Reversibility of renal dysfunction after discontinuation of tenofovir.

OBJECTIVES: The association of tenofovir (TDF) with nephrotoxicity has been a controversial issue. Few studies are published regarding the reversibility of renal dysfunction once TDF is discontinued. Studies have yet to be conducted in an urban, non-white patient population, making this one of the largest studies observing a non-white cohort. The objective of this study was to determine whether the decline in creatinine clearance (CrCL) associated with TDF use is reversible once TDF is discontinued in a non-white patient population. METHODS: This single-center, retrospective, chart review was performed at an urban outpatient HIV clinic. Patients who had been switched from tenofovir to zidovudine or abacavir because of a decline in renal function were included. The primary endpoint was the percentage of patients who regained full, moderate, mild, or no recovery of CrCl. RESULTS: Sixty-two patients were included. The mean age was 50 years old, 82% were black, and 66% were males with a mean baseline CrCL of 76 ml/min. Mean difference in CrCL from baseline to 12 months post-TDF was shown to be -11.34 ml/min. After a 1-year follow up period, 37.5% of patients had a full recovery of their baseline CrCL. An additional 41% of patients achieved a moderate recovery (80%-99% of baseline CrCL) and 17.9% patients had a mild recovery (50%-79% of baseline CrCL). Two patients required dialysis. The percent of patients with an undetectable HIV RNA while on a TDF-containing regimen was 67.1% compared with 74.6% on alternative ART. CONCLUSION: Renal dysfunction caused by TDF was fully reversible in 37.5% of patients. Improvement to at least 50% of baseline was seen in 96.4% of patients. Viral suppression was not compromised when patient was switched from TDF to an alternative nucleoside reverse transcriptase inhibitor.

A detergent-free strategy for the reconstitution of active enzyme complexes from native biological membranes into nanoscale discs.

BACKGROUND: The reconstitution of membrane proteins and complexes into nanoscale lipid bilayer structures has contributed significantly to biochemical and biophysical analyses. Current methods for performing such reconstitutions entail an initial detergent-mediated step to solubilize and isolate membrane proteins. Exposure to detergents, however, can destabilize many membrane proteins and result in a loss of function. Amphipathic copolymers have recently been used to stabilize membrane proteins and complexes following suitable detergent extraction. However, the ability of these copolymers to extract proteins directly from native lipid bilayers for subsequent reconstitution and characterization has not been explored. RESULTS: The styrene-maleic acid (SMA) copolymer effectively solubilized membranes of isolated mitochondria and extracted protein complexes. Membrane complexes were reconstituted into polymer-bound nanoscale discs along with endogenous lipids. Using respiratory Complex IV as a model, these particles were shown to maintain the enzymatic activity of multicomponent electron transporting complexes. CONCLUSIONS: We report a novel process for reconstituting fully operational protein complexes directly from cellular membranes into nanoscale lipid bilayers using the SMA copolymer. This facile, single-step strategy obviates the requirement for detergents and yields membrane complexes suitable for structural and functional studies.

The cell-free integration of a polytopic mitochondrial membrane protein into liposomes occurs cotranslationally and in a lipid-dependent manner.

The ADP/ATP Carrier (AAC) is the most abundant transporter of the mitochondrial inner membrane. The central role that this transporter plays in cellular energy production highlights the importance of understanding its structure, function, and the basis of its pathologies. As a means of preparing proteoliposomes for the study of membrane proteins, several groups have explored the use of cell-free translation systems to facilitate membrane protein integration directly into preformed unilamellar vesicles without the use of surfactants. Using AAC as a model, we report for the first time the detergent-free reconstitution of a mitochondrial inner membrane protein into liposomes using a wheat germ-based in vitro translation system. Using a host of independent approaches, we demonstrate the efficient integration of AAC into vesicles with an inner membrane-mimetic lipid composition and, more importantly, that the integrated AAC is functionally active in transport. By adding liposomes at different stages of the translation reaction, we show that this direct integration is obligatorily cotranslational, and by synthesizing stable ribosome-bound nascent chain intermediates, we show that the nascent AAC polypeptide interacts with lipid vesicles while ribosome-bound. Finally, we show that the presence of the phospholipid cardiolipin in the liposomes specifically enhances AAC translation rate as well as the efficiency of vesicle association and integration. In light of these results, the possible mechanisms of liposome-assisted membrane protein integration during cell-free translation are discussed with respect to the mode of integration and the role of specific lipids.