Robert Santoianni

Tenofovir renal toxicity targets mitochondria of renal proximal tubules

Tenofovir disoproxil fumarate (TDF) is an analog of adenosine monophosphate that inhibits HIV reverse transcriptase in HIV/AIDS. Despite its therapeutic success, renal tubular side effects are reported. The mechanisms and targets of tenofovir toxicity were determined using ‘2 × 2’ factorial protocols, and HIV transgenic (TG) and wild-type (WT) littermate mice with or without TDF (5 weeks). A parallel study used didanosine (ddI) instead of TDF. At termination, heart, kidney, and liver samples were retrieved. Mitochondrial DNA (mtDNA) abundance, and histo- and ultrastructural pathology were analyzed. Laser-capture microdissection (LCM) was used to isolate renal proximal tubules for molecular analyses. Tenofovir increased mtDNA abundance in TG whole kidneys, but not in their hearts or livers. In contrast, ddI decreased mtDNA abundance in the livers of WTs and TGs, but had no effect on their hearts or kidneys. Histological analyses of kidneys showed no disruption of glomeruli or proximal tubules with TDF or ddI treatments. Ultrastructural changes in renal proximal tubules from TDF-treated TGs included an increased number and irregular shape of mitochondria with sparse fragmented cristae. LCM-captured renal proximal tubules from TGs showed decreased mtDNA abundance with tenofovir. The results indicate that tenofovir targets mitochondrial toxicity on the renal proximal tubule in an AIDS model.

Antiretroviral nucleosides, deoxynucleotide carrier and mitochondrial DNA: evidence supporting the DNA pol γ hypothesis

Design:Nucleoside reverse transcriptase inhibitors (NRTIs) exhibit mitochondrial toxicity. The mitochondrial deoxynucleotide carrier (DNC) transports nucleotide precursors (or phosphorylated NRTIs) into mitochondria for mitochondrial (mt)DNA replication or inhibition of mtDNA replication by NRTIs. Transgenic mice (TG) expressing human DNC targeted to murine myocardium served to define mitochondrial events from NRTIs in vivo and findings were corroborated by biochemical events in vitro. Methods:Zidovudine (3′-azido-2′,3′-deoxythymidine; ZDV), stavudine (2′, 3′-didehydro-2′, 3′-deoxythymidine; d4T), or lamivudine ((−)-2′-deoxy-3′-thiacytidine; 3TC) were administered individually to TGs and wild-type (WT) littermates (35 days) at human doses with drug-free vehicle as control. Left ventricle (LV) mass was defined echocardiographically, mitochondrial ultrastructural defects were identified by electron microscopy, the abundance of cardiac mtDNA was quantified by real time polymerase chain reaction, and mtDNA-encoded polypeptides were quantified. Results:Untreated TGs exhibited normal LV mass with minor mitochondrial damage. NRTI monotherapy (either d4T or ZDV) increased LV mass in TGs and caused significant mitochondrial destruction. Cardiac mtDNA was depleted in ZDV and d4T-treated TG hearts and mtDNA-encoded polypeptides decreased. Changes were absent in 3TC-treated cohorts. In supportive structural observations from molecular modeling, ZDV demonstrated close contacts with K947 and Y951 in the DNA pol γ active site that were absent in the HIV reverse transcriptase active site. Conclusions:NRTIs deplete mtDNA and polypeptides, cause mitochondrial structural and functional defects in vivo, follow inhibition kinetics with DNA pol γ in vitro, and are corroborated by molecular models. Disrupted pools of nucleotide precursors and inhibition of DNA pol γ by specific NRTIs are mechanistically important in mitochondrial toxicity.

DECREASED mtDNA, OXIDATIVE STRESS, CARDIOMYOPATHY, AND DEATH FROM TRANSGENIC CARDIAC TARGETED HUMAN MUTANT POLYMERASE γ *

POLG is the human gene that encodes the catalytic subunit of DNA polymerase γ (Pol γ), the replicase for human mitochondrial DNA (mtDNA). A POLG Y955C point mutation causes human chronic progressive external ophthalmoplegia (CPEO), a mitochondrial disease with eye muscle weakness and mtDNA defects. Y955C POLG was targeted transgenically (TG) to the murine heart. Survival was determined in four TG (+/−) lines and wild-type (WT) littermates (−/−). Left ventricle (LV) performance (echocardiography and MRI), heart rate (electrocardiography), mtDNA abundance (real time PCR), oxidation of mtDNA (8-OHdG), histopathology and electron microscopy defined the phenotype. Cardiac targeted Y955C POLG yielded a molecular signature of CPEO in the heart with cardiomyopathy (CM), mitochondrial oxidative stress, and premature death. Increased LV cavity size and LV mass, bradycardia, decreased mtDNA, increased 8-OHdG, and cardiac histopathological and mitochondrial EM defects supported and defined the phenotype. This study underscores the pathogenetic role of human mutant POLG and its gene product in mtDNA depletion, mitochondrial oxidative stress, and CM as it relates to the genetic defect in CPEO. The transgenic model pathophysiologically links human mutant Pol γ, mtDNA depletion, and mitochondrial oxidative stress to the mtDNA replication apparatus and to CM.

Survivin Expression in Ovarian Carcinoma: Correlation with Apoptotic Markers and Prognosis

Survivin is a novel inhibitor of apoptosis commonly detected in tissues during fetal development and in cancer, but not usually in normal tissues. Expression of this protein may be of prognostic significance and therapeutically relevant in many cancers. We assessed survivin expression in ovarian carcinoma, correlating results with expression of other anti-apoptotic (bcl-2, bcl-x, mutant p53) and pro-apoptotic (bax) markers, with prognostic parameters, and prognosis. Paraffin-embedded sections of 49 ovarian carcinoma were immunostained for survivin, bcl-2, bcl-x, bax, and p53. Expression was evaluated in nuclei and cytoplasm, as intensity (0–3+), and percentage of positive cells was scored on a four-tiered system with <10% as negative. Frequency of survivin, bcl-2, bcl-x, bax, and p53 was 73.5%, 36.7%, 93.9%, 77.6%, and 60.4%, respectively. There was significant correlation between nuclear survivin expression and grade (P = .0014), histologic type (P = .0376), and mutant p53 (P = .0414). Survivin expression did not correlate with bcl-2, bcl-x, or bax expression, stage, or overall or disease-free survival. The majority (74%) of ovarian carcinoma show survivin expression, which correlates with poor prognostic parameters (high grade, histologic type, p53 mutation) but not with survival. Therapeutic targeting of survivin in ovarian carcinoma is a future possibility.

Tenofovir renal proximal tubular toxicity is regulated By OAT1 and MRP4 transporters

Tenofovir disoproxil fumarate (TDF) is an oral prodrug and acyclic nucleotide analog of adenosine monophosphate that inhibits HIV-1 (HIV) reverse transcriptase. A growing subset of TDF-treated HIV+ individuals presented with acute renal failure, suggesting tenofovir-associated kidney-specific toxicity. Our previous studies using an HIV transgenic mouse model (TG) demonstrated specific changes in renal proximal tubular mitochondrial DNA (mtDNA) abundance. Nucleosides are regulated in biological systems via transport and metabolism in cellular compartments. In this study, the role(s) of organic anion transporter type 1 (OAT1) and multidrug-resistant protein type 4 (MRP4) in transport and regulation of tenofovir in proximal tubules were assessed. Renal toxicity was assessed in kidney tissues from OAT1 knockout (KO) or MRP4 KO compared with wild-type (WT, C57BL/6) mice following treatment with TDF (0.11 mg/day), didanosine (ddI, a related adenosine analog, 0.14 mg/day) or vehicle (0.1 M NaOH) daily gavage for 5 weeks. Laser-capture microdissection (LCM) was used to isolate renal proximal tubules for molecular analyses. mtDNA abundance and ultrastructural pathology were analyzed. mtDNA abundance in whole kidneys from both KO and WT was unchanged regardless of treatment. Renal proximal tubular mtDNA abundance from OAT1 KO also remained unchanged, suggesting prevention of TDF toxicity due to loss of tenofovir transport into proximal tubules. In contrast, renal proximal tubules from MRP4 KO exhibited increased mtDNA abundance following TDF treatment compared with WT littermates, suggesting compensation. Renal proximal tubules from TDF-treated WT and MRP4 KO exhibited increased numbers of irregular mitochondria with sparse, fragmented cristae compared with OAT1 KO. Treatment with ddI had a compensatory effect on mtDNA abundance in OAT1 KO but not in MRP4 KO. Both OAT1 and MRP4 have a direct role in transport and efflux of tenofovir, regulating levels of tenofovir in proximal tubules. Disruption of OAT1 activity prevents tenofovir toxicity but loss of MRP4 can lead to increased renal proximal tubular toxicity. These data help to explain mechanisms of human TDF renal toxicity.

Survivin expression in hepatocellular carcinoma: correlation with proliferation, prognostic parameters, and outcome

Survivin is a novel inhibitor of apoptosis. It is detected in fetal and neoplastic adult tissue, but not in normal tissues. Several recent studies have shown that survivin not only inhibits apoptosis, but also accelerates cancer cell proliferative activity. Expression of the protein may be of prognostic significance and therapeutic relevance in many cancers. We investigated survivin expression in hepatocellular carcinoma, correlating results with proliferation (MIB-1), prognostic factors, and outcome. Paraffin-embedded sections of 72 hepatocellular carcinoma were immunostained for survivin and MIB-1 using tissue microarray technology. Expression was evaluated in nuclei and cytoplasm as intensity (0–3+), and percentage of positive cells scored on a four-tiered system with less than 10%=negative; 10–25%=1; 26–50%=2; 51–75%=3; and 76–100%=4. Frequency of nuclear survivin expression was 43%. There was a significant correlation between nuclear survivin expression and nuclear grade (P=0.0271), microvascular invasion (P=0.0064), mitotic rate (P=0.0017), and MIB-1 (P=0.0001), as well as local recurrence (P=0.0487), and disease-free survival (P=0.0098). Histologic grade (P=0.0544) and stage (P=0.0548) tended to correlate with survivin expression, which did not correlate with cirrhosis, tumor necrosis, multiple tumors, metastatic disease, or overall survival. Survivin expression correlates with poor prognostic parameters (high nuclear and histologic grade, microvascular invasion, increased proliferation (mitotic count, MIB-1)), local recurrence, and shorter disease-free survival, but does not correlate with overall survival. An important role is suggested for survivin in progression, recurrence, and treatment of hepatocellular carcinoma.

Transgenic expression of the deoxynucleotide carrier causes mitochondrial damage that is enhanced by NRTIs for AIDS

Nucleoside reverse transcriptase inhibitors (NRTIs) are antiretrovirals for AIDS with limiting mitochondrial side effects. The mitochondrial deoxynucleotide carrier (DNC) transports phosphorylated nucleosides for mitochondrial DNA replication and can transport phosphorylated NRTIs into mitochondria. Transgenic mice (TG) that exclusively overexpress DNC in the heart tested DNCs role in mitochondrial dysfunction from NRTIs. Two TG lines were created that overexpressed the human DNC gene in murine myocardium. Cardiac and mitochondrial structure and function were examined by magnetic resonance imaging, echocardiography, electrocardiography, transmission electron microscopy, and plasma lactate. Antiretroviral combinations (HAART) that contained NRTIs (stavudine (2′, 3′-didehydro-2′, 3′-deoxythymidine or d4T)/lamivudine/indinavir; or zidovudine (3′ azido-3′-deoxythymidine or AZT)/lamivudine/indinavir; 35 days) were administered to simulate AIDS therapy. In parallel, a HAART combination without NRTIs (nevirapine/efavirenz/indinavir; 35 days) served as an NRTI-sparing, control regimen. Untreated DNC TGs exhibited normal cardiac function but abnormal mitochondrial ultrastructure. HAART that contained NRTIs caused cardiomyopathy in TGs with increased left ventricle mass and volume, heart rate variability, and worse mitochondrial ultrastructural defects. In contrast, treatment with an NRTI-sparing HAART regimen caused no cardiac changes. Data suggest the DNC is integral to mitochondrial homeostasis in vivo and may relate mechanistically to mitochondrial dysfunction in patients treated with HAART regimens that contain NRTIs.

Aggregation of Actin and Cofilin in Identical Twins with Juvenile-Onset Dystonia

The neuropathology of the primary dystonias is not well understood. We examined brains from identical twins with DYT1‐negative, dopa‐unresponsive dystonia. The twins exhibited mild developmental delays until age 12 years when they began developing rapidly progressive generalized dystonia. Genetic, metabolic, and imaging studies ruled out known causes of dystonia. Cognition was subnormal but stable until the last few years. Death occurred at ages 21 and 22 years. The brains were macroscopically unremarkable. Microscopic examination showed unusual glial fibrillary acidic protein–immunoreactive astrocytes in multiple regions and iron accumulation in pallidal and nigral neurons. However, the most striking findings were 1) eosinophilic, rod‐like cytoplasmic inclusions in neocortical and thalamic neurons that were actin depolymerizing factor/cofilin‐immunoreactive but only rarely actin‐positive; and 2) abundant eosinophilic spherical structures in the striatum that were strongly actin‐ and actin depolymerizing factor/cofilin‐positive. Electron microscopy suggested that these structures represent degenerating neurons and processes; the accumulating filaments had the same dimensions as actin microfilaments. To our knowledge, aggregation of actin has not been reported previously as the predominant feature in any neurodegenerative disease. Thus, our findings may shed light on a novel neuropathological change associated with dystonia that may represent a new degenerative mechanism involving actin, a ubiquitous constituent of the cytoskeletal system.

Murine cardiac mtDNA: effects of transgenic manipulation of nucleoside phosphorylation

Mitochondrial toxicity results from pyrimidine nucleoside reverse transcriptase inhibitors (NRTIs) for HIV/AIDS. In the heart, this can deplete mitochondrial (mt) DNA and cause cardiac dysfunction (eg, left ventricle hypertrophy, LVH). Four unique transgenic, cardiac-targeted overexpressors (TGs) were generated to determine their individual impact on native mitochondrial biogenesis and effects of NRTI administration on development of mitochondrial toxicity. TGs included cardiac-specific overexpression of native thymidine kinase 2 (TK2), two pathogenic TK2 mutants (H121N and I212N), and a mutant of mtDNA polymerase, pol-γ (Y955C). Each was treated with antiretrovirals (AZT-HAART, 3 or 10 weeks, zidovudine (AZT) + lamivudine (3TC) + indinavir, or vehicle control). Parameters included left ventricle (LV) performance (echocardiography), LV mtDNA abundance (real-time PCR), and mitochondrial fine structure (electron microscopy, EM) as a function of duration of treatment and presence of TG. mtDNA abundance significantly decreased in Y955C TG, increased in TK2 native and I212N TGs, and was unchanged in H121N TGs at 10 weeks regardless of treatment. Y955C and I212N TGs exhibited LVH during growth irrespective of treatment. Y955C TGs exhibited cardiomyopathy (CM) at 3 and 10 weeks irrespective of treatment, whereas H121N and I212N TGs exhibited CM only after 10 weeks AZT-HAART. EM features were consistent with cardiac dysfunction. mtDNA abundance and cardiac functional changes were related to TG expression of mitochondrially related genes, mutations thereof, and NRTIs.

GATA-4 and GATA-6 expression in human ovarian surface epithelial carcinoma.

GATA-4 and GATA-6 are zinc finger transcription factors named for their recognition motif and involved in ovarian development and function. GATA factors are strongly expressed and primarily localized within the nuclei of ovarian surface epithelial cells. GATA factors have been previously shown to be expressed in sex-cord stromal ovarian tumors and may contribute to the tumor phenotype. Differential expression of GATA-4 within serous and mucinous ovarian carcinomas has been reported. Using immunohistochemistry, we studied GATA-4 and GATA-6 expression in 50 ovarian surface epithelial carcinomas and examined the relationship to clinicopathologic parameters and outcome. We found that the majority of the carcinomas retained GATA-4 expression, whereas approximately two-thirds of the carcinomas had mislocalization or loss of GATA-6 expression. No statistically significant correlations were found between histologic type, histologic grade, or patient outcome and GATA-4. Cytoplasmic GATA-6 expression tended to correlate with overall survival (P=0.0756). These findings suggest that although GATA factors play a role in ovarian surface epithelial carcinoma oncogenesis, they do not seem to affect clinicopathologic parameters.