ethical considerations This study was approved by the Animal Experiments Local Ethics Committee of Ondokuz Mayıs University (Samsun, Turkey). Animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All animal studies were performed in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.
2.7. Statistical analysis Statistical analyses were conducted using IBM SPSS Base software (IBM Corp., Armonk, NY). Differences in continuous values were evaluated using the Shapiro– Wilk test and were determined to be in accordance with a normal distribution. Repeated measures analysis of variance (ANOVA) was used to estimate variance in cystatin C measurements. Linear regression was used to analyse the change in 10
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cystatin C levels over time in the LdT, LAM and PC groups. Increases in cystatin C concentrations in blood samples obtained 8 days after gentamicin administration and in blood samples obtained at the end of Day 50 were compared by one-way ANOVA with the Tukey test for paired comparisons. The Kruskal–Wallis test was used for variance analysis and the Mann–Whitney U-test was used for pairwise comparisons of histopathological assessments between groups. A P-value of <0.05 was considered statistically significant.
3. Results There were no significant differences in serum cystatin C concentration among the study groups on Days 0 or 8 (P > 0.05). Cystatin C was measured in blood samples collected from the LdT, LAM and PC groups at the beginning of study; the baseline cystatin C concentration was used as the cut-off value in subsequent analyses. Serum cystatin C concentrations in the LdT, LAM and PC groups are presented in Fig. 2. Cut-off values are indicated in the figure by dashed lines.
The mean serum cystatin C concentration and standard error values for the LdT, LAM and PC groups are presented in Table 1.
There was a significant difference in serum cystatin C concentration between the LdT and LAM groups (P = 0.0006) on Day 50. In addition, cystatin C levels differed significantly in the LdT group compared with the PC group (P = 0.0017). However, there was no difference in cystatin C levels between the LAM and PC groups (P > 0.05). 11
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The time required for cystatin C levels to return to the baseline concentration of 2.01 g/mL was ca. 8 days (7.72 days) in the LdT group as measured by the logarithmic formula obtained through regression modelling. Thus, kidney damage peaked on Day 8 and returned to physiological baseline after an additional 8 days.
Although cystatin C values on Day 15 of the LdT group were lower than the baseline value, this difference was not statistically significant (P > 0.05, one-way ANOVA with the Tukey test for paired comparisons).
In the LAM group, cystatin C levels were equivalent to the baseline value of 2.14 g/mL at ca. 29 days after the induction of kidney damage by gentamicin administration on linear formula obtained in the regression model. In this case, the group physiological baseline value was achieved at 21 days after LAM treatment.
In the PC group, cystatin C levels were equivalent to the baseline value of 1.90 g/mL at ca. 59 days after the induction of kidney damage by gentamicin exposure based on the results of linear regression modelling. No antiviral treatment was administered in the PC group. Renal function returned to baseline level at 50 days after the induction of kidney damage by gentamicin in the PC group.
3.1. Histological findings The histological changes are summarised for all experimental groups in Table 2.
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3.1.1. Negative control (NC) group Histopathological examination of the renal cortex of control rats revealed mild (+1) tubulointerstitial inflammatory cell infiltration in one rat, whilst another control rat showed mild (+1) glomerular damage characterised by glomerular basal membrane thickening. The remaining five rats in the control group did not exhibit any histological changes (Figs 3a and 4a; Table 2). The NC group differed significantly from the PS, PC, LAM and LdT groups according to histopathological examination scores.
3.1.2. Pilot study (PS) group necropsied on Day 8 All of the rat kidneys from the early gentamicin-treated PC group showed moderate (+2) focal mesangial proliferation and (+2) acute tubular necrosis. There was mild (+1) tubulointerstitial inflammatory cell infiltration in all cases studied (Fig. 3b; Table 2). The PS group (Day 8) and the PC group (Day 50) were different in terms of glomerular damage, acute tubular necrosis and total score by the classification of AlShabanah et al. [20].
3.1.3. Positive control (PC) group necropsied on Day 50 Kidney sections collected at Week 6 showed moderate (+2) glomerular damage in two rats. Glomerular damage in the remaining five rat kidneys was mild (+1) and included the localised presence of regenerating tubules characterised by basophilic nuclei. In the cortical region, mild (+2) acute tubular necrosis was recorded in two cases. The remaining five rat kidneys exhibited only mild (+1) tubular necrosis. There 13
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was mild (+1) tubulointerstitial inflammatory cell infiltration in all cases studied (Fig. 3c,d; Table 2).
3.1.4. Lamivudine (LAM) group In the LAM group, glomerular damage was mild (+1) in two cases and moderate (+2) in the other five rats. Acute tubular necrosis observed in the cortical region was moderate (+2) in two rats, whilst the remaining five rat kidneys exhibited only mild (+1) tubular necrosis. There was mild (+1) tubulointerstitial inflammatory cell infiltration in five cases, whilst two cases were free of inflammatory cell infiltration (Table 2). There was no significant difference between the PC and LAM groups in terms of glomerular damage, acute tubular necrosis and total histological score.
3.1.5. Telbivudine (LdT) group In the LdT group, glomerular damage was moderate (+2) in one case and mild (+1) in the remaining six cases. Acute tubular necrosis and degeneration were observed in the cortical region and were scored as mild (+1) in four cases, whilst three rat kidneys showed no signs of necrosis or degeneration. There was mild (+1) tubulointerstitial inflammatory cell infiltration in all seven cases (Fig. 3e,f; Table 2).
Periodic acid–Schiff staining of PC, LAM and LdT group kidney sections revealed both glomerular and tubular basal membrane thickening and glomerular mesangial cell proliferation (Fig. 4b,c,d). Histological severity was milder in the LdT group compared with the LAM group in terms of glomerular damage, acute tubular necrosis and total histological score (P < 0.05). Acute tubular necrosis and total histological 14
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score were significantly lower in the LdT group compared with the PC group (P < 0.05). However, there was no difference in glomerular damage and tubulointerstitial inflammatory infiltrates between the LdT and PC groups (Table 2).
The histopathological changes were scored as A in the NC group (Table 2). The score of gentamicin-induced nephrotoxicity on Day 8 of the PS group was C. On day 50, the nephrotoxicity score was B for the PC, LAM and LdT groups. According to these scoring results, it is seen that severe histopathological changes due to nephrotoxicity were formed on Day 8. On Day 50, histopathological changes were alleviated but were not recovered completely. These findings showed that the effect of nephrotoxicity decreased with time but was not completely eliminated.
4. Discussion LdT is a safe and well-tolerated option in HBV treatment [21,22]. It is available as an affordable option in Asia [23]. However, due to the emergence of drug resistance, optimisation strategies for LdT therapy using optimal baseline characteristics and ontreatment early virological response at Week 24 of therapy (HBV-DNA ≥ 300 copies/mL) should be used to reduce the risk of antiviral resistance [22]. The availability of LdT may provide physicians with a wide choice of options to effectively and safely treat patients with CHB, especially those with or at risk of renal impairment [21,24]. A recent meta-analysis including six studies and 1960 patients showed that LdT has a renoprotective effect whereas ETV does not. However, the meta-analysis did not show whether the benefit on renal function outweighs the occurrence of resistance in specific clinical situations [24]. 15
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The present study demonstrates the renoprotective effect of LdT compared with LAM using biochemical and histopathological methods. The observed nephrotoxicity was consistent with the existing literature regarding PS, PC, LAM and LdT treatment [16]. Nephrotoxicity occurred within 8 days and was largely resolved by Day 50 in the LdT, LAM and PC groups. There was no difference in the LAM treatment group compared with the PC group on Day 50 in terms of histological evidence of glomerular damage or acute tubular necrosis. Glomerular damage, acute tubular necrosis and total histological score were significantly reduced in the LdT group compared with the PC and LAM treatments groups. Histopathology and serum cystatin C concentration are strongly associated; LdT treatment was associated with significant improvements in renal recovery, whilst LAM treatment did not enhance the recovery of renal function compared with control treatment.
The change in serum cystatin C concentration between Day 8 and Day 50 was significantly greater in animals treated with LdT compared with the remaining treatment groups. Likewise, there was little evidence of histopathological damage to the kidneys in the LdT group compared with all other groups.
Uteng et al. compared the effects of the CHB drugs LdT, TDF, ETV and ADV on renal function and toxicity in an experimental study. High doses of these NAs were given to rats to evaluate the nephrotoxic effects of NAs. High doses of LdT were not associated with significant renal impairment or renal toxicity as assessed by magnetic resonance imaging (MRI), histology and urine/blood biomarkers [25]. 16
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In the literature, there has not been any study assessing the effect of oral antivirals for CHB on renal function in a nephrotoxicity model except for one poster presentation [25]. Therefore, the current study is the first experimental study to assess positive effects of LdT on renal function of nephrotoxicity in rats.
The guidelines of the European Association for the Study of the Liver (EASL) recommended that patients receiving treatment for CHB with ETV, TDF, LAM or ADV should be closely monitored owing to their potential nephrotoxicity. A minimal decline in renal function has been associated with all NAs, except perhaps for LdT, which appears to improve CLCr [21]. Also, LdT may be a safer treatment option in patients who have an underlying risk of renal impairment, including pregnant women and elderly patients [9,26].
Increases in GFRs have been reported in CHB patients undergoing LdT treatment in a limited subset of clinical studies [9,10,26]. Renal function decreased over time in patients undergoing LAM treatment over a 2-year treatment period in the GLOBE study that included CHB patients. Renal function recovers steadily in most patients, with LdT-treated patients experiencing significant gains in renal function during the second year of treatment [9]. The positive effects of LdT on renal function have been reported in glomerulopathies associated with HBV, haemodialysis patients with residual diuresis, and in HBV-positive renal transplant candidates and recipients [27].
Long-term LdT treatment is effective in improving renal function among patients with decompensated liver cirrhosis, baseline renal failure at age >50 years and history of liver transplantation [9]. Initiation of NAs prophylaxis has been recommended prior to 17
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chemotherapy in patients with hepatocellular carcinoma or in asymptomatic HBV carriers who will be undergoing chemotherapy for any reason. The preference for LdT has grown steadily for patients who will be undergoing chemotherapy protocols that include nephrotoxic agents such as cisplatin. A study conducted by Lin et al. reported that LdT prevents the nephrotoxic effects of cisplatin-based chemotherapy [26]. Similarly, LdT is recommended for CHB patients with diabetic nephropathy [28].
Fung et al. researched the nephrotoxic effects of ADV, TDF, ETV and LdT and the potential nephrotoxic effects of these drugs were compared. ETV has been reported to cause lactic acidosis. Only LdT has been reported to have a potential renoprotective effect [29].
The mechanism for the potential renoprotective effect of LdT is not explained clearly. However, there are some mechanisms offered to explain this effect in the literature, one of which was proposed by Liang et al. The authors proposed that LdT might suppress angiotensin-converting enzyme (ACE) expression. They reported a negative correlation between ACE levels and GFR. Likewise, GFR was significantly increased during long-term LdT treatment [28].
During development of chronic kidney disease, the number of functioning nephrons progressively decreases and the remaining nephrons functionally compensate for the lost ones. Depending both on clinical and experimental observations, Deray et al. hypothesised that LdT stimulates renal repair. LdT might help to repair the injury, and the speed and amount of this recovery may depend on the GFR at the beginning of LdT treatment [30]. LdT does not affect the renal function of normal kidneys and 18
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does not create either structural or functional change in normal rats, suggesting that normally functioning nephrons are not sensitive to LdT and that LdT may have an effect only on injured nephrons. Such a mechanism may involve LdT indirectly interfering with telomerase activity especially among cells driving fibrosis. For example, the telomerase activity of fibroblasts is increased during fibrosis. Suppression of this activity will allow natural recovery by removing devastating stress on injured cells. LdT may also effect mitochondrial function and allow natural recovery via decreasing fibrosis and nephron loss [30]. Promoting recovery of injured cells is suggested to be a mechanism of LdT in inflammation [30].
The main limitation of this study is that the long-term use of NAs in CHB patients is a model of chronic toxicity. However, in the current study an experimental model of acute toxicity was used. A nephrotoxic drug in a single high dose and another drug (either LdT or LAM) was used to search whether it may attenuate the nephrotoxicity. Although this model may be poorly representative of renal toxicity encountered in patients treated for CHB, the renoprotective effect of LdT is promising.
5. Conclusions The data presented in the current study demonstrate the renoprotective effects of LdT in an experimental nephropathy model using both biochemical and histopathological methods. Because of this significant renoprotective effect, LdT may be preferred over other antivirals in patient groups with chronic renal failure, compensated and decompensated liver cirrhosis, liver transplantation, diabetes mellitus, renal failure and patients under nephrotoxic chemotherapy. In patients who 19
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require long-term use, LdT therapy should be carefully monitored for viral resistance and switched to more potent agents. The mechanism for the potential renoprotective effect of LdT is still unclear. Additional comprehensive clinical and molecular studies on this subject are needed.
Funding: This research was supported by the Bozok University Scientific Research Project Units [Project No. 2014TF-A126].
Competing interests: None declared.
Ethical approval: This study was approved by the Animal Experiments Local Ethics Committee of Ondokuz Mayıs University (Samsun, Turkey) [2014/10]. Animal experiments were carried out in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). All animal studies were performed in accordance with the ARRIVE guidelines.
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Fatani AG, et al. Increased urinary losses of carnitine and decreased intramitochondrial coenzyme A in gentamicin-induced acute renal failure in rats. Nephrol Dial Transplant 2010;25:69–76. [21]
European Association for the Study of the Liver. EASL clinical practice
guidelines: management of chronic hepatitis B virus infection. J Hepatol 2012;57:167–85. 23
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[22]
Sarin SK, Kumar M, Lau GK, Abbas Z, Chan HL, Chen CJ, et al. Asian-
Pacific clinical practice guidelines on the management of hepatitis B: a 2015 update. Hepatol Int 2016;10:1–98. [23]
Lui YY, Tsoi KK, Wong VW, Kao JH, Hou JL, Teo EK, et al. Cost-
effectiveness analysis of roadmap models in chronic hepatitis B using tenofovir as the rescue therapy. Antivir Ther 2010;15:145–55. [24]
Wu X, Cai S, Li Z, Zheng C, Xue X, Zeng J, et al. Potential effects of
telbivudine and entecavir on renal function: a systematic review and metaanalysis. Virol J 2016;13:64. [25]
Uteng M, Mahl A, Beckmann N, Piaia A, Ledieu D, Dubost V, et al.
Preclinical assessment on renal function and toxicity of chronic hepatitis B (CHB) drugs telbivudine, entecavir, tenofovir and adefovir. Hepatol Int 2013;7(Suppl 1):139. [26]
Lin CL, Chien RN, Yeh C, Hsu CW, Chang ML, Chen YC, et al.
Significant renoprotective effect of telbivudine during preemptive antiviral therapy in advanced liver cancer patients receiving cisplatin-based chemotherapy: a case–control study. Scand J Gastroenterol 2014;49:1456– 64. [27]
Pipili C, Cholongitas E, Papatheodoridis G. Review article:
nucleos(t)ide analogues in patients with chronic hepatitis B virus infection and chronic kidney disease. Aliment Pharmacol Ther 2014;39:35–49. [28]
Liang KH, Chen YC, Hsu CW, Chang ML, Yeh CT. Decrease of serum
angiotensin converting enzyme levels upon telbivudine treatment for chronic hepatitis B virus infection and negative correlations between the enzyme levels and estimated glomerular filtration rates. Hepat Mon 2014;14:e15074. 24
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[29]
Fung J, Seto WK, Lai CL, Yuen MF. Extrahepatic effects of nucleoside
and nucleotide analogues in chronic hepatitis B treatment. J Gastroenterol Hepatol 2014;29:428–34. [30]
Deray G, Buti M, Gane E, Jia JD, Chan HLY, Craxi A, et al. Hepatitis B
virus infection and the kidney: renal abnormalities in HBV patients, antiviral drugs handling, and specific follow-up. Adv Hepatol 2015;article ID 596829. doi: 10.1155/2015/596829.
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Fig. 1. Study diagram.
Fig. 2. Cystatin C levels in the telbivudine (LdT), lamivudine (LAM) and positive control (PC) groups. The dashed lines indicate the baseline cystatin C concentration used as the cut-off value in subsequent analyses.
Fig. 3. Photomicrographs of renal cortex histology: (a) kidney of the negative control group, showing normal renal parenchyma; (b) kidney of the pilot study group, showing necrosis of the proximal tubular epithelium (arrows); (c,d) kidney of the positive control group, showing necrosis of the proximal tubular epithelium (c) and renal tubules lined with flattened epithelial cells exhibiting hyperchromatic nuclei, consistent with epithelial regeneration (arrows) (d); and (e,f) kidney of the telbivudine group, showing necrosis of the proximal tubular epithelium (e) and interstitial inflammation (arrows) (f). Haematoxylin–eosin, 300.
Fig. 4. Photomicrographs of the renal cortex histology: (a) kidney from the negative control rat showing normal renal parenchyma; (b) kidney from the positive control group, showing glomerular mesangial cell proliferation (arrows); (c) kidney from the lamivudine group, showing the parietal part (arrows) of the Bowman capsule and glomerular basal membrane thickening (arrowheads); and (d) kidney from the telbivudine group, showing tubular basal membrane thickening (arrows). Periodic acid–Schiff, a,b,c, 600; d, 300.
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Table 1 Cystatin C levels in the telbivudine (LdT), lamivudine (LAM) and positive control (PC) groups Day Cystatin C level (mean ± SE) (g/mL) LdT group
LAM group
PC group
0
2.01 ± 0.15
2.14 ± 0.13 1.90 ± 0.11
8
2.82 ± 0.14
2.54 ± 0.15 2.55 ± 0.19
15
1.83 ± 0.99
2.26 ± 0.13 2.49 ± 0.10
22
1.63 ± 0.07
2.21 ± 0.14 2.35 ± 0.16
29
1.41 ± 0.05
2.18 ± 0.15 2.33 ± 0.11
36
1.24 ± 0.02
2.00 ± 0.16 2.24 ± 0.12
43
1.13 ± 0.03
2.07 ± 0.12 2.24 ± 0.09
50
0.99 ± 0.06 a 1.61 ± 0.14 1.92 ± 0.02
SE, standard error. a
Serial measurements of cystatin C levels in the LdT group were lower than those in
the LAM (P = 0.0006) and PC groups (P = 0.0017) on Day 50.
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Table 2 Histological changes in the kidneys of rats in all groups Animal
Glomerular
Acute
Tubulointerstitial
Total
Scoring
No.
injury
tubular
inflammatory
score
system a
necrosis
infiltrates
Negative control (NC) group NC-1
0
0
0
0
A
NC-2
0
0
0
0
A
NC-3
0
0
0
0
A
NC-4
0
0
1
1
A
NC-5
0
0
0
0
A
NC-6
0
0
0
0
A
NC-7
1
0
0
1
A
Median
0 (0–1)
0 (0–1)
0 (0–1)
0 (0–
(range)
1)
Pilot study (PS) group PS-1
2
2
1
5
C
PS-2
2
2
1
5
C
PS-3
2
2
1
5
C
Median
2 (2–2)
2 (2–2)
1 (1–1)
5 (5–
(range)
5)
Positive control (PC) group PC-1
1
1
1
3
B
PC-2
1
2
1
4
B
PC-3
1
1
1
3
B
PC-4
1
2
1
4
B
PC-5
1
1
1
3
B
PC-6
2
1
1
4
B
PC-7
2
1
1
4
B
Median
1 (1–2)
1 (1–2)
1 (1–1)
4 (3–
(range)
4)
Lamivudine (LAM) group 28
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LAM-1
2
1
1
4
B
LAM-2
1
2
0
3
B
LAM-3
2
2
0
4
B
LAM-4
2
1
1
4
B
LAM-5
1
1
1
3
B
LAM-6
2
1
1
4
B
LAM-7
2
1
1
4
B
Median
2 (1–2)
1 (1–2)
1 (0–1)
4 (3–
(range)
4)
Telbivudine (LdT) group LdT-1
1
1
1
3
B
LdT-2
1
1
1
3
B
LdT-3
1
0
1
2
B
LdT-4
1
0
1
2
B
LdT-5
1
0
1
2
B
LdT-6
1
1
1
3
B
LdT-7
2
1
1
4
B
Median
1 (1–2)
1 (0–1)
1 (1–1)
3 (2–
(range) a
4)
A, no nephrotoxicity (0–1); B, mild nephrotoxicity (2–4); C, moderate nephrotoxicity
(5–7); D, severe nephrotoxicity (8–10) [20]. In terms of total score of histological changes: NC vs. PC, LAM and LdT, P < 0.05; PC vs. NC and LdT, P < 0.05; LAM vs. NC and LdT, P < 0.05; and LdT vs NC, PC and LAM, P < 0.05.
29
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S0924-8579(17)30102-4 http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.01.015 ANTAGE 5064
To appear in:
International Journal of Antimicrobial Agents
Received date: Accepted date:
20-4-2016 6-1-2017
Please cite this article as: Cigdem Kader, Mustafa Sunbul, Yavuz Kursad Das, Murat Yarim, Abdulkerim Bedir, Efe Karaca, Mehmet Celikbilek, Resat Ozaras, Telbivudine attenuates gentamicin-induced kidney injury in rats, International Journal of Antimicrobial Agents (2017), http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.01.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Telbivudine attenuates gentamicin-induced kidney injury in rats
Cigdem Kader a, Mustafa Sunbul b, Yavuz Kursad Das c, Murat Yarim d, Abdulkerim Bedir e, Efe Karaca d, Mehmet Celikbilek f, Resat Ozaras g,*
a
Departments of Infectious Diseases and Clinical Microbiology, Bozok University,
School of Medicine, Yozgat, Turkey b
Departments of Infectious Diseases and Clinical Microbiology, Ondokuz Mayıs
University, School of Medicine, Samsun, Turkey c
Department of Pharmacology and Toxicology, Ondokuz Mayıs University, School of
Veterinary Medicine, Samsun, Turkey d
Department of Pathology, Ondokuz Mayıs University, School of Veterinary
Medicine, Samsun, Turkey e
Department of Medical Biochemistry, Ondokuz Mayıs University, School of
Medicine, Samsun, Turkey f
Department of Internal Medicine, Unit of Gastroenterology, Bozok University,
School of Medicine, Yozgat, Turkey g
Department of Infectious Diseases and Clinical Microbiology, Istanbul University,
Cerrahpaşa Medical School, Istanbul, Turkey
* Corresponding author. Tel.: +90 212 414 3095; fax: +90 212 414 3095. E-mail address: [email protected] (R. Ozaras).
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ARTICLE INFO Article history: Received 20 April 2016 Accepted 6 January 2017
Keywords: Telbivudine Chronic hepatitis B Cystatin C Glomerular filtration rate Lamivudine
2
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Highlights Telbivudine (LdT) is not associated with nephrotoxicity in clinical practice. LdT may increase the glomerular filtration rate. Renal function in rats was measured by biochemical and histopathological methods. LdT was associated with significant improvements in renal function in rats in this study.
ABSTRACT Nephrotoxicity has been associated with nucleos(t)ide analogues other than telbivudine (LdT). This study investigated the potential effects of LdT and lamivudine (LAM) on renal function in an experimental rat model of gentamicin-induced acute nephrotoxicity. A total of 28 healthy Wistar albino rats were randomly divided into four experimental groups: negative control; positive control (PC); LdT; and LAM. Nephrotoxicity was induced by gentamicin in the LdT, LAM and PC groups. LdT and LAM were administered to two groups for 6 weeks starting on the ninth day. Blood samples were collected weekly and cystatin C levels were measured by ELISA. Animals were sacrificed on the 50th day and the kidneys were removed for histological examination. Serum cystatin C levels differed significantly between the LdT and LAM groups (P < 0.007) and between the LdT and PC groups (P < 0.001). Renal function was significantly improved in the LdT group at the start of antiviral treatment on Day 8 and at the end of treatment on Day 50 (P = 0.001 and 0.007). Glomerular injury, acute tubular necrosis and total injury score were significantly reduced in the LdT group relative to the PC and LAM groups upon histopathological 3
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examination. LdT was associated with significant improvements in renal function as measured by biochemical and histopathological methods. The acute kidney injury model data should be supported by clinical studies to suggest that LdT treatment may have advantages for patients with underlying chronic kidney disease receiving chronic hepatitis B treatment.
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1. Introduction Nucleos(t)ide analogues (NAs) have been licensed for oral treatment of chronic hepatitis B (CHB) [1] and are the most widely used antiviral drugs in CHB treatment in recent years. NAs inhibit viral DNA polymerase [2]. Although these agents are effective in the suppression of hepatitis B virus (HBV) replication, they cannot fully eliminate the virus from the host. As a result, long-term treatment is required in most patients. NAs are generally safe and well tolerated, although there are concerns of metabolic side effects in some patients with long-term use [3].
Treatment of CHB with NAs is long. Besides renal involvement during the course of hepatitis as an extrahepatic manifestation, long-term use of NAs may be associated with renal failure especially in patients with underlying renal disease, elderly patients and in those using concomitant nephrotoxic drugs. Adefovir (ADV), which nowadays is rarely used, carries a higher risk of nephrotoxicity. Tenofovir (TDF) may induce nephrotoxicity in nearly 15% of patients treated for 2–9 years [2].
NAs are excreted by the kidneys and as a result dosage adjustment according to glomerular filtration rate (GFR) is required [4]. Cohort studies have suggested that renal impairment is more frequently reported in CHB patients treated with ADV. In a longitudinal observational study of 214 patients receiving HBV treatment, the estimated GFR (eGFR) decreased in patients receiving ADV as monotherapy or in a combination but remained stable in patients receiving lamivudine (LAM), TDF or entecavir (ETV) at a median follow-up of 2.4 years. The eGFR decreased in patients with a baseline eGFR of <90 mL/min/1.73 m2 regardless of treatment [5]. Of 641 5
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patients initially randomised in a pivotal study of TDF, 585 patients entered the openlabel phase, of which 10 (1.7%) had an elevation of serum creatinine of ≥0.5 mg/dL above baseline [6]. In another 3-year prospective study of 400 patients receiving TDF treatment, few patients (1.3%) experienced renal adverse reactions, and creatinine clearance (CLCr) remained stable over time. Patients responded favourably when the TDF dose was adjusted for decreased CLCr [7].
In a limited number of clinical studies conducted by different teams worldwide, telbivudine (LdT) has been reported to promote a significant increase in GFR [4,8– 10]. These data suggest that LdT may protect against nephrotoxicity resulting from other conditions. A real-world retrospective study of 587 patients with CHB treated with TDF (n = 170), LdT (n = 184) or ETV (n = 233) showed that eGFR decreased significantly in the TDF group after a mean treatment duration of 17 months, however it increased in the LdT group after a mean treatment duration of 32 months. In the ETV group there was no significant change in eGFR after a mean of 44 months of treatment [11].
In a recent study including 4178 CHB patients treated with NAs, renal functional declined in 706 (16.9%). Age, hypertension, diabetes, history of liver or kidney transplantation, underlying chronic kidney disease and simultaneous administration of diuretics were found to increase the hazard ratio for renal functional decline. The eGFR significantly increased over time in patients receiving LdT or clevudine compared with LAM [12].
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Cystatin C is a low-molecular-weight non-glycosylated protein and a cysteine proteinase inhibitor. It is synthesised continuously at a constant rate in all nucleated cells in the body [13]. Cystatin C is freely filtered through the glomeruli, re-absorbed through tubular epithelial cells and metabolised rapidly in the kidneys [14]. It is used for calculating GFR. More recently, cystatin C has been applied to monitor changes in GFR and has been shown to be more sensitive than traditional measures based on serum creatinine level and CLCr [15]. Gentamicin, an aminoglycoside antibiotic, is frequently used to induce experimental nephrotoxicity [16]. Cystatin C is reported to be the most sensitive test of nephrotoxicity in rats with nephropathy induced by gentamicin and can detect damage earlier than traditional creatinine and blood urea nitrogen assays [17]. Thus, cystatin C was preferred in the current study.
The potential effects of LdT and LAM on renal function were investigated in a model of experimental nephrotoxicity caused by gentamicin exposure. Serum cystatin C levels were measured as an indicator of nephrotoxicity, and histopathological changes in kidney tissue were examined.
2. Materials and methods 2.1. Animals A total of 31 healthy Wistar albino male rats (age 12–16 weeks, weight 260–360 g) were used in the study. Standard chow and water were provided ad libitum during the study period. Animals were housed under a 12-h light/dark cycle.
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2.2. Drugs Gentamicin sulfate was administered by the intraperitoneal (i.p.) route for 8 days at a dose of 80 mg/kg to induce nephropathy [15]. The experimental drugs LdT (Sigma, St Louis, MO) [18] and LAM (Sigma) [19], were dissolved in distilled water and were administered orally by gastric gavage.
2.3. Experimental protocol As a comparator to LdT, LAM was preferred since LAM has sufficient renal safety data, is inexpensive and is easily accessible in many developing and Far East countries. Three rats were used in a preliminary study to verify the nephrotoxicity model [pilot study (PS)]. Rats in the PS group were given 80 mg/kg gentamicin sulfate by i.p. injection once daily for 8 days. Necropsies were performed and nephrotoxicity was determined histopathologically on Day 9.
The principal investigation was initiated after confirming nephrotoxicity in the PS group. The study included four experimental groups as follows: negative control (NC) (i.p. saline only); positive control (PC) (i.p. gentamicin only); LdT (10 mg/kg); and LAM (5 mg/kg).
A total of seven rats were assigned to each group at random. Nephrotoxicity was induced by administering 80 mg/kg gentamicin sulfate to the LdT, LAM and PC groups by i.p. injection for 8 days [16]. Blood samples were collected from the tail vein in all groups at the end of Day 8. A dose of 10 mg/kg LdT was administered to the LdT group [17] and 5 mg/kg LAM was given to the LAM group [19] by gastric 8
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gavage for 6 weeks starting on Day 9. Blood was obtained from the tail vein of rats in the LdT, LAM and PC groups on Days 15, 22, 29, 36, 43 and 50. Blood was collected from the NC group on Day 50. The study protocol is presented in Fig. 1. Rats in all groups were weighed prior to blood collection and LdT and LAM dosing was adjusted according to body weight.
2.4. Serum cystatin C measurement Blood samples were collected into microtubes and were centrifuged at 1000 g for 10 min at room temperature to separate serum for measurement of cystatin C. Serum samples were stored at –80 C for subsequent analysis. Rat serum cystatin C levels were measured quantitatively using a sandwich enzyme-linked immunosorbent assay (ELISA) kit (BioVendor–Laboratorni Medicina a.s., Brno, Czech Republic) according to the manufacturer’s recommendations. Just prior to the assay, serum samples were diluted 500 times with dilution buffer in two steps. The performance of the ELISA kit was validated using low- and high-concentration control materials included by the manufacturer.
2.5. Histopathological examination of kidney tissues PS group rats were sacrificed on Day 8 and rats from the remaining four groups were sacrificed on Day 50. The kidneys from rats in all four groups were excised following necropsy. A semiquantitative evaluation of renal tissues was performed by scoring the degree of damage severity according to previously published criteria [20]. The following parameters were used for the grading of injury: (i) glomerular injury (% 9
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of renal parenchyma involvement): none = 0, <25% of glomeruli exhibiting nonspecific features of injury = +1, 25–50% of glomeruli exhibiting non-specific features of injury = +2, 50–75% of glomeruli exhibiting non-specific features of injury = +3, and >75% of glomeruli exhibiting non-specific features of injury = +4; (ii) acute tubular necrosis (% of renal parenchyma involvement): none = 0, <25% of tubules out of the entire renal parenchyma = +1, 25–50% of tubules out of the entire renal parenchyma = +2, 50–75% of tubules out of the entire renal parenchyma = +3, and >75% of tubules out of the entire renal parenchyma = +4; and (iii) tubulointerstitial inflammatory infiltrates: none = 0, leukocytes confined within the interstitium = +1, and leukocytes infiltrating the interstitium and tubular epithelial cells = +2 [20].
2.6. Ethical considerations This study was approved by the Animal Experiments Local Ethics Committee of Ondokuz Mayıs University (Samsun, Turkey). Animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All animal studies were performed in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.
2.7. Statistical analysis Statistical analyses were conducted using IBM SPSS Base software (IBM Corp., Armonk, NY). Differences in continuous values were evaluated using the Shapiro– Wilk test and were determined to be in accordance with a normal distribution. Repeated measures analysis of variance (ANOVA) was used to estimate variance in cystatin C measurements. Linear regression was used to analyse the change in 10
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cystatin C levels over time in the LdT, LAM and PC groups. Increases in cystatin C concentrations in blood samples obtained 8 days after gentamicin administration and in blood samples obtained at the end of Day 50 were compared by one-way ANOVA with the Tukey test for paired comparisons. The Kruskal–Wallis test was used for variance analysis and the Mann–Whitney U-test was used for pairwise comparisons of histopathological assessments between groups. A P-value of <0.05 was considered statistically significant.
3. Results There were no significant differences in serum cystatin C concentration among the study groups on Days 0 or 8 (P > 0.05). Cystatin C was measured in blood samples collected from the LdT, LAM and PC groups at the beginning of study; the baseline cystatin C concentration was used as the cut-off value in subsequent analyses. Serum cystatin C concentrations in the LdT, LAM and PC groups are presented in Fig. 2. Cut-off values are indicated in the figure by dashed lines.
The mean serum cystatin C concentration and standard error values for the LdT, LAM and PC groups are presented in Table 1.
There was a significant difference in serum cystatin C concentration between the LdT and LAM groups (P = 0.0006) on Day 50. In addition, cystatin C levels differed significantly in the LdT group compared with the PC group (P = 0.0017). However, there was no difference in cystatin C levels between the LAM and PC groups (P > 0.05). 11
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The time required for cystatin C levels to return to the baseline concentration of 2.01 g/mL was ca. 8 days (7.72 days) in the LdT group as measured by the logarithmic formula obtained through regression modelling. Thus, kidney damage peaked on Day 8 and returned to physiological baseline after an additional 8 days.
Although cystatin C values on Day 15 of the LdT group were lower than the baseline value, this difference was not statistically significant (P > 0.05, one-way ANOVA with the Tukey test for paired comparisons).
In the LAM group, cystatin C levels were equivalent to the baseline value of 2.14 g/mL at ca. 29 days after the induction of kidney damage by gentamicin administration on linear formula obtained in the regression model. In this case, the group physiological baseline value was achieved at 21 days after LAM treatment.
In the PC group, cystatin C levels were equivalent to the baseline value of 1.90 g/mL at ca. 59 days after the induction of kidney damage by gentamicin exposure based on the results of linear regression modelling. No antiviral treatment was administered in the PC group. Renal function returned to baseline level at 50 days after the induction of kidney damage by gentamicin in the PC group.
3.1. Histological findings The histological changes are summarised for all experimental groups in Table 2.
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3.1.1. Negative control (NC) group Histopathological examination of the renal cortex of control rats revealed mild (+1) tubulointerstitial inflammatory cell infiltration in one rat, whilst another control rat showed mild (+1) glomerular damage characterised by glomerular basal membrane thickening. The remaining five rats in the control group did not exhibit any histological changes (Figs 3a and 4a; Table 2). The NC group differed significantly from the PS, PC, LAM and LdT groups according to histopathological examination scores.
3.1.2. Pilot study (PS) group necropsied on Day 8 All of the rat kidneys from the early gentamicin-treated PC group showed moderate (+2) focal mesangial proliferation and (+2) acute tubular necrosis. There was mild (+1) tubulointerstitial inflammatory cell infiltration in all cases studied (Fig. 3b; Table 2). The PS group (Day 8) and the PC group (Day 50) were different in terms of glomerular damage, acute tubular necrosis and total score by the classification of AlShabanah et al. [20].
3.1.3. Positive control (PC) group necropsied on Day 50 Kidney sections collected at Week 6 showed moderate (+2) glomerular damage in two rats. Glomerular damage in the remaining five rat kidneys was mild (+1) and included the localised presence of regenerating tubules characterised by basophilic nuclei. In the cortical region, mild (+2) acute tubular necrosis was recorded in two cases. The remaining five rat kidneys exhibited only mild (+1) tubular necrosis. There 13
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was mild (+1) tubulointerstitial inflammatory cell infiltration in all cases studied (Fig. 3c,d; Table 2).
3.1.4. Lamivudine (LAM) group In the LAM group, glomerular damage was mild (+1) in two cases and moderate (+2) in the other five rats. Acute tubular necrosis observed in the cortical region was moderate (+2) in two rats, whilst the remaining five rat kidneys exhibited only mild (+1) tubular necrosis. There was mild (+1) tubulointerstitial inflammatory cell infiltration in five cases, whilst two cases were free of inflammatory cell infiltration (Table 2). There was no significant difference between the PC and LAM groups in terms of glomerular damage, acute tubular necrosis and total histological score.
3.1.5. Telbivudine (LdT) group In the LdT group, glomerular damage was moderate (+2) in one case and mild (+1) in the remaining six cases. Acute tubular necrosis and degeneration were observed in the cortical region and were scored as mild (+1) in four cases, whilst three rat kidneys showed no signs of necrosis or degeneration. There was mild (+1) tubulointerstitial inflammatory cell infiltration in all seven cases (Fig. 3e,f; Table 2).
Periodic acid–Schiff staining of PC, LAM and LdT group kidney sections revealed both glomerular and tubular basal membrane thickening and glomerular mesangial cell proliferation (Fig. 4b,c,d). Histological severity was milder in the LdT group compared with the LAM group in terms of glomerular damage, acute tubular necrosis and total histological score (P < 0.05). Acute tubular necrosis and total histological 14
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score were significantly lower in the LdT group compared with the PC group (P < 0.05). However, there was no difference in glomerular damage and tubulointerstitial inflammatory infiltrates between the LdT and PC groups (Table 2).
The histopathological changes were scored as A in the NC group (Table 2). The score of gentamicin-induced nephrotoxicity on Day 8 of the PS group was C. On day 50, the nephrotoxicity score was B for the PC, LAM and LdT groups. According to these scoring results, it is seen that severe histopathological changes due to nephrotoxicity were formed on Day 8. On Day 50, histopathological changes were alleviated but were not recovered completely. These findings showed that the effect of nephrotoxicity decreased with time but was not completely eliminated.
4. Discussion LdT is a safe and well-tolerated option in HBV treatment [21,22]. It is available as an affordable option in Asia [23]. However, due to the emergence of drug resistance, optimisation strategies for LdT therapy using optimal baseline characteristics and ontreatment early virological response at Week 24 of therapy (HBV-DNA ≥ 300 copies/mL) should be used to reduce the risk of antiviral resistance [22]. The availability of LdT may provide physicians with a wide choice of options to effectively and safely treat patients with CHB, especially those with or at risk of renal impairment [21,24]. A recent meta-analysis including six studies and 1960 patients showed that LdT has a renoprotective effect whereas ETV does not. However, the meta-analysis did not show whether the benefit on renal function outweighs the occurrence of resistance in specific clinical situations [24]. 15
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The present study demonstrates the renoprotective effect of LdT compared with LAM using biochemical and histopathological methods. The observed nephrotoxicity was consistent with the existing literature regarding PS, PC, LAM and LdT treatment [16]. Nephrotoxicity occurred within 8 days and was largely resolved by Day 50 in the LdT, LAM and PC groups. There was no difference in the LAM treatment group compared with the PC group on Day 50 in terms of histological evidence of glomerular damage or acute tubular necrosis. Glomerular damage, acute tubular necrosis and total histological score were significantly reduced in the LdT group compared with the PC and LAM treatments groups. Histopathology and serum cystatin C concentration are strongly associated; LdT treatment was associated with significant improvements in renal recovery, whilst LAM treatment did not enhance the recovery of renal function compared with control treatment.
The change in serum cystatin C concentration between Day 8 and Day 50 was significantly greater in animals treated with LdT compared with the remaining treatment groups. Likewise, there was little evidence of histopathological damage to the kidneys in the LdT group compared with all other groups.
Uteng et al. compared the effects of the CHB drugs LdT, TDF, ETV and ADV on renal function and toxicity in an experimental study. High doses of these NAs were given to rats to evaluate the nephrotoxic effects of NAs. High doses of LdT were not associated with significant renal impairment or renal toxicity as assessed by magnetic resonance imaging (MRI), histology and urine/blood biomarkers [25]. 16
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In the literature, there has not been any study assessing the effect of oral antivirals for CHB on renal function in a nephrotoxicity model except for one poster presentation [25]. Therefore, the current study is the first experimental study to assess positive effects of LdT on renal function of nephrotoxicity in rats.
The guidelines of the European Association for the Study of the Liver (EASL) recommended that patients receiving treatment for CHB with ETV, TDF, LAM or ADV should be closely monitored owing to their potential nephrotoxicity. A minimal decline in renal function has been associated with all NAs, except perhaps for LdT, which appears to improve CLCr [21]. Also, LdT may be a safer treatment option in patients who have an underlying risk of renal impairment, including pregnant women and elderly patients [9,26].
Increases in GFRs have been reported in CHB patients undergoing LdT treatment in a limited subset of clinical studies [9,10,26]. Renal function decreased over time in patients undergoing LAM treatment over a 2-year treatment period in the GLOBE study that included CHB patients. Renal function recovers steadily in most patients, with LdT-treated patients experiencing significant gains in renal function during the second year of treatment [9]. The positive effects of LdT on renal function have been reported in glomerulopathies associated with HBV, haemodialysis patients with residual diuresis, and in HBV-positive renal transplant candidates and recipients [27].
Long-term LdT treatment is effective in improving renal function among patients with decompensated liver cirrhosis, baseline renal failure at age >50 years and history of liver transplantation [9]. Initiation of NAs prophylaxis has been recommended prior to 17
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chemotherapy in patients with hepatocellular carcinoma or in asymptomatic HBV carriers who will be undergoing chemotherapy for any reason. The preference for LdT has grown steadily for patients who will be undergoing chemotherapy protocols that include nephrotoxic agents such as cisplatin. A study conducted by Lin et al. reported that LdT prevents the nephrotoxic effects of cisplatin-based chemotherapy [26]. Similarly, LdT is recommended for CHB patients with diabetic nephropathy [28].
Fung et al. researched the nephrotoxic effects of ADV, TDF, ETV and LdT and the potential nephrotoxic effects of these drugs were compared. ETV has been reported to cause lactic acidosis. Only LdT has been reported to have a potential renoprotective effect [29].
The mechanism for the potential renoprotective effect of LdT is not explained clearly. However, there are some mechanisms offered to explain this effect in the literature, one of which was proposed by Liang et al. The authors proposed that LdT might suppress angiotensin-converting enzyme (ACE) expression. They reported a negative correlation between ACE levels and GFR. Likewise, GFR was significantly increased during long-term LdT treatment [28].
During development of chronic kidney disease, the number of functioning nephrons progressively decreases and the remaining nephrons functionally compensate for the lost ones. Depending both on clinical and experimental observations, Deray et al. hypothesised that LdT stimulates renal repair. LdT might help to repair the injury, and the speed and amount of this recovery may depend on the GFR at the beginning of LdT treatment [30]. LdT does not affect the renal function of normal kidneys and 18
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does not create either structural or functional change in normal rats, suggesting that normally functioning nephrons are not sensitive to LdT and that LdT may have an effect only on injured nephrons. Such a mechanism may involve LdT indirectly interfering with telomerase activity especially among cells driving fibrosis. For example, the telomerase activity of fibroblasts is increased during fibrosis. Suppression of this activity will allow natural recovery by removing devastating stress on injured cells. LdT may also effect mitochondrial function and allow natural recovery via decreasing fibrosis and nephron loss [30]. Promoting recovery of injured cells is suggested to be a mechanism of LdT in inflammation [30].
The main limitation of this study is that the long-term use of NAs in CHB patients is a model of chronic toxicity. However, in the current study an experimental model of acute toxicity was used. A nephrotoxic drug in a single high dose and another drug (either LdT or LAM) was used to search whether it may attenuate the nephrotoxicity. Although this model may be poorly representative of renal toxicity encountered in patients treated for CHB, the renoprotective effect of LdT is promising.
5. Conclusions The data presented in the current study demonstrate the renoprotective effects of LdT in an experimental nephropathy model using both biochemical and histopathological methods. Because of this significant renoprotective effect, LdT may be preferred over other antivirals in patient groups with chronic renal failure, compensated and decompensated liver cirrhosis, liver transplantation, diabetes mellitus, renal failure and patients under nephrotoxic chemotherapy. In patients who 19
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require long-term use, LdT therapy should be carefully monitored for viral resistance and switched to more potent agents. The mechanism for the potential renoprotective effect of LdT is still unclear. Additional comprehensive clinical and molecular studies on this subject are needed.
Funding: This research was supported by the Bozok University Scientific Research Project Units [Project No. 2014TF-A126].
Competing interests: None declared.
Ethical approval: This study was approved by the Animal Experiments Local Ethics Committee of Ondokuz Mayıs University (Samsun, Turkey) [2014/10]. Animal experiments were carried out in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). All animal studies were performed in accordance with the ARRIVE guidelines.
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Fig. 1. Study diagram.
Fig. 2. Cystatin C levels in the telbivudine (LdT), lamivudine (LAM) and positive control (PC) groups. The dashed lines indicate the baseline cystatin C concentration used as the cut-off value in subsequent analyses.
Fig. 3. Photomicrographs of renal cortex histology: (a) kidney of the negative control group, showing normal renal parenchyma; (b) kidney of the pilot study group, showing necrosis of the proximal tubular epithelium (arrows); (c,d) kidney of the positive control group, showing necrosis of the proximal tubular epithelium (c) and renal tubules lined with flattened epithelial cells exhibiting hyperchromatic nuclei, consistent with epithelial regeneration (arrows) (d); and (e,f) kidney of the telbivudine group, showing necrosis of the proximal tubular epithelium (e) and interstitial inflammation (arrows) (f). Haematoxylin–eosin, 300.
Fig. 4. Photomicrographs of the renal cortex histology: (a) kidney from the negative control rat showing normal renal parenchyma; (b) kidney from the positive control group, showing glomerular mesangial cell proliferation (arrows); (c) kidney from the lamivudine group, showing the parietal part (arrows) of the Bowman capsule and glomerular basal membrane thickening (arrowheads); and (d) kidney from the telbivudine group, showing tubular basal membrane thickening (arrows). Periodic acid–Schiff, a,b,c, 600; d, 300.
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Table 1 Cystatin C levels in the telbivudine (LdT), lamivudine (LAM) and positive control (PC) groups Day Cystatin C level (mean ± SE) (g/mL) LdT group
LAM group
PC group
0
2.01 ± 0.15
2.14 ± 0.13 1.90 ± 0.11
8
2.82 ± 0.14
2.54 ± 0.15 2.55 ± 0.19
15
1.83 ± 0.99
2.26 ± 0.13 2.49 ± 0.10
22
1.63 ± 0.07
2.21 ± 0.14 2.35 ± 0.16
29
1.41 ± 0.05
2.18 ± 0.15 2.33 ± 0.11
36
1.24 ± 0.02
2.00 ± 0.16 2.24 ± 0.12
43
1.13 ± 0.03
2.07 ± 0.12 2.24 ± 0.09
50
0.99 ± 0.06 a 1.61 ± 0.14 1.92 ± 0.02
SE, standard error. a
Serial measurements of cystatin C levels in the LdT group were lower than those in
the LAM (P = 0.0006) and PC groups (P = 0.0017) on Day 50.
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Table 2 Histological changes in the kidneys of rats in all groups Animal
Glomerular
Acute
Tubulointerstitial
Total
Scoring
No.
injury
tubular
inflammatory
score
system a
necrosis
infiltrates
Negative control (NC) group NC-1
0
0
0
0
A
NC-2
0
0
0
0
A
NC-3
0
0
0
0
A
NC-4
0
0
1
1
A
NC-5
0
0
0
0
A
NC-6
0
0
0
0
A
NC-7
1
0
0
1
A
Median
0 (0–1)
0 (0–1)
0 (0–1)
0 (0–
(range)
1)
Pilot study (PS) group PS-1
2
2
1
5
C
PS-2
2
2
1
5
C
PS-3
2
2
1
5
C
Median
2 (2–2)
2 (2–2)
1 (1–1)
5 (5–
(range)
5)
Positive control (PC) group PC-1
1
1
1
3
B
PC-2
1
2
1
4
B
PC-3
1
1
1
3
B
PC-4
1
2
1
4
B
PC-5
1
1
1
3
B
PC-6
2
1
1
4
B
PC-7
2
1
1
4
B
Median
1 (1–2)
1 (1–2)
1 (1–1)
4 (3–
(range)
4)
Lamivudine (LAM) group 28
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LAM-1
2
1
1
4
B
LAM-2
1
2
0
3
B
LAM-3
2
2
0
4
B
LAM-4
2
1
1
4
B
LAM-5
1
1
1
3
B
LAM-6
2
1
1
4
B
LAM-7
2
1
1
4
B
Median
2 (1–2)
1 (1–2)
1 (0–1)
4 (3–
(range)
4)
Telbivudine (LdT) group LdT-1
1
1
1
3
B
LdT-2
1
1
1
3
B
LdT-3
1
0
1
2
B
LdT-4
1
0
1
2
B
LdT-5
1
0
1
2
B
LdT-6
1
1
1
3
B
LdT-7
2
1
1
4
B
Median
1 (1–2)
1 (0–1)
1 (1–1)
3 (2–
(range) a
4)
A, no nephrotoxicity (0–1); B, mild nephrotoxicity (2–4); C, moderate nephrotoxicity
(5–7); D, severe nephrotoxicity (8–10) [20]. In terms of total score of histological changes: NC vs. PC, LAM and LdT, P < 0.05; PC vs. NC and LdT, P < 0.05; LAM vs. NC and LdT, P < 0.05; and LdT vs NC, PC and LAM, P < 0.05.
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