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Programme
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Retrieval of urinary albumin by the proximal tubule is achieved by receptor-mediated endocytosis that involves a macromolecular complex that includes megalin/cubulin receptor, the Cl- channel ClC-5, Na-H exchanger isoform 3 (NHE3) and v-H+-ATPase. Defects in this uptake pathway result in increased albumin excretion, albuminuria and eventually nephropathy. Genetic defects in ClC-5 in patients with Dents disease lead to persistent proteinuria. ClC-5 knockout mice also have proteinuria, demonstrating a key role for ClC-5 in this process. ClC-5 is expressed at the apical cell membrane of the proximal tubule and we have been investigating the molecular basis for the role of ClC-5 in albumin uptake. ClC-5 has a large intracellular C-terminus that can potentially act with numerous regulatory proteins. Previously it has been demonstrated that the cell surface expression of ClC-5 can be regulated by an ubiquitin ligase, WWP2. In the current study we showed that Nedd4-2 interacts with ClC-5 and that this interaction is an essential component of constitutive albumin uptake (Hryciw et al., 2004). We also investigated whether serum- and glucocorticoid-inducible kinase (SGK-1) plays a role in this endocytic process.
We first used Glutathione S transferase (GST) fusion pulldowns to show that C-terminus of ClC-5 bound both Nedd4 and Nedd4-2. The Xenopus oocyte expression was then used to show that Nedd4-2 but not Nedd4 reduced ClC-5 currents in a manner that was dependent on an intact proline rich motif containing a tyrosine (PY) in ClC-5. Using luminescence detection of an influenza hemagglutinin-HA-epitope-tagged ClC-5, the decrease in ClC-5 currents was confirmed to be due to a reduction in cell surface levels of ClC-5. Acute exposure of opossum kidney (OK) cells to albumin resulted in a rapid increase in the protein levels of both ClC5 and Nedd4-2 and an increase in proteasome activity. Conversely, inhibition of the proteasome or silencing of endogenous Nedd4-2 in OK cells caused significant decreases in albumin endocytosis. These data indicate that constitutive albumin uptake involves the upregulation of the ubiquitin/proteasome system
In the cortical collecting duct, it is hypothesized that SGK inhibits the action of Nedd4-2 on ENaC. We therefore investigated the role of SGK-1 on albumin uptake in OK cells. Overexpression of wildtype SGK-1 resulted in a significant increase albumin endocytosis 114 ± 3.5%; n = 4; P < 0.05), while overexpression of a kinase dead SGK-1 decreased albumin endocytosis by 81 ± 3%; (n = 4; P < 0.01). These results are consistent with a model in which under normal conditions, the equilibrium between Nedd4-2 is responsible for maintaining normal surface levels of ClC-5. Overexpression of SGK-1 leads to inhibition of Nedd4-2, resulting in more ClC-5 at the cell surface and thereby increasing albumin uptake, with the converse situation arising when with the overexpression of the kinase dead SGK-1. In order to determine whether SGK-1 could alter inhibit the interaction between Nedd4-2 and ClC-5 we then used the Xenopus expression system to determine the effects of SGK-1 on ClC-5 currents. Surprisingly, we found that SGK-1 alone had no effect on the currents carried by ClC-5 and also had no effect on the down regulation of the ClC-5 currents by Nedd4-2.
Our data clearly demonstrate that, similar to the reabsorption of Na+ by the cortical collecting duct, that the constitutive uptake of albumin involves ubiquitin ligases, the proteasome and SGK-1. However, it appears that the effects of SGK-1 do not involve either Nedd4-2 or ClC-5 in OK cells. These data highlight the complex nature of the endocytic process that mediates the retrieval of albumin from the glomerular filtrate.
Hryciw DH, Ekberg J, Lee A, et al. (2004) Journal of Biological Chemistry, 279:54996-5007.