Impairment of endothelial Kir channels in response to elevated glucose is independent of changes in channel expression

Background and Objective. Obesity comprises a complex metabolic pathophysiology often associated with poor glycemic control. Our past studies detailed that impairment of inward rectifying potassium channels (Kir2.1) is a contributing factor to obesity-induced endothelial dysfunction, however, the major obesity-associated factors driving Kir2.1 impairment are unknown. As obesity is often associated with hyperglycemia, we aimed to determine the effect of elevated glucose on endothelial Kir2.1 channel function and expression in vitro. Method. We treated human adipose microvascular endothelial cells (HAMECs) with high glucose (200mg/dL), mannitol (200 mg/dL), or NaCl (300 mOsm/kg) for 72 hours. Control cells were maintained in standard media containing 100 mg/dL glucose. Following treatments, Kir currents were measured using whole-cell patch clamp electrophysiology and Kir2.1 expression was assessed using qPCR, Western immunoblotting, and immunocytochemistry (ICC). ICC was performed on nonpermeabilized HAMECs to detect membrane expression. Statistics: A Kruskal-Wallis one-way analysis of variance (p < 0.05) followed by Dunn Sidak post hoc analysis (*) was used to determine significant differences. Result. HAMECs exposed to elevated glucose exhibited reduced Kir current compared to control. Surprisingly, mannitol induced similar decreases in Kir current. As mannitol originally served as the osmotic control, we next tested the effect of osmotic stress on endothelial Kir current by exposing cells to elevated NaCl, which had no effect. Furthermore, increased glucose or mannitol did not influence channel KCNJ2 gene or total Kir2.1 protein expression, however, preliminary experiments indicate that the membrane expression of Kir2.1 may be increased following exposure to elevated sugars. Discussion and Conclusions. Our findings indicate that glucose and mannitol reduce endothelial Kir currents without affecting channel expression, suggesting that functional impairment of the channels is induced by elevated sugars. This is further supported by preliminary studies that indicate that membrane Kir2.1 expression is increased in response to elevated sugars, suggesting that a possible compensatory trafficking mechanism fails to restore Kir2.1 channel function despite an increased membrane presence. As the observed decreases in Kir channel current are not mediated by an increase in osmotic pressure under these conditions, we aim to next test if damage to the glycocalyx, which is established to occur in obesity and renders endothelial Kir channels dysfunctional, occurs in the presence of elevated sugars. This study was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under award number 2P20GM113125 (Ibra Fancher). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.