The lipid composition of biomembranes influences the properties of the lipid bilayer and that of the proteins. In this study, the lipidome and the lipid/protein ratio of membranes from High Five™ insect cells overexpressing mouse P-glycoprotein was characterized. This provides a better understanding of the lipid environment in which P-glycoprotein is embedded, and thus of its functional and structural properties. The relative abundance of the distinct phospholipid classes and their acyl chain composition was characterized. A mass ratio of 0.57 ± 0.11 phospholipids to protein was obtained. Phosphatidylethanolamines are the most abundant phospholipids, followed by phosphatidylcholines. Membranes are also enriched in negatively charged lipids (phosphatidylserines, phosphatidylinositols and phosphatidylglycerols), and contain small amounts of sphingomyelins, ceramides and monoglycosilatedceramides. The most abundant acyl chains are monounsaturated, with significant amounts of saturated chains. The characterization of the phospholipids by HPLC-MS allowed identification of the combination of acyl chains, with palmitoyl-oleoyl being the most representative for all major phospholipid classes except for phosphatidylserines, which are mostly saturated. A mixture of POPE:POPC:POPS in the ratio 45:35:20 is proposed for the preparation of simple representative model membranes. The adequacy of the model membranes was further evaluated by characterizing their surface potential and fluidity.

The transport of drugs by efflux transporters in biomembranes limits their bioavailability and is a major determinant of drug resistance development by cancer cells and pathogens. A large number of chemically dissimilar drugs are transported, and despite extensive studies, the molecular determinants of substrate specificity are still not well understood. In this work, we explore the role of polar and non-polar interactions on the interaction of a homologous series of fluorescent amphiphiles with the efflux transporter P-glycoprotein. The interaction of the amphiphiles with P-glycoprotein is evaluated through effects on ATPase activity, efficiency in inhibition of [¹²⁵I]-IAAP binding, and partition to the whole native membranes containing the transporter. The results were complemented with partition to model membranes with a representative lipid composition, and details on the interactions established were obtained from MD simulations. We show that when the total concentration of amphiphile is considered, the binding parameters obtained are apparent and do not reflect the affinity for P–gp. A new formalism is proposed that includes sequestration of the amphiphiles in the lipid bilayer and the possible binding of several molecules in P–gp’s substrate-binding pocket. The intrinsic binding affinity thus obtained is essentially independent of amphiphile hydrophobicity, highlighting the importance of polar interactions. An increase in the lipophilicity and amphiphilicity led to a more efficient association with the lipid bilayer, which maintains the non-polar groups of the amphiphiles in the bilayer, while the polar groups interact with P–gp’s binding pocket. The presence of several amphiphiles in this orientation is proposed as a mechanism for inhibition of P-pg function.

Objective: To investigate the potential of acute canagliflozin administration to mitigate acute kidney injury (AKI) and attenuate deleterious pro-inflammatory cytokine release in a clinically relevant swine model of severe renal ischemia reperfusion injury (IRI) induced by hemorrhage and aortic occlusion. Background: Long-term canagliflozin use attenuates renal function decline and reduces AKI in diabetes mellitus and heart failure patients. While several reports indicate prophylactic SGLT2 inhibition prevents AKI in IRI, the efficacy of acute administration on IRI and inflammation is not known. Methods: Female swine (n = 16) underwent controlled hemorrhage of 25% blood volume, followed by 90 min of aortic occlusion at the level of the renal ostia (through resuscitative endovascular balloon occlusion of the aorta). A single 300 mg dose of oral canagliflozin or vehicle (saline) was delivered 5 mins into aortic occlusion. Hemodynamic monitoring, markers of renal function (serum creatinine, blood urea nitrogen, proteinuria, and urinary neutrophil gelatinase-associated lipocalin), and serum cytokine concentrations [including interleukins (ILs): IL-1RA, IL-6, IL-8, IL-10, IL-18, and tumor necrosis factor–alpha] were analyzed after IRI, and during a 6-hour critical care phase. Results: Compared with controls, animals receiving canagliflozin had less severe AKI, improved creatinine clearance, reduced proteinuria, and significantly lower tubular damage as evidenced by histopathology and urinary neutrophil gelatinase-associated lipocalin (NGAL). Furthermore, the pro-inflammatory cytokine IL-6 was markedly attenuated without reduction in anti-inflammatory cytokines (IL-1RA and IL-10). Conclusions: A single dose of canagliflozin administered shortly into ischemic insult mitigates AKI and attenuates harmful pro-inflammatory cytokine release after controlled hemorrhage in a swine model. These findings suggest a potential novel therapeutic role for canagliflozin in mitigating the effects of renal IRI worthy of further investigation.