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Adefovir nephrotoxicity: Possible role of mitochondrial DNA depletion
Adefovir Nephrotoxicity: Possible Role of Mitochondrial DNA Depletion NOZOMU TANJI, MD, PHD, KURENAI TANJI, MD, PHD, NEERAJA KAMBHAM, MD, GLEN S. MARKOWITZ, MD, ALVIN BELL, MD, AND VIVETTE D. D’AGATI, MD This report investigates the pathomechanism of acute renal failure caused by toxic acute tubular necrosis after treatment with the antiretroviral agent adefovir. A 38-year-old white homosexual man with human immunodeficiency virus infection and no history of opportunistic infections was maintained on highly active antiretroviral therapy (HAART), including hydroxyurea, stavudine, indinavir, ritonavir, and adefovir dipivoxil. Histologic examination of the renal biopsy showed severe acute tubular degenerative changes primarily affecting the proximal tubules. On ultrastructural examination, proximal tubular mitochondria were extremely enlarged and dysmorphic with loss and disorientation of their cristae. Functional histochemical stains for mitochondrial enzymes revealed focal tubular deficiency of cytochrome C oxidase (COX), a respiratory chain enzyme partially encoded by mitochondrial DNA (mtDNA), with preservation of succinate dehydrogenase, a respiratory chain enzyme entirely encoded by nuclear DNA (nDNA). Immunoreactivity for COX subunit I (encoded by mtDNA) was weak to undetectable in most tubular epithelial
cells, although immunoreactivities for COX subunit IV and iron sulfur subunit of respiratory complex III (both encoded by nDNA) were well preserved in all renal tubular cells. Single–renal tubule polymerase chain reaction revealed marked reduction of mtDNA in COX-immunodeficient renal tubules. We conclude that adefovir-induced nephrotoxicity is mediated by depletion of mtDNA from proximal tubular cells through inhibition of mtDNA replication. This novel form of nephrotoxicity may serve as a prototype for other forms of renal toxicity caused by reverse transcriptase inhibitors. HUM PATHOL 32:734-740. Copyright © 2001 by W.B. Saunders Company Key words: adefovir, nephrotoxicity, mitochondrial DNA, HIV, acute tubular necrosis. Abbreviations: HIV, human immunodeficiency virus; PCR, polymerase chain reaction; COX, cytochrome C oxidase; SDH, succinate dehydrogenase; PMS, phenazine methosulfate; DAB, 3,3– diaminebenzidine; ADV, adefovir dipivoxil.
Adefovir dipivoxil, the acyclic phosphate compound 9-[2-(bis-pivaloyloxymethyl)-phosphonylmethoxyethyl] adenine, is an effective newer antiretroviral agent for the treatment of infection with the human immunodeficiency virus (HIV).1,2 Its pharmacologic action is to serve as substrate for reverse transcriptase, resulting in premature DNA chain termination.2 Its potent antiretroviral activity is balanced by potential nephrotoxicity, manifesting as acute tubular necrosis and Fanconi syndrome.3 The mechanism of nephrotoxicity has not been elucidated. Severe ultrastructural alterations of the proximal tubular mitochondria suggest a potential mitochondrial toxicity. As a nucleotide analogue, adefovir could serve as substrate for DNA polymerase ␥, which is responsible for the replication of mitochondrial DNA (mtDNA), thereby inhibiting mtDNA replication.2,4 A similar mechanism of cellular toxicity has been implicated in the myopathy caused by a related nucleoside analogue, zidovudine.4,5 To elucidate the putative role of altered mtDNA replication in adefovir-induced nephrotoxicity, we investigated a biopsydocumented case using functional histochemistry for mitochondrial respiratory chain enzymes, immunohis-
tochemistry for mitochondrial respiratory chain proteins and DNA, and single-tubule polymerase chain reaction (PCR) for quantification of mtDNA.
From the Departments of Pathology and Neurology, Columbia University, College of Physicians and Surgeons, New York, NY; and the Department of Medicine, Mountainside Hospital, Montclair, NJ. Accepted for publication March 16, 2001. Address correspondence and reprint requests to Vivette D. D’Agati, MD, Department of Pathology, Columbia University, College of Physicians and Surgeons, 630 West 168th St, New York, NY 10032. Copyright © 2001 by W.B. Saunders Company 0046-8177/01/3207-0010$35.00/0 doi:10.1053/hupa.2001.25586
CASE REPORT A 38-year-old white homosexual man with HIV infection first documented in 1990 but no history of opportunistic infections presented with acute renal failure. Seven months earlier, he had been placed on a regimen of hydroxyurea 500 mg twice daily, stavudine 40 mg twice daily, and adefovir dipivoxil 60 mg daily. At that time, the patient had a CD4 T-cell count of 352 cells/L whole blood, HIV viral load of 3,553 copies/mL, and serum creatinine of 1.0 mg/dL. Three months later, indinavir 400 mg twice daily and ritonavir 200 mg twice daily were added. At the time of presentation with acute renal failure, the patient had a CD4 T-cell count of 363 cells/L whole blood. Urinalysis showed proteinuria, glycosuria, 0 to 2 white blood cells (WBCs) per high-power field (hpf) and 5 to 10 red blood cells (RBCs)/hpf. Laboratory evaluation revealed serum creatinine, 7.6 mg/dL; creatinine clearance, 13 cm3/min; serum albumin, 2.8 g/dL; serum bicarbonate, 19 mmol/L (normal range, 22 to 32 mmol/L); 24-hour urinary protein, 2.5 g/d; and platelet count, 83,000/ mm3. Serum phosphorus and uric acid levels were in the normal range, and serum glucose was borderline elevated at 116 mg/dL (normal range, 70 to 110 mg/dL). The following serologic values were negative or normal: C3, C4, antinuclear antibody, hepatitis B surface antigen, hepatitis C antibody, and antineutrophil cytoplasmic antibody. The patient was hepatitis B core antibody positive. After the biopsy, adefovir was discontinued. At 3.5 months of follow-up, the patient remains dialysis dependent.
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METHODS Standard Renal Biopsy Processing
Single–Renal Tubule PCR
The renal biopsy was processed for light microscopy, immunofluorescence, and electron microscopy using standard techniques. Routine direct immunofluorescence was performed on 3-m cryostat sections of the kidney using polyclonal fluorescein isothiocyanate (FITC)– conjugated antibodies to immunoglobulin (Ig) G, IgM, IgA, C3, C1q, and light chains, fibrinogen, and albumin (Dako, Carpenteria, CA).
Functional Histochemistry for Mitochondrial Respiratory Chain Enzymes Functional histochemical analysis of mitochondrial respiratory chain enzymes was carried out on frozen sections of kidney. Controls consisted of normal kidney and renal biopsy specimens of ischemic acute tubular necrosis and severe toxic acute tubular necrosis secondary to cis-platinum; no controls were HIV positive or receiving other potentially nephrotoxic mediations. Six-micrometer-thick cryostat sections were stained with cytochrome C oxidase (COX) alone, succinate dehydrogenase (SDH) alone, or both in a double-staining procedure, as described elsewhere.6 In brief, SDH reaction solution (pH 7.6) was prepared as follows: 5 mmol/L phosphate buffer (pH 7.4), 5 mmol/L EDTA, 1 mmol/L potassium cyanide, 0.2 mmol/L phenazine methosulfate (PMS), 50 mmol/L succinate, and 1.5 mmol/L nitroblue. In this reaction, nitroblue tetrazolium acts as the electron acceptor and PMS as the intermediate electron donor. COX reaction solution (pH 7.4) was prepared as follows: 5 mmol/L phosphate buffer (pH 7.4), 0.1% 3,3⬘-diaminobenzidine (DAB), 0.1% cytochrome C, 0.02% catalase. In this reaction, DAB acts as electron donor to cytochrome C. Frozen sections were incubated in each solution at 37°C for 30 minutes and 1 hour, respectively. For SDH-COX double staining, the sections were incubated with COX for 30 minutes, followed by SDH for 15 minutes. Sections were then washed with distilled water and mounted in gelatin. SDH positivity is seen as a blue reaction product, whereas COX positivity gives a brown reaction product. In the double-staining procedure, cells with mtDNA dysfunction are observed in blue because of the loss of COX positivity, whereas normal cells are stained blue-brown as a result of the superimposed reactions of SDH and COX.
Immunohistochemistry for Mitochondrial Respiratory Chain Proteins and DNA Immunohistochemical studies were performed on the index case, and all 3 controls (normal kidney, ischemic acute tubular necrosis, and cis-platinum–induced toxic acute tubular necrosis) using antibodies specific for respiratory chain proteins that are encoded by mtDNA and nuclear DNA (nDNA). Serial 4-m-thick paraffin-embedded sections were stained with mouse monoclonal antibody against human COX subunit I (Molecular Probes Inc, Eugene, OR), which is mtDNA encoded; mouse monoclonal antibody against human COX subunit IV (a gift from Dr Bernhard Kadenbach, Phillips Universitat, Marburg, Germany), which is nDNA encoded; and rabbit polyclonal antibody against nDNA-encoded iron sulfur (FeS) subunit of complex III (a gift from Dr Diego Gonzalez-Halphen, Universidad Nacional Autonomade Mexico, Mexico City, Mexico), using an avidin-biotin immunoperoxidase technique, as previously described.6 In addition, immunohistochemical staining for nDNA and mtDNA was performed using mouse monoclonal anti-human DNA (Chemicon International, Temecula, CA).
For single–renal tubule PCR, we adapted the technique of single-fiber PCR designed for individual muscle fibers, as previously described.7 Dissections were performed on 30-mthick frozen sections of the index case that had been double stained by COX and SDH functional histochemical stains. The 30-m-thick segments of tubules, enzymatically COX positive or COX negative, were manually isolated and individually microdissected into an Eppendorf tube, from which total DNA was then extracted.8 Total DNA was measured before PCR, and equal quantities of DNA were amplified for the COX-positive and COX-negative tubules. A 238 – base pair (bp) mtDNA fragment was amplified using the following 2 primers: light strand positions 3116 –3134 and heavy strand positions 3333–3353. Thirty cycles of PCR were performed as follows: denaturation at 94°C for 20 seconds, annealing at 57°C for 20 seconds, and extension at 72°C for 45 seconds, followed by incorporation of [␣-32P]deoxyadenosine triphosphate in the final cycle. After sodium dodecyl sulfate–polyacrylamide gel electrophoresis, the PCR amplification products were assessed semiquantitatively by measuring their respective radioactivity using PhosphoImager (Bio-Rad Laboratories, Hercules, CA).
RESULTS Light Microscopy and Immunofluorescence The renal biopsy included 2 cores of renal cortex and medulla containing 35 glomeruli. Glomeruli appeared histologically normal. The cortical tubules revealed severe and diffuse degenerative changes primarily affecting the proximal nephron (Fig 1A). There was widespread tubular epithelial simplification with luminal ectasia, loss of brush border, cytoplasmic vacuolization, focal multinucleation, apoptosis, and enlarged regenerative nuclei with nucleoli and focal mitotic figures (Fig 1B). Some tubular lumina contained desquamated cellular debris. The interstitium was mildly and diffusely expanded by edema, early fibrosis, and a mild interstitial infiltrate consisting predominantly of lymphocytes and sparse neutrophils, without associated tubulitis. There was no significant tubular atrophy. No tubular microcysts typical of HIV-associated nephropathy and no tubular crystals typical of indinavir toxicity were identified. By immunofluorescence, there was no glomerular or tubulointerstitial staining for IgG, IgM, IgA, C3, C1, , , fibrinogen, or albumin. Electron Microscopy The glomeruli showed no ultrastructural abnormalities and had intact foot processes. The proximal tubules showed widespread and severe alterations including swollen or flattened epithelium with loss or attenuation of brush border, apical blebbing, increased number of phagolysosomes, dilatation of endoplasmic reticulum, sloughing of cytoplasmic fragments into the tubular lumen, and focal apoptosis (Fig 2A). The proximal tubules had striking abnormalities of mitochondrial size, shape, and substructure. The number of tubular mitochondria was not obviously reduced. How-
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FIGURE 1. Light microscopy. (A) Low-power view of the cortex showing diffuse interstitial edema, mild fibrosis, and irregular tubular profiles with dilated lumina and epithelial simplification. The glomeruli are unaffected. (Hematoxylin and eosin; original magnification ⫻80.) (B) High-power view of the proximal tubules shows cytoplasmic vacuolization, enlarged nuclei with nucleoli, and focal apoptotic bodies. There is a sparse interstitial infiltrate without tubulitis. (Hematoxylin and eosin; original magnification ⫻250.)
FIGURE 3. Functional histochemistry for mitochondrial enzymes. (A, C, E) Controls with ischemic and toxic acute tubular necrosis. (B, D, F) Adefovir-induced acute tubular necrosis. The control with toxic acute tubular necrosis has diffuse retention of (A) COX activity and (C) SDH activity. (E) The control with ischemic acute tubular necrosis gives a blue-brown reaction product on double staining for COX and SDH. In the adefovir-induced acute tubular necrosis, there is (B) widespread loss of COX activity and (D) diffuse retention of SDH. (F) Double staining reveals a mosaic pattern with pure blue staining of some cells and blue-brown staining of others. (Original magnifications: A, B ⫻250; C, D ⫻100; E, F ⫻250.)
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FIGURE 2. Electron microscopy. (A) The proximal tubules have apical blebbing with extensive loss of brush border, overhanging intercellular contacts, and binucleation. The mitochondria are densely packed, enlarged, and irregular in shape. (Original magnification ⫻2,000.) (B) On high-power magnification, the mitochondria appear extremely dysmorphic with extensive loss, blunting, and focal clumping of cristae. (Original magnification ⫻10,000.)
ever, many mitochondria were enlarged and dysmorphic, with convoluted contours. The cristae were frequently lost or blunted with focal clumping at the periphery of the mitochondrial inner membrane, producing tight linear arrays (Fig 2B). Functional Histochemistry for Mitochondrial Respiratory Chain Enzymes The controls with ischemic and toxic acute tubular necrosis had preservation of both COX and SDH enzyme activity, despite diffuse tubular injury at the his-
tologic level (Fig 3A, 3C). In the case of adefovirinduced acute tubular necrosis, there was patchy loss of COX enzymatic activity in approximately 50% of proximal tubules (Fig 3B). In contast, SDH enzymatic activity was retained throughout the cortical tubules (Fig 3D). In normal kidney and both acute tubular necrosis controls, double staining for both COX and SDH showed a normal blue-brown reaction product (Fig 3E). In the adefovir case, double staining for both COX and SDH showed a mosaic pattern of blue-brown alternating with pure blue, consistent with focal loss of COX enzymatic activity (Fig 3F). A mosaic pattern was some-
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times observed within a single tubular profile, indicating selective COX loss from individual tubular cells. Immunohistochemistry for Mitochondrial Respiratory Chain Proteins and DNA In normal kidney and the 2 controls with acute tubular necrosis, immunoreactivity against both subunit I and subunit IV of COX was identified diffusely in renal tubular cells (Fig 4A, 4C). In contrast, in the adefovir-induced acute tubular necrosis, the immunoreactivity for subunit I of COX was focally reduced or absent in renal tubular epithelial cells, whereas that for subunit IV was well preserved (Fig 4B, 4D). Immunohistochemistry for FeS (nDNA encoded) showed simi-
lar diffuse renal tubular positivity in the index case and all 3 controls (data not shown). In all 3 controls, staining with anti-DNA antibody showed diffuse intense nuclear positivity and diffuse but weaker cytoplasmic positivity corresponding to a mitochondrial distribution throughout the cortical tubules (Fig 4E). However, in the adefovir case, the cytoplasmic staining of renal tubular cells was reduced overall and focally absent despite retention of intense nuclear staining throughout the tubular epithelium (Fig 4F). Single–Renal Tubule PCR In the adefovir case, COX-negative renal tubules had markedly reduced mtDNA content compared with
FIGURE 4. Immunohistochemical analysis of mitochondrial proteins and DNA. (A, C, E) Control with cis-platinum–induced toxic acute tubular necrosis. (B, D, F) Adefovir-induced acute tubular necrosis. There is (A) diffuse retention of immunoreactivity for COX subunit 1 in the toxic ATN control but (B) widespread loss in adefovir nephrotoxicity. (C, D) Both cases have diffuse retention of COX subunit IV. Immunostaining for DNA shows (E) both nuclear and cytoplasmic positivity, corresponding to mtDNA, in the toxic ATN control and (F) widespread loss of cytoplasmic positivity with retention of nuclear positivity in adefovir nephrotoxicity. (Original magnifications: A-D ⫻250; E, F ⫻400.)
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FIGURE 5. Single-tubule PCR for mtDNA in adefovir-induced acute tubular necrosis. Lanes 1 and 2 represent enzymatically COX-positive tubules, and lanes 4 through 7 represent enzymatically COX-negative tubules microdissected after double labeling for COX and SDH. The PCR products from COX-negative single renal tubules have markedly reduced mtDNA content compared with COX-positive tubules. The size of PCR products is 238 bp (lane 3, molecular marker 100-bp ladder).
COX-positive renal tubules (Fig 5) and compared with tubules from normal kidney controls (data not shown). The level of reduction of mtDNA ranged from 35% to 64% by PhosphoImager analysis. DISCUSSION Modern antiretroviral regimens are designed to target multiple critical steps in the life cycle of HIV.9 A combination of a reverse transcriptase inhibitor (preventing transcription of viral RNA to proviral DNA) and protease inhibitor (preventing the cleavage of viral precursor polypeptides) is commonly used. Adefovir, 9-(2-phosphonomethoxyethyl) adenine, is a nucleotide monophosphate analogue that competes as substrate for reverse transcriptase.2 When integrated into the nascent proviral DNA chain, it causes premature DNA chain termination by blocking incorporation of the subsequent nucleotide. In contrast to better-known nucleoside analogues such as zidovudine, it does not require intracellular monophosphorylation and has a long intracellular half-life, requiring only a single daily administration.1 Its higher affinity for viral reverse transcriptase than cellular DNA polymerase has made it a promising new agent.10 Adefovir dipivoxil (ADV) is a modification of the same drug with additional moieties to improve its bioavailability on oral intake. In a multicenter randomized placebo-controlled study, patients who received ADV had a significant reduction in plasma HIV RNA copies compared with
those in the placebo group.3 Response was seen even in patients resistant to nucleoside analogues zidovudine and lamivudine. Common side effects of ADV included gastrointestinal disturbances such as diarrhea, nausea, vomiting, and dyspepsia, as well as elevated hepatic transaminase levels. The nephrotoxicity of ADV manifests as proximal tubular dysfunction, including elevated serum creatinine and Fanconi syndrome with phosphate wasting.3 Proximal tubular toxicity was documented in 22% to 32% of patients treated with ADV for 48 weeks.3,11 The major route of elimination of ADV is through the kidneys, which may predispose to nephrotoxicity.12 Unfortunately, the plasma levels cannot be used to monitor those at risk for nephrotoxicity because plasma levels do not correlate with intracellular concentrations of the active metabolites.12 The mechanism of renal tubular toxicity is unclear. A recent study implicated an anion transporter-mediated mechanism.13 Our patient had acute renal failure as well as glycosuria and reduced serum bicarbonate, consistent with partial Fanconi syndrome. The histologic appearance of the toxic acute tubular necrosis was typical of that observed for ADV, and there was no evidence of crystalluria to implicate indinavir toxicity. However, because our patient was receiving multiple antiretroviral drugs, including another nucleoside reverse transcriptase inhibitor, stavudine, we cannot exclude the possibility that 1 or more of these agents contributed to the tubular toxicity. The dysmorphic mitochondria seen ultrastructurally throughout the proximal tubules prompted us to investigate mitochondrial enzyme activity and DNA content. Our findings strongly support nephrotoxicity mediated by mtDNA depletion. Mitochondria have their own DNA (mtDNA), which encodes 13 structural proteins or enzymes, 2 ribosomal RNAs, and 22 transfer RNAs. Other mitochondrial structural proteins and enzymes involved in mtDNA replication and repair are encoded by nDNA. Adenosine triphosphate is produced in the mitochondria by the oxidative phosphorylation machinery involving 5 respiratory complexes. Complex II (SDH) of the respiratory chain is entirely encoded by nDNA. The other 4 complexes are encoded partially by mtDNA and partially by nDNA. FeS subunit, belonging to complex III, is encoded by nDNA. Complex IV (COX) of the respiratory chain is composed of 13 subunits. The 3 largest subunits (COX I, II, and III) are encoded entirely by mtDNA and are responsible for the catalytic and proton-pumping activities of the enzyme. The remaining 10 subunits are encoded by nDNA. By staining for SDH, FeS subunit, COX subunit I, and COX subunit IV enzymes, we were able to demonstrate the selective depletion of mtDNA-encoded proteins and enzymatic function, with no identifiable loss of nDNAencoded mitochondrial proteins. These findings support that ADV nephrotoxicity involves depletion of mtDNA. The mtDNA depletion could not be ascribed to a nonspecific effect of acute tubular necrosis because the controls with ischemic and toxic acute tubular ne-
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crosis had retention of mtDNA-encoded proteins and enzyme activity despite a similar severity of tubular damage. Because of limited tissue, Southern blot hybridization could not be performed to confirm the mtDNA depletion. Instead, we used a single-tubule PCR method to quantitate the mtDNA content in affected and unaffected tubules. The method was adapted from the single-fiber PCR technique designed to demonstrate mtDNA deletions in mitochondrial myopathies by quantitating the relative amounts of wild-type and mutant mtDNA in individual muscle fibers.7 Application of single-tubule PCR to the case of adefovir nephrotoxicity showed that the amount of mtDNA in the affected tubules was 35% to 64% of that in unaffected tubules and tubules of normal kidney control. These findings support nephrotoxicity through inhibition of proximal tubular oxidative phosphorylation by impairment of mtDNA replication. Mitochondrial abnormalities caused by other antiretroviral agents have been described previously. Zidovudine, a nucleoside analogue, induces a reversible myopathy with reduced mtDNA in muscle fibers.5,14 The cellular pattern of mtDNA depletion is strikingly similar to that observed in the kidney of adefovir-induced acute tubular necrosis. No renal toxicity has been described secondary to zidovudine, and conversely no myopathy has been reported in association with adefovir therapy. The factors that underlie this tissue-specific injury are unknown but are not likely to be a function of mitochondrial quantity, which is high in both muscle and proximal tubules. Differential toxicity may relate to tissue-specific effects involving routes of drug elimination, subcellular availability of the nucleoside analogues in the target tissue, and the ability of the triphosphate of the antiretroviral nucleoside analogue to serve as alternative substrate for DNA polymerase ␥.4 The high rate of mtDNA turnover, estimated to be on the order of hours, may potentiate toxicity.15 In conclusion, our findings support that ADV-induced nephrotoxicity involves depletion of mtDNA from proximal tubular epithelium, with resultant impairment of cellular oxidative respiration. The high incidence of toxicity has prompted discontinuation of clinical trials for ADV in the treatment of HIV infection.16 However, because of its broad-spectrum activity against other viruses, such as hepatitis B virus, herpesvirus, and cytomegalovirus, ADV continues to be evaluated in clinical trials.17 Potential nephrotoxicity will probably remain an important determinant of drug safety in future applications of this antiviral agent.
Acknowledgment. The authors thank Dr Eduardo Bonilla for helpful discussions and guidance.
REFERENCES 1. Barditch-Crovo P, Toole J, Hendrix CW, et al: Anti-human immunodeficiency virus (HIV) activity, safety, and pharmacokinetics of adefovir dipivoxil (9-[2-(bis-pivaloyloxymethyl)-phosphonylmethoxyethyl] adenine) in HIV-infected patients. J Inf Dis 176:406-413, 1997 2. Cherrington JM, Allen SJW, Bischofberger N, et al: Kinetic interaction of the diphosphates of 9-(2-phosphonylmethoxyethyl)adenine and other anti-HIV active purine congeners with HIV reverse transcriptase and human DNA polymerases ␣, , and ␥. Antiviral Chem Chemother 6:217-221, 1995 3. Kahn J, Lagakos S, Wulfsohn M, et al: Efficacy and safety of adefovir dipivoxil with antiretroviral therapy; a randomized controlled trial. JAMA 282:2305-2312, 1999 4. Brinkman K, ter Hofstede HJM, Burger DM, et al: Adverse effects of reverse transcriptase inhibitors; mitochondrial toxicity as common pathway. AIDS 12:1735-1744, 1998 5. Arnaudo E, Dalakas M, Shanske S, et al: Depletion of muscle mitochondrial DNA in AIDS patients with zidovudine-induced myopathy. Lancet 337:508-510, 1991 6. Sciacco M, Bonilla E: Cytochemistry and immunohistochemistry of mitochondria in tissue section. Methods Enzymol 264:509521, 1996 7. Sciacco M, Gasparo-Rippa P, Vu TH, et al: Study of mitochondrial DNA depletion in muscle by single-fiber polymerase chain reaction. Muscle Nerve 21:1374-1381, 1998 8. Moraes CT, Schon EA: Detection and analysis of mitochondrial DNA and RNA in muscle by in situ hybridization and single-fiber PCR. Methods Enzymol 264:522-539, 1996 9. Mitsuya H, Yarchoan R, Kageyama S, et al: Targeted therapy of human immunodeficiency virus-related disease. FASEB J 5:23692381, 1991 10. Balzarini J, Hao Z, Herdewijn P, et al: Intracellular metabolism and mechanism of antiretrovirus action of 9-(2-phosphonylmethoxyethyl) adenine, a potent anti-human immunodeficiency virus compound. Proc Natl Acad Sci USA 88:1499-1503, 1991 11. Fisher E, Brosgart C, Cohn D, et al: Safety of adefovir dipivoxil and incidence of proximal renal tubular disorder in a placebocontrolled trial in patients with advanced HIV disease. Programs and Abstracts of the 6th Conference on Retroviruses and Opportunistic Infections, Chicago, IL, 1999 (abstr 678) 12. Cundy KC: Clinical pharmacokinetics of the antiviral nucleotide analogues cidofovir and adefovir. Clin Pharmacokinet 36:127143, 1999 13. Cihlar T, Lin DC, Pritchard JB, et al: The antiviral nucleotide analogs cidofovir and adefovir are novel substrates for human and rat renal organic anion transporter 1. Mol Pharmacol 56:570-580, 1999 14. Pezeshkpour G, Illa I, Dalakas MC: Ultrastructural characteristics and DNA immunohistochemistry in human immunodeficiency virus and zidovudine-associated myopathies. HUM PATHOL 22: 1281-1288, 1991 15. Bogenhagen D, Clayton DA: Mouse L cell mitochondrial DNA molecules are selected randomly for replication throughout the cell cycle. Cell 11:718-727, 1977 16. Mellors JW: Adefovir for the treatment of HIV infection. If not now, when? JAMA 283:2355-2356, 1999 17. Gilson RJ, Chopra KB, Newell AM, et al: A placebo-controlled phase I/II study of adefovir dipivoxil in patients with chronic hepatitis B virus infection. J Viral Hepatol 6:387-395, 1995
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cells, although immunoreactivities for COX subunit IV and iron sulfur subunit of respiratory complex III (both encoded by nDNA) were well preserved in all renal tubular cells. Single–renal tubule polymerase chain reaction revealed marked reduction of mtDNA in COX-immunodeficient renal tubules. We conclude that adefovir-induced nephrotoxicity is mediated by depletion of mtDNA from proximal tubular cells through inhibition of mtDNA replication. This novel form of nephrotoxicity may serve as a prototype for other forms of renal toxicity caused by reverse transcriptase inhibitors. HUM PATHOL 32:734-740. Copyright © 2001 by W.B. Saunders Company Key words: adefovir, nephrotoxicity, mitochondrial DNA, HIV, acute tubular necrosis. Abbreviations: HIV, human immunodeficiency virus; PCR, polymerase chain reaction; COX, cytochrome C oxidase; SDH, succinate dehydrogenase; PMS, phenazine methosulfate; DAB, 3,3– diaminebenzidine; ADV, adefovir dipivoxil.
Adefovir dipivoxil, the acyclic phosphate compound 9-[2-(bis-pivaloyloxymethyl)-phosphonylmethoxyethyl] adenine, is an effective newer antiretroviral agent for the treatment of infection with the human immunodeficiency virus (HIV).1,2 Its pharmacologic action is to serve as substrate for reverse transcriptase, resulting in premature DNA chain termination.2 Its potent antiretroviral activity is balanced by potential nephrotoxicity, manifesting as acute tubular necrosis and Fanconi syndrome.3 The mechanism of nephrotoxicity has not been elucidated. Severe ultrastructural alterations of the proximal tubular mitochondria suggest a potential mitochondrial toxicity. As a nucleotide analogue, adefovir could serve as substrate for DNA polymerase ␥, which is responsible for the replication of mitochondrial DNA (mtDNA), thereby inhibiting mtDNA replication.2,4 A similar mechanism of cellular toxicity has been implicated in the myopathy caused by a related nucleoside analogue, zidovudine.4,5 To elucidate the putative role of altered mtDNA replication in adefovir-induced nephrotoxicity, we investigated a biopsydocumented case using functional histochemistry for mitochondrial respiratory chain enzymes, immunohis-
tochemistry for mitochondrial respiratory chain proteins and DNA, and single-tubule polymerase chain reaction (PCR) for quantification of mtDNA.
From the Departments of Pathology and Neurology, Columbia University, College of Physicians and Surgeons, New York, NY; and the Department of Medicine, Mountainside Hospital, Montclair, NJ. Accepted for publication March 16, 2001. Address correspondence and reprint requests to Vivette D. D’Agati, MD, Department of Pathology, Columbia University, College of Physicians and Surgeons, 630 West 168th St, New York, NY 10032. Copyright © 2001 by W.B. Saunders Company 0046-8177/01/3207-0010$35.00/0 doi:10.1053/hupa.2001.25586
CASE REPORT A 38-year-old white homosexual man with HIV infection first documented in 1990 but no history of opportunistic infections presented with acute renal failure. Seven months earlier, he had been placed on a regimen of hydroxyurea 500 mg twice daily, stavudine 40 mg twice daily, and adefovir dipivoxil 60 mg daily. At that time, the patient had a CD4 T-cell count of 352 cells/L whole blood, HIV viral load of 3,553 copies/mL, and serum creatinine of 1.0 mg/dL. Three months later, indinavir 400 mg twice daily and ritonavir 200 mg twice daily were added. At the time of presentation with acute renal failure, the patient had a CD4 T-cell count of 363 cells/L whole blood. Urinalysis showed proteinuria, glycosuria, 0 to 2 white blood cells (WBCs) per high-power field (hpf) and 5 to 10 red blood cells (RBCs)/hpf. Laboratory evaluation revealed serum creatinine, 7.6 mg/dL; creatinine clearance, 13 cm3/min; serum albumin, 2.8 g/dL; serum bicarbonate, 19 mmol/L (normal range, 22 to 32 mmol/L); 24-hour urinary protein, 2.5 g/d; and platelet count, 83,000/ mm3. Serum phosphorus and uric acid levels were in the normal range, and serum glucose was borderline elevated at 116 mg/dL (normal range, 70 to 110 mg/dL). The following serologic values were negative or normal: C3, C4, antinuclear antibody, hepatitis B surface antigen, hepatitis C antibody, and antineutrophil cytoplasmic antibody. The patient was hepatitis B core antibody positive. After the biopsy, adefovir was discontinued. At 3.5 months of follow-up, the patient remains dialysis dependent.
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ADEFOVIR–INDUCED NEPHROTOXICITY (Tanji et al)
METHODS Standard Renal Biopsy Processing
Single–Renal Tubule PCR
The renal biopsy was processed for light microscopy, immunofluorescence, and electron microscopy using standard techniques. Routine direct immunofluorescence was performed on 3-m cryostat sections of the kidney using polyclonal fluorescein isothiocyanate (FITC)– conjugated antibodies to immunoglobulin (Ig) G, IgM, IgA, C3, C1q, and light chains, fibrinogen, and albumin (Dako, Carpenteria, CA).
Functional Histochemistry for Mitochondrial Respiratory Chain Enzymes Functional histochemical analysis of mitochondrial respiratory chain enzymes was carried out on frozen sections of kidney. Controls consisted of normal kidney and renal biopsy specimens of ischemic acute tubular necrosis and severe toxic acute tubular necrosis secondary to cis-platinum; no controls were HIV positive or receiving other potentially nephrotoxic mediations. Six-micrometer-thick cryostat sections were stained with cytochrome C oxidase (COX) alone, succinate dehydrogenase (SDH) alone, or both in a double-staining procedure, as described elsewhere.6 In brief, SDH reaction solution (pH 7.6) was prepared as follows: 5 mmol/L phosphate buffer (pH 7.4), 5 mmol/L EDTA, 1 mmol/L potassium cyanide, 0.2 mmol/L phenazine methosulfate (PMS), 50 mmol/L succinate, and 1.5 mmol/L nitroblue. In this reaction, nitroblue tetrazolium acts as the electron acceptor and PMS as the intermediate electron donor. COX reaction solution (pH 7.4) was prepared as follows: 5 mmol/L phosphate buffer (pH 7.4), 0.1% 3,3⬘-diaminobenzidine (DAB), 0.1% cytochrome C, 0.02% catalase. In this reaction, DAB acts as electron donor to cytochrome C. Frozen sections were incubated in each solution at 37°C for 30 minutes and 1 hour, respectively. For SDH-COX double staining, the sections were incubated with COX for 30 minutes, followed by SDH for 15 minutes. Sections were then washed with distilled water and mounted in gelatin. SDH positivity is seen as a blue reaction product, whereas COX positivity gives a brown reaction product. In the double-staining procedure, cells with mtDNA dysfunction are observed in blue because of the loss of COX positivity, whereas normal cells are stained blue-brown as a result of the superimposed reactions of SDH and COX.
Immunohistochemistry for Mitochondrial Respiratory Chain Proteins and DNA Immunohistochemical studies were performed on the index case, and all 3 controls (normal kidney, ischemic acute tubular necrosis, and cis-platinum–induced toxic acute tubular necrosis) using antibodies specific for respiratory chain proteins that are encoded by mtDNA and nuclear DNA (nDNA). Serial 4-m-thick paraffin-embedded sections were stained with mouse monoclonal antibody against human COX subunit I (Molecular Probes Inc, Eugene, OR), which is mtDNA encoded; mouse monoclonal antibody against human COX subunit IV (a gift from Dr Bernhard Kadenbach, Phillips Universitat, Marburg, Germany), which is nDNA encoded; and rabbit polyclonal antibody against nDNA-encoded iron sulfur (FeS) subunit of complex III (a gift from Dr Diego Gonzalez-Halphen, Universidad Nacional Autonomade Mexico, Mexico City, Mexico), using an avidin-biotin immunoperoxidase technique, as previously described.6 In addition, immunohistochemical staining for nDNA and mtDNA was performed using mouse monoclonal anti-human DNA (Chemicon International, Temecula, CA).
For single–renal tubule PCR, we adapted the technique of single-fiber PCR designed for individual muscle fibers, as previously described.7 Dissections were performed on 30-mthick frozen sections of the index case that had been double stained by COX and SDH functional histochemical stains. The 30-m-thick segments of tubules, enzymatically COX positive or COX negative, were manually isolated and individually microdissected into an Eppendorf tube, from which total DNA was then extracted.8 Total DNA was measured before PCR, and equal quantities of DNA were amplified for the COX-positive and COX-negative tubules. A 238 – base pair (bp) mtDNA fragment was amplified using the following 2 primers: light strand positions 3116 –3134 and heavy strand positions 3333–3353. Thirty cycles of PCR were performed as follows: denaturation at 94°C for 20 seconds, annealing at 57°C for 20 seconds, and extension at 72°C for 45 seconds, followed by incorporation of [␣-32P]deoxyadenosine triphosphate in the final cycle. After sodium dodecyl sulfate–polyacrylamide gel electrophoresis, the PCR amplification products were assessed semiquantitatively by measuring their respective radioactivity using PhosphoImager (Bio-Rad Laboratories, Hercules, CA).
RESULTS Light Microscopy and Immunofluorescence The renal biopsy included 2 cores of renal cortex and medulla containing 35 glomeruli. Glomeruli appeared histologically normal. The cortical tubules revealed severe and diffuse degenerative changes primarily affecting the proximal nephron (Fig 1A). There was widespread tubular epithelial simplification with luminal ectasia, loss of brush border, cytoplasmic vacuolization, focal multinucleation, apoptosis, and enlarged regenerative nuclei with nucleoli and focal mitotic figures (Fig 1B). Some tubular lumina contained desquamated cellular debris. The interstitium was mildly and diffusely expanded by edema, early fibrosis, and a mild interstitial infiltrate consisting predominantly of lymphocytes and sparse neutrophils, without associated tubulitis. There was no significant tubular atrophy. No tubular microcysts typical of HIV-associated nephropathy and no tubular crystals typical of indinavir toxicity were identified. By immunofluorescence, there was no glomerular or tubulointerstitial staining for IgG, IgM, IgA, C3, C1, , , fibrinogen, or albumin. Electron Microscopy The glomeruli showed no ultrastructural abnormalities and had intact foot processes. The proximal tubules showed widespread and severe alterations including swollen or flattened epithelium with loss or attenuation of brush border, apical blebbing, increased number of phagolysosomes, dilatation of endoplasmic reticulum, sloughing of cytoplasmic fragments into the tubular lumen, and focal apoptosis (Fig 2A). The proximal tubules had striking abnormalities of mitochondrial size, shape, and substructure. The number of tubular mitochondria was not obviously reduced. How-
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FIGURE 1. Light microscopy. (A) Low-power view of the cortex showing diffuse interstitial edema, mild fibrosis, and irregular tubular profiles with dilated lumina and epithelial simplification. The glomeruli are unaffected. (Hematoxylin and eosin; original magnification ⫻80.) (B) High-power view of the proximal tubules shows cytoplasmic vacuolization, enlarged nuclei with nucleoli, and focal apoptotic bodies. There is a sparse interstitial infiltrate without tubulitis. (Hematoxylin and eosin; original magnification ⫻250.)
FIGURE 3. Functional histochemistry for mitochondrial enzymes. (A, C, E) Controls with ischemic and toxic acute tubular necrosis. (B, D, F) Adefovir-induced acute tubular necrosis. The control with toxic acute tubular necrosis has diffuse retention of (A) COX activity and (C) SDH activity. (E) The control with ischemic acute tubular necrosis gives a blue-brown reaction product on double staining for COX and SDH. In the adefovir-induced acute tubular necrosis, there is (B) widespread loss of COX activity and (D) diffuse retention of SDH. (F) Double staining reveals a mosaic pattern with pure blue staining of some cells and blue-brown staining of others. (Original magnifications: A, B ⫻250; C, D ⫻100; E, F ⫻250.)
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FIGURE 2. Electron microscopy. (A) The proximal tubules have apical blebbing with extensive loss of brush border, overhanging intercellular contacts, and binucleation. The mitochondria are densely packed, enlarged, and irregular in shape. (Original magnification ⫻2,000.) (B) On high-power magnification, the mitochondria appear extremely dysmorphic with extensive loss, blunting, and focal clumping of cristae. (Original magnification ⫻10,000.)
ever, many mitochondria were enlarged and dysmorphic, with convoluted contours. The cristae were frequently lost or blunted with focal clumping at the periphery of the mitochondrial inner membrane, producing tight linear arrays (Fig 2B). Functional Histochemistry for Mitochondrial Respiratory Chain Enzymes The controls with ischemic and toxic acute tubular necrosis had preservation of both COX and SDH enzyme activity, despite diffuse tubular injury at the his-
tologic level (Fig 3A, 3C). In the case of adefovirinduced acute tubular necrosis, there was patchy loss of COX enzymatic activity in approximately 50% of proximal tubules (Fig 3B). In contast, SDH enzymatic activity was retained throughout the cortical tubules (Fig 3D). In normal kidney and both acute tubular necrosis controls, double staining for both COX and SDH showed a normal blue-brown reaction product (Fig 3E). In the adefovir case, double staining for both COX and SDH showed a mosaic pattern of blue-brown alternating with pure blue, consistent with focal loss of COX enzymatic activity (Fig 3F). A mosaic pattern was some-
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times observed within a single tubular profile, indicating selective COX loss from individual tubular cells. Immunohistochemistry for Mitochondrial Respiratory Chain Proteins and DNA In normal kidney and the 2 controls with acute tubular necrosis, immunoreactivity against both subunit I and subunit IV of COX was identified diffusely in renal tubular cells (Fig 4A, 4C). In contrast, in the adefovir-induced acute tubular necrosis, the immunoreactivity for subunit I of COX was focally reduced or absent in renal tubular epithelial cells, whereas that for subunit IV was well preserved (Fig 4B, 4D). Immunohistochemistry for FeS (nDNA encoded) showed simi-
lar diffuse renal tubular positivity in the index case and all 3 controls (data not shown). In all 3 controls, staining with anti-DNA antibody showed diffuse intense nuclear positivity and diffuse but weaker cytoplasmic positivity corresponding to a mitochondrial distribution throughout the cortical tubules (Fig 4E). However, in the adefovir case, the cytoplasmic staining of renal tubular cells was reduced overall and focally absent despite retention of intense nuclear staining throughout the tubular epithelium (Fig 4F). Single–Renal Tubule PCR In the adefovir case, COX-negative renal tubules had markedly reduced mtDNA content compared with
FIGURE 4. Immunohistochemical analysis of mitochondrial proteins and DNA. (A, C, E) Control with cis-platinum–induced toxic acute tubular necrosis. (B, D, F) Adefovir-induced acute tubular necrosis. There is (A) diffuse retention of immunoreactivity for COX subunit 1 in the toxic ATN control but (B) widespread loss in adefovir nephrotoxicity. (C, D) Both cases have diffuse retention of COX subunit IV. Immunostaining for DNA shows (E) both nuclear and cytoplasmic positivity, corresponding to mtDNA, in the toxic ATN control and (F) widespread loss of cytoplasmic positivity with retention of nuclear positivity in adefovir nephrotoxicity. (Original magnifications: A-D ⫻250; E, F ⫻400.)
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FIGURE 5. Single-tubule PCR for mtDNA in adefovir-induced acute tubular necrosis. Lanes 1 and 2 represent enzymatically COX-positive tubules, and lanes 4 through 7 represent enzymatically COX-negative tubules microdissected after double labeling for COX and SDH. The PCR products from COX-negative single renal tubules have markedly reduced mtDNA content compared with COX-positive tubules. The size of PCR products is 238 bp (lane 3, molecular marker 100-bp ladder).
COX-positive renal tubules (Fig 5) and compared with tubules from normal kidney controls (data not shown). The level of reduction of mtDNA ranged from 35% to 64% by PhosphoImager analysis. DISCUSSION Modern antiretroviral regimens are designed to target multiple critical steps in the life cycle of HIV.9 A combination of a reverse transcriptase inhibitor (preventing transcription of viral RNA to proviral DNA) and protease inhibitor (preventing the cleavage of viral precursor polypeptides) is commonly used. Adefovir, 9-(2-phosphonomethoxyethyl) adenine, is a nucleotide monophosphate analogue that competes as substrate for reverse transcriptase.2 When integrated into the nascent proviral DNA chain, it causes premature DNA chain termination by blocking incorporation of the subsequent nucleotide. In contrast to better-known nucleoside analogues such as zidovudine, it does not require intracellular monophosphorylation and has a long intracellular half-life, requiring only a single daily administration.1 Its higher affinity for viral reverse transcriptase than cellular DNA polymerase has made it a promising new agent.10 Adefovir dipivoxil (ADV) is a modification of the same drug with additional moieties to improve its bioavailability on oral intake. In a multicenter randomized placebo-controlled study, patients who received ADV had a significant reduction in plasma HIV RNA copies compared with
those in the placebo group.3 Response was seen even in patients resistant to nucleoside analogues zidovudine and lamivudine. Common side effects of ADV included gastrointestinal disturbances such as diarrhea, nausea, vomiting, and dyspepsia, as well as elevated hepatic transaminase levels. The nephrotoxicity of ADV manifests as proximal tubular dysfunction, including elevated serum creatinine and Fanconi syndrome with phosphate wasting.3 Proximal tubular toxicity was documented in 22% to 32% of patients treated with ADV for 48 weeks.3,11 The major route of elimination of ADV is through the kidneys, which may predispose to nephrotoxicity.12 Unfortunately, the plasma levels cannot be used to monitor those at risk for nephrotoxicity because plasma levels do not correlate with intracellular concentrations of the active metabolites.12 The mechanism of renal tubular toxicity is unclear. A recent study implicated an anion transporter-mediated mechanism.13 Our patient had acute renal failure as well as glycosuria and reduced serum bicarbonate, consistent with partial Fanconi syndrome. The histologic appearance of the toxic acute tubular necrosis was typical of that observed for ADV, and there was no evidence of crystalluria to implicate indinavir toxicity. However, because our patient was receiving multiple antiretroviral drugs, including another nucleoside reverse transcriptase inhibitor, stavudine, we cannot exclude the possibility that 1 or more of these agents contributed to the tubular toxicity. The dysmorphic mitochondria seen ultrastructurally throughout the proximal tubules prompted us to investigate mitochondrial enzyme activity and DNA content. Our findings strongly support nephrotoxicity mediated by mtDNA depletion. Mitochondria have their own DNA (mtDNA), which encodes 13 structural proteins or enzymes, 2 ribosomal RNAs, and 22 transfer RNAs. Other mitochondrial structural proteins and enzymes involved in mtDNA replication and repair are encoded by nDNA. Adenosine triphosphate is produced in the mitochondria by the oxidative phosphorylation machinery involving 5 respiratory complexes. Complex II (SDH) of the respiratory chain is entirely encoded by nDNA. The other 4 complexes are encoded partially by mtDNA and partially by nDNA. FeS subunit, belonging to complex III, is encoded by nDNA. Complex IV (COX) of the respiratory chain is composed of 13 subunits. The 3 largest subunits (COX I, II, and III) are encoded entirely by mtDNA and are responsible for the catalytic and proton-pumping activities of the enzyme. The remaining 10 subunits are encoded by nDNA. By staining for SDH, FeS subunit, COX subunit I, and COX subunit IV enzymes, we were able to demonstrate the selective depletion of mtDNA-encoded proteins and enzymatic function, with no identifiable loss of nDNAencoded mitochondrial proteins. These findings support that ADV nephrotoxicity involves depletion of mtDNA. The mtDNA depletion could not be ascribed to a nonspecific effect of acute tubular necrosis because the controls with ischemic and toxic acute tubular ne-
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crosis had retention of mtDNA-encoded proteins and enzyme activity despite a similar severity of tubular damage. Because of limited tissue, Southern blot hybridization could not be performed to confirm the mtDNA depletion. Instead, we used a single-tubule PCR method to quantitate the mtDNA content in affected and unaffected tubules. The method was adapted from the single-fiber PCR technique designed to demonstrate mtDNA deletions in mitochondrial myopathies by quantitating the relative amounts of wild-type and mutant mtDNA in individual muscle fibers.7 Application of single-tubule PCR to the case of adefovir nephrotoxicity showed that the amount of mtDNA in the affected tubules was 35% to 64% of that in unaffected tubules and tubules of normal kidney control. These findings support nephrotoxicity through inhibition of proximal tubular oxidative phosphorylation by impairment of mtDNA replication. Mitochondrial abnormalities caused by other antiretroviral agents have been described previously. Zidovudine, a nucleoside analogue, induces a reversible myopathy with reduced mtDNA in muscle fibers.5,14 The cellular pattern of mtDNA depletion is strikingly similar to that observed in the kidney of adefovir-induced acute tubular necrosis. No renal toxicity has been described secondary to zidovudine, and conversely no myopathy has been reported in association with adefovir therapy. The factors that underlie this tissue-specific injury are unknown but are not likely to be a function of mitochondrial quantity, which is high in both muscle and proximal tubules. Differential toxicity may relate to tissue-specific effects involving routes of drug elimination, subcellular availability of the nucleoside analogues in the target tissue, and the ability of the triphosphate of the antiretroviral nucleoside analogue to serve as alternative substrate for DNA polymerase ␥.4 The high rate of mtDNA turnover, estimated to be on the order of hours, may potentiate toxicity.15 In conclusion, our findings support that ADV-induced nephrotoxicity involves depletion of mtDNA from proximal tubular epithelium, with resultant impairment of cellular oxidative respiration. The high incidence of toxicity has prompted discontinuation of clinical trials for ADV in the treatment of HIV infection.16 However, because of its broad-spectrum activity against other viruses, such as hepatitis B virus, herpesvirus, and cytomegalovirus, ADV continues to be evaluated in clinical trials.17 Potential nephrotoxicity will probably remain an important determinant of drug safety in future applications of this antiviral agent.
Acknowledgment. The authors thank Dr Eduardo Bonilla for helpful discussions and guidance.
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