Altered renal proximal tubular endocytosis and histology in mice lacking myosin‐VI

Abstract

Myosin VI (Myo6) is an actin-based molecular motor involved in clathrin-mediated endocytosis that is highly expressed in the renal proximal tubule brush border. We investigated the renal physiological consequences of loss of Myo6 function by performing renal clearance and physiological measurements on Myo6 functional null Snell’s waltzer (sv/sv) and control heterozygous (+/sv) mice. Sv/sv mice showed reduced body weight and elevated blood pressure compared with controls; no differences were observed for glomerular flow rate, urine volume, blood acid-base parameters, and plasma concentrations and urinary excretions of Na+ and K+. To assess the integrity of endocytosis-mediated protein absorption by the kidney, urinary albumin excretion was measured, and the proximal tubular uptake of intravenously injected endocytic marker horseradish peroxidase (HRP) was examined. Albumin excretion was increased nearly 4-fold in sv/sv mice relative to controls. Conversely, HRP uptake was reduced and delayed in proximal tubule cells of the sv/sv kidney observed by electron microscopy at 5 and 30 minutes after injection. Consistent with impaired endocytosis, we also observed defects indicating alterations along the endocytic pathway in sv/sv proximal tubule cells: (1) decreased membrane association of the clathrin adaptor subunit, adaptin beta, and Disabled-2 (Dab2) after sedimentation of renal homogenates and (2) reduced apical vacuole number. In addition, proximal tubular dilation and fibrosis, likely secondary effects of the loss of Myo6, were observed in sv/sv kidneys. These results indicate that Myo6 plays a key role in endocytosis-mediated protein absorption in the mouse kidney proximal tubule.

α-AP-2 Directs Myosin VI-dependent Endocytosis of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels in the Intestine

“…Immunolocalization did not identify labeling for either Dab2 or ␣-AP-2 in the brush border microvillar membranes (except for the overlap of Dab2 at the base of Myo 6 (sv/sv) microvilli) and therefore do not support a role for myosin VI in moving adaptor complexes down the microvilli. Consistent with this finding is the recent observation that the clathrin receptor megalin is also properly localized to the intermicrovillar domain in the proximal tubule of Myo 6 (sv/sv) mice (36). The findings here support a role for myosin VI in 1) securing the intermicrovillar membrane in its normal orientation to promote invagination and coated pit formation and 2) moving the coated vesicle and adaptors away from the plasma membrane to allow internalization of receptors following scission of the invagination.…”

Myosin VI is required for maintenance of brush border structure, composition, and membrane trafficking functions in the intestinal epithelial cell

“…This inter-MV perturbation that can result in MV fusion MV is analogous to that seen in the stereo cilia of the sv/sv hair cell. However, the Myo6 dependent tethering function is not unique to all epithelial cells that express subapical Myo6, since no basal lifting of the BB membrane is observed in the sv/sv kidney proximal tubule epithelial cell [Gotoh et al, 2010]. It is interesting to note that this apparent lifting of the membrane may be dependent on the plus-end directed force exerted on the BB membrane by the major plus-end motor associated with the BB membrane, Myo1a.…”

“…The sv/sv mouse has also been shown to exhibit proteinurea, indicating defects in endocytic uptake of protein from the glomerular filtrate. This was confirmed by demonstrating greatly reduced uptake of lumenal horse radish peroxidase in sv/sv proximal tubules as compared to sv/+ [Gotoh et al, 2010]. …”

Receptor-Mediated Endocytosis in the Proximal Tubule

“…Disruption of F-actin in OK cells using cytochalasins reduced endocytosis of albumin by 50–95% (61, 78). Myosin IIA, myosin VI, and more recently, myosin 9a were implicated in PT endocytosis (79–81), and knockout of Myo9a or myosin VI in mouse models causes LMW proteinuria (79, 81). Megalin interacts with the heavy chain of nonmuscle myosin IIA via its adaptor Dab2, and this interaction appears to be important for endocytosis, as the myosin II inhibitor blebbistatin inhibits lactoferrin uptake in L2 yolk sac cells (80).…”