Immunofluorescent localization of the Rab-GAP protein TBC1D4 (AS160) in mouse kidney

Abstract

TBC1D4 (or AS160) was identified as a Rab-GTPase activating protein (Rab-GAP) that controls insulin-dependent trafficking of the glucose transporter GLUT4 in skeletal muscle cells and in adipocytes. Recent in vitro cell culture studies suggest that TBC1D4 may also regulate the intracellular trafficking of kidney proteins such as the vasopressin-dependent water channel AQP2, the aldosterone-regulated epithelial sodium channel ENaC, and the Na(+)-K(+)-ATPase. To study the possible role of TBC1D4 in the kidney in vivo, we raised a rabbit polyclonal antibody against TBC1D4 to be used for immunoblotting and immunohistochemical studies. In immunoblots on mouse kidney homogenates, the antibody recognizes specific bands at the expected size of 160 kDa and at lower molecular weights, which are absent in kidneys of TBC1D4 deficient mice. Using a variety of nephron-segment-specific marker proteins, immunohistochemistry reveals TBC1D4 in the cytoplasm of the parietal epithelial cells of Bowman's capsule, the thin and thick limbs of Henle's loop, the distal convoluted tubule, the connecting tubule, and the collecting duct. In the latter, both principal as well as intercalated cells are TBC1D4-positive. Thus, with the exception of the proximal tubule, TBC1D4 is highly expressed along the nephron and the collecting duct, where it may interfere with the intracellular trafficking of many renal transport proteins including AQP2, ENaC and Na(+)-K(+)-ATPase. Hence, TBC1D4 may play an important role for the control of renal ion and water handling and hence for the control of extracellular fluid homeostasis.

Akt Substrate of 160 kD Regulates Na+,K+-ATPase Trafficking in Response to Energy Depletion and Renal Ischemia

“…AS160 is expressed at high levels in the renal DCT. 31 Our data indicate that, in the absence of AS160 expression, the relocalization of sodium pump that occurs in response to renal ischemia followed by 24 hours of reperfusion in cells of the DCT is substantially reduced. The absence of AS160 does not reduce the ischemia-induced internalization of the Na,K-ATPase in PTs, consistent with the lack of detectable AS160 expression in proximal segments.…”

“…The absence of AS160 does not reduce the ischemia-induced internalization of the Na,K-ATPase in PTs, consistent with the lack of detectable AS160 expression in proximal segments. 31 Clearly other mechanisms must be involved in driving the substantial internalization of Na,K-ATPase that occurs in response to ischemic injury in PTs.…”

“…AS160 is expressed predominantly in the distal convoluted tubule (DCT). 31 Kidney sections that exhibited calbindinpositive DCT profiles were selected for imaging of Na,KATPase distribution. In control and sham-treated animals, the Na,K-ATPase is localized to the basolateral infoldings of DCT epithelial cells (Figure 7, A and B).…”

“…Because AS160 is not detectably expressed in proximal tubule (PT) epithelial cells, 31 we anticipated that ischemia-induced internalization of the Na,K-ATPase in PT cells would occur to the same extent in WT and AS160 KO mice. Consistent with this hypothesis, we found that the extent of Na,K-ATPase internalization in response to ischemic injury was not appreciably different in PTs of WT versus AS160 KO animals (Supplemental Figure 3).…”

“…The AS160 KO mice had been generated by targeted deletion of part of exon 1 of the AS160 gene as described by Lier et al 31 The 129/SvEv-C57BL/6 TBC1D4-deficient mice were crossed with C57/BL6 WT mice to generate a mixed 129/SvEv-C57BL/6 background. Eight-week-old male AS160 KO mice or WT littermates weighing 20-25 g were anesthetized with ketamine (100 mg/kg) by intraperitoneal injection and, using aseptic techniques, subjected to a laparotomy only (sham surgery) or a laparotomy with bilateral renal pedicle clamping (ischemia) for 30 minutes.…”