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Drug-induced kidney injury (DIKI) is the major cause of acute kidney injury (AKI). Renal proximal tubular epithelial cells (RPTECs) are the primary target sites of DIKI and express transporters involved in renal drug disposition. In the present study, we focused on three-dimensionally cultured human RPTECs (3D-RPTECs) with elevated expression of drug transporters to investigate their utility in DIKI evaluation. Intracellular ATP levels in 3D-RPTECs are reduced by tenofovir and cisplatin that are substrates of an organic anion transporter 1 and an organic cation transporter 2, respectively. In addition, 3D-RPTECs were exposed to 17 and 15 drugs that are positive and negative to RPTEC toxicity, respectively, for up to 28 d. The 20 % decreasing concentration of drugs for ATP amount (EC 20 ) was obtained, and the ratio of EC 20 values and clinical maximum concentration (C max ) ≤100 were used as cut-off value to evaluate potential of DIKI. The sensitivities of 3D-RPTECs were 82.4 % and 88.2 % after 7 d and 28 d of drug exposure, respectively, and the specificities were 100 % and 93.3 %, respectively. The predictive performance of 3D-RPTECs was higher than that of two-dimensional cultured RPTECs and the kidney cell line HK-2. In conclusion, 3D-RPTECs are useful for in vitro evaluation of RPTEC injury by measuring intracellular ATP levels., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The present study was partially supported by Nikkiso Co., Ltd., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Tenofovir alafenamide (TAF) is a new drug from the nucleotide reverse transcriptase inhibitor group approved for the treatment of chronic Hepatitis B in 2016. With this study, we aimed to test whether possible cellular toxicity can be reduced by controlled drug release as a result of loading with chitosan nanoparticles (CHS). We investigated the genotoxic and mitotoxic effects of 45 µM TAF-loaded CHS and TAF-only on HepG2 cells by micronucleus (MN), comet assay, determination of mtDNA quantification, mitochondrial membrane potential (ΔΨm), and ROS levels. Additionally, we compared the samples by RNAseq analyses to reveal the transcriptional responses to each regimen. In terms of genotoxic tests, although MN and comet were found higher in all experimental treatment conditions, the encapsulation of CHS reduced the genotoxicity of TAF. MtDNA level was found to be lower in the TAF treatment, whereas it was higher in CHS and CHS-TAF treatments. The TAF-loaded CHS and TAF treatments had an impaired ΔΨm value. Cellular ROS levels were higher in all treatment conditions. According to the analyses of gene expression patterns; CHS-only changed the expression of relatively few genes (187 genes), while TAF changed the expression of the 1974 genes and TAF-loaded CHS changed the expression of 734 genes. Considering the gene expression numbers, CHS encapsulation of TAF significantly reduced the number of genes that were differentially expressed by TAF-only. Overall, we observed that TAF has genotoxic and mitotoxic effects on HepG2 cells, and upon encapsulation with CHS, its genotoxic and mitotoxic effects were decreased.

During the drug development process, organ toxicity leads to an estimated failure of one-third of novel chemical entities. Drug-induced toxicity is increasingly associated with mitochondrial dysfunction, but identifying the underlying molecular mechanisms remains a challenge. Computational modeling techniques have proven to be a good tool in searching for drug off-targets. Here, we aimed to identify mitochondrial off-targets of the nephrotoxic drugs tenofovir and gentamicin using different in silico approaches (KRIPO, ProBis and PDID). Dihydroorotate dehydrogenase (DHODH) and pyruvate dehydrogenase (PDH) were predicted as potential novel off-target sites for tenofovir and gentamicin, respectively. The predicted targets were evaluated in vitro, using (colorimetric) enzymatic activity measurements. Tenofovir did not inhibit DHODH activity, while gentamicin potently reduced PDH activity. In conclusion, the use of in silico methods appeared a valuable approach in predicting PDH as a mitochondrial off-target of gentamicin. Further research is required to investigate the contribution of PDH inhibition to overall renal toxicity of gentamicin., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: F.G.M.R. is co-inventor on patent EP2010/066792 ‘Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters’ assigned to Radboud University Medical Center and has conflict of interest through commercialization of ciPTEC models via Cell4Pharma. J.A.M.S. is the founding CEO of Khondrion BV, a Radboud University Medical Center spin-out company founded by J.A.M.S., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Aims: Maternal treatment with nucleoside analogues such as telbivudine (LdT) and tenofovir disoproxil fumarate (TDF) has been applied worldwide. However, administration of LdT or TDF during pregnancy may affect the fetal neuromuscular development. We conducted the current study to investigate the histological pathology and transcriptomic changes pertaining to the neuromuscular system of the newborn exposed to LdT or TDF during pregnancy in rodent model., Main Methods: Pregnant C57/BL6 mice were randomly divided into three arms and administered either with LdT solution (0.1 ml, 78 mg/kg/d), TDF solution (0.1 ml, 39 mg/kg/d) or normal saline solution (0.1 ml). Pups in each arm were weighed and sacrificed after birth. Both sides of quadriceps femoris muscle of the newborn were obtained. The histological observation was conducted under light microscope. The transcriptional profiling was analyzed with RNA sequencing (RNA seq)., Key Findings: Four types of morphological abnormalities of the newborn neuromuscular system, being clusters of rhabdomyoblasts, skeletal muscle fibrosis, rhabdomyolysis and necrosis and immature muscle fiber bundles, were noted in both LdT group and TDF group. Moreover, both groups showed significantly decreased gross cross-sectional area of muscle fiber and significantly increased percentage of muscle lesion area. RNA seq identified a total of 164 differentially expressed genes (DEGs) essential to fetal neuromuscular development. These DEGs were involved in calcium regulation, phospholipid activity, muscle cell development, the functioning of mitochondria/endoplasmic reticulum/lysosome/cytoskeleton, the regulation of arachidonic acid and the development of nervous system., Significance: Our findings suggest maternal administration of LdT or TDF lead to abnormal neuromuscular development in offspring mice. Further study should be encouraged to investigate the down-stream signaling pathways., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Purpose: Highly active antiretroviral therapy (HAART) is an accepted treatment option for patients with virus infection. Mounting evidence indicated that persistent HAART treatment is implicated with increased morbidity of HIV-associated neurocognitive disorders (HAND) in patients. Tenofovir disoproxil fumarate (TDF), a novel nucleotide reverse transcriptase inhibitor (NRTI), was used in patients with HIV co-infected with HBV. And it is still a vital first-line antiretroviral compounds in HAART. However, whether persistent treatment with TDF is involved in HAND development remains to be further elucidated. In this study, we aimed to discuss the neurotoxicity of TDF., Methods: We used SH-SY5Y cells and primary neuronal cells to evaluate the neurotoxicity of TDF in vitro. The cytotoxicity of TDF on SH-SY5Y cells and primary neuronal cells was evaluated by the cell viability and LDH levels by MTT assay and LDH kit, respectively. Hoechst 33342 staining, TUNEL assay and flow cytometry were performed to evaluate the cells apoptosis. The intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) production were measured by commercial kits. In addition, the activation level of caspase-3 was evaluated using spectrophotometry and western blotting., Results: Our results showed that TDF treatment significantly induced cell viability and induced apoptosis of SH-SY5Y cells and primary neuronal cells. Furthermore, the ROS levels and MDA productions were significantly up-regulated in nerve cells treated with TDF.  CONCLUSION: Our findings indicated that TDF may induce neuronal cell apoptosis through increasing the intracellular ROS and the expression level of caspase-3, which may be related to the increasing prevalence of HAND., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Tenofovir disoproxil fumarate (TDF) is a drug used in HIV treatment, and several studies have detected its presence in surface water. Furthermore, more information on its environmental impact is needed in the scientific literature. Thus, due to the lack of data on the impact of this drug, and its presence in different waters of the world, this work aimed to evaluate the potential toxicological effects of TDF on the mollusk Biomphalaria glabrata in vivo and in vitro. For in vitro analysis, hemocytes were exposed to different drug concentrations for 1 h and evaluated for feasibility, and phagocytic and metabolic activity. The in vivo analysis consisted of the exposure of groups of five mollusks, in triplicate, at the same drug concentrations for 72 h and 21 days, evaluating mortality, and mollusk and hemolymph behavior. Although the exposure of the mollusk to TDF did not reduce its survival, however it was toxic to its hemocytes. Even if toxicity was identified on the mollusk and its hemocytes initially, further studies should be conducted to understand the effects of this residue on the environment and different life stages of the mollusk because, per the Globally Harmonized System of Classification and Labeling of Chemicals, for aquatic ecosystems, the results obtained were classified as toxic (EC50% 2.65 [1.98; 5.29] mg/L) and could cause unfeasibility in hemocytes at concentrations below 10 mg/l., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Lamivudine and tenofovir disoproxil fumarate act against the replication of hepatitis B and human immunodeficiency viruses via inhibition of the reverse transcriptase enzyme activity, thereby preventing the synthesis of viral DNA. Chronic administration of these drugs has been associated with toxicities, including senescence, oxidative stress and premature death. A study of these toxicities in Drosophila melanogaster, which share 75% genomic similarity with humans could help to develop a pharmacologic intervention., Methods: Susceptibility of D. melanogaster for lamivudine and tenofovir-induced toxicities were investigated. First, flies (≤3 days old) were fed with drugs-supplemented diet at varying concentrations (1mg to 300mg/10-gram diet) or distilled water for seven days to determine LD 50 . Secondly, five groups of 60 flies were fed with four concentrations of test drugs: 2.9mg, 5.82mg, 11.64mg and 23.28mg each per 10-gram diet for 28 days survival and lifespan assays. Then 5-day treatment plan was utilized to determine drugs toxicities on climbing ability and some biomarkers of oxidative stress. Finally, molecular docking was carried out using the Auto-dock vina mode to predict the biological interactions between the test drugs and D. melanogaster acetylcholinesterase (AChE) or glutathione-S-transferase (GST)., Results: The LD 50 of lamivudine or tenofovir was 47.07 or 43.95mg/10g diet, respectively. Each drug significantly (P<0.05) reduced the survival rate, longevity and climbing performance of the flies dose-dependently. These drugs also altered levels of biochemical parameters: AChE, GST, superoxide dismutase (SOD), catalase (CAT), total thiol (T-SH), and malondialdehyde (MDA) of the flies significantly (P<0.05). In silico molecular analysis showed that the test drugs interacted with significantly (P<0.05) higher binding affinities at the same catalytic sites of D. melanogaster GST and AChE compared with substrates (glutathione or acetylcholine)., Conclusion: The significant lamivudine and tenofovir-induced toxicities observed as increased mortality, climbing deficits and compromised antioxidant defence in D. melanogaster demands further research for possible pharmacological intervention., (Copyright © 2022 Académie Nationale de Pharmacie. Published by Elsevier Masson SAS. All rights reserved.)

Background: Tenofovir disoproxil fumarate (TDF) can induce proximal renal tubulopathy (PRT) and necessitate changes in treatment regimen. This prospective study aimed to compare tubular function recovery following early switching versus late switching of TDF in human immunodeficiency virus (HIV)-infected patients with TDF-induced PRT. Methods: For this prospective study, conducted during 2017-2019, we enrolled HIV-1-infected, virologically suppressed adults undergoing TDF-containing combination antiretroviral therapy. Patients were separated into a late-switching group (LSG) and an early-switching group (ESG). The LSG included patients having an estimated glomerular filtration rate (eGFR) decrease ≥25% from the pretreatment level or Fanconi syndrome. The ESG included patients having ≥2 of the following indicators of PRT: fractional excretion of phosphate (FEUP) ≥10%, low tubular maximum reabsorption of phosphate (TmP)/GFR, or uricosuria; fractional uric acid excretion ≥10%; urine protein-creatinine index (UPCI) ≥500 mg/g creatinine, normoglycemic glycosuria, or decrease in eGFR of 15%-24%. Recovery of proximal tubular function at 6 and 12 months after TDF discontinuation was assessed. Complete recovery was defined as normalization of all abnormal tubular markers. Results: Thirty-three HIV-infected patients were enrolled (70% male). Except for tubular function markers, baseline characteristics were not significantly different between the two groups. The proportion of patients having complete recovery was significantly higher in the ESG ( p  = 0.007, log-rank test). FEUP improved significantly in the ESG after TDF discontinuation; improvements of eGFR and UPCI were greater in the LSG. An eGFR change of 10% from baseline was the only independent predictor of failure to achieve complete recovery after switching. After median follow-up of 2.25 years post-trial, sustained recovery of eGFR within 5% of pre-TDF eGFR was achieved only in the ESG. Conclusions: Early-switching of TDF in HIV patients with PRT may allow complete recovery of proximal renal tubular function.

The toxicity of tenofovir alafenamide (TAF) hemifumarate (HF) was evaluated when administered by continuous subcutaneous (s.c.) infusion via an external infusion pump for 28 days to rats and dogs. The toxicokinetics of TAF and two metabolites, tenofovir (TFV) and tenofovir diphosphate (TFV-DP) were also evaluated. After administration of TAF HF in rats and dogs, primary systemic findings supported an inflammatory response that was considered minimal to mild. Gross pathology and histopathologic evaluation of tissue surrounding the s.c. infusion site revealed signs of inflammation, including edema, mass formation, fibrosis, and mononuclear cell inflammation in groups receiving ≥300 μg/kg/day in rats and ≥25 μg/day in dogs. Although these changes were observed in animals receiving vehicle, the severity was greater in animals receiving TAF HF. Changes in the local tissue were considered a TAF HF-mediated exacerbation of an inflammatory response to the presence of the catheter. In rats, systemic and local findings were considered not adverse due to their low severity and reversibility; therefore, the "no observed adverse effect level" (NOAEL) was set at 1,000 μg/kg/day. Because none of the systemic findings were related to systemic exposure to TAF, the systemic NOAEL was set at 250 μg/kg/day in dogs. Due to the severity of the observations noted, a NOAEL for local toxicity could not be established. Although these results might allow for exploration of tolerability and pharmacokinetics of s.c. administered TAF HF in humans, data suggest a local reaction may develop in humans at doses below a clinically relevant dose. IMPORTANCE Human immunodeficiency virus (HIV) infection continues to be a serious global human health issue, with ∼38 million people living with HIV worldwide at the end of 2019. HIV preexposure prophylaxis (PrEP) has introduced the use of antiretroviral therapies as another helpful tool for slowing the spread of HIV worldwide. One possible solution to the problem of inconsistent access and poor adherence to HIV PrEP therapies is the development of subcutaneous (s.c.) depots or s.c. implantable devices that continuously administer protective levels of an HIV PrEP therapy for weeks, months, or even years at a time. We evaluate here the toxicity of tenofovir alafenamide, a potent inhibitor or HIV replication, after continuous s.c. infusion in rats and dogs for HIV PrEP.

Purpose: To evaluate the bioequivalence and safety of two formulations of 25 mg tenofovir alafenamide tablets in Chinese healthy male and female subjects under fed and fasting conditions., Patients and Methods: This was a randomized, open-label, single-center, crossover study consisting of a fasting trial with two periods and a fed trial with four periods. In total, 42 healthy subjects were enrolled in the fasting trial and 32 healthy subjects were enrolled in the fed trial. In each period, blood samples for pharmacokinetic analysis were collected until 72 hours post-dose. The plasma concentrations of tenofovir alafenamide and tenofovir were measured and noncompartmental analysis was used to determine pharmacokinetic parameters. Throughout the entire study, subjects' safety was monitored by assessment of physical examinations, vital signs, 12-lead electrocardiography, clinical laboratory parameters, and treatment emergent adverse events (TEAEs)., Results: Forty subjects completed the fasting trial and 32 subjects completed the fed trial. The 90% confidence intervals (CIs) of the geometric mean ratios for AUC 0-t , AUC 0-∞ , and C max for the two formulations were within 80.00% to 125.00%, which met the bioequivalence acceptance criteria. The study drugs were well tolerated by all subjects., Conclusion: This study demonstrated that the test formulation of 25 mg tenofovir alafenamide tablets was bioequivalent to the formulation marketed under the brand name VEMLIDY ® in healthy Chinese male and female subjects under fasting and fed conditions., Competing Interests: The authors report that there are no conflicts of interest., (© 2021 Li et al.)

Nephrotoxicity is a dose-limiting side effect of long-term use of tenofovir, a reverse transcriptase inhibitor that is used for the treatment of HIV infection and chronic hepatitis B infection. Identifying an agent that prevents tenofovir disoproxil fumarate (TDF)-induced renal injury can lead to its better tolerance, and a more effective treatment can be achieved. The present study is aimed at investigating whether melatonin, a potent antioxidant and anti-inflammatory agent, protects against TDF nephrotoxicity in rats and to determine its cellular targets. Rats were divided into groups and treated as follows. Group I (control): Rats in this group (n = 6) received sterile water only by gavage for 35 days. Group II: Rats (n = 6) in this group received 600 mg/kg body weight TDF in sterile water by gavage for 35 days. Group III: Rats (n = 6) in this group received once daily 20 mg/kg bodyweight melatonin i.p. 2 h before the administration of 600 mg/kg body weight TDF in sterile water by gavage for 35 days. Group IV: Rats were pretreated daily with 20 mg/kg body weight melatonin i.p. 2 h before the administration of sterile water by gavage. All the rats were sacrificed on the 36th day, after overnight fast. Melatonin pretreatment protected the rats against TDF nephrotoxicity both histologically and biochemically. Biochemically, melatonin pretreatment attenuated TDF-induced, oxidative stress, nitrosative stress, mitochondrial pathway of apoptosis, PARP overactivation and preserved proximal tubular function (p < 0.01). This suggests that melatonin may be useful in ameliorating TDF nephrotoxicity.

Antiretroviral therapy (ART) in pregnancy has dramatically reduced HIV vertical transmission rates. Consequently, there is a growing number of children that are HIV exposed uninfected (CHEUs). Studies suggest that CHEUs exposed in utero to ART may experience developmental delays compared to their peers. We investigated the effects of in utero ART exposure on perinatal neurodevelopment in mice, through assessment of developmental milestones. Developmental milestone tests (parallel to reflex testing in human infants) are reflective of brain maturity and useful in predicting later behavioral outcomes. We hypothesized that ART in pregnancy alters the in utero environment and thereby alters developmental milestone outcomes in pups. Throughout pregnancy, dams were treated with boosted-atazanavir combined with either abacavir/lamivudine (ATV/r/ABC/3TC), or tenofovir/emtricitabine (ATV/r/TDF/FTC), or water as control. Pups were assessed daily for general somatic growth and on a battery of tests for primitive reflexes including surface-righting, negative-geotaxis, cliff-aversion, rooting, ear-twitch, auditory-reflex, forelimb-grasp, air-righting, behaviors in the neonatal open field, and olfactory test. In utero exposure to either ART regimen delayed somatic growth in offspring and evoked significant delays in the development of negative geotaxis, cliff-aversion, and ear-twitch reflexes. Exposure to ATV/r/ABC/3TC was also associated with olfactory deficits in male and forelimb grasp deficits in female pups. To explore whether delays persisted into adulthood we assessed performance in the open field test. We observed no significant differences between treatment arm for males. In females, ATV/r/TDF/FTC exposure was associated with lower total distance travelled and less ambulatory time in the centre, while ATV/r/ABC/3TC exposure was associated with higher resting times compared to controls. In utero PI-based ART exposure delays the appearance of primitive reflexes that involve vestibular and sensory-motor pathways in a mouse model. Our findings suggest that ART could be disrupting the normal progress/maturation of the underlying neurocircuits and encourage further investigation for underlying mechanisms., Competing Interests: The authors have no conflicts of interest to disclose in relation to this work. LS reports personal fees from ViiV Healthcare for participation in a Women and Transgender Think Tank. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Objectives: Growing evidence suggested that antiretroviral (ARV) drugs may promote amyloid beta (Aβ) accumulation in HIV-1-infected brain and the persistence of HIV-associated neurocognitive disorders (HANDs). It has also been shown that lipid peroxidation upregulates β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) expression and subsequently promotes Aβ peptide production. In the present study, we examined whether chronic exposure to the anti-HIV drugs tenofovir disoproxil fumarate (TDF) and nevirapine induces lipid peroxidation thereby promoting BACE1 and Aβ generation and consequently impair cognitive function in mice., Methods: TDF or nevirapine was orally administered to female BALB/c mice once a day for 8 weeks. On the 7th week of treatment, spatial learning and memory were assessed using the Morris water maze test. The levels of lipid peroxidation, BACE1, amyloid β 1-42 (Aβ1-42) and Aβ deposits were measured in the hippocampal tissue upon completion of treatment., Results: Chronic administration of nevirapine induced spatial learning and memory impairment in the Morris water maze test, whereas TDF did not have an effect. TDF and nevirapine administration increased hippocampal lipid peroxidation and Aβ1-42 concentration. Nevirapine further upregulated BACE1 expression and Aβ deposits., Conclusion: Our results suggest that chronic exposure to TDF and nevirapine contributes to hippocampal lipid peroxidation and Aβ accumulation, respectively, as well as spatial learning and memory deficits in mice even in the absence of HIV infection. These findings further support a possible link between ARV drug toxicity, Aβ accumulation and the persistence of HANDs.

A key component of antiretroviral therapy (ART) for HIV patients is the nucleoside reverse transcriptase inhibitor (NRTI) is tenofovir. Recent reports of tenofovir toxicity in patients taking ART for HIV cannot be explained solely on the basis of off-target inhibition of mitochondrial DNA polymerase gamma (Polγ). PrimPol was discovered as a primase-polymerase localized to the mitochondria with repriming and translesion synthesis capabilities and, therefore, a potential contributor to mitochondrial toxicity. We established a possible role of PrimPol in tenofovir-induced toxicity in vitro and show that tenofovir-diphosphate incorporation by PrimPol is dependent on the n-1 nucleotide. We identified and characterized a PrimPol mutation, D114N, in an HIV+ patient on tenofovir-based ART with mitochondrial toxicity. This mutant form of PrimPol, targeting a catalytic metal ligand, was unable to synthesize primers, likely due to protein instability and weakened DNA binding. We performed cellular respiration and toxicity assays using PrimPol overexpression and shRNA knockdown strains in renal proximal tubular epithelial cells. The PrimPol-knockdown strain was hypersensitive to tenofovir treatment, indicating that PrimPol protects against tenofovir-induced mitochondrial toxicity. We show that a major cellular role of PrimPol is protecting against toxicity caused by ART and individuals with inactivating mutations may be predisposed to these effects.

Background: Sensory neuropathy (SN) is a common and often painful neurological condition associated with HIV-infection and its treatment. However, data on the incidence of SN in neuropathy-free individuals initiating combination antiretroviral therapies (cART) that do not contain the neurotoxic agent stavudine are lacking., Aims: We investigated the 6-month incidence of SN in ART naïve individuals initiating tenofovir (TDF)-based cART, and the clinical factors associated with the development of SN., Methods: 120 neuropathy-free and ART naïve individuals initiating cART at a single center in Johannesburg, South Africa were enrolled. Participants were screened for SN using clinical signs and symptoms at study enrolment and approximately every 2-months for a period of ~6-months. Diagnostic criteria for symptomatic SN was defined by the presence of at least one symptom (pain/burning, numbness, paraesthesias) and at least two clinical signs (reduced vibration sense, absent ankle reflexes or pin-prick hypoaesthesia). Diagnostic criteria for asymptomatic SN required at least two clinical signs only (as above)., Results: A total of 88% of the cohort completed three visits within the 6-month period. The 6-month cumulative incidence of neuropathy was 140 cases per 1000 patients (95% CI: 80-210) at an incidence rate of 0.37 (95% CI: 0.2-0.5) per person year. Height and active tuberculosis (TB) disease were independently associated with the risk of developing SN (P < .05)., Interpretation: We found that within the first 6 months of starting cART, incident SN persists in the post-stavudine era, with 11 (9%) of individuals developing asymptomatic SN, and 9 (8%) developing symptomatic SN., (© 2019 Peripheral Nerve Society.)

We examined associations of 25-hydroxy vitamin D (25-OHD), tenofovir disoproxil fumarate (TDF), and bone toxicity. We studied TDF/emtricitabine (FTC) HIV pre-exposure prophylaxis (PrEP) in young men who have sex with men (YMSM). Bone toxicity was predefined using bone mineral density/content change from baseline to week 48. Baseline serum 25-OHD was dichotomized as <20 ng/mL (insufficient/deficient) versus ≥20 (sufficient), and week 48 dried blood spot tenofovir diphosphate (TFV-DP) as >700 fmol/punch (protective against HIV acquisition) versus ≤700. Associations were examined by univariate and multivariable logistic regression, reporting crude and adjusted odds ratios (ORs), with 95% confidence intervals (CIs). Of 101 enrolled, 69 had complete bone assessments and 25-OHD; of these, 59 had week 48 TFV-DP data. Median (Q1-Q3) age was 20 (18-21) years; 54% were black/African American. In univariate analysis, participants with baseline 25-OHD <20 ng/mL (OR = 5.4; 95% CI = 1.9-16.5) and blacks (OR = 4.9; 95% CI = 1.7-15.2) had greater odds of bone toxicity than those with 25-OHD ≥20 or other races. TFV-DP was not associated with bone toxicity (OR = 1.6; 95% CI = 0.5-5.5). In multivariable analysis, compared with those with 25-OHD ≥20 and TFV-DP ≤700, those with 25-OHD ≥20 and TFV-DP >700 (OR = 11.5; 95% CI = 1.4-169.6), 25-OHD <20 and TFV-DP ≤700 (OR = 19.4; 95% CI = 3.0-228.7), and 25-OHD <20 and TFV-DP >700 (OR = 32.3; 95% CI = 3.3-653.6) had greater odds of bone toxicity after adjusting for race. In multivariable models, 25-OHD insufficiency, protective TFV-DP concentrations, and black race were significantly associated with bone toxicity after 48 weeks of TDF/FTC PrEP in YMSM. Clinical Trials Registration: NCT01769469.

Introduction: Chronic kidney disease is a significant cause of morbidity and mortality among patients infected with human immunodeficiency virus (HIV). Tenofovir disoproxil fumarate (TDF) is widely used as the part of combination antiretroviral therapy (cART) and may cause renal function impairment., Aim: The primary objective of this analysis was to determine the rate of reversibility of kidney dysfunction and factors correlated with eGFR improvement in patients treated with TDF., Materials and Methods: All patients who discontinued TDF between 2003 and 2015 were screened and included in the study if the reason for withdrawal was nephrotoxicity. Kidney function (eGFR, proteinuria, haematuria) was assessed on treatment and one year after discontinuation. Factors associated with not achieving eGFR recovery one year after discontinuing TDF were assessed., Results: A total of 69 patients out of 1625 screened discontinued TDF due to nephrotoxicity and were included in the analysis. At the end of the study period eGFR (CKD-EPI) improved in 52 (75,4%) patients. The eGFR difference was 11,7 ml/min/1,73m2 (95% CI: 6,0 – 14,5). Two factors were associated with kidney function improvement: the length of TDF treatment and baseline eGFR. Better recovery was observed in patients treated with shorter (difference: 15,6 ml/min/1,73m2, 95% CI: 5,99 – 23,0) and in those with impaired renal function at baseline (difference: 21 ml/min/1,73m2, 95% CI: 11,0 – 27,99)., Conclusions: In majority of patients who discontinue TDF therapy, kidney function improves during oneyear period. The drug withdrawal in case of eGFR deterioration should not be postponed.

Developments in medicine have led to a significant increase in the average human lifespan. This increase in aging is most readily apparent in the case of HIV where antiretroviral therapy has shifted infection from a terminal to a chronic but manageable disease. Despite this advance, patients suffer from co-morbidities best described as an accelerated aging phenotype. A potential contributor is cellular senescence, an aging-associated growth arrest, which has already been linked to other HIV co-morbidities such as lipodystrophies and osteoporosis in response to antiretroviral drugs. We have previously demonstrated that astrocytes senescence in response to antiretroviral drugs. As endothelial cells play a critical role regulating the blood brain barrier (BBB) and senescence could severely impact barrier permeability, we investigate the role of a commonly used combination of HIV reverse transcriptase inhibitors on the senescence program of human umbilical vein endothelial cells (HUVECs). Our studies indicate that HUVECs underwent premature senescence associated with inflammation, oxidative stress and altered eNOS activation. Treated cells had detrimental paracrine effects on astrocytes including paracrine senescence, suggesting that senescent HUVECs could influence astrocytes, which line the other side of the BBB. These results may have implications for HIV-associated neurocognitive disorders (HAND), a set of neurological deficits., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Drug-induced kidney injury in medicinal compound development accounts for over 20% of clinical trial failures and involves damage to different nephron segments, mostly the proximal tubule. Yet, currently applied cell models fail to reliably predict nephrotoxicity; neither are such models easy to establish. Here, we developed a novel three-dimensional (3D) nephrotoxicity platform on the basis of decellularized rat kidney scaffolds (DS) recellularized with conditionally immortalized human renal proximal tubule epithelial cells overexpressing the organic anion transporter 1 (ciPTEC-OAT1). A 5-day SDS-based decellularization protocol was used to generate DS, of which 100- μ m slices were cut and used for cell seeding. After 8 days of culturing, recellularized scaffolds (RS) demonstrated 3D-tubule formation along with tubular epithelial characteristics, including drug transporter function. Exposure of RS to cisplatin (CDDP), tenofovir (TFV), or cyclosporin A (CsA) as prototypical nephrotoxic drugs revealed concentration-dependent reduction in cell viability, as assessed by PrestoBlue and Live/Dead staining assays. This was most probably attributable to specific uptake of CDDP by the organic cation transporter 2 (OCT2), TFV through organic anion transporter 1 (OAT1), and CsA competing for P-glycoprotein-mediated efflux. Compared with 2D cultures, RS showed an increased sensitivity to cisplatin and tenofovir toxicity after 24-hour exposure (9 and 2.2 fold, respectively). In conclusion, we developed a physiologically relevant 3D nephrotoxicity screening platform that could be a novel tool in drug development., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Newer urinary protein kidney safety biomarkers can outperform the conventional kidney functional biomarkers blood urea nitrogen (BUN) and serum creatinine (SCr) in rats. However, there is far less experience with the relative performance of these biomarkers in dogs and nonhuman primates. Here, we report urine protein biomarker performance in tenofovir-treated cynomolgus monkeys and beagle dogs. Tenofovir intravenous daily dosing in monkeys for 2 or 4 weeks at 30 mg/kg/day resulted in minimal to moderate tubular degeneration and regeneration, and tenofovir disoproxil fumarate oral dosing in dogs for 10 days at 45 mg/kg/day resulted in mild to marked tubular degeneration, necrosis, and regeneration. Among biomarkers tested, kidney injury molecule 1 (Kim-1) and clusterin (CLU) clearly outperformed BUN and SCr and were the most reliable in detecting the onset and progression of tenofovir-induced tubular injury. Cystatin C, retinol binding protein 4, β2-microglobulin, neutrophil gelatinase-associated lipocalin, albumin, and total protein also performed better than BUN and SCr and added value when considered together with Kim-1 and CLU. These findings demonstrate the promising utility of these urinary safety biomarkers in monkeys and dogs and support their further evaluation in human to improve early detection of renal tubular injury.

Bone health impairment is a common finding in HIV-infected patients on antiretroviral treatment. High serum parathyroid hormone (PTH) concentration in patients on antiretroviral treatment containing tenofovir disoproxil fumarate (TDF) has been reported. Hyperparathyroidism was not always sustained by a reduction in vitamin D concentration. We thus hypothesized a direct inhibitory effect of TDF on the Calcium-sensing receptor (CaSR), leading to hyperparathyroidism. Human embryonic kidney cells were transfected with CASR wild-type gene or mutated in different sites (N124K, T1051G, C788T, T888M). Cells were grown in standard conditions and the activity of CaSR was assessed after stimulation with CaCl 2 with and without TDF (100 nM-1 μM). We evaluated by western blot phospho-p44/42 ERK expression levels as a marker of CaSR activity. In silico structure models were obtained for wild-type and N124K mutant. Molecular docking with TDF was also evaluated. The stimulation by CaCl 2 and TDF 100 nM led to a decrease of 55% of CaSR activity (P < 0.001), whereas the stimulation by CaCl 2 and TDF 1 μM reduced the activity by 68% (P < 0.001). The decreased CaSR activity was comparable to that observed from known CASR gene inactivating mutations (T1051G, C788T), which inhibit the receptor activity by 56% and 78%, respectively. The TDF inhibits the CaSR activity carrying a gain of function mutation in the intracellular domain (T888M), but it does not influence the activity of the receptor carrying the N124K activating mutation. Our data show that TDF is able to inhibit the activity of CaSR in a dose-dependent manner. Hyperparathyroidism observed in TDF-treated patients may be therefore promoted by the direct effect of the drug on CaSR., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

The application of combination antiretroviral therapy has greatly reduced the death rate from AIDS. However, up to 50% of patients on combination antiretroviral therapy develop HIV-associated neurocognitive disorders (HAND), which is associated with residual neuroinflammation and oxidative injury in the brain. Neural stem cells (NSCs) and progenitors play a vital role in repairing neuronal injuries. Therefore, we hypothesize that combination antiretroviral therapy may adversely affect NSCs/progenitors, contributing to the increasing prevalence of HAND. Here, we show that combined medication of three antiretroviral drugs tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and raltegravir (RAL) affects NSC homeostasis and progenitor proliferation in the mouse dentate gyrus (DG). Our results also show that TDF/FTC/RAL treatment prohibits proliferation and induces apoptosis of cultured mouse neural progenitor cells (NPCs), resulting in a reduction in the viability of NPCs. Moreover, we find that TDF, among the three drugs used in this combination antiretroviral treatment, accounts for most of the effects on neural progenitors. Together, our results offer a mechanistic explanation for the prevalence of HAND in AIDS patients treated with combination antiretroviral therapy.

Highly active anti-retroviral therapy has proved successful in reducing morbidity and mortality associated with HIV infection though it has been linked to increased risk of cardiovascular disease. To date, the direct effects of the anti-retroviral drugs Efavirenz, Tenofovir and Emtricitabine on the vasculature relaxant response have not been elucidated, which impaired may predispose individuals to cardiovascular disease. Increased cellular oxidative stress and overactivation of the DNA repair enzyme poly (ADP-ribose) polymerase (PARP) have been identified as central mediators of vascular dysfunction. The aim of this study was to investigate whether exposure to Efavirenz, Tenofovir or Emtricitabine directly causes endothelial cell dysfunction via overactivation of PARP. Exposure of ex vivo male rat aortic rings or in vitro endothelial cells to Efavirenz but not Tenofovir or Emtricitabine impaired the acetylcholine-mediated relaxant response, increased cellular oxidative stress and PARP activity, decreased cell viability and increased apoptosis and necrosis. Pharmacological inhibition of PARP protected against the Efavirenz-mediated impairment of vascular relaxation and endothelial cell dysfunction. Oestrogen exposure also protected against the Efavirenz-mediated inhibition of the vascular relaxant response, cell dysfunction and increased PARP activation. In conclusion, Efavirenz directly impairs endothelial cell function, which may account for the increased risk of developing cardiovascular complications with anti-retroviral therapy.

Antiretroviral (ARV) treatment may induce metabolic complications in HIV patients on long-term therapy that can affect bone health. In this study, the effects of the ARVs Stavudine (d4T), Tenofovir (TDF) and Lopinavir/ritonavir (LPV/r) on bone metabolism and lipodystrophy were directly compared in rats to negate the consequences of HIV-associated confounding factors. Healthy 12-14-week-old male Wistar rats (n = 40) were divided into four treatment groups and received an oral animal equivalent dose of either Stavudine (6.2 mg/kg/day), TDF (26.6 mg/kg/day), LPV/r (70.8 mg/kg/day) or water (Control 1.5 mL water/day) for a period of 9 weeks. Whole-body DXA measurements, a biomechanical three-point breaking test and histomorphometric analysis were performed on the femurs and tibias at the end of the treatment period. Stavudine monotherapy was found to be associated with decreased femoral bone mineral density that translated into reduced bone strength, whereas histomorphometric analysis demonstrated that Stavudine induces an imbalance in bone metabolism at tissue level, evident in higher resorption (eroded surfaces, osteoclast surfaces and osteoclast number) and lower formation parameters (osteoblast surfaces and osteoid surfaces). This was less clear in the rats treated with either TDF or LPV/r. Furthermore, both Stavudine and TDF treatment resulted in significant bone marrow adiposity, although no significant redistribution of body fat was noted in the treated rats compared to controls. The data from this study suggest that in the absence of HIV-associated factors, LPV/r is less detrimental to bone metabolism compared to Stavudine and TDF.

This study focuses on the antiretrovirals efavirenz (EFV), a non-nucleoside reverse transcriptase inhibitor, and tenofovir disoproxil fumarate (TDF), an oral prodrug of tenofovir analog of adenosine 5'-monophosphate, which belongs to the class of nucleotide reverse transcriptase inhibitors. Both compounds act on the mechanisms of HIV replication, inhibiting the action of reverse transcriptase and thus preventing viral DNA synthesis. The toxic and genotoxic potential of EFV and TDF alone and in combinations {EFV+combivir [zidovudine (AZT)+lamivudine (3TC)] and TDF+3TC} were assessed using the comet assay and the somatic mutation and recombination test (SMART) in Drosophila melanogaster. The results indicate that EFV was toxic at high concentrations and induced genotoxicity using the comet assay, but showed neither mutagenic nor recombinogenic effects using SMART. In combination with combivir, EFV exhibited antagonic genotoxic effects in both tests. Inversely, TDF did not show toxicity but induced genotoxicity at all concentrations tested in both the comet assay and SMART. The prevalence of recombinogenic events in all treatments with TDF alone and in combination with 3TC was detected using SMART. Homologous recombination is an important parameter to be taken into consideration in the evaluation of carcinogenicity of medicines used in antiretroviral therapy regimens, due to the need for lifelong adherence and the unknown effects of long-term treatments., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: In-vivo studies suggest that mitochondria is involved in tenofovir (TFV)-induced renal toxicity, but the underlying mechanisms are still unclear. The aim of the present study was to assess the effects of TFV and its prodrug, TFV disoproxil fumarate, on mitochondrial function and cell survival/viability in a renal proximal tubular cell line., Design and Methods: We evaluated parameters of cellular proliferation/survival (cell count, cell cycle, viability) and mitochondrial function (oxygen consumption, mitochondrial membrane potential, reactive oxygen species production) in NRK-52E cells. Intracellular TFV was measured by HPLC and expression of antioxidant genes was analysed by real-time PCR., Results: Similar intracellular levels of TFV were reached with lower concentrations of the prodrug than of the drug, and correlated directly with a decrease in cell number. Both compounds inhibited proliferation and compromised mitochondrial function by decreasing mitochondrial membrane potential and increasing oxygen consumption and mitochondrial superoxide production. Altered oxidative status was confirmed by the overexpression of antioxidant genes., Conclusions: Intracellular accumulation of TFV induces mitochondrial toxicity in an in-vitro renal model and alters cell proliferation and viability. Our findings call for caution regarding the use of this nucleotide analogue reverse transcriptase inhibitor in patients with other risk factors that compromise mitochondrial function in the kidney.

Rationale: Recently tenofovir disoproxil fumarate (TDF) has been widely used as a first-line therapy for chronic hepatitis B (CHB) infection. Although TDF demonstrates successful viral suppression, the possibility of renal failure and lactic acidosis has been proposed with TDF administration, especially in human immunodeficiency virus co-infected patients. However, TDF induced lactic acidosis has never been reported in CHB mono-infected patients., Patient Concerns: A 59-year-old man received TDF for hepatitis B associated with cirrhosis. After ten days of TDF administration, nausea, vomiting and abdominal pain developed. High anion gap acidosis with elevated lactate level (pH 7.341, pCO2 29.7 mmHg, HCO3- 15.6mmHg, lactate 3.2mmol/L, anion gap 15.4 mEq/L) was developed., Diagnosis: With no infection, normal diagnostic paracentesis, and urinalysis together with high anion gap and increased blood lactate levels suggested lactic acidosis., Interventions: TDF was stopped, and haemodialysis was performed to control lactic acidosis., Outcomes: Although stopping TDF instantly and treating lactic acidosis using hemodialysis, the patient died., Lessons: Although, Fatal lactic acidosis is very rare in TDF patient, however, decompensated cirrhotic patients should be closely observed to keep the possibility of lactic acidosis in mind.

Tenofovir (TFV) is an antiviral drug approved for treating Human Immunodeficiency Virus (HIV) and Hepatitis B. TFV is administered orally as the prodrug tenofovir disoproxil fumarate (TDF) which then is deesterified to the active drug TFV. TFV induces nephrotoxicity characterized by renal failure and Fanconi Syndrome. The mechanism of this toxicity remains unknown due to limited experimental models. This study investigated the cellular mechanism of cytotoxicity using a human renal proximal tubular epithelial cell line (HK-2). HK-2 cells were grown for 48 h followed by 24 to 72 h exposure to 0-28.8 μM TFV or vehicle, phosphate buffered saline (PBS). MTT (MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) and Trypan blue indicated that TFV diminished cell viability at 24-72 h. TFV decreased ATP levels at 72 h when compared to vehicle, reflecting mitochondrial dysfunction. TFV increased the oxidative stress biomarkers of protein carbonylation and 4-hydroxynonenol (4-HNE) adduct formation. Tumor necrosis factor alpha (TNFα) was released into the media following exposure to 14.5 and 28.8 μM TFV. Caspase 3 and 9 cleavage was induced by TFV compared to vehicle at 72 h. These studies show that HK-2 cells are a sensitive model for TFV cytotoxicity and suggest that mitochondrial stress and apoptosis occur in HK-2 cells treated with TFV.

Nephrotoxicity due to tenofovir treatment of HIV patients has been reported. However, the mechanism of tenofovir nephrotoxicity is not clear. NFκB is an important proinflammatory transcription factor that plays a pivotal role in oxidative stress-induced inflammation. We hypothesized that NFκB proinflammatory signalling pathway may play a role in tenofovir induced renal damage. Renal damage was induced in adult male Wistar rats by the oral administration of 600 mg/kg body wt. daily for 5 consecutive weeks. Kidneys were removed and used for histological and biochemical analysis. The protein and mRNA expressions of NFκB and its target genes namely iNOS, COX-2 and TNFα, and its inhibitor IκB-alpha were analysed by immunohistochemical methods, western blot and quantitative RT PCR. NFκBp65 activity was determined by ELISA. The protein and mRNA expressions of NFκB p65, iNOS, COX-2 and TNFα were increased in the kidneys of TDF treated rats. The activity of NFκBp65 was increased by 28 fold in the nuclear fractions of the TDF treated rat kidneys. Pretreatment with melatonin, a NFκB inhibitor attenuated TDF induced renal damage. It is concluded that the activation of NFκB and its downstream proinflammatory target genes iNOS, COX-2, and TNF-α may contribute to the pathophysiology of TDF induced renal damage., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Combining two or more antiretroviral drugs in one medical product is an interesting but challenging strategy for developing topical anti-HIV microbicides. We developed a new vaginal delivery system comprising the incorporation of nanoparticles (NPs) into a polymeric film base - NPs-in-film - and tested its ability to deliver tenofovir (TFV) and efavirenz (EFV). EFV-loaded poly(lactic-co-glycolic acid) NPs were incorporated alongside free TFV into fast dissolving films during film manufacturing. The delivery system was characterized for physicochemical properties, as well as genital distribution, local and systemic 24h pharmacokinetics (PK), and safety upon intravaginal administration to mice. NPs-in-film presented suitable technological, mechanical and cytotoxicity features for vaginal use. Retention of NPs in vivo was enhanced both in vaginal lavages and tissue when associated to film. PK data evidenced that vaginal drug levels rapidly decreased after administration but NPs-in-film were still able to enhance drug concentrations of EFV. Obtained values for area-under-the-curve for EFV were around one log10 higher than those for the free drugs in aqueous vehicle (phosphate buffered saline). Film alone also contributed to higher and more prolonged local drug levels as compared to the administration of TFV and EFV in aqueous vehicle. Systemic exposure to both drugs was low. NPs-in-film was found to be safe upon once daily vaginal administration to mice, with no significant genital histological changes or major alterations in cytokine/chemokine profiles being observed. Overall, the proposed NPs-in-film system seems to be an interesting delivery platform for developing combination vaginal anti-HIV microbicides., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: New drugs against HIV infection are associated with important side effects, including impaired bone health. An important reduction of bone mineral density has been repeatedly described in adult but not in young patients receiving tenofovir disoproxil fumarate (TDF) as part of their antiretroviral regimen. The present study aimed to verify the presence and the characteristics of the adverse effects of TDF on skeletal tissue by using the Danio rerio (zebrafish) model., Methods: TDF administration via microinjection in the yolk has been used for embryo study. Water treatment with TDF and scale analysis by morphological, histological and biochemical experiments have been used for adult animals., Results: TDF administration via microinjection in the yolk did not induce any significant effect on mineralization rate and did not produce deformity of the skeletal structure, or growth or vitality impairment of the embryos. TDF exposure in adult zebrafish induced an age-related reduction of the osteoblastic activity marker alkaline phosphatase (up to -44%) and an increase of the osteoclastic activity marker tartrate-resistant acid phosphatase (up to +57%) in the scales. Old fish treated with TDF exhibited an osteoporotic-like phenotype with resorption area along the edge of the scale., Conclusions: This study demonstrated that in zebrafish, as in humans, TDF shows a catabolic bone effect related to age and underlined the ultimate utility of using teleost fish as a model to assess the effects of drugs on bone tissue.

Chronic HIV treatment with antiretroviral drugs has been associated with adverse health outcomes. Mitochondrial toxicity exhibited by nucleoside reverse transcriptase inhibitors (NRTIs) is pinpointed as a molecular mechanism of toxicity. This study evaluated the effect of NRTIs: Zidovudine (AZT, 7.1 μM), Stavudine (d4T, 4 μM) and Tenofovir (TFV, 1.2 μM), on mitochondrial (mt) stress response, mtDNA integrity and oxidative stress response in human hepatoma cells at 24 and 120 h. Markers for mt function, mt biogenesis, oxidative stress parameters, and antioxidant response were evaluated by spectrophotometry, luminometry, flow cytometry, qPCR and western blots. We found that AZT and d4T reduced mtDNA integrity (120 h, AZT: 76.1%; d4T:36.1%, P < 0.05) and remained unchanged with TFV. All three NRTIs, however, reduced ATP levels (AZT: 38%; d4T: 56.4%; TFV: 27.4%, P = 0.01) and mt membrane potential at 120 h (P < 0.005). Oxidative damage and reactive oxygen species (ROS) were increased by TFV and AZT at 24 h, and by d4T at 120 h (P < 0.05). Antioxidant response molecules and mt biogenesis markers were elevated by all NRTIs, with TFV causing the most significant increase (P < 0.05). Data from this study suggest that AZT, d4T and TFV alter mt function. TFV, however, achieves this independently of mtDNA depletion. Furthermore, AZT exerts toxicity soon after exposure as noted from changes at 24 h and d4T exerts greater toxicity over prolonged exposure (120 h)., (© 2015 Wiley Periodicals, Inc.)