Annexin A2 mediates apical trafficking of renal Na⁺-K⁺-2Cl⁻ cotransporter.

The furosemide-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) is responsible for urine concentration and helps maintain systemic salt homeostasis. Its activity depends on trafficking to, and insertion into, the apical membrane, as well as on phosphorylation of conserved N-terminal serine and threonine residues. Vasopressin (AVP) signaling via PKA and other kinases activates NKCC2. Association of NKCC2 with lipid rafts facilitates its AVP-induced apical translocation and activation at the surface. Lipid raft microdomains typically serve as platforms for membrane proteins to facilitate their interactions with other proteins, but little is known about partners that interact with NKCC2. Yeast two-hybrid screening identified an interaction between NKCC2 and the cytosolic protein, annexin A2 (AnxA2). Annexins mediate lipid raft-dependent trafficking of transmembrane proteins, including the AVP-regulated water channel, aquaporin 2. Here, we demonstrate that AnxA2, which binds to phospholipids in a Ca(2+)-dependent manner and may organize microdomains, is codistributed with NKCC2 to promote its apical translocation in response to AVP stimulation and low chloride hypotonic stress. NKCC2 and AnxA2 interact in a phosphorylation-dependent manner. Phosphomimetic AnxA2 carrying a mutant phosphoacceptor (AnxA2-Y24D-GFP) enhanced surface expression and raft association of NKCC2 by 5-fold upon low chloride hypotonic stimulation, whereas AnxA2-Y24A-GFP and PKC-dependent AnxA2-S26D-GFP did not. As the AnxA2 effect involved only nonphosphorylated NKCC2, it appears to affect NKCC2 trafficking. Overexpression or knockdown experiments further supported the role of AnxA2 in the apical translocation and surface expression of NKCC2. In summary, this study identifies AnxA2 as a lipid raft-associated trafficking factor for NKCC2 and provides mechanistic insight into the regulation of this essential cotransporter.

Pocket-size hand-held cardiac ultrasound as an adjunct to clinical examination in the hands of medical students and junior doctors.

AIMS: While patient history taking and physical examination remain the cornerstones of patient evaluation in clinical practice, there has been a decline in the accuracy of the latter. Pocket-size hand-held echocardiographic (PHHE) devices have recently been introduced and could potentially improve the diagnostic accuracy of both medical students and junior doctors. The amount of training required to achieve optimal results remains a matter of debate. We hypothesized that the use of PHHE after limited training in the form of a tutorial can improve the clinical diagnosis even in the hands of medical students and inexperienced physicians. METHODS AND RESULTS: Five final-year medical students and three junior doctors without prior echocardiographic experience participated in a standardized 2 h PHHE bedside tutorial. Subsequently, they assessed 122 cardiology patients using history, physical examination, ECG and PHHE. Their final clinical diagnosis was compared against that of a consultant clinician's and also expert in echocardiography. A total of 122 PHHE were performed of which 64 (53%) by final-year medical students and 58 (47%) by junior doctors. Mean ± SD for diagnostic accuracy after history, physical examination, and ECG interpretation was 0.49 ± 0.22 (maximum = 1), whereas the addition of PHHE increased its value to 0.75 ± 0.28 (Z = -7.761, P<0.001). When assessing left ventricular systolic dysfunction by means of history and physical examination, specificity was 84.9% and sensitivity only 25.9%, whereas after including findings from PHHE, these figures rose to 93.6 and 74.1%, respectively. CONCLUSION: The use of PHHE after brief bedside training in the form of a tutorial greatly improved the clinical diagnosis of medical students and junior doctors, over and above history, physical examination, and ECG findings.